Methods for identifying LILRB-blocking antibodies

Patent metadata

Field	Value
Publication number	US-11906519-B2
Application number	US-201716321745-A
Country	US
Kind code	B2
Filing date	Jul 27, 2017
Priority date	Jul 29, 2016
Publication date	Feb 20, 2024
Grant date	Feb 20, 2024

Abstract

Provided herein are methods and compositions for the identification of modulators of ApoE-induced LILRB activation. Also provided herein are methods of treating cancer comprising the administration of an inhibitor of ApoE-induced LILRB activation. Also provided are methods of treating autoimmune disease or inhibiting the onset of transplant rejection or treating an inflammatory disorder comprising administering an agonist of ApoE-induced LILRB activation to a subject.

First claim

What is claimed is:

1. A method of identifying a modulator of Leukocyte Immunoglobulin-Like Receptor B4 (LILRB4) activation comprising:
(a) contacting a reporter cell with Apolipoprotein E (ApoE) and a candidate substance, wherein the reporter cell expresses a reporter gene that encodes a detectable reporter operably linked to a promoter regulated by activation of LILRB4; and
(b) detecting a level of LILRB4 activation in the reporter cell, wherein an increase or decrease in the level of LILRB4 activation as compared to a reference level measured from a reporter cell not treated with said candidate substance indicates that the candidate substance is a modulator of LILRB4 activation in the reporter cell.

2. The method of claim 1 , wherein the reporter cell expresses a receptor comprising an extracellular domain of LILRB4.

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

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

5. The method of claim 1 , wherein the receptor is expressed in the cell through a viral expression vector.

6. The method of claim 5 , wherein the viral expression vector is a retroviral expression vector.

7. The method of claim 1 , wherein the level of LILRB4 activation is detected based on the morphology or mobility of the cell.

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

9. The method of claim 8 , wherein the promoter is a nuclear factor of activated T cells (NFAT) promoter.

10. The method of claim 8 , wherein the promoter is a chemokine ligand 2 promoter, a chemokine ligand 4 promoter, a chemokine ligand 5 promoter, an Interleukin 6R promoter, an Interleukin 8 promoter, a glycoprotein 130 promoter, an Oncostatin M promoter, a Tissue Metalloproteinase Inhibitor ½ promoter, a Tumor Necrosis Factor receptor I/II promoter, a urokinase Plasminogen Activator Receptor uPAR promoter or an arginase-1 promoter.

11. The method of claim 8 , wherein the detectable label is a colorometric label, fluorescent label, bioluminescent label, or chemiluminescent label.

12. The method of claim 8 , wherein the detectable label is green fluorescent protein, yellow fluorescent protein, red fluorescent protein, or D-luciferin.

13. The method of claim 8 , wherein the detectable label is GFP.

14. The method of claim 8 , wherein detecting step comprises flow cytometry analysis or quantification of luminescence.

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

16. The method of claim 15 , 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.

17. The method of claim 15 , wherein the antibody is a monoclonal antibody.

18. The method of claim 1 , wherein the reference level is obtained in the reporter cell when it is contacted with only ApoE.

19. The method of claim 1 , wherein the ApoE is recombinant.

20. The method of claim 1 , wherein the ApoE is human ApoE or mouse ApoE.

21. The method of claim 20 , wherein the human ApoE or mouse ApoE is isolated from human serum or mouse serum, respectively.

22. The method of claim 1 , wherein the ApoE is further defined as ApoE2, ApoE3, or ApoE4.

23. The method of claim 1 , wherein an increase in the level of LILRB4 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 LILRB4 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.

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

Assignees

• Univ Texas

Inventors

• Zhang Chengcheng
• An Zhiqiang
• Zhang Ningyan
• Deng mi
• Kim Jaehyup
• Gui Xun

Classifications

• G01N33/5759

  involving compounds localised on the membrane of tumour or cancer cells · CPC title

• G01N33/57505 Primary

  of the blood, e.g. leukaemia · CPC title

• G01N33/575

  for cancer · CPC title

• G01N33/57426 Primary

  Physics · mapped topic

• G01N15/14

  Optical investigation techniques, e.g. flow cytometry · CPC title