Methods for identifying lilrb ligands and blocking antibodies

Patent metadata

Field	Value
Publication number	US-2025172557-A1
Application number	US-202318842528-A
Country	US
Kind code	A1
Filing date	Mar 1, 2023
Priority date	Mar 1, 2022
Publication date	May 29, 2025
Grant date	—

Abstract

Disclosed are the identification of multiple proteins that can bind to LILR receptors. based on which a screening system for LILR antagonist and blocking antibodies has been developed.

First claim

What is claimed is:

1 . A method of identifying a modulator of LILR activation comprising:
(a) contacting a reporter cell with a ligand of LILR and a candidate substance with potential to modulate LILR reporter activation; and (b) detecting a level of LILR reporter activation in the reporter cell, wherein a change in the level of LILR reporter activation as compared to a reference level indicates that the candidate substance is a modulator of LILR activation.

2 . The method of claim 1 , wherein the reporter cell expresses a receptor comprising an extracellular domain of LILR.

3 . The method of claim 1 , wherein the cell is a mouse T-cell hybridoma cell.

4 . The method of claim 2 , wherein the LILR is further defined as LILRA1, LILRA3, LILRB1, LILRB2, LILR3, LILRB4, LILRB5, or LILRA6.

5 . The method of claim 4 , wherein (a) the ligand of LILRA1 is Galectin-1, Galectin-3, Galectin-4, Galectin-7, Galectin-8, Galectin-10, Galectin-12, or Galectin-13, (b) the ligand of LILRA3 is Galectin-1, Galectin-4, Galectin-7, Galectin-8, Galectin-12, or Galectin-13, or (c) the ligand of LILRA6 is Galectin-4 or Galectin-7.

6 . The method of claim 4 , wherein (a) the ligand of LILRB1 is Galectin-4 or Galectin-7, (b) the ligand of LILRB2 is Cystatin SA (CST2), EPH Receptor A4 (EphA4), EPH Receptor A7 (EphA7), Galectin-1, Galectin-2, Galectin-3, Galectin-4, Galectin-5, Galectin-7, Galectin-8, Galectin-12, Galectin-13 or claudin 22 (CLDN22), (c) the ligand of LILRB3 is Galectin-4 or Galectin-7, (d) the ligand of LILRB4 is SCG2, Galectin-4, Galectin-5 or Galectin-7, or (e) the ligand of LILRB5 is EphA4, EphA7, CST2, CLDN22, Galectin-1, Galectin-2, Galectin-3, Galectin-4, Galectin-5, Galectin-7, Galectin-8, or Galectin-12 or elastin.

7 . The method of claim 2 , wherein the receptor further comprises an intracellular domain of paired immunoglobulin-like receptor β (PILRβ).

8 . The method of claim 7 , wherein the ligand of PILRβ is Galectin-7 or Galectin-12.

9 . The method of claim 2 , wherein the receptor is expressed in the cell through a viral expression vector.

10 . The method of claim 9 , wherein the viral expression vector is a retroviral expression vector.

11 . The method of claim 1 , wherein the level of LILR activation is detected based on the morphology or mobility of the cell.

12 . The method of claim 1 , wherein the reporter cell expresses a reporter gene that encodes a protein that emits or generates a detectable label and is operably linked to a promoter regulated by activation of the receptor.

13 . The method of claim 12 , wherein the promoter is a nuclear factor of activated T cells (NFAT) promoter.

14 . The method of claim 12 , wherein the promoter is a CCL2 promoter, a CCL4 promoter, a CCL5 promoter, an IL-6R promoter, an IL-8 promoter, a gpl30 promoter, an OSM promoter, a TIMP-1/2 promoter, a TNF-R1/II promoter, a uPAR promoter or an arginase-1 promoter.

15 . The method of claim 12 , wherein the detectable label is a colorometric label, fluorescent label, bioluminescent label, or chemiluminescent label.

16 . The method of claim 12 , wherein the protein encoded by the reporter gene is GFP, YFP, RFP, or Luciferase.

17 . The method of claim 12 , wherein the detectable label is D-luciferin.

18 . The method of claim 12 , wherein detecting step comprises flow cytometry analysis or quantification of luminescence.

19 . The method of claim 1 , wherein the candidate substance is an antibody.

20 . The method of claim 19 , wherein the antibody is a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab′, a F(ab′)2, a Fv, or a scFv.

21 . The method of claim 19 , wherein the antibody is a monoclonal antibody.

22 . The method of claim 1 , wherein the reference level is obtained in the reporter cell when it is contacted with the ligand of LILRB in the absence of the candidate substance.

23 . The method of claim 1 , wherein an increase in the level of LILRB activation as compared to the reference level indicates that the modulator is an agonist.

24 . The method of claim 1 , wherein a decrease in the level of LILRB activation as compared to the reference level indicates that the modulator is an antagonist.

25 . The method of claim 1 , wherein the candidate substance is linked to a substrate or a cell expressing FcR.

26 . A composition comprising: a candidate LILR modulator;
a ligand of LILR; and a reporter cell that expresses a receptor comprising an extracellular domain of LILR, wherein the reporter cell has a phenotype indicating LILR activation.

27 . The composition of claim 26 , wherein the candidate LILR modulator is an antibody.

28 . The composition of claim 26 , wherein the receptor further comprises an intracellular domain of PILR.

29 . The composition of claim 26 , wherein the reporter cell further comprises a reporter gene that encodes a protein that emits or generates a detectable label and that is operably linked to a promoter regulated by activation of the receptor, such as wherein the protein encoded by the reporter gene is GFP.

30 . The composition of claim 26 , further comprising a cell expressing FcR.

31 . A composition comprising:
a candidate LILR modulator; a cell expressing FcR; and a reporter cell that expresses a receptor comprising an extracellular domain of LILR, wherein the reporter cell has a phenotype indicating LILR activation.

32 . The composition of claim 31 , wherein the candidate LILR modulator is an antibody.

33 . The composition of claim 31 , wherein the receptor further comprises an intracellular domain of PILR.

34 . The composition of claim 31 , wherein the reporter cell further comprises a reporter gene that encodes a protein that emits or generates a detectable label and that is operably linked to a promoter regulated by activation of the receptor.

35 . The composition of claim 34 , wherein the protein encoded by the reporter gene is GFP.

36 . A method of treating cancer in a subject comprising administering an effective amount of an inhibitor of ligand-induced LILR activation to a subject.

37 . The method of claim 36 , wherein the inhibitor of ligand-induced LILR activation is an antibody.

38 . The method of claim 36 , wherein the inhibitor of ligand-induced LILR activation is a protein or peptide.

39 . A method of treating autoimmune disease or inhibiting the onset of transplant rejection or treating an inflammatory disorder in a subject comprising administering an effective amount of an agonist of ligand-induced LILR activation to a subject.

40 . The method of claim 39 , wherein the agonist of ligand-induced LILR activation is an antibody.

41 . The method of claim 39 , wherein the agonist of ligand-induced LILR activation is a protein or peptide.

Assignees

• Univ Texas

Inventors

• Zhang Chengcheng
• An Zhiqiang
• Zhang Ningyan
• He Yubo
• Huang Ryan
• Kim Jaehyup

Classifications

• G01N2500/10

  involving cells · CPC title

• G01N2333/70503

  Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3 · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C12Q1/6897 Primary

  involving reporter genes operably linked to promoters · CPC title

• C07K2317/76

  Antagonist effect on antigen, e.g. neutralization or inhibition of binding · CPC title