Ligand screening and discovery
View Patent ↗Disclosed is a method that includes: (i) providing a plurality of initial nucleic acid cassettes that include: a) a first coding region encoding a first immunoglobulin variable domain, b) a second coding region encoding a second immunoglobulin variable domain, and c) a ribosomal binding site disposed between the first and second coding regions for translation of the second polypeptide in a first expression system, wherein the first and second coding regions are in the same translational orientation; (ii) modifying each nucleic acid cassette of the plurality in a single reaction mixture so that it is functional in a second expression system, wherein the first and second region remain physically attached during the modifying; (iii) introducing each modified nucleic acid cassette into a mammalian cell to produce a mixture of transfected cells; and (iv) expressing each modified nucleic acid cassette in the transfected cells.
1. A method for evaluating display library members, the method comprising:
providing a first plurality of nucleic acid library members, each library member encoding a first immunoglobulin or function fragment thereof that is displayed on the surface of a prokaryotic cell, wherein each cassette comprises a first nucleic acid sequence encoding a first immunoglobulin variable domain, a second nucleic acid sequence encoding a second immunoglobulin variable domain, and a ribosomal binding site disposed between the first and second nucleic acid sequences, wherein the first and second nucleic acid sequences are in the same translational orientation and can be transcribed as a single transcript;
determining an assessment for each library member with respect to a property;
sorting information about the assessments of the library members in a computer database;
filtering the information to identify a subset of the library members; and
reformatting each library member in a single reaction mixture for expression in a mammalian cell, wherein the first and second nucleic acid sequences remain physically attached during the formatting.
2. The method of claim 1 , wherein the display library members comprise phage display library members.
3. The method of claim 1 , wherein the determining comprises a binding assay.
4. The method of claim 1 , wherein the reformatting comprises inserting a segment that comprises an internal ribosome entry site between the first and second nucleic acid sequences.
5. The method of claim 4 , wherein the segment further comprises a signal sequence functional in a mammalian cell.
6. The method of claim 4 , wherein the segment further comprises a polyA addition regulatory sequence.
7. The method of claim 1 , wherein the reformatting comprises attaching the second nucleic acid sequence in frame to a sequence encoding a constant domain that comprises an Fc domain or a cytotoxic domain to form a fusion protein.
8. The method of claim 7 , wherein the reformatting comprises attaching the second nucleic acid sequence in frame to a sequence encoding a constant domain that comprises a cytotoxic domain to form a fusion protein.
9. The method of claim 1 , further comprising introducing reformatted library members into mammalian cells and screening the mammalian cells for production of a polypeptide encoded by a respective reformed library member.
10. The method of claim 7 , wherein the reformatting comprises attaching the second nucleic acid sequence in frame to a sequence encoding a constant domain that comprises an Fc domain to form a fusion protein.