Humanized 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 humanized immunobinder capable of binding to an antigen of interest, wherein the immunobinder comprises:
a variable heavy chain comprising three heavy chain complementarity determining regions (CDRs) and four framework region sequences having at least 90% identity to the four framework region sequences of SEQ ID NO: 4, wherein the framework region sequences have one or more amino acid residues selected from the group consisting of: Threonine (T) at position 24, Valine (V) at position 25, Glycine or Alanine (G/A) at position 56, Lysine (K) at position 82, Threonine (T) at position 84, Valine (V) at position 89 and Arginine (R) at position 108 (AHo numbering), and
a variable light chain comprising three light chain CDRs and a light chain framework sequence,
wherein the heavy chain and light chain CDRs are from an immunobinder 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 humanized immunobinder of claim 1 , wherein the immunobinder is an antibody.
3. The humanized immunobinder of claim 2 , wherein the antibody is a full-length immunoglobulin, Fab or scFv.
4. The humanized immunobinder of claim 1 , wherein the antigen of interest is an integral membrane protein.
5. The humanized immunobinder of claim 4 , wherein the integral membrane protein is a G protein-coupled receptor (GPCR) or an ion channel.
6. The humanized immunobinder of claim 4 , 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.
7. The humanized immunobinder of claim 1 , wherein the antigen of interest is a soluble antigen.
8. The humanized immunobinder of claim 1 , wherein the antigen of interest is expressed from an exogenous gene.
9. The humanized immunobinder of claim 1 , wherein the animal was immunized with said antigen of interest.
10. The humanized immunobinder 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.
11. The humanized immunobinder of claim 10 , wherein the first and/or second sortable label is a fluorescent label.
12. The humanized immunobinder of claim 11 , wherein the fluorescent label is a fluorescent protein or a fluorescent cellular label.
13. The humanized immunobinder of claim 1 , wherein the animal is a rabbit.
14. The humanized immunobinder 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.
15. The humanized immunobinder of claim 1 , wherein the antigen-expressing cell is a yeast or mammalian cell or yeast spheroblast.
16. The humanized immunobinder of claim 1 , wherein the variable light chain framework regions have sequences that have at least 90% identity to the framework region sequences of SEQ ID NO: 2.