Methods for identifying immunobinders of cell-surface antigens
View Patent ↗The invention provides methods for identifying immunobinders, such as scFv antibodies, capable of specifically binding to cell surface antigens, and compositions identified according to said methods.
1. A method for identifying an immunobinder that specifically binds to a cell surface antigen of interest comprising:
(a) providing a plurality of immunobinder-expressing cells comprising a first sortable label;
(b) providing a plurality of antigen-expressing cells comprising a second sortable label, wherein the antigen of interest is displayed at the surface of the antigen-expressing cell;
(c) contacting the antigen-expressing cells with the immunobinder-expressing cells; and
(d) separating from the plurality of immunobinder-expressing cells, one or more immunobinder-expressing cells that can specifically bind to the antigen expressing cells using a cell sorter, wherein the presence of the first and second sortable label in a single cellular complex is indicative of the binding of an immunobinder-expressing cell to an antigen-expressing cell, thereby identifying an immunobinder that binds to a antigen of interest.
2. The method of claim 1 , further comprising clonally isolating the immunobinder-expressing cells obtained in step (d), optionally followed by clonal expansion of the clonally isolated cells.
3. The method of claim 2 , further comprising obtaining the immunobinder-encoding nucleic acid sequence from the isolated immunobinder-expressing cells.
4. The method of claim 2 , further comprising subjecting the isolated immunobinder-expressing cells to a cell-based assay to functionally characterize the immunobinder.
5. The method of claim 1 , wherein the immunobinder is an antibody.
6. The method of claim 5 , wherein the antibody is a mouse, rabbit, chicken, camel, human, humanized, or chimeric antibody.
7. The method of claim 6 , wherein the antibody is a full length immunoglobulin, Fab, Dab, scFv, or Nanobody.
8. The method of claim 1 , wherein the antigen of interest is expressed from an exogenous gene.
9. The method of claim 1 , wherein the antigen of interest is a genetically engineered antigen expressed from an expression vector.
10. The method of claim 1 , wherein the antigen of interest is an integral membrane protein.
11. The method of claim 10 , wherein the integral membrane protein is a GPCR or an ion channel.
12. The method of claim 11 , wherein the GPCR is CXCR2, CXCR1, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAV1.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosin-1-phosphate receptor (S1P1R) or NMDA channel.
13. The method of claim 1 , wherein the first or second sortable label is a fluorescent label.
14. The method of claim 13 , wherein the fluorescent label is a fluorescent protein, an antibody/fluor conjugate, or a fluorescent cellular label.
15. The method of claim 1 , wherein the antigen-expressing cells are yeast cells, yeast spheroblasts or mammalian cells.
16. The method of claim 15 , wherein the antigen-expressing cells are human cells.
17. The method of claim 1 , wherein the immunobinder-expressing cells are yeast or mammalian cells.
18. The method of claim 1 , wherein the immunobinder-expressing cells are B-cells.
19. The method of claim 18 , wherein the B-cells are rabbit 13-cells.
20. The method of claim 18 , wherein the B-cells are isolated from an immunized animal.
21. The method of claim 20 , wherein the animal is immunized by DNA vaccination.
22. The method of claim 1 , wherein the immunobinder-expressing cells comprise an immunobinder expressed from an expression vector.