METHODS OF ANTIBODY PANNING AGAINST TARGET PROTEINS
Disclosed herein are methods for selecting polypeptides that specifically bind to a target protein. In some cases, the methods involve panning a library of polypeptides for target polypeptides that bind to a target protein on the surface of a cell. Additionally, the disclosure provides cell lines expressing target proteins for use with the disclosed methods.
1 . A method of selecting for a polypeptide that selectively binds to a target protein, said method comprising:
(a) contacting a first polypeptide pool comprising a plurality of polypeptides with a first entity that does not comprise said target protein to form a first mixture;
(b) removing said first entity from said first mixture, thereby generating a first depleted polypeptide pool;
(c) contacting said first depleted polypeptide pool with a second entity that comprises said target protein at its surface;
(d) collecting polypeptides that bind to said second entity, thereby generating a target polypeptide pool;
(e) contacting a second polypeptide pool comprising a plurality of polypeptides with the first entity to form a second mixture;
(f) removing the first entity from the second mixture, thereby generating a second depleted polypeptide pool;
(g) contacting said second depleted polypeptide pool with a third entity that does not comprise said target protein, wherein said third entity is the same or different from said first entity;
(h) collecting polypeptides that bind to said third entity, thereby generating an off-target polypeptide pool; and
(i) identifying at least one polypeptide that is present in said target polypeptide pool and is not present in said off-target polypeptide pool, thereby selecting for a polypeptide that selectively binds to said target protein.
2 . The method of claim 1 , further comprising, prior to (i), performing one or more rounds of (a)-(h), each successive round using a target polypeptide pool generated in (d) as a first polypeptide pool in (a), and using an off-target polypeptide pool generated in (h) as a second polypeptide pool in (e).
3 . The method of claim 1 , wherein said identifying of (i) comprises sequencing a polynucleotide tag attached to said at least one polypeptide.
4 . (canceled)
5 . The method of claim 1 , wherein said identifying of (i) comprises sequencing polynucleotide tags attached to said plurality of target polypeptides and sequencing polynucleotide tags to said plurality of off-target polypeptides.
6 . The method of claim 5 , wherein each of said polynucleotide tags is different for each target polypeptide and for each off-target polypeptide.
7 . (canceled)
8 . The method of claim 2 wherein, for each successive round of (a)-(h), said first entity, said second entity, and said third entity are of a different type from a preceding round.
9 . The method of claim 8 , wherein, for each successive round of (a)-(h), said first entity, said second entity, and said third entity are from a different species than from a preceding round.
10 . The method of claim 1 , wherein said first entity comprises a plurality of first entities, said second entity comprises a plurality of second entities, and said third entity comprises a plurality of third entities.
11 - 14 . (canceled)
15 . The method of claim 1 , wherein said target protein is a cell-surface protein, a membrane-bound protein, or a protein engineered to be expressed at a cell surface.
16 . The method of claim 1 , wherein said target protein is a transmembrane protein or an integral membrane protein
17 . The method of claim 16 , wherein said target protein is the transmembrane protein, and wherein said transmembrane protein is a multi-pass transmembrane protein.
18 . The method of claim 1 , wherein said target protein is selected from the group consisting of: a ligand-gated ion channel, a voltage-gated ion channel, and a G protein-coupled receptor (GPCR).
19 - 32 . (canceled)
33 . The method of claim 1 , wherein any one of said first entity, said second entity, and said third entity comprises a whole cell.
34 . (canceled)
35 . (canceled)
36 . The method of claim 1 , wherein said first polypeptide pool, said second polypeptide pool, or both, is an antibody library comprising a plurality of antibodies or antibody fragments.
37 . (canceled)
38 . The method of claim 1 wherein said first polypeptide pool, said second polypeptide pool, or both comprises at least ten polypeptides.
39 - 46 . (canceled)
47 . The method of claim 1 , further comprising, performing one or more affinity maturation steps on said at least one polypeptide to generate a polypeptide with increased affinity for said target protein.
48 - 73 . (canceled)
74 . The method of claim 3 , wherein said sequencing comprises next generation sequencing.
75 . The method of claim 33 , wherein said first entity comprises a plurality of first cells, said second entity comprises a plurality of second cells, and said third entity comprises a plurality of third cells.
76 . The method of claim 33 , wherein, for each successive round of (a)-(h), the whole cell of said first entity, said second entity, and said third entity are different cells than a preceding round.