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US5840479A
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Preparation and use of gene banks of synthetic human antibodies ("synthetic human-antibody libraries")
Assignee: Siemens Healthcare Diagnostics Products GmbH
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Filing: 1994-12-02
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Granted: 1998-11-24
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Expires: 2014-12-02
Abstract
Abstract The invention relates to the preparation and use of gene banks of synthetic human antibodies (huAb) or parts of antibodies which contain the antigen-binding domain. Starting from a huAb framework in a suitable vector, the hypervariable regions of the antibody cDNA are formed by almost "randomly" combined oligonucleotides. Relatively conserved amino acids in the hypervariable regions have here been taken account of in the choice of appropriate nucleotides during the oligonucleotide synthesis and the ratio of the nucleotides used is likewise chosen such that a nonsense codon is to be expected at most in every 89th position. Expression of this synthetic huAb cDNA in microbial expression systems, e.g. in E. coli in the vector pFMT which is described below, thus makes a synthetic huAb library with a comprehensive repertoire for screening using selected antigens available in vitro.
Inventors
- 👤 Melvyn Little
- 👤 Frank Berthold Breitling
- 👤 Thomas Seehaus
- 👤 Stefan Dubel
- 👤 Iris Klewinghaus
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 09:26
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1034; C12N15/1093; C12N15/11; C07; C07K; C07K16/00; C07K16/44; C07K7/00; C07K7/04; C07K7/06; C12N15/62; C12N15/625; C12N15/63; C12N15/65; C12N15/70; C12N9/00; C12N9/88; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/033
Claims Analysis
Total Claims: 57
Claim 1
Independent Claim
100 words
A synthetic human antibody -DNA library obtained by: 1) synthesizing almost random sequences for the antibody hypervariable regions, wherein (a) relatively conserved amino acids in the hypervariable regions have been taken into account in the choice of appropriate nucleotides during the oligonucleotide synthesis and (b) the ratio of the nucleotides used in chosen such that a nonsense codon is to be expected at most in every 89th position; 2) inserting said almost random sequences for the hypervariable regions into a human antibody framework; and 3) incorporating said almost random sequences combined with said human antibody framework into an expression vector.
Claim 2
Dependent Claim
100 words
A synthetic human antibody -DNA library obtained by: 1) synthesizing almost random sequences for the antibody hypervariable regions, wherein (a) relatively conserved amino acids in the hypervariable regions have been taken into account in the choice of appropriate nucleotides during the oligonucleotide synthesis and (b) the ratio of the nucleotides used in chosen such that a nonsense codon is to be expected at most in every 89th position; 2) inserting said almost random sequences for the hypervariable regions into a human antibody framework; and 3) incorporating said almost random sequences combined with said human antibody framework into an expression vector.
Claim 3
Dependent Claim
29 words
The synthetic human antibody-DNA library as claimed in claim 1, wherein the synthesized almost random sequences for the hypervariable regions are amplified before the incorporation in the expression vector.
Claim 4
Dependent Claim
26 words
The synthetic human antibody-DNA library as claimed in claim 1, wherein said almost random sequences are incorporated into antibody variable regions derived from HuVhlys or HuVlls.
Claim 5
Dependent Claim
26 words
The synthetic human antibody-DNA library as claimed in claim 2, wherein said almost random sequences are incorporated into antibody variable regions derived from HuVhlys or HuVlls.
Claim 6
Dependent Claim
18 words
The synthetic human antibody-DNA library of claim 1 wherein the DNA is transfected into a microbial expression system.
Claim 7
Dependent Claim
18 words
The synthetic human antibody-DNA library of claim 2 wherein the DNA is transfected into a microbial expression system.
Claim 8
Dependent Claim
18 words
The synthetic human antibody-DNA library of claim 3 wherein the DNA is transfected into a microbial expression system.
Claim 9
Dependent Claim
18 words
The synthetic human antibody-DNA library of claim 4 wherein the DNA is transfected into a microbial expression system.
Claim 10
Dependent Claim
15 words
The synthetic human antibody-DNA library of claim 5 wherein the microbial expression system is bacterial.
Claim 11
Dependent Claim
15 words
The synthetic human antibody-DNA library of claim 6 wherein the microbial expression system is bacterial.
Claim 12
Dependent Claim
15 words
The synthetic human antibody-DNA library of claim 7 wherein the microbial expression system is bacterial.
Claim 13
Dependent Claim
15 words
The synthetic human antibody-DNA library of claim 8 wherein the microbial expression system is bacterial.
Claim 14
Dependent Claim
14 words
The synthetic human antibody-DNA library of claim 9 wherein the bacteria are E. coli.
Claim 15
Dependent Claim
14 words
The synthetic human antibody-DNA library of claim 10 wherein the bacteria are E. coli.
Claim 16
Dependent Claim
14 words
The synthetic human antibody-DNA library of claim 11 wherein the bacteria are E coli.
Claim 17
Dependent Claim
14 words
The synthetic human antibody-DNA library of claim 12 wherein the bacteria are E. coli.
Claim 18
Dependent Claim
103 words
A process for preparing a synthetic human antibody-DNA library, which comprises: 1) synthesizing almost random sequences for the antibody hypervariable regions, wherein (a) relatively conserved amino acids in the hypervariable regions have been taken into account in the choice of appropriate nucleotides during the oligonucleotide synthesis and (b) the ratio of the nucleotides used in chosen such that a nonsense codon is to be expected at most in every 89th position; 2) inserting said almost random sequences for the hypervariable regions into a human antibody framework; and 3) incorporating said almost random sequences combined with said human antibody framework into an expression vector.
Claim 19
Dependent Claim
103 words
A process for preparing a synthetic human antibody-DNA library, which comprises: 1) synthesizing almost random sequences for the antibody hypervariable regions, wherein (a) relatively conserved amino acids in the hypervariable regions have been taken into account in the choice of appropriate nucleotides during the oligonucleotide synthesis and (b) the ratio of the nucleotides used in chosen such that a nonsense codon is to be expected at most in every 89th position; 2) inserting said almost random sequences for the hypervariable regions into a human antibody framework; and 3) incorporating said almost random sequences combined with said human antibody framework into an expression vector.
Claim 20
Dependent Claim
27 words
The process for preparing a synthetic human antibody-DNA library as claimed in claim 17, wherein the synthesized random sequences are amplified before incorporation in an expression vector.
Claim 21
Dependent Claim
30 words
The process for preparing a synthetic human antibody-DNA library as claimed in claim 17, wherein the almost random sequences are incorporated into antibody variable regions derived from HuVhlys or HuVlls.
Claim 22
Dependent Claim
30 words
The process for preparing a synthetic human antibody-DNA library as claimed in claim 18, wherein the almost random sequences are incorporated into antibody variable regions derived from HuVhlys or HuVlls.
Claim 23
Dependent Claim
31 words
The process as claimed in claim 17, wherein said expression vector further comprises DNA coding for a marker peptide and the desired clones are identified using antibodies against the marker peptide.
Claim 24
Dependent Claim
26 words
The process as claimed in claim 21, wherein said marker peptide is the TAG sequence and the desired clones are identified using the antibody YL 1/2.
Claim 25
Dependent Claim
31 words
The process as claimed in claim 18, wherein said expression vector further comprises DNA coding for a marker peptide and the desired clones are identified using antibodies against the marker peptide.
Claim 26
Dependent Claim
25 words
The process as claimed in claim 23, wherein said marker peptide is the TAG sequence and the desired clones are identified using the antibody YL1/2.
Claim 27
Dependent Claim
20 words
The process for preparing a synthetic human antibody-DNA library of claim 17 wherein the variable regions are incorporated into pFMT.
Claim 28
Dependent Claim
20 words
The process for preparing a synthetic human antibody-DNA library of claim 18 wherein the variable regions are incorporated into pFMT.
Claim 29
Dependent Claim
23 words
The process for preparing a synthetic human antibody-DNA library of claim 17 wherein the expression vector is transfected into a microbial expression system.
Claim 30
Dependent Claim
12 words
The process of claim 27 wherein the microbial expression system is bacterial.
Claim 31
Dependent Claim
11 words
The process of claim 28 wherein the bacteria are E. coli.
Claim 32
Dependent Claim
23 words
The process for preparing a synthetic human antibody-DNA library of claim 18 wherein the expression vector is transfected into a microbial expression system.
Claim 33
Dependent Claim
12 words
The process of claim 30 wherein the microbial expression system is bacterial.
Claim 34
Dependent Claim
11 words
The process of claim 31 wherein the bacteria are E. coli.
Claim 35
Dependent Claim
135 words
A process for obtaining clones secreting specific human antibodies comprising the steps of: a) screening synthetic human antibody-DNA libraries using specific antigens, and b) isolating said clones which secrete the desired specific human antibodies, wherein said specific human antibody-DNA library is obtained by: 1) synthesizing almost random sequences for the antibody hypervariable regions, wherein (a) relatively conserved amino acids in the hypervariable regions have been taken into account in the choice of appropriate nucleotides during the oligonucleotide synthesis and (b) the ratio of the nucleotides used in chosen such that a nonsense codon is to be expected at most in every 89th position; 2) inserting said almost random sequences for the hypervariable regions into a human antibody framework; and 3) incorporating said almost random sequences combined with said human antibody framework into an expression vector.
Claim 36
Dependent Claim
135 words
A process for obtaining clones secreting specific human antibodies comprising the steps of: a) screening synthetic human antibody-DNA libraries using specific antigens, and b) isolating said clones which secrete the desired specific human antibodies, wherein said specific human antibody-DNA library is obtained by: 1) synthesizing almost random sequences for the antibody hypervariable regions, wherein (a) relatively conserved amino acids in the hypervariable regions have been taken into account in the choice of appropriate nucleotides during the oligonucleotide synthesis and (b) the ratio of the nucleotides used in chosen such that a nonsense codon is to be expected at most in every 89th position; 2) inserting said almost random sequences for the hypervariable regions into a human antibody framework; and 3) incorporating said almost random sequences combined with said human antibody framework into an expression vector.
Claim 37
Dependent Claim
44 words
The process for obtaining clones secreting specific human antibodies as claimed in claim 33, wherein the process of obtaining the specific human antibody-DNA library further comprises the step of amplifying the almost random sequences of the hypervariable regions before incorporation into the expression vector.
Claim 38
Dependent Claim
30 words
The process for obtaining clones secreting specific human antibodies as claimed in claim 33, wherein the almost random sequences are incorporated into antibody variable regions derived from HuVhlys or HuVlls.
Claim 39
Dependent Claim
30 words
The process for obtaining clones secreting specific human antibodies as claimed in claim 34, wherein the almost random sequences are incorporated into antibody variable regions derived from HuVhlys or HuVlls.
Claim 40
Dependent Claim
31 words
The process as claimed in claim 33, wherein said expression vector further comprises DNA coding for a marker peptide and the desired clones are identified using antibodies against the marker peptide.
Claim 41
Dependent Claim
26 words
The process as claimed in claim 37, wherein said marker peptide is the TAG sequence and the desired clones are identified using the antibody YL 1/2.
Claim 42
Dependent Claim
31 words
The process as claimed in claim 34, wherein said expression vector further comprises DNA coding for a marker peptide and the desired clones are identified using antibodies against the marker peptide.
Claim 43
Dependent Claim
26 words
The process as claimed in claim 39, wherein said marker peptide is the TAG sequence and the desired clones are identified using the antibody YL 1/2.
Claim 44
Dependent Claim
20 words
The process for obtaining clones secreting specific human antibodies of claim 33 wherein the variable regions are incorporated into pFMT.
Claim 45
Dependent Claim
20 words
The process for obtaining clones secreting specific human antibodies of claim 34 wherein the variable regions are incorporated into pFMT.
Claim 46
Dependent Claim
23 words
The process for obtaining clones secreting specific human antibodies of claim 33 wherein the expression vector is transfected into a microbial expression system.
Claim 47
Dependent Claim
12 words
The process of claim 43 wherein the microbial expression system is bacterial.
Claim 48
Dependent Claim
11 words
The process of claim 44 wherein the bacteria are E. coli.
Claim 49
Dependent Claim
23 words
The process for obtaining clones secreting specific human antibodies of claim 34 wherein the expression vector is transfected into a microbial expression system.
Claim 50
Dependent Claim
12 words
The process of claim 46 wherein the microbial expression system is bacterial.
Claim 51
Dependent Claim
11 words
The process of claim 47 wherein the bacteria are E. coli.
Claim 52
Dependent Claim
22 words
The synthetic human antibody-DNA library as claimed in claim 1 wherein the expression vector further comprises DNA coding for a marker peptide.
Claim 53
Dependent Claim
18 words
The synthetic human antibody-DNA library as claimed in claim 49 wherein the marker peptide is the TAG sequence.
Claim 54
Dependent Claim
22 words
The synthetic human antibody-DNA library as claimed in claim 2 wherein the expression vector further comprises DNA coding for a marker peptide.
Claim 55
Dependent Claim
18 words
The synthetic human antibody-DNA library as claimed in claim 51 wherein the marker peptide is the TAG sequence.
Claim 56
Dependent Claim
18 words
The synthetic human antibody-DNA library as claimed in claim 2 wherein the variable regions are incorporated into pFMT.
Claim 57
Dependent Claim
18 words
The synthetic human antibody-DNA library as claimed in claim 53 wherein the variable regions are incorporated into pFMT.
References Cited (Backward Citations)
WO1988006630A1
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2001-11-23 | 2002-05-25 | 2002-11-24 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2005-11-23 | 2006-05-25 | 2006-11-24 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2009-11-23 | 2010-05-25 | 2010-11-24 | $7700.00 | Window Closed / Lapsed |