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1. (NZ545936) Method for linking sequences of interest
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Claims

1. A method of linking a plurality of non-contiguous nucleotide sequences of interest said method comprising:

a) amplifying, in a multiplex molecular amplification procedure, nucleotide sequences of interest using a template derived from an isolated single cell; and

b) effecting linkage of the nucleotide sequences of interest amplified in step a), wherein the nucleotide sequences of interest comprise variable region encoding sequences and the linkage generates a cognate pair of variable region encoding sequences.

2. The method according to claim 1, wherein the nucleotide sequences of interest comprise immunoglobulin variable region encoding sequences and the linkage generates a cognate pair of a light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence.

3. The method according to claim 1 , wherein the nucleotide sequences of interest comprise T cell receptor variable region encoding sequences and the linkage generates a cognate pair constituted of an alpha chain variable region encoding sequence associated with a beta chain variable region encoding sequence or a gamma chain variable region encoding sequence associated with a delta chain variable region encoding sequence.

4. The method according to any one of the preceding claims, wherein said multiplex molecular amplification procedure is a multiplex RT-PCR amplification.

5. The method according to claim 4, wherein said multiplex RT-PCR amplification is a two step process comprising a separate reverse transcription (RT) step prior to the multiplex PCR amplification.

6. The method according to claim 4, wherein said multiplex RT-PCR amplification is performed in a single step comprising initially adding all the components necessary to perform both reverse transcription (RT) and multiplex PCR amplification into a single vessel. 5 59J¾7

7. The method according to any one of the preceding claims, wherein the single cell, or the population of genetically different cells is obtained from a lymphocyte containing cell fraction.

8. The method according to claim 13, wherein said single cell is expanded in order to obtain a population of isogenic cells prior to the reverse transcription step of the multiplex molecular amplification procedure.

9. The method according to any one of the preceding claims, wherein said linkage of the nucleotide sequences of interest is performed in the same vessel as the multiplex molecular amplification.

10. The method according to any one of claims 4 to 9, wherein said linkage of the nucleotide sequences of interest is effected in association with the multiplex PCR amplification, utilizing a multiplex overlap-extension primer mix.

11. The method according to any one of the preceding claims, wherein said linkage of the nucleotide sequences of interest is effected by ligation.

12. The method according to any one of the preceding claims, wherein an additional molecular amplification, utilizing a primer mix adapted for amplifying the linked nucleic acid sequences of interest, is performed.

13. The method according to claim 10, wherein the multiplex overlap-extension primer mix comprises primer sets wherein at least one primer set member of each primer set comprises an overlap-extension tail capable of hybridizing to the overlap-extension tail of a primer set member of a second primer set.

14. The method according to claim 10 or 13, wherein the multiplex overlap-extension primer mix comprise:

a) at least one CL or JL primer complementary to the sense strand of an

immunoglobulin light chain region encoding sequence;

b) at least one VL 5' primer or VLL primer complementary to the antisense strand of an immunoglobulin light chain variable region encoding sequence or light chain


variable region leader sequence, and capable of forming a primer set with the primer(s) in step a);

c) at least one CH or ¾ primer complementary to the sense strand of an

immunoglobulin constant heavy chain domain encoding sequence or a heavy chain joining region encoding sequence; and

d) at least one VH 5' primer or VHL primer complementary to the antisense strand of an immunoglobulin heavy chain variable region encoding sequence or heavy chain variable region leader sequence, and capable of forming a primer set with the primer(s) in step c)

15. The method according to claim 14, wherein the primers of step b) are VLL primers with at least 90% sequence identity with the gene-specific region of SEQ ID 93 to 98, and the primers of step d) are VHL primers with at least 90% sequence identity with the gene-specific region of SEQ ID 86 to 92.

16. A method of producing a library of cognate pairs comprising linked variable region encoding sequences, said method comprising:

a) providing a lymphocyte-containing cell fraction from a donor;

b) optionally enriching for a particular lymphocyte population from said cell fraction; c) obtaining a population of isolated single cells, comprising distributing cells from said cell fraction individually into a plurality of vessels; and

d) amplifying and effecting linkage of the variable region encoding sequences contained in said population of isolated single cells, according to a method of any one of claims 1 to 15.

17. The method according to claim 16 wherein the individual isolated single cell in the population of single cells is expanded to a population of isogenic cells, prior to performing amplification and linkage (step d).

18. The method according to any one of claims 7, 16 or 17, wherein the lymphocyte- containing cell fraction constitutes whole blood, bone marrow, mononuclear cells, or white blood cells.


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20. The method according to any one of claims 7 or 16 to 19, wherein the lymphocyte-containing cell fraction or B lymphocyte linage is enriched for plasma cells.

21. The method according to any one of claims 7 or 16 to 20, wherein cells from the lymphocyte-containing cell fraction, B lymphocyte linage or plasma cells are enriched for antigen-specificity.

22. The method according any one of claims 7 or 16 to 18 , wherein the lymphocyte-containing cell fraction is enriched for cells of the T lymphocyte lineage.

23. The method according any one of claims 7 or 16 to 18 , wherein antigen-specific T cells are generated by stimulation of the lymphocyte-containing cell fraction.

24. The method according to any one of the preceding claims, further comprising inserting the linked nucleotide sequences or a library of cognate pairs into a vector.

25. The method according to claim 24, wherein said vector is selected among cloning vectors, shuttle vectors, display vectors or expression vectors.

26. The method according to claim 24 or 25, wherein the linked nucleotide sequences or the individual members of the library of cognate pairs comprise an immunoglobulin heavy chain variable region encoding sequence associated with light chain variable region encoding sequence and said sequences are inserted in-frame into a vector already containing sequences encoding one or more immunoglobulin constant domains or fragments thereof.

27. The method according to claim 24 or 25, wherein the linked nucleotide sequences or the individual members of the library of cognate pairs comprise a T cell receptor a chain variable region encoding sequence associated with a β chain variable region encoding sequences and said sequences are inserted in-frame into a vector already containing sequences encoding one or more T cell receptor constant domains or fragments thereof.

28. The method according to any one of claims 16 to 27, further comprising creating a sub-library by selecting a subset of cognate pairs of linked variable region sequences that 5 59|§>0

encode binding proteins with a desired target specificity, generating a library of target-specific cognate pairs of variable region encoding sequences.

29. The method according to any one of claims 26 to 28, further comprising transferring said cognate pair or library of target-specific cognate pairs of variable region encoding sequences to a mammalian expression vector.

30. The method according to claim 29, wherein the mammalian expression vector encodes one or more constant region domains selected from human immunoglobulin classes IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, IgM, kappa light chain or lambda light chain or from human T cell receptor alpha, beta, delta and/or gamma chains.

31. The method according to one of claims 24 to 30, further comprising the steps:

a) introducing a vector encoding a segment of linked nucleotide sequences into a host cell;

b) cultivating said host cells under conditions adapted for expression; and

c) obtaining the protein product expressed from the vector inserted into said host cell.

32. The method according to claim 31 wherein said protein product is a monoclonal antibody comprising a cognate pair of a light chain variable region associated with a heavy chain variable region.

33. The method according to claim 31 , wherein said polypeptide product is a monoclonal T cell receptor comprising a cognate pair of an alpha chain variable region associated with a beta chain variable region.

34. A library of cognate pairs consisting of linked variable region encoding sequences, where each individual cognate pair is inserted into a vector capable of expressing said cognate pair, and wherein the cognate pairs in said library are derived from a population of isolated single cells.

35 The library according the claim 34, wherein said library comprises at least 10 different

INTELLECTUAL PROPERTY

cognate pairs. OFFICE OF N.Z.

23 MAR 2009

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36. The library according to claim 34, wherein said cognate pairs of variable region encoding sequences are obtained by the method according to claim 16 or any claim dependent thereon.

37. The library according to any one of claims 34-36, wherein the individual cognate pairs comprise an immunoglobulin light chain variable region encoding sequence associated with a heavy chain variable region encoding sequence.

38. The library according to claim 37, wherein said individual cognate pairs encode a full-length antibody selected from human immunoglobulin classes IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4 or IgM.

39. A library of cognate pairs consisting of linked variable region encoding sequences, wherein individual members of said cognate pairs comprise a TcR alpha chain variable region encoding sequence associated with a beta chain variable region encoding sequence or a gamma chain variable region encoding sequence associated with delta chain variable region encoding sequence.

40. The library according claim 36, wherein said individual members encode a full-length TcR.

41. The library of cognate pairs of linked variable region encoding sequences according to any one of claims 34 to 40, wherein each individual member has been selected for its ability to encode a protein exhibiting a desired binding specificity directed against a particular target.

42. The library of cognate pairs according to claim 37 or 38, where antibodies expressible from said library are capable of reacting with, or binding to Tetanus toxin.

43. A population of host cells comprising a library according to any one of claims 34 to 42, wherein said cells do not include human cells in vivo.

44. The population of host cells according to claim 43, wherein the cells are mammalian cells.

[INTELLECTUA PROPERTY OFFICE OF N.Z

2 3 MAR 2009

R EC E I V E D


45. A recombinant polyclonal protein expressed from host cells according to claim 43 or 44.

46. The recombinant polyclonal antibody according to claim 45, wherein the individual antibody members are full-length, and the constant heavy chain region is chosen from the human immunoglobulin classes IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, or IgE, and the constant light chain region is chosen from human lambda or kappa chains.

47. A recombinant polyclonal immunoglobulin or fragments thereof capable of reacting with or binding to Tetanus Toxin, wherein the immunoglobulin is produced by the method of any of claims 1 to 33.

48. The recombinant polyclonal immunoglobulin according to claim 47, wherein the


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53. A pharmaceutical composition according to claim 51 or 52 for use as a medicament.

54. A method according to claim 1, substantially as herein described with reference to any one of Examples 1 to 14 and/or Figures 1 to 25 and/or the Sequence Listing thereof.

55. A library according to claim 34, substantially as herein described with reference to any one of Examples 1 to 14 and/or Figures 1 to 25 and/or the Sequence Listing thereof.