JP2000080100A - Human monoclonal antibody against parathyroid hormone-related protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain (a part of) a human monoclonal antibody against a parathyroid hormone-related protein which is completely free from the antigenicity to human body, free from side effect and useful for the treatment of hypercalcemia, malignant tumor, septicemia, hypophosphatemia or the like since the antibody has reactivity with (a part of) the human parathyroid hormone-related protein. SOLUTION: This antibody has reactivity with (a part of) a human parathyroid hormone-related protein and has a property among (i) suppressive activity on the intracellular increase of cAMP accompanied by the stimulation with a parathyroid hormone-related protein, (ii) suppressive activity on the release of calcium from bone and (iii) suppressive activity on the increase of blood calcium. Further, the human monoclonal antibody has reactivity with a partial amino acid sequence of a human parathyroid hormone-related protein having an amino acid sequence of either formula I or formula II, and a bonding velocity constant Ka and a dissociation velocity constant Kd between the antibody and the human parathyroid hormone-related protein are >=1.0×103 (l/M.sec) and <=1.0×10-3 (l/sec), respectively.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human parathyroid hormone-related protein (Parathyroid Hormone-Related Protein).
The present invention relates to a human monoclonal antibody reactive to ein, PTH-related protein (PTHrP) or a part thereof, a cell producing the human monoclonal antibody, and a pharmaceutical composition comprising the human monoclonal antibody.

[0002]

2. Description of the Related Art The serum calcium concentration of a normal mammal is strictly maintained at about 9 to 10 mg / 100 ml (about 2.5 mM), and the calcium concentration is determined to be calcium homeostasis (calcium) in a living body.
homeostasis). If this value drops below 50%, tetany (tonicity) will occur, and if it rises by 50%, consciousness will be turbid, in each case life threatening. To maintain this calcium homeostasis, the duodenum plays a role as a calcium uptake organ, the bone plays a role as a calcium storage organ, and the kidney plays a role as a calcium excretion organ. Further, such calcium regulation is controlled by various hormones collectively referred to as “calcium-regulating hormones”. Representative hormones include active vitamin D [1α, 25 (OH) ₂ D ₃ ]. ,
Parathyroid Hormone (PTH), Calcitonin and PTHrP (Parathyroid Hormone-Related Pro
tein, PTH-related Protein, PTHrP).

[0003] Bone plays an important role not only as a supporting tissue of a living body and as a motor organ, but also as a storage organ for calcium, a constituent component thereof. To perform such a function, bone tissue repeats its formation (osteogenesis) and resorption (bone resorption) for a lifetime.
Osteoblasts derived from mesenchymal cells play a major role in bone formation, and osteoclasts derived from hematopoietic cells play a major role in bone resorption. Osteogenesis is a mechanism that involves the formation of osteoid by bone organic matter (bone matrix protein such as type I collagen) produced by osteoblasts present on the osteogenic surface, followed by calcification. On the other hand, in bone resorption, osteoclasts attach to the bone surface, absorb calcium into cells via acid secretion and ion transport, and excrete the absorbed calcium to the bone marrow, thereby sending calcium into the blood. Mechanism. The bone defect absorbed by the osteoclasts is repaired by osteoblastic bone formation. Such a series of phenomena is called bone remodeling, and the remodeling replaces old bone with new bone, thereby maintaining the strength of the whole bone and maintaining calcium homeostasis.

[0004] The generation of osteoblasts and osteoclasts (differentiation, induction, and proliferation from immature cells) that play an important role in such bone remodeling and calcium homeostasis include:
Various factors (bone metabolism regulating hormone, bone formation control cytokine, bone resorption control cytokine, etc.) are involved.
Examples of the bone metabolism regulating hormone include vitamin A, vitamin D, parathyroid hormone (PTH), parathyroid-related protein (PTHrP), calcitonin, estrogen, and prostaglandin.

[0005] Parathyroid hormone (PTH) is the most important hormone in maintaining calcium homeostasis. Human PTH is composed of a prepropeptide (-31--1) consisting of a 31 amino acid sequence followed by an 84 amino acid sequence (PTH
(1-84)), and the structure necessary and sufficient for the expression of the biological activity of PTH is a region of 1-34 on the N-terminal side of PTH (1-84) (PTH (1-3
4)) (Proc. Natl. Acad. Sci. USA.,
Vol.68, p.63, 1971; Endocrinology, Vol.93, p.134
9, 1973; Peptide and Protein Reviews, Vol. 2, p. 20
9, 1984). The hormonal action of PTH is exerted by specifically binding to PTH / PTHrP receptors (PTH and PTHrP share the same receptor) present in the cell membrane of bone and kidney.

[0006] The most important effect of PTH is the maintenance of calcium homeostasis, that is, the decrease in blood calcium concentration immediately promotes the secretion of PTH from the parathyroid gland,
In bone, it acts on osteoblasts (eg, osteoblast activation of osteoclasts, production of bone organic degrading enzymes, etc.), promotes osteoclastic resorption, mobilizes calcium from bone,
In the kidney, it promotes calcium reabsorption in the distal tubule. On the other hand, when the blood calcium ion concentration increases, the secretion of PTH from the parathyroid gland is immediately suppressed, and the amount of calcium supplied to the extracellular fluid is reduced (Brown,
EM, Homeostatic mechanisms regulating extracellu
lar and intracellular calcium metabolism, in The p
arathyroids, p.19, 1994, Raven press, New York).

[0007] A hormone that has recently attracted attention as an important bone metabolism regulating hormone like parathyroid hormone (PTH) is parathyroid hormone-related protein (PTHrP). Suggestions for the presence of PTHrP date back to the 1930s, and in patients with hypercalcemia associated with malignant tumors, their serum biochemical findings, such as hypercalcemia and hypophosphatemia, are primary endogenous PTH excess states. It was known to closely resemble hyperparathyroidism. 1941, ectopic due to malignancy
Production of PTH suggests the development of hypercalcemia,
The existence of a TH-like substance was assumed (New Engl. J. Med., Vo
l.225, p.789, 1941). In the 1980s, the PTH-like substance was isolated and identified from various tumor cells as another substance that is different from PTH but induces PTH-like action through the same receptor as PTH (Proc. Natl. Acad. Sci. USA, Vol.
80, p. 1454, 1983; J. Clin. Invest., Vol. 72, p. 151
1, 1983).

[0008] There are three isoforms of human PTHrP, one of which is a pre-propeptide (-36--1) consisting of 36 amino acid sequences followed by a 139 amino acid sequence (PTHr
P (1-139)), and the other two are PTHrP (1 139) with additional 2 and 34 amino acids respectively extended at the C-terminus of the same sequence as the prepropeptide and the subsequent PTHrP (1-139). -141) and PTHrP (1-173). That is, these three isoforms are completely identical in their prepro sequence and the subsequent amino acid sequence up to 1-139. Human PTHrP has high amino acid homology with mouse and rat PTHrP, and particularly has extremely high homology at amino acids 1-111 (Crit. Rev. Biochem. Mol. Biol., VoI.
l.26, No.3-4, p.377, 1991).

The N-terminal 1-13 amino acid portion of PTHrP is PTH
And relatively high homology (8 amino acids are identical)
Although the amino acid sequence of the 14-34 amino acid portion is specific to PTHrP, its higher-order structure is similar to that of the 14-34 amino acid portion of PTH. In addition to these structural properties,
Since the 1-34 amino acid portion of PTHrP shows almost the same biological activity as PTH, the 1-34 amino acid portion of PTHrP is called a PTH-like region (Endocrine Rev., Vo
l.12, p.110, 1991; Endocrinology, Vol.125, p.2215,
1989). On the other hand, since the amino acid sequence at the C-terminal side from the amino acid portion 1-34 is unique to PTHrP and does not show homology with PTH, it is called a PTH-unlike region. As described above, PTHrP shares a receptor with PTH and binds to a PTH / PTHrP receptor. From these series of characteristics, PTHrP is a parathyroid hormone-related protein (PTH-related protein, PTH-like p
rotein, PTHrP) (Proc. Natl.
Acad. Sci. USA, Vol. 84, p. 5048, 1987; Science, Vo.
l.237, p.893, 1987; Biochem Biophys. Res. Comm., V
ol.146, p.672, 1987; J. Clin. Invest., Vol.80, p.18
03, 1987; Crit. Rev. Biochem. Mol. Biol., Vol. 26,
No.3-4, p.377, 1991).

[0010] PTHrP has been discovered as a causative substance of hypercalcemia associated with malignant tumors. I have. From previous studies, P
THrP has been shown to be expressed physiologically in a wide range of tissues of adult animals and fetuses. In adult animals, skin, kidney, bone, synovium, brain, peripheral nerves, choroid plexus, stomach, bone marrow, parathyroid, adrenal glands, endocrine organs such as pancreatic islets of Langerhans and pituitary gland, vascular smooth muscle, striated muscle, Expression is found in tissues such as placenta, bladder, vas deferens, seminiferous tubules, urethra, and mammary gland. Also, in fetal mammals, cartilage and bones with endochondral ossification, skin, hair follicles, bronchi, pharynx, intestine, thyroid, parathyroid, adrenal, smooth muscle, striated muscle, amniotic membrane, chorion, and Expression is seen in the choroid plexus.

Although the physiological function of PTHrP still remains unclear, it is a factor that regulates cell growth and differentiation in a paracrine and autocrine manner through binding to PTH / PTHrP receptors. Some things are becoming clear. Previous studies have shown that PTHrP has PTH-like effects, non-PTH
It has been elucidated and / or suggested that it has various effects such as a TGF-β-like effect, an osteoclast inhibitory effect and the like.

Among these, the PTH-like action of PTHrP has been relatively well studied, and the structure required for the expression of its biological activity is maintained by a region containing at least the 1-34 amino acid portion of PTHrP. . With respect to the PTHrP's PTH-like action, PTHrP (1-34) is approximately equivalent to PTH (1-34) and molecules longer than PTHrP (1-34) (e.g., PTHrP (1-84), PT
HrP (1-108) and PTHrP (1-141)) (J. Biol. Chem., Vol. 264, p. 14806, 1989). PTHrP
In the 1-34 amino acid portion of, like PTH (1-34), PTH / PTH
At least two receptor binding sites for the rP receptor (1-
6 and 25-34 amino acid portions). Examples of the PTHrP-like action of PTHrP include, for example, the action of adenylate cyclase (AC) or phospholipase C (PL) on bone in osteoblasts.
C) to stimulate osteoclastic bone resorption, and in the kidney, to promote cyst AMP (cAMP) and phosphorus excretion and to promote calcium reabsorption (Crit. Rev. B
iochem. Mol. Biol., Vol. 26, No. 3-4, p. 377, 199
1).

On the other hand, since PTHrP is deeply involved in the regulation of bone metabolism as described above, there have been reports suggesting that PTHrP is associated with bone metabolism-related diseases and abnormal calcium kinetics. Particular attention has been paid to hypercalcemia associated with (malignant tumor), bone metastasis of cancer, and the relationship between rheumatism and PTHrP. It is known that various abnormalities of calcium metabolism and bone lesions are associated with cancer (malignant tumors). Representative symptoms are hypercalcemia associated with cancer and bone lesions associated with bone metastasis of cancer. is there.

[0014] Hypercalcemia (malignancy-associated hypercarcemia (MAH)) associated with cancer (malignant tumor) is
Paraneoplast, a paraneoplast syndrome often seen clinically
ic syndrome). The most frequent case of patients with hypercalcemia is primary hyperparathyroidism (primary hy
perparathyroidism (HPT), which usually follows a chronic course. On the other hand, most MAHs are progressive and severe, and rapid initiation of treatment requires a patient's quality of life (QOL).
e) It is effective for improvement. MAH is roughly classified into the following two types. One is malignant humoral hypercalcemia (humo) caused by the systemic action of humoral factors produced from tumors.
ral hypercarcemia of malignancy (HHM)) and local osteolysis caused by enhanced bone resorption (bone destruction, osteolysis) by direct invasion of the tumor into the bone (bone metastasis). Hypercalcemia (local osteolytic
hypercarcemia (LOH)). LOH is mainly based on extensive bone metastasis of cancer and is considered to be secondary to bone lesions. On the other hand, HHM accounts for nearly 90% of hypercalcemia associated with cancer (N. Engl. J. Med., Vol. 300,
p.1377, 1980), whose primary causative agent is secreted by tumors
It is being clarified that this is due to the action of PTHrP (Am.
J. Clin. Pathol., Vol. 105, p. 487, 1996).

According to the above classification, HHM should have no, if any, bone metastasis of cancer, but hypercalcemia in which bone metastasis of widespread cancer and large production of PTHrP are simultaneously observed. Disease (a case with both LOH and HHM) is also seen clinically. Also, in LOH, PTHrP produced by the tumor may contribute to local bone destruction (osteolysis). further,
There is also a rare but benign tumor-producing hypercalcemia due to PTHrP (Am. J. Clin. Pathol., Vol. 105,
p.487, 1996). HHM due to PTHrP production is found in various cancers across all tissues, especially squamous cell carcinoma (lung, esophagus, cervix, vulva, skin, head, neck), renal cancer, bladder cancer, ovarian cancer, and There are many reports on adult T-cell leukemia (ATL) (N. Engl. J. Med., Vol. 322, p. 1106, 1
990; J. Clin. Endocrinol. Metab., Vol. 73, 1309, 19
91).

LOH is often found in myeloma, breast cancer, and lymphoma. As described above, LOH is directly caused by extensive infiltration of tumor cells into bone and bone destruction. However,
The involvement of PTHrP in LOH is being revealed. Regarding myeloma, along with TNF-β, IL-1β and IL-6, the involvement of PTHrP has also been suggested (Am. J. Hemato
l, Vol. 45, p. 88, 1994), and elevated PTHrP in blood has been reported in patients with multiple myeloma with hypercalcemia (Ann. Intern. Med., Vol. 111, p. .807, 1989, Budayr
AA et al.). Extensive bone metastases are often seen in breast cancer, with about 4
Hypercalcemia occurs in 0% of patients, and high expression of PTHrP is found in bone metastases of breast cancer (Cancer Res., Vol.
51, p.3059, 1991). In addition, a comparison of the expression of PTHrP in primary lesions and bone metastases in breast cancer patients suggests that PTHrP is particularly high in bone, and that PTHrP production by cancer cells is promoted in a bone microenvironment (J. Bone). Miner. Re
s., Vol. 7, p. 971, 1992). About 8 in ATL patients
It is said that 0% is accompanied by hypercalcemia, and the expression of PTHrP reaches a high level in proportion to the disease state, and it has been shown that it is correlated with the degree of hypercalcemia,
It has been suggested that PTHrP is deeply involved in one of the causes of ATL (Leukemia, Vol. 8, p. 1708, 1994).

[0017] Bone metastasis of cancer and PTHrP, which are the main causes of LOH
As described above, it has been reported that the expression of PTHrP in primary lesions and bone metastases of breast cancer patients is particularly high in bone, as described above (J. Bone Miner. Res., V.
ol. 7, p. 971, 1992), and it has been reported that bone destructive metastasis of human breast cancer cells in nude mice is suppressed by anti-PTHrP antibody (J. Clin. Invest., Vol.
98, p.1544, 1996; Cancer Res., Vol.56, p.4040, 199
6; International Patent Application Publication No. WO 96/22790). For the relationship between PTHrP and rheumatoid arthritis (Rheumatoid Arthritis (RA)) and osteoarthritis (Osteoarthritis (OA)), see R
High levels of PTHrP expression have been observed in the synovial fluids of both A and OA patients (J. Bone
Miner. Res., Vol. 12, p. 847, 1997).

On the other hand, as treatment for hypercalcemia,
Conventionally, infusions, diuretics, corticosteroids, and calcitonin preparations have been used. In recent years, bisphosphonate-based compounds (eg, pamidronate and the like) having a strong bone resorption inhibiting action have been used. Further, as described above, based on the findings showing the relationship between hypercalcemia and PTHrP, attempts have been made to treat hypercalcemia using an antibody against PTHrP (Japanese Patent Application Publication No. -
No. 228089). In addition, based on the knowledge of the relationship between PTHrP and bone metastasis and osteolysis of cancer as described above, a series of cancer bone metastasis using antibodies against PTHrP, a series of cancer-related osteolysis and cancer cell proliferation, etc. Attempts to prevent and treat symptoms have also been made (International Patent Application Publication WO 96/22790).

Based on the close relationship between cancer (malignant tumor) and hypercalcemia and the relationship between them and PTHrP, treatment of hypercalcemia with the aforementioned bisphosphonate compounds Of cancer-related symptoms and anti-PTHrP antibody treatment, that is, a combination of bisphosphonate compounds and anti-PTHrP antibody to prevent a series of cancer-related symptoms and hypercalcemia Attempts have also been made to treat them (the aforementioned International Patent Application Publication WO96 / 22790). Such treatment is based on the finding that PTHrP is a bone metabolism-regulating hormone with a strong PTH-like bone resorbing action, but recently PTHrP has been transformed by bacterial toxins (endotoxins) Sepsis (sepsis) and Systemic Inflammatory Response Syndrome (SIRS)
For example, there has been an attempt to treat sepsis and SIRS using an antibody against PTHrP, based on the finding that it is involved in the pathogenesis of inflammation.

Hypophosphatemia seen in patients with hypercalcemia due to malignant tumors is considered to be due to the effect of PTHrP on inhibiting resorption of phosphorus in the kidney / promoting phosphorus excretion. Attempts have also been made to treat hypophosphatemia using E. coli (WO 98/13388). Furthermore, the relationship between PTHrP and teeth composed of calcium as well as bone has been studied, and PTHrP is involved in tooth development and various diseases associated with teeth (alveolar pyorrhea, gingivitis, etc.) (Anatomical Journal, Vol. 68, No. 6, p. 726, 1993; Journal of the Japan Society for Bone Metabolism, Vol. 14, No. 2, p. 334, 1996; Japanese Society for Developmental Biology 30 times) Kaname, p.118, 1997).

In the treatment of patients with the above-mentioned antibodies, it goes without saying that a monoclonal antibody against human PTHrP is used. Monoclonal antibodies against human PTHrP include non-human mammal-derived monoclonal antibodies such as mouse monoclonal antibodies and rat monoclonal antibodies produced by immunizing non-human mammals such as mice and rats with human PTHrP protein or a partial peptide thereof. Many are known (Clin. Chem., Vol. 3
7, No. 10, p. 1781, 1991; J. Immunol. Methods, Vol. 1
46, p.33-42, 1992; Clin. Chem., Vol.37, No.5, p.67.
8, 1991; J. Immunol. Methods, Vol. 127, p. 109, 199
0; J. Bone Min. Res., Vol. 8, No. 7, p. 849, 1993; U.S. Pat. No. 5,217,896; International Patent Application Publication WO 97/0431
No. 2). In addition, production of a chimeric antibody comprising a variable region of a mouse monoclonal antibody and a constant region of a human immunoglobulin has been reported (International Patent Application Publication WO98 / 1).
No. 3388). However, production of a human-derived monoclonal antibody against human PTHrP and attempts to treat various diseases using the human monoclonal antibody have not yet been reported.

[0022]

SUMMARY OF THE INVENTION Monoclonal antibodies derived from non-human mammals such as mice and rats reported so far have been used in serum or tissues of mammals including humans.
It may be useful for use in in vivo tests with non-human mammals for detecting the expression of PTHrP in vitro or confirming the therapeutic effect of the monoclonal antibody on diseases. However, when such a non-human mammal-derived monoclonal antibody is administered to a patient (human body), the administered mouse or rat monoclonal antibody is foreign to the human body (has immunogenicity). Therefore, due to the immune mechanism of foreign body elimination of the human body, the monoclonal antibody is not only rapidly eliminated, but also in the process, an antibody against the administered monoclonal antibody is produced in the HAMA immune response (Huma
n Anti-Mouse / Murine Antibody Immune Response is elicited. Due to the mechanism of this HAMA, repeated administration of a non-human mammal-derived antibody such as the mouse monoclonal antibody not only neutralizes the administered monoclonal antibody and diminishes its effect, but also causes severe allergy and the like. Side effects are caused.

As an attempt to reduce such a HAMA immune response, recently, a partial amino acid sequence of an antibody derived from a non-human mammal such as a mouse monoclonal antibody has been modified by genetic engineering into an amino acid sequence derived from a human immunoglobulin. Techniques for reducing the immunogenicity of a human body have been used. One is a mouse / human chimeric antibody in which the constant region of a mouse monoclonal antibody is replaced with the constant region of a human immunoglobulin, and the other is the three complementarity determining regions (hypervariable regions) of the hypervariable region of a mouse monoclonal antibody. -determining residu
e; humanized antibody (humani) in which all sequences except CDR1, CDR2, CDR3) are replaced with sequences derived from human immunoglobulin
zed anitbody, CDR-grafted antibody).

However, even such a recombinant antibody is a foreign substance to a human body as long as a partial sequence derived from a non-human mammal such as a mouse remains in its structure, and has a HAMA immune response. A similar HACA immune response (Huma
n AntiChimeric Antibody Immune Response and HAHA immune response (Human AntiHumanized Antibody Immune Response)
se) is caused not a little. Therefore, in order to use the antibody as a pharmaceutical, an antibody that does not cause such side effects due to HAMA, that is, a human-derived antibody (human antibody) is most desirable.

[0025] Human PTHrP is also used without any side effects.
Various diseases or conditions as described above caused by THrP production (hypercalcemia, bone metastasis of cancer, osteolysis, bone destruction,
Diseases of the oral cavity such as rheumatoid arthritis, osteoarthritis), teeth, periodontal and gingival tissues (alveolar pyorrhea, gingivitis, periodontal disease, etc.), and various other diseases or symptoms (sepsis (sepsis)
is), systemic inflammatory response syndrome (SIRS), hypophosphatemia, etc.), and it is strongly desired to provide a human monoclonal antibody applicable to prevention or treatment.

[0026]

SUMMARY OF THE INVENTION The present inventors have developed human PTHrP to meet the above-mentioned clinical needs.
As a result of intensive studies on the production of human monoclonal antibodies against, a transgenic mouse capable of producing human-derived antibodies was produced using genetic engineering technology, and the transgenic mice were immunized with human PTHrP or a part thereof. By using the method of Koehler and Milstein et al. (Nature, Vol. 256, p. 495, 1975) commonly used in the production of monoclonal antibodies, various properties (antigen specificity, antigen affinity, neutralization) Activity and cross-reactivity) and biological activity (PTHrP-dependent intracellular cAMP increase inhibitory activity, PTHrP-dependent inhibitory activity on calcium release from bone, bone resorption inhibitory activity, osteolysis inhibitory activity, etc.) For the first time in the world to produce various human monoclonal antibodies against That is, the human monoclonal antibody of the present invention has no antigenicity to humans, and is a major problem in the treatment of antibody drugs consisting of antibodies derived from non-human mammals such as conventional mouse-derived antibodies. Since it does not cause any side effects, the value of the antibody as a drug is dramatically increased.

That is, the present invention is as follows. (1) A human monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof. (2) The human monoclonal antibody according to the above (1) or a part thereof, wherein the human monoclonal antibody has the properties described in any of the following (a) to (c): Has an inhibitory effect on intracellular cAMP elevation associated with thyroid hormone-related protein stimulation; (b) has an inhibitory effect on calcium release from bone caused by parathyroid hormone-related protein stimulation; or (C) acts to suppress an increase in blood calcium associated with stimulation of parathyroid hormone-related protein. (3) The method according to (1), wherein the human monoclonal antibody has reactivity with a partial amino acid sequence of a human parathyroid hormone-related protein having any one of the following amino acid sequences (a) and (b): The described human monoclonal antibodies or parts thereof: (a) AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA; or (b) AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRAT. (4) The immunoglobulin class of the human monoclonal antibody is IgG2.
Or the human monoclonal antibody according to any one of (3) to (3). (5) The human according to any one of (1) to (4) above, wherein the human monoclonal antibody is a monoclonal antibody derived from a transgenic non-human mammal capable of producing a human antibody. Monoclonal antibodies or parts thereof. (6) The human monoclonal antibody or a part thereof according to (5), wherein the transgenic non-human mammal is a transgenic mouse. (7) The binding rate constant (ka) between the human monoclonal antibody and the human parathyroid hormone-related protein is 1.0 × 1
The human monoclonal antibody or a part thereof according to any one of the above (1) to (6), which has a numerical value of not less than 0 ³ (1 / M.Sec). (8) The dissociation rate constant (kd) between the human monoclonal antibody and the human parathyroid hormone-related protein is 1.0 × 1
The human monoclonal antibody or a part thereof according to any one of the above (1) to (6), which has a numerical value of 0 ^-3 (1 / Sec) or less. (9) The dissociation constant (Kd) between the human monoclonal antibody and the human parathyroid hormone-related protein is 1.0 × 10
^-7 (M) The human monoclonal antibody or a part thereof according to any one of the above (1) to (6), which has the following numerical value. (10) When the binding rate constant (ka) is 1.0 × 10 ⁴ (1 / MS
ec) The human monoclonal antibody or a part thereof according to the above (7), wherein the value is not less than (ec). (11) The dissociation rate constant (kd) is 1.0 × 10 ⁻⁴ (1 / Se
c) The human monoclonal antibody or a part thereof according to the above (8), which has the following numerical values. (12) The human monoclonal antibody or a part thereof according to (9), wherein the dissociation constant (Kd) is a value of 1.0 × 10 ⁻⁸ (M) or less. (13) The human monoclonal antibody or a part thereof according to (12), wherein the dissociation constant (Kd) is a value of 1.0 × 10 ⁻⁹ (M) or less. (14) The human monoclonal antibody or a part thereof according to (13), wherein the dissociation constant (Kd) is a value of 1.0 × 10 ⁻¹⁰ (M) or less. (15) The binding rate constant (ka) between the human monoclonal antibody and the human parathyroid hormone-related protein is 1.0
The human monoclonal antibody or a part thereof according to the above (2) or (3), which has a numerical value of not less than × 10 ³ (1 / M.Sec). (16) The dissociation rate constant (kd) between the human monoclonal antibody and the human parathyroid hormone-related protein is 1.0
The human monoclonal antibody or a part thereof according to the above (2) or (3), which has a numerical value of × 10 ⁻³ (1 / Sec) or less. (17) The dissociation constant (Kd) between the human monoclonal antibody and the human parathyroid hormone-related protein is 1.0 × 10
^-7 (M) The human monoclonal antibody or a part thereof according to the above (2) or (3), which has the following numerical value. (18) The binding rate constant (ka) is 1.0 × 10 ⁴ (1 / MS
(ec) The human monoclonal antibody or a part thereof according to the above (15), wherein the value is not less than (ec). (1
(9) The human monoclonal antibody or a part thereof according to (16), wherein the dissociation rate constant (kd) is a value of 1.0 × 10 ⁻⁴ (1 / Sec) or less. (20)
The human monoclonal antibody or a part thereof according to the above (17), wherein the dissociation constant (Kd) is a value of 1.0 × 10 ⁻⁸ (M) or less. (21) The human monoclonal antibody or a part thereof according to (20), wherein the dissociation constant (Kd) is a value of 1.0 × 10 ⁻⁹ (M) or less. (22) The human monoclonal antibody or a part thereof according to (21), wherein the dissociation constant (Kd) is a value of 1.0 × 10 ⁻¹⁰ (M) or less. (23) A cell producing a human monoclonal antibody reactive to a human parathyroid hormone-related protein or a part thereof. (24) The cell according to (23), wherein the cell is a B cell derived from a transgenic non-human mammal capable of producing a human antibody. (25) The cell according to (24), wherein the transgenic non-human mammal is a transgenic mouse. (26) The cell described above, wherein the cell is a hybridoma obtained by fusing a B cell derived from a transgenic non-human mammal capable of producing a human antibody with a myeloma cell derived from a mammal. The cell according to 23). (27) The cell according to (26), wherein the transgenic non-human mammal is a transgenic mouse. (28) The cell according to (2) above, wherein the cell is a hybridoma identified by International Accession No. FERM BP-6390.
The cell according to 7). (29) the cell is a DNA encoding a heavy chain of a human monoclonal antibody reactive with a human parathyroid hormone-related protein or a part thereof, or a DNA encoding a light chain thereof, or both of them; D
The cell according to (23), wherein the cell is a transformed cell transformed by introducing NA into the cell. (30) A human monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof, the human monoclonal antibody being produced from a hybridoma identified by International Accession No. FERM BP-6390 or the human monoclonal antibody or a part thereof. A human monoclonal antibody having substantially the same properties as a human monoclonal antibody, or a part thereof. (31) A monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof, wherein the light chain variable region of the monoclonal antibody is described in any of the following (a) or (b): (A) from the group consisting of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 An amino acid sequence of amino acids 21 to 119 in the amino acid sequence described in any one selected SEQ ID NO; or (b) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, and SEQ ID NO: 2
An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of amino acids 21 to 119 in the amino acid sequence described in any one of SEQ ID NOs selected from the group consisting of 2; A monoclonal antibody having an amino acid sequence comprising: or a part thereof. (32) A monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof, wherein the heavy chain variable region of the monoclonal antibody is any one of the following (a) to (d): (A) SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 3
6, amino acid numbers 20 to 117 in the amino acid sequence described in any one of SEQ ID NOs selected from the group consisting of SEQ ID NOs: 38 and 42; (b) SEQ ID NO: 40 (C) SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 3
6, one or several amino acids are deleted in the amino acid sequence of amino acids 20 to 117 in the amino acid sequence described in any one of SEQ ID NOs selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 42 (D) one or several amino acids are deleted, substituted or added in the amino acid sequence of amino acids 20 to 115 in the amino acid sequence described in SEQ ID NO: 40; A monoclonal antibody or a part thereof having an amino acid sequence comprising: (33) V region, D region and J region DNAs encoding the heavy chain of the human monoclonal antibody are V3-30,
The human monoclonal antibody or a part thereof according to any one of the above (1) to (22), which is derived from DN1 and JH6. (34) The V region, D region and J region DNAs encoding the heavy chain of the human monoclonal antibody are VH4.16,
The human monoclonal antibody or a part thereof according to any one of the above (1) to (22), which is derived from DA1 and JH6. (35) The DNA according to (1) to (22), wherein the DNAs of the V region and the J region encoding the light chain of the human monoclonal antibody are derived from DPK15 and Jκ3, respectively.
Or the human monoclonal antibody or a part thereof. (36) The V region, D region and J region DNAs encoding the heavy chain of the human monoclonal antibody are V3-30,
DNAs of V region and J region derived from DN1 and JH6 and encoding the light chain of the human monoclonal antibody were expressed by DP, respectively.
The human monoclonal antibody or a part thereof according to any one of the above (1) to (22), which is derived from K15 and Jκ3. (37) The V region, D region and J region DNAs encoding the heavy chain of the human monoclonal antibody are VH4.16,
DNAs of the V region and the J region derived from DA1 and JH6 and encoding the light chain of the human monoclonal antibody were expressed by DP, respectively.
The human monoclonal antibody or a part thereof according to any one of the above (1) to (22), which is derived from K15 and Jκ3. (38) The above (1) to (22) or (3)
0) A pharmaceutical composition comprising the human monoclonal antibody or a part thereof according to any one of (37) and a pharmaceutically acceptable carrier. (39) The human monoclonal antibody or a part thereof according to any one of (2), (3), (15) to (22), or (30) to (37), and pharmaceutically A pharmaceutical composition comprising an acceptable carrier. (40) The pharmaceutical composition according to (39), wherein the pharmaceutical composition is used for treating a disease caused by release of calcium from bone in a parathyroid hormone-related protein-dependent manner. (41) The pharmaceutical composition according to the above (39), wherein the pharmaceutical composition is used for treating hypercalcemia. (42) The pharmaceutical composition according to the above (39), wherein the pharmaceutical composition is used for suppressing or preventing osteolysis. (43) The pharmaceutical composition according to (3), wherein the pharmaceutical composition is used for treating rheumatoid arthritis or osteoarthritis.
The medical composition according to 9). (44) The pharmaceutical composition according to (39), wherein the pharmaceutical composition is used for inhibiting or preventing cancer metastasis to bone. (45) The pharmaceutical composition according to the above (39), wherein the pharmaceutical composition is used for suppressing or preventing the growth of cancer cells present in bone tissue. (46) The pharmaceutical composition according to (39), wherein the pharmaceutical composition is used for treating a disease caused by local production of a parathyroid hormone-related protein. (47) The above (39), wherein the pharmaceutical composition is used for treating a condition caused by a primary local cancer.
A pharmaceutical composition according to claim 1. (48) The pharmaceutical composition according to (47), wherein the symptom is a symptom selected from the group consisting of pain, nerve compression, hypercalcemia, fracture, and cachexia. (49) The above-mentioned (3), wherein the pharmaceutical composition is used for prolonging the life of a patient suffering from primary local cancer.
The pharmaceutical composition according to 9). (50) The pharmaceutical composition according to (3), wherein the pharmaceutical composition is used for treating a disease in teeth, periodontal or gingival.
The pharmaceutical composition according to 9). (51) The pharmaceutical composition according to (38), wherein the pharmaceutical composition is used for treating sepsis (sepsis) or systemic inflammatory response syndrome (SIRS). (52) The pharmaceutical composition according to the above (39), wherein the pharmaceutical composition is used for treating hypophosphatemia.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail by clarifying the meaning of words used in the present invention.
"Mammal" in the present invention, human, cow, goat,
Rabbit, mouse, rat, hamster, guinea pig, etc., preferably human, rabbit, rat, hamster or mouse, and particularly preferably human, goat, cow, rat or mouse. Three letters or one letter of an alphabet used to represent an amino acid in the present specification or the drawings mean the following amino acids, respectively. (Gly / G) glycine, (Ala /
A) Alanine, (Val / V) valine, (Leu / L) leucine, (Ile / I) isoleucine, (Ser / S) serine, (Th
(r / T) threonine, (Asp / D) aspartic acid, (Glu
/ E) glutamic acid, (Asn / N) asparagine, (Glu /
Q) Glutamine, (Lys / K) lysine, (Arg / R) arginine, (Cys / C) cysteine, (Met / M) methionine,
(Phe / F) phenylalanine, (Tyr / Y) tyrosine,
(Trp / W) tryptophan, (His / H) histidine,
(Pro / P) Proline.

The term “parathyroid hormone-related protein (PTH)” used in the present invention refers to “parathyroid hormone-related protein (PTH).
-related protein, PTHrP) ； Parathyroid hormone-like
The “protein (PTH-like protein, PTHLP)” is a human parathyroid hormone-related protein (PTHrP) having the structure as described above and having the biological activity and / or function as exemplified above. There are three isoforms of human PTHrP, and specifically,
PTHrP (1-139) having the amino acid sequence of amino acids 1 to 139 of the amino acid sequence described in the above, and the alanine (Ala) at position 141 of the amino acid sequence of amino acids 1 to 141 of SEQ ID NO: 1 is a hittidine (His) PTHrP (1-141) having the amino acid sequence of SEQ ID NO: 1, and PTHrP (1-17) having the amino acid sequence of amino acids 1 to 173 of SEQ ID NO: 1.
3). All of these isoforms are included in the human parathyroid hormone-related protein referred to in the present invention (Cr
itical Reviews in Biochemistry and Molecular Biolo
gy, Vol.26, p.377-395, 1991; Bone Science, "Bone formation and bone resorption and their regulators (Vol.2)", p.322,
(Fig. 11.34, 1995, Hirokawa Shoten (published)).

The term "part of the human parathyroid hormone-related protein" as used in the present invention means any partial sequence of the amino acid sequence of the "human parathyroid hormone-related protein" defined above. Has a partial sequence of human PTHrP having 5 to 100 amino acid residues, more specifically, a partial sequence of human PTHrP having 5 to 50 amino acid residues, more specifically 5 to 40 amino acids A partial sequence of human PTHrP having residues is included. Preferably, the site required for human PTHrP to exert its biological function (eg, various active sites such as a region containing PTHrP (1-34) that exerts a PTH-like effect) or human PTHrP Sites that bind or interact with (PTHrP (1-6)
A partial sequence of human PTHrP including a receptor binding site such as PTHrP (25-34).

Specifically, for example, a partial polypeptide containing at least PTHrP (1-34) (amino acid numbers 1 to 34 of the amino acid sequence shown in SEQ ID NO: 1) which is considered to have a PTH-like action is exemplified. Can be In addition, PT as detected in serum of patients with malignant hypercalcemia
HrP (1-74) (amino acid numbers 1 to 74 of SEQ ID NO: 1) and P
THrP (109-138) (amino acids 1 to 138 of SEQ ID NO: 1)
(N. Engl. J. Med., Vol. 322, p. 1106, 19).
89). In addition, PTHrP undergoes post-translational modification with Arg37,
It is known that PTHrP starting with a38 is also produced (J.
Biol. Chem., Vol. 267, p. 18236, 1992), and the present invention also includes post-translationally modified PTHrP resulting from processing at such various post-translational modification sites. In addition, various regions that are responsible for the non-PTH-like action of PTHrP are also included in the “part,” for example, PTHrP (75-85) (Exp.
Physiol., Vol. 75, p. 605, 1990), PTHrP (1-36) which is thought to have a TGFβ-like effect (J. Clin. Invest.,
Vol.83, p.1057, 1989) and osteostatin (oste), which is a partial sequence of PTHrP and has an osteoclast-suppressing action.
ostatin), a PTHrP (107-111) and a region PT including the region and also thought to have osteoclast inhibitory action
HrP (107-139) (Endocrinology, Vol.129, p.3424, 199
1) and the like.

Further, the "parathyroid hormone-related protein" and "part thereof" in the present invention include the "human monoclonal antibody" of the present invention described later in which the primary structure (amino acid sequence) of the natural protein as described above is included. Having human PT
As long as it has reactivity with HrP or a part thereof, a human PTHrP derivative having an amino acid sequence substantially identical to the primary structure of the natural protein and a part thereof are also included.

As used herein, the term "human PTHrP derivative having substantially the same amino acid sequence" refers to a naturally occurring human PTHrP derivative.
Several amino acids, preferably 1 to 10 amino acids, particularly preferably 1 to 5 amino acids in the amino acid sequence have substitution, deletion and / or A protein having a modified amino acid sequence, and a protein having an amino acid sequence in which several amino acids, preferably 1 to 10 amino acids, and particularly preferably 1 to 5 amino acids have been added to the amino acid sequence. I do. Furthermore, the case of a plurality of combinations of such substitution, deletion, modification and addition may be used.

The human PTHrP in the present invention is produced by a method known in the art, such as a chemical synthesis method or a cell culture method, or a modification method thereof as appropriate, in addition to the gene recombination technique. be able to. In addition, the partial sequence of human PTHrP can be produced by a genetic recombination technique or a chemical synthesis method according to a known method or a modification method thereof known in the technical field described below, or isolated by a cell culture method. Human PTHrP can be produced by appropriately cleaving it with a proteolytic enzyme or the like.

The "human monoclonal antibody" in the present invention is a human monoclonal antibody reactive with "human parathyroid hormone-related protein" or "part thereof" as defined above. Specifically, it is a human monoclonal antibody having the characteristics described in any one of the inventions (1) to (22) or (30) to (37). For example, various human monoclonal antibodies having various properties described in Examples described later can be mentioned. The "human monoclonal antibody" of the present invention includes a heavy chain having an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of each of the heavy and / or light chains constituting the antibody. A monoclonal antibody consisting of a chain and / or a light chain is also encompassed, but the "several amino acids" herein means a plurality of amino acids, specifically 1 to 10 amino acids, and is preferably Is one to five amino acids. Amino acid partial modification (deletion, substitution, insertion, addition) as described above in the amino acid sequence of the PTHrP or monoclonal antibody of the present invention.
Can be introduced by partially modifying the nucleotide sequence encoding the amino acid sequence. This partial modification of the base sequence can be performed by a known site-directed mutagenesis method (Sites
pecific mutagenesis) by a conventional method (Proc. Natl. Acsd. Sci. USA, Vol. 81, p.
5662-5666, 1984).

Here, "human monoclonal antibody" refers to the variable region (Variable region) of the heavy chain (H chain) constituting the immunoglobulin and the constant region (Constant region) of the H chain.
Region) and all regions including the light chain (L chain) variable region and the L chain constant region are human immunoglobulins derived from genes encoding human immunoglobulins. L
The chain includes a human κ chain or a human λ chain. The human monoclonal antibody of the present invention includes IgG (IgG1,
G2, IgG3, IgG4), IgM, IgA (IgA1, IgA2), I
Includes monoclonal antibodies having any immunoglobulin class and isotype, such as gD or IgE. Preferably, it is IgG or IgM, and more preferably, IgG. Particularly preferably IgG2 or
IgG4.

The human monoclonal antibody of the present invention can be produced, for example, by the following production method. That is, for example, a human parathyroid hormone-related protein (including natural, recombinant, synthetic, and cell culture supernatants) as defined above, or a part thereof, or a substance suitable for increasing the antigenicity of an antigen (For example, KLH (keyhole
limpet hemocyanin), if necessary, together with Freund's adjuvant (Freund's Adjuvant), if necessary, a transgenic non-human mammal (preferably a genetically engineered one) to produce a human antibody as described below. Human antibody-producing transgenic mouse)
Immunize. Polyclonal antibodies can be obtained from serum obtained from the immunized transgenic animal. In addition, a monoclonal antibody is prepared by preparing a hybridoma from the antibody-producing cells obtained from the immunized animal and a myeloma cell line (myeloma cell) having no autoantibody-producing ability, cloning the hybridoma, and immunizing a mammal. It is produced by selecting a clone producing a monoclonal antibody showing specific affinity for the antigen.

More specifically, it can be produced as follows. That is, human parathyroid hormone-related proteins as defined above (natural, recombinant, synthetic,
Cell culture supernatant, or a part thereof, or a substance suitable for enhancing the antigenicity of an antigen (for example, KLH (key
hole limpet hemocyanin)) and, if necessary, Freund's adjuvant (Fr
eund's Adjuvant), subcutaneously, intramuscularly, intravenously, and hood of a transgenic non-human mammal (preferably a transgenic mouse producing human antibodies) that has been genetically engineered to produce human antibodies as described below. Immunization is performed by one or several injections or transplantation into the pad or intraperitoneal cavity. Usually, 1 to 10 immunizations are performed about every 1 to 14 days after the initial immunization, and antibody producing cells are obtained from the immunized animal about 1 to 5 days after the final immunization. The number of immunizations and the time interval can be appropriately changed depending on the nature of the immunogen used.

The preparation of a hybridoma secreting a monoclonal antibody can be carried out according to the method of Koehler and Milstein et al. (Nature, Vol. 256, p. 495-497, 1975) and a modification method analogous thereto. That is, spleen obtained from the transgenic non-human mammal immunized as described above, lymph nodes, bone marrow or tonsils, etc., preferably antibody-producing cells contained in lymph nodes or spleen, preferably mice, rats, It is prepared by cell fusion with myeloma cells having no autoantibody-producing ability derived from mammals such as guinea pig, hamster, rabbit or human, more preferably mouse, rat or human.

Examples of myeloma cells used for cell fusion include mouse-derived myeloma cells P3 / X63-AG8.653
(ATCC No .: CRL 1580), P3 / NS1 / 1-Ag4-1 (NS-
1), P3 / X63-Ag8.U1 (P3U1), SP2 / 0-Ag14 (Sp2 /
O, Sp2), NSO, PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag.2.3., Human-derived myeloma U-2
66AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used. Screening of a hybridoma clone producing a monoclonal antibody is carried out by culturing the hybridoma, for example, in a microtiter plate, and examining the reactivity of the culture supernatant of a well in which proliferation has been observed with the immunizing antigen used in the above-mentioned immunization, for example. The measurement can be performed by an enzyme immunoassay such as RIA or ELISA.

For production of a monoclonal antibody from a hybridoma, the hybridoma can be cultured in vitro and isolated from the culture supernatant. It can also be cultured in vivo in mouse, rat, guinea pig, hamster, rabbit, or the like, preferably mouse or rat, more preferably mouse ascites, and isolated from ascites.

Further, a gene encoding a human monoclonal antibody is cloned from the hybridoma, and the gene is incorporated into an endogenous gene using a transgenic animal production technique.
It is also possible to prepare goats, sheep or pigs and obtain a large amount of a monoclonal antibody derived from the antibody gene from the milk of the transgenic animal (Nikkei Science, April 1997, pages 78 to 84). page).
When culturing hybridomas in vitro, the hybridomas are grown, maintained, and stored according to various conditions such as the characteristics of the cell type to be cultured, the purpose of the test and research, and the culture method, and the monoclonal antibody is produced in the culture supernatant. It is possible to use any known nutrient medium derived from or prepared from a known nutrient medium or a known basal medium.

As a basic medium, for example, Ham 'medium, MC
Low calcium medium such as DB153 medium or low calcium MEM medium and MCD104 medium, MEM medium, D-MEM medium, R
High calcium medium such as PMI1640 medium or RD medium, A
SF104 medium (trademark), EX-CELL620 medium (trademark) or H
YBRIDOMA-SFM medium (trademark) and the like, and the basic medium can contain, for example, serum, hormones, cytokines, and / or various inorganic or organic substances depending on the purpose. Monoclonal antibody isolation and purification is performed by using the above-mentioned culture supernatant or ascites fluid with saturated ammonium sulfate,
Euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or D
E52, etc.) and affinity column chromatography such as an anti-immunoglobulin column or a protein A column.

The "part of the monoclonal antibody" in the present invention means a part of the human monoclonal antibody as defined above, and specifically, F (ab ') ₂ , Fab', Fab, Fv
(Variable fragment of antibody), sFv, dsF
v (disulphide stabilised Fv) or dAb (singl
e domain antibody) (Expert Opinion on Therapeutic Patents (Exp.
Opin. Ther. Patents), Volume 6, Issue 5, 441-456
P. 1996).

Here, “F (ab ′) ₂ ” and “Fab ′” are
An antibody produced by treating an immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, and digested before and after a disulfide bond existing between two H chains in a hinge region. Means fragment. For example, when an IgG is treated with papain, it is cleaved upstream of a disulfide bond existing between two H chains in the hinge region to form an L consisting of V _L (light chain variable region) and C _L (light chain constant region). Chain and V _H (H
Two homologous antibody fragments in which an H chain fragment consisting of a chain variable region) and C _H γ1 (the γ1 region in the H chain constant region) are linked by a disulfide bond at the C-terminal region can be produced. Each of these two homologous antibody fragments is called Fab '. When IgG is treated with pepsin, it is cleaved downstream of a disulfide bond existing between two H chains in the hinge region to produce an antibody fragment slightly larger than the two Fab's connected by the hinge region. Can be. F (ab ')
_Two .

"Binding rate constant (ka)" in the present invention
The term means a value indicating the strength (degree) of binding of the monoclonal antibody to the target antigen, which is calculated based on the antigen-antibody reaction kinetics. The “dissociation rate constant (kd)” means a value indicating the strength (degree) of dissociation of the monoclonal antibody from the target antigen, calculated based on the kinetics of the antigen-antibody reaction. The “dissociation constant (Kd)” is the “dissociation rate constant (kd)
d)) divided by the “binding rate constant (ka)” value. These constants are used as indices indicating the affinity of the monoclonal antibody for the antigen and the neutralizing activity of the antigen. The constant can be analyzed according to various methods, and a commercially available measurement kit, Biacor
Using eX (manufactured by Amersham Pharmacia) or a similar kit, analysis can be easily performed according to the instruction manual and experimental operation method attached to the kit. The ka value, kd value and Kd value determined using the kit are expressed in units of 1 / M.Sec, 1 / Sec and M (mol), respectively. The monoclonal antibodies tested show that the higher the ka value, the stronger the antigen binding activity, and the Kd
The larger the value, the stronger the neutralizing activity.

The human monoclonal antibody of the present invention includes a human monoclonal antibody having a ka value, a kd value or a Kd value as shown in the following (1) to (3). (1) Numerical value of binding rate constant (ka) to human parathyroid hormone-related protein is 1.0 × 10 ³ (1 / M.Sec) or more, preferably 1.0 × 10 ⁴ (1 / M.Sec) or more. A human monoclonal antibody having reactivity to the human parathyroid hormone-related protein or a part thereof. (2) The dissociation rate constant (kd) with human parathyroid hormone-related protein is 1.0 × 10 ⁻³ (1 / Sec) or less, preferably 1.
A human monoclonal antibody having a value of 0 × 10 ⁻⁴ (1 / Sec) or less and reactive with human parathyroid hormone-related protein or a part thereof. (3) the dissociation constant of the human parathyroid hormone related protein (Kd) is, 1.0 × 10 ^{- 7} (M) or less, preferably 1.0 × 10
^-8 (M) or less, more preferably 1.0 × 10 ^-9 (M) or less, still more preferably 1.0 × 10 ^-10 (M) or less human parathyroid hormone-related protein or a portion thereof A human monoclonal antibody.

More specifically, for example, the ka value (1 / M.Se
About c), about 1.9 × 10^FourMore than human monoclonal anti
Body, more specifically about 1.9 x 10^FourTo about 1.5
× 10^FiveHuman monoclonal antibodies. kd value
About (1 / Sec), about 9.5 × 10 ^-FiveThe following human monochrome
Null antibodies, more specifically about 6.0 × 10^-6No
Approx.9.5 x 10^-FiveHuman monoclonal antibodies
You. About Kd value (M), about 8.4 × 10^-TenThe following Hitomo
Noclonal antibodies, more specifically, about 1.
1 × 10^-Ten~ 8.4 × 10^-TenHuman monoclonal antibodies
I can do it. In addition, each value of the above-mentioned ka, kd, and Kd
Is slightly different from the error range, depending on the measurement conditions.
Is expected to occur, but the index
Generally, there is almost no change. In the present invention,
The term "cells that produce noclonal antibodies"
Any cell that produces a clear human monoclonal antibody
To taste. Specifically, for example, the following (1) to (3)
Cells described in any of them can be mentioned. (1) Human PTHrP, a part thereof or a cell that secretes the PTHrp
Transgenics capable of producing human antibodies in cells, etc.
Obtained by immunizing a non-human mammal.
Human PTHrP or a part thereof
Monoclonal antibody derived from the non-human mammal
Internal antibody producing B cells. (2) sucking the antibody-producing B cells thus obtained
Obtained by cell fusion with myeloma cells
Hybridoma (fused cells). (3) the monoclonal antibody producing B cells or monochrome
Monoclonal isolated from a hybridoma producing an internal antibody
The gene encoding the lonal antibody (the gene encoding the heavy chain)
Either the gene encoding the gene or the light chain,
Or both genes) and the B cells and hybrid
Monoclonal antibody obtained by transforming non-mammary cells
Transgenic somatic cells (genetically modified cells). Here, the monoclonal antibody-producing form described in (3) above
Transformed cells (genetically modified cells) are as described in (1) above.
Produced by the B cells or the hybridoma of (2)
Genetic recombination producing recombinants of clonal antibodies
Means cell.

The human antibody-producing transgenic mouse used as an animal to be immunized in preparing the human monoclonal antibody of the present invention can be produced according to a method already reported (Nature Genetics, Vol. 7, p. 13-21, 199).
4; Nature Genetics, Vol.15, p.146-156, 1997;
JP-A-4-504365; JP-T-Hei 7-509137; Nikkei Science, June, pages 40 to 50, 1995; International Application Publication WO 94/25585; Nature, Vol.
p.856-859, 1994; and JP-T-Hei 6-500233). Specifically, for example, it can be manufactured by using a method including the following steps.

(1) The mouse endogenous immunoglobulin heavy chain gene functions by substituting at least a part of the mouse endogenous immunoglobulin heavy chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination. Producing knockout mice that have been specifically inactivated. (2) By replacing at least a part of the mouse endogenous immunoglobulin light chain locus with a drug resistance marker gene (such as a neomycin resistance gene) by homologous recombination, the mouse endogenous immunoglobulin light chain gene (particularly κ
A knockout mouse in which the chain gene is functionally inactivated. (3) Yeast artificial chromosome,
YAC) a step of producing a transgenic mouse in which a desired region of a human immunoglobulin heavy chain locus is integrated into a mouse chromosome using a vector capable of carrying a large gene such as a vector. (4) A human immunoglobulin light chain (particularly κ
Chain) producing a transgenic mouse in which the desired region of the locus has been integrated into the mouse chromosome. (5) By breeding the knockout mouse and transgenic mouse of (1) to (4) in any order, both the mouse endogenous immunoglobulin heavy chain locus and the mouse endogenous immunoglobulin light chain locus function. Producing a transgenic mouse that has been specifically inactivated and has both the desired region of the human immunoglobulin heavy chain locus and the desired region of the human immunoglobulin light chain locus integrated on the mouse chromosome.

In the knockout mouse, the locus cannot be rearranged (rearranged) by substituting an appropriate region of the mouse endogenous immunoglobulin locus by homologous recombination with an exogenous marker gene (such as a neomycin resistance gene). Can be produced by inactivating as described above. Inactivation using the homologous recombination includes, for example, positive negative selection (Positive negative selection).
A method called Negative Selection (PNS) can be used (Nikkei Science, May, p.52-62, 199).
Four). For functional inactivation of the immunoglobulin heavy chain locus, for example, a J region or a C region (eg, a Cμ region)
Can be achieved by introducing obstacles to some of them. Functional inactivation of an immunoglobulin light chain (for example, a κ chain) can be achieved, for example, by introducing a disorder into a part of the J region or the C region, or a region including a region spanning the J region and the C region. It is.

The transgenic mouse can be prepared by a conventional method such as that generally used in the production of transgenic animals (for example, the latest manual for animal cell experiments, L.I.
(See C Publishing, Chapter 7, pp. 361-408, 1990). Specifically, for example, an HPRT-negative (lacking the hypoxanthine guanine phosphoribosyltransferase gene) ES cell (embryonic stem cell) derived from a normal mouse blastcyst is ligated to the human immunoglobulin heavy chain. The gene encoding the locus or light chain locus or a part thereof and the yeast containing the YAC vector into which the HPRT gene has been inserted are fused by spheroplast fusion. ES cells in which the exogenous gene has been integrated on the mouse endogenous gene are selected by the HAT selection method. Next, the selected ES cells are microinjected into fertilized eggs (blastocysts) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12,
pp. 7380-7384, 1980; U.S. Patent No. 4,873,191).
The blastocysts are implanted into the uterus of another normal mouse as a foster mother. Thus, a chimeric transgenic mouse is born from the foster parent mouse. By crossing the chimeric transgenic mouse with a normal mouse, a heterotransgenic mouse is obtained. The heterogeneic
By crossing transgenic mice,
According to Mendel's law, homogenic transgenic mice are obtained.

The "pharmaceutical composition" of the present invention comprises a pharmaceutically acceptable carrier, ie, an excipient, a diluent, a bulking agent, a disintegrant, using the human monoclonal antibody of the present invention or a part thereof as an active ingredient. A pharmaceutical composition together with one or more of a stabilizer, a preservative, a buffer, an emulsifier, a fragrance, a coloring agent, a sweetener, a thickener, a flavoring agent, a solubilizing agent or other additives, and a tablet, a pill. Preparations, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions,
It can be administered orally or parenterally in the form of an emulsion or syrup.

In particular, in the case of an injection, 0.1 μg antibody / ml carrier to 10 mg antibody / ml in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection.
It can be produced by dissolving or suspending to a carrier concentration. The injection thus produced is administered to a human patient in need of treatment at a rate of 1 μg to 100 mg, preferably 50 μg to 50 mg, per day per 1 kg of body weight in a single administration. It can be administered once to several times. As a form of administration, intravenous injection,
Medically appropriate administration forms such as subcutaneous injection, intradermal injection, intramuscular injection, intraperitoneal injection, peritoneal injection, cerebrospinal injection, or local injection can be exemplified. Intravenous injection or local injection is preferred, but local injection into the site of cancer metastasis, bone disease (osteolysis site, bone destruction site, fracture site, etc.) or oral cavity (periodontal, gingival, gum, etc.) is also possible. Preferred Embodiment 1
One.

The monoclonal antibody or the pharmaceutical composition of the present invention can be applied to the treatment or prevention of various diseases or conditions possibly caused by PTHrP. The disease or symptom includes a disease caused by release of calcium from bone in a parathyroid hormone-related protein-dependent manner (such as hypercalcemia), a malignant tumor (kidney cancer, lung cancer, gastric cancer, breast cancer, pharyngeal cancer, esophageal cancer) , Tongue cancer, prostate cancer, bladder cancer,
Hypercalcemia associated with malignant lymphoma, skin cancer, thyroid cancer, testicular cancer, liver cancer, pancreatic cancer, colorectal cancer, rectal cancer, urothelial cancer, etc., rheumatoid arthritis, osteoarthritis, cancer (squamous cell carcinoma) , Adenocarcinoma cells, melanoma cells, osteosarcoma cells, neuroblastoma cells, blood cancer cells, etc.) bone metastasis, osteolysis, bone destruction,
Proliferation of cancer cells present in bone tissue, disease caused by local production of parathyroid hormone-related protein, symptoms caused by primary local cancer (pain, nerve compression, hypercalcemia, fracture and cachexia) ), Diseases in the teeth, periodontal or gingival tissues (alveolar pyorrhea, gingivitis, periodontal disease, etc.), sepsis (sepsis), systemic inflammatory response syndrome (SIRS), and hypophosphatemia (hypophosphatemic kuru Disease, hypophosphatemic vitamin D-resistant rickets, etc.). Further, the human monoclonal antibody of the present invention can be applied to prolong the life of patients suffering from primary local cancer.

The human monoclonal antibody reactive with human PTHrP of the present invention and a pharmaceutical composition thereof are used as a therapeutic agent for hypercalcemia and diseases associated with bone, tooth and calcium metabolism disorders. Bisphosphonate (bisphosphonate) -based compounds (eg, pamidronate, etidronate, risedronate, pyrophosphate, clodronate, tiludronate, alendronate) having a strong bone resorption inhibiting action Salt, BM21.095
5, YM-175, CGP42446, etc.) or a hydrate thereof can be used to prevent and treat a combination of cancer-related symptoms and hypercalcemia. Administration of the bisphosphonate compound is, for example, a body weight of 70
A dose of about 5 to 25 mg / day can be used for an adult of kg, but an appropriate dose can be set depending on the weight, age and health of the patient.

The therapeutic or prophylactic effect of the monoclonal antibody or the pharmaceutical composition of the present invention can be examined by an in vitro test or an in vivo test using a known disease model animal according to a conventional method. The inhibitory effect on calcium release from bone in a parathyroid hormone-related protein-dependent manner is that parietal bones of neonates born from pregnant mice to which ⁴⁵ Ca was administered were cultured in the presence of PTHrP and an anti-PTHrP monoclonal antibody. ⁴⁵ released into
It can be examined by measuring the amount of Ca with a liquid scintillation counter.

The therapeutic effect on hypercalcemia due to malignant tumors was measured in the same manner as previously reported, ie, anti-PTHrP monoclonal antibody was administered to nude mice transplanted with PTHrP-producing human tumor cells, and the calcium ion concentration in blood was measured. (J. Bone and Min. Res., Vo
l.8, p.849-860, 1993). Therapeutic effects on osteolysis associated with malignant tumors include, for example, anti-PTHrP monoclonal antibody in nude mice injected (or transplanted) into the left ventricle of the heart with estrogen-independent human breast cancer cells MDA-MB-231 or melanoma cells A375. And the degree of osteolysis (osteopathy)
Can be examined by visual inspection by X-ray examination (J. Clin. Invest., Vol. 98, p. 1544-1549, 1996; C
ancer Res., Vol. 52, p. 5395-5399, 1992; J. Bone Min.
Res., Vol. 8, Suppl. 1, No. 92, 1993).

The therapeutic effect on cancer bone metastasis can be simultaneously examined by X-ray examination using the previously reported model (J. Clin. Invest., Vol. 98, p. 1544-1549, 199).
6). The therapeutic effects on rheumatoid arthritis and osteoarthritis (Osteoarthritis) can be examined using various arthropathy models detailed in the previous report (Biopharmaceutical Science Experimental Account, “12 Inflammation and Allergy”). ", I-2, p.153-193, 1993, Hirokawa Shoten).

The therapeutic effect on sepsis (sepsis) or systemic inflammatory response syndrome (SIRS) was determined in mice pre-administered with an anti-PTHrP monoclonal antibody by one of the causes of sepsis.
LPS (lipopolysaccharide), which is one of the components of bacteria, is administered, and its inflammatory response and lethality can be analyzed and observed (International Patent Application Publication WO96 / 39184).
No.). The therapeutic effect against hypophosphatemia seen in patients with hypercalcemia and other malignant tumors was determined by administering anti-PTHrP monoclonal antibody to nude mice that had induced hypercalcemia by transplanting PTHrP-producing human tumor cells. It can be examined by measuring the concentration of phosphorus contained in urine before and after administration of the antibody and blood collected through a cannula inserted into the bladder (International Patent Application Publication WO98 / 13388).

[0061]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but it goes without saying that the present invention is not limited to only the embodiments described in the Examples.

Example 1 Preparation of Transgenic Mice Producing Human Antibodies Based on the genetic engineering techniques commonly used in the production of knockout mice and transgenic mice described above, the immunoglobulin class was changed to Ig according to the method already reported.
Transgenic mice producing human antibodies producing human immunoglobulins that are G2 / κ were produced (Nature Genet
ics, Vol. 7, p. 13-21, 1994; Nature Genetics, Vol. 1
5, p.146-156, 1997; Japanese Patent Publication No. 4-504365;
No. 509137; Nikkei Science, June, 40th to 5th
0, 1995; International Application Publication WO 94/25585.
Publication, Nature, Vol. 368, p. 856-859, 1994; and Japanese Patent Publication No. 6-500233. This human antibody-producing transgenic mouse was used in Examples described later.

Example 2 Preparation of Human PTHrP Partial Peptide <2-1> Preparation of Human PTHrP (1-34) -KLH Sequence of N-terminal 1-34 of human PTHrP Amino acids 1 to 34, Peptide Institute (manufactured by)
Into the conjugation buffer (5 ml (0.1 M MES, 0.9 M
(NaCl, pH 4.7) to prepare 3.2 mg) and KLH
(keyhole limpet hemocyanin, 20mg / 2ml H ₂ O, Pierce
(PIERCE)) and mix with EDC (1-Ethyl-3- (3-dimet).
hylaminopropyl) carbodiimide (35 mg) was added and reacted at room temperature with stirring for 3 hours. The reaction solution was dialyzed three times against a phosphate buffer (300 ml for 3 hours, 600 ml for 15 hours, 500 m
lH for 2 hours), wash with phosphate buffer, PTHrP (1-34) -K
An LH conjugate (2.7 mg / ml) was prepared. P obtained
THrP (1-34) -KLH was used as an antigen (immunogen) in immunization of human antibody-producing mice described in the Examples below.

<2-2> Preparation of human PTHrP (1-39) -Cys-KLH The C-terminal of the sequence of human PTHrP 1-39 (PTHrP (1-39), amino acids 1 to 39 of SEQ ID NO: 1) The sequence PTHrP (1-39) -Cys (5 mg, Peptide Research Laboratories, Inc.) with cysteine added to the peptide was dissolved in phosphate buffer (1 ml) and maleimide-activated KLH (10 mg / ml, PIERCE) And add at room temperature 3.
The reaction was performed for 5 hours. The reaction solution is dialyzed against phosphate buffer, and P
THrP (1-39) -Cys-KLH conjugate (1.65 mg / ml) was prepared. The obtained PTHrP (1-39) -Cys-KLH was used as an antigen (immunogen) in the immunization of human antibody-producing mice described in the Examples below.

Example 3 Preparation of Human Monoclonal Antibody Against Human PTHrP The preparation of the monoclonal antibody in this example was carried out using the Cell Engineering Handbook of Experimental Medicine (separate volume) (edited by Toshio Kuroki et al.
It was prepared according to general methods as described in Yodosha, pp. 66-74, 1992) and an introduction to monoclonal antibody experimental procedures (written by Tamto Ando et al., Published by Kodansha, 1991). Human PTHrP as immunogen was prepared in Example 2
PTHrP (1-34) -KLH or PTHrP (1-39) -Cys-KLH prepared in
Was used. The animals to be immunized were prepared in the examples.
Transgenic mice producing human antibodies producing IgG2 / κ human immunoglobulin were used. The cell culture operation was performed using a multiwell microplate.

PTHrP (1-34) -KLH (50 μg / animal) was added to each of the human antibody-producing transgenic mice (30 mice).
Complete Freund's Adjuvant
Adjuvant) and the first (day 0) immunization. Every week after the initial immunization, the same antigen (the same amount, but without complete Freund's adjuvant) was boosted at least four times by injection in the footpad, and further, on the 4th and 3rd days of obtaining spleen cells and lymph node cells described below.
The day before, PTHrP (1-34) (50 μg / animal) was similarly immunized. The spleen and lymph nodes were surgically obtained from each animal, and the spleen cells and lymph node cells collected from each tissue were used as mouse myeloma P3 / X63-AG8.653 (ATCC No .: CRL158).
0) and polyethylene glycol 4000 or polyethylene glycol 1500 (Boehri
Nger Mannheim) to produce a large number of hybridomas. Hybridoma culture was performed using 10% fetal calf serum (Fetal Calf Serum, F
HAT containing EX-CELL6 containing CS) and aminopterin
This was performed by culturing in a 20-HSF medium (manufactured by JRH Bioscience). Selection (screening) of a hybridoma clone producing an anti-human PTHrP human monoclonal antibody and characterization of a human monoclonal antibody produced by each hybridoma were performed by measuring by ELISA described later.

Example 4 Screening of Hybridomas Producing Human Monoclonal Antibodies by ELISA The following four types of ELISA were used to determine whether the human immunoglobulin heavy chain (hIgH) and human immunoglobulin light chain κ
A number of hybridomas producing human monoclonal antibodies having specific reactivity with human PTHrP were obtained (Tables 1 and 2). In addition, in any of the following examples including this example, and in the tables or figures shown as the test results in the examples, each of the human anti-human
Hybridoma clones producing the PTHrP monoclonal antibody were named using symbols. The following hybridoma clones represent the parent clones: 1A12, 12A7, 1B3, 1B.
4, 4B4, 5B12, 1C1, 1C11, 5C5, 1D2, 2D10, 15D9, 5E
6, 16E12, 2F8, 11F11, 13F7, 1G7, 2G4, 3G4, 4G4, 16
G5, and 15H7. Here, the clone 16G5 is a clone obtained by immunizing PTHrP (1-39) -Cys-KLH in the above-mentioned Example. The hybridoma clones subcloned from each of the parent clones were named by adding an additional number after the parent clone name. 1D5 and 1D11 are mouse anti-human PTHrP monoclonal antibodies used as controls in the ELISA described below. ID5 is a mouse monoclonal antibody specific for human PTHrP (1-34), and ID11 is human PTHrP (37-6
Mouse monoclonal antibody specific for 7) (Cli
n. Chem., Vol. 37, No. 10, p. 1781, 1991; J. Immunol.
Methods, Vol. 146, p. 33-42, 1992; Clin. Chem., Vo.
l.37, No.5, p.678, 1991; J. Immunol. Methods, Vol.
127, p.109, 1990). ELISA for both ID5 and 1D11
Is positive. In addition, PTHrP-dependent intracellular cAM described later
In an inhibition test for an increase in P, ID5 has inhibitory activity, but ID11 has no inhibitory activity.

[0068]

[Table 1]

[0069]

[Table 2]

One of them, hybridoma clone 1B3-9-16, was internationally deposited on June 16, 1998 with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of the Ministry of International Trade and Industry (International Deposit No. FERM BP-6390). ).

<4-1> Human immunoglobulin heavy chain (IgH)
Of a monoclonal antibody having human PTHrP (1-34) and ovalbumin (OVA, manufactured by Sigma) (human PTHrP (1-34) -OVA; 25-5
(0 μg / ml, 50 μl / well) was added to each well of a 96-well ELISA microplate (manufactured by SUMILON), incubated at room temperature for 2 hours, and human PTHrP (1-34) -OVA was adsorbed to the microplate. . Then, discard the supernatant and add 0.05% Tween
After washing three times with a phosphate buffer solution containing 20 (PBS-T), a blocking reagent (100 μl, PBS-T containing 0.5% bovine serum albumin (BSA)) was added to each well, and the mixture was incubated at room temperature for 1.5 hours. The site where PTHrP (1-34) -OVA was not bound was blocked. Each well was washed three times with PBS-T. In this way, each well is filled with human PTHrP (1-34) -OVA
A microplate coated with was prepared.

To each well, the culture supernatant (50 μl) of each hybridoma was added, and reacted at room temperature for 2 hours.
Each well was washed three times with PBS-T. Then, a goat anti-human IgG (Fc) antibody labeled with biotin (Biotin) (EY Labo
ratories) diluted 1,000 times with PBS-T (50μ
1 / well) was added to each well and incubated for 1 hour at room temperature. After washing the microplate three times with PBS-T, streptavidin-β-galactosidase (Streptoavidi) diluted 10,000-fold with PBS-T containing ₂ mM MgCl ₂ was used.
n-β-galactosidase, 50 μl / well, Gibco BRL)
Was added to each well and incubated for 1 hour at room temperature.

After washing the microplate four times with PBS-T, the substrate buffer (100 mM NaCl, 1 mM MgCl ₂ , 10 mM phosphate buffer (containing Na and K), BSA (1 mg / ml)) (PH 7.
5 mg / ml of 4-methyl-umbelliferyl-β-D-galactoside (4-Methyl-umbelliferyl-β-D
-galactoside, 100 μl / well, Sigma) was added to each well and incubated at room temperature for 20 minutes. To each well, 2M Na ₂ CO ₃ (21.2 g / 200 ml, 50 μl / well) was added to stop the reaction. Fluorescence intensity at a wavelength of 460 nm (excitation: 355 nm) is measured using a fluoroscan microplate reader.
(Labsystems / Dainipponsha). As a control anti-human PTHrP monoclonal antibody, human PTHrP (1-3
Mouse monoclonal antibody 1D5 (100ng) specific to 4)
/ ml) (Clin. Chem., Vol. 37, No. 10, p. 1781, 1991;
J. Immunol. Methods, Vol. 146, p. 33-42, 1992; Clin.
Chem., Vol. 37, No. 5, p. 678, 1991; J. Immunol. Met
hods, Vol. 127, p. 109, 1990). In this control test, a biotin-labeled goat anti-mouse IgG (H + κ) antibody (American Corex) was used as the biotin-labeled antibody.

<4-2> Human immunoglobulin light chain κ (IgL
Detection of Monoclonal Antibody Having κ) Conjugate (Human PTHrP (1-34) -OVA; 1.25) composed of human PTHrP (1-34) and ovalbumin (OVA, manufactured by Sigma)
μg / well) with a 96-well microplate for ELISA (Nunc
-Immuno Plate, Catalog No. # 439454), and incubated at room temperature for 2 hours to allow PTHrP (1-34) to adsorb to the microplate. Next, the supernatant was discarded, and after washing with a phosphate buffer (PBS), a blocking reagent (200 μl, a buffer containing 0.5% OVA) was added to each well, and the mixture was incubated at room temperature for 2 hours. ) The site not bound by -OVA was blocked. Fill each well with 0.05% T
Washing was performed three times with phosphate buffer (PBS, 200 μl) containing ween20. In this manner, each well was prepared using human PTHrP (1-3
4) A microplate coated with -OVA was prepared.

After adding the culture supernatant (50 μl) of each hybridoma to each well and reacting for 2 hours, each well was filled with a phosphate buffer solution (200 μl) containing 0.05% Tween 20.
And washed three times. Next, peroxidase (P
goat anti-human Igκ antibody (1,000 fold diluted, 50 μl / well, manufactured by PROTOS, catalog No. 726) labeled with eroxidase), and incubated at room temperature for 1 hour. After washing the microplate three times with a phosphate buffer containing 0.05% Tween 20, a substrate buffer (100 μl / well; citrate / phosphate buffer (pH 5.0, 50 ml), o-phenylenediamine (O-Phenylenediamine) , OPD; 20 mg) and 30% aqueous hydrogen peroxide (15 μl) were added to each well, and incubated at room temperature for 20 minutes.

Next, 2 M sulfuric acid (50 μl) was added to each well to stop the reaction. The absorbance at a wavelength of 490 nm was measured with a fluoroscan microplay reader (Labsystems / Dainippon). As a control anti-human PTHrP monoclonal antibody, mouse monoclonal antibody 1D5 (100 ng / ml) specific to human PTHrP (1-34) (Clin. Chem., V
ol. 37, No. 10, p. 1781, 1991; J. Immunol. Methods, V
ol. 146, p.33-42, 1992; Clin. Chem., Vol.37, No.5,
p.678, 1991; J. Immunol. Methods, Vol.127, p.109,
1990). In this control test, a goat anti-mouse Igκ antibody (Southern Biotechnology) was used as a secondary antibody.
Was used.

<4-3> Human PTHrP using N-Bio-PTHrP
Goat anti-human IgG (Fc) antibody (2.4μ) diluted in phosphate buffer
g / ml, 50 μl / well, manufactured by Organon Teknika)
To each well of a 96-well microplate (Nunc) for incubation, and incubated at room temperature for 2 hours to remove goat anti-human IgG (F
c) The antibody was adsorbed on the microplate. Next, the supernatant was discarded, and the well was washed three times with a phosphate buffer solution (PBS-T) containing 0.05% Tween 20, and then a blocking reagent (200 μl, PBS-T containing 0.5% bovine serum albumin (BSA)) was added to each well. In addition, the mixture was incubated at room temperature for 2 hours to block a site where no goat anti-human IgG (Fc) antibody was bound.

Each well was washed three times with PBS-T (200 μl of phosphate buffer containing 0.05% Tween 20). Thus, a microplate in which each well was coated with a goat anti-human IgG (Fc) antibody was prepared. For each well,
The culture supernatant (50 μl) of each hybridoma diluted with the blocking reagent was added, and reacted at room temperature for 2 hours. Then, each well was washed three times with PBS-T. Next, human PT whose amino terminal (N-terminal) is labeled with biotin (Biotin)
A blocking reagent diluted solution (100 ng / ml, 50 μl / well) of HrP (1-34) (hereinafter sometimes referred to as N-Bio-PTHrP (1-34); manufactured by Peninsula Laboratories) was added to each well. And incubated at room temperature for 2 hours.

After the incubation, the microplate was washed three times with PBS-T, and then streptavidin-β diluted 10,000 times with a blocking reagent containing ₂ mM MgCl _2.
-Galactosidase (Streptoavidin-β-galactosidas
e, 50 μl / well, Gibco BRL) was added to each well and incubated for 1 hour at room temperature. After washing the microplate three times with PBS-T, the substrate buffer (100 mM NaC
l, 5 mg / ml 4-mL diluted with 1 mM MgCl ₂ , 10 mM phosphate buffer (containing Na and K), BSA (1 mg / ml) (pH 7.0)
Methyl-umbelliferyl-β-D-galactoside (4
-Methyl-umbelliferyl-β-D-galactoside, 100 μl / well, manufactured by Sigma) was added to each well and incubated at room temperature for 20 minutes. Each well contains 2M Na ₂ CO ₃ (21.2
g / 200 ml, 50 μl / well) was added to stop the reaction. wavelength
Fluorescence intensity at 460 nm (excitation: 355 nm) was measured using a fluoroscan microplate reader (Labsystems / Dainippon).
Was measured. As a control anti-human PTHrP monoclonal antibody, mouse monoclonal antibody 1D5 (100 ng / ml) specific to human PTHrP (1-34) (Clin. Chem., Vol. 3)
7, No. 10, p. 1781, 1991; J. Immunol. Methods, Vol. 1
46, p.33-42, 1992; Clin. Chem., Vol.37, No.5, p.67.
8, 1991; J. Immunol. Methods, Vol. 127, p. 109, 199
0) was used. In this control test, a goat anti-mouse IgG (Fc) antibody (Organon Tekni
ka company) was used.

<4-4> Human PTHrP using C-Bio-PTHrP
Of N-Bio-PTHrP (1-34) used in the above Example <4-3>, instead of C-Bio-PTHrP ( 1-39) The procedure was the same as in Example <4-3>, except that -Cys (bonded to biotin via the SH group of Cys) was used as the labeling antigen.

Example 5 Confirmation of cross-reactivity Human PTHrP (1-34) -OVA used in Example <4-2> above
And instead of goat anti-human Igκ antibody, human PTH (1-34), respectively
(SEQ ID NO: 2) and a peroxidase (Peroxidas) which performs the same operation except that a goat anti-human IgH (Fc) antibody is used.
e) The cross-reactivity of human anti-human PTHrP monoclonal antibodies produced by the various hybridomas obtained in the above Examples with human PTH was examined by labeled ELISA. For comparison, each human anti-human PTHrP monoclonal antibody
The reactivity to THrP was determined by a peroxidase-labeled ELISA that performed the same procedure except that a goat anti-human IgH (Fc) antibody was used instead of the goat anti-human Igκ antibody used in Example <4-2>. It was measured. As shown in Table 3, all human anti-human PTHrP monoclonal antibodies were human PTHrP
It showed specific reactivity only to human PTH and no reactivity to human PTH.

[0082]

[Table 3]

Example 6 Purification of Monoclonal Antibody A human monoclonal antibody was purified from the culture supernatant of each of the hybridomas obtained in Example 4 as follows for use in the in vitro test described below. Table 4 shows the antibody production (μg / ml) of each hybridoma.

[0084]

[Table 4]

10% fetal calf serum (Fetal Calf Serum,
EX-CEL containing HAT containing FCS) and aminopterin
L-620-HSF medium supernatant of each hybridoma culture cultured in (JRH Bioscience Inc.) (30-40 ml / 50 ml tube) was added, the adsorption buffer (20 mM of KH ₂ PO _4, 180 mM of Na ₂ HP
O ₄ and 154mM of NaCl, pH 7.6) and recombinant Protein A
(RProtein A Fast Flow, 0.2ml, IgG adsorption amount: 35mg / ml
Gel, manufactured by Pharmacia) and stirred at 15 ° C. for 5 hours to convert the antibody contained in each culture supernatant into Prot.
Adsorbed on ein A. Centrifuge each tube (3,000
After centrifugation for 10 min), discard the centrifugal supernatant and buffer (20 mM
aPi, 150 mM NaCl, pH 6.0) (10 ml) was added to elute bovine IgG contained in the culture supernatant. Centrifugation (3,000
After centrifugation at 10 min., the centrifugal supernatant (bovine IgG was eluted) was discarded, and the buffer solution (20 mM NaPi, 150 mM NaCl, pH 6.
0) (10 ml) was added, followed by centrifugation (3,000 rpm,
10 minutes). After the centrifugation, the centrifugal supernatant was discarded, and the centrifugation residue was subjected to buffer solution (20 mM NaPi, 150 mM NaCl, pH 6.0) (0.65 mM
l) was added twice and mixed, and then transferred to a 1.5 ml microtube.

The following procedure was repeated once or three times for each microtube to obtain a purified human anti-human PTHrP monoclonal antibody. Each microtube was centrifuged (14,000 rpm, 2 minutes), the supernatant was discarded, and the elution buffer (77 mM Na ₂ HPO ₄ , 67 mM citric acid, 150 mM NaCl, pH 3.
8) (0.9 ml) was added, and after centrifugation, it was further centrifuged (1400 rpm,
2 minutes). After filtering the centrifugal supernatant with a filter, a neutralization buffer (500 mM Na ₂ HPO ₄ , 50 mM KH ₂ PO ₄ , pH 8.7) (0.1
or 0.2 ml) to obtain an eluted fraction. The obtained eluted fraction was dialyzed against a phosphate buffer, and human anti-human PTHrP
A purified product of the monoclonal antibody was obtained.

Example 7 Inhibition of PTHrP-Dependent Increase in Intracellular cAMP by a Human Anti-human PTHrP Monoclonal Antibody As a PTH-like action of PTHrP, for example, in bone, it acts on osteoblasts and adenylate cyclase (AC) Activates phospholipase C (PLC) to induce osteoclastic bone resorption.
P) and the action of promoting phosphorus excretion and promoting calcium reabsorption (Crit. Rev. Biochem. Mol. B
iol., Vol.26, No.3-4, p.377, 1991). cAMP plays a role as a second messenger having a role of transmitting extracellular information (first messenger) such as hormones and neurotransmitters acting on the cell surface into the cell. Depends on signals from various first messengers to increase its production and control subsequent reactions in the cell.

In this test, the various human anti-human
The presence or absence of the activity of a PTHrP monoclonal antibody to functionally control the action of human PTHrP was examined using the inhibitory effect on PTHrP-dependent increase in intracellular cAMP production as an index. Contains RPMI1640 medium containing 10% fetal calf serum (FCS)
Rat osteosarcoma cell line UM in 24-well microtiter plate
R106 (ATCC CRL-1661) (1 × 10 ⁴ cells / well) was inoculated and cultured for 3 days. After washing each well with RPMI1640 medium, a modified RPMI1640 medium (0.1% in 100 ml of PPMI1640 medium)
100 mg of bovine serum albumin (BSA), 1 mM IBMX (cAMP
It has a degrading enzyme inhibitory effect. Sigma) at a concentration of 1 ml / well and incubated at 37 ° C. for 20 minutes.
The medium was made up of human PTHrP (10 ng / ml) and each human anti-human PT
Replaced with RPMI1640 medium containing 10% FCS containing HrP monoclonal antibody (0.3, 1.0 or 5.0 μg / ml) (pre-culture for 1 hour at room temperature with addition of PTHrP and antibody, 0.5 ml / well).
Cultured at 20 ° C for 20 minutes. Then, after removing the medium, the extract was recovered by adding cAMP to 95% ethanol (0.25 ml / well) containing 3 mM hydrochloric acid and treating at 4 ° C. for 2 hours. Was injected. After drying each supernatant sample, cAMP detection EIA kit (Amersha
The amount of cAMP contained in each culture supernatant sample was measured according to the experimental procedure attached to the kit using the kit (manufactured by m Corporation).

The results obtained when the same culture was carried out using a medium containing only human PTHrP (1-34) without any human anti-human PTHrP monoclonal antibody was used as a control. As a control anti-human PTHrP monoclonal antibody, human PTHrP
Mouse monoclonal antibody 1D5 specific for (1-34)
(Positive control) and human PTHrP (37-67)
Mouse monoclonal antibody 1D11 (negative control) (Clin. Chem., Vol. 37, No. 10, p.
1781, 1991; J. Immunol. Methods, Vol. 146, p. 33-42,
1992; Clin. Chem., Vol. 37, No. 5, p. 678, 1991;
Immunol. Methods, Vol. 127, p. 109, 1990) was used.
The results are shown in FIGS.

From the test results, it was found that any human anti-human PT
It was confirmed that the HrP monoclonal antibody had an activity to significantly suppress the PTHrP-dependent increase in intracellular cAMP and to functionally suppress the action of human PTHrP.

Example 8 Inhibition of PTHrP-dependent Bone Resorption by a Human Anti-human PTHrP Monoclonal Antibody As described above, PTHrP has a PTH-like action similar to PTH, that is, it acts on osteoblasts in bone (osteoblast). Activates osteoclasts by cells, produces bone organic degrading enzymes, etc.) Has bone resorption activity that promotes osteoclastic resorption and mobilizes calcium from bone (Brown, EM, Homeostatic
mechanisms regulating extracellular and intracellu
lar calcium metabolism, in The parathyroids, p.19,
1994, Raven press, New York). In this study, the presence or absence of the activity of the various human anti-human PTHrP monoclonal antibodies obtained above that functions to control the action of human PTHrP was examined using the PTHrP-dependent inhibitory effect on bone resorption as an index. did.

On the 15th day of pregnancy, ⁴⁵ Ca radioisotope (50 μCi / animal) was subcutaneously administered to ICR mice (4 to 8 mice / antibody, manufactured by Charles River Japan). Seven days after administration, the left and right parietal bones of the newborn (6 to 7 days old) are aseptically removed,
The cells were pre-cultured in a BG-Jb medium (Gibco BRL) for 24 hours. Next, the medium was discarded, and the sequence of the N-terminal 1-34 of human PTHrP (PTH
rP (1-34), amino acid numbers 1-34 of SEQ ID NO: 1, Peptide Institute (manufactured by) (10 ng / ml) and each human anti-human PTHrP monoclonal antibody (0.1, 1.0, 10 or 100 μl)
g / ml) and a BG-Jb medium (Gibco BRL) was added and cultured. After 48 hours of culture, the same concentration of human PTHrP (1-34) as above
The medium was replaced with a medium containing a human anti-human PTHrP monoclonal antibody, and the cells were further cultured for 48 hours. After the culture, the amount of ⁴⁵ Ca contained in each of the medium and the parietal bone (dissolved by treatment with 2N hydrochloric acid at 60 ° C. for 24 hours) was measured using a liquid scintillation counter.

The results obtained when the same culture was performed using a medium containing only human PTHrP (1-34) without any human anti-human PTHrP monoclonal antibody was used as a control. As a control anti-human PTHrP monoclonal antibody, human PTHrP
Mouse monoclonal antibody 1D5 specific for (1-34)
(Positive control) and human PTHrP (37-67)
Mouse monoclonal antibody 1D11 (negative control) (Clin. Chem., Vol. 37, No. 10, p.
1781, 1991; J. Immunol. Methods, Vol. 146, p. 33-42,
1992; Clin. Chem., Vol. 37, No. 5, p. 678, 1991;
Immunol. Methods, Vol. 127, p. 109, 1990) was used.
The results are shown in FIGS. Table 5 shows the antibody concentration (IC ₅₀ ) that inhibits bone resorption by 50%.

[0094]

[Table 5]

Example 9 Measurement of Affinity and Neutralizing Activity of Monoclonal Antibody for Antigen The binding rate constant (ka) of the human monoclonal antibody to the various human parathyroid hormone-related proteins prepared in the above Examples was determined. ), The dissociation rate constant (kd) and the dissociation constant (Kd) were measured using a commercially available measurement kit, BiacoreX (manufactured by Amersham Pharmacia). The operations described below other than the immobilization of the antigen on the sensor chip were performed according to the instruction manual attached to the kit and the experimental operation method. As the human thyroid hormone-related protein immobilized on the sensor chip, human PTHrP (1-39) -Cys prepared in the above example was used. The antigen is immobilized on the sensor chip by attaching the human PTHrP (1--) to the carboxyl group of carboxymethyl dextran (CM) coated on the surface of the sensor chip via a linker.
39) Mercapto group of cysteine residue of -Cys (SH
Group).

The flow cell 1 (Flow Cell 1) attached to the kit
1) In 0.01M HEPES buffer (0.15M NaCl, 3mM EDTA
And 0.005% of surfactant P20. pH 7.4) at a flow rate of 5 μl / min, and 100 μl of 0.05 M NHS (N-Hydroxysuccinimide)
/0.2M EDC (N-Ethyl-N '-(dimethylaminopropyl) carbod
iimido) was added to activate the carboxyl groups of the CM coated on the sensor chip surface. Then 100
μl of 80 mM PDEA (2- (2-pyridinyldithio) ethaneamine)
A /0.1 M borate buffer (pH 8.5) was added to introduce an SS group that undergoes a SH group reaction. In addition, 8 μl of 1 μg / ml human PT
HrP (1-39) -Cys / 10mM sodium acetate buffer (pH5.0)
Was added to immobilize human PTHrP (1-39) -Cys on the sensor chip. Immobilized human PTHrP (1-39) -Cy
The amount of s was 45 RU (resonance unit). The unreacted SS group was 100 μl of 50 mM cysteine / 1 M NaCl / 0.1.
Blocking was performed by adding 1 M sodium formate buffer (pH 4.3).

[0097] Flow cell 2 (Flow
Cell 2) was capped as above using cysteine instead of human PTHrP (1-39) -Cys. A phosphate buffer solution was flowed through the flow cell at a flow rate of 30 μl / min, and the purified human monoclonal antibody derived from the following hybridoma clone (20 to 60 μg / ml, 60 μl) or the control monoclonal antibody described below was added. <Clone> 2F8-10-3, 1C1-3, 1B3-9-16, 15H7-8-3, 1
6E12-6, 5B12-16-12, 4B4-6-21, 2G4-12-20, and 1B4-1
0-13 <Control antibody> Mouse monoclonal antibody 1D5 specific to human PTHrP (1-34) (Clin. Chem., Vol. 37, No. 10, p. 1)
781, 1991; J. Immunol. Methods, Vol. 146, p. 33-42,
1992; Clin. Chem., Vol. 37, No. 5, p. 678, 1991; J. I
mmunol. Methods, Vol. 127, p. 109, 1990) The measurement was performed under the standard conditions of 2 minutes for the binding phase and 15 minutes for the dissociation phase, and a sensorgram was obtained. Based on the sensorgram data obtained, analysis software (BI
Aevaluation 3.1) was used to calculate the association rate constant (ka), dissociation rate constant (kd), and dissociation constant (Kd).
Table 6 shows the results. All the monoclonal antibodies had extremely high antigen affinity and antigen neutralizing activity.

[0098]

[Table 6]

Example 10 Determination and Analysis of Gene Sequence and Amino Acid Sequence cDNA encoding the variable region of a heavy chain constituting a human monoclonal antibody against various human parathyroid hormone-related proteins prepared in the above Examples The sequences and the cDNA sequences encoding the variable and constant regions of the light chain were determined as follows, and the structural features of the genes were analyzed. The sequence of the sequence analysis in this example is schematically shown in FIG. The following hybridoma producing a human monoclonal antibody against the human parathyroid hormone-related protein prepared in the above example (each about 5 × 10 ⁷ cells) was cultured, followed by centrifugation,
The precipitate is collected and -80 until the extraction of PolyA + RNA described below.
Stored at ° C. <Hybridoma clone> 15H7-8-
3, 16E12-6, 1B3-9-16, 1B4-10-13, 1C1-3, 2F8-10-3,
2G4-12-20, 3G4-3, 4B4-6-21, and 5B12-16-12.

Extraction and purification of PolyA ⁺ RNA from each hybridoma were performed using a commercially available FastTrack 2.0 kit (manufactured by INVITROGEN).
Was used as follows. Said frozen cells,
The cells were lysed in a cell lysis buffer (Lysis Buffer), disrupted by POLYTRON, and solubilized. After the lysate was incubated at 45 ° C., Oligo (dT) cellulose was added, and the mixture was gently shaken for about 1 hour. Next, Oligo (dT) cellu
After washing the lose, PolyA ⁺ RNA was eluted with Ellution Buffer. The eluted PolyA ⁺ RNA is ethanol precipitated and 20 μl
Was dissolved in Tris-EDTA buffer solution. The concentration of the resulting PolyA + RNA was determined by measuring the absorbance at a wavelength of 260 nm. Using the obtained PolyA ⁺ RNA as a template, commercially available Maratho
n RACE using cDNA Amplification Kit (CLONTECH)
-CDNA was synthesized by a conventional method using PCR (see “Gene amplification
PCR Method, Basics and New Developments ”, 1992, 2nd printing, published by Kyoritsu Shuppan Co., Ltd., p.13-15). That is, using PolyA ⁺ RNA (1 to 5 μg) purified from each hybridoma as a template,
1st strand cDNA and 2nd strand cDNA were sequentially synthesized.
The cDNA was subjected to extraction once each using phenol / chloroform / isoamino alcohol and chloroform. Next, the cDNA was precipitated with ethanol and ligated to the adapter DNA (SEQ ID NO: 43). Using the resulting DNA reaction product diluted 1/250 as a template, PCR was performed by a conventional method using synthetic primers to prepare cDNAs encoding the antibody heavy chain and the antibody light chain, respectively. For the PCR relating to the antibody heavy chain, the primer described in SEQ ID NO: 44 was used. The primer described in SEQ ID NO: 45 was used for PCR relating to the antibody light chain.

Each PCR product was fractionated by agarose gel electrophoresis, and DNA was recovered. Determination of the nucleotide sequence of each of the obtained cDNAs was performed using commercially available DyeTerminator Cycle Sequencing.
FS Kit (PE-Applied Biosystems) and PRISM377 DNA
This was performed using a Sequencer (manufactured by PE-Applied Biosystems). The sequencing primer for this sequencing is
The primers used in the aforementioned PCR were used. Further, an appropriate sequencing primer was prepared from the obtained sequence, and the reaction was further performed. A cDNA sequence encoding a variable region of a heavy chain of a human monoclonal antibody against a human parathyroid hormone-related protein produced by each of the above hybridomas, a cDNA sequence encoding a variable region and a constant region of a light chain, The amino acid sequence deduced from each cDNA sequence is shown in the sequence listing as follows.

<Clone 15H7-8-3> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 23 (signal sequence: base numbers 1 to 57, V region: base numbers 58 to 351, N region: base numbers 352 to 352) 354, D region: 355 to 369, N region: 370 to 373, J region: 374 to 429) Amino acid sequence: SEQ ID NO: 24 (signal sequence: amino acids 1 to 19, variable region: including amino acids 20 to 117) (Variable region and constant region of light chain) DNA sequence: SEQ ID NO: 3 (Signal sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: base number 398 To 717) amino acid sequence: SEQ ID NO: 4 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 119) <clone 16E12-6> (variable region of heavy chain) DNA sequence: SEQ ID NO: 25 ( Signal sequence: base number 1 To 57, V region: base numbers 58 to 351; N region: base numbers 352 to 354; D region: 355 to 369; N region: 370 to 373; J region: 374 to 429) Amino acid sequence: SEQ ID NO: 26 (signal) Sequence: amino acids 1 to 19, variable region: includes amino acids 20 to 117 (variable region and constant region of light chain) DNA sequence: SEQ ID NO: 5 (signal sequence: base numbers 1 to 60, V region: base number) 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 6 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 119) < Clone 1B3-9-16> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 27 (signal sequence: base numbers 1 to 57, V region: base numbers 58 to 351, N region: base numbers 352 to 354, D region : 355 to 369, N area: 370 to 373, J area : 374 to 429) Amino acid sequence: SEQ ID NO: 28 (signal sequence: amino acids 1 to 19, variable region: including amino acids 20 to 117) (light chain variable region and constant region) DNA sequence: SEQ ID NO: 7 (signal Sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 8 (signal sequence: amino acid numbers 1 to 20) <Clone 1B4-10-13> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 29 (Signal sequence: Nucleotide numbers 1 to 57, V region: Nucleotide numbers 58 to 119) 351, N region: base numbers 352 to 354, D region: 355 to 369, N region: 370 to 373, J region: 374 to 429) Amino acid sequence: SEQ ID NO: 30 (signal sequence: amino acids 1 to 19, variable region) : Amino acid number (Including the variable region and constant region of the light chain) DNA sequence: SEQ ID NO: 9 (signal sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 10 (signal sequence: including amino acids 1 to 20, variable region: including amino acids 21 to 119) <clone 1C1-3> (variable region of heavy chain) DNA Sequence: SEQ ID NO: 31 (signal sequence: base numbers 1 to 57, V region: base numbers 58 to 351, N region: base numbers 352 to 354, D region: 355 to 369, N region: 370 to 373, J region: 374 to 429) Amino acid sequence: SEQ ID NO: 32 (signal sequence: amino acids 1 to 19, variable region: including amino acids 20 to 117) (light chain variable region and constant region) DNA sequence: SEQ ID NO: 13 (signal sequence : Base number 1 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 14 (signal sequence: amino acid numbers 1 to 20, variable region: amino acid number) <Clone 2F8-10-3> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 33 (signal sequence: base numbers 1 to 57, V region: base numbers 58 to 351, N region: base) Nos. 352 to 354, D region: 355 to 369, N region: 370 to 373, J region: 374 to 429) Amino acid sequence: SEQ ID NO: 34 (signal sequence: amino acids 1 to 19, variable region: amino acids 20 to 117) (Variable region and constant region of light chain) DNA sequence: SEQ ID NO: 11 (signal sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: Base numbers 398 to 717) amino acid sequence: SEQ ID NO: 12 (Signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 119) <Clone 2G4-12-20> (heavy chain variable region) DNA sequence: SEQ ID NO: 35 (signal sequence: base Nos. 1 to 57, V region: base numbers 58 to 351; N region: base numbers 352 to 354; D region: 355 to 369; N region: 370 to 373; J region: 374 to 429) Amino acid sequence: SEQ ID NO: 36 (Signal sequence: amino acids 1 to 19, variable region: including amino acids 20 to 117) (Light chain variable region and constant region) DNA sequence: SEQ ID NO: 15 (signal sequence: base numbers 1 to 60, V region: Base numbers 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 16 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 119) <Claw 3G4-3> (variable region of heavy chain) DNA sequence: SEQ ID NO: 37 (signal sequence: base numbers 1 to 57, V region: base numbers 58 to 351, N region: base numbers 352 to 354, D region: 355 to 369, N region: 370 to 373, J region: 374 to 429) Amino acid sequence: SEQ ID NO: 38 (including signal sequence: amino acids 1 to 19, variable region: amino acids 20 to 117) (light chain variable region and (Constant region) DNA sequence: SEQ ID NO: 17 (signal sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: sequence No. 18 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 119) <clone 4B4-6-21> (heavy chain variable region) DNA sequence: SEQ ID NO: 39 (signal sequence: base number 1 to 57, V region: base Issue 58 to 347, N region: nucleotide number 348 to 349, D region: 350 to 356, N regions 357,
J region: 358 to 417) Amino acid sequence: SEQ ID NO: 40 (signal sequence: amino acids 1 to 19, variable region: including amino acids 20 to 115) (light chain variable region and constant region) DNA sequence: SEQ ID NO: 19 (Signal sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base numbers 360 to 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 20 (signal sequence: amino acid number 1 <Clone 5B12-16-12> (Variable region of heavy chain) DNA sequence: SEQ ID NO: 41 (signal sequence: base numbers 1 to 57, V region: base number) 58 to 351; N region: base numbers 352 to 354; D region: 355 to 369; N region: 370 to 373; J region: 374 to 429) Amino acid sequence: SEQ ID NO: 42 (signal sequence: amino acid numbers 1 to 19; Variable area: A (The variable region and the constant region of the light chain) DNA sequence: SEQ ID NO: 21 (signal sequence: base numbers 1 to 60, V region: base numbers 61 to 359, J region: base number 360) To 397, C region: base numbers 398 to 717) Amino acid sequence: SEQ ID NO: 22 (signal sequence: amino acids 1 to 20, variable region: including amino acids 21 to 119)

Based on the determined DNA sequences, a library of human immunoglobulin variable region genes, V BASE Sequence (Immunol. Today, Vol. 1), prepared by Tomlinson et al.
6, No. 5, p. 237-242, 1995). As a result, regarding the heavy chain V region, nine clones except the clone 4B4-6-21 were composed of the same _VH segment V3-30. Clone 4B4-6-21 was composed of segment V4.16. For the heavy chain D region, 9 except clone 4B4-6-21
The clone was composed of the same D segment DN1. Clone 4B4-6-21 was composed of segment DA1. As for the heavy chain J region, all 10 clones were composed of the same J segment JH6. For the light chain V region, all 10 clones have the same Vκ segment.
It consisted of DPK15. For the light chain J region, 10
All clones consisted of the same Jκ segment Jκ3. For the light chain C region, all 10 clones were composed of CK. Furthermore, as shown in the sequence listing, the cDNA sequences encoding the heavy chains of the ten types of human monoclonal antibodies have a region between the V region and the D region and a region between the D region and the D region.
There was an N region (N-addition) between the region and the J region.

[0104] Example 11 antibody fragments F (ab ') ₂ and antibody fragments Fab various human anti-human PTHrP monoclonal antibody prepared as Preparation aforementioned F (ab') ₂ and Fab,
It can be prepared as follows. Monoclonal antibody (5m
g / ml) in 20 mM sodium acetate buffer (pH 3.5) and incubate at 37 ° C. for 30 minutes. Then, insolubilized pepsin (1 ml, Pierce) is added and incubated at 37 ° C. for 12 hours while rotating with a rotator. The reaction solution is collected, centrifuged (3,000 rpm, 10 minutes), and the supernatant is collected. The protein A affinity chromatography is performed as follows according to the protocol of a protein A column kit (manufactured by Amersham). Add binding buffer to the centrifuged precipitate and centrifuge (3,000 rpm, 10 minutes)
And collect the supernatant. The supernatant collected by two centrifugations is collected, an equal volume of binding buffer is added, and the pH is adjusted to 8.9 by the addition of 1N sodium hydroxide. The mixed solution is added to the protein A column equilibrated with the binding buffer, and then washed twice with the binding buffer (5 ml) to collect an elution fraction. The obtained eluted fraction is dialyzed (4 ° C., 24 hours) against 5 mM phosphate buffer (2 L, pH 6.8).

For further purification, high performance liquid chromatography (HPLC) is performed using a hydroxyapatite column (manufactured by Bio-Rad). The solution obtained by dialysis was added to the hydroxyapatite column,
After a phosphate buffer of M is flowed for 15 minutes, a linear gradient elution is carried out with a phosphate buffer of 5 mM to 0.4 M. The eluate is collected by a fraction collector, the absorbance at 280 nm is measured, and the fraction containing F (ab ') ₂ is collected. The obtained fraction is dialyzed against a phosphate buffer (2 L) (4 ° C., 24 hours) to obtain a purified monoclonal antibody F (ab ′) 2. In addition to the above method,
It can also be prepared by the following alternative method. That is, the monoclonal antibody is mixed and reacted with commercially available microbeads to which pepsin is bound, and the supernatant is recovered by centrifugation.
The supernatant is added to commercially available microbeads to which protein A is bound (eg, Pierce), and the eluate is collected. This eluate is used as an F (ab ') ₂ solution.

Example 12 Therapeutic Effect of PTHrP-Induced Hypercalcemia by Anti-Human PTHrP Human Monoclonal Antibody As described above, PTHrP has a PTH-like effect similar to PTH, that is, it acts on osteoblasts in bone. (Activation of osteoclasts by osteoblasts, production of bone organic degrading enzymes, etc.) Has bone resorption activity that promotes osteoclastic resorption and mobilizes calcium from bone (Brown, EM, Homeostatic
mechanisms regulating extracellular and intracellu
lar calcium metabolism, in The parathyroids, p.19,
1994, Raven press, New York). However, as described above, in cancer patients, excessive PTHrP production causes hypercalcemia, such as tumor-dependent hypercalcemia, which is often caused by PTHrP secreted in large quantities by tumor cells. In this test, the therapeutic effect of various human anti-human PTHrP monoclonal antibodies obtained above on hypercalcemia was examined using hypercalcemia model mice artificially produced by administering PTHrP.

[0107] Blood was collected from the fundus of each normal CRJ ICR mouse (5-week-old, male, about 300, Charles River, Japan), which had been fasted overnight, and 10 mice were determined by measuring the blood calcium concentration. Into groups. Blood calcium concentration was measured using a Type 634 automatic Ca ²⁺ / PH analyzer. 3 hours after blood collection from the fundus, the mice (10 mice per group)
Each of the human anti-human PTHrP monoclonal antibodies (2F8-1
0-3, 1B3-9-16, 15H7-8-3, 5B12-16-12 or 4B4-6-2
One of one. Concentration: 3, 10 or 30 mg / kg), or a mouse anti-human PTHrP monoclonal antibody 1D5 (concentration: 1 or 3 mg / kg) as a positive control was administered intravenously.
Immediately thereafter, the sequence of the N-terminal 1-34 of human PTHrP (PTHrP
(1-34), amino acids 1 to 34 of SEQ ID NO: 1, Peptide Institute (manufactured by) (0.1 μg / body) were subcutaneously administered. The P
One hour after the administration of THrP, blood was collected from the fundus of each mouse, and the calcium concentration in the blood was measured in the same manner as described above. The following control experiment was performed in the same manner as described above. (1) A case in which a phosphate buffer solution is intravenously administered 3 hours after blood collection from the fundus, and a physiological saline solution is subcutaneously administered immediately thereafter. (2) A case in which a phosphate buffer solution is intravenously administered 3 hours after blood collection from the fundus, and the PTHrP (0.1 μg / body) is administered subcutaneously immediately thereafter. The results are shown in FIGS. This test showed that any of the human anti-human PTHrP monoclonal antibodies of the present invention had significant inhibitory and therapeutic effects on PTHrP-induced hypercalcemia.

Example 13 Therapeutic Effect of Paraneoplastic Malignant Hypercalcemia with Anti-human PTHrP Human Monoclonal Antibody Paraneoplastic Syndrome (para) frequently observed in cancer patients
A typical example of neoplastic syndrome is malignancy-associated hypercarcemia (MAH). Many of the MAHs are progressive and severe. MAH is a malignant humoral hypercalcemia (humo) caused by the systemic effects of humoral factors produced by tumors.
ral hypercarcemia of malignancy (HHM)) and local osteolytic hypercalcemia (local osteolytic) caused by increased bone resorption (bone destruction, osteolysis) due to direct tumor invasion into bone (bone metastasis) hypercarcemia
(LOH)). LOH is mainly based on extensive bone metastasis of cancer and is thought to be secondary to bone lesions, but PTHrP produced by tumors has local bone destruction (osteolysis)
In some cases. HHM, on the other hand, accounts for nearly 90% of hypercalcemia associated with cancer (N. Engl. J. Me.
d., Vol. 300, p. 1377, 1980), and its main causative agent is being clarified by the action of PTHrP secreted by tumors (Am. J. Clin. Pathol., Vol. 105, p. .487, 19
96). HHM due to PTHrP production is found in a variety of cancers across all tissues, especially squamous cell carcinomas (lung, esophagus,
Cervix, vulva, skin, head, neck), kidney cancer, bladder cancer,
There are many reports on ovarian cancer and adult T-cell leukemia (ATL) (N. Engl. J. Med., Vol. 322, p. 1106, 1990; J. Cli).
n. Endocrinol. Metab., Vol. 73, 1309, 1991).

In this test, the various human anti-human
The therapeutic effect of PTHrP monoclonal antibody on paraneoplastic malignant hypercalcemia (MAH) was examined using MAH model mice artificially created by transplanting tumor cells into living bodies. BALB / C-nu mice (4 weeks old, male, 5 to 7 mice in each group, manufactured by CLEA Japan) were each treated with a human oral squamous cell carcinoma-derived cell line HOSO (Hasina Rifat et al., 54th Annual Meeting of the Japanese Cancer Society) P.349, 1995, and Hasina Rifat et al., 55th Annual Meeting of the Cancer Society of Japan, p.184, 1996) at about 6 × 10 ⁶ cells /
It was implanted subcutaneously with the culture solution at the concentration of the head. About 4 to 5 months after transplanting the cells, blood was collected from the fundus of each mouse, and the calcium concentration in the blood was measured using a type 634 automatic Ca ²⁺ / PH analyzer. Blood calcium level is 1.
Mice (7 mice) with an increase of 3 mmol / l or more were selected, and to each mouse, an anti-human PTHrP human monoclonal antibody 4B4-6-21 (3 mg / kg) dissolved in a phosphate buffer was intravenously administered. . At 1, 3 and 6 days after the antibody administration, blood was collected from the fundus of each mouse, and the blood calcium concentration was measured.

The following control experiment was performed in the same manner as described above. (1) In the above-described test, mice which were subcutaneously transplanted with the culture solution only (not containing the cancer cell line) on the same day as the subcutaneous transplantation of the cancer cell line and subcutaneously transplanted for about 4 to 5 months, were given only phosphate buffer solution ( (An anti-human PTHrP human monoclonal antibody 4B4-6-21 is not included) in the tail vein. (2) The same cancer cell line HOSO (same concentration) as in the above test
About 4 to 5 months after subcutaneous transplantation of blood
The mice (6 mice) increased to 1.3 mmol / l or more were treated with phosphate buffer alone (anti-human PTHrP human monoclonal antibody 4B4-6-2).
1) is administered in the tail vein. The results are shown in FIG. This test showed that the human anti-human PTHrP monoclonal antibody of the present invention had a significant inhibitory and therapeutic effect on paraneoplastic malignant hypercalcemia.

[0111]

The present invention provides, for the first time in the world, a human monoclonal antibody against human PTHrP that has not yet been provided to society. Human anti-human of the present invention
The human monoclonal antibody disclosed as one embodiment of the PTHrP monoclonal antibody has various properties (antigen specificity,
Antigen affinity, neutralizing activity, etc.) and biological activity (PTHrP-dependent inhibitory activity on intracellular cAMP elevation, PTHrP-dependent inhibitory activity on calcium (Ca) release from bone, inhibitory activity on bone resorption, inhibitory activity on osteolysis, etc. ). The human monoclonal antibody of the present invention has no antigenicity to humans, and has been a major problem in the treatment of antibody drugs consisting of antibodies derived from non-human mammals such as conventional mouse-derived antibodies HAMA, HACA Alternatively, since no side effect is caused by HAHA, the value of the antibody as a drug is dramatically increased.

That is, the monoclonal antibody of the present invention has an activity of functionally controlling the biological activity of human PTHrP, and does not cause side effects such as allergy and rejection which are major problems of conventional antibody drugs. Therefore, the human monoclonal antibody against human PTHrP of the present invention or a pharmaceutical composition thereof is extremely useful for the treatment or prevention of the following various diseases or conditions possibly caused by PTHrP. The disease or symptom includes a disease caused by release of calcium from bone in a parathyroid hormone-related protein-dependent manner (such as hypercalcemia), a malignant tumor (kidney cancer, lung cancer, gastric cancer, breast cancer, pharyngeal cancer, esophageal cancer) , Tongue cancer, prostate cancer, bladder cancer, malignant lymphoma, skin cancer, thyroid cancer, testicular cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, urothelial cancer, etc.) Osteoarthritis, bone metastasis of cancer (squamous cell carcinoma cells, adenocarcinoma cells, melanoma cells, osteosarcoma cells, neuroblastoma cells, blood cancer cells, etc.), osteolysis, bone destruction, cancer present in bone tissue Diseases caused by cell proliferation, local production of parathyroid hormone-related protein, symptoms caused by primary local cancer (such as pain, nerve compression, hypercalcemia, fractures and cachexia),
Diseases in the teeth, periodontal or gingiva (eg, alveolar purulence, gingivitis, periodontal disease, etc.), sepsis (sepsis), systemic inflammatory response syndrome (SIRS), and hypophosphatemia (hypophosphatemic rickets,
Hypophosphatemic vitamin D-resistant rickets). The anti-human PTHrP human monoclonal antibodies of the present invention are also useful in prolonging the life of patients suffering from primary local cancer.

Further, the human monoclonal antibody reactive with human PTHrP of the present invention and its pharmaceutical composition are used as therapeutic agents for hypercalcemia and diseases related to bone, tooth and calcium metabolism disorders. Bisphosphonate (bisphosphonate) -based compounds (eg, pamidronate, etidronate, risedronate, pyrophosphate, clodronate, tiludronate, alendronate) having a strong bone resorption inhibiting action Salt, BM21.095
5, YM-175, CGP42446, etc.) or a hydrate thereof is also useful in the prevention and treatment of a combination of cancer-related symptoms and hypercalcemia.

[0114]

[Sequence List] SEQUENCE LISTING <110> Japan Tobacco, Inc. <120> Human Monoclonal Antibody For Parathyroid Hormone-Related Protein <130> J98-0142 <140> <141> <150> JP P1998-188196 <151> 1998-06-17 <150> JP P1998-196729 <151> 1998-06-26 <160> 45 <170> PatentIn Ver. 2.0 <210> 1 <211> 209 <212> PRT <213> Homo sapiens <220> <221> PROPEP <222> (1) .. (36) <400> 1 Met Gln Arg Arg Leu Val Gln Gln Trp Ser Val Ala Val Phe Leu Leu 1 5 10 15 Ser Tyr Ala Val Pro Ser Cys Gly Arg Ser Val Glu Gly Leu Ser Arg 20 25 30 Arg Leu Lys Arg Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly 35 40 45 Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile 50 55 60 Ala Glu Ile His Thr Ala Glu Ile Arg Ala Glu Ser Glu Val Ser Pro 65 70 75 80 Asn Ser Lys Pro Ser Pro Asn Thr Lys Asn His Pro Val Arg Phe Gly 85 90 95 Ser Asp Asp Glu Gly Arg Tyr Leu Thr Gln Glu Thr Asn Lys Val Glu 100 105 110 Thr Tyr Lys Glu Gln Pro Leu Lys Thr Pro Gly Lys Lys Lys Lys Ala 115 120 125 Lys Pro Gly Lys Arg Lys Glu Gln Glu Lys Lys Lys Arg Arg Thr Arg 130 135 140 Ser Ala Trp Leu Asp Ser Gly Val Thr Gly Ser Gly Leu Glu Gly Asp 145 150 155 160 His Leu Ser Asp Thr Ser Thr Thr Ser Leu Glu Leu Asp Ser Arg Thr 165 170 175 Ala Leu Leu Trp Gly Leu Lys Lys Lys Lys Glu Asn Asn Arg Arg Thr 180 185 190 His His Met Gln Leu Met Ile Ser Leu Phe Lys Ser Pro Leu Leu Leu 195 200 205 Leu <210> 2 <211> 34 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1) .. (34) <400> 2 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe <210> 3 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (467) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (492) <223> This base has not yet been determined.The codon containing this base encodes Phe or Leu. <400> 3 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg act cag tnt cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Xaa Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc att tcc tgc agg ttt agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Phe Ser Gln Ser 35 40 45 ctc ctg cat agt aat gga aac aac tat ttg gat tgg tac ctg cag aag 192 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ttc ctg atc tat ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Phe Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct cta caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tnt gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Xaa Val Val Cys Leu 145 150 155 160 ctg aat aac ttn tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Xaa Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtt tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 4 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (156) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (164) <223> This amino acid has not yet been determined and is possibly Phe or Leu. <400> 4 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Xaa Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Phe Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Phe Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Xaa Val Val Cys Leu 145 150 155 160 Leu Asn Asn Xaa Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Gl u Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 5 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <400> 5 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg act cag tct cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc acc atc tcc tgc agg tcc agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Thr Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg cat aga aat gga aac aac tat ttg gat tgg ttc ttg cag aag 192 Leu Leu His Arg Asn Gly Asn Asn Tyr Leu Asp Trp Phe Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ctc ctg atc tat ttg ggc tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggc gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa ctc agc aga gtg gag gct gag gat gtt ggg ctt tat tac 336 Thr Leu Lys Leu Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr 100 105 110 tgc atg caa gct cta caa att cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Leu Gln Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 6 <211> 239 <212> PRT <213> Homo sapiens <400> 6 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Thr Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Arg Asn Gly Asn Asn Tyr Leu Asp Trp Phe Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Leu Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr 100 105 110 Cys Met Gln Ala Leu Gln Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Gl u Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 7 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (74) <223> This base has not yet been determined.The codon containing this base encodes either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (252) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Pro. <400> 7 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg ant cag tnt cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg cat agt aat gga aac aat tat ttg gat tgg tat ctg cag aag 192 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cta cag ctc ctg atc tat ttg ggc tct aat cgg gcc 240 Pro Gly Gln Ser Leu Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc ccn gac agg ttc agt ggc agt gga tcagg gat ttt 288 Ser Gly Val Xaa Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gtt gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Val Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct tta caa act cca ttc act ttc ggc cct gag acc aaa 384 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Glu Thr Lys 115 120 125 gtg gat ttc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Phe Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtt tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln210 215 220 ggc ctg agt tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 8 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (25) <223> This amino acid has not yet been determined and is possibly either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (84) <223> This amino acid has not yet been determined and is possibly Pro. <400> 8 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Leu Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Xaa Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Val Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Glu Thr Lys 115 120 125 Val Asp Phe Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr Gl u Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 9 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (230) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (252) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Pro. <220> <221> unsure <222> (349) <223> This base has not yet been determined.The codon containing this base either one of Leu, Ile or Val. <400> 9 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg act cag tct cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg cat agt aat gga aac aat tat ttg gat tgg tat ctg cag aag 192 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cta cag ctc ctg atc tat ttg ggc tnt aat cgg gcc 240 Pro Gly Gln Ser Leu Gln Leu Leu Ile Tyr Leu Gly Xaa Asn Arg Ala 65 70 75 80 tcc ggg gtc ccn gac agg ttc agt ggc agt gga tca gat ttt 288 Ser Gly Val Xaa Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gtt gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Val Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct nta caa act cca ttc act ttc ggc cct gag acc aaa 384 Cys Met Gln Ala Xaa Gln Thr Pro Phe Thr Phe Gly Pro Glu Thr Lys 115 120 125 gtg gat ttc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Phe Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 10 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (77) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (84) <223> This amino acid has not yet been determined and is possibly Pro. <220> <221> unsure <222> (117) <223> This amino acid has not yet been determined and is possibly either one of Leu, Ile or Val. <400> 10 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Leu Gln Leu Leu Ile Tyr Leu Gly Xaa Asn Arg Ala 65 70 75 80 Ser Gly Val Xaa Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Val Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Xaa Gln Thr Pro Phe Thr Phe Gly Pro Glu Thr Lys 115 120 125 Val Asp Phe Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr G lu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 11 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (74) <223> This base has not yet been determined.The codon containing this base encodes either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (349) <223> This base has not yet been determined.The codon containing this base encodes either one of Leu, Ile or Val. <220> <221> unsure <222> (437) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (648) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Val. <400> 11 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg ant cag tnt cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 gtc act cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg cat agt aat gga aac aac tat ttg gat tgg tac ctg cag aag 192 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ttc ctg atc tat ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Phe Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct nta caa att cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Xaa Gln Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tnt gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Xaa Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtn tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Xaa Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 12 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (25) <223> This amino acid has not yet been determined and is possibly either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (117) <223> This amino acid has not yet been determined and is possibly either one of Leu, Ile or Val. <220> <221> unsure <222> (146) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (216) <223> This amino acid has not yet been determined and is possibly Val. <400> 12 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Phe Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Xaa Gln Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Xaa Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr G lu Lys His Lys Xaa Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 13 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (465) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Ala. <400> 13 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 ggt tcc agt ggg gat att gtg atg act cag tcc cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg cat agt aat ggg aat aac tat ttg gat tgg tac ctg cag aag 192 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ctc ctg atc tat ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggcca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc agg gtg gag gct gag gat gtg ggg att tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr 100 105 110 tgc atg caa gct cta caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcn tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Xaa Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 14 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (155) <223> This amino acid has not yet been determined and is possibly Ala. <400> 14 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr 100 105 110 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Xaa Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr G lu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 15 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (14) <223> This base has not yet been determined.The codon containing this base encodes either one of Val, Ala, Asp or Gly. <220> <221> unsure <222> (39) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Leu. <220> <221> unsure <222> (74) <223> This base has not yet been determined.The codon containing this base encodes either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (648) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Val. <400> 15 atg agg ctc cct gnt cag ctc ctg ggg ctg cta atg ctn tgg gtc tct 48 Met Arg Leu Pro Xaa Gln Leu Leu Gly Leu Leu Met Xaa Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg ant cag tnt cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctt cat agt aat gga tac aac tat ttg gat tgg ttc ctg cag aag 192 Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Phe Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ctc ctg atc tat ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggcca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct cta caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtn tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Xaa Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 16 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (5) <223> This amino acid has not yet been determined and is possibly either one of Val, Ala, Asp or Gly. <220> <221> unsure <222> (13) <223> This amino acid has not yet been determined and is possibly Leu. <220> <221> unsure <222> (25) <223> This amino acid has not yet been determined and is possibly either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (216) <223> This amino acid has not yet been determined and is possibly Val. <400> 16 Met Arg Leu Pro Xaa Gln Leu Leu Gly Leu Leu Met Xaa Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Phe Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr G lu Lys His Lys Xaa Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 17 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> V_region <222> (398) .. (717) <220> <221> unsure <222> (7) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Leu, Ile or Val. <220> <221> unsure <222> (11) <223> This base has not yet been determined.The codon containing this base encodes either one of Leu, Pro, His or Arg. <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (349) <223> This base has not yet been determined.The codon containing this base encodes either one of Leu, Ile or Val. <400> 17 atg agg ntc cnt gct cag ctc ctg ggg ctg cta atg ttc tgg gtc tct 48 Met Arg Xaa Xaa Ala Gln Leu Leu Gly Leu Leu Met Phe Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg act cag tnt cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Xaa Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc att tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg cat agt aat gga aac aac tat ttg gat tgg tac ctg cag aag 192 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ttc ctg atc tat ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Phe Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct nta caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Xaa Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 18 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (3) <223> This amino acid has not yet been determined and is possibly either of Phe, Leu, Ile or Val. <220> <221> unsure <222> (4) <223> This amino acid has not yet been determined and is possibly either one of Leu, Pro, His or Arg. <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (117) <223> This amino acid has not yet been determined and is possibly either one of Leu, Ile or Val. <400> 18 Met Arg Xaa Xaa Ala Gln Leu Leu Gly Leu Leu Met Phe Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Thr Gln Xaa Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu His Ser Asn Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Phe Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Xaa Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr G lu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 19 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (74) <223> This base has not yet been determined.The codon containing this base encodes either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (637) <223> This base has not yet been determined.The codon containing this base encodes either one of Gln, Lys or Glu. <220> <221> unsure <222> (643) <223> This base has not yet been determined.The codon containing this base encodes either one of Gln, Lys or Glu. <220> <221> unsure <222> (657) <223> This base has not yet been determined.The codon cintaining this base encodes Cys or Trp. <220> <221> unsure <222> (659) <223> This base has not yet been determined.The codon containing this base either one of Val, Ala, Glu or Gly. <220> <221> unsure <222> (665) <223> This base has not yet been determined.The codon containing this base encodes either one of Ile, Thr, Asn or Ser. <400> 19 atg agg ctc cct gct cag ctc ctg ggg ctg cta atg ctc tgg gtc tct 48 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 gga tcc agt ggg gat att gtg atg ant cag tnt cca ctc tcc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc atc tcc tgc agg tct agt cag agc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 ctc ctg aat agt aat gga tac aac tat ttc gat tgg tac ctg cag aag 192 Leu Leu Asn Ser Asn Gly Tyr Asn Tyr Phe Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tct cca cag ctc ctg atc tat ttg ggt tct aat cgg gcc 240 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggcca gat ttt 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa act cta caa act cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Thr Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 aac aag gac agc acc tac agc ctc agc agc acc ctg acg gtg agc aaa Asn Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Val Ser Lys 195 200 205 gca gaa tac gag naa cac naa gtt tac gcc tgn gna gtc anc cat cag 672 Ala Glu Tyr Glu Xaa His Xaa Val Tyr Ala Xaa Xaa Val Xaa His Gln 210 215 220 ggc ctg agc tcg ccc gtc aca aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 20 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (25) <223> This amino acid has not yet been determined and is possibly either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (213) <223> This amino acid has not yet been determined and is possibly either one of Gln, Lys or Glu. <220> <221> unsure <222> (215) <223> This amino acid has not yet been determined and is possibly either one of Gln, Lys or Glu. <220> <221> unsure <222> (219) <223> This amino acid has not yet been determined and is possibly Cys or Trp. <220> <221> unsure <222> (220) <223> This amino acid has not yet been determined and is possibly either one of Val, Ala, Glu or Gly. <220> <221> unsure <222> (222) <223> This amino acid has not yet been determined and is possibly either one of Ile, Thr, Asn or Ser. <400> 20 Met Arg Leu Pro Ala Gln Leu Leu Gly Leu Leu Met Leu Trp Val Ser 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Leu Ser Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 35 40 45 Leu Leu Asn Ser Asn Gly Tyr Asn Tyr Phe Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Thr Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Asn Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Val Ser Lys 195 200 205 Ala Glu Tyr G lu Xaa His Xaa Val Tyr Ala Xaa Xaa Val Xaa His Gln 210 215 220 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 21 <211> 720 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (720) <220> <221> sig_peptide <222> (1) .. (60) <220> <221> V_region <222> (61) .. (397) <220> <221> C_region <222> (398) .. (717) <220> <221> unsure <222> (1) .. (2) <223> These bases have not yet been determined.The codon containing these bases highly possibly encodes Met. <220> <221> unsure <222> (7) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Leu, Ile or Val. <220> <221> unsure <222> (11) .. (12) <223> These bases have not yet been determined.The codon containing these bases encodes either one of Leu, Pro, His, Gln or Arg. <220> <221> unsure <222> (14) <223> This base has not yet been determined.The codon containing this base encodes either one of Val, Ala, Asp or Gly. <220> <221> unsure <222> (31) <223> This base has not yet been determined.The codon containing this base encodes either Leu, Ile or Val. <220> <221> unsure <222> (47) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (74) <223> This base has not yet been determined.The codon containing this base encodes either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (80) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (87) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Leu. <220> <221> unsure <222> (88) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Leu, Ile or Val. <220> <221> unsure <222> (349) <223> This base has not yet been determined.The codon containing this base encodes either one of Leu, Ile or Val. <220> <221> unsure <222> (648) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Val. <220> <221> unsure <222> (693) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Thr. <400> 21 nng agg ntc cnn gnt cag ctc ctg ggg ctg nta atg ctc tgg gtc tnt 48 Xaa Arg Xaa Xaa Xaa Gln Leu Leu Gly Leu Xaa Met Leu Trp Val Xaa 1 5 10 15 gga tcc agt ggg gat att gtg atg ant cag tnt cca ctn ntc ctg ccc 96 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Xaa Xaa Leu Pro 20 25 30 gtc acc cct gga gag ccg gcc tcc atc tcc tgt agg tct ggt cag cgc 144 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Gly Gln Arg 35 40 45 ctc ctg cat aga aat gga aac acc tat ttg gat tgg tac ctg cag aag 192 Leu Leu His Arg Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 cca ggg cag tcc cca cag ctc ctg atc tat ttg ggt tct gat cgg gcc 240 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asp Arg Ala 65 70 75 80 tcc ggg gtc cct gac agg ttc agt ggc agt gga tca ggcca gat ttc 288 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 aca ctg aaa atc agc aga gtg gag gct gag gat gtt ggg gtt tat tac 336 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 tgc atg caa gct nta caa att cca ttc act ttc ggc cct ggg acc aaa 384 Cys Met Gln Ala Xaa Gln Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 gtg gat atc aaa cga act gtg gct gca cca tct gtc ttc atc ttc ccg 432 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 cca tct gat gag cag ttg aaa tct gga act gcc tct gtt gtg tgc ctg 480 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg aag gtg gat 528 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 aac gcc ctc caa tcg ggt aac tcc cag gag agt gtc aca gag cag gac 576 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 agc aag gac agc acc tac agc ctc agc agc acc ctg acg ctg agc aaa 624 Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 gca gac tac gag aaa cac aaa gtn tac gcc tgc gaa gtc acc cat cag 672 Ala Asp Tyr Glu Lys His Lys Xaa Tyr Ala Cys Glu Val Thr His Gln 210 215 220 ggc ctg agc tcg ccc gtc acn aag agc ttc aac agg gga gag tgt tag 720 Gly Leu Ser Ser Pro Val Xaa Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240 <210> 22 <211> 239 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (1) <223> This amino acid has not yet been determined and is possibly Met. <220> <221> unsure <222> (3) <223> This amino acid has not yet been determined and is possibly either of Phe, Leu, Ile or Val. <220> <221> unsure <222> (4) <223> This amino acid has not yet been determined and is possibly either one of Leu, Pro, His, Gln or Arg. <220> <221> unsure <222> (5) <223> This amino acid has not yet been determined and is possibly either one of Val, Ala, Asp or Gly. <220> <221> unsure <222> (11) <223> This amino acid has not yet been determined and is possibly either one of Leu, Ile or Val. <220> <221> unsure <222> (16) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (25) <223> This amino acid has not yet been determined and is possibly either one of Ile, Thr, Asn or Ser. <220> <221> unsure <222> (27) <223> This amino acid has not yet been determined and is possibly either one of Phe, Ser, Tyr or Cys. <220> <221> unsure <222> (29) <223> This amino acid has not yet been determined and is possibly Leu. <220> <221> unsure <222> (30) <223> This amino acid has not yet been determined and is possibly either of Phe, Leu, Ile or Val. <220> <221> unsure <222> (117) <223> This amino acid has not yet been determined and is possibly either one of Leu, Ile or Val. <220> <221> unsure <222> (216) <223> This amino acid has not yet been determined and is possibly Val. <220> <221> unsure <222> (231) <223> This amino acid has not yet been determined and is possibly Thr. <400> 22 Xaa Arg Xaa Xaa Xaa Gln Leu Leu Gly Leu Xaa Met Leu Trp Val Xaa 1 5 10 15 Gly Ser Ser Gly Asp Ile Val Met Xaa Gln Xaa Pro Xaa Xaa Leu Pro 20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Gly Gln Arg 35 40 45 Leu Leu His Arg Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys 50 55 60 Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asp Arg Ala 65 70 75 80 Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 85 90 95 Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr 100 105 110 Cys Met Gln Ala Xaa Gln Ile Pro Phe Thr Phe Gly Pro Gly Thr Lys 115 120 125 Val Asp Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 130 135 140 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 145 150 155 160 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 165 170 175 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 180 185 190 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 195 200 205 Ala Asp Tyr G lu Lys His Lys Xaa Tyr Ala Cys Glu Val Thr His Gln 210 215 220 Gly Leu Ser Ser Pro Val Xaa Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 23 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 23 atg gaa ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg ggg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt acc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg ttt gat gga agt cat aaa tac tat gca 240 Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser His Lys Tyr Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct ata 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 100 105 110 tat tac tgt gcg aga cac agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 24 <211> 143 <212> PRT <213> Homo sapiens <400> 24 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser His Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 25 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 25 atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt agc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gaa tgg gtg gca gtt ata tgg ttt gat gga agt aag aaa tac tat gca 240 Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser Lys Lys Tyr Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tca aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tac tgt gcg aga cac agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 26 <211> 143 <212> PRT <213> Homo sapiens <400> 26 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser Lys Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 27 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <220> <221> unsure <222> (1) .. (2) <223> These bases have not yet been determined.The codon containing these bases highly possibly encodes Met. <220> <221> unsure <222> (7) .. (9) <223> These bases have not yet been determined. <220> <221> unsure <222> (14) <223> This base has not yet been determined.The codon containing this base encodes either one of Met, Thr, Lys or Arg. <220> <221> unsure <222> (18) <223> This base has not yet been determined.The codon containing this base encodes Ser or Arg. <220> <221> unsure <222> (28) <223> This base has not yet been determined.The codon containing this base encodes either Phe, Leu, Ile or Val. <220> <221> unsure <222> (32) <223> This base has not yet been determined.The codon containing this base encodes either one of Val, Ala, Asp or Gly. <400> 27 nng gag nnn ggg ang agn tgg gtc ttc ntc gnt gct cta aga aga ggt 48 Xaa Glu Xaa Gly Xaa Xaa Trp Val Phe Xaa Xaa Ala Leu Arg Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt acc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg tat gat gga agt aat caa tac tat gca 240 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac aga ctg aga gcc gag gac acg gct gtg 336 Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tac tgt gcg aga cat agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 28 <211> 143 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (1) <223> This amino acid has not yet been determined and is possibly Met. <220> <221> unsure <222> (3) <223> This amino acid has not yet been determined. <220> <221> unsure <222> (5) <223> This amino acid has not yet been determined and is possibly either one of Met, Thr, Lys or Arg. <220> <221> unsure <222> (6) <223> This amino acid has not yet been determined and is possibly Ser or Arg. <220> <221> unsure <222> (10) <223> This amino acid has not yet been determined and is possibly either of Phe, Leu, Ile or Val. <220> <221> unsure <222> (11) <223> This amino acid has not yet been determined and is possibly either one of Val, Asp, Arg or Gly. <400> 28 Xaa Glu Xaa Gly Xaa Xaa Trp Val Phe Xaa Xaa Ala Leu Arg Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 29 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <220> <221> unsure <222> (7) .. (9) <223> These bases have not yet been determined. <220> <221> unsure <222> (22) <223> This base has not yet been determined.The codon containing this base encodes either one of Phe, Leu, Ile or Val. <220> <221> unsure <222> (32) <223> This base has not yet been determined.The codon containing this base encodes either one of Val, Ala, Asp or Gly. <400> 29 atg gag nnn ggg agg agc tgg ntc ttc gtc gnt gct cta aga aga ggt 48 Met Glu Xaa Gly Arg Ser Trp Xaa Phe Val Xaa Ala Leu Arg Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt acc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg tat gat gga agt aat caa tac tat gca 240 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac aga ctg aga gcc gag gac acg gct gtg 336 Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tac tgt gcg aga cat agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 30 <211> 143 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (3) <223> This amino acid has not yet been determined. <220> <221> unsure <222> (8) <223> This amino acid has not yet been determined and is possibly either of Phe, Leu, Ile or Val. <220> <221> unsure <222> (11) <223> This amino acid has not yet been determined and is possibly either one of Val, Ala, Asp or Gly. <400> 30 Met Glu Xaa Gly Arg Ser Trp Xaa Phe Val Xaa Ala Leu Arg Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Gln Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 31 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 31 atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ttg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca acg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 agt gac tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg tat gat gga agc cat aaa ttc tat gca 240 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser His Lys Phe Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tat tgt gcg aga cac agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc cca ggg acc acg gtc acc gtc tca 429 Gly Met Asp Val Trp Gly Pro Gly Thr Thr Val Val Thr Val Ser Ser 130 135 140 <210> 32 <211> 143 <212> PRT <213> Homo sapiens <400> 32 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe 35 40 45 Ser Asp Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser His Lys Phe Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Pro Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 33 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 33 atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt acc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg tat gat gga agt tat aaa tac tat gca 240 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Tyr Lys Tyr Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tac tgt gcg aga cac agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 34 <211> 143 <212> PRT <213> Homo sapiens <400> 34 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Tyr Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 35 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 35 atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt agc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg tat gat gga agt tat aaa atc tat gca 240 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Tyr Lys Ile Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg aat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg 336 Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tat tgt gcg aga cac agc agt ggc tgg tat gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc accgt tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 36 <211> 143 <212> PRT <213> Homo sapiens <400> 36 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Tyr Lys Ile Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Asn Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 37 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 37 atg gaa ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gtg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg ggg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt acc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg ttt gat gga agt cat aaa tac tat gca 240 Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser His Lys Tyr Tyr Ala 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct ata 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 100 105 110 tat tac tgt gcg aga cac agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 38 <211> 143 <212> PRT <213> Homo sapiens <400> 38 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Thr Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Phe Asp Gly Ser His Lys Tyr Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 39 <211> 417 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (417) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (417) <220> <221> N_region <222> (348) .. (349) <220> <221> N_region <222> (357) <220> <221> unsure <222> (231) <223> This base has not yet been determined.The codon containing this base highly possibly encodes Tyr. <400> 39 atg aaa cat ctg tgg ttc ttc ctt ctc ctg gtg gca gct ccc aga tgg 48 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 gtc ctg tcc cag gtg cag ctg cagag tcg ggc cca gga ctg gtg aag 96 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 cct tcg gag acc ctg tcc ctc acc tgc act gtc tct ggt ggc tcc atc 144 Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 agt agt tac tac tgg agc tgg atc cgg cag ccc cca ggg aag gga ctg 192 Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 gag tgg att ggg tat atc tat tac agt ggg agc acc aan tac aac ccc 240 Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Xaa Tyr Asn Pro 65 70 75 80 tcc ctc aag agt cga gtc acc ata tca gtg gac acg tcc aag aac cag 288 Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 ttc tcc ctg aag ctg agc tct gtg acc gct gcg gac acg gcc gtt tat 336 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 tac tgt gcg agc gca gta acc tac tac tac tac tac ggt ttg gac gtc 384 Tyr Cys Ala Ser Ala Val Thr Tyr Tyr Tyr Tyr Tyr Gly Leu Asp Val 115 120 125 tgg ggc caa ggg acc acg gtc acc gtc tcc tca 417 Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 <210> 40 <211> 139 <212> PRT <213> Homo sapiens <220> <221> unsure <222> (77) <223> This amino acid has not yet been determined and is possibly Tyr. <400> 40 Met Lys His Leu Trp Phe Phe Leu Leu Leu Val Ala Ala Pro Arg Trp 1 5 10 15 Val Leu Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys 20 25 30 Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile 35 40 45 Ser Ser Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu 50 55 60 Glu Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Xaa Tyr Asn Pro 65 70 75 80 Ser Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln 85 90 95 Phe Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr 100 105 110 Tyr Cys Ala Ser Ala Val Thr Tyr Tyr Tyr Tyr Tyr Gly Leu Asp Val 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 <210> 41 <211> 429 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (429) <220> <221> sig_peptide <222> (1) .. (57) <220> <221> V_region <222> (58) .. (429) <220> <221> N_region <222> (352) .. (354) <220> <221> N_region <222> (370) .. (373) <400> 41 atg gag ttt ggg ctg agc tgg gtt ttc ctc gtt gct ctt tta aga ggt 48 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 gtc cag tgt cag gtg cag ctg gg gag tct ggg gga ggc gtg gtc cag 96 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 cct ggg agg tcc ctg aga ctc tcc tgt gca gcg tct gga ttc acc ttc 144 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 agt agc tat ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg ctg 192 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 gag tgg gtg gca gtt ata tgg tat gat gga agt aat aaa tac tat gta 240 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val 65 70 75 80 gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac 288 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 acg ctg tat ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg 336 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 tat tac tgt gcg aga cac agc agt ggc tgg tac gag gac tac tac tac 384 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tca 429 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Thr Val Ser Ser 130 135 140 <210> 42 <211> 143 <212> PRT <213> Homo sapiens <400> 42 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Val 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg His Ser Ser Gly Trp Tyr Glu Asp Tyr Tyr Tyr 115 120 125 Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Val Ser Ser 130 135 140 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized adaptor sequence <220> <221> misc_difference <222> (1) .. (27) <400> 43 ccatcctaat acgactcact atagggc 27 <210> 44 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized primer sequence <220> <221> primer_bind <222> (1) .. (25) <400> 44 ccagggccgc tgtgctctcg gaggt 25 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Artificially synthesized primer sequence <220> <221> primer_bind <222> (1) .. (23) <400> 45 gggggtcagg ctggaactga gga 23

"Sequence List Free Text" SEQ ID NO: 3 Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (467) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (492) Other Information: This base has not yet been determined. Codons containing this base encode Phe or Leu. SEQ ID NO: 4 Location: (27) Other information: This amino acid has not been determined yet. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (156) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (164) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Phe or Leu. SEQ ID NO: 7 Location: (74) Other information: This base has not been determined yet. Codons containing this base encode any of Ile, Thr, Asn or Ser. Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (252) Other information: This base has not yet been determined. Codons containing this base are likely to encode Pro. SEQ ID NO: 8 Location: (25) Other information: This amino acid has not been determined yet. Also,
The amino acid can be any of Ile, Thr, Asn or Ser. Location: (27) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (84) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Pro. SEQ ID NO: 9 Location: (230) Other information: This base has not been determined yet. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (252) Other information: This base has not yet been determined. Codons containing this base are likely to encode Pro. Location: (349) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Ile or Val. SEQ ID NO: 10 Location: (77) Other information: This amino acid has not been determined yet. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (84) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Pro. Location: (117) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Leu, Ile or Val. SEQ ID NO: 11 Location: (74) Other information: This base has not been determined yet. Codons containing this base encode any of Ile, Thr, Asn or Ser. Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (349) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Ile or Val. Location: (437) Other Information: This base has not been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (648) Other information: This base has not yet been determined. Codons containing this base are likely to encode Val. SEQ ID NO: 12 Location: (25) Other information: This amino acid has not been determined yet. Also,
The amino acid can be any of Ile, Thr, Asn or Ser. Location: (27) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (117) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Leu, Ile or Val. Location: (146) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (216) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Val. SEQ ID NO: 13 Location: (465) Other information: This base has not been determined yet. Codons containing this base are likely to encode Ala. SEQ ID NO: 14 Location: (155) Other information: This amino acid has not been determined yet. Also,
The amino acid can be Ala. SEQ ID NO: 15 Location: (14) Other information: This base has not been determined yet. The codon containing this base encodes any of Val, Ala, Asp or Gly. Location: (39) Other information: This base has not yet been determined. Codons containing this base are likely to encode Leu. Location: (74) Other information: This base has not yet been determined. Codons containing this base encode any of Ile, Thr, Asn or Ser. Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (648) Other information: This base has not yet been determined. Codons containing this base are likely to encode Val. SEQ ID NO: 16 Location: (5) Other information: This amino acid has not been determined yet. Also,
The amino acid can be any of Val, Ala, Asp or Gly. Location: (13) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Leu. Location: (25) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Ile, Thr, Asn or Ser. Location: (27) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Phe, Ser, Tyr or Cys. Location: (216) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Val. SEQ ID NO: 17 Location: (7) Other information: This base has not been determined yet. The codon containing this base encodes any of Phe, Leu, Ile or Val. Location: (11) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Pro, His or Arg. Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (349) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Ile or Val. SEQ ID NO: 18 Location: (3) Other information: This amino acid has not been determined yet. Also,
The amino acid can be any of Phe, Leu, Ile or Val. Location: (4) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Leu, Pro, His or Arg. Location: (27) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (117) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Leu, Ile or Val. SEQ ID NO: 19 Location: (74) Other information: This base has not been determined yet. Codons containing this base encode any of Ile, Thr, Asn or Ser. Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (637) Other information: This base has not yet been determined. The codon containing this base encodes any of Gln, Lys or Glu. Location: (643) Other Information: This base has not yet been determined. The codon containing this base encodes any of Gln, Lys or Glu. Location: (657) Other information: This base has not yet been determined. Codons containing this base encode Cys or Trp. Location: (659) Other Information: This base has not yet been determined. The codon containing this base encodes any of Val, Ala, Glu or Gly. Location: (665) Other information: This base has not yet been determined. Codons containing this base encode any of Ile, Thr, Asn or Ser. SEQ ID NO: 20 Location: (25) Other information: This amino acid has not been identified yet. Also,
The amino acid can be any of Ile, Thr, Asn or Ser. Location: (27) Other information: This amino acid has not yet been identified. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (213) Other information: This amino acid has not yet been identified. Also,
The amino acid can be any of Gln, Lys or Glu. Location: (215) Other information: This amino acid has not yet been identified. Also,
The amino acid can be any of Gln, Lys or Glu. Location: (219) Other information: This amino acid has not yet been identified. Also,
The amino acid can be Cys or Trp. Location: (220) Other information: This amino acid has not yet been identified. Also,
The amino acid can be any of Val, Ala, Glu or Gly. Location: (222) Other information: This amino acid has not yet been identified. Also,
The amino acid can be Ile, Thr, Asn or Ser. SEQ ID NO: 21 Location: (1) .. (2) Other information: This base has not been determined yet. Codons containing this base are likely to encode Met. Location: (7) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Leu, Ile or Val. Location: (11) .. (12) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Pro, His, Gln or Arg. Location: (14) Other information: This base has not yet been determined. The codon containing this base encodes any of Val, Ala, Asp or Gly. Location: (31) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Ile or Val. Location: (47) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (74) Other information: This base has not yet been determined. Codons containing this base encode any of Ile, Thr, Asn or Ser. Location: (80) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Ser, Tyr or Cys. Location: (87) Other information: This base has not yet been determined. Codons containing this base are likely to encode Leu. Location: (88) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Leu, Ile or Val. Location: (349) Other information: This base has not yet been determined. The codon containing this base encodes any of Leu, Ile or Val. Location: (648) Other information: This base has not yet been determined. Codons containing this base are likely to encode Val. Location: (693) Other information: This base has not yet been determined. Codons containing this base are likely to encode The. SEQ ID NO: 22 Location: (1) Other information: This amino acid has not been determined yet. Also,
The amino acid can be Met. Location: (3) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Leu, Ile or Val. Location: (4) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Leu, Pro, His, Gln or Arg. Location: (5) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Val, Ala, Asp or Gly. Location: (11) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Leu, Ile or Val. Location: (16) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (25) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Ile, Thr, Asn or Ser. Location: (27) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Ser, Tyr or Cys. Location: (29) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Leu. Location: (30) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Leu, Ile or Val. Location: (117) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Leu, Ile or Val. Location: (216) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Val. Location: (231) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Thr. SEQ ID NO: 27 Location: (1) .. (2) Other information: This base has not been determined yet. Codons containing this base are likely to encode Met. Location: (7) .. (9) Other information: This base has not yet been determined. Location: (14) Other information: This base has not yet been determined. The codon containing this base encodes any of Met, Thr, Lys or Arg. Location: (18) Other information: This base has not yet been determined. The codon containing this base encodes Ser or Arg. Location: (28) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Leu, Ile or Val. Location: (32) Other information: This base has not yet been determined. The codon containing this base encodes any of Val, Ala, Asp or Gly. SEQ ID NO: 28 Location: (1) Other information: this amino acid has not been determined yet. Also,
The amino acid can be Met. Location: (3) Other information: This amino acid has not yet been determined. Location: (5) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Met, Thr, Lys or Arg. Location: (6) Other information: This amino acid has not yet been determined. Also,
The amino acid can be Ser or Arg. Location: (10) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Leu, Ile or Val. Location: (11) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Val, Asp, Arg or Gly. SEQ ID NO: 29 Location: (7) .. (9) Other information: This base has not been determined yet. Location: (22) Other information: This base has not yet been determined. The codon containing this base encodes any of Phe, Leu, Ile or Val. Location: (32) Other information: This base has not yet been determined. The codon containing this base encodes any of Val, Ala, Asp or Gly. SEQ ID NO: 30 Location: (3) Other information: this amino acid has not been determined yet. Location: (8) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Phe, Leu, Ile or Val. Location: (11) Other information: This amino acid has not yet been determined. Also,
The amino acid can be any of Val, Ala, Asp or Gly. SEQ ID NO: 39 Location: (231) Other information: This base has not been determined. Codons containing this base are likely to encode Tyr. SEQ ID NO: 40 Location: (77) Other information: This amino acid has not been determined yet. Also,
The amino acid can be Tyr. SEQ ID NO: 43 Other information: Description of artificial sequence: Adapter sequence artificially synthesized SEQ ID NO: 44 Other information: Description of artificial sequence: Primer sequence artificially synthesized SEQ ID NO: 45 Other information: Description of artificial sequence: artificially synthesized primer sequence

[0116]

[Brief description of the drawings]

FIG. 1 is a graph showing the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 1 or 5 μg / ml. In the horizontal axis, “PTHr
`` P (+) '' is PTHrP without any monoclonal antibody
The results in the test using the medium containing only (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and PTHrP (1-34)
Are not shown).

FIG. 2 shows the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 0.3 or 1.0 μg / ml. In the horizontal axis, "PT
`` HrP (+) '' does not contain any monoclonal antibodies
The results of the test using a medium containing only P (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and
Are not shown).

FIG. 3 is a graph showing the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 0.3 or 1.0 μg / ml. In the horizontal axis, "PT
`` HrP (+) '' does not contain any monoclonal antibodies
The results of the test using a medium containing only P (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and
Are not shown).

FIG. 4 is a graph showing the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 1 or 5 μg / ml. In the horizontal axis, “PTHr
`` P (+) '' is PTHrP without any monoclonal antibody
The results in the test using the medium containing only (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and PTHrP (1-34)
Are not shown).

FIG. 5 is a view showing the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 0.3 or 1.0 μg / ml. In the horizontal axis, "PT
`` HrP (+) '' does not contain any monoclonal antibodies
The results of the test using a medium containing only P (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and
Are not shown).

FIG. 6 shows the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 0.3 or 1.0 μg / ml. In the horizontal axis, "PT
`` HrP (+) '' does not contain any monoclonal antibodies
The results of the test using a medium containing only P (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and
Are not shown).

FIG. 7 shows the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 0.3 or 1.0 μg / ml. In the horizontal axis, "PT
`` HrP (+) '' does not contain any monoclonal antibodies
The results of the test using a medium containing only P (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and
Are not shown).

FIG. 8 shows the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on PTHrP-dependent increase in intracellular cAMP. The vertical axis indicates the amount of cAMP produced, and the horizontal axis indicates the clone name of each monoclonal antibody at an antibody concentration of 0.3 or 1.0 μg / ml. In the horizontal axis, "PT
`` HrP (+) '' does not contain any monoclonal antibodies
The results of the test using a medium containing only P (1-34) are shown. "PTHrP (-)" indicates that only the medium (antibody and
Are not shown).

FIG. 9 shows various anti-human Ps against the bone resorption promoting action of PTHrP.
The figure which shows the inhibitory effect of THrP human monoclonal antibody. The vertical axis shows the inhibition rate (%), and the horizontal axis shows the antibody concentration. n indicates the number of mice tested. Each value is an average value (± SEM) of the test using a plurality of mice.

FIG. 10 is a graph showing the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on the bone resorption promoting effect of PTHrP.
The vertical axis shows the inhibition rate (%), and the horizontal axis shows the antibody concentration. n indicates the number of mice tested. Each value is an average value (± SEM) of the test using a plurality of mice.

FIG. 11 is a graph showing the inhibitory effect of various anti-human PTHrP human monoclonal antibodies on the bone resorption promoting effect of PTHrP.
The vertical axis shows the inhibition rate (%), and the horizontal axis shows the antibody concentration. n indicates the number of mice tested. Each value is an average value (± SEM) of the test using a plurality of mice.

FIG. 12 is a diagram schematically showing a procedure for determining a DNA sequence encoding each of a heavy chain and a light chain of an anti-human PTHrP human monoclonal antibody.

FIG. 13: Anti-human PTHrP human monoclonal antibody 2F8-10-
FIG. 3 is a view showing the therapeutic effect of PTHrP-induced hypercalcemia possessed by 3.

FIG. 14: Anti-human PTHrP human monoclonal antibody 1B3-9-1
FIG. 6 shows the therapeutic effect of 6 on PTHrP-induced hypercalcemia.

FIG. 15: Anti-human PTHrP human monoclonal antibody 15H7-8-
FIG. 3 is a view showing the therapeutic effect of PTHrP-induced hypercalcemia possessed by 3.

FIG. 16: Anti-human PTHrP human monoclonal antibody 5B12-16
FIG. 12 shows the therapeutic effect of P-12 on PTHrP-induced hypercalcemia.

FIG. 17: Anti-human PTHrP human monoclonal antibody 4B4-6-2
FIG. 1 shows the therapeutic effect of 1 on PTHrP-induced hypercalcemia.

FIG. 18: Anti-human PTHrP human monoclonal antibody 4B4-6-2
FIG. 2 shows the therapeutic effect of paraneoplastic malignant hypercalcemia possessed by 1.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61K 31/00 635 43/00643 A61K 39/395 39/395 DN C12N 5/10 C12P 21/08 15/02 ZNA C12N 5/00 B C12P 21/08 15/00 ZNAC // (C12N 5/10 C12R 1:91) (C12N 15/02 ZNA C12R 1:91) (C12P 21/08 C12R 1 : 91) (72) Inventor Masafumi Kamata 1-13-2, Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa Japan F-term in the Pharmaceutical Research Laboratory, Tobacco Inc. 4B024 AA01 AA11 BA44 BA80 CA04 DA02 EA04 GA03 GA12 GA18 HA01 HA15 4B064 AG01 AG27 CA06 CA10 CA19 CA20 CC24 CE12 DA01 DA13 4B065 AA91X AA93Y AB01 AB05 BA02 BA04 BA08 BD15 CA24 CA25 CA44 CA46 4C085 AA14 BB07 CC02 EE01 GG01 GG02 GG03 GG04 G G06 4H045 AA11 AA30 BA10 BA18 BA19 BA41 CA40 DA45 DA76 EA21 EA22 EA24 EA25 EA27 EA28 FA70 FA74 GA10 GA15 GA24 GA26

Claims (52)

[Claims] 1. A human monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof. 2. The human monoclonal antibody according to claim 1, wherein the human monoclonal antibody has the property described in any one of the following (a) to (c) or a part thereof: (a) (B) acts to suppress the increase in intracellular cAMP caused by stimulation of parathyroid hormone-related protein; (b) acts to suppress calcium release from bone caused by stimulation of parathyroid hormone-related protein; Or (c) acts to suppress an increase in blood calcium accompanying stimulation of parathyroid hormone-related protein. 3. The method according to claim 1, wherein the human monoclonal antibody has reactivity with a partial amino acid sequence of a human parathyroid hormone-related protein having any one of the following amino acid sequences (a) and (b): Or a part thereof: (a) AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA; or (b) AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRAT. 4. The human monoclonal antibody or a part thereof according to claim 1, wherein the immunoglobulin class of the human monoclonal antibody is IgG2. 5. The human antibody according to claim 1, wherein the human monoclonal antibody is a monoclonal antibody derived from a transgenic non-human mammal capable of producing a human antibody. Human monoclonal antibodies or parts thereof. 6. The human monoclonal antibody or a part thereof according to claim 5, wherein the transgenic non-human mammal is a transgenic mouse. 7. The binding rate constant (ka) between the human monoclonal antibody and a human parathyroid hormone-related protein is as follows:
The human monoclonal antibody or a part thereof according to any one of claims 1 to 6, wherein the value is 1.0 × 10 ³ (1 / M.Sec) or more. 8. The dissociation rate constant (kd) between the human monoclonal antibody and a human parathyroid hormone-related protein is as follows:
The human monoclonal antibody or a part thereof according to any one of claims 1 to 6, wherein the value is 1.0 × 10 ⁻³ (1 / Sec) or less. 9. The dissociation constant (Kd) between the human monoclonal antibody and a human parathyroid hormone-related protein is 1.0.
2. A numerical value of not more than × 10 ⁻⁷ (M).
A human monoclonal antibody according to any one of claims 6 to 6, or a part thereof. 10. The binding rate constant (ka) is 1.0 × 10 ⁴
8. A numerical value not less than (1 / M.Sec).
Or the human monoclonal antibody thereof. 11. The dissociation rate constant (kd) is 1.0 × 10
^{9. The method according} to claim 8, wherein the value is not more than ^-4 (1 / Sec).
Or the human monoclonal antibody thereof. 12. The dissociation constant (Kd) is 1.0 × 10 ⁻⁸ (M)
The human monoclonal antibody or a part thereof according to claim 9, wherein the numerical value is as follows. 13. The dissociation constant (Kd) is 1.0 × 10 ⁻⁹ (M)
The human monoclonal antibody or a part thereof according to claim 12, which has the following numerical values. 14. The dissociation constant (Kd) is 1.0 × 10
^14. The human monoclonal antibody or a part thereof according to claim 13, which has a numerical value of ^-10 (M) or less. 15. A binding rate constant (ka) between the human monoclonal antibody and a human parathyroid hormone-related protein.
Is a numerical value of 1.0 × 10 ³ (1 / M.Sec) or more, the human monoclonal antibody according to claim 2 or 3, or a part thereof. 16. The dissociation rate constant (kd) between the human monoclonal antibody and a human parathyroid hormone-related protein.
Is a numerical value of 1.0 × 10 ⁻³ (1 / Sec) or less, the human monoclonal antibody according to claim 2 or 3, or a part thereof. 17. The dissociation constant (Kd) between the human monoclonal antibody and a human parathyroid hormone-related protein is 1.
3. A numerical value of 0 × 10 ⁻⁷ (M) or less.
Or the human monoclonal antibody according to claim 3 or a part thereof. 18. The binding rate constant (ka) is 1.0 × 10 ⁴
2. A numerical value equal to or more than (1 / M.Sec).
6. The human monoclonal antibody according to 5, or a part thereof. 19. The dissociation rate constant (kd) is 1.0 × 10
^4. A numerical value of ^-4 (1 / Sec) or less.
7. The human monoclonal antibody according to 6, or a part thereof. 20. The dissociation constant (Kd) is 1.0 × 10 ⁻⁸ (M)
The human monoclonal antibody or a part thereof according to claim 17, which has the following numerical values. 21. The dissociation constant (Kd) is 1.0 × 10 ⁻⁹ (M)
21. The human monoclonal antibody or a part thereof according to claim 20, which has the following numerical values. 22. The dissociation constant (Kd) is 1.0 × 10
22. The human monoclonal antibody or part thereof according to claim 21, which has a numerical value of ^-10 (M) or less. 23. A cell producing a human monoclonal antibody reactive to a human parathyroid hormone-related protein or a part thereof. 24. The cell, wherein the cell is derived from a transgenic non-human mammal capable of producing human antibodies.
The cell according to claim 23, which is a cell. 25. The cell according to claim 24, wherein the transgenic non-human mammal is a transgenic mouse. 26. The cell according to claim 26, wherein said cell is derived from a transgenic non-human mammal capable of producing human antibodies.
24. A hybridoma obtained by fusing a cell with a myeloma cell derived from a mammal.
The cell according to 1. 27. The cell according to claim 26, wherein the transgenic non-human mammal is a transgenic mouse. 28. The cell according to claim 28, wherein the cell is international deposit number FERM BP-6390.
The cell according to claim 27, which is a hybridoma identified by: 29. The cell according to claim 29, wherein the cell is a DNA encoding a heavy chain of a human monoclonal antibody reactive with a human parathyroid hormone-related protein or a part thereof, or a DNA encoding a light chain thereof, or The cell according to claim 23, which is a transformed cell transformed by introducing both DNAs into the cell. 30. A human monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof, wherein the human monoclonal antibody is produced from a hybridoma identified by International Accession No. FERMBP-6390 or A human monoclonal antibody having substantially the same properties as the human monoclonal antibody, or a part thereof. 31. A monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof, wherein the light chain variable region of the monoclonal antibody is one of the following (a) or (b): (A) Consists of SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 An amino acid sequence of amino acids 21 to 119 in any one of the SEQ ID NOs selected from the group; or (b) SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 2
An amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of amino acids 21 to 119 in the amino acid sequence described in any one of SEQ ID NOs selected from the group consisting of 2; A monoclonal antibody having an amino acid sequence comprising: or a part thereof. 32. A monoclonal antibody or a part thereof reactive to a human parathyroid hormone-related protein or a part thereof, wherein the heavy chain variable region of the monoclonal antibody is one of the following (a) to (d): (A) SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 3
6, amino acid numbers 20 to 117 in the amino acid sequence described in any one of SEQ ID NOs selected from the group consisting of SEQ ID NOs: 38 and 42; (b) SEQ ID NO: 40 (C) SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 3
6, one or several amino acids are deleted in the amino acid sequence of amino acids 20 to 117 in the amino acid sequence described in any one of SEQ ID NOs selected from the group consisting of SEQ ID NO: 38 and SEQ ID NO: 42 (D) one or several amino acids are deleted, substituted or added in the amino acid sequence of amino acids 20 to 115 in the amino acid sequence described in SEQ ID NO: 40; A monoclonal antibody or a part thereof having an amino acid sequence comprising: 33. The V region, D region and J region DNA encoding the heavy chain of said human monoclonal antibody
3-30, derived from DN1 and JH6.
23. A human monoclonal antibody according to any one of claims 22 to 22, or a part thereof. 34. The DNA of the V region, D region and J region encoding the heavy chain of the human monoclonal antibody is VH
4.16, derived from DA1 and JH6
23. A human monoclonal antibody according to any one of claims 22 to 22, or a part thereof. 35. The DNA of the V region and the J region encoding the light chain of the human monoclonal antibody are DPK15 and JK, respectively.
3. The method according to claim 1, wherein said compound is derived from κ3.
3. The human monoclonal antibody or part thereof according to any one of 2. 36. The DNA of the V region, D region and J region encoding the heavy chain of the human monoclonal antibody, respectively,
3-30, DNA of V region and J region derived from DN1 and JH6 and encoding the light chain of the human monoclonal antibody,
The human monoclonal antibody or a part thereof according to any one of claims 1 to 22, wherein the human monoclonal antibody is derived from DPK15 and Jκ3, respectively. 37. The DNA of the V region, D region and J region encoding the heavy chain of the human monoclonal antibody is VH
4.16, DNA of V region and J region derived from DA1 and JH6 and encoding the light chain of the human monoclonal antibody,
The human monoclonal antibody or a part thereof according to any one of claims 1 to 22, wherein the human monoclonal antibody is derived from DPK15 and Jκ3, respectively. 38. A pharmaceutical composition comprising the human monoclonal antibody or a part thereof according to any one of claims 1 to 22 or 30 to 37, and a pharmaceutically acceptable carrier. . 39. The human monoclonal antibody according to claim 2, claim 3, claim 15 to claim 22 or claim 30 or claim 37, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising: 40. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for treating a disease caused by the release of calcium from bone in a parathyroid hormone-related protein-dependent manner.
A pharmaceutical composition according to claim 1. 41. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for treating hypercalcemia. 42. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for inhibiting or preventing osteolysis. 43. The medical composition according to claim 39, wherein the pharmaceutical composition is used for treating rheumatoid arthritis or osteoarthritis. 44. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for suppressing or preventing cancer metastasis to bone. 45. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for suppressing or preventing the growth of cancer cells present in bone tissue. 46. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for treating a disease caused by local production of a parathyroid hormone-related protein. 47. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for treating a condition caused by a primary local cancer. 48. The pharmaceutical composition according to claim 47, wherein the symptom is a symptom selected from the group consisting of pain, nerve compression, hypercalcemia, fracture and cachexia. 49. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for prolonging the life of a patient suffering from primary local cancer. 50. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for treating a disease in a tooth, periodontal or gingival. 51. The pharmaceutical composition according to claim 38, wherein the pharmaceutical composition is used for treating sepsis or systemic inflammatory response syndrome (SIRS). 52. The pharmaceutical composition according to claim 39, wherein the pharmaceutical composition is used for treating hypophosphatemia.