Methods for producing members of specific binding pairs

• US 5,858,657 A
• Filed: 06/07/1995
• Issued: 01/12/1999
• Est. Priority Date: 05/15/1992
• Status: Expired due to Term

• Current Assignee: Cambridge Antibody Technology Ltd (Astrazeneca PLC), Medical Research Council (Government of United Kingdom)
• Original Assignee: Cambridge Antibody Technology Ltd (Astrazeneca PLC), Medical Research Council (Government of United Kingdom)
• Inventors: Smith, Andrew John Hammond, Griffiths, Andrew David, Winter, Gregory Paul, Johnson, Kevin Stuart
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US08/480,006
• Time in Patent Office: 1,315 Days
• Field of Search: 435/172.3, 435/252.33, 435/69.4, 435/69.6, 435/91.1
• US Class Current: 435/6.14
• CPC Class Codes: C07K 16/18 against material from animals or humans
• 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 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• 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

The invention provides methods and kits for producing specific binding pairs (sbp) members. Populations of polypeptide chain components of sbp members are combined to form libraries of sbps displayed by secreted replicable genetic display packages (rgdp). At least one of the polypeptide chains is expressed as a fusion with a component of an rgdp which thereby displays that polypeptide chain at the surface of rgdp. At least one population of polypeptide chains is expressed from nucleic acid which is capable of being packaged using a component of an rgdp, whereby the genetic material of rgdps produced encodes a polypeptide chain. The methods enable production of libraries of multimeric sbp members from a very large number of possible combinations. In one embodiment of the invention a method employs "chain shuffling" in the production of sbp members of desired specificity for a counterpart sbp member. Selection procedures are also described.

Claims

1. A method of producing specific binding pair (sbp) members comprising a first polypeptide chain and a second polypeptide chain, which method comprises:

introducing into host cells;

(i) first vectors comprising nucleic acid encoding a genetically diverse population of said first polypeptide chain fused to a component of a secreted replicable genetic display package (rgdp) for display of said polypeptide chains at the surface of rgdps; and

(ii) second vectors comprising nucleic acid encoding a genetically diverse population of said second polypeptide chain;

said first vectors being packaged in infectious rgdp'"'"'s and their introduction into host cells being by infection into host cells harboring said second vectors;

or

said second vectors being packaged in infectious rgdp'"'"'s and their introducing into host cells being by infection into host cells harboring said first vectors; and

expressing said first and second polypeptide chains within the host cells to form a library of said sbp members displayed by rgdps, at least one of said populations being expressed from nucleic acid that is capable of being packaged using said rgdp component, whereby the genetic materials of each said rgdp encodes a polypeptide chain of the sbp member displayed at its surface.

2. A method according to claim 1 wherein at least one of said populations is expressed from a phage vector.

3. A method according to claim 1 or claim 2 wherein at least one of said populations is expressed from a phagemid vector, the method including using a helper phage or a plasmid expressing complementing phage genes, to help package said phagemid genome, and said component of the rgdp is a capsid protein therefore.

4. A method according to claim 1 wherein either or both of the populations of said first and second chains polypeptide chains is derived from a repertoire selected from the group consisting of:

(i) the repertoire of rearranged immunoglobulin genes of an animal immunized with a complementary sbp member;

(ii) the repertoire of rearranged immunoglobulin genes of an animal not immunized with a complementary sbp member;

(iii) the repertoire of artificially rearranged immunoglobulin gene or genes;

(iv) a repertoire of an immunoglobulin homolog gene or genes;

(v) a repertoire of sequences derived from a germ-line immunoglobulin gene or genes;

(vi) a repertoire of an immunoglobulin gene or genes artificially mutated by the introduction of one or more point mutations; and

(vii) a mixture of any of (i), (ii), (iii), (iv), (v) and (vi).

5. A method according to claim 1 wherein each said first and second polypeptide chain is expressed from nucleic acid which is packaged as a rgdp using said component fusion product, whereby encoding nucleic acid for both said first and second polypeptide chains is packaged in respective rgdps.

6. A method according to claim 1 which comprises introducing vectors encoding a population of said first polypeptide chains into host organisms which express a population of said second polypeptide chains in free form, or introducing vectors encoding a population of said second polypeptide chains in free form into host organisms which express a population of said first polypeptide chains.

7. A method according to claim 1 wherein said second polypeptide chains are each expressed as a fusion with a component of a rgdp which thereby displays said second polypeptide chains at the surface of rgdps.

8. A method according to claim 1 wherein nucleic acid encoding a said first or second polypeptide chain is linked downstream to a viral capsid protein through a suppressible translational stop codon.

9. A method according to claim 1 wherein the rgdp is a bacteriophage, the host is a bacterium, and said component of the rgdp is a capsid protein for the bacteriophage.

10. A method according to claim 9 wherein the phage is a filamentous phage.

11. The method according to claim 10 wherein the phage is selected from the group consisting of the class 1 phages, Fd, M13, f1, If1, Ike, ZJ/Z, Ff and the class II phages xf, Pf1 and Pf3.

12. A method according to claim 10 wherein the first polypeptide chains are expressed as fusions with the gene III capsid protein of phage fd or its counterpart in another filamentous phage.

13. A method according to claim 12 wherein the first polypeptide chains are each inserted in the N-terminal region of the mature capsid protein downstream of a secretory leader peptide.

14. A method according to claim 9 wherein the host is E.coli.

15. A method according to claim 1 wherein rgdps formed by said expression are selected or screened to provide an individual sbp member or a mixed population of said sbp members associated in their respective rgdps with nucleic acid encoding a polypeptide chain thereof.

16. A method according to claim 15 wherein the rgdps are selected by affinity with a member complementary to said sbp member.

17. A method according to claim 16 which comprises recovering any rgdps bound to said complementary sbp member by washing with an eluant.

18. A method according to claim 17 wherein the eluant contains a molecule which competes with said rgdp for binding to the complementary sbp member.

19. A method according to claim 16 wherein the rgdp is applied to said complementary sbp member in the presence of a molecule which competes with said package for binding to said complementary sbp member.

20. A method according to claim 15 wherein nucleic acid derived from a selected or screened rgdp is used to express said sbp member or a fragment or derivative thereof in a recombinant host organism.

21. A method of preparing an individual specific binding pair member, a mixed population of specific binding pair 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 15; 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.

22. A method of preparing nucleic acid encoding an individual specific binding pair member, a mixed population of specific binding pair 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 15; 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.

23. A method of producing multimeric specific binding pair (sbp) members, which method comprises

(i) causing or allowing intracellular recombination between (a) first vectors comprising nucleic acid encoding a population of a fusion of a first polypeptide chain of a specific binding pair member and a component of a secreted replicable genetic display package (rgdp) 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 polypeptide fusion and a said second polypeptide chain and capable of being packaged using said rgdp component;

and

(ii) 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.

24. A method according to claim 23 wherein the intracellular recombination is promoted by inclusion in the vectors of sequences at which site-specific recombination will occur.

25. A method according to claim 24 wherein said resultant recombinant vector comprises nucleic acid encoding a single chain Fv region derivative of an immunoglobulin resulting from recombination between first and second vectors.

26. A method according to claim 24 wherein the sequences at which site-specific recombination will occur are loxP sequences obtainable from coliphage P1, and site-specific recombination is catalysed by Cre-recombinase, also obtainable from coliphage P1.

27. A method according to claim 26 wherein the Cre-recombinase used is expressible under the control of a regulatable promoter.

28. A method according to claim 27 wherein said bacterial host is a PolA strain of E.coli or of another grain-negative bacterium.

29. A method according to claim 23 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, and the intracellular recombination takes place in a bacterial host which replicates plasmids preferentially over phages or phagemids, or which replicates phages or phagemids preferentially over plasmids.

30. A method according to claim 23 wherein nucleic acid from one or more rgdp'"'"'s is taken and used in a further method to obtain an individual sbp member or a mixed population of sbp members, or polypeptide chain components thereof, or encoding nucleic acid therefor.

31. A method of producing one or a selected population of multichain polypeptide members of a specific binding pair (sbp members) specific for a counterpart specific binding pair member of interest, which method comprises the following steps:

(i) expressing from a vector in recombinant host organism cells a genetically diverse population of a first polypeptide chain of said multichain protein, fused to a component of a replicable genetic display package (rgdp) which thereby displays said polypeptide chains at the surface of rgdps;

(ii) combining said population with a unique or restricted population of second polypeptide chains of said multichain sbp members, not being expressed from the same vector as said population of first polypeptide chains, said combining forming a library of said multichain sbp members displayed by rgdps, said genetically diverse population being expressed from nucleic acid which is capable of being packaged using said rgdp component, whereby the genetic material of each said rgdp encodes a said first polypeptide chain;

(iii) selecting by affinity with said counterpart sbp member of interest multichain sbp members specific for said counterpart sbp member associated in their respective rgdps with nucleic acid encoding a said first polypeptide chain thereof;

(iv) combining said first polypeptide chains of multichain sbp members selected in step (iii) with a genetically diverse population of second polypeptide chains of multichain sbp members, the said second polypeptide chains being fused to a component of a rgdp which thereby displays them at the surface of rgdps, the said combining in this step (iv) forming a library of multichain sbp members from which one or more multichain sbp members specific for said counterpart sbp member are selectable by affinity with it.

32. A method according to claim 31 wherein said multichain sbp members are antibodies, or other members of the immunoglobulin family, or binding fragments thereof.

36. A method according to claim 32 comprising an additional step (v) wherein humanized antibodies for said antigen are selected by affinity with said antigen.

33. A method according to claim 33 wherein each of said second polypeptide chains of steps (i) and (ii) comprises a variable domain derived from a non-human animal antibody specific for the antigen of interest.

34. A method according to claim 33 wherein said second polypeptide chains of steps (i) and (ii) are chimaeric, comprising a human antibody domain.

35. A method according to claim 34 wherein said human antibody domain comprises Cy1.

37. A method of producing one or a selected population of multichain polypeptide members of a specific binding pair (sbp members) comprising a first polypeptide chain and a second polypeptide chain, and specific for a counterpart specific binding pair member of interest;

which method comprises;

(i) introducing into host cells;

(A) first vectors comprising nucleic acid encoding a genetically diverse population of said first polypeptide chain fused to a component of a secreted replicable genetic display package (rgdp) for display of said polypeptide chains at the surface of rgdps; and

(B) second vectors comprising nucleic acid encoding a unique or restricted population of said second polypeptide chain;

said first vectors being packaged in infectious rgdp'"'"'s and their introduction into host cells being by infection into host cells harboring said second vectors;

or

said second vectors being packaged in infectious rgdp'"'"'s and their introducing into host cells being by infection into host cells harboring said first vectors;

(ii) expressing said first and second polypeptide chains within the host cells to form a library of said multichain sbp members displayed by rgdps;

said genetically diverse population of first polypeptide chains being expressed from nucleic acid that is capable of being packaged using said rgdp component, whereby the genetic material of each said rgdp encodes a first polypeptide chain of the sbp member displayed at its surface;

(iii) selecting by affinity with said counterpart sbp member of interest multichain sbp members specific for said counterpart sbp member associated in their respective rgdps with nucleic acid encoding a said first polypeptide chain thereof;

(iv) combining said first polypeptide chains of multichain sbp members selected in step (iii) with a genetically diverse population of second polypeptide chains of multichain sbp members, the said second polypeptide chains being fused to a component of a rgdp which thereby displays them at the surface of rgdps, the said combining in this step (iv) forming a library of multichain sbp members from which one or more multichain sbp members for said counterpart sbp member of interest are selectable by affinity with it.

38. A method according to claim 37 wherein rgdps formed by said expression are selected or screened to provide an individual sbp member or a mixed population of said sbp members associated in their respective rgdps with nucleic acid encoding a polypeptide chain thereof.

39. A method of preparing an individual specific binding pair member, a mixed population of specific binding pair 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 38; 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.

40. A method of preparing nucleic acid encoding an individual specific binding pair member, a mixed population of specific binding pair 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 38; 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.