JP4527389B2 - Method for assessing the biological activity of a ligand binding inhibitor - Google Patents

Description

  The present invention relates to a method for evaluating the biological activity of a ligand binding inhibitor. More specifically, the present invention relates to a method for evaluating the biological activity of a ligand binding inhibitor using the binding amount between a ligand binding protein expressed on a cell membrane and a ligand as an index.

In the development of pharmaceuticals, it is an important issue to establish a bioactivity measurement method used for prescription design, quality design and quality control of drug substances and their preparations. The biological activity measurement method is simple and highly accurate and reproducible, and a method that can explain the action mechanism of a pharmaceutical is desirable (see, for example, Non-Patent Document 1). Until now, the binding activity between a ligand and a ligand-binding protein has been measured using a radioimmunoassay using a radioisotope (RI), an enzyme-labeled solid phase immunoassay (ELISA) using chemiluminescence, or the like.

In the case of measuring the binding activity of a ligand and a receptor, an evaluation system using cells is desirable in order to evaluate the binding activity of the ligand via the receptor in the native state. In such an evaluation system using cells, a measurement method using RI is usually used. For example, Cytostar ^™ -T (manufactured by Amersham Pharmacia) using the SPA method measures the amount of binding between the ligand and the cell membrane receptor using a special 96-well plate containing an RI-labeled ligand and a scintillator on the bottom. In this method, an RI-labeled ligand is first reacted with cells that are attached and cultured on a plate. When the RI-labeled ligand binds to the cell membrane receptor and is taken into the cell, the scintillator on the bottom of the plate is excited, and the amount of ligand binding is measured from the amount of luminescence (see, for example, Non-Patent Document 2). However, this method has an upper limit on the amount of RI used, requires dedicated facilities / equipment and dedicated equipment, and has safety problems such as treatment of radioactive pollutants.

On the other hand, the chemiluminescence method using an enzyme-labeled antibody can evaluate the binding activity between a ligand and a ligand-binding protein without using RI. For example, Patent Document 1 discloses that an antigen or antibody against an ALP-labeled measurement object is caused to undergo an antigen-antibody reaction with the measurement object in a sample; phosphomonoesterase bound or not bound to the measurement object is quantified by chemiluminescence. A method for quantifying the measurement object is described (see Patent Document 1). However, this method does not consider an evaluation system using living cells, so it is not necessarily a measurement method that reflects the interaction between the ligand binding protein expressed on the cell membrane and the ligand in the native state. .

By the way, parathyroid hormone related peptide (Parathyroid Hormone related Peptide,
Hereinafter referred to as “PTHrP”. ) Is a peptide that promotes bone resorption of osteoclasts and calcium resorption in renal tubules to induce clinical symptoms such as neoplastic hypercalcemia and hypophosphatemia. It is known that the action of PTHrP is exerted by PTHrP binding to PTH / PTHrP receptors mainly present in bone and kidney and activating a plurality of intracellular signal transduction systems. Therefore, anti-PTHrP antibodies that inhibit the action of PTHrP are highly expected as pharmaceuticals.

  Conventionally, the biological activity of anti-PTHrP antibodies has been evaluated using as an index the enhancement of intracellular cAMP production by PTHrP receptor binding (see, for example, Non-Patent Document 3). This method detects intracellular reactions and sufficiently reflects receptor binding activity, but a simpler measurement method is desirable.

JP 7-53525 A Mire-Sluis A.R., “Progress in the use of biological assay during the development of biotechnology products” Pharmaceutical Research 2001, 18 (9): p1239-1246 Mark Harvey et al., “The Use of Cytostar-TTM Scintillating Microtitre Plates for Monitoring Receptor Binding Using Live Cells.” Poster presented at 2nd International Conference on Microplate Technologies, Virginia, USA (1997) Sato K., et al., “Passive immunization with anti-parathyroid hormone-related protein monoclonal antibody markedly prolongs survival time of hypercalcemic nude mice bearing transplanted human PTHrP-producing tumors.” J. Bone Miner. Res. 1993; 8 (7 ): p849-860

  It is an object of the present invention to quantify the binding activity between a ligand and a native ligand-binding protein expressed on a cell membrane with high sensitivity and high accuracy without using RI, thereby enabling more accurate detection of a ligand binding inhibitor. To provide an evaluation method.

  As a result of intensive studies to solve the above problems, the present inventors have completed a method for quantifying the amount of ligand binding by binding a ligand-enzyme fusion protein to cells that highly express the ligand binding protein. It was. Then, by using this method, it was found that the biological activity of a ligand binding inhibitory substance via a ligand binding protein in a native state can be evaluated with high sensitivity and high accuracy, and the present invention has been completed.

  That is, the present invention relates to a method for quantitatively evaluating the biological activity of a ligand binding inhibitor using the binding amount of a ligand binding protein expressed on a cell membrane and a ligand-enzyme fusion protein as an index.

  In the above method, the amount of binding between the ligand binding protein and the ligand-enzyme fusion protein can be determined using, for example, chemiluminescence via the enzyme in the ligand-enzyme fusion protein.

  In the above method, it is preferable to use a ligand-binding protein-expressing cell as the ligand-binding protein expressed on the cell membrane.

For example, the method of the present invention can be carried out by sequentially performing the following steps.
1) A ligand-enzyme fusion protein is reacted with a solid phase-immobilized ligand-binding protein-expressing cell in the presence of a test substance;
2) After removing the free ligand-enzyme fusion protein, the enzyme substrate is added and the amount of luminescence is measured;
3) Quantitatively evaluate the biological activity of the test substance as a ligand binding inhibitor based on the measured amount of luminescence.

In certain embodiments, the ligand binding protein is a ligand receptor. The ligand binding inhibitor is a ligand antibody or an agonist or antagonist for the ligand receptor.
In the method of the present invention, as a ligand-binding protein-expressing cell, a recombinant cell in which a ligand-binding protein is forcibly highly expressed can be preferably used.

Furthermore, it is preferable to use human placental alkaline phosphatase as the enzyme to be bound to the ligand.

One embodiment of the present invention includes a method in which the ligand is PTHrP, the ligand binding protein is a PTH / PTHrP receptor, and the ligand binding inhibitor is an anti-PTHrP antibody.

The present invention also provides a kit for quantitatively evaluating the biological activity of a ligand binding inhibitor used in the method of the present invention. The kit includes at least the following 1) to 3). 1) Ligand-bound protein-expressing cell 2) Ligand-enzyme fusion protein 3) Ligand binding inhibitor The kit of the present invention is necessary for evaluating the biological activity of a ligand binding inhibitor in addition to the above essential components. Other reagents may be included.

A preferred embodiment of the kit includes a kit including the following 1) to 3).
1) Immobilized PTH / PTHrP receptor-expressing cells 2) PTHrP-human placental alkaline phosphatase fusion protein 3) Anti-PTHrP antibody Using this kit, the biological activity of PTHrP binding inhibitors can be evaluated quantitatively. Can do.

  According to the present invention, the binding activity between a ligand and a native ligand-binding protein expressed on a cell membrane can be quantified with high sensitivity and high accuracy without using RI. Thereby, the biological activity of the ligand binding inhibitor can be more accurately evaluated.

Hereinafter, the present invention will be described in detail.
1. Method for Quantitative Evaluation of Ligand Binding Inhibitor Biological Activity The method of the present invention quantitatively evaluates the biological activity of a ligand binding inhibitor using the binding amount of a ligand binding protein expressed on a cell membrane and a ligand-enzyme fusion protein as an index. It is a method to evaluate.

1.1 Explanation of Terms (1) Ligand “Ligand” used in the present invention is not particularly limited, and examples thereof include bioactive peptides such as peptide hormones, growth factors, and cytokines, fibronectin, collagen, laminin, cadherin, and NCAM. Cell adhesion molecules such as antigens, antibodies, and the like.

(2) Ligand-enzyme fusion protein In the present invention, the ligand is used as a “ligand-enzyme fusion protein” bound to an enzyme protein. The enzyme bound to the ligand is not particularly limited as long as it enables detection of the ligand, and examples thereof include peroxidase, β-D-galactosidase, glucose oxidase, tyrosinase, acid phosphatase, alkaline phosphatase and the like. . In particular, in the method of the present invention using cells, placental alkaline phosphatase is preferably used in that it is hardly affected by intracellular enzymes. This is because endogenous alkaline phosphatase and peroxidase are present in the cells, and human placental alkaline phosphatase is optimal for quantification without being affected by these endogenous enzymes.

  The ligand-enzyme fusion protein can be easily prepared using a known gene recombination technique. For example, first, a gene encoding a ligand peptide and a gene encoding an enzyme protein are each cloned. Each cloned gene is digested with a restriction enzyme, and fragments thereof are ligated and incorporated into an appropriate vector. The vector to be used is not particularly limited as long as it can express the ligand-enzyme fusion protein in the cell to be introduced. Note that the fusion protein does not need to include the entire region of the ligand peptide, but may include a specific region necessary for binding to the ligand binding protein in the ligand peptide.

Next, suitable cells such as CHO cells are transformed with the expression vector. The obtained recombinant cells are repeatedly passaged and selected to select cells capable of stably expressing the fusion protein.

  Once the fusion protein-producing recombinant cells are obtained in this way, the cells can be cultured under appropriate conditions, and the desired fusion protein can be purified and obtained from the culture supernatant. The fusion protein may be purified using a known means such as an affinity column or gel filtration.

(3) Ligand-binding protein expressed on cell membrane In the present invention, "ligand-binding protein expressed on cell membrane" refers to the above-mentioned ligand, that is, a biologically active peptide such as peptide hormone, growth factor, or cytokine. Cell membrane receptor, fibronectin, collagen, laminin, cadherin, and other on-cell membrane adhesion molecules to which cell adhesion molecules such as NCAM bind, or antigens or antibodies.

(4) Ligand-binding protein-expressing cell In the present invention, a cell expressing the ligand-binding protein is used as the ligand-binding protein expressed on the cell membrane. It is preferable that the cell highly expresses the ligand binding protein on its cell membrane. As such a cell, a recombinant cell in which a gene encoding the ligand-binding protein is introduced into an appropriate cell and forcibly highly expressed can be preferably used.

(5) Ligand binding inhibitory substance In the present invention, the “ligand binding inhibitory substance” is a substance that can inhibit the binding between a ligand and a ligand-binding protein, for example, various antibodies that inhibit the binding between a ligand and a receptor, An agonist, an antagonist, etc. can be mentioned.

1.2 Implementation Process Specifically, the method of the present invention can be implemented by sequentially performing the following processes.
1) A ligand-enzyme fusion protein is reacted with a solid phase-immobilized ligand-binding protein-expressing cell in the presence of a test substance;
2) After removing the free ligand-enzyme fusion protein, the enzyme substrate is added and the amount of luminescence is measured;
3) Quantitatively evaluate the biological activity of the test substance as a ligand binding inhibitor based on the measured amount of luminescence.

(1) Immobilization of ligand-binding protein-expressing cells Ligand-binding protein-expressing cells can be immobilized on an appropriate solid phase to facilitate separation of free ligand-enzyme fusion proteins (B / F separation). preferable. The solid phase used is not particularly limited, and examples thereof include a 96-well plate and microbeads.

(2) B / F separation Next, the immobilized cells are reacted with the ligand-enzyme fusion protein in the presence of the test substance to remove the free ligand-enzyme fusion protein. Since the binding between the ligand and the ligand binding protein is usually affected by pH, the reaction is performed in an appropriate buffer capable of maintaining a pH suitable for binding. The type of the buffer is appropriately selected from the buffers having a buffering action in the appropriate pH range described above depending on the binding characteristics of the ligand to be used and the ligand binding protein.

  The reaction time is set so that the ligand and the ligand-binding protein can sufficiently bind. Since the reaction time is affected by the reaction temperature, an appropriate reaction temperature and reaction time are appropriately set according to the binding characteristics of the ligand to be used and the ligand binding protein.

(3) Measurement of chemiluminescence amount After removing the free ligand-enzyme fusion protein, an enzyme substrate is added to the reaction system and the chemiluminescence amount via the enzyme is measured.

  What is necessary is just to select an enzyme substrate suitable for the enzyme to be used. For example, in the case of peroxidase, a hydrogen donor such as 3- (4-Hydroxyphenyl) propoinic acid, 4-Hydroxyphenylacetic acid, 1,2-Phenylenedediamine, or 3,3 ′, 5,5′-Tetramethylbenzidine, or luminol A luminescent substrate can be used. For β-D-galactosidase, 4-methylumbelliferyl-β-D-garactoside, 2-nitrophenyl-β-D-garactoside, or 2-nitrophenyl-β-D-garactoside is used. In the case of alkaline phosphatase, 4-methylumbelliferylphosphate, NADP, 4-nitrophenylphosphate, AMPPD, or the like can be used.

  Enzyme substrates such as those described above are well known in the art and are commercially available. In addition, measurement of chemiluminescence by reaction between the substrate and the enzyme can be easily performed based on a well-known method. The amount of chemiluminescence thus measured reflects the amount of ligand bound to the ligand binding protein expressed on the cell membrane.

(4) Quantitative evaluation of biological activity The biological activity of a ligand binding inhibitor of a test substance is evaluated based on the amount of ligand (ligand-enzyme fusion protein) bound to the ligand binding protein on the cell membrane in the presence of the test substance. To do. Preferably, the addition concentration of the test substance is changed variously, and the biological activity of the test substance as a ligand binding inhibitor is evaluated from the relationship between the concentration and the amount of ligand binding.

2. Method for Evaluating Biological Activity of Anti-PTHrP Antibody Hereinafter, as a preferred embodiment of the present invention, human PTHrP-human placental alkaline phosphatase (hereinafter referred to as SEAP) fusion protein and PTH / PTHrP receptor high expression cell A method for measuring the biological activity of an anti-PTHrP antibody using the binding amount (activity) as an index will be described. The anti-PTHrP antibody can be produced, for example, according to the method described in WO98 / 13388. .

2.1 Preparation of ligand-enzyme fusion protein
The PTHrP-SEAP fusion protein (ligand-enzyme fusion protein) is produced, for example, as follows.

First, according to a known method, a gene encoding PTHrP is cloned. Similarly, a gene encoding SEAP is cloned. The cloned PTHrP and SEAP genes are
A PTHrP-SEAP fusion protein expression vector is prepared by cleaving and ligating with an appropriate restriction enzyme such as EcoRI / NotI and incorporating into an appropriate expression vector. Here, the PTHrP gene fragment is preferably a gene fragment encoding a fragment containing the amino acid sequence from the 1st position to the 34th position in the PTHrP (1-34): PTHrP peptide. This is because if the length between the receptor binding site of PTHrP and the enzyme is too long, the PTHrP site is susceptible to cleavage, and apparently the binding activity to the receptor decreases.

Next, an appropriate cell, such as a CHO cell, is transformed with the expression vector. The obtained recombinant cells are repeatedly passaged and selected to select cells that can stably express the PTHrP-SEAP fusion protein.

If a PTHrP-SEAP fusion protein-producing recombinant cell is thus obtained, the cell can be subsequently cultured, and the PTHrP-SEAP fusion protein can be purified and obtained from the culture supernatant. The PTHrP-SEAP fusion protein may be purified using an affinity column or the like.

2.2 Cells that highly express ligand-binding protein
PTH / PTHrP receptor high-expressing cells can be prepared by introducing a gene encoding a PTH / PTHrP receptor into an appropriate cultured cell by the same method as described above. If necessary, the gene is integrated under the control of a highly active promoter and introduced into a cell. The obtained cells can be established as a PTH / PTHrP receptor high expression cell line by repeated selection and passage.

  Examples of such cells include HkRk-B7 cells and HkRk-B28 cells (both human PTH receptor transfected LLC-PK1 cells: introduced from Massachusetts General Hospital, Takasu K, Guo J, and Bringhurst FR. J Bone Miner Res 1999; 14 (1): 11-20).

2.3 Evaluation Method of Biological Activity of Ligand Binding Inhibitor The biological activity of the anti-PTHrP antibody (ligand binding inhibitor) was determined using the above-mentioned HkRk-B7 cell or HkRk-B28 cell, PTH / PEAP fusion protein using PTHrP-SEAP fusion protein. Can be assessed by PTHrP receptor binding assay.

As for the PTH / PTHrP receptor high-expressing cells, in the aforementioned HkRk-B7 and HkRk-B28 cells, the expression level of PTH / PTHrP receptor is higher in HkRk-B7 cells than in HkRk-B28 cells, and the amount of PTHrP-SEAP binding Therefore, the assay of the present invention is preferable.

The PTH / PTHrP receptor high-expressing cells (HkRk-B7 cells or HkRk-B28 cells) are seeded in a 96-well plate, cultured in 10% FCS-DMEM medium, and then the medium is changed to a binding buffer. As the buffer to be used, 50 mM Tris-HCl (pH 7.7), 100 mM NaCl, 2 mM CaCl ₂ , 5 mM
KCl, 5% horse serum, 0.5% FCS, 1% BSA-HBSS, etc. are preferred. The number of seeded cells is preferably about 5 × 10 ⁴ cells / well in the case of HkRk-B7 cells and HkRk-B28 cells.

  The PTHrP-SEAP fusion protein and anti-PTHrP antibody are added here for the binding reaction, but the addition amount of the PTHrP-SEAP fusion protein must be set so as not to be oversaturated with respect to the receptor. In consideration of sufficient detection with the receptor, the preferred addition amount of the PTHrP-SEAP fusion protein is 1.25 to 10 nM, particularly around 5 nM.

The reaction time between PTH / PTHrP receptor high-expressing cells, PTHrP-SEAP fusion protein, and anti-PTHrP antibody requires about 30 to 60 minutes at 16 ° C. to room temperature. For sufficient binding to the receptor, the reaction time should be 30 minutes or more at room temperature and 60 minutes or more at 16 ° C.

After completion of the binding reaction, free PTHrP-SEAP fusion protein is removed, a buffer containing a luminescent enzyme substrate is added, and the resulting luminescence is measured. The measurement needs to be performed within a range in which the receptor binding inhibitory activity of PTHrP by the anti-PTHrP antibody can be quantified. Such a range can be set based on a change in the amount of PTHrP binding measured in advance by changing the concentration of the anti-PTHrP antibody.

3. Kit for Evaluation of Ligand Binding Inhibitor The present invention also provides a kit for quantitatively evaluating the biological activity of a ligand binding inhibitor used in the method of the present invention. The kit includes at least the following 1) to 3). 1) Ligand-binding protein-expressing cell 2) Ligand-enzyme fusion protein 3) Ligand-binding inhibitor Substance-bound ligand-binding protein-expressing cell and ligand-enzyme fusion protein, which are essential components of the kit of the present invention The ligand binding inhibitor is as described in 1.1 and 1.2.

  In addition to these essential components, the kit may contain other reagents and the like required for evaluating the biological activity of the ligand binding inhibitor. Examples of such reagents include 3- (4-Hydroxyphenyl) propoinic acid, 4-Hydroxyphenylacetic acid, 1,2-Phenylenedediamine, 3,3 ′, 5,5′-Tetramethylbenzidine, luminol, 4-methylumbelliferyl- Enzyme substrates such as β-D-garactoside, 2-nitrophenyl-β-D-garactoside, or 2-nitrophenyl-β-D-garactoside, 4-methylumbelliferylphosphate, NADP, 4-nitrophenylphosphate, and AMPPD, reaction buffers, etc. Is included.

Preferable examples of the kit include a kit for evaluating the biological activity of a PTHrP binding inhibitor. The kit includes at least the following 1) to 3).
1) immobilized PTH / PTHrP receptor-expressing cells 2) PTHrP-human placental alkaline phosphatase fusion protein 3) anti-PTHrP antibody Using the kit of the present invention, the biological activity of a ligand binding inhibitor can be detected with high sensitivity. Highly accurate and quantitative evaluation is possible.

4). Others In the method of the present invention, the ligand-binding protein interacts with a ligand or a ligand-binding inhibitor in a native state expressed on the cell membrane. Moreover, a measurement method based on chemiluminescence via an enzyme has sensitivity comparable to RI and exhibits excellent reproducibility. Therefore, by using the method of the present invention, it is possible to accurately screen for an agonist or antagonist for a ligand receptor in a state close to physiological conditions.

  Moreover, the expression cloning of a receptor can also be performed by utilizing the evaluation method of the present invention. Furthermore, it is possible to screen for low molecular weight compounds that act on receptors and other ligands, and to evaluate and screen the interaction between adhesion molecules on the surface of cell membranes and the biological activities of various compounds that act on them.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

[Example 1] Preparation of PTHrP-SEAP and confirmation of physical properties Cloning of PTHrP (1-34) and PTHrP (1-141) genes The DNA sequences of human PTHrP (1-34) and human PTHrP (1-141) are expressed in human large cell carcinoma cell line LC-6 (LC- 6-JCK cell (purchased from Central Research Laboratory for Experimental Animals). First, total RNA was extracted from LC-6 cells using ISOGEN (Nippon Gene), and cDNA was synthesized using this RNA as a template with the following reverse primer of PTHrP (1-34) or PTHrP (1-141). . The obtained cDNA was subjected to PCR using the template as a template and the primer and PTHrP forward primer to amplify the target gene sequence. For cloning purposes, the EcoRI site was linked to the forward primer and the XbaI site was linked to the 5 ′ side of the reverse primer.
Primer (Amersham Pharmacia):
PTHrP forward: 5'-CCGGAATTCCACCATGCAGCGGAGACTGGTTCAGC-3 '(SEQ ID NO: 1)
PTHrP (1-34) reverse: 5'-CGCTCTAGAAGCTGTGTGGATTTCTGCGATC-3 '(SEQ ID NO: 2)
PTHrP (1-141) reverse: 5'-CGCTCTAGAATGCCTCCGTGAATCGAGCTC-3 '(SEQ ID NO: 3)

  Next, the obtained PCR product was cleaved with EcoRI / XbaI and inserted into the EcoRI / XbaI site of the cloning vector pBluescriptII SK (+) (pBS). For the resulting vectors pBS-PTHrP (1-34) and pBS-PTHrP (1-141), the inserted DNA sequence was confirmed using a DNA sequencer.

2. Cloning of SEAP gene
The DNA sequence of SEAP (secreted human placental alkaline phosphatase) is pSEAP (Applied
(Manufactured by Biosystems). To introduce a linker (5 amino acids) with PTHrP on the N-terminal side of SEAP and (His) ₆ tag on the C-terminal side, the SEAP forward primer from the 5 'side, EcoRI site, XbaI site, 5 The -amino acid linker was linked to the reverse primer from the 5 'side as NotI site, (His) ₆ tag as an adaptor. PCR was performed using pSEAP as a template using these primers, and the product was digested with EcoRI / NotI and inserted into the EcoRI / NotI site of pBluescriptII SK (+). The obtained vector pBS-SEAP was confirmed for the inserted DNA sequence using a DNA sequencer. Primer (Amersham Pharmacia):
SEAP forward: 5'-CCGGAATTCGCTCTAGAAGCTCCGGAATCATCCCAGTTGAGGAGGAGAAC-3 '(SEQ ID NO: 4)
SEAP reverse: 5'-CGCGCGGCCGCTCAGTGATGGTGATGGTGATGACCCGGGTGCGCGGCGTCGGT-3 '(SEQ ID NO: 5)

3. Construction of pCHOI-PTHrP (1-34) -SEAP and pCHOI-PTHrP (1-141) -SEAP
pBS-PTHrP (1-34) and pBS-PTHrP (1-141) were cleaved with EcoRI / XbaI to prepare PTHrP (1-34) fragment and PTHrP (1-141) fragment. Further, pBS-SEAP was cleaved with XbaI / NotI to prepare a SEAP fragment. The PTHrP fragment, SEAP fragment, and EcoRI / NotI-cut pCHOI (see Toshihiko Ohtomo et al., BBRC 1999; see p258-585) were ligated, and pCHOI-PTHrP (1-34) -SEAP and pCHOI-PTHrP (1-141 ) -SEAP was prepared (FIG. 1). The prepared vector was confirmed for the inserted DNA sequence using a DNA sequencer.

4). Preparation of cells stably expressing PTHrP-SEAP
The PTHrP-SEAP expression vector pCHOI-PTHrP (1-34) -SEAP and pCHOI-PTHrP (1-141) -SEAP were partially digested with the restriction enzyme PvuI. 10 μg of this partially digested expression vector and 1 × 10 ⁷ cells / mL
0.8 mL of CHO cells were added to a 0.4 cm cuvette for electroporation, and voltage was applied under conditions of 1.5 kV and 25 μF using Gene Pulser (manufactured by BIO-RAD).
The cells were cultured at 37 ° C.-5% CO ₂ , and the expression level of the fusion protein was compared using the SEAP activity as an index, and clones with high activity were selected.

  In addition, for the purpose of examining conditions (medium and additives) for expressing PTHrP-SEAP, PTHrP-SEAP-expressing CHO cells were cultured in a T25 flask for 2 days, and the medium was serum-free medium (CHO-S-SFM II), Alternatively, it was replaced with a serum-free medium containing protease inhibitor (0.1% protease inhibitor-CHO-S-SFM II: manufactured by GIBCO) and cultured for 2 days, and its SEAP activity was evaluated.

5). Purification of PTHrP-SEAP fusion protein
PTHrP (1-34) -SEAP and PTHrP (1-141) -SEAP were purified using a His-tag affinity purification kit (TALON Metal Affinity Resins Purification Kit: manufactured by CLONTECH). The SEAP activity in the sample was measured using Phospha-Light System (Applied Biosystems). A calibration curve was prepared from the SEAP concentration and luminescence of the control enzyme, and the SEAP content of the sample was determined. The PTHrP-SEAP concentration in the sample was calculated from the following formula.

PTHrP-SEAP (nmol / L) = alkaline phosphatase concentration (ng / mL) / 57953 ^* x 1000
* 57953 shows the calculated molecular weight results of human placental alkaline phosphatase precursor (535 amino acid) in FIG.

6). PTHrP activity of PTHrP-SEAP fusion protein
Whether the PTHrP-SEAP fusion protein retained the function as PTHrP was examined by examining 1) PTH / PTHrP receptor binding activity and 2) cAMP-inducing activity.
1) PTH / PTHrP receptor binding activity
HkRk-B7 cells (Takasu K, Guo J, and Bringhurst FR. J Bone Miner Res 1999; 14 (1): 11-20: introduced from Massachusetts General Hospital) are used in 10% FCS-DMEM medium (GIBCO) The cells were cultured at 37 ° C. and 5% CO ₂ , detached with Trypsin-EDTA, and prepared to 4 × 10 ⁶ cells / mL. 50 μL of this cell suspension was added to a 1.5 mL tube, and 50 μL each of PTHrP (1-34) -SEAP or PTHrP (1-141) -SEAP was added. Separately, a sample containing PTHrP (1-34) 1 μmol / L added to PTHrP-SEAP was added to the cell suspension.

After allowing to stand at room temperature for 30 min, the PTHrP sample was bound to the PTH / PTHrP receptor on the HkRk-B7 cell membrane and then washed 3 times with PBS to remove the unbound PTHrP sample. PTHrP-SEAP bound to the receptor was detected using the Phospha-Light System (Applied Biosystems). The cells were suspended in 150 μL of 1 × Dilution buffer, heat-treated at 65 ° C. for 30 min, cooled on ice, and added to a 96-well black plate at 50 μL / well. Add Assay Buffer 50 μL / well and leave at room temperature for 5 minutes. Add Reaction Buffer 50 μL / well containing CSPD Substrate and leave at room temperature for 20 minutes, then measure the luminescence using TopCount (manufactured by Packard). did.

  As a result, binding of PTHrP (1-34) -SEAP to the PTH / PTHrP receptor was detected, and this binding was inhibited to about 1/10 by addition of PTHrP (1-34) (1 μmol / L). In contrast, PTHrP (1-141) -SEAP did not detect binding to the PTH / PTHrP receptor (FIG. 3).

2) cAMP-inducing activity
HkRk-B7 cells were prepared to 2.5x10 ⁵ cells / mL using 10% FCS-DMEM medium, added to a 96-well plate at 100 μL / well, and cultured for 1 day at 37 ° C and 5% CO ₂ Thereafter, PBS was added at 100 μL / well for washing.

Anti-PTHrP antibody (Lot No. 99B01: WO98 / 13388) was prepared at 560 μg / mL with 10% FCS-DMEM.
PTHrP (1-34) -SEAP, PTHrP (1-141) -SEAP, or PTHrP (1-34) was prepared with 2 mmol / L IBMX-10% FCS-DMEM. Mix equal amounts of anti-PTHrP antibody and PTHrP sample, and add this to the plate.
μL / well was added.

  The plate was allowed to stand at room temperature for 30 min, then 2 x Lysis buffer 50 μL / well was added and left at room temperature for 30 min, and the cAMP content was measured using an HTRF Cyclic AMP kit (manufactured by CIS bio international).

The cAMP standard was prepared by diluting the cAMP maximum calibrator attached to the kit with 1 × Lysis buffer. Add 1 x Lysis buffer (Negative control, Positive control), sample, and cAMP standard twice to a 384 well black plate at 10 μL / well, and add 5 μL / well of anti-cAMP Ab-cryptate conjugate and cAMP-XL665 conjugate. 5 μL / well was added. However, only negative control was added 5 μL / well of distilled water instead of cAMP-XL665 conjugate. After leaving the plate at room temperature for 3 hr, the fluorescence intensity at 665 nm and 615 nm was measured by ARVO HTS 1420 Multilabel Counter (manufactured by Wallac Bertol Japan). For each sample, an average value measured twice was used as a measured value of fluorescence intensity. Moreover, DF / DF max% was calculated | required with the following formula about the measurement result of the sample and cAMP standard.
Ratio = 665 nm / 615 nm x 10000
Delta R = (Sample Ratio − Negative Ratio)
Delta F% = Delta R / Negative Ratio x 100
DF / DF max% = Delta F% of Sample / Delta F% of Positive control x 100

Thus, a calibration curve was created from the cAMP standard cAMP concentration and DF / DF max%, and the amount of cAMP contained in the sample was determined.
As a result, the amount of cAMP produced in HkRk-B7 cells increased with the addition of PTHrP (1-34). His-tag purified PTHrP (1-34) -SEAP and PTHrP (1-141) -SEAP induced cAMP production, but PTHrP (1-34)
The activity was low. Moreover, the cAMP induction activity by PTHrP (1-34) and PTHrP-SEAP was suppressed in the presence of an anti-PTHrP antibody (Lot No. 99B01: WO98 / 13388) (FIG. 4).

7). SDS-PAGE of PTHrP-SEAP
Culture supernatant of PTHrP-SEAP expressing cells and His-tag purified sample were separated by reducing SDS-PAGE. Samples and molecular weight markers were prepared with Laemmli Sample Buffer (manufactured by BIO RAD) + 5% 2-mercaptoethanol, and reduced and denatured at 95 ° C. for 5 min. This was applied to 8% Tris-Glycine gel and electrophoresed at 40 mA (const.) × about 1.5 hr. After completion of the electrophoresis, the gel was stained with CBB to detect the protein.

As a result, a large number of bands were detected in the culture supernatant containing PTHrP-SEAP, but 71 kD (PTHrP (1-34) -SEAP) or 78 kD (PTHrP (1-141)) in the His-tag purified sample. -SEAP) detected a main band. However, minor bands such as about 30 kD and about 180 kD were detected in the purified His-tag sample in addition to the main band (FIG. 5).

8). N-terminal amino acid sequence analysis of PTHrP-SEAP
The N-terminal amino acid sequence of PTHrP-SEAP expressed in CHO cells was analyzed by entrusting it to Apro Science Corporation. As a result, in PTHrP (1-34) -SEAP, there are two types of proteins: Nla-terminal protein with Ala-Val-Ser-Glu-His and Arg-Phe-Phe-Leu-His. Mainly detected. In PTHrP (1-141) -SEAP, a protein having the Ser-Ala-Trp-Leu-Asp sequence was mainly detected.

  From the amino acid sequence of PTHrP-SEAP, it was speculated that PTHrP (1-34) -SEAP contains the target protein and PTHrP (21-34) -SEAP lacking the PTHrP (1-20) residue . In addition, it was estimated that PTHrP (1-141) -SEAP mainly includes PTHrP (109-141) -SEAP lacking PTHrP (1-108).

[Example 2] PTH / PTHrP receptor binding assay and examination of its conditions Test Method The procedure of PTH / PTHrP receptor binding assay using PTHrP-SEAP prepared in Example 1 and a commercially available SEAP detection kit: Phospha-Light System (Applied Biosystems) is shown below. In the following assay, 50 mmol / L Tris-HCl (pH 7.7), 100 mmol / L NaCl, 2 mmol / L CaCl ₂ , 5 mmol / L KCl, 5% Horse serum was used as the buffer. , 0.5% FCS was used.

1) Put 10% FCS-DMEM (100 μL / well) in a 96-well plate, seed the cells at 5x10 ⁵ cells / mL cell, and incubate at 37 ° C and 5% CO ₂ for 1 day. Discard the medium, wash with buffer (200 μL / well), add a sample solution (50 μL / well) containing PTHrP-SEAP and anti-PTHrP antibody, and react at room temperature for 30 minutes.
2) After washing 3 times with buffer (200 μL / well), dilute with 1 x Dilution buffer-0.02% Triton X-100 (150 μL / well), leave at 65 ° C for 30 minutes, then cool on ice, 96 wells Transfer to plate.
3) Add Assay Buffer (50 mL / well) attached to the kit, react at room temperature for 5 minutes, add Reaction Buffer (50 mL / well) containing CSPD and Emerald, react for 15-20 minutes, taking measurement.

2. Examination of conditions The optimum conditions for quantifying the biological activity of anti-PTHrP antibodies were examined using the above test method. As PTHrP-SEAP, PTHrP (1-34) -SEAP prepared in Example 1 was used.
(1) PTH / PTHrP receptor-expressing cell line
As PTH / PTHrP receptor-expressing cells, HkRk-B7 and HkRk-B28 cells (both human PTH receptor transfected LLC-PK1 cell (Takasu K, Guo J, and Bringhurst FR. J Bone Miner Res 1999; 14 (1)): 11-20): introduced from Massachusetts General Hospital) using LLC-PK1 cells (PTH / PTHrP receptor (-)) as a control. As a result, the binding of PTHrP-SEAP to HkRk-B28 was about 40% of the binding to HkRk-B7 (FIG. 9). This was considered to depend on the expression level of PTH / PTHrP receptor (HkRk-B7; 950 × 10 ³ / cell, HkRk-B28; 280 × 10 ³ / cell) in each cell. From the viewpoint of detection sensitivity in the PTH / PTHrP receptor binding assay, it seemed preferable to select HkRk-B7 cells with high expression levels of PTH / PTHrP receptors and high PTHrP-SEAP binding levels.

(2) Number of seeded cells When seeded on a 96-well plate at 2.5 × 10 ⁴ cells / well, seeded at 5 × 10 ⁴ cells / well instead of confluent after 1 day culture, almost after 1 day culture It was confluent. Therefore, it was determined that the seeding number was 5 × 10 ⁴ cells / well that would become confluent after 1 day of culture.

(3) Reaction Time / Temperature First, when the reaction temperature was 4 ° C. and the receptor binding amount of PTHrP-SEAP after 2 hours was measured, receptor-specific binding was not detected. Therefore, when the reaction conditions were changed to room temperature-30 min, receptor-specific binding was detected (FIG. 6). When the reaction conditions were set at 16 ° C.-60 min, PTH / PTHrP receptor-specific binding could be detected as at room temperature-30 min. From the above results, it was revealed that the reaction temperature is important in the receptor binding assay by PTHrP-SEAP. In addition, receptor-specific binding of PTHrP-SEAP was detectable from 16 ° C to room temperature. Moreover, it was detectable in 30-60 min at room temperature. Therefore, it was judged that it was appropriate to set the reaction conditions at room temperature and 30 min.

(4) Buffer
When the binding reaction between PTH / PTHrP receptor and PTHrP was compared with 10% FCS-DMEM, Binding buffer, and 1% BSA-HBSS medium, the binding amount was approximately Two times higher (Figure 7). Therefore, it was considered appropriate to select Binding buffer or 1% BSA-HBSS. On the other hand, when the adsorptivity of PTHrP-SEAP to the container was examined, it was confirmed that 1% BSA-HBSS was significantly higher than 10% FCS-DMEM and Binding buffer. Based on the above, it was determined that selecting a binding buffer as a binding buffer was appropriate.

(5) PTHrP-SEAP addition amount
When detecting the amount of inhibition of binding by an anti-PTHrP antibody in the PTH / PTHrP receptor binding assay, the amount of label added should be set so as not to be supersaturated with respect to the receptor. When the amount of PTHrP (1-34) -SEAP added was examined at 1.25 to 10 nmol / L, the amount bound to the receptor increased depending on the concentration of the label (Fig. 8). It was considered not saturated. PTHrP-SEAP specifically bound to PTH / PTHrP receptor-expressing cells at each concentration. Therefore, it was determined that the addition amount of PTHrP (1-34) 2 -SEAP should be 5 nmol / L which can detect the binding sufficiently and the receptor is not saturated.

(6) Amount of anti-PTHrP antibody added The conditions for quantifying the PTHrP-SEAP binding inhibitory activity of the anti-PTHrP antibody under the conditions set above were examined. The binding of PTHrP-SEAP was inhibited depending on the concentration of anti-PTHrP antibody added. From FIG. 9, PTHrP PTH / PTHrP by anti-PTHrP antibody in the range of anti-PTHrP antibody concentration 0.125 to 1.0 μg / mL
It was considered that the binding inhibitory activity to the receptor could be quantified.

(7) Specificity of measurement sample
The amount of PTHrP-SEAP binding when a protein not specifically binding to PTHrP was added to the PTH / PTHrP receptor binding assay system was examined. When human IgG was added as a protein having no specific binding ability, PTHrP-SEAP binding was not inhibited in the range of 0.125 to 10 μg / mL (FIG. 10). From this, it was confirmed that the anti-PTHrP antibody inhibits the binding to the receptor by specifically binding to PTHrP-SEAP.

[Example 3] Comparison of PTH / PTHrP receptor binding assay and cAMP assay
The reproducibility of the anti-PTHrP antibody biological activity evaluation method using the PTH / PTHrP receptor binding assay or the cAMP assay was evaluated. Each test method was measured four times on different measurement days, and from the results, accuracy and indoor reproduction accuracy were calculated using 4-parameter fitting, and efficacy ratio was calculated using parallel line assay.

1. Test method (1) PTH / PTHrP receptor binding assay
Prepare HkRk-B7 cells with 10% FCS-DMEM medium to 5x10 ⁵ cells / mL.
Each microliter / well was added to a 96-well plate and cultured at 37 ° C. and 5% CO _{2 for} 1 day.

  PTHrP (1-34) -SEAP was prepared to 10 nmol / L with the above binding buffer. A standard anti-PTHrP antibody (Lot No. 99B01: WO98 / 13388) was prepared at 0.13, 0.25, 0.50, 1.0, 2.0, and 4.0 μg / mL with Binding buffer, respectively. A sample anti-PTHrP antibody (manufactured by CBSI) was prepared in a binding buffer at 0.25, 0.50, 1.0, and 2.0 μg / mL, respectively.

After culturing HkRk-B7 cells for 1 day, 200 μL / well of binding buffer was added once to wash the plate. Anti-PTHrP antibody and PTHrP (1-34) -SEAP (10 nmol / L) prepared at each concentration were mixed in equal amounts and added to 3 wells at 50 μL / well.

The plate was allowed to stand at room temperature for 30 min, and then binding buffer was added three times at 200 μL / well for washing. PTHrP-SEAP bound to the receptor was detected using the Phospha-Light System (Applied Biosystems). 1 x Dilution buffer-0.02% Triton X-100 was added at 150 μL / well, the plate was heat-treated at 65 ° C for 30 min, cooled on ice, and the sample was transferred to a 96-well black plate at 50 μL / well x duplicate . Assay buffer 50 μL / well was added to this and allowed to stand at room temperature for 5 min, then Reaction buffer 50 μL / well was further added and allowed to stand at room temperature for 15 to 20 min, and the amount of luminescence was measured using ARVO. The average value of duplicate was defined as the binding amount (RLU) of each sample.

(2) cAMP assay
HkRk-B7 cells were prepared at 5 × 10 ⁵ cells / mL using 10% FCS-DMEM medium. This cell suspension was added to a 96-well plate at 100 μL / well and cultured at 37 ° C. under 5% CO _{2 for} 1 day. PTHrP (1-34)
Was prepared to 2 nmol / L with 10% FCS-DMEM.
A standard anti-PTHrP antibody (Lot No. 99B01: WO98 / 13388) was prepared at 0.50, 1.0, 2.0, 4.0, 8.0, and 16 μg / mL with 2 mmol / L IBMX-10% FCS-DMEM, respectively. sample Anti-PTHrP antibody (CBSI
Were prepared with 2 mmol / L IBMX-10% FCS-DMEM to 1.0, 2.0, 4.0, and 8.0 μg / mL, respectively.
HkRk-B7 cells were cultured for 1 day and then washed once with PBS at 100 μL / well. An equal amount of anti-PTHrP antibody and PTHrP (1-34) (2 nmol / L) prepared at each concentration were mixed and added to 3 wells at 50 μL / well.

Let the plate stand at room temperature for 30 min, then add 2 x Lysis buffer at 50 μL / well.
For 30 min at room temperature. 20 μL of each sample was diluted with 60 μL of 1 × Lysis buffer, and cAMP content was measured using HTRF Cyclic AMP kit.

2. Analysis result
The results of 4-parameter fitting are shown in Table 1 (PTH / PTHrP receptor binding assay) and Table 2 (cAMP).
Assay).

1) The accuracy was 98.0 to 10% (PTH / PTHrP receptor binding assay) and 95.2 to 111% (cAMP assay), and no significant difference was observed between the two test methods.
2) Indoor reproduction system The indoor reproduction accuracy is ~ 8.35% (PTH / PTHrP receptor binding assay, anti-PTHrP antibody 0.25-1.0 μg / mL), ~ 17.1% (cAMP assay, anti-PTHrP antibody 2.0 μg / mL), The PTH / PTHrP receptor binding assay was considered to be more accurate.
3) parallel line assay
The results of parallel line assay are shown in Table 3. When analysis was performed using three anti-PTHrP antibody concentrations (0.13, 0.25, 0.50 μg / mL) in the PTH / PTHrP receptor binding assay, non-parallelism was significant in one out of four measurements. In the measurement results of three times when the parallel line test was established, the efficacy ratio averaged 1.19, CV (%) 6.27%, and the 95% confidence interval of the efficacy ratio was 1.06 to 1.35.

When the cAMP assay was analyzed with 3 anti-PTHrP antibody concentrations (1.0, 2.0, 4.0 μg / mL), non-parallelism became significant at 2/4. Therefore, when the anti-PTHrP antibody concentration was analyzed as 4 concentrations (0.5, 1.0, 2.0, 4.0 μg / mL), 1/4 was significant in non-parallelism. At this time,
The efficacy ratio averaged 0.939, CV (%) 27.5%, and the 95% confidence interval for efficacy ratio was 0.661-1.34.

From the analysis results of the parallel line assay, the PTH / PTHrP receptor binding assay had a smaller coefficient of variation in potency ratio and the width of the 95% confidence interval of potency ratio than the cAMP assay. When the analysis was performed using the parallel line assay, the test could fail in any of the test methods. When expressing the biological activity of an anti-PTHrP antibody as an efficacy ratio, it was considered preferable to measure it using a PTH / PTHrP receptor binding assay with a small coefficient of variation and a 95% confidence interval.

3. Conclusion The PTH / PTHrP receptor binding assay as a method for evaluating the biological activity of anti-PTHrP antibodies was superior in terms of indoor reproducibility and potency compared to the cAMP assay.

[Example 4] Biological activity measurement of accelerated anti-PTHrP antibody (PTH / PTHrP receptor binding assay and cAMP)
Assay comparison)
1. Test Method 1) PTH / PTHrP Receptor Binding Assay The test was performed according to the methods described in Examples 2 and 3.
2) cAMP assay
HkRk-B7 cells were prepared at 5 × 10 ⁵ cells / mL using 10% FCS-DMEM medium. This cell suspension was added to a 96-well plate at 100 μL / well and cultured at 37 ° C. under 5% CO _{2 for} 1 day.

PTHrP (1-34) was prepared to 2 nmol / L with 2 mmol / L IBMX-10% FCS-DMEM.
The standard anti-PTHrP antibody was prepared with 10% FCS-DMEM to 0.50, 1.0, 2.0, 4.0, and 8.0 μg / mL, respectively. The sample anti-PTHrP antibody was prepared with 10% FCS-DMEM to 0.50, 1.0, 2.0, 4.0, and 8.0 μg / mL, respectively.

  HkRk-B7 cells were cultured for 1 day and then washed once with 100 μL / well of PBS. A standard or sample anti-PTHrP antibody prepared at each concentration was mixed with PTHrP (1-34) (2 nmol / L) in an equal amount, and this was added at 50 μL / well in triplicate.

The plate was allowed to stand at room temperature for 30 min, 2 x Lysis buffer was added at 50 μL / well, and the plate was left at room temperature for 30 min. 20 μL of each sample was diluted with 60 μL of 1 × Lysis buffer, and cAMP content was measured using HTRF Cyclic AMP kit.
For cAMP standard, dilute the cAMP maximum calibrator supplied with the kit with 1 x Lysis buffer,
Prepared.

To a 384 well black plate, 1 × Lysis buffer (Negative control, Positive control), a sample, and cAMP standard were added at 10 μL / well in duplicate. To this, anti-cAMP Ab-cryptate conjugate was added at 5 μL / well and cAMP-XL665 conjugate was added at 5 μL / well. However, only negative control was added 5 μL / well of distilled water instead of cAMP-XL665 conjugate. After leaving the plate at room temperature for 3 hr, the fluorescence intensity at 665 nm and 620 nm was measured. The average value of the duplicate of each sample was used as a measurement value of fluorescence intensity.
About the measurement result of the sample and cAMP standard, DF / DF max% was calculated | required with the following formulas.

  A calibration curve was created from the cAMP concentration of cAMP standard and DF / DF max%, and the amount of cAMP contained in the sample was determined.

2. analysis method
The test results were analyzed as follows using "Parallel line test and efficacy ratio" of SAS program.
1) PTH / PTHrP receptor binding assay Efficacy ratio of anti-PTHrP antibody accelerated product (sample: Initial, 50 ° C-1M) to anti-PTHrP antibody standard (standard: anti-PTHrP antibody original, Lot No. BG00102) and 95% The confidence interval was determined by parallel line test from the anti-PTHrP antibody concentration (μg / mL) and the binding amount (RLU) for each of three concentrations of 0.13, 0.25 and 0.50 (μg / mL) of standard and sample.

  The efficacy ratio and 95% confidence interval of the anti-PTHrP antibody accelerated product (sample: 50 ° C-2M, 50 ° C-3M) to the anti-PTHrP antibody standard product (standard: anti-PTHrP antibody substance, Lot No. BG00102) is standard 0.13 , 0.25, 0.50 (μg / mL) and samples 0.18, 0.35, 0.71 (μg / mL), respectively, were determined from the anti-PTHrP antibody concentration (μg / mL) and the amount of binding (RLU) by a parallel line test.

2) cAMP assay Accelerated anti-PTHrP antibody (sample: Initial, 50 ° C-1M, 50 ° C-2M, 50 ° C-3M) against anti-PTHrP antibody standard (standard: anti-PTHrP antibody, Lot No. BG00102) Efficacy ratio and 95% confidence interval are parallel from anti-PTHrP antibody concentration (μg / mL) and cAMP amount (pg / well) for 4 concentrations of 0.50, 1.0, 2.0, and 4.0 (μg / mL) for standard and sample, respectively. Obtained by line test.

3. Measurement results Using the PTH / PTHrP receptor binding assay and cAMP assay set as described above, the activity of the anti-PTHrP antibody accelerated product (50 ° C-1M, 50 ° C-2M, 50 ° C-3M) and its initial activity was measured 4 times. Measured one by one. About the obtained result, the potency ratio and 95% confidence interval for the anti-PTHrP antibody standard were calculated by parallel line test. In both test methods, the parallel line test was established for all samples, and the potency ratio could be calculated. The results are shown in Table 4 (PTH / PTHrP receptor binding assay) and Table 5 (cAMP assay).

  The potency ratio of the anti-PTHrP antibody accelerated product Initial calculated by the PTH / PTHrP receptor binding assay was 1.07, and the potency ratio of the anti-PTHrP antibody accelerated product 50 ° C.-1M was 1.00, and no significant difference was observed. The efficacy ratios of the anti-PTHrP antibody accelerated products 50 ° C.-2M and 50 ° C.-3M were 0.801 and 0.629, respectively, and the efficacy ratio tended to decrease with time. On the other hand, the efficacy ratio of the anti-PTHrP antibody accelerated product Initial calculated by cAMP assay is 0.983, and the efficacy ratio of the anti-PTHrP antibody accelerated product 50 ° C.-1M is 0.981, as in the PTH / PTHrP receptor binding assay. There was no significant difference in potency ratio between Initial and 50 ℃ -1M. The efficacy ratios of the accelerated anti-PTHrP antibody products 50 ° C-2M and 50 ° C-3M were 0.747 and 0.521, respectively, and the activity tended to decrease over time.

When comparing the two test methods, the efficacy ratios of the anti-PTHrP antibody accelerated products 50 ° C-1M, 50 ° C-2M, and 50 ° C-3M were calculated to the same degree. A decrease could be detected.
The indoor reproducibility (CV%) of potency ratio measured 4 times for each test method was about 8% for the PTH / PTHrP receptor binding assay and about 22% for the cAMP assay.

  According to the present invention, the binding activity between a ligand and a native ligand-binding protein expressed on a cell membrane can be quantified with high sensitivity and high accuracy without using RI. That is, by using the method and kit of the present invention, it is possible to accurately screen for an agonist or antagonist for a ligand receptor and to measure biological activity.

  Therefore, the evaluation method of the present invention includes the expression cloning of receptors, screening of ligands such as low molecular weight compounds acting on the receptors, the interaction between adhesion molecules on the cell membrane surface and the biological activities of various compounds acting on them. It can be used for evaluation and screening.

FIG. 1 shows the structures of pCHOI-PTHrP (1-34) -SEAP (1A) and pCHOI-PTHrP (1-141) -SEAP (1B). FIG. 2 is a graph showing purification of PTHrP-SEAP using a His-tag affinity column. FIG. 3 is a graph showing the PTH / PTHrP receptor binding activity of PTHrP-SEAP. FIG. 4 is a graph showing cAMP induction (HkRk-B7 cells) by PTHrP-SEAP. FIG. 5 shows the electrophoresis results of PTHrP (1-34) -SEAP and PTHrP (1-141) -SEAP. FIG. 6 is a graph showing the effect of temperature in the PTH / PTHrP receptor binding assay. FIG. 7 is a graph showing the effect of buffer in the PTH / PTHrP receptor binding assay. FIG. 8 is a graph showing the PTH / PTHrP receptor binding activity of PTHrP (1-34) -SEAP. FIG. 9 is a graph showing the effect of anti-PTHrP antibody concentration in the PTH / PTHrP receptor binding assay. FIG. 10 is a graph showing specific inhibition of PTH / PTHrP receptor binding by anti-PTHrP antibodies.

SEQ ID NO: 1-description of artificial sequence: primer SEQ ID NO: 2-description of artificial sequence: primer SEQ ID NO: 3- description of artificial sequence: primer SEQ ID NO: 4- description of artificial sequence: primer SEQ ID NO: 5- description of artificial sequence: primer

Claims (9)

Using the amount of PTH / PTHrP receptor expressed on the cell membrane and PTHrP - human placental alkaline phosphatase fusion protein as an index, the biological activity of the PTHrP binding inhibitor is 98.0 to 108% as a true and indoor reproduction. A method of evaluating at 8.35% or less as a property. The binding amount of the PTH / PTHrP receptor and PTHrP - human placental alkaline phosphatase fusion protein is determined by measuring the amount of chemiluminescence mediated by human placental alkaline phosphatase in the PTHrP - human placental alkaline phosphatase fusion protein. The method of claim 1. As PTH / PTHrP receptor expressed on the cell membrane, using a PTH / PTHrP receptor-expressing cells, according to claim 1 or 2 wherein. The method of claim 1 comprising the following steps:
1) reacting PTHrP - human placental alkaline phosphatase fusion protein with immobilized PTH / PTHrP receptor- expressing cells in the presence of a test substance;
2) After removing free PTHrP - human placental alkaline phosphatase fusion protein, human placental alkaline phosphatase substrate is added and the amount of luminescence is measured;
3) Based on the measured amount of luminescence, the biological activity of the test substance as a PTHrP binding inhibitor is evaluated as 98.0 to 108% as the trueness and 8.35% or less as the indoor reproducibility. PTHrP binding inhibition substance is an agonist or antagonist of PTHrP antibody or against PTHrP receptor, The method according to any one of claims 1 to 4. PTH / PTHrP receptor expression cell is a PTH / PTHrP recombinant cell receptor forced high expression method according to any one of claims 1-5. The method according to any one of claims 1 to 6 , wherein the PTHrP binding inhibitor is an anti-PTHrP antibody. A kit for evaluating the biological activity of a PTHrP binding inhibitor containing 98.0 to 108% as true and 8.35% or less as indoor reproducibility, including the following 1) to 3).
1) immobilized PTH / PTHrP receptor- expressing cells 2) PTHrP - human placental alkaline phosphatase fusion protein 3) PTHrP binding inhibitor The kit according to claim 8 , wherein the PTHrP binding inhibitor is an anti-PTHrP antibody.

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