Methods for identifying lilrb-blocking antibodies

Patent metadata

Field	Value
Publication number	US-2024101667-A1
Application number	US-202118255745-A
Country	US
Kind code	A1
Filing date	Dec 2, 2021
Priority date	Dec 3, 2020
Publication date	Mar 28, 2024
Grant date	—

Abstract

Provided herein are methods and compositions for the identification of modulators of LILRB3 activation. Also provided herein are methods of treating cancer comprising the administration of an inhibitor LILRB3 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 LILRB3 activation to a subject.

First claim

1 . A method of identifying a modulator of LILRB3 activation comprising:
(a) contacting a reporter cell with galectin-4 and a candidate substance, wherein said reporter cell expresses a chimeric receptor having an external domain of LILRB3; and (b) detecting a level of receptor activation in the reporter cell, wherein a change in the level of receptor activation as compared to a reference level indicates that the candidate substance is a modulator of LILRB3 activation.

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

3 . The method of claim 1 , wherein the receptor comprises an intracellular domain of paired immunoglobulin-like receptor β (PILRβ).

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

5 . The method of claim 4 , wherein the viral expression vector is a retroviral expression vector.

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

7 . The method of claim 6 , wherein the promoter is a nuclear factor of activated T cells (NFAT) promoter.

8 . The method of claim 6 , wherein the promoter is an inducible promoter, a tissue specific promoter or a constitutive promoter.

9 . The method of claim 6 , wherein the detectable label is a colorometric label, fluorescent label, bioluminescent label, or chemiluminescent label.

10 . The method of claim 6 , wherein the detectable label is GFP, YFP, RFP, or D-luciferin.

11 . The method of claim 6 , wherein detecting step comprises flow cytometry analysis or quantification of luminescence.

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

13 . The method of claim 12 , 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.

14 . The method of claim 12 , wherein the antibody is a monoclonal antibody.

15 . The method of claim 1 , wherein the reference level is obtained in the reporter cell when it is contacted with galectin-4.

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

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

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

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

20 . A composition comprising:
a candidate LILRB3 modulator; galectin-4; and a reporter cell that expresses a chimeric receptor having an extracellular domain of LILRB3, wherein the reporter cell has a phenotype indicating receptor activation.

21 . An isolated monoclonal antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) variable region (VH) and a light chain (LC) variable region (VL) comprising clone-paired CDR sequences as set forth in Tables 3 and 4; and variants thereof wherein one or more of the HC-CDRs and/or LC-CDRs has one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

22 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 , wherein the isolated monoclonal antibody is a murine, a rodent, a rabbit, a chimeric, humanized, or human antibody.

23 . The isolated monoclonal antibody or an antigen-binding fragment thereof of claim 21 , wherein the antigen-binding fragment is a recombinant ScFv (single chain fragment variable) antibody, Fab fragment, F(ab′)2 fragment, or Fv fragment.

24 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 , wherein the isolated monoclonal antibody is a human antibody.

25 . The isolated monoclonal antibody or an antigen-binding fragment thereof of claim 21 , wherein the VH and VL chains have amino acid sequences at least 90% or 95% identical to clone-paired sequences of Appendices II and IV, respectively.

26 . The isolated monoclonal antibody or an antigen-binding fragment thereof of claim 21 , wherein the VH and VL chains are encoded by nucleic acid sequences at least 80% or 90% identical to clone-paired sequences of Appendices I and III, respectively.

27 . The isolated monoclonal antibody or an antigen-binding fragment thereof of claim 21 , wherein the VH and VL chains have amino acid sequences identical to clone-paired sequences of Appendices II and IV, respectively.

28 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 wherein the VH and VL chains are encoded by nucleic acid sequences identical to clone-paired sequences of Appendices I and III, respectively.

29 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 , wherein the isolated monoclonal antibody is a humanized antibody.

30 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 , wherein the antibody is a chimeric antibody.

31 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 , which induces the activation of LILRB3.

32 . The isolated monoclonal antibody or an antigen binding fragment thereof of claim 21 , which suppresses the activation of LILRB3.

33 . An isolated monoclonal antibody or an antigen binding fragment thereof, which competes for the same epitope with the isolated monoclonal antibody or an antigen-binding fragment thereof according to claim 21 .

34 . A pharmaceutical composition comprising the isolated monoclonal antibody or an antigen-binding fragment thereof according to claim 21 , and a pharmaceutically acceptable carrier.

35 . An isolated nucleic acid that encodes the isolated monoclonal antibody according to any one of claim 21 .

36 . A vector comprising the isolated nucleic acid of claim 35 .

37 . A host cell comprising the vector of claim 36 .

38 . The host cell of claim 37 , wherein the host cell is a mammalian cell.

39 . The host cell of claim 37 , wherein the host cell is a CHO cell.

40 . A hybridoma or engineered cell encoding and/or producing the isolated monoclonal antibody according to claim 21 .

41 . A process of producing an antibody, comprising culturing the host cell of claim 37 under conditions suitable for expressing the antibody, and recovering the antibody.

42 . A chimeric antigen receptor (CAR) protein comprising an antigen-binding fragment according to claim 21 .

43 . An isolated nucleic acid that encodes a CAR protein of claim 42 .

44 . A vector comprising the isolated nucleic acid of claim 43 .

45 . An engineered cell comprising the isolated nucleic acid of claim 43 .

46 . The engineered cell of claim 45 , wherein the cell is a T cell, NK cell, or macrophage.

47 . A method of treating or amel

Assignees

• Univ Texas

Inventors

• Zhang Chengcheng
• Wu Guojin
• Kim Jaehyup
• Chen Heyu
• Deng mi
• An Zhiqiang
• Zhang Ningyan
• Huang Ryan

Classifications

• G01N33/575

  for cancer · CPC title

• G01N2333/705

  Assays involving receptors, cell surface antigens or cell surface determinants · CPC title

• G01N2333/4724

  Lectins · CPC title

• G01N2500/02

  Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) · CPC title

• G01N33/5023

  on expression patterns · CPC title