CA2127797A1 - Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virus - Google Patents
Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virusInfo
- Publication number
- CA2127797A1 CA2127797A1 CA002127797A CA2127797A CA2127797A1 CA 2127797 A1 CA2127797 A1 CA 2127797A1 CA 002127797 A CA002127797 A CA 002127797A CA 2127797 A CA2127797 A CA 2127797A CA 2127797 A1 CA2127797 A1 CA 2127797A1
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- Prior art keywords
- antibodies
- idiotypic
- cell line
- under deposit
- hcv
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
- C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
- C07K16/4216—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-viral Ig
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
- C07K16/109—Hepatitis C virus; Hepatitis G virus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
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- Biochemistry (AREA)
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- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Hematology (AREA)
- General Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Communicable Diseases (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to monoclonal antibodies specifically immunoreactive with hepatitis C viral antigens, cell lines secreting said antibodies, an immunodiagnostic reagent comprising the antibodies, and a method and test-kit for the detection of HCV.
The present invention further relates to anti-idiotypic antibodies, cell lines secreting these anti-idiotypic antibodies, immunodiagnostic reagents comprising said anti-idiotypic antibodies and a test-kit for the detection of anti-HCV antibodies in a sample, using anti-idiotypic antibodies.
The present invention further relates to anti-idiotypic antibodies, cell lines secreting these anti-idiotypic antibodies, immunodiagnostic reagents comprising said anti-idiotypic antibodies and a test-kit for the detection of anti-HCV antibodies in a sample, using anti-idiotypic antibodies.
Description
W094/14974 ~l~ 7 7 9 7 PCT~ ~3/03707 Title: Monoclonal antibodies and anti-idiotypic antibodies to Hepatitis C virus.
The present invention relates to ~onoclonal antibodies specifically immunoreactive with : hepatitis C viral antigens, cell lines secreting said antibodies, an immunodiagnostic reagent comprising the antibodies, and a method and test-kit for the detection of HCV.
The present invention further relates to anti-idiotypic antibodies, cell lines secret;ing said anti-idiotypic antibodies, im~unodiagnostic reagents comprising said a~ti-idioty~ic antibodies and a test-kit for the detection of anti-HCV anti~odies in a sample, usinq anti-idiotypic antibodias~
Hepatitis C virus (HCV) is a 9.4-kb, single stranded polyad~nylated RNA virus which has been recognized as one of the causative agents of NANB
hepatitis ~Non-A, Non-B). It causes acute and chronic liver disease and is implicated in hepat~c~llular carcinoma.
It can be distinguîshed from other forms of viral-associated liver diseases, including those caused by known hepatitis viruses, i.e., hepatitis A virus (HAV), hepatitis B virus (H8V), and hepatitis delta virus ~HDV), as well as the hepatitis induced ~y cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Evidence based on hydrophobicity plots and sequence homologies suggests tha~ H~V may be distantly related to the f~mily Flaviviridae fHouahton _~L et al~.
Hcna~ylg~y_ 14 ~81, l9~l~. Non-A, Non-B Hepatitis -~
was first identified in transfused individuals.
Transmission from man to chimpanzee and serial CONFIRMATION COPY
7 !3 7 W094/14974 PCT~3/03707 -.
passage in chimpanzees pr~vided evidence that Non-A, Non-B Hepatitis is due to a transmissible infectious agent or agents.
Epidemiologic evidence is suggestive that three types of Non-A, Non-B Hepatitis exist: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring ~community acquired3 type. The viral. genome of ~CV encodes a polyprotein of approximately 3010 amino acids that undergoes extensive posttran~la ional processing. The viral structural region is loca~ed upstream from t:he nonstructural region and putatively includes a highly cons~rved l9-kDa nucleocapsid protein, and two extensi~ely glycosylated envelope polypeptides, ~p 33 (El~ and gp72 (~2/NSl).
Recent studies indicate that substantial sequence heterogeneity exists among virtually all HCV
isolates in the N-terminal region of E2/NSl, suggesting that this region of the HCV envelope may be under strong immune selection. A variety of presumed nonstructural proteins are processed f rom the remainder of the HCV polyprotein, including a me~brane-bound 23-kDa proteinj- NS2, and a soluble protein of approximat~ly 60 kDa, NS3, which corresponds to the viral helicase and ~ay contain a N-terminal serine protease ~omain, currently thought to be inYolved in the processing of the NS proteins. The function of the NS4 protein is presentl~ unknown, but it comprises the 5-1-1 fra~ment that contains im~unodominant antibody binding sites (Kuo G~ et al. ! ~ci~nce 244:362. 1491~ ~er~no . A. et al.J.Immunol.~ 147:2692) ; NS5 contains the viral replicase. Clinical studies have shown that, following exposure to HCV, antibodies to conserved regions of the viral nucleoprotein and - WO94/14974 ~12 7 7 9 7 PCT~3/03707 NS3 may appear several weeks before seroconversion to anti-clOO-3, a recombinant protein encompassing the C-terminus of NS3 and part of the NS4 protein. Thus, serological assays incorporating the highly-conserved HCV
nucleocapsid protein as well as NS3 are likely to become useful ~iagnostic markers of acute HCV
infection.
It is an object of the present invention to provide novel monoclonal antibodies that specifically react with the NS3 protein of the HCV virus and are particularly useful in immunodiagnostic tests for the detection of the presence or absence of HCV in clinical specimen.
The present inv~ntion provide~ monoclonal antibodies which bind to an epitope of the NS3 protein of hepatitis C virus, which epitope i~
specifically recognized by monoclonal antibodies secreted by the Epstein-Barr virus-transformed hum~n ly~phocyte-B cell line deposited at the European Collection of Ani~al Cell Cultures, Porton Down (UK~ under deposit No. g21~1609.
A preferred monoclonal antibody accor~ing to the invention is ~ecreted by the ~p~tein-Barr virus-transform~d human lymphocyte-B cell line depo~ited at the European Collection of ~nimal Cell Cultures, Porton Down (UK~ under deposit No.
92121609.
Peptides comprising the NS-3 epitope recognized by the monoclonal antibodies according to this invention are described in the co-pending and co-owned Patent Application No.
PCT/EP93/03478, the contents of which are incorporated herein by reference.
WO94/14974 ~ l ~ 7 7 9 7 PCT~3/03707 . 4 The above cell line tldS been deposited at the ECACC~ on 16 December 1992, under the terms and conditions of the Budapest treaty, 1977.
Cell lines capable of excreting these monoclonal antibodies are also part of the present invention.
The preparation of cell lines producing monoclonal antibodies may occur by, for example, tran~for~ation with Epstein-Barr Virus, the ~hler and Milstein technique (Kohler nnd Mil~tein devised the techniques that resulted in the formation monoclonal antibody-producing hybrido~s (G. K~hler and C. Mil~tein, 1975, Nature ~5~:495 497; 1976, Eur. J. I~unol. 6:511-519)), or a direct transformation t~chnique of ~-lymphocyt~s with oncogenic DN~, nr a dir~ct fusion of ~um~n ~-lymphocytes with a fusion partner being eith~r a hu~an or a mouse-hu~an hybrid myeloma cell line, or a direct fusion of an EBV-tranæformed B cell line with said ~yeloma cell ~ines.
A preferred cell line according to the invention is an Epstein-Barr virus-tran~form~d human lymphocyte-B clone cell line capable of excreting monoclonal antibodies which bind to an epitope on the NS3 protein of hepatitis C virus, which cell line is deposited at the European Collection of Animal Cell ~ultures, Porton Down (UK), under deposit No. 9212160~.
T~e Epstein Barr virus (EBV) is capable of transforming and immortalizing human ~-lymphocytes. With the aid of the Epstein Bar virus immortalized human ~-lymphocytes can be .~ wo94ll4s74 ~ ~ 7 7 ~ 7 PCT~3/03707 obtained without the need of a myeloma partner cell. The Epstein Barr virus can be obtained from a variety of sources. The most common used source of EBV is the B95-8 marmoset cell line. The B95-8 cell line spontaneously releases Epstein Barr virus into the medium.
A variety of cell types has been suggested to provide good feeder layers for the cloning of EBV transformed cells. The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
PB~Cs conæist of monocytes, T lymphocy~es and B-lympho~ytes (5-1~%). Because not all B-lymphocytes will provide antibodies of the ri.ght specificity, it is ad~antageous to enric~ PBMCs for appropriate cells. T~ prevent the generation of ~ytotoxic T-cells against ~BV-transfor~ed cells, ~-lymphocytes can be removed, prior to ~BV
infection (with, for example, the supernatant from the EBV-productive B95-8 cell line), by treating the cells with wa~hed sheep red blood cells ( ~ I~ a -Dr~içle g;~U~çhn, Edi~or ~a~ a~
Enql~n~L ~9~. C~ 4). -~5 Immortalized B cell lines according to the invention were considered monoclonal if they satisfied the following require~ents~
1) Stability of antibody secretion over time (~6 months), 2) Secretion of only one XgG (H and L) functional molecule, 3) Specificity and stability of antibody secretion following at least 2 sequential subcloning procedures ~100% of growing colonies ecreting IgG with specificity and genetic phenotype identical to the parental line).
The present invention relates to ~onoclonal antibodies specifically immunoreactive with : hepatitis C viral antigens, cell lines secreting said antibodies, an immunodiagnostic reagent comprising the antibodies, and a method and test-kit for the detection of HCV.
The present invention further relates to anti-idiotypic antibodies, cell lines secret;ing said anti-idiotypic antibodies, im~unodiagnostic reagents comprising said a~ti-idioty~ic antibodies and a test-kit for the detection of anti-HCV anti~odies in a sample, usinq anti-idiotypic antibodias~
Hepatitis C virus (HCV) is a 9.4-kb, single stranded polyad~nylated RNA virus which has been recognized as one of the causative agents of NANB
hepatitis ~Non-A, Non-B). It causes acute and chronic liver disease and is implicated in hepat~c~llular carcinoma.
It can be distinguîshed from other forms of viral-associated liver diseases, including those caused by known hepatitis viruses, i.e., hepatitis A virus (HAV), hepatitis B virus (H8V), and hepatitis delta virus ~HDV), as well as the hepatitis induced ~y cytomegalovirus (CMV) or Epstein-Barr virus (EBV). Evidence based on hydrophobicity plots and sequence homologies suggests tha~ H~V may be distantly related to the f~mily Flaviviridae fHouahton _~L et al~.
Hcna~ylg~y_ 14 ~81, l9~l~. Non-A, Non-B Hepatitis -~
was first identified in transfused individuals.
Transmission from man to chimpanzee and serial CONFIRMATION COPY
7 !3 7 W094/14974 PCT~3/03707 -.
passage in chimpanzees pr~vided evidence that Non-A, Non-B Hepatitis is due to a transmissible infectious agent or agents.
Epidemiologic evidence is suggestive that three types of Non-A, Non-B Hepatitis exist: the water-borne epidemic type; the blood or needle associated type; and the sporadically occurring ~community acquired3 type. The viral. genome of ~CV encodes a polyprotein of approximately 3010 amino acids that undergoes extensive posttran~la ional processing. The viral structural region is loca~ed upstream from t:he nonstructural region and putatively includes a highly cons~rved l9-kDa nucleocapsid protein, and two extensi~ely glycosylated envelope polypeptides, ~p 33 (El~ and gp72 (~2/NSl).
Recent studies indicate that substantial sequence heterogeneity exists among virtually all HCV
isolates in the N-terminal region of E2/NSl, suggesting that this region of the HCV envelope may be under strong immune selection. A variety of presumed nonstructural proteins are processed f rom the remainder of the HCV polyprotein, including a me~brane-bound 23-kDa proteinj- NS2, and a soluble protein of approximat~ly 60 kDa, NS3, which corresponds to the viral helicase and ~ay contain a N-terminal serine protease ~omain, currently thought to be inYolved in the processing of the NS proteins. The function of the NS4 protein is presentl~ unknown, but it comprises the 5-1-1 fra~ment that contains im~unodominant antibody binding sites (Kuo G~ et al. ! ~ci~nce 244:362. 1491~ ~er~no . A. et al.J.Immunol.~ 147:2692) ; NS5 contains the viral replicase. Clinical studies have shown that, following exposure to HCV, antibodies to conserved regions of the viral nucleoprotein and - WO94/14974 ~12 7 7 9 7 PCT~3/03707 NS3 may appear several weeks before seroconversion to anti-clOO-3, a recombinant protein encompassing the C-terminus of NS3 and part of the NS4 protein. Thus, serological assays incorporating the highly-conserved HCV
nucleocapsid protein as well as NS3 are likely to become useful ~iagnostic markers of acute HCV
infection.
It is an object of the present invention to provide novel monoclonal antibodies that specifically react with the NS3 protein of the HCV virus and are particularly useful in immunodiagnostic tests for the detection of the presence or absence of HCV in clinical specimen.
The present inv~ntion provide~ monoclonal antibodies which bind to an epitope of the NS3 protein of hepatitis C virus, which epitope i~
specifically recognized by monoclonal antibodies secreted by the Epstein-Barr virus-transformed hum~n ly~phocyte-B cell line deposited at the European Collection of Ani~al Cell Cultures, Porton Down (UK~ under deposit No. g21~1609.
A preferred monoclonal antibody accor~ing to the invention is ~ecreted by the ~p~tein-Barr virus-transform~d human lymphocyte-B cell line depo~ited at the European Collection of ~nimal Cell Cultures, Porton Down (UK~ under deposit No.
92121609.
Peptides comprising the NS-3 epitope recognized by the monoclonal antibodies according to this invention are described in the co-pending and co-owned Patent Application No.
PCT/EP93/03478, the contents of which are incorporated herein by reference.
WO94/14974 ~ l ~ 7 7 9 7 PCT~3/03707 . 4 The above cell line tldS been deposited at the ECACC~ on 16 December 1992, under the terms and conditions of the Budapest treaty, 1977.
Cell lines capable of excreting these monoclonal antibodies are also part of the present invention.
The preparation of cell lines producing monoclonal antibodies may occur by, for example, tran~for~ation with Epstein-Barr Virus, the ~hler and Milstein technique (Kohler nnd Mil~tein devised the techniques that resulted in the formation monoclonal antibody-producing hybrido~s (G. K~hler and C. Mil~tein, 1975, Nature ~5~:495 497; 1976, Eur. J. I~unol. 6:511-519)), or a direct transformation t~chnique of ~-lymphocyt~s with oncogenic DN~, nr a dir~ct fusion of ~um~n ~-lymphocytes with a fusion partner being eith~r a hu~an or a mouse-hu~an hybrid myeloma cell line, or a direct fusion of an EBV-tranæformed B cell line with said ~yeloma cell ~ines.
A preferred cell line according to the invention is an Epstein-Barr virus-tran~form~d human lymphocyte-B clone cell line capable of excreting monoclonal antibodies which bind to an epitope on the NS3 protein of hepatitis C virus, which cell line is deposited at the European Collection of Animal Cell ~ultures, Porton Down (UK), under deposit No. 9212160~.
T~e Epstein Barr virus (EBV) is capable of transforming and immortalizing human ~-lymphocytes. With the aid of the Epstein Bar virus immortalized human ~-lymphocytes can be .~ wo94ll4s74 ~ ~ 7 7 ~ 7 PCT~3/03707 obtained without the need of a myeloma partner cell. The Epstein Barr virus can be obtained from a variety of sources. The most common used source of EBV is the B95-8 marmoset cell line. The B95-8 cell line spontaneously releases Epstein Barr virus into the medium.
A variety of cell types has been suggested to provide good feeder layers for the cloning of EBV transformed cells. The most commonly used are peripheral blood mononuclear cells (PBMC) and fibroblasts.
PB~Cs conæist of monocytes, T lymphocy~es and B-lympho~ytes (5-1~%). Because not all B-lymphocytes will provide antibodies of the ri.ght specificity, it is ad~antageous to enric~ PBMCs for appropriate cells. T~ prevent the generation of ~ytotoxic T-cells against ~BV-transfor~ed cells, ~-lymphocytes can be removed, prior to ~BV
infection (with, for example, the supernatant from the EBV-productive B95-8 cell line), by treating the cells with wa~hed sheep red blood cells ( ~ I~ a -Dr~içle g;~U~çhn, Edi~or ~a~ a~
Enql~n~L ~9~. C~ 4). -~5 Immortalized B cell lines according to the invention were considered monoclonal if they satisfied the following require~ents~
1) Stability of antibody secretion over time (~6 months), 2) Secretion of only one XgG (H and L) functional molecule, 3) Specificity and stability of antibody secretion following at least 2 sequential subcloning procedures ~100% of growing colonies ecreting IgG with specificity and genetic phenotype identical to the parental line).
4 ~ ~ 7 7 9 7 PCT~3/03707 .~.
The monoclonal antibodies according to the invention are extremely suitable to be used in so-called immuno-assays in order to detect HCV or S HCV-fragments in a test sample~
An il~munodia~nostic reagent comprising the antibodies according to the invention and a test kit for the detection of HCV in a sample are also part of the present invention.
Depending on the assay system employed, 1:he im~unochemical reaction that takes place can ~ a so-called sandwich reaction, an agglutination reaction, a co~petition reaction or an inhibition reaction.
The following assay syste~s are illustra~ive only and ~re not intended to limit the invention.
Carrying out, for instance, a s~ndwich reaction for t~e d~tection of HCV in a test sample the test kit to be used comprises a monoclonal antibody according to the invention coated on a solid support, for example the inner wall of a microtest we}l, and eith~r a labelled monoclonal antibsdy or fragment therecf as conjugate.
Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a ~embrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal RQl or metal compound as sol particle, a carrier protein such as BSA or KLH.
Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound or other sol as sol particle. A
~27~97 -- WOg4/14974 PCT~3/03707 further example of an immuno assay that can be used for the detection of HCV is a inhibition assay using human monoclonal antibodies as labelled reagent. The binding of this reagent to antigen on a solid phase can be competed by antibodies in the test sample.
As already mentioned monoclonal antibodies according to the invention are very suitable in diagnosi~, while those antibodies which are 1~ neutralizing are very useful in pas~ive immunotherapy.
The monoclonal antibodi~s according to the invention can also be used to raise anti- ~:
idiotypic antibodies.
Anti-idiotypic antibodies are antibodies directed to the variable part of immuno~lobulins.
A sub-population of anti-idiotypic antibodies is known as "anti-idiotypic B" or "internal i~agesn.
These anti-idiotypic ~ antibodias have either a structural or a three dimensional resemblance with the antigen ~Y~h~9sL_ F.G~M~ ~ al.
~ ). Thi~ type of anti-idiotypic antibodies is wide~y used-a~ a ~accine against înfectious diseases in animal models (~içrnaux ~.R~ . 1:~55~ 5,~ Z-~L~i~lLL2~). For use in a~says the anti-idiotypic antibodies can b~ raised in large amounts.
Techniques for raising anti-idiotypic antibodies are known in the art. The following example is illustrative only and is not intended to limit the invention; anti-idiotypic antibodies according to the invention can be obtained by immunizing BALB/c mice with monoclonal antibodies, coupled to KLH with qlutaraldehyde W094/14g74 ~1 ~ 7 r~ 9 7 PCT~3/03707 according to standard litera~ure procedures, mixed with Freund's complete adjuvant. The spleen cells of these mice can be immortalized and the thus obtained hybridomas can be screened for anti-idiotypic antibody production. Screening of the hybridomas can be performed, for example but not limited to, by binding a HCV peptide to a solid phase (wells of microtiter plates) and incubating the solid phase with culture 1~ sup*rnatant of growing hybridomas and the ~onoclonal antibodies coupled to Horse Radish Peroxidase (HRP) according to our invention. The presence of anti-idiotypic antibodies in culture æupernatant will then be indicated by inhibition of the binding o~ the monoclonal antibodies according to our invention and the HCV peptide coated on the solid phase.
Anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the prasent invention.
Anti-idiotypic antibodies, especially representing the internal image, can ~e uæed in mimicking antigan in a diagnostic test. Beside the use in diagnostic tests anti-idiotypic antibodies are also useful for prevention and/or treatment of Non-A, Non-8 Hepatitis, as well ~s for the elucidation of important epitopic regions of HCV-antigens.
Preferred anti-idiotypic antibodies . according to the present invention are the anti-idîotypic antibodies secreted by the cell lines ~hich are deposited at the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK) - W094/14974 ~12 7 7 ~ 7 PCT~3/03707 under deposit No. 93122307 (HCVID.OT2A), under deposit No. 93122308 (HCVID.OT2B), under deposit No. 93122309 (HCVID.OT2C), under deposit No.
93122310 (HCVID.OT2D), under deposit No. 93122311 (HCVID.OT2E), under ~aposit No~ 93122312 (HCVID.OT2I), under deposit No. 93122313 (HCVID.OT2J), under deposit No. 93122314 (HCVID.OT2M), under deposit No. 93122315 (HCVID.OT2P), under deposit No. 93122316 (HCVID.OT2Q), under deposit No. 93122317 ~HCVID.OT3A~, under ~eposit No. 931~.2318 (HCVID.OT3D), under deposit No. 93122319 (HCVID.OT3E), under deposit No. 9312.2320 (HCVID.OT3H), under deposit No~ 93122321 ~HCVID.OT3L), and under deposit ~o~ 93122322 (HCVID.OT30).
Anti-idiotypic antibodies according to the in~ention preferably are anti-idiotypic antibodies capable of competinq with an epitope for t~e binding to t~e monoclonal antibody according to the invention, where the epitope is the epitope recognized by t~e monoclonal antibodies secreted by the Epstein-Barr `virus-transformed human lymphocyte-~ cell line deposited with the European Collection of Animal Cell Cultures (ECACC~, Porton Down (UK), under deposit No. 921~1609.
Immunodiagnostic reagents comprising the anti-idiotypic antibodies according to the inventi~n, and a test kit for the detection of anti-HCV antibodies in a sample using an im~unodiagnostic reagent comprising anti-idiotypic antibodies are also part of the present invention.
W094/~4974 ~1 2 ~ 7 9 7 PCT~3/03707 Immortalized cell lines capable of secreting anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the present S invention.
The preparation of cell lines producing anti-idiotypic antibodies may occur by, for example, transformation with ~pstein-Barr Vi~.us, 1.0 the K~hler and Milstein ~echni~ue (K~hler and Milstein d~vised the techniques that resultedl in the formation mono~lonal antibody-produc:ing hybridomas (G. K~hler and C. Milstein, 1975, Nature 256:495-497: 1976, Eur. J. I~munol. 6:511-519)~, or a direct transformation technique of ~-lymphocytes with oncogenic DNA, or a direct :`
fusion of human B-lymphocytes with a fusion p~rtner being either a hu~an or a mou~e-human hybrid myelo~a cell line, or a direct fusion of an EBV-transformed B ~ell line with said myeloma cell lines.
Preferred cell lines according to the invention are deposited at the European ~5 Collection of Animal Cell Cultures, Porton Down (~K~. HCVID.OT2A under deposit No. 93122307, HCVID.OT2B under deposit No. 9312230fi, HCVID.OT2C
under deposit No. 93122309, HCVID~OT2D under deposit No. 93122310, HCVID~OT2E under deposit No. g3122311, HCVID.OT2I under deposit ~o.
93122312, HCVID.OT2J under deposit No. 93122313, HCVID.OT2M under deposit No. 93122314, HCVID.OT2P
under deposit No. 93122315, HCVID.OT2~ under deposit No. 93122316, HCVID.OT3A under deposit No. 93122317, HCVID.OT3D under deposit No.
93122318, HCVID.OT3E under deposit No. 93122319, HCVID.OT3H under deposit No. 93122320, HCVID.OT3L
~1~7~!37 - ~W094l14974 PCT~3103707 under deposit No. 93122321, and HCVID.OT30 un~er deposit No. 93122322.
An immunodiagnostic reagent comprising the anti-idiotypic antibodies according to the invention and a test kit for the detection of HCV
in a sample are also part of the present invention.
The present invention is further exemplified by the following examples:
Example ~ illustrates the way in which a cell line, producing ~onoclonal antibodies, accvrding to the invention may be derived.
Example 2 further exemplifies ~he specific ~-immune reactivity of monoclonal antibodies according to the invention~
Exa~ple 3 exe~plifies the production of anti-idiotypic antibodies and the~ specific ability o these anti-idiotypic antibodies to mi~ic a immuno~ominant NS-3 epitope.
wo 94~14974 ~ ~ ~ 7 7 ~ 7 PCT/E~3103707 EX~MPLES
E ~ PLE l: EE~Paration of_B-cell line clone Peripheral blood mononuclear cells (PBMC) s were obtained from one patient with chronic HCV
infection and circulating anti~odies to the NS3 and nucleocapsid proteins of HCV but not to clOO-3/NS4(HCV 1st generation antibody test, Ortho Diagnostic Systems, ~aritan, NJ) as shown by reactivity of the patient's serum with a com~ercial assay that incorporates a recombinant protein designated c22-3, the viral nucleoprotein, and the recombinant nonstructllral protein c200 encoded by the NS3 and NS4 regions of the HCV genome ~Ortho HCV ELISA Test Syste~, 2nd generation~.
P8~C were resuspended in RP~I ~6~0 m~dium containing 10% heat-inactivated pooled hu~an serum, supplemented with 10% human endothelial 2~ culture supernatant (HEC5) + 20 U~l rIL6 and cultured in 25 cm2 flasks at the concentration of 4 x lOE6/~l in the presen~e of recombinant NS3 protein (Organon Teknika) at the concentration of 25, l25, and 625 ng/ml for 6 days at 37 C in 5%
C02.
After washing, cells were enriched in lymphocytes-B by removing lymphocytefi-T with S-(2-amino-ethyl~-isothiouronium-bromide-hydrobromide treated sheep red blood cells and infected with supernatant from the EBV-productive B95-8 cell line as described in ~erL~Q A- ~ al.
J.ImmunQL.. 147. ~2692. l99l.
After overnight incubation, cells were seeded at a density of 3* 104/well in U-bottom g6-well microtiter plates. After 20-30 days in culture, cultures secreting antibodies reacti~
with solid phase NS3 protein were subcultured at ~ 127797 W094114974 PCT~ ~3/03707 I2-50 cells/well in U-bottom wells with 3000 R-irradiated allogeneic PBMC as feeder cells. B
cell lines were subcultured at least twice hefore further characterization.
IgG heavy and 1ight (L) chains were determined as described in Cerino A et al.
J~nmunol.. 147, p2692. 1~1. Briefly, B cell line supernatants were incubated on NS3 coated wells for 1 hour at 37 C and, after w~shing, ~:~
hu~an IgG subclass-specific murine mAb were adcted for 1 hour ~ollowed by a peroxida e-conjugat:ed rabbit anti-mouse Ig for another hour. To determine IgG L chains, peroxidase-conjugat:ed rabbit anti-human Ig kappa- or lambda-chain w~re used in a direct assay. In both c~ses the reaotion was develope~ with orthophenylen-diamine/2 HCl as substrate.
Results:
Significant anti NS3 production was obtained after in vitro stimulation with 25 and 125 ng/ml of rNS3, whereas no increase in Ab production, compared with control cultures~ was found using the highest Ag concentration. A total o 480 wells w~re seeded with cells deri~ed froD
cultures at 25 ad 125 ng~ml of NS3: 3 of these cell lines secreted significant levels ~A492>1) of anti~NS3 and were stable for a time sufficient to allow further characterization. One cell line, HCVHU.OT3 (deposited at the ECACC under deposit No. 92121609), satisfied the criteria for monoclonality. Indeed such line has been subcultured twice yielding clone~ secreting identical amounts of specific antibody and has been kept in continuous culture for ~ 6 months.
Further~ore, clone HCVHU.0~3 produced IgGl (k) exclusively.
W094/14974 ~12 7 7 9 7 PCT~3/03707 .~ 14 EXAMPLE 2: Testina on anti NS3 ~roduction o~
B-çell lines.
The specificity of oligoclonal and monoclonal IgG-containing supernatants was further tested with the following reagents:
1) A recombinant purified HCV nucleoprotein expressed in E.coli (Organon Teknika).
2) A recom~inant purified NS3 prot~ein expressed in E.coli (Organon Tekn ka).
3) A recombinant purified NS-5 prot,ein expressed in E.coli.
4) Recombinant immunoblot assay (RIBA II
generation, Ortho Diagnostics). This is a nitrocellulose-based assay that includes 4 reco~binant HCV antigens:
- c100-3, derived from NS-4 protein, - 5-1-1, a 42-aminoacid frag~ent of c100-3, - c33c, derived from t~e NS3 protain, - c22-3, derived from the viral nucleo-protein .
Humarl superoxide dismutase (SOD) is also present on nitrocellulose strips as a control.
Results.
Supernatant from clone HCVHU.OT3 was analyæed for pecificity in the above described way.
Figure 1 illustrates the specific binding capacity of antibodies according to the invention. The binding of the antibodies according to the invention (HCVHU.OT3) ~o different HCV derived proteins is compared with the binding of anti~odies specif ic for HCV core and NS4 proteins respectively. As can be seen ~om Figure l, the monoclonal antibodies accordin~ to the invention tHCVHU.OT3) recognized W094/14974 ~12 7 7 9 7 PCT~3103707 a reco~binant NS3 prctein preparation and qa~e a cl~ar positive reaction in Ortho II generation assay, whereas no binding to recombinant HCV core and NS-5 proteins could be documented. Analysis of HCVHU.OT3 supernatant by recombinant im~unoblot (RIBA II generation) revealed clear binding to the c33c polypeptide only, further attesting to the specificity of the mAb as can be seen from figure 2, where lane 2 represents an 1~ antibody according to the invention (HCVHU.OT3).
~i~77!37 `--W094/14974 PCT~3tO3707 EXAMPLE 3 _ Producti~n of ~ti~ typic antibod~
Monoclonal human IgG (HCVHU.OT3) was purified from the culture supernatant of the cell-line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under ~.
deposit No. 92121609. This IgG was conjugated to KLH (OosterLaken ~A,M. _ et al~
lG Scandin~vi~n_ Journal ~f Immunoloav. 31. 1!~2) mixed with Complete Freunds adjuvant or Quil. A
and injected into 4 mice and 4 ralts.
Immunizations were performed according to the scheme in table 1. After 2 immunizations the animal with the highest serum titer, one ~0~5e and one rat, was used for making monoclonal antibodie~, essentially as described in Methodse_ 163. ~3._ lg~ an~ ~ourn~L Q~
ImmunQl~ical Methn~sL 152.... S9. 1992). Clon~6 secreting putative anti-idiotypic antibadies were also detected with t~e a~say describ~d in ~igure 3. In this way 16 clones w~re obtained that reacted well in this assay. The clones secreting putative anti-idiotypic antib~dies were cultured and subcloned to 100% clonality according to standard procedures (~hl~r~ G. and ~ einL ~.
~
Hybrido~a ~echnolo~Y in ~he Bio5~Qn~es and n~Y~5~L~a_el3-qwelve of the monoclonal antibodies were of mouse origin, 6 were of rat origin. Isotypes of the different antibodies are listed in table 2.
. WO 94/14974 ~1~ ( ( J ~ PCT/EP93/03707 _ ~ o C--~ C~
C
.4 ~ ~ ~ r t~ ~ ~ ~1 ~ 1 ~ H --~ ~ I O C~
~ ~ ~ Cl~ a ~ a . ~
~0 X ~ ~ 0 J~ ~ ~ J~ 3 I ~ .~ ~: ~ O,C
o o ~ ~ o 0 .
~:,C O O ~ O O :~
1 C ~a InO J4 ~ .:
J~
* ~
~ ~ ~ ~ ~ ~, ~ ~ ~ 3 N ~ .C ~
:~ ~ ~ ~ . . ~ . . ~ ~ ~ ~ 8 a c~ a a ~
o .N N ~ ~ o O ~ ~ O ~ O ~ 0 t o O
~ .. o o ~ ~ o o ~ ~ ~ N 8 ~ t~
o ~ ~ I ~o_ ~ X X JJ
.
_ N _ ~ æ U O g ~D O a ~ m ~ ~ ~ ~ a , ~ ~ ~ ~ 0 :~ o o o o c ~ ~ ~ ~ ~' o ~
~ ~ ~ ~: æ ~ x ~
E~ ~ . _ _ WO 94ll4974 ~ ~ ~ 7 7 9 7 PCTIEP93/03707 Ta~le ?
Llst o~ monoclon~ll anti-idiotypic antibodies directed tG HCY
Hu. OT3 Mou~e Isot Q
_ _ ,YP _ :
H~V~D . OT2A IgG1 (~; ) HCV~O . OT2B IgG 1 ( K ) HCVID . OT2C IgG1 tK ) HC'JID ~ OT2D I~Gl ~K ) ~CVID . OT;2E I5~GI tK~
HCVID . OT2I Igt;1 (K) HCVID . OT2J IgG1 (K) HCYID . QT211 ~gG1 (~;) ::
HCYID.0~2P Io~2A lK) ~CVID ~ OT2Q ~G2A (~C) _ ~ _ .,.
Rat ~
HC~ID . 0~3A IgG I ~ :HCVII~ . OT3D IgG2A :
HCVID . OT3E IgG2A
HCVID. OT3H IgG~A
HCVID . OT3L ¦tgG~
HClJID . 0~3Q ~IgG2B
~27797 WOg4/14974 PCT~3/03707 The cell lines were grown in hi~her cell densities and cultured for 5 days in serumfree medium. Antibodies were purified from the culture supernatant using a Protein A-SepharoseR column.
Purification was perfor~ed according to the manufacturers instructions (Pharmacia-LKB).
From two of the rat monoclonal antibodies no pure anti-idiotypic antibody could be obtained (HCVHU.OT3H and HCVHU.OT3E)~
The anti-idiotypic antibodies were further analysed for their ability to mimic the NS-3 epitope recognized by the h~man monoclonal antibody HCVHU.OT3. For that purpose the purified monoclonal antibodies were conjugated to HRP with 1~ the Actizyme-peroxidase kit (Zymed laboratories).
The proceduxe was performed according to the manufacturers instructions.
The procedure to analy~e the ability to mimic the NS-3 epitope is further illustrated hereunder.
C33-B-galactosidase (containing amino acids 1192 to 1457 of the HCV geno~e) was immobilized on a 96-well ~icro ELISA plate acaording to standard procedures (1 ~g/ml in 0.05~ carbonate bufer, pH=9.6, incubated overnight at 4 C.).
Serum sa~ples of mice or rats or culture supernatant of the cells to be screened for the production of anti-idiotypic antibodies was mixed with HC~ OT3-HRP and incubated at 37 C for 1 hour. Thereafter the mixture was brought to the well coated with the C33-B-galactosidase. Binding of the anti-idiotypic antibodies to HCVHU.0~3 was detected after extensive washing with PBS
: 35 supplemented with 0.05 % qween 20R. If anti-idiotypic antibodies had bound to HCVHU.OT3-HRP, the signal decreased to background value.
'~127~97 WO94114974 PCT~ ~3/03707 With this material a sandwich assay was developed consisting at one hand of one of the monoclonal anti-idiotypic antibodies i~mobilized in a well of a 96-well micro ELISA plate and on the other hand of the same monoclonal antibody labelled with HRP in solution. (Figure 4).
The assay procedure to validate putative anti-idiotypic antibodies is further illustrated hereunder.
Putative anti-idiotypic antibody was -.
immobilized on a 96-well micro ELISA plate according to standard procedures (lO ~g/ml, 0.05M
carbo~ate buffer, pH=9.6, incubation overnight at C)- ..
Serum dilutions or dilutions of HC~HU.OT3 were added to the wells follnwed by l hour incubation at 37 C.
After extensive washing with P8S
~upplemented wi~h 0.05% Tween 20R ~4 times), HRP
labelled putative anti-idiotypic antibody was added.
Thereafter the plate was incu~ated for l hour at 37 C, followed by washing (4 times) with PBS with O.05% Tween 20~.
Thereafter substrate for HRP was added.
. Using the assay described in figure ~, further evidence was obtained for the ~pecificity of the monoclonal anti-idiotypic antibodies. The anti-idiotypic antibodies were able to discriminat~ between H~VHU.OT3 and an irrelevant human monoclonal antibody of the same isotype as HCVHU.OT3. In figure 5 representative examples of this specificity are shown.
- 2127797 :
.WO94/14974 PCT~3/03707 In the same assay (figure 5) it was established whether the anti-idiotyic antibodies were able to specifically bind to antibodies in serum from human individuals infected with HCV.
For that purpose a panel of human sera was sel~cted, known to contain antibodies against the HCV NS-3 antigen as determined by the commercial RIBA as~ay (Chiron Corporation Emmeryville).
In figure 5, reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant huoan monoclonal antibody wi~h the same subclass as HCVHU.0~3 is shown as shaded bars. Other ~onoclonal anti-idiotypic antibodies gave comparable results.
The ability of the anti-idiotypic antibodies prepared as described ab~ve were indeed able to mi~ic the immunodominant NS-3 epitopa as defined by the human monoalonal HCVHU.OT3, is exemplified in tabl~ 3.
Table 3 shows the reactivity of human sera from HCV infected patients and normal controls with anti-idiotypic antibodies HCVID.OT2A and HCVID.OT2I.
WO 94/~4974 ~ ~ 2 ~ 7 9 7 PCT/EP93/03707 To.ble 3 R~activ~y ef human ~er~ frc)m HCV inrected p~ti~nt~ ~nd nor~l c,~ntrols with ~r~ti-id~otypic antibodie~ HCV~D.OT2A ~Ind MCVID.OT2I.
~ ~-- --G6rulc lD 511ClOO C33 ~:2? TOT.OD450'R.R.~' OD4~0' N.R.~-A537 2 4 4 4 + 0.~210.99 0.620 1.1 A590 4 4 4 q t O . 599 l . 41 0 . 90o 1. 64 A598 4 4 4 4 + O . ~22 1 . 23 0 . 724 1. 31 ~599 1 2 3 3 t 1 . 302 ~ . 06 1 . 565 2 . 99 A732 1 0 4 4 ~ O . 46~ 1. 09 0 . 691 1. 25 A73~ 4 1 4 4 ~ l . 650 3 . 68 Z . 203 3 . 98 al~55 Z 0 4 4 ~ O . 474 1 . 12 û . 990 ~ . ~9 UOO~ 3 4 4 ~ ~ i .0312 . 191 .556 2.7i3 U013 O O 1 4 ~ 0.52Q1.~5 O.E~04 1.45 U033 4 3 4 4 ~ 1.0722.52 1.900 3.43 U036 4 4 ~I 4 ~ O . 57'` i . 35 0 . ~63 1 . S6 U038 2 2 3 4 ~ 0.7841.65 ~.0~9 1.~8 ~J043 2 ~ ~ O + o.~iO31.~ 0.9S3 i.72 C463 O O 4 0 ind . 13 . 676 1. 59 0 . a66 1. 8~
C~65 O ~- 3 ~- ind . O . 500 1.18 0 . 717 1. 25 C466 O G 4 0ind. 0.~95 l.6,1 ~l.101 1.99 C5J2 _ j ~ O 4 ~ nd . 0 . 990 2 . 3S 1 .187 2 . 14 Nor~a 1 Contrcl~ ~ ~ _ _... ~
H227 o D C O - O . 24'5 0 . 5~ 0 . 3~2 0 . 69 N202 O O O O - O . 20~ 0 . 4~ 0 . 325 0 . 5~
N203 j O O O C - O . 207 0 . ~5~ 0 . 3~0 0 . hO
N206 1 0 0 0 O _ O . 239 0 . 56 0 . 369 0 . t;7 N?07 1 0 0 0 0 - 0.173 0.4~0.292 0.53 ~20~ O O - o.zo~ 0.510.307 O.~IS
N211 1 0 0 O O - O . 209 0 . ~ O . 4~S 0 . B2 ~21~ O O ~ - 0.156 0.3~0.21~ 0.4~
N213 1 0 0 0 0 - 0~2lB 0.510.37~ 0.6B
^ OD - optlcdl den~ity ~t 450 ~n ~ N R - non~l ~ed r~ponse . c~lcul~t~d a3 OD4SO s:~f ~rum sample divided by c:ut of ~ v~ i ue .
~:ut off v~lue is ~ver~ge nor~nal :~erum (n~O) ~ 3 ti~ !~t~ntlard deviat ion .
.- WO94/14974 ~ 1 2 f 7 9 7 PC~ ~3/03707 Figures:
Figure l is a graph illustrating the binding specificity of monoclonal antibody HCVHU~OT3 for the NS3 protein.
Figure 2 is a photograph of a recombinant im~unoblot assay. ~he characters A-G represent the following proteins;
l~ A: High Ig control B: 5-l l (NS-4) C: c100-3 ~NS-4) D: c33c (NS-3) E: c22-3 (core) F: superoxide dismutase G: low Ig contL ol .
Lane l represents negative control serum, lane 2 represents an anti NS-3 monoclon~l antibody ~HCVHU.OT3), lane 3 represents an anti-core monoclonal ~HCVHU.OT2), and lane 4 represents polyclonal serum from a ~CV-infected patient.
Figure 3 represents a scrsening procedure used for detection of anti-idiotypic antib~dies against human monoclonal antibody HCVHU.OT3 in seru~ and in culture supernatant of ~onoclonal antibody producing cells.
Figure 4 represents an assay procedure used to validate putative anti-idiotypic antibodies which emerged from the screening procedure descri~ed in figure 3.
W094114974 ~ 7 9 7 PCT~3/03707 Figure 5 represents a sandwich assay (as shown in figure 4) to determine the specificity of 6 different anti-idiotypic antibodies (2A, 2B, 2I, 2P, 2Q and 3A) for HCVHU.OT3.
Reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant human monoclonal antibody with the same subclass as HCVHU.OT3 is shown as shaded bars. Other monoclonal anti-idistypic antibodies gave comparable results.
SU~3STITUTE SHEET
The monoclonal antibodies according to the invention are extremely suitable to be used in so-called immuno-assays in order to detect HCV or S HCV-fragments in a test sample~
An il~munodia~nostic reagent comprising the antibodies according to the invention and a test kit for the detection of HCV in a sample are also part of the present invention.
Depending on the assay system employed, 1:he im~unochemical reaction that takes place can ~ a so-called sandwich reaction, an agglutination reaction, a co~petition reaction or an inhibition reaction.
The following assay syste~s are illustra~ive only and ~re not intended to limit the invention.
Carrying out, for instance, a s~ndwich reaction for t~e d~tection of HCV in a test sample the test kit to be used comprises a monoclonal antibody according to the invention coated on a solid support, for example the inner wall of a microtest we}l, and eith~r a labelled monoclonal antibsdy or fragment therecf as conjugate.
Supports which can be used are, for example, the inner wall of a microtest well or a cuvette, a tube or capillary, a ~embrane, filter, test strip or the surface of a particle such as, for example, a latex particle, an erythrocyte, a dye sol, a metal RQl or metal compound as sol particle, a carrier protein such as BSA or KLH.
Labelling substances which can be used are, inter alia, a radioactive isotope, a fluorescent compound, an enzyme, a dye sol, metal sol or metal compound or other sol as sol particle. A
~27~97 -- WOg4/14974 PCT~3/03707 further example of an immuno assay that can be used for the detection of HCV is a inhibition assay using human monoclonal antibodies as labelled reagent. The binding of this reagent to antigen on a solid phase can be competed by antibodies in the test sample.
As already mentioned monoclonal antibodies according to the invention are very suitable in diagnosi~, while those antibodies which are 1~ neutralizing are very useful in pas~ive immunotherapy.
The monoclonal antibodi~s according to the invention can also be used to raise anti- ~:
idiotypic antibodies.
Anti-idiotypic antibodies are antibodies directed to the variable part of immuno~lobulins.
A sub-population of anti-idiotypic antibodies is known as "anti-idiotypic B" or "internal i~agesn.
These anti-idiotypic ~ antibodias have either a structural or a three dimensional resemblance with the antigen ~Y~h~9sL_ F.G~M~ ~ al.
~ ). Thi~ type of anti-idiotypic antibodies is wide~y used-a~ a ~accine against înfectious diseases in animal models (~içrnaux ~.R~ . 1:~55~ 5,~ Z-~L~i~lLL2~). For use in a~says the anti-idiotypic antibodies can b~ raised in large amounts.
Techniques for raising anti-idiotypic antibodies are known in the art. The following example is illustrative only and is not intended to limit the invention; anti-idiotypic antibodies according to the invention can be obtained by immunizing BALB/c mice with monoclonal antibodies, coupled to KLH with qlutaraldehyde W094/14g74 ~1 ~ 7 r~ 9 7 PCT~3/03707 according to standard litera~ure procedures, mixed with Freund's complete adjuvant. The spleen cells of these mice can be immortalized and the thus obtained hybridomas can be screened for anti-idiotypic antibody production. Screening of the hybridomas can be performed, for example but not limited to, by binding a HCV peptide to a solid phase (wells of microtiter plates) and incubating the solid phase with culture 1~ sup*rnatant of growing hybridomas and the ~onoclonal antibodies coupled to Horse Radish Peroxidase (HRP) according to our invention. The presence of anti-idiotypic antibodies in culture æupernatant will then be indicated by inhibition of the binding o~ the monoclonal antibodies according to our invention and the HCV peptide coated on the solid phase.
Anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the prasent invention.
Anti-idiotypic antibodies, especially representing the internal image, can ~e uæed in mimicking antigan in a diagnostic test. Beside the use in diagnostic tests anti-idiotypic antibodies are also useful for prevention and/or treatment of Non-A, Non-8 Hepatitis, as well ~s for the elucidation of important epitopic regions of HCV-antigens.
Preferred anti-idiotypic antibodies . according to the present invention are the anti-idîotypic antibodies secreted by the cell lines ~hich are deposited at the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK) - W094/14974 ~12 7 7 ~ 7 PCT~3/03707 under deposit No. 93122307 (HCVID.OT2A), under deposit No. 93122308 (HCVID.OT2B), under deposit No. 93122309 (HCVID.OT2C), under deposit No.
93122310 (HCVID.OT2D), under deposit No. 93122311 (HCVID.OT2E), under ~aposit No~ 93122312 (HCVID.OT2I), under deposit No. 93122313 (HCVID.OT2J), under deposit No. 93122314 (HCVID.OT2M), under deposit No. 93122315 (HCVID.OT2P), under deposit No. 93122316 (HCVID.OT2Q), under deposit No. 93122317 ~HCVID.OT3A~, under ~eposit No. 931~.2318 (HCVID.OT3D), under deposit No. 93122319 (HCVID.OT3E), under deposit No. 9312.2320 (HCVID.OT3H), under deposit No~ 93122321 ~HCVID.OT3L), and under deposit ~o~ 93122322 (HCVID.OT30).
Anti-idiotypic antibodies according to the in~ention preferably are anti-idiotypic antibodies capable of competinq with an epitope for t~e binding to t~e monoclonal antibody according to the invention, where the epitope is the epitope recognized by t~e monoclonal antibodies secreted by the Epstein-Barr `virus-transformed human lymphocyte-~ cell line deposited with the European Collection of Animal Cell Cultures (ECACC~, Porton Down (UK), under deposit No. 921~1609.
Immunodiagnostic reagents comprising the anti-idiotypic antibodies according to the inventi~n, and a test kit for the detection of anti-HCV antibodies in a sample using an im~unodiagnostic reagent comprising anti-idiotypic antibodies are also part of the present invention.
W094/~4974 ~1 2 ~ 7 9 7 PCT~3/03707 Immortalized cell lines capable of secreting anti-idiotypic antibodies reactive with the monoclonal antibodies according to the invention, as described above, are part of the present S invention.
The preparation of cell lines producing anti-idiotypic antibodies may occur by, for example, transformation with ~pstein-Barr Vi~.us, 1.0 the K~hler and Milstein ~echni~ue (K~hler and Milstein d~vised the techniques that resultedl in the formation mono~lonal antibody-produc:ing hybridomas (G. K~hler and C. Milstein, 1975, Nature 256:495-497: 1976, Eur. J. I~munol. 6:511-519)~, or a direct transformation technique of ~-lymphocytes with oncogenic DNA, or a direct :`
fusion of human B-lymphocytes with a fusion p~rtner being either a hu~an or a mou~e-human hybrid myelo~a cell line, or a direct fusion of an EBV-transformed B ~ell line with said myeloma cell lines.
Preferred cell lines according to the invention are deposited at the European ~5 Collection of Animal Cell Cultures, Porton Down (~K~. HCVID.OT2A under deposit No. 93122307, HCVID.OT2B under deposit No. 9312230fi, HCVID.OT2C
under deposit No. 93122309, HCVID~OT2D under deposit No. 93122310, HCVID~OT2E under deposit No. g3122311, HCVID.OT2I under deposit ~o.
93122312, HCVID.OT2J under deposit No. 93122313, HCVID.OT2M under deposit No. 93122314, HCVID.OT2P
under deposit No. 93122315, HCVID.OT2~ under deposit No. 93122316, HCVID.OT3A under deposit No. 93122317, HCVID.OT3D under deposit No.
93122318, HCVID.OT3E under deposit No. 93122319, HCVID.OT3H under deposit No. 93122320, HCVID.OT3L
~1~7~!37 - ~W094l14974 PCT~3103707 under deposit No. 93122321, and HCVID.OT30 un~er deposit No. 93122322.
An immunodiagnostic reagent comprising the anti-idiotypic antibodies according to the invention and a test kit for the detection of HCV
in a sample are also part of the present invention.
The present invention is further exemplified by the following examples:
Example ~ illustrates the way in which a cell line, producing ~onoclonal antibodies, accvrding to the invention may be derived.
Example 2 further exemplifies ~he specific ~-immune reactivity of monoclonal antibodies according to the invention~
Exa~ple 3 exe~plifies the production of anti-idiotypic antibodies and the~ specific ability o these anti-idiotypic antibodies to mi~ic a immuno~ominant NS-3 epitope.
wo 94~14974 ~ ~ ~ 7 7 ~ 7 PCT/E~3103707 EX~MPLES
E ~ PLE l: EE~Paration of_B-cell line clone Peripheral blood mononuclear cells (PBMC) s were obtained from one patient with chronic HCV
infection and circulating anti~odies to the NS3 and nucleocapsid proteins of HCV but not to clOO-3/NS4(HCV 1st generation antibody test, Ortho Diagnostic Systems, ~aritan, NJ) as shown by reactivity of the patient's serum with a com~ercial assay that incorporates a recombinant protein designated c22-3, the viral nucleoprotein, and the recombinant nonstructllral protein c200 encoded by the NS3 and NS4 regions of the HCV genome ~Ortho HCV ELISA Test Syste~, 2nd generation~.
P8~C were resuspended in RP~I ~6~0 m~dium containing 10% heat-inactivated pooled hu~an serum, supplemented with 10% human endothelial 2~ culture supernatant (HEC5) + 20 U~l rIL6 and cultured in 25 cm2 flasks at the concentration of 4 x lOE6/~l in the presen~e of recombinant NS3 protein (Organon Teknika) at the concentration of 25, l25, and 625 ng/ml for 6 days at 37 C in 5%
C02.
After washing, cells were enriched in lymphocytes-B by removing lymphocytefi-T with S-(2-amino-ethyl~-isothiouronium-bromide-hydrobromide treated sheep red blood cells and infected with supernatant from the EBV-productive B95-8 cell line as described in ~erL~Q A- ~ al.
J.ImmunQL.. 147. ~2692. l99l.
After overnight incubation, cells were seeded at a density of 3* 104/well in U-bottom g6-well microtiter plates. After 20-30 days in culture, cultures secreting antibodies reacti~
with solid phase NS3 protein were subcultured at ~ 127797 W094114974 PCT~ ~3/03707 I2-50 cells/well in U-bottom wells with 3000 R-irradiated allogeneic PBMC as feeder cells. B
cell lines were subcultured at least twice hefore further characterization.
IgG heavy and 1ight (L) chains were determined as described in Cerino A et al.
J~nmunol.. 147, p2692. 1~1. Briefly, B cell line supernatants were incubated on NS3 coated wells for 1 hour at 37 C and, after w~shing, ~:~
hu~an IgG subclass-specific murine mAb were adcted for 1 hour ~ollowed by a peroxida e-conjugat:ed rabbit anti-mouse Ig for another hour. To determine IgG L chains, peroxidase-conjugat:ed rabbit anti-human Ig kappa- or lambda-chain w~re used in a direct assay. In both c~ses the reaotion was develope~ with orthophenylen-diamine/2 HCl as substrate.
Results:
Significant anti NS3 production was obtained after in vitro stimulation with 25 and 125 ng/ml of rNS3, whereas no increase in Ab production, compared with control cultures~ was found using the highest Ag concentration. A total o 480 wells w~re seeded with cells deri~ed froD
cultures at 25 ad 125 ng~ml of NS3: 3 of these cell lines secreted significant levels ~A492>1) of anti~NS3 and were stable for a time sufficient to allow further characterization. One cell line, HCVHU.OT3 (deposited at the ECACC under deposit No. 92121609), satisfied the criteria for monoclonality. Indeed such line has been subcultured twice yielding clone~ secreting identical amounts of specific antibody and has been kept in continuous culture for ~ 6 months.
Further~ore, clone HCVHU.0~3 produced IgGl (k) exclusively.
W094/14974 ~12 7 7 9 7 PCT~3/03707 .~ 14 EXAMPLE 2: Testina on anti NS3 ~roduction o~
B-çell lines.
The specificity of oligoclonal and monoclonal IgG-containing supernatants was further tested with the following reagents:
1) A recombinant purified HCV nucleoprotein expressed in E.coli (Organon Teknika).
2) A recom~inant purified NS3 prot~ein expressed in E.coli (Organon Tekn ka).
3) A recombinant purified NS-5 prot,ein expressed in E.coli.
4) Recombinant immunoblot assay (RIBA II
generation, Ortho Diagnostics). This is a nitrocellulose-based assay that includes 4 reco~binant HCV antigens:
- c100-3, derived from NS-4 protein, - 5-1-1, a 42-aminoacid frag~ent of c100-3, - c33c, derived from t~e NS3 protain, - c22-3, derived from the viral nucleo-protein .
Humarl superoxide dismutase (SOD) is also present on nitrocellulose strips as a control.
Results.
Supernatant from clone HCVHU.OT3 was analyæed for pecificity in the above described way.
Figure 1 illustrates the specific binding capacity of antibodies according to the invention. The binding of the antibodies according to the invention (HCVHU.OT3) ~o different HCV derived proteins is compared with the binding of anti~odies specif ic for HCV core and NS4 proteins respectively. As can be seen ~om Figure l, the monoclonal antibodies accordin~ to the invention tHCVHU.OT3) recognized W094/14974 ~12 7 7 9 7 PCT~3103707 a reco~binant NS3 prctein preparation and qa~e a cl~ar positive reaction in Ortho II generation assay, whereas no binding to recombinant HCV core and NS-5 proteins could be documented. Analysis of HCVHU.OT3 supernatant by recombinant im~unoblot (RIBA II generation) revealed clear binding to the c33c polypeptide only, further attesting to the specificity of the mAb as can be seen from figure 2, where lane 2 represents an 1~ antibody according to the invention (HCVHU.OT3).
~i~77!37 `--W094/14974 PCT~3tO3707 EXAMPLE 3 _ Producti~n of ~ti~ typic antibod~
Monoclonal human IgG (HCVHU.OT3) was purified from the culture supernatant of the cell-line deposited at the European Collection of Animal Cell Cultures, Porton Down (UK) under ~.
deposit No. 92121609. This IgG was conjugated to KLH (OosterLaken ~A,M. _ et al~
lG Scandin~vi~n_ Journal ~f Immunoloav. 31. 1!~2) mixed with Complete Freunds adjuvant or Quil. A
and injected into 4 mice and 4 ralts.
Immunizations were performed according to the scheme in table 1. After 2 immunizations the animal with the highest serum titer, one ~0~5e and one rat, was used for making monoclonal antibodie~, essentially as described in Methodse_ 163. ~3._ lg~ an~ ~ourn~L Q~
ImmunQl~ical Methn~sL 152.... S9. 1992). Clon~6 secreting putative anti-idiotypic antibadies were also detected with t~e a~say describ~d in ~igure 3. In this way 16 clones w~re obtained that reacted well in this assay. The clones secreting putative anti-idiotypic antib~dies were cultured and subcloned to 100% clonality according to standard procedures (~hl~r~ G. and ~ einL ~.
~
Hybrido~a ~echnolo~Y in ~he Bio5~Qn~es and n~Y~5~L~a_el3-qwelve of the monoclonal antibodies were of mouse origin, 6 were of rat origin. Isotypes of the different antibodies are listed in table 2.
. WO 94/14974 ~1~ ( ( J ~ PCT/EP93/03707 _ ~ o C--~ C~
C
.4 ~ ~ ~ r t~ ~ ~ ~1 ~ 1 ~ H --~ ~ I O C~
~ ~ ~ Cl~ a ~ a . ~
~0 X ~ ~ 0 J~ ~ ~ J~ 3 I ~ .~ ~: ~ O,C
o o ~ ~ o 0 .
~:,C O O ~ O O :~
1 C ~a InO J4 ~ .:
J~
* ~
~ ~ ~ ~ ~ ~, ~ ~ ~ 3 N ~ .C ~
:~ ~ ~ ~ . . ~ . . ~ ~ ~ ~ 8 a c~ a a ~
o .N N ~ ~ o O ~ ~ O ~ O ~ 0 t o O
~ .. o o ~ ~ o o ~ ~ ~ N 8 ~ t~
o ~ ~ I ~o_ ~ X X JJ
.
_ N _ ~ æ U O g ~D O a ~ m ~ ~ ~ ~ a , ~ ~ ~ ~ 0 :~ o o o o c ~ ~ ~ ~ ~' o ~
~ ~ ~ ~: æ ~ x ~
E~ ~ . _ _ WO 94ll4974 ~ ~ ~ 7 7 9 7 PCTIEP93/03707 Ta~le ?
Llst o~ monoclon~ll anti-idiotypic antibodies directed tG HCY
Hu. OT3 Mou~e Isot Q
_ _ ,YP _ :
H~V~D . OT2A IgG1 (~; ) HCV~O . OT2B IgG 1 ( K ) HCVID . OT2C IgG1 tK ) HC'JID ~ OT2D I~Gl ~K ) ~CVID . OT;2E I5~GI tK~
HCVID . OT2I Igt;1 (K) HCVID . OT2J IgG1 (K) HCYID . QT211 ~gG1 (~;) ::
HCYID.0~2P Io~2A lK) ~CVID ~ OT2Q ~G2A (~C) _ ~ _ .,.
Rat ~
HC~ID . 0~3A IgG I ~ :HCVII~ . OT3D IgG2A :
HCVID . OT3E IgG2A
HCVID. OT3H IgG~A
HCVID . OT3L ¦tgG~
HClJID . 0~3Q ~IgG2B
~27797 WOg4/14974 PCT~3/03707 The cell lines were grown in hi~her cell densities and cultured for 5 days in serumfree medium. Antibodies were purified from the culture supernatant using a Protein A-SepharoseR column.
Purification was perfor~ed according to the manufacturers instructions (Pharmacia-LKB).
From two of the rat monoclonal antibodies no pure anti-idiotypic antibody could be obtained (HCVHU.OT3H and HCVHU.OT3E)~
The anti-idiotypic antibodies were further analysed for their ability to mimic the NS-3 epitope recognized by the h~man monoclonal antibody HCVHU.OT3. For that purpose the purified monoclonal antibodies were conjugated to HRP with 1~ the Actizyme-peroxidase kit (Zymed laboratories).
The proceduxe was performed according to the manufacturers instructions.
The procedure to analy~e the ability to mimic the NS-3 epitope is further illustrated hereunder.
C33-B-galactosidase (containing amino acids 1192 to 1457 of the HCV geno~e) was immobilized on a 96-well ~icro ELISA plate acaording to standard procedures (1 ~g/ml in 0.05~ carbonate bufer, pH=9.6, incubated overnight at 4 C.).
Serum sa~ples of mice or rats or culture supernatant of the cells to be screened for the production of anti-idiotypic antibodies was mixed with HC~ OT3-HRP and incubated at 37 C for 1 hour. Thereafter the mixture was brought to the well coated with the C33-B-galactosidase. Binding of the anti-idiotypic antibodies to HCVHU.0~3 was detected after extensive washing with PBS
: 35 supplemented with 0.05 % qween 20R. If anti-idiotypic antibodies had bound to HCVHU.OT3-HRP, the signal decreased to background value.
'~127~97 WO94114974 PCT~ ~3/03707 With this material a sandwich assay was developed consisting at one hand of one of the monoclonal anti-idiotypic antibodies i~mobilized in a well of a 96-well micro ELISA plate and on the other hand of the same monoclonal antibody labelled with HRP in solution. (Figure 4).
The assay procedure to validate putative anti-idiotypic antibodies is further illustrated hereunder.
Putative anti-idiotypic antibody was -.
immobilized on a 96-well micro ELISA plate according to standard procedures (lO ~g/ml, 0.05M
carbo~ate buffer, pH=9.6, incubation overnight at C)- ..
Serum dilutions or dilutions of HC~HU.OT3 were added to the wells follnwed by l hour incubation at 37 C.
After extensive washing with P8S
~upplemented wi~h 0.05% Tween 20R ~4 times), HRP
labelled putative anti-idiotypic antibody was added.
Thereafter the plate was incu~ated for l hour at 37 C, followed by washing (4 times) with PBS with O.05% Tween 20~.
Thereafter substrate for HRP was added.
. Using the assay described in figure ~, further evidence was obtained for the ~pecificity of the monoclonal anti-idiotypic antibodies. The anti-idiotypic antibodies were able to discriminat~ between H~VHU.OT3 and an irrelevant human monoclonal antibody of the same isotype as HCVHU.OT3. In figure 5 representative examples of this specificity are shown.
- 2127797 :
.WO94/14974 PCT~3/03707 In the same assay (figure 5) it was established whether the anti-idiotyic antibodies were able to specifically bind to antibodies in serum from human individuals infected with HCV.
For that purpose a panel of human sera was sel~cted, known to contain antibodies against the HCV NS-3 antigen as determined by the commercial RIBA as~ay (Chiron Corporation Emmeryville).
In figure 5, reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant huoan monoclonal antibody wi~h the same subclass as HCVHU.0~3 is shown as shaded bars. Other ~onoclonal anti-idiotypic antibodies gave comparable results.
The ability of the anti-idiotypic antibodies prepared as described ab~ve were indeed able to mi~ic the immunodominant NS-3 epitopa as defined by the human monoalonal HCVHU.OT3, is exemplified in tabl~ 3.
Table 3 shows the reactivity of human sera from HCV infected patients and normal controls with anti-idiotypic antibodies HCVID.OT2A and HCVID.OT2I.
WO 94/~4974 ~ ~ 2 ~ 7 9 7 PCT/EP93/03707 To.ble 3 R~activ~y ef human ~er~ frc)m HCV inrected p~ti~nt~ ~nd nor~l c,~ntrols with ~r~ti-id~otypic antibodie~ HCV~D.OT2A ~Ind MCVID.OT2I.
~ ~-- --G6rulc lD 511ClOO C33 ~:2? TOT.OD450'R.R.~' OD4~0' N.R.~-A537 2 4 4 4 + 0.~210.99 0.620 1.1 A590 4 4 4 q t O . 599 l . 41 0 . 90o 1. 64 A598 4 4 4 4 + O . ~22 1 . 23 0 . 724 1. 31 ~599 1 2 3 3 t 1 . 302 ~ . 06 1 . 565 2 . 99 A732 1 0 4 4 ~ O . 46~ 1. 09 0 . 691 1. 25 A73~ 4 1 4 4 ~ l . 650 3 . 68 Z . 203 3 . 98 al~55 Z 0 4 4 ~ O . 474 1 . 12 û . 990 ~ . ~9 UOO~ 3 4 4 ~ ~ i .0312 . 191 .556 2.7i3 U013 O O 1 4 ~ 0.52Q1.~5 O.E~04 1.45 U033 4 3 4 4 ~ 1.0722.52 1.900 3.43 U036 4 4 ~I 4 ~ O . 57'` i . 35 0 . ~63 1 . S6 U038 2 2 3 4 ~ 0.7841.65 ~.0~9 1.~8 ~J043 2 ~ ~ O + o.~iO31.~ 0.9S3 i.72 C463 O O 4 0 ind . 13 . 676 1. 59 0 . a66 1. 8~
C~65 O ~- 3 ~- ind . O . 500 1.18 0 . 717 1. 25 C466 O G 4 0ind. 0.~95 l.6,1 ~l.101 1.99 C5J2 _ j ~ O 4 ~ nd . 0 . 990 2 . 3S 1 .187 2 . 14 Nor~a 1 Contrcl~ ~ ~ _ _... ~
H227 o D C O - O . 24'5 0 . 5~ 0 . 3~2 0 . 69 N202 O O O O - O . 20~ 0 . 4~ 0 . 325 0 . 5~
N203 j O O O C - O . 207 0 . ~5~ 0 . 3~0 0 . hO
N206 1 0 0 0 O _ O . 239 0 . 56 0 . 369 0 . t;7 N?07 1 0 0 0 0 - 0.173 0.4~0.292 0.53 ~20~ O O - o.zo~ 0.510.307 O.~IS
N211 1 0 0 O O - O . 209 0 . ~ O . 4~S 0 . B2 ~21~ O O ~ - 0.156 0.3~0.21~ 0.4~
N213 1 0 0 0 0 - 0~2lB 0.510.37~ 0.6B
^ OD - optlcdl den~ity ~t 450 ~n ~ N R - non~l ~ed r~ponse . c~lcul~t~d a3 OD4SO s:~f ~rum sample divided by c:ut of ~ v~ i ue .
~:ut off v~lue is ~ver~ge nor~nal :~erum (n~O) ~ 3 ti~ !~t~ntlard deviat ion .
.- WO94/14974 ~ 1 2 f 7 9 7 PC~ ~3/03707 Figures:
Figure l is a graph illustrating the binding specificity of monoclonal antibody HCVHU~OT3 for the NS3 protein.
Figure 2 is a photograph of a recombinant im~unoblot assay. ~he characters A-G represent the following proteins;
l~ A: High Ig control B: 5-l l (NS-4) C: c100-3 ~NS-4) D: c33c (NS-3) E: c22-3 (core) F: superoxide dismutase G: low Ig contL ol .
Lane l represents negative control serum, lane 2 represents an anti NS-3 monoclon~l antibody ~HCVHU.OT3), lane 3 represents an anti-core monoclonal ~HCVHU.OT2), and lane 4 represents polyclonal serum from a ~CV-infected patient.
Figure 3 represents a scrsening procedure used for detection of anti-idiotypic antib~dies against human monoclonal antibody HCVHU.OT3 in seru~ and in culture supernatant of ~onoclonal antibody producing cells.
Figure 4 represents an assay procedure used to validate putative anti-idiotypic antibodies which emerged from the screening procedure descri~ed in figure 3.
W094114974 ~ 7 9 7 PCT~3/03707 Figure 5 represents a sandwich assay (as shown in figure 4) to determine the specificity of 6 different anti-idiotypic antibodies (2A, 2B, 2I, 2P, 2Q and 3A) for HCVHU.OT3.
Reaction with HCVHU.OT3 is shown as black bars, reaction with an irrelevant human monoclonal antibody with the same subclass as HCVHU.OT3 is shown as shaded bars. Other monoclonal anti-idistypic antibodies gave comparable results.
SU~3STITUTE SHEET
Claims (15)
1. Monoclonal antibody which binds to an epitope of the NS3 protein of hepatitis C virus, which epitope is recognized by monoclonal antibodies secreted by the Epstein-Barr virus transformed human lymphocyte-B cell line, deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
2. Monoclonal antibody according to claim. 1, secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No.
92121609.
92121609.
3. Immortalized cell line capable of excreting antibodies according to claim 1.
4. Cell line deposited at the ECACC under deposit No. 92121609.
5. Method for the detection of hepatitis C
virus in a sample comprising contacting the sample with a monoclonal antibody according to claim 1 or 2, and detecting immune complexes formed between the monoclonal antibody and a hepatitis C antigen.
virus in a sample comprising contacting the sample with a monoclonal antibody according to claim 1 or 2, and detecting immune complexes formed between the monoclonal antibody and a hepatitis C antigen.
6. Immunodiagnostic reagent comprising a monoclonal antibody according to claim 1 or 2.
7. Test kit for the detection of hepatitis C
virus in a sample, comprising an immunodiagnostic reagent according to claim 6.
virus in a sample, comprising an immunodiagnostic reagent according to claim 6.
8. Anti-idiotypic antibody reactive with the antibody according to claim 1.
9. Anti-idiotypic antibody according to claim 8, secreted by the hybridoma cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 93122307, No. 93122308, No. 93122309, No. 93122310, No. 93122311, No. 93122312, No.
93122313, No. 93122314, No. 93122315, No.
93122316, No. 93122317, No. 93122318, No.
93122319, No. 93122320, No. 93122321 or No.
93122322.
93122313, No. 93122314, No. 93122315, No.
93122316, No. 93122317, No. 93122318, No.
93122319, No. 93122320, No. 93122321 or No.
93122322.
10. Anti-idiotypic antibody according to claim 8 or 9, capable of competing with an epitope for the binding to the monoclonal antibody according to claim 1, where the epitope is the epitope recognized by the monoclonal antibodies secreted by the Epstein-Barr virus-transformed human lymphocyte-B cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 92121609.
11. Immortalized cell line capable of secreting anti-idiotypic antibodies according to any of the claims 8-10.
12. Cell line deposited with the European Collection of Animal Cell Cultures (ECACC), Porton Down (UK), under deposit No. 93122307, No.
93122308, No. 93122309, No. 93122310, No.
93122311, No. 93122312, No. 93122313, No.
93122314, No. 93122315, No. 93122316, No.
93122317, No. 93122318, No. 93122319, No.
93122320, No. 93122321 or No. 93122322.
93122308, No. 93122309, No. 93122310, No.
93122311, No. 93122312, No. 93122313, No.
93122314, No. 93122315, No. 93122316, No.
93122317, No. 93122318, No. 93122319, No.
93122320, No. 93122321 or No. 93122322.
13. Method for the detection of anti-HCV
antibodies in a sample, comprising contacting the sample with an anti-idiotypic antibody according to any of the claims 8-10, and detecting immune complexes formed between the anti-idiotypic antibody and anti-HCV antibodies.
antibodies in a sample, comprising contacting the sample with an anti-idiotypic antibody according to any of the claims 8-10, and detecting immune complexes formed between the anti-idiotypic antibody and anti-HCV antibodies.
14. Immunodiagnostic reagent comprising an anti-idiotypic antibody according to any of the claims 8-10.
15. Testkit for the detection of anti-HCV
antibodies in a sample comprising an immunodiagnostic reagent according to claim 14.
antibodies in a sample comprising an immunodiagnostic reagent according to claim 14.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP92204107.4 | 1992-12-29 | ||
| EP92204107 | 1992-12-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2127797A1 true CA2127797A1 (en) | 1994-07-07 |
Family
ID=8211185
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002127797A Abandoned CA2127797A1 (en) | 1992-12-29 | 1993-12-27 | Monoclonal antibodies and anti-idiotypic antibodies to hepatitis c virus |
Country Status (8)
| Country | Link |
|---|---|
| EP (1) | EP0628084A1 (en) |
| JP (1) | JPH07504333A (en) |
| KR (1) | KR950700423A (en) |
| AU (1) | AU682335B2 (en) |
| CA (1) | CA2127797A1 (en) |
| FI (1) | FI943524A0 (en) |
| WO (1) | WO1994014974A1 (en) |
| ZA (1) | ZA939756B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6822563B2 (en) | 1997-09-22 | 2004-11-23 | Donnelly Corporation | Vehicle imaging system with accessory control |
| JP3217600B2 (en) * | 1994-07-12 | 2001-10-09 | 株式会社先端生命科学研究所 | Immunoassay for non-A non-B hepatitis virus-related antigen, monoclonal antibody used therein, and hybridoma producing this antibody |
| JP2002171972A (en) * | 1996-10-14 | 2002-06-18 | Chemo Sero Therapeut Res Inst | Hepatitis kanen virus epitope |
| DE10112748A1 (en) * | 2001-03-14 | 2002-09-19 | Transmit Technologietransfer | Invention relating to HCV diseases |
| EP1377832A2 (en) * | 2001-04-03 | 2004-01-07 | Immune Network Ltd. | Anti-idiotypic antibody and its use in diagnosis and therapy of hepatitis c virus related diseases |
| EP1504276B1 (en) | 2002-05-03 | 2012-08-08 | Donnelly Corporation | Object detection system for vehicle |
| US7526103B2 (en) | 2004-04-15 | 2009-04-28 | Donnelly Corporation | Imaging system for vehicle |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0423239A4 (en) * | 1988-07-06 | 1992-05-20 | Genelabs Incorporated | Post-transfusion, non-a, non-b hepatitis virus and antigens |
-
1993
- 1993-12-27 CA CA002127797A patent/CA2127797A1/en not_active Abandoned
- 1993-12-27 KR KR1019940703025A patent/KR950700423A/en not_active Application Discontinuation
- 1993-12-27 EP EP94904191A patent/EP0628084A1/en not_active Withdrawn
- 1993-12-27 JP JP6507580A patent/JPH07504333A/en active Pending
- 1993-12-27 AU AU58346/94A patent/AU682335B2/en not_active Expired - Fee Related
- 1993-12-27 WO PCT/EP1993/003707 patent/WO1994014974A1/en not_active Application Discontinuation
- 1993-12-29 ZA ZA939756A patent/ZA939756B/en unknown
-
1994
- 1994-07-27 FI FI943524A patent/FI943524A0/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU682335B2 (en) | 1997-10-02 |
| AU5834694A (en) | 1994-07-19 |
| FI943524A (en) | 1994-07-27 |
| WO1994014974A1 (en) | 1994-07-07 |
| KR950700423A (en) | 1995-01-16 |
| FI943524A0 (en) | 1994-07-27 |
| EP0628084A1 (en) | 1994-12-14 |
| JPH07504333A (en) | 1995-05-18 |
| ZA939756B (en) | 1994-08-18 |
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