EP0368684B1
Expired / Lapsed
EP
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Cloning immunoglobulin variable domain sequences.
Assignee: Medical Research Council
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Filing: 1989-11-13
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Granted: 1994-03-09
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Expires: 2009-11-13
Abstract
Abstract The present invention relates to single domain ligands derived from molecules in the immunoglobulin (Ig) superfamily, receptors comprising at least one such ligand, methods for cloning, amplifying and expressing DNA sequences encoding such ligands, preferably using the polymerase chain reaction, methods for the use of said DNA sequences in the production of Ig-type molecules and said ligands or receptors, and the use of said ligands or receptors in therapy, diagnosis or catalysis.
Inventors
- 👤 Gregory Paul Winter
- 👤 Detlef Güssow
- 👤 Elizabeth Sally Ward
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-23 17:10
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/575; C07K14/655; C07K16/00; C07K16/46; C07K16/461; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6876; A; A61; A61K; A61K38/00; C07K2317/00; C07K2317/20; C07K2317/24
Claims Analysis
Total Claims: 34
Claim 1
Independent Claim
115 words
A method of cloning sequences (target sequences) each containing a sequence encoding at least part of an immunoglobulin variable domain, which method comprises providing a sample repertoire of nucleic acid containing target sequences, and using forward and back primers in the process of copying and cloning of the target sequences for expression of a repertoire of proteins each comprising at least part of an immunoglobulin variable domain, the forward primer being specific for a sequence at or adjacent the 3' end of the sense strand of each of the target sequences, the back primer being specific for a sequence at or adjacent the 3' end of the antisense strand of each of the target sequences.
Claim 2
Independent Claim
115 words
A method of cloning sequences (target sequences) each containing a sequence encoding at least part of an immunoglobulin variable domain, which method comprises providing a sample repertoire of nucleic acid containing target sequences, and using forward and back primers in the process of copying and cloning of the target sequences for expression of a repertoire of proteins each comprising at least part of an immunoglobulin variable domain, the forward primer being specific for a sequence at or adjacent the 3' end of the sense strand of each of the target sequences, the back primer being specific for a sequence at or adjacent the 3' end of the antisense strand of each of the target sequences.
Claim 3
Dependent Claim
179 words
A method according to claim 1 which method comprises: (a) providing a sample repertoire of double-stranded nucleic acid containing target sequences; (b) causing the two strands of the doubled-stranded nucleic acid to be separated; (c) annealing to the sample a forward and a back oligonucleotide primer, the forward primer being specific for a sequence at or adjacent the 3' end of the sense strand of each of the target sequences, the back primer being specific for a sequence at or adjacent the 3' end of the antisense strand of each of the target sequences, under conditions which allow the primers to hybridize specifically to the nucleic acid; (d) treating the annealed sample with a DNA polymerase enzyme in the presence of deoxynucleoside triphosphates under conditions which cause primer extension to take place, thereby producing double-stranded nucleic acid; (e) repeating steps (b) to (d), thereby producing some double-stranded DNA (product DNA) containing only the target sequences; (f) cloning product DNA into expression vectors for expression of a repertoire of proteins each comprising at least part of an immunoglobulin variable domain.
Claim 4
Dependent Claim
20 words
A method according to claim 2 wherein steps (b) to (d) are repeated a plurality of times before step (f).
Claim 5
Dependent Claim
178 words
A method according to claim 1, which comprises: (a) providing a repertoire of mRNA; (b) annealing to the mRNA an oligonucleotide primer specific for a sequence at or adjacent the 3' end of each of the target sequences on the sense strands, under conditions which allow the primer to hybridize specifically to the nucleic acid; (c) treating the primer-annealed mRNA with a polymerase enzyme in the presence of deoxynucleoside triphosphates under conditions which cause primer extension to take place, thereby producing antisense cDNA; (d) annealing to the cDNA an oligonucleotide primer specific for a sequence at or adjacent the 3' end of each of the target sequences on the antisense strands, under conditions which allow the primer to hybridize specifically to the nucleic acid; (e) treating the primer-annealed cDNA with a polymerase enzyme in the presence of deoxynucleoside triphosphates under conditions which cause primer extension to take place, thereby producing double-stranded DNA (product DNA); (f) cloning product DNA into expression vectors for expression of a repertoire of proteins each comprising at least part of an immunoglobulin variable domain.
Claim 6
Dependent Claim
134 words
A method according to claim 4 wherein, after step (e) the following steps are performed before step (f): (i) causing the two strands of the product DNA to be separated; (ii) annealing to the separated strands a forward and a back oligonucleotide primer, the forward primer being specific for a sequence at or adjacent the 3' end of the sense strand of each of the target sequences, the back primer being specific for a sequence at or adjacent the 3' end of the antisense strand of each of the target sequences, under conditions which allow the primers to hybridize specifically to the nucleic acid; (iii) treating the annealed sample with a DNA polymerase enzyme in the presence of deoxynucleoside triphosphates under conditions which cause primer extension to take place, thereby producing double-stranded nucleic acid.
Claim 7
Independent Claim
52 words
A method according to any one of claims 1 to 5 wherein the back primer is specific for a sequence at or adjacent the 3' end of the antisense strand of the sequences which are contained in the target sequences and which each encode at least part of an immunoglobulin variable domain.
Claim 8
Independent Claim
23 words
A method according to any one of claims 1 to 6 wherein the sample repertoire of double-stranded nucleic acid is derived from lymphocytes.
Claim 9
Dependent Claim
23 words
A method according to claim 7 wherein the lymphocytes are derived from an animal or human mounting an immune response to an antigen.
Claim 10
Dependent Claim
18 words
A method according to claim 7 wherein the lymphocytes are derived from a patient with an auto-immune disease.
Claim 11
Dependent Claim
21 words
A method according to claim 1 wherein the sample repertoire of nucleic acid is derived from rearranged immunoglobulin variable region genes.
Claim 12
Dependent Claim
16 words
A method according to claim 1 wherein the sample repertoire of nucleic acid is genomic DNA.
Claim 13
Dependent Claim
21 words
A method according to claim 1 wherein the sample repertoire of nucleic acid is derived from unrearranged immunoglobulin variable region genes.
Claim 14
Independent Claim
27 words
A method according to any one of claims 1 to 12 wherein the target sequence contains a sequence encoding a variable domain from an immunoglobulin heavy chain.
Claim 15
Dependent Claim
29 words
A method according to claim 13 wherein the product DNA is inserted into an expression vector for expression of single domain ligands selectable by their binding affinity for antigen.
Claim 16
Independent Claim
34 words
A method according to any one of claims 1 to 13 wherein product DNA is inserted into an expression vector for expression of antibodies or antibody fragments selectable by their binding affinity for antigen.
Claim 17
Independent Claim
24 words
A method according to any one of claims 1 to 13 wherein the product DNA is inserted into an expression vector for expression alone.
Claim 18
Independent Claim
29 words
The method of any one of claims 1 to 13 wherein the product DNA is inserted into an expression vector for expression in combination with a complementary variable domain.
Claim 19
Independent Claim
32 words
A method according to any one of claims 1 to 13 wherein the product DNA is inserted into an expression vector already containing sequences encoding one or more constant domains for expression.
Claim 20
Independent Claim
26 words
A method according to any one of claims 1 to 13 wherein the product DNA is inserted into an expression vector for expression as fusion proteins.
Claim 21
Independent Claim
26 words
A method according to any one of claims 1 to 13 wherein the product DNA is inserted into an expression vector for expression with peptide tags.
Claim 22
Independent Claim
52 words
A method according to any one of claims 1 to 13 wherein product DNA containing sequences encoding at least immunoglobulin heavy chain variable domains and product DNA containing sequences encoding at least immunoglobulin light chain variable domains are inserted into expression vectors for expression of a combinatorial repertoire of complementary variable domains.
Claim 23
Dependent Claim
27 words
A method according to claim 21 wherein the product DNA is inserted into an expression vector already containing sequences encoding one or more constant domains for expression.
Claim 24
Dependent Claim
19 words
A method according to claim 21 wherein product DNA is inserted into expression vectors for expression as fusion proteins.
Claim 25
Dependent Claim
21 words
A method according to claim 21 wherein the product DNA is inserted into an expression vector for expression with peptide tags.
Claim 26
Independent Claim
22 words
A method according to any one of claims 1 to 24 wherein the forward and back primers are provided as single oligonucleotides.
Claim 27
Independent Claim
22 words
A method according to any one of claims 1 to 24 wherein the forward primers are supplied as a mixture of oligonucleotides.
Claim 28
Independent Claim
22 words
A method according to any one of claims 1 to 24 wherein the back primers are supplied as a mixture of oligonucleotides.
Claim 29
Independent Claim
23 words
A method according to any one of claims 1 to 27 wherein each primer includes a sequence encoding a restriction enzyme recognition site.
Claim 30
Dependent Claim
24 words
A method according to claim 28 wherein the restriction enzyme recognition site is located in the sequence which is annealed to the nucleic acid.
Claim 31
Dependent Claim
25 words
A method according to claim 1 wherein the back primer is a general primer useful for cloning a desired antibody specificity from a specific species.
Claim 32
Dependent Claim
34 words
A method according to claim 1 wherein the back primer is a mixture of primers having a variety of sequences designed to be complementary to the various families of VH, Vk or V sequences.
Claim 33
Dependent Claim
20 words
An expression library comprising a repertoire of nucleic acid sequences each encoding at least part of an immunoglobulin variable domain.
Claim 34
Dependent Claim
20 words
An expression library comprising a repertoire of nucleic acid sequences each encoding at least part of an immunoglobulin variable domain.
References Cited (Backward Citations)
WO1988001649A1
Cited
WO1988006630A1
Cited
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