Patent EA-003637-B1 (EA)
Title: Novel methods for the identification of ligand and target biomolecules
Inventor: HALKIER TORBEN
IPC: C12Q1/68
Filed: 1999-07-16
Abstract: 1. A method for identifying a modulator in the form of a biologically active polypeptide fragment which is capable of detectably modulating in vivo, a phenotypic trait in a cell, the method comprising the steps of: (a) preparing a pool of expression vectors, each vector of said pool containing at least one member from a library of randomly modified nucleotide sequences derived from a parent nucleotide sequence encoding a parent peptide which in vivo directly modulates activity of a known protease, wherein the randomly modified nucleotide sequences comprise an invariable part encoding a scaffold portion of the parent peptide, said scaffold portion serving to stabilize said polypeptide fragment and being stable towards proteolytic attack and/or being insensitive to a reducing environment, and random nucleotides, (b) transforming a population of substantially identical cells with said vectors of said pool so as to obtain transformed cells, (c) culturing said transformed cells under conditions facilitating expression of said randomly modified nucleotide sequences, (d) examining said transformed cells and isolating transformed cell(s) wherein the preselected phenotypic trait is modulated thereby indicating that the expression product of said randomly modified nucleotide sequence is biologically active, and (e) identifying the modulator by determining said randomly modified nucleotide sequence of said vector present in cell(s) isolated in step (d) and/or determining the amino acid sequence of the expression product encoded by said randomly modified nucleotide sequence. 2. The method according to claim 1, wherein the substantially identical cells are prokaryotic cells. 3. The method according to claim 1, wherein the substantially identical cells are eukaryotic cells. 4. The method according to claim 3, wherein the eukaryotic cells are selected from the group consisting of fungal cells, protozoan cells, animal cells, and plant cells. 5. The method according to claim 4, wherein the animal cells are selected from the group consisting of mammalian cells, arthropod cells such as insect cells, avian cells, and piscine cells. 6. The method according to any of the preceding claims, wherein the transformed cells examined in step (d) predominantly carries one single copy of the vector. 7. The method according to claim 6, wherein transformation step (b) is performed under such conditions that the cells transformed are predominantly or at most transformed with one single vector from said pool, or wherein, prior to carrying out step (d), cells being transformed with more than one vector from said pool are substantially excluded from the further steps. 8. The method according to any one of the preceding claims, wherein the random nucleotides are introduced in part(s) of the parent nucleotide sequence which encode(s) the active site(s) of the parent peptide, or the part (s) which encode (s) structure(s) interfering with the active site(s). 9. The method according to any one of the preceding claims, wherein the invariable part of the nucleotide sequence encodes truncated parts of the scaffold portion of the parent peptide sufficient to maintain stability. 10. The method according to any of the preceding claims, wherein the invariable part of the parent nucleotide sequence encodes a peptide which is free from disulfide bridges. 11. The method according to any one of claims 1-9, wherein the invariable part of the parent nucleotide sequence encodes a peptide having disulfide bridges. 12. The method according to any one of the preceding claims, wherein the random nucleotides are introduced in the form of an insertion or a substitution into the parent nucleotide sequence, optionally in combination with deletion(s) in the parent nucleotide sequence. 13. The method according to claim 12, wherein the number of random nucleotides which are introduced is in the range from 3 to about 100. 14. The method according to any one of the preceding claims, wherein the random nucleotides are nucleotide sequences and/or are single random nucleotides introduced at specific sites in the parent nucleotide sequence. 15. The method according to any one of the preceding claims, wherein the random nucleotides are selected from the group consisting of synthetic, completely random deoxyribonucleotides; synthetic random DNA sequences, wherein limitation on randomization of some nucleotides is introduced so as to limit the number of available sequences and/or to avoid undesired stop codons and/or to facilitate introduction of post-translational modifications of expressed peptide(s); synthetic random DNA sequences as in (1) or (2) coupled to a sequence encoding a purification tag; and CDR encoding nucleotide sequences isolated from a library of immune-competent cells raised against an antigen. 16. The method according to claim 15, wherein the CDR encoding nucleotide sequences encode CDR-3 peptide sequences. 17. The method according to any one of claims 14-16, wherein the random nucleotides are prepared by random codon synthesis where defined DNA codons are synthesized in a random order. 18. The method according to claim 17, wherein the relative amount of synthesized codons ensure that all encoded amino acids will be present with substantially the same frequency in the total of encoded polypeptide fragments. 19. The method according to any one of the preceding claims, wherein the random nucleotides are introduced into the expression vector by the principle of site directed PCR-mediated mutagenesis. 20. The method according to any one of the preceding claims, wherein the parent peptide is an inhibitor of activity of the known protease. 21. A method according to claim 20, wherein the inhibitor is selected from the group consisting of a BPTI/Kunitz family protease inhibitor, a serpin family protease inhibitor, a Kazal family protease inhibitor, a soybean trypsin inhibitor (Kunitz) family protease inhibitor, a potato inhibitor I family member, a Bowman-Birk family protease inhibitor, a squash inhibitor family member, a wap-type 'Four-disulfide Core' proteinase inhibitor, a hirudin family protease inhibitor, a factor Xa inhibitor, an Ascaris trypsin inhibitor family member, a cystatin family protease inhibitor, a calpain family cysteine protease inhibitor, a tissue inhibitor of metalloproteinases family member, a carboxypeptidase A inhibitor, a metallocarboxypeptidase inhibitor, and an angiotensin-converting enzyme inhibitor. 22. The method according to claim 21, wherein the parent peptide is a potato inhibitor family I member. 23. The method according to claim 22, wherein the parent peptide is chymotrypsin inhibitor 2A (CI-2A). 24. The method according to any of the preceding claims wherein the substantially identical cells are mammalian cells and the vector is selected from the group consisting of a retroviral vector, a vaccinia virus vector, an adenoviral vector, an adeno associated virus (AAV) vector, a herpes simplex virus (HSV) vector, an alpha virus vector, and a Semliki forest virus vector. 25. The method according to claim 24, wherein the vector is retroviral. 26. The method according to claim 25, wherein the retroviral vector is derived from retrovirus selected from the group consisting of Avian Leukosis-Sarcoma Virus (ALSV), Mammalian type C, Mammalian type B, and Lentivirus, and optionally modified with heterologous cis-acting elements. 27. The method according to claim 25 or 26, wherein the retroviral vector has non-identical ends. 28. The method according to claim 27, wherein the non-identical ends contain non-identical promoters. 29. The method according to any one of claims 25-28, wherein the retroviral vector contains a heterologous promoter replacing the viral promoter in the 5'-LTR, such as a CMV promoter, an RSV promoter, an SV-40 promoter, a TK promoter, an MT promoter, or an inducible system such as Tet or Ecdysone. 30. The method according to any one of claims 25-29, wherein step (a) is carried out by 1) transfecting suitable packaging cells with vectors which comprise the randomly modified nucleotide sequences and which are integratable in virions produced by said packaging cells, 2) culturing said transfected packaging cells in a culture medium under conditions which facilitate production by the packaging cells of virions containing the randomly modified nucleotide sequences, 3) recovering and optionally concentrating said virions, and 4) transducing said substantially identical cells with the virions. 31. The method according to claim 30, wherein the packaging cells are selected from the group consisting of PE501, Bosc23, Psi2, GP+E86, PhoenixEco, PA317, GP+AM12, DA(ampho), Bing, FLYA13, ProPak, CRIP, PsiAM, Phoenix-Ampho, PG13, H9 (293GPG), and EcoPack. 32. The method according to any one of claims 25-31, wherein the virions are pseudotyped retrovirus produced by an ecotropic packaging cell line so as to confer broad tropism to the virions produced thereby, or wherein an ecotropic receptor has been introduced into the substantially identical cells so as to allow transduction with ecotropic virions. 33. The method according to claim 32, wherein the ecotropic receptor has been introduced in the substantially identical cells by means of transduction. 34. The method according to any of the preceding claims wherein the randomly modified nucleotide sequences are coupled to a nucleotide sequence encoding at least one fusion partner. 35. The method according to claim 34, wherein the fusion partner serves to facilitate expression and/or purification/isolation and/or further stabilization of the expression product. 36. The method according to claim 35, wherein the fusion partner includes a purification tag such as His6 tag, myc tag, BSP biotinylation target sequence, of BirA, flu tag, lacZ, and GST. 37. The method according to claim 34 or 35, wherein the fusion partner is a sorting signal or a targeting sequence. 38. The method according to claim 37, wh