Production of anti-self antibodies from antibody segment repertoires and displayed on phage

• US 6,555,313 B1
• Filed: 05/16/2000
• Issued: 04/29/2003
• Est. Priority Date: 12/02/1991
• Status: Expired due to Fees

• Current Assignee: MedImmune Ltd. (Astrazeneca PLC)
• Original Assignee: Cambridge Antibody Technology Ltd (Astrazeneca PLC), Medical Research Council (Government of United Kingdom)
• Inventors: Hoogenboom, Hendricus Renerus Jacobus Mattheus, Marks, James David, Griffiths, Andrew David, Winter, Gregory Paul, McCafferty, John, Grigg, Geoffrey Walter
• Primary Examiner(s): Ponnaluri, Padmashri
• Application Number:
  US09/572,392
• Time in Patent Office: 1,078 Days
• Field of Search: 435/5, 435/6, 435/69.1, 435/69.7, 435/91.1, 435/235.1, 435/320.1, 435/DIG.1, 435/DIG.2, 435/DIG.4, 435/DIG.34, 435/DIG.37, 435/DIG.47, 435/DIG.14, 435/DIG.15, 530/387.1, 530/387.3, 530/867, 536/23.1, 536/23.4, 536/23.53
• US Class Current: 506/9
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: B01J 2219/00718 Type of compounds synthesised
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/241 Tumor Necrosis Factors
• CPC Class Codes: C07K 16/26 against hormones ; against hormone releasing or inhibiting factors
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 16/2812 against CD4
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/2866 against receptors for cytokines, lymphokines, interferons
• CPC Class Codes: C07K 16/3007 Carcino-embryonic Antigens
• CPC Class Codes: C07K 16/3092 against tumour-associated mucins
• CPC Class Codes: C07K 16/34 against blood group antigens
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 16/4208 against an idiotypic determinant on Ig
• CPC Class Codes: C07K 16/4241 against anti-human or anti-animal Ig
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: C07K 16/462 Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/24 : containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/55 : Fab or Fab'
• CPC Class Codes: C07K 2317/622 : Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• CPC Class Codes: C07K 2319/02 : containing a signal sequence
• CPC Class Codes: C07K 2319/41 : containing a Myc-tag
• CPC Class Codes: C07K 2319/735 : containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
• CPC Class Codes: C12N 15/1037 : Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C40B 40/02 : 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
• CPC Class Codes: Y10S 530/867 : involving immunoglobulin or antibody produced via recombinant dna technology

Abstract

Methods are disclosed for the production of human self-antibodies and antibody fragments, which bind human antigens. Methods comprise providing a library of replicable genetic display packages (rgdps), such as filamentous phage, each rgdp displaying at its surface a member of a specific binding pair which is an antibody or antibody fragment, and each rgdp containing nucleic acid sequence derived from a species of mammal. The nucleic acid sequence in each rgdp encodes a polypeptide chain which is a component part of the sbp member displayed at the surface of that rgdp. Human antibodies or antibody fragments are selected by binding with human antigens. The displayed antibody fragments may be scFv, Fd, Fab or any other fragment which has the capability of binding to and is a human antigen. Nucleic acid libraries used may be derived from V-gene sequences of unimmunised humans. Part or all of the nucleic acid may be derived from oligonucleotide synthesis.

Claims

1. A method of obtaining a member of a specific binding pair, the specific binding pair member being an antibody or antibody fragment and having an antigen binding site comprising an antibody light chain variable region and an antibody heavy chain variable region, the antigen binding site having binding specificity for an antigen which is a human self antigen, the method comprising:

(a) providing a library of filamentous bacteriophage, each filamentous bacteriophage displaying at its surface a specific binding pair member, and each filamentous bacteriophage containing nucleic acid with sequence derived from human species unimmunized with said human self antigen and encoding a polypeptide chain which is a component part of the specific binding pair member displayed at the surface of that filamentous bacteriophage; and

(b) selecting, by binding with said human self antigen, one or more specific binding pair members with binding specificity for said human self antigen.

2. A method according to claim 1 wherein said providing a library of filamentous bacteriophage comprises:

combining (i) a first polypeptide chain component part of a specific binding member fused to a component of a filamentous bacteriophage which thereby displays said first polypeptide chain component part of population thereof at the surface of filamentous bacteriophage on expression in a recombinant host cell organism, or a population of such a first polypeptide chain component part fused to said component of a filamentous bacteriophage, with (ii) a second polypeptide chain component part of a specific binding pair member or a population of such a second polypeptide chain component part, to form a library of specific binding pair members displayed at the surface of filamentous bacteriophage;

at least one of said first or second polypeptide chain component part or populations thereof being encoded by nucleic acid which is packaged using said component of a filamentous bacteriophage.

3. A method according to claim 1 wherein said providing a library of filamentous bacteriophage comprises:

combining (i) nucleic acid which encodes a first polypeptide chain component of a specific binding pair member fused to a component of a filamentous bacteriophage or a population of such a first polypeptide chain component part fused to a component of a filamentous bacteriophage, with (ii) nucleic acid encoding a second polypeptide chain component part of a specific binding pair member of a population thereof, to form a library of nucleic acid, nucleic acid of said library being packaged using said component of a filamentous bacteriophage;

expressing in a recombinant host organism said first polypeptide chain component part fused to a component of a filamentous bacteriophage or population thereof and said second polypeptide chain component part of a specific binding pair member or a population thereof, to produce a library of filamentous bacteriophage each displaying at its surface a specific binding pair member and containing nucleic acid encoding a first and a second polypeptide chain component part of the specific binding pair member displayed at its surface.

4. A method according to claim 1 wherein each said specific binding pair member displayed at the surface of a filamentous bacteriophage is an antibody fragment comprising a V_H domain and a V_L domain.

5. A method according to claim 2 wherein both said first and second polypeptide chain component parts or populations thereof are expressed from nucleic acid capable of being packaged using said component of a filamentous bacteriophage.

6. A method according to claim 1 wherein each said specific binding pair member displayed at the surface of a filamentous bacteriophage is an scFv antibody fragment.

7. A method according to claim 2 wherein said second polypeptide chain component part or population thereof is encoded by nucleic acid separate from nucleic acid encoding said first polypeptide chain component part or population.

8. A method according to claim 3 wherein said second polypeptide chain component part or population thereof is encoded by a nucleic acid separate from nucleic acid encoding said first polypeptide chain component part or population.

9. A method according to claim 1 wherein each said specific binding pair member displayed at the surface of a filamentous bacteriophage is an Fab antibody fragment.

10. A method according to claim 1 wherein the nucleic acid is derived from rearranged V genes of an unimmunised human.

11. A method according to claim 1 wherein the nucleic acid is derived from a library prepared by artificial or synthetic recombination of V-gene sequences.

12. The method of claim 1 wherein the nucleic acid is synthetic.

13. A method according to claim 11 wherein said V gene sequences are germ line V-gene sequences.

14. A method according to claim 1 wherein specific binding pair members selected in (b) are displayed at the surface of filamentous bacteriophage are selected or screened to provide an individual filamentous bacteriophage displaying a specific binding pair member or a mixed population of said filamentous bacteriophage, with each filamentous bacteriophage containing nucleic acid encoding the specific binding pair member or a polypeptide chain thereof which is displayed at its surface.

15. Method according to claim 1 wherein nucleic acid which encodes a selected or screened specific binding pair member and which is derived from a filamentous bacteriophage which displays at its surface a selected or screened specific binding pair member is used to express a specific binding pair member or a fragment thereof with binding specificity for said human self antigen or derivative of said specific binding pair member in a recombinant host organism.

16. A method according to claim 15 wherein nucleic acid from one or more filamentous bacteriophage is taken and used to provide encoding nucleic acid in a further method to obtain an individual specific binding pair member or a mixed population of specific binding pair members, or encoding nucleic acid therefor.

17. A method according to claim 16 wherein the expression end product is modified to produce a derivative thereof.

18. The method of claim 15 wherein said derivative of said specific binding pair member is selected from the group consisting of specific binding pair members having additions, specific binding pair members having deletions, specific binding pair members having substitutions, and specific binding pair members having insertions of amino acids.

19. The method of claim 15 wherein said derivative comprises the addition of a polypeptide to said specific binding pair member, wherein the polypeptide is selected from the group consisting of Fc fragments, enzymes and fluoresceins.

20. The method of claim 18 wherein said derivative comprises the addition of a polypeptide to said specific binding pair member, wherein the polypeptide is selected from the group consisting of Fc fragments, enzymes and fluoresceins.

21. A method according to claim 1 wherein said nucleic acid is derived from mRNA of peripheral blood lymphocytes of an individual who is without an autoimmune disease wherein antibodies produced are specific for said self antigen.

22. The method according to claim 21 wherein said peripheral blood lymphocytes secrete IgM antibody.