Methods for identifying immunobinders of cell-surface antigens

Abstract

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 a B-cell clone that specifically binds to an antigen of interest comprising:
 (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 , further comprising isolating a nucleic acid molecule encoding an immunobinder from the B-cell clone that binds to an antigen of interest. 
     
     
         3 . The method of  claim 2 , further comprising producing an immunobinder capable of binding to an antigen of interest, wherein the immunobinder is produced by introducing the nucleic acid sequence into an expression environment such that the encoded immunobinder is produced. 
     
     
         4 . An immunobinder that binds to an antigen of interest produced by the method of  claim 3 . 
     
     
         5 . The immunobinder of  claim 4 , wherein the immunobinder is an antibody. 
     
     
         6 . The immunobinder of  claim 5 , wherein the antibody is a mouse, rabbit, chicken, camel, human, humanized, or chimeric antibody. 
     
     
         7 . The immunobinder of  claim 5 , wherein the antibody is a full length immunoglobulin, Fab, Dab, scFv, or Nanobody. 
     
     
         8 . The immunobinder of  claim 4 , wherein the antigen of interest is an integral membrane protein. 
     
     
         9 . The immunobinder of  claim 8 , wherein the integral membrane protein is a GPCR or an ion channel. 
     
     
         10 . The immunobinder of  claim 8 , 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. 
     
     
         11 . The method of  claim 1 , wherein the antigen of interest is an integral membrane protein. 
     
     
         12 . The method of  claim 11 , wherein the integral membrane protein is a GPCR or an ion channel. 
     
     
         13 . The method of  claim 11 , 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. 
     
     
         14 . 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. 
     
     
         15 . The method of  claim 14 , wherein the first and/or second sortable label is a fluorescent label. 
     
     
         16 . The method of  claim 15 , wherein the fluorescent label is a fluorescent protein or a fluorescent cellular label. 
     
     
         17 . The method of  claim 1 , wherein the animal is a rabbit. 
     
     
         18 . 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. 
     
     
         19 . The method of  claim 1 , wherein the antigen-expressing cell is a yeast or mammalian cell or yeast spheroblast.