Method for tapping the immunological repertiore

• US 6,291,161 B1
• Filed: 09/04/1992
• Issued: 09/18/2001
• Est. Priority Date: 05/16/1989
• Status: Expired due to Term

• Current Assignee: Catalyst Assets LLC, The Scripps Research Institute, 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
• Primary Examiner(s): KETTER, JAMES S
• Application Number:
  US07/941,762
• Time in Patent Office: 3,301 Days
• Field of Search: 435/91, 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 2317/56 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 coexpression vector capable of expressing a V_H and V^L containing molecule of predetermined specificity, which method comprises the steps:

(a) synthesizing a V_H-coding gene library containing a plurality of different V_H-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-coding sequences

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

(b) synthesizing a V_L-coding gene library containing a plurality of different 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_L-coding sequences

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

(c) joining in operable combination V_H-coding sequences from said V_H-coding gene library with V_L-coding sequences from said V_L-coding gene library into expression vectors to form cexpression vectors so as to be able to coexpress a V_H-coding sequence and a V_L-coding sequence from each vector, whereby a diverse library is formed,

(d) selecting from said diverse library at least one coexpression vector capable of producing V_H and V_L containing molecules 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 said compound capable of binding to said a V_H and V_L containing molecule.

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 selected from the group consisting of lambda and lambda zap II phage.

7. A coexpression vector prepared according to method of claim 1.

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

9. A method of producing a V_H and V_L containing molecule of pre-determined specificity, said method comprising the steps:

isolating a coexpression vector according to claim 1,

transforming a host cell with said expression vector,

isolating a V_H and V_L containing molecule encoded by said vector from said host cell.

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

11. A method of producing an isolated coexpression vector capable of expressing a V_H and V_L containing 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-coding sequences

(ii) amplifying a plurality of V_H-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-coding sequences under conditions permitting hybridization to occur, wherein the number of primer sequences is less than the number of unique V_H-coding sequences present in the polynucleotide containing composition,

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

(iv) amplifying a plurality of 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_L-coding sequences under conditions permitting hybridization to occur, wherein the number of primer sequences is less than the number of unique V_L-coding sequences present in the polynucleotide containig composition,

(v) joining in operable combination V_H-coding sequences from said V_H-coding library with V_L-coding sequences from said V_L-coding library into expression vectors so as to be able to coexpress a V_H-coding sequence and a V_L-coding sequence from each vector, whereby a diverse library is formed, and

(vi) selecting from said diverse library at least one coexpression vector capable of producing V_H and V_L containing molecules having the desired specificity.

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 said compound capable of binding to said a V_H and V_L containing molecule.

14. The method of claim 12, wherein said amplification steps are by polymerase chain reaction.

15. The method of claim 11, wherein said expression vector is a phage expression vector.

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

17. A coexpression vector prepared according to method of claim 11.

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

19. A method of producing a V_H and V_L containing molecule of pre-determined specificity, said method comprising the steps:

isolating a coexpression vector according to claim 11,

transforming a host cell with said expression vector,

isolating a V_H and V_L containing molecule encoded by said vector from said host cell.

20. A method according to claim 11, wherein said a V_H and V_L containing molecule of predetermined specificity is a catalytic antibody and said selecting step comprises the step of adding a substrate for a reaction catalyzed by said catalytic antibody.

21. A method of producing an isolated coexpression vector capable of expressing a V_H and V_L containing molecule of predetermined specificity, which method comprises the steps:

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

(i) adding a first primer, wherein said first primer is capable of hybridizing to a primer binding site adjacent to a plurality of V_H and V_L coding sequences, and said coding sequences are present in a polynucleotide containing composition that comprises a plurality of different V_H-coding sequences and 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 and V_L coding sequences and said second primer binding site is not adjacent to said first primer binding site

(iii) amplifying said plurality of V_H and V_L coding sequences

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

(c) selecting from said diverse library at least one coexpression vector capable of producing V_H and V_L containing molecules 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 said compound capable of binding to said a V_H and V_L containing molecule.

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 selected from the group consisting of lambda and lambda zap II phage.

26. The method of claim 25, wherein said amplifying is by polymerase chain reaction is a Lambda Zap phage.

27. A coexpression vector prepared according to method of claim 21.

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

29. A method of producing a V_H and V_L containing molecule of pre-determined specificity, said method comprising the steps:

isolating a coexpression vector according to claim 21,

transforming a host cell with said expression vector,

isolating a V_H and V_L containing molecule encoded by said vector from said host cell.

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

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

(a) synthesizing a V_H-coding gene library containing a plurality of different V_H-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-coding sequences

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

(b) synthesizing a V_L-coding gene library containing a plurality of different 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_L-coding sequences

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

(c) joining in operable combination V_H-coding sequences from said V_H-coding gene library with V_L-coding sequences from said V_L-coding gene library into expression vectors so as to be able to coexpress a V_H-coding sequence and a V_L-coding sequence from a single each vector is selected from the group consisting of lambda and lambda zap II phage, 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 31, wherein said expression vector is a phage expression vector.

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

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

38. A genetic library according to claim 31, said library capable of expressing a plurality of different combinations of V_H and V_L encoding sequences.

39. A method of producing a genetic library encoding a plurality of diverse V_H and 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-coding sequences

(ii) amplifying a plurality of V_H-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-coding sequences under conditions permitting hybridization to occur, whereby a plurality of amplified V_H-coding sequences are produced and said amplified V_H-coding sequences form a V_H-coding library,

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

(iv) amplifying a plurality of 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_L-coding sequences under conditions permitting hybridization to occur, whereby a plurality of amplified V_L-coding sequences are produced and said amplified V_L-coding sequences form a V_L-coding library,

(v) joining in operable combination V_H-coding sequences from said V_H-coding gene library with V_L-coding sequences from said V_L-coding gene library into expression vectors so as to be able to coexpress a V_H-coding sequence and a V_L-coding sequence from a single 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-coding sequences 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 selected form the group consisting of lambda and lambda zap II phage.

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

47. A genetic library according to claim 39, said library capable of expressing a plurality different combinations of V_H and V_L encoding sequences.

48. A method of producing a genetic library encoding a plurality of diverse V_H and 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 and V_L coding sequences,

adding a second primer, wherein said second primer is capable of hybridizing to a second Primer biding site adjacent to a plurality of V_H and V_L coding sequences and said second conserved nucleotide sequence is not adjacent to said first conserved nucleotide sequence,

amplifying a plurality of V_H and V_L coding sequences by a method of amplification employing said first and second primers, whereby a V_H-coding library and a V_L-coding library are formed,

joining in operable combination V_H-coding sequences from said V_H-coding gene library with V_L-coding sequences from said V_L-coding gene library into expression vectors so as to be able to coexpress a V_H-coding sequence and a V_L-coding sequence from each vector, whereby genetic libray encoding a plurality of diverse V_H and V_L sequences 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 selected from the group consisting of lambda and lambda zap II phage.

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

54. A genetic library according to claim 53, said library capable of expressing a plurality of different V_H and V_L sequence combinations.

58. The method of claim 52, wherein said amplifying is by polymerase chain reaction.

55. An expression library comprising a plurality of nucleic acid sequences for expressing a plurality of at least 10⁴ proteins each comprising a functional portion of an immunoglobulin heavy chain variable domain (V_H domain) and a functional portion of an immunoglobulin light chain variable domain (V_L domain).

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

57. 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.