ILT3 ligand
The present invention provides antibodies and antigen-binding fragments thereof that bind ILT3, ILT3 ligand (i.e., PI16) or a complex between ILT3 and the ILT3 ligand. Methods for determining whether ILT3 and the ILT3 ligand bind together or whether a substance agonizes or antagonizes such binding are also provided.
1. A complex between ILT3 and ILT3 ligand wherein the ILT3 ligand is peptidase inhibitor 16 (PI16), wherein the ILT3 or ILT3 ligand or both are detectably labeled, bound to a metal surface, bound to a recombinant antibody or antigen-binding fragment thereof, or bound to a solid insoluble substrate.
2. The complex of claim 1 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, or wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
3. The complex of claim 1 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, and wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
4. The complex of claim 1 , wherein the ILT3 is detectably labeled with a FRET (fluorescence resonance energy transfer) acceptor and ILT3 ligand is detectably labeled with a FRET donor; or
ILT3 is detectably labeled with a FRET donor and ILT3 ligand is detectably labeled with a FRET acceptor; and wherein the complex is determined to form if FRET between the acceptor and donor is detected.
5. A method for determining whether a substance agonizes or antagonizes binding between ILT3 and ILT3 ligand, the method comprising:
contacting ILT3 ligand with ILT3 in vitro in the presence of the substance and determining whether the ILT3 ligand and the ILT3 bind to each other to form a complex,
wherein the substance is determined to agonize said binding if more of the complex is determined in the presence of the substance than in the absence of the substance and wherein the substance is determined to antagonize said binding if less of the complex is determined in the presence of the substance than in the absence of the substance; and
wherein the ILT3 ligand is PI16.
6. The method of claim 5 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, or wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
7. The method of claim 5 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, and wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
8. A method for determining whether a substance agonizes or antagonizes binding between ILT3 and ILT3 ligand, the method comprising:
contacting ILT3 ligand with ILT3 in vitro in the presence of the substance and determining whether the ILT3 ligand and the ILT3 bind to each other to form a complex,
wherein the ILT3 is bound to a gold or silver metal surface on the surface of a glass slide, is contacted with the ILT3 ligand, and the metal is illuminated through the glass with polarized light wherein, if a change in the refractive index of medium in close vicinity to the metal surface illuminated with the light is detected, then the complex is determined to have formed between the ILT3 and the ILT3 ligand; or
the ILT3 ligand is bound to a gold or silver metal surface on the surface of a glass slide, is contacted with the ILT3 and the metal is illuminated through the glass with polarized light wherein, if a change in the refractive index of medium in close vicinity to the metal surface illuminated with the light is detected, then the complex is determined to have formed between the ILT3 and the ILT3 ligand;
wherein the substance is determined to agonize said binding between ILT3 and ILT3 ligand if more of the complex is determined in the presence of the substance than in the absence of the substance and wherein the substance is determined to antagonize said binding between ILT3 and ILT3 ligand if less of the complex is determined in the presence of the substance than in the absence of the substance; and
wherein the ILT3 ligand is PI16.
9. The method of claim 8 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, or wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
10. The method of claim 8 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, and wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
11. A method for determining whether a substance agonizes or antagonizes binding between ILT3 and ILT3 ligand, the method comprising:
contacting ILT3 ligand with ILT3 in vitro in the presence of the substance and determining whether the ILT3 ligand and the ILT3 bind to each other form a complex,
wherein the ILT3 ligand is expressed on the surface of a macrophage cell and the ILT3 is fused to a cytosolic domain of CD3 zeta and is expressed on the surface of a T-cell that comprises an NFAT promoter operably linked to a reporter gene and wherein binding between ILT3 and ILT3 ligand is determined if reporter gene expression in the T-cell is detected,
wherein the substance is determined to agonize said binding between ILT3 and ILT3 ligand if more of the complex is determined in the presence of the substance than in the absence of the substance and wherein the substance is determined to antagonize said binding between ILT3 and ILT3 ligand if less of the complex is determined in the presence of the substance than in the absence of the substance; and
wherein the ILT3 ligand is PI16.
12. The method of claim 11 , wherein the reporter gene is luciferase.
13. The method of claim 11 , wherein the T-cell is a Jurkat cell.
14. The method of claim 11 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, or wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
15. The method of claim 11 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, and wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
16. A method for determining whether a substance agonizes or antagonizes binding between ILT3 and ILT3 ligand, the method comprising:
contacting ILT3 ligand with ILT3 in the presence of the substance in vitro and determining whether the ILT3 ligand and the ILT3 bind to each other to form a complex,
wherein the formation of the complex is determined by binding the ILT3 with an antibody or antigen-binding fragment thereof which is immobilized to a solid phase, removing the bound ILT3 from the solid phase, and determining the presence of ILT3 ligand bound to the ILT3, or
wherein the formation of the complex is determined by binding the ILT3 ligand with an antibody or antigen-binding fragment thereof which is immobilized to a solid phase, removing the bound ILT3 ligand from the solid phase, and determining the presence of ILT3 bound to the ILT3 ligand; and
wherein the substance is determined to agonize said binding between ILT3 and ILT3 ligand if more of the complex is determined in the presence of the substance than in the absence of the substance and wherein the substance is determined to antagonize said binding between ILT3 and ILT3 ligand if less of the complex is determined in the presence of the substance than in the absence of the substance; and
wherein the ILT3 ligand is PI16.
17. The method of claim 16 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, or wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.
18. The method of claim 16 , wherein the ILT3 comprises amino acids 22-259 of SEQ ID NO:1, and wherein the ILT3 ligand comprises SEQ ID NO: 42, 43, or 44.