ANTIGEN BINDING MOLECULES AND METHODS OF SCREENING THEREOF
Described herein are methods of generating a library of cells expressing a plurality of polypeptides or recombinant polypeptides activated by an antigen and methods of panning said library of cells against a target antigen. The methods can be utilized for screening a library of chimeric antigen receptors reactive to a target antigen.
1 . A method of screening a library of cells comprising:
a) contacting a plurality of cells with a target antigen; the plurality of cells comprising a library of recombinant polypeptides, wherein each recombinant polypeptide comprises an antigen binding domain, a transmembrane domain, an activation domain or an inhibition domain, and a first detectable marker, and wherein the antigen binding domain differs among the plurality of cells;
b) selecting cells that display expression of a first T cell activation marker, thereby producing a first subset of activated cells;
c) contacting the subset of activated cells with a plurality of cells not expressing the target antigen; and
d) selecting cells of the subset of activated cells that do not display expression of a second T cell activation marker, thereby producing a subset of low-background binding, activated cells.
2 . The method of claim 1 , further comprising contacting the subset of low-background, activated cells with the target antigen and selecting cells from the low-background, activated cells that display activation of a third T cell activation marker, thereby producing a subset of high antigen-binding, low-background binding activated cells.
3 . (canceled)
4 . (canceled)
5 . The method of claim 1 , wherein the target antigen is immobilized to a solid support.
6 . The method of claim 5 , wherein the solid support is a bead.
7 . The method of claim 5 , wherein the solid support is a column.
8 . The method of claim 1 , wherein the target antigen is a soluble antigen.
9 . The method of claim 5 , wherein the target antigen is conjugated to a detectable moiety.
10 . (canceled)
11 . The method of claim 1 , wherein the recombinant polypeptide comprises a detectable tag.
12 . (canceled)
13 . The method of claim 1 , wherein the first T cell activation marker, the T cell second activation marker, and the third T cell activation marker are the same.
14 . The method of claim 1 , wherein the plurality of cells further comprises a nucleic acid sequence encoding a reporter polypeptide.
15 . The method of claim 14 , wherein the reporter nucleic acid comprises a reporter gene under the control of an immune cell promoter, and wherein the second T cell activation marker or the third T cell activation marker comprises the reporter polypeptide.
16 . The method of claim 14 , wherein the reporter polypeptide comprises a fluorescent protein or a luciferase protein.
17 . The method of claim 15 , wherein the immune cell promoter comprises a nuclear factor κB (NFκB) promoter or a Nuclear factor of activated T-cells (NFAT) promoter.
18 . (canceled)
19 . (canceled)
20 . The method of claim 1 , wherein the first T cell activation marker comprises an endogenous T cell activation marker.
21 . The method of claim 1 , wherein the second T cell activation marker or the third T cell activation marker comprises an endogenous T cell activation marker.
22 . The method of claim 1 , wherein the first T cell activation marker is selected from the group consisting of CD69, CD25, and a combination thereof.
23 . The method of claim 1 , wherein the antigen binding domain comprises a single-chain variable fragment (scFv).
24 . The method of claim 1 , wherein each recombinant polypeptide comprises two or more different antigen binding domains.
25 . The method of claim 24 , wherein one or more of the two or more different antigen binding domains binds to CD3.
26 - 78 . (canceled)
79 . The method of claim 1 , wherein the library of recombinant polypeptides comprises at least 1×10 5 different antigen binding domains.