GB2299084A - Preparation of antibodies involving murine erythroleukaemia (MEL) cells - Google Patents
Preparation of antibodies involving murine erythroleukaemia (MEL) cells Download PDFInfo
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- GB2299084A GB2299084A GB9605619A GB9605619A GB2299084A GB 2299084 A GB2299084 A GB 2299084A GB 9605619 A GB9605619 A GB 9605619A GB 9605619 A GB9605619 A GB 9605619A GB 2299084 A GB2299084 A GB 2299084A
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- antibodies
- mel
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- mel cells
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- 208000036566 Erythroleukaemia Diseases 0.000 title claims description 5
- 241001529936 Murinae Species 0.000 title claims description 5
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000013604 expression vector Substances 0.000 claims abstract description 8
- 239000013598 vector Substances 0.000 claims abstract description 6
- 210000003200 peritoneal cavity Anatomy 0.000 claims abstract description 3
- 238000001890 transfection Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 34
- 238000002347 injection Methods 0.000 abstract description 4
- 239000007924 injection Substances 0.000 abstract description 4
- 230000007910 cell fusion Effects 0.000 abstract description 2
- 210000004698 lymphocyte Anatomy 0.000 abstract description 2
- 230000003393 splenic effect Effects 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract 3
- 239000003124 biologic agent Substances 0.000 abstract 1
- 238000010348 incorporation Methods 0.000 abstract 1
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- 102000004169 proteins and genes Human genes 0.000 description 12
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- 241001465754 Metazoa Species 0.000 description 5
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000007499 fusion processing Methods 0.000 description 2
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- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
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- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 1
- 101100504320 Caenorhabditis elegans mcp-1 gene Proteins 0.000 description 1
- 208000003643 Callosities Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
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- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
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- 239000000427 antigen Substances 0.000 description 1
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- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
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- 230000003185 calcium uptake Effects 0.000 description 1
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- 239000002975 chemoattractant Substances 0.000 description 1
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- 229930182817 methionine Natural products 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 210000002955 secretory cell Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
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- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
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- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70542—CD106
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Cell Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
A process for the generation in a mammalian host of antibodies to a biological agent comprises incorporation of MEL cells comprising DNA encoding said agent into said host, allowing the MEL cells to express and secrete said agent, allowing said host to raise antibodies thereto, and isolating said antibodies from said host. The agent may be incorporated into the MEL cells via tansfection with a vector, preferably the pEV expression vector. The MEL cells may be introduced into the host by injection, preferably in the form of a bolus, either subcutaneously or into the peritoneal cavity, thereby to yield polyclonal antibodies. Splenic lymphocytes from the host may be used to produce, via cell fusion, cell lines, from which monoclonal antibodies may be isolated.
Description
PROCESS
The present invention relates to methods for the generation of antibodies. In particular it relates to the use of live, secretory cells in such methods.
Available methods for the production of antibodies to a biological of interest are involved and have certain disadvantages. In particular they require the production of sufficient purified material to carry out the immunisation of animals. This may involve the use of cell cultures or tissues, fermentation broths, protein isolation and extraction from gels, affinity chromatography and other procedures.
We have now found that antibodies can be successfully raised in mice which have been injected with transfected murine erythroleukaemia (MEL) cells. This was gratifying since it was unclear whether or not the cells expressing the recombinant protein, or the expressed protein itself would remain in the tissue for sufficient time for a significant immune response to occur. Failure to observe tumour formation in animals which had been injected with the recombinant cells indicated that the cells at least were being rapidly cleared from the animal tissues.
Therefore in a first aspect of the invention we provide a method for the generation in a mammalian host of antibodies to a biological of interest which method comprises incorporating murine erythroleukaemia (MEL) cells comprising DNA encoding the biological of interest into the mammalian host, allowing the MEL cells to express and secrete the biological of interest, allowing the host to raise antibodies thereto, and isolating said antibiodies from the host.
The mammalian host is conveniently a non-human animal such as a rat or a mouse.
preferably a mouse.
The biological of interest comprises any convenient protein to which antibodies may be raised. In particular it may be a protein to which is difficult to raise or purify antibodies using presently available technology. Such difficulties may also arise by virtue of the tissue or species of origin, for example proteins of human origin. It is conveniently introduced into the murine erythroleukaemia (MEL) cells via transfection with a vector.
Convenient vectors include the pEV expression vector. The pEV expression vector is a modified version of the previously reported MEL expression vector pGSE1417/EC3 (Needham et al., Nucleic Acids Research, 1992, 2n (5), 997-1003). pEV was generated as follows:
The EcoRI site in the thymidine kinase promoter, the BglII site in the neomycin resistance gene and the NotI site in the LCR ofpGSE1417 (20) were sequentially deleted by restriction enzyme digestion followed by T4 polymerase treatment and religation to generate pDGSE1417. The ClaI-Asp718 fragment from pEC3 (1) was ligated between the
ClaI and Asp718 sites of pDGSE1417 to generate pEV.
DNA encoding the biological of interest is introduced into the vector using conventional molecular biology techniques for example as disclosed by Maniatis d al in
Molecular Cloning, A Laboratory Manual.
The transfected MEL cells are conveniently incorporated into the mammalian host by injection, for example in the form of a bolus to be introduced subcutaneously or into the peritoneal cavity.
The polyclonal antibodies are conveniently harvested from serum obtained from the mammal. The use of splenic lymphocytes from animals immunised in this way can, via cell fusion, give rise to monoclonal antibody producing cell lines. Such polyclonal and monoclonal antibodies represent further and independent aspects of the invention.
The invention will now be illustrated but not limited by reference to the following
Examples:
Example 1
Generation of anti-VCAM antibodies using recombinant MEL cell immunisation.
Modlficatlon of VCAM-I cDNA:
VCAM-I cDNA (Lobb et al., Biochem. and Biophys. Res. Corns., 1991, 178 (3), p1498-1504) was obtained from British Biotechnology and was modified by polymerase chain reaction (PCR) procedures to:1) Remove unwanted 5' non-coding sequence.
2) Include a consensus translation initiation (C.T.I.) sequence immediately upstream of the translational start site (5' methionine).
(N.B. In addition this sequence was omitted for the purpose of baculovirus expression).
3) Terminate translation at the amino acid prior to the start of the transmembrane region (by introducing a translational stop codon at amino acid 72).
4) Include restriction enzyme sites at each end of the VCAM-I cDNA to enable sub-cloning into a range of expression vectors.
MEL cell Expression of sVCAM-1: The modified sVCAM-I cDNA was transferred into the MEL cell expression vector pEV (Needham et al., 1995, Protein Expression and Purification, 6 (2), 1995). MEL
C88 cells (Deisseroth et al, Cell, 1978, 15, 55-63) were transfected with the pEV/sVCAM-I expression construct. Six single cell clones and a population representing > 200 clones were isolated, induced and tested for sVCAM-I expression by VCAM-I enzyme linked immunosorbent assay (ELISA). Initial ELISA results indicated that sVCAM-I protein was expressed at between 1 and Smg/ml. One clone (* 12) which expressed sVCAM-l at Smg/ml was selected for further studies.
Antlbodv Generation.
MEL/sVCAM-I clone *12 cells expressing were washed free from any residual culture medium using phosphate buffered saline (PBS) and then centrifuged to a soft pellet using a bench-top centrifuge. The resultant slurry was diluted with fresh PBS to give a final concentration of 5xl07 cells per 100uls. 100uls of cell suspension was injected as a subcutaneous bolus into the posterior flank of each of a group of 12 week old female Balb/C mice. This dose was repeated after 21 days and again after a further 14 days. Blood samples were taken on days 35 and 65.
Solid phase enzyme linked immunosorbent assay (ELISA) of pooled serum samples using purified recombinant VCAM protein as antigen gave indicated 50% binding titres of 1:1400 at 35 days and 1:6000+ at 65 days indicating that anti-VCAM polyclonal antibodies had been generated in the test animals.
12 weeks after the 3rd dose, the mice were each given a booster injection of 20ugs of purified recombinant VCAM protein contained in 100uls of PBS. 4days later the mice were humanely culled and splenectomised and the splenocytes from 4 of their spleens used in fusion experiments with the NSO myeloma cell line (Methods in Enzymology, 1981, 73B:3). 23 16 culture wells were set up following the fusion process and after biochemical selection using HAT medium (Littlefield, Experimental Cell Research, 1966, 411, 190) and growth in culture 95 of these produced growing monoclonal cell colonies. Supernatant culture medium which was positive for anti-VCAM activity as judged by ELISA was produced by 16 of the 95. 12 of the 16 were recloned, by limiting dilution and 5 of the 12 produced clones which were postive for anti-VCAM activity.
Examples of the positive clones were grown up to produce frozen stocks of the cell lines and exhausted supernatant from these cell lines was tested by ELISA and Western Blot analysis to confirm the continuance of monoclonal antibody activity.
Example 2
Generation of monoclonal anti-Monocyte Chemoattractant protein 1 (MCP 1) antibodies by recombinant MEL cell immunisation Modification of MCP-l cDNA: MCP-I cDNA was isolated and modifed for expression as reported by Needham et al., Protein Expression and Purification, 1996, 7 173-182.
Mel cell expression MCP-I cDNA
The modified MCP-I cDNA was transferred into the MEL cell expression vector pEV (Needham et al., 1995, Protein Expression and Purification, 6 (2). 1995). MEL C88 cells (Deisseroth et al, Cell, 1978, 15, 55-63) were transfected with the pEV/MCP-1 expression construct. Six single cell clones and a population representing > 200 clones were isolated, induced and tested for MCP-I expression by Northern blot, calcium flux and chemotaxis assays and by mass spectrometry (Needham et al., Protein Expression and
Purification 1996, 7, 173-182). Results indicated that MCP-I protein was expressed at between 1 and Smg/ml. One clone (*5) which expressed MCP-I at > 5mg/ml was selected for further studies.
Antibodv Generation:
This was performed exactly as for VCAM in Example 1 except that for antibody generation, the rest period between the last MEL cell injection and the pre-fusion booster was reduced to six weeks. Also 480 culture wells were set up after the fusion process and 35 of these produced growing monoclonal cell colonies. These eventually gave rise to 3 monoclonal antibody producing lines.
Claims (8)
1. A method for the generation in a mammalian host of antibodies to a biological of interest which method comprises incorporating murine erythroleukaemia (MEL) cells comprising DNA encoding the biological of interest into the mammalian host, allowing the
MEL cells to express and secrete the biological of interest, allowing the host to raise antibodies thereto, and isolating said antibodies from the host.
2. A method as claimed in claim 1 wherein the mammalian host is a non-human animal.
3. A method as claimed in claim 1 wherein DNA encoding the biological of interest is introduced into the MEL cells via transfection with a vector.
4. A method as claimed in claim 3 wherein the vector is the pEV expression vector.
5. A method as claimed in any one of the previous claims wherein the MEL cells are injected into the mammalian host.
6. A method as claimed in claim 5 wherein the MEL cells are injected in the form of a bolus to be introduced subcutaneously or into the peritoneal cavity.
7. Polyclonal antibodies produced using a method as claimed in any one of the previous claims.
8. Monoclonal antibodies produced using a method as claimed in any of claims 1-6.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GBGB9505777.4A GB9505777D0 (en) | 1995-03-22 | 1995-03-22 | Process |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB9605619D0 GB9605619D0 (en) | 1996-05-22 |
| GB2299084A true GB2299084A (en) | 1996-09-25 |
| GB2299084B GB2299084B (en) | 1997-03-26 |
Family
ID=10771639
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GBGB9505777.4A Pending GB9505777D0 (en) | 1995-03-22 | 1995-03-22 | Process |
| GB9605619A Expired - Fee Related GB2299084B (en) | 1995-03-22 | 1996-03-18 | Preparation of antibodies to protein secreted by murine erythroleukaemia (MEL) cells |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GBGB9505777.4A Pending GB9505777D0 (en) | 1995-03-22 | 1995-03-22 | Process |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0817848A1 (en) |
| JP (1) | JPH11502116A (en) |
| GB (2) | GB9505777D0 (en) |
| WO (1) | WO1996029410A1 (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9027917D0 (en) * | 1990-12-21 | 1991-02-13 | Ici Plc | Expression systems |
| GB9105532D0 (en) * | 1991-03-15 | 1991-05-01 | Imutran Ltd | Antibody production |
| CA2169635C (en) * | 1993-08-26 | 2002-11-12 | Dennis A. Carson | Method, compositions and devices for administration of naked polynucleotides which encode biologically active peptides |
-
1995
- 1995-03-22 GB GBGB9505777.4A patent/GB9505777D0/en active Pending
-
1996
- 1996-03-18 GB GB9605619A patent/GB2299084B/en not_active Expired - Fee Related
- 1996-03-18 EP EP96906864A patent/EP0817848A1/en not_active Withdrawn
- 1996-03-18 WO PCT/GB1996/000616 patent/WO1996029410A1/en not_active Application Discontinuation
- 1996-03-18 JP JP8528176A patent/JPH11502116A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| Protein Purification and Expression 1995,6,124-131 * |
| Protein Purification and Expression 1996,7,173-182 * |
Also Published As
| Publication number | Publication date |
|---|---|
| GB9605619D0 (en) | 1996-05-22 |
| GB2299084B (en) | 1997-03-26 |
| EP0817848A1 (en) | 1998-01-14 |
| WO1996029410A1 (en) | 1996-09-26 |
| JPH11502116A (en) | 1999-02-23 |
| GB9505777D0 (en) | 1995-05-10 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 732E | Amendments to the register in respect of changes of name or changes affecting rights (sect. 32/1977) | ||
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 20100318 |