Intercellular labeling of ligand-receptor interactions

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.

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.   
     
     
         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 4 , wherein the first cell is a T cell, and the second cell is a B cell, or vice versa. 
     
     
         6 . 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.   
     
     
         7 . The intercellular labeling method of  claim 1 , wherein the detectable label is biotin or a fluorescent dye. 
     
     
         8 . The intercellular labeling method of  claim 1 , wherein the sortase is a sortase A. 
     
     
         9 . The intercellular labeling method of  claim 8 , wherein the sortase is a mutant sortase A that exhibits improved catalytic activity as compared to the wild-type counterpart. 
     
     
         10 . The intercellular labeling method of  claim 9 , wherein the mutant 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. 
     
     
         11 . The intercellular labeling method of  claim 10 , wherein the mutant sortase A contains mutations P94S, D160N, and K196T. 
     
     
         12 . 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. 
     
     
         13 . The intercellular labeling method of  claim 12 , wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2). 
     
     
         14 . The intercellular labeling method of  claim 1 , wherein the sortase acceptor peptide is an oligoglycine. 
     
     
         15 . The intercellular labeling method of  claim 14 , wherein the oligoglycine consists of 1-5 glycine residues. 
     
     
         16 . 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. 
     
     
         17 . The intercellular labeling method of  claim 16 , wherein the first cell is a B cell, and the second cell is a T cell. 
     
     
         18 . The intercellular labeling method of  claim 1 , wherein the first polypeptide, the second polypeptide, or both further comprise a protein tag. 
     
     
         19 . The intercellular labeling method of  claim 1 , wherein the method is performed in vitro. 
     
     
         20 . The intercellular labeling method of  claim 1 , wherein the method is performed in vivo. 
     
     
         21 . The intercellular labeling method of  claim 20 , wherein the first cell, the second cell, or both are endogenous cells of a transgenic animal. 
     
     
         22 . The intercellular labeling method of  claim 21 , wherein the first cell is an endogenous cell of a transgenic animal, and the second cell is constructed in vitro and transferred into the same transgenic animal, or vice versa. 
     
     
         23 . The intercellular labeling method of  claim 21 , wherein transgenic animal is a transgenic mouse, rat, or rabbit. 
     
     
         24 . The intercellular labeling method of  claim 20 , wherein the first cell, the second cell, or both are constructed in vitro and transferred into a 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 21 , wherein the peptide comprising the sortase recognition sequence is administered to the transgenic animal or the subject. 
     
     
         27 . The intercellular labeling method of  claim 20 , wherein the contacting step is carried out in a germinal center. 
     
     
         28 . 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 indicates that the compound is a modulator of the receptor-ligand pair. 
     
     
         29 . 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. 
     
     
         30 . The intercellular labeling method of  claim 29 , wherein the APC cell is a B cell, a dendritic cell, a macrophage, or a B cell. 
     
     
         31 . The intercellular labeling method of  claim 29 , wherein the APC is engineered to further express a polypeptide encoded by a member of a cDNA library. 
     
     
         32 . The intercellular labeling method of  claim 29 , wherein step (i) is performed by providing a plurality of APCs which collectively express polypeptides encoded by the cDNA library; and wherein step (iii) is performed by contacting the plurality of the APCs with the T cell in the presence of the sortase substrate. 
     
     
         33 . The intercellular labeling method of  claim 32 , further comprising isolating the labeled APCs produced in step (iii). 
     
     
         34 . The intercellular labeling method of  claim 33 , further comprising identifying the member of the cDNA library that is expressed in the labeled APCs for determining antigen specificity of the TCR expressed on the T cell. 
     
     
         35 . A kit for intercellular labeling, comprising:
 (i) a first cell expressing a first polypeptide on its surface, the first polypeptide comprising a sortase acceptor peptide, which is located at the N-terminus of the first polypeptide; and   (ii) a second cell expressing a second polypeptide on its surface, the second polypeptide comprising a sortase or an active fragment thereof.   
     
     
         36 . The kit of  claim 35 , 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. 
     
     
         37 . The kit of  claim 35 , wherein the first cell, the second cell, or both are immune cells. 
     
     
         38 . The kit of  claim 37 , wherein the first cell, the second cell, or both are T cells, B cells, dendritic cells, macrophages, or natural killer cells. 
     
     
         39 . The kit of  claim 38 , wherein the first cell is a T cell and the second cell is a B cell, or vice versa. 
     
     
         40 . The kit of  claim 36 , 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.   
     
     
         41 . The kit of  claim 35 , wherein the sortase is a sortase A. 
     
     
         42 . The kit of  claim 35 , wherein the sortase is a mutant sortase A that exhibits improved catalytic activity as compared to the wild-type counterpart. 
     
     
         43 . The kit of  claim 42 , wherein the mutant sortase A 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. 
     
     
         44 . The kit of  claim 43 , wherein the mutant sortase A contains mutations P94S, D160N, and K196T. 
     
     
         45 . The kit of  claim 35 , wherein the sortase acceptor peptide is an oligoglycine. 
     
     
         46 . The kit of  claim 45 , wherein the oligoglycine consists of 1-5 glycine residues. 
     
     
         47 . The kit of  claim 46 , wherein the first polypeptide comprises CD40 and the N-terminal sortase acceptor, which is oligoglycine GGGGG (SEQ ID NO: 3), and the second polypeptide comprises CD40L, which is fused to the sortase at the C-terminus. 
     
     
         48 . The kit of  claim 47 , wherein the first cell is a B cell, and the second cell is a T cell, or vice versa. 
     
     
         49 . The kit of  claim 35 , wherein the first polypeptide, the second polypeptide, or both further comprise a protein tag. 
     
     
         50 . The kit of  claim 35 , wherein the kit further comprises a sortase substrate comprising a sortase recognition sequence, wherein the sortase substrate is associated with a detectable label. 
     
     
         51 . The kit of  claim 50 , wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X is any amino acid residue. 
     
     
         52 . The kit of  claim 51 , wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2). 
     
     
         53 . The kit of  claim 35 , 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. 
     
     
         54 . The kit of  claim 53 , wherein the APC cell is a B cell, a dendritic cell, a macrophage, or a B cell. 
     
     
         55 . The kit of  claim 53 , wherein the APC is engineered to further express a polypeptide encoded by a member of a cDNA library. 
     
     
         56 . The kit of  claim 55 , wherein (i) contains a plurality of APCs which collectively express polypeptides encoded by the cDNA library. 
     
     
         57 . The kit of  claim 53 , further comprising a sortase substrate comprising a sortase recognition sequence, wherein the sortase substrate is associated with a detectable label. 
     
     
         58 . The kit of  claim 57 , wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X is any amino acid residue. 
     
     
         59 . The kit of  claim 58 , wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2). 
     
     
         60 . A non-human animal, comprising:
 (i) a first cell expressing a first polypeptide on its surface, the first polypeptide comprising a sortase acceptor peptide, which is located at the N-terminus of the first polypeptide;   (ii) a second cell expressing a second polypeptide on its surface, the second polypeptide comprising a sortase, or   (iii) both (i) and (ii).   
     
     
         61 . The non-human animal of  claim 60 , 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. 
     
     
         62 . The non-human animal of  claim 60 , wherein the animal is a transgenic animal, in which a gene encoding the first polypeptide, a gene encoding the second polypeptide, or both are inserted into the genome of the animal. 
     
     
         63 . The non-human animal of  claim 60 , wherein the animal is a transgenic animal, in which a nucleic acid sequence encoding the sortase acceptor peptide is inserted into the endogenous locus encoding the one member of the ligand-receptor pair for expression of the first polypeptide. 
     
     
         64 . The animal of  claim 60 , wherein the animal is a transgenic animal, in which a nucleic acid sequence encoding the sortase is inserted into the endogenous locus encoding the other member of the ligand-receptor pair for expression of the second polypeptide. 
     
     
         65 . The non-human animal of  claim 60 , wherein the animal is a transgenic animal, in which the gene encoding the first polypeptide is inserted into the genome of the animal, and the second cell that expresses the second polypeptide is constructed in vitro and transferred into the animal. 
     
     
         66 . The non-human animal of  claim 60 , wherein the animal is a transgenic animal, in which the gene encoding the second polypeptide is inserted into the genome of the animal and the first cell that expresses the first polypeptide is constructed in vitro and transferred into the animal. 
     
     
         67 . The non-human animal of  claim 60 , wherein the transgenic animal is a transgenic mouse, rat, or rabbit. 
     
     
         68 . The non-human animal of  claim 60 , wherein both the first cell and the second cell are constructed in vitro and transferred into the animal. 
     
     
         69 . The non-human animal of  claim 60 , wherein the first cell, the second cell, or both are immune cells. 
     
     
         70 . The non-human animal of  claim 69 , wherein the first cell, the second cell, or both are T cells, B cells, dendritic cells, macrophages, or natural killer cells. 
     
     
         71 . The non-human animal of  claim 70 , wherein the first cell is a T cell, and the second cell is a B cell, or vice versa. 
     
     
         72 . The non-human animal of  claim 61 , 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.   
     
     
         73 . The non-human animal of  claim 60 , wherein the sortase is a sortase A. 
     
     
         74 . The non-human animal of  claim 60 , wherein the sortase is a mutant sortase A that exhibits improved catalytic activity as compared to the wild-type counterpart. 
     
     
         75 . The non-human animal of  claim 74 , wherein the mutant sortase A 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. 
     
     
         76 . The non-human animal of  claim 75 , wherein the mutant sortase A contains mutations P94S, D160N, and K196T. 
     
     
         77 . The non-human animal of  claim 60 , wherein the sortase acceptor peptide is an oligoglycine. 
     
     
         78 . The non-human animal of  claim 77 , wherein the oligoglycine consists of 1-5 glycine residues. 
     
     
         79 . The non-human animal of  claim 60 , wherein the first polypeptide comprises CD40 and the N-terminal acceptor peptide, which is GGGGG (SEQ ID NO: 3), and the second polypeptide comprises CD40L, which is fused to the sortase at the C-terminus. 
     
     
         80 . The non-human animal of  claim 79 , wherein the first cell is a B cell and the second cell is a T cell. 
     
     
         81 . The non-human animal of  claim 60 , wherein the non-human animal is a transgenic non-human mammal that comprises one or more human immunoglobulin genes or a portion thereof. 
     
     
         82 . The non-human animal of  claim 81 , wherein the non-human mammal is a transgenic mouse or transgenic rat. 
     
     
         83 . The non-human animal of  claim 60 , wherein the non-human mammal comprises a humanized immune system. 
     
     
         84 . The non-human animal of  claim 60 , wherein the non-human animal is a transgenic mouse expressing a fusion polypeptide comprising sortase A and CD40L. 
     
     
         85 . The non-human animal of  claim 60 , wherein the non-human animal is a transgenic mouse expressing a fusion polypeptide comprising GGGGG (SEQ ID NO: 3) and CD40, wherein the GGGGG (SEQ ID NO: 3) fragment is located at the N-terminus of the fusion polypeptide. 
     
     
         86 . A nucleic acid comprising a nucleotide sequence that encodes a polypeptide comprising a sortase and a member of a ligand-receptor pair. 
     
     
         87 . The nucleic acid of  claim 86 , wherein the sortase is a sortase A. 
     
     
         88 . The nucleic acid of  claim 87 , wherein the sortase A is a mutant of a wild-type sortase A, which has improved catalytic activity as compared to the wild-type counterpart. 
     
     
         89 . The nucleic acid of  claim 88 , wherein the mutant 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. 
     
     
         90 . The nucleic acid of  claim 89 , wherein the mutant contains mutations of P94S, D160N, and K196T. 
     
     
         91 . The nucleic acid of  claim 86 , wherein the polypeptide further comprises a protein tag. 
     
     
         92 . A vector comprising the nucleic acid of  claim 86 . 
     
     
         93 . The vector of  claim 92 , wherein the vector is an expression vector. 
     
     
         94 . A host cell comprising the vector of  claim 92 . 
     
     
         95 . A method for identifying a B cell expressing a high affinity B cell receptor (BCR) to an antigen, the method comprising:
 (i) providing a mammal that comprises (a) a plurality of B cells expressing a first polypeptide on its surface, the first polypeptide comprising a sortase acceptor peptide, which is located at the N-terminus of the first polypeptide, and (b) a plurality of T cells each expressing a second polypeptide on its surface, the second polypeptide comprising a sortase or an active fragment thereof;   (ii) administering to the mammal an effective amount of a sortase substrate comprising a sortase recognition sequence, wherein the sortase substrate is associated with a detectable label;   (iii) isolating lymphocytes from a germinal center of the animal; and   (iv) identifying a B cell that is conjugated to the detectable label, wherein the B cell thus identified expresses a high affinity BCR to an antigen.   
     
     
         96 . The method of  claim 95 , wherein the first polypeptide is a fusion polypeptide comprising the sortase acceptor peptide and one member of a receptor-ligand pair, the member being a B cell surface protein; 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, the other member being a T cell surface receptor. 
     
     
         97 . The method of  claim 95 , wherein the mammal is a transgenic mammal, in which a gene encoding the first polypeptide, a gene encoding the second polypeptide, or both are inserted into the genome of the mammal. 
     
     
         98 . The method of  claim 95 , wherein the mammal is a transgenic animal, in which a nucleic acid sequence encoding the sortase acceptor peptide is inserted into the endogenous locus encoding the one member of the ligand-receptor pair for expression of the first polypeptide. 
     
     
         99 . The method of  claim 95 , wherein the mammal is a transgenic animal, in which a nucleic acid sequence encoding the sortase is inserted into the endogenous locus encoding the other member of the ligand-receptor pair for expression of the second polypeptide. 
     
     
         100 . The method of  claim 95 , wherein the mammal is a transgenic mammal, in which the gene encoding the first polypeptide and the gene encoding the second polypeptide are both inserted into the genome of the mammal. 
     
     
         101 . The method of  claim 95 , wherein the mammal is a transgenic mammal, in which the gene encoding the first polypeptide is inserted into the genome of the mammal and expressed on naïve B cells and the plurality of T cells that expresses the second polypeptide are constructed in vitro and transferred into the mammal. 
     
     
         102 . The method of  claim 95 , wherein the transgenic mammal is a transgenic mouse, rat, or rabbit. 
     
     
         103 . The method of  claim 95 , wherein the sortase is a sortase A. 
     
     
         104 . The method of  claim 95 , wherein the sortase is a mutant sortase A that exhibits improved catalytic activity as compared to the wild-type counterpart. 
     
     
         105 . The method of  claim 104 , wherein the mutant of sortase A 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. 
     
     
         106 . The method of  claim 105 , wherein the mutant sortase A contains mutations P94S, D160N, and K196T. 
     
     
         107 . The method of  claim 95 , wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X is any amino acid residue. 
     
     
         108 . The method of  claim 107 , wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2). 
     
     
         109 . The method of  claim 95 , wherein the sortase acceptor peptide is an oligoglycine. 
     
     
         110 . The method of  claim 109 , wherein the oligoglycine consists of 1-5 glycine residues. 
     
     
         111 . The method of  claim 95 , wherein the mammal is immunized with an antigen of interest. 
     
     
         112 . The method of  claim 95 , the method further comprising isolating one or more nucleic acid encoding at least a portion of a heavy chain variable region, at least a portion of a light chain variable region, or both of the BCR from the B cell that is conjugated to the detectable label. 
     
     
         113 . The method of  claim 112 , wherein the at least a portion of the heavy chain variable region, the at least a portion of the light chain variable region, or both encode at least one complementarity determining region of the BCR. 
     
     
         114 . The method of  claim 113 , further comprising sequencing the at least a portion of the heavy chain variable region, the at least a portion of the light chain variable region, or both. 
     
     
         115 . The method of  claim 95 , the method further comprising producing a hybridoma cell derived from the B cell that is conjugated to the detectable label, wherein the hybridoma cell produces high affinity antibodies to the antigen. 
     
     
         116 . The method of  claim 115 , further comprising culturing the hybridoma cell for producing the antibodies. 
     
     
         117 . The method of  claim 95 , wherein the non-human mammal is a transgenic non-human mammal that comprise one or more human immunoglobulin genes, or a portion thereof. 
     
     
         118 . The method of  claim 117 , wherein the non-human mammal is a transgenic mouse or transgenic rat. 
     
     
         119 . The method of  claim 95 , wherein the non-human mammal comprises a humanized immune system. 
     
     
         120 . A method for identifying a binding partner of a protein of interest, the method comprising:
 (i) providing a first population of cells expressing a plurality of polypeptides, each of which expresses a sortase acceptor peptide and a candidate protein;   (ii) providing a second population of cells expressing a sortase or an active fragment thereof, and the protein of interest;   (iii) contacting the first population of cells with the second population of cells in the presence of a sortase substrate comprising a sortase recognition sequence, wherein the peptide is associated with a detectable label;   (iv) detecting labeling of cells in the first population of cells; and   (iv) identifying a binding partner of the protein of interest, wherein a candidate protein is a binding partner of the protein of interest, if the cell that expresses a polypeptide comprising the candidate protein is labeled in step (iii).   
     
     
         121 . The method of  claim 120 , wherein in (i), the sortase acceptor peptide and the candidate protein are covalently linked to form a fusion polypeptide. 
     
     
         122 . The method of  claim 120 , wherein in (ii), the sortase or the active fragment thereof and the protein of interest are covalently linked to form a fusion polypeptide. 
     
     
         123 . The method of  claim 122 , wherein the protein of interest is a receptor of an immune cell. 
     
     
         124 . The method of  claim 120 , wherein the immune cell is a T cell, a B cell, a dendritic cell, a macrophage, or a natural killer cell. 
     
     
         125 . The method of  claim 120 , wherein the detectable label is biotin or a fluorescent dye. 
     
     
         126 . The method of  claim 120 , wherein the sortase is a sortase A. 
     
     
         127 . The method of  claim 120 , wherein the sortase is a mutant sortase A that exhibits improved catalytic activity as compared to the wild-type counterpart. 
     
     
         128 . The method of  claim 127 , wherein the mutant sortase A 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. 
     
     
         129 . The method of  claim 128 , wherein the mutant sortase A contains mutations P94S, D160N, and K196T. 
     
     
         130 . The method of  claim 120 , wherein the sortase recognition sequence is LPXTG (SEQ ID NO: 1), in which X is any amino acid residue. 
     
     
         131 . The method of  claim 130 , wherein the sortase recognition sequence is LPETG (SEQ ID NO: 2). 
     
     
         132 . The method of  claim 120 , wherein the sortase acceptor peptide is an oligoglycine. 
     
     
         133 . The method of  claim 132 , wherein the oligoglycine consists of 1-5 glycine residues. 
     
     
         134 . The method of  claim 120 , wherein the polypeptide comprising the protein of interest, the polypeptides comprising the candidate proteins, or both further comprise a protein tag. 
     
     
         135 . The method of  claim 120 , wherein the method is performed in vitro.