# US 11,906,519 B2 · Methods for identifying LILRB-blocking antibodies

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- Number: US 11,906,519 B2
- Document ID: US11906519B2
- Status: Granted
- Kind: B2
- Application number: 16/321,745
- Filed: 2017-07-27
- Published: 2021-11-11
- Granted: 2024-02-20
- Earliest claimed: 2016-07-29
- Assignees: Board of Regents, The University of Texas System
- Applicants: The Board of Regents of the University of Texas System
- Inventors: Chengcheng Zhang; Zhiqiang An; Ningyan Zhang; Mi Deng; Jaehyup Kim; Xun Gui
- Web page: https://patentgrep.com/patent/US11906519B2

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- G01N 33/57505: of the blood, e.g. leukaemia
- A61P 35/02: specific for leukemia
- G01N 15/14: Optical investigation techniques, e.g. flow cytometry
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 21/6456: {Spatial resolved fluorescence measurements; Imaging}
- G01N 33/4915: {using flow cells (flow cytometry G01N15/14)}
- G01N 33/5023: {on expression patterns}
- G01N 33/575: for cancer
- G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- G01N 15/01: specially adapted for biological cells, e.g. blood cells (investigating sedimentation of particle suspensions in blood G01N15/05)
- G01N 2015/1006: {for cytology}
- G01N 2021/6439: {with indicators, stains, dyes, tags, labels, marks}
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 2)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

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.

### Claim 11 (depends on 8)

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

### Claim 12 (depends on 8)

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

### Claim 13 (depends on 8)

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

### Claim 14 (depends on 8)

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

### Claim 15 (depends on 1)

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

### Claim 16 (depends on 15)

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.

### Claim 17 (depends on 15)

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

### Claim 18 (depends on 1)

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

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 1)

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

### Claim 21 (depends on 20)

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

### Claim 22 (depends on 1)

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

### Claim 23 (depends on 1)

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.

### Claim 24 (depends on 1)

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.

### Claim 25 (depends on 1)

The method of claim 1, wherein the candidate substance is linked to a substrate.

### Claim 26 (depends on 1)

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