Methods for producing recombinant vectors

• US 5,962,255 A
• Filed: 12/05/1994
• Issued: 10/05/1999
• Est. Priority Date: 03/24/1992
• Status: Expired due to Term

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Cambridge Antibody Technology Ltd (Astrazeneca PLC), Medical Research Council (Government of United Kingdom)
• Inventors: Waterhouse, Peter Michael, Nissim, Ahuva, Smith, Andrew John Hammond, Griffiths, Andrew David, Winter, Gregory Paul, Johnson, Kevin Stuart, Williams, Samuel Cameron
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): Wai, Thanda
• Application Number:
  US08/350,260
• Time in Patent Office: 1,765 Days
• Field of Search: 435/69.1, 435/172.1, 435/172.3, 435/320.1, 435/252.3, 435/252.33, 435/91.41, 935/22, 935/23, 935/24
• US Class Current: 435/69.1
• CPC Class Codes: B01J 2219/00718 Type of compounds synthesised
• 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 16/40 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 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 2319/41 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)

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. A method of producing recombinant vectors, which method comprises:

causing or allowing recombination between (a) first vectors comprising nucleic acid encoding a population of a 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. The method according to claim 1 wherein the recombination takes place in vitro.

3. The method 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.

4. The method according to claim 3 wherein the recombination takes place in a bacterial host which replicates the recombinant vector preferentially over the first vectors and the second vectors.

5. The method according to claim 4 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.

6. The method according to claim 5 wherein said bacterial host is a PolA strain of E. coli or of another gram-negative bacterium.

7. The method according to claim 1 wherein said first vectors each encode a polypeptide fusion of a said first polypeptide chain and a component of a replicable genetic display package (rgdp) 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 rdgps using said rgdp component.

8. The method according to claim 7 comprising expressing said polypeptide fusions and said second polypeptide chains, producing rgdps 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.

9. The method of claim 7 wherein said rgdps are secreted bacteriophage.

10. A method for preparing specific binding pair (sbp) member or members or polypeptide chain components thereof comprising the steps of:

(i) obtaining nucleic acid from one or more rgdps produced by a method according to claim 8; and

(ii) producing by expression from the nucleic acid obtained in step (i) an individual specific binding pair member, a mixed population of specific binding pair members, or polypeptide chain components thereof.

11. A method of producing nucleic acid encoding a specific binding pair (sbp) member or members or polypeptide chain components thereof comprising the steps of:

(i) obtaining nucleic acid from one or more rgdps produced by a method according to claim 8; and

(ii) producing from the nucleic acid obtained in step (i) nucleic acid which encodes an individual specific binding pair member, a mixed population of specific binding pair members, or polypeptide chain components thereof.

12. The method according to claim 1 wherein recombination is intracellular.

13. The method according to claim 1 wherein said resulting recombinant vector comprises nucleic acid encoding a single chain sbp member resulting from recombination between first and second vectors.

14. The method 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.

15. The method according to claim 1 wherein one of said first and second site-specific recombination sequences is coliphase P1 loxP and the second site-specific recombination sequence is a mutant loxP sequence.

16. The method 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.

17. A method according to claim 13 wherein said resulting recombinant vectors encode a fusion of said single chain sbp member and a component of a replicable genetic display package (rgdp) and wherein said recombinant vectors are capable of being packaged into replicable genetic display packages using said replicable genetic display package component.

18. A method according to claim 17 comprising expressing 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.

19. The method of claim 17 wherein said replicable genetic display packages are secreted bacteriophage.

20. A method for preparing specific binding pair (sbp) member or members or polypeptide chain components thereof comprising the steps of:

(i) obtaining nucleic acid from one or more rgdps produced by a method according to claim 18; and

(ii) producing by expression from the nucleic acid obtained in step (i) an individual specific binding pair member, a mixed population of specific binding pair members, or polypeptide chain components thereof.

21. A method of producing nucleic acid encoding a specific binding pair (sbp) member or members or polypeptide chain components thereof comprising the steps of:

(i) obtaining nucleic acid from one or more rgdps produced by a method according to claim 18; and

(ii) producing from the nucleic acid obtained in step (i) nucleic acid which encodes an individual specific binding pair member, a mixed population of specific binding pair members, or polypeptide chain components thereof.

22. The method 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 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 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.

23. The method according to claim 22 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.

24. The method according to claim 22 wherein said population of a second polypeptide chain of a specific binding pair member is genetically diverse.

25. The method according to claim 22 wherein said first, second and third 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.

26. A method according to claim 22 wherein said first vectors each encode a fusion of a said first polypeptide chain and a component of a replicable genetic display package (rgdp) 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.

27. A method according to claim 26 comprising expressing 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.

28. The method of claim 26 wherein said replicable genetic display packages are secreted bacteriophage.

29. A method for preparing specific binding pair (sbp) member or members or polypeptide chain components thereof comprising the steps of:

(i) obtaining nucleic acid from one or more rgdps produced by a method according to claim 27; and

(ii) producing by expression of the nucleic acid obtained in step (i) an individual specific binding pair member, a mixed population of specific binding pair members, or polypeptide chain components thereof.

30. A method of producing nucleic acid encoding a specific binding pair (sbp) member or members or polypeptide chain components thereof comprising the steps of:

(i) obtaining nucleic acid from one or more rgdps produced by a method according to claim 27; and

(ii) producing from the nucleic acid obtained in step (i) nucleic acid which encodes an individual specific binding pair member, a mixed population of specific binding pair members, or polypeptide chain components thereof.

31. A method of producing recombinant vectors 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.

32. A method according to claim 31 wherein the recombination takes place intracellularly.

33. The method according to claim 31 wherein the first vectors comprise nucleic acid encoding a genetically diverse population of sbp members.

34. The method according to claim 31 wherein the second vectors comprise nucleic acid for expression of the sbp member following recombination.

35. The method according to claim 31 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.

36. The method according to claim 31 wherein one of said first and second site-specific recombination sequences is coliphase P1 and the second site-specific recombination sequence is a mutant loxP sequence.

37. The method according to claim 31 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.

38. The method according to claim 31 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 recombination 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.

39. The method according to claim 38 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.

40. The method according to claim 38 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 said 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.

41. The method according to claim 38 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.