Methods for producing members of specific binding pairs

• US 6,492,160 B1
• Filed: 06/25/1998
• Issued: 12/10/2002
• Est. Priority Date: 05/15/1991
• Status: Expired due to Fees

• Current Assignee: Cambridge Antibody Technology Ltd (Astrazeneca PLC)
• Original Assignee: Cambridge Antibody Technology Ltd (Astrazeneca PLC), Medical Research Council (Government of United Kingdom)
• Inventors: Nissim, Ahuva, Smith, Andrew John Hammond, Williams, Samuel Cameron, Waterhouse, Peter Michael, Griffiths, Andrew David, Winter, Gregory Paul, Johnson, Kevin Stuart
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US09/104,337
• Time in Patent Office: 1,629 Days
• Field of Search: 435/235.1, 435/320.1, 435/6, 530/387.3, 530/388.1
• US Class Current: 435/235.1
• CPC Class Codes: B01J 2219/00718 Type of compounds synthesised
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• 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 16/28 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 16/34 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 16/44 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 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• 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 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: C12N 15/73 : Expression systems using phage (lambda) regulatory sequences
• 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: G01N 33/53 : Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 33/6857 : Antibody fragments

Abstract

Methods, recombinant host cells and kits are disclosed-for the production of members of specific binding pairs (sbp), e.g. antibodies, using display on the surface of secreted replicable genetic display packages (rgdps), e.g. filamentous phage. To produce a library of great diversity recombination occurs between first and second vectors comprising nucleic acid encoding first and second polypeptide chains of sbp members respectively, thereby producing recombinant vectors each encoding both a first and a second polypeptide chain component of a sbp member. The recombination may take place in vitro or intracellularly and may be site-specific, e.g. involving use of the loxP sequence and mutants thereof. Recombination may take place after prior screening or selecting for rgdps displaying sbp members which bind complementary sbp member of interest.

Claims

1. Recombinant vectors produced by a method which comprises:

causing or allowing recombination between (a) first vectors comprising nucleic acid encoding a population of first polypeptide chain of a specific binding pair member; and

(b) second vectors comprising nucleic acid encoding a population of a second polypeptide chain of a specific binding pair member, at least one of said populations being genetically diverse, the recombination resulting in recombinant vectors each of which comprises nucleic acid encoding a said first polypeptide chain and a said second polypeptide chain, the recombination being promoted by inclusion in said first and second vectors of sequences at which site-specific recombination occurs, each of the first vectors and each of the second vectors including a first site-specific recombination sequence and a second site-specific recombination sequence different from the first, site-specific recombination taking place preferentially between first site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector.

2. Recombinant vectors according to claim 1 wherein said first vectors each encode a polypeptide fusion of said first polypeptide chain and a component of a replicable genetic display package and the recombination results in recombinant vectors each of which comprises nucleic acid encoding a said polypeptide fusion and a said second polypeptide chain, and wherein said recombinant vectors are capable of being packaged into replicable genetic display packages using said replicable genetic display package component.

3. Recombinant vectors according to claim 1 wherein the recombination is intracellular.

4. Recombinant vectors according to claim 1 comprising nucleic acid encoding a single chain sbp member resulting from recombination between first and second vectors.

5. Recombinant vectors according to claim 1 wherein said first polypeptide chain is an immunoglobulin heavy chain variable region and said second polypeptide chain is an immunoglobulin light chain variable region, or said first polypeptide chain is an immunoglobulin light chain variable region and said second polypeptide chain is an immunoglobulin heavy chain variable region.

6. Recombinant vectors according to claim 1 wherein one of said first and second site-specific recombination sequences is loxP and the second site-specific recombination sequence is a mutant coliphage P1 loxP sequence.

7. Recombinant vectors according to claim 1 wherein the site-specific recombination sequences are selected from the group consisting of loxP, loxP 511, loxP 1, loxP 2, loxP 3 and loxP 4, whose sequences are shown in Table 8.

8. Recombinant vectors according to claim 1 wherein each first vector includes a first site-specific recombination sequence , a second site-specific recombination sequence different from the first and a third site-specific recombination sequence different from the first and second and which has a frequency of recombination with the first site-specific recombination sequence which is low compared with the frequency of recombination between first site-specific recombination and a frequency of recombination w with the second d site-specific recombination sequence which is low compared with the frequency of recombination between second site-specific recombination sequence which is low compared with the frequency of recombination between second site-specific recombination sequences, and each second vector includes a said first and a said second site-specific recombination sequence, site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector.

9. Recombinant vectors according to claim 8 wherein following said site-specific recombination, resultant recombinant vectors are recombined with third vectors including a said first and a said third site-specific recombination sequence, site-specific recombination taking place preferentially between first site-specific recombination sequences on different vectors and between third site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a third site-specific recombination sequence on the same vector.

10. Recombinant vectors according to claim 9 wherein said population of a second polypeptide chain of a specific binding pair member is genetically diverse.

11. Recombinant vectors according to claim 8 wherein said first, second and third site-specific recombination sequences are selected from the group consisting of loxP, loxP 511, loxP 2, loxP 3, and loxP 4, whose sequences are shown in Table 8.

12. Recombinant vectors according to claim 1 wherein the recombination takes place in vitro.

13. Recombinant vectors according to claim 1 wherein the first vectors are phages or phagemids and the second vectors are plasmids, or the first vectors are plasmids and the second vectors are phages or phagemids.

14. Recombinant vectors according to claim 8 wherein the recombination takes place in a bacterial host which replicates the recombinant vector preferentially over the first vectors and the second vectors.

15. Recombinant vectors according to claim 14 wherein the recombination takes place in a bacterial host which replicates plasmids preferentially over phages or phagemids, or which replicates phages or phagemids preferentially over plasmids.

16. Recombinant vectors according to claim 15 wherein said bacterial host is a PolA strain of E. coli or of another gram-negative bacterium.

17. Recombinant vectors according to claim 2 wherein said replicable genetic display packages are secreted bacteriophage.

18. Recombinant vectors according to claim 4 wherein said resulting recombinant vectors encode a fusion of said single chain sbp member and a component of a replicable genetic display package and wherein said recombinant vectors are capable of being packaged into replicable genetic display packages using said replicable genetic display package components.

19. Recombinant vectors according to claim 18 wherein said replicable genetic display packages are secreted bacteriophage.

20. Recombinant vectors according to claim 8 wherein said first vectors each encode a fusion of a said first polypeptide chain and a component of a replicable genetic display package and the recombination results in recombinant vectors each of which comprises nucleic acid encoding a said fusion and a said second polypeptide chain, and wherein said recombinant vectors are capable of being packaged into replicable genetic display packages using said replicable genetic display package component.

21. The method of claim 20 wherein said replicable genetic display packages are secreted bacteriophage.

33. Replicable genetic display packages produced by a method comprising expressing from recombinant vectors according to claim 2, said polypeptide fusions and said second polypeptide chains, producing replicable genetic display packages which display at their surface said first and second polypeptide chains and which each comprise nucleic acid encoding a said first polypeptide chain and a said second polypeptide chain.

34. Replicable genetic display packages produced by a method comprising expressing from recombinant vectors according to claim 18 said fusions, producing replicable genetic display packages which display at their surface said single chain sbp members and which each comprise nucleic acid encoding a said single chain sbp member.

35. Replicable genetic display packages produced by a method comprising expressing from recombinant vectors according to claim 20 said fusions and said second polypeptide chains, producing replicable genetic display packages which display at their surface said first and second polypeptide chains and which each comprise nucleic acid encoding a said single chain sbp member.

22. Recombinant vectors produced by a method comprising:

causing or allowing recombination between (a) first vectors comprising nucleic acid encoding a specific binding pair (sbp) member and (b) second vectors, the vectors comprising site-specific recombination sequences and the site-specific recombination sequences of the first vectors flanking the nucleic acid encoding a specific binding pair member, each of the first vectors and each of the second vectors including a first site-specific recombination sequence and a second site-specific recombination sequence different from the first, site-specific recombination taking place preferentially between first site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector, thereby producing recombinant vectors.

23. Recombinant vectors according to claim 22 wherein the recombination takes place intracellularly.

24. Recombinant vectors according to claim 22 wherein the first vectors comprise nucleic acid encoding a genetically diverse population of sbp members.

25. Recombinant vectors according to claim 22 wherein the second vectors comprise nucleic acid for expression of the sbp member following recombination.

26. Recombinant vectors according to claim 22 wherein the second vectors comprise nucleic acid for expression of a fusion of the sbp member and a component of a replicable genetic display package following recombination.

27. Recombinant vectors according to claim 22 wherein one of said first and second site-specific recombination sequences is loxP and the second site-specific recombination sequence is a mutant coliphage P1 loxP sequence.

28. Recombinant vectors according to claim 22 wherein the site-specific recombination sequences are selected from the group consisting of loxP, loxP 511, loxP 1, loxP 2, loxP 3, and loxP 4, whose sequences are shown in Table 8.

29. Recombinant vectors according to claim 22 wherein nucleic acid encoding the sbp member in each first vector is flanked by a first site-specific recombination sequence and a second site-specific recombination sequence different from the first, and wherein the sbp member comprises two polypeptide chains and nucleic acid encoding each polypeptide chain is separated by a third site-specific recombination sequence different from the first and second, the third site-specific recombinant sequence having a frequency of recombination with the first site-specific recombination sequence which is low compared with the frequency of recombination between first site-specific recombination sequences and a frequency of recombination with the second site-specific recombination sequence which is low compared with the frequency of recombination between second site-specific recombination sequences, and wherein each second vector includes a said first and a said second site-specific recombination sequence, site-specific recombination taking place preferentially between first site-specific recombination sequences on different vectors and between second site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a second site-specific recombination sequence on the same vector.

30. Recombinant vectors according to claim 29 wherein the site-specific recombination sequences are selected from the group consisting of loxP, loxP 511, loxP 1, loxP 2, loxP 3, and loxP 4, whose sequences are shown in Table 8.

31. Recombinant vectors according to claim 29 wherein the first vectors are provided by recombination between (i) vectors comprising nucleic acid encoding a first polypeptide chain flanked by two site-specific recombination sequences wherein one is a said first site-specific recombination sequence and the other is a said third site-specific recombination sequence and (ii) vectors comprising nucleic acid enclosing a second polypeptide chain flanked by two site-specific recombination sequences wherein one is a third site-specific recombination sequence and the other is a said second site-specific recombination sequence and further comprising a said first site-specific recombination sequence, recombination taking place preferentially between first site-specific recombination sequences on different vectors and between third site-specific recombination sequences on different vectors compared with a first site-specific recombination sequence and a third site-specific recombination sequence on the same vector.

32. Recombinant vectors according to claim 29 wherein the site-specific recombination sequences are selected from the group consisting of loxP, loxP 511, loxP 1, loxP 2, loxP 3, and loxP 4, whose sequences are shown in Table 8.

36. Replicable genetic display packages produced by a method comprising expressing from recombinant vectors according to claim 26 said fusions, producing replicable genetic display packages which display at their surface a specific binding pair member and which each comprise nucleic acid encoding a specific binding pair member.