WO1989006694A1 - Process for selection of proteinaceous substances which mimic growth-inducing molecules - Google Patents
Process for selection of proteinaceous substances which mimic growth-inducing molecules Download PDFInfo
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- WO1989006694A1 WO1989006694A1 PCT/US1989/000138 US8900138W WO8906694A1 WO 1989006694 A1 WO1989006694 A1 WO 1989006694A1 US 8900138 W US8900138 W US 8900138W WO 8906694 A1 WO8906694 A1 WO 8906694A1
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- 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
- C07K14/575—Hormones
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
Definitions
- This invention is directed to methods of producing novel biologically active proteinaceous substances which can be used as drugs, vaccines, diagnostic agents or similar treatments.
- the invention is more particularly directed to methods of selecting novel biologically active proteinaceous substances produced by recombinant DNA technique which mimic growth-inducing molecules of interest.
- Background of the invention Contemporary recombinant DNA techni-ques give scientists the capacity to generate large numbers of novel DNA sequences, RNA sequences, peptides, polypeptides and proteins.
- Some of these novel molecules by mimicing their natural counterparts, have practical, biologically active properties and can be used as the basis for novel drugs, novel vaccines, possible tailored treatments for autoimmune diseases and new diagnostic reagents as well as other uses.
- SUBSTITUTE SHEE ⁇ Some types of tumors have been found to proliferate by means of an autocrine mechanism or growth stimulating feedback loop. With this mechanism, a cell which has a receptor for a growth-inducing molecule will b stimulated to grow and divide by producing and secreting that same growth-inducing molecule which then binds to the receptor on or in the same cell. For example, some neoplastic transformation of cells by oncogenes and carcinogens is believed to result in the production of growth factors. Some Of these growth factors are able to bind to the receptors on the cell that secretes them, and to induce behavior indicative of a transformed phenotype which grows and divides uncontrollably. These findings have demonstrated an autocrine mechanism of transformation whereby production and secretion of growth factors by the cell which then bind to its own receptors by that factor leads to uncontrolled stimulation of the cell's own growth
- This invention is directed to methods for selecting proteinaceous substances which mimic growth- inducing molecules.
- the proteinaceous substances can be peptides, polypeptides or proteins.
- the choice of growth- inducing molecule is arbitrary, since the method of the invention is not restricted to any particular type of growth-inducing molecule. Specific examples include epidermal growth factor (EGF), dexamethasone and antigens.
- EGF epidermal growth factor
- dexamethasone dexamethasone
- Antigens include epidermal growth factor (EGF), dexamethasone and antigens.
- the proteinaceous substances are the translation products of the synthetic genes in the expression vector. Novel peptides, polypeptides or proteins which mimic the growth-inducing molecule will bind to the receptor for the molecule, thus closing the autocrine loop and stimulating cell growth and division. This process allows the rapid and easy detection of cells which secrete a peptide, polypeptide or protein with the desired growth- inducing property. Rapid, easy detection is essential when dealing with large numbers of molecules which potentially have growth-inducing activity.
- the invention is also directed to methods of selecting cells which produce a proteinaceous substance which mimics a growth-inducing molecule.
- Cells which require a growth-inducing molecule to stimulate cell growth and division and which cells have been transformed by recombinant DNA technique to produce proteinaceous substances not normally produced by the cells are supplied with a growth medium deficient in the growth-inducing molecule. The cells are then grown in the medium and the subset of cells which grow and divide are then isolated.
- the proteinaceous substances are the translation products of the synthetic genes in the expression vector.
- the invention further relates to processes for selecting synthetic genes which code for a proteinaceous substance which mimics a growth-inducing molecule.
- Expression vectors which contain synthetic genes which cod for a proteinaceous substance are used to transform cells which require a growthrinducing molecule to stimulate cell growth and division thereby causing the cells to produce a proteinaceous substance which is the translation product o the synthetic genes in the expression vector.
- the transformed cells are grown in a growth medium deficient i the growth-inducing molecule.
- the subset of cells which grow and divide are then isolated.
- the subset of cells containing an expression vector comprising synthetic genes which mimic the growth-inducing molecule of interest are generally the only cells which grow and divide and can be easily harvested.
- synthetic genes refers to DNA sequences which are the result of manipulation by laboratory techniques.
- natural gene refers to DNA sequences as they are found in situ in a chromosome.
- Synthetic gene include stochastic (random) or partially stochastic DNA sequences as well as DNA sequences which are composed of parts of a natural DNA sequence and stochastic DNA sequences or particular nucleotides.
- synthetic gene also includes DNA sequences composed of a plurality o DNA sequences derived from natural genes.
- recombinant DNA technique refers to the techniques of producing synthetic genes, inserting the DNA sequences into expression vectors, and transforming cells with expression vectors. Illustrations of the recombinant DNA technique used in the invention are set forth in detai below in the section entitled Recombinant DNA Technique which has two subsections, A.Production of Synthetic Genes and B. Transformation of Cells.
- linkers which can be ligated in random order and generate codons for all twenty amino acids without generating stop codons in any reading frame or in either orientation
- linkers suitable for use in the invention include Clal, Ndel, Hindlll, Sail, Pstl and EcoRI. Equimolar or other mixtures of the linkers are ligated together using T4-DNA ligase (Boehringer and Mannheim Biochemicals) using the procedure of Maniatis et al. , Molecular Cloning, A Laboratory Manual, (Cold Spring Harbor Laboratory, 1982), which is specifically incorporated herein by reference.
- the ligated linkers are partially digested with one or several of the restriction enzymes for any of the linkers used to make the sequences such as EcoRI enzyme (Boehringer and Mannheim Biochemicals) according to the manufacturer's instructions.
- the digested mixture is then separated on a gel such as a 0.7% agarose gel using a standard borate buffer system according to the method of Maniatis (see reference above) .
- the fragments of desired size range(s) of DNA sequences are cut from the gel and isolated according to the method of Maniatis et al. , (see above reference) .
- the isolated fragments are then cloned into a restriction enzyme cleavage site or sites of an expression vector that causes secretion from the cell of the peptide or protein which is the translation product of the synthetic genes which have been cloned into the expression vector.
- the expression vector pUCDS3 constructed according to the method in Stern et al. , Science 235: 231-235, (1987) which is specifically incorporated herein by reference, can be used in the invention.
- This expression vector is constructed by modifying pFBl which consists of a partial Sau 3A fragment containing the Moloney murine leukemia virus long terminal repeat (LTR) cloned into the Bam HI site of pUC13.
- LTR Moloney murine leukemia virus long terminal repeat
- the Hpa I-Bam HI fragment containing the SV40 polyadenylation site was provided with synthetic Hpa I-Sal I and Bam HI-Hind III adapters and cloned into pUC8 adjacent to the EGF gene.
- the Eco Rl-Hine III fragment containing the EGF gene and polyadenylation site was then cloned into pUCDS2 to produce pUCDS3.
- the expression vector is modified by removing the fragment containing the epidermal growth factor (EGF) gene using restriction enzymes Sail and EcoRI.
- the expression vector is then purified by methods known in the art.
- the ligated linkers are partially digested using Sail and EcoRI restriction enzymes and the fragments are purified and isolated as described above.
- a signal peptide can be cloned into expression vectors without a signal peptide in a position adjacent to the synthetic genes coding for the peptides or proteins to cause the cell to secrete the peptides or proteins using methods known in the art.
- Signal peptides such as a mouse immunoglobulin heavy-chain signal peptide or the random sequences of Kaiser et al.. Science 235: 312-317, (1987) may be used in the invention.
- the ligated linkers should be partially digested with the same restriction enzymes used in the expression vector to create the site for the synthetic genes so that the ends of the ligated linkers will fit the restriction enzyme sites. However, if the ligated linkers have not been partially digested in this way, the ends of the fragments can be modified using other cloning procedures known in the art for these and other synthetic genes such as blunt end ligation.
- the library of synthetic genes cloned into the expression vectors is then inserted into cells which lack a growth-inducing molecule.
- Cells can be transfected by methods such as the calcium phosphate coprecipitation method of Wigler et al.. Cell jLl: 223-232 (1977) ; the polyethylene glycol 6000 calcium phosphate treatment method of Sutherland and Bennett, Cancer Research 44(7) : 2769-2772, (1984), or Sutherland et al.. Proc. Natl. Acad. Sci. USA 82(2) : 2399- 2403, (1985); or the electroporation method of Toneguzzo et al.. Mol. Cell Biol. 6(2) : 703-706, (1986) or Potter et al., Proc. Natl. Acad. Sci 8 7161-7165, (1984), all of which are specifically incorporated herein by reference. Cell Culture
- Cells which need a growth-inducing molecule to stimulate cell growth and division are suitable for use in the invention.
- Human diploid fibroblast cells such as WI- 38, a cell line which requires the addition of exogenous growth factors to stimulate cell growth and division or B lymphocytes are exemplary for use in this invention. These cells are transformed with expression vector which includes synthetic genes as provided above. In the case of WI-38, the cells are then grown according to the method of
- Proteinaceous substance which mimics the growth-inducing molecule not included in the medium.
- This proteinaceous substance is the translation product of the synthetic genes which were inserted into the cell via the expression vector.
- These cells are isolated and the proteinaceous substance characterized using methods for identifying peptides, polypeptides and proteins known in the art. Methods used to identify the proteinaceous substance will vary accordin to the type of proteinaceous substance. It is within the scope of the invention to select proteinaceous substances which mimic arbitrary antigens, i.e. the selection method can be used to search for proteinaceous mimics of any antigen of interest.
- synthetic genes inserted into an expression vector are inserted into B lymphocytes, or other immune system cells, with antibody receptor molecules on their surface against the same external antigenic determinant. If a proteinaceous substance produced by a B lymphocyte which is the translation product of the synthetic genes mimics the antigen, this proteinaceous substance will bind to antibodies on that B lymphocyte which are functioning as receptors. This binding of the proteinaceous substance which mimics the antigen should stimulate the lymphocyte to divide.
- the proteinaceous substances which mimic the growth-inducing molecule of interest are believed to be useful as drugs, vaccines, diagnostics or similar purposes. It is also within the scope of the invention to select novel genes which produce the proteinaceous substances which mimic the growth-inducing molecule of interest. After a proteinaceous substance has been found which mimics the growth-inducing molecule of interest, the expression vector which carries the genes for the proteinaceous substance is removed from the cells which produce the proteinaceous substance. Then the expression vector is analyzed by methods known in the art to determine the sequence of DNA bases which codes for the proteinaceous substance.
- the base sequence and, correspondingly, the proteinaceous substance can be altered by methods known in the art in order to improve the ability of the proteinaceous substance to mimic the growth-inducing molecule of interest or otherwise to improve the practice of the embodiments of this invention.
- the altered gene is inserted into an expression vector which is then inserted into a cell which lacks a growth-inducing molecule necessary for cell growth and division.
- the selection procedure which has been previously described for selecting a proteinaceous substance which mimics a growth-inducing molecule is then followed.
Abstract
A method of selecting proteinaceous substances produced by recombinant DNA technique which mimic an arbitrary growth-inducing molecule is disclosed. Also disclosed is a method of selecting cells which produce proteinaceous substances which mimic an arbitrary growth-inducing molecule. A process for selecting synthetic genes which codes for a proteinaceous substance which mimics a growth-inducing molecule is also provided. The proteinaceous substances produced by this method are believed to be useful as novel drugs, vaccines, diagnostic agents or similar purposes.
Description
Process For Selection Of Proteinaceous Substances Which Mimic Growth-Inducing Molecules
Field of the Invention
This invention is directed to methods of producing novel biologically active proteinaceous substances which can be used as drugs, vaccines, diagnostic agents or similar treatments. The invention is more particularly directed to methods of selecting novel biologically active proteinaceous substances produced by recombinant DNA technique which mimic growth-inducing molecules of interest. Background of the invention Contemporary recombinant DNA techni-ques give scientists the capacity to generate large numbers of novel DNA sequences, RNA sequences, peptides, polypeptides and proteins. Some of these novel molecules, by mimicing their natural counterparts, have practical, biologically active properties and can be used as the basis for novel drugs, novel vaccines, possible tailored treatments for autoimmune diseases and new diagnostic reagents as well as other uses. Although large numbers of novel DNA sequences, RNA sequences, peptides, polypeptides and proteins such as those generated by Ballivet and Kauffman, U.S. application 942,630 filed 6/17/85, which is specifically incorporated herein by reference, can be produced, not all novel molecules produced by recombinant DNA technique are biologically active. Consequently, there exists a need for processes for quickly and easily screening those cells which produce biologically active molecules.
SUBSTITUTE SHEEΓΓ
Some types of tumors have been found to proliferate by means of an autocrine mechanism or growth stimulating feedback loop. With this mechanism, a cell which has a receptor for a growth-inducing molecule will b stimulated to grow and divide by producing and secreting that same growth-inducing molecule which then binds to the receptor on or in the same cell. For example, some neoplastic transformation of cells by oncogenes and carcinogens is believed to result in the production of growth factors. Some Of these growth factors are able to bind to the receptors on the cell that secretes them, and to induce behavior indicative of a transformed phenotype which grows and divides uncontrollably. These findings have demonstrated an autocrine mechanism of transformation whereby production and secretion of growth factors by the cell which then bind to its own receptors by that factor leads to uncontrolled stimulation of the cell's own growth
Stern et al. (Science 235: 321-324, 1987) constructed in a vector a novel oncogene which expressed human epidermal growth factor (EGF) . This expression vector was transformed into a population of cells which ha EGF receptors on their surfaces. The translation product of this novel oncogene was secreted from each transformed cell and induced focus formation by binding to the cell's own EGF receptor. Thus focus formation is the consequence of autocrine loop closure and chronic autocrine stimulation.
In view of the foregoing, it is desired to provide rapid, easy methods for selecting proteinaceous substances which mimic growth-inducing molecules, methods for selecting cells which produce proteinaceous substances which mimic growth-inducing molecules and methods for selecting synthetic genes which code for proteinaceous substances which mimic growth-inducing molecules.
Summary of the Invention
This invention is directed to methods for selecting proteinaceous substances which mimic growth- inducing molecules. The proteinaceous substances can be peptides, polypeptides or proteins. The choice of growth- inducing molecule is arbitrary, since the method of the invention is not restricted to any particular type of growth-inducing molecule. Specific examples include epidermal growth factor (EGF), dexamethasone and antigens. Cells which require a growth-inducing molecule to stimulate cell growth and division and which have been transformed by recombinant DNA technique to secrete proteinaceous substances not normally secreted by the cells are supplied with a growth medium deficient in the growth-inducing molecule and the cells are then grown in the medium. The subset of cells which grow and divide are then collected and the proteinaceous substance produced by the subset of cells is then isolated. In preferred embodiments of the invention the proteinaceous substances are the translation products of the synthetic genes in the expression vector. Novel peptides, polypeptides or proteins which mimic the growth-inducing molecule will bind to the receptor for the molecule, thus closing the autocrine loop and stimulating cell growth and division. This process allows the rapid and easy detection of cells which secrete a peptide, polypeptide or protein with the desired growth- inducing property. Rapid, easy detection is essential when dealing with large numbers of molecules which potentially have growth-inducing activity. The invention is also directed to methods of selecting cells which produce a proteinaceous substance which mimics a growth-inducing molecule. Cells which require a growth-inducing molecule to stimulate cell growth and division and which cells have been transformed by recombinant DNA technique to produce proteinaceous substances not normally produced by the cells are supplied with a growth medium deficient in the growth-inducing molecule. The cells are then grown in the medium and the
subset of cells which grow and divide are then isolated. In preferred embodiments of the invention the proteinaceous substances are the translation products of the synthetic genes in the expression vector. The invention further relates to processes for selecting synthetic genes which code for a proteinaceous substance which mimics a growth-inducing molecule. Expression vectors which contain synthetic genes which cod for a proteinaceous substance are used to transform cells which require a growthrinducing molecule to stimulate cell growth and division thereby causing the cells to produce a proteinaceous substance which is the translation product o the synthetic genes in the expression vector. The transformed cells are grown in a growth medium deficient i the growth-inducing molecule. The subset of cells which grow and divide are then isolated. The subset of cells containing an expression vector comprising synthetic genes which mimic the growth-inducing molecule of interest are generally the only cells which grow and divide and can be easily harvested.
Detailed Description of the Invention
The term synthetic genes refers to DNA sequences which are the result of manipulation by laboratory techniques. The term natural gene refers to DNA sequences as they are found in situ in a chromosome. Synthetic gene include stochastic (random) or partially stochastic DNA sequences as well as DNA sequences which are composed of parts of a natural DNA sequence and stochastic DNA sequences or particular nucleotides. The term synthetic gene also includes DNA sequences composed of a plurality o DNA sequences derived from natural genes.
The term recombinant DNA technique refers to the techniques of producing synthetic genes, inserting the DNA sequences into expression vectors, and transforming cells with expression vectors. Illustrations of the recombinant DNA technique used in the invention are set forth in detai below in the section entitled Recombinant DNA Technique
which has two subsections, A.Production of Synthetic Genes and B. Transformation of Cells.
Recombinant DNA Technique Although a large number of methodologies may be employed in the practice of the invention, the following are illustrative of certain, preferred embodiments. A. Production of Synthetic Genes
Commercially available, prepared restriction enzyme linkers which can be ligated in random order and generate codons for all twenty amino acids without generating stop codons in any reading frame or in either orientation (e.g. octameric linkers purchased from New England Biolabs) are used in the invention. Examples of linkers suitable for use in the invention include Clal, Ndel, Hindlll, Sail, Pstl and EcoRI. Equimolar or other mixtures of the linkers are ligated together using T4-DNA ligase (Boehringer and Mannheim Biochemicals) using the procedure of Maniatis et al. , Molecular Cloning, A Laboratory Manual, (Cold Spring Harbor Laboratory, 1982), which is specifically incorporated herein by reference. After ligation is complete, the ligated linkers are partially digested with one or several of the restriction enzymes for any of the linkers used to make the sequences such as EcoRI enzyme (Boehringer and Mannheim Biochemicals) according to the manufacturer's instructions. The digested mixture is then separated on a gel such as a 0.7% agarose gel using a standard borate buffer system according to the method of Maniatis (see reference above) . The fragments of desired size range(s) of DNA sequences are cut from the gel and isolated according to the method of Maniatis et al. , (see above reference) . The isolated fragments are then cloned into a restriction enzyme cleavage site or sites of an expression vector that causes secretion from the cell of the peptide or protein which is the translation product of the synthetic genes which have been cloned into the expression vector.
For example, the expression vector pUCDS3 constructed according to the method in Stern et al. ,
Science 235: 231-235, (1987) which is specifically incorporated herein by reference, can be used in the invention. This expression vector is constructed by modifying pFBl which consists of a partial Sau 3A fragment containing the Moloney murine leukemia virus long terminal repeat (LTR) cloned into the Bam HI site of pUC13. This expression vector was constructed by J. Morgenstern and H. Land of the Whitehead Institute. The single Eco Rl site of pUCl was destroyed by cleaving with EcoRI, filling in, and religating to produce pFBl RI. The Nde I-Pst I fragment of immunoglobulin heavy chain complementary DNA clone 17.2.25 of Loh et al.. Cell 3_3_: 85, (1983), which is specifically incorporated herein by reference, which contains sequences endcoding the signal peptide, was modified by oligonucleotide-directed mutagenesis according to the method of Dalbadie-McFarland, et al. f Proc. Natl. Acad. Sci. U.S.A. 79.: 6409, (1982) which is specifically incorporated herein by reference, to create an Eco Rl site. The fragment excised by cleavage at the Ava II site upstream from the coding sequences and at the newly created Eco Rl site was joined to pFB RI cleaved with Sal I and Hind III by using Ava II-pseudo-Sal I and Eco Rl-Hind III adapters to yield pUCDS2. A chemically synthesized gene encoding human EGF, prepared according to the method of Hare et al.. J. Cell Biochem. Suppl. 8A: 87, (1984), which is specifically incorporated herein, was provided with an Eco Rl site and upstream sequences encoding the first three a ino acids of immunoglobulin 17.2.25 and subcloned into pUC8. The Hpa I-Bam HI fragment containing the SV40 polyadenylation site was provided with synthetic Hpa I-Sal I and Bam HI-Hind III adapters and cloned into pUC8 adjacent to the EGF gene. The Eco Rl-Hine III fragment containing the EGF gene and polyadenylation site was then cloned into pUCDS2 to produce pUCDS3. The expression vector is modified by removing the fragment containing the epidermal growth factor (EGF) gene using restriction enzymes Sail and EcoRI. The expression vector is then purified by methods known in the art.
The ligated linkers are partially digested using Sail and EcoRI restriction enzymes and the fragments are purified and isolated as described above. The fragments are then ligated into the purified expression vector using T4-DNA ligase. Other expression vectors which cause the cell to secrete from the cell the peptide or protein coded by the synthetic gene are also useful in the invention. A signal peptide can be cloned into expression vectors without a signal peptide in a position adjacent to the synthetic genes coding for the peptides or proteins to cause the cell to secrete the peptides or proteins using methods known in the art. Signal peptides such as a mouse immunoglobulin heavy-chain signal peptide or the random sequences of Kaiser et al.. Science 235: 312-317, (1987) may be used in the invention.
The ligated linkers should be partially digested with the same restriction enzymes used in the expression vector to create the site for the synthetic genes so that the ends of the ligated linkers will fit the restriction enzyme sites. However, if the ligated linkers have not been partially digested in this way, the ends of the fragments can be modified using other cloning procedures known in the art for these and other synthetic genes such as blunt end ligation. The library of synthetic genes cloned into the expression vectors is then inserted into cells which lack a growth-inducing molecule. B. Transformation of Cells
Cells can be transfected by methods such as the calcium phosphate coprecipitation method of Wigler et al.. Cell jLl: 223-232 (1977) ; the polyethylene glycol 6000 calcium phosphate treatment method of Sutherland and Bennett, Cancer Research 44(7) : 2769-2772, (1984), or Sutherland et al.. Proc. Natl. Acad. Sci. USA 82(2) : 2399- 2403, (1985); or the electroporation method of Toneguzzo et al.. Mol. Cell Biol. 6(2) : 703-706, (1986) or Potter et al., Proc. Natl. Acad. Sci 8 7161-7165, (1984), all of which are specifically incorporated herein by reference.
Cell Culture
Cells which need a growth-inducing molecule to stimulate cell growth and division are suitable for use in the invention. Human diploid fibroblast cells such as WI- 38, a cell line which requires the addition of exogenous growth factors to stimulate cell growth and division or B lymphocytes are exemplary for use in this invention. These cells are transformed with expression vector which includes synthetic genes as provided above. In the case of WI-38, the cells are then grown according to the method of
Phillips and Cristofalo, Experimental Cell Research 134: 297-302, (1981), except that the medium does not include the growth-inducing molecule which is being sought.
Cells which grow and divide in this medium are then presumed to be synthesizing a proteinaceous substance which mimics the growth-inducing molecule not included in the medium. This proteinaceous substance is the translation product of the synthetic genes which were inserted into the cell via the expression vector. These cells are isolated and the proteinaceous substance characterized using methods for identifying peptides, polypeptides and proteins known in the art. Methods used to identify the proteinaceous substance will vary accordin to the type of proteinaceous substance. It is within the scope of the invention to select proteinaceous substances which mimic arbitrary antigens, i.e. the selection method can be used to search for proteinaceous mimics of any antigen of interest. In this case, synthetic genes inserted into an expression vector, such as the system described above, are inserted into B lymphocytes, or other immune system cells, with antibody receptor molecules on their surface against the same external antigenic determinant. If a proteinaceous substance produced by a B lymphocyte which is the translation product of the synthetic genes mimics the antigen, this proteinaceous substance will bind to antibodies on that B lymphocyte which are functioning as receptors. This binding of the proteinaceous substance
which mimics the antigen should stimulate the lymphocyte to divide.
The proteinaceous substances which mimic the growth-inducing molecule of interest are believed to be useful as drugs, vaccines, diagnostics or similar purposes. It is also within the scope of the invention to select novel genes which produce the proteinaceous substances which mimic the growth-inducing molecule of interest. After a proteinaceous substance has been found which mimics the growth-inducing molecule of interest, the expression vector which carries the genes for the proteinaceous substance is removed from the cells which produce the proteinaceous substance. Then the expression vector is analyzed by methods known in the art to determine the sequence of DNA bases which codes for the proteinaceous substance. Once the base sequence of the gene coding for the proteinaceous substance is known, the base sequence and, correspondingly, the proteinaceous substance can be altered by methods known in the art in order to improve the ability of the proteinaceous substance to mimic the growth-inducing molecule of interest or otherwise to improve the practice of the embodiments of this invention. In the first case, the altered gene is inserted into an expression vector which is then inserted into a cell which lacks a growth-inducing molecule necessary for cell growth and division. The selection procedure which has been previously described for selecting a proteinaceous substance which mimics a growth-inducing molecule is then followed.
Claims
1. A process for selecting a proteinaceous substance which mimics a growth-inducing molecule comprising the steps of: a. providing cells which require a growth- inducing molecule to stimulate cell growth and division and which have been transformed by recombinant DNA technique to secrete proteinaceous substances not normally secreted by said cells; b. supplying said cells with growth medium deficient in said growth inducing molecule; c. growing said cells in said medium; d. collecting a subset of said cells which grow and divide; and e. isolating said proteinaceous substance from said subset of said cells.
2. The process of claim 1 wherein said proteinaceous substance is the translation product of synthetic genes.
3. The process of claim 1 wherein said proteinaceous substance is a peptide.
4. The process of claim 1 wherein said proteinaceous substance is a protein.
5. The process of claim 1 wherein said growth- inducing molecule is epidermal growth factor.
6. The process of claim 1 wherein said growth- inducing molecule is dexamethasone.
7. The process of claim 1 wherein said growth- inducing molecule is an antigen.
8. The process of claim 1 wherein said cells are a human diploid fibroblast.
9. The process of claim 8 wherein said human diploid fibroblast is WI-38.
10. The process of claim 1 wherein said cells are immune system dells.
11. The process of claim 10 wherein said immune system cells are B lymphocytes.
12. A process of selecting cells which produce a proteinaceous substance which mimics a growth-inducing molecule, said proteinaceous substance being the translation product of synthetic genes introduced into said cells by a recombinant DNA technique, comprising the steps of: a. providing cells which require a growth- inducing molecule to stimulate cell growth and division and which cells have been transformed by recombinant DNA technique to produce proteinaceous substances not normally secreted by said cells; b. supplying said cells with a growth medium deficient in said growth-inducing molecule; c. growing said cells in said medium; and d. isolating a subset of said cells which grow and divide.
13. The process of claim 12 wherein said proteinaceous substance is the translation product of synthetic genes.
14. The method of claim 12 wherein said proteinaceous substance is a peptide.
15. The method of claim 12 wherein said proteinaceous substance is a protein.
16. The process of claim 12 wherein said growth- inducing molecule is epidermal growth factor.
17. The process of claim 12 wherein said growth- inducing molecule is dexamethasone.
18. The process of claim 12 wherein said growth- inducing molecule is an antigen.
19. The process of claim 12 wherein said cells are a human diploid fibroblast.
20. The process of claim 19 wherein said human diploid fibroblast is WI-38.
21. The process of claim 12 wherein said cells are immune system cells.
22. The process of claim 21 wherein said immune system cells are B lymphocytes.
23. A process for selecting synthetic genes which code for a proteinaceous substance which mimics a growth- inducing molecule comprising the following steps: (a) . providing expression vectors which contain synthetic genes which code for a proteinaceous substance; (b) . transforming cells which require a growth- inducing molecule to stimulate cell growth and division to include said expression vector thereby causing said cells to produce a proteinaceous substance which is the translation product of said synthetic genes in said expression vector; (c) . supplying said transformed cells with growth medium deficient in said growth-inducing molecule;
(d) . growing said transformed cells in said growth medium;
(d) . isolating a subset of said cells which grow and divide;
(e) . removing said expression vector from said subset of said cells which grow and divide; and
(f) . analyzing said synthetic genes in said expression vector.
24. The process of claim 23 wherein said proteinaceous substance is a peptide.
25. The process of claim 23 wherein said proteinaceous substance is a protein.
26. The process of claim 23 wherein said growth- inducing molecule is epidermal growth factor.
27. The process of claim 23 wherein said growth- inducing molecule is dexamethasone.
28. The process of claim 23 wherein said growth- inducing molecule is an antigen.
29. The process of claim 23 wherein said cells are a human diploid fibroblast.
30. The process of claim 29 wherein said human diploid fibroblast is WI-38.
31. The process of claim 23 wherein said cells are immune system cells.
32. The process of claim 31 wherein said immune system cells are B lymphocytes.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14446888A | 1988-01-15 | 1988-01-15 | |
US144,468 | 1988-01-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1989006694A1 true WO1989006694A1 (en) | 1989-07-27 |
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Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1989/000138 WO1989006694A1 (en) | 1988-01-15 | 1989-01-13 | Process for selection of proteinaceous substances which mimic growth-inducing molecules |
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WO (1) | WO1989006694A1 (en) |
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