Method for tapping the immunological repertoire

• US 6,291,158 B1
• Filed: 08/21/1992
• Issued: 09/18/2001
• Est. Priority Date: 05/16/1989
• Status: Expired due to Term

• Current Assignee: Catalyst Assets LLC, UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom), Stratagene California, The Scripps Research Institute
• Inventors: Lerner, Richard A., Sorge, Joseph A., Winter, Gregory P., Riechmann, Lutz, Huse, William D.
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US07/933,958
• Time in Patent Office: 3,315 Days
• Field of Search: 435/172.3, 435/6, 435/235.1, 435/320.1, 435/69.6, 435/252.33, 435/489, 536/23.53
• US Class Current: 435/5
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL

Abstract

The present invention relates to a method for isolating from the immunological gene repertoire a gene coding for a receptor having the ability to bind a preselected ligand. Receptors produced by the gene isolated by the method, particularly catalytic receptors, are also contemplated.

Claims

1. A method of producing an isolated expression vector capable of expressing a V_H or V_L polypeptide of predetermined specificity, which method comprises the steps:

(a) synthesizing a V_H or V_L-coding gene library containing a plurality of different V_H or V_L-coding DNA sequences, by a method comprising the steps;

(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different V_H or V_L-coding sequences

(ii) amplifying a plurality of V_H or V_L-coding sequences in said polynucleotide containing composition, using amplification primers

(iii) joining in operable combination V_H or V_L-coding sequences from said V_H or V_L-coding gene library into expression vectors so as to be able to express a V_H or V_L-coding sequence, whereby a diverse library is formed,

(b) selecting from said diverse library at least one expression vector capable of producing V_H or V_L polypeptides having the desired specificity.

2. A method according to claim 1, wherein said polynucleotide containing composition was isolated from blood cells comprising rearranged variable region genes.

3. A method according to claim 2, wherein said blood cells were isolated from an animal that was immunologically exposed to a compound that is capable of binding to said V_H or V_L polypeptide.

4. The method of claim 1, wherein said amplification steps are by polymerase chain reaction.

5. The method of claim 1, wherein said expression vector is a phage expression vector.

6. The method of claim 5 wherein said expression vector is a lambda phage selected from the group consisting of Lambda Zap and Lambda Zap II.

7. An expression vector prepared according to the method of claim 1.

8. A method according to claim 1, wherein said diverse library is capable of expressing a plurality of different V_H or V_L coding sequences.

9. A method of producing a V_H or V_L polypeptide of pre-determined specificity, said method comprising the steps:

isolating an expression vector according to claim 1,

transforming a host cell with said expression vector,

isolating a V_H or V_L polypeptide encoded by said vector from said host cell.

10. A method according to claim 1, wherein said V_H or V_L polypeptide of predetermined specificity is a catalytic polypeptide and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic polypeptide.

(iv) selecting from said diverse library at least one expression vector capable of producing a V_H or V_L polypeptide having the desired specificity.

11. A method of producing an isolated expression vector capable of expressing a V_H or V_L polypeptide molecule of predetermined specificity, which method comprises the steps:

(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of V_H or V_L-coding sequences

(ii) amplifying a plurality of V_H or V_L-coding sequences from said polynucleotide containing composition by a method of amplification comprising the step of

adding primer sequences capable of hybridizing upstream and downstream from a plurality of said V_H or V_L-coding sequences under conditions permitting hybridization to occur, wherein the number of said primer sequences added is less than the number of unique V_H or V_L-coding sequences present in said polynucleotide containing composition.

12. A method according to claim 11, wherein said polynucleotide containing composition was isolated from blood cells comprising rearranged variable region genes.

13. A method according to claim 12, wherein said blood cells were isolated from an animal that was immunologically exposed to a compound that is capable of binding to said V_H or V_L polypeptide.

14. The method of claim 11, wherein said amplification steps are by polymerase chain reaction.

15. The method of claim 14, wherein said expression vector is a phage expression vector.

16. The method of claim 15, wherein said expression vector is a lambda phage selected from the group consisting of Lambda Zap and Lambda Zap II.

17. An expression vector prepared according to the method of claim 11.

18. A method according to claim 11, wherein said diverse library is capable of expressing a plurality of different V_H or V_L coding sequences.

19. A method of producing a V_H or V_L polypeptide of pre-determined specificity, said method comprising the steps:

isolating an expression vector according to claim 11,

transforming a host cell with said expression vector,

isolating a V_H or V_L polypeptide encoded by said vector from said host cell.

20. A method according to claim 19, wherein said V_H or V_L polypeptide of predetermined specificity is a catalytic polypeptide and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic polypeptide.

21. A method of producing an isolated expression vector capable of expressing a V_H or V_L polypeptide molecule of predetermined specificity, which method comprises the steps:

(a) producing a V_H or V_L-coding genetic library, by a method comprising the steps,

(i) adding a first primer, wherein said first primer is capable of hybridizing to a first primer binding site adjacent to a plurality of V_H or V_L coding regions, and said coding sequences are present in a polynucleotide containing composition that comprises a plurality of different V_H or V_L-coding sequences,

(ii) adding a second primer to said nucleotide containing composition, wherein said second primer is capable of hybridizing to a second primer binding site adjacent to a plurality of V_H or V_L coding regions and said second primer binding site is not adjacent to said first conserved nucleotide sequence, amplifying a plurality of Vh or Vl coding sequences,

(b) joining in operable combination, V_H or V_L-coding sequence from said V_H or V_L-coding gene library with expression vectors so as to be able to express a V_H or V_L-coding sequence from each vector, whereby a diverse library is formed,

(c) selecting from said diverse library at least one expression vector capable of producing V_H or V_L polypeptides having the desired specificity.

22. A method according to claim 21, wherein said polynucleotide containing composition was isolated from blood cells comprising rearranged variable region genes.

23. A method according to claim 22, wherein said blood cells were isolated from an animal that was immunologically exposed to a compound that is capable of binding to said V_H or V_L polypeptide.

24. The method of claim 21, wherein said expression vector is a phage expression vector.

25. The method of claim 24 wherein said expression vector is a lambda phage selected from the group consisting of Lambda Zap and Lambda Zap II.

26. The method of claim 21, wherein said amplifying is by polymerase chain reaction.

27. An expression vector prepared according to the method of claim 21.

28. A method according according to claim 21, wherein said diverse library is capable of expressing a plurality of different V_H or V_L encoding sequences.

29. A method of producing a V_H or V_L polypeptide of pre-determined specificity, said method comprising the steps:

isolating an expression vector according to claim 21,

transforming a host cell with said expression vector,

isolating a V_H or V_L polypeptide encoded by said vector from said host cell.

30. A method according to claim 21, wherein said V_H or V_L polypeptide of predetermined specificity is a catalytic polypeptide and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic polypeptide.

31. A method of producing a genetic library encoding a plurality of diverse V_H or V_L encoding polynucleotide sequences, which method comprises the steps:

(a) synthesizing a V_H or V_L-coding gene library containing a plurality of different V_H or V_L-coding DNA sequences by a method comprising the steps;

(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of different V_H or V_L-coding sequences, and

(ii) amplifying a plurality of V_H or V_L-coding sequences in said polynucleotide containing composition, using amplification primers and

(b) joining in operable combination V_H or V_L-coding sequences from said V_H or V_L-coding gene library with expression vectors so as to be able to express a V_H or V_L-coding sequence from each vector, whereby a diverse library is formed.

32. A method according to claim 31, wherein said polynucleotide containing composition was isolated from blood cells comprising rearranged variable region genes.

33. A method according to claim 32, wherein said blood cells were isolated from an animal that was immunologically exposed to a compound of interest.

34. The method of claim 31, wherein said amplification steps are by polymerase chain reaction.

35. The method of claim 34, wherein said expression vector is a phage expression vector.

36. The method of claim 35, wherein said expression vector is a lambda phage selected from the group consisting of Lambda Zap and Lambda Zap II.

37. A genetic library produced according to the method of claim 31.

38. A genetic library according to claim 37, said library capable of expressing a plurality of different V_H or V_L coding sequences.

39. A method of producing a genetic library encoding a plurality of diverse V_H or V_L sequences, said method comprising the steps:

(i) preparing a polynucleotide containing composition, wherein at least a portion of the polynucleotides in said composition comprise a plurality of V_H or V_L-coding sequences

(ii) amplifying a plurality of V_H or V_L-coding sequences from said polynucleotide containing composition by a method of amplification comprising the step of

adding primer sequences capable of hybridizing upstream and downstream from a plurality of said V_H or V_L-coding sequences under conditions permitting hybridization to occur, whereby a plurality of amplified V_H or V_L-coding sequences are produced,

(iii) joining in operable combination V_H or V_L-coding sequences from said V_H or V_L-coding gene library with expression vectors so as to be able to express a V_H or V_L-coding sequence from each vector, whereby a diverse library is formed.

40. A method according to claim 39, wherein the number of said primer sequences added is less than the number of unique V_H or V_L-coding sequences present in said polynucleotide containing composition.

41. A method according to claim 39, wherein said polynucleotide containing composition was isolated from blood cells comprising rearranged variable region genes.

42. A method according to claim 41, wherein said blood cells were isolated from an animal that was immunologically exposed to a compound of interest.

43. The method of claim 39, wherein said amplification steps are by polymerase chain reaction.

44. The method of claim 39, wherein said expression vector is a phage expression vector.

45. The method of claim 44, wherein said expression vector is a lambda phage selected from the group consisting of Lambda Zap and Lambda Zap II.

46. A genetic library produced by the method of claim 39.

47. A genetic library according to claim 46, said library capable of expressing a plurality of different V_H or V_L coding sequences.

48. A method of producing a genetic library encoding a plurality of diverse V_H or V_L sequences, said method comprising the steps:

adding a first primer, wherein said first primer is capable of hybridizing to a first primer binding site adjacent to a plurality of V_H or V_L-coding regions,

adding a second primer, wherein said second primer is capable of hybridizing to a second primer binding site adjacent to a plurality of V_H or V_L-coding regions and said second primer binding site is not adjacent to said first conserved nucleotide sequence amplifying the Vh or Vl coding regions, and, and

joining in operable combination said V_H or V_L-coding sequences from said V_H or V_L-coding gene library with expression vectors so as to be able to express a V_H or V_L-coding sequence from each vector, whereby a diverse library is formed.

49. A method according to claim 48, wherein said polynucleotide containing composition was isolated from blood cells comprising rearranged variable region genes.

50. A method according to claim 49, wherein said blood cells were isolated from an animal that was immunologically exposed to a compound of interest.

51. The method of claim 48, wherein said expression vector is a phage expression vector.

52. The method of claim 51 wherein said expression vector is a lambda phage selected from the group consisting of Lambda Zap and Lambda Zap II.

53. The method of claim 48, wherein said amplifying is by polymerase chain reaction.

54. A genetic library prepared according to method of claim 48.

55. An expression library comprising a plurality of nucleic acid sequences for expressing at least a plurality of 10⁴ proteins each comprising a functional portion of an immunoglobulin variable domain.

56. An expression library according to claim 55 wherein the immunoglobulin variable domain is selected from the group consisting of an immunoglobulin heavy chain variable domain (V_H domain) and an immunoglobulin light chain variable domain (V_L domain).

57. An expression library according to claim 55 wherein the plurality of nucleic acid sequences is derived from peripheral blood lymphocytes.

58. An expression library according to claim 55 wherein the plurality of nucleic acid sequences is prepared from lymphocytes of an animal immunized with an antigen.