Patent Application
20080075712
Herein below, the present invention will be specifically described with reference to Examples, but it is not to be construed as being limited thereto.
Expression vectors for a soluble receptor, in which the C terminal of the extracellular region of either human ARI chain or AR2 chain was tagged with FLAG (ARIFLAG, AR2FLAG) or His6 (ARI His, AR2His), were introduced into CHO cells separately and purified from culture supernatants using affmity columns. The expression vector for a chimeric molecule comprising the extracellular region of human ARI chain and the intracellular region of G-CSF receptor was introduced into mouse proB cell line Ba/F3 to establish a high expression cell line. A high expression cell line was similarly established for a chimeric molecule comprising the extracellular region of human AR2 chain and the intracellular region of G-CSF receptor. The cells were individually used to intraperitoneally immunize BALB/c. ARI His or AR2His was intravenously injected three days before excising the spleen.
(a) Panning of Phage Library
PolyA(+)RNA was extracted from the spleen of an immunized mouse, and scFv was synthesized by RT-PCR to construct a phagemid library expressing scFv as a fusion protein with gene3 of fl phage (J. Immun. Methods, 201, (1997), 35-55). The E. coli library (2 x 109 cfu) was inoculated into 50 mL of 2x YTAG (2x TY containing 100 pg/mL ampicillin and 2% glucose), and cultured at 37° C till OD 600 reached 0.4 to 0.5.4 x 101 ofhelperphage VCSM13 was added to the culture, which was left to stand at 37° C for 15 minutes for infection. The infected cells were cultured at 26° C for 10 hours, following addition of 450 niL of 2x YTAG and 25 jL of 1 mol/L IPTG. The culture supernatant was collected by centrifugation, mixed with 100 mL of PEG-NaCl (10% polyethylene glycol 8000, 2.5 mol/L NaCI), and left to stand at 4° C for 60 minutes. Phage was precipitated by centrifugation at 10,800x g for 30 minutes, and the precipitate was suspended in 40 mL of water, mixed with 8 niL of PEG-NaCi, and left to stand at 4° C for 20 minutes. Phage was precipitated by centrifugation at 10,800x g for 30 minutes, and suspended in 5 mL of PBS. ARI FLAG and AR2FLAG prepared in Example 1 were labeled with biotin using No-Weigh Premeasured NHS-PEO4-Biotin Microtubes (Pierce). 100 pmol of biotin labeled ARIFLAG or AR2FLAG was added to the phage library and contacted with the antigen for 60 minutes. 600 jL of Streptavidin MagneSphere (Promega) washed with 5% M- PBS (PBS containing 5% skim milk) added for binding for 15 minutes. Beads were washed with I mL PBST (PBS containing 0.1 % Tween-20) and PBS three times each. The beads were suspended in 0.8 mL of 0.1 mol/L glycine/HCI (pH 2.2) for 5 minutes to elute the phage. The phage solution thus collected was neutralized by adding 2 mol/L Tris (45 liL), added to 10 mL of XLl-Blue in logarithmic growth phase (OD 600 =0.4 to 0.5), and left to stand for 30 minutes at 37° C for infection. The mixture was spread on a 2x YTAG plate and cultured at 30° C. Colonies were collected, inoculated into 2x YTAG, and cultured at 37° C until OD 600 =0.4 to 0.5. IPTG (1 mol/L; 5 pL) and helper phage VCSM13 (10 pfu) were added to the culture solution (10 mL), and the mixture was left to stand at 37° C for 30 minutes. The cells were collected by centrifugation, resuspended in 2x YTAG (100 mL) containing kanamycin (25 jig/mL), and cultured at 30° C for 10 hours. The culture supernatant was collected by centrifugation, mixed with of PEG-NaCI (20 nmL), and left to stand at 4° C for 20 minutes. Phage was precipitated by centrifugation at 10,800x g for 30 minutes, and suspended in PBS (2 mL), and provided for the subsequent panning. Beads were washed five times each for PBST and PBS at the second panning. Clones producing AR chain binding phages were selected by ELISA, from E.coli that could infect the eluted phages.
(b) Phage ELISA
The above-described single colony was inoculated into 2x YTAG (150 jL) and cultured at 30° C overnight. After 5 lL of this culture was inoculated into 2x YTAG (500 JL) and cultured at 37° C for 2 hours, helper phage (2.5 x 109 pfu) and 2x YTAG (100 JIL) containing 1 mol/L IPTG (0.3 ,L) was added, and the culture was then left to stand at 37° C for 30 minutes. After subsequent overnight culture at 30° C, the centrifuged supernatant was subjected to ELISA. StreptaWell 96 microtiter plate (Roche) was coated over night with PBS (100 [L) containing 1.0 gg/mL biotin-labeled ARIFLAG or AR2FLAG. After washing with PBST to remove the antigen, the reaction was blocked with 200 lL of 2% (w/v) M-PBS over night. After removal of 2% (w/v) M-PBS, the culture supernatant was added therein and left to stand for 40 minutes for antibody binding. After washing, the bound phage was detected with an HRP-bound anti-M13 antibody (Amersham Pharmacia Biotech) diluted with 2% (w/v) M-PBS, and BM blue POD substrate (Roche). The reaction was stopped by adding sulfuric acid, and the A450 value was measured.
(c) Sequence Determination and Clone Selection
The scFv region was amplified by PCR using primers PBG3-FI (5-CAGCTATGAAATACCTATTGCC -3/SEQ ID NO: 27) and PBG3-Rl (5-CTTTTCATAATCAAAATCACCGG-3/SEQ ID NO: 28) from the phage solution of an ELISA positive clone, and its nucleotide sequence was determined. A PCR reaction solution (20 lL) containing 1 lL phage solution, 2 pL 10 x KOD Dash buffer solution, 10 Fmol/L primer (0.5 jL each), and 0.3 lL KOD Dash polymerase (TOYOBO, 2.5 U/PL) was amplified on a Perkin Elmer 9700 via 30 cycles of 96° C., 10 seconds, 55° C, 10 seconds, and 72° C, 30 seconds. After PCR, 3 lL of ExoSAP-IT (Amersham) was added to 5 lL of the reaction solution, and incubated at 37° C for 20 minutes, then at 80° C for 15 minutes. This sample was reacted with primer PBG3-F2 (5-ATTGCCTACGGCAGCCGCT -3/SEQ ID NO:29) or PBG3-R2 (5′-AAATCACCGGAACCAGAGCC -3′/SEQ ID NO:30) using a BigDye Terminator Cycle Sequencing kit (Applied Biosystems), and electrophoresed on an Applied Biosystems PRISM 3700 DNA Sequencer. For each of the anti-ARI chain and anti-AR2 chain, 45 clones with CDR3 amino acid sequences different from those predicted from the nucleotide sequences were selected.
For expression as scFv-CH1-Fc, an expression vector pCAGGss-g4CH hetero IgG4, where scFv can be inserted between a human signal sequence (driven by promoter CAGG) and the intron -CHl-Fc (human IgG4 cDNA) via an SfiI site, was constructed. For expression as a heteromolecule, amino acid substitutes that are substituted at the CH3 site of IgG4 were produced based on the knobs-into-holes of IgGl (Ridgway JB et al. Protein Engineering 1996, 9: 617-621). Type A is a substitute with Y349C and T366W substitutions, and type B is a substitute with E356C, T366S, L368A, and Y407V substitutions. The substitution of -ppcpScp- to -ppcpPcp- was introduced into the hinge region of both types. Type A was constructed with a human IL-3 signal sequence (pCAGG-IL3ss-g4CHPa) and type B with a human IL-6 signal sequence (pCAGG-IL6ss-g4CHPb). PCR products of the scFv region of the clones selected based on the nucleotide sequences were SfiI treated, then the anti-ARl chain clone was subdloned into pCAGG-IL3ss-g4CHPa and the anti-AR2 chain clone was subcloned into pCAGG-IL3ss-g4CHPb. Expression vectors for a total of 2025 combinations (anti-ARI chain and anti-AR2 chain clones 45 x 45) were used to transfect HEK 293 cells using lipofectamine 2000, and three days later, culture supernatants were collected.
(a) Ba/F3 Growth Assay
BaF3-ARG was established by introducing expression vectors for chimeric molecules comprising the extracellular region of ARI chain or AR2 chain and the intracellular region of G- CSF receptor into Ba/F3 cells, which proliferate in a mouse IL-3-dependent manner. BaF3-ARG proliferated IFNoc-dependently. After three washes, 0.1 ImL medium containing the sample and 1X103 cells per well was seeded to a 96-well plate. After four days of culture, 10 liL of Cell Count Reagent SF (Nacalai Tesque) was added and incubated at 37° C for two hours, and then A450 was measured.
(b) Daudi Cell Proliferation Inhibition Assay
Daudi cells are a human B cell line with high sensitivity towards IFN. 6.25 x 103 cells per well were seeded to a 96-well plate in 0.1 ImL medium containing the sample. After four days of culture, 10 pL of Cell Count Reagent SF (Nacalai Tesque) was added and incubated at 37° C for two hours, and then A450 was measured.
(c) Sequences of Ligand Function-Substituting Bispecific Antibodies
Amino acid sequences of the variable regions of the antibodies selected by the above screening method are described as SEQ ID NOs: I to 26. Correlation between the name of each antibody and the SEQ ID NO is shown in the above Table 1.
(d) Reporter Gene Assay Using ISRE
40 Rg of pISRE-Luc was added to 3 niL of OPTI-MEM I and 100 IL DMRIE-C (Invitrogen), stirred, and left to stand at room temperature for 20 minutes. This was added to 8 x 106 human K562 cells prepared in 2 mL OPTI-MEM I, and after four hours of culturing at 37° C, 10 mL of 15%FCS-RPMI1640 was added and the cells were cultured overnight. The next day, K562 collected by centrifugation was resuspended in 10.5 mL of 10% FCS-RPMI1640 and seeded to a 96-well flat bottom plate at 70 ,L/well.
Bispecific scFv-CH in the culture supernatants of HEK293 cells introduced with the antibody gene was adjusted to a concentration of 12.5 ng/mL with reference to IgG and a series of 5-fold dilutions were made. Alternatively, culture supernatants of COS7 cells expressing bispecific IgG were diluted 2-fold and a series of 5-fold dilutions were made. These were added to cells introduced with a reporter plasmid at 30 EL/well. For the positive control wells, a series of 5-fold dilutions of IFN-a 2a were dispensed at 30 EL/well. After culturing at 37° C for 24 hours, 50 lL/mL of a Bright-Glo Luciferase Assay System (Promega) was added and left to stand at room temperature for 10 minutes, and luciferase activity was determined with Analyst HT (LJL) (
5-1. Immunization and preparation of hybridomas Eight BALB/c mice (male, 6 weeks old when immunization was initiated (Charles River, Japan)) and five MRL/lpr mice (male, 6 weeks old when immunization was initiated (Charles River, Japan)) were immunized with Factor IXap (Enzyme Research Laboratories, Inc.) as described below. As an initial immunization, Factor IXap (40 jg/head) emulsified with FCA (Freund's complete adjuvant H37 Ra (Difco laboratories)) was subcutaneously administered. Two weeks later, Factor IXap (40 pig/head) emulsified with FIA (Freund's incomplete adjuvant (Difco laboratories)) was subcutaneously administered. Afterward, three to seven booster immunizations were performed at one week intervals. After the titer of a plasma antibody against Factor IXap was confirmed to be elevated by ELISA (Enzyme linked immunosorbent assay) described in 5-2, Factor IXap (40 ig/head) diluted in PBS(-) (phosphate buffered saline free of calcium ion and magnesium ion) was intravenously administered as a fmal immunization. Three days after the final immunization, mice spleen cells were fused with mouse myeloma cells P3X63Ag8U.1 (referred to as P3U1, ATCC CRL-1597) by a standard method using PEG1500 (Roche Diagnostics). Fused cells suspended in RPMI1640 medium (Invitrogen) containing 10% FBS (Invitrogen) (hereinafter referred to as 1 o%FBS/kPMI 1640) were seeded in a 96-well culture plate, and 1, 2, 3, and 5 days after the fusion, the medium was replaced with a HAT selection medium (10% FBS/RPMI1640 / 2% HAT 50x concentrate (Dainippon Pharmaceutical Co. Ltd) / 5% BM-Condimed H1 (Roche Diagnostics)-to selectively culture hybridomas. Using the supernatants collected on the 8,h or 9th day after fusion, Factor IXa-binding activity was measured by ELISA described in 5-2 to select hybridomas having Factor IXa-binding activity. Subsequently, the activity of neutralizing Factor IXa enzymatic activity was measured by the method described in 5-3 to select hybridomas that do not have Factor IXa-neutralizing activity. Hybridomas were cloned twice by performing limiting dilutions in which one cell is seeded in each well of a 96-well culture plate. Single colony cells confimed by microscopic observation were subjected to ELISA and neutralization activity assay as described in 5-2 and 5-3 was performed for clone selection. Ascites containing the cloned antibody was prepared by the method described in 5-4, and the antibody was purified from the ascites. The purified antibody was unable to extend APTT (activated partial thromboplastin time) and this was confirmed by the method described in 5-5. 5-2. Factor IXa ELISA Factor IXap was diluted to 1 jg/mL with a coating buffer (100 mM sodium bicarbonate, pH 9.6, 0.02% sodium azide) and distributed in Nunc-Immuno plate (Nunc-Immuno 96 MicroWellm plates MaxiSorpm (Nalge Nunc International)) at 100 gL/well. Then, the plate was incubated at 4° C overnight. After washing the plate with PBS(-) containing Tween(R) 20 thrice, it was blocked with a diluent buffer (50 mM Tris-HCl, pH 8.1, 1% bovine serum albumin, 1 mM MgCl2, 0.15 M NaCl, 0.05% Tween(R) 20, 0.02% sodium azide) at room temperature for 2 hours. After removal of the buffer, a diluent buffer-diluted mouse antiserum or hybridoma culture supernatant was added at 100 ElL/well, and incubated at room temperature for 1 hour. After washing the plate thrice, alkaline phosphatase-labeled goat anti-mouse IgG (H+L) (Zymed Laboratories) which had been diluted to 1/2000 with the diluent buffer was added at 100 gL/well, and incubated at room temperature for 1 hour. After washing the plate six times, a colorimetric substrate Blue-Phosm Microwell Phosphatase Substrate (Kirkegaard & Perry Laboratories) was added at 100 gL/well, and incubated at room temperature for 20 minutes. After adding the Blue- Phosm Stop Solution (Kirkegaard & Perry Laboratories) (100 gL/well), absorbance at 595 nm was measured with a Model 3550 Microplate Reader (Bio-Rad Laboratories). 5-3. Measurement of Factor IXa neutralizing activity Phospholipid (Sigma-Aldrich) was dissolved in distilled water for injection, and ultrasonicated to prepare a phospholipid solution (400 gg/mL). Tris buffered saline containing 0.1% bovine serum albumin (hereinafter abbreviated as TBSB) (40 gL), 30 ng/mL Factor IXa, (Enzyme Research Laboratories) (10 EL), 4004g/mL phospholipid solution (5 PL), TBSB containing 100 mM CaCl2 and 20 mM MgCl2 (5 ,L), and hybridoma culture supernatant (10 [L) were mixed in a 96-well plate, and incubated at room temperature for 1 hour. To this mixed solution, 50 gg/mL Factor X (Enzyme Research Laboratories) (20 [L) and 3 U/mL Factor VIII (American diagnostica) (10 gL) were added and reacted at room temperature for 30 minutes. The reaction was stopped by adding 0.5 M EDTA (10 gL). After addition of an S-2222 solution (50 gL; Chromogenix) and incubation at room temperature for 30 minutes, the absorbance was measured at measurement wavelength 405 nm and reference wavelength 655 nm on a Model 3550 Microplate Reader (Bio-Rad Laboratories, Inc.). 5-4. Ascites preparation and antibody purification Ascites of the established hybridomas was produced according to standard procedures. That is, the hybridoma was cultured in vitro (2 x 106) and transplanted into the peritoneal cavity of a BALB/c mouse (male, 5 to 7 weeks old at the time experiment was started, Japan Charles River) or BALB/c nude mouse (female, 5 to 6 weeks old at the time experiment was started, Japan Charles River and Japan CLEA), which was intraperitoneally administered twice with pristane (2,6,10,14-tetramethylpentadecane, WAKO Pure Chemical Industries) in advance. One to four weeks after the transplantation, ascites was collected from the mouse with an inflated abdomen.
The antibody was purified from the ascites using a Protein G Sepharosem 4 Fast Flow 5 column (Amersham Biosciences). The ascites was diluted 2-fold with a binding buffer (20 mM sodium acetate, pH 5.0) and applied to the column, which had been washed with 10 column volumes of the binding buffer. The antibody was eluted with 5 column volumes of an elution buffer (0.1 M glycine-HCI, pH 2.5), and neutralized with a neutralizing buffer (1 M Tris-HCl, pH 9.0). The resulting solution was concentrated using a CentriprepTm 10 (Millipore), and the 10 solvent was replaced with TBS (50 mM Tris-buffered saline). The antibody concentration was calculated from the absorbance at 280 nm with A (1%, 1 cm) =13.5. Absorbance was measured with DU-650 (Beckman Coulter). 5-5. Measurement of APTT (Activated Partial Thromboplastin Time) 15 APTT was measured with a CR-A (Amelung)-connected KC 1OA (Amelung). A mixture of the TBSB-diluted antibody solution (50 jiL), standard human plasma (Dade Behring) (50 pL), and APTT reagent (Dade Behring) (50 lL) was warmed at 37° C for 3 minutes. To this mixture, 20 mM CaCl2 (Dade Behring) (50 [tL) was added to start a coagulation reaction, and the coagulation time was measured. 20
Eight BALB/c mice (male, 6 weeks old when immunization was initiated, Japan Charles River) and five MRL/lpr mice (male, 6 weeks old when immunization was initiated, Japan 25 Charles River) were immunized with Factor X (Enzyme Research Laboratories) as described below. As an initial immunization, Factor X (40 gg/head) emulsified with FCA was subcutaneously administered. Two weeks later, Factor X (20 or 40 gg/head) emulsified with FIA was subcutaneously administered. Subsequently, three to six booster immunizations were given at one week intervals. After the titer of a plasma antibody against Factor X was confirmed 30 to be elevated by ELISA as described in 6-2, Factor X (20 or 40 fg/head) diluted in PBS (-) was administered intravenously as a final immunization. Three days after the fmal immunization, mouse spleen cells were fused with mouse myeloma P3U1 cells, according to a standard method using PEG1500. Fused cells suspended in 10% FBS/RPMI1640 medium were seeded in a 96- well culture plate, and hybridomas were selectively cultured by replacing the medium with a 35 HAT selection medium 1, 2, 3 and 5 days after the fusion. Binding activity toward Factor X was measured by ELISA described in 6-2, using the culture supernatant collected on the 8th day after fusion. Hybridomas having Factor X-binding activity were selected, and their activities to neutralize Factor Xa enzymatic activity were measured by the method described in 6-3. Hybridomas that do not have a neutralizing activity toward Factor Xa were cloned by performing limiting dilution twice. Ascites containing the cloned antibody was prepared by the method described in 5-4, and the antibody was purified from the ascites. The purified antibody was unable to extend APTT and this was confirmed by the method described in 5-5. 6-2. Factor X ELISA Factor X was diluted to 1 jg/iL with a coating buffer, and dispersed into Nunc-Immuno plate at 100 tL/well. Then the plate was incubated at 4° C overnight. After washing the plate with PBS (-) containing Tween (R) 20 thrice, it was blocked with a diluent buffer at room temperature for 2 hours. After removal of the buffer, a diluent buffer-diluted mouse antiserum or hybridoma culture supernatant was added to the plate, and incubated at room temperature for 1 hour. After washing the plate thrice, alkaline phosphatase-labeled goat anti-mouse IgG (H+L) which had been diluted to 1/2000 with the diluent buffer was added, and incubated at room temperature for 1 hour. After washing the plate six times, a colorimetric substrate Blue-PhosTm Microwell Phosphatase Substrate (Kirkegaard & Perry Laboratories)was added at 100 JAL/well, and incubated at room temperature for 20 minutes. After adding Blue-PhosTm Stop Solution (Kirkegaard & Perry Laboratories) (100 EL/well), absorbance at 595 nm was measured with a Model 3550 Microplate Reader (Bio-Rad Laboratories). 6-3. Measurement of Factor Xa-neutralizing activity Hybridoma culture supernatant diluted to 1/5 with TBSB (10 iL) was mixed with 40 JL of TBCP (TBSB containing 2.78 mM CaCI2 and 22.2 ,uM phospholipids (phosphatidyl choline:phosphatidyl serine =75:25, Sigma-Aldrich) containing 250 pg/mL Factor Xa (Enzyme Research Laboratories), and incubated at room temperature for 1 hour. To this mixed solution, TBCP (50 lL) containing prothrombin (Enzyme Research Laboratories) (20 jg/mL) and 100 ng/mL activated coagulation factor V (Factor Va (Haematologic Technologies)) were added, and reacted at room temperature for 10 minutes. The reaction was stopped by adding 0.5 M EDTA (10 liL). To this reaction solution, 1 mM S-2238 solution (Chromogenix) (50 liL) was added, and after incubation at room temperature for 30 minutes, absorbance at 405 nm was measured with a Model 3550 Microplate Reader (Bio-Rad Laboratories).
7-1. Preparation of Antibody Variable Region-Encoding DNA Fragments from Hybridomas
From the hybridomas that produce anti-F.IXa antibody or anti-F.X antibody, total RNA was extracted using the QIAGEN(R) RNeasy( Mini Kit (QIAGEN) according to the method described in the instruction manual. The total RNA was dissolved in sterile water (40 EL). Single-stranded cDNA was synthesized by RT-PCR using the SuperScript cDNA synthesis system (Invitrogen) with the purified RNA (1 to 2 pig) as template, according to the method described in the instruction manual. 7-2. PCR amplification of antibody H chain variable region and sequence analysis As primers for amplifying the mouse antibody H chain variable region (VH) cDNA, an HB primer mixture and HF primer mixture described in the report by Krebber et al. (J. Immunol. Methods 1997; 201: 35-55) were prepared. Using 0.5 gL each of the 100 gM HB primer mixture and 100 gM HF primer mixture, a reaction solution (25 gL) (cDNA solution prepared in 7-1 (2.5 EL), KOD plus buffer (TOYOBO), 0.2 mM dNTPs, 1.5 mM MgCl2, 0.75 units DNA polymerase KOD plus (TOYOBO)) was prepared. Using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer), PCR was performed according to amplification efficiency of the cDNA fragments, either under conditions A (3 min heating at 98° C followed by 32 cycles of reaction (98° C, 20 sec, 58° C, 20 sec, and 72° C, 30 sec in one cycle)) or conditions B (3 min heating at 94° C followed by 5 cycles of reaction (94° C, 20 sec, 46° C, 20 sec, and 68° C, 30 sec in one cycle) and 30 cycles of reaction (94° C, 20 sec, 58° C, 20 sec, and 72° C, 30 sec in one cycle)). After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. Amplified fragments of the desired size (about 400 bp) were purified using a QlAquick Gel Extraction Kit (QIAGEN) according to the methods described in the attached instruction manual, and eluted with sterile water (30 lIL). Nucleotide sequences of the DNA fragments were determined using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on a DNA sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), according to the method described in the attached instruction manual. Sequence groups determined by this method were comparatively analyzed using an analytical software, GENETYX-SV/RC Version 6.1 (Genetyx), and DNAs with different sequences were selected. 7-3. Preparation of antibody variable region DNA fragments for cloning The following procedure was performed to add restriction enzyme Sfi I cleavage sites for cloning to both termini of the antibody variable region amplification fragments.
To amplify the VH fragments added with an Sfi I cleavage site (Sfi I-VH), a primer (primer VH-5′ end) in which the primer HB (Gly4Ser)2-linker sequence was replaced with a sequence containing Sfi I cleavage site (SEQ ID NO: 31) was prepared. Using 0.5 pL each of the 10 liM sequence-specific primer VH-5′ end and 10 gM primer scfor (J. Immunol. Methods 1997; 201: 35-55), a reaction solution (20 pL) (purified solution of VH cDNA amplification fragment prepared in 7-2 (1 EL), KOD plus buffer (TOYOBO), 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5 units DNA polymerase KOD plus (TOYOBO)) was prepared. Using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer), PCR was performed according to amplification efficiency of the cDNA fragments, either under conditions A (3 min heating at 98° C followed by 32 cycles of reaction (98° C, 20 sec, 58° C, 20 sec. and 72° C, 30 sec in one cycle)) or conditions B (3 min heating at 94° C followed by 5 cycles of reaction (94° C, 20 sec, 46° C, 20 sec, and 68° C, 30 sec in one cycle) and 30 cycles of reaction (94° C, 20 sec, 58° C, 20 sec, and 72° C, 30 sec in one cycle)). After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. Amplified fragments of the desired size (about 400 bp) were purified using a QlAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction manual, and eluted with sterile water (30 EL).
To amplify the mouse antibody L chain variable region (VL) cDNA fragments, 0.5 PL each of the 100 μM LB primer mixture and 100 pM LF primer mixture described in the report by Krebber et al. (J. Immunol. Methods 1997; 201: 35-55) was first used, and a reaction solution (25 pL) (cDNA solution prepared in 7-1 (2.5 pL), KOD plus buffer (TOYOBO), 0.2 mM dNTPs, 1.5 mM MgCI2, 0.75 units DNA polymerase KOD plus (TOYOBO)) was prepared. Using a thermal cycler GeneAmp PCR system 9700 (Parkin Elner), PCR was performed according to amplification efficiency of the fragments, under conditions of 3 minutes heating at 94° C. followed by 5 cycles of reaction (94° C, 20 sec, 46° C, 20 sec, and 68° C., 30 sec in one cycle) and 30 cycles of reaction (94° C, 20 sec, 58° C, 20 sec, and 72° C., 30 sec in one cycle). After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. Amplified fragments of the desired size (about 400 bp) were purified using the QlAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction manual, and eluted with sterile water (30 liL). The fragments are in a state in which the primer LF-derived (Gly4Ser)3-linker sequence is added to their C termini. In order to add an Sfi I cleavage site to the C termini of the fragments, a primer (primer VL-3′ end) in which the primer LF (Gly4Ser)3-linker sequence was replaced with a sequence having Sfi I cleavage site (SEQ ID NO: 32) was prepared. To amplify the VL fragments added with an Sfi I cleavage site (Sfi I-VL), 0.5 pL each of the 10 IM VL-3′ end primer mixture and 10 liM scback primer was used, and a reaction mixture (20 pL) (purified solution of VL cDNA amplification fragment (1 pL), KOD plus buffer (TOYOBO), 0.2 mM dNTPs, 1.5 mM MgCI2, 0.5 units DNA polymerase KOD plus (TOYOBO)) was prepared. PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) under conditions of 3-minutes heating at 94° C followed by 5 cycles of reaction (94° C, 20 sec, 46° C, 20 sec, and 68° C, 30 sec in one cycle) and 30 cycles of reaction (94° C, 20 sec, 58° C, 20 sec, and 72° C, 30 sec in one cycle). After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis. Amplified fragments of the desired size (about 400 bp) were purified using the QlAquick Gel Extraction Kit (QIAGEN) by the method described in the attached instruction manual, and eluted with sterile water (30 EL).
The purified Sfi I-VH and Sfi I-VL fragments were digested with Sfi I (Takara Bio) at 50° C overnight in a reaction solution prepared according to the method described in the attached instruction manual. Subsequently, the reaction solution was purified using a QIAquick PCR Purification Kit (QIAGEN) by the method described in the attached instruction manual, and eluted with Buffer EB (30 AL) included in the kit. 7-4. Human IgG4-Mouse chimera bispecific IgG antibody expression plasmid When producing the bispecific IgG antibody of interest, the knobs-into-holes technique of IgGl (Ridgway et al., Protein Eng. 1996; 9: 617-621) was referred to when preparing IgG4 with an amino acid-substituted CH3 portion to form heteromolecules for each H chain. Type a (IgG4ya) is substituted with Y349C and T366W, and type b (IgG4yb) is substituted with E356C, T366S, L368A, and Y407V. Further, a substitution (-ppcpScp- ->-ppcpPcp-) was also introduced at the hinge regions of both types. Almost all the H chains become heteromolecules by this technique; however, this does not necessarily apply to L chains, and the formation of unnecessary antibody molecules may affect subsequent activity measurements. Therefore, to separately express the arms of each antibody molecule (called HL molecule), which have different specificities, and efficiently form the type of bispecific IgG antibody of interest within cells, those that are inducible by different drugs were used as the expression vectors for each HL molecule.
As an expression vector for an arm of the antibody molecule (called right arm HL molecule for convenience), pcDNA4-g4H or pcDNA4-g4L (
As an expression vector for the antibody molecule's other arm (called left arm HL molecule for convenience), pIND-g4H or pIND-g4L (
In each of the ampicillin-resistant transformants thus obtained, insertion of the fragment of interest was confirmed by colony PCR method using primers that sandwich the inserted fragment. First, for the anti-F.IXa antibody chimeric H chain or L chain expression vector, a 21 - mer CMVF primer (SEQ ID NO: 36) which anneals to the CMV forward priming site upstream of the insertion site, and an 18-mer BGHR primer (SEQ ID NO: 37) which anneals to the BGH reverse priming site downstream of the insertion site were synthesized (Sigma Genosys). For the anti-F.X antibody chimeric H chain or L chain expression vector, a 24-mer EcdF primer (SEQ ID NO: 38), which anneals to the upstream of the insertion site and an 18-mer BGHR primer (SEQ ID NO: 37) which anneals to the BGH reverse priming site downstream of the insertion site were synthesized (Sigma Genosys). For colony PCR, a reaction solution (20 JIL) (0.2 VL primer (10 ELM), KOD dash buffer (TOYOBO), 0.2 mM dNTPs, and 0.75 units DNA polymerase KOD dash) (TOYOBO)) was prepared. To this reaction solution, cells of the transformant strain were added in appropriate amounts and PCR was performed. PCR was performed using a thermal cycler GeneAmp PCR system 9700 (Parkin Elmer) under conditions of 1 minute heating at 96° C followed by 30 cycles of reaction (96° C, 10 sec, 55° C, 10 sec, and 72° C, 30 sec in one cycle). After PCR, the reaction solution was subjected to 1% agarose gel electrophoresis, and clones from which amplification fragments of the desired size were obtained were selected. The PCR product was treated with an ExoSAP-IT (Amersham Biosciences) to inactivate excess primers and dNTPs according to the attached instruction manual. Nucleotide sequences of the DNA fragments were determined using a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) on a DNA sequencer ABI PRISM 3100 Genetic Analyzer (Applied Biosystems), according to the method described in the attached instruction manual. Sequence groups determined by the present method were analyzed with an analytical software, GENETYX- SV/RC Version 6.1 (Genetyx). For VH, clones of interest having no insertion, deletion, or mutation were selected. For VL, different from the P3U1 -derived pseudo VL gene used in hybridomas, clones of interest having no insertion, deletion, or mutation were selected.
From the clones of interest, the respective plasmid DNAs were isolated by using a QlAprep Spin Miniprep Kit (QIAGEN), and then dissolved in sterile water (100 JIL). Anti- F.IXa antibody chimeric H chain expression vector, anti-F.IXa antibody chimeric L chain expression vector, anti-F.X antibody chimeric H chain expression vector, and anti-F.X antibody chimeric L chain expression vector were named pcDNA4-g4IXaHn, pcDNA4-g4IXaLn, pIND- g4XHn, and pIND-g4XLn, respectively. Each plasmid solution was stored at 4° C till use. [Example 8] Expression of chimera bispecific antibodies in animal cells 8-1. Preparation of DNA solutions Expression of the antibody's right arm HL molecule expression vectors (pcDNA4- g4IXaHn and pcDNA4-g4IXaLn) is induced by tetracycline. In the absence of tetracycline, Tet repressor-encoding plasmid pcDNA6/TR (Invitrogen) is required to completely suppress their expressions. Further, expression of the left arm antibody HL molecule expression vectors (pIND-g4XHn and pIND-g4XLn) was induced by an insect hormone ecdysone analogue (ponasterone A). This requires plasmid pVgRXR (Invitrogen) which encodes the ecdysone receptor and retinoid X receptor that react with ponasterone A and induce expression. Therefore, for the transfection of animal cells, a mixture of six types of plasmid DNAs in total was prepared. For 1 mL of cell culture, pcDNA4-g4IXaHn, pcDNA4-g4IXaLn, pIND-g4XHn and pIND- g4XLn (218.8 ng each), as well as pcDNA6/TR and pVgRXR (1312.5 ng each) were used. 8-2 Transfection of animal cells A HEK293H strain (Invitrogen) derived from human fetal renal cancer cells was suspended in DMEM medium (Invitrogen) containing 10% FCS (MOREGATE), plated onto each well of a 12-well plate for cell adhesion at a cell density of 5 x 1 cells/mL, and cultured in a CO2 incubator (37° C, 5% CO2). The plasmid DNA mixture prepared in 8-1 was added to a mixed solution of transfection reagent Lipofectamine 2000 (7 jL; Invitrogen) and Opti-MEM I medium (250 liL; Invitrogen) and left to stand at room temperature for 20 minutes. This mixed solution was added to cells in each well, and the cells were incubated in a C02 incubator (37° C, 5% CO2) for four to five hours. 8-3 Induction of bispecific IgG antibody expression After the medium was removed by suction from the above transfected cell cultures, I mL CHO-S-SFM-II (Invitrogen) medium containing 1 gg/mL tetracycline (WAKO Pure Chemical Industries) was added, and primary expression of the antibody's right arm HL molecule was induced by culturing the cells in a C02 incubator (37° C, 5% CO2) for one day. Subsequently, the medium was removed by suction, and the cells were washed once with 1 mL of CHO-S-SFM-II medium, and cultured in a C02 incubator (37° C, 5% CO2) for 2 or 3 days following the addition of 1 mL of CHO-S-SFM-II medium containing 5 gM ponasterone A (Invitrogen), and secondary expression of the antibody's left arm HL molecule was induced for secretion of the bispecific IgG antibody into the medium. The collected culture supematant was centrifuged (approximately 2000g, 5min, room temperature) to remove the cells, and concentrated as needed by Microcon(R) YM-50 (Millipore). The samples were stored at 4° C till use. [Example 9] Quantification of human IgG concentration Goat affinity purified antibody to human IgG Fc (Cappel) was prepared at 1 gg/mL with a coating buffer, and solid-phased onto a Nunc-Immuno plate. After blocking with a diluent buffer (D.B.), samples of culture supernatants appropriately diluted with D.B. were added. As a standard for calculating the antibody concentration, human IgG4 (humanized anti-TF antibody, see WO 99/51743) diluted with D.B. in a 2-fold dilution series with 11 levels from 1000 ng/mL was similarly added. After three washes, alkaline phosphatase goat anti-human IgG (Biosource International) was added for reaction. After five washes, the plate was color developed using the Sigma 104(R) phosphatase substrate (Sigma-Aldrich) as a substrate, and the absorbance at 405 nm was measured on an absorbance reader Model 3550 (Bio-Rad Laboratories) with a reference wavelength of 655 nm. Using the Microplate Manager III (Bio-Rad Laboratories) software, human IgG concentration in the culture supernatant was calculated from the standard curve. [Example 10] F.VIIIa (activated coagulation factor VIII)-mimetic activity assay The F.VIIIa-mimetic activity of a bispecific antibody was assessed by the following enzymatic assay. The following reactions were all performed at room temperature. A mixed solution of 40 jL Factor IX (3.75 jg/mL; Enzyme Research Laboratories) and 10 lL of the antibody solution was incubated in a 96-well plate for one hour. Then, 10 gL Factor XIa (10 ng/mL; Enzyme Research Laboratories), 201L Factor X (50 jg/mL; Enzyme Research Laboratories), 5 lL phospholipid (400 tg/mL; see Example 5-3), and 15 lL TBSB containing 5mM CaCl2 and I mM MgCl2 (hereinafter abbreviated as TBSB-S) were added to initiate the enzymatic reaction. After one hour, the reaction was stopped by adding 10 VL of 0.5M EDTA.
After adding a colorimetric substrate solution (50 pL) to each well, absorbance at 405 nm (reference wave length 655 nm) was measured at 0 and 30 minutes with a Model 3550 Microplate Reader (Bio Rad Laboratories). The F.VIIIa-mimetic activity was presented as a value obtained by subtracting the value of absorbance change in 30 minutes without antibody addition from that with the antibody addition (see
TBSB was used as a solvent for phospholipids, while TBSB-S was used as a solvent for Factor Xla, Factor IX, and Factor X. The colorimetric substrate solution was a 1:1 mixture of “Tesutochimu” colorimetric substrate S-2222 (Chromogenix) dissolved according to the attached instruction manual and a polybrene solution (0.6 mg/L hexadimethrine bromide (Sigma)).
Further, the concentration dependency of XB12/SB04's F.VIIIa-mimetic activity, which was the highest among all, was measured (
To elucidate whether a bispecific antibody corrects the coagulation ability of hemophilia A blood, effects of the bispecific antibody on activated partial thromboplastin time (APTT) were examined using F.VIII-deficient plasma. A mixed solution comprising an antibody solution at various concentrations (50 pL), F.VIII-deficient plasma (50 liL; Biomerieux) and APTT reagent (50 lL; Dade Behring) was warmed at 37° C for 3 minutes. Coagulation reaction was initiated by adding 20 mM CaCl2 (50 VL; Dade Behring) to the above-described mixture. The time required for coagulation was measured with CR-A (Amelung)-connected KC 1 OA (Amelung) (
Further, XB 12/SB04, which showed the highest coagulation time-shortening activity, was measured for its concentration dependency (
The culture supematant (10 mL) obtained by the method described in Example 8 was concentrated to 1 mL with Centricon(R) YM-50 (Millipore). To this concentrate, 10% BSA (10 ItL), 1% Tween(R) 20 (10 tL), and rProtein A Sepharosem Fast Flow (Amersham Biosciences) (100 pL) were added, and the solution was mixed by overturning at 4° C overnight. The solution was transferred to an Ultrafree(R)-MC 0.22 gm filter cup (Millipore), and after washing with TBS containing 0.01% Tween(R) 20 (500 SAL) thrice, the rProtein A Sepharosem resin was suspended in 100 ;L of 0.01% Tween(R) 20 (pH 2.0) containing 10 mM HCI, and left to stand for 3 minutes. Then, the antibody was eluted, and the eluate was immediately neutralized with the addition of 5 pL IM Tris-HCI, pH 8.0. Using the Microplate Manager III (Bio-Rad Laboratories) software, the human IgG concentration was calculated from the standard curve. The antibody. concentration was quantified according to Example 9.
The present invention provides bispecific antibodies that have the effect of functionally substituting for ligands of heteromolecule-comprising receptors.
The present invention also provides bispecific antibodies that recognize both an enzyme and its substrate, and which functionally substitute for a cofactor that enhances the enzymatic activity.
The bispecific antibodies according to the present invention are thought to have high stability in blood and low antigenicity. Thus, it is greatly expected that they will become pharmaceuticals.
This application is the National Stage of International Application No. PCT/JP2003/013123, filed on Oct. 14, 2003.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP03/13123 | 10/14/2003 | WO | 00 | 4/30/2007 |
