WO2023116796A1

WO2023116796A1 - A method for cell-cell interaction labeling and detection of antigen-specific t cells

Info

Publication number: WO2023116796A1
Application number: PCT/CN2022/140859
Authority: WO
Inventors: Peng Chen; Jie Li; Hongyu Liu
Original assignee: Peking University; Nanjing University
Priority date: 2021-12-23
Filing date: 2022-12-22
Publication date: 2023-06-29

Abstract

Provided is a method for cell-cell interaction labeling and detection of antigen-specific T cells. Further provided is a method comprising: contacting a first cell with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule, inducing binding of said detectable label to said first cell when said first cell is within an effective proximity range of said sensitizing molecule.

Description

A method for cell-cell interaction labeling and detection of antigen-specific T cells

BACKGROUND OF THE INVENTION

Cell-cell interactions are essential in numerous biological processes, including immune responses, embryonic development and neuronal signaling. A technique for monitoring the dynamics of cell-cell interactions is required for researchers to better understand these biological processes. To date, there are limited methods for monitoring cell-cell interaction in vitro and in vivo. A generalizable, robust and cost-effective method for detection of cell-cell interaction is required.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a method for identifying a first cell from a sample, comprising:

(a) contacting said sample with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule,

(b) inducing binding of said detectable label to said first cell when said first cell is within an effective proximity range of said sensitizing molecule,

(c) detecting the presence of the cell which is bound with said detectable label as an indication of the presence of said first cell,

wherein, said second cell is capable of specifically recognizing and/or binding said first cell.

In one aspect, the present disclosure provides a method for detecting cell-to-cell interaction, comprising:

(a) contacting a first cell with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule,

(c) detecting the presence of the cell which is bound with said detectable label as an indication of the presence of the cell-to-cell interaction of said first cell and said second cell,

In one aspect, the present disclosure provides a method for intracellular labeling, comprising:

In one aspect, the present disclosure provides a method for treating a tumor comprising administering to a subject in need thereof a TSA reactive T cell identified by, or labelled by the method of the present application.

In one aspect, the present disclosure provides a TSA reactive T cell identified by, or labelled by the method of the present application, for use in treating a tumor.

In one aspect, the present disclosure provides a use of a TSA reactive T cell identified by, or labelled by the method of the present application in the manufacture of a medicament/composition for the treatment of a tumor.

In one aspect, the present disclosure provides a method for identifying a tumor-specific antigen (TSA) -specific T cell receptors (TCRs) comprising identifying a TSA reactive T cell by the method of the present application.

In one aspect, the present disclosure provides a method for expanding (TSA) reactive T cells comprising performing the method of the present application, and expanding said the TSA-specific T cells.

In one aspect, the present disclosure provides a method for determining the relative binding affinity for an antigen of TCR respectively expressed on a T cell comprising performing the method of the present application, and quantifying the level of detectable label bound to said T cell.

In one aspect, the present disclosure provides a modified cell, which is bound with a sensitizing molecule.

In one aspect, the present disclosure provides a composition, comprising a modified cell of the present application。

In one aspect, the present disclosure provides a kit for intracellular labeling a target cell, comprising

(a) a modified cell of the present application and/or a composition of the present application, and

(b) a detectable label,

wherein, said modified cell is capable of specifically recognizing and/or binding said target cell.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “figure” and “FIG. ” herein) , of which:

FIG. 1 illustrates DBF-LPETGG-based CCI Labeling Tool for Cell-Cell Interaction Labeling.

FIGs. 2A-2C illustrate structure of DBF-LPETGG and Biotin-PEG2-NH ₂.

FIG. 3 illustrates synthesis steps of DBF-LPETGG.

FIGs. 4A-4C illustrate mechanism of DBF-LPETGG-mediated Proximity Labeling.

FIGs. 5A-5D illustrate DBF-LPETGG Displaying on Cell Surface.

FIGs. 6A-6B illustrate results of Optimization of DBF-LPETGG Labeling Wavelength.

FIG. 7A illustrates results of Biotin probe screening. FIG. 7B illustrates structure of Biotin probes.

FIGs. 8A-8D illustrate results of Optimization of DBF-LPETGG Labeling Conditions.

FIGs. 9A-9C illustrate Cell Viability After DBF-LPETGG Displaying and Labeling.

FIGs. 10A-10B illustrate iDC-ova-OT-I T Cell Model System.

FIGs. 11A-11B illustrate Specificity of DBF-LPETGG in Probing DC-T Cell Interactions.

FIG. 12 illustrates CCI-dependent Labeling of DBF-LPETGG.

FIGs. 13A-13D illustrate Quantitative analysis of cell-cell interaction.

FIG. 14 illustrates Workflow for Mouse TSA-specific T Cells Capture.

FIG. 15 illustrates CD8+ TILs Capture in B16-ova.

FIG. 16 illustrates Source Expansion of TSA-specific T Cells-from TILs and dLN.

FIGs. 17A-17B illustrate Correlation Between Biotin Signal and TSA-specific Markers.

FIGs. 18A-18B illustrate TSA-specific T Cells Detection in E0771 TILs and dLN.

FIGs. 19A-19B illustrate Antigen specificity of Captured Bio+ T Cells.

FIG. 20 illustrates Workflow for TSA-specific T Cells Capture in Human Samples.

FIGs. 21A-21B illustrate Cell Validation Before Labeling-67#Bladder Cancer Sample.

FIG. 22 illustrates 67#Bladder Cancer Sample TSA-specific T Cells Capture in TILs, LN and PBMC.

FIG. 23 illustrates Bladder Cancer Sample Labeling Summary.

FIG. 24 illustrates 5#Lung Cancer Sample TSA-specific CD8+ T Cells Capture in TILs, LN and PBMC. FIG. 25 illustrates 5#Lung Cancer Sample TSA-specific CD4+ T Cells Capture in TILs, LN and PBMC.

FIG. 26 illustrates Lung Cancer Sample Labeling Summary.

FIGs. 27A-27D illustrate Synthesis of DBF-LPETGG.

FIGs. 28-29 illustrate Results of Biotin probe screening.

FIG. 30 illustrates Results of pulse-chase assay.

FIGs. 31-32 illustrate Results of spatial-controlled intercellular labeling in interacting synapse.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

It must be noted that as used herein and in the appended claims, the singular forms “a” , “an” , and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the peptide” includes reference to one or more peptides and equivalents thereof, e.g., polypeptides, known to those skilled in the art, and so forth.

The terms "peptide, " "polypeptide" and "protein" are used interchangeably to refer to an isolated polymer of amino acid residues, and are not limited to a minimum length unless otherwise defined. Peptides, oligopeptides, dimers, multimers, and the like, are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically and isolated from the natural environment, produced using recombinant technology, or produced synthetically typically using naturally occurring amino acids. In some aspects, the polypeptide or protein is a "modified polypeptide" comprising non-naturally occurring amino acids. In some aspects, the polypeptides comprise a combination of naturally occurring and non-naturally occurring amino acids, and in some embodiments, the peptides comprise only non-naturally occurring amino acids. The term "peptide" as used herein encompasses native peptides (either degradation products, synthetically synthesized peptides or recombinant peptides) and 30 peptidomimetics (typically, synthetically synthesized peptides) , as well as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body or more capable of penetrating into cells. Such modifications include, but are not limited to, N-terminus modification, C-terminus modification, peptide bond modification, backbone modification, and/or side chain modification.

The term "polynucleotide, " or "nucleotide" as used herein, refer generally to linear polymers of natural or modified nucleosides, including deoxyribonucleosides, ribonucleosides, 15 alpha-anomeric forms thereof, and the like, usually linked by phosphodiester bonds or analogs thereof ranging in size from a few monomeric units, e.g., 2-4, to several hundreds of monomeric units. When a polynucleotide is represented by a sequence of letters, such as "ATGC, " it will be understood that the nucleotides are in 5'->3' order from left to right. Polynucleotide as used herein also includes a basic, sugar-phosphate or sugar-phosphorothioate polymers.

The term "detectable label" as used herein, refer generally to a molecule that can be distinguished from other molecules based on one or more physical, chemical or optical differences among the molecules being separated, including but not limited to, electrophoretic mobility, molecular weight, shape, solubility, pKa, hydrophobicity, charge, charge/mass ratio, polarity or the like. In one aspect, molecular tags in a plurality or set differ in electrophoretic mobility and optical detection characteristics and can be separated by electrophoresis. In another aspect, molecular tags in a plurality or set may differ in molecular weight, shape, solubility, pKa, hydrophobicity, charge, polarity and can be separated by normal phase or reverse phase HPLC, ion exchange HPLC, capillary electrochromatography, mass spectroscopy, gas phase chromatography or like technique.

The terms "sample" refers to a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue sample may be solid tissue as from a fresh, frozen, and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; or cells from any time in gestation or development of the subject. The tissue sample may contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics or the like. Cells may be fixed in a conventional manner (e.g., formalin-fixed, paraffin-embedding (FFPE) ) .

The term "sensitizing molecule " as used herein, refer generally to a group that produces an active species that is capable of inducing a linkage, for example, by oxidation. Preferably, the active species is a chemical species that exhibits short-lived activity so that its linkage-inducing effects may be only in the proximity of the site of its generation.

A method for identifying a first cell from a sample, comprising:

(a) contacting said sample with a second cell in the presence of a detectable label, wherein said second cell may be bound with a sensitizing molecule,

(b) inducing binding of said detectable label to said first cell when said first cell may be within an effective proximity range of said sensitizing molecule,

(c) detecting the presence of the cell which may be bound with said detectable label as an indication of the presence of said first cell,

wherein, said second cell may be capable of specifically recognizing and/or binding said first cell.

A method for detecting cell-to-cell interaction, comprising:

(a) contacting a first cell with a second cell in the presence of a detectable label, wherein said second cell may be bound with a sensitizing molecule,

(c) detecting the presence of the cell which may be bound with said detectable label as an indication of the presence of the cell-to-cell interaction of said first cell and said second cell,

A method for intracellular labeling, comprising:

For example, wherein said first cell may express a first molecule on cell surface, said second cell may express a second molecule on cell surface, and said first molecule may be capable of specifically recognize and/or binding said second molecule.

For example, wherein said first molecule may comprise peptide, protein, glycoprotein, steroid, lipid and/or lipoprotein.

For example, wherein said second molecule may comprise peptide, protein, glycoprotein, steroid, lipid and/or lipoprotein.

The first cell

For example, wherein said first cell may comprise immune cell.

For example, wherein said first cell may comprise B cell, T cell, lymphoid cell, dendritic cell, granulocyte, macrophage, monocyte and/or natural killer cell.

For example, wherein said first cell may comprise a tumor-specific cell.

For example, wherein said first cell may comprise a tumor-specific antigen (TSA) reactive T cell.

For example, wherein said TSA may be derived from a tumor selected from the group consisting of bladder tumor, melanoma; breast tumor, pancreatic tumor, lung tumor, kidney tumor, ovarian tumor, colon tumor, lymphoma, leukemia, skin tumor, and prostate tumor.

The first molecule

For example, wherein said first molecule may comprise a TCR (T cell receptor) .

The sample

For example, wherein said sample may comprise a cell population.

For example, wherein said sample may comprise a population of tumor infiltrating lymphocytes (TILs) , a population of lymph node cells, a population of peripheral blood mononuclear cells, and/or a population of circulating T cells.

The second cell

For example, wherein said second cell may comprise immune cell.

For example, wherein said second cell may comprise B cell, T cell, lymphoid cell, dendritic cell, granulocyte, macrophage, monocyte and/or natural killer cell.

For example, wherein said second cell may comprise an antigen-presenting cell.

For example, wherein said second cell may comprise a dendritic cell.

For example, wherein said second cell may be primed with one or more tumor antigens.

For example, wherein said second cell may be primed with tumor lysate, and said tumor lysate may comprise one or more tumor antigens.

The second molecule

For example, said second molecule may comprise peptide-MHC.

For example, wherein said first cell and said second cell may be specifically recognizing the same antigen.

For example, wherein said first cell and said second cell may be activated by the same antigen.

The detectable label

For example, wherein said detectable label may be selected from the group consisting of a small molecule, a polynucleotide, and a polypeptide.

For example, wherein said detectable label may be selected from the group consisting of an antibody, a chemical marker, a biological marker and a probe. For example, wherein said detectable label may be fluorophore.

For example, wherein said detectable label may comprise biotin and/or fluorophore.

For example, wherein said detectable label may be selected from the group consisting of biotin-NH ₂ (BAm) , biotin-alcohol (BAl) , biotin-mercaptan (BMe) , biotin-phenol (BP) , biotin-aniline (BAn) and biotin-naphthylamine (BNap) . For example, wherein said detectable label may be biotin-NH ₂ (BAm) .

The sensitizing molecule

For example, wherein said sensitizing molecule may comprise a molecule capable of inducing production of singlet oxygen.

For example, wherein said effective proximity range of said sensitizing molecule may be from 10 nm to 200 nm. For example, wherein said effective proximity range of said sensitizing molecule may be from 10 nm to 200 nm, 50 nm to 200 nm, 100 nm to 200 nm, 10 nm to 100 nm, 50 nm to 100 nm, or 10 nm to 50 nm.

For example, wherein said sensitizing molecule may be bound to the cell surface of said second cell.

For example, wherein said sensitizing molecule may be covalently bound to the cell surface of said second cell.

For example, wherein said sensitizing molecule may be bound to an amino acid and/or a saccharide on the cell surface of said second cell.

For example, wherein said sensitizing molecule may be bound to said second cell through a linker.

For example, wherein said linker may comprise a cleavable linker and/or a non-cleavable linker.

For example, wherein said linker may comprise (GGGGS) m, wherein m may be 0 or more. For example, wherein m may be 0, 1, 2, 3, 4, 5, 10, 20, or 50.

For example, wherein said sensitizing molecule may be bound to said second cell surface via glycosylation reaction, sulfhydryl-reactive crosslinker reaction, amine-reactive crosslinker reaction and/or transpeptidation.

For example, wherein a concentration of 10 μM to 500 μM of said sensitizing molecule may be bound to said second cell surface via a reaction. For example, wherein a concentration of 10 μM to 500 μM, 50 μM to 500 μM, 100 μM to 500 μM, 200 μM to 500 μM, 10 μM to 200 μM, 50 μM to 200 μM, 100 μM to 200 μM, 10 μM to 100 μM, 50 μM to 100 μM, or 10 μM to 50 μM of said sensitizing molecule may be bound to said second cell surface via a reaction. For example, wherein a concentration of 100 μM of said sensitizing molecule may be bound to said second cell surface via a reaction.

For example, wherein said reaction may be selected from the group consisting of glycosylation reaction, sulfhydryl-reactive crosslinker reaction, amine-reactive crosslinker reaction and transpeptidation.

For example, wherein the duration of said reaction may be about 1 minute to about 20 minutes. For example, wherein the duration of said reaction may be about 1 minute to about 20 minutes, about 5 minutes to about 20 minutes, about 10 minutes to about 20 minutes, about 1 minute to about 10 minutes, about 5 minutes to about 10 minutes or about 1 minutes to about 5 minutes.

For example, wherein said sensitizing molecule may comprise an organic molecule, an inorganic molecule, and/or a complex thereof.

For example, wherein said sensitizing molecule may comprise a polymer.

For example, wherein said sensitizing molecule may comprise a photosensitizer.

For example, wherein said sensitizing molecule can induce singlet oxygen production.

For example, wherein said sensitizing molecule may be selected from the group consisting of dye, aromatics, porphyrin, metalloporphyrin, phthalocyanine, metal phthalocyanine, macrocycles, tetrapyrrole, transition metal complex, and semiconductor.

For example, wherein said sensitizing molecule may be selected from the group consisting of rose bengal, fluorescein, Dibromofluorescein (DBF) , methylene blue, napthalene, anthracene, biphenyl, octaethylporphine (OEP) , metal OEP, tetraphenylporphine (TPP) , metal TPP, metal phthalocyanine tetrasulfonate (PcTS) , texaphyrin, complex of Ruthenium, complex of Osmium, complex of Iridium, complex of Chromium, and complex of Platinumm, TiO ₂, and ZnO.

For example, wherein said sensitizing molecule may comprise DBF.

For example, the sensitizing molecule could be:

For example, the linker could be (GGGGS) m, wherein m could be 0 or more.

For example, wherein the contacting of step (a) may be performed under light condition.

For example, wherein the light intensity may be about 6.14 mW/cm ², 12.57 mW/cm ², and/or 17.17 mW/cm ².

For example, wherein the wavelength of light may be from 450 nm to 520 nm. For example, wherein the wavelength of light may be 450 nm, 480 nm or 520 nm. For example, wherein the wavelength of light may be 520 nm.

For example, wherein the duration of light may be about 5 min to 10 min. For example, wherein the duration of light may be about 1 minute to about 20 minutes, about 5 minutes to about 20 minutes, about 10 minutes to about 20 minutes, about 1 minute to about 10 minutes, about 5 minutes to about 10 minutes or about 1 minutes to about 5 minutes.

For example, wherein inducing binding of said detectable label to said first cell of step (b) may be switched on/off via photo irradiation. For example, wherein inducing binding of said detectable label to said first cell of step (b) may be controlled by light source removement or light source providing. For example, when the light source is removed, said inducing binding of said detectable label to said first cell may pause. For example, when the light source is provided, said inducing binding of said detectable label to said first cell may start. For example, the light may be provided as a pulse manner. For example, Light irradiation may not be provided at the certain minutes followed by re-provided at the certain minutes. For example, Light irradiation may be paused at the second and fifth minutes followed by regained at the third and sixth minutes.

For example, wherein the number ratio of the second cells and the cells of said sample of step (a) may be from about 1: 5 to about 5: 1. For example, wherein the number ratio of the second cells and the cells of said sample of step (a) may be about 1: 5, about 1: 2, about 1: 1, about 2: 1, or about 5: 1.

For example, wherein step (b) may comprise enriching for said first cells.

For example, wherein enriching for said first cells may be via Fluorescence-activated cell sorting (FACS) .

For example, further may comprise determining whether the first cell comprise a marker indicative of TSA reactivity.

For example, wherein enriching for the first cells comprising a marker indicative of TSA reactivity may be via FACS.

For example, wherein said marker may comprise PD-1 expression; CD39 expression, LAG3 expression, and/or TIM3 expression.

For example, further comprising enriching for said first cell comprising CD8 ⁺ and/or CD4 ⁺expression.

For example, the labeling may occur in the interaction region of said first cell and said second cell. For example, the labeling may occur in the interaction synapse of said first cell and said second cell.

For example, wherein the magnitude of the detectable label present on the first cell may be indicative of the binding affinity of a T cell receptor expressed on the surface of the T cell for the TSA.

For example, wherein the magnitude of the detectable label present on the first cell may be indicative of the enriched first cell’s ability to kill other cells expressing the TSA and/or of the enriched the first cell’s ability to become activated in the presence of the TSA.

For example, the enriched or labeled cells may be sequenced.

For example, further comprising expanding the enriched cells for patient specific immune cell therapy.

For example, the method of the present application may comprise

(b) inducing binding of said detectable label to said first cell when said first cell is within an effective proximity range of said sensitizing molecule, wherein said first cell may comprise a tumor-specific antigen (TSA) reactive T cell, wherein said first molecule may comprise a TCR (T cell receptor) , wherein said second cell may comprise a dendritic cell primed with one or more tumor antigens, said second molecule may comprise peptide-MHC,

For example, the method of the present application may comprise

(a) contacting said sample with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule, wherein the contacting of step (a) may be performed under light condition, wherein the wavelength of light may be from 450 nm to 520 nm,

For example, the method of the present application may comprise

(a) contacting said sample with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule, wherein the contacting of step (a) may be performed under light condition, wherein the duration of light may be about 5 min to 10 min,

A method for identifying a tumor-specific antigen (TSA) -specific T cell receptors (TCRs) comprising identifying a TSA reactive T cell by the method of the present application.

A method for expanding (TSA) reactive T cells comprising performing the method of the present application, and expanding said the TSA-specific T cells.

A method for determining the relative binding affinity for an antigen of TCR respectively expressed on a T cell comprising performing the method of the present application, and quantifying the level of detectable label bound to said T cell.

A modified cell, which may be bound with a sensitizing molecule.

The modified cell of the present application, wherein said sensitizing molecule may comprise a molecule capable of inducing production of singlet oxygen.

The modified cell of the present application, wherein said sensitizing molecule may be bound to the cell surface of a cell.

The modified cell of the present application, wherein said sensitizing molecule may be covalently bound to the cell surface of said second cell.

The modified cell of the present application, wherein said sensitizing molecule may be bound to an amino acid and/or a saccharide on the cell surface of said second cell.

The modified cell of the present application, wherein said sensitizing molecule may be bound to said second cell through a linker.

The modified cell of the present application, wherein said linker may comprise a cleavable linker and/or a non-cleavable linker.

The modified cell of the present application, wherein said sensitizing molecule may be bound to said second cell surface via glycosylation reaction, sulfhydryl-reactive crosslinker reaction, amine-reactive crosslinker reaction and/or transpeptidation.

The modified cell of the present application, wherein said sensitizing molecule may comprise an organic molecule, an inorganic molecule, and/or a metal complex.

The modified cell of the present application, wherein said sensitizing molecule may comprise a polymer.

The modified cell of the present application, wherein said sensitizing molecule may comprise a photosensitizer.

The modified cell of the present application, wherein said sensitizing molecule may be selected from the group consisting of dye, aromatics, porphyrin, metalloporphyrin, phthalocyanine, metal phthalocyanine, macrocycles, tetrapyrrole, transition metal complex, and semiconductor.

The modified cell of the present application, wherein said sensitizing molecule may be selected from the group consisting of rose bengal, fluorescein, DBF, methylene blue, napthalene, anthracene, biphenyl, octaethylporphine (OEP) , metal OEP, tetraphenylporphine (TPP) , metal TPP, metal phthalocyanine tetrasulfonate (PcTS) , texaphyrin, complex of Ruthenium, complex of Osmium, complex of Iridium, complex of Chromium, and complex of Platinumm, TiO ₂, and ZnO.

The modified cell of the present application, wherein said sensitizing molecule may comprise DBF.

For example, the sensitizing molecule could be:

For example, the linker could be (GGGGS) m, wherein m could be 0 or more.

A composition, comprising a modified cell of the present application。

A kit for intracellular labeling a target cell, comprising

(b) a detectable label,

wherein, said modified cell may be capable of specifically recognizing and/or binding said target cell.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair (s) ; kb, kilobase (s) ; pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i.m., intramuscular (ly) ; i.p., intraperitoneal (ly) ; s.c., subcutaneous (ly) ; and the like.

Example 1

Proximity Labeling Tool

Firstly, the present application establishes a DBF-based Proximity Labeling tool (DBF-LPETGG) for CCI (Cell-Cell Interaction) Labeling. As shown in Fig 1.

DBF-LPETGG is a small molecule photosensitizer and can be displayed on the cell surface by SrtA-mediated cell surface ligation without genetic modification. After non-genetic displaying on cell surface, the DBF-LPETGG-mediated Labeling can label substrate Biotin-amine on nearby surface proteins within 5-10 min induced by 520 nm light.

This method could be applied to cell-cell interaction labeling and further to capture TSA-specific T cells.

Example 2

Synthesis and Mechanism

Structure of DBF-LPETGG, Mass spectrometry that confirms the molecular weight of synthesized DBF-LPETGG is correct, and structure of Biotin probe are shown in Fig 2A-2C.

Synthesis steps are shown in Fig 3, which is synthesized by common methods. For example,

Synthesis of DBF-LPETGG

To synthesize these compounds as below and Figs. 27A-27D.

Synthesis of DBF

Bromine solution: Sodium hydroxide (2.8 g, 70 mmol) was added to 30 ml of water and cooled at 0℃. Then, bromine (1.4 ml) was added slowly to this solution.

Fluorescein solution: 5 (6) -carboxyfluorescein (3.6 g, 9.6 mmol) was dissolved in alcohol (5 mL) and cooled at 0℃. The mixture was added to 100 mL of water and 2 mL of a 50 wt%aqueous solution of sodium hydroxide.

Bromine solution was added slowly to fluorescein solution with stirring over 30 min. The reaction was quenched with phosphoric acid (5.4 mL, 85%aqueous) . The crude products were precipitated and collected by filtration. The orange–red solid was dried and used without further purification.

Synthesis of Ac ₂-DBF

DBF (2.8 g, 5.1 mmol) was dissolved in 25 mL of anhydrous acetic anhydride and 1.2 mL of dry pyridine. The mixture was stirred overnight at 80℃. When the reaction was completed, the solvent was removed on a rotary evaporator, and the crude residue was redissolved in ethyl acetate (50 mL) , washed with saturated aqueous ammonium chloride (2 × 50 mL) , dried over anhydrous Na ₂SO ₄ and concentrated in vacuo. The pale-yellow solid was used without further purification.

Synthesis of Ac ₂-DBF-NHS

Ac ₂-DBF (ca. 3.8 g) was dissolved in 33 mL of dry dichloromethane and cooled at 0℃. The N-hydroxysuccinimide (1.2 eq. ) and N, N'-Diisopropylcarbodiimide (1.5 eq. ) were added to the mixture, and the reaction was allowed to reach room temperature for 24 h. When the reaction was completed, the solvent was vaporized under reduced pressure, and the resulting mixture was purified by column chromatography and eluted with toluene and EtOAc (5: 1) to give the product as a white solid (751 mg, 11%yield over three steps) .

Synthesis of DBF-LPETGG

(GGGGS) ₂-LPETGG peptide (120 mg, 0.1 mmol) and DIPEA (26 μl, 0.15 mmol) were dissolved in DMSO/H ₂O (1: 1, 5 mL) and cooled at 0℃. Then, the Ac ₂-DBF-NHS (21 mg, 0.03 mmol) was added. The mixture was stirred at room temperature for 6 h. When the reaction was completed, the resulting mixture was subjected to RP-HPLC using a linear AB gradient (0.5%CH ₃CN/min) at a flow rate of 0.25 mL/min, where eluent A was 0.05%aqueous TFA and eluent B was 0.05%TFA in CH ₃CN. The solvent was rotary vaporized to give DBF-LPETGG as a red solid (27.2 mg, 56%) . purity: >95%. The purity level of product was determined by the reversed-phase HPLC, which was performed on an ACQUITY UPLC I-Class SQD 2 (Waters) system with a BEH C18 Acquity column (1.7 mm, 2.1×100 mm) . The mobile phase was acetonitrile and water. Samples were eluted with a 6 min linear gradient (5%-95%acetonitrile, 0.3 mL/min) directed to the mass detector. Molecular mass of DBF-LPETGG was detected as 1716.22 Da.

The mechanism of DBF-LPETGG-mediated Proximity Labeling is shown in Fig 4A-4C. The R group of an amino acid and/or a saccharide on the cell surface could be bound with NH ₂ in the presence of a detectable label, like DBF.

For example, the sensitizing molecule could be:

For example, the linker could be (GGGGS) m, wherein m could be 0 or more.

Example 3

The present application uses an mgSrtA-mediated cell surface ligation method to display DBF-LPETGG on DC. After optimization of DBF-LPETGG concentration, it can be successfully displayed, and the background Adhesion is relatively low. As shown in Fig 5A-5D.

The present application uses DBF-LPETGG self-labeling assay to optimize the labeling condition. After displaying DBF-LPETGG on cell surface, the substrate biotin-NH ₂ is added in the culture medium and irradiated 10 minutes with different wavelength light.

Firstly, the Reaction conditions of DBF-LPETGG is optimized.

As shown in Fig 6A-6B. Next, to test labeling ability of various nucleophilic biotin after photooxidation, six biotin-conjugated substrates were chosen, which included from primary aliphatic amine, alcohol, mercaptan to aromatic phenol, aniline, and naphthylamine. Different biotin probes were added into cell culture followed by 10 minutes 520 nm irradiation, biotin signal was detected on cell surface. During test, biotin-alcohol (BAl) showed little labeling signal on BMDC cell surface, while a higher labeling background was observed when biotinylated by biotin-mercaptan (BMe) , biotin-aniline (BAn) and biotin-naphthylamine (BNap) , which hindered the following application. Labeling efficiency of biotin-phenol (BP) was relatively lower than biotin-amine (BAm) . To the end, Biotin-NH ₂ (BAm) was chosen as the best probe for biotin-tagging on cell surface. As shown in Fig 7A-7B.

Biotin probe screening

For biotin probe screening, six altered nucleophilic biotin probes (100 μM) were added to 96-well plates containing DBF-functionalized iDCs or unmodified iDCs respectively, and no probes were added to the blank group. iDCs were irradiated under 450 nm (12.57 mW/cm ²) , 480 nm (17.17 mW/cm ²) and 520 nm light LEDs or under dark conditions for 10 min followed by direct supernatant removal after 450 × g centrifugation for 3 min. Then, the cells were treated and analyzed according to the general present application-induced cell surface biotinylation protocol.

When using DBF-modified BMDC as the model cell, biotin-alcohol (BAl) showed no labeling signal under all three wavelengths, while there existed higher background in the groups of biotin-mercaptan (BMe) , biotin-phenol (BP) , biotin-aniline (BAn) and biotin-naphthylamine (BNap) without photocatalyst, which hindered the further applications. By contrast, biotin-NH ₂ (BAm) showed a significant labeling under 480 nm and 520 nm irradiation, with a lower labeling background at 520 nm. Although BP also induced a low background under 520 nm irradiation, the labeling efficiency is not as high as BAm. Noteworthy, this application performed a parallel screening test on probes and light wavelengths on bovine serum albumin (BSA) protein, which showed different results, suggesting that cell surface-labeling conditions should be screened in situ rather than on proteins due to the complexed environment on cell surface. As shown in Figs 28-29. Taken together, the biotin-NH ₂ (BAm) probe and 520 nm wavelength were chosen as our optimal condition for proximity labeling on cell surface.

Then the concentration of substrate and reaction duration is optimized. Data showed that the best labeling condition is 100-200 Micromolar and 5-10 min. Moreover, concentration titration of Biotin-NH2 and irradiation duration test further concluded that 100 μM biotin-NH ₂ and 5-10 minutes irradiation exhibited the best labeling condition. As shown in Fig 8A-8D.

Example 4

Cell Viability After DBF-LPETGG Displaying and Labeling

After DBF-LPETGG displaying and biotin labeling, the percentage of living cells was calculated by Zombie Aqua ^TM Fixable Viability Kit (biolegend) . DBF-LPETGG displaying and biotin labeling did not impact cell activity. Labeled Bio+/-OT-I T cells were sorting and culture in the present of 500 IU/mL IL-2, cell number was calculated by flow cytometry. DBF-LPETGG-induced intercellular labeling did not impact cell activity. As shown in Fig 9A-9C.

Example 5

Next, label CCI is performed in model system. In the iDC-ova-OT-I model system, there are three parts. CD45.1 DC is differentiated for mouse BM in the presence of GM-CSF, and the OT-I T cell is from spleen of CD45.2 Transgenic mice. OT-I T cell contain OT-I TCR on the CD8+ T cells that can specially recognize ova SIINFEKL peptide. so, the present application present SIINFEKL peptide on the DC, and during coculture OT-I T cell can specifically interact with iDC-ova through a pMHC-TCR-dependent interaction. After displaying DBF-LPETGG on the DC, iDC-ova is cocultured with OT-I T cells for 2h, and label Biotin during the stable interaction time point, then use flow cytometric analysis to analyze the result. To use CD3 for T cells and CD45.1 for DC to distinguish different cells types, and analysis the Biotin signal on each of them. As shown in Fig 10A-10B.

The GP33 is a non-specific peptide and the unprimed group is the negative control. From the labeling results, it can be seen that the method can specifically label OT-I T cells, the labeling efficiency can reach to 43%, and other labeling background is much lower. As shown in Fig 11A-11B.

After interacting, T cell will be activated, CD69 is a T cell activation marker, and can be the cell-cell interaction marker in this model. This application increased the ratio of DC to T cells, and it can be seen that during the ratio increasing, more CD69+ T cell can be labeled. It shows that DBF-LPETGG-based cell capture is CCI-dependent. As shown in Fig 12.

Next, the SIINFEKL peptide is changed to mimic different Strength of CCI. To choose some SIINFEKL variants, they can mediate iDC-ova-OT-I in different EC50. To choose three concentrations to test the correlation between labeling efficiency and CCI Strength. From the data it can be seen that the change tendency of labeling efficiency is the same as that of CD69 in both of the three concentrations. So that labeling efficiency can reflect the Strength of CCI. As shown in Fig 13A-13D.

Pulse-chase assay

The pulse-chase assay has been frequently used to characterize the temporal control of photocatalytic system. This application characterized this ability of the present application on cell surface and demonstrated that singlet oxygen-induced cell labeling could be rapidly switched on/off via photo irradiation (Fig. 30) . Pulse-chase assay of antigen-specific OT-Ⅰ T cells capture. Light irradiation was paused at the second and fifth minutes followed by regained at the third and sixth minutes. Labeling cannot continue after light source removement, while can restarted when green-light irradiation again.

spatial-controlled intercellular labeling in interacting synapses

To examine the labeling region on interacting cells, this application performed confocal imaging experiment on ex vivo DC-T cell clusters after the present application and found that the biotin signals were more likely to occur in the interaction synapse (Figs. 31-32) .

Example 6

After demonstrated that the method can specifically labeling CCI, the tool is used to capture TSA-specific T cells in mouse system. DC is an Antigen presenting cell that can phagocytose and present antigen by themselves. So, after putting tumor to single cell suspension and isolating tumor cells and TILs. The tumor cells are made into tumor lysates which naturally contain Neoantigen. Add tumor lysates to DC, DC can present neoantigen epitope on the MHC, so primed DC can be acted as a Living Tetramer to capture T cell that can recognize Neoantigen epitopes. As shown in Fig 14.

First, the capture is done in one of mouse tumor model B16-ova. After select the CD8+ T cells in B16-ova TILs, it can be seen that the CD8+ TSA-specific T cell can be capture by our method. and the capture efficiency is up to 37.5%. TIL represents Tumor infiltrating lymphocyte. As shown in Fig 15A-15B.

After successfully capture CD8+ TILs, it is tried to expand the source of TSA-specific T cells. It is tried to capture in draining LN, and not only for CD8+ T cells, but for CD4+ T cells. Again, the method can simultaneously capture CD8+ and CD4+ T cells in TILs and dLN, which expanded the source of TSA-specific T cells. As shown in Fig 16.

And all the labeled cells were PD-1+ and CD39+, Further demonstrated that labeled cells were TSA-specific. PD-1 and CD39 were reported as markers of TSA-specific T cells. As shown in Fig 17A-17B.

Then, the same capture is done in E0771, one of the mouse triple negative breast cancer cell lines. Again, the same results are got. TSA-specific CD8 ⁺ and CD4 ⁺ T cells were simultaneously captured in E0771 TILs and dLN. As shown in Fig 18A-18B.

ELISPOT data further proved that Captured CD8+ and CD4+ TILs were TSA-reactive T cells. As shown in Fig 19A-19B.

Example 7

After successfully capture TSA-specific T cells in Mouse tumor, the method can be tested in human clinical samples. As shown in Fig 20.

So, the tool is used to capture TSA-specific T cells in human bladder cancer sample. In the Human sample capture workflow, DC is differentiated from CD14+ Monocyte which from PBMC and in the presence of IL4 and GM-CSF, and during DC differentiation, TILs and LN are expanded for about 7 to 8 days. After expansion, it can get enough T cells for labeling.

First the cell validation is done before labeling. For the immature DC, it showed CD11c+, CD86+, CD80-, CD83-Phenotype, and after maturation, The level of these markers will be further increased. For the T cells after expansion, it could make sure that there are enough T cells for labeling and T cells viability. As shown in Fig 21A-21B.

For Bladder cancer sample. Take 67#Sample as an example, it shows that the method can also be used in human samples and TSA-specific CD8+ and CD4+ T cells were simultaneously captured. Moreover, the method can not only detect TSA-specific T cells in TILs, but also can capture in LN and PBMC, in which abundance of TSA-specific T cells were much lower. As shown in Fig 22.

Summary results of bladder cancer sample labeling are shown in Fig 23.

Next, as for Lung cancer, which is the most important cancer in some area, and many immunotherapy methods have been carried out in lung cancer. As an example, it shows that our method can also be used in human samples and TSA-specific CD8+ and CD4+ T cells were simultaneously captured in TILs, LN and PBMC. As shown in Fig 24 and 25.

Summary results of Lung cancer sample labeling are shown in Fig 26.

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

A method for identifying a first cell from a sample, comprising:

(a) contacting said sample with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule,

(b) inducing binding of said detectable label to said first cell when said first cell is within an effective proximity range of said sensitizing molecule,

(c) detecting the presence of the cell which is bound with said detectable label as an indication of the presence of said first cell,

wherein, said second cell is capable of specifically recognizing and/or binding said first cell.
A method for detecting cell-to-cell interaction, comprising:

(a) contacting a first cell with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule,

(b) inducing binding of said detectable label to said first cell when said first cell is within an effective proximity range of said sensitizing molecule,

(c) detecting the presence of the cell which is bound with said detectable label as an indication of the presence of the cell-to-cell interaction of said first cell and said second cell,

wherein, said second cell is capable of specifically recognizing and/or binding said first cell.
A method for intracellular labeling, comprising:

(a) contacting a first cell with a second cell in the presence of a detectable label, wherein said second cell is bound with a sensitizing molecule,

(b) inducing binding of said detectable label to said first cell when said first cell is within an effective proximity range of said sensitizing molecule,

wherein, said second cell is capable of specifically recognizing and/or binding said first cell.
The method of any one of claims 1-3, wherein said first cell expresses a first molecule on cell surface, said second cell expresses a second molecule on cell surface, and said first molecule is capable of specifically recognize and/or binding said second molecule.
The method of any one of claims 1-4, wherein said first molecule comprises peptide, protein, glycoprotein, steroid, lipid and/or lipoprotein.
The method of any one of claims 1-5, wherein said second molecule comprises peptide, protein, glycoprotein, steroid, lipid and/or lipoprotein.
The method of any one of claims 1-6, wherein said first cell comprises immune cell.
The method of any one of claims 1-7, wherein said first cell comprises B cell, T cell, lymphoid cell, dendritic cell, granulocyte, macrophage, monocyte and/or natural killer cell.
The method of any one of claims 1-8, wherein said first cell comprises a tumor-specific cell.
The method of any one of claims 1-9, wherein said first cell comprises a tumor-specific antigen (TSA) reactive T cell.
The method of claim 10, wherein said TSA is derived from a tumor selected from the group consisting of bladder tumor, melanoma; breast tumor, pancreatic tumor, lung tumor, kidney tumor, ovarian tumor, colon tumor, lymphoma, leukemia, skin tumor, and prostate tumor.
The method of any one of claims 4-11, wherein said first molecule comprises a TCR (T cell receptor) .
The method of any one of claims 1-12, wherein said sample comprises a cell population.
The method of any one of claims 1-13, wherein said sample comprises a population of tumor infiltrating lymphocytes (TILs) , a population of lymph node cells, a population of peripheral blood mononuclear cells, and/or a population of circulating T cells.
The method of any one of claims 1-14, wherein said second cell comprises immune cell.
The method of any one of claims 1-15, wherein said second cell comprises B cell, T cell, lymphoid cell, dendritic cell, granulocyte, macrophage, monocyte and/or natural killer cell.
The method of any one of claims 1-16, wherein said second cell comprises an antigen-presenting cell.
The method of any one of claims 1-17, wherein said second cell comprises a dendritic cell.
The method of any one of claims 1-18, wherein said second cell is primed with one or more tumor antigens.
The method of any one of claims 1-19, wherein said second cell is primed with tumor lysate, and said tumor lysate comprises one or more tumor antigens.
The method of any one of claims 4-20, said second molecule comprises peptide-MHC.
The method of any one of claims 1-21, wherein said first cell and said second cell are specifically recognizing the same antigen.
The method of any one of claims 1-22, wherein said first cell and said second cell are activated by the same antigen.
The method of any one of claims 1-23, wherein said detectable label is selected from the group consisting of a small molecule, a polynucleotide, and a polypeptide.
The method of any one of claims 1-24, wherein said detectable label is selected from the group consisting of an antibody, a chemical marker, a biological marker and a probe.
The method of any one of claims 1-25, wherein said detectable label comprises biotin and/or fluorophore.
The method of any one of claims 1-26, wherein said detectable label is selected from the group consisting of biotin-NH ₂ (BAm) , biotin-alcohol (BAl) , biotin-mercaptan (BMe) , biotin-phenol (BP) , biotin-aniline (BAn) and biotin-naphthylamine (BNap) .
The method of any one of claims 1-27, wherein said sensitizing molecule comprises a molecule capable of inducing production of singlet oxygen.
The method of any one of claims 1-28, wherein said effective proximity range of said sensitizing molecule is from 10 nm to 200 nm.
The method of any one of claims 1-29, wherein said sensitizing molecule is bound to the cell surface of said second cell.
The method of any one of claims 1-30, wherein said sensitizing molecule is covalently bound to the cell surface of said second cell.
The method of any one of claims 1-31, wherein said sensitizing molecule is bound to an amino acid and/or a saccharide on the cell surface of said second cell.
The method of any one of claims 1-32, wherein said sensitizing molecule is bound to said second cell through a linker.
The method of claim 33, wherein said linker comprises a cleavable linker and/or a non- cleavable linker.
The method of any one of claims 1-34, wherein said sensitizing molecule is bound to said second cell surface via glycosylation reaction, sulfhydryl-reactive crosslinker reaction, amine-reactive crosslinker reaction and/or transpeptidation.
The method of any one of claims 1-35, wherein a concentration of 10 μM to 500 μM of said sensitizing molecule is bound to said second cell surface via a reaction.
The method of claim 36, wherein said reaction is selected from the group consisting of glycosylation reaction, sulfhydryl-reactive crosslinker reaction, amine-reactive crosslinker reaction and transpeptidation.
The method of any one of claims 35-37, wherein the duration of said reaction is about 1 minute to about 20 minutes.
The method of any one of claims 1-38, wherein said sensitizing molecule comprises an organic molecule, an inorganic molecule, and/or a complex thereof.
The method of any one of claims 1-39, wherein said sensitizing molecule comprises a polymer.
The method of any one of claims 1-40, wherein said sensitizing molecule comprises a photosensitizer.
The method of any one of claims 1-41, wherein said sensitizing molecule is selected from the group consisting of dye, aromatics, porphyrin, metalloporphyrin, phthalocyanine, metal phthalocyanine, macrocycles, tetrapyrrole, transition metal complex, and semiconductor.
The method of any one of claims 1-42, wherein said sensitizing molecule is selected from the group consisting of rose bengal, fluorescein, Dibromofluorescein (DBF) , methylene blue, napthalene, anthracene, biphenyl, octaethylporphine (OEP) , metal OEP, tetraphenylporphine (TPP) , metal TPP, metal phthalocyanine tetrasulfonate (PcTS) , texaphyrin, complex of Ruthenium, complex of Osmium, complex of Iridium, complex of Chromium, and complex of Platinumm, TiO ₂, and ZnO.
The method of any one of claims 1-43, wherein said sensitizing molecule comprises DBF.
The method of any one of claims 1-44, wherein the contacting of step (a) is performed under light condition.
The method of claim 45, wherein the light intensity is about 6.14 mW/cm ², 12.57 mW/cm ², and/or 17.17 mW/cm ².
The method of any one of claims 45-46, wherein the wavelength of light is from 450 nm to 520 nm.
The method of any one of claims 45-47, wherein the duration of light is about 5 min to 10 min.
The method of any one of claims 1-48, wherein the number ratio of the second cells and the cells of said sample of step (a) is from about 1: 5 to about 5: 1.
The method of any one of claims 1-49, wherein inducing binding of said detectable label to said first cell of step (b) is controllable by light source removement or light source providing.
The method of any one of claims 1-50, wherein step (b) comprises enriching for said first cells.
The method of any one of claims 1-51, wherein enriching for said first cells is via Fluorescence-activated cell sorting (FACS) .
The method of any one of claims 1-52, further comprises determining whether the first cell comprises a marker indicative of TSA reactivity.
The method of any one of claims 1-53, further comprises enriching for the first cells comprising a marker indicative of TSA reactivity via FACS.
The method of any one of claims 53-54, wherein said marker comprises PD-1 expression; CD39 expression, LAG3 expression, and/or TIM3 expression.
The method of any one of claims 1-55, further comprising enriching for said first cell comprising CD8 ⁺ and/or CD4 ⁺ expression.
The method of any one of claims 1-56, wherein the magnitude of the detectable label present on the first cell is indicative of the binding affinity of a T cell receptor expressed on the surface of the T cell for the TSA.
The method of any one of claims 1-57, wherein the magnitude of the detectable label present on the first cell is indicative of the enriched first cell’s ability to kill other cells expressing the TSA and/or of the enriched the first cell’s ability to become activated in the presence of the TSA.
The method of any one of claims 1-58, further comprising expanding the enriched cells for patient specific immune cell therapy.
A method for treating a tumor comprising administering to a subject in need thereof a TSA reactive T cell identified by, or labelled by the method of any one of claims 1-59.
A method for identifying a tumor-specific antigen (TSA) -specific T cell receptors (TCRs) comprising identifying a TSA reactive T cell by the method of any one of claims 1-59.
A method for expanding TSA reactive T cells comprising performing the method of any one of claims 1-59, and expanding said the TSA-specific T cells.
A method for determining the relative binding affinity for an antigen of TCR respectively expressed on a T cell comprising performing the method of any one of claims 1-59, and quantifying the level of detectable label bound to said T cell.
A modified cell, which is bound with a sensitizing molecule.
The modified cell of claim 64, wherein said sensitizing molecule comprises a molecule capable of inducing production of singlet oxygen.
The modified cell of any one of claims 64-65, wherein said sensitizing molecule is bound to the cell surface of a cell.
The modified cell of any one of claims 64-66, wherein said sensitizing molecule is covalently bound to the cell surface of said modified cell.
The modified cell of any one of claims 64-67, wherein said sensitizing molecule is bound to an amino acid and/or a saccharide on the cell surface of said modified cell.
The modified cell of any one of claims 64-68, wherein said sensitizing molecule is bound to said modified cell through a linker.
The modified cell of claim 69, wherein said linker comprises a cleavable linker and/or a non-cleavable linker.
The modified cell of any one of claims 64-70, wherein said sensitizing molecule is bound to said modified cell surface via glycosylation reaction, sulfhydryl-reactive crosslinker reaction, amine-reactive crosslinker reaction and/or transpeptidation.
The modified cell of any one of claims 64-71, wherein said sensitizing molecule comprises an organic molecule, an inorganic molecule, and/or a metal complex.
The modified cell of any one of claims 64-72, wherein said sensitizing molecule comprises a polymer.
The modified cell of any one of claims 64-73, wherein said sensitizing molecule comprises a photosensitizer.
The modified cell of any one of claims 64-74, wherein said sensitizing molecule is selected from the group consisting of dye, aromatics, porphyrin, metalloporphyrin, phthalocyanine, metal phthalocyanine, macrocycles, tetrapyrrole, transition metal complex, and semiconductor.
The modified cell of any one of claims 64-75, wherein said sensitizing molecule is selected from the group consisting of rose bengal, fluorescein, DBF, methylene blue, napthalene, anthracene, biphenyl, octaethylporphine (OEP) , metal OEP, tetraphenylporphine (TPP) , metal TPP, metal phthalocyanine tetrasulfonate (PcTS) , texaphyrin, complex of Ruthenium, complex of Osmium, complex of Iridium, complex of Chromium, and complex of Platinumm, TiO ₂, and ZnO.
The modified cell of any one of claims 64-76, wherein said sensitizing molecule comprises DBF.
A composition, comprising a modified cell of any one of claims 64-77.
A kit for intracellular labeling a target cell, comprising

(a) a modified cell of any one of claims 64-77 and/or a composition of claim 78, and

(b) a detectable label,

wherein, said modified cell is capable of specifically recognizing and/or binding said target cell.