CA2198899A1 - Methods for producing antibody libraries using universal or randomized immunoglobulin light chains - Google Patents

Methods for producing antibody libraries using universal or randomized immunoglobulin light chains

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Publication number
CA2198899A1
CA2198899A1 CA002198899A CA2198899A CA2198899A1 CA 2198899 A1 CA2198899 A1 CA 2198899A1 CA 002198899 A CA002198899 A CA 002198899A CA 2198899 A CA2198899 A CA 2198899A CA 2198899 A1 CA2198899 A1 CA 2198899A1
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CA
Canada
Prior art keywords
oligonucleotide
immunoglobulin
light chain
gene
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CA002198899A
Other languages
French (fr)
Other versions
CA2198899C (en
Inventor
Carlos F. Barbas
Dennis R. Burton
Richard A. Lerner
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Scripps Research Institute
Original Assignee
Carlos F. Barbas
Dennis R. Burton
Richard A. Lerner
The Scripps Research Institute
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Publication of CA2198899A1 publication Critical patent/CA2198899A1/en
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Publication of CA2198899C publication Critical patent/CA2198899C/en
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Abstract

The present invention describes methods for producing antibody libraries, and particularly for increasing antibody library diversity by inducing mutagenesis within the CDR regions of immunoglobulun heavy or light chains that are displayed on the surface of filamentous phage particles comprising the library. The invention also describes oligonucleotides useful for increasing the library diiversity, and universal light chains useful in the library production methods.

Claims (37)

1. An oligonucleotide useful as a primer for inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene, said oligonucleotide having 3' and 5' termini and comprising:
a) a nucleotide sequence at said 3' terminus capable of hybridizing to a first framework region of an immunoglobulin gene;
b) a nucleotide sequence at said 5' terminus capable of hybridizing to a second framework region of an immunoglobulin gene; and c) a nucleotide sequence between said 3' and 5' termini according to the formula:
[NNK]n, wherein N is independently any nucleotide, K is G or T, n is 3 to about 24, said 3' and 5' terminal nucleotide sequences having a length of about 6 to 50 nucleotides, or an oligonucleotide having a sequence complementary thereto.
2. The oligonucleotide of claim 1 wherein said 5' terminus has the nucleotide sequence 5' -TATACTGTCAGCAGTAT-3' (SEQ ID NO
26) or 5' -GATTTTGCAGTGTATTACTGTCAGQGTAT-3' (SEQ ID NO 27), or an oligonucleotide having a sequence complementary thereto.
3. The oligonucleotide of claim 1 wherein said 3' terminus has the nucleotide sequence 5' -ACTTTCGGCGGAGGGACQAGGTGGAG-3' (SEQ ID NO 28) or 5' -ACTTTCGGCGGAGGGACC-3' (SEQ ID NO 29), or an oligonucleotide having a sequence complementary thereto.
4. The oligonucleotide of claim 1 wherein n is 4, 5, 6, 10 or 16.
5. The oligonucleotide of claim 1 wherein said immunoglobulin is human.
6. The oligonucleotide of claim 1 wherein said CDR is CDR3.
7. The oligonucleotide of claim 1 according to the formula: 5' -GATTTTGCAGTGTATTACTGT [NNK] 10TTCGGCGGAGGGACCAAGGTGGAG-3' (SEQ ID NO 12), or an oligonucleotide having a sequence complementary thereto.
8. An oligonucleotide useful as a primer for inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene, said oligonucleotide having 3' and 5' termini and comprising:
a) a nucleotide sequence at said 3' terminus capable of hybridizing to a first framework region of an immunoglobulin gene;
b) a nucleotide sequence at said 5' terminus capable of hybridizing to a second framework region of an immunoglobulin gene; and c) a nucleotide sequence between said 3' and 5' termini according to the formula:
[MNN]n, wherein N is independently any nucleotide, M is A or C, n is 3 to about 24, said 3' and 5' terminal nucleotide sequences having a length of about 6 to 50 nucleotides, or an oligonucleotide having a sequence complementary thereto.
9 . The oligonucleotide of claim 8 wherein said 5' terminus has the nucleotide sequence 5' -GTTCCACCTTGGTCCCTTGGCCGAA-3' (SEQ
ID NO 30), or an oligonucleotide having a sequence complementary thereto.
10. The oligonucleotide of claim 8 wherein said 3' terminus has the nucleotide sequence 5' -ACAGTAGTACACTGCAAAATC-3' (SEQ ID
NO 31), or an oligonucleotide having a sequence complementary thereto.
11. The oligonucleotide of claim 8 wherein n is 8, 10 or 16.
12. The oligonucleotide of claim 8 wherein said immunoglobulin is human.
13. The oligonucleotide of claim 8 wherein said CDR is CDR3.
14. A method for producing an antibody combining site in a polypeptide comprising inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene which comprises amplifying a CDR portion of the immunoglobulin gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide, said oligonucleotide having 3' and 5' termini and comprising:
a) a nucleotide sequence at said 3' terminus capable of hybridizing to a first framework region of an immunoglobulin gene;
b) a nucleotide sequence at said 5' terminus capable of hybridizing to a second framework region of an immunoglobulin gene; and c) a nucleotide sequence between said 3' and 5' termini according to the formula:
[NNK]n, wherein N is independently any nucleotide, K is G or T, and n is 3 to about 24, said 3' and 5' terminal nucleotide sequences having a length of about 6 to 50 nucleotides, or an oligonucleotide having a sequence complementary thereto.
15. The method of claim 14 wherein said 5' terminus has the nucleotide sequence 5'-TATACTGTCAGCAGTAT-3' (SEQ ID NO 26) or 5'-GATTTTGCAGTGTATTACTGTCAGCAGTAT-3' (SEQ ID NO 27), or an oligonucleotide having a sequence complementary thereto.
16 . The method of claim 14 wherein said 3' terminus has the nucleotide sequence 5' -ACTTTCGGCGGAGGGACCAAGGTGGAG-3' (SEQ ID NO
28) or 5' -ACTTTCGGCGGAGGGACC-3' (SEQ ID NO 29), or an oligonucleotide having a sequence complementary thereto.
17. The method of claim 14 wherein n is 4, 5, 6, 10 or 16.
18. The method of claim 14 wherein said immunoglobulin is human.
19. The method of claim 14 wherein said CDR is CDR3.
20. The method of claim 14 according to the formula:
5'-GATTTTGCAGTGTATTACTGT [NNK]10TTCGGCGGAGGGCCAAGGTGGAG-3' (SEQ ID NO
12), or an oligonucleotide having a sequence complementary thereto.
21. The method of claim 14 wherein said immunoglobulin light chain gene includes a sequence having the sequence characteristics of the light chain shown in SEQ ID NO 2 or in SEQ
ID NO 62.
22. The method of claim 14 wherein said immunoglobulin light chain gene has the sequence characteristics of the light chain gene in ATCC Accession No. 75408.
23. The method of claim 14 that further comprises the steps of:
a) isolating the amplified CDR to form a library of mutagenized immunoglobulin light chain genes;
b) expressing the isolated library of mutagenized light chain genes in combination with one or more heavy chain genes to form a combinatorial antibody library of expressed heavy and light chain genes; and c) selecting species of said combinatorial antibody library for the ability to bind a preselected antigen.
24. The method of claim 23 wherein said one or more immunoglobulin heavy chain genes is a library of heavy chain genes.
25. A method for producing an antibody combining site in a polypeptide comprising inducing mutagenesis in a complementarity determining region (CDR) of an immunoglobulin light chain gene which comprises amplifying a CDR portion of the immunoglobulin gene by polymerase chain reaction (PCR) using a PCR primer oligonucleotide, said oligonucleotide having 3' and 5' termini and comprising:
a) a nucleotide sequence at said 3' terminus capable of hybridizing to a first framework region of an immunoglobulin gene;
b) a nucleotide sequence at said 5' terminus capable of hybridizing to a second framework region of an immunoglobulin gene; and c) a nucleotide sequence between said 3' and 5' termini according to the formula:
[MNN]n, wherein N is independently any nucleotide, M is A or C, n is 3 to about 24, said 3' and 5' terminal nucleotide sequences having a length of about 6 to 50 nucleotides, or an oligonucleotide having a sequence complementary thereto.
26. The method of claim 25 wherein said 5' terminus has the nucleotide sequence 5'-GTTCCACCTTGGTCCCTTGGCCGAA-3' (SEQ ID NO
30), or an oligonucleotide having a sequence complementary thereto.
27. The method of claim 25 wherein said 3' terminus has the nucleotide sequence 5'-ACAGTAGTACACTGCAAAATC-3' (SEQ ID NO 31), or an oligonucleotide having a sequence complementary thereto.
28. The method of claim 25 wherein n is 8, 10 or 16.
29. The method of claim 25 wherein said immunoglobulin is human.
30. The method of claim 25 wherein said CDR is CDR3.
31. The method of claim 25 wherein said immunoglobulin light chain gene includes a sequence having the sequence characteristics of the light chain shown in SEQ ID NO 2 or in SEQ
ID NO 62.
32. The method of claim 25 wherein said immunoglobulin light chain gene has the sequence characteristics of the light chain gene in ATCC Accession No. 75408.
33. The method of claim 25 that further comprises the steps of:
a) isolating the amplified CDR to form a library of mutagenized immunoglobulin light chain genes;
b) expressing the isolated library of mutagenized light chain genes in combination with one or more heavy chain genes to form a combinatorial antibody library of expressed heavy and light chain genes; and c) selecting species of said combinatorial antibody library for the ability to bind a preselected antigen.
34. The method of claim 33 wherein said one or more immunoglobulin heavy chain genes is a library of heavy chain genes.
35. A method for producing a heterodimeric immunoglobulin molecule having immunoglobulin variable domain heavy and light chain polypeptides comprising the steps of:
a) combining an immunoglobulin variable domain light chain gene that includes a sequence having the sequence characteristics of the light chain shown in SEQ ID NO 2 or 62 with one or more immunoglobulin variable domain heavy chain genes to form a combinatorial immunoglobulin heavy and light chain gene library, said combining comprising operatively linking said light chain gene with one of said heavy chain genes in a vector capable of co-expression of said heavy and light chain genes;
b) expressing the combinatorial gene library to form a combinatorial antibody library of expressed heavy and light chain polypeptides; and c) selecting species of said combinatorial antibody library for the ability to bind a preselected antigen.
36. The method of claim 35 wherein said immunoglobulin light chain gene has the sequence characteristics of the light chain gene in ATCC Accession No. 75408.
37. The method of claim 35 wherein said one or more immunoglobulin heavy chain genes is a library of heavy chain genes.
CA2198899A 1994-09-02 1995-09-01 Methods for producing antibody libraries using universal or randomized immunoglobulin light chains Expired - Lifetime CA2198899C (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/300,386 1994-09-02
US08/300,386 US5667988A (en) 1992-01-27 1994-09-02 Methods for producing antibody libraries using universal or randomized immunoglobulin light chains
PCT/US1995/011235 WO1996007754A1 (en) 1994-09-02 1995-09-01 Methods for producing antibody libraries using universal or randomized immunoglobulin light chains

Publications (2)

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CA2198899A1 true CA2198899A1 (en) 1996-03-14
CA2198899C CA2198899C (en) 2010-02-23

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US (2) US5667988A (en)
EP (1) EP0779933B1 (en)
JP (1) JPH10504970A (en)
AT (1) ATE236992T1 (en)
AU (1) AU706343B2 (en)
CA (1) CA2198899C (en)
DE (1) DE69530305T2 (en)
DK (1) DK0779933T3 (en)
ES (1) ES2196077T3 (en)
PT (1) PT779933E (en)
WO (1) WO1996007754A1 (en)

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