Methods for producing members of specific binding pairs

• US 5,871,907 A
• Filed: 03/31/1994
• Issued: 02/16/1999
• Est. Priority Date: 05/15/1991
• 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:
  US08/150,002
• Time in Patent Office: 1,783 Days
• Field of Search: 435/320.1, 435/235.1, 435/172.3, 435/6, 435/7.1, 435/7.2, 435/91.1, 435/257.33, 435/69.1
• US Class Current: 506/9
• 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 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• 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

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) a first polypeptide chain comprising an antibody heavy chain variable domain and a second polypeptide chain comprising an antibody light chain variable domain, or

(b) a first polypeptide chain comprising an antibody light chain variable domain and a second polypeptide chain comprising an antibody heavy chain variable domain;

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 first 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 rdgps and said introducing of said first vectors into host cells being by infection into host cells harboring said second vectors, or

said second vectors being packaged in infectious rgdps and said introducing of said second vectors 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 population being expressed from nucleic acid that is packaged using rgdp component, whereby each said rgdp contains genetic material which 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 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 kit for use in carrying out a method according to claim 1, said kit having the following components in addition to components required for carrying out the method:

(i) a vector having the following features;

(a) an origin of replication for single-stranded bacteriophage, (b) a restriction site for insertion of nucleic acid encoding or a polypeptide component of an sbp member, (c) said restriction site being in the 5'"'"' end region of the mature coding sequence of a phage capsid protein, and (d) with a secretory leader sequence upstream of said site which directs a fusion of the capsid protein and sbp polypeptide to the periplasmic space of a bacterial host; and

(ii) another vector, having some or all of the features (a), (b), (c) and (d) of the vector described in (i).

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 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).

9. A method according to claim 1 wherein said sbp members displayed by rgdps are scFv molecules.

10. 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.

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

12. The method according to claim 11 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.

13. A method according to claim 11 or claim 12 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.

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

15. A method according to claim 10 wherein the host is E. coli.

16. 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.

17. A method of preparing an individual specific binding pair member, a mixed population of specific binding pair member, 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 16; 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.

18. 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 16; 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.

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

20. A method according to claim 19 which comprises recovering any rgdps bound to said complementary sbp member by washing with an eluant.

21. A method according to claim 19 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.

22. A method according to claim 20 wherein the eluant contains a molecule which compete with said rgdp for binding to the complementary sbp member.

23. A method according to any one of claims 16 or 19-22 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.

24. A method of producing one or a selected population of multichain polypeptide members of a specific binding pair (sbp members) comprising:

(a) a first polypeptide chain comprising an antibody heavy chain variable domain and a second polypeptide chain comprising an antibody light chain variable domain, or

(b) a first polypeptide chain comprising an antibody light chain variable domain and a second polypeptide chain comprising an antibody heavy chain variable domain,

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 first 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 rgdps and said introducting of said first vectors into host cells being by infection into host cells harboring said second vectors, or

said second vectors being packaged in infectious rgdps and said introducing of said second vectors 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 packaged using said rgdp component, whereby each said rgdp contains genetic material which 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 of said multichain sbp members;

(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 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 said counterpart sbp member of interest.

25. A method according to claim 24 wherein said selectable sbp members are scFv molecules.

26. A method according to claim 25 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.

27. A method according to claim 24 wherein said multichain sbp members are antibodies, or other members of the immunoglobulin family, or binding fragments thereof.

28. A method according to claim 27 comprising an additional step 5 wherein humanized antibodies for said antigen are selected by affinity with said antigen.

29. A method according to claim 27 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.

30. A method according to claim 29 wherein said second polypeptide chains of (i) and (ii) are chimaeric, comprising a human antibody domain.

31. A method according to claim 30 wherein said human antibody domain comprises Cy1.