PL190351B1 - Antibodies against human protein being affiliated to parathyroid hormone - Google Patents

Abstract

1. A chimeric chain L, covering region C of chain L of a human antibody and region V of chain L of mouse monoclonal antibody, against a human protein related to parathormone. It is characterised in that region V of chain L covers an amino acid sequence as showed in Sequence ID No. 45. 3. A chimeric chain H, covering region C of chain H of a human antibody and region V of chain H of mouse monoclonal antibody, [acting] against a human protein related to parathormone. It is characterised in that region V of chain H covers an amino acid sequence as showed in Sequence ID No. 46. 4. A chimeric chain H indicated in patent claim 3. It is characterised in that chain Cγ1 constitutes region C.

Description

The present invention relates to a chimeric L chain, a chimeric H chain, a chimeric monoclonal antibody, a polypeptide, a humanized antibody L chain, a humariized H chain, a humanized antibody, DNA, a recombinant vector, a transformant, a method of producing a chimeric antibody, a method of producing a humanized antibody, a pharmaceutical composition, factor.

The present invention relates to human / murine antibodies comprising a variable region (V region) of a murine monoclonal antibody against a parathyroid hormone related protein and a constant region (C region) of a human antibody; a humanized antibody in which the complementarity determining regions have been grafted into the human antibody

190 351 V regions of the light chain (L chain) and heavy chain (H chain) of a murine monoclonal antibody against a parathyroid hormone-related protein, the L and H chains of said antibody, as well as a polypeptide containing the V region forming the H and L chain of said antibody

The present invention also relates to a DNA containing the base sequence encoding the above-mentioned antibody, in particular its V region and DNA encoding the L and H chain containing the V region. The present invention further relates to a recombinant vector comprising said DNAs and a host transformed with said vector.

Further, the present invention relates to processes for the production of chimeric and humanized antibodies against PTHrP. Further, the present invention relates to a pharmaceutical composition and an agent containing an anti-PTHrP antibody as an active ingredient for reducing hypercalcemia or improving the condition of hypophosphatemia.

The basis of the invention

Tumor-associated hypercalcemia is a serious, complicated symptom, occurring in 5 to 20% of all cancer patients, and is considered to be the final symptom of cancer, possibly fatal if left untreated. Controlling hypercalcemia can significantly affect a patient's prognosis and QOL (quality of life); and therefore will play a clinically significant role.

In general, hypercalcemia of malignant neoplastic disease patients is generally classified into HHM (hormonal neoplastic hypercalcemia) based on hormonal tumor agents that induce bone resorption and 10h (local osteolytic hypercalcemia) based on the local action of factors that are transmitted to or infiltrate the bone. In the case of hHm, hypercalcemia is believed to be caused by bone resorption and the breakdown of bone tissue resulting in increased calcium flux combined with a decreased ability of the kidneys to excrete calcium (S. Wada and N. Nagata, Internal Medicine, 69, 644-648)

Hypercalcaemia is considered symptomatic when serum calcium levels exceed 12 mg / dL, non-specific symptoms such as anorexia (lack of appetite), nausea and vomiting seen in the early stages of the disease in cancer patients. When hypercalcemia worsens, a reduction in the concentrating capacity of the urine due to damage to the distal renal tubule leads to excessive urination (polyuria), anorexia and nausea accompanied by dehydration associated with insufficient water intake.

Among the hormonal factors causing malignant hypercalcemia, Moseley J.M. et al. found a parathyroid hormone-related protein (referred to herein as "PTHrP") which is substantially parathyroid hormone (PTH) -like as the humoral inducer of HHM: Proc. Natl.Acad. Sci. USA (1987), 84, 5048-5052.

Subsequently, the gene encoding PTHrP was isolated (Suva LJ et al., Science (1987) 237, 893), and due to the alternative splicing of the gene and the fact that different fragments were present in the blood as a result of the degradation of the total PTHrP molecule, it became clear that there were three human PTHrP species having 139, 141 and 173 amino acids: Baba H. Clinical Calcium (1995) 5, 229-223. In the PTHrP molecule, 8 amino acids of the 13th amino acid N-terminus are identical to PTH, and it can also be concluded that the amino acid site from position 14 to position 34 has a spatial structure similar to PTH; thus, PTH and PTHrP bind to the common PTI ι / PTHrP receptor by at least the N-terminal region; Jueppner H. et al., Science (1991) 254, 1024-1026; Abou-Samra, A-B. et al, Proc. Natl. Acad. Sci. USA (1992) 89,2732-2736.

It has been reported that PTHrP is produced by a variety of neoplastic tissues, and it has become apparent that it is produced not only in tumors, PTHrP is also produced from prenatal to maturity in a variety of normal tissues including skin, central nervous system, uterus, and placenta. , lactating mammary gland, thyroid, parathyroid, adrenal gland, liver, kidney and bladder Burtis WJ., Clin Chem. (1992) 38,2171-2183; Stewart A.F. & Broadus A.E., J. Clin. Endocrinol (1991) 71, 1410-1414. Subsequently, PTHrP is believed to play an important metabolic role

190 351 regulate calcium metabolism, which is kept high from fetal to neonatal period, unlike the mother's body

It is known that PTH / PTHrP receptors are predominantly present in bone and kidney (C. Shigeno, Clinical Calcium (1995) 5, 355-359) and the binding of PTHrP to the receptors activates numerous intracellular signaling systems. One is adenylyl cyclase and the other is phospholipase C. Activation of adenylate cyclase increases the concentration of intracellular cAMP activating protein kinase A. Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphonate, resulting in the formation of inositol 1,4,5-tphosphonate and diacylglycerol. The G protein is involved in these signaling pathways: Coleman, D.T. et al., Biochemical mechanisms of parathyroid hormane action. In "The parathyroids" (Bilezkian J.P et al), Raven Press, New York (1994) p. 239.

Through these signaling pathways, PTHrP induces hypercalcemia, hypophosphataemia as observed in HHM, reduces the renal phosphate uptake capacity, increases renal cAMP secretion, and the like.

Thus, it is becoming clear that PTHrP is closely associated with hypercalcemia in malignant neoplasms. Calcitonin, steroid compounds, indomethacin, inorganic phosphorus salts, bisphosphonates and the like are used in the treatment of tumor-related hypercalcemia, as well as fluid replacement. However, these agents may show a reduction in their effects with continued administration, some serious side effects, or a slowing down of the occurrence of their pharmacological effects; hence, the use of agents or drugs showing better therapeutic effects and fewer side effects is highly anticipated

On the other hand, a new approach to the treatment of cancer-related hypercalcemia reported by Kukreja S.C. et al., is administering to athymic mice implanted with human lung or laryngeal cancer cells with PTHrP neutralizing serum to induce hypercalcemia, which induced a reduction in calcium and urinary cAMP levels J Clin. Invest. (1988) 82, 1798-1802. Kanji Sato et al. report that the administration of nude mice transplanted with a human tumor producing PTHrP with antibodies to PTHiP (1-34) reduces hypercalcemia and significantly prolongs the survival time of the mice: J. Bone & Mine. Res. (1993) 8, 849-860. Further disclosed Japanese Patent Application Publication No. 4-228089 discloses murine / human chimeric antibodies against human PTHrP (1-34). WO 96/22790 also discloses murine / human antibodies but no specific sequence is shown.

Mouse monoclonal antibodies are highly immunogenic (sometimes referred to as "antigenic") in humans, limiting the therapeutic clinical value of murine monoclonal antibodies in humans. For example, a mouse antibody may be metabolized as a foreign substance when administered to humans; thus, the half-life of the murine antibody is relatively short in humans and its expected effects are not sufficiently expressed. Subsequently, the level of human anti-mouse antibodies (HAMA) rising in response to administration of the murine antibody can cause an immune response that is burdensome and dangerous for the patient, such as serum sickness and other allergic reactions. Therefore, murine monoclonal antibodies cannot often be served to people

To overcome these problems, methods are developed to reduce the immunogenicity of non-human antibodies, for example, mouse-derived antibodies. One of these methods is to produce 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. Since the resulting chimeric antibody has the entire variable region of the original murine antibody, it is expected that the antibody is a chimeric antibody will bind to an antigen with the same specificity as the original murine antibody. Subsequently, such a chimeric antibody has a significantly reduced proportion of the amino acid sequence derived from the non-human animal, and thus is expected to have lower immunogenicity as compared to the original murine antibody.

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Although the chimeric antibody binds to an antigen identically to the original murine antibody exhibiting lower immunogenicity, it may elicit some immune responses to the murine variable region. LoBuglio A.F et al., Proc Natl Acad. Sci USA, 86, 4240-4224, 1989

The second way to reduce the immunogenicity of murine antibodies is still more complicated, but a further greater reduction in the potential immunogenicity of murine antibodies is expected. In this method, only the complementarity determining regions (CDRs) of the murine antibody variable region are grafted into the human variable region to "re-engineer" the human variable region. If necessary, a partial amino acid sequence of the CDR supporting framework in the murine antibody variable region can be grafted into human variable region to create a CDR structure in the re-engineered human variable region more similar to that of the original murine antibody.

Then, these humanized, redesigned variable regions are fused to the human constant regions. In the finally redesigned humanized antibody, only the CDRs and very small FR portions are the portions derived from the non-human amino acid sequence. The CDRs are composed of a hypervariable amino acid sequence and do not display any species-specific sequence. Thus, humanized antibodies containing murine CDRs no longer possess greater immunogenicity than naturally occurring human antibodies containing human CDRs.

With regard to humanized antibodies, further reference is made to Riechmann L. et al., Nature, 332, 323-327, 1988; Verhoeye M. et al., Science, 239, 1534-1536, 1988; Kettleborough C.A. et al., Protein Engng., 4, 773-783,1991; Maeda H. et al, Human Antibodies and Hybridoma, 2, 124-134, 1991; Gorman S.D. et al., Proc. Natl. Acad. Sci. USA, 88,4181-4185,1991; Tempest P.r. et al., Bio / Technology, 9, 266-271,1991; What M.S. et al., Proc. Natl Acad. Sci. USA, 88, 2869-2873,1991; Carter P. et al., Proc. Natl Acad Sci. USA, 89, 4285-4289,1992, Co M.S. and m., J. Immunol., 148, 1149-1154, 1992; and Sato K. et al., Cancer Res., 53, 851-856, 1993.

Although, as previously mentioned, humanized antibodies are expected to be useful for therapeutic purposes, no humanized antibodies to PTHrP are known, nor even suggested in the references above. Then there are no standardized means generally applicable to any antibody in the method of making humanized antibodies; various means and methods are required to generate a humanized antibody having sufficient binding and neutralizing activity for a specific antigen: see, for example, Sato, K. et al., Cancer Res, 53, 851-856, 1993.

Disclosure of the Invention

It is an object of the present invention to provide a human / murine antibody comprising the variable region (V region) of a murine monoclonal antibody against PTHrP and a constant region (C region) of a human antibody; a humanized antibody in which the regions determining the complementarity of the light chain (L chain) and heavy chain (H chain) V regions of a mouse anti-PTHrP monoclonal antibody have been grafted into the human antibody; the L and H chains of said antibody; as well as a polypeptide comprising the V region forming the H and L chains of said antibody.

Another object of the present invention is to provide a DNA containing the base sequence encoding the above-mentioned antibody, in particular its V region and DNA encoding the L and H chain containing the V region. Yet another object of the present invention is to provide a recombinant vector comprising said DNA and a host transformed with said vector. It is further an object of the present invention to provide methods for the production of chimeric and humanized anti-PTHrP antibodies. Yet another object of the present invention is to provide an anti-PTHrP antibody having a significant neutralizing activity. Yet another object of the present invention is to provide a pharmaceutical composition and an agent containing as

An anti-PTHrP antibody active ingredient for the reduction of hypercalcemia or the improvement of hypophosphatemia or alkalosis.

As a result of vigorous research intended to achieve the aforementioned ccls, the inventors have succeeded in having an antibody in which the immunogenicity of murine monoclonal antibodies against PTHrP is reduced in humans, and thus the objects of the present invention have been achieved.

The present invention relates to obtaining a chimeric L chain comprising the C region of a human antibody and the V region of a murine monoclonal antibody against PTHrP. The V region of the L chain comprises one that contains one amino acid sequence as shown in Id. Seq. No. 45 and the C region including the CX region.

The present invention relates to obtaining a chimeric H chain comprising the C region of the H chain of a human antibody and the V region of the H chain of the H chain of a murine monoclonal antibody against PTHrP. The V region of the H chain comprises one that contains one amino acid sequence as shown in Id. Seq. No. 46 and region C including the region of Cyl.

Further, 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 comprises a polypeptide comprising the V region of the L chain of a humanized antibody comprising the framework regions 1 to 4 of the V region of the L chain of a human antibody and the complementarity determining regions 1 to 3 of the V region of the L chain of a murine monoclonal antibody against PTHrP. The complementarity determining regions 1 to 3 respectively include those containing the amino acid sequences as shown in Id. Seq. No. 59-61; Framework regions 1 to 3 include those derived from human antibody HSU03868 framework 1 to 3, respectively, and framework 4 including that derived from human antibody S25755 framework 4, or framework regions 1 to 3 including identical regions to corresponding framework regions 1 to 3 of the human antibody HSU03868 and a framework 4 region comprising an identical region to the framework 4 region of the human antibody S25755.

The term "identical" means that the framework regions of a human antibody used in a humanized antibody may have deletions, the replaced and / or added amino acid (s) required to form complementarity determining regions of the murine monoclonal antibody such that the humanized antibody should have an activity equivalent to that of has a mouse monoclonal antibody.

Thus, the present invention relates to a polypeptide comprising the V region of the L chain of a humanized antibody in which, in the framework regions, amino acids 36 and 49 are tyrosine and aspartic acid, respectively as recommended by Kabat (Kabat EA et al, US Dept. Health and Human Services, US Government Printing Offices, 1991).

The present invention also relates to a polypeptide comprising the V region of the L chain of a humanized antibody comprising the amino acid sequence as shown in any one of Id. Seq '. no. 48-51.

Further, the present invention relates to a polypeptide comprising the V region of the L chain of a humanized antibody, wherein in the framework regions amino acids 45 and 87 are, according to Kabat, lysine and isoleucine, respectively.

Further, the present invention relates to a polypeptide comprising the V region of the L chain of a humanized antibody comprising an amino acid sequence as shown in any one of Id. Seq '. no. 52-55

The present invention further relates to a polypeptide comprising the V region of the H chain of a humanized antibody comprising the framework regions 1 and 4 of the V region of the H chain of a human antibody and the complementarity determining regions 1 to 3 of the V region of the H chain of a mouse anti-human PTHrP monoclonal antibody Complementarity regions 1 to 3 include such regions. which suitably contain the amino acid sequences as shown in SEQ ID NO. Nos. 62-64, framework regions 1 to 4 including such

190 351 regions that are derived from human subgroup III framework 1-4 human antibody (Human Subgroup III (HSG IE), Kabat EA et al., US Dept Health and Human Services, US Government Printing Offices, 1991), more in particular those that are derived from the framework regions 1 to 4 of the human antibody S31679, respectively, or those that are identical to the framework regions 1 to 4 of the human antibody S31679, respectively

Also, the present invention relates to a polypeptide comprising the V region of the H chain of a humanized antibody comprising the amino acid sequence as shown in Fig. Seq. no 56

The present invention also relates to a L chain of a humanized anti-PTHrP antibody comprising a polypeptide comprising the V region of the L chain of said humanized antibody and a polypeptide comprising the C region of the L chain of a human antibody. The C region includes the CX region, the framework regions 1 to 3 containing such regions that are identical to the framework 1 to 3 respectively of the human antibody HSU03868, the framework 4 containing the region that is identical to the framework 4 of the human antibody S25755, and the amino acid sequence of the defining regions. a complementarity of 1 to 3 including those represented by Id, respectively. Seq. no. 59-61.

Further, the present invention also relates to the H chain of a humanized anti-PTHrP antibody comprising polypeptides comprising the H chain C region and the H chain V region of said human antibody. The C region comprises the Cyl region, framework regions 1 to 4 containing such regions that are derived from framework regions 1 to 4 derived from a human antibody belonging to HSGIII, complementarity determining regions 1 to 3 which contain the amino acid sequences shown in Id. Seq. no. 62-64

The present invention further relates to a DNA comprising a base sequence encoding the V region of the L chain or the V region of the H chain of a murine monoclonal antibody against human PTHrP. The V region of the L chain and the V region of the H chain comprises those regions which respectively comprise the amino acid sequence shown in Id. Seq. No. 45-46, a DNA comprising the base sequence encoding the V region of the L chain comprising the sequence represented by Id. SEQ ID NO: 65 and a DNA comprising the base sequence encoding the V region of the H chain comprising the sequence represented by Id. Seq. no 57.

According to the invention, the DNA comprises base sequences encoding the polypeptides defined above.

Further, the present invention also relates to DNA encoding said chimeric L and H chains. The DNA encoding said L chain includes, for example, DNA comprising a base sequence as shown in SEQ ID NO. No. 65, and DNA encoding said H chain including the base sequence as shown in Id. Seq. no 57.

Further, the present invention also relates to DNA comprising base sequences encoding the V region of the L chain or the V region of the H chain of said humanized antibody. The DNA comprising the base sequence encoding the V region of the L chain comprises one that comprises the base sequence as shown in either Id. Seq. Nos. 66-74, and a DNA comprising the base sequence encoding the V region of the H chain which comprises the base sequence as represented by Id. Seq. no 58.

The present invention also relates to DNA for the V region of the L chain of a humanized antibody comprising a base sequence encoding an amino acid sequence as shown in any one of Id. Seq. no. 47-55. Said DNA includes the base sequence shown in either of the Seq .IDs. no. 66-74.

Further, the present invention relates to DNA for the V region of the H chain of a humanized antibody encoding an amino acid sequence as shown in Id. SEQ ID NO: 56. Said DNA comprises the base sequence as shown in any one of Id. Seq No. 58.

The present invention relates to a recombinant vector comprising one of the said DNAs.

190 351

The present invention further relates to a host cell transformed with said recombinant vector.

The present invention also relates to a method of producing a chimeric or humanized antibody against a human parathyroid-related protein comprising culturing said transformants and collecting chimeric and humanized antibodies against a human parathyroid-related protein from the resulting culture.

Further, the present invention also relates to a pharmaceutical composition or an agent for reducing hypercalcemia or improving the condition of hypophosphatemia, comprising as an active ingredient said humanized antibody. Elevated blood calcium levels are caused by malignant tumors, and hypophosphatemia is often seen in patients suffering from tumor-associated hypercalcemia. Thus, the antibody of the present invention can be used to treat malignant tumors or to reduce the symptoms of hypercalcemia or hypophosphatemia. Preferably, the hypophosphatemia is vitamin D resistant hypophosphatemia. D. Malignancies include, but are not limited to, at least one selected from the group consisting of cancers of the pancreas, lung, pharynx, larynx, tongue, gingiva, esophagus, stomach, bile ducts, breast, kidney, bladder, uterus and prostate, and malignant lymphoma. The factor for inhibiting hypercalcemia of the present invention can be used in any malignant tumor which causes hypercalcemia.

The present invention will be described in detail below.

1. Production of mouse monoclonal antibodies against PTThP

Murine monoclonal antibodies to PTHpP can be made by preparing hybridomas by cell fusion between sprheak cells and antibody-producing cells derived from animals immunized with antigen, and by selecting clones of the obtained hybridomas that produce antibodies that specifically inhibit PTHrP activity.

(1) Preparation of antigens

The PTHrP used to immunize animals contains peptides having all or part of the amanacid PTHpP sequence produced by recnmbinnwayegn DNA technology or by chemical synthesis, PTHrP is derived from supernatants of hypercalcemic tumor cells. For example, the peptide [PTHrP (1-34) J comprising amino acids 1 to 34 of known PTHzP (Kemp BE et al., Science 91987) 238, 1568-1570) can be included as an antigen. Human PTHrP (1-34) has an amino acid sequence as shown in Fig. Seq. op 75

The resulting PTHpP was combined with a carrier protein such as l ^ zeog ^^^, followed by the addition of an adjuvant. Any aaruwayt can be added, including complete and incomplete Freuyaa aiquantc (2) [.mmuyieation and harvesting of antibody producing cells

The above-formed antigen is administered to mammals such as mice, rats, horses, monkeys, rabbits, goats, or sheep. Immunization can be performed by any of the known methods, including intravenous, subcutaneous, and intraperitoneal injection. Intervals between immunization injections are not particularly defined and may be from several days to several weeks, preferably from 4 to 21 liters.

Two or three days after the final Immunization, antibody producing cells are harvested. Antibody producing cells include spleen, lymph node and peripheral blood cells; splease cells are generally used. The size of a single dose of the antigen used for imaging is 100 µg per mouse.

(3) Determination of the antibody titer

In order to determine the immune napnvreazl level of the animals and to select hybridomas of interest from the cell fused cells, the antibody level in the blood of the immune cells or the antibody titer in the supernatant of the antibody producing cells is measured.

190 351

Methods for determining antibodies are known and include EIA (enzyme immunoassay), RIA (radioimmunoassay), and ELISA (enzyme-linked immunosorbent assay).

(4) Cell fusion

The myeloma cells used for fusion with the antibody producing cells include cell lines from a variety of animals such as mice, rats and humans, and are usually available to those skilled in the art. Cell lines that are useful and used are those that are drug resistant, unable to survive in a selective medium such as HAT in an unfused state, and survive in fused state. 8-azaguanine resistant cell lines are generally used that they do not have hypoxanthine-guanine-phosphoribosyltransferase and cannot grow in hypoxanthine-aminopterin-thymidine (HAT) medium.

Suitable myeloma cells that can be used include a variety of known cell lines such as P3 (P3x63Ag8 653) (J. Immunol. (1979) 123: 1548-1550); P3x63Ag8U 1 (Current Topics in Microbiology and Immunology (1978) 81: 1-7); NS-1 (Kohler G and Milstein C, Eur. J. Immunol. (1976) 6: 511-519); MPC-11 (Margulies D.H. et al., Cell (1976) 8.405-415); SP2 / 0 (Shulman, M. et al., Nature (1978) 276: 269-270), FO (de St. Groth, S.F. et al., J. Immunol. Methods (1980) 35: 1-21); S194 (Trowbridge I.S., J. Exp. Med. (1978) 148.313-323) and R210 (Galfre G et al., Nature (1979) 277: 131-133).

The antibody producing cells may be obtained from spleen cells, lymph node cells, or the like. The spleen, lymph node or similar tissue is extracted or removed from any of the above-mentioned animals and then crushed the tissues. The resulting crushed tissue material is suspended in a medium or buffer such as PBS, DMEM or ROM11640, filtered through a steel sieve or the like, and centrifuged in to obtain the desired antibody producing cells.

Subsequently, said myeloma cells and antibody producing cells were cell fused.

Cell fusion can be performed by bringing the myeloma cells and antibody producing cells into contact at a ratio of 1: 1 to 110 in animal cell culture medium such as MEM, DMEM, or RPME-1640 in the presence of a fusion accelerator at a temperature of 30 ° C. to 37 ° C for 1 to 15 minutes. Any fusion accelerator or virus can be used, such as polyethylene glycol with an average molecular weight of 1000 to 6000, polyvinyl alcohol or Sendai virus to accelerate cell fusion. The fusion of antibody producing cells and myeloma cells can also be performed in a commercial cell fusion apparatus that uses electrical stimulation such as electroporation.

(5) Selection and cloning of hybrids

Hybridomas of interest are selected from cells after cell fusion, e.g. by a method involving selective cell growth on specific media

Thus, the cell suspension is diluted with an appropriate medium and plated on microtiter plates. A selective medium such as HAT medium is added to each well and incubated, during which the selective medium is carefully replaced with a new one

Thus, cultured cells are harvested as hybridomas.

These hybridomas are then screened by limiting dilution, fluorescence activated cell sorter or other methods. Finally, hybridomas that produce monoclonal antibodies are obtained.

(6) Collection of monoclonal antibodies

Methods for harvesting monoclonal antibodies from the resulting hybridomas include conventional cell culture and methods for creating peritoneal exudate.

In a cell culture method, hybridomas are grown in an animal cell culture medium, such as RPMI-1640 medium containing 10 to 20% fetal bovine serum, MEM medium, or serum free medium, under standard conditions (i.e. 37 ° C, 5% CO2). for 2 to 14 days, antibodies are collected from the supernatant.

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In the peritoneal exudate method, hybridomas are implanted intraperitoneally into the same mammalian species as the mammal from which the myeloma cells are derived, so that the hybridomas grow abundantly. After 1 to 4 weeks, the exudative fluid or serum is collected.

When it is necessary to purify the antibodies obtained by these methods, known methods such as ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography are selected or combined.

2. Whoistnunwunc: Chimeric Antibodies (1) Cloning of DNA containing the base sequence encoding the V region of a murine monoclonal antibody against human PTHrP.

(i) Preparation of mRNA

To clone DNA containing a base sequence encoding the V region of a murine monoclonal antibody against PTHrP, the collected hybridomas are treated in a conventional manner, for example, with guanidine ultracentrifugation (Chirgwin JM et al., Biochemistry (1979) 18, 5294-5299) or by the AGPC method (Chomczyński P. et al., Analytical Biochemistry (1987) 162, 156-159) to obtain total RNA from which mRNA is obtained using an Oligo (dT) -cellulose spin column attached to the mRNA purification kit (Pharmacia). The Quick Prep mRNA purification kit (Pharmacia AB) can also be used without the need to extract total RNA.

(u) Preparation and amplification of cDNA

From the mRNA obtained as in (i) above, each cDNA of the V regions of the L and H chains is synthesized using reverse transcriptase. In cDNA synthesis, an Oligo-dt primer or other suitable primer that hybridizes to the C region of the L or H chain, for example, an MHC2 primer having a base sequence as shown in Id. Seq. No. 1.

In cDNA synthesis, said mRNA and primer are mixed and the reaction is performed in the presence of reverse transcriptase at e.g. 52 ° C for 30 minutes.

The cDNA amplification of both L and H chains can be performed by PCR (polymerase chain reaction) based on the 5'-RACE method (Frohman MA et al, Proc. Natl. Acad. Sci USA, 85, 8998-9002, 1988; Belyavsky A. and in., Nucleic Acids Res, 17, 2919-2932, 1989) using the 5'-Ampli FINDER RACE kit (CLONTECH Inc.). Thus, the Ampli FINDER Anchor (SEQ ID NO: 42) is attached to the 5 'end of the cDNA synthesized above and PCR is performed for DNa containing the base sequences encoding the L and H chain V regions (here, DNA containing the base sequence encoding the V region) L chain is sometimes simply referred to simply as "DNA for the V region of the L chain" or DNA encoding the V region of the L chain. This also applies to the V region of the H chain, C region, etc.).

A primer that can be used to amplify the DNA of the L chain V region includes, for example, an anchor primer (SEQ ID NO: 2) and primers made of conserved sequences of the LA chain constant region (C λ region) of murine antibodies, such as an MLC primer having base sequence as shown in Id. Seq. ID NO: 4. A primer that can be used to amplify the DNA of the H chain V region includes, for example, an anchor primer (SEQ ID NO: 2) and an MHC-G1 primer (SEQ ID NO: 3) (ST Jones et al. ., Biotechnology, 9, 88, 1991).

(iii) DNA purification and base sequence determination

The PCR products are subjected to agarose gel electrophoresis according to standard procedures for cutting the DNA fragments of interest, which are then recovered, purified, and ligated to vector DNA.

DNA purification can be performed using commercially available kits such as GENECLEAN II; BIO1O1. A known DNA vector which can be used here for piζχπυΰζ, υιπα 11 agnicniu in unn, j νοι 11α ruau puvi 7 αιυυ ijiucdviipi.

Said DNAs and vector DNA are ligated using a known ligation kit (Takara Shuzo) to obtain a recombinant vector. The resulting recombinant vector is introduced into e.g. Escherich coli JM109 and ampicillin resistant colonies are selected, so that the DNA vector is prepared by known methods: J. Sambrook et al., "Molecular Cłoning", Cold Spring Harbor Laboratory Press, 1989. After digestion vector DNA restriction enzyme (s) by known methods such as the dideoxy method: J. Sambrook

190 351 et al., "Molecular Cloning", Cold Spring Harbor Laboratory Press, 1989, the base sequence of the desired DNA is determined. An automated sequencing device (DNA Sequencer 373A; ABI Inc.) can be used in the present invention.

(iv) Regions determining complementarity

The V regions of the L and H chains form the antigen-binding site and their overall structure shares some similarity with one another. Four parts of their framework regions (FRs) are linked by three hypervariable regions, or complementarity determining regions (CDRs). The amino acid sequence of the FR is relatively well conserved, while the variability of the amino acid sequence of the CDR region is very large: Kabat E.A. et al., "Sequence of Proteins of Immunological Interest," US Dept. Health and Human Services, 1983.

Multiple portions of the four FRs have a β-sheet structure with the result that the three CDRs form a loop. CDRs sometimes have a partial β sheet structure. Thus, the three CDRs are held in very close proximity to each other by the FRs, creating an antigen-binding site including three CDRs in the paired regions.

Accordingly, to find mutual homology, the CDR regions can be found by comparing the amino acid sequences of the variable regions of the mouse anti-PTHrP monoclonal antibody with each other and the antibody amino acid sequence database prepared by Kabat et al. ("Sequence of Proteins of Immunological Mterest" US Dept. Health and Human Services, 1983).

(2) Constructing an antibody expression vector, chimeric

First, the DNA fragments encoding the V regions of the L and H chain of a mouse monoclonal antibody are cloned (here sometimes the L and H chain of an antibody is called "mouse L chain" etc. for murine antibodies and "human H chain" etc. for human antibodies), DNA encoding murine V regions and DNA encoding human antibody constant regions are ligated and expressed to generate chimeric antibodies against human PTHrP.

Typical methods for producing chimeric antibodies involve ligating the mouse leader sequence and V region sequences present in the cloned cDNA to an already existing human antibody C region encoding sequence in an expression vector of a mammalian cell. Alternatively, the murine leader sequence and the V region sequence already present in the cloned cDNA are ligated to the coding sequence of the human antibody C region, followed by ligation into a mammalian cell expression vector.

The polypeptide comprising the C region of a human antibody can be any of the C regions of the L or H chain of a human antibody including, for example, Cyl, Cy2, Cy3 or Cy4 of human H chains or CX or Ck L chains.

To produce a chimeric antibody, first, two expression vectors are constructed, such that an expression vector is constructed containing the DNA encoding the mouse L chain V region and the human L chain C region under control of an expression control region such as an enhancer / promoter system and an expression vector containing DNA Coding the V region of the murine H chain and the C region of the human H chain under the Control of an expression Control region such as an enhancer / promoter system. Subsequently, host cells such as mammalian cells are co-transformed with these expression vectors and the transformed cells are cultured in vitro and in vivo to produce chimeric antibodies; see, for example, WO91 / 16928.

Alternatively, a mouse leader sequence present in the cloned cDNA and the DNA encoding the mouse L chain V region and the human L chain C region as well as the mouse leader sequence present in the cloned / m cDNA and ngn / v encoding DNA is inserted into the single expression vector. ncucKa a, i romn-n C, ι ^ ύΙκιππ Jłńcucłh W

AlUijIU '* · N' »A i-3 A -.AAAW ^ AWAA V AAAJ L> IV £, V HA * XVUvi» UAAA AW ^ AWAA AU »Vk * _j AAA νφ V» 4 ΜΧΧ V U.Vi It * AA (see, for example, WO94 / 11523) and the resulting vector is used to transform host cells; then, the transformed host cells are cultured in vitro and in vivo to produce the desired chimeric antibodies.

(i) Production of the H chain of a chimeric antibody

A vector for expressing the H chain of a chimeric antibody can be obtained by inserting a cDNA containing a base sequence encoding the V region of the mouse H chain

190 351 (also referred to herein as "H chain V region cDNA") to a suitable expression vector containing genomic DNA comprising a base sequence encoding a human H chain C region (also referred to herein as "H chain C region genomic DNA") or cDNA encoding this region (also referred to herein as "H chain C region cDNA"). The H chain C region includes, for example, the Cyl, Cy2, Cy3, or Cy4 regions.

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

Expression vectors having genomic DNA encoding the H chain C region, and particularly those that encode the Cyl region, include, for example, HEF-PMhgy 1 (WO92 / 19759) and DHFR-AE-RVh-PMl-f (WO92 / 19759).

Once the cDNA encoding the murine H chain V region has been inserted into these expression vectors, a suitable base sequence can be inserted into the cDNA by PCR. For example, PCR may be performed using a PCR primer that is designed such that the DNA of interest has a recognition sequence for a suitable restriction enzyme at its 5 'end and a Kozak consensus sequence immediately before the start codon to improve transcription efficiency, as well as a PCR primer. which is designed such that the cDNA in question has a recognition sequence for a useful restriction enzyme at its 3 'end and a binding donor site to properly bind the primary genomic DNA transcription products to allow the mRNA to introduce these appropriate base sequences into the expression vector.

After the constructed cDNA encoding the murine H chain V region is treated with the appropriate restriction enzyme (s), it is inserted into the given expression vector to create a chimeric H chain expression vector containing genomic DNA encoding the H chain C region (Cyl region) .

(i-b) Construction of a chimeric H chain expression vector containing cDNA comprising a base sequence encoding an H chain.

Expression vectors having a cDNA encoding the H chain C region, such as the Cy 1 region, can be constructed as follows: mRNA is prepared from CHO cells into which the DHFR-AE-RWi-PM1-f expression vector has been introduced (see WO92 / 19759) containing DNA encoding the V region of the H chain of humanized PM1 antibody and genomic DNA of the C region of the H chain of human antibody (N Takahashi et al., Cell, 29, 671-679 91982)) and the RV1-PMla expression vector (see WO92 / 19759) including genomic DNA encoding the V region of the L chain of a humanized PM1 antibody and genomic DNA of the C region of the Lk chain of a human antibody; The cDNA encoding the V region of the H chain of the humanized PM1 antibody and the cDNA encoding the C region of the H chain (Cyl) of the human antibody are cloned by RT-PCR and ligated into an animal cell expression vector that has been treated with the appropriate restriction enzyme (s) in to construct the desired expression vector.

When the cDNA encoding the murine H chain V region is directly ligated to a cDNA encoding the C 1 region of the H chain of a human antibody, the corresponding base sequences can be PCR inserted into a fragment containing the cDNA encoding the H chain V region. For example, PCR can be performed using a PCR primer that is designed such that said cDNA sequence has a recognition sequence at the 5 'end for a useful restriction enzyme and a Kozak consensus sequence immediately before the start codon in order to improve transcription efficiency, as well as PCR, which is designed such that the cDNA concerned has a recognition sequence for a useful restriction enzyme restriction enzyme at its 3 'end to ligate directly into the C1 region of the H chain so as to insert these appropriate base sequences into the given cDNA.

Thus, the constructed cDNA encoding the murine H chain V region is treated with the appropriate restriction enzyme (s), ligated to the cDNA encoding the given C C region of the H chain, and incorporated into an expression vector such as pCOS1 or pCHO1 to build the vector an expression cDNA encoding a chimeric H.

190 351 (ii) Production of the L chain of a chimeric antibody

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

(ii-a) Construction of an expression vector containing a cDNA encoding a chimeric LA chain

Once an expression vector containing cDNA encoding the murine L chain V region has been constructed, a suitable base sequence can be inserted into said expression vector by PCR. For example, PCR may be performed using a PCR primer that is designed such that the cDNA in question has a sequence recognized by a useful restriction enzyme at its 5 'end and a Kozak consensus sequence to improve transcription efficiency, as well as a PCR primer that is designed to do so. that the cDNA in question has a recognition sequence for a suitable restriction enzyme at its 3 'end in order to directly ligate the C11 region of the H chain to introduce the appropriate base sequences into said cDNA.

The entire base sequence of the cDNA encoding the C region of the human Ι! Λ chain, can be synthesized using a DNA synthesizer or constructed using the PCR method. It is known that the C region of the human Lλ chain has at least four different isotypes and each isotype can be used to construct an expression vector For example, based on a homology search with the Lλ chain C regions of cloned murine monoclonal antibodies, a Mcg + Ke + Oz isotype expression vector was found and used for a fragment of the human L λ chain C region (accession no. Χ57819) (PDariavach et al. , Proc. Natl. Acad. Sci. USA, 84, 9074-9078, 1987). To build a cDNA such as Mcg + Ke + Oz- of a known C region of the human Τλ chain, the following four primers were designed as shown in Id. Seq '. no. 11-14. The primers MBClHGP1 (SEQ ID NO: 11) and MBClHGP3 (SEQ ID NO: 13) have the sense DNA sequence, and the primers MBC1HGP2 (SEQ ID NO: 12) and MBClHGP4 (SEQ ID NO: 14) have the anti-sense DNA sequence, wherein each primer has from 20 to 23 bp of the complementary sequence at each end thereof.

MBClHGPS (SEQ ID NO: 15) and MBClHGPR (SEQ ID NO: 16) are called external primers, have homologous sequences of zMBCIHGP1 and MBClHGP4, respectively, and each have sequences recognized by the respective restriction enzymes. Four primers were combined by PCR to synthesize the full-length cDNA, and external primers were added to amplify the cDNA. Linking by PCR means that MBClHGP1 and MBClHGP2 or MBClHGP3 and MBClHGP4 are joined by their complementary sequences to synthesize the MBClHGP1-MBClHGP2 fragment and the MBClHGP3-MBClHGP4 fragment and each fragment is re-attached by its complementary CD sequence to complement the CD sequence of the human Lλ chain.

The thus constructed cDNA encoding the C region of the human chain and the above constructed cDNA of the murine L chain V region can be ligated between the appropriate restriction enzyme sites and incorporated into an expression vector such as pCOS1 or pCHO1 to construct an expression vector containing the cDNA encoding the LA chain of the chimeric antibody.

(ii-b) Construction of an expression vector containing a cDNA encoding a chimeric Lk chain.

Once an expression vector containing cDNA encoding the murine L chain V region has been constructed, a suitable base sequence can be inserted into said expression vector by PCR. For example, PCR may be performed using a PCR primer that is designed such that the cDNA in question has a recognition sequence for the appropriate restriction enzyme at its 5 'end and a Kozak consensus sequence to improve transcription efficiency, as well as a PCR primer that is designed to do so. that the cDNA in question has a recognition sequence for it

190 351 of a restriction enzyme at its 3 'end in order to insert these appropriate base sequences into said cDNA.

DNA encoding the human Lk chain C region that can be ligated to DNA encoding the murine L chain V region can be constructed from, for example, HEF-PM1k-gk containing genomic DNA (see WO92 / 19759).

Recognition sequences for the appropriate restriction enzymes can be inserted using a PCR method, into the 5 'and 3' ends of the DNA encoding the Lk chain C region and the DNA encoding the mouse L chain V region constructed as above, and the DNA encoding the Lk chain C region can be ligated between each other and incorporated into an expression vector such as pCOS1 or pCHO1 to construct an expression vector containing the cDNA encoding the Lk chain of the chimeric antibody.

3. Production of humanized antibodies (1) Search for homology with human antibodies.

In order to generate humanized antibodies in which the CDR of the murine monoclonal antibody is grafted into the human antibody, it is desirable to have a high homology between the FR of the murine monoclonal antibody and the FR of the human antibody. Accordingly, the V regions of the L and H chains of the murine monoclonal antibody against PTHrP are compared and the V regions of all known antibodies, the structure of which was determined using the Protein Data Bank. They are further simultaneously compared to the human antibody subgroups (HSG: Human Subgroups) classified by Kabat et al. based on antibody FR lengths, amino acid homology and the like: Kabat E A. et al, US Dep. Health and Human Services, US Government Printing Offices, 1991

The human H chain V regions can be classified into HSG I to III according to the HSG Classification of Kabat et al. and the V regions of the H chain of the murine monoclonal antibody against PTHrP share 82.7% homology with the consensus sequence of HSG III. On the other hand, the V regions of the human LZ chain can be classified into HSG I to IV according to the HSG classification of Kabat et al. and the V regions of the LZ chain of the murine monoclonal antibody against PTHrP do not share high homology with the consistent sequences of the V regions of a human LZ chain belonging to any of the subgroups.

When a murine monoclonal antibody against human PTHrP is humanized, it is desirable to use to build a humanized antibody the V region of the human H chain belonging to HSG III and having the greatest homology, or the V region of a human H chain having an FR structure which corresponds to the structural formula (Chothia C. and in., J Mol. Biol., 196, 901-917, 1987) as the V region of the human H chain. Furthermore, if there is no consensus sequence with a high degree of homology in the subsets of the V regions of the human LZ chain, it is desirable to use it to build a humanized the antibody of the V region of the human LZ chain with the highest degree of homology registered with the Protein Data Bank.

(2) Designing a DNA encoding the V region of a humanized antibody

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

In the present invention, a humanized antibody of the FR of the V region of a human antibody having a homology greater than 80% with the V region of the murine antibody can be used in a humanized antibody. The FR of the H chain V region as a fragment of an identical FR may include FRs derived from those belonging to the HI subgroup such as S31.679: NBRF-PDB, Cusmier AM. et al, Eur J Immunol., 23, 110-118, 1993. Then, the FR of the V region of the L chain as nlpimnum no. n-o rrmonf 11 UgjlllVllU

X X <X- from the human antibody HSU03868 (GEN-BANK, Deftos M. et al., Scand. J. Immunol., 39, 95-103, 1994) and FR4 derived from human antibody S25755 (NBRF-PDB).

The human antibody S31679 was cloned from a human fetal liver cDNA library, while the human antibody HSU03868 was cloned as the new gene for the human L Z chain V region.

190 351 (3) Preparation of polypeptides containing the V region of a humanized antibody

In a humanized antibody of the present invention, the C region and the framework regions (FR) of the V region of a given antibody are derived from humans and the complementarity determining regions (CDRs) of the V region are derived from mice (Figure 1). A polypeptide containing the V region of a humanized antibody of the present invention can be produced by a PCR method in a method called CDR grafting as long as the dNa fragment of the human antibody is available as a template. "CDR grafting" refers to a method by which a DNA fragment encoding a mouse-derived CDR is produced and exchanged with a CDR of a human antibody as a template.

If a DNA fragment of a human antibody cannot be used as a template, the base sequence registered in the database can be synthesized in a DNA synthesizer, and the DNA of the humanized antibody V region can be produced by PCR. Further, as long as only the amino acid sequence is registered in the database, the entire base sequence can be deduced from the amino acid sequence based on the basic knowledge of codon usage in building antibodies as described by Kabat E.A. et al., US Dep. Health and Human Services, US Government Printing Offices, 1991 This base sequence is synthesized in a DNA synthesizer, and the DNA of the humanized antibody V region can be made by PCR and introduced into an appropriate host followed by expression for production of the desired polypeptide.

The general procedures for CDR grafting by PCR are described below, while a human antibody DNA fragment is available as template.

(i) CDR Transplantation

We assume that the DNA encoding the V region comprises DNA encoding FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 which are linked together as shown in Figure 2.

First, mouse-derived DNA fragments corresponding to the respective CDRs are synthesized. CDRs 1 to 3 are synthesized from the base sequence of the previously cloned murine H and L chain V regions. The graft primers B and E are synthesized such that primer B should have a sequence hybridizing to the mouse CDR1 and human FR2 antibody in the sense, and the primer E should have a sequence hybridizing with CDR1 and human FR1 antibody in the antisense direction (Fig. 2 (1)). Similarly, graft primers C and F and primers D and G are synthesized. Then the corresponding primers called "outer primers" and corresponding to A and H in Figure 2 (1), which can hybridize to regions upstream of FR1 and downstream of FR4, are also synthesized. . The isolation and extraction of the graft primers can be performed according to known procedures: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989.

Then, the first PCR is performed using graft primer E and outer primer A, grafting primers B and F, grafting primers C and G as well as grafting primer D and outer primer H resulting in the formation of AE, BF fragments, respectively. , CG and DH (Fig. 2 (2)).

Since the regions upstream of graft primer B and part of the region downstream of graft primer E have been designed to overlap (The same applies to graft primer C and F as well as D and G), these fragments can anneal by PCR to the appropriate complementary sequences by reaction under appropriate temperature conditions and combine to form DNA having a length from A to H. After the DNA fragment encoding the V region is obtained, the outer primers A and H can be added and the next PCR reaction can be performed to obtain DNA encoding the V region of a humanized antibody in which FRs 1 to 4 are from human and CDRs 1 to 3 are from mice. This DNA can then be incorporated into an appropriate host for expression, and thus the desired polypeptide (Fig 2 (3)).

190 351 (ii) THE CONSTRUCTION OF DNA AND THE VARIABLE CKDPREEJNO JOINING THE EG REGION OF THE ANIMATED H CHAIN

In omeJsecm wcyalaeOu, the entire base sequence of the DNA encoding the V region of the H chain of a human antibody that has been used as a template for the humanized antibody can be extracted in the DNA syytetceeree and constructed by PCR, although this DNA is not available in the natural environment.

The Regin V H chain of the murine mnynOlnnalnegn antibody against human PTHrP shows high homology with S31679 belonging to the human subgroup III. To incorporate this human antibody as a template for constructing a DNA encoding the V region of the humanized H chain, for example, four primers as shown in Id. Seq No. 23-26. The primers MBClHGP1 (SEQ ID NO: 23) and MBClHGP3 (SEQ ID NO: 24) have the sense DNA sequences, aMBClHGP2 (SEQ ID NO: 25) and MBC1HGP4 (SEQ ID NO: 26) have anti-sense DNA sequences. They were made to have a 15 to 21 bp complement sequence at each end.

The outer primers MBClHVS1 (SEQ ID NO 27) and MBClHVR1 (SEQ ID NO 28) have napvedin homologous sequences from MBClHGP1 andMBClHGP4 and each have a sequence recognized by the respective restriction enzymes. Four primers were connected by PCR to the full-length cDNA esynthetwayla and external primers were added to amplify the cDNA. each fragment is re-linked through its complementary sequence to extract the full-length DNA of the V region of the humanized H chain.

The C region of the H chain of a human antibody can be any C region of a human H chain, for example, Cyl, Cy2, Cy3, or Cy4.

The DNA of each H chain H chain of a human antibody constructed as described above can be ligated to the DNA of any C region of the H chain of a human antibody, for example, to the Cyl region of a human H chain. As mentioned in the section "Production of the H chain of a chimeric antibody", the DNA of the H chain V region can be treat with a suitable restriction enzyme and ligate to DNA encoding the human H chain C region under the control of an expression control region such as the wzmahmace / promoter system to generate an expression vector containing the DNA of the humanized H chain V region and human H chain C region.

(in) Construction of DNA and expression vector encoding the V region of the humanized I chain,

In the present invention, the entire base sequence of the DNA encoding the V region of the L chain of the human antibody that was used as a template for the humaiznwaoegn antibody can be escatetated into DNA scitalization and constructed by PCR, although the DNA of the L chain V region is inaccessible as is the case with DNA encoding the V region chain H.

To construct the humanized L chain V region DNA using the JaOn template of the human SU03868 antibody displaying the largest hnmnIngga with the V region of the L chain of the murine mnnOlnyalyegn anti PTHrP antibody, four primers were used as for example shown in SEQ ID No. op 29-32. The primers MBC1LGP1 (SEQ ID NO: 29) and MBC1LGP3 (SEQ ID NO: 30) have the sense DNA sequences, aMBClLGP2 (SEQ ID NO: 31) and MBC1LGP4 (SEQ ID NO: 32) have Antcsensnwye DNA sequences. They are designed to have 15 to 21 bp of the complementary sequence on each of their l <nn oAmr

The outer primers MBC1LVS1 (SEQ ID NO 33) and MBC1LVR1 (SEQ ID NO 34) have sequences homologous to MBC1LGP1 and MBC1LGP4, respectively, and each have a sequence recognized by the respective restriction enzymes. Four primers were connected by PCR to the full-length cDNA and external primers were added to amplify the cDNA. "PCR Linking" here includes the attachment of MBC1LGP1 and MB-C1LGP3 or MBC1LGP2 and MBC1LGP4 by their complementary sequences

190 351 to synthesize the MBC1HLP1-MBC1HLP3 fragment and the MBC1LGP2MBC1LGP4 fragment and each fragment is re-linked through its complementary sequence to synthesize the full length DNA of the humanized H chain V region

The C region of the L chain of a human antibody can be any C region of a human L chain, for example CA or Ck.

The DNA of the V region of the L chain of a humanized antibody constructed as described above can be ligated to the DNA of each C region of the L chain of a human antibody, for example the CA region of a human L chain. DNA of the V region of the L chain can be treated with an appropriate restriction enzyme and ligated to DNA encoding the L chain region The C of human LA chain, under the control of an expression control region such as an enhancer / promoter system to generate an expression vector comprising DNA of the V region of a humanized L chain and the C region of a human LA chain.

Even though a polypeptide containing the humanized antibody V region can be produced as described above, it is not clearly clear whether or not this polypeptide will exhibit antibody activity such as binding or neutralizing activity against its antigen. Especially for the L chain, since the V region of the L chain of the murine monoclonal antibody to human PTHrP is derived from the very rare V λχ gene, it may be necessary to check for activity or absence by linking to a humanized H chain and expressing it in an animal cell such as COS- 7.

An effective way to explain which FRs in the V region of a humanized antibody may be responsible for the binding and neutralizing activity of a humanized antibody can be to construct a hybrid V region (Othomo T. et al., Molecular Immunology, 32, 407-416, 1995) and confirm efficacy. In order to clarify which amino acid of the V region of the L chain of the humanized antibody of the present invention should be mutated to obtain one region having activity, DNA is constructed in which a fragment of the FR region of the humanized antibody is recombined with a fragment of the FR region derived from the mouse, and each region is evaluated. in terms of humanization.

As shown in Figure 3, an antibody having a polypeptide containing a recombinant V region in which FR1 and FR2 are derived from a human antibody and FR3 and FR4 are derived from a murine antibody (such an antibody having a recombinant fragment is referred to as a "hybrid antibody") is produced. a hybrid antibody in which only FR1 is human and a hybrid antibody in which only FR2 is human. Each of the DNAs encoding these hybrid antibodies is inserted into the hybrid vector and the hybrid antibodies are temporarily expressed to check for the presence of antibody activity.

Using this method, the inventors examined polypeptides containing the V regions of the L chain responsible for antigen binding and neutralizing actions, and finally found that specific amino acids in FR2 and FR3 should be replaced.

Thus, in the present invention, a polypeptide has been produced comprising the V region in which such amino acid (s) is / are mutated (e.g., listed).

First, the above-mentioned CDR grafting method has prepared a polypeptide comprising a V region having an amino acid sequence as a basis into which the amino acid mutation (s) is introduced. This basic polypeptide comprises the amino acid sequence as shown in Id. Seq. No. 47 and is referred to as version a (in table 1).

Then, different fragment variants in which one or more FR amino acids have been mutated are prepared from version a as the basis.

Introducing a mutation can be accomplished by designing an oligonucleotide primer (mutagenic primer) encoding the amino acid that is the desired mutation and performing PCR using that primer.

Thus, polypeptides containing V regions (versions b to t) are produced in which the specific amino acid (s) in FR2 and FR3 is / are mutated (s) (b to t in Table 1)

190 351

Table 1

MBC IL PEP FRl 1 2 3 123456789012345678901234567890 EYOLYESGGDLYKPGGSLKLSCAASGFTFS CDF 12345 SYGMS S31679 * ***** QVQLVESGGGVVQPGRSLRLSCAASGFTFS SYAMH hMBCl-H. pep SYGMS

MBC Η. PEP

S31679 hMBCl-H. pep

FR2

67890123456789 WIRQTPDKRLEWVA * ♦ ♦ Ψ

WVRQAPGKGLEWVA

CDR2

6

012A3456789012345

TISSGGSYTYYPDSYKG

YISYDGSNKYYADSYKG

TISSGGSYTYYPDSYKG

FR3 CDR3 FR4 7 8 9 10 11 67890123456789012ABC345678901234 567890Λ12 34567890123 MBC H. PEP RFTISRDNAKNTLYLOMSSLKSEDTAMFYCAR OTTMTYFAY WGOGTLYTYSA * ***** * S31679 RFTISRDNSKNTLYLOMNSLRAEDTAYYYCAR ESRGDY WGQGTLVTVSS hMBCl-H. pep OTTMTYFAY

FRl CDR1 FR2 CDR2 1 2 3 4 8 12345678901234567890123 4667A8901234 867800123468789 01234ABCDS6 MBC L. PEP QLVLTQSSS-ASFSLCASAKLTC TLSSOHSTYTIE YYOOOPLKPPKYYMD LKOOGSHSTCD * ♦ ♦ * ***** HSU03868 hMBCl-L. pep and QLVLTQSPS-ASASLGASVKLTC TLSSGHSSYAIA TLSSOHSTYTIE WHOOOPEKGPRYLMK LNSOGSHSKGD LKODGSHSTGD

b - - -p-d d

e f

h i

j k

tn n

about

P q

-Y- -Y- -YK—— -YKD -YKV— -YKY-0 -YD -YV— -YYD -Y-κ- -YK — D -γ- _D -YKYD -YYD 190 351 Table 1

FR3 CDR3 FR4 6 7 8 9 10 11 78901234567890123456789012345678 9012345ABCD67 890123456A7890 UBC L PEP GIPDRFSGSSSGADRYLSISNIOPEDEAMYIC 4 4 44 4 4 4 GVGBTIKEQPVYV FGGGTKVTVLGQP HSU03868 hMBCl-L pep GIPDRFSGSSSGAERYLTISSLOSEDEADYYC OTtGTGI GVGOT1KEQPVYV -L-

a b

c d

a f

g h

and j

k α

n o

P q

r s

vol

-j_

-1_ p-1_

IIIIII-LLLL- _L -LL- _L -LLLL- _L -LLLLLL-LDNA encoding any of the versions of the V region of the L chain of a humanized antibody constructed as above can be ligated to the DNA of any of the C regions of the L chain of a human antibody, such as the CA region. The human L chain is thus treated with an appropriate restriction enzyme and ligated to DNA encoding the human LA chain C region under the control of an expression control region such as an enhancer / promoter system to construct an expression vector comprising DNA encoding each version of the humanized V region. antibodies and DNA encoding the C region of the human LA chain.

The DNA encoding the V region of the H chain of a humanized antibody and the C region of a human H chain as previously constructed and the DNA encoding the V region of the humanized L chain and the C region of the human L chain as disclosed in WO94 / 11523 may be inserted into a single expression vector, these vectors may be be used to transform host cells, and the transformed host cells can be cultured in vivo and in vitro to produce the desired humanized antibody.

4. Production of chimeric and humanized antibodies

In order to produce chimeric and humanized antibodies, the two expression vectors mentioned above must be prepared. Thus, with respect to a chimeric antibody, an expression vector is constructed containing DNA encoding the mouse H chain V region and the human H chain C region under the control of an expression control region such as an enhancer / promoter system and an expression vector comprising DNA encoding the mouse L chain V region and the C region of the human L chain under the control of an expression knockout region such as an enhancer / promoter system. With respect to a humanized antibody, an expression vector is constructed containing DNA encoding the V region of a humanized H chain and a C region of a human H chain under the control of an expression control region such as an enhancer / promoter system and an expression vector comprising DNA encoding the V region of a humanized L chain and the C region of a human L chain under the control of an expression control region such as an enhancer / promoter system.

190 351

Then, host cells such as mammalian cells are co-transformed with these expression vectors and the resulting cells are transformed to produce chimeric or humanized antibodies in vitro and in vivo (see, for example, WO91 / 16928).

Alternatively, DNA encoding the H chain V and C regions and DNA encoding the L chain V and C regions can be ligated into a single vector and transformed into appropriate host cells for antibody production. Thus, in expressing a chimeric antibody, a single expression vector as disclosed in e.g. WO94 / 11523 is inserted DNA encoding a murine leader sequence present in a cloned cDNA, a murine H chain V region and a human H chain C region, as well as DNA encoding a murine sequence. leader, the mouse L chain V region and the human L chain C region. In the expression of a humanized antibody, a single expression vector as disclosed in e.g. WO94 / 11523 is inserted DNA encoding a humanized H chain V region and a human H chain C region and DNA encoding a region The V region of the humanized L chain and the C region of the human L chain. Such a vector is used to transform host cells, and the transformed cells are cultured to produce chimeric or humanized antibodies of interest in vitro and in vivo.

Chimeric or humanized antibodies of interest produced by culturing transformants transformed with DNA encoding said chimeric or humanized antibodies can be isolated from the extracellular and intracellular environment and purified to homogeneity.

The isolation and purification of the chimeric and humanized antibodies of interest according to the present invention can be performed using a protein A agarose column, but also can be performed by any method used in the isolation and purification of conventional proteins, and thus are not limited. For example, a variety of chromatographic methods, ultracentrifugation, desalting, and dialysis may optionally be selected or combined to isolate and purify chimeric or humanized antibodies.

Any expression system can be used to produce chimeric or humanized antibodies against human PTHrP of the present invention. For example, eukaryotic cells including animal cells such as established mammalian cell lines, fungal and mold cells, yeast cells, prokaryotic cells including bacterial cells such as Escherichia cells. coli. Preferably, the chimeric or humanized antibody of the present invention is expressed in mammalian cells such as COS and CHO cells.

Any conventional promoter useful for expression in mammalian cells can be used. For example, it is preferable to use the human cytomegalovirus (HCMV) immediate early promoter. Examples of expression vectors containing the HCMV promoter include HCMV-VH-HCyl and HCMV-VL-HCK derived from pSV2neo (WO92 / 19759).

Additionally, promoters for expression of the gene in host cells may be used in the present invention, including viral promoters such as the retrovirus, mouse salivary gland mixed tumor virus, adenovirus and simian virus (SV) promoter, and mammalian cell-derived promoters such as the human factor-factor promoter. 1α polypeptide chain extension (HEF-1a). For example, the SV40 promoter can be used simply according to the method of Mulhgan et al. (Nature, 277,108,1979); the method of Mizushima S. et al. (Nusleid Acids research, 18, 5322, 1990) easily used with the HEF-kx promoter.

An origin of replication useful herein includes those derived from SV40, mouse salivary gland mixed tumor virus, adenovirus, or bovine wart virus (BPV). Further, the expression vector may include a gene for APH (3 ') phosphotransferase II or I (neo), thymidine kinase (TK), E. coli xanthine guanine phosphoribosyltransferase (Ecogpt), or dihydrophohic acid salt reductase (DHFR) as a selective marker of increasing numbers. gene copies in the host cell system.

190 351

5. Assessing antigen binding and neutralizing activity of chimeric and humanized antibodies (1) Determination of antibody concentration

The concentration of the obtained purified antibody can be performed by an ELISA method.

ELISA plates for determining antibody concentration are prepared as follows: 100 μί of goat IgG antibody prepared at a concentration of e.g. 1 µg / ml is immobilized in each well of a 96-well ELISA plate (e.g. Maxisorp, NUNC). After blocking with 200 μί with dilute buffer (e.g. 50 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween20, 0.02% NaN3, 1% Bovine Serum Albumin (BSA), pH 7, 2) The diluted COS-7 or CHO cell supernatant expressing the chimeric, hybrid or humanized antibody was added to each well, or purified chimeric, hybrid or humanized antibody was added, 100 μί of alkaline phosphatase conjugated goat anti-human antibody was added and then added 1 mg / ml substrate solution (Sigma 104, p-mtrophenylphosphoric acid, SIGMA), followed by measuring absorbance at 405 nm with a microplate reader (Bio Rad). Purified Hu IgGIZ, (The Binding Site) can be used as a standard to determine concentrations.

(2) Determination of antigen binding capacity

ELISA plates for determining antigen binding capacity are prepared as follows: 100 μί of human PTHrP prepared at 1 µg / ml is immobilized in each well of the 96-well ELISA plate. After blocking with 200 μί with diluted buffer, the diluted COS-7 or CHO cell supernatant expressing a chimeric, hybrid or humanized antibody was gradually added to each well, or purified chimeric, hydribre or humanized antibody was added, 100 μί of goat anti-alkaline phosphatase-conjugated antibody was added to each well. human IgG and then 1 mg / ml of substrate solution (Sigma 104, p-mtrophenylphosphoric acid, SIGMA) was added and the absorbance at 405 nm was measured with a microplate reader (Bio Rad).

(3) Determination of the neutralizing activity

Determination of the neutralizing activity of murine, chimeric and humanized antibodies can be performed, for example, using cells of the ROŚ 17 / 2.8-5 cell line of rat bone sarcoma (Sato K. et al., Acta Endocrinology, 116, 113-120, 1987). ROSI7 / 2.8-5 cells are stimulated with 4 mM hydrocortisone to stimulate the PTH / PTHrP receptor. The cAMP-degrading enzyme is inhibited by 1 mM isobutyl-1-methyl xanthine (ΙΒΜΧ, SIMGA). A mouse chimeric or humanized antibody whose neutralizing activity is determined is mixed with an equal amount of PTHrP (1-34) and the resulting mixture of each antibody and PTHrP (1-34) is added to each well. The neutralizing capacity of the murine chimeric or humanized antibodies can be determined by measuring the amount of cAMP produced by cells of the ROSI7 / 2.8-5 rat bone sarcoma cell line in response to stimulation with PTHrP.

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

In the present invention, the kinetics of the interaction between PTHrP and an anti-PTHrP antibody can be analyzed using a variety of means and procedures. association constants. Analysis using a surface plasma resonance sensor called BLACORE will be described below as one example.

The rhodium-rhodium structure of BIACORE includes a light source, a prism, a sensor and a micropass. Practically, the ligand is immobilized at the tip of the cartridge-type sensor into which the test substance is injected. When there is any affinity between them, the associated amount is measured by an optical method.

The principle of detection is a phenomenon called surface plasma resonance. A flash of light is introduced into the space between the glass and the metal film so that all its reflection appears, a flash of light is used at a certain angle to

190 351 excitation of the surface plasmon and attenuation. The angle varies depending on the changes in the concentration of the solution in contact with the metal film (sensor). BIACORE explores these changes.

In BIACORE, these changes are called the resonance signal (SPR signal) t a 0.1 degree change is 1000 RU (resonance units). 1000 RU corresponds to a binding change of approximately 1 ng of protein on a thin gold sensor with an area of 1 mm ² . For a protein, the 50 RU (50 pg) change can be fully measured.

The measured signals are transferred to a binding curve called a sensorogram by a computer connected to BIACORE, which is drawn on the computer screen in real time: Natsume T. et al. (1995) Expenmental medicine, 13, 563-569; Karlsson R. et al., (1991) J. Immunol. Methods 145, 229-240.

6. Pharmaceutical composition ΐ a factor that reduces the Ιήρο ^ Ι ^ ικίς collects an antibody against PTHrP or a humanized antibody as an active ingredient

The therapeutic effect of a humanized antibody on PTHrP can be confirmed by administering the antibody to PTHrP or a humanized antibody to an animal with symptoms of hi ^^^ ζώ and measuring the hlpyrkylcymia index. Hypophosphytymia is frequently observed in symptomatic animals and patients suffering from hyperkalcymia, and the antibodies of the lower invention may also be used to reduce the symptoms of hipoCosCatimil.

The antibody used in the present invention is an anti-PTHrP antibody, including human, chimeric and or humanized anti-PTHrP antibody with constant dissociation rate, constant dissociation rate, and constant association rate. The antibody will neutralize PTHrP activity by binding to PTHrP and preferably comprises humanized antibody # 23-57-137-1. The method of producing humanized antibody # 23-57-137-1 will be described in Examples 1 to 3.

The antibody used in the present invention can be purified to a high purity state by any combination of conventional purification methods such as desalting, gel filtration using HPLC, etc., affinity chromatography using a Protein A column, etc. Recognition of PTHrP by these purified antibodies can be accurately confirmed by any of the conventional methods. immunoassay tests such as Radioimmunoassay (RIA), Enzyme Immunoassay (ELA, ELISA) or ImmunoCluoric Testing.

Animals showing signs of hypercalcemia that can be used include an animal model prepared in such a way that PTHrP-producing tumor cells were implanted in a test animal with impaired or suppressed immune defense. Neoplastic cells preferably of human origin include, for example, PAN-7 human pancreatic carcinoma cells. An immunocompromised or suppressed animal that is implanted with tumor cells includes nude or SCID mice. The reduction in hypercalcemia can be assessed by looking at blood calcium levels, weight loss, or a decrease in physical activity per unit time and the degree of improvement.

The pharmaceutical composition and the hypercalcymia-reducing agent contain as a component an antibody or humanized antibody to PTHrP according to the invention, and can be administered parenterally, systemically or locally. For example, intravenous injection may be selected, including intravenous infusion, intramuscular injection, intraperitoneal injection, or subcutaneous injection. The method of administration can be properly chosen according to the age of the patient and the severity of the disease. Effective single dose of iris ilzracn f) A1 rlo 1 ΟΠΠ mrr no ριλτόπι mol

AIVlVk) U * WW X WV X \ J W * XV * ikg) VI yŁAłl U V / X «Xli

LU V / X xlcx. L nivi rum, c naty νίΠΑν

The figure for figure can be from 5 to 10,000 g / body, preferably from 50 to 1,000 g / body.

A pharmaceutical composition and a hypyrcalcemia reducing agent containing an antibody or humanized antibody to PTHrP as an active ingredient may further contain pharmaceutically acceptable carriers and / or additives depending on the route of administration. Examples of such carriers and additives may include water, pharmaceutically acceptable

190 351 organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, poly (sodium acrylate), sodium argiman, water-soluble dextran, sodium carboxymethyl starch, pectin, methyl cellulose, ethyl cellulose, xanthylcellulose , casein, gelatin, agar, diglycerin, glycerin, propylene glycol, polyethylene glycol, petroleum jelly, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), manntol, sorbitol, lactose and surfactants acceptable as pharmaceutical additives. The additives used may be suitably selected from the above both individually and in combination and are not limited thereto.

The antibody of the present invention can be widely used in hypercalcemia associated with various neoplasms (malignant neoplasms). These tumors are not particularly limited and include not only individual tumors, but also combinations and accumulations of tumors. Neoplasms may include, for example, tumors of the pancreas, lung, pharynx, larynx, tongue, gums, esophagus, stomach, bile ducts, breast, kidney, bladder, uterus and prostate, and malignant lymphoma.

Brief description of the drawings

Figure 1 is a diagram showing an antibody of the invention;

Figure 2 is a CDR "grafting" scheme;

Figure 3 is an illustration of the determination of the FR and CDR of the V region;

Figure 4 is a graph showing the results of measuring the antigen-binding activity of antibodies.

Figure 5 is a graph showing the results of measuring the antigen-binding activity of antibodies.

Figure 6 is a graph showing the results of measuring the antigen-binding activity of antibodies.

Figure 7 is a graph showing the results of measuring the antigen-binding activity of antibodies;

Figure 8 is a graph showing the results of measuring the antigen-binding activity of antibodies;

Figure 9 is a graph showing the results of measuring the antigen-binding activity of antibodies.

Figure 10 is a graph showing the results of measuring the antigen-binding activity of antibodies;

Figure 11 is a graph showing the results of measuring the antigen-binding activity of antibodies.

Figure 12 is a graph showing the results of measuring neutralization activity by humanized antibodies;

Figure 13 is a graph showing the results of measuring neutralization activity by humanized antibodies;

Figure 14 is a graph showing the results of measuring neutralization activity by humanized antibodies;

Figure 15 is a graph showing the effectiveness of the antibodies of the invention in a hypercalcemic animal model;

Figure 16 is a graph showing the effectiveness of the antibodies of the invention in a hypercalcemic animal model;

Figure 17 is a graph showing the effectiveness of the antibodies of the invention in a hypercalcemic animal model;

Figure 18 is a graph showing the effectiveness of the antibodies of the invention in a hypercalcemic animal model;

Figure 19 is a graph from a sensor showing PTHrP locking at the end of the sensor.

Figure 20 is a graph showing the results of a kinetic analysis of an antibody of the invention.

190 351

Figure 21 is a graph showing the results of a kinetic analysis of an antibody of the invention;

Figure 22 is a graph showing the results of a kinetic analysis of an antibody of the invention.

Figure 23 is a graph showing the results of a liquid analysis of an antibody of the invention;

Figure 24 is a graph showing the results of the kinetic analysis of an antibody of the invention.

Figure 25 is a graph showing the results of a study of the effect of a humanized antibody of the invention on fraOhyjae phosphate yield;

Figure 26 is a graph showing the results of a study of the effect of a humated antibody of the invention on plasma phosphate concentration;

Figure 27 is a photograph showing the widespread symptoms of a murine hlperkaJcemll model induced by administration of an anti-PTHrP antibody of the invention (live animal morphology);

Figure 28 is a photograph showing the wreath symptoms of the kharceae of a mouse model of hipeocalcemia induced by administration of an anti-PTHrP antibody of the invention (live animal morphology);

Figure 29 is a graph showing the change over time of the spontaneous activity of an animal with a model hlperkaJemela after administration of an anti-PTHrP antibody of the invention compared to a control animal administered with saline;

Figure 30 is a graph showing the change over time in body temperature of an animal with model 1ι ^ ιΌ3ΐοοιοίί) after administration of an anti-PTHrP antibody of the invention compared with a saline-administered control animal; and

Figure 31 is a graph showing the change over time in pH of an animal with a model hicercalhemia after administration of an anti-PTHrP antibody of the invention compared to a control animal that was given the fieanJngrhea salt.

Pose

The invention will now be described in more detail below with reference to the following examples which are not intended to limit the scope of the invention.

Namesieyia example 1

Generation of a hybridoma producing the murine οόμ ^ οαΙ ^ against PTHrP (1-34)

Hybridomas capable of producing an anti-human PTHrH (1-34) mnnncaleal antibody # 23-57-154 and # 23-57-137-1 were prepared according to the method described by Kanji Sato (Sato et al., J. Bone Miner. Res. 8: 849-860,1993).

PTHrP (1-34) (Peniysula) was used as the immunogen, to which tyoenglnbuhn was conjugated as a carrier protein using carbodiimide (Ι ^ ϊοο). PTHrP (1-34) coupled with tcpenglobuliya aiaJiznwayn to obtain a solution with a protein content of 2 µl / γο1. The resulting solution was mixed with Freund ^ (ϋβ ^) adjuvant in a ratio of 1 · 1 to form an emulsion. For mouse immunity, the emulsion was injected into 16 female BALB / c mice subcutaneously in the back and intraperitoneally at an amount equal to 100 g / lyCse. The injection was repeated 11 times. For aaiuwait, complete Freundd adjuvant was used for the first ^ οιτο ^^ ι and for knleaachh lmmuyleacal was used for incomplete Freund ^ adruwaat ^.

In each of lmmumznwaycch mice were tested for antibody titer in the serum in the following sposóbOd each mouse koew collected from the tail vein and centrifuged to obtain serum diluted serum RIA buffer, mixed with PTHpP (1-34) zyakowayym ¹²⁵ I and subjected to assay the binding activity. Mice found satisfactory. high titers of antibodies were injected intraperitoneally with PTHrP (1-34) without protein carrier at a dose of 50 pg / mouse for final rmmuyrzacar.

Three days after the final rmmuilization, the mice were sacrificed and their spleens excised. Subsequently, the scleynhctc was fused to the P3x63Ag8U.l myeloma cell line according to the known art.

By conventional method, using 50% polyethylene glycol 4000, the resulting fused cells were inoculated into 85 wells of a 96-well plate at 2x10 / well. Screening of the hybridoma was performed using the HAT medium as follows.

The screening was performed by detecting the presence of antibodies recognizing PTHrP in the supernatant with respect to wells where cell growth was observed in HAT medium by the RIA method on a solid medium. Hybridomas were harvested from wells, in which the binding capacity of antibodies to PTHrP was found. The hybridomas obtained in this way were suspended in an RPMI 1640 positive containing 15% FCS with the addition of OPI (Sigma), and then diluted by limiting dilution, thus obtaining two types of hybridoma clones. # 23-57-154 and # 23-57-137-1 showing strong binding ability to PTHrP (1-34).

Hybridoma clone # 23-57-154, which was designated "mouse-mouse hybridoma # 23-57-154, was deposited under the Budapest Treaty on August 15, 1996, at the National Institute of Bioscience and Human-technology, Agency for Industrial Science and Technology, Japan. (1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken, Japan), under accession number FERM BP-5631.

Example 1

Cloning of DNA encoding the V region of a murine monoclonal antibody against human PTHrP (1-34)

Cloning of the DNA encoding the V region of the mouse anti-human PTHrP (1-34) monoclonal antibody obtained above, # 23-57-137-1, was performed as follows.

(1) Preparation of mRNA mRNA was prepared from hybridoma # 23-57-137-1 using Quick Prep mRNA Purification Kit (Pharmacia Biotech) as follows.

Hybridoma # 23-57-137-1 cells obtained as above were homogenized with extraction buffer and mRNA extracted using oligo (dI) -Cellulose Spun Column, according to kit manufacturer's instructions. The extraction solution was subjected to ethanol precipitation to obtain mRNA precipitates. The mRNA precipitates were dissolved in the elution buffer.

(2) Preparation and amplification of cDNA of the gene encoding the V region of mouse H chain.

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

The DNA encoding the V region of the H chain of a murine monoclonal antibody against human PTHrP was cloned by 5'-RACE (Frohman et al., Proc. Natl. Acad. Sci. USA, 85.8998-9002, Belavsky et al., Nucl Acids Res. 17: 2919 -2932, 1989). This method was performed using the 5'-Ampli FINDER RACE kit (Clonetech) according to the manufacturer's instructions. In this method, the primer used to synthesize the cDNA was an MHC2 primer (SEQ ID NO: 1) that could be hybridized to the C region of mouse H chain. Approximately 2 µg of mRNA as prepared above, which was a template, was mixed with 10 pmol of MHC2 primer. The resulting mixture was reacted with reverse transcriptase at 52 ° C for 30 minutes to form a cDNA that was complementary to the mRNA.

The product was mixed with 6N NaOH to hydrolyze the mRNA (at 65 ° C for 30 minutes), and then subjected to ethanol precipitation to isolate cDNA precipitates. The cDNA thus isolated was ligated with an Ampli FINDER Anchor (SEQ ID NO: 42) at the end of 5 'by reacting with T4 RNA ligase at 37 ° C for 6 hours and additionally at room temperature for 16 hours. As primers for the PCR amplification of cDNA, an anchor primer (SEQ ID NO: 2) and an MHC-G1 primer (SEQ ID NO: 3) were used (Jones et al., Biotechnology 9:88, 1991).

PCR solution 50 lb of the resulting tvrn cnsobhip awunrred IOimMΛ ^ rio_Cl ^ 1 β, λ mM KCl, 0.25 mM dNTP (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl ₂ , 2.5 TaKaRa Taq units (Takara Shuzo ), 10 pmol anchor primer and 1 µl cDNA reaction mixture ligated with MHC-GI primer and Ampli FINDER Anchor primer that was covered with 50 µl mineral oil. 30 cycles of PCR were performed using a Thermal Cycler Model 480J (Perkin Elmer) and a temperature cycle of 45 seconds at 94 ° C, 45 seconds at 60 ° C and 2 minutes at 72 ° C

190 351 (ii) cDNA cloning for the V region of the L chain of antibody # 23-57-137-1

The DNA encoding the V region of the L chain of the murine monoclonal antibody against human PTHrP was cloned by the 5'RACE method (Frohman et al., Proc. Natl. Acad. Sci. USA, 85.8998-9002; BelavsKy et al., Nuci. Acids Res. 17: 2919 -2932, 1989). This method was performed using the 5'-Ampli FINDER RACE kit (Clonetech) according to the manufacturer's instructions. In this method, the primer used to synthesize the cDNA was an oligo-dT primer that could be annealed to the C region of mouse H chain. Approximately 2 μg of mRNA as obtained above which was template was mixed with 10 pmol MHC2 primer. The resulting mixture was reacted with reverse transcriptase at 52 ° C for 30 minutes to form a cDNA that was complementary to the mRNA. The product was mixed with 6N NaOH to hydrolyze mRNA (at 65 ° C for 30 minutes) and then subjected to ethanol precipitation to isolate cDNA precipitates. The thus isolated cDNA was ligated to an Ampli FINDER Anchor at the 5 'end by reacting with T4 RNA ligase at 37 ° C for 6 hours and additionally at room temperature for 16 hours.

As primers for PCR amplification of the cDNA, an MLC primer (SEQ ID No. 4) was used, designed based on the conserved sequence of the C region of the λ chain, mouse L chain, and then synthesized on a 394 DNA / RNA Synthesizer (ABI) machine. The PCR solution (100 µl) used in this method contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTP (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl ₂ , 2 , 5 units of AmpliTaq (PerKinElmer), 50 pmoles of Anchor primer and 1 µl of a cDNA reaction mixture ligated with an MLC primer (SEQ ID No. 4) and an Ampli FINDER Anchor primer that was covered with 50 µl of mineral oil. 35 cycles of PCR were performed using a Thermal Cycler Model 480J machine (PerKin Elmer) and a temperature cycle of 45 seconds at 94 ° C, 45 seconds at 60 ° C and 2 minutes at 72 ° C.

(3) Purification and fragmentation of the PCR product

Each of the amplified DNA fragments described above was separated by electrophoresis on a NuSieve GTG 3% agarose gel (FMc Bio. Products). For each of the H chain V regions and L chain V regions, a gel fraction containing a DNA fragment of approximately 550 bp in size, respectively. Each of the obtained gel fractions was subjected to DNA purification using the GENECLEAN II kit (BIO101) according to the manufacturer's instructions. The purified DNA was precipitated from the solution with ethanol and then dissolved in 20 µl of a solution containing 10 mM Tris-HCL (pH 7.4) and 1 mM EDTA. 1 µl of the DNA solution prepared in this way was digested with the restriction enzyme XmaL (New England Biolabs) at 37 ° C for one hour and additionally with the enzyme EcoRI (Takara Shuzo) at 37 ° C for one hour. The digestive mixture was extracted with phenol and chloroform, followed by ethanol precipitation to collect DNA.

In this way, DNA encoding the H chain V region and DNA encoding the L chain V region were obtained, both of which had an EcoRI recognition sequence at the 5 'end and a XmaI recognition sequence at the 3' end.

Each of the EcoRI-Xmal DNA fragments containing the DNA encoding the H chain V region and the DNA encoding the mouse L chain V region, respectively, was reacted with the vector pUC19, which was digested with EcoRI and XmaI at 16 ° C for one hour using DNA ligation kit ver 2 ( Takara Shuzo) according to the manufacturer's instructions. The ligation mixture (10 µl) was added to 100 µl of the solution containing E. coli JM109 competent cells (Nippon Gene). The cell mixture was left for 15 minutes on ice at 42 ° C for a minute and then on ice for a minute. Cells will be mixed with 300 µl SOC medium (Molecular Cloning: A Laboratory Manual, SambrooK et al., Cold Spring Harbor Laboratory Press, 1989) and incubated at 37 ° C for 30 minutes. The resulting cell suspension was smeared on LB or 2xYT medium with agar (Molecular Cloning: A Laboratory Mirnual, Sambrook et al. Cod Sprng Harbor L aborat Giy Press, 1989) supplemented with 100 or 50 pg / ml ampicillin, 0.1 mM. IPTG and 20 µg / ml X-gal and incubated at 37 ° C overnight. In this way, E. coli transformants were produced.

Transformants were grown overnight in 2 ml LB or 2xYT medium supplemented with 100 or 50 µg / ml ampicillin at 37 ° C and plasmid DNA prepared using Plasmid Extracter PI-100® (Karabou) or QIAprep Spin Plasmid Kit (QIAGEN). The plasmid DNA obtained in this way was verified for the DNA sequence.

190 351 (4) Sequencing of a cDNA encoding the V region of a murine antibody

The sequence of the coding region of the cDNA carried by the plasmid was determined using the DNA Sequencer 373A machine (ABI, Perkin Elmer) using the Dye Terminator Cycle Sequencing Kit (Perkin Elmer). This DNA sequence was determined by confirming the base sequence in both directions using M13 Primer M4 primers (Takara Shuzo) (Seq ID No 5) and M13 Primer RV (Takara shuzo) (Seq ID No 6).

The plasmids thus obtained that contained the cDNA encoding the murine H chain V region and the cDNA encoding the murine L chain V region derived from hybridoma # 23-57-137-1 were designated "MBC1HO4" or "MBC1L24", respectively.

The sequences (including the appropriate amino acid sequences) of the DNA encoding the H chain V region and the DNA encoding the L chain V region of murine antibody # 23-57-137-1 (carried by MBC1HO4 and MBC1H24, respectively) are shown in Id. Seq. Nos. 57 and 65. Both polypeptides for the V fragment of the H chain and the V fragment of the L chain were translated starting from base 58 (encoding glutamine) in the DNA sequences shown as SEQ ID NO. Nos. 57 and 65. The amino acid sequences for the V region of the H chain and the V region of the L chain are shown in Id. Seq. 46 and 45.

E coli containing the MBClHO4 plasmid and E. coli containing the MBClL24 plasmid were designated Escherichia coli JM 109 (MBClHO4) "and" Escherichia coli JM 109 (MBClL24) ", respectively. These E. coli strains were deposited under the terms of the Budapest Treaty on August 15, 1996, at the National Institute of Bioscience and Human-technology, Agency for Industrial Science and Technology, Japan (1-3, Higashi 1-chome, Tsukuba-shi, Ibaragi-ken , Japan), under accession number FERM BP-5628 for Escherichia coli JM109 (MBClHO4) and FERM BP-5627 for Escherichia coli JM109 (MBC1L24), respectively (5) Determination of the CDRs of the murine monoclonal antibody # 23-57-137-1 against human PTHrP.

The general patterns of the H chain V region and the L chain V region are similar to each other. That is, both structures have four framework regions connected by three hypervariable regions (i.e., complementarity ensuring regions (CDRs)). The amino acid sequences of the framework regions are relatively conserved, while the amino acid sequences of the CDR regions show extremely strong mutagenicity (Kabat et al., Sequence of Proteins of Immunological Interest, US Dept. Health and Human Services, 1983).

Based on the above facts, the CDRs were determined by looking for amino acid sequence homology of the V region of a murine monoclonal antibody by reference to the antibody amino acid sequence database established by Kabat et al.

The amino acid sequences of CDRs 1-3 in the V region of the L chain are shown in Id. Seq. Nos. 59-61, and the amino acid sequences of CDRs 1-3 of the V region of the H chain are shown in Id. Seq. no. 62-64

Table 2

Region V Seq Id no CDR1 CDR2 CDR3 The V region of the H chain 57 31-35 50-66 99-107 The V region of the L chain 65 23-34 50-60 93-105

Example 2

Construction of a chimeric antibody (1) Construction of the H chain of a chimeric antibody (i) Construction of a V regpon of the H chain

The cloned cDNA encoding the mouse H chain V region was modified by PCR to fuse it with an expression vector carrying genomic DNA for the C 1 region of human H chain. The primer used "downstream", MBC1-S1 (SEQ ID NO: 7) was designed

190 351 such that it could hybridize to DNA encoding the 5 'end region of the V region Stroke sequence and had both Kozak consensus sequences (Kozak et al., J. Mol. Biol 196'947-950, 1987) and HindIII hypervariables. The upstream primer MBC1-a (SEQ ID NO: 8) was designed to hybridize to DNA encoding the 3 'region of the J region and to have both splice donor sequences and the BamHI recognition sequence. PCR reaction was performed using TaKaRa Ex Taq and the attached buffer. The PCR solution (50 µl) consisted of 0.07 µg of plasmid MBClHO4 as template DNA, 50 pmoles of MBC1-a and 50 pmoles of MBC1-S1 as primers, 2.5 units of TaKaRa Ex Taq and 0.25 µm of dNTP in buffer that was coated with 50 µl of mineral oil. 35 cycles of PCR were performed using a Thermal Cycler Model 480J machine (Perkin Elmer) and a temperature cycle of 1 minute at 94 ° C, a minute at 55 ° C and 2 minutes at 72 ° C. Each of the amplified DNA fragments obtained was separated by electrophoresis on a 3% NuSieve GTG agarose gel (FMC Bio Products).

Then, an agarose gel fragment containing a 437 bp DNA fragment was excised and the DNA fragment purified using GENECLEAN II (BIO101) according to the manufacturer's instructions. The purified DNA was collected by ethanol precipitation and dissolved in 20 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mole of EDTA. 1 µl of the resulting DNA solution was digested with BamHI and HindIII (Takara Shuzo) restriction enzymes at 37 ° C for 1 hour. The digestive mixture was extracted with phenol and chloroform, and then ethanol precipitated to collect DNA.

The obtained HindIII-BamHI DNA fragment that contained the DNA encoding the V region of the murine H chain was cloned into the pUC19 vector that had been digested with HindIII and BamHI. The resulting plasmid was sequenced on a DNA Sequencer 373A machine (Perkin Elmer) using the M13 Pnmer M4 and M13 Primer RV primers and the Dye Terminator Cycle Sequencing Kit (Perkin-Elmer). A plasmid containing DNA with the correct base sequence encoding the V region of murine H chain from hybridoma # 23-57-137-1 and having a HindIII recognition sequence and a Kozak sequence in the 5 'region and a BamHI recognition sequence at the 3' end, "MBC1H / pUC19".

(ii) KonstKionv ^^^ i ^ ie the V region of the H chain used for the production of the murine human chimeric H chain type c DNA

DNA encoding the V region of the murine H chain constructed as described above was modified by PCR to fuse it with the cDNA of the Cy1 region of the human H chain. The MBC1HVS2 reverse primer (SEQ ID NO: 9) used to modify the V regions of the H chain was designed such that replaced the second amino acid (ie, asparagine) of the coding sequence with the initial portion of the V region leader sequence with glycine and included the consensus sequence Kozak) Kozak et al., J. Mol. Biol. 196: 947-950,187) and HindIII and EcoRI recognition sequences. The forward primer MBC1HVR2 (SEQ ID NO: 10) used to modify the V region of the H chain was designed to hybridize to the DNA sequence encoding the 3 'region of the J region, encoding the 5' region of the C region and containing the sequence recognized by ApaI and SmaI .

The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo) and the included buffer. The PCR solution (50 (.1) included 0.6 µg of plasmid MBC1H / pUC19 as DNA template, 50 pmoles MBC1HVS2 and 50 pmoles MBC1HVR2 as primers, 2.5 U TaKaRa Ex Taq and 0.25 mM dNTP in buffer, which was covered with 50 [mu] l mineral oil. 30 cycles of PCR were performed using a cycle of 1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C. The amplified PCR fragments were separated by agarose gel electrophoresis using 1% Sea agarose Kem GTG (FMC Bio. Products). Then, a fragment of the gel ^^ VR; miięy oiagom / tu k / ansi wicmusni njf up w — i Ltdguicui 17ΑΝ / Λ. BLUbUjć ^ c

GENECLEAN II (BIO101) according to the manufacturer's instructions. The purified DNA fragments were ethanol precipitated and dissolved in 20 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.

pl of the resulting DNA solution was digested with restriction enzymes EcoRI and SmaI (Takara Shuzo) at 37 ° C for 1 hour. The digestive mixture was extracted with phenol and chloroform and precipitated with ethanol to recover DNA. Received DNA fragments

190 351

EcoRI-SmaI that contained the DNA encoding the Pegins V of the murine Olnynwayn H chain into the pUC19 vector, which precapnwaan by digestion with EcoRI and SmaI. The resultant sequencing plasmid on a Seguencer 373A DNA device (Perkin Elmer) using the M13 Primer M4 and M13 Primer RV primers and the Dye Terminator Cycle Sequencing Kit (PerCin Elmer) Plasmid that contained DNA with the correct base sequence coding for the V region of the murine H chain from the hybridoma # 23-57-137-1 and has the rnzcnznaway sequence through HmdIII and the Kozak sequence at the 5 'end and the ApaI and SmaI recognition sequence at the 3' end are designated "MBC1Hv / pUC19".

(in) Construction of an expression vector for the H chain of a chimeric antibody cDNA containing the region's C lude01egn antibody H chain was prepared as follows mRNA was prepared from CHO cells into which the DHFR-AE-RVh-PM-1-f expression vector wcrnwaaznnn (see WO 92 / 19759) encoding the geanmnwe DNA of the V region of the H chain of humanized PM1 antibody and the C region of the H chain of human IgG1 antibody and the expression vector RV1-PM1a (see WO 92/19759) encoding gennmnwe DNA of the V region of the L chain of humanized antibody PM1 and the C region of the κ chain of human antibody . Using the thus obtained mRNA, the Olnanwaan cDNA containing the V regins of the H chain of the humanized PM1 antibody and the Cyl regrnn of the human antibody by PCR followed by clnynwayn to the plasmid pUC19 in the HindIn-BamHI site. The Olnynwayc plasmid was tested for DNA sequence and the correct plasmid was designated "pRVh-PMlfcDNA".

DHFR-AE-RVh-PM-1-f expression vector which had a deletion of the HmdIII site between the Sv40 promoter and the DHFR gene, and an EcoRI site between the EF-1a promoter and the V region of the H chain of humanized PM1 antibody wctwnpknown to construct an expression vector for cDNA containing the V regrno of the H chain of a humanized PM1 antibody and the Cyl region of a human antibody.

The obtained cRVh-PM1f-cDNA plasmid BamHI travin, blunted with the Kunowa fragment, and further with the HiridIn travinine to obtain a blunt ended HmdIII-BamHI fPagment. This HindIn-BamHI fragment was ligated with the site of HmdIII and EcoRI of the DHFR-A-PME-1 expression vector -f, which was digested with HmdIII and BamHI to construct an RVh-PM1f-cDNA expression vector containing the cDNA encoding the V regino of the H chain of the humanized PM1 antibody and pegrno Cyl of the human antibody.

The RVh-PM1f-cDNA expression vector contained the cDNA encoding the H chain regino of the PM1 antibody, and the Cyl regrna of the luazkiegn antibody was digested with ApaI and BamHI, and a DNA fragment containing the H chain regino C was extracted. . The plasmid thus produced was designated "MBC1HcDNA / pUC19". This plasmid contained the cDNA encoding the V regrno of the H chain of the murine antibody and the re / n of Cyl of the human antibody and has an EcoRI and HindIII recognition sequence at the 5 'end and a BamHI recognition sequence at the 3' end.

The plasmid MBOHcDNApUC? Was digested with EcoRI and BamHI to obtain DNA encoding the H chain of the chimeric antibody. The resulting DNA fragment was inserted into the expression vector pCOS1, which was tpawrnon EcoRI and BamHI. The expression vector for the chimeric antibody was designated "MBC1HcDNA / pCOS1". Subsequently, the sOnostounwaon expression vector pCOS1 using HEF-PMh-gG1 (see WO 92/19759) by removing the antibody from my gene using EcoRI and SmaI and then ligating with the EhnRI-NntI-BamHI (Takara Shuzo) Plasmid StcnjLzeola Adapter For expression in CHO, plasmid MDCtHcDNApUC19 was digested with EcoRI and BamHI to obtain DNA encoding the H chain of the chimeric antibody which was then crvwenenone of the pCHO1 expression plasmid which was digested with EcoRI and BamHI. Expression plasmid for the hhimers'he.oego nenacenon antibody "MBC1HcDNApCHO1". The expression vector pCHO1 sknostPunwaon using DHFR-AE-rvH-PM-1-f (see WO92 / 19759) by removing the antibody gene from the oregn using EcoRI and SmaI digestion followed by Co ligation of the EcnRI-NntI-BamHI Adapter (Takara Shuzo).

190 351 (2) Construction of L light chain C region (i) Generation of cloning vector

To construct the pUC19 vector containing the human L chain C region, the vector pUC19 with the HindIII site removed was generated. 2 pg of vector pUC19 was digested with 20 µl of a reaction solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl 2, 1 mM DTT, 100 mM KCl, 8 U HindIII (Takara Shuzo) at 37 ° C for 1 hour. The resulting digestive mixture was extracted with phenol and chloroform, and then subjected to ethanol precipitation to collect the desired DNA.

The collected DNA was reacted in 50 µl of a reaction solution consisting of 50 mM Tns-HCl (pH 7.5), 10 mM MgCl, 1 mM DTT, 100 mM NaCl, 0.5 mM dNTP and 6 U Klenow fragment (Gibco BRL) at room for 20 minutes, resulting in blunt DNA ends. The reaction mixture was extracted with phenol and chloroform, and then subjected to ethanol precipitation to collect the desired DNA.

Vector DNA was reacted in 10 µl of a reaction solution consisting of 50 mM Tris-HCl (pH 7.6), 10 mM MgCT, 1 mM ATP, 1 mM DTT, 5% polyethylene glycol 8000, and 0.5 U T4 DNA ligase (Gibco BRL ) at 16 ° C for 2 hours to religate the vector DNA. 5 µl of the reaction solution was added to 100 µl of the solution containing E. coli JM 109 competent cells (Nippon Gene). The cell mixture was left for 30 minutes on ice, at 42 ° C for one minute, then for one minute on ice. Cells were mixed with 500 µl SOC medium and incubated at 37 ° C for one hour. The resulting cell suspension was smeared over 2xYT medium with agar (supplemented with 50 µg / ml ampicillin) which was applied with X-gal on the surface (Molecular Cloning: A Laboratory Manual, Sambrook, et al. Cold Spring Harbor Laboratory Press, 1989) and incubated at 37 ° C overnight. In this way, E. coli transformants were produced.

Transformants were grown overnight in 2xYT medium supplemented with 50 µg / ml ampicillin at 37 ° C. Plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN). The plasmid DNA thus obtained was digested with HindIII. A plasmid found to have a HindIII site is designated "pUC19AHindIII" (ii) Construction of DNA encoding the C region of the X chain, the human L chain

It is known that the C region of the X chain of the L chain of a human antibody comprises at least the four isotypes Mcg + Ke + Oz-, Mcg-Ke-Oz-, Mcg-Ke-Oz + and Mcg-Ke + Oc- (Dariavah et al., Proc. Natl. Acad. Sci. USA 84: 9074-9078, 1987). A search for the C region of the X chain of the L chain of a human antibody homologous to the C region of the X chain of the murine L chain # 23-57-137-1 was performed based on the EMBL database. As a result, the isotype Mcg + Ke + Oz- (Accession No. (57819 (Dariavah et al., Proc. Natl. Acad. Sci. USA 84: 9074-9078, 1987) of the human antibody X chain was found to have the highest homology with the C region X chain of mouse # 23-57-137-1 L chain, sharing 64.4% amino acid sequence homology and 73.4% DNA sequence homology.

Next, construction of DNA encoding the C region of the X chain of the L chain of the human antibody was performed by PCR. All primers were synthesized using the 394 DNA / RNA Synthesizer (ABI) machine. The primers HLAMB1 (SEQ ID NO: 11) and HLAMB3 (SEQ ID NO: 13) with sense DNA sequence, and HLAMB2 (SEQ ID NO: 12) and HLAMB4 (SEQ ID NO: 14) with the antisense DNA sequence were synthesized. each primer contained a complementary sequence of 20-23 bp at both ends.

The outer primers of HLAmBs (SEQ ID NO: 15) and HLAMBR (SEQ ID NO: 16) had a complementary sequence to the primers HLAMB1 and HLAMB4, respectively, but contained sequences recognized by EcoRI, HindIII and BlnI and EcoRI, respectively. In the first t-oLnii rpolzoiP Τ-ΓΤ Δ NAR 1 _ΐ-ΤΤ and Ϊ-ΓΤ ΔΚΛΤί ^ -Τ-ΤΤ ΔΑΛΏζΙ T ^ Q of the reaction, both results were mixed in equal amounts and combined with another PCR reaction. External HLAMBS and HLAMBR primers were added to the resulting product. The reaction mixture was subjected to a third PCR reaction to amplify full-length DNA.

PCR reactions were performed using TaKaRa Ex Taq (Takara Shuzo) according to the manufacturer's instructions. The first PCR reaction used either 100 µl of a reaction solution consisting of 5 pmol HLAMB1, 0.5 pmol HLAMB2, and 5 U TaKaRa Ex Taq (Takara Shuzo), or

190 351 reaction solution containing 0.5 pmoles of HLAMB 4 and 5 U TaKaRa Ex Taq (Takara Shuzo) overlaid with 50 μ) of mineral oil and carried out 5 cycles of PCR reaction using a program of 1 minute at 94 ° C, 1 minute at 60 ° C and 1 minute at 72 ° C. In the second PCR reaction, a mixture of 50 μL of each of the reaction solutions was used, overlayed with 50 μL of mineral oil, and three cycles of PCR were performed using a program of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C. . The third PCR reaction used a reaction solution to which 50 pmoles of each of the HLAMBS and HLAMBR outer primers were added, and 30 cycles of PCR were performed using a program of 1 minute at 94 ° C, 1 minute at 60 ° C and 1 minute at 72 ° C.

The DNA fragment obtained from the third PCR reaction was electrophoresed with 3% low freezing point agarose (NuSieve GTG, FMC), harvested and gel purified using the GENECLEAN II kit (BIO1 01) according to the manufacturer's instructions.

The thus obtained DNA fragment was digested with 20 µl of a reaction solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl, 1 mM DTT, 100 mm NaCl, and 8 U EcoRI (Takara Shuzo) at 37 ° C for one hour. The digestive solution was extracted with phenol and chloroform and then ethanol precipitated to give DNA. DNA was collected and dissolved in 8 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA).

0.8 µg of plasmid pUC19 AHindIII was digested with EcoRI in the same manner as described above. The digestive solution was extracted with phenol and chloroform, followed by ethanol precipitation to give pUC19 AHindIII. The plasmid was harvested and dissolved in 50 μl of a solution containing 50 mM Tris-HCl (pH 9.0), 1 mM MgCE and alkaline phosphatase (E coli C75, Takara Shuzo) at 37 ° C for 30 minutes to dephosphorylate the plasmid (i.e. BAP treatment). The reaction solution was subjected to phenol and chloroform extraction and ethanol precipitation to obtain DNA. The digestive solution was extracted with phenol and chloroform, and then ethanol precipitated to obtain DNA. DNA was collected and dissolved in 10 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA).

μ! BAP-treated pUC10 AHindIII plasmid was mixed with 4 µl of the DNA obtained from the above-described PCR reactions, ligated together using the DNA ligation kit ver. 2 (Takara Shuzo). The resulting plasmid was introduced into E. coli JM 109 competent cells to prepare transformants. Transformants were grown overnight in 2 ml of 2xYT medium containing 50 µg / m! ampicillin. Plasmid was purified from cells using the QLAprep Spin Plasmid Kit (QLAGEN).

With respect to the plasmid described above, the DNA sequence was confirmed. The 373A DNA Sequencer (ABI) device and the primers "M13 Primer M4" and "M13 Primer RV" (Takara Shuzo) were used to determine the DNA sequence. As a result, the cloned DNA was found to have a 12 bp deletion. The plasmid containing the DNA was designated "c AA / pUC19". Subsequently, the HCLMS (SEQ ID NO: 17) and HCLMR (SEQ ID NO: 18) primers were made to generate a portion and the correct DNA was reconstructed using a PCR method.

A first PCR was performed using the C AA / pUC19 plasmid containing the deleted DNA as template, followed by the HLAMBS and HCLMS primers or the HCLMS and HLAMB4 primers. Each of the PCR reaction products was purified. In the second PCR reaction, the PCR products were combined together. The outer primers HLAMBS and HLAMB4 were added to the product, followed by a third PCR reaction to amplify the full-length DNA.

In the first PCR reaction, 100 μl of reaction solutions containing 0.1 μg of cAA / pUC19 as a template were used, 50 pmol of each of the HLAMBS and HCLMR primers or 50 pmol of each of the HCLMS and HLAMB4 primers and 5 U TaKaRa Ex Taq (Takara Shuzo) were used, 50 µl of mineral oil was overlayed and 30 cycles of PCR were performed using a cycle of 1 minute at 94 ° C, 1 minute at 60 ° C and 1 minute at 72 ° C.

The PCR products HLAMBS-HCLMR (236 bp) and HCLMS-HLAMB4 (147 bp) were electrophoresed using 3% low freeze point agarose to isolate the DNA fragment. The DNA fragment was harvested and gel purified using the GENECLEAN II kit (BIO1O1). In the second PCR reaction, 20 μl of a reaction solution containing 40 ng of the purified DNA fragment and 1 U TaKaRa Ex Taq (Takara Shuzo) was used, overlayed with 25 μl

190 351 mineral oil and 5 cycles of PCR were performed using a cycle of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C.

In the third PCR reaction, 100 μΐ of the reaction solution containing 2 μΐ of the reaction solution obtained in the second PCR reaction, 50 pmol of each of the outer primers HLAMBS and HLAMB4 and 5 U TaKaRa Ex Taq (Takara Shuzo) were used, 50 μΐ of mineral oil was layered and 30 cycles were performed. PCR reactions using a cycle of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C, yielding a 357 bp DNA fragment (third PCR product). The DNA fragment was electrophoresed using 3% low freeze point agarose to isolate the DNA fragment. The DNA fragment was collected and gel purified using the GENECLEAN II kit (BIO101).

0.1 µg of the DNA fragment was digested with EcoRI and then cloned into the plasmid pUC19 AHind III, which had been treated with BAP. The resulting plasmid was introduced into E. coli JM109 competent cells to obtain transformants. The transformants thus prepared were grown overnight in 2 ml of 2xYT medium containing 50 µg / ml of ampicillin. Plasmid was purified from the cell fraction using the QIAgen Spin Plasmid Kit (QUIAGEN).

The DNA sequence of the obtained plasmid was confirmed using M13 Primer M4 (Takara Shuzo) and M13 Primer RV (Takara Shuzo) primers using a DNA Sequencer 373A device (ABI; Perkin Elmer). The plasmid containing the correct DNA sequence without deletion was designated "CX / pUC19".

(iii) Construction of DNA encoding the human L chain, κ chain C region

The DNA fragment encoding the C region of the κ chain of the L chain was cloned from the plasmid HEF-PMlk-gk (WO 92/19759) by PCR. The HKAPS primer (SEQ ID NO: 19) was designed to contain EcoRI, Hmdin and BlnI recognition sequences, and the HKAPA reverse primer (SEQ ID NO: 20) was designed to contain an EcoRI recognition sequence by both of which were synthesized on a 394 DNA / RNA Synthesizer (ABI) machine.

The PCR reaction was performed using 100 µl of a reaction solution containing 0.1 µg of HEF-PMlkgk plasmid as template, 50 pmol of each of the HKAPS and HKAPA primers, and 5 U TaKaRa Ex Taq, overlaid with 50 µl of mineral oil. 30 cycles of PCR reactions were performed using a cycle of 1 minute at 94 ° C, 1 minute at 60 ° C, and 1 minute at 72 ° C. The amplified fragments in a 360 bp PCR reaction were separated by agarose gel electrophoresis using 3% low temperature agarose. clotting and the DNA fragment was purified using GENECLEAN II (BIO101).

The DNA fragment was digested with EcoRI and cloned into the plasmid pUC19 AHindUI which had been treated with BAP. The resulting plasmid was introduced into E. coli competent cells, JM 109, and grown overnight in 2xYT medium supplemented with 50 µg / ml ampicillin at 37 ° C. Plasmid DNA was prepared using the QIAprep Spin Plasmid Kit (QIAGEN).

The plasmid DNA thus obtained was sequenced with the M13 Primer M4 and the M13 Primer RV using a 373A DNA Sequencer (ABI). A plasmid with a confirmed correct base sequence was designated "CK / pUC19" (3) Construction of the L chain expression vector of a chimeric antibody

The L chain of chimeric antibody # 23-57-137-1 was constructed. DNA encoding the V region of the L chain of chimeric antibody # 23-57-137-1 was ligated at the Hin <4TTT and Blnl sites immediately upstream of the human antibody C region of each of the CA / pUC19 iCx / pUC19 plasmids, thereby obtaining the pUC19 DNA containing vectors encoding the V region of the L chain and the C region of the κ chain of the L chain of chimeric antibody # 23-57-137-1. Each of the resulting vectors was digested with EcoRI by excising the EcoRI DNA encoding the L chain of the chimeric antibody, and the DNA was cloned into the HEF expression vector.

DNA encoding the V region of the L chain of antibody # 23-57-137-1 was cloned from plasmid MBC1L24 by PCR. The primers were individually synthesized using 394 DNA / RNA Synthesizer (ABI). The reverse primer MBCCHL1 (SEQ ID NO: 21) was designed to contain Kozak consensus sequences (Kozak et al., J. Mol. Biol. 196: 947-950, 1987) and the MBCCHL3 primer (SEQ ID NO. 22) was designed to contain BglI and EcoRI recognition sequences.

190 351

A PCR reaction was performed using 100 µl of a reaction solution consisting of 10 µM Tns-HCl (pH 8.3), 50 mM KCl, 0.25 mM dNTP (dATP, dGTP, dCTP, dTTP), 1.5 mM MgCl 2, 2.5 units AmpliTaq (Perkln-Elmyr), 0.1 µg MBC1L24, 50 pmol of each of the MBCCHL1 and MBCCHL3 primers, which was covered with 50 µl of mineral oil. 30 cycles of PCR were performed using a temperature cycle of 45 seconds at 94 ° C, 45 seconds at 60 ° C and 2 minutes at 72 ° C

The 444 bp DNA fragment was electrophoresed on an agarose gel using 3% Low Freezing Point agarose and the DNA fragment was purified using GENECLEAN II (BIO 101). The purified DNA fragment was dissolved in 20 µl of a solution containing 10 µM Tris-HCl (pH 7.4) and 1 mM EDTA. 1 µl of PCR product was digested in 20 µl of a reaction solution consisting of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2, 1 mM DTT, 50 mM NaCl, 8 U HindIII (Takara Shuzo) and 8 U EcoRI at 37 ° C for one hour. The digestive solution was extracted with phenol and chloroform, and then precipitated with ethanol to give DNa. DNA was collected and dissolved in 8 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA).

pg of plasmid pUC19 was digested with HindIII and EcoRI in the same manner as described above. The digestive solution was extracted with phenol and chloroform and then with ethanol to give a digested plasmid. The plasmid was treated with BAP (alkaline phosphatase, E. coli C75; TaKara Shuzo), extracted with phenol and chloroform, and clarified with ethanol. DNA was collected and dissolved in 10 µl of a solution containing 10 µM Tris-HCl (pH 7.4) and 1 mM EDTA.

pl of BAP treated plasmid pUC10 was mixed with 4 µl of PCR product for ligation using DNA ligation kit ver. 2 (Takara Shuzo). The obtained plasmid was introduced into E. coli JM109 (N ^ pon Gyzy) competent cells as described above to obtain trazsCorzazt. The thus produced trαzformins were mounted overnight on 2xYT medium containing 50 µg / ml ampicillin at 37 ° C. The resulting trazformαzt was cultured overnight in 2 ml of 2xYT medium containing 50 µg / ml ampicillin at 37 ° C. Plasmid was purified from the cell fraction using the piAgen Spin Plaszid Kit (QUIAGEN). After the DNA sequence was determined, the plasmid was confirmed to be the correct DNA sequence and designated "CHL / pUC19".

pg Each of the CZ / pUC19 and Ck / pUC19 plasmids was digested in 20 µl of a reaction solution consisting of 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U HindIII, and 2 U BlnI at 37 ° C for one hour. The digestive solution was extracted with phenol and chloroform, followed by precipitation with ethanol to obtain DNA. DNA was treated with BAP at 37 ° C for 30 minutes, followed by phenol and chloroform extraction followed by ethanol extraction. The product was dissolved in 10 µl of a solution consisting of 10 mM TrisHCl (pH 7.4) and 1 mM EDTA µl of CHL / pUC19 plasmid, which contained DNA encoding the V region of the L chain of antibody # 23-57-137-1, digested with HindIII and BlnI as described above way. The 409 bp DNA fragment was electrophoresed using 3% low Freezing Point agarose and harvested and purified using the GENECLEAN II kit. The DNA fragment was dissolved in 10 µl of a solution consisting of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.

pl DNA for the V region of the L chain was cloned into 1 µl of the BAP CZ / pUC19 or ck / pUC19 tractor plasmids and introduced into competitive El cells. coli, JM 109 to give trαzsformantj '. Transformites were grown overnight in 3 ml of 2xYT medium containing 50 µg / ml of ampicillin. Plasmid was purified from cells using the QIAprep Spin Plasmid Kit (QIAGEN). The plasmid thus obtained was designated "MBC1T (Z) /" pUC19 "or" MBC1L (K) / pUC 19 ".

and / · * / · * O T

MBC1L (Z -) / pUC19 and MBC1L (k) / pUC19 were digested with Ec Electrophoresed using 3% Low Clotting Point agarose. A 743 bp DNA fragment was isolated and gel purified using the GENECLEAN II kit (BIO 101). then dissolved in 10 µl of a solution containing 10 mM Tris-HCL (pH 7.4)

Each . - - j from plazzids • r ..........

from _- zM EDTA

With 2.7 pg of the expression vector, the HEF-PM1K-gk plasmid was digested with EcoRI and extracted with phenol and chloroform, followed by precipitation with ethanol to give DNA. Fragment

190 351

DNA was treated with BAP followed by electrophoresis with 1% low freeze point agarose, and 6561 bp DNA was purified from the gel using the GENECLEAN II kit (BlO101). DNA was collected and dissolved in 10 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.

The HEF vector was prepared and treated with BAP, and 2 µl of the BAP treated HEF vector was mixed with 3 µl of the EcoRI fragment of either MbC1 (λ) / pUC19 or MBC1L (k) / pUC19 for ligation. The ligation product was introduced into competent E. coli JM109 cells to obtain transformants. Transformants were grown overnight in 2 ml of 2xYT medium containing 50 µg / ml of ampicillin. Plasmid was purified from cells using the QIAprep Spin Plasmid Kit (QLAGEN).

Plasmid DNA purified in this way was digested in 20 µl of a solution containing 20 mM Tris-HCl (pH 8.5), 10 mM MgCl ₂ , 1 mM DTT, 100 mM KCl, 8 U HindIII (TaKara Shuzo) and 2 U Pvul (Takara Shuzo) at 37 ° C for one hour. In this digestion reaction, it was assumed that if the DNA fragment was inserted into the vector in the correct orientation, a 5104/2195 bp fragment would be obtained, while if the DNA fragment was inserted into the vector in the reverse orientation, this would result in a fragment of 4387/2926 bp in size. Based on these assumptions, plasmids in which the fragment was inserted in the correct orientation are designated "MBC1L (() / neo" and "MBC1L (K) / neo".

(4) Transfection of COS-7 cells

The expression plasmids produced as above were transiently expressed using COS-7 cells to test the chimeric antibody for binding neutralizing activity against the antigen.

Transient expression of the chimeric antibody was achieved by combining plasmids MBC1HcDNA / pCOSl and MBC1L (Zj / noo or plasmids MB3CIHcDNA // pCOSl MBBC1L (K) / neo using a Gene Pulser (B ^ RaT) device to co-transfect each of the plasmids into COS- 7. That is, to 0.8 ml of a cell suspension in which COS-7 cells were suspended in PBS (-) at a density of 1x10 ⁷ K cells / ml, 10 pg of each of the plasmid DNAs was added. The resulting solution was pulsed at 1500 V and a capacity of pF causing elecroporation After 10 minutes at room temperature, cells were suspended in DMEM medium containing 2% Ultra Low IgG fetal calf serum (Gibco), and then cultured using a 10 cm dish in a CO ₂ incubator. After culturing for 72 hours, the culture supernatant was collected and centrifuged to remove cellular debris and provided as sample for ELISA.

In this procedure, purification of chimeric antibodies from the culture supernatant of COS-7 cells was performed using the Affigel Protein A MAPSII kit (Bio Rad) according to the manufacturer's instructions.

(5) ELISA test (i) Determination of antibody concentration

An ELISA plate for determining the antibody concentration was prepared as follows. The wells of a 96-wellKownj ELISA plate (MaxisOTp, NUNC) were coated with 100 µl of a solution containing Goat anti-human IgG (TAGO) prepared in coating buffer (0.1 M NaHCOj, 0.02 NaNj) at a concentration of 1 µg / ml, and then blocked with 200 µl of dilution buffer (50 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN ₂ , 1% Bovine Serum Albumin (BSA); pH 7 , 2). COS-7 Cell culture supernatant expressing chimeric antibodies or purified chimeric antibodies in gradual dilutions was added to each of the wells. After incubation at room temperature for one hour and washing with PBS-Tween 20, 100 µl of an alkaline phosphatase-conjugated goat anti-human IgG (TAGO) solution was added to each of the wells. After incubating, and at room temperature for one hour, and washing with PBS-Tween 20, 1 mg / ml of substrate solution ("Sigma 104", p-n -trophenylphosphoric acid, Sigma) was added to each well. The absorbance of the solution was measured at 405 nm using a Microplate Reader (Bio Rad). Purified Hu IgG1 ((The Binding Site) was used as standard.

(ii) Determining the antigen-binding capacity

An ELISA plate for determining antibody binding was prepared as follows

190 351

Each well of a 96-well A plate was coated with 100 µl of a solution containing human PTHrP (1-34) (Peptide Research Institute) prepared in 1 µg / ml buffer and then blocked with 200 µl of blocking buffer. The culture supernatant of COS-7 cells in which the chimeric antibodies or the purified chimeric antibodies were expressed was added in stepwise dilution to each well. After incubation at room temperature and washing with PBS-Tween 20, 100 µl of a solution of goat anti-human IgG conjugated with alkaline phosphatase (TAGO) was added to each well. After incubation at room temperature and washing with PBS-Tween 20, 1 mg was added to each of the wells. / ml of substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, Sigma) The absorbance of the solution was measured at 405 nm using a Microplate Reader (Bio Rad).

As a result, the chimeric antibody was found to bind human PTHrP (1-34) as well as to have the correct structure of the cloned murine antibody V region (Figure 4). It has also been found that there are no differences in the binding capacity of PTHrP (1-34) between a chimeric antibody in which the C region of the L chain has an X chain and one in which the C region of the L chain has a k chain. Hence, the C region of the L chain was constructed. a humanized antibody using the X chain of the L chain of a humanized antibody (6) Establishing a stably transformed CHO cell line

To establish stable transformants for the chimeric antibody, the expression plasmid described above was introduced into CHO cells (DXB11).

Establishment of stable transformants of the chimeric antibody was performed using a combination of CHO cell expression plasmids, MBS1HcDNA / pCHO1 and MBC1L ^) / neo or CHO cell expression plasmids MBC1HcDNApCHO1 and MBC1L (k) / neo. These plasmid combinations were individually co-transfected into CHO cells by electroporation using a Gene Pulser (Bio Rad). Each of the expression vectors was cleaved with the restriction enzyme PvuI to obtain linear DNA. The resulting DNA was collected by phenol and chloroform extraction followed by ethanol precipitation. The expression plasmids produced in this way were electroporated. 10 µg of each plasmid DNA was added to 0.8 ml of a cell suspension containing CHO cells in PBS (-) at a density of 1x10 ⁷ cells / ml. The resulting mixture was pulsed at 1500 V and a capacity of 25 µF. After ten minutes at room temperature, the cells were suspended in MEM-α medium (Gibco) supplemented with 10% fetal calf serum (Gibco). The resulting suspension was grown in three 96-well plates (Falcon) in a CO2 incubator. The day after the start of the culture, the medium was replaced with a selective medium (MEM-α medium supplemented with 10% fetal calf serum (Gibco) and 500 pg / ml geneticin (G418 sulfate, Gibco) without ribonucleosides or deoxyribonucleosides. After replacing the selective medium with fresh medium. , before and after two weeks of culture, cells were observed under a microscope. When cell growth was observed, cells were tested for the amount of produced antibodies in an ELISA test. Among the cells were selectively collected those that produced larger amounts of antibodies

Up-scaling of the stable transformant cultures for the established antibodies was performed in roller bottles using MEM medium supplemented with 2% Ultra Low IgG1 fetal calf serum without ribonucleosides and deoxyribonucleosides. On culture days 3 and 4, the culture supernatants were harvested and filtered through a 0.2 µm culture filter (Millipore) to remove cell debris.

Subsequently, purification of chimeric antibodies from the supernatant of CHO cells was performed using Poros Protein A Column (PerSeptive Biosystems) on ConSep LC100 (Millipore) according to the manufacturer's instructions. Purified chimeric antibodies were provided as samples for determining the neutralizing activity and for testing the efficacy of a hyperxlcemic animal model. The concentration and binding activity of the purified chimeric anti-antigen antibodies were determined by the same system

ELISA.

190 351

Example 3

Construction of human antibodies (1) Construction of the H chain of a humanized antibody (i) Construction of the V region of the H chain

The H chain of antibody # 23-57-137-1 was generated by CDR grafting using PCR. For the production of the H chain of humanized antibody # 23-57-137-1 (version "a") with FRs derived from human antibody S31679 (NMRF-PDB ;ędziinier et al., Eur J. Immunol 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 (containing the sense DNA sequence) and MBC1HGP2 (SEQ ID NO: 25) and MBC1HGP4 (SEQ ID NO: SEQ ID NO: 26) (containing the DNA antisense sequence) which all included a 15-21 bp complementary sequence at both ends, and the outer primers MBC1HVS1 (SEQ ID NO: 27) and MBC1HVR1 (SEQ ID NO: NO). 28), including homology with CDR grafting primers MBC1HGP1 and MBC1HGP4, respectively

The CDR grafting primers MBC1HGP1, MBC1HGP3, MBC1HGP2 and MBC1HGP4 were isolated using urea-denatured polyacrylamide gel (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, 1989) and extracted from the gel fractions by crushing and soaking (Molecular Cloning Laboratory: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) as follows.

nmol of each of the CDR grafting primers was isolated with a 6% denatured polyacrylamide gel to obtain a DNA fragment. From the resulting DNA fragments, fragments of the desired size were identified by UV irradiation on a silica gel plate, and then collected by crushing and soaking. The product was dissolved in 20 μl of a solution consisting of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo). The reaction solution (100 μl used in the PCR reaction included 1 μl of each of the aforementioned CDR grafting primers MBC1HGP1, MBC1HGP3, MBC1HGP2 and MBC1HGP4, 0.25 mM dNTP and 2.5 U TaKaRa Ex Taq in buffer. Five cycles of PCR were performed using a cycle of a cycle. consisting of 1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C. The outer primers MBC1HVS1 and MCB1HVR1 were added to the reaction product at 50 pmol each. Using this reaction mixture, an additional 30 cycles of PCR were performed using the same temperature cycle The DNA fragment amplified in this way was isolated by agarose gel electrophoresis with 4% NuSieve GTG agarose (FMC Bio. Products).

The agarose fragment containing the 421 bp DNA fragment was excised and the DNA fragment purified using the GENECLEAN II kit (BIO101) according to the manufacturer's instructions. The DNA fragment thus purified was ethanol precipitated and dissolved in 20 μl of a solution comprising 10 mM Tris-HCl (pH 7.4) pil mM EDTA. The reaction mixture was used to clone the DNA fragment into plasmid pUC19, which had been prepared by digestion with BamHI and HindIU, and then the base sequence of the plasmid was determined. The plasmid with the correct sequence was designated "hMGCHv / pUC19".

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

For the cDNA ligation of the C C1 region of the humanized H chain, the V region of the H chain was constructed according to the above step by PCR. In this method, a reverse primer MBC1HVS2 was designed to hybridize to a sequence encoding the 5 'region of the V-region leader sequence and to possess the Kozak sequence (Kozak et al., J. Mol. Biol. 196: 947-950, 1987) and HindIU and EcoRI recognition sequences. The MBC1HVR2 primer used to modify the DNA of the H chain V region was designed to hybridize to both DNA sequences encoding the 3 'region of the J region and the DNA sequence encoding the 5' region of the C region and bearing the sequences recognized by ApaI and SmaI.

The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo) and the supplied bufom The reaction solution used for the PCR reaction included 0.4 μg hMBCHv / pUC19 as template DNA, 50 pmol of each of MBC1HVS2 and MBC1HVR2 as primers, 2.5 U TaKaRa Ex Taq and 0.25 mM dNTP in buffer. 30 cycles of PCR reactions were performed using a cycle of 1 minute at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C. Amplified

The 190,351 PCR fragments were separated by agarose gel electrophoresis using 3% NuSieve agarose (FMC Bio. Products).

Then, the gel fragment containing the 456 bp DNA fragment was excised and the DNA fragment purified using GENECLEAN II (BIO101) according to the manufacturer's instructions. The purified DNA fragments were ethanol precipitated and dissolved in 20 µl of a solution containing 10 mM Tris-HCl (pH 7.4) µM EDTA . The PCR reaction mixture was used to clone the DNA fragment into plasmid pUC19, which had been prepared by digesting plasmid EcoRI and SmaI; then the DNA sequence of the resulting plasmid was determined. Plasmid DNA which contains the DNA encoding the H chain V region from hybridoma # 23-57-137-1 and contains EcoRI and HindIII recognition sequences and a Kozak sequence at the 5 'end and the sequences recognized by ApaI and SmaI at the 3' end are labeled " hMBC1Hv / pUC19 ".

(2) Construction of a humanized antibody H chain expression vector

The RVh-PM1f-cDNA plasmid containing the H chain cDNA sequence 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.

The DNA fragment was introduced into the hMBC1Hv / pUC19 plasmid, which was prepared by digesting the ApaI and BamHI plasmids. The plasmid prepared in this way was designated "hMBC1HcDNApUC19". This plasmid contained DNA encoding the V region of the H chain of humanized antibody # 23-57-137-1 and DNA encoding the Cy1 region of the C chain of the H chain, and had EcoRI and HindIII recognition sequences in the 5 'region and BamHI recognition sequences in the 3' region. the corresponding amino acid sequence of the humanized H chain version "a" in plasmid hMBC1HcDNA / pUC19 is shown in SEQ ID NO. No. 58 and Id. Seq No. 56

The hMBC1HcDNA / pUC19 plasmid was digested with EcoRI and BamHI to give a DNA fragment containing the base sequence encoding the H chain. The DNA fragment was introduced into an expression plasmid pCOS1 which had been prepared by digesting EcoRI and BamHI plasmids. The expression plasmid for the humanized antibody obtained in this way was designated "hMBC 1 HcDNA / pCOS 1"

To generate a plasmid for expression in CHO cells, the hMBC1HcDNApUC19 plasmid was digested with EcoRI and BamHI to give a DNA fragment containing the base sequence encoding the H chain. The DNA fragment was introduced into an expression plasmid pCHO1 which had been prepared by digesting plasmids with EcoRI and BamHI. The expression plasmid for the humanized antibody obtained in this way was designated "hMBC1HcDNApCHO1" (3) Construction of the hybrid L chain variable region (i) Generation of the hybrid antibody FR1.2 / FR3.4

DNA encoding the L chain was constructed in which FR regions were recombined with regions from the humanized antibody and murine (chimeric) antibody, and the region examined for its suitability for humanization. In this step, a hybrid antibody containing FR1 and FR2 derived from human antibody and FR3 and FR4 derived from mouse antibody was generated by using the Af1II restriction site present in CDR2 pg of each of the MBC 1 L (X) / neo and hMBC1L (k) neo plasmids. digested in 100 µl of a reaction solution consisting of 10 mM Tris-HCl (pH 7.5), 10 mM MgCF, 1 mM DTT, 50 mM NaCl, 0.01% (w / v) BSA and 10 U AfHI (Takara Shuzo ) at 37 ° C for one hour. The reaction solution was electrophoresed on a 2% low freezing point agarose gel and DNA fragments of 6282 bp (referred to as "cl") and 1022 bp (referred to as h1 ") were collected and purified from the SC GEEJECEAAN II (BIOIO.).

pg of c1 and h1 fragments were subjected to BAP treatment. The DNA fragment was extracted with phenol and chloroform, collected by ethanol precipitation, dissolved in 10 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.

pl of BAP treated c 1 and h1 DNA fragments were mixed with 4 µl of h2 and c2 DNA fragments for ligation (at 4 ° C overnight). The ligation product was introduced into E coli JM109 competent cells to obtain transformants. Transformants were grown in 2 ml of 2xYT

190 351 containing 50 pg / ml ampicillin From the cell reaction, the plasmid was purified using the αIAprep Spis Plasmid Kit (QIAGEN).

Plasmid DNA was digested in 20 µl of a reaction solution consisting of 10 mM TrisHCl (pH 7.5), 10 mM MgCL, 1 mM DTT, 2 U ApaLI (Takaoa Shuzo) and 8 U BamHI (Takara Shuzo) or HiodIII (Takaoa Shuzo) for one hour at 37 ° C. At this stage, the erdeotcfrknwaon plasmid is expected that if the c1-h2 fragment has been correctly ligated into the plasmid, it should give ApaLI fragments of 5560/1246/498 bp, and a BamHI / HioCni digestion of 7134/269 bp fragments.

Expression vector encoding the hybrid L chain antibody human FRJ, 2 / mouse FR3,4 neoacennn "h / mMBClL (λ) / nen". On the other hand, it was possible not to obtain clone h1-c1. StąC, recombination of the pUC vector was performed, followed by Olnanwaare Co of the HEF vector. The plasmid hMBC1Lak / pUC19 was used as the DNA template, Itozyp containing DNA encoding the V region of the H chain of a humanized antibody without acid substitutions, and the plasmid hMBC1LdZ / pUC19, which iNA / O ncUajhcy e / V gion of the Latin L-amino acid substitutions containing amino acid substitutions position 91 in FR3 (ie amiynoacid or 87 as defined by Kabat), tyrosine by ienJeucca.

pl OażCego from plasmids MBC1L (X) / pUC19, hMBC1LaX / pUC19 and hMBC1LCX / pUC19 trawlnon in 30 µl of nbeam_aqhegn reaction solution 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mMDTT, 50 mM NaCl, 0.01 % (w / v) BSA, 16UHmdIII and 4UAfHI at 37 ° C for 1 hour The reaction solution was electrotreated with a 2% low freezing point agarain gel; and then the following DNA fragments were purified using the GENECLEAN II kit (BIO1-1): 215 bp DNA fragment from MBdL (Z) / pUC19 plasmid (referred to as "c2") or 3218 bp DNA fragment from each / oz of irMBC1Lak / pUC19 plasmids and hMBC1LCZ / pUC19MBC (nOoeśJaac here as ha1 'and hdP).

Each of the Ca1 'and hd1 fragments' cnddayn h / ation with fOa / c2' mentation and was introduced into competent E. coli JM 109 ntozcm_aqi traysfoomanty cells. Cndnwaon traosformants in 2 ml of 2xYT medium containing 50 pg / ml of ampricJmy. Plasmid was purified from the cell fraction using the QIAprep Spio Plasmid Kit (QIAGEN). The resulting plasmid DNA containing the haF and hd1 'fragment was designated "m / hMBC1LaX / pUC19' 'and" 014 ^ / 030 ^ 0- / pUC19 ".

Each of these plasmids "m / hMBC1Lak / pUC19" and "m / hMBC1Laλ / pUCI9" were digested with EcoRI Foa / 743 bp DNA metothesis, electrophoresed using a 2% low coagulation point agaro gel and then harvested and gel purified using GENECLEAN II kit (BIO101). The product was dissolved in 20 µl of a solution containing 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA.

pl of the DNA fragment was mixed with 1 pl of the above-mentioned traOtnwaoegn BAP HEF vector for ligation. The ligation product was introduced into Co competent E. coli JM109 cells to obtain traosformants. Hndnwaan transfamants in 2 ml of 2xYT medium containing 50 µl of ampicillin From the cell fraction, the plasmid was purified using the Q1Acrep Spio Plasmid Kit (QiAGEN).

Plasmid DNA was purified and digested in 20 µl of nbeam reaction solution 20 mM Tris-HCl (41 8.5), 10 mM MgCl 2, 1 mM DTT, 100 mM KCl, 8 U HindIII (Takara Shuzo) and 2 U PvcI (Takaoa Shuzo) at 37 ° C by pig. At this stage, the plasmid DNA was identified based on the expectation that, if the DNA foagmeot was inserted into the plasmid in the correct orientation, digestion should result in 5104/2195 bp fragments, while the DNA fragment was inserted into the plasmid in the reverse orientation, digesting the DNA fragment 4378 / 2926 bp. The obtained plasmids were expression vectors plotting lah ^ ena ^ lj Οΰ ^ ΐ o 0 ^^ tOyOry esy jogu oiyaiu u c.1,1 ivnuuz, Ricgu rKj, -t ·, cuic ijziiauzuntp as expression vectors "m / hMBCILak / neo" and "M / hMBClLdk / neo".

(ii) The result of the two! habη / docanC FR P / FR2

The FR1 / FR2 hybrid antibody was prepared in the same manner as described above using the SoaBI restriction site present in CDR1.

pg of each of the MBC.1L plasmids (Lyneo and mMBC1L (I) / neo travrnnn in 20 µl of a reaction solution consisting of 10 mM Tris-HCl (pH 7.9), 10 mM MgCL, 1 mM DTT,

190 351 mM NaCl, 0.01% (w / v) BSA and 6U SnaBI (Takara Shuzo) at 37 ° C for 1 hour The reaction product was further digested in 50 µl of a reaction solution comprising 20 mM Tris-HCl (pH 8, 5), mM MgCl 2, 1 mM DTT, 100 mM KCl, 1.0.01% (w / v) BSA and 6 U Pvul at 37 ° C for one hour.

The reaction product was electrophoresed using a 1.5% low freezing point agarose gel, and DNA fragments of 4955 bp and 2349 bp were collected and purified using the GENECLEAN II kit (BIO101). DNA fragments obtained from plasmid MBC1L (X) / neo were labeled "ml" (4955 bp) and "m2" (2349 bp), while DNA fragments obtained from plasmid h / inMBCT I .. (L) / neo were labeled '(4955 bp) ) and "bm2" (2349 bp)

Each of the DNA fragments was dissolved in 40 µl of a solution containing 10 mM Tris-HCl (pH 7.4) µl mM EDTA.

p1 of each of the ml and hm1 fragments were ligated to 4 µl of each of the hm2 and m2 fragments, and then introduced into competent E. coli cells, JM109 obtain;. c transformants. Transformants were grown in 2 ml of 2xYT medium containing 50 pg / ml ampicillin. From the cell fraction, the plasmid was purified using the QIAprep Spin Plasmid Kit (QLAGEN)

Plasmid DNA was digested in 20 µl of a reaction solution containing 10 mM TrisHCl (pH 7.5), 10 mM MgCF, 1 mm dTt and 8U ApaI (Takara Shuzo) or 2 U ApalI (Takara Shuzo) at 37 ° C for 1 hour

The plasmids (m1-hm2 and hm1-m2) were identified based on the fact that when the DNA fragments were joined in the plasmid in the correct orientation, digesting the plasmid (m1-hm2) with ApaI and ApaLI should yield a 7304 bp fragment and 5560 / fragments, respectively 1246/498 bp, and digestion of plasmid (hm1-m2) with ApaI and ApaLI should yield 6538/766 bp fragments and a 3535/2025 / 1C46) fragment of 498 bp, respectively.

The expression vector encoding the human FR1 / mouse FR2,3,4 hybrid antibody? Chain was designated "hmmMBClL (λ) / neo", aa) vector) → Jkprreyyn → y encoding the L pz chain of the mouse FR1 / human FSZ / mouse FR3.4 hybrid antibody was designated "MhmMBClL (λ yneo") (4) Construction of a humanized antibody L chain

The L chain of humanized antibody # 23-57-137-1 was generated by CDR grafting using a PCR method. Ie. the L chain of humanized antibody # 23-57-137-1 (version "a") was prepared which contained FR1, FR2, and FR3 derived from human antibody HSU03868 (Gen-Bank, Deftos et al., Scand. J. Immunol. 39.95- 103, 1994) and FR4 derived from the human antibody S25755 (NBRF-PDB) using six types of primers

CDR grafting primers MBC1LGP1 (SEQ ID NO 29) and MBC1LGP3 (SEQ ID NO 30) with erneJ DNA sequence, CDR grafting primers MBC1LGP2 (SEQ ID NO 31) iMBC1LGP4 (SEQ ID NO 32) ), both with an antenna sequence, all CDR grafting primers having a complementary sequence of 15-21 bp at both ends; the outer primers MBC1LVSI (SEQ ID NO: 33) and MBC1L ^/ R1 (SEQ ID NO: 34) having homology with the CDR grafting primers MBC1LGP1 and MBC1LGP4, respectively.

The CDR grafting primers MBC1LGP1, MBC1LGP3, MBC1LGP2, andMBC1LGP4 were isolated using urea-enriched poly amide gel (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) and extracted from the gel fraction by crushing and cloning by crushing. Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989).

nmol of each of the CDR grafting primers was isolated with a 6% denatured polyacrylamide gel to obtain DNA tragment. From the resulting DNA fragments, fragments of the desired size were identified by UV irradiation on a silica gel plate, and then collected by crushing and soaking. The product was dissolved in 20 µl of a solution consisting of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA

The PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo) with a buffer. The reaction solution (100 µl) used in the PCR reaction comprised 1 µl of each

190 351 CDR grafting primers MBC1LGP1, MBC1LGP3, MBC1LGP2 and MBC! LGP4, 0.25 mM dNTP and 2.5 U TaKaRa Ex Taq in buffer. Five cycles of PCR reactions were performed using a cycle of 1 minute at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C. The outer primers MBC1LVS1 and MBC1LVR1 at 50 pmol were added to the reaction product. An additional 30 cycles of PCR were run using this reaction mixture using the same temperature cycle. The DNA fragment amplified in this way was isolated by agarose gel electrophoresis with 4% NuSieve GTG agarose (FMC Bio. Products).

The agarose fragment containing the 421 bp DNA fragment was excised and the DNA fragment purified using the GENECLEAN II kit (BIO101) according to the manufacturer's instructions. The DNA fragment thus purified was ethanol precipitated and dissolved in 20 μί of a solution consisting of 10 mM Tris-HCl (pH 7.4) and 1 mM EDTA. The reaction mixture was used to clone the DNA fragment into plasmid pUC 19, which had been prepared by digestion with BamHI and HindIII, and then the base sequence of the plasmid was determined. The plasmid with the correct sequence was designated "hMBCL / pUC19". However, in this plasmid it was found that the amino acid at position 104 (amino acid number 96 of Kabat) of CDR4 was replaced with arginine. In order to correct this amino acid for tyrosine, the MBC1LGP10R primer (SEQ ID NO: 35) was designed. Then, a PCR reaction was performed using TaKaRa Ex Taq (Takara Shuzo) with buffer. The reaction solution (100 μί) used for the PCR reaction contained 0.6 μg of the hMBCL / pUC19 plasmid as template DNA, 50 pmol of each of the MBC1LVS1 iMBCILGPlOR primers, 2.5 U TaKaRa Ex Taq (Takara Shuzo) and 0.25 mM dNTP in buffer on which mineral oil has been deposited. 30 cycles of PCR reactions were performed using a temperature cycle involving 1 minute at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C. The DNA fragment amplified in this way was isolated by electrophoresis on a NuSieve GTG 3% agarose gel (FMC Bio. Products).

A 421 bp DNA fragment was excised and purified using the Geneclean II kit according to the manufacturer's instructions. The PCR reaction mixture was used to clone the DNA fragment into plasmid pUC19, which had been digested with BamHI and HindIII.

The plasmid DNA sequence was determined using the M13 Primer M4 and the M13 Primer RV. As a result, it was confirmed that the plasmid had the correct sequence. The plasmid was then digested with Hindin and Blnl, and a 416 bp DNA fragment was isolated by electrophoresis on a 1% agarose gel. The DNA fragment was purified using the Genecleanll kit according to the manufacturer's instructions, and then inserted into a CX / pUC plasmid that had been digested with HindIII and Blnl. The resulting plasmid was designated "hMBClLaX. / pUC19 ". This plasmid was digested with EcoRI to obtain a DNA fragment encoding the humanized L chain. The DNA fragment was inserted into plasmid pCOS1 such that the humanized L chain start codon was located downstream of the EF1? Promoter. The resulting plasmid was designated "hMBClLaλ / pCOS1". The DNA sequence (including the corresponding amino acid sequence) of the "a" version of the humanized L chain is shown in Id. Seq. No. 66. The amino acid sequence of version "a" is shown in SEQ ID NO. no 47.

Version "b" was prepared using PCR mutagenic introduction techniques. Version "b" was designed such that it replaced the amino acid at position 43, glycine (amino acid # 43 as defined by Kabat) with proline and amino acid at position 49, lysine (amino acid No. 49 as defined by Kabat) with aspartic acid. The PCR reaction was performed using the hMBClLak / pUC19 plasmid as template and the mutagenic MBC1LGP5R primer (SEQ ID NO: 36) and the MBC1LVS1 primer. The DNA fragment was digested with BamHI and HindIII, and the digested fragment was cloned into the BamHI-HmdlEI site of pUC19. After sequencing, plasmid DNA was obtained by digesting with HindIII and ΑίΙΠ, and the resulting fragment was ligated with the plasmid hMBC! La, X / pUC19, which had been digested with HindIII and ΑΠΙΙ

The obtained plasmid was designated "hMBClLbX / pUC19". This plasmid DNA was digested with EcoRI to obtain a DNA fragment containing the DNA encoding the humanized L chain. The DNA fragment was inserted into the pCOS1 plasmid such that the start codon for the humanized L chain was located downstream of the EF1? Promoter. The plasmid thus obtained was designated "hMBClLbX / pUC19".

190 351

Version "c" was generated using PCR mutagenic insertion. Version "c" was designed to replace the amino acid at position 84, serine (amino acid No. 80 as defined by Kabat) with proline. The PCR reaction was performed using the hMBC 1 Ln / JpUC19 plasmid as a template for muragrinic MBC1LGP6S (SEQ ID NO: 37) and the M13 Primer Rv. The DNA fragment was digested with BamHI and HindIII, and the digested fragment was cloned into the BamHI-HindIII site of pUC19. After sequencing, plasmid DNA was obtained by digestion with BstPI and Akr51HI. The obtained plasmid was designated "hMBC1Lyλ / pUC19". This plasmid DNA was digested with EcoRI to obtain a DNA fragment containing the DNA encoding the humanized L chain. The DNA fragment was inserted into plasmid pCOS 1 such that the start codon for the humanized L chain was located downstream of the EF1a promoter. The plasmid thus obtained was designated "hMBC1Lc // pCOS1"

Versions "d", "e", and "f" were also produced using the PCR mutageric insertion technique. The "d", "e" and "f" versions are each designed to replace the amino acid at position 91, tyrosine (amino acid # 87 as defined by Kabat) with an isoleucc version of "a", "b" and "b", respectively. y "For each version" d "," e "and" fo, a PCR reaction was performed using the plasmid hMBCHa / l / pUClO (<1So wrrsi id), hMBOEbX / pUCi9 (for version e) and hMBClLci / pUCH (for version f) as template and mutagenic primer MBC1LGP11R (SEQ ID NO: 38) and M-S1 primer (SEQ ID NO: 44). After sequencing, the DNA fragment was digested with BamHI and HindIII, followed by a subclqquark into pUC19, which was prepared by digesting pUC19 with BamHI and HindIII. After sequencing, the plasmid was digested with HindIII and BlnI, and the digested fragment was cloned into the BamHI-HindIII site into the C // pUC19 plasmid. The plasmids thus obtained were marked. kkdspedmo. "HMBC1d // pUC19", "hMBC1Leλ / pPC19" and "hMBU1Lfλ / pUU19". Each of the plasmids was digested with EcoRI to obtain a DNA fragment encoding the humanized L chain. The DNA fragment was inserted into the EcoRI site of the pCOS1 plasmid in such a way that the start codon for the humanized L chain was located below the EF1a promoter. The plasmids thus obtained were designated "hMBC1Ld /", respectively. / pCOS1 "," hMBC 1Le // pCOS1 "and" hMBC1Lf // pCOS1 "

The "g" and "h" versions were also produced using the mutagenic introduction technique by PCR. The "g" and "h" versions are each designed to replace the amino acid at position 36, histedena (amino acid # 36 as defined by Kabat) with the "a" and "d" versions of isoleucene, respectively. For each version "g" and "h", PCR reactions were performed using the hMBU1Lαλ / pUC19 plasmid as template ^{1 of} the mutagenic MBC1LGP9R primer (SEQ ID NO: 39) and the M13 Primer Rv primer. After the Sonovam sequences, the DNA fragment was digested with BamHI and HindIII and the digested fragment was cloned into a BamHI-HindIII C // pUC19 site. Using this plasmid as a template, PCR was performed with the MBC1LGP13R (SEQ ID NO: 40) iMBC1LVS1 site. The obtained PCR fragment was digested with ApaI and HindIII and inserted into plasmids hMBC1La // pUC19 and hMBC1Ld // pUC19, which had been digested with ApaI and HindIII. The DNA sequence of the obtained plasmids was determined. The plasmids with the confirmed sequence were designated "hMBC1Lg // pUC19" and "hMBC1Lh // pUC19", respectively. Each of these plasmids was digested with EcoRI to obtain a DNA fragment encoding the humanized L chain. The DNA fragment was introduced into the EcoRI site of the pCOS1 plasmid such that the start codon for the humanized L chain was located downstream of the EF1a promoter. The plasmids obtained in this way were designated "hMBC1Lg // - pCOS1" and "hMBC 1Lhλ / pUOSl", respectively.

There are also versions of the poisoning »fooVi

UUV1-> - ----- mutagenic treatment by PCR. A PCR reaction was performed using the hMBC''1 Ea / JpUJd 9 ^ Εο maCric plasmid and primed muragrmzrneko MBC1LGΡlSS (SeOw. No. 41) and the V1RV (/) primer (SEQ ID nn 43). The resulting fragment was digested with ApaI and BlnI and cloned into the plasmid hMBC1Lg // pUC19 which had been digested with ApaI and BlanI. The DNA sequence of these plasmids was determined and the clones into which the mutation was introduced were selected. The plasmids obtained were designated as "hMBC1Lx // pUC19 (x = i, j, k, 1, m, n or o), respectively"

190 351

Each of these plasmids was digested with EcoRI to obtain a DNA fragment encoding the humanized L chain. The DNA fragment was inserted into the EcoRI site of the pCOS1 plasmid such that the start codon for the humanized L chain was located downstream of the EF1a promoter. The plasmids thus obtained were designated "hMBC1LxZ / pCOS1. (x = i, j, k, l, m, z or o) "DNA sequences (including corresponding amlzoKayy sequences) of versions, j", "l", "m" and "o" are shown in Id. Seq. Nos. 67, 68, 69 and 70, the amino acid sequences of these versions are shown in Id. Seq. 48, 49, 50 and 51.

The "p", "q", "r", "s" and "t" versions are a modified form of the "i", j "," z "," l "and" o "versions, respectively, wherein the amino acid at position 87, tyrosine has been replaced with isoleucine. These versions were generated by using the Aor51MI restriction site in FR3 to replace version "h" with versions "i, j", "z", "l" and "o" as follows. From the expression plasmid hMBC1 Εχλ / pCOS 1 (x = i, j, m, 1 afoo o), Eczema yys1yκkvJZvy Aor1 1HI (114 pp) containing CDR3, part of FR3 and all FR4 was removed. The remainder of the expression plasmid was ligated to the Aor51HI restriction fragment (514 bp) containing CDR3, part of fR3, and all of FR4 such that the amino acid at position 91, tyrosine, was replaced with isoleucine (amino acid 87 in Kabat numbering). The sequences of the resulting plasmids were determined and the cloze of each of the versions "i", j "," z "," l "and" o "were selected, in which amino acid 91, tyrosine, was replaced with iso | yucca (amino acid 87 in Kabat numbering). The versions corresponding to the versions "i", j "," m "," l "and" o "are marked with" p "," q "," s "," r "and" t "respectively, and the plasmids for these The versions are marked "hMBC1LxZ / pCOS1 (x = p, q, s, r or t), respectively. The DNA sequences (including the corresponding and amino acid sequences) of the "q", "r", "s" and "t" versions are shown in Id. SeKw. Nos. 71, 72, 73, and 74. Amiaokway sequences of these versions are shown as Id. Seq. no.52, 53, 54 and 55.

The hMBC1EqE / pCOS plasmid is also HintUII and EcoRI, αι ^^ ρή and is related to pkzzid pUC19 by digestion of plasmids HianIII and EcoRI. The plasmid thus obtained was designated "hMBC1LqZ / pUC19".

Positions of replaced amino acids in each variant of the Kyzαnc L chain huzylzca nc in Table 3 below

Table 3

Version 36 43 45 47 49 80 87 1 2 3 4 5 6 7 8 and b P. D c P. d AND e P. D AND f P. AND g Y h Y T. and 1 Y K. J. Y K. D k Y K. V

190 351 c d table 3

1 2 3 4 5 6 7 8 1 Y K. V D m Y D n Y V 0 Y V D P. Y K. AND q Y K. D AND r Y D AND s Y K. V D AND vol Y V D AND

In the table above 3, capital letters represent the following amino acids Y tyrosine; P proline, K lysine, V valine, D aspartic acid and I isoleucine

The E. coli strain containing the hMBC1HcDNA / pUC19 plasmid and the E. coli strain containing the hMBC1Lq ^ Ap ^ l ^ C19 plasmid was labeled "Eccerrichia coli JMK) 9 (WBBClHcDNA / pUC19ł" i, Escherichia coli JM 109 (hMBC1Iql) a / pUCenanach a Of the Buaapesthen Treaty August 15, 1996, at the National Institute of B ^ cience and Humao-teahnology, Agency for Industnal Science and Technology Japan (1-3, Higashi 1-chome, Tsukuba-shi, ibaragi-ken, Japan), under accession number FERM BP-5629 for Escherichia coli JM109 (hMBC1HcDNA / pUB19) and FERM BP-5630 for Escherichia coli JM109 (hMBClLqλ / pUC19).

(5) Transfection into COS-7 cells

The expression plasmids described above were transiently expressed in COS-7 cells to determine the antigen-binding activity and the neuralizing activity of the hybrid antibody and humanized antibody # 23-57-137-1. For the transient expression of the? Chain of the hybrid antibody, each of the following plasmid combinations was knatransfected into COS-7 cells by electroporation using a Gene Pulser (Bio Rad):

hMBC1HcDNA / pCOS1 ih / iMBSaLGneneo; MBdHcDNA / pCOS1 imhMffiCLLaλ / neo; hMBC1HcDNA / pCOS1 r m / hMBC1LdA / neor hMBB1Laλ / n <n> and hMBClHcDNA / pCOS1 and hmmMBC1L (A) / neo; and hMBB1HcDNA / pBOS1 and mhmMBC1L (λ) / oen. For this purpose, 10 µg of each of the µlmLUowyah DNA was added to 0.8 ml of the cell suspension in which the COS-7 cells were suspended in PBS (-) at a density of 1x10 µl cells / ml. The resulting solution was pulsed at 1500 V and 2 μF, causing electroporation. After 10 minutes at room temperature, cells were suspended in DMEM medium containing 2% Ultra Low IgG fetal calf serum (Gibco) and then cultured using a 10 cm dish in a CO2 incubator. After culturing for 72 hours, the culture was harvested and centrifuged to remove cell debris. and provided as sample for ELISA.

For the transient expression of humanized antibody # 23-57-137-1, a combination of plasmids (iMBC 1 HcDNA / pCCSS and hM BC1LxA actor / pCOS 1 pr = a -1 and radioactive to COS-7 cells using a Gene Pulser (Bio Rad) device in the same method as described above for the hybrid antibody The obtained culture supernatant was provided as a sample for the ELISA test.

In this procedure, purification of chimeric antibodies from the COS-7 cell culture supernatant was performed using the Affigel Protein A MAPSII kit (Bio Rad) according to the manufacturer's instructions.

190 351 (6) ELISA Test (i) Determination of antibody concentration

An ELISA plate for determining the antibody concentration was prepared as follows. Wells of a 96-rtudcieokowes ELISA plate (Maxisorp, NUNC) were coated with 100 µl of a solution containing goat anti-human IgG (TAGO) prepared in coating buffer (0.1 M NaHCO3, 0.02 NaNs) at a concentration of 1 µg / ml, and then blocked with 200 µl of dilution buffer (50 mM Tris-HCl, 1 mM MgCl ₂ , 0.1 M NaCl, 0.05% Tween 20, 0.02% NaN3, 1% Bovine Serum Albumin (BSA); pH 7, 2). COS-7 cell culture supernatant expressing either the chimeric antibodies or the purified chimeric antibodies in stepwise dilutions was added to each well. After incubation at room temperature for one hour and washing with PBS-Tween 20, 100 µl of an alkaline phosphatase-conjugated goat anti-human IgG (TAGO) solution was added to each of the wells. After incubating at room temperature for one hour and washing with PBS-Tween 20, 1 mg / ml of substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, Sigma) was added to each of the wells. The absorbance of the solution was measured at 405 nm using a Microplate Reader (Bio Rad). Purified Hu IgG1X, (The Binding Site) was used as standard.

(ii) Determining the antigen-binding capacity

An ELISA plate for determining antibody binding was prepared as follows. Each well of a 96-well plate was coated with 100 µl of a solution containing human PTHrP (1-34) (Peptide Research Institute) prepared in 1 µg / ml buffer, and then blocked with 200 µl of blocking buffer. The culture supernatant of COS-7 cells in which the chimeric antibodies or the purified chimeric antibodies were expressed was added in stepwise dilution to each well. After incubation at room temperature and washing with PBS-Tween 20, 100 µl of an alkaline phosphatase-conjugated goat anti-human IgG (TAGO) solution was added to each well. After incubation at room temperature and washing with PBS-Tween 20, 1 mg / ml of substrate solution ("Sigma 104", p-nitrophenylphosphoric acid, Sigma) was added to each of the wells. The absorbance of the solution was measured at 405 nm using a Microplate Reader (Bio Rad).

(7) Proof of activity (i) Study of humanized H chain

The antibody comprising the humanized H chain version "a" and the chimeric L chain was found to have the same level of PTHrP binding activity as the chimeric antibody (see Figure 5). This result suggests that the humanization of the H chain V region was achieved satisfactorily with the "a" version. Hence, version "a" of the humanized H chain was provided as the H chain of the humanized antibody in the following experiment (ii) Hybrid Antibody Activity (ii-a) FR1.2 / FR3.4 Hybrid Antibody

When the L chain was h / mMBC1Ld (L), the antibody had no antigen binding activity. However, when the L chain of the hybrid antibody was m / hMBC1Lak or m / hMBC1Ld λ, the antibody displayed the same level of antigen binding activity as chimeric antibody # 23-57-137-1 (Figure 6). These results indicate that FR3 and FR4 are useful for a humanized antibody, but there is an amino acid residue that needs to be replaced in FR1 and FR2 (ii-b) FR1 / FR2 hybrid antibody

Gd

DryGowego was uuuiunBC1L (/ v), antibody mc wyc / ΓΟΓΊ T SA \

........... iryi I V »-łrł> · iuiivuvnvm JLj p / A £ ^ VV1» ł C1U1U llj 'showed antigen-binding activity. However, when the L chain of the hybrid antibody was hmmMBC11 # U), this antibody showed the same level of cc activity and antigen binding as chimeric antibody # 23-57-137-1 (Figure 7). These results suggest that FR1 is useful for a humanized antibody, but there are amino acid residues (s) that need to be replaced in FR2.

190 351 (iii) Humane-mollusc antibody activity

Humanized antibody with L-chain versions "a" to "t" was tested for antigen-binding activity. As a result, it was found that humanized antibodies with L-chains of versions J "," l ", m", "o", "q "," R "," s ", and" t "showed the same level of PTHrP binding activity as the chimeric antibody (Figs. 8-11) (8) Determe stably transformed CHO cell union

To establish stable transformants for the chimeric antibody, the expression plasmid described above was introduced into CHO cells (DXB11)

Establishment of stable tranformαnts of the chimeric antibody was carried out using a combination of CHO cell expression plasmids, hMBC1HcDNA>'pCHO1 and hMBClLmk / pCOS1; hMBC1HcDNA) pCHO1 and hMBC1Lqk / pCOSl; iMBBC lHcDNA / pCHO1 and hMBC1L ^ / pCOS1. These plasmid combinations were individually co-transfected into CHO cells by the rleetroporacJl method using a Gene Pulser (Bio Rad). Each of the expression vectors was cleaved with the restriction enzyme PvuI to obtain linear DNA. The resulting DNA was collected by phenol and chloroform extraction followed by ethanol precipitation. The expression plasmids produced in this way were subjected to elretroporation. 10 µg of each plasmid DNA was added to 0.8 ml of cell suspension containing CHO cells in PBS (-) at a density of 1x10 ⁷ cells / ml. The resulting mixture was pulsed at 1500 volts and 25 pF. After ten minutes at room temperature, the cells were suspended in MEM-α medium (Gibco) supplemented with 10% fetal calf serum (Gibco). The resulting suspension was grown in three 96-well plates (Falcon) in a CTT incubator. The next day after the start of culture, the medium was replaced with selective medium (MEM medium supplemented with 10% fetal calf serum (Gibco) and 500 pg / ml grnrtcccnc (G418 sulfate), Gibco without ribonucleosides or deoxyribonuk ^^ and ^^ w After replacing the selective medium with fresh medium, cells were observed under a microscope before and after two weeks of culture. When cell growth was observed, cells were examined for the amount of produced antibodies in an ELISA test. Among the cells were selectively collected those that produced higher amounts of antibodies.

Up-scaling of the stable transformant cultures for the established antibodies was performed in roller bottles using MEM medium supplemented with 2% Ultra Low IgG1 fetal calf serum without rcbonuclosides and deokyribonuclosides. On days 3 and 4 of culture, culture supernatants were harvested and filtered through a 0.2 µm culture filter (Milliporr) to remove cell debris. Subsequently, purification of chimeric antibodies from the supernatant of CHO cells was performed using a Poros Protein A Column (PerSeptive Bioscetems) on ConSrp LC100 (Millipore) according to the manufacturer's instructions. Purified chimeric antibodies were provided as samples for the determination of neutralizing activity and for the effectiveness of the hypercalcrimic animal model. The concentration and binding activity of the purified chimeric anti-antigen antibodies were determined by the same ELISA system.

Example 4

Determination of neutralizing activity

Determination of the neutralizing activity of the murine antibody, the chimeric antibody and the humanized antibody was performed using rat ROS17 / 2.8-5 epipenic cells. ROS17 / 2.8-5 cells were grown in Ham'aF-12 medium (Gibco) supplemented with 10% fetal calf serum (Gibco) using a CO2 incubator. ROS17 / 2.8-5 cells were inoculated into each of the wells of a 96-well plate at a density of 10 µl / well and grown for 1 day. The culture medium was replaced with media F1 and Gi.'c x - x

o)) with dcdaeklnm 4 ιμΠ yyroekGlzyjnuu 1 10θ) ι srtHvCcy GWCj ciclcze ', After culturing for 3 to 4 days, the cells were washed with 260 µl Ham's F-12 medium (Gibco), followed by the addition of 80 µl Ham ^ F- medium 12 with the addition of 1 mM izdbutclo-1-mrtcldkeantync (IBMX, Sigma), 10% fetal calf serum and 10 mM HEPES. The resulting mixture was incubated at 37 ° C for 30 minutes.

The murine antibody, the chimeric antibody and the humanized antibody tested for neutralizing activity were previously diluted in the following groups: [10 pg / ml,

Iz o U o rv »'o X XCX1XA XX

190 351

3.3 gg / ml, 1.1 gg / ml and 0.37 gg / ml], [10 gg / ml, 2 gg / ml, 0.5 gg / ml and 0.01 gg / ml] and [10 gg / ml, 5 gg / ml, 1.25 gg / ml, 0.63 gg / ml and 0.31 gg / ml] Each of the diluted antibody samples was mixed with an equivalent volume of 4 ng / ml PTHrP (1-34) 80 g of the resulting solution was added to each of the wells. The final concentration of each antibody became a quarter of the concentration of the above antibody, and the concentration of PTHrH (1-34) was 1 ng / ml. 10 minutes after treatment at room temperature, culture supernatants were removed and the residue was washed with PBS three times. From the above, cAMP was extracted from the cells with 10 g of 0.3% HHC-95% ethaol, and then a ppzz donated using urzdhema wwaeer et aapirator to remove HCL-ethanol. The residue was dissolved in 120 g of EIA buffer included with the cAMP EIA kit (Cayman Chemical's) to extract cAMP. CAMP levels areosob using the cAMP EIA Kit (Caym ^ Chemicals) according to the manufacturer's instructions. As a result, it was found that among humanized antibodies having L-chain versions exhibiting the same level of antigen-binding activity as the chimeric antibody, those having L-chain versions of "q", "r", "s", and "t" in which tyrosine at position 91 replaced with isoleucine, they showed the neutralizing activity closest to that of the chimeric antibody, and especially those with the "q" L chain version showed the strongest neutralizing activity (Figures 12 to 14).

Example 5

Pharmacological efficacy study in an animal model with hypercalcemia. (1)

The therapeutic efficacy against hypercalcnm11 anti-PTHrP antibodies of chimeric and humanized L-chain versions of "m", "r" and "q" was investigated using an animal model of hlpnzKalcnmll (human neoplastic tumor).

Nude mice transplanted with human PAN-7 pancreatic carcinoma (purchased from the Central Institute for Experimental Animals) were used as an animal model of hlperkslcemil. It is known that nude mice which have been transplanted with human PAN-7 pancreatic carcinoma show a decreased blood calcium concentration as the tumor grows in size and develop hyperKalcnmls, which is accompanied, for example, by a reduction in body weight and spontaneous activity. In this example, the therapeutic effect of the chimeric antibody and the humanized antibody of the invention on hypnzKalcnmll induced by PAN-7 human pancreatic cancer was investigated by measuring body weight and blood calcium concentration of test animals.

Passage of PAN-7 human pancreatic carcinoma was performed with BA-BZc-nu / nu (Nippon Charles River) in vivo. For pharmacological efficacy studies, 5-week mbwn male BALB / c mice (Nippon Charles River) were purchased and treated for one week, and when they reached 6 weeks of age, they were prepared for experiment. A model of hypercalcemia in mice was generated and the groups were divided as follows. Passive pancreatic cancer PAN-7 was excised and then finely cut into 3 mm Cubes. The resulting tumor fragments are transplanted subcutaneously into mice in the amount of one per mouse. Two to three weeks after transplantation, when it was confirmed that the tumor volume of each mouse had become large enough, the blood calcium levels and body weight were established in the individual groups, and the mice were used as an animal model of hypercalcemia.

The study of therapeutic efficacy against hypercalcemia was performed as follows. Each pbhsob hypocalcinoma mice through the tail vein with a single dose of a chimeric or humanized L chain version "m" or "r" antibody against pohaob PTHrP in an amount of 10 to 30 g / mouse. A single dose of the humanized L-chain antibody in the "q" version was administered to each of the hypocalcinmic mice at a dose of 20 or 60 gg per mouse. On days 1, 4 or 7, and 11 after pbhsolk, each mouse had its blood calcium levels measured and its body weight was measured to measure the effectiveness of the fszmaKblogicznee antibody. Tumor volume was determined by measuring the greater diameter (a mm) and the minor diameter (b mm) of the tumor and calculating using both diameters according to the Galanf equation [ab2 / 2]. The blood calcium concentration was calculated as the ionized calcium concentration in whole blood by collecting blood from each mouse through the orbital plexus, using a tube and placing the sample in an automatic 643 Automatic Ca2 + / pH ιοι ^ ζ device (CIBA-CORNING).

190 351

As a result, it was found that administration of chimeric antibody and huzanizowaavch antibodies with L-chain versions "z", "r" and "q" lead to rapid improvement in body weight and blood calcium concentration and to long-term improvement of the patient's condition. This result demonstrates that the chimeric and humanized antibodies of the invention are useful in the treatment of tumor associated hyperyrkαlcemia (see Figures 15 and 16).

Example 6

Study of the effectiveness of pharzecology on an animal model of hypercalcymia (2)

Using an animal model of hipyrcalcemil (human tumor transplanted into nude mouse), a chimeric and humanized antibody with "q" version of the L chain against PTHrP was tested for therapeutic efficacy against hlpyrkalcemil as follows.

The study of therapeutic efficacy against hlpycalcemia was performed as follows. Each mouse with the hyricalcymia model was dosed with a single dose of the chimeric or humanized version "q" of the L chain against PTHrP via the tail vein at a dose of 10 or 30 µg / mouse. On days 1, 3, 7, and 11 after the administration, the blood of each mouse was tested for the concentration of output and the body weight was tested to determine the pharmacological efficacy of the antibodies. Blood calcium concentration was calculated as whole blood calcium zjozlzoąαaygd concentration by collecting blood from each mouse through the orbital plexus; using a tube and placing the sample on an automatic 643 Automatic Cy2 / rH anNero instrument (CIBA-CORNING).

As a result, it was found that in an animal model of hypercalcemia with human pancreatic cancer PAN-7, administration of a chimeric or humanized antibody with the "q" version of the L chain leads to a sharp improvement in body weight and blood calcium concentration, and to preserve the improvement in the condition of the subject by longer time. This result demonstrates that the chimeric and humanized antibodies of the invention are useful for the treatment of tumor associated hypercalcemia (see Figure 17).

Example 7

Pharacological effectiveness study on a model of animals with hypercalcemia (3)

The therapeutic efficacy against hypercalcemia of chimeric and humanized L-chain "q" version antibodies against PTHrP was investigated using an animal model of hypercalcemia (human LC-6 lung carcinoma transplanted into nude vice).

As an animal model of hypercalcemia, nude mice were used to transplant human LC-6 lung cancer (purchased from the Central Institute for Experimyatαl Animals). It is known that nude mice transplanted with LC-6 human lung cancer show an increased concentration of calcium in the blood and with increasing size. the tumor develops hypercalcemia accompanied, for example, by weight loss and intestinal activity

In this example, the therapeutic effect of the chimeric antibody and the humanized antibody of the invention on human LC-6 lung cancer induced hypercalcosis was investigated by measuring body weight and blood calcium concentration of test animals.

Passage of human LC-6 lung carcinoma was carried out by BALB / c-nu / nu (Nippoz Charles River) in vivo. For pharmacological efficacy testing, r-tvgonnlde male BALB / c Charles River mice were purchased and aliquoted for one week, and were prepared for experiment when they were 6 weeks old. A model of hypercalcemia in mice was generated and the groups were divided as follows. The pyrolysis of the LC-6 lung was excised and then finely dissected into 3 mm Cubes. The resulting tumor fragments were subcutaneously implanted with one mouse per mouse. Two to three weeks after transplantation, when it was confirmed that the tumor volume in each mouse had become large enough, the blood bath concentration and body weight of the individual σηιη were averaged, and the mvszv was used for ν ΐ ui ^- ------ σ-- -j --- X '- ~ --- j - j - ---- - ~ j - animal model hirerkalcymll.

The study of therapeutic efficacy against hypercalcymia was performed as follows. A single dose of the chimeric or humanized L chain version "q" antibody against PTHrP at 10 or 30 pg per mouse was administered to each hipyrkαlcislrzai mouse via the tail vein. On day 1, 3, 6 or 10, the blood calcium levels were measured after each mouse was given

190 351 and the body weight was tested to measure the pharmacological efficacy of the antibodies. The blood calcium concentration was calculated as the whole blood concentration of sedimented calcium by collecting blood from each mouse through the orbital plexus, using a tube and placing the sample in the 643 Automatic Ca2 + / pH analyzer (CIBA-CORNING).

As a result, it was found that the administration of the chimeric antibody and the humanized antibodies with the L chain version "q" led to a dramatic improvement in body weight and blood calcium concentration and to long-term improvement in the patient's condition. This result demonstrates that the chimeric and humanized antibodies of the invention are useful for the treatment of tumor associated hypercalcemia (see Figure 18).

Example 8

Kinetic analysis of the interaction between PTHrP and anti-PTHrP antibody using BIACORE

In this experiment, a kinetic analysis of the antigen-antibody interaction was carried out using BIACORE PTHrP (1-34 + Cys) was used as antigen and adsorbed at the tip of a sensor specific for C-ends. Purified antibodies of different concentrations were used as an analytical sample. From the obtained rheosorogram, the kinetic parameters (constant "Kass" binding and the dysocSaśJi constant "1 ^") For the kinetic analysis, the literature used as reference was "Kinetic analysis of monoclonal antibody αotlgeo ioteraśtioor with a new bioreoror based analytical system" Karlsson et al., (1991) J Immunol Methods 145.229- 240.

(1) Locking PTHrP (1-34 + C) on the sensor tip

PTHrP (1-34 + C) was adsorbed on the tip of the CM5 sensor (Pharmacia)

As the working buffer, HBS (10 mM HEPES, pH 7.4; 0.15 M NaCl; 3.4 mM EDTA; 0.005% Surfactant P20) was used at a flow of 5 µl / mm. The carboxymethyldextran carboxyl groups at the tip of the CM5 sensor were activated by an injection of 100 µl 0.05 M N-hydroxyruccinimide (HNS) / 0.2 M N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and a 100 µl injection of 80 mM 2- (2-pyridyllothio) ethanammy (PDEA) / 0.1 M borate buffer (pH 8.5) and additional injection of 10 µl 5 µg / ml PTHrP (1-34 + C) / 10 mM sodium acetate buffer pH 5.0) to adsorb the C-ends of PTHrP (1-34 + C) specifically on Cys residues. Then, 100 µl of 50 mM (L) -cysteine / 1 M NaCl / 0.1 M sodium formate buffer (pH 4.3) was injected to block overactive groups. Then, 10 µl of 0.1 M glycine-HCl buffer (pH 2.5) and 10 µl of 10 mM HCl were injected to wash out non-covalently bound substances. The amount of PTHrP (1-34 + C) so immobilized was 226.4 RU (resonance units) (see Fig. 19).

(2) Interaction between immobilized PHHrP (1-34 + C) and purified mouse anti-PTHrP antibody

HBS was used as the working buffer at a flow of 20 µl / mmut. Antibody-producing hybridomas were injected into the peritoneal cavity of Balb / c mice, and after a few weeks the exudate was collected and loaded onto a protein A column to purify the antibodies. Purified antibody # 23-57-137-1 was designated "MBC" and purified antibody 3F5 was designated "3F5". These antibodies were diluted with HBS at a range of concentrations of 1.25, 2.5, 5, 10, and 20 pg / ml.

During the analysis, 40 µl of the antibody solution was injected for 2 minutes to obtain the binding phase, and then HBS was injected for 2 minutes to give the dyrocJaśsi phase. After the nozzle was finished, 10 µl of 10 mM HCl was injected to clean the sensor tip. The analysis was performed using this loosening-dissociation cycle. - purification and injection of various antibody solutions to obtain a rheororogram.

V »U111V) mi

DTEJ-D / I iluonylia and lHU ^ r

O - C) and cleaned up!

U, humamated anti-PTHrP antibody

HBS was used as the working buffer at a flow of 20 µl / mmut. The antibody produced in CHO cells was purified on a protein A column. The purified chimeric antibody was labeled "chMBC" and the pure antibodies of the m and q versions were labeled "hMBCm" and "hMBCq", respectively. These antibodies were diluted with HBS at a series of concentrations of 1.25, 2.5, 5, 10 and 20 pg / ml.

190 351

During the analysis, 40 µl of the antibody solution was injected for 2 minutes to form a binding phase and then HBS was injected for 2 minutes to create a dissoy phase. After the derocjaySi was completed, 10 µΐ 10 mM HCl was injected to clean the tip of the sensor. The analysis was performed using this binding-csayJi-sanding cycle and different antibody solutions were injected to give sensorognsm.

(4) Kinetic interaction analysis

The data file was read and the comparison of the reaction patterns was performed by overlapping the reaction regions of interest (Figures 20-24). In each of Figures 20-24, lines sequentially mark the top of the data for antibody concentrations of 1.25, 2.5, 5, 10, and 20 µg / ml. Further, the kinetic analysis of the interaction was performed using the analysis software specifically designed for BLACORE "BIAevαluatikr 2 1" (Pharmayia), which allows the calculation of kinetic parameters (binding constant "km" and constant derkySscJi "kam") by cutting the curve (Tables 4 and 5)

Table 4

Kinetic Parameters of MBC and 3F5

MBC 3F5 kum [1 / s] 7.38x10 5 1.22x10'2 km [1 / Ms] 7.23x105 6.55x105 KD [M] 1.02x10 '° 1.86x10 ^sec

Table 5

Kinetic parameters of humanized and chimeric antibodies

yhH-yh / hMBCm hMBCq 1 ^ [1 / s] (xl0 ⁴ ) 1.66 3.160 2.32 Cash [1Ms] (x10 ⁶ ) 1.24 0.883 1.03 KD [M] (x10 · ¹⁰⁾ 1.34 3.580 2.25

In this experiment, the analysis type 4 model (BIAevaluation 2.1 Software HandtOok, A1-A5) was used to determine the binding constant.

Example 9

Inhibition of phosphorus secretion in a model of tumor associated hypercalcemia

Tumor-associated hyperkalaemia (HHM) is a disease caused by PTHnP deficiency and, as is known, PTHnP accelerates bone nessorption and reorphism. Calcium is present in the kidney and tubule, which causes hyperkalemia to develop. On the other hand, with regard to phosphorus, PTHrP inhibits the phosphorus deficiency in the kidney and renal tubule resulting in an elimination effect and thus patients with clinical HHM often develop hypkfkrfaemia. The effect of a humanized anti-PTHnP antibody on phosphorus secretion in the kidney was investigated using a rat model of tumor associated hyperkalaemia.

As an animal model, nude rats injected with a micro human LC-6 lung (purchased from the Central Institute by Experimental Animals) were used. It is known that nude rats transplanted with human LC-6 lung cells show an increased concentration of calcium in the blood as the tumor grows in size and develop hyperkaliemis accompanied by, for example, reduction in body weight and spontaneous activity. Using this animal model, the effect of a humanized anti-PTHrP antibody according to

The invention of the invention on phosphate secretion in the kidney by the renal clearance method based on fractionated phosphate secretion.

Passage of human LC-6 lung carcinoma was performed using BALB / c-ou / ou (Nippon Kurea) ir vivo. For the pharmacological efficacy study of cnkupinoo 5atygnUoiowf, male F344N / Jal-rou (Nippon Kurea) rats were acclimated for one week, and when they reached 6 weeks of age, they were prepared for experiment

A model of tumor associated hypercalcemia in rats was generated as follows. Passed LC-6 lung cancer was dissected and then finely sectioned into 3 mm cubes. The resulting tumor fragments were implanted subcutaneously into rats in an amount of one per rat About 30 days after transplantation, when it was confirmed that the tumor volume in each of the mice had become large enough (3000 mm ³ ), the presence of hypercalcemia associated with malignant tumors was confirmed in rats based on the calcium concentration in blood and body weight

The study of therapeutic efficacy against hypercalcemia was performed as follows (1) Renal Clearance Method

Animals with a coifpathetic tumor-associated hypercalcemia model using pfotzbarbital (Nembutal, Dainippon Pharmaaeurianl Co., Ltd) were mounted on a heated mat at 37 ° C and a catheter (polyethylene tube, PE50, Nippon Becton Dickmson) was inserted into the bladder for collection. urine. The animal then introduced a catheter (polyethylene tube, PE10, Nippon Becton Dickinson) into the femoral vein, followed by an infusion solution (0.7% inulin, 5% mnoltnl, 0.2% pentnbnrbital, and 0.9% sodium chloride) with flow 2 ml / hour by an iofusion pump (Tfrufusino syringe pump; sTC-525; Terumo). After equilibration for 50 minutes, urine was collected five times at 20-ml intervals (ie, period 1 to period 5) to obtain urine samples. At an intermediate time point during urine collection, approximately 0.25 ml of blood was collected from the jugular vein using a hfparinized syringe.

(2) Administration of the antibody

During the clearance test, at the time point when the 2nd period of urine collection started, the humanized anti-PTHrP antibody was intravenously administered to the animal at a dose of 1 mg / ml / kg (3) Ozsnczaoif concentrations of inulin and phosphorus in urine and blood

Urine samples obtained from period 1 to 5 were measured in terms of their volume, and then their inulin and phosphorus concentrations were determined. The blood samples obtained as described above were subjected to refrigerated centrifugation to obtain a plasma sample, which was used to determine the concentration of inulin and phosphorus in the plasma. Inulin determinations were carried out by the anti-salt method (Roe et al., J. Biol. Chem. 178, 839-845, 1949) and the phosphorus determination was carried out on a Hitachi Automalc Analyzer model 7170 with oUac, γIlmkifm for the determination of inorganic phosphorus, Autosera IP ( Danchi Pure Chemicals) according to the manual (method Ph ^ sl ^ -SabaioNa).

(4) close to the clearance of inulin, phosphorus and fractionated phosphorus excretion

Inulin clearance (Cin), phosphorus clearance (Cp) and fractionated phosphorus excretion were calculated according to the following formula.

Calculation of inulin clearance (Cin) 'cin = Uin V / Pin where Cin ozoacon inulin clearance (ml / kg / min); Uin ocoaaon concentration of inulin in urine (mg / ml), V is the amount of urine per unit time (ml / kg / min); and Pin is the concentration of mulma in the blood (mg / ml).

Ohllacaoif phosphorus klireos (Cp) '

Cp = Up V / Pp where Cp is the clearance of phosphorus (ml / kg / min); Up is the concentration of phosphorus in the urine (mg / ml), V is the amount of urine per unit time (ml / kg / min); and Pp ocoaccn blood phosphorus concentration (mg / ml)

Calculation of fractionated phosphorus excretion (FEp):

FEp = Cp / Cm

190 351 wherein FEp is fraction of phosphorus secretion; Cio stands for ΟΗοΌδ moulin and Cp stands for phosphorus klireos. Four-animal test. Ozoacinone results as mean ± standard error

The results of the fractional phosphate excretion and the concentration of phosphate in the eye are shown in Figs. 25 and 26

Figuoa 25 is a graph illustrating the relationship of phosphorus excretion (phosphoocclusion / inulin clearance) versus Co Olireos periods (1 oko = 20 minutes). Treated with pozehi_kn PTHrP antibodies (1 mg / kg) cndaan (i.v.) at the start of period 2.

Figure 26 is a graph showing plasma phosphorus concentration versus Olireos periods (1 period = 20 miout). Humanized anti-PTHrP antibody (1 mg / Og) was administered (i v.) At the start of period 2.

From these results, it was concluded that the fractional phosphorus excretion of the antibody (i.e., from period 2 to period 5) was markedly inhibited by pre-antibody excretion (i.e., period 1). In other words, administration of a neutralizing antibody to an individual developing hiceofnsfatemla, which leads to an acceleration of phosphorus excretion (FEp> 0.2), restores the subject's phosphorus excretion to normal ebJlenyegn Co levels (fractionated phosphorus excretion = 1-EFp> 0.8%) and as a result, the observed human phosphorus concentration results in an anrmatic. These results suggest the precautionary nature of the antibodies of the invention to treat the accelerated phosphorus excretion and hlceofnsfatemrr caused by the presence of PTHrP.

Since PTHrP is a substrate for the growth of hrcerkalhemra accompanying malignant disease, the possibility of increased phosphorus excretion and reduction of phosphorus in tissue scnwnanwaaegn by PTHrP can be predicted. Thus, it is believed that different degrees of hypnfnsphatemia-related disease such as kozc_rha hrcnfnsfate [οιζοα and vitamin D-resistant rickets, scnwnanwaoe are mainly due to an increase in urinary phosphorus excretion and hence the antibodies of the invention be useful for treating these diseases.

Example 10

ZmyreJseeole of various symptoms of onwnt_noami-related hicercalhemia

It is known that on_ntwnraml scn_ndn_ana-related hypercalcemia is the presence of PTHrP, which is produced by onwnt_ór ooaz, with PTHoP curing bone resnocence and κ ^ φ ^ wapoia in the kidneys and renal tubules, leading to Co hyperOaJhemir. Further, in a patient suffering from ciceocalcemia, symptoms of klrniheochh, such as weakness, loss of consciousness, general weakness, hcdrocsaa, nausea and vomiting (aonreOsI) are observed. The effect of anti-PTHrP antibody on these symptoms ΟΕο ^ οϊ was investigated using an animal model of hlcerkalhemia in the human system transplanted into an oagą mouse and onwnt_nou humanOregn transferred to a nude rat.

As in the animal model of hypercalcemia, a nude mouse and a οα / ιϊ / ^ oa rat that were transplanted into LC-6 human lung cancer (purchased from Central Institute for Expeoimeatal Aaimals) were used. Nude mice and oa / and aa rats that are transplanted in the LC-6 lung oaOa tract show an increased concentration of calciferous void with an increase in tumor volume, leading to hypercalcemia associated with decreased body temperature and body weight

The enhancement effect of anti-PTHrP antibodies on tumor-associated nbaa_c hrcerOaJhemri was investigated using the human / oaOa LC-6 aa mouse lung transplant system, and the vomi ^ Ο ^ αμ photographed. The effect of the antibody oa improvement of spontaneous activity, body temperature and baaaon anorexia using the LC _- 6 oa mouse lung cancer transplantation system,

Zmmejseeore of kJiaiceoccC symptoms associated with hyperOalcemia

Passage of lung cancer LC-6 run with BALB / c-ou / au Charles Rrver) in vivo. For the pharmacological efficacy study, 5th / nd male BALB / c mice (Nlpcna Charles River) and akJimatyenwaan were purchased for one week, and when they reached the age of 6 coec / ntnwaan them for the experiment.

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A model of hypercalcemia in mice was generated and the pbdzinlbob group was as follows. Lung cancer LC-6 was dissected and then finely cut into 3 mm cubes. The resulting tumor fragments were implanted subcutaneously in mice in an amount of one per mouse. Two to three weeks after transplantation, when it was confirmed that each mouse's tumor volume had become large enough, the blood calcium concentration and body weight of the individual groups were averaged, and the mice were used as an animal model of hlpercalcnmll.

Tumor volume was determined by measuring the larger diameter (a mm) and a smaller diameter (b mm) of the tumor and calculating using the both diameters of zdwosoia Galanfa [ab ^2/2].

The Blood calcium concentration was calculated as the concentration of total blood collected calcium by collecting blood from each mouse through the orbital plexus, using a tube and placing the sample in an automatic 643 Automatic Caa ⁺ / pH s / sIcz ™ instrument (CIBACORNING)

The study of therapeutic efficacy of the oa and hypercalcemic antibody was performed as follows. Mouse anti-PTHrP antibody was administered to each mouse via the tail vein on days 27, 30, 34 and 37 after tumor transplantation at 100 (µg / mouse). To generate Control, saline was administered in place of the antibody in place of the antibody. Same method On day 41 after tumor transplantation, a typical mouse was selected from each antibody-treated and control group, and a picture of it was selected with the normal mouse.

As a result, in a hypoxicemic animal model transplanted with human LC-6 lung cancer, even though the antibody was administered to mice (pbkszaoee in the center of Figures 27 and 28) bearing the same tumor load as Control mice (shown on the right in Figures 27 and 28). ), showed ooe the same appearance as normal mice (shown on the left side of Figures 27 and 28). The result suggests that administration of anti-PTHrP antibodies produces an improvement in clinical symptoms (Figures 27 and 28).

2. Improvement of the decrease in spontaneous hlpezkslcemla activity

Passage of human LC-6 lung carcinoma was performed with BALB / c-ou / ou (Nippon Kurea) in vivo. For the pharmacological efficacy study zskuplbob 5-tcgoholbwe male F344N / Jcl-mu (Nippoo Kurea) and aKllmstyzbwaoo rats were eaten for a week and were prepared for experiment when they reached 6 weeks of age.

A model of tumor associated hypercalcemia in rats was generated as follows. Passive LC-6 lung cancer was excised, then a 3 mm Ankle was finely dissected. The resulting tumor fragments were subcutaneously implanted in rats in an amount of one rat. About 30 days after transplantation, when it was confirmed that the tumor volume in each mouse had become large enough, the presence of hypercalcemia associated with malignant tumors was confirmed in rats based on blood calcium concentration and body weight.

Blood calcium concentrations were calculated as the zJboizbwaongb calcium concentration in whole blood by collecting blood from each mouse through the orbital plexus, using a tube, and placing the sample in the 643 Automatic Cchi / pH analyzer (CIBA-CORNING).

(1) The method of determination of antispasmodic activity

Determination of the robotics activity was carried out using the ANIMEX activity meter of the SE type (Farad, Electronics, Sweden), which was placed in a specific position in the Pollution Cage in which the individual animals (Poennln and Feeding) were kept. The device was designed to measure the amount of movement in the rat. Using this device, the amount of movement was recorded as a unit over a specified period of time. The measurement was carried out for 13 hours (from 7 one day to the next day and the results are presented as a number of a day.

(2) Administration of the antibody

Hi 'iał pzzniióKb PTHrP ^ 1U1V LZA Z-iWl r ł 1VX_ / X XX XI X

M / Arlni trnwn 1 z- »-, bdsósmo To each of the rats,

Which developed hlpnrKalcemlp, through the tail vein as an amount control 5mg / 0.5ml / Kg. Saline was administered to my group of rats in the same way. The measurement was performed with a change from Control rats to test rats

Postbase measurement in milking 0 (i.e. the day immediately preceding the administration of antibodies), 2, 4, 7 and 14 for antibodies administered to rats, and on days 1, 3, 5, 8 and 15 for Control rats

190 351

As a result, nir control rats showed changes or decreases in epnthenic locomotor activity during the study period, while rats given the antibody showed an increase in spontaneous activity after 4 days of dosing (Figure 29).

Reduction of the decrease in body trmprreturc associated with hyprcalcrmia

Passage of human LC-6 lung carcinoma and preparation of an animal model of tumor-associated hypercalcuria were performed in the manner of the caretaker in step 2.

(1) A method of measuring body temperature

Body temperature measurement was performed using a digital thermometer by anesthetizing one of the animals with pentobarbitalrm (Nembutal Damippon Phermacrutlcel Co Lid), and a temperature probe was inserted into the rectum.

(2) Administration of the antibody

Humanized PTHrp was administered to each of the model hyperealcemic rats in the caudal vein at a daily dose of 1 mg / ml / kg. For control, other rats were administered saline intravenous through the caudal vein. Further, the temperature of normal rats that were administered nir with antibodies was also measured. The body temperature of the rats were measured on day 0 (i.e. the day of administration) 1, 2, and 3 after antibody administration with respect to the antibody-treated rats, control and ndrmelncch rats.

As a result, normal rats showed changes in body temperature (34.2-34.4 ° C for the duration of the test, while rats with model tumor-induced hypercalcaemia induce a reduction in body temperature of about 2 ° C in parallel with normal rats. humanized anti-PTHrP antibody was administered to model rats, it was confirmed that the model hypercal and tumor associated rats equilibrate their temperature to the same level as normal rats three days after administration. tr results indicate that the humanized anti-PTHrP antibody of the invention is effective to increase body temperature in an animal model of hypercalcaemia associated with malignant tumors (Fig. 30)

Increasing food intake

Passage of human lung cancer LC-6 and production of animals with hypercalcemia modrm were performed as described above in Chapter 2. Model animals were divided into two groups by averaging blood calcium concentration and body weight for each group, and mice were used in the following experiments.

(1) Measure the amount of food ingested

In the study okrrsir, rats were individually placed in metabolic cages and fed and watered. For each animal, the amount of food ingested was determined as the amount (g) per 24 hours (starting at 9:00 am to 9:00 am the next day). The determination was carried out by measuring the total weight of the food container at 9:00 a.m. on a given day and at 9:00 a.m. the following day calculating the difference.

(2) Administration of the antibody

Humanized anti-PTHrP antibody was administered to the rats with model hyperrealemia (HHM rats) described above, via the tail vein at a dose of 5 mg / 0.5 ml / kg. Control rats were administered saline in the same manner. The saline solution was administered to normal rats in the same manner. For all antibody-treated rats, control rats and normal rats, food intake calculations were performed on day 0 (i.e. the day before administration to the day of administration), dma 1 (i.e. from the day of administration to the next day) , day 3 (ie the period from the third day after administration to the next day) and day 5 (ie the period from five days after administration to the next day).

A small amount of food ingested by

In the "unmikl mum ykyvtcx" v uinvni hlprrkelcrmic rats (5-9 rats) had an average of 8.11 g, while normal rats ingested an average of 12.06 g, illustrating a clear reduction in the amount consumed by the hypercalmic rats. When the humanized anti-PTHrP antibody was administered to hypercalcemic rats, although nir changes were observed in the amount consumed by control rats, the amount consumed in the rats dosed with the antibody returned to the level of normal rats. These results indicate that the food is humanized

The anti-PTHrP antibody of the invention is effective in improving the reduction in the amount consumed by rats with a model hypercalcymla (Table 6).

Table 6

Effect on consumption

Animal Individual Administration Amount consumed by an individual (g) day 0 day 1 day 3 day 5 Normal rat 1 salt 13.70 16.7 18.63 18.71 2 salt 14.27 15.3 19.55 19.39 3 salt 9.83 15.5 20.72 19.88 4 salt 10.42 15.04 20.28 22.03 Rat HHM 5 salt 10.77 14.24 12.66 11.82 6 salt 6.99 8.92 2.59 14.8 Rat HHM 7 Antibody against PTHrP 7.46 17.65 22.52 17.99 8 Antibody against PTHrP 12.00 12.38 20.94 23.10 9 Antibody against PTHrP 3.35 16.65 20.36 21.89

Administration of salt (saline) 0.5 ml / kg, via the tail vein, i

Administration of the antibody. 5 mg / 0.5 ml / kg, via the tail vein

From the above results, we conclude that the chimeric and humanized antibodies of the invention are useful as agents for ameliorating various clinical symptoms of tumor associated hypercalcemia.

5. Improvement in blood pH reduction induced by hypercalceosis

Passage of human lung cancer LC-6 and production of animals with the hypercalcemia of the trauma model as described above in Chapter 2. Model animals were divided into two groups, averaging the concentration of blood drink and the body weight of the individual groups.

(1) Determining the pH of the blood

Blood collected from test animals using a hypotermic syringe using the robierαmy method Blood from the heart - introduced into the 643 Automatic Ca2 ⁺ / pH analyzer (CIBA-CORNING) to determine the pH of the blood sample (2) Administration of the antibody

The humanized anti-PTHrP antibody was administered to the rats with the model hypercalcemia (HHM rats) described above, via the tail vein at a dose of 5 mg / O.L / Kg (z = 3). Coatrolavz rats were administered saline in the same way (n = 2). In cnniysiyaiu for all rats treated with the antibody, control rats, the blood pH was determined on day 0 (ie the day of administration), on day 1 and on day 7. pcnyac results as the mean of the obtained pH values. _

As a result, before the administration of the antibody, the blood pH in the rats with the model hypercalcemia was 7.49 (while in the normal rats 7.4 ± 0.02), which means that the rats

190 351 models developed metabolic alkalosis. While the humanized anti-PTHnP antibody of the invention was administered to the model rats, although the control rats showed no improvement in the pH value, the higher and ^ c 'treated rats returned to near the pH of the normal rats seven days after the administration of the antibody. Metabolic alkalosis, known to be caused by inhibition of the excretion of bicarbonate ion (HCO <) in the kidney, has been reported as one of the clinical manifestations of tumor associated hyperkalaemia (HHM). While administering a humanized anti-PTHrP antibody of the invention normalizes blood pH in the hypencalyemil model, it is suggested that the antibody can reduce the metabolic alkalosis found in HHM (Figure 31).

From the results described above, it has been shown that the chimeric and humanized antibodies of the invention are useful as agents in reducing the clinical symptoms of tumor associated hyperkaleemia.

Possible industrial use

With reference to the present invention, chimeric or humanized antibodies against PTHrP are provided. These antibodies have poor antigenicity in humans and are therefore useful as agents in the treatment of hyperxlyemia, hypophosphatemia, etc.

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GTCTGAATTC AAGCTTAGTA CTTGGCCAGC CCAAGGCCAA CCCCACGGTC ACCCTGTTCC CGCCCTCCTC TGAGGAGCTC CAAGCCAACA AGGCCACACT AGTGTGTCT (2) INFORMATION FOR SEQUENCE IDENTIFIER NO .: 12:

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GGTTTGGTGG TCTCCACTCC CGCCTTGACG GGGCTGCCAT CTGCCTTCCA GGCCACTGTC

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GGAGTGGAGA CCACCAAACC CTCCAAACAG AGCAACAACA AGTACGGGGC CAGCAGCTAC

CTGAGCCTGA CGCCCGAGCA GTGGAAGTCC CACAGAAG (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 14:

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TGTTGAATTC TTACTATGAA

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GTCTAAGCTT CCACCATGGG GTTTGGGCTG AGCTGGGTTT TCCTCGTTGC ΤΟΠΤΓΑΑΟΑ GGTGTCCAGT GTCAGGTGCA GCTGGTGGAG TCTGGGGGAG GCGTGGTCCA GCCTGGGAGG

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ACCATTAGTA GTGGTGGTAG TTACACCTAC TATCCAGACA GTGTGAAGGG GCGATTCACC

ATCTCCAGAG ACAATTCCAA GAACACGCTG TATCTGCAAA TGAACAGCCT GAGAGCTGAG

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CAAGACATGC CATAGCTACT GAAGGTGAAT CCAGAGGCTG CACAGGAGAG TCTCAGGGAC

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ACAAAGCTTC CACCATGGGC TGGACTCCTC TCTTCTTCTT CTTTGTTCTT CATTGCTCAG

GTTCTTTCTC CCAGCTTGTG CTGACTCAAT CGCCCTCTGC CTCTGCCTCC CTGGGAGCCT

CGGTCAAGCT CAC

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AGCAAGATCG AAGCCACAGC ACAGGTCATG GGATTCCTGA TCGCTTCTCA GGCTCCAGCT CTGGGGCTGA GCGCTACCTC ACCATCTCCA GCCTCCAGTC TGAGGATGAG GCTGACTA (2) INFORMATION FOR SEQUENCE IDENTIFIER: 31:

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rrPTrrrTTr γδτγττωγττ ΔΛΓ.ΤΓΓΓΑΤΓ ΑΑΓ.ΤΑΓΓΓιΑΟ GGCCCTTCTC TGGCTGCTGC

Vlu l W'- 'A 1 KZ l <W * t * 4 A - ------ TGATGCCATT CAATGGTGTA CGTACTGTGC TGACTACTCA AGGTGCAGGT GAGCTTGACC

GAGGCTCC

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(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 114 base pairs (B) TYPE: nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLES :: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi ) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 32:

CTTGGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CCCTCACAAA TTGTTCCTTA ATTGTATCAC CCACACCACA GTAATAGTCA GCCTCATCCT CAGA (2) INFORMATION DJLA LDENTIFICCATOERA NO: 33:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) STRETCHITY: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (ii) DESCRIPTION SEQUENCE: SEQUENCE ID. NO: 33:

ACAAAGCTTC caccatg (2) DETAILS FOR THE IDENTIFICATION OF THE CATEGORY AND SEQUENCE IDENTIFICATION NO: 34:

(i) CHARACTERISTICS OF THE SEQUENCES:

190 351 (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDNESS: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA ( xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 34:

CTTGGATCCG GGCTGACCT (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 35:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 75 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 35:

CTTGGATCCG GGCTGACCTA GGAOGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA TTGTTCCTTA ATTGT (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 36:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 43 base pairs (B) TYPE: nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA

190 351 (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 36:

AAAGGATCCT TAAGATCCAT CAAGTACCGA GGGGGCTTCT CTG (2) INFORRBATION DILA IDENTIFICATION CATHORY AND SEQUENCE NO: 37:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 46 base pairs (B) TYPE: Nucleic acid (C) THREAD: Single.

(D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER. NO: 37:

ACAAAGCTTA GCGCTACCTC ACCATCTCCA GCCTCCAGCC TGAGGA (2) DILA INFORRBATION IDENTIFICATION CATHORY NO: 38:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 111 base pairs (B) TYPE: nucleic acid (C) THREADITY: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 38:

CTTCGATCCG GGCTGACCTA GGACGGTCAG TTTGGTCCCT CCGCCGAACA CGTACACAAA

TTCTTCCTTA ATTGTATCAC CCACACCACA GATATAGTCA GCCTCATCCT C # χ · \ \ -r »7» z-, -t-λ ν- »ττ» -rr> riXTmr nv m -n nnrzr.Tnnn * tt (z; x ujljllHAu. JlnujljllHAu. .jn jc α & ΛΗωΊυυι.

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 42 base pairs (B) TYPE: Nucleic acid

190 351 (C) THREADNESS: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER. NO: 39:

CTTCTCTGGC TGCTGCTGAT ACCATTCAAT GGTGTACGTA CT (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 40:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 26 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 40:

CGAGGGCCCT TCTCTGGCTG CTGCTG (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 41:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 35 base pairs (B) TYPE: Nucleic acid (C) STRANDNESS: Single (D) TOPOLOGY: Linear (ii) TYPE OF PARTICLE: Other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 41:

GAGAAGGGCC CTARGTACST GATGRAWCTT AAGCA (2) INFORMATION FOR SEQUENCE ID NO: 42:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 35 base pairs

190 351 (B) TYPE: nucleic acid (C) THREAD: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER. NO: 42:

CACGAATTCA CTATCGATTC TGGAACCTTC AGAGG (2) INFORMATION FOR SEQUENCE ID NO: 43:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 18 base pairs (B) TYPE: nucleic acid z.

(C) THREADNESS: single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid (A) DESCRIPTION: / desc = "synthetic DNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER. NO: 43:

GGCTTGGAGC TCCTCAGA (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 44:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 20 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Single (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: other nucleic acid

ODTQ _: νχ. a. KZ // dnc n -

O X r »·» ł- ł— τ z »▼ r" synthetic r \ kT7 \ rr DNA.

GACAGTGGTT CAAAGTTTTT (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 44:

190 351 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 45:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 45:

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

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

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

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

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

190 351

70 75

Tyr Leu Cheese Ile Cheese Asn Ile Gln Pro Glu Asp Glu Ala Het Tyr

85 90

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

100 105

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

110

115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 4 6 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 46:

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

10 15

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

25 30

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

40 45

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

55 60

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

190 351

70 75

Lys Asn Thr Leu Tyr L ^^ u Gin Met Ser Ser Leu Lys Ser Glu Asp

85 90

Thr Ala Mtet Phe Tyr Cys Ala Arg Gln hhr hhr Met Thr Tyr Phh

110 115

Ala Tyr Trp GGy GGn Gly Thr Leu Val Thr Val Ser Ala

110 115 (2) INFORMATION DULA OF SEiQUENCE IDENTIFIER NIR: 4 7:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 116 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (s) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 47:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser All Ser I.lu Gly

10 15

Ha Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gil His Ssr Thr

25 30

Tyr Thr Ile Glu Trp His Gln Gln Gln Pro Glu LLy Giy Pro Arg

40 45

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

55 60

Gly Iie Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arr

190 351

70 75

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

85 90

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

100 105

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly

110 115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 48 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 48:

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

10 15

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

25 30

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

40 45

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

55 60

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

190 351

Tyr Leu Thr Ile Cheese Cheese Leu Gln

Tyr Cys Gly Val G1y Asp Thr Ue

Phe Gly Gly Gly Thr Lys Leu Tlm

110

70 75 Cheese Glu Asp Glu Ala Asp Tyr 85 99 Lys Glu Main Flie Val Tyr Vvl 110 115 Val Leu Gly Main Pro 111

(2) IDFFRRMATION FOR THE IDENTIFICATION OF THE SEICING IDENTIFICATION NO: 49:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (E) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 49:

Gln Leu Val Leu Hu · Gln Pro Cheese Ala Ser Ala Ser Leu Gly

10 15

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

25 3

Tyr Thr Ile Glu Tri) Tyr G In Gln Gln IPo Glu Lys Gly FPo Lys

40 45

Tyr Val Met Asp eeu ys s Gln Asp Gis Ser H is Ser ThG G ly / Asi?

55 6

Gly Ile Pro Tsp Ar. Phe S er r ly Ser SSr Ser Sir Ala Glu Ara

190 351

70 75

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

85 90

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

100 105

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

110 115 (2) INFORMATION DIA IDENTIFHKATO1R SEIKFENCE NO: 50 (i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE protein (s) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 50:

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

10 15

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

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

55 60

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

190 351

70 75

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

85 90

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

100 105

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

110 115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 51:

(x> CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 51:

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

10 15

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

25 30

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

40 45

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

55 60

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

190 351

70 75

Tyr Leu Thr Ile Cheese Leu Gln Cheese Glu Asg GIa Ala Asp Tyr

85 07

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

170 107

Phe Gly Gly Gly Thr Lys Leu The lal Leu Gly Gln Pro

007 018 (2) INFORMATION FOR THE IDENTIFICATION OF THE SEEKIENCE CATHORY NO:: 552 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 52:

Main Lee Val Leu Thr Main Cheese Pro Cheese Ala Cheese Ala Cheese Leu Gly 1 5 70 10 Ala Cheese Val Lys Dream TSt Css SIil Gene i5en rer Main His Cheese Tlnr 22 25 thirty Tyr Thr How much G lu Trp Tyr Main Main Main Pro Glu Lys Gly tPro Lys 38 70 45 T _ 1___ LSG AND f 1 n " Ολ ». at; > - TK · »· r. and " AND iyi mc m nap ¹ LLU tear O vxn utop we ner HU wr HIM. MA / <IO | A 87 85 Gly How much Pro Ace And AAg PPe PSr Gly Cheese Cheese Cheese Gly Ala Glu Prg

190 351

70 75

Tyr Leu Thr Ile Cheese Leu Gln Cheese Glu Asp Glu Ala Asp Tjr

85 90

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

100 100

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

110 110 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 53:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (11) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 53:

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

10 10

Ala Ser Val Lys Leu Thr Cys Thr Leu err err dn Hss Ser TTh

25 30

Tyr Thr Ile Glu Trp Tyr Gln Gln Gln roo du Lys Gly FP-o AAr

40 45

Tyr Leu Met Asp Leu Lys Gln Asp Gly Ser Hss Ser Thr Gly Aas

55 66

Gly Ile Pro Asp Arg Phe Ser Gly Ser Srr Ser lly Ala Glu AAr

190 351 ⁶⁵ 70 75

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

85 90

Ile Cys Gly Val Gly Asp Thr Ile Lys Glu Gln Phe Val Tyr Val 100 105

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

110

115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 54 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 54:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 15 10 15

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

25 30

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

40 45

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

55 60

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

190 351

70 75

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

85 90

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

100 105

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

110 115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 55:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 55:

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly 15 10 15

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

25 30

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

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 Cheese Gly Cheese Cheese Ser Gly Ala Glu Arg

190 351

65 70 75 Tyr 1 eti Thr How much Cheese Cheese l.eu Main Cheese Glu Asp Glu Ala Asp Tyr 80 85 90 How much Cys Gly Val Gly Asp Ihr How much Lys Glu Main Phe Val Tyr Val 95 100 105 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Main Pro 110 115

(2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 56 ii) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 118 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 56:

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

10 15

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

25 30

Cheese Tyr Gly Met Cheese 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

190 351

70 75

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

85 90

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

100 105

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

110 115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 57:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 411 bp (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: CDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 57:

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

Met Asn Phe Gly Leu Cheese 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 HA 90

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

5 10

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

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

190 351

15 twenty 25 TTC ACT rrc AGT AGC TAT GGC ATG TCT TGG ATT CGC CAG ACT CCA 180 Phe Thr Phe Cheese Cheese Tyr Gly Underworld Cheese Trp How much Arg Main Thr Pro thirty 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 How much Cheese Cheese Gly Gly Cheese 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 Cheese Val Lys Gly Arg Phe Thr How much Cheese 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 Gin Underworld Cheese Cheese Leu 75 80 85 AAG TCT GAG GAC ACA GCC ATG πτ TAC TGT GCA AGA CAG ACT ACT 360 Lys Cheese Glu Asp Thr Ala Underworld Phe Tyr Cys Ala Arg Main Thr Thr 90 95 100 ATG ACT TAC TTT GCT TAC TGG GGC CAA GGG ACT CTG GTC ACT GTC 405 Underworld Thr Tyr Phe Ala Tyr Trp Gly Main Gly Thr Leu Val Thr Val

105 110 115

TCT ca 411

Ala cheese (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 58 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 base pairs (B) TYPE: nucleic acid (C) STRETCHITY: double i γί \ mr> nr \ T r \ r * T τ \ · Ή η-ΐ ζιτ.τϋ (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 58:

190 351

ATG GGG TTT GGG CTC agc TGG GIT CTC CTC CTT GCT crr TTT CAG 45 Underworld Gly Phe Gly Leu Cheese Tnp Val Phe Leu Val Ala Leu Leu Arg; -15 -10 -5 GGT GTC CAG TCT GAG GTC CAG CTC GTC Gd TCT OH GGA GGC GTC 90 Gly Val Main Cys Main Val Main Leu Vvl Glu Cheese Gly Glyy Gly Vvl 1 5 10 GTC CAG CCT GGG AtH TCC CTC AGA crc TCC TCT GCA GCC TCT GGA 135 Val Main Pro Gly Arg Cheese iLiU Arg Leu Cheese Cys Ala Ala Cheese Glyr 15 twenty 25 TTC ACC TTC ACT AdC TAT GGC ATC TCT TK GTC OGC GAG GGT CCA 180 Phe Thr Phe Cheese Cheese Tyr Glyy Underworld Cheese Trp V £ ^ l Arg Main Ala Pro thirty 35 40 GGC AAG GGG γτγ ' GAG TCG CTG GGA AĆCC ATT AGT ACT GGT GlT AGT 225 Gly Lys dy Leu Glu Trp Val Ala Hu How much Cheese Cheese Gly Glyy Cheese 45 50 55 TAC ACC TAC TAT CCA GAC AdT GTC AAG GGG CGA TTC ACC ATC TCC 270 Tyr Thr Tyr Tyr Pro Asp Cheese Val Lys Gly Arg Phe Tlhr How much Cheese 60 65 70 AGA GAC AAT TCC AAG AlAC ACH crc TAT CTG CAA ATC AAC AGC CTC 315 Arg Asp AAn Cheese Lys Asn Background * Leu Tyr Leu Main Underworld Asn Cheese Leu 75 80 85 AGA CCT GGG GAC ACG GCT GTC TAT TAC TCT GCG AGA GAG ACT ACT 360 Arg Ala du Asp Thr Ala Val Tyr Tyr Cys Ala Arg Main Tlhr Thr 90 95 100 ATG ACT TTA TTT GCT TAC TGG ax CAG GGA ACC CTC GTC AGC GTC 405 Underworld Thr Ttt Phe Ala Tyr Trp Gly Main Gly Thr Funnel Vvł Ttr Vv1

TCC TCA 411

Cheese Cheese

190 351 (2) INFORMATION DRA SEQUENCE IDENTIFIER NO: 59:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH): 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 59:

Lys Ala Ser Gln Asp Val Asn Thr Ala Val Ala (2) DJL INFORMATION SEQUENCE IDENTIFIER NO .: 60:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: peeptide (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 60:

Ser Ala Ser Asn Tyr Thr (2) SEQUENCE IDENTIFIER INFORMATION NO: 61:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH): 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) R0t ^ 2 ^ A ^ J MOLECULES :: peptide (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 61:

Gln Gln His Tyr Ser Thr Pro Phe Thr (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 62:

(l) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

190 351 (ii) TYPE OF PARTICLE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 62:

Pro Tyr Trp Met Gln (2) SEQUENCE IDENTIFIER ELA INFORMATION NO: 63:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 16 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: peptide (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 63:

Cheese Ile Phe Gly Asp Gly Asp Thr Arp Tyr

Gln Lys Phe Lys Gly cheese (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 64:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID. NO: 64:

Gly Leu Arp Arp Gly Gly Tyr Tyr Phe Asp

Tyr

190 351 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 65 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER 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

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

190 351

CAG CAC ACT ACG TCC CCC Aπ AAA TCC ATT CCAG (CAr <C1G CCC CTC 180 Main His Cheese Thr Tyr hhr How much G1u rsp Τργ Main Gin Main Prr Lli thirty 35 40 AAG CCT CCT AAG AAT TTG TGG ATT TTT AGC CM GAT GCA AG) CCA 225 Lys Pro Pro Lys Τργ aal Mt t Asp eu u Ts s Main Asp Gly Cheese His 45 50 55 AGC ACA GGT GAT GGG ATT CCT GAT CCC TCC TCT GCA TGC AG) TTC 270 Cheese Thr Gly Asp Hy He Poo ssp rgg hee S ^ ip Gly Cheese Cheese Cheese 60 65 70 GGT GCT GAT CGC ACC TTT GCC TTT CCC ACC ATC CAG <CCA GAA GAT 315 Gly Ala Asp Arg Tpt euu err lle Srr snn How much Main ! Pp3 Glu Asp 75 80 85 GAA GCA ATG TAC TTC GCT GCT GGG GTT GAT ACA ATT AAG GAA CCA. 360 Glu Ala Underworld Tyr U e Ts s ly y a _a i G1 y ss p Ήττ How much Lys Glu Gin 90 95 100 TTT GTG TAT GTT TTC GGG GGT cgg AOC AAG {^ HC ACT GTC CTA GIT 405 Phe Val Tyr Val hee Gly Hy Hy TPr Tss Val Thr Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 66:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 405 bp (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (li) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO; 66:

190 351

ATG GCC TGG AC * CCT CTC Underworld Ala Trp Tlur Pio Llu -15 GGT TCT TTC TCC CCA GCT Gly Cheese Phe Cheese Main Glu 1 GCC TCC CTG GG / A GCC TCC Ala Cheese Leu Gly Ala Cheese 15 CAG CAC AGT ATC TAC (ACC Main His Cheese TItu Yo yo '(Here thirty AAG GGC CCT CTC TAC (CCG Lys Gly Pro Arg Tyr Llu 45 AGC ACA GGT GAT (Gd ATT * Cheese Thr Gly Asp Gly How much 60 GGG GCT GAG GCC TAC CTC Gly Ala Glu Arg You Glu 75 GAG GCT GAC TAT TAC TCT Glu Ala Asp Tyr T (uo Cys

Phe Phe Val

GTG CTC ACT CfAA TCG

Val Leu (Thr Gln Siar

GTC AAG CCC AGC (TGC

Val Lys Leu Thr Cys

GTT GAA TCd CAT CAG

Ile Glu Trp His Gln (35

GTG AAA CTT AAG CAA

Met Lys Leu Lys Gln

GCT GAT (CGC TTC TCA

Pro Asp Arg Phe Ser

A <GG AITG TCC ATC CTC

Thr Ile Cheese Cheese Leu

GGT GTC GGT GAT ACA

Gly Val Gly Asp Thr

CTT CAT TCRC TCA 45

Leu His Cys Ser

TCC TCT (GCC CCC 90

Pro Ser Ala S®r

AGC TTG AGT AGT 135

Tut Leu Cheese Cheese

CAG CAG CCA GAG 118)

Gln Gln Pro Glu (GAT GGA ATC (GAC 225

Asp Gly Ser His (GGC TCC ATC TCT 270

Gly Cheese Cheese Cheese

CAG TCT GAG GAT 315

Gln Cheese Glu Asp

ATT AAG GAA CAA 360

How Much Lys Glu Gln

190 351

95 HW

TTT GTG TAC GTG TTC GiGGGGACGGi ACC AAA CTG ACC GTC CTA GGT 405

Phe Val Tyr Val Phe Gly Gly Gly 'Πιΐ' Lys Leu Thr Val Leu Gly

105 111 115 (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 67:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 67:

ATG GCC TGG ACT CCT CTC TTC TTC TIC ΤΤΤ (ΤΓΤ CTT CAT 'TGC TCA 45 Underworld Ala Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Cheese -15 -10 -5 GGT TCT TTC TCC CAG CTT GTG CTG ACT CM TCG αχ HERE (GCC TTC 90 Gly Cheese Phe Cheese Main LeL Val Leu Thr Main Cheese FPo Cheese Ala Cheese 1 5 10 GCC TCC CTG GGA GCC TCG GTC AAG CCC AOC THIS AOC rrc AGT AGT 133 Ala Cheese Leu Gly A1a Cheese VaV Lys Llu Here* Cys Πη- L ^ u Cheese & 3Γ 15 twenty 22 CAG CAC AGT ACT: TAC ACC ATT GM THIS TAT CAG CAG CAG (CA GAG 180 Main His Cheese Thr TyT Thr How much GGu Ttt Ie GGn GGn TGu Ti ^ o GGu

190 351

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

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

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 Cheese Cheese Leu Gln Cheese Glu Asp

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

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) INFORMATION FOR SEQUENCE IDENTIFIER NO: 68:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 411 bp (B) TYPE: Nucleic acid (C) THREADITY: double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 68:

190 351

ATG GCC TGG GCT CCT ccc CTT TCC ITC πτ GIT CTT CAT TCC TCA 45 Underworld Ala Trp Thr Per Cuu CPe Phe Phe Phe Val Luju His Cys Cheese -15 -10 -5 GGT TCT TTC TCC CAG CTT CGG CTC ACC ' CM TCG GAC 'TCT dC IGCT 90 Gly Cheese Phe eer Pin Llu Inch Leu Tr Main Cheese Fer> Cheese Ala Cheese 1 5 10 GCC TCC CTG GGA HIM CGO CTC AAG CTC AGC rac ACCC TTC AGT AGT 115 Ala Cheese Leu Gly Ala Cheese Vv1 Cys Leu Tugr Cys Thr Leu Cheese Cheese 15 twenty 25 CAG CAC AGT A (C CAC A (X AUT CM Tm TAT String CAG GAG OC \ GAG 180 Main His Cheese TCr Tjr 'Crr How much Glu Tnp Tyr Main Main Main CPo Glu thirty 35 40 AAG GGC CCT TAG TAC GTC aat; G / AT CCT AAG CM GAT GGA Ad CAC 225 Lys Gly Pro Τ> τ Val Underworld Asp Ll3U Lys Main AND;? Gly Cheese His 45 50 55 AGC ACA GGT GAT CGG ATT CCT GAT cgc TTC TCA GGC rac AGC iccr 270 Cheese Thr Gly Asp Gly How much pro Asp Arg Phe Cheese Gly Cheese Cheese Cheese 60 65 70 GGG GCT GAG CGC TAC CTC ACC ATC TOC AGC CTC CAG ΤΤΑΤ G ^ G GAT 315 Gly Ala Glu uArg Tjr Leu TCrr How much Cheese Cheese Leu Main Cheese Glu Asp 75 80 85 GAG GCT GAC TAT TAC TCT GUT CTG GGT GfæT ACA ATT MG GM CM 360 Glu Ala Asp Ter Tyr Cys Gly Val Gly Asp Thr How much Lys Glu Main 90 95 100 TTT GTG TAC GTG TCC GGC GGA GGC CCC AM TTC CCC GAC CTC GGC 005

190 351

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

105 110 115

CAG CCC 411

Gln Pro (2) DILA ΙΟΕΝΤΥΓΙΚΤΟΚ SEFKJENCE INFO NO: 69 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to uRNAA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 69:

ATG GCC TGG ACT CCC CCC TTC TTC TTC ΤΓΓ GIT CTT CAT TGC TCA 45

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

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTC ACT CM TOG (TC 1CT GCC TCT 90

Gly Cheese Phe Cheese Gln Leu Val Hu · Gln Aug: FPro Cheese Ala Ser

5 10

GCC TCC CTG GGA CCC TCC GTC MG CTC AG3 TOS ACC TTC ACT AGT 135

Ala Cheese Leu Gly Alei Cheese Val Lys Leu Ήτ ’Cys Hu bej Cheese Cheese

20 25

CAG CAC AGT ACG TAC MC ATT GM TCGG TAT (CAG CIG OlG OCl GAG 180

Gln His Ser Thr Tyr Thr He Glu Trp Tjyr Gln Gln Gln FPo Glu

190 351

35 40

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

Lys Gly Pro Arg TTr Leu Met Asp Leu Lys Gln Asp Gly Ser C is

55 55

AGC ACA GGT GAT GGG ATTCCT GAT (GC TCC 'CA GGCC T (CC AAG CTC 270

Ser Thr Gly Asp GGy Ile Pro Asp Arg Re Ser Gly Ser Ser C er

65 70

GGG GCT GCG CGC TTA CTC ACC ATC TCC AGC CTC CAG TCT GGA CGA 315

Gly Ala Glu Arg Tti Leu Thr Ile Cheese Cheese Leu Gln Cheese Gil Cap

80 85

GAG GCT GAC TCT TTC TGT OTT GTG (OT GCT ACC ATT GCG GGC CCC 360

Glu Ala Asp Tyr Tir Cys Gly Val Gly Asp Thr Ile Lys Gil Cll

95 100

TTT GTG TCC GTO TTCGGC GGA G (G ACC AAC CTG ACC GTC CT 'CGC 405

Phe Val Tyr Val Phe Gly Gly Gl.y Thr Lys Leu Thr Val Lee G ^

105 111 115

CAG CCC 411

Gln Pro (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 70:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 70:

100

190 351

ATG GCC TGG ACTCCTCTC TTC TTC TTC TTT GTT CTTCATTGC TCA 45

Met Ala Trp Background Pro Leu Phe Phe The The Tal Teu Tis Tas Ter

-15 -10 -5 ^ Τ TAT CTT TOCCAT (ATT GTG CAT ACA CAG TTA CAA TTA CGA TTA 90

Gly Sur hhr Ser Gln Ten Val LerTGe GGl Ter Thr Ter da Ter

5 10

GAC TAA ATG GGA GTT; (GAT GTC AAG CAT ATA TTG CTT TTC CGG TTG 135

Ala Srr Luu Gly Ala Ser Val Ler Ter TTe Tas TTr Ter Ter Ter

20 25

ATG AAA TGT ACAT ^ / ^ (T AOC ATT GGT TTG TTT CAG CAT CAT TTC CGG 110

Gln His err Thr T (y TTuc He Glu Ter Ter GL · CL · GL · Thr GGl

35 40

ATG GGA AAG A (TG TAC GTG ATG GAT CAG CTG CAT C ^ T * GGA CGG: GATT 225 eys Gly hro Arg Tisr V ^ 1 Met Asp Ler Ces GG ^ Cap GGl Ger Gis

50 55

AGA TAT GGC GAT ATT (AAT GAT (AGT CTC TCA (^^ TTA! AAC TTA 270 err CUr Gly Asp Gly Ile ihro Asp (Ur Che Ger Gly Ger Ser Ger

65 70

GAG GAG CGC tttq CCC ACC ATC TCC AGC CTCT CAG TCT GAG CAT 315

Gly Gla Glu Arg Tyr Leu Thr Ile Cheese Ser Leu GGn Ser Glu (Gp>

80 85

GAG GCT GTA TCT "TAC TGT GCT CTG CG ^ T GAT A (TT AGTT (TTG G / GT CTG \ 360

Glu Ala Gsp T (r Tyr Cys Gly Val Gly Asp TTer Ile Lys Glu Gln

95 1 (0)

TCC GCG GTA GTCTTC GGC GGA ΟΓ (GAT (GAT CAT (GXT GTC CATA 005

190 351

101

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 110 115

CAG CCC 411

Gln Pro (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 71:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 71:

ATG GCC TGG ACT CCT CTC TTC TTC TTC ΕΠ 'GTT CTT CAT GGC TCA 45

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

-15 -10 -5

GOT TCT TTC TCC CAG CU GTG CTG ACT CAA TGG CCC TCT GCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu Thr Gln Ser Poo Ser / Ha Ser

5 10

GCC TCC CTG GGA GCC TCG GTC AAG CTC ACC GGC ACC TGG AGT AGT 135

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

20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CGG CAG CGG CCA GAG 1 ^ 0

Gln His Ser Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln (Pro G1g

102

190 351

35 44

TTG GGC CCT TAG TTC CTG ART GAT επ ’AAG CAA GAT GGA ATG CAA 225

Lys GIt Ppo Lys Tyr eeu Mtt ssp Leu Lys G1n ssp Hy Sse His

50 55

AGC TCT GG 'GTT GCG ATT CCT GAT ^- CCG TTC TCA GGC TCC AGCTCT C70

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

65 70

GGG GCT GAG CGC TAC CTCACCATC TCC AGC CTC CAG TCT GAG GAT TT5

Gly Tla Glu Arg Ttp Iuu TPr He Ιργ Ιργ Iuu Gln Ιργ GGg Atp

80 85

GTG GCT GTC TTT TTC TGTGGTGTG GGT GAT ATCt ATT AAG GAA CGT T60

Glu Tla Asp Tyr Ile Ts s Gly aal Gly ssp Thr He Tss GGg GL

95 100

TH GTG TAC GTG TTC GGC GGA GGG ACC AAA CTG ACC GTT CTA CCT GO5

Phe VaI Tyr VaG Phe GGy GGy Gly Thr Lys Leu Thr Val Lli Gly

105 110 115

CAG CCC 411

GGn Pro (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 72 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) THREADITY: Double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 72:

190 351

103

ATG GCC TGTACT CCC CCT GTC GTC GTT GTT GTT FAT CAT 'TCC TCA 45

Met Ala Tit TTt Gir Cte Phh Phh FPih Phh Val Leu His Cci GSr

-15 -10 -5

GGT TAA TTT TCC CCC CTT CTG FAT ACT CM TCG GACA FTCT CGAC FTA 90

Gly Cheese PhP rSr dn CLr Val Leu FTt Gln Cheese FPr »Seer Ala Cer

5 10

GAA TAC CTGGGA GCC GTC GTC MG CTC ACC 'TGA AGA GIC AGT AGT 113

Ala Ser Leu Gly AAa 'Sr Val Lys Llu FTt · Cys Tt Leu Ser Ser

20 22

AGG CCC AGG ACC TAAA AAC GATT GM GTC TAT GAG GAG CC ^ G CCC GAG 118

Gln His Srr Thr Tyr Thr ILe Glu Trp Tyr Gln Gln Gln Pro Glu

35 40

CCG GGC CCTAGG CAC CCG AAT GAT FCT MG CM GAT GGiA FGGC FCAA 225

Lys Gly Pro Arg Tyr Leu Mrt Asp Leu Lys Gln Asp Gly Srr His

50 55

GGC CCC GGT GAT CGG AAT GCT GAT GGA TTC TCA GGC TGC AGC TCT 270

Ser Thr Gly Asp Gly IIe FPo Asp Mg Phe Ser Gly Ser Ser Ser

65 70

Gd GCT GAdCC CAC CTC ACC ATC TGC AGC CTC CAG TCT GGG GGT 315

Gly Ala Glu Arg Tyr Leu Thr Ile Cheese Ser Leu Gln Ser Glu Gpp

80 85

GAG GCT GAC TAT AATA TCT GGT GTC GGT GAT ACA ATT MG GM CAA 3 ^ 0

Glu Ala Asp Tyr Ilr Cys Gly Val Gly Asp Thr 11g Ipg Hg lin

915 IG)

TTT GTC TAC GTC GTT (FGGC FHA Gd ACC GACA CTG ACC GTC CTA GGC -435

104

190 351

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

50 55

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

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

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 Cheese Cheese Leu Gln Cheese Glu Asp

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

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) INFORMATION FOR SEQUENCE IDENTIFIER NO: 74 (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 411 base pairs (B) TYPE: Nucleic acid (C) STRANDNESS: Double (D) TOPOLOGY: linear (ιί) TYPE OF PARTICLE: cDNA to mRNA (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 74:

190 351

105

Phe Val Tyo Val Phe Gly Gly Gly Tho Lys Leu Tho Val Leu Gly

105 110 115

CAG CCC 411

Gln Pro (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 73:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 411 bases (B) TYPE: Nucleic acid (C) THREADITY: double (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: cDNA to IRV (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER NO: 73:

ATG GCC TGG ACT CCT CTC TTC TTC TTC TTT CIG CTT CAA TGC TCA 45

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

-15 -10 -5

GGT TCT TTC TCC CAG CTT GTG CTG ACT CAAACGCCC TCT GCCC TCT 90

Gly Ser Phe Ser Gln Leu Val Leu TUv Gln ner Pro Ser Ala. Cheese

5 11

GCC TO CTG GGA GCC TCG GTC AAG CTC ACC CGCACC TTG AGT AGT 135

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

20 25

CAG CAC AGT ACG TAC ACC ATT GAA TGG TAT CAG CAG CAG CCA GAG 100

Gln His Svv Thr Tyr Thv Ile Glu Trp TyrGln Gln Gln JPv> Glu

106

190 351

ATG GCC TGG ACT CCT CTC TTCTTC TTCTTTGTT CTT CAT TGC TCA 45

Met Ala Trp Thr Pro Ltu PTe PTe PTe PTe Vd Leu Tis Tcs Ter

-15 -10 -5

GGC CCT TTC TCC CAA CCT GTG TTG ACC CAG TCG CCC TCT TCC TTC TC

GLy Ser Phe Ser Gln Uru VtL Uru Thr GLn Ser Oro Ser Ala Ser

5 10

GTT TCT CTG GGA TCO TO TCCACGGCCTTeTTCTTC TTT TAG TTG 135

ALc Cheese Ueu GLy AIa. Cts rd Lye Ltu UTt Cts Tłu- Tup Tsp Tsp

20 25

ATG CTC TGC GTC TAC TCAC ATT GMTCG TATCAG (C ^ TTA TCC dG 180

GLn His Ser The Tjt Che I Te G lu TjtiTits G ln Gln du Tor du

35 40

TAG TCT CTG GGG TAC GTT ATT GATCTTAAG C / CGGAT Td TTC TCG 225

Uys GLy Oro Teg Tjtt Val Met Asp Leu Lys G ln du Tsp Tis

50 55

AGC TCA TCC GAC GGG ATT CCT GAT CTCTTC TCA (GGA TTC TTC TCT 270

See The GLy Tsp Gty I le Pro A τρ Arg Phe S er Gly Ser Ter Ter

65 70

GTC GCT GTG AGA TAC CTC AGA ATCTCCATCCTTCAG TTC TGG TAT 315

GLy GLc GLu Teg Tjt- Leu Thr I le S er S er Leu Gln Ser du TTl

80 85

GAG TCT GAC CAT ATT TGT GGT GTG TCT GATACA ATT TH TM TCT 360

GLu ALc Asp Cyc Ile Cc: TT ty VvT du AglTTt Ile Les du dn

95 100

TTC GTG TGT dG TTCGGCTCAGTC ACC AAACCT ACC GTC CTA TTC 405

190 351

107

Phe Val Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 105 HO US

CAG CCC 411

Gln Pro (2) INFORMATION FOR SEQUENCE IDENTIFIER NO: 75:

(i) CHARACTERISTICS OF THE SEQUENCES:

(A) LENGTH: 34 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF PARTICLE: peptide (xi) SEQUENCE DESCRIPTION: SEQ IDENTIFIER 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

25 30

Ile His Thr Ala

108

190 351 to (2)

FIG. 2

Hj-ndlU θ

D BamHI

FRi CDR1 FR2 COR2 FR3 CDR3 FR4 (A-E)

F.

First PCR reaction (B-F) (C-G) (D-H)

Connection

S Second PCR reaction (3)

190 351

109

FIG. 3 v region

FRl FR2 FR3 FR4 CDR1 Bil CDR2 Sil CDR3 Plasmid Activity H. H. m m h / mMBClL (l) - m m H. H m / hMBClLU) + H. m m m hmmMBClL (l) + m H. m m mhmMBClL (λ) -

H: FR Human antibodies m: FR Murine antibodies

110

190 351

FIG. 4

Determination of Antigen Binding Activity

FROM. 405/620

Antibody concentration (ng / ml)

190 351

111

FROM. 405/620

FIG.5

112

190 351

FROM. 405/620

FIG. 6

Determination of Antigen Binding Activity

Antibody concentration (ng / ml)

190 351

113

FROM. 405/620

FIG. 7

114

190 351

FROM. 405/620

FIG. 8

Determination of Antigen Binding Activity

Antibody concentration (ng / ml)

190 351

115

O. D. 405/620

FIG. 9

Antibody concentration (ng / ml)

116

190 351

FROM. 405/620

FIG.10

Determination of Antigen Binding Activity

Antibody concentration (ng / ml)

190 351

117

FROM. 405/620

FIG.11

Determination of Antigen Binding Activity

Antibody concentration (ng / ml)

118

190 351

C-AMP concentration (pmole / m3)

FIG. 1 2

Neutralizing activity of Humanized Antibody against PTHrP (1-34)

Antibody concentration (pg / ml) hMBC j hMBC m hMBC o hMBC r chMBC # 23-57-137-1

190 351

119

FIG. 1 3

The neutralizing activity of the Humanized Antibody

chMBC hMBC I hMBCs hMBC t

Antibody concentration ^ g / ml)

120

190 351

FIG.14

Neutralizing activity of Humanized Antibody against PTHrP (1-34)

chMBC hMBC q hMBC r

Antibody concentration ^ g / ml)

121

190 351

(6) E (e and Jeżcie

Days After Transplant (Days)

122

190 351

N

E.

OJ

-at ABOUT WHAT ABOUT C3 this about CM <D £ ABOUT m ABOUT ω σ ABOUT ABOUT ABOUT .2 2 m tn -C JZ 2 2 CQ AT about x: .c Q-

Effect of Chimeric Antibody and Humanized Antibody on Animal Model of Hypercalcemia (Nude Mice Carrying Human Pancreatic Cancer PAN-7)

(6) E} EJQ JEZSlQ

Days After Transplant (Days)

123

190 351

Days After Transplant (Days)

124

190 351

N CZ) > » P. 03 Sat X about π ABOUT AT b. ca c about 2 at

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X

ABOUT

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S u

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n σ cr

O O ca ca S z * z · o

£ '?

Cd

b.

CU .S2 c5

Ό

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T.

c

Q

FIG. 18

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about

about.

N

ΙΛ <D

0.

ABOUT

CL

Ξ (S) Err3! 0 ·> ^Β ? ^δ ! 0

190 351

125

IN

CU

N

ABOUT

about ABOUT ABOUT ABOUT ABOUT ABOUT ABOUT about about about ABOUT ABOUT ABOUT ABOUT ABOUT ABOUT about about about ABOUT ABOUT ABOUT about ABOUT ABOUT about about CN ABOUT 00 CD N- CN about 00 CN CN CN x— X ™ x— X " χ—

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CZ) W Φ <D>>. Z 2 Z 2

Γ- CM m ABOUT What ABOUT CM r- Tt t— Τ- about 7 about 1 Tt 1 T. Ο 1 γ- Ol this Tt 00 00 ι- CM What WHAT about CM ο ' CM X— What CM ABOUT p 00 Ol What CD WHAT Tt you χΙ- 00 WHAT this 00 LO Ο tn CM cn r- t— this about Ie- What »- CM CM Tt CM CM χ ^- Τ- V— x ~ x ~ about Ο about about ABOUT p tri tri tri tri tri tri this CM this about What χί- cn CM this What Tt Ο ex What this cn tn tn 00 What X ^- CM What tn tn CM CM CM CM CM CM

6063 5.0 12383.6 0.13 0.00 No 226.4 10mM-HCI-10ul

PTHrP mobilization sensorogram (1-34 + C) at the sensor tip

126

190,351 m

ro

Ό $

Φ 'c _ Φ o N Φ

Φ- N ** —ł ω x 3

- = o c tn cm uo o

about

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QQ

about ABOUT What s 75 E. ro and_ about o> about What L_ ABOUT ω ω c fl) ω (0 What g> N </) ABOUT fl) about c about ro CM ~ σ ro 72 ro 2

FIG. 20

ABOUT about ABOUT ABOUT about ABOUT o about ABOUT about ABOUT ABOUT about ABOUT about about m- CM ABOUT 00 CD xr CM CM

(na) efo ^ eey

190 351

127

3F5

ABOUT

ABOUT

LO o

about

N o

what about

about

CN

C \ J o

LL.

about ABOUT ABOUT ABOUT about about o about about ABOUT ABOUT ABOUT about about about about Ν ' CN ABOUT WHAT What CN CN

(ny) efo ^ esy

WHAT

What ro

N o

3F5 sensorgraph overlap

128

190 351

FIG. 22 chMBC

and a \ »λΓλιιΛΛ \ a kAa?

z—>

w <n ro

N

ABOUT

Overlapping chMBC sensorogram

190 351

129

O r ° LO

FIG. 23 hMBCm

Overlapping chMBC sensorogram

about about ABOUT ABOUT about about o ABOUT about about ABOUT ABOUT about about about ABOUT TC CN ABOUT 00 What TC CN CN

\ ino \ a

130

190 351

ABOUT

ABOUT

LO

ABOUT

O xr o

about what

FIG. 24 hMBCq

about about ABOUT ABOUT about about o ABOUT about about ABOUT ABOUT about about about ABOUT CM ABOUT 00 What 'M' CM CM

(na) Φ> | ΒθΗ o

o cm (Λ (0

N o

HMBCq sensorgraph overlap

190 351

131

FIG. 25

Effect on fractionated phosphate excretion

Fractionated phosphate excretion

Period (1 period: 20 minutes; mean value ± standard error)

132

190 351

0.070

FIG. 26

Effect on plasma phosphate levels

Plasma phosphate concentration (mg / ml)

0.065

0.060

0.055

Humanized Antibody against PTHrP

0.050 ^L 0

J_I_I_I_L

2 3 4 5

Period (1 period: 20 minutes; mean value ± standard error)

190 351

133

FIG. 27

134

190 351

FIG. 28

190 351

135

FIG. 29

Influence on spontaneous movement

Days

136

190 351

FIG.30

Effect on body temperature

Body temperature (° C)

J I-1-1

3 4

Normal rat Antibody against PTHrP

Control

Days

190 351

137

FIG. 31

Effect on blood pH Blood pH

7.52

7.50

7.48

7.46

7.44

7.42

HHM Control

HHM Antibody against PTHrP

Days

138

190 351

FIG. 1

FR (framework region)

Heavy chain (H chain)

Publishing Department of the UP RP. Mintage 50 copies. Price PLN 6.00.

Claims (49)

Patent claims 1 A chimeric L chain comprising the C region of the L chain of a human antibody and the V region of the L chain of a murine monoclonal antibody against a human parathyroid hormone-related protein, characterized in that the V region of the L chain comprises an amino acid sequence as shown in Seq. No .: 45 The chimeric L chain of claim 1, wherein the C region is the CX chain A chimeric H chain comprising the C region of the H chain of a human antibody and the V region of the H chain of a mouse monoclonal antibody against a human parathyroid hormone-related protein, characterized in that the V region of the H chain comprises an amino acid sequence as shown in Id. Seq. No .: 46. 4. A chimeric H chain according to claim 3, wherein the C region is a Cyl chain A chimeric monoclonal antibody against a human parathyroid hormone-related protein comprising a chimeric L chain as defined in claim 1 and a chimeric H chain as defined in claim 1. 3 6. A polypeptide comprising the V region of the L chain of a humanized anti-human parathyroid-related protein, characterized in that the framework regions 1 to 3 of the V region of the L chain are identical to the framework regions 1 to 3 of the human antibody HSU03868 and the framework region 4 of the V region of the chain L is identical to region 4 of the human antibody S25755, and the complementarity determining regions 1 to 3 of the V region of the L chain include the amino acid sequences shown in Id. Seq. No: from 59 to 61. The polypeptide of claim 7 6. The process of claim 6, wherein in the framework regions amino acids 36 and 49 as defined by Kabat are tyrosine and aspartic acid, respectively. The polypeptide of claim 8 The method of claim 7, comprising an amino acid sequence as shown in one of Id. Seq. No: from 48 to 51. The polypeptide of claim 9 The process of claim 6, wherein in the framework regions amino acids 45 and 87 as defined by Kabat are lysine and isoleucine, respectively. The polypeptide of claim 10 9. The method of claim 9, comprising an amino acid sequence as shown in one of Id. Seq. No: from 52 to 55. A polypeptide comprising the V region of the H chain of a humanized antibody against a human parathyroid-related protein, characterized in that the framework regions 1 to 4 of the V region of the H chain are identical to the framework regions 1 to 4 of a human m subgroup antibody, and the complementarity regions 1 to The 3 H chain V regions include the amino acid sequences as shown in Id. Seq. No: from 62 to 64 The polypeptide of claim 12 The method of claim 11, wherein the human antibody is the human antibody S31679. The polypeptide of claim 13 The method of claim 11, wherein it comprises the amino acid sequence as shown in SEQ ID NO: 56. The L chain of a humanized anti-human parathyroid hormone-related protein comprising a polypeptide comprising the C region of the L chain of a human antibody and a polyptide according to claim 1. 6. 190 351 15. The humanized antibody L chain of claim 1. 14. The process of claim 14, wherein the C region is the CX chain, the framework regions 1 to 3 are identical to the framework regions 1 to 3 of the human antibody HSU03868, respectively, and the framework region 4 is identical to the framework region 4 of the human antibody S25755, and the regions defining complementarity 1 to 3 include the amino acid sequences as shown in Id. Seq. No '59 to 61. The H chain of a humanized anti-human parathyroid hormone-related protein comprising a polypeptide comprising the C region of the H chain of a human antibody and a polypeptide according to claim 1. 11. 17. Chain II of a humanized antibody according to claim 1 According to claim 16, wherein the C region is a Cyl chain, the framework regions 1 to 4 are derived from regions 1 to 4 of the human HSGIII antibody, respectively, and the complementarity determining regions 1 to 3 include the amino acid sequences as shown in Id, respectively. Seq ^; . No .: 62 to 64. 18. A humanized antibody against a human parathyroid hormone-related protein comprising the L chain of a humanized antibody of claim 1; And the H chain of a humanized antibody according to claim 14 or 15, and 16 or 17. 19. A DNA comprising a base sequence encoding the V region of the L chain of a murine monoclonal antibody against a human parathyroid hormone related protein, characterized in that the V region of the L chain comprises an amino acid sequence as shown in Id. Sequence No. 45. 20. A DNA comprising a base sequence encoding the V region of the L chain of a murine monoclonal antibody against a human parathyroid hormone related protein characterized in that the base sequence encoding the V region of the L chain is shown in Id. Seq. No .: 65 21. A DNA comprising a base sequence encoding the V region of the H chain of a murine monoclonal antibody against a human parathyroid hormone related protein, characterized in that the V region of the H chain comprises the amino acid sequence as shown in SEQ ID NO. No. 46 22. A DNA comprising a base sequence encoding the H chain V region of a murine monoclonal antibody against human parathyroid hormone related protein, characterized in that the base sequence encoding the H chain V region is shown in Id. Seq. No: 57. 23. DNA encoding a chimeric L chain as defined in claim 1 1. 24. DNA according to claim 1 23, which encodes a chimeric L chain comprising a base sequence as shown in Id. Seq. No .: 65. A DNA encoding a chimeric H chain as defined in claim 1 3. 26. DNA according to claim 1 The method of claim 25, which encodes a chimeric H chain comprising a base sequence as shown in Id. Sequence No .: 57. 27. A DNA comprising a base sequence encoding a polypeptide of claim 1. 6. 28. DNA according to claim 28 27, characterized in that it comprises the base sequence as shown in one of Id. Seq. No: 66 to 74. 29. A DNA comprising a base sequence encoding a polypeptide of claim 1 11. 30. DNA according to claim 29, characterized in that it comprises a base sequence as shown in Id. Seq. No .: 58. 31. DNA encoding the chimeric L chain of the humanized antibody of claim 1 14. DNA encoding the chimeric L chain of the humanized antibody of claim 1 15. 33. DNA for the L chain of a humanized antibody comprising a base sequence encoding an amino acid sequence as shown in one of Id. Seq · '. No. 47 to 55 34. The DNA of claim 33, wherein the DNA for the L chain of the humanized antibody comprises a base sequence as shown in one of Id. Seq. No: 66 to 74. 190 351 DNA encoding the H chain of the humanized antibody of claim 16 36. A DNA encoding the H chain of the humanized antibody of claim 1; 17 37 The DNA for the humanized H chain comprises a base sequence encoding an amino acid sequence as shown in Id. Seq. No: 56. 38. DNA according to claim 1 37, characterized in that the DNA for the H chain of the humanized antibody comprises a base sequence as shown in Id. Seq. No .: 58. 39. A recombinant vector comprising a DNA as defined in any one of claims 1-7. 19, 20, 21, 22, 23, 25, 27, 29, 31, 32, 33, 35, 36, 37. 40. A host cell transformed with the recombinant vector according to claim 40. 39. 41. A method of producing a chimeric antibody against a human parathyroid hormone-related protein comprising culturing transformants transformed with an expression vector comprising the DNA of one of claims 1 to 4. 19, 20, 23 and an expression vector comprising a dNa as defined in any one of claims 21, 22, 25; and harvesting from the resulting culture a chimeric antibody against the human parathyroid hormone-related protein. A method of producing a humanized antibody against a human parathyroid hormone-related protein comprising culturing transformants transformed with an expression vector comprising the DNA of one of claims 1 to 4. 27, 31, 32, 33, 34 and an expression vector comprising DNA as defined in one of claims 29, 35, 36, 37, and harvesting from the resultant culture the humanized antibody against human parathyroid hormone-related protein. 43. A pharmaceutical composition wherein the active ingredient is a humanized antibody of any one of claims 1-3. 18. An agent for inhibiting hypercalcemia, wherein the active ingredient is a humanized antibody according to claim 1. 18. An agent for inhibiting tumor associated hypercalcemia comprising a humanized antibody of claim 1 as an active ingredient. 18. 46. The agent for inhibiting hypercalcemia according to claim 46. The method of claim 45, characterized in that the malignant neoplasm is at least one selected from the group consisting of pancreatic, lung, pharyngeal, laryngeal, tongue, gum, esophagus, stomach, biliary, breast, kidney, bladder, uterine and prostate cancer, and lymphoma. malicious. An agent for the amelioration of a hypophosphataemic patient, characterized in that it comprises as active agent a humanized antibody according to claim 1. 18. 48. The factor of claim 48 47, characterized in that the hypophosphatemia is hypophosphatemic rickets. 49. The factor for ameliorating a hypophosphataemic patient according to claim 49. 47 characterized in that the hypophosphatemia is vitamin D resistant hypophosphatemia.