Antibody Against Secreted N-Terminal Peptide of GPC3 Present in Blood or C-Terminal Peptide of GPC3
20170233462 · 2017-08-17
Assignee
Inventors
- Hiroyuki Aburatani (Tokyo, JP)
- Yutaka Midorikawa (Tokyo, JP)
- Kiyotaka Nakano (Shizuoka, JP)
- Iwao Ohizumi (Shizuoka, JP)
- Yukio Ito (Tokyo, JP)
- Susumu Tokita (Tokyo, JP)
Cpc classification
Y02A90/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07K2317/732
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
A61P43/00
HUMAN NECESSITIES
C07K2317/34
CHEMISTRY; METALLURGY
C07K17/00
CHEMISTRY; METALLURGY
International classification
C07K17/00
CHEMISTRY; METALLURGY
Abstract
Disclosed is an antibody against a secreted form of GPC3 capable of detecting a secreted form of glypican 3 (GPC3) in a test sample. It is possible to determine whether a subject suffers from cancer, in particular hepatoma. Also disclosed is an antibody against GPC as well as a cell disrupting agent and an anti-cancer agent comprising the same, which can disrupt cells, in particular cancer cells.
Claims
1-2. (canceled)
3. An isolated monoclonal antibody that binds an N-terminal peptide of GPC3, wherein the N-terminal peptide consists of amino acid residues 1-374 or amino acid residues 1-358 as set forth in SEQ ID NO:4.
4-5. (canceled)
6. The antibody according to claim 1 which is immobilized to an insoluble support.
7. The antibody according to claim 1 which is labeled with a labeling material.
8-18. (canceled)
19. An isolated monoclonal antibody-that binds a C-terminal peptide of GPC3, wherein the C-terminal peptide consists of amino acid residues 359-563 as set forth in SEQ ID NO:4, wherein the antibody has ADCC or CDC activity.
20. The antibody according to claim 19 which is a recombinant antibody.
21. The antibody according to claim 19 which is a humanized antibody.
22. The antibody according to claim 21, wherein the constant region of the heavy chain of the humanized antibody is selected from the group consisting of Cγ1, Cγ2, Cγ3, and Cγ4.
23. The antibody according to claim 21, wherein the constant region of the light chain of the humanized antibody is Cκ or Cλ.
24. A method for inducing cytotoxicity comprising contacting a cell with the antibody according to claim 19.
25. The method according to claim 24, wherein the cell is a cancer cell.
26. A method for treating cancer comprising administering a monoclonal antibody that binds a C-terminal peptide of GPC3, wherein the C-terminal peptide consists of amino acid residues 359-563 as set forth in SEQ ID NO:4, and wherein the antibody has ADCC or CDC activity.
27. The method according to claim 26, wherein the cancer is selected from the group consisting of hepatoma, lung cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer, and lymphoma.
28. The method according to claim 27, wherein the cancer is a hepatoma.
29. The method according to claim 26, wherein the antibody is a recombinant antibody.
30. The method according to claim 29, wherein the recombinant antibody is a humanized antibody.
31. The method according to claim 30, wherein the constant region of the heavy chain of the humanized antibody is selected from the group consisting of Cγ1, Cγ2, Cγ3, and Cγ4.
32. The method according to claim 30, wherein the constant region of the light chain of the humanized antibody is Cκ or Cλ.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0125]
[0126]
[0127]
[0128]
[0129]
[0130]
[0131]
[0132]
[0133]
[0134]
[0135]
BEST MODE FOR CARRYING OUT THE INVENTION
[0136] The invention is now specifically described in the following Examples. However, the invention is not limited by the Examples.
[0137] In the Examples described in this specification, the following materials were used.
[0138] As expression vectors of the soluble form of GPC3 and the soluble form of the GPC3 core protein, pCXND2 and pCXND3 prepared by integrating the DHFR gene and the neomycin-resistant gene in pCAGGS were used.
[0139] DXB11 was purchased from ATCC. For culturing, 5% FES (CISCO BRL CAT #10099-141, Lot #AO275242/Minimum Essential Medium Alpha medium (αMEM (+)) (GIBCO BRL CAT #12571-071)/1% Penicillin Streptomycin (GIBCO BRL CAT #15140-122) was used. For selection of stable cell line of DXB11 expressing each protein, 500 μg/mL Geneticin (GIBCO BRL CAT #10131-027)/5% FES/α MEM without ribonucleotides and deoxyribonucleotides (GIBCO BRL CAT #12561-056) (αMEM(−))/PS was used alone or with supplemented with MTX to a final concentration of 25 nM.
[0140] HepG2 was, purchased from ATCC and maintained in 10 FBS/Dulbecco's modified Eagle medium (DMEM) (GIBCO BRL CAT #11995-065)/PS.
[0141] The hybridoma was maintained in 10 FBS/MI1640/1 HAT media supplement (SIGMA CAT #H-0262)/0.5×BM-Condimed H1 Hybridoma cloning supplement (Roche CAT #1088947)
EXAMPLE 1
[0142] Cloning and Expression Analysis of Human GPC3 (GPC3) cDNA Cloning of Full-Length cDNA Encoding Human Glypican 3 (GPC3 Hereinafter)
[0143] The full-length cDNA encoding human GPC3 was amplified by PCR, using as a template a first strand cDNA prepared from a colon cancer cell line Caco2 by a general method and Advantage 2 kit (Clontech Cat. No. 8430-1). Specifically, 50 μl of a reaction, solution containing Caco2-derived cDNA of 2 μl, 1 μl of a sense primer (SEQ ID NO: 1), 1 μl of an antisense primer (SEQ ID NO: 2), 5 1.4,1 of Advantage2 10× PCR buffer, 8 μl of dNTP mix (1.25 mM) and 1.0 μl of Advantage polymerase Mix was subjected to 35 cycles of 94° C. for one minute, 63° C. for 30 seconds and 68° C. for 3 minutes. The amplified product from the PCR (inserted in TA vector pGEM-T easy using pGEM-T Easy Vector System I (Promega Cat No. A1360)) was sequenced using ABI3100 DNA sequencer to confirm that cDNA encoding the full-length human GPC3 was isolated. The sequence represented by SEQ ID NO: 3 indicates the nucleotide sequence of the human GPC3 gene, while the sequence represented by SEQ ID NO: 4 indicates the amino acid sequence of human GPC3 protein.
TABLE-US-00001 SEQ ID NO: 1: GATATC-ATGGCCGGGACCGTGCGCACCGCGT SEQ ID NO: 2: GCTAGC-TCAGTGCACCAGGAAGAAGAAGCAC
Expression Analysis of Human GPC3 mRNA Using GeneChip
[0144] mRNA expression was analyzed in 24 cases with hepatoma lesions (well-differentiated cancer: WD; moderately differentiated cancer: MD; poorly differentiated cancer: PD) 16 hepatoma cases with non-dancer lesions (hepatitis lesion: CH, cirrhosis lesion: LC), 8 cases with normal liver: NL (informed consent acquired; available from Tokyo University, School of Medicine and Saitama Cancer Center), using GeneChip UG-95A Target (Affymetrix). Specifically, total RNA was prepared using ISOGEN (Nippon Gene) from the individual tissues from which 15 μg each of total RNA was used for gene expression analysis according to the Expression Analysis Technical Manual (Affymetrix).
[0145] As shown in
EXAMPLE 2
Preparation of Anti-GPC3 Antibody
Preparation of the Soluble Form of Human GPC3
[0146] As a material for preparing anti-GPC3 antibody, the soluble for a of the GPC3 protein lacking the hydrophobic region on the C-terminal side was prepared.
[0147] Using a plasmid DNA containing the complete full-length human GPC3 cDNA supplied from Tokyo University, Advanced Technology Institute, a plasmid DNA for expressing the soluble form of the GPC3 cDNA was constructed. PCR was conducted using a downstream primer (5′-ATA GAA TTC CAC CAT GGC CGG GAC CGT GCG C-3′) (SEQ ID NO: 5) designed to remove the hydrophobic region on the C-terminal side (564-580 amino acid), and an upstream primer (5′-ATA GGA TCC CTT CAG CGG GGA ATG AAC GTT C-3′) (SEQ ID NO.6) with the EcoRI recognition sequence and the Kozak's sequence having been added. The resulting PCR fragment (1711 bp) was cloned is pCXND2-Flag. The prepared expression plasmid DNA was introduced in a CHO cell line DXB11. Selection with 500 μg/ml Geneticin resulted in a CHO line highly expressing the soluble fear of GPC3.
[0148] Using a 1700-cm.sup.2 roller bottle, the CHO line highly expressing the soluble form of GPC3 was cultured at a large scale, and the culture supernatant was collected for purification. The culture supernatant was applied to DEAE Sepharose Fast Flow (Amersham CAT #17-0709-01), washed, and eluted with a buffer containing 500 mM NaCl. Subsequently, the product was affinity purified using Anti Flag M2 agarose affinity gel (SIGMA CAT #A-2220) and eluted with 200 μg/mL Flag peptide. After concentration with Centriprep-10 (Millipore Cat #4304), the Flag peptide was removed by gel filtration with Superdex 200 HR 10/30 (Amersham CAT #17-1088-01). Finally, the product was concentrated using DEAE Sepharose Fast Flow column, and eluted with PBS (containing 500 mM NaCl) containing no Tween 20 for replacement of the buffer.
Preparation of the Soluble Form of Human GPC3 Core Protein
[0149] Using the wild type human GPC3 cDNA as template, cDNA was prepared by assembly PCR, where Ser 495 and Ser 509 were substituted with Ala. A primer was designed in such a fashion that His tag might be added to the C terminus. The resulting cDNA was cloned in pCXND3 vector. The prepared expression plasmid DNA was introduced in a DXB11 line, followed by selection with 500 μg/mL Geneticin, to obtain the CHO line highly expressing the soluble form of the GPC3 core protein.
[0150] A large scale cultivation was done with a 1700-cm.sup.2 roller bottle, and the culture supernatant was collected for purification. The supernatant was applied to Q sepharose Fast Flow (Amersham CAT #17-0510-01), washed, and eluted with a phosphate buffer containing 500 mM NaCl. Subsequently, the product was affinity purified using Chelating Sepharose Fast Flow (Amersham CAT #17-0575-01), and eluted with a gradient of 10-150 mM imidazole. Finally, the product was concentrated with Q sepharose Fast Flow and eluted with a phosphate buffer containing 500 mM NaCl.
[0151] SDS polyacrylamide gel electrophoresis showed a smear-like band of 50 to 300 kDa and a band of about 40 kDa.
[0152] So as to remove antibodies against heparan sulfate in the following screening for hybridoma, the soluble form of the GPC3 core protein where a heparan sulfate-addition signal sequence Ser 495 and Ser 509 were substituted with Ala. CHO cell line highly expressing the protein was prepared as above, and the culture supernatant was affinity purified utilizing the His-tag. SDS polyacrylamide gel electrophoresis showed three bands of 70 kDa, 40 kDa and 30 kDa. Amino acid sequencing indicated that the band of 30 kDa was the C-terminal fragment of GPC3. The C-terminal fragment starts from serine 359 or from valine 375. Thus, it was anticipated that GPC3 received some enzymatic cleavage. The reason why the band of 30 kDa was not observed in the GPC3 of heparan sulfate-added type was that the fragment formed the smear-like band due to the addition of heparan sulfate. It is a novel finding that GPC3 receives enzymatic cleavage at a specific amino acid sequence, but the biological meaning thereof has not yet been elucidated.
[0153] The inventors made an assumption on the basis of the results that GPC3 on the membrane even in hepatoma patients would be cleaved and secreted as the soluble form in blood. Compared with AFP as a hepatoma marker, the expression of the gene of GPC3 was found higher in hepatoma patients at earlier stages (
Preparation of Anti-GPC3 Antibody
[0154] Because the homology of human GPC3 with mouse GPC3 is as high as 94% at the amino acid levels, it was considered that it might be difficult to obtain the anti-GPC3 antibody by the immunization of normal mouse with human GPC3. Thus, MRL/lpr mouse with autoimmune disease was used as an animal to be immunized. Five MRL/lpr mice (CRL) were immunized with the soluble form of GPC3. For the first immunization, the immunogen protein was adjusted to 100 μg/animal and was then emulsified using FCA (Freund's complete adjuvant (H37 Ra), Difco (3113-60), Becton Dickinson (cat #231131)), which was then subcutaneously administered to the mice. Two weeks later, the protein was adjusted to 50 μg/animal and emulsified with FIA (Freund's incomplete adjuvant. Difco (0639-60), Becton Dickinson (cat #263910)) for subcutaneous administration to the mice. At one week interval since then, booster was carried out in total of 5 times. For final booster, the protein was diluted with PBS to 50 μg/animal, which was administered in the caudal vein. By ELISA using an immunoplate coated with the GPC3 core protein, it was confirmed that the serum antibody titer against GPC3 was saturated. A mouse myeloma cell P3U1 and mouse splenocyte were mixed together to allow for cell fusion in the presence of PEG1500 (Roche Diagnostics, cat #783641) The resulting mixture was inoculated in a 96-well culture plate. From the next day, hybridoma was selected with the HAT medium, the culture supernatant was screened by ELISA. Positive clones were subjected to monocloning by limited dilution method. The resulted monoclone was cultured at an enlarged scale and the culture supernatant was collected. The screening by ELISA was done using the binding activity to the GPC3 core protein as a marker to obtain six clones of an anti-GPC3 antibody with a strong binding potency.
[0155] The antibody was purified using Hi Trap Protein G HP AmershamCAT #17-0404-01). The supernatant from the hybridoma culture was applied directly to a column, washed with a binding buffer (20 mM sodium phosphate pH 7.0) and eluted with an elution buffer (0.1 M glycine-HCl, pH 2.7). The eluate was collected into a tuba containing a neutralization buffer (1 M Tris-HCl, pH 9.0) for immediate neutralization. After body fractions were pooled, the resulting pool was dialyzed against 0.05% Tween 20/PBS overnight and for a whole day for buffer replacement. NaN.sub.3 was added to the purified antibody to 0.02%. The antibody was stored at 4° C.
Analyst of Anti-GPC3 Antibody
[0156] The antibody concentration was assayed by mouse IgG sandwich ELISA using goat anti-mouse IgG (gamma) (ZYMED CAT #62-6600) and alkali phosphatase-goat anti-mouse IgG (gamma) (ZYMED CAT #62-6622), along with a commercially available purified mouse IgG1 antibody (ZYMED CAT #02-6100) as a standard.
[0157] The isotyping of the anti-GPC3 antibody was done with ImmunoPure Monoclonal. Antibody Isotyping Kit II (PIERCE CAT #37502) by the method according to the attached manual. The results of the isotyping indicated that all of the antibodies were of IgG1 type.
[0158] By western blotting using the GPC3 core protein, the epitopes of the anti-GPC3 antibody were classified. The soluble form of the GPC3 core protein was applied to 10% SDS-PAGE mini (TEFCO CAT #01-075) at 100 ng/lane for electrophoresis (60 V for 30 min; 120 V for 90 min) and subsequently transferred on Immobilon-P (Millipore CAT #IPVH R85 10) using Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (BIO-RAD) (15 V for 60 min). After the membrane was gently rinsed with TBS-T (0,05% Tween 20, TBS), the membrane was shaken with 5 skim milk-containing TBS-T for one hour (at ambient temperature) or overnight (at 4° C.). After shaking with TBS-T for about 10 minutes each anti-GPC3 antibody diluted with 1% skim milk-containing TBS-T to 0.1 to 10 μg/ml was added for one-hour with shaking. The membrane was rinsed with TBS-T (10 minutes×three times) and shaken with HRP-anti-mouse IgG antibody (Amersham CAT #NA 931) diluted to 1.1000 with 1% skim milk-containing TBS-T for one hour, and rinsed with TBS-T (10 minutes×three time). ECL-Plus (Amersham RPN 2132) was used for chromogenic reaction. Hyperfilm ECL (Amersham CAT #RPN 2103K) was used for detection.
EXAMPLE 3
Detection of the Secreted Form of GPC3
Mouse Xenograft Model
[0159] 3,000,000 human hepatoma HepG2 cells were transplanted under the abdominal skin in 6-weeks female SCID mice (Fox CHASE C. B-17/Icr-scid Japan Clair) and nude mice (BALB/cA Jcl-nu, Japan Clair). 53 days later when tumor was sufficiently formed, whole blood was drawn out from the posterior cava of HopG2-transplanted SCID mice #1, 3, and 4. Plasma was prepared in the presence of EDTA-2Na and aprotinin (Nigro Neotube vacuum blood tube, NIPRO, NT-EA0205) and stored at −20° C. until assay date. In the case of the HepG2-transplanted SCID mouse #2, whole blood was taken 62 days after HepG2 transplantation. In the case of the HepG2-transplanted nude mice #1 and #2, whole blood was taken 66 days after HepG2 transplantation. As a control, plasma was prepared from normal SCID mouse of the same age by the same procedures.
Sandwich ELISA
[0160] So as to detect the secreted form of GPC3 in blood, sandwich ELISA system of GPC3 was constructed. M6B1 was used as an antibody to be coated in a 96-well plate. M18D4 labeled with biotin was used as an antibody detecting GPC3 bound to M6B10 Fox chromogenic reaction, AMPAK of DAKO was used for achieving high detection sensitivity.
[0161] A 96-well immunoplate was coated with the anti-GPC3 antibody diluted with a coating buffer (0.1 M NaHCO.sub.3, pH 9.6, 0.02 w/v NaN.sub.3) to obtain a concentration of 10 μg/mL, and incubated at 4° C. overnight. On the next day, the plate was rinsed three times with 300 μl/well of rinse buffer (0.05 v/v % Tween 20, PBS) and 200 μl of dilution buffer (50 mM Tris-HCl, pH 8.1, 1 mM MgCl.sub.2, 150 mM NaCl,0.05 v/v % Tween 20, 0.02 w/v % NaN.sub.3, 1 w/v % BSA) was added for blocking. After storage for several hours at ambient temperature or at 4° C. overnight, mouse plasma or the culture supernatant appropriately diluted with a dilution buffer was added and incubated at ambient temperature for one hour. After rinsing with RB at 300 μl/well three times, the biotin-labeled anti-GPC3 antibody diluted with a dilution buffer to 10 μg/mL was added, and incubated at ambient temperature for one hour. After rinsing with RB at 300 μl/well three times, AP-streptoavidin (ZYMED) diluted to 1/1000 with a dilution buffer was added, and incubated at ambient temperature for one hour. After rinsing with the rinse buffer at 300 μl/well five times, AMPAK (DAKO CAT #K6200) was added for chromogenic reaction according to the attached protocol, and the absorbance, was measured with a microplate reader.
[0162] For biotinylation of the antibody, Biotin Labeling Kit (CAT #1 418 165) of Roche was used. A spreadsheet software GlaphPad PRISM (GlaphPad software Inc. ver. 3.0) was used to calculate the concentration of the soluble form of GPC3 in a sample.
[0163] Using the purified soluble form of GPC3, a standard curve was prepared. Consequently, a system with a detection limit of several nanogams/mL could be constructed.
TABLE-US-00002 TABLE 1 Tumor volume M6B01 (N)- M19B11 (N)- M6B1(N)- M13B3(C)- M13B3(C)- (mm.sup.3) M1BD4(N) M18D4(N) BioM3C11(C) BioM18D4(N) BioM3B8(C) Culture supernatant of HepG2 1190 1736 224 234 <1 HepG2-transplanted SCID mouse #1 2022 65.4 76.9 <10 <10 <10 HepG2-transplanted SCID mouse #2 1706 71.7 94.8 <10 <10 <10 HepG2-transplanted SCID mouse #3 2257 90.3 113.9 <10 <10 <10 HepG2-transplanted SCID mouse #4 2081 87.3 107.3 <10 15.0 <10 HepG2-transplanted nude mouse #1 1994 58.7 53.6 19.7 35.5 102.2 HepG2-transplanted nude mouse #2 190 & 549 22.9 33.6 <10 11.5 40.6 Normal SCID mouse #1 0 <10 <10 <10 <10 <10 Normal SCID mouse #2 0 <10 <10 <10 <10 <10 Normal SCID mouse #3 0 <10 <10 <10 <10 <10
Structure of Secreted Form of GPC3
[0164] It was examined whether or not the blood-secreted GPC3 has the structure of the N-terminal fragment as preliminarily assumed. In case that the secreted form of GPC3 was the N-terminal fragment it is considered that the secreted form of GPC3 will not be detected by sandwich ELISA with a combination of an antibody recognizing the N terminus and an antibody recognizing the C terminus. Using three types of each antibody recognizing the N-terminal fragment and each antibody recognizing the C-terminal fragment, sandwich ELISA systems with various combinations were constructed.
EXAMPLE 4
Preparation of Anti-GPC3 Mouse-Human Chimera Antibody
[0165] Using total RNA extracted from a hybridoma producing an antibody capable of binding to human GPC3 (human GPC3-antibody recognizing C-terminus: M3C11, M1E07; human GPC3-antibody recognizing N terminus: M19B11, M18D04, M51309, M10D02), the cDNA of variable region of the antibody was amplified by RT-PCR. The total RNA was extracted from the hybridoma of 1×10.sup.7 cells, using RNeasy Plant Mini Kits (manufactured by QIAGEN). Using 1 μg of the total RNA and also using SMART RACE cDNA Amplification Kit (manufactured by CLONTECH), a synthetic oligonucleotide MHC-IgG1 (SEQ ID NO:7) complementary to the mouse IgG1 constant region sequence or a synthetic oligonucleotide kappa (SEQ ID NO:8) complementary to the nucleotide sequence of the mouse κ chain constant region, a 5′-terminal fragment of the gene was amplified. The reverse-transcription was done at 42° C. for one hour and 30 minutes. 50 μl of the PCR solution contained 5 μl of 10×Advantage 2 PCR Buffer, 5 μl of 10×Universal Primer A Mix, 0.2 mM dNTPs (dATP, dGTP, dCTP, dTTP), 1 μl of Advantage 2 Polymerase Mix (all manufactured by CLONTECH), 2.5 μl of the reverse-transcription product, and 10 pmole of the synthetic oligonucleotide MHC-IgG1 or kappa. After the initial temperature at 94° C. for 30 seconds, a cycle of 94° C. for 5 seconds and 72° C. for 3 minutes was repeated five times; a cycle of 94° C. for 5 seconds, 70° C. for 10 seconds and 72° C. for 3 minutes was repeated five times; and a cycle of 94° C. for 5 seconds for 10 seconds and 72° C. for 3 minutes was repeated 25 times. Finally, the reaction product was heated at 72° C. for 7 minutes. After the individual PCR products were purified from agarose gel using QIAquick Gel Extraction Kit (manufactured by QIAGEN), the products were cloned in pGEM-T Easy vector (manufactured by Promega), and the nucleotide sequence was determined.
[0166] Then the sequences of the variable regions of the H chain and L chain were linked to the constant regions of the human H chain and L chain. PCR was done using a synthetic oligonucleotide complementary to the 5′-terminal nucleotide sequence of the H chain variable region of each antibody and having the Kozak's sequence and a synthetic oligonucleotide complementary to the 3′-terminal nucleotide sequence and having an NheI site. The resulting PCR products were cloned In a pB-CH vector with the human IgG1 constant region inserted in pBluescript KS+ vector (manufactured by TOYOBO). The mouse H chain variable region and the human H chain (γ1 chain) constant region are liked together via the NheI site. The prepared H chain gene fragment was cloned in an expression vector pCXND3. The scheme the construction of the vector pCXND3 is described below. So as to divide the gene encoding the antibody H chain and the vector sequence from DHFR-ΔE-rvH-PMl-f (see WO 92/19759) the vector was digested at the restriction enzyme EcoRI/SmaI sites to recover only the vector sequence. Subsequently, the vector sequence was cloned in EcoRI-NotI-BamHI adaptor (manufactured by Takara Shuzo Co., Ltd.). This vector was designated as pCHOl. A region from pCHOl expressing the DHFR gene was cloned in pCXN at the restriction enzyme HindiIII site (Niwa et al., Gene 1991: 108: 193-200). The resulting vector was designated as pCXND3. The nucleotide sequences of the H chains of the anti-GPC3 mouse-human chimera antibodies (M3C11, M1E07, M19811, M18D04) contained in each plasmid are shown as SEQ ID NOS: 9, 11, 13 and 15, respectively. The amino acid sequences thereof are shown as SEQ ID NOS: 10, 12, 14, and 16, respectively. Additionally, PCR was done using a synthetic oligonucleotide complementary to the 5′-terminal nucleotide sequence of the L chain variable region of each antibody and having the Kozak's sequence and a synthetic oligonucleotide complementary to the 3′-terminal nucleotide sequence and having a BsiWI site. The resulting PCR products were cloned in a pB-CL vector, where the human kappa chain constant region was preliminarily inserted in pBluescript KS+ vector (manufactured by TOYOBO). The human L chain variable region and the constant region were linked together via the BsiWI site. The prepared L chain gene fragment was cloned in an expression vector pUCAG. The vector pUCAG is a vector prepared by digesting pCXN (Niwa et al., Gene 1991: 108: 193-200 with restriction enzyme BamHI to obtain a 2.6-kbp fragment, which is then cloned into the restriction enzyme BamHI site of pUC19 vector (manufactured by TOYOBO) The nucleotide sequences of the L chains of the anti-GPC3 mouse-human chimera antibodies (M3C11, M1E07, M19B11, M18D04) contained in each plasmid are shown as SEQ ID NOS: 17, 19, 21 and 23, respectively. The amino acid sequences thereof are shown as SEQ ID NOS: 18, 20, 22 and 24, respectively.
[0167] So as to prepare an expression vector of the anti-GPC3 mouse-human chimera antibody, a gene fragment obtained by digesting the pUCAG vector having the L chain gene fragment inserted therein with restriction enzyme HindIII (manufactured by Takara Shuzo Co., Ltd.) was cloned into the restriction enzyme cleavage site of pCXND3 having the H chain gene inserted therein. The plasmid will express the neomycin-resistant gene, the DHFR gene and the anti-GPC3 mouse-human chimera antibody gene in animal cells.
[0168] A CHO-based cell line for stable expression (DG44 line) was prepared as follows. The gene was introduced by electroporation method using Gene PulserII (manufactured by Bio Rad) 25 μg of each expression vector of the anti-GPC3 mouse-human chimera antibody and 0.75 ml of CHO cells (1×10.sup.7 cells/ml) suspended in PBS were mixed together, and cooled on ice for 10 minutes, which was then transferred into a cuvette and received a pulse at 1.5 kV and 25 μFD. After a recovery time at ambient temperature for 10 minutes, the cells treated by the electroporation were suspended in 40 mL of a CHO-S-SFMII culture medium (manufactured by Invitrogen) containing 1×HT supplement (manufactured by Invitrogen). A 50-fold dilution was prepared using the same culture medium, and added at 100 μl/well in a 96-well culture plate. After culturing in a CO.sub.2 incubator (5% CO.sub.2) for 24 hours, Geneticin (manufactured by Invitrogen) was added to 0.5 mg/mL, and continued cultivation for 2 weeks. The IgG in the culture supernatant from the wells of colonies of a Geneticin resistance transformant cell was assayed by the following concentration assay method. A cell line with high productivity was expanded at an enlarged scale. The cell line stably expressing the anti-GPC3 mouse-human chimera antibody was cultured in a large-scale culturing and the culture supernatant was collected.
[0169] The IgG concentration in the culture supernatant was assayed by human IgG sandwich ELISA using Goat Anti-human IgG (manufactured by BIOSORCE) and Goat Anti-human IgG alkaline phosphatase conjugated (manufactured by BIOSORCE) and compared with the commercially available purified human IgG (manufactured by Cappel).
[0170] Each anti-GPC3 mouse-human chimera antibody was purified using Hi. Trap Protein G HP (manufactured by Amersham). A culture supernatant of a CHO cell line producing the anti-GPC3 mouse-human chimera antibody was directly applied to a column and eluted with elution buffer (0.1 M glycine-HCl, pH 2.7). Eluate a collected into a tube containing a neutralization buffer (1 M Tris-HCl, pH 9.0) for immediate neutralization. Antibody fractions were pooled and dialyzed against 0.05 Tween 20/PBS overnight and for a whole day to replace the buffer NaN.sub.3 was added to the purified antibody to 0.02 and stored at 4° C.
EXAMPLE 5
Preparation of a CHO Cell Line Stably Expressing the Full Length GPC3
[0171] Human GPC3 cDNA was obtained by digesting pGEM-T Easy vector with the full-length human GPC3 cDNA cloned therein with rest lotion enzyme EcoRI (manufactured by Takara Shuzo C., Ltd.) and cloned in an expression vector pCOS2. The scheme of the construction of the vector pCOS2 is described below. So as to divide the gene of the antibody H chain of DHFR-ΔE-rvH-PM1-f (see WO 92/19759) from the vector, the vector was digested at the restriction enzyme EcoRI/SmaI sites, to recover only the vector sequence. Subsequently, the vector sequence was cloned in EcoRI-NotI-BamHI adaptor (manufactured by Takara Shuzo Co., Ltd.). This vector a designated as pCHO1. A region from pCHO1 expressing the DHFR gene was removed, into which the sequence of the neomycin resistant gene in HEF-VH-gγl (Sato et al., Mol. Immunol. 1994: 31: 371-381) was inserted. The vector was designated as pCOS2.
[0172] A cell line stably expressing the full-length human GPC3 was prepared as follows, 10 of the full-length human GPC3 gene expressing vector and 60 μl of SuperFect (manufactured by QIAGEN) were mixed together, to form a complex, which was then added to a CHO cell line DXB11 to introduce the gene. After culturing in a CO.sub.2 incubator (5% CO.sub.2) for 24 hours, αNEM (manufactured by GIBCO BRL) containing Geneticin (manufactured by Invitrogen) to a final concentration of 0.5 mg/mL and 10% PBS (manufactured by GIBCO BRL) was used to start selection. The resulting Geneticin-resistant colonies were collected and cell cloning was done by limited dilution method. Individual cell clones were solubilized to confirm the expression of the full-length human GPC3 by western blotting using the anti-GPC3 antibody. A cell strain stably expressing human GPC3 was obtained.
EXAMPLE 6
[0173] ADCC Assay Using PBMC Derived from Human Peripheral Blood
(1) Preparation of Human PBMC
[0174] Peripheral blood was collected from normal subjects with heparinized syringes and diluted to 2 fold with PBS (−), and overlaid on Picoll-Paque™ PLUS (Amersham Pharmacia Biotech AB). This was centrifuged (500×g, 30 minutes, 20° C.) and collected the intermediate layer as a mononuclear cell fraction. After rinsing three times, the resulting fraction was suspended in 10% FBS/RPMI to prepare a human PBMC solution.
(2) Preparation of Target Cell
[0175] HepG2 cell cultured in 10% FBS/RPMI 1640 culture medium was detached from the dish using trypsin-EDTA (Invitrogen Corp) divided in each well at 1×10.sup.4 cells/well in a U-bottom 96-well plate (Falcon), and cultured for 2 days. After culturing, 5.55 MBq of chromium-51 was added and the cells were incubated in a 5% CO.sub.2 gas incubator at 37° C. for one hour. The resulting cells were rinsed once with the culture medium, to which 50 μl of 10% FBS/RPMI 1640 culture medium was added to prepare a target cell.
(3) Chromium Release Test (ADCC Activity)
[0176] 50 μl of an antibody solution prepared to each concentration was added to the target cell on ice for 15 minutes. Subsequently, 100 Ill of a human PBMC solution was added (5×10.sup.5 cells/well), and incubated in a 5% CO.sub.2 gas incubat at 37° C. for 4 hours. After incubation, the plate was centrifuged and the radioactivity in 100 μl of the culture supernatant was counted with a gamma counter. The specific chromium release ratio was determined by the following formula:
Specific chromium release ratio (%)=(A−C)×100/(B−C)
[0177] “A” represents the mean radioactivity value (cpm) in each well; “B” represents the mean radioactivity value (cpm) in a well where 100 of aqueous 2% NP-40 solution (Nonidet P-40, Code No. 252-23, Nakarai Tesque) and 50 μl of 10% FBS/RPMI culture medium were added to the target cell; and “C” represents the mean radioactivity value (cpm) in a well where 150 μl of 10% FBS/RPMI culture medium was added to the target cell. The test was done in triplicate to calculate the mean of the ADCC activity (%) and the standard error.
[0178] The results are shown in
EXAMPLE 7
Assay of Compliment-Dependent Cytotoxic Activity (CDC Activity)
(1) Preparation of Human Albumin Veronal Buffer (HAVB)
[0179] 12.75 g of NaCl (superior grade; Wako Pure Chemical Industries Ltd.), 0.5625 g of Na-barbital (superior grade; Wako Pure Chemical Industries, Ltd.), and 0.8625 g of barbital (superior grade; Wako Pure Chemical Industries, Ltd.) were dissolved in Milli Q water to 200 L, and autoclaved (121° C., 20 minutes). 100 mL of autoclaved warm Milli Q water was added. Then, it was confirmed that the resulting mixture was at pH 7.43 (pH 7.5 recommended) This was defined as 5×Ver seal Buffer a 0.2205 g of CaCl.sub.2-2H.sub.2O (superior grade; Wako Pure Chemical Industries, Ltd.) was dissolved in 50 mL of Milli water to 0.03 mol/L. The resulting solution was defined as CaCl.sub.2 solution. 1.0165 g of MgCl.sub.2-6H.sub.2O (superior grade Wako Pure Chemical Industries. Ltd.) was dissolved in 50 mL of Milli Q water to 0.1 mol/L. The resulting solution was defined as MgCl.sub.2 solution. 100 mL of 5×Veronal Buffer, 4 mL of human serum albumin (Buminate.sup.R 25%, 250 mg/mL of human serum albumin concentration, Baxter), 2.5 mL of the CaCl.sub.2 solution, 2.5 mL of the MgCl.sub.2 solution, 0.1 g of KCl (superior grade; Wako Pure Chemical Industries, Ltd.) and 0.5 g of glucose (D (+)-glucose anhydrous glucose, superior grade; Wako Pure Chemical Industries, Ltd.) were dissolved in Milli Q water to 500 mL. This was defined as HAVB. After filtration and sterilization, the resulting solution was stored at a set temperature of 5° C.
(2) Preparation of Target Cell
[0180] CHO cell expressing GPC3 on the cell membrane as prepared in Example 4 was cultured in alpha-MEM nucleic acid (+) culture medium (GIBCO) supplemented with 10 FBS and 0.5 mg/mL Geneticin (GIBCO), detached from the dish using a cell dissociation buffer (Invitrogen Corp), and divided at 1×10.sup.4 cells/well in each well of a 96-well flat bottom plate (Falcon), for culturing for 3 days. After culturing, 5.55 MBq of chromium-51 was added, and incubated in a 5% CO.sub.2 gas incubator at 37° C. for one hour. The resulting cell was rinsed twice with HAVB, to which 50 μl of HAVB was added to prepare a target cell.
(3) Chromium Release Test (CDC Activity)
[0181] Each chimera antibody was diluted with HAVB to prepare an antibody solution of 40 μg/mL. The antibody solution was added in a 50 μl-portion to the target cell, which was then left on ice for 15 minutes. Subsequently, baby rabbit compliment (Cedarlane) diluted with HAVE was added in 100 μl portions to each well to a final concentration of 30% (final antibody concentration of 10 μg/mL), and incubated in a 5% CO.sub.2 gas incubator at 37° C. for 90 minutes. After centrifugation of the plate, a 100 μl portion of the supernatant was recovered from each well, and the radioactivity was measured with a gamma counter. The specific chromium release ratio was determined by the following formula:
Specific chromium release ratio (%)=(A−C)×100/(B−C)
[0182] “A” represents the mean radioactivity value (cpm) in each well; “B” represents the mean radioactivity value (cpm) in a well where 100 μl of aqueous 2% NP-40 solution (Nonidet P-40, Code No. 252-23, Nakarai Tesque) and 50 μl of HAVB were added to the target cell; and “C” represents the mean radioactivity value (cpm) in well where 150 μl of HAVB was added to the target cell. The test was done in triplicate to calculate the mean of the CDC activity (%) and the standard error.
[0183] The results are shown in
INDUSTRIAL APPLICABILITY
[0184] As shown in the Examples, it was suggested such that a portion of GPC3 highly expressed in hepatoma cells may exist as a secreted form in blood. Because the gene expression of GPC3 is observed at an earlier stage than that of AFP, a hepatoma marker, GPC3 detection is expected to be useful for cancer diagnosis. It is observed that GPC3 is expressed in cancer cell lines other than hepatoma cell lines, such as lung cancer, colon cancer, breast cancer, prostate cancer, pancreatic cancer and lymphoma. Accordingly, GPC3 is possibly applicable to the diagnosis of cancers other than hepatoma.
[0185] Additionally, it is also suggested that a secreted form of GPC3 in blood predominantly comprises the N-terminal fragment of about 40 kDa, which is observed in the soluble form of the GPC3 core protein. This indicates that antibodies recognizing the N-terminal fragment are useful as the antibody for use in such diagnosis. In addition, if antibodies recognizing the C-terminal fragment with the ADCC activity and/or the CDC activity are used for treating hepatoma, the antibodies can efficiently reach hepatoma cell without being trapped by the secreted form of GPC3 present in blood. Thus, such antibodies are useful as agents for disrupting cancer cells and as anti-cancer agents.
[0186] The contents of all the publications listed in this specification are entirely included in the specification. Additionally, a person skilled in the art will readily understand that various modifications and variations of the invention are possible without departure from the technical scope and inventive range described in the attached claims. It is intended that the invention also encompasses such modifications and variations.