METHOD OF MODULATING THE ACTIVITY OF FUNCTIONAL IMMUNE MOLECULES
20170240647 · 2017-08-24
Assignee
Inventors
- Nobuo HANAI (Machida-shi, JP)
- Kazuyasu NAKAMURA (Machida-shi, JP)
- Emi HOSAKA (Machida-shi, JP)
- Motoo YAMASAKI (Machida-shi, JP)
- Kazuhisa UCHIDA (Machida-shi, JP)
- Toyohide SHINKAWA (Machida-shi, JP)
- Susumu IMABEPPU (Machida-shi, JP)
- Yutaka KANDA (Machida-shi, JP)
- Naoko YAMANE (Machida-shi, JP)
- Hideharu Anazawa (Chiyoda-ku, JP)
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
A61P31/00
HUMAN NECESSITIES
C07K16/2866
CHEMISTRY; METALLURGY
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K2317/73
CHEMISTRY; METALLURGY
G01N33/53
PHYSICS
A61P37/06
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The invention relates to a method for controlling the activity of an immunologically functional molecule, such as an antibody, a protein, a peptide or the like, an agent of promoting the activity of an immunologically functional molecule, and an immunologically functional molecule having the promoted activity.
Claims
1-4. (canceled)
5. A method for producing an antibody composition, said method comprising: (1) introducing a vector comprising a nucleotide sequence encoding an antibody into a host cell; (2) culturing the antibody-producing host cell; and (3) recovering the antibody from the culture supernatant, wherein said antibody composition comprises antibody molecules having complex-type and/or hybrid-type N-glycoside-linked sugar chain in a Fc region, wherein all of said antibody molecules have two N-glycoside-linked sugar chains in which fucose is not bound (F0).
6. The method according to claim 5, wherein said antibody molecules comprise N-glycoside-linked sugar chains in which at least one N-acetylglucosamine is bound to a mannose at the non-reducing end of the following structure: ##STR00007##
7. The method according to claim 5, wherein said antibody molecules comprise the complex-type N-glycoside-linked sugar chains in which two N-acetylglucosamine are respectively bound to two mannoses at the non-reducing end of the following structure: ##STR00008##
8. The method according to claim 5, wherein said antibody molecules have an increased antibody dependent cellular cytotoxicity (ADCC).
9. The method according to claim 5, wherein said antibody molecules are molecules of a human antibody, a humanized antibody, a chimeric antibody or a human CDR-grafted antibody.
10. The method according to claim 5, wherein said antibody molecules are molecules of an IgG class antibody.
Description
BRIEF EXPLANATION OF THE DRAWINGS
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BEST MODE FOR CARRYING OUT THE INVENTION
Example 1
Production of Anti-Ganglioside GD3 Human Chimeric Antibody:
[0218] 1. Construction of Tandem Expression Vector, pChiLHGM4, for Anti-Ganglioside GD3 Human Chimeric Antibody
[0219] A plasmid, pChi641LGM40, was constructed by ligating a fragment of about 4.03 kb containing an L chain CDNA, obtained by digesting an L chain expression vector, pChi641LGM4 (J. Immunol. Methods, 167, 271 (1994)) for anti-ganglioside GD3 human chimeric antibody (hereinafter referred to as “anti-GD3 chimeric antibody”) with restriction enzymes, MulI (manufactured by Takara Shuzo) and SalI (manufactured by Takara Shuzo), with a fragment of about 3.40 kb containing a G418-resistant gene and a splicing signal, obtained by digesting an expression vector pAGE107 (Cytotechnology, 3, 133 (1990)) for animal cell with restriction enzymes, MulI (manufactured by Takara Shuzo) and SalI (manufactured by Takara Shuzo), using DNA Ligation Kit (manufactured by Takara Shuzo), and then transforming E. coli HB101 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 19B9) with the ligated product using DNA Ligation Kit (manufactured by Takara Shuzo).
[0220] Next, a fragment of about 5.68 kb containing an L chain cDNA, obtained by digesting the constructed plasmid pChi641LGM40 with a restriction enzyme, ClaI (manufactured by Takara Shuzo), blunt-ending it using DNA Blunting Kit (manufactured by Takara Shuzo) and further digesting it with MluI (manufactured by Takara Shuzo), was ligated with a fragment of about 8.40 kb containing an H chain cDNA, obtained by digesting an anti-GD3 chimeric antibody H chain expression vector, pChi641HGM4 (J. Immunol. Methods, 167, 271 (1994)) with a restriction enzyme, XhoI (manufactured by Takara Shuzo), blunt-ending it using DNA Blunting Kit (manufactured by Takara Shuzo) and further digesting it with MluI (manufactured by Takara Shuzo), using DNA Ligation Kit (manufactured by Takara Shuzo), and then E. coli HB101 (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab. Press, New York, 1989) was transformed with the ligated product to thereby construct a tandem expression vector, pChi641LRGM4, for anti-GD3 chimeric antibody.
2. Production of Cells Stably Producing Anti-GD3 Chimeric Antibody
[0221] Using the tandem expression vector, pChi641LHGM4, for anti-GD3 chimeric antibody constructed in the item 1 of Example 1, cells capable of stably producing an anti-GD3 chimeric antibody were prepared as described below.
(1) Production of Producer Cell Using Rat Myeloma YB2/0 Cell
[0222] After introducing 5 μg of the anti-GD3 chimeric antibody expression vector, pChi641LHGM4, into 4×10.sup.6 cells of rat myeloma YB2/0 by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 40 ml of RPMI1640-FBS(10) (RPMI1640 medium containing 10% FBS (manufactured by GIBCO BRL)) and dispensed in 200 μl/well into a 96 well culture plate (manufactured by Sumitomo Bakelite). Twenty-four hours after culturing at 37° C. in a 5% CO.sub.2 incubator, G418 was added to a concentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from respective well in which colonies of transformants showing G418 resistance were formed and growth of colonies was observed, and the antigen binding activity of the anti-GD3 chimeric antibody in the supernatant was measured by the ELISA shown in the item 3 of Example 1.
[0223] Regarding the transformants in wells in which production of the anti-GD3 chimeric antibody was observed in culture supernatants, in order to, increase amount of the antibody production using a DHFR gene amplification system; each of them was suspended in the RPMI1640-FBS(10) medium containing 0.5 mg/ml of G418 and 50 nM DHFR inhibitor, methotrexate (hereinafter referred to as “MTX”; manufactured by SIGMA) to give a density of 1 to 2×10.sup.5 cells/ml, and the suspension was dispensed in 2 ml into wells of a 24 well plate (manufactured by Greiner). Transformants showing 50 nM MTX resistance were induced by culturing at 37° C. for 1 to 2 weeks in a 5% CO.sub.2 incubator. The antigen binding activity of the anti-GD3 chimeric antibody in culture supernatants in wells in which growth of transformants was observed was measured by the ELISA shown in the item 3 of Example 1. Regarding the transformants in wells in which production of the anti-GD3 chimeric antibody was observed in culture supernatants, the MTX concentration was increased to 100 nM and then to 200 nM, and a transformant capable of growing in the RPMI1640-FBS(10) medium containing 0.5 mg/ml of G418 and 200 nM MTX and of producing the anti-GD3 chimeric antibody in a large amount was finally obtained by the same method as described above. The obtained transformant was made into a single cell (cloning) by limiting dilution twice.
[0224] The obtained anti-GD3 chimeric antibody-producing transformed cell clone 7-9-51 has been deposited on Apr. 5, 1999, as FERM BP-6691 in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba, Ibaraki, Japan).
(2) Production of Producer Cell Using CHO/DG44 Cell
[0225] After introducing 4 μg of the anti-GD3 chimeric antibody expression vector, pChi641LHGM4, into 1.6×10.sup.6 cells of CHO/DG44 by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 10 ml of IMDM-FBS(10) (IMDM medium containing 10% FBS and 1× concentration of HT supplement (manufactured by GIBCO BRL)) and dispensed in 200 μl/well into a 96 well culture plate (manufactured by Iwaki Glass). Twenty-four hours after culturing at 37° C. in a 5% CO.sub.2 incubator, G418 was added to a concentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from respective well in which colonies of transformants showing G418 resistance were formed and growth of colonies was observed, and the antigen binding activity of the anti-GD3 chimeric antibody in the supernatant was measured by the ELISA shown in the item 3 of Example 1.
[0226] Regarding the transformants in wells in which production of the anti-GD3 chimeric antibody was observed in culture supernatants, in order to increase amount of the antibody production using a DHFR gene amplification system, each of them was suspended in an IMDM-dFBS(10) medium (IMDM medium containing 10% dialyzed fetal bovine serum (hereinafter referred to as “dFBS”; manufactured by GIBCO BRL)) containing 0.5 mg/ml of G418 and 10 nM MTX to give a density of 1 to 2×10.sup.5 cells/ml, and the suspension was dispensed in 0.5 ml into wells of a 24 well plate (manufactured by Iwaki Glass). Transformants showing 10 nM MTX resistance were induced by culturing at 37° C. for 1 to 2 weeks in a 5% CO.sub.2 incubator. Regarding the transformants in wells in which their growth was observed, the MTX concentration was increased to 100 nM, and a transformant capable of growing in the IMDN-dPBS(10) medium containing 0.5 mg/ml of G418 and 100 nM MTX and of producing the anti-GD3 chimeric antibody in a large amount was finally obtained by the same method as described above. The obtained transformant was made into a single cell (cloning) by limiting dilution twice.
(3) Production of Producer Cell Using Mouse Myeloma NS0 Cell
[0227] After introducing 5 μg of the anti-GD3 chimeric antibody expression vector pChi64ILHGM4 into 4×10.sup.6 cells of mouse myeloma NS0 by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 40 ml of EX-CELL302-FBS(10) (EX-CELL302 medium containing 10% FBS and 2 mM L-glutamine (hereinafter referred to as “L-Gln”; manufactured by GIBCO BRL)) and dispensed in 200 μl/well into a 96 well culture plate (manufactured by Sumitomo Bakelite). Twenty-four hours after culturing at 37° C. in a 5% CO.sub.2 incubator, G418 was added to a concentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from respective well in which colonies of transformants showing G418 resistance were formed and growth of colonies was observed, and the antigen binding activity of the anti-GD3 chimeric antibody in the supernatant was measured by the ELISA shown in the item 3 of Example 1.
[0228] Regarding the transformants in wells in which production of the anti-GD3 chimeric antibody was observed in culture supernatants, in order to increase amount of the antibody production using a DHFR gene amplification system, each of them was suspended in an EX-CELL302-dFBS(10) medium (EX-CELL302 medium containing 10% dFBS and 2 mM L-Gln) containing 0.5 mg/ml of G418 and 50 nM MTX to give a density of 1 to 2×10.sup.5 cells/ml, and the suspension was dispensed in 2 ml; into wells of a 24 well plate (manufactured by Greiner). Transformants showing 50 nM MTX resistance were induced by culturing at 37° C. for 1 to 2 weeks in a 5% CO.sub.2 incubator. The antigen binding activity of the anti-GD3 chimeric antibody in culture supernatants in wells in which growth of transformants was observed was measured by the ELISA shown in the item 3 of Example 1. Regarding the transformants in wells in which production of the anti-GD3 chimeric antibody was observed in culture supernatants, the MTX concentration was increased to 100 nM and then to 200 nM, and a transformant capable of growing in the EX-CELL302-dFBS(10) medium containing 0.5 mg/ml of G418 and 200 nM MTX and of producing the anti-GD3 chimeric antibody in a large amount was finally obtained by the same method as described above. The obtained transformant was made into a single cell (cloning) by limiting dilution twice.
3. Measurement of Binding Activity of Antibody to GD3 (ELISA)
[0229] The binding activity of the antibody to GD3 was measured as described below.
[0230] In 2 ml of ethanol solution containing 10 μg of dipalmitoylphosphatidylcholine (manufactured by SIGMA) and 5 μg of cholesterol (manufactured by SIGMA), 4 nmol of GD3 was dissolved. Into each well of a 96 well plate for ELISA (manufactured by Greiner), 20 μl of the solution (40 pmol/well in final concentration) was dispensed, followed by air-drying, 1% bovine serum albumin (hereinafter referred to as “BSA”; manufactured by SIGMA)-containing PBS (hereinafter referred to as “1% BSA-PBS”) was dispensed in 100 μl/well, and then the reaction was carried out at room temperature for 1 hour for blocking remaining active groups. After discarding 1% BSA-PBS, a culture supernatant of a transformant or a diluted solution of a human chimeric antibody was dispensed in 50 μl/well to carry out the reaction at room temperature for 1 hour. After the reaction, each well was washed with 0.05% Tween 20 (manufactured by Wake Pure Chemical Industries)-containing PBS (hereinafter referred to as “Teen-PBS”), a peroxidase-labeled goat anti-human IgG (H & L) antibody solution (manufactured by American Qualex) diluted 3,000 times with 1% BSA-PBS was dispensed in 50 μl/well as a secondary antibody solution, and then the reaction was carried out at room temperature for 1 hour. After the reaction and subsequent washing with Tween-PBS, ABTS substrate solution (a solution prepared by dissolving 0.55 g of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2) and adding 1 μl/ml of hydrogen peroxide to the solution just before use) was dispensed in 50 μl/well for color development, and then absorbance at 415 nm (hereinafter referred to as “OD415”) was measured.
4. Purification of Anti-GD3 Chimeric Antibody
(1) Culturing of YB2/0 Cell-Derived Producer Cell and Purification of Antibody
[0231] The anti-GD3 chimeric antibody-producing transformed cell clone obtained in the above item 2 (1) of Example 1 was suspended in the Hybridoma-SFM Medium containing 0.2% BSA, 200 nM MTX and 100 nM triiodothyronine (hereinafter referred to as “T3”; manufactured by SIGMA) to give a density of 3×10.sup.5 cells/ml and cultured using a 2.0 liter capacity spinner bottle (manufactured by Iwaki Glass) under agitating at a rate of 50 rpm. Ten days after culturing at 37° C. in a temperature-controlling room, the culture supernatant was recovered. The anti-GD3 chimeric antibody was purified from the culture supernatant using a Prosep-A (manufactured by Bioprocessing) column in accordance with the manufacture's instructions. The purified anti-GD3 chimeric antibody was named YB2/0-GD3 chimeric antibody.
(2) Culturing of CHO/DG44 Cell-Derived Producer Cell and Purification of Antibody
[0232] The anti-GD3 chimeric antibody-producing transformed cell clone obtained in the above item 2(2) of Example 1 was suspended in the EX-CELL302 medium containing 3 mM L-Gln, 0.5% fatty acid concentrated solution (hereinafter referred to as “CDLC”; manufactured by GIBCO BRL) and 0.3% Pluronic F68 (hereinafter referred to as “PF68”; manufactured by GIBCO BRL) to give a density of 1×10.sup.4 cells/ml, and the suspension was dispensed in 50 ml into 175 mm.sup.2 flasks (manufactured by Greiner). Four days after culturing at 37° C. in a 5% CO.sub.2 incubator, the culture supernatant was recovered. The anti-GD3 chimeric antibody was purified from the culture supernatant using a Prosep-A (manufactured by Bioprocessing) column in accordance with the manufacture's instructions. The purified anti-GD3 chimeric antibody was named CHO/DG44-GD3 chimeric antibody.
(3) Culturing of NS0 Cell-Derived Producer Cell and Purification of Antibody
[0233] The anti-GD3 chimeric antibody-producing transformed cell clone obtained in the above item 2(3) of Example 1 was suspended in the EX-CELL302 medium containing 2 mM L-Gln, 0.5 mg/ml of G418, 200 nM MTX and 1% FBS, to give a density of 1×10.sup.6 cells/ml, and the suspension was dispensed in 200 ml into 175 mm.sup.2 flasks (manufactured by Greiner). Four days after culturing at 37° C. in a 5% CO.sub.2 incubator, the culture supernatant was recovered. The anti-GD3 chimeric antibody was purified from the culture supernatant using a Prosep-A (manufactured by Bioprocessing) column in accordance with the manufacture's instructions. The purified anti-GD3 chimeric antibody was named NS0-GD3 chimeric antibody (302). Also, the transformed cell clone was suspended in the GIT medium containing 0.5 mg/ml of G418 and 200 nM MTX to give a density of 3×10.sup.5 cells/ml, and the suspension was dispensed in 200 ml into 175 mm.sup.2 flasks (manufactured by Greiner). Ten days after culturing at 37° C. in a 5% CO.sub.2 incubator, the culture supernatant was recovered. The anti-GD3 chimeric antibody was purified from the culture supernatant using a Prosep-A (manufactured by Bioprocessing) column in accordance with the manufacture's instructions. The purified anti-GD3 chimeric antibody was named NS0-GD3 chimeric antibody (GIT).
(4) Culturing of SP2/0 Cell-Derived Producer Cell and Purification of Antibody
[0234] The anti-GD3 chimeric antibody-producing transformed cell clone described in Japanese Published Unexamined Patent Application No. 304989/93 was suspended in the GIT medium containing 0.5 mg/ml of G418 and 200 nM MTX to give a density of 3×10.sup.5 cells/ml and the suspension was dispensed in 200 ml into 175 mm.sup.2 flasks (manufactured by Greiner). Eight days after culturing at 37° C. in a 5% CO.sub.2 incubator, the culture supernatant was recovered. The anti-GD3 chimeric antibody was purified from the culture supernatant using a Prosep-A (manufactured by Bioprocessing) column in accordance with the manufacture's instructions. The purified anti-GD3 chimeric antibody was named SP2/0-GD3 chimeric antibody.
5. Analysis of the Purified Anti-CD3 Chimeric Antibodies
[0235] In accordance with a known method (Nature, 227, 680, 1970), 4 μg of each of the five anti-GD3 chimeric antibodies produced by and purified from respective animal cells, obtained in the above item 4 of Example 1, was subjected to SDS-PAGE to analyze the molecular weight and purification degree. The results are shown in
Example 2
Activity Evaluation of Anti-GD3 Chimeric Antibody:
1. Binding Activity of Anti-GD3 Chimeric Antibodies to GD3 (ELISA)
[0236] The activity of the five purified anti-GD3 chimeric antibodies obtained in the above item 4 of Example 1 to bind to GD3 (manufactured by Snow Brand Milk Products} was measured by the ELISA shown in the item 3 of Example 1.
2. In Vitro Cytotoxic Activity (ADCC Activity) of Anti-GD3 Chimeric Antibody
[0237] In order to evaluate in vitro cytotoxic activity of the five purified anti-GD3 chimeric antibodies obtained in the above item 4 of Example 1, the ADCC activity was measured in accordance with the following method.
(1) Preparation of Target Cell Solution
[0238] A human melanoma cultured cell line G-361 (ATCC CRL 1424) was cultured using the RPMI1640-FBS(10) medium to prepare. 1×10.sup.6 cells, and the cells were radioisotope-labeled by reacting them with 3.7 MBq equivalents of a radioactive substance Na.sub.2.sup.51CrO.sub.4 at 37° C. for 1 hour. After the reaction, the cells were washed three times through their suspension in the RPMI1640-FBS(10) medium and centrifugation, re-suspended in the medium and then allowed to stand at 4° C. for 30 minutes in ice for spontaneous dissolution of the radioactive substance. After centrifugation, the precipitate was adjusted to 2×10.sup.5 cells/ml by adding 5 ml of the RPMI1640-FBS (10) medium and used as the target cell solution.
(2) Preparation of Effector Cell Solution
[0239] From a healthy person, 50 ml of vein blood was collected, and gently mixed with 0.5 ml of heparin sodium (manufactured by Takeda Pharmaceutical). The mixture was centrifuged to isolate a mononuclear cell layer using Lymphoprep (manufactured by Nycomed Pharma AS) in accordance with the manufactures instructions. After washing with the RPMI1640-FDS(10) medium by centrifugation three times, the resulting precipitate was re-suspended to give a density of 2×10.sup.6 cells/ml using the medium and used as the effector cell solution.
(3) Measurement of ADCC Activity
[0240] Into each well of a 96 well U-shaped bottom plate (manufactured by Falcon), 50 of the target cell solution prepared in the above (1) (1×10.sup.4 cells/well) was dispensed. Next, 100 μl of the effector cell solution prepared in the above (2) was added thereto (2×10.sup.5 cells/well, the ratio of effector cells to target cells becomes 20:1). Subsequently, each of the anti-GD3 chimeric antibodies was added to give a final concentration from 0.0025 to 2.5 μg/ml, followed by reaction at 37° C. for 4 hours. After the reaction, the plate was centrifuged, and the amount of .sup.51Cr in the supernatant was measured using a 7-counter. The amount of spontaneously released .sup.51Cr was calculated by the same operation using only the medium instead of the effector cell solution and the antibody solution and measuring the amount of .sup.51Cr in the supernatant. The amount of total released .sup.51Cr was calculated by the same operation using only the medium instead of the antibody solution and adding 1 N hydrochloric acid instead of the effector cell solution, and measuring the amount of .sup.51Cr in the supernatant. The ADCC activity was calculated from the following equation.
[0241] The results are shown in
Example 3
Production of Anti-Human Interleukin 5 Receptor a Chain Human CDR-Grafted Antibody:
1. Production of Cells Stably Producing Anti-Human Interleukin 5 Receptor a Chain Human CDR-Grafted Antibody
(1) Production of Producer Cell Using Rat Myeloma YB2/0 Cell
[0242] Using the anti-human interleukin 5 receptor a chain human CDR-grafted antibody (hereinafter referred to as “anti-hIL-5Rα CDR-grafted antibody”) expression vector, pKANTEX1259HV3LV0, described in WO 97/10354, cells capable of stably producing anti-hIL-5Rα CDR-grafted antibody were prepared as described below.
[0243] After introducing 5 μg of the anti-hIL-5Rα CDR-grafted antibody expression vector, pKANTEX1259HV3LV0, into 4×10.sup.6 cells of rat myeloma YB2/0 by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 40 ml of RPMI1640-FBS(10) and dispensed in 200 μl/well into a 96 well culture plate (manufactured by Sumitomo Bakelite). Twenty-four hours after culturing at 37° C. in a 5% CO.sub.2 incubator, G418 was added to give a concentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from respective well in which colonies of transformants showing G418 resistance were formed and growth of colonies was observed, and the antigen binding activity of the anti-hIL-5Rα CDR-grafted antibody in the supernatant was measured by the ELISA shown in the item 2 of Example 3.
[0244] Regarding the transformants in wells in which production of the anti-hIL-5Rα CDR-grafted antibody was observed in culture supernatants, in order to increase amount of the antibody production using a DHFR gene amplification system, each of the them was suspended in the RPMI1640-FBS(10) medium containing 0.5 mg/ml of G418 and 50 nM MTX to give a density of 1 to 2×10.sup.5 cells/ml, and the suspension was dispensed in 2 ml into wells of a 24 well plate (manufactured by Greiner). Transformants showing 50 nM MTX resistance were induced by culturing at 37° C. for 1 to 2 weeks in a 5% CO.sub.2 incubator. The antigen binding activity of the anti-hIL-5Rα CDR-grafted antibody in culture supernatants in wells in which growth of transformants was observed was measured by the ELISA shown in the item 2 of Example 3. Regarding the transformants in wells in which production of the anti-hIL-5Rα CDR-grafted antibody was observed in culture supernatants, the MTX concentration was increased to 100 nM and then to 200 nM, and a transformant capable of growing in the RPMI1640-FBS (10) medium containing 0.5 mg/ml of G418 and 200 nM MTX and of producing the anti-hIL-5Rα CDR-grafted antibody in a large amount was finally obtained in the same manner as described above. The obtained transformant was made into a single cell (cloning) by limiting dilution twice. The obtained anti-hIL-5Rα CDR-grafted antibody-producing transformed cell clone No. 3 has been deposited on Apr. 5, 1999, as FERM BP-6690 in National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology (Higashi 1-1-3, Tsukuba, Ibaraki, Japan).
(2) Production of Producer Cell Using CHO/dhfr.SUP.− Cell
[0245] After introducing 4 μg of the anti-hIL-5Rα CDR-grafted antibody expression vector, pKANTEX1259HV3LV0, described in WO 97/10354 into 1.6×10.sup.6 cells of CHO/dhfr.sup.− by electroporation (Cytotechnology, 3, 133 (1990)), the cells were suspended in 10 ml of IMDM-FBS(10) and dispensed in 200 μl/well into a 96 well culture plate (manufactured by Iwaki Glass). Twenty-four hours after culturing at 37° C. in a 5% CO.sub.2 incubator, G418 was added to give a concentration of 0.5 mg/ml, followed by culturing for 1 to 2 weeks. The culture supernatant was recovered from respective well in which colonies of transformants showing G418 resistance were formed and growth of colonies was observed, and the antigen binding activity of the anti-hIL-5Rα CDR-grafted antibody in the supernatant was measured by the ELISA shown in the item 2 of Example 3.
[0246] Regarding the transformants in wells in which production of the anti-hIL-5Rα CDR-grafted antibody was observed in culture supernatants, in order to increase amount of the antibody production using a DHFR gene amplification system, each of the transformants was suspended in an IMDM-dFBS(10) medium containing 0.5 mg/ml of G418 and 10 nM MTX to give a density of 1 to 2×10.sup.5 cells/ml, and the suspension was dispensed in 0.5 ml into wells of a 24 well plate (manufactured by Iwaki Glass). Transformants showing 10 nM MTX resistance were induced by culturing at 37° C. for 1 to 2 weeks in a 5% CO.sub.2 incubator. Regarding the transformants in wells in which their growth was observed, the MTX concentration was increased to 100 nM and then to 500 nM, and a transformant capable of growing in the IMDM-dFBS(10) medium containing 0.5 mg/ml of G418 and 500 nM MTX and of producing the anti-hIL-5Rα CDR-grafted antibody in a large amount was finally obtained in the same manner as described above. The obtained transformant was made into a single cell (cloning) by limiting dilution twice.
(3) Production of Producer Cell Using Mouse Myeloma NS0 Cell
[0247] An anti-hIL-5Rα CDR-grafted antibody expression vector was prepared is accordance with the method of Yarranton et al. (BIO/TECHNOLOGY, 10, 169 (1992)) and using the antibody H chain and L chain cDNA on the anti-hIL-5Rα CDR-grafted antibody expression vector, pKANTEX1259HV3LV0, described in WO 97/10354, and NS0 cell was transformed to obtain a transformant capable of producing the anti-hIL-5Rα CDR-grafted antibody in a large: amount. The obtained transformant was made into a single cell (cloning) by limiting dilution twice.
2. Measurement of Binding Activity of Antibody to hIL-5Rα (ELISA)
[0248] The binding activity of the antibody to hIL-5Rα was measured as described below.
[0249] A solution was prepared by diluting the anti-hIL-5R a mouse antibody, KM1257, described in WO 97/10354 with PBS to give a concentration of 10 μg/ml, and 50 μl of the resulting solution was dispensed into each well of a 96 well plate for ELISA (manufactured by Greiner), followed by reaction at 4° C. for 20 hours. After the reaction, 1% BSA-PBS was dispensed in 100 μl/well, and then the reaction was carried out at room temperature for hour for blocking remaining active groups. After discarding 1% BSA-PBS, a solution prepared by diluting the soluble hIL-5Rα described in WO 97/10354 with 1% BSA-PBS to give a concentration of 0.5 μg/ml was dispensed in 50 μl/well, followed by reaction at 4° C. for 20 hours. After the reaction, each well was washed with Tween-PBS, culture supernatants of transformants or diluted solutions of a purified human CDR-grafted antibodies were dispensed in 50 μg/well to carry out the reaction at room temperature for 2 hours. After the reaction, each well was washed with Tween-PBS, a peroxidase-labeled goat anti-human IgG (H & L) antibody solution (manufactured by American Qualex) diluted 3,000 times with 1% BSA-PBS was dispensed in 50 μl/well as a secondary antibody solution, followed by reaction at room temperature for 1 hour. After the reaction and subsequent washing with Tween-PBS, ABTS substrate solution (a solution prepared by dissolving 0.55 g of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt in 1 liter of 0.1 M citrate buffer (pH 4.2) and adding 1 μl/ml of hydrogen peroxide to the solution just before use) was dispensed in 50 μl/well for color development, and then the absorbance at OD415 was measured.
3. Purification of Anti-hIL-5Rα CDR-Grafted Antibody
(1) Culturing of YB2/0 Cell-Derived Producer Cell and Purification of Antibody
[0250] The anti-hIL-5Rα CDR-grafted antibody-producing transformed cell clone obtained in the above item 1(1) of Example 3 was suspended in the GIT medium containing 0.5 mg/ml of G418 and 200 nM MTx to give a density of 3×10.sup.5 cells/ml and dispensed in 200 ml into 175 mm.sup.2 flasks (manufactured by Greiner). Eight days after culturing at 37° C. in a 5% CO.sub.2 incubator, the culture supernatant was recovered. The anti-hIL-5Rα CDR-grafted antibody was purified from the culture supernatant using ion exchange chromatography and a gel filtration method. The purified anti-hIL-5Rα CDR-grafted antibody was named YB2/0-hIL-5RCDR antibody.
(2) Culturing of CHO/dhfr.SUP.− Cell-Derived Producer Cell and Purification of Antibody
[0251] The anti-hIL-5Rα CDR-grafted antibody-producing transformed cell clone obtained in the above item 1(2) of Example 3 was suspended in the EX-CELL302 medium containing 3 mM L-Gln, 0.5% CDLC and 0.3% PF68 to give a density of 3×10.sup.5 cells/ml and cultured using a 4.0 liter capacity spinner bottle (manufactured by Iwaki Glass) under agitating at a rate of 100 rpm. Ten days after culturing at 37° C. in a temperature-controlling room, the culture supernatant was recovered. The anti-hIL-5Rα CDR-grafted antibody was purified from the culture supernatant using ion exchange chromatography and a gel filtration method. The purified anti-hIL-5Rα CDR-grafted antibody was named CHO/d-hIL-5RCDR antibody.
(3) Culturing of NS0 Cell-Derived Producer Cell and Purification of Antibody
[0252] The anti-hIL-5Rα CDR-grafted antibody-producing transformed cell clone obtained in the above item 1(3) of Example 3 was cultured in accordance with the method of Yarranton at al. (BIO/TECHNOLOGY, 10, 169 {1992)) and then a culture supernatant was recovered. The anti-hIL-5Rα CDR-grafted antibody was purified from the culture supernatant using ion exchange chromatography and the gel filtration method. The purified anti-hIL-5R a CDR-grafted antibody was named NS0-hIL-5RCDR antibody.
4. Analysis of Purified Anti-hIL-5Rα CDR-Grafted Antibodies
[0253] In accordance with a known method (Nature, 227, 680, (1970)), 4 μg of each of the three anti-hIL-5Rα CDR-grafted antibodies produced by and purified from respective animal cells, obtained in the above item 3 of Example 3, was subjected to SDS-PAGE to analyze the molecular weight and purification degree. The results are shown in
Example 4
Activity Evaluation of Anti-hIL-5Rα CDR-Grafted Antibody:
[0254] 1. Binding Activity of Anti-hIL-5Rα CDR-Grafted Antibody to hIL-5Rα (ELISA)
[0255] The activity of the three purified anti-hIL-5Rα CDR-grafted antibodies obtained in the above item 2 of Example 3 to bind to hIL-5Rα was measured by the ELISA shown in the item 2 of Example 3.
2. In Vitro Cytotoxic Activity (ADCC Activity) of Anti-hIL-5Rα CDR-Grafted Antibody
[0256] In order to evaluate in vitro cytotoxic activity of the three purified anti-hIL-5Rα CDR-grafted antibodies obtained in the above item 3 of Example 3, the ADCC activity was measured in accordance with the following method.
(1) Preparation of Target Cell Solution
[0257] A mouse T cell line CTLL-2(h5R) expressing the hIL-5Rα chain and 13 chain described in WO 97/10354 was cultured using the RPMI1640-FBS(10) medium to prepare a 1×10.sup.6 cells/0.5 ml suspension, and the cells were radioisotope-labeled by reacting them with 3.7 MBq equivalents of a radioactive substance Na.sub.2.sup.51CrO.sub.4 at 37° C. for 1.5 hours. After the reaction, the cells were washed three times through their suspension in the RPMI1640-FBS(10) medium and centrifugation, re-suspended in the medium and then allowed to stand at 4° C. for 30 minutes in ice for spontaneous dissolution of the radioactive substance. After centrifugation, the precipitate was adjusted to 2×10.sup.5 cells/ml by adding 5 ml of the RPMI1640-FBS(10) medium and used as the target cell solution.
(2) Preparation of Effector Cell Solution
[0258] From a healthy person, 50 ml of vein blood was collected and gently mixed with 0.5 ml of heparin sodium (manufactured by Takeda Pharmaceutical). The mixture was centrifuged to separate a mononuclear cell layer using Polymorphprep (manufactured by Nycomed Pharma AS) and in accordance with the manufacture's instructions. After washing with the RPMI1640-FBS(10) medium by centrifugation three times, the resulting cells were re-suspended to give a density of 9×10.sup.6 cells/ml using the medium and used as the effector cell solution.
(3) Measurement of ADCC Activity
[0259] Into each well of a 96 well U-shaped bottom plate (manufactured by Falcon), 50 μl of the target cell solution prepared in the above (1) (1×10.sup.4 cells/well) was dispensed. Next, 100 μl of the effector cell solution prepared in the above (2) was dispensed (9×10.sup.5 cells/well, the ratio of effector cells to target cells becomes 90:1). Subsequently, each of the anti-hIL-5Rα CDR-grafted antibodies was added to give a final concentration from 0.001 to 0.1 μg/ml, followed by reaction at 37° C. for 4 hours. After the reaction, the plate was centrifuged, and the amount of .sup.51Cr in the supernatant was measured using a 7-counter. The amount of spontaneously released .sup.51Cr was calculated by the same operation using only the medium instead of the effect cell solution and the antibody solution and measuring the amount of .sup.51Cr in the supernatant. The amount of total released .sup.51Cr was calculated by the same operation using only the medium instead of the antibody solution and adding 1 N hydrochloric acid instead of the effector cell solution, and measuring the amount of .sup.51Cr in the supernatant.
[0260] The ADCC activity was calculated from the following equation.
[0261] The results are shown in
3. In Vivo Activity Evaluation of Anti-hIL-5Rα CDR-Grafted Antibody
[0262] In order to evaluate in vivo activity of the three purified anti-hIL-5Rα CDR-grafted antibodies obtained in the above item 3 of Example 3, the inhibition activity in an hIL-5-induced eosinophilia increasing model of Macaca faseicularis was examined in accordance with the following method.
[0263] The hIL-5 (preparation method is described in WO 97/10354) was administered to Macaca faseicularis under the dorsal skin at a dose of 1 μg/kg, starting on the first day and once a day for a total of 14 times. Each anti-hIL-5Rα CDR-grafted antibody was intravenously administered at a dose of 0.3 mg/kg one hour before the hIL-5 administration on the day zero. An antibody-non-added group was used as the control. In the antibody-administered groups, three animals of Macaca faseicularis were used in each group (No. 301, No. 302, No. 303, No. 401, No. 402, to 403, No. 501, No. 502 and No. 503), and two animals (No. 101 and No. 102) were used in the antibody-non-added group. Starting 7 days before commencement of the administration and until 42 days after the administration, about 1 ml of blood was periodically collected from a saphena or a femoral vein, and the number of eosinophils in 1 μl of peripheral blood was measured. The results are shown in
[0264] Based on the above results, it is expected that an antibody having high ADCC activity is useful also in the clinical field for various diseases in human.
Example 5
Analysis of ADCC Activity-Increasing Sugar Chain:
1. Preparation of 2-Aminopyridine-Labeled Sugar Chain (PA-Treated Sugar Chain)
[0265] The humanized antibody of the present invention was acid-hydrolyzed with hydrochloric acid to remove sialic acid. After hydrochloric acid was completely removed, the sugar chain was cut off from the protein by hydrazinolysis (Method of Enzymology, 83, 263, 1982). Hydrazine was removed, and N-acetylation was carried out by adding an ammonium acetate aqueous solution and acetic anhydride. After freeze-drying, fluorescence labeling with 2-aminopyridine was carried out (J. Biochem., 95, 197 (1984)). The fluorescence-labeled sugar chain (PA-treated sugar chain) was separated as an impurity using Surperdex Peptide HR 10/30 Column (manufactured by Pharmacia). The sugar chain fraction was dried using a centrifugal concentrator and used as a purified PA-treated sugar chain.
2. Reverse Phase HPLC Analysis of PA-Treated Sugar Chain of Purified Anti-hIL-5Rα CDR-Grafted Antibody
[0266] Using respective anti-hIL-5Rα CDR-grafted antibody PA-treated sugar chains prepared in the above item 1 of Example 5, reverse phase HPLC analysis was carried out by CLC-ODS column (manufactured by Shimadzu). An excess amount of α-L-fucosidase (derived from bovine kidney, manufactured by SIGMA) was added to the PA-treated sugar chain for digestion (37° C., 15 hours), and then the products were analyzed by reverse phase HPLC (
TABLE-US-00001 TABLE 1 Antibody-producing cell α-1,6-Fucose-linked sugar chain (%) YB2/0 47 NS0 73
[0267] About 47% of the anti-hIL-5RCDR-grafted antibody produced by the YB2/0 cell and about 73% of the anti-hIL-5RCDR-grafted antibody produced by the NS0 cell were sugar chains having α-1,6-fucose. Thus, sugar chains having no α-1,6-fucose were more frequent in the antibody produced by the YB2/0 cell in comparison with the antibody produced by the NS0 cell.
3. Analysis of Monosaccharide Composition of Purified Anti-hIL-5Rα CDR-Grafted Antibody
[0268] Sugar chains of anti-hIL-5Rα CDR-grafted antibodies produced by the YB2/0 cell, NS0 cell and CHO/d cell were hydrolyzed into monosaccharides by acid hydrolysis with trifluoroacetic acid, and monosaccharide composition analysis was carried out using BioLC (manufactured by Dionex).
[0269] Among N-glycoside-linked sugar chains, there are 3 mannose units in one sugar chain in the complex type N-glycoside-linked sugar chain. A relative ratio of each monosaccharide obtained by calculating the number of mannose as 3 is shown in Table 2.
TABLE-US-00002 TABLE 2 Antibody- ADCC activity producer cell Fuc GlcNAc Gal Man (%)* YB2/0 0.60 4.98 0.30 3.00 42.27 NS0 1.06 3.94 0.66 3.00 16.22 CHO/dhFr.sup.− 0.85 3.59 0.49 3.00 25.73 0.91 3.80 0.27 3.00 *Antibody concentration: 0.01 μg/ml
[0270] Since the relative ratios of fucose were in an order of YB2/0<CHO/d<NS0, the sugar chain produced in the antibody produced by YB2/0 cell showed the lowest fucose content as also shown in the present results.
Example 6
Sugar Chain Analysis of Antibody Produced by CHO/dhfr.SUP.− .Cell:
[0271] PA-treated sugar chains were prepared from purified anti-hIL-5Rα CDR-grafted antibody produced by CHO/dhfr.sup.− cell, and reverse phase HFLC analysis was carried out using CLC-ODS column (manufactured by Shimadzu) (
Example 7
Separation of High ADCC Activity Antibody:
[0272] The anti-hIL-5Rα CDR-grafted antibody produced by rat myeloma YB2/0 cell was separated using a lectin column which binds to sugar chains having fucose. HPLC was carried out using LC-6A manufactured by Shimadzu at a flow rate of 1 ml/min and at room temperature as the column temperature. After equilibration with 50 mM Tris-sulfate buffer (pH 7.3), the purified anti-hIL-5R a CDR-grafted antibody was injected and then eluted by a linear density gradient (60 minutes) of 0.2 M α-methylmannoside (manufactured by Nakalai Tesque). The anti-hit-5Rα CDR-grafted antibody was separated into non-adsorbed fraction and adsorbed fraction. When the non-adsorbed fraction and a portion of the adsorbed fraction were sampled and their binding activity to hIL-5Rα was measured, they showed similar binding activity (
Example 8
Determination of Transcription Product of α1,6-Fucosyltransferase (FUT8) Gene in Host Cell Line:
[0273] (1) Preparation of Single-Stranded cDNA Derived from Various Cell Lines
[0274] Chinese hamster ovary-derived CHO/DG44 cell was suspended in the IMDM medium (manufactured by Life Technologies) supplemented with 10% FES (manufactured by Life Technologies) and 1× concentration of HT supplement (manufactured by Life Technologies) and inoculated into a T75 flask for adhesion cell culture (manufactured by Grainer) at a density of 2×10.sup.5 cells/ml. Also, the rat myeloma-derived YB2/0 cell was suspended in the RPMI1640 medium (manufactured by Life Technologies) supplemented with 10% FBS (manufactured by Life Technologies) and 4 mM glutamine (manufactured by Life Technologies) and inoculated into a T75 flask for suspension cell culture (manufactured by Greiner) at a density of 2×10.sup.5 cells/mL. These cells were cultured′ at 37° C. in a 5% CO.sub.2 incubator, and 1×10.sup.7 cells of each host cell were recovered on the 1st, 2nd, 3rd, 4th and 5th day to extract total RNA using RNAeasy (manufactured by QUIAGEN).
[0275] The total, RNA was dissolved in 45 μl of sterile water, mixed with 0.5 U/μl of RQ1 RNase-Free DNase (manufactured by Promega) and 5 μl of attached 10× Dnase buffer and 0.5 μl of RNasin Ribonuclease inhibitor (manufactured by Promega), followed by reaction at 37° C. for 30 minutes. After the reaction, the total RNA was again purified using RNAeasy (manufactured by QUIAGEN). and dissolved in 50 μl of sterile water.
[0276] According to SUPERSCRIPT™ Preamplification System for First Strand cDNA Synthesis (manufactured by Life Technology), 3 μg of the obtained total RNA each was subjected to a reverse transcription reaction in a 20 μl system using oligo(dT) as a primer to, thereby synthesize cDNA. A solution of 1× concentration of the solution after the reverse transcription reaction was used for cloning of FUT8 and β-actin derived from each host cell, and a solution after the reverse transcription reaction further diluted 50 times with water was used for the determination of the transcription quantity of each gene using the competitive PCR, and each of the solutions was stored at −80° C. until use.
(2) Preparation of Respective cDNA Partial Fragments of Chinese Hamster FUT8 and Rat FUT8
[0277] Respective cDNA partial fragments of Chinese hamster FUT8 and of rat FUT8 were obtained as described below. First, primers (shown in SEQ ID NO:1 and SEQ ID NO:2) specific for nucleotide sequences common in a human FUT8 cDNA (Journal of Biochemistry, 121, 626 (1997)) and a swine FUT8 cDNA (Journal of Biological Chemistry, 271, 27810 (1996)) were designed.
[0278] Next, using a DNA polymerase ExTaq (manufactured by Takara Shuzo), 25 μl of a reaction solution constituted by ExTaq buffer (manufactured by Takara Shuzo), 0.2 mm dNTPs, 0.5 μM of each of the above specific primers (SEQ ID NO:1 and SEQ ID NO:2), and 1 μl of each of the cDNA derived from CHO cell and the cDNA derived from YB2/0 cell, each obtained on the 2nd day of culturing in (1), was prepared, and polymerase chain reaction (PCR) was carried out. The PCR was carried out under conditions in which, after heating at 94° C. for 1 minute, a cycle consisting of reactions at 94° C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 2 minutes is repeated 30 cycles and then the reaction solution is heated at 72° C. for 10 minutes. Each specific amplified fragment of 979 bp obtained by the PCR was connected to a plasmid pCR2.1 using TOPO TA Cloning Kit (manufactured by Invitrogen) to obtain a plasmid containing respective cDNA partial fragment of Chinese hamster FUT8 or rat FUT8 (CHFT8-pCR2.1 or YBFT8-pCR2.1).
[0279] The nucleotide sequence of each cDNA obtained was determined using DNA Sequencer 377 (manufactured by Parkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by Parkin Elmer) to confirm that the obtained cDNAS encode open reading frame (ORF) partial sequences of Chinese hamster FUT8 and rat FUT8 (shown in SEQ ID NOs:3 and 4).
(3) Preparation of Chinese Hamster 13-Actin and Rat 13-Actin cDNA
[0280] Since it is considered that the β-actin gene is constantly transcribed in each cell and its transcription quantity is almost the same among cells, transcription quantity of the β-actin gene is determined as a standard of the efficiency, of synthesis reaction of cDNA derived from respective cells.
[0281] Chinese hamster β-actin and rat β-actin were obtained by the following method. First, a forward primer (shown in SEQ ID NO:5) specific for a common sequence containing a translation initiation codon and reverse primers (shown in SEQ ID NO:6 and SEQ ID NO:7) specific for the respective sequence containing a translation termination codon were designed from a Chinese hamster β-actin genomic sequence (GenBank, U20114) and a rat β-actin genomic sequence (Nucleic Acid Research, 11, 1759 (1983)).
[0282] Next, using a DNA polymerase, KOD (manufactured by TOYOBO), 25 μl of a reaction solution constituted by KOD buffer #1 (manufactured by TOYOBO), 0.2 mM dNTPs, 1 mm MgCl.sub.2, 0.4 μM of each of the above gene specific primers (SEQ ID NO:5 and SEQ ID NO:6, or SEQ ID NO:5 and SEQ ID NO:7), 5% DMSO, and 1 μl of each of the cDNA derived from CHO cell and the cDNA derived from YB2/0 cell, each obtained on the 2nd day of culturing in (1), was prepared, and polymerase chain reaction (PCR) was carried out. The PCR was carried out under a condition in which, after heating at 94° C. for 4 minutes, a cycle consisting of reactions at 98° C. for 15 seconds, 65° C. for 2 seconds and 74° C. for 30 seconds is repeated 25 cycles. The 5′-terminal of each specific amplified fragment of 1,128 bp obtained by the PCR was phosphorylated using MEGALABEL (manufactured by Takara Shuzo) and then digested with a restriction enzyme, EcoRV, and the resulting fragment (2.9 Kb) was connected to pBluescript II, (KS(+) (manufactured by Stratagene) using Ligation High (manufactured by TOYOBO) to obtain a plasmid containing an ORF full length of respective cDNA of Chinese hamster β-actin or rat β-actin (CHAc-pBS or YBAc-pBS).
[0283] The nucleotide sequence of the respective cDNA obtained was determined using DNA Sequencer 377 (manufactured by Parkin Elmer) and BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by Parkin Elmer) to confirm that they respectively encode ORF full length sequences of cDNA of Chinese hamster β-actin and rat β-actin.
(4) Preparation of Standard and Internal Sequence Control
[0284] In order to measure the quantity of mRNA transcription from the FUT8 gene in producer cells, a calibration curve was firstly prepared.
[0285] As the FUT8 standard to be used in the calibration curve, plasmids, CHFT8-pCR2.1 and YBFT8-pCR2.1, obtained in (2) by inserting respective cDNA partial fragments of Chinese hamster FUT8 or rat FUT8 into pCR2.1 were digested with a restriction enzyme, EcoRI, and the resulting DNA fragments were used after making them into linear chains.
[0286] As the internal control to be used in the PUTS determination, among CHFT8-pCR2.1 and YBFT8-pCR2.1, CHFT8d-pCR2.1 and YBFT8d-pCR2.1 obtained by deleting 203 bp between ScaI-HindIII of internal nucleotide sequences of Chinese hamster FUT8 or rat FUT8 were digested with the restriction enzyme, EcoRI, and the resulting DNA fragments were used after making them into linear chains.
[0287] As the standard of the quantity of mRNA transcribed from the β-actin gene in producer cells, plasmids CHAc-pBS and YBAc-pBS obtained in (3) by integrating the ORF full length of respective cDNAs of Chinese hamster β-actin and rat β-actin into pBluescript II KS(+) were respectively digested, the former with HindIII and PstI and the latter with HindIII and KpnI, and the resulting DNA fragments were used by making them into linear chains.
[0288] As the internal control for the determination of β-actin, among CHAc-pBS and YBAc-pBS, CHAcd-pBS and YBAcd-pBS obtained by deleting 180 bp between DraIII-DraIII of internal nucleotide sequences of Chinese hamster β-actin and rat β-actin were digested, the former with HindIII and PstI and the latter with HindIII and KpnI, and the resulting DNA fragments were used after making them into linear chains.
(5) Determination of Transcription Quantity by Competitive RT-PCR
[0289] First, a primer set (shown in SEQ ID NOs:8 and 9) common sequence-specific for internal sequences of ORF partial sequences of Chinese hamster FUT8 and rat FUT8 obtained in (2) was designed.
[0290] Next, PCR was carried out using a DNA polymerase ExTaq (manufactured by Takara Shun) in 20 μl in total volume of a reaction solution constituted by ExTaq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 μM of each of the above: gene specific, primers (SEQ ID NO:8 and SEQ ID NO:9), 5% DMSO, and 5 μl of a 50 times diluted solution of each of the cDNAs derived from respective host cell lines obtained in (1) and 5 μl (10 fg) of the plasmid for internal control. The PCR was carried out by heating at 94° C. for 3 minutes and then repeating 30 cycles using reactions at 94° C. for 1 minute, 60° C. for 1 minute and 72° C. for 1-minute as one cycle.
[0291] The β-actin transcription product was determined as described below. Primer sets gene-specific for internal sequences of the ORF full lengths of Chinese hamster β-actin. and rat β-actin obtained in (3) (the former are shown in SEQ ID NO:10 and SEQ ID NO:11, and the latter in SEQ ID NO:12 and SEQ ID NO:13) were respectively designed.
[0292] Next, PCR was carried out using a DNA polymerase ExTaq (manufactured by Takara Shuzo) in 20 μl in total volume of a reaction solution constituted by ExTaq buffer (manufactured by Takara Shuzo), 0.2 mM dNTPs, 0.5 μM of the above gene specific primers (SEQ ID NO:10 and SEQ ID NO:11, or SEQ ID NO:12 and SEQ ID NO:13), 5% DMSO, and 5 μl of a 50 times diluted solution of each of the cDNAs derived from respective host cell lines obtained in (1) and 5 μl (1 μg) of the plasmid for internal control. The PCR was carried out by heating at 94° C. for 3 minutes and then repeating 17 cycles using reactions at 94° C. for 30 seconds, 65° C. for 1 minute and 72° C. for 2 minutes as one cycle.
TABLE-US-00003 TABLE 3 Size (bp) of PCR amplified product Target gene *Primer set Target Competitor FUT8 F: 5′-GTCCATGGTGA 638 431 TCCTGCAGTGTGG-3′ (SEQ ID NO: 8) R: 5′-CACCAATGATA TCTCCAGGTTCC-3′ (SEQ ID NO: 9) B-Actin F: 5′-GATATCGCTGC 789 609 (Chinese GCTCGTTGTCGAC-3′ hamster) (SEQ ID NO: 10) R: 5′-CAGGAAGGAAG GCTGGAAAAGAGC-3′ (SEQ ID NO: 11) B-Actin F: 5′-GATATCGCTGC 789 609 (rat) GCTCGTCGTCGAC-3′ (SEQ ID NO: 12) R: 5′-CAGGAAGGAAG GCTGGAAGAGAGC-3′ (SEQ ID NO:13) *F: forward primer, R: reverse primer
[0293] Determinative PCR was carried out using the primer sets shown in Table 3. As a result, the DNA fragment having the size shown in the target column of Table 3 was amplified from the respective gene transcription product and the corresponding standard, and the DNA fragment having the size shown in the competitor column of Table 3 was amplified from the corresponding internal control.
[0294] After 7 μl of the solution after PCR was subjected to 1.75% agarose gel electrophoresis, the gel was stained with SYBR Green I Nucleic Acid Gel Stain (manufactured by Molecular Probes). The quantity of the amplified DNA fragments was measured by calculating luminescence strength of each of the amplified DNA fragments using FluorImager SI (manufactured by Molecular Dynamics).
[0295] Furthermore, PCR was carried out by changing the amount of the standard plasmid prepared in (4) to 0.1 fg, 1 fg, 5 fg, 10 fg, 50 fg, 100 fg and 500 fg, instead of the cell-derived cDNA, and the amount of amplified products was determined. A calibration curve was prepared by plotting the measured values against the amounts of standard plasmid.
[0296] Using this calibration curve, the amount of cDNA of the gene of interest in each cell was calculated from the amount of the amplified product when the total cDNA derived from each cell was used as the template, and the amount was defined as the mRNA transcription quantity in each cell.
[0297] Amounts of the FUT8 transcription product in each host cell line in using rat FUT8 sequences as the standard and internal control are shown in
[0298] Also, the FUT8 transcription quantity is shown in Table 4 as a relative value to the amount of β-actin transcription product.
TABLE-US-00004 TABLE 4 Culture days Cell line Day 1 Day 2 Day 3 Day 4 Day 5 CHO 2.0 0.90 0.57 0.52 0.54 YB2/0 0.07 0.13 0.13 0.05 0.02
[0299] While the FUT8 transcription quantity of the YB2/0 cell line was about 0.1% β-actin, that of the CHO cell line was from 0.5 to 2%.
[0300] Based on the above results, it was shown that the amount of the FUT8 transcription product in the YB2/0 cell line was significantly smaller than that in the CHO cell line.
INDUSTRIAL APPLICABILITY
[0301] The present invention relates to a sugar chain which controls the activity of an immunologically functional molecule, such as an antibody, a protein, a peptide or the like, as well as an antibody, a protein or a peptide having the sugar chain. The present invention further relates to methods for the production of the sugar chain and an antibody, a protein or a peptide having the sugar chain, as well as a diagnostic agent, a preventive agent and a therapeutic agent which contain these products as an active ingredient.