Methods for producing 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 of producing an immunobinder capable of binding to an antigen of interest, wherein the immunobinder is produced by introducing a nucleic acid sequence into a host cell such that the immunobinder is produced, wherein the nucleic acid sequence encodes the immunobinder, and wherein the immunobinder-encoding nucleic acid sequence is derived from a B-cell clone identified by:
(a) isolating B-cells from an animal;
(b) contacting the B-cells with antigen-expressing cells expressing the antigen of interest or an artificial cell-like body displaying the antigen of interest; and,
(c) isolating one or more complexes in which a B-cell is binding to an antigen-expressing cell or said artificial cell-like body displaying the antigen of interest, wherein the isolating is accomplished using a cell sorter, and wherein the B-cell in an isolated complex is identified as a B-cell clone that binds to the antigen of interest.
2. The method of claim 1 , wherein the immunobinder is an antibody.
3. The method of claim 2 , wherein the antibody is a mouse, rabbit, chicken, camel, rat, hamster, sheep, goat, human, humanized, or chimeric antibody.
4. The method of claim 3 , wherein the antibody is a full-length immunoglobulin, Fab, Dab, scFv, or Nanobody.
5. The method of claim 1 , wherein the antigen of interest is an integral membrane protein.
6. The method of claim 5 , wherein the integral membrane protein is a GPCR or an ion channel.
7. The method of claim 5 , wherein the integral membrane protein 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.
8. The method of claim 1 , wherein the antigen of interest is a soluble antigen.
9. The method of claim 1 , wherein the antigen of interest is expressed from an exogenous gene.
10. The method of claim 1 , wherein the animal was immunized with said antigen of interest.
11. The method of claim 1 , wherein the B-cells are labeled with a first sortable label, and the antigen-expressing cells or the artificial cell-like body displaying the antigen of interest are labeled with a second sortable label.
12. The method of claim 11 , wherein the first and/or second sortable label is a fluorescent label.
13. The method of claim 12 , wherein the fluorescent label is a fluorescent protein or a fluorescent cellular label.
14. The method of claim 1 , wherein the animal is a rabbit.
15. The method of claim 1 , wherein the artificial cell-like body is a bead coated with the antigen of interest, a liposome or a single-layer membrane body.
16. The method of claim 1 , wherein the antigen-expressing cell is a yeast or mammalian cell or yeast spheroblast.