WO1996029410A1 - In vivo generation of antibodies against antigens secreted in a host by transfected mel cells - Google Patents
In vivo generation of antibodies against antigens secreted in a host by transfected mel cells Download PDFInfo
- Publication number
- WO1996029410A1 WO1996029410A1 PCT/GB1996/000616 GB9600616W WO9629410A1 WO 1996029410 A1 WO1996029410 A1 WO 1996029410A1 GB 9600616 W GB9600616 W GB 9600616W WO 9629410 A1 WO9629410 A1 WO 9629410A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- host
- mel
- antibodies
- biological
- interest
- Prior art date
Links
- 239000000427 antigen Substances 0.000 title description 3
- 102000036639 antigens Human genes 0.000 title description 3
- 108091007433 antigens Proteins 0.000 title description 3
- 238000001727 in vivo Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims abstract description 19
- 208000036566 Erythroleukaemia Diseases 0.000 claims abstract description 5
- 241001529936 Murinae Species 0.000 claims abstract description 5
- 239000013604 expression vector Substances 0.000 claims description 7
- 239000013598 vector Substances 0.000 claims description 5
- 210000003200 peritoneal cavity Anatomy 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 208000031637 Erythroblastic Acute Leukemia Diseases 0.000 abstract 1
- 208000021841 acute erythroid leukemia Diseases 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 30
- 108090000623 proteins and genes Proteins 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 11
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 9
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 5
- 241000699670 Mus sp. Species 0.000 description 4
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 description 4
- 102100023543 Vascular cell adhesion protein 1 Human genes 0.000 description 4
- 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
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- 230000000694 effects Effects 0.000 description 3
- 238000002649 immunization Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 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
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- 239000006228 supernatant Substances 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 101000668058 Infectious salmon anemia virus (isolate Atlantic salmon/Norway/810/9/99) RNA-directed RNA polymerase catalytic subunit Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 101710091439 Major capsid protein 1 Proteins 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
- 238000001042 affinity chromatography Methods 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 230000003185 calcium uptake Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
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- 239000006285 cell suspension Substances 0.000 description 1
- 239000002975 chemoattractant Substances 0.000 description 1
- 230000035605 chemotaxis Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
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- 229930182817 methionine Natural products 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
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- 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
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- 238000000164 protein isolation Methods 0.000 description 1
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- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000003393 splenic effect Effects 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 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
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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
Definitions
- the present invention relates to methods for the generation of antibodies.
- it relates to the use of live, secretory cells in such methods.
- 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.
- MEL murine erythroleukaemia
- a method for the generation in a mammalian host of antibodies to a biological of interest 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.
- MEL murine erythroleukaemia
- 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.
- 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.
- MEL murine erythroleukaemia
- 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, __ (5), 997-1003). pEV was generated as follows :-
- the EcoRI site in the thymidine kinase promoter, the Bglll site in the neomycin resistance gene and the NotI site in the LCR of pGSE1417 (20) were sequentially deleted by restriction enzyme digestion followed by T4 polymerase treatment and religation to generate pDGSE1417.
- the Clal-Asp718 fragment from pEC3 (1) was ligated between the Clal 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 si al in Molecular Cloning, A Laboratory Manual.
- 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.
- 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.
- Example 1 The invention will now be illustrated but not limited by reference to the following Examples: Example 1
- VCAM-1 cDNA Modification of VCAM-1 cDNA
- VCAM-1 cDNA (Lobb et al., Biochem. and Biophys. Res. Comms.. 1991, US (3), pl498-1504) was obtained from British Biotechnology and was modified by polymerase chain reaction (PCR) procedures to
- C.T.I. consensus translation initiation sequence immediately upstream of the translational start site (5' methionine).
- MEL cell Expression of sVCAM-1 The modified sVCAM-1 cDNA was transferred into the MEL cell expression vector pEV (Needham et al., 1995, Protein Expression and Purification, _ (2), 1995). MEL C88 cells (Deisseroth et al. Cell, 1978, L5_, 55-63) were transfected with the pEV/sVCAM-1 expression construct. Six single cell clones and a population representing >200 clones were isolated, induced and tested for sVCAM-1 expression by VCAM-1 enzyme linked immunosorbent assay (ELISA). Initial ELISA results indicated that sVCAM-1 protein was expressed at between 1 and 5mg/ml. One clone (* 12) which expressed sVCAM-1 at 5mg/ml was selected for further studies.
- ELISA VCAM-1 enzyme linked immunosorbent assay
- MEL/sVCAM-1 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 centifuge. The resultant slurry was diluted with fresh PBS to give a final concentration of 5x107 cells per lOOuls. lOOuls 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.
- PBS phosphate buffered saline
- mice 12 weeks after the 3rd dose, the mice were each given a booster injection of 20ugs of purified recombinant VCAM protein contained in lOOuls 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 NS0 myeloma cell line (Methods in Enzymology, 1981 , 73B:3). 2316 culture wells were set up following the fusion process and after biochemical selection using HAT medium (Littlefield, Experimental Cell Research, 1966. 41 1, 190) and growth in culture, 95 of these produced growing monoclonal cell colonies.
- HAT medium Littlefield, Experimental Cell Research, 1966. 41 1, 190
- 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.
- MCP1 monoclonal anti-Monocyte Chemoattractant protein- 1
- MCP-1 cDNA was isolated and modifed for expression as reported by Needham et al., Protein Expression and Purification, 1996, 2, 173-182.
- the modified MCP-1 cDNA was transferred into the MEL cell expression vector pEV (Needham et al., 1995, Protein Expression and Purification, £ (2), 1995).
- MEL C88 cells (Deisseroth et al, Cell, 1978, ___, 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-1 expression by Northern blot, calcium flux and chemotaxis assays and by mass spectrometry (Needham et al.. Protein Expression and Purification, 1996, 2, 173-182). Results indicated that MCP-1 protein was expressed at between 1 and 5mg/ml.
- One clone (*5) which expressed MCP-1 at >5mg/ml was selected for further studies.
- Antibody 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.
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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
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP96906864A EP0817848A1 (en) | 1995-03-22 | 1996-03-18 | In vivo generation of antibodies against antigens secreted in a host by transfected mel cells |
JP8528176A JPH11502116A (en) | 1995-03-22 | 1996-03-18 | In vivo formation of antibodies to antigens secreted in the host by transfected MEL cells |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9505777.4 | 1995-03-22 | ||
GBGB9505777.4A GB9505777D0 (en) | 1995-03-22 | 1995-03-22 | Process |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US08913790 A-371-Of-International | 1997-09-19 | ||
US10/193,378 Continuation US20020182685A1 (en) | 1995-03-22 | 2002-07-11 | In vivo generation of antibodies against antigens secreted in a host by transfected MEL cells |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996029410A1 true WO1996029410A1 (en) | 1996-09-26 |
Family
ID=10771639
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1996/000616 WO1996029410A1 (en) | 1995-03-22 | 1996-03-18 | In vivo generation of antibodies against antigens secreted in a host by transfected mel cells |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP0817848A1 (en) |
JP (1) | JPH11502116A (en) |
GB (2) | GB9505777D0 (en) |
WO (1) | WO1996029410A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992011380A1 (en) * | 1990-12-21 | 1992-07-09 | Imperial Chemical Industries Plc | Expression systems |
WO1992016645A1 (en) * | 1991-03-15 | 1992-10-01 | Imutran Limited | PROTEIN PRODUCTION FROM IMMORTAL CELLS GROWN $i(IN VIVO) |
WO1995005853A1 (en) * | 1993-08-26 | 1995-03-02 | The Regents Of The University Of California | 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 WO PCT/GB1996/000616 patent/WO1996029410A1/en not_active Application Discontinuation
- 1996-03-18 EP EP96906864A patent/EP0817848A1/en not_active Withdrawn
- 1996-03-18 JP JP8528176A patent/JPH11502116A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992011380A1 (en) * | 1990-12-21 | 1992-07-09 | Imperial Chemical Industries Plc | Expression systems |
WO1992016645A1 (en) * | 1991-03-15 | 1992-10-01 | Imutran Limited | PROTEIN PRODUCTION FROM IMMORTAL CELLS GROWN $i(IN VIVO) |
WO1995005853A1 (en) * | 1993-08-26 | 1995-03-02 | The Regents Of The University Of California | Method, compositions and devices for administration of naked polynucleotides which encode biologically active peptides |
Non-Patent Citations (5)
Title |
---|
C. NEWTON ET AL.: "Cloning and expression in murine erythroleukemia cells: The soluble forms of the type I and type II tumor necrosis factor receptors fused to an immunogenic affinity tag.", PROTEIN EXPRESSION AND PURIFICATION, vol. 5, no. 5, October 1994 (1994-10-01), SAN DIEGO, CA, USA, pages 449 - 457, XP000574795 * |
M. NEEDHAM ET AL.: "Further development of the locus control region/murine erythroleukemia expression system: High level expression and characterization of recombinant human calcitonin receptor.", PROTEIN EXPRESSION AND PURIFICATION, vol. 6, no. 2, April 1995 (1995-04-01), SAN DIEGO, CA, USA, pages 124 - 131, XP000574794 * |
M. NEEDHAM ET AL.: "High level expression of human MCP-1 using the LCR/MEL expression system.", PROTEIN EXPRESSION AND PURIFICATION, vol. 7, no. 2, March 1996 (1996-03-01), SAN DIEGO, CA, USA, pages 173 - 182, XP000574796 * |
M. NEEDHAM ET AL.: "LCR/MEL: A versatile system for high-level expression of heterologous proteins in erythroid cells.", NUCLEIC ACIDS RESEARCH, vol. 20, no. 5, 11 March 1992 (1992-03-11), OXFORD, GB, pages 997 - 1003, XP002007371 * |
R. BRINES ET AL.: "Polyclonal activation of immature B cells by preactivated T cells: the role of IL-4 and CD40 ligand.", INTERNATIONAL IMMUNOLOGY, vol. 5, no. 11, November 1993 (1993-11-01), OXFORD, GB, pages 1445 - 1450, XP000574797 * |
Also Published As
Publication number | Publication date |
---|---|
GB9605619D0 (en) | 1996-05-22 |
GB2299084B (en) | 1997-03-26 |
EP0817848A1 (en) | 1998-01-14 |
JPH11502116A (en) | 1999-02-23 |
GB2299084A (en) | 1996-09-25 |
GB9505777D0 (en) | 1995-05-10 |
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