Kit, method and use thereof for detecting cell-cell interactions in a sample

Abstract

The present invention provides an in vitro method for detecting cell-cell interactions and related kits and uses, wherein the method comprises: (a) at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically different; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; The first and second secondary binders are conjugated or fused to DNA sequences, wherein the DNA sequences are different and connected to form a circle and are amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binder is labeled with a FRET donor and the second secondary binder is fused to an enzyme, which reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich portion on the surface of a molecule adjacent to the enzyme; wherein the method comprises: i. contacting an isolated sample containing cells with the at least two primary binders; ii. contacting the sample with the at least two secondary binders; iii. performing a washing step; iv. detecting the interaction between the secondary binders.

Description

Technical Field

[0001] The present invention relates to a kit and method for detecting cell-cell interactions, particularly in fixed samples. The present invention further provides quantification of cell-cell interactions. Specifically, the present invention relates to detecting the interaction between two molecules expressed on two different cells in close proximity. For example, the present invention relates to the interaction at the cellular level between immune checkpoint proteins presented on the cell surface of different cells in close proximity, such as the interaction between PD-1 and PD-L1 / PD-L2.

[0002] The present invention relates to the use of coincidence assays (such as proximity ligation assays, coincidence detection or (Fluorescence) resonance energy transfer (FRET)) to improve the detection of cell-cell interactions in samples. More specifically, the present invention relates to a dual-site method in which two sites are located on different molecules (e.g., proteins) expressed on different cells. The method and kit can be used to determine whether checkpoint protein inhibitors are likely to be effective in treating cancer.

[0003] The present invention also relates to kits and methods for predicting and identifying patients likely to respond to cancer treatments (such as inhibition of the PD-1:PD-L1 / PD-L2 pathway), as well as stratifying patients who will not respond to such treatments based on their responder / non-responder profiles and providing for determining treatment options for patients.

[0004] The present invention can be used to analyze tumor samples and can be applied to the diagnosis, monitoring, stratification and treatment of cancer patients and drug development in the field of cancer. Background Art

[0005] Cell-cell communication is essential for the function of multicellular organisms. Modulation, overstimulation, or disruption of cell-cell signaling can lead to a variety of disease states. Consequently, research efforts have focused on examining and elucidating cell-cell interactions to (a) identify new therapeutic targets, (b) guide patient selection, and (c) monitor therapeutic efficacy in a variety of disease states.

[0006] One area where the detection and quantification of cell-cell interactions is crucial is examining the interactions between immune and non-immune cells in maintaining a healthy immune system. In this context, disrupted or aberrant interactions can lead to a variety of disease states, ranging from overactive immune responses to inflammatory diseases and tumors that result in an underactive immune response.

[0007] Indeed, understanding, measuring, and quantifying the ability of tumor cells to evade immune cell-mediated destruction by binding to and inhibiting T cells has become a major concern. Failure to understand this is believed to be one of the main reasons that has limited the therapeutic application of inhibitory T cell-tumor cell interactions to modulating specific T cell-tumor cell protein interactions.

[0008] Cancer immunotherapy research has sought to overcome the ability of tumor cells to evade immune-mediated destruction and stimulate an individual's immune system to target tumor cells.

[0009] One of the most promising approaches to overcome the ability of tumor cells to evade immune cell-mediated destruction is to block immune checkpoint markers. Immune checkpoints refer to a number of immunosuppressive pathways that are crucial for maintaining self-tolerance and regulating the severity of immune responses to minimize unnecessary damage to non-pathogenic tissues. It is known in the art that tumor cells hijack these immunosuppressive checkpoint pathways, serving as a primary mechanism for escaping destruction by T cells specific for tumor antigens. The inhibitory interaction between T cells and tumor cell surface checkpoint proteins is therefore an attractive target for cancer therapy.

[0010] In fact, it has been shown that blocking T cells: Therapeutic antibodies that inhibit the interaction of tumor cell checkpoints successfully reduce tumor volume in clinical settings. However, due to the absence of available methods or assays to detect which immune checkpoints will become promising targets for cancer treatment, these treatments are limited to very few immune checkpoints. In addition, there may be a large number of checkpoint interactions that may exist that have not yet been discovered. Obviously, it is necessary to allow the detection of cell-cell interactions (such as T cells: tumor cell checkpoint interactions).

[0011] A well-characterized immune checkpoint protein interaction is the binding of the programmed cell death 1 (PD-1) receptor (also known as cluster of differentiation (CD) 279) to either of the ligands programmed cell death ligand 1 or 2 (PD-L1 or 2), also known as CD274 or CD273, respectively. PD-1 is expressed on the surface of activated T cells, while PD-L1 / 2 is expressed on antigen-presenting cells (such as dendritic cells and macrophages) as well as tumor cells.

[0012] The combination of PD-1 and PD-L1 / 2 generates an inhibitory signal in T cells that stops or limits T cell proliferation and cytokine production. The PD-1: PD-L1 / 2 interaction ensures that the immune system is activated at the right time, thereby minimizing potential autoimmunity to the greatest extent possible. Cancer tumor cells utilize the PD-1: PD-L1 / PD-L2 checkpoint pathway to provide a mechanism to avoid detection and subsequent T cell-mediated destruction. Blocking PD-1: PD-L1 / PD-L2 therapy has shown unprecedented persistent tumor response rates in a variety of cancer types (Ribas et al., Clin. Cancer Res; 20(19)2014).

[0013] Another well-characterized immune checkpoint protein interaction is the binding of cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152, to either cluster of differentiation ligand 80 (CD80) or 86 (CD86), also known as B7-1 and B7-2, respectively. CTLA-4 is expressed on activated T cells, while CD80 and CD86 are expressed on antigen-presenting cells, such as dendritic cells or macrophages. The binding of CTLA-4 to CD80 / 86 generates an inhibitory signal on T cells that stops or limits T cell proliferation and cytokine production. The CTLA-4 / CD80 / 86 interaction ensures that the immune system is activated at the right time, thereby minimizing potential autoimmunity. Cancer tumor cells utilize the CTLA-4 / CD80 / 86 checkpoint pathway, providing a mechanism to avoid detection by T cells and subsequent destruction by them. Therapies such as ipilimumab, a monoclonal antibody that blocks CTLA-4 / CD80 / 86, lead to increased immune system activation and tumor regression in multiple cancer types (Buchbinder, EI & Desai, A. American Journal of Clinical Oncology; 39(1) 2016).

[0014] The assessment of cell-cell interactions has not been widely reported, partly due to the lack of available methods / techniques that allow detection of cell-cell interactions in fixed samples. Typically, conventional proteomics applied to tumor samples relies on immunohistochemistry (IHC) to determine relative protein content, or relies on extraction and processing, such as enzyme-linked immunosorbent assay (ELISA). Although conventional IHC minimizes manipulation and retains tissue organization, it is limited in its specificity (single-site determination). ELISA-type methods can bring specificity (two-site determination) but require processing, which brings challenges to sample quantity and destroys spatial information. Current systems are only used to detect interactions between molecules in solution (soluble recombinant proteins) or single cells. For example, the interaction of PD-1:PD-L1 / PD-L2 has been previously studied by FRET, but only in solutions using recombinant or purified proteins in the context of the assay.

[0015] WO 2014 / 140554 describes a method for detecting molecules in a sample using a combination of fluorescence resonance energy transfer (FRET) and a tyramide activation system (TSA) in a dual-site detection method. However, the use of this type of method is limited to detecting and quantifying two sites on the same protein or two sites on different proteins under the background of the same cell. Therefore, current detection methods are limited in their specificity and sensitivity, and cannot detect and quantitatively interact at the cellular level.

[0016] Previous methods known in the art relied on qualitative intensity-based assessments to detect cell-cell interactions by detecting co-localization of overlapping immune signals and manually counting the number of overlapping signals and estimating the ratio of number of signals: number of nuclei.

[0017] There remains a need to provide sensitive and specific methods for detecting and quantifying interactions between molecules (e.g., two proteins) present on the cell surfaces of two separate cells, particularly immune checkpoint protein interactions, to aid in the development of improved and effective methods for identifying and treating cancer patients.

[0018] The object of the present invention is to provide an improved method and kit for detecting cell-cell interactions between two molecules expressed on two different cells in fixed samples (such as tissue sections), in particular immune checkpoint protein interactions on the surface of different cells (such as PD-1:PD-L1 / PD-L2, major histocompatibility complex (MHC) I-II:T cell receptor (TCR) / CD8 / CD3 or CTLA-4 / CD80 / 86). Another object of the present invention is to provide a kit and method for predicting and identifying patients who are likely to respond to cancer treatments (such as inhibition of PD-1:PD-L1 / PD-L2, MHC I-II:TCR / CD8 / CD3 or CTLA-4 / CD80 / 86), and stratifying patients who do not respond to such treatments based on the patient's responder / non-responder profile and providing treatment options for the patient. Summary of the Invention

[0019] The present invention provides a method for detecting and quantifying cell-cell interactions. Specifically, the present invention relates to methods for detecting and quantifying interactions between molecules on different cells, such as protein-protein interactions, wherein each protein is on the surface of different cells, i.e., in a spanning manner. Specifically, the present invention relates to detecting and quantifying immune checkpoint protein interactions, such as the interaction between PD-1 and PD-L1 or PD-L2, or the interaction between CTLA-4 or CD28 and CD80 or 86, or the interaction between MHC class I or MHC class II peptides and TCR, CD83, CD3, and combinations thereof, at the cellular level.

[0020] According to the present invention, there is provided an in vitro method for detecting cell-cell interactions, the method comprising:

[0021] at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0022] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0023] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0024] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0025] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0026] The method comprises:

[0027] (a) contacting an isolated sample containing cells with the at least two primary binding agents;

[0028] (b) contacting the sample with the at least two secondary binding agents;

[0029] (c) performing a washing step;

[0030] (d) detecting the interaction between the secondary binding agents.

[0031] In the methods, kits and uses of the present invention, the interaction between the secondary binding agents can be detected by detecting the emitted fluorescence.

[0032] In the methods, kits and uses of the present invention, the interaction between the secondary binding agents can be detected by detecting the altered fluorescence behavior. Preferably, the detection of the altered fluorescence behavior is time-resolved.

[0033] In the methods, kits and uses of the present invention, the first cell and the second cell may be the same type of cell.

[0034] In the methods, kits and uses of the present invention, the first cell and the second cell may be different types of cells, ie, not the same type of cells.

[0035] In the methods, kits and uses of the present invention, the isolated sample may be a fixed cell sample.

[0036] In the methods, kits and uses of the present invention, the isolated sample may be a fixed tumor cell sample.

[0037] In the methods, kits and uses of the present invention, the first molecule and the second molecule can be proteins, preferably endogenous proteins. Preferably, the proteins are immune checkpoint proteins.

[0038] In the methods, kits and uses of the present invention, the first molecule may be PD-1 and the second molecule may be PD-L1 or PD-L2.

[0039] In the methods, kits and uses of the present invention, the first molecule may be CTLA-4 or CD28 and the second molecule may be CD80 or CD86.

[0040] In the methods, kits and uses of the present invention, the first molecule may be an MHC class I or II peptide and the second molecule may be selected from the group consisting of TCR, CD8, CD3 and combinations thereof.

[0041] In the methods, kits and uses of the present invention, the at least two primary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds, antibodies or antigen-binding fragments thereof, or a combination thereof.

[0042] In the methods, kits and uses of the present invention, the at least two secondary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds or antibodies or antigen-binding fragments thereof or a combination thereof.

[0043] In the methods, kits and uses of the present invention, at least one of the secondary binding agents may be an antibody scaffold, an antibody or an antigen binding fragment. In the methods, kits and uses of the present invention, at least two of the secondary binding agents may be an antibody scaffold, an antibody binding fragment or an antigen binding fragment.

[0044] In the methods, kits and uses of the present invention, the antibody or antigen-binding fragment may be a Fab fragment, a scFv fragment or a combination thereof.

[0045] In the methods, kits and uses of the present invention, the antibody scaffold can be an adnectin, affibodies, affilin, anticalin, atrimer, avimer, bicyclic peptide, centyrin, cysteine ​​knot (Cys-knot), DARPin, fynomer, Kunitz domain, obodies and Tn3.

[0046] In the methods, kits and uses of the present invention, the primary binding agent may be unlabeled.

[0047] In the methods, kits and uses of the invention, the first primary binding agent may be a murine binding agent and at least one additional primary binding agent may be a rabbit binding agent.

[0048] In the methods, kits and uses of the invention, the first primary binding agent may bind to PD-1 and the at least one additional primary binding agent may bind to PD-L1 or PD-L2.

[0049] In the methods, kits and uses of the invention, the first primary binding agent may bind to PD-1 on a first cell and the at least one additional primary binding agent may bind to PD-L1 or PD-L2 on a second cell.

[0050] In the method, kit and use of the present invention, the method can detect the binding of PD-1 to PD-L1 or PD-L2.

[0051] In the methods, kits and uses of the present invention, the first molecule may be CTLA-4 or CD28 and the second molecule may be CD80 or CD86.

[0052] In the methods, kits and uses of the invention, the first primary binding agent may bind to CTLA-4 or CD28 on a first cell and the at least one additional primary binding agent may bind to CD80 or CD86 on a second cell.

[0053] In the method, kit and use of the present invention, the method can detect the binding of CTLA-4 or CD28 to CD80 or CD86.

[0054] In the methods, kits and uses of the present invention, the first molecule may be an MHC class I or II peptide and the second molecule may be selected from the group consisting of TCR, CD8, CD3 and combinations thereof.

[0055] In the methods, kits and uses of the invention, the first primary binding agent may bind to an MHC class I or MHC class II peptide and the second primary binding agent may bind to TCR, CD83, CD3 and combinations thereof on the second cell.

[0056] In the methods, kits and uses of the present invention, the methods can detect the binding of MHC class I or MHC class II peptides to TCR, CD83, CD3 and combinations thereof.

[0057] In the methods, kits and uses of the present invention, the first cell may be a lymphocyte, preferably wherein the first cell may be a T cell.

[0058] In the methods, kits and uses of the present invention, the second cell may be a non-lymphocyte, preferably wherein the second cell may be an antigen presenting cell.

[0059] In the methods, kits and uses of the present invention, the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte can be detected.

[0060] In the methods, kits and uses of the present invention, the interaction between CTLA-4 or CD28 on a first lymphocyte and CD80 or CD86 on a second non-lymphocyte can be detected.

[0061] In the methods, kits and uses of the present invention, the interaction between an MHC class I or II peptide on a first lymphocyte and a TCR, CD8, CD3 or a combination thereof on a second non-lymphocyte can be detected.

[0062] In the methods, kits and uses of the invention, the first secondary binding agent may be an anti-murine binding agent and at least one additional secondary binding agent may be an anti-rabbit binding agent.

[0063] In the methods, kits and uses of the present invention, the FRET donor may be selected from the group consisting of ORG 488, GFP, fluorescein, IAEDANS, EDANS, BODIPY FL, ATTO488 and combinations thereof.

[0064] In the methods, kits and uses of the present invention, the FRET acceptor may be selected from the group consisting of ALX 594, mRFP, tetramethylrhodamine, fluorescein, dabcyl, BODIPY FL, QSY 7, QSY 9 and combinations thereof.

[0065] In the methods, kits and uses of the present invention, the enzyme may be selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases and ligases.

[0066] In the methods, kits and uses of the present invention, the enzyme may be selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

[0067] In the methods, kits and uses of the present invention, the enzyme may be selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and β-galactosidase.

[0068] In the methods, kits and uses of the present invention, the substrate may be tyramide.

[0069] In the methods, kits and uses of the present invention, the first cell may be a T cell and the second cell may be a tumor cell.

[0070] In the methods, kits and uses of the present invention, at least two primary binding agents may be contacted with each other simultaneously or sequentially.

[0071] In the methods, kits and uses of the present invention, at least two secondary binding agents may be contacted with each other simultaneously or sequentially.

[0072] In the methods, kits and uses of the present invention, at least two primary binding agents can be contacted with the sample simultaneously with at least two secondary antibodies.

[0073] In the methods, kits and uses of the present invention, at least two primary binding agents may be contacted with the sample before at least two secondary binding agents.

[0074] In the methods, kits and uses of the present invention, a washing step may be performed after contacting the at least two primary binding agents with the sample and before contacting the at least two secondary binding agents with the sample.

[0075] In the method, kit and use of the present invention, the method may further comprise the step of quantifying the interaction between the first site on the first cell and the second site on the second cell.

[0076] In the methods, kits and uses of the invention, the first cell may be a lymphocyte and the second cell may be a non-lymphocyte cell type.

[0077] In the method, kit and use of the present invention, the method can detect the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte.

[0078] In the methods, kits and uses of the present invention, the first molecule may be located on the cell surface of a first cell and the second molecule may be located on the cell surface of a second cell.

[0079] In the methods, kits and uses of the present invention, at least two primary binding agents may bind to the first or second molecule in such a manner that a checkpoint inhibitor or activator may bind to the first or second molecule simultaneously or sequentially.

[0080] In some methods, kits, and uses of the invention, at least two primary binding agents do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[0081] The present invention also provides use of the aforementioned in vitro method for detecting the interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell.

[0082] The present invention further provides a kit for use in an in vitro method for detecting cell-cell interactions, the kit comprising:

[0083] a) at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0084] b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary antibody; and wherein:

[0085] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0086] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0087] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0088] c) instructions for performing a method comprising:

[0089] i. contacting the isolated sample containing cells with the at least two primary binding agents;

[0090] ii. contacting the sample with the at least two secondary binding agents;

[0091] iii. performing a washing step;

[0092] iv. detecting the interaction between the secondary binding agents.

[0093] In the kit of the present invention, instructions for detecting the interaction between the secondary binding agents can be detected by detecting emitted fluorescence.

[0094] In the kit of the present invention, instructions for detecting the interaction between the secondary binding agents can be detected by detecting altered fluorescence behavior.

[0095] In the kit of the present invention, detection of the altered fluorescence behavior may be time-resolved.

[0096] In the kit of the present invention, the first cell and the second cell may be cells of the same type.

[0097] In the kit of the present invention, the first cell and the second cell may be different types of cells, ie, not the same type of cells. In the kit of the present invention, the isolated sample may be a fixed cell sample.

[0098] In the kit of the present invention, the isolated sample may be a fixed tumor cell sample.

[0099] In the kit of the present invention, the first molecule and the second molecule can be proteins, preferably endogenous proteins. Preferably, the proteins are immune checkpoint proteins.

[0100] In the kit of the present invention, the first molecule may be PD-1 and the second molecule may be PD-L1 or PD-L2.

[0101] In the kit of the present invention, the first molecule may be CTLA-4 or CD28 and the second molecule may be CD80 or CD86.

[0102] In the kit of the present invention, the first molecule may be an MHC class I or II peptide and the second molecule may be selected from the group consisting of TCR, CD8, CD3, and combinations thereof.

[0103] In the kit of the present invention, the at least two primary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds, antibodies or antigen-binding fragments thereof, or a combination thereof.

[0104] In the kit of the present invention, the at least two secondary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds or antibodies or antigen-binding fragments thereof or a combination thereof.

[0105] In the kits of the present invention, at least one of the secondary binding agents may be an antibody scaffold, an antibody or an antigen binding fragment. In the methods, kits and uses of the present invention, at least two secondary binding agents may be an antibody scaffold, an antibody binding fragment or an antigen binding fragment.

[0106] In the kit of the present invention, the antibody or antigen-binding fragment may be a Fab fragment, a scFv fragment, or a combination thereof.

[0107] In the kit of the present invention, the antibody scaffold can be an adnectin, affibodies, affilin, anticalin, atrimer, avimer, bicyclic peptide, centyrin, cysteine ​​knot, DARPin, fynomer, Kunitz domain, obodies and Tn3.

[0108] In the kit of the present invention, the primary binding agent may be unlabeled.

[0109] In the kits of the invention, the first primary binding agent may be a murine binding agent and at least one additional primary binding agent may be a rabbit binding agent.

[0110] In the kits of the invention, the first primary binding agent may bind to PD-1 and at least one additional primary binding agent may bind to PD-L1 or PD-L2.

[0111] In the kit of the present invention, the first primary binding agent may bind to CTLA-4 or CD28 and the second primary binding agent may bind to CD80 or CD86.

[0112] In the kits of the present invention, the first primary binding agent may bind to an MHC class I or II peptide and the second primary binding agent may bind to TCR, CD8, CD3, and combinations thereof.

[0113] In the kits of the invention, the first secondary binding agent may be an anti-murine binding agent and at least one additional binding agent may be an anti-rabbit binding agent.

[0114] In the kit of the present invention, the FRET donor may be selected from the group consisting of ORG 488, GFP, fluorescein, IAEDANS, EDANS, BODIPY FL, ATTO488, and combinations thereof.

[0115] In the kit of the present invention, the FRET acceptor may be selected from the group consisting of ALX 594, mRFP, tetramethylrhodamine, fluorescein, dabcyl, BODIPY FL, QSY 7, QSY 9, and combinations thereof.

[0116] In the kit of the present invention, the enzyme may be selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases and ligases.

[0117] In the kit of the present invention, the enzyme may be selected from the group consisting of peroxidase, oxidase, phosphatase, esterase and glycosidase. Preferably, the enzyme may be selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and β-galactosidase.

[0118] In the kit of the present invention, the substrate may be tyramide.

[0119] In the kit of the present invention, the first cell is a T cell and the second cell is a tumor cell.

[0120] In the kits of the invention, the instructions may provide for contacting at least two primary binding agents with each other simultaneously or sequentially.

[0121] In the kits of the invention, the instructions may provide for contacting at least two secondary binding agents with each other simultaneously or sequentially.

[0122] In the kits of the invention, the instructions may provide for contacting the at least two primary binding agents and the at least two secondary binding agents with the sample simultaneously.

[0123] In the kits of the invention, the instructions may provide for contacting the at least two primary binding agents with the sample before the at least two secondary binding agents.

[0124] In the kits of the invention, the instructions may provide for performing a washing step after contacting the at least two primary binding agents with the sample and before contacting the at least two secondary binding agents with the sample.

[0125] In the kits of the present invention, the instructions provide for the method further comprising the step of quantifying the interaction between a first site on a first cell and a second site on a second cell.

[0126] In the kit of the invention, the first cell may be a lymphocyte and the second cell may be a non-lymphocyte cell type.

[0127] In the kit of the present invention, the instructions may provide a method for detecting the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte.

[0128] In the kit of the present invention, the first molecule may be located on the cell surface of a first cell and the second molecule may be located on the cell surface of a second cell.

[0129] In the kit of the present invention, at least two primary binding agents can be bound to the first molecule or the second molecule in such a manner that the checkpoint inhibitor or activator can be bound to the first molecule or the second molecule simultaneously or sequentially.

[0130] In some aspects of the kits of the invention, the at least two primary binding agents do not inhibit binding of the inhibitor or activator to the first molecule or the second molecule.

[0131] The present invention also provides the use of the aforementioned kit of the present invention in an in vitro method for detecting cell-cell interactions. Preferably, the use of the kit is in an in vitro coincidence assay for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell.

[0132] The present invention further provides a method for selecting a patient suffering from cancer for treatment, the method comprising:

[0133] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0134] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0135] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0136] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0137] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0138] The method comprises:

[0139] (a) contacting an isolated tumor cell sample from the patient with the at least two primary binding agents;

[0140] (b) contacting the sample with the at least two secondary binding agents;

[0141] (c) performing a washing step;

[0142] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; wherein:

[0143] a. Where the intended therapy comprises a checkpoint activator targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[0144] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy; or

[0145] ii. if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0146] b. wherein the intended therapy comprises a test that targets at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0147] In the case of checkpoint inhibitors:

[0148] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0149] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy.

[0150] In the method of the present invention, the first checkpoint target molecule may be PD-1 and the second checkpoint target molecule may be PD-L1, and wherein when determining the fluorescent signal score:

[0151] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-PD-1, anti-PD-1:L1 or anti-PD-L2 binding agent; or

[0152] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-PD-1, anti-PD-1:L1 or anti-PD-L2 binding agent.

[0153] In the method of the present invention, the first checkpoint target molecule may be CTLA-4 or CD28 and the second checkpoint target molecule may be CD80 or CD86, and wherein the fluorescent signal fraction is determined:

[0154] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-CTLA-4 / CD28 binding agent or an anti-CD80 / 86 binding agent; or

[0155] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-CTLA-4 / CD28 binding agent or an anti-CD80 / 86 binding agent.

[0156] In the methods of the present invention, the first checkpoint target molecule can be an MHC class I or II peptide and the second checkpoint target molecule can be TCR, CD8, CD3, and combinations thereof, and wherein the fluorescent signal fraction is determined:

[0157] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-MHC class I or II binding agent or an anti-TCR / CD8 / CD3 binding agent; or

[0158] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-MHC class I or II binding agent or an anti-TCR / CD8 / CD3 binding agent.

[0159] In some aspects of the methods of the invention, the patient has not previously received cancer therapy or the patient has not previously received therapy targeting at least one of the checkpoint target molecules.

[0160] In the methods of the present invention, if the total score is between 0.5% and 5%, this may indicate that the patient will not respond to a therapy targeting at least one of the checkpoint target molecules.

[0161] In the methods of the present invention, if the total score is between 5% and 10%, this may indicate that the patient is likely to respond to a therapy targeting at least one of the checkpoint target molecules.

[0162] In the methods of the present invention, if the total score is greater than 10%, this may indicate that the patient will respond to a therapy targeting at least one of the checkpoint target molecules.

[0163] In the methods of the present invention, if the total score is between 0.5% and 5%, this may indicate that the patient will respond to a therapy targeting the MHC class I / II-TCR / CD8 / CD3 interaction.

[0164] In the methods of the present invention, if the total score is between 5% and 10%, this may indicate that the patient is likely to respond to a therapy targeting the MHC class I / II-TCR / CD8 / CD3 interaction.

[0165] In the methods of the present invention, if the total score is greater than 10%, this may indicate that the patient will not respond to a therapy targeting the MHC class I / II-TCR / CD8 / CD3 interaction.

[0166] In the methods of the present invention, the total fraction may be a FRET efficiency percentage.

[0167] In the methods of the present invention, at least two primary binding agents may be bound to the first or second molecule in such a manner that a checkpoint inhibitor or activator may be bound to the first or second molecule simultaneously or sequentially.

[0168] In some aspects of the methods of the invention, the at least two primary binding agents do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[0169] The present invention also provides use of the aforementioned method of the present invention in selecting a patient suffering from cancer for treatment, wherein:

[0170] a. Where the intended therapy comprises a checkpoint activator targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[0171] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy; or

[0172] ii. if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0173] b. When the intended therapy comprises a checkpoint inhibitor targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[0174] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0175] ii. If the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy.

[0176] The present invention further provides an in vitro coincidence assay method for detecting whether a checkpoint activator or inhibitor effectively inhibits or modulates a tumor response, the method comprising:

[0177] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0178] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary antibody; and wherein:

[0179] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0180] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0181] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0182] The method comprises:

[0183] (a) contacting the isolated tumor cell sample with the at least two primary binding agents;

[0184] (b) contacting the sample with the at least two secondary binding agents;

[0185] (c) performing a washing step;

[0186] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0187] (e) contacting the sample with the checkpoint activator or inhibitor;

[0188] (f) detecting any changes in the interactions between the secondary binding agents, wherein:

[0189] a. In cases where the checkpoint molecule is an inhibitor:

[0190] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[0191] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[0192] b. In the case where the checkpoint molecule is an activator:

[0193] i. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[0194] ii. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[0195] In the methods of the present invention, the checkpoint inhibitor may be an anti-PD-1 binding agent, and the first primary binding agent may bind to PD-1 and the second primary binding agent may bind to PD-L1 or PD-L2.

[0196] In the methods of the invention, the checkpoint inhibitor can be an anti-CTLA-4 or CD28 binding agent, and the first primary binding agent can bind to CTLA-4 or CD28 and the second primary binding agent can bind to CD80 or CD86.

[0197] In the method of the present invention, the check point inhibitor can be an anti-MHC class I or II binding agent, and the first primary binding agent can be combined with MHC class I or II and the second primary binding agent can be combined with TCR, CD8, CD3 and a combination thereof. In the method of the present invention, the check point inhibitor can be selected from the group consisting of: Ipilimumab, Nivolumab (nivolumab) (BMS-936558, DX 1106 or ONO-4538), Pembrolizumab (iambrolizumab) (rambrolizumab (iambrolizumab) or MK-3475), pidilizumab (pidilizumab) (CT-Q11), ED-0680 (AMP-514), AMP-224, BMS-936559 (MDX-1105), ED 4736, MPDL3280A (RG7448), MSB0010718C and fragments and salts thereof.

[0198] In the methods of the invention, an increase in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% may indicate that the checkpoint activator will be effective.

[0199] In the methods of the present invention, a decrease in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% may indicate that the checkpoint inhibitor will be effective.

[0200] In the methods of the present invention, at least two primary binding agents may be bound to the first or second molecule in such a manner that a checkpoint inhibitor or activator may be bound to the first or second molecule simultaneously or sequentially.

[0201] In some aspects of the methods of the invention, the at least two primary binding agents do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[0202] The present invention also provides the use of the in vitro coincidence assay of the present invention for detecting whether a checkpoint inhibitor or activator will effectively inhibit or modulate a tumor response,

[0203] in:

[0204] a. In cases where the checkpoint molecule is an inhibitor:

[0205] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[0206] ii. If the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective;

[0207] or

[0208] b. In the case where the checkpoint molecule is an activator:

[0209] i. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[0210] ii. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[0211] The present invention further provides a kit for use in an in vitro coincidence assay method for detecting whether a checkpoint inhibitor or activator effectively inhibits or modulates a tumor response, the kit comprising:

[0212] (a) at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0213] (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0214] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0215] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0216] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0217] (c) instructions for performing a method comprising:

[0218] a. contacting the isolated tumor cell sample with the at least two primary binding agents;

[0219] b. contacting the sample with the at least two secondary binding agents;

[0220] c. performing a washing step;

[0221] d. detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0222] e. contacting the sample with the checkpoint activator or inhibitor;

[0223] f. detecting any changes in the interactions between said secondary binding agents, wherein:

[0224] Where the checkpoint molecule is an inhibitor:

[0225] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[0226] ii. If the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective;

[0227] or

[0228] Where the checkpoint molecule is an activator:

[0229] iii. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[0230] iv. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[0231] In the kit of the present invention, instructions for detecting the interaction between the secondary binding agents can be detected by detecting emitted fluorescence.

[0232] In the kit of the present invention, instructions for detecting the interaction between the secondary binding agents can be detected by detecting altered fluorescence behavior.

[0233] In the kit of the present invention, detection of the altered fluorescence behavior may be time-resolved.

[0234] In the kit of the present invention, the first cell and the second cell may be cells of the same type.

[0235] In the kit of the present invention, the first cell and the second cell may be different types of cells, ie, not the same type of cells. In the kit of the present invention, the isolated sample may be a fixed cell sample.

[0236] In the kit of the present invention, the isolated sample may be a fixed tumor cell sample.

[0237] In the kit of the present invention, the first molecule and the second molecule can be proteins, preferably endogenous proteins. Preferably, the proteins are immune checkpoint proteins.

[0238] In the kit of the present invention, the first molecule may be PD-1 and the second molecule may be PD-L1 or PD-L2.

[0239] In the kit of the present invention, the first molecule may be CTLA-4 or CD28 and the second molecule may be CD80 or CD86.

[0240] In the kit of the present invention, the first molecule may be an MHC class I or II peptide and the second molecule may be selected from the group consisting of TCR, CD8, CD3, and combinations thereof.

[0241] In the kit of the present invention, the at least two primary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds, antibodies or antigen-binding fragments thereof, or a combination thereof.

[0242] In the kit of the present invention, the at least two secondary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds or antibodies or antigen-binding fragments thereof or a combination thereof.

[0243] In the kits of the present invention, at least one of the secondary binding agents may be an antibody scaffold, an antibody or an antigen binding fragment. In the methods, kits and uses of the present invention, at least two secondary binding agents may be an antibody scaffold, an antibody binding fragment or an antigen binding fragment.

[0244] In the kit of the present invention, the antibody or antigen-binding fragment may be a Fab fragment, a scFv fragment, or a combination thereof.

[0245] In the kits of the present invention, the antibody scaffold can be an adnectin, affibodies, affilin, anticalin, atrimer, avimer, bicyclic peptide, centyrin, cysteine ​​knot, DARPin, fynomer, Kunitz-type domain, Obodies and Tn3.

[0246] In the kit of the present invention, the primary binding agent may be unlabeled.

[0247] In the kits of the invention, the first primary binding agent may be a murine binding agent and at least one additional primary binding agent may be a rabbit binding agent.

[0248] In the kits of the invention, the first primary binding agent may bind to PD-1 and at least one additional primary binding agent may bind to PD-L1 or PD-L2.

[0249] In the kit of the present invention, the first primary binding agent may bind to CTLA-4 or CD28 and the second primary binding agent may bind to CD80 or CD86.

[0250] In the kits of the present invention, the first primary binding agent may bind to an MHC class I or II peptide and the second primary binding agent may bind to TCR, CD8, CD3, and combinations thereof.

[0251] In the kit of the invention, the first secondary binding agent may be an anti-murine binding agent and at least one additional secondary binding agent may be an anti-rabbit binding agent.

[0252] In the kit of the present invention, the FRET donor may be selected from the group consisting of ORG 488, GFP, fluorescein, IAEDANS, EDANS, BODIPY FL, ATTO488, and combinations thereof.

[0253] In the kit of the present invention, the FRET acceptor may be selected from the group consisting of ALX 594, mRFP, tetramethylrhodamine, fluorescein, dabcyl, BODIPY FL, QSY 7, QSY 9, and combinations thereof.

[0254] In the kit of the present invention, the enzyme may be selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases and ligases.

[0255] In the kit of the present invention, the enzyme may be selected from the group consisting of peroxidase, oxidase, phosphatase, esterase and glycosidase. Preferably, the enzyme may be selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and β-galactosidase.

[0256] In the kit of the present invention, the substrate may be tyramide.

[0257] In the kit of the present invention, the first cell is a T cell and the second cell is a tumor cell.

[0258] In the kits of the invention, the instructions may provide for contacting at least two primary binding agents with each other simultaneously or sequentially.

[0259] In the kits of the invention, the instructions may provide for contacting at least two secondary binding agents with each other simultaneously or sequentially.

[0260] In the kits of the invention, the instructions may provide for contacting the at least two primary binding agents and the at least two secondary binding agents with the sample simultaneously.

[0261] In the kits of the invention, the instructions may provide for contacting the at least two primary binding agents with the sample before the at least two secondary binding agents.

[0262] In the kits of the invention, the instructions may provide for performing a washing step after contacting the at least two primary binding agents with the sample and before contacting the at least two secondary binding agents with the sample.

[0263] In the kits of the present invention, the instructions provide for the method further comprising the step of quantifying the interaction between a first site on a first cell and a second site on a second cell.

[0264] In the kit of the invention, the first cell may be a lymphocyte and the second cell may be a non-lymphocyte cell type.

[0265] In the kit of the present invention, the instructions may provide a method for detecting the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte.

[0266] In the kit of the present invention, the instructions may provide a method for detecting the interaction between CTLA-4 or CD28 on a first lymphocyte and CD80 or 86 on a second non-lymphocyte.

[0267] In the kit of the present invention, the instructions may provide a method for detecting the interaction between an MHC class I or II peptide on a first lymphocyte and a TCR, CD8, CD3, or a combination thereof on a second non-lymphocyte.

[0268] In the kit of the present invention, the first molecule may be located on the cell surface of a first cell and the second molecule may be located on the cell surface of a second cell.

[0269] In the kit of the present invention, at least two primary binding agents can be bound to the first molecule or the second molecule in such a manner that the checkpoint inhibitor or activator can be bound to the first molecule or the second molecule simultaneously or sequentially.

[0270] In some aspects of the kits of the invention, the at least two primary binding agents do not inhibit binding of the inhibitor or activator to the first molecule or the second molecule.

[0271] The present invention also provides use of the aforementioned kit of the present invention in an in vitro coincidence assay method for detecting whether a checkpoint inhibitor or activator effectively inhibits or modulates a tumor response, wherein:

[0272] Where the checkpoint molecule is an inhibitor:

[0273] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[0274] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[0275] Where the checkpoint molecule is an activator:

[0276] iii. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[0277] iv. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[0278] The present invention further provides an in vitro method for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the method comprising:

[0279] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0280] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0281] (iv) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0282] (v) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0283] (vi) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0284] The method comprises:

[0285] (a) contacting an isolated tumor cell sample obtained from the patient prior to treatment comprising the checkpoint activator or inhibitor with at least two primary binding agents;

[0286] (b) contacting the sample with the at least two secondary binding agents;

[0287] (c) performing a washing step;

[0288] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0289] (e) repeating steps (a) to (d) using an isolated tumor cell sample obtained from the patient during the treatment comprising the checkpoint activator or inhibitor in step (a);

[0290] (f) comparing the total fluorescence fractions between the samples, wherein:

[0291] a. Where the therapy comprises a checkpoint inhibitor:

[0292] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[0293] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[0294] b. Where the therapy comprises a checkpoint activator:

[0295] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[0296] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[0297] In the methods of the present invention, the checkpoint inhibitor may be an anti-PD-1 or PD-L1 binding agent, and the first primary binding agent may bind to PD-1 and the second primary binding agent may bind to PD-L1 or PD-L2. The first primary agent and the second primary agent may be different from the checkpoint inhibitor and may not bind to the same epitope as the checkpoint inhibitor.

[0298] In the methods of the invention, the checkpoint inhibitor can be an anti-CTLA-4 or CD28 binding agent, and the first primary binding agent can bind to CTLA-4 or CD28 and the second primary binding agent can bind to CD80 or CD86.

[0299] In the methods of the present invention, the checkpoint inhibitor can be selected from the group consisting of: nivolumab (BMS-936558, DX 1106 or ONO-4538), pembrolizumab (rambrolizumab or MK-3475), pidilizumab (CT-Q11), ED-0680 (AMP-514), AMP-224, BMS-936559 (MDX-1105), ED 4736, MPDL3280A (RG7448), MSB0010718C, and fragments and salts thereof.

[0300] In the methods of the present invention, an increase in the total score of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% may indicate that a therapy comprising a checkpoint activator will be effective.

[0301] In the methods of the present invention, a decrease in the total score of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% may indicate that a therapy comprising a checkpoint inhibitor will be effective.

[0302] In the methods of the present invention, at least two primary binding agents may be bound to the first or second molecule in such a manner that a checkpoint inhibitor or activator may be bound to the first or second molecule simultaneously or sequentially.

[0303] In some aspects of the methods of the invention, the at least two primary binding agents do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[0304] The present invention also provides use of the aforementioned method of the present invention for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, wherein:

[0305] a. Where the therapy comprises a checkpoint inhibitor:

[0306] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[0307] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[0308] b. Where the therapy comprises a checkpoint activator:

[0309] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[0310] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[0311] The present invention further provides a kit for use in an in vitro method of determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the kit comprising:

[0312] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0313] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0314] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0315] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0316] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0317] Instructions for performing a method comprising:

[0318] (a) contacting an isolated tumor cell sample obtained from the patient prior to treatment comprising the checkpoint activator or inhibitor with the at least two primary binding agents;

[0319] (b) contacting the sample with the at least two secondary binding agents;

[0320] (c) performing a washing step;

[0321] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0322] (e) repeating steps (a) to (d) using an isolated tumor cell sample obtained from the patient during the treatment comprising the checkpoint activator or inhibitor in step (a);

[0323] (f) comparing the total fluorescence fractions between the samples, wherein:

[0324] a. Where the therapy comprises a checkpoint inhibitor:

[0325] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[0326] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[0327] b. Where the therapy comprises a checkpoint activator:

[0328] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[0329] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[0330] The present invention also provides use of the aforementioned kit of the present invention in an in vitro method for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, wherein:

[0331] a. Where the therapy comprises a checkpoint inhibitor:

[0332] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[0333] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[0334] b. Where the therapy comprises a checkpoint activator:

[0335] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[0336] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[0337] The present invention further provides an in vitro coincidence assay method for identifying whether a molecule of interest is a checkpoint activator or a checkpoint inhibitor, the method comprising:

[0338] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0339] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0340] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0341] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0342] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0343] The method comprises:

[0344] (a) contacting the isolated tumor cell sample with the at least two primary binding agents;

[0345] (b) contacting the sample with the at least two secondary binding agents;

[0346] (c) performing a washing step;

[0347] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0348] (e) contacting the sample with the molecule of interest;

[0349] (f) detecting any changes in the interactions between the secondary binding agents, wherein:

[0350] a. If the total score increases between steps (d) and (f), then the molecule is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or

[0351] b. If the total score decreases between steps (d) and (f), then the molecule is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or

[0352] c. If the total score does not change between steps (d) and (f), then the molecule is neither a checkpoint activator nor a checkpoint inhibitor for at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0353] In the methods of the present invention, an increase in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% may indicate that the molecule of interest is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0354] In the method of the present invention, a decrease in the total score between steps (d) and (f) of 5% to 0.5%, preferably 10% to 5%, more preferably greater than 10% may indicate that the molecule of interest is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0355] In the methods of the present invention, at least two primary binding agents may bind to the first molecule or the second molecule in such a way that the molecule of interest may bind to the first molecule or the second molecule simultaneously or sequentially.

[0356] In some aspects of the methods of the invention, the at least two primary binding agents do not inhibit binding of the molecule of interest to the first molecule or the second molecule.

[0357] The present invention also provides an in vitro method for determining whether a patient with cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, the method comprising:

[0358] at least two primary binding agents, wherein a first primary binding agent binds to PD-1 on a first cell and a second primary binding agent binds to PD-L1 or PD-L2 on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and

[0359] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0360] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0361] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0362] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0363] The method comprises:

[0364] (a) contacting an isolated tumor cell sample obtained from the patient with the at least two primary binding agents;

[0365] (b) contacting the sample with the at least two secondary binding agents;

[0366] (c) performing a washing step;

[0367] (d) detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein:

[0368] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or

[0369] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the use of blockade

[0370] Therapeutic responses to agents that target the PD-1:PD-L1 / PD-L2 pathway.

[0371] In the methods of the present invention, the sample may be a fixed tumor cell sample.

[0372] In the method of the present invention, the method may:

[0373] (i) performing a biological sample obtained from the patient prior to treatment to guide a decision on whether to treat with a single agent that blocks the PD-1:PD-L1 / PD-L2 pathway or with a combination therapy of an agent that blocks the PD-1:PD-L1 / PD-L2 pathway and at least one additional anti-tumor agent; and

[0374] (ii) performing a biopsy on at least one biological sample obtained from the patient during treatment to monitor the subject's response to the current treatment regimen and guide the decision of whether to choose treatment with single-agent PD-1:PD-L1 / PD-L2 blockade therapy or combination therapy.

[0375] The present invention also provides use of the aforementioned in vitro method of the present invention for determining whether a patient suffering from cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, wherein:

[0376] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or

[0377] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway.

[0378] The present invention further provides a kit for use in an in vitro method of determining whether a patient suffering from cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, the kit comprising:

[0379] at least two primary binding agents, wherein a first primary binding agent binds to PD-1 on a first cell and a second primary binding agent binds to PD-L1 or PD-L2 on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and

[0380] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0381] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0382] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0383] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; and

[0384] Instructions for performing a method comprising:

[0385] a. contacting an isolated tumor cell sample obtained from said patient with said at least two primary binding agents;

[0386] b. contacting the sample with the at least two secondary binding agents;

[0387] c. performing a washing step;

[0388] d. detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein:

[0389] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or

[0390] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the use of blockade

[0391] Therapeutic responses to agents that target the PD-1:PD-L1 / PD-L2 pathway.

[0392] In the kit of the present invention, the sample may be a fixed tumor cell sample.

[0393] In the kit of the present invention, the instructions for performing the method may further include instructions for performing the following methods:

[0394] (i) performing the method on a biological sample obtained from the patient prior to treatment to guide a decision on whether to treat with a single agent that blocks the PD-1:PD-L1 / PD-L2 pathway or with a combination therapy of an agent that blocks the PD-1:PD-L1 / PD-L2 pathway and at least one additional anti-tumor agent; and

[0395] (ii) performing the method on at least one biological sample obtained from the patient during treatment to monitor the subject's response to the current treatment regimen and guide the decision of whether to select treatment with single-agent PD-1:PD-L1 / PD-L2 blockade therapy or combination therapy.

[0396] The present invention also provides use of the aforementioned kit of the present invention in an in vitro method for determining whether a patient suffering from cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, wherein:

[0397] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or

[0398] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway.

[0399] The present invention further provides an in vitro coincidence assay method for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, the method comprising:

[0400] a first fusion protein and a second fusion protein, wherein each fusion protein comprises a detection domain, a recognition domain, and a linker domain;

[0401] The detection domain comprises a DNA binding domain and is capable of cooperating with another detection domain to bind to a cognate specific nucleotide sequence;

[0402] The recognition domain is capable of binding to a target molecule;

[0403] The linker domain is fused to the detection domain at one end and to the recognition domain at the other end;

[0404] The detection domain, recognition domain, and linker domain are heterologous to each other;

[0405] The method comprises the following steps:

[0406] (i) contacting a sample with the first and fusion proteins;

[0407] (ii) incubating to allow binding;

[0408] (iii) removing unbound fusion protein;

[0409] (iv) contacting the sample with a nucleic acid comprising the homology-specific nucleotide sequence;

[0410] (v) incubating to allow heterotrimer binding of said nucleic acid; and

[0411] (vi) detecting nucleic acid bound to the sample;

[0412] Wherein, if the nucleic acid is detected in step (vi), this indicates that both target molecules are present in the sample at the same time.

[0413] In the methods, kits and uses of the present invention, the interaction between the secondary binding agents can be detected by detecting the emitted fluorescence.

[0414] In the methods, kits and uses of the present invention, the interaction between the secondary binding agents can be detected by detecting the altered fluorescence behavior. Preferably, the detection of the altered fluorescence behavior is time-resolved.

[0415] In the methods, kits and uses of the present invention, the first cell and the second cell may be the same type of cell.

[0416] In the methods, kits and uses of the present invention, the first cell and the second cell may be different types of cells, ie, not the same type of cells.

[0417] In the methods, kits and uses of the present invention, the isolated sample may be a fixed cell sample.

[0418] In the methods, kits and uses of the present invention, the isolated sample may be a fixed tumor cell sample.

[0419] In the methods, kits and uses of the present invention, the first molecule and the second molecule can be proteins, preferably endogenous proteins. Preferably, the proteins are immune checkpoint proteins.

[0420] In the methods, kits and uses of the present invention, the first molecule may be PD-1 and the second molecule may be PD-L1 or PD-L2.

[0421] In the methods, kits and uses of the present invention, the first molecule may be CTLA-4 or CD28 and the second molecule may be CD80 or CD86.

[0422] In the methods, kits and uses of the present invention, the first molecule may be an MHC class I or II peptide and the second molecule may be selected from the group consisting of TCR, CD8, CD3 and combinations thereof.

[0423] In the methods, kits and uses of the present invention, the at least two secondary binding agents may be selected from the group consisting of whole immunoglobulins, antibody scaffolds or antibodies or antigen-binding fragments thereof or a combination thereof.

[0424] In the methods, kits and uses of the present invention, at least one of the secondary binding agents may be an antibody scaffold, an antibody or an antigen binding fragment. In the methods, kits and uses of the present invention, at least two of the secondary binding agents may be an antibody scaffold, an antibody binding fragment or an antigen binding fragment.

[0425] In the methods, kits and uses of the present invention, the antibody or antigen-binding fragment may be a Fab fragment, a scFv fragment or a combination thereof.

[0426] In the methods, kits and uses of the present invention, the antibody scaffold can be an adnectin, affibodies, affilin, anticalin, atrimer, avimer, bicyclic peptide, centyrin, cysteine ​​knot, DARPin, fynomer, Kunitz-type domain, obodies and Tn3.

[0427] In the methods, kits and uses of the present invention, the primary binding agent may be unlabeled.

[0428] In the methods, kits and uses of the invention, the first primary binding agent may be a murine binding agent and at least one additional primary binding agent may be a rabbit binding agent.

[0429] In the methods, kits and uses of the invention, the first primary binding agent may bind to PD-1 and the at least one additional primary binding agent may bind to PD-L1 or PD-L2.

[0430] In the methods, kits and uses of the invention, the first primary binding agent may bind to CTLA-4 or CD28 and the second primary binding agent may bind to CD80 or CD86.

[0431] In the methods, kits and uses of the invention, the first primary binding agent may bind to an MHC class I or II peptide and the at least one additional primary binding agent may bind to TCR, CD8, CD3 or a combination thereof.

[0432] In the methods, kits and uses of the invention, the first secondary binding agent may be an anti-murine binding agent and at least one additional secondary binding agent may be an anti-rabbit binding agent.

[0433] In the methods, kits and uses of the present invention, the FRET donor may be selected from the group consisting of ORG 488, GFP, fluorescein, IAEDANS, EDANS, BODIPY FL, ATTO488 and combinations thereof.

[0434] In the methods, kits and uses of the present invention, the FRET acceptor may be selected from the group consisting of ALX 594, mRFP, tetramethylrhodamine, fluorescein, dabcyl, BODIPY FL, QSY 7, QSY 9 and combinations thereof.

[0435] In the methods, kits and uses of the present invention, the enzyme may be selected from the group consisting of oxidoreductases, hydrolases, lyases, transferases, isomerases and ligases.

[0436] In the methods, kits and uses of the present invention, the enzyme may be selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

[0437] In the methods, kits and uses of the present invention, the enzyme may be selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and β-galactosidase.

[0438] In the methods, kits and uses of the present invention, the substrate may be tyramide.

[0439] In the methods, kits and uses of the present invention, the first cell may be a T cell and the second cell may be a tumor cell.

[0440] In the methods, kits and uses of the present invention, at least two primary binding agents may be contacted with each other simultaneously or sequentially.

[0441] In the methods, kits and uses of the present invention, at least two secondary binding agents may be contacted with each other simultaneously or sequentially.

[0442] In the methods, kits and uses of the present invention, at least two primary binding agents may be contacted with the sample simultaneously with at least two secondary binding agents.

[0443] In the methods, kits and uses of the present invention, at least two primary binding agents may be contacted with the sample before at least two secondary binding agents.

[0444] In the methods, kits and uses of the present invention, a washing step may be performed after contacting the at least two primary binding agents with the sample and before contacting the at least two secondary binding agents with the sample.

[0445] In the method, kit and use of the present invention, the method may further comprise the step of quantifying the interaction between the first site on the first cell and the second site on the second cell.

[0446] In the methods, kits and uses of the invention, the first cell may be a lymphocyte and the second cell may be a non-lymphocyte cell type.

[0447] In the method, kit and use of the present invention, the method can detect the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte.

[0448] In the methods, kits and uses of the invention, the first cell may be a lymphocyte and the second cell may be a non-lymphocyte cell type.

[0449] In the method, kit and use of the present invention, the method can detect the interaction between CTLA-4 or CD28 on a first lymphocyte and CD80 or CD86 on a second non-lymphocyte.

[0450] In the methods, kits and uses of the invention, the first cell may be a lymphocyte and the second cell may be a non-lymphocyte cell type.

[0451] In the method, kit and use of the present invention, the method can detect the interaction between an MHC class I or II peptide on a first lymphocyte and a TCR, CD8, CD3 or a combination thereof on a second non-lymphocyte.

[0452] In the methods, kits and uses of the present invention, the first molecule may be located on the cell surface of a first cell and the second molecule may be located on the cell surface of a second cell.

[0453] In the methods, kits and uses of the present invention, at least two primary binding agents may bind to the first or second molecule in such a manner that a checkpoint inhibitor or activator may bind to the first or second molecule simultaneously or sequentially.

[0454] In some methods, kits, and uses of the invention, at least two primary binding agents do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[0455] The skilled person will appreciate that the optional features outlined above may be applied in combination with the methods, uses and kits of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0456] Non-limiting embodiments of the present invention will now be further described with reference to the accompanying drawings, in which:

[0457] Figure 1 Shown are changes in PD-1:PD-L1 interactions obtained by TSA-FRET in patients treated with anti-RTK drugs and binding agents that block PD-1.

[0458] Figure 2 Shown are changes in PD-1:PD-L1 interaction obtained by TSA-FRET.

[0459] Figure 3 Representative intensity and lifetime images of PD-1 alone and PD-1:PD-L1 in the presence and absence of blocking monoclonal binders are shown.

[0460] Figure 4 Shown are PD-1 and PD-L1 interactions quantified by immunoFRET (FRET).

[0461] Figure 5 Shown are changes in CTLA-4:CD80 interactions in single cells in the absence or presence of the anti-CTLA-4 antibody ipilimumab.

[0462] Figure 6 Shown are CTLA-4 and CD80 interactions quantified by immunoFRET (iFRET).

[0463] Figure 7 Shown are changes in CTLA-4-CD80 interaction in metastatic melanoma tissue in the absence or presence of the anti-CTLA-4 antibody ipilimumab (A) and quantification of each result for each sample (B).

[0464] Figure 8 Shown is the PD-1:PD-L1 interaction in primary renal cell carcinoma tissue.

[0465] Figure 9 Shows the immune Use of an i-FRET (interferon-French Resonance Energy Transfer) assay to determine the interaction between programmed death receptor-1 (PD-1) and programmed death-ligand 1 (PD-L1) on clear cell renal cell carcinoma (ccRCC) tissue. DETAILED DESCRIPTION

[0466] The present invention provides methods, kits, and uses for coincidence assays for detecting interactions between molecules at the cellular level, including FRET, FRET with amplification (e.g., TSA-FRET), proximity ligation, and coincidence detection. Specifically, the claimed invention provides for the first time the detection and quantification of the interaction between two proteins that are each on the cell surface of different cells, i.e., in a spanning manner. The present invention provides for the detection and / or quantification of the interaction between two proteins expressed on different cells.

[0467] The present invention provides an in vitro coincident assay method for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, the method comprising:

[0468] at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0469] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0470] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0471] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0472] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0473] The method comprises:

[0474] (a) contacting an isolated sample containing cells with the at least two primary binding agents;

[0475] (b) contacting the sample with the at least two secondary binding agents;

[0476] (c) performing a washing step;

[0477] (d) detecting the interaction between the secondary binding agents.

[0478] The present invention also provides use of the above in vitro coincidence assay method for detecting the interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell.

[0479] The present invention further provides a kit for use in the above in vitro assay method for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell.

[0480] The present invention further provides a method for selecting a patient suffering from cancer for treatment, the method comprising:

[0481] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0482] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0483] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0484] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0485] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0486] The method comprises:

[0487] (a) contacting an isolated tumor cell sample from the patient with the at least two primary binding agents;

[0488] (b) contacting the sample with the at least two secondary binding agents;

[0489] (c) performing a washing step;

[0490] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; wherein:

[0491] a. When the intended therapy comprises a checkpoint targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule

[0492] In the case of activators:

[0493] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy; or

[0494] ii. if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0495] b. wherein the intended therapy comprises a test that targets at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0496] In the case of checkpoint inhibitors:

[0497] (iii) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0498] (iv) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy.

[0499] The present invention also provides use of the above in vitro coincidence determination method for selecting patients with cancer for treatment, and a kit for use therein.

[0500] In an additional aspect, the present invention provides an in vitro coincidence assay for detecting whether a checkpoint activator or inhibitor effectively inhibits or modulates a tumor response, the method comprising:

[0501] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0502] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0503] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0504] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0505] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0506] The method comprises:

[0507] (a) contacting the isolated tumor cell sample with the at least two primary binding agents;

[0508] (b) contacting the sample with the at least two secondary binding agents;

[0509] (c) performing a washing step;

[0510] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0511] (e) contacting the sample with the checkpoint activator or inhibitor;

[0512] (f) detecting any changes in the interactions between the secondary binding agents, wherein:

[0513] a. In cases where the checkpoint molecule is an inhibitor:

[0514] i. If the total score decreases between steps (d) and (f), it indicates or predicts that the checkpoint inhibitor is effective;

[0515] or

[0516] ii. if the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint inhibitor is ineffective; or

[0517] b. In the case where the checkpoint molecule is an activator:

[0518] i. If the total score increases between steps (d) and (f), it indicates or predicts that the checkpoint activator is effective;

[0519] or

[0520] ii. If the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint activator is ineffective.

[0521] The present invention also provides use of the above in vitro coincidence assay method for detecting whether a checkpoint activator or inhibitor effectively inhibits or regulates tumor response, and a kit for use therein.

[0522] In an additional aspect, the invention provides an in vitro method of determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the method comprising:

[0523] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0524] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0525] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0526] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0527] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0528] The method comprises:

[0529] (a) contacting an isolated tumor cell sample obtained from the patient prior to treatment comprising the checkpoint activator or inhibitor with at least two primary binding agents;

[0530] (b) contacting the sample with the at least two secondary binding agents;

[0531] (c) performing a washing step;

[0532] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0533] (e) repeating steps (a) to (d) using an isolated tumor cell sample obtained from the patient during the treatment comprising the checkpoint activator or inhibitor in step (a);

[0534] (f) comparing the total fluorescence fractions between the samples, wherein:

[0535] a. Where the therapy comprises a checkpoint inhibitor:

[0536] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[0537] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[0538] b. Where the therapy comprises a checkpoint activator:

[0539] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[0540] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[0541] The present invention also provides use of the above in vitro method for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, and kits for use therein.

[0542] The present invention further provides an in vitro coincidence assay method for identifying whether a molecule of interest is a checkpoint activator or a checkpoint inhibitor, the method comprising:

[0543] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0544] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0545] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0546] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0547] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0548] The method comprises:

[0549] (a) contacting the isolated tumor cell sample with the at least two primary binding agents;

[0550] (b) contacting the sample with the at least two secondary binding agents;

[0551] (c) performing a washing step;

[0552] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0553] (e) contacting the sample with the molecule of interest;

[0554] (f) detecting any changes in the interactions between the secondary binding agents, wherein:

[0555] a. If the total score increases between steps (d) and (f), then the molecule is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or

[0556] b. If the total score decreases between steps (d) and (f), then the molecule is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or

[0557] c. If the total score does not change between steps (d) and (f), then the molecule is not a checkpoint activator or a checkpoint inhibitor thereof for at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0558] The present invention also provides the use of the above in vitro method for identifying whether a molecule of interest is a checkpoint activator or a checkpoint inhibitor, and a kit for use therein.

[0559] The present invention further provides an in vitro method of determining whether a patient suffering from cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, the method comprising:

[0560] at least two primary binding agents, wherein a first primary binding agent binds to PD-1 on a first cell and a second primary binding agent binds to PD-L1 or PD-L2 on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and

[0561] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0562] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0563] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0564] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0565] The method comprises:

[0566] (a) contacting an isolated tumor cell sample obtained from the patient with the at least two primary binding agents;

[0567] (b) contacting the sample with the at least two secondary binding agents;

[0568] (c) performing a washing step;

[0569] (d) detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein:

[0570] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or

[0571] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway.

[0572] The present invention also provides the use of the above in vitro method for determining whether a patient with cancer will respond to an agent that blocks the PD-1:PD-L1 / L2 pathway, and kits for use therein.

[0573] The present invention further provides an in vitro coincident assay method for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, the method comprising:

[0574] a first fusion protein and a second fusion protein, wherein each fusion protein comprises a detection domain, a recognition domain, and a linker domain;

[0575] The detection domain comprises a DNA binding domain and is capable of cooperating with another detection domain to bind to a cognate specific nucleotide sequence;

[0576] The recognition domain is capable of binding to a target molecule;

[0577] The linker domain is fused to the detection domain at one end and to the recognition domain at the other end;

[0578] The detection domain, recognition domain, and linker domain are heterologous to each other;

[0579] The method comprises the following steps:

[0580] (i) contacting a sample with the first fusion protein and the second fusion protein;

[0581] (ii) incubating to allow binding;

[0582] (iii) removing unbound fusion protein;

[0583] (iv) contacting the sample with a nucleic acid comprising the homology-specific nucleotide sequence;

[0584] (v) incubating to allow heterotrimer binding of said nucleic acid; and

[0585] (vi) detecting nucleic acid bound to the sample;

[0586] Wherein, if the nucleic acid is detected in step (vi), this indicates that both target molecules are present in the sample at the same time.

[0587] The methods and kits of the present invention provide coincident assays for detecting interactions between molecules at the cellular level, including FRET, FRET with amplification (eg, TSA-FRET), proximity ligation, and coincident detection.

[0588] The present invention also provides use of the above in vitro method for detecting the interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, and a kit for use therein.

[0589] The methods, kits and uses of the present invention have the advantage of providing for the first time the detection and quantification of the interaction of two proteins each on the cell surface of different cells (ie in a spanning manner) in a low-cost, versatile and robust method with improved signal / noise ratio.

[0590] The novel uses of the present invention can be particularly useful in addressing the challenges of studying protein-protein interactions, preferably endogenous protein interactions, at the cellular level, where proteins are expressed on different cells, and in particular to allow detection and quantification of interactions between immune checkpoint inhibitors expressed on lymphocytes and non-lymphocyte cell types.

[0591] The present invention can therefore produce an improved method for identifying the efficacy of checkpoint inhibitors and for determining whether a patient is likely to respond to cancer therapies, such as anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD28, anti-CD80, anti-CD86, anti-MHC class I, anti-MHC class II, anti-TCR, anti-CD8 and / or anti-CD3 therapies. The present invention also provides an improved method for stratifying patients for responses to cancer therapies (such as anti-PD-1, anti-PD-L1, anti-PD-L2, anti-CTLA-4, anti-CD28, anti-CD80, anti-CD86, anti-MHC class I, anti-MHC class II, anti-TCR, anti-CD8 and / or anti-CD3 therapies) to help provide patients with more targeted cancer therapies.

[0592] definition

[0593] The present invention can utilize the use of a variety of "coincidence assays" that are capable of detecting two binding agents (e.g., antibodies) in close proximity. Exemplary coincidence assays of the present invention include (Fluorescence) resonance energy transfer (FRET), FRET with amplification (e.g., tyramide signal amplification FRET (TSA-FRET)), proximity ligation assay (PLA), bimolecular fluorescence complementation (BiFC), enzyme fragment complementation, and coincident bioassays involving oligonucleotide capture using recombinant protein biosensors produced in bacterial expression systems.

[0594] A "proximity ligation assay" refers to an assay that is capable of detecting the coincidence of two binding agents in close proximity. Different antigens are detected by species-distinct primary binding agents, which are bound by species-specific secondary binding agents covalently modified with ring-forming oligonucleotides. The ring-forming oligonucleotides can be ligated and used as templates for rolling circle amplification and fluorescence detection. Alternatively, species-distinct primary binding agents can be directly ligated to the ring-forming oligonucleotides, which can be ligated and used as templates for rolling circle amplification and fluorescence detection. These methods can detect antibody distances of up to about 40 nm. Preferably, these methods can detect antibody distances of up to about 28 nm.

[0595] "Coincidence biodetection" refers to a method for detecting the coincidence of two target molecules. The method uses two fusion proteins, each of which comprises a detection domain, a recognition domain, and a linker domain. The detection domain may comprise a DNA binding domain and is capable of cooperating with other detection domains to bind to a homologous specific nucleotide sequence. The recognition domain is capable of binding to a target molecule. The linker domain is fused to the detection domain at one end and to the recognition domain at the other end. The detection domain, recognition domain, and linker domain are heterologous to each other. The method involves the following steps: (i) contacting the sample with the fusion protein; (ii) incubating to allow binding; (iii) removing unbound fusion protein; (iv) contacting the sample with a nucleic acid comprising a homologous specific nucleotide sequence; (v) incubating to allow heterotrimer binding of the nucleic acid; and (vi) detecting the nucleic acid bound to the sample. If the nucleic acid is detected in step (vi), this indicates that two target molecules are simultaneously present in the sample. Such methods are disclosed in WO / 2011 / 161420, which is incorporated herein by reference in its entirety.

[0596] In some embodiments, the present invention relates to a method for the detection of a single target protein. The method comprises the steps of: 1) detecting a single target protein of a plurality of affinity probes (a fusion protein with a recognition module) and 2) detecting a single target protein of a plurality of affinity probes (a fusion protein with a recognition module) in a plurality of target proteins. The method comprises the steps of detecting a single target protein of a plurality of affinity probes (a fusion protein with a recognition module) and 3) detecting a single target protein of a plurality of affinity probes (a fusion protein with a recognition module) in a plurality of target proteins. The method comprises the steps of ... The length of the linker can be varied to adjust the distance between the target epitopes, as detailed above. The following diagram illustrates the principle of overlapping bioassays:

[0597]

[0598] "(Fluorescence) resonance energy transfer (FRET)" can also be used in coincident assays that are capable of detecting two binding agents (e.g., antibodies) in close proximity. FRET can be used in the methods of the invention by itself or in combination with amplification, such as an enzyme activation system (e.g., tyramide signal amplification (TSA)).

[0599] FRET is a photophysical process in which energy is transferred from an excited FRET (donor) fluorophore to a neighboring FRET (acceptor) fluorophore via nonradiative dipole-dipole interactions. The efficiency of energy transfer varies inversely with the sixth power of the distance separating the donor and acceptor fluorophores, limiting the distance over which FRET can occur to 9 nm. Therefore, FRET is a "chemical ruler" for measuring molecular proximity.

[0600] The following diagram illustrates the principle of FRET:

[0601]

[0602] FRET makes it possible to measure the interaction (association or dissociation) between two proteins in close proximity (<10 nm) labeled with a pair of fluorescent dyes.

[0603] A "FRET donor" is a chromogenic or fluorescent substrate that has a shorter excitation / emission wavelength than the FRET acceptor. In the figure above, the FRET donor is cyan fluorescent protein (CFP).

[0604] A "FRET acceptor" is a chromogenic or fluorescent substrate that has a longer excitation / emission wavelength than the FRET donor. In the figure above, the FRET acceptor is yellow fluorescent protein (YFP).

[0605] When the emission spectrum of the donor overlaps with the excitation spectrum of the acceptor, the donor chromophore (FRET donor) excites the acceptor molecule (FRET acceptor). FRET donor and FRET acceptor molecule need to be in close proximity (less than 10nm). When the distance between the donor and the acceptor is less than 10nm, the excitation of the acceptor occurs, thereby providing a measurable fluorescence reporter signal. In addition to detecting the reporter signal, it is also possible to quantitatively report the signal. This method can be used to determine the distance between the donor and the acceptor chromophore. This method can also be used to measure protein-protein interactions, protein-lipid interactions, protein-DNA interactions, and protein conformational changes, such as the conformation and post-translational modification state of individual proteins.

[0606] FRET efficiency (E) is the quantum yield of the energy transfer transition, that is, the fraction of energy transfer events that occur per FRET donor excitation event:

[0607]

[0608] where k ET k ET is the energy transfer rate, k f k f is the radiation decay rate and k i k i is the rate constant for any other de-excitation pathway.

[0609] The FRET efficiency depends on many physical parameters, including the distance between the donor and acceptor, the spectral overlap of the FRET donor emission spectrum and the FRET acceptor absorption spectrum, and the relative orientation of the FRET donor emission dipole moment and the FRET acceptor absorption dipole moment.

[0610] The EE depends on the separation distance rr between the FRET donor and the FRET acceptor, where the inverse sixth order is attributed to the dipole-dipole coupling mechanism:

[0611]

[0612]

[0613] R0R0 is the pair of FRET donor and FRET acceptor Distance. This is the distance at which the energy transfer efficiency is approximately 50%.

[0614] Donors of the present invention include ORG488, GFP, luciferin, IAEDANS, EDANS, BODIPYFL, and ATTO488.

[0615] Acceptors of the invention include ALX594, mRFP, fluorescein, tetramethylrhodamine, dabcyl, BODIPYFL, and the QSY7 and QSY9 dyes.

[0616] Combinations of the above donor and acceptor pairs are encompassed by the present invention.

[0617] Other exemplary donor-acceptor pairs include cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP), YFP-CFP, ORG488-ALX594, and ATTO488-ALX594.

[0618] The donor and acceptor can be of two different types (heterologous FRET) or of the same type (homologous FRET). In the case of homologous FRET, spectral differences are not used to detect and measure FRET. Instead, the difference in anisotropy between the light that excites the donor and acceptor and the emitted light can be detected and measured. Exemplary methods for detecting homologous FRET include FRET anisotropy imaging. Quantitative anisotropy levels (polarization differences between the excitation and emission beams) provide an indication of how many FRET events have occurred.

[0619] Table 1 below provides exemplary FRET donor and acceptor pairs and their typical R0 values:

[0620] FRET donor FRET acceptors <![CDATA[R0(A)]]> Fluorescein Tetramethylrhodamine 55 IAEDANS Fluorescein 46 EDANS Dabcyl 33 Fluorescein Fluorescein 44 BODIPYFL BODIPYFL 57 Fluorescein QSY7 and QSY9 dyes 61

[0621] There are several ways to measure FRET efficiency by monitoring changes in fluorescence emitted by the donor or acceptor. These methods are well known to those skilled in the art, such as sensitized emission, photobleaching FRET and lifetime measurements, and are encompassed by the present invention.

[0622] Exemplary uses of FRET include: determining the structure and conformation of proteins; determining the distribution and assembly of protein complexes; determining receptor / ligand interactions; immunoassays; enzymatic assays; probing single molecule interactions; determining the structure and conformation of nucleic acids; real-time PCR assays and SNP detection; detecting nucleic acid hybridization; primer extension assays for detecting mutations; automated DNA sequencing; determining the distribution and transport of lipids; membrane fusion assays; membrane potential sensing; indicators of cyclic AMP and calcium; and detecting and quantifying Akt activation in tumors, such as breast tumors.

[0623] "Coincident FRET" or "two-site" FRET describes a method in which a single protein is simultaneously labeled at two different sites with a donor and acceptor pair, and FRET between them is detected.

[0624] An "enzyme activation system" refers to an enzyme system in which at least one enzyme is coupled to a member of a specific binding pair by any means known to those skilled in the art. For example, the enzyme can be conjugated or fused to the specific binding pair. In the present invention, the specific binding pair can be located on an antibody. In certain embodiments, the specific binding pair is located on an antibody selected from the group consisting of a first primary antibody, a second primary antibody, a first secondary antibody and a second secondary antibody, and combinations thereof.

[0625] The enzyme reacts with the conjugate comprising the detectably labeled substrate by itself or in combination with a second enzyme to form an activated conjugate. The activated conjugate is combined with an acceptor (e.g., electron-rich part) on the molecular surface of the adjacent enzyme. The combination can be via covalent bonding. The activated conjugate can be deposited anywhere where the acceptor (e.g., electron-rich part) of the activated conjugate is found. The acceptor (e.g., electron-rich part) on the molecular surface cannot react with the enzyme activation system. Therefore, only when the detectably labeled substrate has been activated by enzyme to form an activated conjugate, the detectably labeled substrate is combined with the acceptor (e.g., electron-rich part). In the absence of an enzyme, the detectably labeled substrate does not form an activated conjugate.

[0626] The detectably labeled substrate of the conjugate can comprise one or more components. In one embodiment, the detectably labeled substrate comprises one component comprising a binding site for a receptor (e.g., an electron-rich moiety) and a detectable label. In another embodiment, the substrate comprises two components; one component can comprise a binding site for a receptor (e.g., an electron-rich moiety) and be detectably labeled. The other component can comprise a component that prevents or interferes with binding to the receptor (e.g., an electron-rich moiety) until the enzyme activates the conjugate.

[0627] The term "detectably labeled" means that the substrate is coupled directly or indirectly to the detectable label.

[0628] The substrate can be detectably labeled using methods well known to those of ordinary skill in the art.

[0629] For indirect labeling, a substrate can be coupled to the unlabeled first member of the specific binding pair. After the activated conjugate is activated and binds to an acceptor (e.g., an electron-rich portion), the first member of the specific binding pair can react with the second member of the specific binding pair, which is coupled to a detectable label. Alternatively, before the activated conjugate is activated and binds to an acceptor (e.g., an electron-rich portion), the first member of the specific binding pair can be pre-reacted with the second member of the specific binding pair, which is coupled to a reporter molecule.

[0630] In the present invention, detectable label can comprise FRET acceptor or FRET donor.In a preferred embodiment, detectable label comprises FRET acceptor.In aspects of the present invention some, substrate comprises junket amide.In some embodiments, the substrate that detectably labels comprises junket amide with FRET donor label.In a preferred embodiment, the substrate that detectably labels comprises junket amide with FRET acceptor label.

[0631] The enzyme can be selected from oxidoreductases, hydrolases, lyases, transferases, isomerases, ligases, and combinations thereof. In certain embodiments, the enzyme is selected from peroxidases, oxidases, phosphatases, esterases, glycosidases, and combinations thereof. In a preferred embodiment, the enzyme is selected from horseradish peroxidase, glucose oxidase, alkaline phosphatase, beta-galactosidase, and combinations thereof.

[0632] The term "activated conjugate" refers to a conjugate comprising a detectably labeled substrate that is specific for an enzyme activation system and has been activated by an enzyme of the system. Upon activation, the activated conjugate can bind to a receptor (e.g., an electron-rich moiety) on the surface of a molecule adjacent to the enzyme. The sample is subjected to reaction conditions sufficient to cause enzyme-catalyzed substrate activation to form an activated conjugate.

[0633] Reaction conditions sufficient to cause enzyme catalysis substrate activation are well known to those skilled in the art. With regard to tyramide signal amplification (TSA), the enzyme employed is catalase and the detectably labeled substrate is detectably labeled tyramide. Reaction conditions require the presence of hydrogen peroxide so that catalase catalyzes the activation of detectably labeled tyramide to form an activated conjugate containing a detectably labeled tyramide group. In a preferred embodiment, the detectable label is a FRET acceptor or a FRET donor. In a particularly preferred embodiment, the detectable label is ALX594 or ORG488. In other preferred embodiments, the detectable label is ALX594 or ATTO488.

[0634] In a preferred embodiment, the substrate of detectable labeling comprises junket amides and enzyme (for example horseradish peroxidase) activation junket amides with FRET acceptor labeling to form activated conjugate, this activated conjugate comprises the junket amide group of high reactivity, short life with FRET acceptor link coupling, and described group can be covalently coupled with the part that is rich in electrons on the molecule of contiguous enzyme.In a preferred embodiment, the residue that is rich in electrons is tyrosine residues.Molecular surface can be protein or nucleotide sequence.

[0635] The term "amplification" refers to the amplification of the reporter signal provided by the detectable label of the detectably labeled substrate caused by the combination of the conjugate containing the detectably labeled substrate activated by the enzyme activation system and the electron-rich portion on the molecular surface of the adjacent enzyme. In the present invention, the reporter signal can include fluorescence. In some embodiments, the reporter signal includes fluorescence emitted by a FRET acceptor or a FRET donor. In a preferred embodiment, the reporter signal includes fluorescence emitted by a FRET acceptor.

[0636] The enzyme activation system of the present invention can be applied to the first primary antibody, the second primary antibody, the first secondary antibody, the second secondary antibody, or a combination thereof, provided that the system is applied to at least one of the first secondary antibody and the second secondary antibody.

[0637] "Proximity" in the context of an enzyme activation system means that the receptor is positioned within close proximity to the enzyme. For example, the distance between the enzyme and the receptor can be about 2 to 9 nm or less than a 100 kDa globular protein (preferably, 2 to 9 nm, 2 to 7 nm, 2 to 6 nm, 2 to 5 nm, 2 to 4 nm, or 2 to 3 nm or less than 90 kDa, less than 80 kDa, less than 70 kDa, less than 60 kDa, less than 50 kDa, less than 40 kDa, less than 30 kDa, less than 20 kDa, or less than 10 kDa).

[0638] A specific embodiment of single enzyme activation system of the present invention is tyrosine amide signal amplification (TSA) system.This system utilizes the catalytic activity of horseradish peroxidase, the tyrosine amide of this horseradish peroxidase activation reporter molecule coupling is to form activated conjugate, this activated conjugate comprises highly reactive, short-lived tyrosine amide group, and described group can be covalently coupled with the tyrosine residue on the molecular surface of contiguous horseradish peroxidase.Molecular surface can be protein or nucleic acid residue.

[0639] In certain aspects, a cell sample is obtained from a patient before or during treatment. "Obtained from a subject before treatment" can mean obtained from a patient who has previously been treated for cancer, e.g., previously treated with an anti-tumor agent, or can mean obtained from a patient who has not previously been treated for cancer (i.e., a naive patient).

[0640] In some aspects, a "cell sample" is a tumor cell sample. The cell sample is preferably a fixed cell sample. The cell sample can be a tumor cell sample, including surrounding patient tissue (e.g., macrophages, T cells, B cells, etc.). The cell sample can be a tumor biopsy.

[0641] The term "tumor cell" includes cancer cells. This can include primary tumor cells, secondary (metastatic) tumor cells, solid tumors and related patient tissues (e.g., macrophages, T cells, B cells, etc.). A tumor sample can be, for example, a tumor biopsy or surgical incision obtained from a patient. Typically, a tumor sample comprises an invasive tumor margin. A tumor sample can be obtained from a metastatic lesion. The sample can comprise peripheral blood. The patient can be a patient suspected of having metastatic cancer. Examples of cancer include, but are not limited to, melanoma, lung cancer (including, for example, non-small cell lung cancer), breast cancer, head and neck cancer, and urothelial carcinoma. Other examples of cancer include adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain stem glioma, cerebellar astrocytoma, ependymoma, carcinoid tumor, cancer of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer, germ cell tumor, extragonadal trophoblastic tumor, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cavity cancer, liver cancer, small cell lung cancer, lymphoma, cutaneous T-cell lymphoma , Hodgkin's and non-Hodgkin's lymphoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloid and extramyeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell tumor, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma, salivary gland cancer, Sézary syndrome, skin cancer, Kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, malignant thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0642] The term "anti-PD-1 antibody" or "anti-PD-L1 antibody" refers to an antibody that targets the programmed death 1 (PD-1) molecule or its ligands PD-L1 and PD-L2, such as by disrupting the interaction between PD-1 and PD-L1 or PD-L2, blocking the activation of PD-1 regardless of whether PD-1 interacts with PD-L1 or PD-L2, or blocking the PD-L1 or PD-L2 signaling pathway that is triggered when PD-L1 or PD-L2 interacts with PD-1.

[0643] The term "anti-PD-1" or "anti-PD-L1 therapy" means a therapeutic strategy that targets programmed death 1 (PD-1) molecules or their ligands PD-L1 and PD-L2, such as by disrupting a current interaction established between PD-1 and PD-L1 or PD-L2, or blocking future interactions between PD-1 and PD-L1 or between PD-1 and PD-L2, blocking the activation of PD-1 regardless of whether PD-1 interacts with PD-L1 or PD-L2, or blocking the PD-L1 or PD-L2 signaling pathway triggered when PD-L1 or PD-L2 interacts with PD-1. Examples of anti-PD-1 immunotherapeutics include, but are not limited to, pembrolizumab, nivolumab, and pidilizumab. Examples of anti-PD-L1 immunotherapeutics include, but are not limited to, BMS-936559 and atezolizumab.

[0644] The term "anti-CTLA-4" or "anti-CD80 / 86" means a therapeutic strategy that targets the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) molecule or its ligands CD80 or CD86, such as by disrupting the interaction between CTLA-4 and CD80 or CD86, blocking the activation of CTLA-4 regardless of whether CTLA-4 interacts with CD80 or CD86, or blocking the CD80 or CD86 signaling pathway that is triggered when CD80 or CD86 interacts with CTLA-4.

[0645] The term "anti-CTLA-4 therapy" or "anti-CD80 / 86 therapy" means a therapeutic strategy that targets the cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) molecule or its ligand, such as by disrupting a current established interaction between CTLA-4 and CD80 or CD86, or blocking future interactions between CTLA-4 and CD80 or between CTLA-4 and CD86, blocking activation of CTLA-4 regardless of whether CTLA-4 interacts with CD80 or CD86, or blocking the CD80 or CD86 signaling pathway triggered when CD80 or CD86 interacts with CTLA-4. Examples of anti-CTLA-4 immunotherapeutics include, but are not limited to, ipilimumab.

[0646] The terms "anti-MHC class I," "anti-MHC class II," or "anti-TCR" refer to a therapeutic strategy that targets the T cell receptor (TCR) or its ligand MHC class I or MHC class II, such as by disrupting the interaction between the TCR and MHC class I or MHC class II, blocking activation of the TCR regardless of whether the TCR interacts with MHC class I or MHC class II, or blocking the TCR signaling pathway that is triggered when MHC class I or MHC class II interacts with the TCR.

[0647] The terms "anti-TCR therapy," "anti-MHC class I therapy," or "anti-MHC class II therapy" refer to a therapeutic strategy that targets a T cell receptor molecule of its ligand MHC class I or MHC class II, such as by disrupting a currently established interaction between the TCR and MHC class I or MHC class II, or blocking future interactions between the TCR and MHC class I or MHC class II, blocking activation regardless of interaction with MHC class I or MHC class II, or blocking the MHC class I or MHC class II signaling pathway that is triggered upon interaction of MHC class I or MHC class II with the TCR.

[0648] "Treating" or "treatment" a disease in a patient means (1) preventing the development of symptoms or the disease in a person or animal that does not yet exhibit symptoms of the disease; and / or (2) inhibiting the disease or arresting its progression; and / or (3) ameliorating or causing regression of the disease or symptoms associated with the disease.

[0649] "Immunologically different" in the context of binding agents means that the binding agents are produced in different host species or different isotypes from the same species. In one embodiment of the invention, the primary binding agents are a first antibody and a second antibody, wherein the first primary antibody is produced in a different host species than the second primary antibody, or the first primary antibody is a first isotype from a species and the second primary antibody is a second isotype from the same species, wherein the first isotype and the second isotype are different. Exemplary host species include mouse, rat, rabbit, goat, camel, sheep, or horse. For example, the first primary antibody can be produced in mouse and the second primary antibody can be produced in rabbit. This enables the first secondary binding agent to be a universal anti-mouse antibody (labeled with a donor) and the second secondary binding agent to be a universal anti-rabbit antibody (conjugated to an enzyme). This provides a universal high-throughput methodology. In a preferred embodiment, the first primary antibody is an anti-PD-1 mouse antibody and the second primary antibody is an anti-PD-L1 or PD-L2 rabbit antibody. In other preferred embodiments, the first primary antibody is an anti-CTLA-4 or CD28 mouse antibody and the second primary antibody is an anti-CD80 or CD86 rabbit antibody. In another preferred embodiment, the first primary antibody is an anti-MHC class I or II mouse antibody and the second primary antibody is an anti-TCR, CD8, CD3 and / or combinations thereof rabbit antibody.

[0650] "Binding agent" refers to any molecule capable of binding to another molecule and may necessarily include, but is not limited to, whole immunoglobulins, antibody scaffolds, and antibodies or antigen-binding fragments.

[0651] "Antibody" is used in the broadest sense and specifically encompasses whole immunoglobulins as well as antibodies or antigen-binding fragments thereof, such as variable domains. Exemplary whole immunoglobulins include full-length and native antibodies, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), chimeric antibodies.

[0652] "Monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic determinant (also called an epitope). Monoclonal antibodies can be prepared by any technique or method known in the art, including hybridoma methods, recombinant DNA methods, and isolation from phage antibody libraries.

[0653] In contrast, "polyclonal antibodies" are typically heterogeneous populations of immunoglobulin isotypes and / or classes, and also exhibit a variety of epitope specificities.

[0654] A "chimeric antibody" refers to a type of monoclonal antibody in which a portion or the entire amino acid sequence of one or more regions or domains of the heavy and / or light chain is identical, homologous, or a variant thereof to the corresponding sequence in a monoclonal antibody from another species or belonging to another immunoglobulin class or isotype, or from a consensus sequence. Chimeric antibodies include fragments of such antibodies, provided that the antibody exhibits the desired biological activity of its parent antibody, such as binding to the same epitope.

[0655] An "antibody or antigen-binding fragment" refers to a portion of a full-length antibody that retains the variable regions or functional capabilities of the parent antibody, such as specific epitope binding. The antibodies or antigen-binding fragments of the present invention have conserved epitopes in the constant regions for secondary recognition. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibodies, and other multispecific antibodies formed from antibody fragments.

[0656] Antibody scaffolds are non-natural antigen-binding proteins, peptides, or antibody fragments. Antibody scaffolds include adnectins, affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, centyrins, cysteine ​​knots, DARPins, fynomers, Kunitz-type domains, obodies, and Tn3.

[0657] "Fab fragment" refers to the fragment - the antigen-binding fragment, which is the region of an antibody that binds to an antigen. It is composed of one constant domain and one variable domain in each of the heavy and light chains. These domains form the antigen-binding site at the amino terminus of the fragment. The two variable domains bind to an epitope on their specific antigen. The Fab fragment of the present invention has a conserved epitope in the constant region that is recognized by a secondary antibody.

[0658] Methods for preparing Fab fragments are well known to the skilled person, for example the enzyme papain can be used to cleave a whole immunoglobulin into two Fab fragments and one Fc fragment.

[0659] The Fab fragment can be further cleaved to form F(ab')2 and Fab' fragments using methods known to those skilled in the art. For example, the enzyme pepsin can be used to cleave the Fab fragment below the hinge region to produce a F(ab')2 fragment and a pFc' fragment. Alternatively, the enzyme IdeS (immunoglobulin degrading enzyme from Streptococcus pyogenes, trade name FabRICATOR) can be used to cleave IgG in a sequence-specific manner at neutral pH to produce F(ab')2 fragments. The F(ab')2 fragment can be separated into two Fab' fragments, for example, by mild reduction.

[0660] "Single-chain Fv" or "scFv" antibody fragments are fragments comprising the variable domain of the heavy chain of an antibody (V H ) and light chain variable domain (V L ) single chain Fv variants, wherein the domains are present in a single polypeptide chain and are capable of recognizing and binding antigens. The scFv fragments of the present invention have conserved epitopes in the constant region for secondary antibody recognition. The scFv polypeptide optionally contains a V H With V L A polypeptide linker between the domains that enables the scFv to form the desired three-dimensional structure for antigen binding.

[0661] Methods for producing scFv are well known to those skilled in the art. For example, isolated V H and V L The chains can be fused together. scFv is approximately half the size of a Fab fragment but retains the original specificity of the parent antibody.

[0662] "Diabodies" are small antibody fragments with two antigen-binding sites. Each fragment contains V H domain, the V H Domain and V L Connect to form V H -V L or V L -V H By using a linker that is too short to allow pairing between the two domains on the same chain, the linked V H -V L The domains pair with the complementary domains of another chain and create two antigen-binding sites.

[0663] A "linear antibody" is one comprising a pair of tandem Fd fragments (V H -C H 1-V H -C H 1), these fragments form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[0664] The antibodies, antibody scaffolds and antibodies or antigen-binding fragments thereof of the present invention may be labeled. In a preferred embodiment, the tag is FLAG.

[0665] "Primary binding agent" refers to any molecule that is capable of binding to another molecule and may necessarily include, but is not limited to, whole immunoglobulins, antibody scaffolds, and antibodies or antigen-binding fragments. For example, a primary binding agent may be an antibody, antibody scaffold, or an antibody or antigen-binding fragment thereof that binds to a first site on a molecule such as a protein, DNA, or lipid. A primary binding agent has binding specificity for a site on a molecule. The first primary binding agent binds to a first site on a molecule such as a protein.

[0666] In a preferred embodiment, the first site is PD-1. The second primary binding agent binds to a second site on a molecule (such as a protein). In a preferred embodiment, the second site is PD-L1.

[0667] In other preferred embodiments, the first site is CTLA-4 or CD28. The second primary binding agent binds to a second site on a molecule (such as a protein). In a preferred embodiment, the second site is CD80 or CD86.

[0668] In another preferred embodiment, the first site is an anti-MHC class I or II peptide. The second primary binding agent binds to a second site on a molecule (such as a protein). In a preferred embodiment, the second site is TCR, CD8, CD3 and / or a combination thereof.

[0669] In preferred embodiments, the primary binding agent is unlabeled. In other embodiments, the primary binding agent can be labeled. For example, the label can be a tag, such as a FLAG tag.

[0670] In embodiments of the present invention, the primary binding agent can be a whole immunoglobulin, an antibody scaffold, or an antibody or an antigen-binding fragment thereof. Combinations of the above are also envisioned. Preferred antibody fragments are Fab fragments or scFv fragments. For example, in the methods of the present invention, both the first and second primary antibodies can be whole immunoglobulins. Alternatively, both the first and second primary antibodies can be antibodies or antigen-binding fragments. In some embodiments, the first and second primary antibodies are Fab fragments, scFv fragments, or a combination thereof. Alternatively, the first and second primary antibodies can be whole immunoglobulins and the second primary antibody can be antibodies or antigen-binding fragments, or the second primary antibody can be antibodies or antigen-binding fragments and the second primary antibody can be whole immunoglobulins. In some embodiments, the primary antibody can be labeled (e.g., with a FLAG tag) and the secondary antibody can have binding specificity for the tag (e.g., anti-FLAG).

[0671] "Secondary binding agent" refers to any molecule capable of binding to another molecule and may necessarily include, but is not limited to, whole immunoglobulins, antibody scaffolds, and antibodies or antigen-binding fragments thereof. For example, a secondary binding agent may be an antibody, antibody scaffold, or antibody or antigen-binding fragment thereof that binds to a primary binding agent (such as a first or second primary binding agent) or a tag (such as a FLAG tag) on ​​a primary binding agent.

[0672] In embodiments of the present invention, the secondary binder can be a whole immunoglobulin, an antibody scaffold, or an antibody or antigen-binding fragment thereof. Combinations of the above are also contemplated. Preferred antibody fragments are Fab fragments or scFv fragments. In particularly preferred embodiments, at least one secondary binder is an antibody, antibody scaffold, or antigen-binding fragment. In preferred embodiments, both the first primary binder and the second primary binder are antibodies, antibody scaffolds, or antigen-binding fragments. In some embodiments, the first secondary binder and the second secondary binder are Fab fragments, scFv fragments, or combinations thereof. Alternatively, the first secondary binder can be an antibody, antibody scaffold, or antigen-binding fragment and the second secondary binder can be a whole immunoglobulin, or the first secondary binder can be a whole immunoglobulin and the second secondary binder can be an antibody, antibody scaffold, or antigen-binding fragment. In some embodiments, neither the first secondary binder nor the second secondary binder is a whole immunoglobulin. The present invention was found to not work with some secondary binders in which both are whole immunoglobulins, presumably due to conformational issues that result in the FRET donor and FRET acceptor being closer than the distance required for FRET to occur (>10 nm). This was found to be the case regardless of the order in which the primary and secondary binders were administered.

[0673] In some embodiments, the enzyme is conjugated or fused to a secondary binding agent. In preferred embodiments, the enzyme is conjugated or fused to a second secondary binding agent. In embodiments where the second secondary binding agent is a scFv fragment, the scFv fragment can be recombinantly fused to the enzyme.

[0674] In the present invention, the first site is different from the second site to enable FRET detection between different sites. In a preferred embodiment, the first site and the second site are on the same molecule. In a particularly preferred embodiment, the first site and the second site are on the same protein. In other embodiments, the first site and the second site are on different molecules, such as different proteins in a complex. Preferably, the protein detected in the isolated sample is an endogenous protein.

[0675] An "isolated" sample is a biological sample that has been isolated from a subject, such as an isolated tumor sample. A biological sample may include organs, tissues, cells, and / or fluids.

[0676] The term "subject" refers to any animal, particularly animals classified as mammals (including humans), domestic and farm animals, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the subject is a human.

[0677] A "washing step" is used in the context of the present invention in its usual sense in immunohistochemistry to mean washing the sample with an acceptable solution, such as a saline solution. For example, a washing step may be used to remove any unbound binding agent from a previous step or to remove any detectably labeled substrate that has not yet been activated to form an activated conjugate.

[0678] "Checkpoint proteins" are known in the art. Under normal physiological conditions, when the immune system responds to pathogenic infections, immune checkpoints prevent autoimmunity and protect tissues from damage. The expression of checkpoint proteins can be abnormally regulated by tumors as part of the immune resistance mechanism. Immune checkpoint proteins can be activators or inhibitors of immune checkpoint pathways and can be used to treat various cancers by providing anti-tumor immune responses or inflammatory diseases to enhance the resolution of immune responses. Specifically, checkpoint protein inhibition or activation can provide an amplification of antigen-specific T cell responses. Exemplary checkpoint proteins include programmed cell death protein 1 (PD-1), PD-1 ligand (PD-L1), PD2 ligand (PD-L2), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), B7 family proteins, TNF family proteins, CD40L, adenosine A2a receptor (A2aR), B7-related protein 1 (B7RP1), B and T lymphocyte attenuator (BTLA), galectin 9 (GAL9), herpes virus entry mediator (HVEM), inducible T cell co-stimulatory molecule (ICOS), interleukin (IL), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), transforming growth factor-β (TGF-β), T cell membrane protein 3 (TIM3) and MHC I or II class peptide. A preferred checkpoint target is PD-1. PD-L1 is also a preferred checkpoint target. CTLA-4 is a preferred checkpoint target. MHC I or II class peptide is a preferred checkpoint target. LAG3, B7-H3, B7-H4, and TIM3 are also preferred checkpoint targets.

[0679] CTLA-4, PD-1, LAG3, and TIM3 are inhibitory receptors. PD-1, B7-H3, and B7-H4 are inhibitory ligands. All are in clinical development for the treatment of various cancers.

[0680] CTLA-4 is expressed only on T cells, where it primarily regulates the magnitude of the early stages of T cell activation. Primarily, CTLA-4 counteracts the activity of the T cell co-stimulatory receptor CD28. CD28 and CTLA-4 share the same ligands CD80 (also known as B7.1) and CD86 (also known as B7.2).

[0681] PD-1 is an immune checkpoint receptor that limits the activity of T cells in peripheral tissues and limits autoimmunity during inflammatory responses to infections. When T cells become activated, PD-1 expression is induced. When engaged by one of its two ligands, PD-L1 or PD-L2 (B7 family members), PD-1 inhibits kinases involved in T cell activation. PD-L1 can also interact with CD80.

[0682] Membrane-bound ligands of the B7 family bind both co-stimulatory and inhibitory receptors.All B7 family members and their known ligands belong to the immunoglobulin superfamily.

[0683] Tumor necrosis factor (TNF) family members that bind to cognate TNF receptor family molecules represent a second family of regulatory ligand-receptor pairs. These receptors primarily deliver co-stimulatory signals when engaged by their cognate ligands.

[0684] Another major category of signals that regulate T cell activation comes from soluble cytokines in the microenvironment. The communication between T cells and APCs is bidirectional. In some cases, this occurs when the ligand itself conducts signals to the APC. In other cases, activated T cells upregulate ligands (such as CD40L) that engage cognate receptors on APCs. A2aR, adenosine A2a receptor; B7RP1, B7-related protein 1; BTLA, B and T lymphocyte attenuation factor; GAL9, galectin 9; HVEM, herpes virus entry mediator; ICOS, inducible T cell co-stimulatory molecule; IL, interleukin; KIR, killer cell immunoglobulin-like receptor; LAG3, lymphocyte activation gene 3; PD-1, programmed cell death protein 1; PDL, PD-1 ligand; TGFβ, transforming growth factor-β; TIM3, T cell membrane protein 3.

[0685] "Checkpoint target molecule" refers to a molecule, such as a protein, that is targeted by the overlapping assay method of the present invention. As explained above, a "checkpoint" is an immune checkpoint that prevents autoimmunity and protects tissues from damage when the immune system responds to pathogenic infection. The expression of checkpoint proteins can be abnormally regulated by tumors as part of an immune resistance mechanism. Exemplary checkpoint target molecules include programmed cell death protein 1 (PD-1), PD-1 ligand (PD-L1), PD2 ligand (PD-L2), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), B7 family proteins, TNF family proteins, CD40L, adenosine A2a receptor (A2aR), B7-related protein 1 (B7RP1), B and T lymphocyte attenuator (BTLA), galectin 9 (GAL9), herpes virus entry mediator (HVEM), inducible T cell co-stimulatory molecule (ICOS), interleukin (IL), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), transforming growth factor-β (TGF-β), T cell membrane protein 3 (TIM3) and MHC I or II class peptide. A preferred checkpoint target is PD-1. PD-L1 is also a preferred checkpoint target. CTLA-4 is a preferred checkpoint target. MHC I or II class peptide is a preferred checkpoint target. LAG3, B7-H3, B7-H4, and TIM3 are also preferred checkpoint targets.

[0686] "Check point activators" means molecules, such as proteins, that activate immune checkpoint pathways. Exemplary check point activators include those from TNF receptors and B7-CD28 superfamilies, including CD40 (TNFSFR5) agonists, GITR (glucocorticoid-induced tumor necrosis factor receptor; TNFSFR18) stimulators, OX40 (CD134; TNFSFR4) agonists, 4-1BB (CD137; TNFSFR9) agonists, CD27 (TNFSFR7) agonists, ICOS (inducible costimulatory molecules) molecule agonists, Trail receptor agonists and / or HVEM (herpes virus invasion mediator) receptor agonists. Preferred check point activators include OX-40, GITR and / or 4-1BB agonists.

[0687] "Checkpoint inhibitors" means molecules that inhibit immune checkpoint pathways, such as proteins. Exemplary checkpoint inhibitors include anti-PD-1 binding agents, anti-PD-L1 binding agents, anti-PD-L2 binding agents, anti-CTLA-4 binding agents, anti-MHC class I binding agents and / or anti-MHC class II binding agents. Exemplary anti-CTLA-4 binding agents are ipilimumab. Exemplary anti-PD-1 binding agents include nivolumab (BMS-936558, DX 1106 or ONO-4538) and pembrolizumab (rambrolizumab or MK-3475).

[0688] Exemplary checkpoint inhibitors include those selected from the group consisting of ipilimumab, nivolumab (BMS-936558, DX 1106, or ONO-4538), pembrolizumab (rambrolizumab or MK-3475), pidilizumab (CT-Q11), ED-0680 (AMP-514), AMP-224, BMS-936559 (MDX-1105), ED 4736, MPDL3280A (RG7448), MSB0010718C, and fragments and salts thereof.

[0689] Method of the present invention

[0690] The method of the present invention can be used to detect and quantify cell-cell interactions, in particular the interaction of two proteins each on the cell surface of different cells.Preferably, the protein detected in the isolated sample is an endogenous protein.

[0691] Advantageously, the methods and kits of the present invention can quantitatively calculate FRET by measuring the reduction in the lifetime of the donor chromophore in the presence of an acceptor chromophore, thereby measuring cell-cell interactions. The present invention therefore provides an average measurement of the total amount of interaction between a molecule expressed on a first cell and a second molecule expressed on a second cell in a sample. This results in an improved dynamic range of measurable interactions, which can be used in a clinical setting to stratify cancer patients into groups to determine patients who will respond well to therapy, and also to stratify the effectiveness of a therapy (e.g., a novel therapy) that regulates the binding of a cell to another cell.

[0692] An exemplary use of the methods of the invention is to examine the interaction of PD-1 with PD-L1 or PD-L2, which are immune checkpoint proteins identified as playing a role in tumor progression in various cancers.

[0693] Another exemplary use of the methods of the present invention is to examine the interaction of CTLA-4 or CD28 with CD80 or CD86, which are also immune checkpoint proteins identified as playing a role in tumor progression in various cancers.

[0694] Another exemplary use of the methods of the present invention is in examining the interaction of an MHC peptide (e.g., MHC class I or class II) with a TCR (or CD8 or CD3). Specifically, the methods of the present invention can be used to detect and / or quantify the interaction between a member of a TCR complex on a T cell (e.g., any member of a TCR or CD8 or CD3 complex) and an MHC class I or class II complexed with an antigenic peptide.

[0695] The methods of the invention can also be used to examine interactions between other immune checkpoint proteins, such as those exemplified herein.

[0696] The methods of the present invention are sensitive, quantitative and allow the localization of (altered) molecular pathways using conventional binding agents. Such methods facilitate the detection of pharmacodynamic markers and promote the discovery / development of new small molecule inhibitors.

[0697] The methods of the present invention preferably utilize coincidence assays.

[0698] The method of the present invention can be used in combination with detection by fluorescence lifetime imaging microscopy (FLIM).Time-resolved FRET can provide this information in single cells.

[0699] Two-site FRET without amplification

[0700] In one aspect, the invention uses two-site FRET without amplification.

[0701] In the non-amplified dual-site FRET method of the present invention, the method uses at least two primary binding agents that are bound to different target molecules and are immunologically different, and at least two secondary binding agents that are bound to their corresponding primary binding agents and are labeled with a FRET donor and a FRET acceptor, respectively. Specifically, the first primary binding agent is bound to a first molecule (e.g., a checkpoint target protein) on a first cell and the second primary binding agent is bound to a second molecule (e.g., another or different checkpoint target protein) on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically different. The first secondary binding agent is labeled with a FRET donor and is bound to the first primary binding agent; and the second secondary binding agent is labeled with a FRET acceptor and is bound to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent.

[0702] In embodiments where the FRET donor on the bound first secondary binding agent is in sufficiently close proximity (less than or equal to 10 nm) to the FRET acceptor on the second secondary binding agent, a positive FRET signal can be detected.

[0703] In cases where the FRET donor on the bound first secondary binding agent is not in close enough proximity (greater than 10 nm) to the FRET acceptor on the second secondary binding agent, the FRET signal will be reduced or absent.

[0704] In the absence of either or both of the first and second molecules in the sample, no FRET signal will be detected.

[0705] In one example, primary whole immunoglobulins anti-PD-1 (mouse) and anti-PD-L1 (T308) (rabbit) were contacted with a fixed tumor cell sample from a cancer patient. Secondary Fab fragments anti-mouse ORG488 and anti-rabbit ALX594 were contacted with the sample and bound to the anti-PD-1 (mouse) and anti-PD-L1 (T308) (rabbit) antibodies, respectively. When PD-1 and PD-L1 were in close proximity (less than or equal to 10 nm) on the cell surface of different cells, a positive FRET signal was generated between ORG488 and ALX594. The FRET signal can be detected in a time-resolved manner by multi-frequency domain FLIM (mFD-FLIM).

[0706] Two-site FRET with amplification

[0707] In another aspect, the invention provides a method employing at least two primary binding agents, at least two secondary binding agents, and a conjugate (eg, two-site FRET with amplification, such as two-site TSA-FRET).

[0708] The first primary binding agent binds to a first molecule (e.g., a checkpoint target protein) on a first cell and the second primary binding agent binds to a second molecule (e.g., another or different checkpoint target protein) on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct.

[0709] The first secondary binding agent is labeled with a fluorescence resonance energy transfer (FRET) donor and binds to the first primary binding agent; and the second secondary binding agent is conjugated to an enzyme and binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent.

[0710] The conjugate comprises a FRET acceptor and a substrate specific for an enzyme, wherein when the substrate reacts with the enzyme, the conjugate is activated, which binds to an electron-rich moiety on the surface of a molecule adjacent to the enzyme.

[0711] The methods of the invention may comprise the steps of contacting a cell sample with at least two primary binding agents; contacting the sample with at least two secondary binding agents; performing a wash step; contacting the sample with a conjugate; and detecting any FRET signal generated by the FRET acceptor.

[0712] Using the methods of the present invention, if the first molecule and the second molecule are in close spatial proximity (less than or equal to 9 nm), it is possible to detect a FRET signal even if the first molecule and the second molecule are expressed at low levels.

[0713] In the methods of the present invention, at least two primary binding agents are contacted with a cell sample. The at least two primary binding agents can be contacted with the sample simultaneously or sequentially. Thus, the first primary binding agent can be contacted with the sample first and then the second primary binding agent. Alternatively, the second primary binding agent can be contacted with the sample first and then the third primary binding agent. When the first and second primary binding agents are contacted with the sample sequentially, optional washing steps can be performed between the sequential steps.

[0714] The first primary binding agent binds to any first molecule on a first cell present in the sample and the second primary binding agent binds to any second molecule on a second cell present in the sample. An optional wash step removes any unbound primary binding agent.

[0715] The at least two secondary binding agents can be contacted with the cell sample simultaneously with the at least two primary binding agents or the at least two primary binding agents can be contacted with the sample before the at least two secondary binding agents. The at least two secondary binding agents can be contacted with the sample simultaneously with each other or sequentially with each other. Thus, the first secondary binding agent can be contacted with the sample first and then the second secondary binding agent can be contacted with the sample. Alternatively, the second secondary binding agent can be contacted with the sample first and then the first secondary binding agent can be contacted. When the first secondary binding agent and the second secondary binding agent are contacted with the sample sequentially with each other, an optional washing step can be performed between the sequential steps. In embodiments where the at least two primary binding agents are contacted with the sample before the at least two secondary binding agents, an optional washing step can be performed between the administration of the at least two primary binding agents and the administration of the at least two secondary binding agents.

[0716] The first secondary binding agent binds to the first primary binding agent and the second primary binding agent binds to the second primary binding agent. An optional washing step removes any unbound secondary binding agent or any secondary binding agent that has bound to a primary binding agent that has not yet bound to the first or second site (i.e., unbound primary binding agent).

[0717] After the at least two primary binding agents and the at least two secondary binding agents are contacted with the sample, a washing step is performed before the conjugate is contacted with the sample. The washing step removes any binding agent (primary or secondary) that has not yet bound to its target (e.g., the first site, the second site, the first primary binding agent, or the second primary binding agent). The washing step in the method of the present invention can be performed using a saline solution or another suitable solution. The conditions used in the washing step are well known to those of ordinary skill in the art.

[0718] In some aspects, after the washing step, the conjugate is contacted with the sample. In the case where the second primary binding agent has been combined with the second molecule and the second secondary binding agent has been combined with the second primary binding agent, the substrate of the conjugate and the enzyme reaction conjugated with the second secondary binding agent are to form an activated conjugate. The activated conjugate will be combined with the electron-rich part on the molecular surface (e.g., protein surface) of the adjacent enzyme. The enzyme can activate a variety of conjugates, thereby providing a variety of activated conjugates and the combination of the electron-rich part on the molecular surface of the adjacent enzyme. This amplifies the number of conjugates containing the activation of the FRET acceptor combined near the second site.

[0719] Any FRET signal generated by the FRET acceptor is detected.

[0720] In embodiments where the FRET donor on the bound first secondary antibody is in sufficiently close proximity (less than or equal to 10 nm) to the FRET acceptor on the bound activated conjugate, a positive FRET signal can be detected.

[0721] In cases where the FRET donor on the bound primary secondary antibody is not in close enough proximity (greater than 10 nm) to the FRET acceptor on the bound activated conjugate, the FRET signal will be reduced or absent.

[0722] In the absence of either or both of the first and second molecules in the sample, no FRET signal will be detected.

[0723] In another aspect of the present invention, an enzyme activation system can be applied to a first secondary binder as well as a second secondary binder. This advantageously amplifies both the FRET donor signal and the FRET acceptor signal. In this aspect, the first secondary binder is conjugated to the enzyme in place of the FRET donor. The method further employs a second conjugate comprising a FRET donor and a substrate specific for the enzyme, wherein when the substrate reacts with the enzyme, a second activated conjugate is formed that binds to an electron-rich moiety on the surface of a molecule adjacent to the enzyme. This substrate does not react with the enzyme conjugated to the second secondary binder.

[0724] The methods of the present invention are adjusted accordingly. For example, the methods may include the steps of contacting the sample with at least two primary binding agents; contacting the sample with at least two secondary binding agents; performing a washing step; contacting the sample with a first conjugate having specificity for an enzyme conjugated to the first secondary binding agent and a second conjugate having specificity for an enzyme conjugated to the second secondary binding agent; and detecting any FRET signal generated by the FRET acceptor. The first conjugate may be applied to the second conjugate simultaneously or sequentially. For example, the first conjugate may be contacted with the sample first and then the second conjugate may be contacted with the sample. Alternatively, the second conjugate may be contacted with the sample first and then the first conjugate. When the first conjugate and the second conjugate are contacted with the sample sequentially with each other, an optional washing step may be performed between the sequential steps.

[0725] In embodiments where the FRET donor on the bound first activated conjugate is in sufficiently close proximity (less than or equal to 10 nm) to the FRET acceptor on the bound second activated conjugate, a positive FRET signal can be detected.

[0726] In cases where the FRET donor on the bound first activated conjugate is not in close enough proximity (greater than 9 nm) to the FRET acceptor on the bound second activated conjugate, the FRET signal will be reduced or absent.

[0727] In the absence of either or both of the first and second molecules in the sample, no FRET signal will be detected.

[0728] In one aspect of the invention, the primary binding agent is unlabeled. For example, the primary binding agent is not labeled with a FRET donor or a FRET acceptor. This has the advantage that the methods of the invention can provide a high-throughput, general method that does not rely on generating primary binding agents with individual binding specificities labeled with a FRET donor or a FRET acceptor, which is both time-consuming and expensive.

[0729] The primary binder can be labeled. For example, the primary binder can be labeled with a FRET donor or a FRET acceptor. In this regard, the secondary binder can be omitted. Alternatively, a labeled primary binder can be used in combination with a primary binder-secondary binder pair and a conjugate. For example, a first primary binder labeled with a FRET acceptor can be used in combination with a second primary binder that is bound by a second secondary binder conjugated to an enzyme and a conjugate. In this case, the first secondary binder can be omitted. Alternatively, a first primary binder bound by a first secondary binder labeled with a FRET acceptor can be used in combination with a second primary binder conjugated to an enzyme and a conjugate. In this case, the second secondary binder can be omitted. In these embodiments, an enzyme activation system can be applied to the first primary binder and the second primary / secondary binder. This advantageously amplifies the FRET donor signal and the FRET acceptor signal. In this regard, the first primary binder is conjugated to an enzyme in place of a FRET donor. The method further employs a second conjugate comprising a FRET donor and a substrate specific for the enzyme, wherein when the substrate reacts with the enzyme, a second activated conjugate is formed that binds to an electron-rich moiety on the surface of a molecule adjacent to the enzyme. The substrate does not react with the enzyme conjugated to the second primary / secondary binder.

[0730] The methods of the present invention are adjusted accordingly. For example, the methods may include the steps of contacting the sample with at least two primary binding agents; optionally contacting the sample with at least one secondary binding agent; performing a washing step; contacting the sample with a first conjugate having specificity for an enzyme conjugated to the first primary / secondary binding agent and a second conjugate having specificity for an enzyme conjugated to the second primary / secondary binding agent; and detecting any FRET signal generated by the FRET acceptor. The first conjugate may be applied to the second conjugate simultaneously or sequentially. For example, the first conjugate may be contacted with the sample first and then the second conjugate may be contacted with the sample. Alternatively, the second conjugate may be contacted with the sample first and then the first conjugate. When the first conjugate and the second conjugate are contacted with the sample sequentially with each other, optional washing steps may be performed between the sequential steps.

[0731] In embodiments where the FRET donor on the bound first activated conjugate, first primary binding agent, or first secondary binding agent is in sufficiently close proximity (less than or equal to 10 nm) to the FRET acceptor on the bound second activated conjugate, a positive FRET signal can be detected.

[0732] In cases where the FRET donor on the bound first activated conjugate, first primary binding agent, or first secondary binding agent is not in close enough proximity (greater than 10 nm) to the FRET acceptor on the bound second activated conjugate, the FRET signal will be reduced or absent.

[0733] In the absence of either or both of the first and second molecules in the sample, no FRET signal will be detected.

[0734] In some embodiments, the methods of the invention employ more than two primary binding agents.

[0735] In some embodiments, the methods of the invention employ more than two secondary binding agents.

[0736] The samples of the present invention include isolated biological samples, isolated cells and tissue sections. In a preferred embodiment, the sample is a breast tumor sample, including a breast tumor tissue section.

[0737] Advantageously, the secondary binding agent employed in the present invention can be an antibody or antigen binding fragment, such as a Fab fragment or scFv fragment, rather than a whole immunoglobulin. The secondary binding agent can be a combination of a Fab fragment, an antibody scaffold, and a whole immunoglobulin (Fab fragment mixture). It has been found that embodiments of the present invention employing an antibody or antigen binding fragment (e.g., with a size in the range of 50 kDa to 100 kDa) or a Fab fragment mixture as a secondary binding agent are particularly effective. Particular advantages are that the distance between the FRET donor and the FRET acceptor chromophore is reduced, FRET efficiency is improved, and the FRET donor is easy to penetrate tissue and bind to its target. In addition, their inherent specificity is further enhanced due to the fact that they lack an Fc region, thereby significantly reducing any background generated by nonspecific binding to endogenous Fc receptors. This is particularly advantageous when the two target sites are on the same molecule.

[0738] The combination of an enzyme activation system and the FRET method of the present invention improves FRET efficiency, particularly in two-site FRET. Previously, it was expected that the size of the system would increase the distance between the FRET donor and the FRET acceptor, leading to a loss of FRET. However, the inventors discovered that the method of the present invention provides an improved signal-to-noise ratio, as well as a low-cost, versatile, and robust high-throughput method.

[0739] Advantageously, the enzyme activation system increases the detection of low expressed proteins, preferably endogenous proteins, and also allows dilution of the primary binder, which in turn reduces non-specific interactions and thus increases specificity.

[0740] The method of the present invention has the advantage of significantly increasing sensitivity without increasing background.

[0741] In one example, primary whole immunoglobulins anti-PD-1 (mouse) and anti-PD-L1 (T308) (rabbit) were contacted with fixed tumor cell samples from cancer patients. Secondary Fab fragments anti-mouse ORG488 and anti-rabbit HRP were contacted with the sample and bound to the anti-PD-1 (mouse) and anti-PD-L1 (T308) (rabbit) antibodies, respectively. After a wash step, tyramide (TSA)-ALX594 was applied to the sample. HRP catalyzed the activation of multiple copies of TSA-ALX594. The resulting short-lived tyramide group was covalently coupled to electron-rich residues adjacent to the HRP, which deposited multiple copies of ALX594 adjacent to the PD-L1 target site. The short half-life of the tyramide group minimized diffusion-related losses in the localization of the ALX594 signal. A positive FRET signal was generated between ORG488 and ALX594 when PD-1 and PD-L1 were in close proximity (less than or equal to 10 nm) on the cell surface of different cells. FRET signals can be detected in a time-resolved manner by multi-frequency-domain FLIM (mFD-FLIM).

[0742] In one aspect, the present invention relates to a highly sensitive quantitative coincidence assay. In certain embodiments, two-site TSA-FRET combines immunofluorescence tyramide signal amplification (TSA) with a Fab fragment secondary antibody conjugate to maximize sensitivity and specificity.

[0743] mFD-FLIM can be automated using an "interpolation" algorithm. This small instrument automatically distinguishes regions of interest (ROIs) within cells and tumors, allowing for unbiased selection of specific ROIs.

[0744] In an embodiment of the present invention, small-scale automated mFD-FLIM can be used in combination with two-site TSA-FRET to easily detect the activation of PD-1 and / or PD-L1 / PD-L2 in tumor samples. The method can be routinely used to inform prognosis, prediction, and diagnosis of immune checkpoint inhibitors.

[0745] Advantageously, the method of the present invention combines the spatiotemporal and quantitative properties of time-resolved FRET detected by multi-frequency domain FLIM (mFD-FLIM) with the sensitivity of the tyramide signal amplification (TSA) system.

[0746] In another example, a primary full anti-CTLA-4 or anti-CD28 immunoglobulin (mouse) and a primary full anti-CD80 or anti-CD86 immunoglobulin (rabbit) are contacted with a fixed tumor cell sample from a cancer patient. Secondary Fab fragments anti-mouse ORG488 and anti-rabbit HRP are contacted with the sample and bind to anti-CTLA-4 / CD28 (mouse) and anti-CD80 / CD86 (rabbit) antibodies, respectively. After a wash step, tyramide (TSA)-ALX594 is applied to the sample. HRP catalyzes the activation of multiple copies of TSA-ALX594. The resulting short-lived tyramide group is covalently coupled to an electron-rich residue adjacent to the HRP, which deposits multiple copies of ALX594 adjacent to the CD80 / CD86 target site. The short half-life of the tyramide group minimizes diffusion-related losses in the localization of the ALX594 signal. When CTLA-4 / CD28 and CD80 / CD86 are in close proximity (less than or equal to 10 nm) on the cell surface of different cells, a positive FRET signal is generated between ORG488 and ALX594. The FRET signal can be detected in a time-resolved manner by multi-frequency domain FLIM (mFD-FLIM).

[0747] In another example, a primary full anti-MHC class I or II peptide immunoglobulin (mouse) and a primary full anti-TCR, CD8 or CD3 immunoglobulin (rabbit) are contacted with a fixed tumor cell sample from a cancer patient. Secondary Fab fragments anti-mouse ORG488 and anti-rabbit HRP are contacted with the sample and bind to anti-MHC class I / II peptide (mouse) and anti-TCR, CD8 or CD3 (rabbit) antibodies, respectively. After a wash step, tyramide (TSA)-ALX594 is applied to the sample. HRP catalyzes the activation of multiple copies of TSA-ALX594. The resulting short-lived tyramide group is covalently coupled to an electron-rich residue adjacent to the HRP, which deposits multiple copies of ALX594 adjacent to the TCR / CD8 / CD3 target site. The short half-life of the tyramide group minimizes diffusion-related losses in the localization of the ALX594 signal. In the case of close proximity (less than or equal to 10 nm) of the MHC class I / II peptide and TCR / CD8 / CD3 on the cell surface of different cells, a positive FRET signal is generated between ORG488 and ALX594. The FRET signal can be detected in a time-resolved manner by multi-frequency domain FLIM (mFD-FLIM). The skilled person will appreciate that the claimed methods, uses, and kits can be applied to other immune checkpoint target pairs to detect and / or quantify interactions between immune checkpoint targets at the cellular level.

[0748] Proximity Connection

[0749] In another aspect, the present invention provides a method employing at least two primary binders and at least two secondary binders conjugated or fused to DNA sequences. The DNA sequence conjugated or fused to the first secondary binder can be different from the DNA sequence conjugated or fused to the second secondary binder. The DNA sequences are linked by rolling circle DNA amplification to form a ring, and are bound by an externally applied fluorescently labeled DNA probe complementary to the amplified circular DNA sequence.

[0750] The additional DNA probe can be labeled with a detectable label, such as a fluorescent label. Other detectable labels are also envisioned, such as HRP and chromophores.

[0751] The first primary binding agent binds to a first molecule (e.g., a checkpoint target protein) on a first cell and the second primary binding agent binds to a second molecule (e.g., another or different checkpoint target protein) on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct.

[0752] The first secondary binding agent is conjugated or fused to a first DNA sequence and binds to the first primary binding agent; and the second secondary binding agent is conjugated or fused to a second DNA sequence and binds to the second primary binding agent. The first DNA sequence and the second DNA sequence may be different. The first secondary binding agent does not bind to the second primary binding agent, and the second secondary binding agent does not bind to the first primary binding agent.

[0753] DNA sequences are ligated to form circles, amplified by rolling circle DNA amplification, and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence.

[0754] More specifically, a secondary binding agent (referred to as a PLA probe) directed against the constant region of a corresponding primary binding agent binds to the corresponding primary binding agent. A first secondary binding agent (referred to as a first PLA probe) directed against the constant region of a first primary binding agent binds to the first primary binding agent. A second secondary binding agent (referred to as a second PLA probe) directed against the constant region of a second primary binding agent binds to the second primary binding agent. The first PLA probe and the second PLA probe have different short DNA strands attached to them. If the first PLA probe and the second PLA probe are in close proximity (up to about 40 nm; preferably up to about 28 nm), that is, if two target cells of interest are in close proximity, the DNA strands can be ligated and then amplified in the presence of appropriate substrates and enzymes. Ligation is determined by the distance between the PLA probes, such that ligation occurs when the probes are in close proximity (up to about 40 nm; preferably up to about 28 nm). Once ligated, amplification occurs in the presence of appropriate substrates and enzymes. Such substrates and enzymes are known in the art.

[0755] The rolling circle DNA synthesis step can result in a hundreds-fold amplification of the DNA circle, which, when in contact with a fluorescently labeled oligonucleotide probe that binds to the amplified DNA, can produce high-intensity fluorescence, for example, as detected by fluorescence microscopy.

[0756] The method of the present invention may include the following steps: contacting a cell sample with at least two primary binding agents; contacting the sample with at least two secondary binding agents; performing a washing step; performing rolling circle DNA amplification; contacting the sample with a fluorescently labeled DNA probe that is complementary to the amplified circular DNA sequence; and detecting any fluorescent signal generated.

[0757] Using the methods of the present invention, it is possible to detect a fluorescent signal if the first molecule and the second molecule are in close proximity (less than about 40 nm; preferably less than about 28 nm).

[0758] In the methods of the present invention, at least two primary binding agents are contacted with a cell sample. The at least two primary binding agents can be contacted with the sample simultaneously or sequentially. Thus, the first primary binding agent can be contacted with the sample first and then the second primary binding agent. Alternatively, the second primary binding agent can be contacted with the sample first and then the third primary binding agent. When the first and second primary binding agents are contacted with the sample sequentially, optional washing steps can be performed between the sequential steps.

[0759] The first primary binding agent binds to any first molecule on a first cell present in the sample and the second primary binding agent binds to any second molecule on a second cell present in the sample. An optional wash step removes any unbound primary binding agent.

[0760] The at least two secondary binding agents can be contacted with the cell sample simultaneously with the at least two primary binding agents or the at least two primary binding agents can be contacted with the sample before the at least two secondary binding agents. The at least two secondary binding agents can be contacted with the sample simultaneously with each other or sequentially with each other. Thus, the first secondary binding agent can be contacted with the sample first and then the second secondary binding agent can be contacted with the sample. Alternatively, the second secondary binding agent can be contacted with the sample first and then the first secondary binding agent can be contacted. When the first secondary binding agent and the second secondary binding agent are contacted with the sample sequentially with each other, an optional washing step can be performed between the sequential steps. In embodiments where the at least two primary binding agents are contacted with the sample before the at least two secondary binding agents, an optional washing step can be performed between the administration of the at least two primary binding agents and the administration of the at least two secondary binding agents.

[0761] The first secondary binding agent binds to the first primary binding agent and the second primary binding agent binds to the second primary binding agent. An optional washing step removes any unbound secondary binding agent or any secondary binding agent that has bound to a primary binding agent that has not yet bound to the first or second site (i.e., unbound primary binding agent).

[0762] After contacting the at least two primary binding agents and the at least two secondary binding agents with the sample, a washing step is performed before contacting the conjugate with the sample. The washing step removes any binding agent (primary or secondary) that has not yet bound to its target (e.g., the first site, the second site, the first primary antibody, or the second primary binding agent). The washing step in the method of the present invention can be performed using a saline solution or another suitable solution. The conditions used in the washing step are well known to those of ordinary skill in the art.

[0763] Rolling circle DNA amplification is performed. DNA sequences are linked to form a ring and then amplified by rolling circle DNA amplification. The amplified DNA is then contacted with an externally fluorescently labeled DNA probe complementary to the amplified circular DNA sequence. An optional washing step is performed. The bound probe is then detected via its detectable label.

[0764] The rolling circle DNA amplification step amplifies the number of circular DNA sequences bound by the externally added fluorescently labeled DNA probe, indicating the presence of target molecule interaction. Any detectable signal generated is detected.

[0765] In embodiments where the first target molecule is in sufficiently close proximity (less than about 40 nm; preferably less than about 28 nm) to the second target molecule, a positive detectable (fluorescent) signal can be detected.

[0766] In cases where the first target molecule is not in sufficiently close proximity (greater than about 40 nm; preferably greater than about 28 nm) to the second target molecule, the detectable (fluorescent) signal will be reduced or absent.

[0767] In the absence of either or both of the first and second molecules in the sample, no detectable (fluorescent) signal will be detected.

[0768] The methods of the present invention may be adapted accordingly. For example, the methods may include the following steps: contacting the sample with at least two primary binding agents; contacting the sample with at least two secondary binding agents; performing a wash step; performing rolling circle DNA amplification; contacting the sample with an external fluorescently labeled DNA probe complementary to the amplified circular DNA sequence; and detecting any detectable (fluorescent) signal generated.

[0769] In some embodiments, the methods of the invention employ more than two primary binding agents.

[0770] In some embodiments, the methods of the invention employ more than two secondary binding agents.

[0771] Advantageously, the secondary binding agent employed in the present invention may be an antibody or antigen binding fragment, such as a Fab fragment or scFv fragment, rather than a whole immunoglobulin. The secondary binding agent may be a combination of a Fab fragment, an antibody scaffold, and a whole immunoglobulin (Fab fragment mixture). Embodiments of the present invention employing antibodies or antigen binding fragments (e.g., with a size in the range of 50 kDa to 100 kDa) or a Fab fragment mixture as the secondary binding agent have been found to be particularly effective. Particular advantages are reduced target distance and improved fluorescence efficiency, ease of penetrating tissue and binding to its target. In addition, their inherent specificity is further enhanced by the fact that they lack an Fc region, thereby significantly reducing any background generated by nonspecific binding to endogenous Fc receptors.

[0772] The method of the present invention has the advantage of significantly increasing sensitivity without increasing background.

[0773] In one example, a primary whole immunoglobulin anti-PD-1 (mouse) and anti-PD-L1 (T308) (rabbit) were contacted with a fixed tumor cell sample from a cancer patient. Secondary Fab fragments anti-mouse fused to a DNA sequence and anti-rabbit fused to another DNA sequence were contacted with the sample and bound to the anti-PD-1 (mouse) and anti-PD-L1 (T308) (rabbit) antibodies, respectively.

[0774] DNA sequences are linked to form a ring and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe complementary to the amplified circular DNA sequence, wherein PD-1 and PD-L1 are in close proximity (less than about 40 nm, preferably less than about 28 nm) on the cell surface of different cells. The fluorescent signal can be detected by fluorescence microscopy. The present invention relates to a highly sensitive quantitative coincidence assay.

[0775] In another example, a primary full anti-CTLA-4 or anti-CD28 immunoglobulin (mouse) and a primary full anti-CD80 or anti-CD86 immunoglobulin (rabbit) are contacted with a fixed tumor cell sample from a cancer patient. Secondary Fab fragments anti-mouse fused to a DNA sequence and anti-rabbit fused to another DNA sequence are contacted with the sample and bind to the anti-CTLA-4 / CD28 (mouse) and anti-CD80 / CD86 (rabbit) antibodies, respectively.

[0776] DNA sequences are linked to form a ring and amplified by rolling circle DNA amplification, and bound by an external fluorescently labeled DNA probe complementary to the amplified circular DNA sequence, wherein CTLA-4 / CD28 and CD80 / CD86 are in close proximity (less than about 40 nm, preferably less than about 28 nm) on the cell surface of different cells. The fluorescent signal can be detected by fluorescence microscopy. The present invention relates to a highly sensitive quantitative coincidence assay.

[0777] In another example, a primary full anti-MHC class I or II peptide immunoglobulin (mouse) and a primary full anti-TCR, CD8 or CD3 immunoglobulin (rabbit) are contacted with a fixed tumor cell sample from a cancer patient. A secondary Fab fragment anti-mouse fused to a DNA sequence and anti-rabbit fused to another DNA sequence are contacted with the sample and bind to the anti-MHC class I / II peptide (mouse) and anti-TCR / CD8 / CD3 (rabbit) antibodies, respectively.

[0778] DNA sequences are linked to form a ring and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe complementary to the amplified circular DNA sequence, wherein the MHC class I / II peptide and TCR / CD8 / CD3 are in close proximity (less than about 40 nm, preferably less than about 28 nm) on the cell surface of different cells. The fluorescent signal can be detected by fluorescence microscopy. The present invention relates to a highly sensitive quantitative coincidence assay.

[0779] Although the method of the present invention can be used with PLA, it was found that this method can only measure distances in the range of 20 nm to 40 nm. PLA distances less than 20 nm cannot be measured.

[0780] In contrast, the two-site TSA FRET method of the present invention is able to measure distances in the range of 10 nm or less. Such methods also allow for quantitative rather than merely qualitative assays with general application (not limited to specific cancer types or specific cell-cell interactions).

[0781] Coincidence detection

[0782] In another aspect, the present invention uses coincident detection methods to detect the interaction between two target molecules presented on the surface of separate cells.

[0783] The method uses two fusion proteins, wherein each fusion protein comprises a detection domain, a recognition domain and a linker domain. The detection domain may comprise a DNA binding domain and can cooperate with other detection domains to bind to a homologous specific nucleotide sequence. The recognition domain can bind to a target molecule. The linker domain is fused to the detection domain at one end and to the recognition domain at the other end. The detection domain, the recognition domain and the linker domain are heterologous to each other. The method involves the following steps: (i) contacting the sample with the fusion protein; (ii) incubating to allow binding; (iii) removing unbound fusion protein; (iv) contacting the sample with a nucleic acid comprising a homologous specific nucleotide sequence; (v) incubating to allow heterotrimer binding of the nucleic acid; and (vi) detecting the nucleic acid bound to the sample. If the nucleic acid is detected in step (vi), this indicates that two target molecules are present in the sample at the same time. This type of method is disclosed in WO / 2011 / 161420, which is incorporated herein by reference in its entirety.

[0784] The present invention provides an in vitro coincident assay method for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, the method comprising:

[0785] a first fusion protein and a second fusion protein, wherein each fusion protein comprises a detection domain, a recognition domain, and a linker domain;

[0786] The detection domain comprises a DNA binding domain and is capable of cooperating with other detection domains to bind to homologous specific nucleotide sequences;

[0787] The recognition domain is capable of binding to a target molecule;

[0788] The linker domain is fused to the detection domain at one end and to the recognition domain at the other end;

[0789] The detection domain, recognition domain, and linker domain are heterologous to each other;

[0790] The method comprises the following steps:

[0791] (i) contacting a sample with the first and fusion proteins;

[0792] (ii) incubating to allow binding;

[0793] (iii) removing unbound fusion protein;

[0794] (iv) contacting the sample with a nucleic acid comprising the homology-specific nucleotide sequence;

[0795] (v) incubating to allow heterotrimer binding of said nucleic acid; and

[0796] (vi) detecting nucleic acid bound to the sample;

[0797] Wherein, if the nucleic acid is detected in step (vi), this indicates that both target molecules are present in the sample at the same time.

[0798] In one example, the first fusion protein detects PD-1 and the second fusion protein detects PD-L1. In another example, the first fusion protein binds CTLA-4 or CD28 and the second fusion protein binds CD80 or CD86. In another example, the first fusion protein binds an MHC class I or II peptide and the second fusion protein binds TCR, CD8, or CD3.

[0799] Selecting and stratifying cancer patients

[0800] The present invention provides methods for selecting patients with cancer for treatment. The methods provide for coincidence assays (including FRET, FRET with amplification (e.g., TSA-FRET), proximity ligation, and coincidence detection) for use in methods for selecting patients for cancer treatment. The methods provide for detecting molecular interactions at the cellular level to guide patient selection.

[0801] The present invention provides a method for selecting a patient suffering from cancer for treatment, the method comprising:

[0802] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0803] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0804] (iv) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0805] (v) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0806] (vi) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0807] The method comprises:

[0808] (e) contacting an isolated tumor cell sample from the patient with the at least two primary binding agents;

[0809] (f) contacting the sample with the at least two secondary binding agents;

[0810] (g) performing a washing step;

[0811] (h) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; wherein:

[0812] a. When the intended therapy comprises a checkpoint targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule

[0813] In the case of activators:

[0814] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy; or

[0815] ii. if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0816] b. wherein the intended therapy comprises a test that targets at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0817] In the case of checkpoint inhibitors:

[0818] (v) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[0819] (vi) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy.

[0820] The present invention also provides use of the above in vitro coincidence determination method for selecting patients with cancer for treatment, and a kit for use therein.

[0821] The patient may not have previously received cancer therapy, or may have previously received cancer therapy, but for a target molecule different from the target molecule (e.g., a checkpoint target protein) for which the patient was selected in the aforementioned methods.

[0822] If the total score is between 0.5% and 5%, this indicates that the patient will not respond to a therapy targeting at least one of the checkpoint targets. If the total score is between 5% and 10%, this indicates that the patient is likely to respond to a therapy targeting at least one of the checkpoint targets. If the total score is greater than 10%, this indicates that the patient will respond to a therapy targeting at least one of the checkpoint targets. The total score can be a FRET efficiency percentage.

[0823] In some methods, at least two primary binding agents bind to the first or second molecule in such a manner that the checkpoint inhibitor or activator can bind to the first or second molecule simultaneously or sequentially. In some methods, at least two primary binding agents do not inhibit binding of the checkpoint inhibitor or activator to the first or second molecule.

[0824] Tumor cell samples can be obtained from patients before or after treatment for different target molecules.

[0825] The cell sample is preferably a fixed cell sample. Tumor cell samples can include primary tumor cells, secondary (metastatic) tumor cells, solid tumors and related patient tissues (e.g., macrophages, T cells, B cells, etc.). The cell sample can be a tumor biopsy or surgical incision obtained from the patient. Typically, the tumor sample comprises an invasive tumor margin. The tumor sample can be obtained from a metastatic lesion. The sample can comprise peripheral blood.

[0826] The patient can be a patient suspected of having metastatic cancer. The example of cancer includes but is not limited to melanoma, lung cancer (including for example non-small cell lung cancer), breast cancer, head and neck cancer, urothelial carcinoma. Other examples of cancer include adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain stem glioma, cerebellar astrocytoma, ependymoma, carcinoid tumor, unknown primary cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing's family tumor (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, gastric cancer, germ cell tumor, extragonadal trophoblastic tumor, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, liver cancer, small cell lung cancer, lymphoma, cutaneous T cell lymphoma, leukemia ... Cancer, Hodgkin's and non-Hodgkin's lymphoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell tumor, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma, salivary gland cancer, Sézary syndrome, skin cancer, Kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, malignant thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0827] In one example, a first fusion protein binds a first checkpoint protein target on a first cell and a second fusion protein detects a second checkpoint protein target on a second cell, such that the fusion proteins detect a cell-cell interaction.

[0828] Exemplary checkpoint target molecules include programmed cell death-1 (PD-1) receptor, PD-ligand 1 (PD-L1), PD-ligand 2 (PD-L2), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), B7 family proteins, tumor necrosis factor (TNF) family proteins, cluster of differentiation 40L (CD40L), adenosine A2a receptor (A2aR), B7-related protein 1 (B7RP1), B and T lymphocyte attenuator (BTLA), galectin 9 (GAL9), herpes virus entry mediator (HVEM), inducible T cell co-stimulatory molecule (ICOS), interleukin (IL), killer cell immunoglobulin-like receptor (KIR), lymphocyte activation gene 3 (LAG3), transforming growth factor-β (TGF-β), T cell membrane protein 3 (TIM3) and MHC class I or II peptide. A preferred checkpoint target is PD-1. PD-L1 or PD-L2 are also preferred checkpoint targets. CTLA-4 is a preferred checkpoint target. MHC class I or II peptides are preferred checkpoint targets. LAG3, B7-H3, B7-H4, and TIM3 are also preferred checkpoint targets.

[0829] In one example, the first fusion protein binds to PD-1 and the second fusion protein binds to PD-L1 or PD-L2. In determining the fluorescent signal score:

[0830] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-PD-1, anti-PD-L1 or PD-L2 antibody; or

[0831] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-PD-1, anti-PD-L1 or PD-L2 antibody.

[0832] In another example, the first fusion protein binds CTLA-4 or CD28 and the second fusion protein binds CD80 or CD86. In determining the fluorescent signal score:

[0833] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-CTLA-4, anti-CD28, anti-CD80, or anti-CD86 antibody; or

[0834] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-CTLA-4, anti-CD28, anti-CD80, or anti-CD86 antibody.

[0835] In another example, the first fusion protein binds to an MHC class I or II peptide and the second fusion protein binds to TCR, CD8, or CD3. In determining the fluorescent signal score:

[0836] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-MHC class I, anti-MHC class II, anti-TCR, anti-CD8, or anti-CD3 antibody; or

[0837] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-MHC class I, anti-MHC class II, anti-TCR, anti-CD8 or anti-CD3 antibody.

[0838] In an additional aspect, the present invention provides an in vitro coincidence assay for detecting whether a checkpoint activator or inhibitor effectively inhibits or modulates a tumor response, the method comprising:

[0839] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0840] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0841] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0842] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0843] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0844] The method comprises:

[0845] (a) contacting the isolated tumor cell sample with the at least two primary binding agents;

[0846] (b) contacting the sample with the at least two secondary binding agents;

[0847] (c) performing a washing step;

[0848] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0849] (e) contacting the sample with the checkpoint activator or inhibitor;

[0850] (f) detecting any changes in the interactions between the secondary binding agents, wherein:

[0851] a. In cases where the checkpoint molecule is an inhibitor:

[0852] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[0853] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[0854] b. In the case where the checkpoint molecule is an activator:

[0855] i. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[0856] ii. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[0857] The present invention also provides use of the above in vitro coincidence assay method for detecting whether a checkpoint activator or inhibitor effectively inhibits or regulates tumor response, and a kit for use therein.

[0858] Exemplary checkpoint inhibitors include anti-PD-1 binding agents, anti-PD-L1 binding agents, anti-PD-L2 binding agents, anti-CTLA-4 binding agents, anti-MHC class I and / or anti-MHC class II binding agents. An exemplary anti-CTLA-4 antibody is ipilimumab. Exemplary anti-PD-1 binding agents include nivolumab (BMS-936558, DX 1106, or ONO-4538) and pembrolizumab (rambrolizumab or MK-3475).

[0859] Other exemplary checkpoint inhibitors include those selected from the group consisting of ipilimumab, nivolumab (BMS-936558, DX 1106, or ONO-4538), pembrolizumab (rambrolizumab or MK-3475), pidilizumab (CT-Q11), ED-0680 (AMP-514), AMP-224, BMS-936559 (MDX-1105), ED 4736, MPDL3280A (RG7448), MSB0010718C, and fragments and salts thereof.

[0860] Exemplary checkpoint activators include those from TNF receptors and B7-CD28 superfamilies, including CD40 (TNFSFR5) agonists, GITR (glucocorticoid-induced tumor necrosis factor receptor; TNFSFR18) stimulators, OX40 (CD134; TNFSFR4) agonists, 4-1BB (CD137; TNFSFR9) agonists, CD27 (TNFSFR7) agonists CD27 (TNFSFR7) agonists, ICOS (inducible costimulatory molecules) molecule agonists, Trail receptor agonists and / or HVEM (herpes virus invasion mediator) receptor agonists. Preferred checkpoint activators include OX-40, GITR and / or 4-1BB agonists.

[0861] In the above method, an increase in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the checkpoint activator will be effective. A decrease in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the checkpoint inhibitor will be effective.

[0862] In the above methods, at least two primary binding agents can bind to the first molecule or the second molecule in such a manner that the check point inhibitor or activator can bind to the first molecule or the second molecule simultaneously or sequentially. In some aspects, the at least two primary binding agents do not inhibit the binding of the check point inhibitor or activator to the first molecule or the second molecule.

[0863] In an additional aspect, the invention provides an in vitro method of determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the method comprising:

[0864] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0865] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0866] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0867] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0868] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0869] The method comprises:

[0870] (a) contacting an isolated tumor cell sample obtained from the patient prior to treatment comprising the checkpoint activator or inhibitor with at least two primary binding agents;

[0871] (b) contacting the sample with the at least two secondary binding agents;

[0872] (c) performing a washing step;

[0873] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0874] (e) repeating steps (a) to (d) using an isolated tumor cell sample obtained from the patient during the treatment comprising the checkpoint activator or inhibitor in step (a);

[0875] (f) comparing the total fluorescence fractions between the samples, wherein:

[0876] a. Where the therapy comprises a checkpoint inhibitor:

[0877] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[0878] ii. if the total score does not change, then it indicates or predicts that the therapy is ineffective; or

[0879] b. Where the therapy comprises a checkpoint activator:

[0880] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[0881] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[0882] The present invention also provides use of the above in vitro method for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, and kits for use therein.

[0883] The present invention further provides an in vitro method of determining whether a patient suffering from cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, the method comprising:

[0884] at least two primary binding agents, wherein a first primary binding agent binds to PD-1 on a first cell and a second primary binding agent binds to PD-L1 or PD-L2 on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and

[0885] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0886] (iv) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0887] (v) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0888] (vi) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0889] The method comprises:

[0890] (d) contacting an isolated tumor cell sample obtained from the patient with the at least two primary binding agents;

[0891] (e) contacting the sample with the at least two secondary binding agents;

[0892] (f) performing a washing step;

[0893] (d) detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein:

[0894] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or

[0895] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the use of blockade

[0896] Therapeutic responses to agents that target the PD-1:PD-L1 / PD-L2 pathway.

[0897] The method may:

[0898] (iii) performing a biopsy on a biological sample obtained from the patient prior to treatment to guide the decision to treat with a single agent that blocks the PD-1:PD-L1 / PD-L2 pathway or with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway and at least one other anti-PD-1 agent.

[0899] Tumor agents are treated with combination therapy; and

[0900] (iv) performing a biopsy on at least one biological sample obtained from the patient during treatment to monitor the subject's response to the current treatment regimen and guide the decision to select treatment with single-agent PD-1:PD-L1 / PD-L2 blockade therapy or combination therapy.

[0901] In the above method, a decrease in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the PD-1:PD-L1 / PD-L2 pathway will be effective.

[0902] The above methods can also be applied to other agents that block immune checkpoint pathways or activate immune checkpoint pathways. Exemplary checkpoint inhibitors and activators are described above. For example, the agent can block CTLA-4, CD28, CD80 and / or CD86. The agent can block MHC class I peptides, MHC class II peptides, TCR, CD8 and / or CD3.

[0903] Where the agent is a checkpoint inhibitor, a decrease in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the agent will be effective.

[0904] Where the agent is a checkpoint activator, an increase in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the agent will be effective.

[0905] In the above methods, at least two primary binding agents can bind to the first molecule or the second molecule in such a manner that the check point inhibitor or activator can bind to the first molecule or the second molecule simultaneously or sequentially. In some aspects, the at least two primary binding agents do not inhibit the binding of the check point inhibitor or activator to the first molecule or the second molecule.

[0906] The sample used in the above method can be a fixed tumor cell sample. In some aspects, a cell sample is obtained from a patient before or during treatment. The sample can be obtained from a patient who has previously been treated for cancer, such as a patient who has previously been treated with an anti-tumor agent, or can be obtained from a patient who has not previously been treated for cancer (i.e., an untreated patient).

[0907] Cell samples can include primary tumor cells, secondary (metastatic) tumor cells, solid tumors and related patient tissues (e.g., macrophages, T cells, B cells, etc.). Tumor samples can be, for example, tumor biopsies or surgical incisions obtained from patients. Typically, tumor samples include invasive tumor margins. Tumor samples can be obtained from metastatic lesions. Samples can include peripheral blood vessels. Patients can be suspected patients with metastatic cancer. Examples of cancer include, but are not limited to, melanoma, lung cancer (including, for example, non-small cell lung cancer), breast cancer, head and neck cancer, and urothelial carcinoma. Other examples of cancer include adrenocortical carcinoma, anal cancer, bladder cancer, blood cancer, brain stem glioma, cerebellar astrocytoma, ependymoma, carcinoid tumor, cancer of unknown primary, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, extrahepatic bile duct cancer, Ewing family of tumors (PNET), extracranial germ cell tumor, eye cancer, intraocular melanoma, gallbladder cancer, stomach cancer, germ cell tumor, extragonadal trophoblastic tumor, hypopharyngeal cancer, islet cell carcinoma, kidney cancer (renal cell carcinoma), laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer, liver cancer, small cell lung cancer, lymphoma, cutaneous T-cell lymphoma, and leukemia. Cancer, Hodgkin's and non-Hodgkin's lymphoma, multiple myeloma and other plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, epithelial ovarian cancer, ovarian germ cell tumor, pancreatic cancer, islet cell carcinoma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pituitary cancer, plasma cell tumor, prostate cancer, rhabdomyosarcoma, rectal cancer, renal cell carcinoma, salivary gland cancer, Sézary syndrome, skin cancer, Kaposi's sarcoma, melanoma, small intestine cancer, soft tissue sarcoma, stomach cancer, testicular cancer, thymoma, malignant thyroid cancer, urethral cancer, uterine cancer, sarcoma, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0908] Approach to selecting a checkpoint inhibitor or activator

[0909] The present invention further provides an in vitro coincidence assay method for identifying whether a molecule of interest is a checkpoint activator or a checkpoint inhibitor, the method comprising:

[0910] at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct;

[0911] at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein:

[0912] (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor;

[0913] (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[0914] (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[0915] The method comprises:

[0916] (a) contacting the isolated tumor cell sample with the at least two primary binding agents;

[0917] (b) contacting the sample with the at least two secondary binding agents;

[0918] (c) performing a washing step;

[0919] (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction;

[0920] (e) contacting the sample with the molecule of interest;

[0921] (f) detecting any changes in the interactions between the secondary binding agents, wherein:

[0922] a. If the total score increases between steps (d) and (f), then the molecule is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or

[0923] b. If the total score decreases between steps (d) and (f), then the molecule is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or

[0924] c. If the total score does not change between steps (d) and (f), then the molecule is not a checkpoint activator or a checkpoint inhibitor thereof for at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0925] An increase of 0.5% to 5%, preferably 5% to 10%, and more preferably greater than 10% in the total score between steps (d) and (f) indicates that the molecule of interest is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule. A decrease of 5% to 0.5%, preferably 10% to 5%, and more preferably greater than 10% in the total score between steps (d) and (f) indicates that the molecule of interest is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

[0926] In the above methods, at least two primary binding agents may bind to the first molecule or the second molecule in such a way that the molecule of interest may bind to the first molecule or the second molecule simultaneously or sequentially.

[0927] In some aspects, the at least two primary binding agents do not inhibit binding of the molecule of interest to the first molecule or the second molecule.

[0928] The present invention also provides the use of the above in vitro method for identifying whether a molecule of interest is a checkpoint activator or a checkpoint inhibitor, and a kit for use therein.

[0929] Example

[0930] The present invention is described in more detail with reference to the following non-limiting examples, which are provided in order to more fully illustrate the invention but should not be construed as limiting the scope thereof.

[0931] The methods of the present invention can be used to quantify changes in donor lifespan.

[0932] Example 1 - Analysis of fixed tissue sections by high-throughput frequency-domain fluorescence lifetime imaging microscopy (f-FLIM) Two-site TSA-FRET

[0933] For PD-1: PD-L1 interaction studies, FFPE sections were covered and heated with Tris-EDTA buffer and then incubated for an additional 10 minutes. The slides were then cooled for 20-30 minutes, washed with PBS for 5 minutes, and washed twice. The slides were marked with a PAP pen to prevent liquid from leaking from the tissue in the next step. Each time liquid was added to the tissue, enough liquid was added to completely cover the tissue. Endogenous peroxidase was quenched by adding an endogenous peroxidase inhibitor for 30 minutes at room temperature, and the tissue was then washed with PBS. The tissue was then blocked for 60 minutes using (10 mg / ml) BSA in PBS at room temperature.

[0934] The tissue sections were then divided into two conditions: donor only (D) and donor and recipient (D / A). The two primary antibodies used were mouse anti-PD-1 and rabbit anti-PD-L1 diluted 1:100 and 1:500, respectively, in 1% BSA / PBS buffer. The donor only condition was incubated with mouse anti-PD-1, while the donor and recipient condition was incubated with both mouse anti-PD-1 and rabbit anti-PD-L1. The primary antibody incubation was allowed to proceed overnight at 4°C.

[0935] After incubation, the tissue sections were washed twice with PBS + 0.02% Tween 20 to remove the non-specific binding of the primary antibody. The sections labeled with mouse anti-PD-1 (donor only) were further incubated with anti-mouse FAB-ATTO488 (1: 100). The sections labeled with mouse anti-PD-1 and rabbit anti-PD-L1 were incubated with anti-mouse FAB-ATTO488 (1: 100) and anti-rabbit FabHRP (1: 200). The anti-rabbit FabHRP antibody was then reacted with tyramide-Alexa 594 and amplified therewith. The tissue was incubated at room temperature for two hours and then washed twice with PBS. The slide was then incubated with tyramide buffer at room temperature for 20 minutes, followed by washing twice with PBS. The slide was then mounted.

[0936] Three patients with different types of stage IV melanoma were treated with anti-receptor tyrosine kinase (RTK) drugs and an antibody that blocks PD-1.

[0937] Tissue samples were obtained from three patients with the following characteristics:

[0938] MM14 (82-year-old woman with stage 4 mucosal melanoma).

[0939] o BRAF wild-type.

[0940] o cKIT mutants.

[0941] o KIT is a receptor tyrosine kinase that undergoes a hyperactive mutation.

[0942] oNo active treatment.

[0943] oPatients were previously treated with nilotinib, a tyrosine kinase inhibitor.

[0944] oThe tissue is a lymph node metastasis.

[0945] MM17 (61-year-old woman with stage 4 cutaneous melanoma).

[0946] o BRAF wild-type.

[0947] oNo effective treatment

[0948] oPrevious treatment with ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1).

[0949] oThe tissue is a lymph node metastasis.

[0950] MM19 (49-year-old woman with stage 4 cutaneous melanoma).

[0951] o BRAF wild-type.

[0952] o NRAS mutants.

[0953] o NRAS is an oncogene that causes overactivation of the MAPK pathway.

[0954] oUntreated.

[0955] o Tissues are lymph node metastasis and transverse colon metastasis.

[0956] The results are Figure 1 and Figure 2 Shown in. Figure 1 and Figure 2 Figure 2 shows changes in the PD-1:PD-L1 interaction obtained by TSA-FRET in patients treated with anti-RTK and PD-1 blocking antibodies. Figure 1 This is indicated by a decrease in lifetime in the presence of the receptor (PD-L1) fluorophore ALX 594.

[0957] Figure 2 A box plot is provided showing the median FRET efficiency and highlighting the changes in PD-1:PD-L1 interactions. P values ​​indicate highly significant differences in FRET efficiency. Each data point represents the area with the highest receptor (PD-1) concentration on the provided tissue. The results show that the method of the present invention allows for quantification of molecular interactions in tissues, such as PD-1 and PD-L1 interactions, and that differences in molecular interactions can be detected after drug treatment.

[0958] Example 2 - Dual site analysis in cells by high-throughput frequency-domain fluorescence lifetime imaging microscopy (f-FLIM) TSA-FRET

[0959] The following assay was used to determine the interaction between PD-1 and PD-L1 in cells. Experiments were performed in the presence of anti-PD-1 antibodies using 8-well plates as shown in Table 1.

[0960]

[0961]

[0962] Table 1: PD-1:PD-L1 blocking assay in 8-well plates in the presence and absence of 25 μg / ml anti-PD-1 antibodies

[0963] PD-L1 APC / CHO-K1 cells were added to an 8-well plate and incubated in a 37°C, 5% CO2 incubator for 19 hours. The culture medium was removed, and 25 μg / ml of blocking anti-PD-1 antibody was added to half of the plate, while assay buffer was added to the remaining portion. PD-1 effector cells (Jurkat) were added and incubated in a 37°C, 5% CO2 incubator for 22 hours.

[0964] Unbound cells were removed and the plates were washed twice with PBS. Cells were fixed with 4% PFA, washed again twice with PBS, and stored in PBS at 4° C. Cells were not permeabilized with any detergent prior to antibody treatment.

[0965] FRET solution:

[0966] Endogenous peroxidase was quenched using an endogenous peroxidase inhibitor for 30 minutes at room temperature.The wells were washed twice with PBS, blocked with 1% BSA for 1 hour at room temperature and washed twice with PBS.

[0967] The wells were incubated overnight at 4°C with 80 μl of primary antibody against PD-1 (1:100) in 1% BSA and, for donor / acceptor conditions, with primary antibody against PD-L1 (1:500). The plates were washed twice with 0.02% PBS-Tween.

[0968] The donor-only condition was labeled with secondary anti-mouse FabATTO 488 (1:100), while the donor / acceptor condition was labeled with secondary anti-mouse FabATTO 488 (1:100) and anti-rabbit Fab-HRP (1:200). The Fab fragment was incubated for 2 hours under both conditions. The plate was washed twice with 0.02% PBS-Tween and the tyramide signal was amplified for the amplification of the receptor. The slide was then mounted. Monoclonal blocking antibodies that promote interaction destruction were used as negative controls.

[0969] Time-resolved FRET acquisition:

[0970] An automated multi-frequency high-throughput lifetime imaging microscope was created by modifying a multi-frequency domain FLIM lifetime imaging microscope from Lambert Instruments.

[0971] The FRET efficiency (Ef) was determined using the following formula: Ef (%) 1 / 4[[1(tDA / tD)]100]; where (when FRETtD / A<<tD); tD is the donor lifetime and tD / A is the donor plus acceptor lifetime.

[0972] Data acquisition was performed using a 60× oil immersion objective (N / A 1.49), and the donor lifetime and intensity of ATTO 488 were determined using a modulated 473 nm laser beam at 40 MHz with an exposure time of 70 ms and a threshold of 35% at 70 mW peak power. For acceptor intensity acquisition, a mercury source with a TRITC excitation / emission filter was used with an 8× neutral density filter for 1 ms.

[0973] Data Analysis:

[0974] The results are Figure 3 and Figure 4 Shown in. Figure 3 The figure above shows that in the presence of the receptor PD-L1, the donor lifetime is reduced from 1.39 to 1.19 ns. This lifetime reduction is attributed to the interaction of the two proteins. In the presence of the blocking antibody, the lifetime of the donor (with the receptor) is 1.29 ns. Therefore, the donor lifetime is not reduced to the same extent in the presence of the blocking antibody.

[0975] FRET efficiency was calculated based on the change in donor lifetime in the presence of acceptor with and without blocking antibody and plotted as a boxplot distribution as Figure 4 shown.

[0976] Figure 4 This study demonstrates that the PD-1:PD-L1 interaction can be quantified by FRET. The data demonstrate highly significant differences between the receptor-ligand interaction and its inhibition by a blocking antibody. Each point on the boxplot represents a region of interest containing an average of five cells. P values ​​were determined using the nonparametric Mann-Whitney test.

[0977] The results show that the method of the present invention allows for the quantification of molecular interactions in cells, such as PD-1 and PD-L1 interactions, and that differences in molecular interactions can be detected after drug treatment.

[0978] Example 3 - i-FRET Assay for the Interaction between CTLA-4 and CD80 in Cell Culture

[0979] The Promega Blockade Bioassay (CS186907) protocol, originally designed to measure antibody blocking of the cytotoxic T lymphocyte antigen 4 and cluster of differentiation 80 interaction (CTLA-4-CD80) by luminescence, has been adapted for use in the i-FRET protocol.

[0980] Plate preparation

[0981] Jurkat cells expressing CTLA-4 provided by Promega Blockade Bioassay were seeded at 100 μl / well. 8-well plates. Anti-CTLA-4 antibody (ipilimumab) obtained from Qualasept was added to half of the wells to obtain a final concentration of 100 μl / ml (Table 1). 100 μl of Raji cells expressing CD80 were added to each well and the plate was incubated at 37°C and 5% CO2 for 19 hours. Unbound cells were removed by washing with PBS and fixed with 4% paraformaldehyde (PFA) for 12 minutes. PFA was removed and all wells were washed with PBS.

[0982]

[0983] Table 2. 8-well plate design. Wells 1-4 represent donor-only conditions and wells 5-8 represent donor / recipient conditions. Culture medium was added to wells 1, 2, 5, and 6, and 100 μl / ml of the anti-CTLA-4 antibody Ipilimumab was added to wells 3, 4, 7, and 8.

[0984] Primary antibodies for staining with anti-CTLA-4 and anti-CD80

[0985] Pierce endogenous peroxidase inhibitor from Thermo Fisher Scientific was added to each well and incubated at room temperature for 30 minutes. The cells were washed with PBS, incubated with 1% (10 mg / ml) bovine serum albumin (BSA) for 1 hour and washed again with PBS. Primary antibody staining was performed using mouse monoclonal anti-CTLA-4 and rabbit polyclonal anti-CD80 obtained from Abcam and MyBioSource respectively. Both were diluted in BSA (1: 100). Anti-CTLA-4 was used to mark only donor conditions (1-4) and anti-CTLA-4 and anti-CD80 were used to mark donor / acceptor conditions (5-8). The plate was incubated overnight at 4°C, then washed twice (PBST) with 0.02% Tween20 in PBS and washed once with PBS.

[0986] Secondary staining with anti-mouse Fab-ATTO488 and anti-rabbit Fab-HRP

[0987] Secondary Fab ATTO488 was diluted (1:15) with 1% BSA and added to the donor well and donor / acceptor wells (1-8). The Fab ATTO488 conjugate contains 4.1 molecules of ATTO488 per molecule of Fab fragment protein. Secondary Fab HRP was diluted (1:200) with 1% BSA and added only to the donor / acceptor wells (5-8). The plate was incubated for 2 hours, then washed twice with 0.02% PBST and once with PBS.

[0988] Tyramide amplification

[0989] Alexa594 conjugated tyramide was diluted in amplification buffer (1:100) in the presence of 0.15% H2O2. In this mixture, 100 μl was added to each donor / acceptor well (5-8) and the plate was incubated in the dark for 20 minutes. To remove the tyramide, the wells were washed twice with PBST and once with PBS. 5 μl of Prolong Diamond anti-fading mounting medium was added to each well and fixed with a cover glass.

[0990] CTLA-4-CD80 interaction determined using i-FRET

[0991] use Resonance energy transfer (FRET) was used to assess the CTLA-4-CD80 interaction between cells. CTLA-4 and CD80 intensities were determined using a modulated 473 nm laser beam at 40 MHz and a mercury source, respectively. CTLA-4 intensity, CD80 intensity, and lifetime are plotted in Figure 2. Figure 5 When comparing donor-only to donor / acceptor wells, a greater reduction in donor lifetime (τ) was observed in the no-treatment condition compared to the 100 μl ipilimumab condition.

[0992] Compared with the 100 μl / ml ipilimumab condition, the FRET efficiency value under the 0 μl / ml ipilimumab condition ( Figure 6 ) were significantly (***) higher (p=0.00044, p=0.00024). There were no significant differences in FRET values ​​between wells without Ipilimumab (p=0.054).

[0993] in conclusion

[0994] 1) Cell-cell contacts can be quantified by i-FRET using the CTLA-4-CD80 pair.

[0995] 2) FRET efficiency is significantly reduced in the presence of ipilimumab compared to untreated cells.

[0996] 3) The FRET efficiency values ​​decreased under 100 μl / ml Ipilimumab conditions, strongly suggesting that the interaction observed in the absence of Ipilimumab is due to a specific interaction between CTLA-4 and CD80.

[0997] Example 4 - iFRET Assay for the Interaction between CTLA-4 and CD80 in Metastatic Melanoma Tissue

[0998] Metastatic melanoma tissue was immunized- Resonance Energy Transfer (i-FRET) assay, which determines the interaction of cytotoxic T-lymphocyte-associated protein-4 (CTLA-4) with cluster of differentiation 80 (CD80). Samples were obtained from different patients as detailed in Table 3 (below).

[0999] method

[1000] Antigen retrieval

[1001] Antigen retrieval was performed using PTLink at Fastbase Laboratories, after which 1-2 drops of Pierce endogenous peroxidase inhibitor were added to each slide to quench endogenous peroxidase and incubated for 30 minutes. 300 μl of 10 mg / ml bovine serum albumin (BSA) diluted in phosphate-buffered saline (PBS) was then added to each slide to prevent nonspecific primary antibody binding. Both incubations were performed in a humidified tray.

[1002] Primary antibody staining

[1003] Primary antibodies were stained using mouse monoclonal anti-CTLA-4 and rabbit polyclonal anti-CD80. Both were diluted in 1% BSA in PBS (1:100). Anti-CTLA-4 was used to label the donor-only condition, while anti-CTLA-4 and anti-CD80 were used to label the donor / recipient condition. 150 μl was added to each slide and incubated overnight.

[1004] Secondary antibody staining

[1005] The donor slide was incubated with anti-mouse Fab-ATTO488 (1:15) final dilution and the donor acceptor slide was incubated with anti-mouse Fab ATTO488 (1:15) + anti-rabbit Fab-HRP (1:200). The dilution was prepared using 1% BSA in PBS. 150 μl was added to each slide and then incubated for 2 hours at room temperature in a humidified tray.

[1006] Tyramide labeling

[1007] Alexa 594 conjugated tyramide was diluted (1:100) in amplification buffer in the presence of 0.15% H2O2. Of this mixture, 150 μl was added to each donor / acceptor slide and incubated in the dark at room temperature for 20 minutes. 150 μl of Prolong Diamond antifade mounting medium was added to each slide and mounted using a coverslip.

[1008] Hematoxylin and eosin staining

[1009] Additional slides of the corresponding samples were stained with hematoxylin and eosin (H&E). H&E staining allows for pathological analysis to identify areas of immune cell infiltration. The selected areas are the focus of i-FRET analysis in this assay.

[1010] Table 3 shows the patient background of the samples used for this assay, including genetic background, details of past and active treatments, and sample source.

[1011]

[1012]

[1013] Table 3. Patient background

[1014] Figure 6 showed that the interaction between CTLA-4 and CD80 in metastatic melanoma tissue can be measured by FRET.

[1015] Example 5 - Detection of cell-cell contacts in renal cell carcinoma using i-FRET and the PD-1 / PD-L1 pair

[1016] Immunization of renal cell carcinoma tissue Resonance energy transfer (i-FRET) assay, which determines that programmed death receptor-1 (PD-1) interacts with programmed death ligand 1 (PD-L1) and thus determines cell-cell contacts. Samples were obtained from different patients and included a variety of renal cell carcinomas, namely clear cell renal cell carcinoma (ccRCC), papillary renal cell carcinoma (PRCC), and chromophobe renal cell carcinoma (ChRCC). All samples were primary tumors.

[1017] method

[1018] Antigen retrieval

[1019] Antigen retrieval was performed using PTLink at Fastbase Laboratories, after which 1-2 drops of Pierce endogenous peroxidase inhibitor were added to each slide to quench endogenous peroxidase and incubated for 30 minutes. 300 μl of 10 mg / ml bovine serum albumin (BSA) diluted in phosphate-buffered saline (PBS) was then added to each slide to prevent nonspecific primary antibody binding. Both incubations were performed in a humidified tray.

[1020] Primary antibody staining

[1021] Primary antibody staining was performed using mouse monoclonal anti-PD-1 and rabbit monoclonal anti-PD-L1 diluted (1:100) and (1:500), respectively, in 1% BSA in PBS. Anti-PD-1 was used to label the donor-only condition, while anti-PD-1 and anti-PD-L1 were used to label the donor / acceptor condition. 150 μl was added to each slide and incubated overnight.

[1022] Secondary antibody staining

[1023] The donor slide was incubated with anti-mouse Fab-ATTO488 (1:15) final dilution and the donor acceptor slide was incubated with anti-mouse Fab-ATTO488 (1:15) + anti-rabbit Fab-HRP (1:200). The dilution was prepared using 1% BSA in PBS. 150 μl was added to each slide and then incubated for 2 hours at room temperature in a humidified tray.

[1024] Tyramide labeling

[1025] Alexa 594 conjugated tyramide was diluted in amplification buffer (1:100) in the presence of 0.15% H2O2. Of this mixture, 150 μl was added to each donor / acceptor slide and incubated in the dark at room temperature for 20 minutes. 150 μl of Prolong Diamond antifade mounting medium was added to each slide and mounted using a coverslip.

[1026] The results of the above examples are Figure 7 Shown in.

[1027] Example 6 - Detection of cell-cell interactions in ccRCC tissue using i-FRET and PD-1 / PD-L1 pair

[1028] Immunofluorescence staining of clear cell renal cell carcinoma (ccRCC) tissue Resonance energy transfer (i-FRET) assay, which determines the interaction of programmed death receptor-1 (PD-1) with programmed death ligand 1 (PD-L1) and thus cell-cell interactions. Samples were obtained from Cruces Hospital, Bilbao. Prior to the assay, samples were either PD-L1+ / + or PD-L1- / -.

[1029] method

[1030] Antigen retrieval

[1031] Antigen retrieval was performed using PTLink at FASTBASE SOLUTIONS laboratories, after which 1-2 drops of Pierce endogenous peroxidase inhibitor were added to each slide to quench endogenous peroxidase and incubated for 30 minutes. 300 μl of 10 mg / ml bovine serum albumin (BSA) in phosphate-buffered saline (PBS) was then added to each slide to prevent nonspecific primary antibody binding. Both incubations were performed in a humidified tray.

[1032] Primary antibody labeling

[1033] Primary antibody labeling was performed using mouse monoclonal anti-PD-1 and rabbit monoclonal anti-PD-L1 diluted (1:100) and (1:500), respectively, in 1% BSA in PBS. Anti-PD-1 was used to label the donor-only condition, while anti-PD-1 and anti-PD-L1 were used to label the donor / acceptor condition. 150 μl was added to each slide and incubated overnight.

[1034] Secondary antibody labeling

[1035] The donor slide was incubated with anti-mouse Fab-ATTO488 (1:15) final dilution and the donor acceptor slide was incubated with anti-mouse Fab-ATTO488 (1:15) + anti-rabbit Fab-HRP (1:200). The dilution was prepared using 1% BSA in PBS. 150 μl was added to each slide and then incubated for 2 hours at room temperature in a humidified tray.

[1036] Tyramide labeling

[1037] Alexa 594 conjugated tyramide was diluted in amplification buffer (1:100) in the presence of 0.15% H2O2. Of this mixture, 150 μl was added to each donor / acceptor slide and incubated in the dark at room temperature for 20 minutes. 150 μl of Prolong Diamond antifade mounting medium was added to each slide and mounted using a coverslip.

[1038] The results of the above examples are Figure 8The results are presented in Figure 2 and demonstrate that i-FRET determines the specific interaction of PD-1 with PD-L1 in invasive areas with low PD-L1 expression. This is not the case when using PLA, where the proximity of PD-1 and PD-L1 cannot be detected at low PD-L1 expression. Furthermore, i-FRET can quantify the heterogeneity of invasive areas. The precision of i-FRET can identify outliers that may be excluded by other methods for therapeutic treatment.

[1039] Example 7 - Two-site TSA-FRET in fixed tissue sections analyzed by high-throughput frequency-domain fluorescence lifetime imaging microscopy (f-FLIM).

[1040] T cells recognize antigens through the interaction of the T cell receptor (TCR) with peptides embedded in MHC molecules (pMHC) on the surface of antigen-presenting cells (APCs). Cytotoxic T lymphocyte TCRs recognize epitopes presented on MHC class I molecules on the surface of cells in the body, distinguishing between 'self' antigens and foreign antigens (virus-infected cells), as well as neoantigens presented by tumor cells. Helper T cell TCRs recognize epitopes presented on MHC class II molecules on the surface of antigen-presenting immune cells. TCRs on other T cell types also interact with MHC-like molecules from the CD1d family—for example, the interaction between the semi-invariant TCR expressed by invariant NKT cells and the CD1d-lipid complex, and the interaction between the TCR expressed by mucosal-associated invariant T cells (MAIT cells) and the MHC-like molecule MR1 (Bhati et al., 2013). TCRs interact with CD3 molecules to form the TCR complex. CD3 contains three different polypeptide chains: γ, ε, and δ. Depending on the type of T cell expressing the TCR, the TCR complex may further interact with CD4 or CD8 molecules.

[1041] In the methods, kits and uses of the present invention, the first molecule can be a TCR or other member of the TCR complex, including CD3γ, CD3ε and CD3δ or CD8 or CD4 and combinations thereof 1 and the second molecule can be an antigen-loaded MHC class I, MHC class II or MHC-like molecule (such as CD1d or MR).

[1042] To date, measurement of TCR-pMHC interactions has been accomplished by FRET using recombinant pMHC complexes (Huang et al., 2010) or MHC conjugated to fluorescent peptides (Axmann et al., 2015). However, methods for measuring TCR-pMHC interactions endogenously expressed on fixed tissues are lacking.

[1043] In the methods, kits and uses of the present invention, the first molecule may be an endogenously expressed TCR or other member of the TCR complex, including CD3γ, CD3ε and CD3δ or CD8 or CD4 and combinations thereof and the second molecule may be an endogenously expressed antigen-loaded MHC class I, MHC class II or MHC-like molecule (such as CD1d or MR1).

[1044] For TCR-pMHC interaction studies, FFPE sections were coverslipped and heated with Tris-EDTA buffer and then processed according to the method described in Example 1.

[1045] The tissue sections are then divided into two conditions: donor only (D) and donor and recipient (D / A). The two primary antibodies used are, for example, mouse anti-TCR and rabbit anti-MHC diluted 1:100 and 1:500, respectively, in 1% BSA / PBS buffer. The donor only condition is incubated with mouse anti-TCR, while the donor and recipient condition is incubated with both mouse anti-TCR and rabbit anti-MHC.

[1046] In this specification, “comprises” means “including or consisting of” and “comprising” means “including or consisting of.”

[1047] The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific form or in terms of means for performing the disclosed functions, or methods or processes for obtaining the disclosed results, may, where appropriate, be used alone or in any combination of such features to realize the invention in its various forms.

[1048] The present invention will now be defined by reference to the following clauses.

[1049] 1. An in vitro coincidence assay for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, the method comprising:

[1050] at least two primary antibodies, wherein the first primary antibody binds to the first molecule on the first cell and the second primary antibody binds to the second molecule on the second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1051] at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1052] (i) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is labeled with a FRET acceptor;

[1053] (ii) the first secondary antibody and the second secondary antibody are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[1054] (iii) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[1055] The method comprises:

[1056] (a) contacting the isolated sample containing cells with the at least two primary antibodies;

[1057] (b) contacting the sample with the at least two secondary antibodies;

[1058] (c) performing a washing step;

[1059] (d) detecting the interaction between the secondary antibodies.

[1060] 2. The method according to clause 1, wherein the interaction between the secondary antibodies is detected by detecting emitted fluorescence.

[1061] 3. The method according to clause 1, wherein the interaction between the secondary antibodies is detected by detecting altered fluorescence behavior.

[1062] 4. The method according to clause 3, wherein detecting the altered fluorescence behavior is time-resolved.

[1063] 5. The method of clause 1, wherein the first cell and the second cell are the same type of cell.

[1064] 6. The method of clause 1, wherein the first cell and the second cell are not the same type of cell.

[1065] 7. The method according to clause 1, wherein the isolated sample is a fixed cell sample.

[1066] 8. The method according to clause 7, wherein the isolated sample is a fixed tumor cell sample.

[1067] 9. The method according to any preceding clause, wherein the first molecule and the second molecule are proteins, preferably endogenous proteins.

[1068] 10. The method of clause 9, wherein the protein is an immune checkpoint protein.

[1069] 11. The method according to clause 9 or 10, wherein the first molecule is PD-1 and the second molecule is PD-L1 or PD-L2.

[1070] 12. The method according to clause 9 or 10, wherein the first molecule is CTLA-4 or CD28 and the second molecule is CD80 or CD86.

[1071] 13. The method according to clause 9 or 10, wherein the first molecule is selected from MHC class I or II peptides and the second molecule is selected from TCR, CD8, CD3 and combinations thereof.

[1072] 14. The method according to any preceding clause, wherein the at least two primary antibodies are selected from the group consisting of whole immunoglobulins, antibodies or antigen-binding fragments thereof, or a combination thereof.

[1073] 15. The method of any preceding clause, wherein at least one of the secondary antibodies is an antibody or antigen-binding fragment.

[1074] 16. The method of any preceding clause, wherein the at least two secondary antibodies are antibodies or antigen-binding fragments.

[1075] 17. The method of any preceding clause, wherein the antibody or antigen-binding fragment is a Fab fragment, a scFv fragment, or a combination thereof.

[1076] 18. The method of any preceding clause, wherein the primary antibody is unlabeled.

[1077] 19. The method according to any preceding clause, wherein the first primary antibody is a murine antibody and the at least one additional primary antibody is a rabbit antibody.

[1078] 20. The method according to clause 11, wherein the first primary antibody binds to PD-1 and the at least one additional primary antibody binds to PD-L1 or PD-L2.

[1079] 21. The method of clause 12, wherein the first primary antibody binds to CTLA-4 or CD28 and the second primary antibody binds to CD80 or CD86.

[1080] 22. The method according to clause 13, wherein the first primary antibody binds to an MHC class I or II peptide and the at least one additional primary antibody binds to TCR, CD8, CD3, or a combination thereof.

[1081] 23. The method according to clause 19, wherein the first secondary antibody is an anti-murine antibody and the at least one additional secondary antibody is an anti-rabbit antibody.

[1082] 24. The method according to any of the preceding clauses, wherein the FRET donor is selected from the group consisting of: ORG 488, GFP, fluorescein, IAEDANS, EDANS, BODIPY FL, ATTO488, and combinations thereof.

[1083] 25. The method of any of the preceding clauses, wherein the FRET acceptor is selected from the group consisting of ALX 594, mRFP, tetramethylrhodamine, fluorescein, dabcyl, BODIPY FL, QSY 7, QSY 9, and combinations thereof.

[1084] 26. The method of any preceding clause, wherein the enzyme is selected from the group consisting of an oxidoreductase, a hydrolase, a lyase, a transferase, an isomerase, and a ligase.

[1085] 27. The method according to clause 26, wherein the enzyme is selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

[1086] 28. The method according to clause 27, wherein the enzyme is selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and beta-galactosidase.

[1087] 29. A method according to any of the preceding clauses, wherein the substrate is tyramide.

[1088] 30. The method of any of the preceding clauses, wherein the first cell is a T cell and the second cell is a tumor cell.

[1089] 31. The method according to any preceding clause, wherein the at least two primary antibodies are contacted with each other simultaneously or sequentially.

[1090] 32. The method according to any preceding clause, wherein the at least two secondary antibodies are contacted with each other simultaneously or sequentially.

[1091] 33. The method according to clauses 1 to 31, wherein the at least two primary antibodies and the at least two secondary antibodies are contacted with the sample simultaneously.

[1092] 34. The method according to clauses 1 to 31, wherein the at least two primary antibodies are contacted with the sample before the at least two secondary antibodies.

[1093] 35. The method according to clause 34, wherein a washing step is performed after contacting the at least two primary antibodies with the sample and before contacting the at least two secondary antibodies with the sample.

[1094] 36. The method of any preceding clause, further comprising the step of quantifying the interaction between a first site on the first cell and a second site on the second cell.

[1095] 37. A method according to any preceding clause, wherein the first cell is a lymphocyte and the second cell is a non-lymphocyte type.

[1096] 38. The method of clause 37, wherein the method detects the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte.

[1097] 39. The method of any preceding clause, wherein the first molecule is located on the cell surface of the first cell and the second molecule is located on the cell surface of the second cell.

[1098] 40. The method according to any preceding clause, wherein the at least two primary antibodies bind to the first or second molecule in such a way that a checkpoint inhibitor or activator can bind to the first or second molecule simultaneously or sequentially.

[1099] 41. The method of any preceding clause, wherein the at least two primary antibodies do not inhibit binding of a checkpoint inhibitor or activator to the first molecule or the second molecule.

[1100] 42. Use of an in vitro coincidence assay according to any preceding clause for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell.

[1101] 43. A kit for use in an in vitro coincident assay for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell, the kit comprising:

[1102] a) at least two primary antibodies, wherein the first primary antibody binds to the first molecule on the first cell and the second primary antibody binds to the second molecule on the second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1103] b) at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1104] (i) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is labeled with a FRET acceptor;

[1105] (ii) the first secondary antibody and the second secondary antibody are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[1106] (iii) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[1107] c) instructions for performing a method comprising:

[1108] i. contacting the isolated sample containing cells with the at least two primary antibodies;

[1109] ii. contacting the sample with the at least two secondary antibodies;

[1110] iii. performing a washing step;

[1111] iv. detecting the interaction between the secondary antibodies.

[1112] 44. The kit for use according to clause 43, wherein the instructions for detecting the interaction between the secondary antibodies are for detecting by detecting emitted fluorescence.

[1113] 45. The kit for use according to clause 43, wherein the instructions for detecting the interaction between the secondary antibodies are for detecting by detecting altered fluorescence behavior.

[1114] 46. ​​A kit for use according to clause 45, wherein detecting said altered fluorescence behaviour is time resolved.

[1115] 47. A kit for use according to clause 43, wherein the first cell and the second cell are of the same type of cell.

[1116] 48. A kit for use according to clause 43, wherein the first cell and the second cell are not the same type of cell.

[1117] 49. A kit for use according to clause 43, wherein the isolated sample is a fixed cell sample.

[1118] 50. A kit for use according to clause 49, wherein the isolated sample is a fixed tumor cell sample.

[1119] 51. A kit for use according to any preceding clause, wherein said first molecule and said second molecule are proteins, preferably endogenous proteins.

[1120] 52. A kit for use according to clause 51, wherein the protein is an immune checkpoint protein.

[1121] 53. A kit for use according to clause 51 or 52, wherein the first molecule is PD-1 and the second molecule is PD-L1 or PD-L2.

[1122] 54. A kit for use according to clause 51 or 52, wherein the first molecule is CTLA-4 or CD28 and the second molecule is CD80 or CD86.

[1123] 55. A kit for use according to clause 51 or 52, wherein the first molecule is an MHC class I or II peptide and the second molecule is selected from the group consisting of TCR, CD8, CD3 and combinations thereof.

[1124] 56. A kit for use according to any preceding clause, wherein the at least two primary antibodies are selected from the group consisting of whole immunoglobulins, antibodies or antigen-binding fragments thereof, or a combination thereof.

[1125] 57. A kit for use according to any preceding clause, wherein at least one of the secondary antibodies is an antibody or antigen-binding fragment.

[1126] 58. A kit for use according to any preceding clause, wherein the at least two secondary antibodies are antibodies or antigen-binding fragments.

[1127] 59. A kit for use according to any preceding clause, wherein the antibody or antigen-binding fragment is a Fab fragment, a scFv fragment or a combination thereof.

[1128] 60. A kit for use according to any preceding clause, wherein the primary antibody is unlabelled.

[1129] 61. A kit for use according to any preceding clause, wherein the first primary antibody is a murine antibody and the at least one further primary antibody is a rabbit antibody.

[1130] 62. A kit for use according to clause 53, wherein the first primary antibody binds to PD-1 and the at least one additional primary antibody binds to PD-L1 or PD-L2.

[1131] 63. A kit for use according to clause 54, wherein the first primary antibody binds to CTLA-4 or CD28 and the second primary antibody binds to CD80 or CD86.

[1132] 64. The kit for use according to clause 55, wherein the first primary antibody binds to an MHC class I or II peptide and the second primary antibody binds to TCR, CD8, CD3, and combinations thereof.

[1133] 65. A kit for use according to clause 61, wherein the first secondary antibody is an anti-murine antibody and the at least one further secondary antibody is an anti-rabbit antibody.

[1134] 66. A kit for use according to any preceding clause, wherein the FRET donor is selected from the group consisting of ORG 488, GFP, fluorescein, IAEDANS, EDANS, BODIPY FL, ATTO488 and combinations thereof.

[1135] 67. A kit for use according to any preceding clause, wherein the FRET acceptor is selected from the group consisting of ALX 594, mRFP, tetramethylrhodamine, fluorescein, dabcyl, BODIPY FL, QSY 7, QSY 9 and combinations thereof.

[1136] 68. A kit for use according to any preceding clause, wherein the enzyme is selected from the group consisting of an oxidoreductase, a hydrolase, a lyase, a transferase, an isomerase, and a ligase.

[1137] 69. A kit for use according to clause 68, wherein the enzyme is selected from the group consisting of peroxidases, oxidases, phosphatases, esterases and glycosidases.

[1138] 70. A kit for use according to clause 69, wherein the enzyme is selected from the group consisting of horseradish peroxidase, glucose oxidase, alkaline phosphatase and beta-galactosidase.

[1139] 71. A kit for use according to any one of the preceding clauses, wherein the substrate is tyramide.

[1140] 72. A kit for use according to any preceding clause, wherein the first cell is a T cell and the second cell is a tumor cell.

[1141] 73. A kit for use according to any preceding clause, wherein the instructions provide for contacting the at least two primary antibodies simultaneously or sequentially with each other.

[1142] 74. A kit for use according to any preceding clause, wherein the instructions provide for contacting the at least two secondary antibodies simultaneously or sequentially with each other.

[1143] 75. A kit for use according to clauses 43 to 73, wherein the instructions provide for contacting the at least two primary antibodies and the at least two secondary antibodies with the sample simultaneously.

[1144] 76. A kit for use according to clauses 43 to 73, wherein the instructions provide for contacting the at least two primary antibodies with the sample before the at least two secondary antibodies.

[1145] 77. A kit for use according to clause 76, wherein the instructions provide for performing a washing step after contacting the at least two primary antibodies with the sample and before contacting the at least two secondary antibodies with the sample.

[1146] 78. A kit for use according to any preceding clause, wherein the instructions provide a method further comprising the step of quantifying the interaction between a first site on the first cell and a second site on the second cell.

[1147] 79. A kit for use according to any preceding clause, wherein the first cell is a lymphocyte and the second cell is a non-lymphocyte type.

[1148] 80. A kit for use according to clause 79, wherein the instructions provide a method for detecting the interaction between PD-1 on a first lymphocyte and PD-L1 or PD-L2 on a second non-lymphocyte.

[1149] 81. A kit for use according to any preceding clause, wherein the first molecule is located on the cell surface of the first cell and the second molecule is located on the cell surface of the second cell.

[1150] 82. A kit for use according to any preceding clause, wherein the at least two primary antibodies bind to the first or second molecule in such a way that a checkpoint inhibitor or activator can bind to the first or second molecule simultaneously or sequentially.

[1151] 83. A kit for use according to any preceding clause, wherein said at least two primary antibodies do not inhibit binding of an inhibitor or activator to said first molecule or said second molecule.

[1152] 84. Use of a kit according to any one of clauses 43 to 83 in an in vitro coincident assay for detecting an interaction between a first molecule expressed on a first cell and a second molecule expressed on a second cell.

[1153] 85. A method of selecting a patient suffering from cancer for treatment, the method comprising:

[1154] at least two primary antibodies, wherein the first primary antibody binds to a first checkpoint target molecule on a first cell and the second primary antibody binds to a second checkpoint target molecule on a second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1155] at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1156] (i) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is labeled with a FRET acceptor;

[1157] (ii) the first secondary antibody and the second secondary antibody are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[1158] (iii) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[1159] The method comprises:

[1160] (a) contacting an isolated tumor cell sample from the patient with the at least two primary antibodies;

[1161] (b) contacting the sample with the at least two secondary antibodies;

[1162] (c) performing a washing step;

[1163] (d) detecting the interaction between the secondary antibodies by measuring the total fluorescence fraction; wherein:

[1164] a. wherein the intended therapy comprises a checkpoint activator targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[1165] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy; or

[1166] ii. if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[1167] b. wherein the intended therapy comprises a checkpoint inhibitor targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[1168] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[1169] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy.

[1170] 86. The method of clause 75, wherein the first checkpoint target molecule is PD-1 and the second checkpoint target molecule is PD-L1 or PD-L2, and wherein in determining the fluorescent signal score:

[1171] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an anti-PD-1, anti-PD-L1 or anti-PD-L2 antibody; or

[1172] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an anti-PD-1, anti-PD-L1 or PD-L2 antibody.

[1173] 87. The method of clause 86 or 87, wherein the patient has not previously received cancer therapy or wherein the patient has not previously received therapy targeting at least one of the checkpoint target molecules.

[1174] 88. A method according to clauses 86 to 88, wherein if the total score is 0.5% to 5%, this indicates that the patient will not respond to therapy targeting at least one of the checkpoint target molecules.

[1175] 89. The method of clauses 86 to 88, wherein if the total score is 5% to 10%, this indicates that the patient is likely to respond to therapy targeting at least one of the checkpoint target molecules.

[1176] 90. The method of clauses 86 to 88, wherein if the total score is greater than 10%, this indicates that the patient will respond to therapy targeting at least one of the checkpoint target molecules.

[1177] 91. A method according to clauses 86 to 90, wherein the total fraction is a FRET efficiency percentage.

[1178] 92. The method according to clauses 86 to 91, wherein the at least two primary antibodies bind to the first or second molecule in such a manner that the checkpoint inhibitor or activator can bind to the first or second molecule simultaneously or sequentially.

[1179] 93. The method of clauses 86 to 92, wherein the at least two primary antibodies do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[1180] 94. Use of a method according to clauses 86 to 93 for selecting a patient suffering from cancer for treatment, wherein:

[1181] a. wherein the intended therapy comprises a checkpoint activator targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[1182] i. if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy; or

[1183] ii. if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[1184] b. wherein the intended therapy comprises a checkpoint inhibitor targeting at least one of the first checkpoint target molecule and the second checkpoint target molecule:

[1185] (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to the intended therapy; or

[1186] (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to the intended therapy.

[1187] 95. An in vitro coincidence assay for determining whether a checkpoint activator or inhibitor effectively inhibits or modulates a tumor response, the method comprising:

[1188] at least two primary antibodies, wherein the first primary antibody binds to a first checkpoint target molecule on a first cell and the second primary antibody binds to a second checkpoint target molecule on a second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1189] at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1190] (i) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is labeled with a FRET acceptor;

[1191] (ii) the first secondary antibody and the second secondary antibody are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[1192] (iii) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[1193] The method comprises:

[1194] (a) contacting the isolated tumor cell sample with the at least two primary antibodies;

[1195] (b) contacting the sample with the at least two secondary antibodies;

[1196] (c) performing a washing step;

[1197] (d) detecting the interaction between the secondary antibodies by measuring the total fluorescence fraction;

[1198] (e) contacting the sample with the checkpoint activator or inhibitor;

[1199] (f) detecting any changes in the interaction between said secondary antibodies, wherein:

[1200] a. Where the checkpoint molecule is an inhibitor:

[1201] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[1202] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[1203] b. wherein the checkpoint molecule is an activator:

[1204] i. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[1205] ii. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[1206] 96. The method of clause 95, wherein the checkpoint inhibitor is an anti-PD-1 antibody, and the first primary antibody binds to PD-1 and the second primary antibody binds to PD-L1 or PD-L2.

[1207] 97. The method of clause 95 or 96, wherein the checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab (BMS-936558, DX 1106, or ONO-4538), pembrolizumab (rambrolizumab or MK-3475), pidilizumab (CT-Q11), ED-0680 (AMP-514), AMP-224, BMS-936559 (MDX-1105), ED4736, MPDL3280A (RG7448), MSB0010718C, and fragments and salts thereof.

[1208] 98. A method according to clauses 95 to 97, wherein an increase in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the checkpoint activator will be effective.

[1209] 99. A method according to clauses 95 to 97, wherein a decrease in the total score between steps (d) and (f) of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the checkpoint inhibitor will be effective.

[1210] 100. The method according to clauses 95 to 99, wherein the at least two primary antibodies bind to the first or second molecule in such a manner that the checkpoint inhibitor or activator can bind to the first or second molecule simultaneously or sequentially.

[1211] 101. The method of clauses 95 to 100, wherein the at least two primary antibodies do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[1212] 102. Use of an in vitro coincidence assay according to any one of clauses 95 to 101 for determining whether a checkpoint inhibitor or activator will effectively inhibit or modulate a tumor response,

[1213] in:

[1214] a. Where the checkpoint molecule is an inhibitor:

[1215] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[1216] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[1217] b. wherein the checkpoint molecule is an activator:

[1218] iii. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[1219] iv. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[1220] 103. A kit for use in an in vitro coincidence assay for detecting whether a checkpoint inhibitor or activator effectively inhibits or modulates a tumor response, the kit comprising:

[1221] (a) at least two primary antibodies, wherein the first primary antibody binds to a first checkpoint target molecule on a first cell and the second primary antibody binds to a second checkpoint target molecule on a second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1222] (b) at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1223] (i) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is labeled with a FRET acceptor;

[1224] (ii) the first secondary antibody and the second secondary antibody are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[1225] (iii) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[1226] (c) instructions for performing a method comprising:

[1227] a. contacting the isolated tumor cell sample with the at least two primary antibodies;

[1228] b. contacting the sample with the at least two secondary antibodies;

[1229] c. performing a washing step;

[1230] d. detecting the interaction between the secondary antibodies by measuring the total fluorescence fraction;

[1231] e. contacting the sample with the checkpoint activator or inhibitor;

[1232] f. detecting any changes in the interaction between said secondary antibodies, wherein:

[1233] Wherein the checkpoint molecule is an inhibitor of:

[1234] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[1235] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[1236] Wherein the checkpoint molecule is an activator:

[1237] iii. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[1238] iv. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[1239] 104. Use of the kit of clause 83 in an in vitro coincident assay for detecting whether a checkpoint inhibitor or activator effectively inhibits or modulates a tumor response, wherein:

[1240] Wherein the checkpoint molecule is an inhibitor of:

[1241] i. If the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or

[1242] ii. if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or

[1243] Wherein the checkpoint molecule is an activator:

[1244] iii. If the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or

[1245] iv. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

[1246] 105. An in vitro method for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the method comprising:

[1247] at least two primary antibodies, wherein the first primary antibody binds to a first checkpoint target molecule on a first cell and the second primary antibody binds to a second checkpoint target molecule on a second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1248] at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1249] (i) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is labeled with a FRET acceptor;

[1250] (ii) the first secondary antibody and the second secondary antibody are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA amplification, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or

[1251] (iii) the first secondary antibody is labeled with a FRET donor and the second secondary antibody is fused to an enzyme that reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme;

[1252] The method comprises:

[1253] (a) contacting an isolated tumor cell sample obtained from the patient prior to treatment comprising the checkpoint activator or inhibitor with at least two primary antibodies;

[1254] (b) contacting the sample with the at least two secondary antibodies;

[1255] (c) performing a washing step;

[1256] (d) detecting the interaction between the secondary antibodies by measuring the total fluorescence fraction;

[1257] (e) repeating steps (a) to (d) using an isolated tumor cell sample obtained from the patient during the treatment comprising the checkpoint activator or inhibitor in step (a);

[1258] (f) comparing the total fluorescence fractions between the samples, wherein:

[1259] a. Where the therapy comprises a checkpoint inhibitor:

[1260] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[1261] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[1262] b. Wherein said therapy comprises a checkpoint activator:

[1263] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[1264] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[1265] 106. The method of clause 105, wherein the checkpoint inhibitor is an anti-PD-1 antibody, and the first primary antibody binds to PD-1 and the second primary antibody binds to PD-L1 or PD-L2.

[1266] 107. The method of clause 105 or 106, wherein the checkpoint inhibitor is selected from the group consisting of nivolumab (BMS-936558, DX 1106, or ONO-4538), pembrolizumab (rambrolizumab or MK-3475), pidilizumab (CT-Q11), ED-0680 (AMP-514), AMP-224, BMS-936559 (MDX-1105), ED4736, MPDL3280A (RG7448), MSB0010718C, and fragments and salts thereof.

[1267] 108. The method of clauses 105 to 107, wherein an increase in the total score of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the therapy comprising the checkpoint activator will be effective.

[1268] 109. The method of clauses 105 to 107, wherein a reduction in the total score of 0.5% to 5%, preferably 5% to 10%, more preferably greater than 10% indicates that the checkpoint inhibitor-containing therapy will be effective.

[1269] 110. The method according to clauses 105 to 109, wherein the at least two primary antibodies bind to the first or second molecule in such a way that the checkpoint inhibitor or activator can bind to the first or second molecule simultaneously or sequentially.

[1270] 111. The method of clauses 105 to 110, wherein the at least two primary antibodies do not inhibit binding of the checkpoint inhibitor or activator to the first molecule or the second molecule.

[1271] 112. Use of a method according to clauses 105 to 111 for determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, wherein:

[1272] c. Wherein said therapy comprises a checkpoint inhibitor:

[1273] i. If the total score decreases, it indicates or predicts that the therapy is effective; or

[1274] ii. if the total score does not change, indicating or predicting that the therapy is ineffective; or

[1275] d. wherein the therapy comprises a checkpoint activator:

[1276] i. If the total score increases, it indicates or predicts that the therapy is effective; or

[1277] ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

[1278] 113. A kit for use in an in vitro method of determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the kit comprising:

[1279] at least two primary antibodies, wherein the first primary antibody binds to a first checkpoint target molecule on a first cell and the second primary antibody binds to a second checkpoint target molecule on a second cell and wherein the first primary antibody and the second primary antibody are immunologically different;

[1280] at least two secondary antibodies, wherein a first secondary antibody binds to the first primary antibody; and a second secondary antibody binds to the second primary antibody, wherein the first secondary antibody does not bind to the second primary antibody and the second secondary antibody does not bind to the first primary antibody; and wherein:

[1281] (i) the first second...

Claims

1. An in vitro coincidence assay for determining whether a checkpoint activator or inhibitor effectively inhibits or modulates a tumor response, the method comprising: at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent, and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor, and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binder and the second secondary binder are conjugated or fused to DNA sequences, wherein the DNA sequences are different and connected to form a circle and are amplified by rolling circle DNA and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binding agent is labeled with a FRET donor, and the second secondary binding agent is fused to an enzyme, the enzyme reacts with the conjugate to form an activated conjugate comprising a FRET acceptor and a substrate specific for the enzyme, the activated conjugate binding to an electron-rich moiety on a surface of a molecule adjacent to the enzyme; The method comprises: (a) contacting the isolated tumor cell sample with the at least two primary binding agents; (b) contacting the sample with the at least two secondary binding agents; (c) performing a washing step; (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; (e) contacting the sample with the checkpoint activator or inhibitor; (f) detecting any changes in the interactions between the secondary binding agents, wherein: a. In cases where the checkpoint molecule is an inhibitor: i. If the total score decreases between steps (d) and (f), then it indicates or predicts that the checkpoint inhibitor is effective; or ii. if the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint inhibitor is ineffective; or b. In the case where the checkpoint molecule is an activator: i. If the total score increases between steps (d) and (f), it indicates or predicts that the checkpoint activator is effective; or ii. If the total score does not change between steps (d) and (f), it indicates or predicts that the checkpoint activator is ineffective.

2. The method according to claim 1, wherein The checkpoint inhibitor is an anti-PD-1 binding agent, and the first primary binding agent binds to PD-1 and the second primary binding agent binds to PD-L1 or PD-L2.

3. The method according to claim 1, wherein The checkpoint inhibitor is an anti-CTLA-4 or CD28 binding agent, and the first primary binding agent binds to CTLA-4 or CD28, and the second primary binding agent binds to CD80 or CD86.

4. The method of claim 1, wherein the checkpoint inhibitor is an anti-MHC class I or II peptide binder, and the first primary binder binds to an MHC class I or II peptide, and the second primary binder binds to TCR, CD8, CD3, and combinations thereof.

5. The method according to claim 2, wherein: The anti-PD-1 binding agent is selected from the group consisting of nivolumab, pidilizumab, AMP-224, BMS-936559, ED 4736, MPDL3280A, MSB0010718C and pembrolizumab.

6. The method according to claim 5, wherein: The nivolumab is BMS-936558, DX 1106 or ONO-4538, the pembrolizumab is rambrolizumab or MK-3475, and the pidilizumab is CT-Q1 1 or ED-0680.

7. The method according to claim 6, wherein: The ED-0680 is AMP-514.

8. The method according to claim 5, wherein The BMS-936559 is MDX-1105.

9. The method according to claim 5, wherein: The MPDL3280A is RG7448.

10. The method according to claim 3, wherein: The anti-CTLA-4 binding agent is ipilimumab.

11. The method according to any one of claims 1 to 4, wherein: An increase of 0.5% to 5% in the total score between steps (d) and (f) indicates that the checkpoint activator will be effective.

12. The method according to any one of claims 1 to 4, wherein: An increase of 5% to 10% in the total score between steps (d) and (f) indicates that the checkpoint activator will be effective.

13. The method according to any one of claims 1 to 4, wherein: An increase of greater than 10% in the total score between steps (d) and (f) indicates that the checkpoint activator will be effective.

14. The method according to any one of claims 1 to 4, wherein: A decrease in the total score of 0.5% to 5% between steps (d) and (f) indicates that the checkpoint inhibitor will be effective.

15. The method according to any one of claims 1 to 4, wherein: A 5% to 10% decrease in the total score between steps (d) and (f) indicates that the checkpoint inhibitor will be effective.

16. The method according to any one of claims 1 to 4, wherein: A decrease of greater than 10% in the total score between steps (d) and (f) indicates that the checkpoint inhibitor will be effective.

17. The method according to any one of claims 1 to 4, wherein: The at least two primary binding agents bind to the first checkpoint target molecule or the second checkpoint target molecule in such a manner that the checkpoint inhibitor or activator binds to the first checkpoint target molecule or the second checkpoint target molecule simultaneously or sequentially.

18. The method of any one of claims 1 to 4, wherein the at least two primary binding agents do not inhibit binding of the check point inhibitor or activator to the first check point target molecule or the second check point target molecule.

19. Use of an in vitro coincidence assay according to any one of claims 1 to 4 for detecting whether a checkpoint inhibitor or activator will effectively inhibit or modulate a tumor response, in: a. In cases where the checkpoint molecule is an inhibitor: i. If the total score decreases between steps (d) and (f), it indicates or predicts that the checkpoint inhibitor is effective; or ii. if the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint inhibitor is ineffective; or b. In the case where the checkpoint molecule is an activator: i. If the total score increases between steps (d) and (f), it indicates or predicts that the checkpoint activator is effective; or ii. If the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint activator is ineffective.

20. A kit for use in an in vitro coincidence assay for detecting whether a checkpoint inhibitor or activator effectively inhibits or modulates a tumor response, the kit comprising: (a) at least two primary binders, wherein: a first primary binding agent binds to a first checkpoint target molecule on a first cell, and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent, and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA, and are bound by an external fluorescently labeled DNA probe, wherein the external fluorescently labeled DNA probe is complementary to the amplified DNA sequence; or (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; (c) Instructions for performing a method comprising: a. contacting the isolated tumor cell sample with the at least two primary binding agents; b. contacting the sample with the at least two secondary binding agents; c. performing a washing step; d. detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; e. contacting the sample with the checkpoint activator or inhibitor; f. detecting any changes in the interactions between said secondary binding agents, wherein: Where the checkpoint molecule is an inhibitor: i. If the total score decreases between steps (d) and (f), then it indicates or predicts that the checkpoint inhibitor is effective; or ii. if the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint inhibitor is ineffective; or Where the checkpoint molecule is an activator: iii. If the total score increases between steps (d) and (f), then it indicates or predicts that the checkpoint activator is effective; or iv. If the total score does not change between steps (d) and (f), then it indicates or predicts that the checkpoint activator is ineffective.

21. Use of the kit according to claim 20 in an in vitro coincidence assay for detecting whether a checkpoint inhibitor or activator effectively inhibits or modulates a tumor response, wherein: Where the checkpoint molecule is an inhibitor: (i) if the total score decreases between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is effective; or (ii) if the total score does not change between steps (d) and (f), indicating or predicting that the checkpoint inhibitor is ineffective; or Where the checkpoint molecule is an activator: (iii) if the total score increases between steps (d) and (f), indicating or predicting that the checkpoint activator is effective; or (iv) If the total score does not change between steps (d) and (f), then indicating or predicting that the checkpoint activator is ineffective.

22. A kit for use in an in vitro method of determining whether a therapy comprising a checkpoint activator or inhibitor is effective in a patient, the kit comprising: at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA, and are bound by an external fluorescently labeled DNA probe, wherein the external fluorescently labeled DNA probe is complementary to the amplified DNA sequence; or (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; Instructions for performing the following methods: (a) contacting an isolated tumor cell sample obtained from the patient prior to treatment comprising the checkpoint activator or inhibitor with the at least two primary binding agents; (b) contacting the sample with the at least two secondary binding agents; (c) performing a washing step; (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; (e) repeating steps (a) to (d) using an isolated tumor cell sample obtained from the patient during the treatment comprising the checkpoint activator or inhibitor in step (a); (f) comparing the total fluorescence fractions between the samples, wherein: a. Where the therapy comprises a checkpoint inhibitor: i. If the total score decreases, it indicates or predicts that the therapy is effective; or ii. if the total score remains unchanged, indicating or predicting that the therapy is ineffective; or b. Where the therapy comprises a checkpoint activator: i. If the total score increases, it indicates or predicts that the therapy is effective; or ii. If the total score does not change, it indicates or predicts that the therapy is ineffective.

23. An in vitro coincidence assay method for identifying whether a molecule of interest is a checkpoint activator or a checkpoint inhibitor, the method comprising: at least two primary binding agents, wherein a first primary binding agent binds to a first checkpoint target molecule on a first cell and a second primary binding agent binds to a second checkpoint target molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent, and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (iv) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (v) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (vi) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; The method comprises: (a) contacting the isolated tumor cell sample with the at least two primary binding agents; (b) contacting the sample with the at least two secondary binding agents; (c) performing a washing step; (d) detecting the interaction between the secondary binding agents by measuring the total fluorescence fraction; (e) contacting the sample with the molecule of interest; (f) detecting any changes in the interactions between the secondary binding agents, wherein: a. If the total score increases between steps (d) and (f), then the molecule is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or b. If the total score decreases between steps (d) and (f), then the molecule is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule; or c. If the total score does not change between steps (d) and (f), then the molecule is neither a checkpoint activator nor a checkpoint inhibitor for at least one of the first checkpoint target molecule and the second checkpoint target molecule.

24. The method according to claim 23, wherein An increase of 0.5% to 5% in the total score between steps (d) and (f) indicates that the molecule of interest is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

25. The method according to claim 23, wherein An increase of 5% to 10% in the total score between steps (d) and (f) indicates that the molecule of interest is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

26. The method according to claim 23, wherein An increase of greater than 10% in the total score between steps (d) and (f) indicates that the molecule of interest is a checkpoint activator of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

27. The method according to claim 23, wherein A decrease in the total score of 5% to 0.5% between steps (d) and (f) indicates that the molecule of interest is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

28. The method according to claim 23, wherein A decrease in the total score of 10% to 5% between steps (d) and (f) indicates that the molecule of interest is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

29. The method according to claim 23, wherein A decrease of greater than 10% in the total score between steps (d) and (f) indicates that the molecule of interest is a checkpoint inhibitor of at least one of the first checkpoint target molecule and the second checkpoint target molecule.

30. The method according to any one of claims 23 to 29, wherein The at least two primary binding agents bind to the first checkpoint target molecule or the second checkpoint target molecule in such a way that the molecule of interest binds to the first checkpoint target molecule or the second checkpoint target molecule simultaneously or sequentially.

31. The method according to any one of claims 23 to 29, wherein: The at least two primary binding agents do not inhibit binding of the molecule of interest to the first checkpoint target molecule or the second checkpoint target molecule.

32. A kit for use in an in vitro method of determining whether a patient with cancer will respond to an agent that blocks the PD-1:PD-L1 / PD-L2 pathway, the kit comprising: At least two primary binders, wherein a first primary binding agent binds to PD-1 on a first cell and a second primary binding agent binds to PD-L1 or PD-L2 on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; and Instructions for performing the following methods: a. contacting an isolated tumor cell sample obtained from said patient with said at least two primary binding agents; b. contacting the sample with the at least two secondary binding agents; c. performing a washing step; d. detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein: (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway; or (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an agent that blocks the PD-1:PD-L1 / PD-L2 pathway.

33. The kit according to claim 32, wherein The sample is a fixed tumor cell sample.

34. The kit according to any one of claims 32 to 33, wherein The instructions for performing the method further include instructions for performing the method on: (i) performing the method on a biological sample obtained from the patient prior to treatment to guide a decision on whether to treat with a single agent that blocks the PD-1:PD-L1 / PD-L2 pathway or with a combination therapy of an agent that blocks the PD-1:PD-L1 / PD-L2 pathway and at least one additional anti-tumor agent; and (ii) performing the method on at least one biological sample obtained from the patient during treatment to monitor the patient's response to the current treatment regimen and guide the decision of whether to select treatment with single-agent PD-1:PD-L1 / PD-L2 blockade therapy or combination therapy.

35. A kit for use in an in vitro method of determining whether a patient suffering from cancer will respond to an agent that blocks the CTLA-4 / CD28-CD80 / CD86 pathway, the kit comprising: at least two primary binding agents, wherein a first primary binding agent binds to CTLA-4 or CD28 on a first cell and a second primary binding agent binds to CD80 or CD86 on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; and Instructions for performing the following methods: a. contacting an isolated tumor cell sample obtained from said patient with said at least two primary binding agents; b. contacting the sample with the at least two secondary binding agents; c. performing a washing step; d. detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein: (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the CTLA-4 / CD28-CD80 / CD86 pathway; or (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an agent that blocks the CTLA-4 / CD28-CD80 / CD86 pathway.

36. The kit of claim 35, wherein the sample is a fixed tumor cell sample.

37. The kit of any one of claims 35 to 36, wherein the instructions for performing the method further comprise instructions for performing the method: (i) performing the method on a biological sample obtained from the patient prior to treatment to guide a decision on whether to treat with a single agent that blocks the CTLA-4 / CD28-CD80 / CD86 pathway or with a combination therapy that blocks the CTLA-4 / CD28-CD80 / CD86 pathway and at least one additional anti-tumor agent; and (ii) performing the method on at least one biological sample obtained from the patient during treatment to monitor the patient's response to the current treatment regimen and guide the decision of whether to select treatment with single-agent CTLA-4 / CD28-CD80 / CD86 blockade therapy or combination therapy.

38. A kit for use in an in vitro method of determining whether a patient suffering from cancer will respond to an agent that blocks the MHC class I / II-TCR / CD8 / CD3 pathway, the kit comprising: at least two primary binding agents, wherein a first primary binding agent binds to an MHC class I or II peptide on a first cell and a second primary binding agent binds to a TCR, CD8, CD3, and combinations thereof on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically distinct; and at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent; and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent and the second secondary binding agent are conjugated or fused to DNA sequences, wherein the DNA sequences are different and are linked to form a circle and amplified by rolling circle DNA, and are bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is fused to an enzyme, the enzyme reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich moiety on a molecular surface adjacent to the enzyme; and Instructions for performing the following methods: a. contacting an isolated tumor cell sample obtained from said patient with said at least two primary binding agents; b. contacting the sample with the at least two secondary binding agents; c. performing a washing step; d. detecting the interaction between the secondary binding agents by measuring the fraction of the total fluorescent signal, wherein: (i) if the total score is less than or equal to a threshold score, then the total score indicates or predicts that the patient will not respond to treatment with an agent that blocks the MHC class I / II-TCR / CD8 / CD3 pathway; or (ii) if the total score is greater than a threshold score, then the total score indicates or predicts that the patient will respond to treatment with an agent that blocks the MHC class I / II-TCR / CD8 / CD3 pathway.

39. The kit according to claim 37, wherein The sample is a fixed tumor cell sample.

40. The kit according to any one of claims 38 to 39, wherein The instructions for performing the method further include instructions for performing the method on: (i) performing the method on a biological sample obtained from the patient prior to treatment to guide a decision on whether to treat with a single agent that blocks the MHC class I / II-TCR / CD8 / CD3 pathway or with a combination therapy of an agent that blocks the MHC class I / II-TCR / CD8 / CD3 pathway and at least one additional anti-tumor agent; and (ii) performing the method on at least one biological sample obtained from the patient during treatment to monitor the patient's response to the current treatment regimen and guide the decision of whether to select treatment with single-agent MHC class I / II-TCR / CD8 / CD3 blockade therapy or combination therapy.

41. An in vitro coincidence assay for detecting an interaction between a first checkpoint target molecule expressed on a first cell and a second checkpoint target molecule expressed on a second cell, the method comprising: a first fusion protein and a second fusion protein, wherein each fusion protein comprises a detection domain, a recognition domain, and a linker domain; The detection domain comprises a DNA binding domain and is capable of cooperating with another detection domain to bind to a cognate specific nucleotide sequence; The recognition domain is capable of binding to a target molecule; The linker domain is fused to the detection domain at one end and to the recognition domain at the other end; The detection domain, recognition domain, and linker domain are heterologous to each other; The method comprises the following steps: (i) contacting a sample with the first and fusion proteins; (ii) incubating to allow binding; (iii) removal of unbound fusion protein; (iv) contacting the sample with a nucleic acid comprising the homology-specific nucleotide sequence; (v) incubating to allow heterotrimer binding of said nucleic acid; and (vi) detecting nucleic acid bound to the sample; Wherein, if the nucleic acid is detected in step (vi), this indicates that both target molecules are present in the sample at the same time.

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