Intercellular labeling of ligand-receptor interactions

Patent metadata

Field	Value
Publication number	US-10053683-B2
Application number	US-201514875140-A
Country	US
Kind code	B2
Filing date	Oct 5, 2015
Priority date	Oct 3, 2014
Publication date	Aug 21, 2018
Grant date	Aug 21, 2018

Abstract

An sortase-mediated intercellular labeling method allowing for tracking ligand-receptor interaction both in vitro and in vivo; and uses thereof for tracking molecule interactions both in vitro and in vivo, identifying modulators of ligand-receptor interaction, identifying potential binding partners of a protein of interest, identifying B cells expressing high affinity B cell receptors to antigens, and identifying the antigen to which a T cell of interest binds.

First claim

What is claimed is:

1. An intercellular labeling method comprising:
(i) providing a first cell expressing a first polypeptide on the surface of the first cell, the first polypeptide comprising a sortase acceptor peptide, which is located at the N-terminus of the first polypeptide;
(ii) providing a second cell expressing a second polypeptide on the surface of the second cell, the second polypeptide comprising a sortase or an active fragment thereof; and
(iii) contacting the first cell with the second cell in the presence of a sortase substrate comprising a sortase recognition sequence, wherein the sortase substrate is associated with a detectable label;
wherein upon interaction between the first cell and the second cell, the sortase or the active fragment thereof links the sortase substrate to the first polypeptide, thereby labeling the first cell expressing the first polypeptide, and wherein the first polypeptide the second polypeptide, or both, are fusion polypeptides.

2. The intercellular labeling method of claim 1 , wherein the first polypeptide is a fusion polypeptide comprising the sortase acceptor peptide and one member of a receptor-ligand pair; and wherein the second polypeptide is a fusion polypeptide comprising the sortase or the active fragment thereof and the other member of the receptor-ligand pair.

3. The intercellular labeling method of claim 1 , wherein the first cell, the second cell, or both are immune cells.

4. The intercellular labeling method of claim 3 , wherein the first cell, the second cell, or both are T cells, B cells, dendritic cells, macrophages, or natural killer cells.

5. The intercellular labeling method of claim 2 , wherein the receptor-ligand pair is selected from the group consisting of:
CD40 and CD40L,
CD80 and CD28,
CD80 and CTLA4,
CD86 and CD28,
CD86 and CTLA4
PD-1 and PD-L1,
PD-1 and PD-L2, and
ICOS and ICOSL.

6. The intercellular labeling method of claim 1 , wherein the detectable label is biotin or a fluorescent dye.

7. The intercellular labeling method of claim 1 , wherein the sortase is a sortase A.

8. The intercellular labeling method of claim 7 , wherein the sortase is a mutant sortase A that comprises one or more mutations of P94R or P94S, S102C, A104H, E105D, K138P, K152I, D160K or D160N, K162H, T164N, D165A, K173E, I182V, K190E, and K196S or K196T.

9. The intercellular labeling method of claim 1 , wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X is any amino acid residue.

10. The intercellular labeling method of claim 1 , wherein the sortase acceptor peptide is an oligoglycine.

11. The intercellular labeling method of claim 1 , wherein the contacting step (iii) is performed in vitro.

12. The intercellular labeling method of claim 1 , wherein the contacting step (iii) is performed in vivo.

13. The intercellular labeling method of claim 2 , wherein the contacting step is performed in the presence of a candidate compound, and the method further comprises assessing whether the candidate compound modulates the interaction between the two members of the receptor-ligand pair, wherein a change of the labeling of the first cell in the presence of the candidate compound as compared with the labeling of the first cell in the absence of the candidate compound indicates that the compound is a modulator of the receptor-ligand pair.

14. The intercellular labeling method of claim 1 , wherein the first cell is an antigen-presenting cell (APC) that expresses a MHC class I molecule, a MHC class II molecule, or both; and the second cell is a T cell that expresses a T cell receptor (TCR) molecule.

15. The intercellular labeling method of claim 14 , wherein the APC is engineered to further express a polypeptide encoded by a member of a cDNA library.

16. The intercellular labeling method of claim 14 , wherein step (i) is performed by providing a plurality of APCs which collectively express polypeptides encoded by a cDNA library; wherein step (iii) is performed by contacting the plurality of the APCs with the T cell in the presence of the sortase substrate; and wherein the polypeptides encoded by the cDNA library are in addition to the first polypeptide that comprises the sortase acceptor peptide.

17. The intercellular labeling method of claim 16 , further comprising isolating the labeled APCs produced in step (iii).

18. The intercellular labeling method of claim 4 , wherein the first cell is a T cell, and the second cell is a B cell, or wherein the first cell is a B cell, and the second cell is a T cell.

19. The intercellular labeling method of claim 8 , wherein the mutant sortase A contains mutations P94S, D160N, and K196T.

20. The intercellular labeling method of claim 9 , wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2).

21. The intercellular labeling method of claim 10 , wherein the oligoglycine consists of 1-5 glycine residues.

22. The intercellular labeling method of claim 2 , wherein the first polypeptide comprises CD40 and the N-terminal sortase acceptor peptide, which is oligoglycine GGGGG (SEQ ID NO: 3), and the second polypeptide comprises CD40L, which is fused to the sortase or the active fragment thereof.

23. The intercellular labeling method of claim 12 , wherein the first cell, the second cell, or both are endogenous cells of a transgenic mouse, rat, or rabbit.

24. The intercellular labeling method of claim 12 , wherein the first cell, the second cell, or both are constructed in vitro and transferred into a non-human subject.

25. The intercellular labeling method of claim 24 , wherein the subject is a mouse, a rabbit, a rat, or a monkey.

26. The intercellular labeling method of claim 12 , wherein the substrate comprising the sortase recognition sequence is administered to a non-human subject.

27. The intercellular labeling method of claim 12 , wherein the contacting step is carried out in a germinal center.

28. The intercellular labeling method of claim 14 , wherein the APC cell is a B cell, a dendritic cell, a macrophage, or a B cell.

29. The intercellular labeling method of claim 17 , further comprising identifying the member of the cDNA library that is expressed in the labeled APCs.

30. The intercellular labeling method of claim 13 , wherein a decrease of the labeling of the first cell in the presence of the candidate compound as compared to the level of labeling of the first cell in the absence of the candidate compound indicates that the compound inhibits the interaction between the ligand and receptor.

31. The intercellular labeling method of claim 13 , wherein an increase of the labeling of the first cell in the presence of the candidate compound as compared to the level of labeling of the first cell in the absence of the candidate compound indicates that the compound enhances the interaction between the ligand and receptor.

Assignees

• Whitehead Inst Biomedical Res

Inventors

• Pasqual Giulia
• Victora Gabriel

Classifications

• A01K2267/03

  Animal model, e.g. for test or diseases · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• A01K67/027

  New or modified breeds of vertebrates · CPC title

• A01K2217/072

  maintaining or altering function, i.e. knock in · CPC title

• C12N9/52 Primary

  derived from bacteria {or Archaea} · CPC title