Method for detecting the expression or clustering of cell surface moieties

Abstract

The present disclosure relates to methods for detecting and / or quantifying the expression of at least a first cell surface moiety and a second cell surface moiety in a patient sample, and for detecting and / or quantifying the clustering of at least a first cell surface moiety with a second cell surface moiety in the sample, wherein the sample is exposed to molecules having binding specificity for at least the first and second cell surface moieties. The disclosure further relates to methods for predicting a subject's responsiveness to such binding molecules, methods for determining the efficacy of such binding molecules, methods for confirming the mode of action of such binding molecules, methods for treating a subject, and methods for screening one or more test agents for the ability to induce clustering of a first cell surface moiety with a second cell surface moiety.

Description

[Technical Field]

[0001] This disclosure preferably relates to a method for detecting and / or quantifying the expression or clustering of at least a first cell surface portion and a second cell surface portion in a patient's tumor sample. In certain embodiments, the method of this disclosure can be used to predict whether a patient is likely to benefit from treatment with a binder that binds to both cell surface portions, or to confirm the mode of action of such a binder. [Background technology]

[0002] The development of therapeutic antibodies for cancer treatment has increased rapidly in recent years. Diagnostic assays are used to assess whether a particular patient will benefit from treatment with a specific drug for various types of cancer, and the results can be predicted to be likely to be safe and / or effective. One category of diagnostic assays that has been used in conjunction with biological agents or macromolecule therapeutics involves testing the expression levels of antigens targeted by the biological agent in a patient's tissue sample. For example, a tissue biopsy may be taken from the patient's tumor and subjected to a quantitative assay. Examples of such quantitative assays include immunohistochemistry (IHC), dual insights hybridization assays, chromogenic insights hybridization (CISH) assays, and fluorescence insights hybridization (FISH) assays.

[0003] IHC (Intracellular Health Concentration) was a standard testing method for evaluating HER2 expression, for example, in breast cancer. IHC testing utilizes specific monoclonal or polyclonal antibodies that bind to the HER2 protein on the cell surface. The addition of a secondary tagged antibody with reporter function, followed by an enzymatic reaction, yields a signal proportional to the amount of HER2 protein present. However, despite guidelines for grading and scoring IHC expression levels, inter-laboratory variability cannot be completely prevented.

[0004] FISH, CISH, and silver-enhanced Insights hybridization assays quantify the number of gene copies per cell using single or dual probe techniques. FISH has become a widely accepted platform, for example, in HER2 trials. However, FISH assays are expensive, labor-intensive, and require fluorescence microscopy and advanced training. Bright-field Insights hybridization assays, such as CISH and silver-enhanced Insights hybridization, do not require fluorescence microscopy and are less expensive.

[0005] Another development is an assay validated as a method for measuring total HER2, HER2 homodimer, or p95HER2 expression in breast cancer, which is a proximity-based assay designed to quantify protein expression, dimerization, and protein-protein interactions (detailed in Diagn Mol Pathol 2009;18:11-21;Shi et al.). [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] To date, the art has lacked a robust method to demonstrate the ability of two or more antigens to cluster in the presence of multispecific agents, such antigens not typically associate under somatic conditions. Here, we demonstrate such an assay and exemplary applications thereof. [Means for solving the problem]

[0007] The inventors have developed an assay for detecting and quantifying the expression levels of CD137, a cell surface parenchyma expressed on T cells, and PD-L1, a cell surface parenchyma expressed on tumor cells. This makes it possible to predict whether a particular patient is likely to respond to and benefit from a treatment that binds to these two cell surface parenchyma. In certain embodiments, other applicable methods may be used, such as the assay used herein, which is based on measuring the signal produced when the two cell surface parenchyma are present in the same sample. In this context, the signal may be the presence or absence of a signal. In certain embodiments, both such readouts provide information about the expression levels of the cell surface parenchyma. The method can also be used to detect and / or quantify the expression levels of any two or more cell surface parenchyma that can be bound by certain drugs, such as multispecific agents, such as bispecific or trispecific antibodies.

[0008] The inventors have further developed assays used for the detection and quantification of CD137 and the clustering of CD137 with PD-L1. CD137 and PD-L1 do not form homogeneous receptor-ligand pairs, and they do not spontaneously cluster or form direct protein-protein interactions. However, when targeted by a drug that binds simultaneously to both cell surface regions, such as a multispecific drug like a bispecific or triplicate antibody, these two cell surface regions are brought into close proximity to each other, thereby producing a detectable signal. Similarly, when targeted by a multivalent drug that binds to at least two of the same cell surface regions, such as a monospecific bivalent drug like a monospecific antibody, or a multispecific drug like a bispecific or triplicate antibody, at least two cell surface regions are brought into close proximity to each other, thereby producing a detectable signal.

[0009] In certain embodiments, the disclosure is based on the therapeutic use of multispecific agents, such as bispecific or trispecific antibodies, that simultaneously bind to two or more target antigens on the cell surface of cells from tumor cells and / or the immune system. The multispecific agent, together with it, induces the clustering of two or more target antigens. Thus, the clustering of two or more antigens occurs only in the presence of the multispecific agent, or at an increased level compared to the absence of the multispecific agent.

[0010] In certain embodiments, this allows for the assessment of whether the drug being administered to a patient actually binds simultaneously to two cell surface regions and exhibits its expected mode of action. In certain embodiments, other applicable methods can be used, such as the assay used herein, which are based on measuring the signal produced when two cell surface regions are in close proximity. In this context, the signal may be the presence or absence of the assay's reporter or feature. In certain embodiments, both such readouts (presence or loss of the reporter) provide information about the proximity of the cell surface regions. This method can also be used to detect and / or quantify the clustering of any two or more cell surface regions that can simultaneously bind to a particular drug, such as a multispecific drug, such as a bispecific or trispecific antibody.

[0011] In a particular embodiment, the present disclosure is a method for detecting and / or quantifying the presence of clustering of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being The method involves bringing a sample in which first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions, into contact with a first binding molecule that specifically binds to the first cell surface portion and a second binding molecule that specifically binds to the second cell surface portion, wherein, as long as the first and second cell surface portions are not in close proximity to each other, at least one of the first binding molecule and the second binding molecule contains an undetectable molecular tag, and The present invention relates to a method for detecting the presence or absence of molecular tags and for detecting the presence of clustering of a first cell surface portion and a second cell surface portion in a sample.

[0012] This disclosure also relates to a method for detecting and / or quantifying the expression of at least two cell surface parenchyma in a sample, including a first cell surface parenchyma and a second cell surface parenchyma, wherein the method is The method involves contacting a tumor biopsy sample derived from a subject with cancer with at least one binding molecule that detects a first cell surface portion and at least one binding molecule that detects a second cell surface portion, A binding molecule that detects a first cell surface region and a binding molecule that detects a second cell surface region, including a molecular tag, are brought into contact. and The present invention relates to a method for detecting the expression of a first cell surface region and a second cell surface region in a sample by detecting and / or quantifying the presence or absence of a molecular tag.

[0013] This disclosure further relates to a method for predicting the response of a subject, in particular a cancer patient, to a drug or a plurality of drugs that bind to a first cell surface portion and a second cell surface portion, in particular the portion expressed on immune effector cells and the portion expressed on tumor cells, the method being - To detect and / or quantify the expression levels of a first cell surface region and a second cell surface region in a biological sample derived from the target, - To determine whether the expression levels of the first and second cell surface regions in the target sample are above or below the threshold level, and The present invention relates to a method that includes predicting that if the expression levels of a first cell surface region and a second cell surface region in a target sample are above a threshold level, the target is likely to respond to a drug or a combination of drugs that bind to the first cell surface region and the second cell surface region.

[0014] This disclosure further relates to a method for treating a subject with cancer who is in need of treatment, the method being - Predicting the response of a subject, particularly a subject with cancer, to a drug or multiple drugs that bind to the first and second cell surface portions described herein, and The present invention relates to a method comprising administering a drug or a group of drugs that bind to a first cell surface portion and a second cell surface portion to a target that is likely to respond.

[0015] The Disclosure further relates to a method for determining the efficacy of a drug, wherein the drug comprises a binding molecule having at least a binding domain that specifically binds to a first cell surface portion and a binding domain that specifically binds to a second cell surface portion, and the method comprises detecting and / or quantifying the clustering of the first cell surface portion with the second cell surface portion in a biological sample of a subject being treated with the drug, as described herein.

[0016] This disclosure further relates to a method for confirming the mode of action of a drug, wherein the drug comprises a binding molecule having at least a binding domain that specifically binds to a first cell surface portion and a binding domain that specifically binds to a second cell surface portion, and the method comprises detecting and / or quantifying the clustering of the first cell surface portion with the second cell surface portion in a biological sample of a subject being treated with the drug, as described herein.

[0017] This disclosure further relates to a method for treating subjects in need of treatment, in particular subjects having cancer, the method being - A drug that binds to the first and second cell surface regions to treat the target that requires treatment. -The present invention relates to a method, including, as described herein, of analyzing the efficacy or mode of action of a drug.

[0018] The present disclosure further provides a method for screening one or more test agents for their ability to induce clustering of a first cell surface moiety with a second cell surface moiety, the method comprising: - contacting one or more test cell cultures with a test agent, wherein the test cell cultures comprise cells expressing a first cell surface moiety and cells expressing a second cell surface moiety, detecting the level of clustering of the first and second cell surface moieties as described herein, and comparing the level of clustering with the level of clustering detected for clustering in a control cell culture that has not been contacted with the test agent or a reference agent, wherein the control cell culture comprises the first cell surface moiety and the second cell surface moiety. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1] Schematic diagram of the concept of the VeraTag® assay used according to certain embodiments of the present disclosure. The VeraTag® assay format shown in this figure utilizes two primary antibodies that bind to one or two specific antigens, one of the primary antibodies containing a tag and the other containing a cleavage-inducing moiety (scissors symbol). Upon photoactivation, when the cleavage-inducing moiety is released from one of the antibodies, it induces cleavage of the tag from the other antibody. The signal produced by the tag is then measured by capillary electrophoresis (CE). However, the disclosure presented herein is not limited to the assay format presented in this figure. [Figure 2]This is a schematic diagram of an example of a VeraTag® assay using a primary antibody. In this diagram, the first and second cell surface portions are different cell surface portions. However, the disclosure also includes embodiments in which the first and second cell surface portions are the same. A) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, wherein the first binding molecule comprises a first molecular tag bound to the first binding molecule via a cleavable linker, the second binding molecule comprises a cleavage-inducing portion (scissors symbol), the third binding molecule comprises a second molecular tag bound to the third binding molecule via a cleavable linker, and the fourth binding molecule comprises a cleavage-inducing portion (scissors symbol). B) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, wherein the second binding molecule includes a first molecular tag bound to the second binding molecule via a cleavable linker, the first binding molecule includes a cleavage-inducing portion (scissors symbol), the fourth binding molecule includes a second molecular tag bound to the fourth binding molecule via a cleavable linker, and the third binding molecule includes a cleavage-inducing portion (scissors symbol). C) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, wherein the first binding molecule includes a first molecular tag bound to the first binding molecule via a cleavable linker, the second binding molecule includes a cleavage-inducing portion (scissors symbol), the fourth binding molecule includes a second molecular tag bound to the fourth binding molecule via a cleavable linker, and the third binding molecule includes a cleavage-inducing portion (scissors symbol).D) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, wherein the second binding molecule includes a first molecular tag bound to the second binding molecule via a cleavable linker, the first binding molecule includes a cleavage-inducing portion (scissors symbol), the third binding molecule includes a second molecular tag bound to the third binding molecule via a cleavable linker, and the fourth binding molecule includes a cleavage-inducing portion (scissors symbol). [Figure 3]This is a schematic diagram of an example of a VeraTag® assay using two primary and secondary antibodies for each target. In this diagram, the first and second cell surface portions are different cell surface portions. However, the disclosure also includes embodiments in which the first and second cell surface portions are the same. A) First and second binding molecules that specifically bind to the first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to the second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first and third binding molecules each contain first and second molecular tags bound to them via a cleavable linker, the fifth binding molecule binds to the second binding molecule and contains a cleavage-inducing portion (scissors symbol), and the sixth binding molecule binds to the fourth binding molecule and contains a cleavage-inducing portion (scissors symbol). B) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second and fourth binding molecules each contain first and second molecular tags bound to them via a cleavable linker, the fifth binding molecule binds to the first binding molecule and contains a cleavage-inducing portion (scissors symbol), and the sixth binding molecule binds to the third binding molecule and contains a cleavage-inducing portion (scissors symbol). C) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first and third binding molecules include cleavage-inducing portions (scissors symbol), the fifth binding molecule includes a first molecular tag that binds to the second binding molecule and is bound to the fifth binding molecule via a cleavable linker, and the sixth binding molecule includes a second molecular tag that binds to the fourth binding molecule and is bound to the sixth binding molecule via a cleavable linker.D) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second and fourth binding molecules include a cleavage-inducing portion (scissors symbol), the fifth binding molecule includes a first molecular tag that binds to the first binding molecule and is bound to the fifth binding molecule via a cleavable linker, and the sixth binding molecule includes a second molecular tag that binds to the third binding molecule and is bound to the sixth binding molecule via a cleavable linker. E) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first binding molecule includes a first molecular tag bound thereto via a cleavable linker, the fourth binding molecule includes a second molecular tag bound thereto via a cleavable linker, the fifth binding molecule is bound to the second binding molecule and includes a cleavage-inducing portion (scissors symbol), and the sixth binding molecule is bound to the third binding molecule and includes a cleavage-inducing portion (scissors symbol). F) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first binding molecule includes a first molecular tag bound thereto via a cleavable linker, the third binding molecule includes a cleavage-inducing portion (scissors symbol), the fifth binding molecule is bound to the second binding molecule and includes a cleavage-inducing portion (scissors symbol), and the sixth binding molecule is bound to the fourth binding molecule and includes a second molecular tag bound thereto via a cleavable linker.G) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first binding molecule includes a first molecular tag bound thereto via a cleavable linker, the fourth binding molecule includes a cleavage-inducing portion (scissors symbol), the fifth binding molecule is bound to the second binding molecule and includes a cleavage-inducing portion (scissors symbol), and the sixth binding molecule is bound to the third binding molecule and includes a second molecular tag bound thereto via a cleavable linker. H) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second binding molecule includes a first molecular tag bound thereto via a cleavable linker, the third binding molecule includes a second molecular tag bound thereto via a cleavable linker, the fifth binding molecule binds to the first binding molecule and includes a cleavage-inducing portion (scissors symbol), and the sixth binding molecule binds to the fourth binding molecule and includes a cleavage-inducing portion (scissors symbol). I) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second binding molecule includes a first molecular tag bound thereto via a cleavable linker, the third binding molecule includes a cleavage-inducing portion (scissors symbol), the fifth binding molecule is bound to the first binding molecule and includes a cleavage-inducing portion (scissors symbol), and the sixth binding molecule is bound to the fourth binding molecule and includes a second molecular tag bound thereto via a cleavable linker.J) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second binding molecule includes a first molecular tag bound thereto via a cleavable linker, the fourth binding molecule includes a cleavage-inducing portion (scissors symbol), the fifth binding molecule is bound to the first binding molecule and includes a cleavage-inducing portion (scissors symbol), and the sixth binding molecule is bound to the third binding molecule and includes a second molecular tag bound thereto via a cleavable linker. K) A first and second binding molecule that specifically binds to a first portion expressed on the cell surface, a third and fourth binding molecule that specifically binds to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first binding molecule includes a cleavage-inducing portion (scissors symbol), the third binding molecule includes a second molecular tag bound to it via a cleavable linker, the fifth binding molecule includes a first molecular tag bound to the second binding molecule via a cleavable linker, and the sixth binding molecule includes a cleavage-inducing portion (scissors symbol) bound to the fourth binding molecule. L) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first binding molecule includes a cleavage-inducing portion (scissors symbol), the fourth binding molecule includes a second molecular tag bound to it via a cleavable linker, the fifth binding molecule includes a first molecular tag bound to the second binding molecule via a cleavable linker, and the sixth binding molecule includes a cleavage-inducing portion (scissors symbol) bound to the third binding molecule.M) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the first and fourth binding molecules include a cleavage-inducing portion (scissors symbol), the fifth binding molecule includes a first molecular tag bound to the second binding molecule via a cleavable linker, and the sixth binding molecule includes a second molecular tag bound to the third binding molecule via a cleavable linker. N) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second binding molecule includes a cleavage-inducing portion (scissors symbol), the third binding molecule includes a second molecular tag bound to it via a cleavable linker, the fifth binding molecule includes a first molecular tag bound to the first binding molecule via a cleavable linker, and the sixth binding molecule includes a cleavage-inducing portion (scissors symbol) bound to the fourth binding molecule. O) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second binding molecule includes a cleavage-inducing portion (scissors symbol), the fourth binding molecule includes a second molecular tag bound to it via a cleavable linker, the fifth binding molecule includes a first molecular tag bound to the first binding molecule via a cleavable linker, and the sixth binding molecule includes a cleavage-inducing portion (scissors symbol) bound to the third binding molecule. P) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second and third binding molecules include cleavage-inducing portions (scissors symbol), the fifth binding molecule includes a first molecular tag bound to the first binding molecule via a cleavable linker, and the sixth binding molecule includes a second molecular tag bound to the fourth binding molecule via a cleavable linker. [Figure 4]This is a schematic diagram of an example of a VeraTag® assay using two primary antibodies and two secondary antibodies for each target. In this diagram, the first and second cell surface portions are different cell surface portions. However, this disclosure also includes embodiments in which the first and second cell surface portions are the same. A) First and second binding molecules that specifically bind to the first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to the second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, wherein the fifth binding molecule binds to the first binding molecule and includes a first molecular tag bound thereto via a cleavable linker, the seventh binding molecule binds to the second binding molecule and includes a cleavage-inducing portion (scissors symbol), the sixth binding molecule binds to the third binding molecule and includes a second molecular tag bound thereto via a cleavable linker, and the eighth binding molecule binds to the fourth binding molecule and includes a cleavage-inducing portion (scissors symbol). B) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, wherein the fifth binding molecule binds to the first binding molecule and includes a cleavage-inducing portion (scissors symbol), the seventh binding molecule binds to the second binding molecule and includes a first molecular tag bound to it via a cleavable linker, the sixth binding molecule binds to the third binding molecule and includes a cleavage-inducing portion (scissors symbol), and the eighth binding molecule binds to the fourth binding molecule and includes a second molecular tag bound to it via a cleavable linker.C) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, wherein the fifth binding molecule includes a first molecular tag bound to the first binding molecule via a cleavable linker, the seventh binding molecule includes a cleavage-inducing portion (scissors symbol) bound to the second binding molecule, the sixth binding molecule includes a cleavage-inducing portion (scissors symbol) bound to the third binding molecule, and the eighth binding molecule includes a second molecular tag bound to the fourth binding molecule via a cleavable linker. D) First and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, wherein the fifth binding molecule binds to the first binding molecule and includes a cleavage-inducing portion (scissors symbol), the seventh binding molecule binds to the second binding molecule and includes a first molecular tag bound to it via a cleavable linker, the sixth binding molecule binds to the third binding molecule and includes a second molecular tag bound to it via a cleavable linker, and the eighth binding molecule binds to the fourth binding molecule and includes a cleavage-inducing portion (scissors symbol). [Figure 5]This is a schematic diagram of an example of a VeraTag® assay format using one primary antibody for each target and one secondary antibody against one of the primary antibodies. This format uses DTT-mediated release of molecular tags. In this diagram, the first and second cell surface portions are different cell surface portions. However, the disclosure also includes embodiments in which the first and second cell surface portions are the same. A) A first binding molecule that specifically binds to the first portion expressed on the cell surface, a second binding molecule that specifically binds to the second portion expressed on the cell surface, and a third binding molecule that binds to the first binding molecule, wherein the second binding molecule contains the first molecular tag bound to it via a cleavable linker, and the third binding molecule contains the second molecular tag bound to it via a cleavable linker. B) A first binding molecule that specifically binds to a first portion expressed on the cell surface, a second binding molecule that specifically binds to a second portion expressed on the cell surface, and a third binding molecule that binds to the second binding molecule, wherein the first binding molecule includes a first molecular tag bound thereto via a cleavable linker, and the third binding molecule includes a second molecular tag bound thereto via a cleavable linker. [Figure 6]This is a schematic diagram of an example of a VeraTag® assay using one primary antibody for each target to detect and / or quantify target clustering. In this diagram, the first and second cell surface regions are different cell surface regions expressed on different cells. However, this disclosure also includes embodiments in which the first and second cell surface regions are present on the same cell. Also in this diagram, the molecule having binding specificity to the first and second cell surface regions is a bivalent bispecific antibody. However, this disclosure also includes situations in which the molecule having binding specificity to the cell surface region is a multivalent bispecific antibody or a multispecific antibody, such as a triplicate or quadruplicate antibody. A) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby causing the first and second cell surface portions to be in close proximity to each other; a first binding molecule that specifically binds to the first cell surface portion; and a second binding molecule that specifically binds to the second cell surface portion, wherein the first binding molecule includes a molecular tag attached thereto via a cleavable linker, and the second binding molecule includes a cleavage-inducing portion (scissors symbol). B) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby causing the first and second cell surface portions to be in close proximity to each other; a first binding molecule that specifically binds to the first cell surface portion; and a second binding molecule that specifically binds to the second cell surface portion, wherein the second binding molecule includes a molecular tag attached thereto via a cleavable linker, and the first binding molecule includes a cleavage-inducing portion. [Figure 7]This is a schematic diagram of an example of a VeraTag® assay form using one primary antibody for each target and a secondary antibody against one of the primary antibodies for detecting and / or quantifying target clustering. In this diagram, the first and second cell surface regions are different cell surface regions expressed on different cells. However, this disclosure also includes embodiments in which the first and second cell surface regions are present on the same cell. Also in this diagram, the molecule having binding specificity to the first and second cell surface regions is a bivalent bispecific antibody. However, this disclosure also includes situations in which the molecule having binding specificity to the cell surface region is a multivalent bispecific antibody or a multispecific antibody, such as a triplicate or quadruplicate antibody. A) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby causing the first and second cell surface portions to be in close proximity to each other; a first binding molecule that specifically binds to the first cell surface portion; a second binding molecule that specifically binds to the second cell surface portion; and a third binding molecule, wherein the first binding molecule includes a molecular tag attached thereto via a cleavable linker, and the third binding molecule binds to the second binding molecule and includes a cleavage-inducing portion (scissors symbol). B) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby causing the first and second cell surface portions to be in close proximity to each other; a first binding molecule that specifically binds to the first cell surface portion; a second binding molecule that specifically binds to the second cell surface portion; and a third binding molecule, wherein the second binding molecule includes a molecular tag attached thereto via a cleavable linker, and the third binding molecule binds to the first binding molecule and includes a cleavage-inducing portion (scissors symbol). C) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby bringing the first and second cell surface portions into close proximity; a first binding molecule that specifically binds to the first cell surface portion; a second binding molecule that specifically binds to the second cell surface portion; and a third binding molecule, wherein the first binding molecule includes a cleavage-inducing portion (scissors symbol), and the third binding molecule includes a molecular tag that binds to the second binding molecule and is attached to it via a cleavable linker.D) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby causing the first and second cell surface portions to be in close proximity to each other; a first binding molecule that specifically binds to the first cell surface portion; a second binding molecule that specifically binds to the second cell surface portion; and a third binding molecule, wherein the second binding molecule includes a cleavage-inducing portion (scissors symbol), and the third binding molecule includes a molecular tag that binds to the first binding molecule and is attached to it via a cleavable linker. [Figure 8]This is a schematic diagram of the form of a VeraTag® assay using one primary antibody and two secondary antibodies against the primary antibody for each target to detect and / or quantify target clustering. In this diagram, the first and second cell surface regions are different cell surface regions expressed on different cells. However, this disclosure also extends to locations where the first and second cell surface regions reside on the same cell. Also in this diagram, the molecule having binding specificity to the first and second cell surface regions is a bivalent bispecific antibody. However, this disclosure also includes situations where the molecule having binding specificity to the cell surface region is a multivalent bispecific antibody or a multispecific antibody, such as a triplicate or quadruplicate antibody. A) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby bringing the first and second cell surface portions into close proximity; a first binding molecule that specifically binds to the first cell surface portion; a second binding molecule that specifically binds to the second cell surface portion; a third binding molecule; and a fourth binding molecule, wherein the third binding molecule binds to the first binding molecule and includes a molecular tag attached thereto via a cleavable linker; and the fourth binding molecule binds to the second binding molecule and includes a cleavage-inducing portion (scissors symbol). B) A bispecific antibody (100) that binds to a first cell surface portion and a second cell surface portion, thereby bringing the first and second cell surface portions into close proximity; a first binding molecule that specifically binds to the first cell surface portion; a second binding molecule that specifically binds to the second cell surface portion; a third binding molecule; and a fourth binding molecule, wherein the third binding molecule is bound to the first binding molecule and includes a cleavage-inducing portion (scissors symbol); and the fourth binding molecule is bound to the second binding molecule and includes a molecular tag attached thereto via a cleavable linker. [Figure 9]This figure shows the PD-L1 expression level in relative peak area (RPA) as measured using the VeraTag® assay. Sample A: Cell pellet prepared by incubation with an anti-CD137 positive control antibody; Sample B: Cell pellet prepared by incubation with a bispecific antibody that binds to CD137 and PD-L1; Sample C: Cell pellet prepared by incubation with a negative control antibody that binds to RSV. [Figure 10] This figure shows the CD137 expression level in relative peak area (RPA) as measured using the VeraTag® assay. Left: Incubation with anti-CD137 assay antibody BBK2, Right: Incubation with anti-CD137 assay antibody M127. Sample A: Cell pellet prepared by incubation with anti-CD137 positive control antibody, Sample B: Cell pellet prepared by incubation with bispecific antibody binding to CD137 and PD-L1, Sample C: Cell pellet prepared by incubation with negative control antibody binding to RSV. [Figure 11] This graph shows the clustering of CD137 cells in relative peak area (RPA) as measured using the VeraTag® assay. Left: Incubation with anti-CD137 assay antibody BBK2; Right: Incubation with anti-CD137 assay antibody M127. Sample A: Cell pellet prepared by incubation with a negative control antibody that binds to RSV; Sample B: Cell pellet prepared by incubation with a bispecific antibody that binds to CD137 and PD-L1; Sample C: Cell pellet prepared by incubation with an anti-CD137 positive control antibody. [Figure 12]This figure shows the PD-L1-CD137 clustering in relative peak area (RPA) as measured using the VeraTag® assay. Left: Incubation with anti-CD137 assay antibody BBK2; Right: Incubation with anti-CD137 assay antibody M127. Sample A: Cell pellet prepared by incubation with a negative control antibody that binds to RSV; Sample B: Cell pellet prepared by incubation with a bispecific antibody that binds to CD137 and PD-L1; Sample C: Cell pellet prepared by incubation with an anti-CD137 positive control antibody. The level of clustering in this assay is compared to the level measured in an isotype-controlled experiment (ITC). [Figure 13] A - The assay described herein for detecting the expression of a receptor (antigen 1) present on a cell membrane; B - The proximity assay described herein for detecting clustering of receptors (antigen 1) on the same cell; and C - The proximity assay described herein for detecting clustering of receptors (antigen 1) on one cell having a receptor (antigen 2) on another cell, thereby forming an immunological synapse. [Modes for carrying out the invention]

[0020] In one embodiment, the disclosure provides a method for detecting and / or quantifying the expression of at least a first cell surface parenchyma and a second cell surface parenchyma in a sample, the method comprising detecting signals produced when the first and second cell surface parenchyma are expressed in the same sample. In a particular embodiment, preferably, two different signals are produced so that the expression of each cell surface parenchyma can be quantified. Alternatively, in a particular embodiment, the signal-producing parenchyma are selected such that the combined signal provides information regarding the expression level of each cell surface parenchyma.

[0021] The signal can be produced in a variety of ways, including, but not limited to, providing different fluorescent molecular tags to the first and second cell surface regions, preferably different chromogenic molecular tags to the first and second cell surface regions, preferably different radioactive molecular tags to the first and second cell surface regions, and preferably different isotopically pure metal chelating molecular tags to the first and second cell surface regions. Quenching is another method that can be used. In certain embodiments, different fluorophores are preferably used for each cell surface region to allow measurement of the decrease in fluorescence intensity of one fluorophore or signal from the signal-releasing agent caused by interaction with a second quencher.

[0022] The methods described herein can be carried out in different forms. In one form, the herein is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, wherein the method is a) Contacting the sample with at least one binding molecule for detecting a first cell surface region and at least one binding molecule for detecting a second cell surface region, The at least one binding molecule for detecting a first cell surface region and the at least one binding molecule for detecting a second cell surface region each include a molecular tag, and optionally, the molecular tag bound to the binding molecule for detecting the first cell surface region is different from the molecular tag bound to the binding molecule for detecting the second cell surface region, and b) A method is provided which includes detecting the presence or absence of a molecular tag, or measuring its amount, to detect and / or quantify the expression of a first cell surface portion and a second cell surface portion in a sample.

[0023] In another form, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface parenchyma in a sample, including a first cell surface parenchyma and a second cell surface parenchyma, the method being a) Contacting the sample with at least two binding molecules for detecting a first cell surface region and at least two binding molecules for detecting a second cell surface region, Preferably, one of the binding molecules for detecting a first cell surface region and one of the binding molecules for detecting a second cell surface region include a molecular tag bound thereto via a cleavable linker, and optionally, another of the binding molecules for detecting a first cell surface region and another of the binding molecules for detecting a second cell surface region include a cleavage-inducing portion, b) Optionally, induce cleavage of molecular tags, and c) A method is provided which includes detecting the presence or absence of a molecular tag, or measuring its amount, to detect and / or quantify the expression of a first cell surface portion and a second cell surface portion in a sample.

[0024] In certain embodiments, the methods according to the present disclosure may be used to measure the co-expression of at least two different cell surface regions in a single sample, preferably a patient sample. Therefore, this is particularly useful for predicting a patient's, preferably cancer patient's, response to treatment with agents or multiple agents that bind to at least two different cell surface regions. Examples of such agents include, for example, multispecific antibodies.

[0025] In certain embodiments, the methods used in this disclosure include the VeraTag® assay. The VeraTag® assay is well known in the art and is described, for example, in WO2017 / 161030 and the references cited therein, which are incorporated herein in their entirety.

[0026] The VeraTag® assay can be performed in different forms. One form is a proximity assay using two primary binding molecules for each target region, where one primary binding molecule to each target region contains a molecular tag and the other contains a cleavage-inducing region.

[0027] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a1) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, A first binding molecule includes a first molecular tag bonded thereto via a cleavable linker, a second binding molecule includes a cleavage-inducing portion, a third binding molecule includes a second molecular tag bonded thereto via a cleavable linker, and a fourth binding molecule includes a cleavage-inducing portion, by contact, or a2) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, A second binding molecule includes a first molecular tag bonded to it via a cleavable linker, the first binding molecule includes a cleavage-inducing portion, a fourth binding molecule includes a second molecular tag bonded to it via a cleavable linker, and a third binding molecule includes a cleavage-inducing portion, by contact, or a3) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, A first binding molecule includes a first molecular tag bonded thereto via a cleavable linker, a second binding molecule includes a cleavage-inducing portion, a fourth binding molecule includes a second molecular tag bonded thereto via a cleavable linker, and a third binding molecule includes a cleavage-inducing portion, by contact, or a4) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, A second binding molecule includes a first molecular tag bonded to it via a cleavable linker, the first binding molecule includes a cleavage-inducing moiety, a third binding molecule includes a second molecular tag bonded to it via a cleavable linker, and a fourth binding molecule includes a cleavage-inducing moiety. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of the first and second molecular tags, and c) A method is provided which includes detecting the presence or absence of released first and second molecular tags to detect and / or quantify the expression of the first and second cell surface regions in a sample.

[0028] Another form is a proximity assay that uses two primary binding molecules for each target site, and a secondary binding molecule for one of the primary binding molecules.

[0029] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a1) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, The first and third binding molecules each include first and second molecular tags bonded to them via a cleavable linker, the fifth binding molecule is bonded to the second binding molecule and includes a cleavage-inducing portion, and the sixth binding molecule is bonded to the fourth binding molecule and includes a cleavage-inducing portion, by contact, or a2) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, The second and fourth binding molecules each include first and second molecular tags bonded to them via a cleavable linker, the fifth binding molecule is bonded to the first binding molecule and includes a cleavage-inducing portion, and the sixth binding molecule is bonded to the third binding molecule and includes a cleavage-inducing portion, by contact, or a3) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contacting, or, where the first and third binding molecules include cleavage-inducing portions, the fifth binding molecule includes a first molecular tag bonded to the second binding molecule via a cleavable linker, and the sixth binding molecule includes a second molecular tag bonded to the fourth binding molecule via a cleavable linker, or a4) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contacting the second and fourth binding molecules, which include cleavage-inducing portions, the fifth binding molecule, which is bound to the first binding molecule and a first molecular tag bound to it via a cleavable linker, and the sixth binding molecule, which is bound to the third binding molecule and includes a second molecular tag bound to it via a cleavable linker, or a5) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, A first binding molecule includes a first molecular tag bonded thereto via a cleavable linker, a fourth binding molecule includes a second molecular tag bonded thereto via a cleavable linker, a fifth binding molecule is bonded to the second binding molecule and includes a cleavage-inducing portion, and a sixth binding molecule is bonded to the third binding molecule and includes a cleavage-inducing portion, by contact, or a6) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contacting a first binding molecule which includes a first molecular tag bonded thereto via a cleavable linker, a third binding molecule which includes a cleavage-inducing portion, a fifth binding molecule which is bonded to a second binding molecule which includes a cleavage-inducing portion, and a sixth binding molecule which is bonded to a fourth binding molecule which includes a second molecular tag bonded thereto via a cleavable linker, or a7) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contacting a first binding molecule which includes a first molecular tag bonded thereto via a cleavable linker, a fourth binding molecule which includes a cleavage-inducing portion, a fifth binding molecule which is bonded to a second binding molecule which includes a cleavage-inducing portion, and a sixth binding molecule which is bonded to a third binding molecule which includes a second molecular tag bonded thereto via a cleavable linker, or a8) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, A second binding molecule includes a first molecular tag bonded to it via a cleavable linker, a third binding molecule includes a second molecular tag bonded to it via a cleavable linker, a fifth binding molecule is bonded to the first binding molecule and includes a cleavage-inducing portion, and a sixth binding molecule is bonded to the fourth binding molecule and includes a cleavage-inducing portion, by contact, or a9) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contact, or a10) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contacting a second binding molecule which includes a first molecular tag bonded to it via a cleavable linker, a fourth binding molecule which includes a cleavage-inducing portion, a fifth binding molecule which is bonded to the first binding molecule which includes a cleavage-inducing portion, and a sixth binding molecule which is bonded to the third binding molecule which includes a second molecular tag bonded to it via a cleavable linker, or a11) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, A first binding molecule includes a cleavage-inducing portion, a third binding molecule includes a second molecular tag bonded to it via a cleavable linker, a fifth binding molecule is bonded to the second binding molecule and includes a first molecular tag bonded to it via a cleavable linker, and a sixth binding molecule is bonded to the fourth binding molecule and includes a cleavage-inducing portion, by contact, or a12) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, A first binding molecule includes a cleavage-inducing portion, a fourth binding molecule includes a second molecular tag bonded to it via a cleavable linker, a fifth binding molecule is bonded to the second binding molecule and includes a first molecular tag bonded to it via a cleavable linker, and a sixth binding molecule is bonded to the third binding molecule and includes a cleavage-inducing portion, by contact, or a13) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, Contacting, or a14) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, A second binding molecule includes a cleavage-inducing portion, a third binding molecule includes a second molecular tag bonded to it via a cleavable linker, a fifth binding molecule is bonded to the first binding molecule and includes a first molecular tag bonded to it via a cleavable linker, and a sixth binding molecule is bonded to the fourth binding molecule and includes a cleavage-inducing portion, by contact, or a15) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, A second binding molecule includes a cleavage-inducing portion, a fourth binding molecule includes a second molecular tag bonded to it via a cleavable linker, a fifth binding molecule is bonded to the first binding molecule and includes a first molecular tag bonded to it via a cleavable linker, and a sixth binding molecule is bonded to the third binding molecule and includes a cleavage-inducing portion, by contact, or a16) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, The second and third binding molecules include cleavage-inducing moieties, the fifth binding molecule includes a first molecular tag bonded to the first binding molecule via a cleavable linker, and the sixth binding molecule includes a second molecular tag bonded to the fourth binding molecule via a cleavable linker. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of the first and second molecular tags, and c) A method is provided which includes detecting the presence or absence of released first and second molecular tags to detect and / or quantify the expression of the first and second cell surface regions in a sample.

[0030] Another form is a proximity assay using two primary binding molecules for each target region, and a secondary binding molecule for each of the primary binding molecules, where one of the secondary binding molecules for each target region contains a molecular tag and the other contains a cleavage-inducing region.

[0031] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a1) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, A fifth binding molecule is bound to the first binding molecule and includes a first molecular tag bound thereto via a cleavable linker; a seventh binding molecule is bound to the second binding molecule and includes a cleavage-inducing portion; a sixth binding molecule is bound to the third binding molecule and includes a second molecular tag bound thereto via a cleavable linker; and an eighth binding molecule is bound to the fourth binding molecule and includes a cleavage-inducing portion, by contact, or a2) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, Contacting a fifth binding molecule which is bound to the first binding molecule and includes a cleavage-inducing portion, a seventh binding molecule which is bound to the second binding molecule and includes a first molecular tag bound to it via a cleavable linker, a sixth binding molecule which is bound to the third binding molecule and includes a cleavage-inducing portion, and an eighth binding molecule which is bound to the fourth binding molecule and includes a second molecular tag bound to it via a cleavable linker, or a3) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, Contacting a fifth binding molecule which is bound to the first binding molecule and includes a first molecular tag attached thereto via a cleavable linker, a seventh binding molecule which is bound to the second binding molecule and includes a cleavage-inducing portion, a sixth binding molecule which is bound to the third binding molecule and includes a cleavage-inducing portion, and an eighth binding molecule which is bound to the fourth binding molecule and includes a second molecular tag attached thereto via a cleavable linker, or a4) Contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, A fifth binding molecule is bound to the first binding molecule and includes a cleavage-inducing moiety; a seventh binding molecule is bound to the second binding molecule and includes a first molecular tag bound to it via a cleavable linker; a sixth binding molecule is bound to the third binding molecule and includes a second molecular tag bound to it via a cleavable linker; and an eighth binding molecule is bound to the fourth binding molecule and includes a cleavage-inducing moiety. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of the first and second molecular tags, and c) A method is provided which includes detecting the presence or absence of released first and second molecular tags to detect and / or quantify the expression of the first and second cell surface regions in a sample.

[0032] Another form is DTT-mediated release, a method for detecting and / or quantifying the expression of at least a first cell surface portion and a second cell surface portion in a sample. a) Contacting the sample with at least one binding molecule for detecting a first cell surface region and at least one binding molecule for detecting a second cell surface region, The invention comprises at least one binding molecule for detecting a first cell surface region and at least one binding molecule for detecting a second cell surface region, each comprising a molecular tag bound thereto via a cleavable linker. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of molecular tags, and c) detecting the presence or absence of released molecular tags to detect and / or quantify the expression of a first cell surface portion and a second cell surface portion in the sample.

[0033] In one form, the DTT-mediated release assay uses a primary binding molecule for each target portion containing a molecular tag.

[0034] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a) Contacting the sample with a first binding molecule that specifically binds to a first portion expressed on the cell surface, and a second binding molecule that specifically binds to a second portion expressed on the cell surface, The first binding molecule includes a first molecular tag attached to it via a cleavable linker. The second binding molecule includes a second molecular tag attached to it via a cleavable linker. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of the first and second molecular tags, and c) A method is provided which includes detecting the presence or absence of released molecular tags to detect and / or quantify the expression of a first cell surface portion and a second cell surface portion in a sample.

[0035] Another form is a DTT-mediated release assay that uses primary and secondary binding molecules for each target region, where the secondary binding molecule contains a molecular tag.

[0036] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a) Contacting the sample with a first binding molecule that specifically binds to a first portion expressed on the cell surface, a second binding molecule that specifically binds to a second portion expressed on the cell surface, a third binding molecule that specifically binds to the first binding molecule, and a fourth binding molecule that specifically binds to the second binding molecule, A third binding molecule contains a first molecular tag bound to it via a cleavable linker. A fourth binding molecule includes a second molecular tag bonded to it via a cleavable linker. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of the first and second molecular tags, and c) A method is provided which includes detecting the presence or absence of released molecular tags to detect and / or quantify the expression of a first cell surface portion and a second cell surface portion in a sample.

[0037] Another form is a DTT-mediated release assay that uses a primary binding molecule for each target site, and a secondary binding molecule that binds to one of the primary binding molecules and contains a molecular tag.

[0038] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the expression of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a1) Contacting the sample with a first binding molecule that specifically binds to a first portion expressed on the cell surface, a second binding molecule that specifically binds to a second portion expressed on the cell surface, and a third binding molecule that specifically binds to the first binding molecule, The second binding molecule includes a first molecular tag bonded to it via a cleavable linker. A third binding molecule includes a second molecular tag bonded to it via a cleavable linker, or a2) Contacting the sample with a first binding molecule that specifically binds to a first portion expressed on the cell surface, a second binding molecule that specifically binds to a second portion expressed on the cell surface, and a third binding molecule that specifically binds to the second binding molecule, The first binding molecule includes a first molecular tag attached to it via a cleavable linker. A third binding molecule includes a second molecular tag bonded to it via a cleavable linker. The molecular tag bound to the binding molecule that detects the first cell surface region is different from the molecular tag bound to the binding molecule that detects the second cell surface region, and is brought into contact with it. b) Inducing the cleavage of the first and second molecular tags, and c) A method is provided which includes detecting the presence or absence of released molecular tags to detect and / or quantify the expression of a first cell surface portion and a second cell surface portion in a sample.

[0039] In each of the above-described examples in which primary and secondary bonding molecules are used, one or more bonding molecules may be present between the primary and secondary bonding molecules.

[0040] The method according to this disclosure may include a combination of a proximity assay and a DTT-mediated release assay, wherein a first cell surface portion is detected using the proximity assay and a second cell surface portion is detected using the DTT-mediated release assay.

[0041] The method described herein can be used to measure the co-expression of at least two different cell surface regions in a single sample. The knowledge obtained therefrom can be used to predict the response of a patient, particularly a cancer patient, to a drug or multiple drugs that bind to two different cell surface regions.

[0042] Therefore, the present disclosure relates to a method for predicting the response of a patient, particularly a cancer patient, to a drug or a plurality of drugs that bind to at least a first cell surface portion and a second cell surface portion, in particular a portion expressed on immune effector cells and a portion expressed on tumor cells, the method being a) Using the method described herein, to detect the expression levels of a first cell surface portion and a second cell surface member in a biological sample derived from a target, particularly from a target tumor, b) Determining whether the expression levels of the first and second cell surface regions in the sample are above or below the threshold level, and c) A method is provided which includes predicting that a subject is likely to respond to a drug or a combination of drugs that binds to the first and second cell surface regions if the expression levels of the first and second cell surface regions in the subject sample are above a threshold level. The drug or combination of drugs includes drugs as further defined herein. This method is also referred to herein as the “prediction method”.

[0043] This disclosure also relates to a method for treating subjects in need of treatment, in particular subjects having cancer, the method being a) Using the above prediction method, predict the response of a target to a drug or multiple drugs that bind to the first cell surface portion and the second cell surface portion, and b) A method is provided comprising administering a drug or a group of drugs bound to a first cell surface portion and a second cell surface to a subject that is likely to respond according to predictions. The drug or a group of drugs includes drugs as further defined herein.

[0044] This disclosure relates to a subject, in particular a subject having cancer, and to a combination of agents or agents that bind to a first cell surface portion and a second cell surface portion, for the treatment of the subject, a) Using the above prediction method, predict the response of a target to a drug or multiple drugs that bind to the first cell surface portion and the second cell surface portion, and b) Further providing a drug or a group of drugs comprising administering a drug or a group of drugs bound to a first cell surface portion and a second cell surface to a subject that is likely to respond according to predictions. The drug or a group of drugs includes drugs as further defined herein.

[0045] This disclosure further provides a method for detecting and / or quantifying the presence of clustering of at least two cell surface parenchyma in a sample, including a first cell surface parenchyma and a second cell surface parenchyma, the method comprising detecting the presence or absence of a signal, the signal being undetectable unless the first and second cell surface parenchyma are in close proximity to each other in a sample exposed to a drug having binding specificity to at least the first and second cell surface parenchyma. This method can be used to indicate whether two or more cell surface parenchyma are in close proximity to each other. If two or more cell surface parenchyma are in close proximity to each other, they are considered to cluster if they produce the signal described herein. The proximity of at least two cell surface parenchyma is caused or induced by a drug having binding specificity to at least two cell surface parenchyma, such as a multispecific antibody. Therefore, in certain embodiments, this method may also be defined as comprising detecting a signal produced when the first and second cell surface parenchyma are simultaneously bound by a drug having binding specificity to at least two cell surface parenchyma.

[0046] The method according to this disclosure can be carried out by different means, including different forms of proximity assays. One means of carrying out this method involves a quench of a signal from a fluorophore bound to a binding molecule that detects one of the cell surface parts, the quench being caused by a quencher bound to a binding molecule that detects another of the cell surface parts. Another means of carrying out this method involves a signal produced by interference of fluorophores bound to binding molecules, in which one of the cell surface parts is detected together with another different fluorophore bound to a binding molecule that detects another of the cell surface parts.

[0047] In one form of proximity assay, two primary binding molecules are used, one for each target region, with one primary binding molecule containing a molecular tag and the other primary binding molecule containing a cleavage-inducing region.

[0048] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the presence of clustering of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a1) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion and a second binding molecule that specifically binds to the second cell surface portion, A first binding molecule includes a molecular tag attached thereto via a cleavable linker, and a second binding molecule includes a cleavage-inducing portion, by contact, or a2) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion and a second binding molecule that specifically binds to the second cell surface portion, The second binding molecule includes a molecular tag attached thereto via a cleavable linker, and the first binding molecule includes a cleavage-inducing portion, and b) Inducing the cleavage of molecular tags, and c) A method is provided which includes detecting the presence or absence of released molecular tags to detect and / or quantify the clustering of a first cell surface portion with a second cell surface portion in a sample.

[0049] In another form, a primary binding molecule is used for each target site, and a secondary binding molecule is used for one of the primary binding molecules, and a primary binding molecule not bound by a secondary binding molecule contains a molecular tag, and the secondary binding molecule contains a cleavage inducer, or a primary binding molecule not bound by a secondary binding molecule contains a cleavage inducer, and the secondary binding molecule contains a molecular tag.

[0050] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the presence of clustering of at least two cell surface portions in a sample, including a first cell surface portion together with a second cell surface portion, the method being a1) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion, a second binding molecule that specifically binds to the second cell surface portion, and a third binding molecule, A first binding molecule includes a molecular tag attached to it via a cleavable linker, and a third binding molecule is attached to the second binding molecule and includes a cleavage-inducing portion, by contact, or a2) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion, a second binding molecule that specifically binds to the second cell surface portion, and a third binding molecule, A second binding molecule includes a molecular tag attached to it via a cleavable linker, and a third binding molecule is attached to the first binding molecule and includes a cleavage-inducing portion, by contact, or a3) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion, a second binding molecule that specifically binds to the second cell surface portion, and a third binding molecule, The first binding molecule includes a cleavage-inducing portion, and the third binding molecule is bound to the second binding molecule and includes a molecular tag attached to it via a cleavable linker, or a4) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion, a second binding molecule that specifically binds to the second cell surface portion, and a third binding molecule, The second binding molecule includes a cleavage-inducing portion, and the third binding molecule is bound to the first binding molecule and includes a molecular tag attached to it via a cleavable linker, b) Inducing the cleavage of molecular tags, and c) A method is provided which includes detecting the presence or absence of released molecular tags to detect and / or quantify the clustering of a first cell surface portion with a second cell surface portion in a sample.

[0051] In another form, a primary binding molecule is used for each target site, and a secondary binding molecule is used for both primary binding molecules, one of which contains a molecular tag and the other contains a cleavage-inducing site.

[0052] Therefore, in one embodiment, the present disclosure is a method for detecting and / or quantifying the presence of clustering of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, the method being a1) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion, a second binding molecule that specifically binds to the second cell surface portion, a third binding molecule, and a fourth binding molecule, A third binding molecule is bound to the first binding molecule and includes a molecular tag attached to it via a cleavable linker, and a fourth binding molecule is bound to the second binding molecule and includes a cleavage-inducing portion, by contact, or a2) A sample in which the first and second cell surface portions have been exposed to a drug having binding specificity to at least the first and second cell surface portions is brought into contact with a first binding molecule that specifically binds to the first cell surface portion, a second binding molecule that specifically binds to the second cell surface portion, a third binding molecule, and a fourth binding molecule, A third binding molecule is bound to the first binding molecule and includes a cleavage-inducing portion, and a fourth binding molecule is bound to the second binding molecule and includes a molecular tag attached to it via a cleavable linker, causing contact. b) Inducing the cleavage of molecular tags, and c) A method is provided which includes detecting released molecular tags to detect and / or quantify the clustering of a first cell surface portion with a second cell surface portion in a sample.

[0053] In each of the above-described examples in which primary and secondary bonding molecules are used, one or more bonding molecules may be present between the primary and secondary bonding molecules.

[0054] This disclosure relates to a method for detecting and / or quantifying the presence of clustering of at least two cell surface portions in a sample, including a first cell surface portion and a second cell surface portion, wherein the method is The method involves contacting a sample in which the first and third cell surface portions are exposed to a drug having binding specificity to at least the first and third cell surface portions with a first binding molecule that specifically binds to the first cell surface portion and a second binding molecule that specifically binds to the second cell surface portion, wherein at least one of the first and second binding molecules contains a molecular tag that is not detected unless the first and second cell surface portions are in close proximity to each other, and The present invention further provides a method that includes detecting the presence or absence of molecular tags to detect the presence of clustering of a first cell surface portion and a second cell surface portion in a sample.

[0055] In this context, at least the first and third cell surface portions are preferably different cell surface portions. In a particular embodiment, the first cell surface portion is CD137 or another co-stimulatory molecule, and the third cell surface portion is a tumor-associated portion or immune checkpoint portion, preferably a portion on another cell such as PD-L1. The first and second cell surface portions are preferably the same cell surface portion. In a particular embodiment, the first and second cell surface portions are CD137.

[0056] The methods according to the present disclosure can be used to measure the clustering of two or more different cell surface regions in a single sample, and in particular can be used in accordance with the disclosure when the clustering is induced by a drug having binding specificity to two or more different cell surface regions. In certain embodiments, the knowledge obtained therefrom can be used to determine whether treatment with a drug having binding specificity to two or more different cell surface regions is effective, and this determination is based on confirmation of the simultaneous binding of the drug having binding specificity to two different cell surface regions to those regions.

[0057] A drug may be any single portion capable of simultaneously binding to at least two cell surface regions. In certain embodiments, a drug may include at least a binding domain that specifically binds to a first cell surface region and a binding domain that specifically binds to a second cell surface region. For example, suitable drugs may include binding molecules such as antibodies, including, for example, multispecific antibodies such as bispecific and tripspecific antibodies, antibody fragments, molecules containing antibody-derived domains, and fusion proteins. A drug may also be referred to as a medicinal substance.

[0058] Accordingly, the present disclosure provides a method for determining the efficacy of a drug, wherein the drug comprises at least a binding domain that specifically binds to a first cell surface portion and a binding domain that specifically binds to a second cell surface portion, and the method comprises detecting and / or quantifying the clustering of the first cell surface portion with the second cell surface portion in a biological sample of a subject being treated with the drug, by using the method according to the present disclosure. The drug is a drug as further defined herein. The method is also referred to herein as the “monitoring method”.

[0059] This disclosure also provides a method for confirming the mode of action of a drug, wherein the drug comprises at least a binding domain that specifically binds to at least a first cell surface portion and a binding domain that specifically binds to a second cell surface portion, and the method comprises detecting and / or quantifying the clustering of the first cell surface portion with the second cell surface portion in a biological sample of a subject being treated with the drug, by using the method according to this disclosure. The drug is a drug as further defined herein. The mode of action is, for example, the simultaneous binding of the drug to the first and second cell surface portions. In this context, the first and second cell surface portions are preferably different cell surface portions, and the drug is a multispecific antibody. Another mode of action is, for example, the clustering of two or more cell surface portions. In this context, in one embodiment, at least the first and second cell surface portions are the same, and the drug is a monospecific antibody; in another embodiment, at least the first and second cell surface portions are different cell surface portions, and the drug is a multispecific antibody. This method is also referred to herein as the “confirmation method”. For example, it is possible to determine whether a multispecific antibody having specificity for a drug, preferably an immune effector cell, preferably a cell surface portion expressed on CD137 or any other immune effector cell costimulatory protein, and for tumor cells, preferably a cell surface portion expressed on PD-L1 or any other tumor-associated portion or immune checkpoint, induces clustering of two or more of the cell surface portions expressed on one or more immune effector cells, preferably CD137 or any other immune effector cell costimulatory protein.

[0060] This disclosure relates to a method for treating subjects in need of treatment, particularly subjects with cancer, and the method is a) Treating the target with a drug that binds to the first and second cell surface regions. b) Further methods are provided, including analyzing the efficacy of a drug using a monitoring method described herein, or confirming the mode of action of a drug using a confirmation method described herein. A drug is a drug as further defined herein. In certain embodiments, the method may further include continuing or adapting treatment based on the results of the analysis or confirmation, and adapting includes, but is not limited to, increasing or decreasing the dose and / or increasing or decreasing the frequency of administration of the drug, or discontinuing treatment.

[0061] This disclosure relates to a drug that binds to a first cell surface portion and a second cell surface portion for use in the treatment of a subject, in particular a subject having cancer, and the treatment thereof is a) Treating the target with a drug that binds to the first and second cell surface regions. b) Further providing a drug comprising analyzing the efficacy of the drug using a monitoring method described herein, or confirming the mode of action of the drug using a confirmation method described herein. A drug is a drug as further defined herein. In certain embodiments, treatment may further include continuing or adapting treatment based on the results of the analysis or confirmation, and adapting includes, but is not limited to, increasing or decreasing the dose and / or increasing or decreasing the frequency of administration of the drug, or discontinuing treatment.

[0062] The method described herein can be used to further screen one or more test agents for their ability to induce clustering of at least one cell surface portion with a second cell surface portion.

[0063] Therefore, the present disclosure is a method for screening one or more test agents for their ability to induce clustering of at least a first cell surface portion with a second cell surface portion, the method being a) Contacting one or more test cell cultures with a test drug, The test cell culture includes cells expressing at least a first cell surface region and a second cell expressing a second cell surface region, and is brought into contact with the culture. b) Using the method according to the present disclosure to detect the level of clustering of the first and second cell surface portions, and c) Comparing the level of clustering detected in step b) with the level of clustering detected in a control cell culture that has not been in contact with the test drug or the reference drug, The method further provides a control cell culture comprising a first cell surface portion and a second cell surface portion. In certain embodiments, the method may further include selecting a test agent that induces a level of clustering equal to or higher than the level of clustering in the control cell culture.

[0064] Using this method, novel, additional, or alternative binding molecules with binding specificity to two or more different cell surface regions, in addition to those already known, can be identified. For example, this method can be used to identify additional or alternative binding molecules with binding specificity to CD137 or any other immune effector cell costimulatory region, and to PD-L1 or any other tumor-associated or immune checkpoint region.

[0065] This disclosure further provides kits of parts. In certain embodiments, the kit includes binding molecules that specifically bind to at least first and second cell surface portions in accordance with the methods described herein. In one embodiment, the kit of parts includes binding molecules, at least two binding molecules that specifically bind to first and second cell surface portions, wherein optionally one of the binding molecules includes a molecular tag bound thereto via a cleavable linker, and the other binding molecule includes a cleavage-inducing portion; and instructions for bringing a patient sample into contact with at least two binding molecules to optionally induce cleavage of the molecular tag, and measuring the signal induced by bringing the patient sample into contact with at least two binding molecules.

[0066] The following is a further description of the features of the methods described herein. For clarity and conciseness, features are described herein as part of the same or distinct embodiments, but it will be understood that the scope of this disclosure may include embodiments having all or some combinations of the described features.

[0067] The methods described herein can be used to detect the expression of a first cell surface portion and a second cell surface portion. The disclosure further provides a method that can be used to indicate whether the two cell surface portions are in close proximity to each other. The detection method used, which involves the release of a molecular tag from a binding molecule bound to one of the cell surface portions, also enables the quantification of the expression of the first and second cell surface portions (or their complex).

[0068] In certain embodiments, the method according to this disclosure enables the detection and / or quantification of first and second cell surface regions in a single sample. For this purpose, a molecular tag bound to an antibody that binds to the first cell surface region is different from a molecular tag bound to an antibody that binds to the second cell surface region. In certain embodiments, this method can be used to determine whether the first and second cell regions are co-expressed in a particular sample.

[0069] The sample in the method according to certain embodiments of this disclosure may be, but is not limited to, a tissue sample, a blood sample, or a cultured cell. Preferably, the sample is a tissue sample, blood sample, or cultured cell derived from a subject or patient. The tissue sample derived from a subject or patient may be a fresh sample, a formalin-fixed paraffin-embedded (FFPE) sample, or other fixed sample. This method is particularly useful for detecting and / or quantifying first and second cell surface portions (clusters) in tumor biopsy samples derived from a subject with cancer.

[0070] In certain embodiments, the first cell surface portion and the second cell surface portion are preferably different portions and may be expressed on the same cell type, the same cell, or different cells, such as tumor cells or immune cells. In certain embodiments, the first cell surface portion and the second cell surface portion are preferably expressed on different cell types.

[0071] In certain embodiments, the methods of the present disclosure are useful for any application where the objective is to measure the co-expression of two or more cell surface regions, and / or to determine the clustering of two or more cell surface regions on the same or separate cells.

[0072] In certain embodiments, at least one of two cell surface regions is preferably expressed on an immune effector cell, in particular an NK cell, T cell, B cell, monocyte, macrophage, dendritic cell, or neutrophil, preferably a T cell.

[0073] In certain embodiments, at least one of two cell surface regions is expressed on cells that may be of tumor or immune cell origin, such as tumor cells, B cells, myeloid cells, dendritic cells, or neutrophils, for example.

[0074] In certain embodiments, one of at least two cell surface parenchyma is expressed on immune effector cells, particularly NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophils, preferably T cells, and the other of the at least two cell surface parenchyma is expressed on cells that may be of tumor or immune cell origin, such as, for example, tumor cells, B cells, myeloid cells, dendritic cells, or neutrophils.

[0075] In certain embodiments, the immune effector cells may be NK cells, T cells, B cells, monocytes, macrophages, dendritic cells, or neutrophils, preferably T cells.

[0076] In certain embodiments, the immunoeffector cell costimulatory moiety may be CD137, OX40, GITR, CD27, CD28, ICOS, CD40L, or LIGHT, preferably CD137.

[0077] In certain embodiments, the immune checkpoint moiety or tumor-related moiety can be selected from, but is not limited to, PD-L1, PD-L2, B7-H3, B7-H4, TIM3, CD47, or CD70, preferably PD-L1.

[0078] The first and second cell surface portions can be any cell surface portions that cluster in response to a drug, bringing both cell surface portions into close proximity to each other. In certain embodiments, the first and second cell surface portions may be the same, and this method is used to detect and / or quantify the clustering, e.g., dimerization or trimerization, of these cell surface portions in response to a drug. This is illustrated herein, for example, for the clustering of at least two CD137 molecules.

[0079] In a particular embodiment, the first cell surface portion is CD137, and the second cell surface portion is PD-L1.

[0080] Methods for measuring HER2 homodimers, as well as HER1 / HER2 heterodimers, HER2 / HER3 heterodimers, HGF-c-Met complexes, HER3-PI3K complexes, and PD-1-PD-L1 complexes are not part of this disclosure.

[0081] Accordingly, this disclosure provides a method for detecting and / or quantifying the expression of at least two cell surface parenchyma in a sample, including a first cell surface parenchyma and a second cell surface parenchyma, as described herein, such that the first and second cell surface parenchyma are not HER2, HER1 and HER2, HER2 and HER3, HGF and c-Met, HER3 and PI3K, or PD-1 and PD-L1.

[0082] This disclosure also provides a method for detecting and / or quantifying the presence of clustering of at least two cell surface parenchyma in a sample, including a first cell surface parenchyma and a second cell surface parenchyma, as described herein, such that the first and second cell surface parenchyma are not HER2, HER1 and HER2, HER2 and HER3, HGF and c-Met, HER3 and PI3K, or PD-1 and PD-L1.

[0083] In certain embodiments of the method of this disclosure, the sample comes into contact with binding molecules that bind to first and second cell surface portions. These are also referred to as assay binding molecules.

[0084] In certain forms of the methods described herein, two binding molecules are used for each part. This disclosure refers to two binding molecules that bind to a first cell surface part as the first and second binding molecules. Two binding molecules that bind to a second cell surface part are referred to herein as the third and fourth binding molecules. The numbers used when referring to these binding molecules indicate that the binding molecules are different from one another in terms of binding specificity and / or to facilitate visualization of examples of useful assay forms. The numbers do not refer to any particular order or required presence of one or more of the binding molecules. Also, in forms using a primary binding molecule and one or more secondary binding molecules, other binding molecules may be present between the primary binding molecule, i.e., the binding molecule that binds directly to the cell surface part, and the secondary binding molecule, i.e., the binding molecule containing a molecular tag or cleavage-inducing moiety.

[0085] In certain forms of the methods of this disclosure, the two binding molecules that bind to a first cell surface portion are binding molecules that bind to different epitopes and are selected so as not to interfere with each other's binding to the first cell surface portion. Similarly, the two binding molecules that bind to a second cell surface portion are binding molecules that bind to different epitopes and are selected so as not to interfere with each other's binding to the second cell surface portion. The first, second, third, and / or fourth binding molecules can bind to the extracellular domain of the cell surface portion, but can also bind to the intracellular domain of the cell surface portion. Combinations of these are also possible. For example, two binding molecules that bind to a first cell surface portion may be directed toward its extracellular domain, while two binding molecules that bind to a second cell surface portion may bind to its intracellular domain, or one of the binding molecules that bind to the first cell surface portion may bind to its extracellular domain and the other to its intracellular domain, and the same applies to the second cell surface portion, or one of the binding molecules that bind to the first cell surface portion may bind to its extracellular domain and the other to its intracellular domain, while both binding molecules that bind to the second cell surface portion may bind to its extracellular or intracellular domain, or vice versa.

[0086] In certain embodiments, one primary binding molecule is used for each portion. This disclosure refers to two primary binding molecules that bind to a first cell surface portion and a second cell surface portion, respectively, as a first and second binding molecule. The numbers used when referring to these binding molecules indicate that the binding molecules are different from each other in terms of binding specificity and / or to facilitate visualization of examples of useful assay forms. The numbers do not indicate any particular order or required presence of one or more of the binding molecules. Nor do they indicate that they are the same as the first and second binding molecules referred to in relation to other embodiments of this disclosure. Furthermore, in forms using a primary binding molecule and one or more secondary binding molecules, other binding molecules may be present between the primary binding molecule, i.e., the binding molecule that directly binds to the cell surface portion, and the secondary binding molecule, i.e., the binding molecule containing a molecular tag or cleavage-inducing portion.

[0087] In a particular embodiment, the two binding molecules that bind to the first and second cell surface regions are binding molecules that bind to different cell surface regions. The first and second binding molecules can bind to the extracellular domain of the cell surface region, or they can bind to the intracellular domain of the cell surface region. Combinations of these are also possible. For example, the binding molecule that binds to the first cell surface region may bind to its extracellular domain, while the binding molecule that binds to the second cell surface region may bind to its intracellular domain, or vice versa.

[0088] The binding molecule used in certain embodiments of the methods of this disclosure is preferably an antibody or an antigen-binding fragment thereof. In this context, the first and second cell surface portions can be considered antigens. Antibodies or antigen-binding fragments thereof that specifically bind to antigens are known in the art and are available for a number of different antigens. They are commercially available or readily manufactured. Such antibodies or antigen-binding fragments typically bind to antigens but otherwise do not exhibit biological functions such as, for example, blocking the interaction between the antigen and its ligand or inducing cytotoxic activity.

[0089] In certain forms of the methods of this disclosure, one of the primary binding molecules to each cell surface region includes a molecular tag or a cleavage-inducing moiety. In certain forms of the methods of this disclosure, one of the primary binding molecules includes a molecular tag, and the other includes a cleavage-inducing moiety. Such molecular tags or cleavage-inducing moieties can be conjugated to primary binding molecules, in particular antibodies, using standard techniques of the art. In some cases, it may be difficult to conjugate a molecular tag or cleavage-inducing moiety to a particular binding molecule. In such cases, a secondary binding molecule, usually an antibody, to which the molecular tag or cleavage-inducing moiety conjugates can be used. Such secondary binding molecules, including molecular tags or cleavage-inducing moieties, are typically directed to the Fc region of a primary binding molecule and are generally commercially available.

[0090] A molecular tag can be any molecular portion that can be detected, e.g., a molecule. In a particular case, upon release, the molecular portion provides a measurable signal. A molecular tag may be selected based on one or more of its properties that distinguish it from other portions, including, but not limited to, electrophoretic mobility, molecular weight, shape, solubility, pKa, hydrophobicity, charge, charge / mass ratio, and polarity. Differences in at least one of these properties allow for the separation of molecular tags in assays that measure multiple cell surface portions in a single sample. In a particular embodiment, the molecular tag includes a detection portion, e.g., but not limited to, fluorescent labels, colorimetric labels, radioactive labels, or electrochemical labels. Exemplary fluorescent dyes include water-soluble rhodamine dyes, fluorescein, 4,7-dichlorofluorescein, benzoxanthene dyes, and energy transfer dyes, as disclosed in the following reference: Handbook of Molecular Probes and Research Reagents, 8 thed. (2002), Molecular Probes, Eugene, Oreg., WO 2001 / 32783, U.S. Patent Application Publications 2002-0081616 and 2002-0086985, and Lee et al., 1997, Nucleic Acids Research 25:2816-2822. Examples of preferred molecular tags include, but are not limited to, VeraTag® reporter molecules. VeraTag® reporter molecules are well known in the art. Preferred molecular tags include, for example, VeraTag® reporter molecules Pro11 and Pro125. Pro11 is an example of a photo-emitting tag, and Pro125 is an example of a DTT-emitting tag. Other VeraTag® molecules are described, for example, in U.S. Patent Publication Nos. 2004-0166529, 2004-0126818, 2003-0013126, 2005-0079565, and 2011-0180408, each of which and the references cited herein are incorporated herein by reference in whole.

[0091] The cleavage-inducing portion can be any portion that can directly or indirectly induce the cleavage of the molecular tag from the binding molecule to which the molecular tag is bound via a cleavable linker. In certain embodiments, the cleavage-inducing portion is, for example, a portion that produces an active species that can cleave the cleavable linker. Exemplary active species include singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals, phenoxy radicals, superoxide, etc. Exemplary quenchers of active species that cause oxidation include polyenes, carotenoids, vitamin E, vitamin C, tyrosine, histidine, and the amino acid pyrrole N-conjugates of glutathione. See, for example, Beutner et al., 2000, Meth. Enzymol. 319:226-241. One example involves contacting biotin conjugated to a binding molecule with streptavidin conjugate methylene blue and exposing it to light, resulting in the release of singlet oxygen that can cleave the cleavable linker.

[0092] In certain assay configurations, molecular tags are bound to a binding molecule via cleavable linkers. These cleavable linkers may include, but are not limited to, linkers that can be cleaved by singlet oxygen, hydrogen peroxide, or DTT. Linkers that can be cleaved by DTT are SS linkers. Cleavable bonds may also include bonds that are unstable to agents acting on the entire reaction mixture, such as base-instability bonds, photocleavable bonds, reduction-cleavable bonds, oxidation-cleavable bonds, acid-instability bonds, and peptide bonds that can be cleaved by certain proteases. References describing many such bonds include Greene and Wuts, 1991, Protective Groups in Organic Synthesis, Second Edition, John Wiley & Sons, New York; Hennenson, 1996, Bioconjugate Techniques, Academic Press, New York; and U.S. Patent Application Publication 2003-0119059.

[0093] The cleavage of the molecular tag from the binding molecule can be induced by methods known in the art, including but not limited to the use of photoinduction and DTT-mediated emission. Photoinduction induces the activation of a photoabsorbing molecule that converts molecular oxygen to singlet oxygen when activated by light. Inducing cleavage by using DTT involves DTT-induced cleavage of a disulfide linker that is cleavable by reduction and links the molecular tag to the binding molecule.

[0094] In certain embodiments, the signal measured by the method of the Disclosure is the amount of released molecular tag. In certain forms of the method of the Disclosure, at least two different molecular tags are used. In certain embodiments, these can be separated before detection by electrophoresis or methods based on differences in molecular weight, shape, solubility, pKa, hydrophobicity, charge, charge / mass ratio, and polarity, including, but not limited to, methods known in the art. The detection method depends on the molecular tag.

[0095] In the context of this disclosure, “proximity” means that the binding molecule containing the bound molecular tag and the binding molecule containing the cleavage-inducing portion are within a distance that allows for the cleavage of the molecular tag induced by the cleavage-inducing portion. In the VeraTag® assay, this is approximately 1000 nm, preferably within approximately 20–200 nm or 30–100 nm of each other. Other ranges for proximity apply depending on the nature of the molecular tag used and can be readily determined by those skilled in the art, and are information provided by the suppliers of commercially available tags, quenchers, and reporter portions. Information required by those skilled in the art to apply a particular proximity assay in this disclosure is available in the art. For example, Nathan P. 2020, Assay Guidance Manual, Compound-Mediated Assay Interferences in Homogenous Proximity Assays (which is incorporated herein by reference in its entirety) provides, among other things, information on donor and acceptor fluorophores that may be used in FRET assays, including a description of the optimal distance between the donor and acceptor fluorophores (see, for example, Tables 2 and 3).

[0096] The method described herein can be used to measure the co-expression of two different cell surface parenchyma in a single sample. The knowledge obtained therefrom can be used to determine a treatment plan for a patient. For example, if two cell surface parenchyma are co-expressed in a patient's tissue or blood sample, it can be decided to treat the patient with a drug or multiple drugs that target these two cell surface parenchyma. One example of such a situation is when a tumor biopsy sample from a cancer patient shows co-expression of two tumor-associated antigens on tumor cells. The patient can then be successfully treated with one or more drugs that bind to these tumor-associated antigens, interfere with the signaling pathway of the tumor-associated antigens, and / or induce T cell-mediated tumor cell killing. If a patient's tumor sample does not show co-expression of such tumor-associated antigens, the treating physician may determine that the patient is unlikely to benefit from such treatment. Another situation is, for example, when a tumor biopsy shows co-expression of tumor-associated antigens on tumor cells and antigens expressed on immune effector cells. This indicates, for example, the presence of immune effector cells such as T cells and / or NK cells in the tumor microenvironment. Such patients may benefit from treatment with agents that bring immune cells close to tumor cells and / or activate immune effector cells so that tumor cells are selectively killed. Therefore, in certain embodiments, the methods of the present disclosure may be used to predict a patient, preferably a cancer patient, response to treatment with agents or multiple agents that bind to two different cell surface regions.

[0097] An example of a drug that binds to two different cell surface regions is, for example, a multispecific antibody. Such a multispecific antibody may be a bispecific or triplicate antibody, or its antigen-binding fragment, that binds simultaneously to both cell surface regions. Such a multispecific antibody may exhibit monovalent binding to both cell surface regions, such that the multispecific antibody contains a single antigen-binding fragment for each cell surface region. The cell surface regions may be either the first and second cell surface regions disclosed herein.

[0098] Specific examples of multispecific antibodies that bind to two different cell surface regions and are relevant to the usefulness of the methods of this disclosure are multispecific antibodies that bind to PD-L1 on tumor cells and CD137 on T cells. Such multispecific antibodies may include a CD137-binding domain comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 12, 16, 19, 23, 27, 30, 34, 38, 42, 45, 48, or 52, where one, two, or three amino acid substitutions may be permitted. In a particular embodiment, the CD137-binding domain comprises a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 12, 16, 19, 23, 27, 30, 34, 38, 42, 45, 48, or 52. The CD137 binding domain may further comprise heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 21, 25, 32, 36, 40, 44, or 50, allowing for one, two, or three amino acid substitutions, and / or further comprise heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 11, 15, 22, 26, 29, 33, 37, 41, 47, or 51, allowing for one, two, or three amino acid substitutions. In certain embodiments, the CD137-binding domain may include a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 21, 25, 32, 36, 40, 44, or 50, and / or a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 11, 15, 22, 26, 29, 33, 37, 41, 47, or 51. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred CD137-binding domains include combinations of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 2, 3, and 4; 6, 7, and 8; 10, 11, and 12; 14, 15, and 16; 18, 3, and 19; 21, 22, and 23; 25, 26, and 27; 10, 29, and 30; 32, 33, and 34; 36, 37, and 38; 40, 41, and 42; 44, 41, and 45; 2, 47, and 48; or 50, 52, and 52. The CD137-binding domain of such a multispecific antibody may have one of SEQ ID NOs: 1, 5, 9, 13, 17, 20, 24, 28, 31, 35, 39, 43, 46, or 49, or may include a heavy chain variable region having at least 80%, 85%, 90%, 95%, or 99%, preferably 95%, sequence identity with respect to those framework regions. In certain embodiments, the CD137-binding domain of such a multispecific antibody also includes a CH1 domain. Any CH1 domain may be used. An example of a preferred CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

[0099] The multispecific antibody may further include a PD-L1 binding domain comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs. 56, 58, 61, 72, 76, 80, 84, 88, 91, 95, 99, 102, or 106, allowing for one, two, or three amino acid substitutions therein. In a particular embodiment, the PD-L1 binding domain comprises a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs. 56, 58, 61, 72, 76, 80, 84, 88, 91, 95, 99, 102, or 106. The PD-L1 binding domain may further comprise a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs. 54, 60, 65, 68, 70, 74, 78, 82, 86, 90, or 93, allowing for one, two, or three amino acid substitutions, and / or further comprise a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs. 55, 3, 63, 66, 71, 75, 79, 83, 87, 94, 98, 101, 105, or 108, allowing for one, two, or three amino acid substitutions. In certain embodiments, the PD-L1 binding domain includes a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs: 54, 60, 65, 68, 70, 74, 78, 82, 86, 90, or 93, and / or a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs: 55, 3, 63, 66, 71, 75, 79, 83, 87, 94, 98, 101, 105, or 108. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred PD-L1 binding domains are: SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 58; SEQ ID NO: 60, SEQ ID NO: 3, and SEQ ID NO: 61; SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 56; SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 56; SEQ ID NO: 68, SEQ ID NO: 55, and SEQ ID NO: 56; SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72; SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; SEQ ID NO: 82, sequence number Includes sequences 83 and 84; 86, 87, and 88; 90, 79, and 91; 93, 94, and 95; 68, 55, and 56; 70, 98, and 99; 93, 101, and 102; 74, 105, and 106; or combinations of HCDR1, HCDR2, and HCDR3 of sequences 86, 108, and 88. The PD-L1 binding domain of such a multispecific antibody may have one of SEQ ID NOs: 53, 57, 59, 62, 64, 67, 69, 73, 77, 81, 85, 89, 92, 96, 97, 100, 103, 104, or 107, or may include a heavy chain variable region having at least 80%, 85%, 90%, 95%, 99%, preferably 95%, sequence identity with respect to those framework regions. In certain embodiments, the PD-L1 binding domain of such a multispecific antibody also includes a CH1 domain. Any CH1 domain may be used. An example of a preferred CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

[0100] In certain embodiments, the multispecific antibody may include any combination of CD137 and PD-L1 binding domains disclosed herein, see, for example, Table 1. One such multispecific antibody is MCLA-145.

[0101] In certain embodiments, the multispecific antibody may further include any light chains. A preferred example of a light chain is a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 113, and including a light chain variable region that allows for one, two, or three amino acid substitutions. In certain embodiments, the light chain variable region includes a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 113. The light chain variable region may further include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 111, and / or a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 112, and / or including a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 112. In certain embodiments, the light chain variable region includes a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 111, and / or a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 112. The light chain variable region of a multispecific antibody may include a light chain variable region having SEQ ID NO: 110 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with respect to its framework region. In certain embodiments, the light chain of such a multispecific antibody also includes a CL domain. Any CL domain may be used. An example of a preferred CL domain is provided by the amino acid sequence provided as SEQ ID NO: 115.

[0102] In certain embodiments, the multispecific antibody may further include an Fc region or a portion thereof. Such an Fc region may include any modifications known in the art, such as, but are not limited to, modifications to eliminate or reduce Fc effector function, and / or modifications to promote heterodimerization of different CH3 domains. Any Fc region may be used. Examples of preferred Fc regions are provided by amino acid sequences provided as SEQ ID NOs: 116-120.

[0103] The method according to this disclosure can be used to detect clustering of at least two different cell surface regions in a single sample, and in particular, can be used in accordance with this disclosure when the clustering is induced by a drug having binding specificity to at least two different cell surface regions. The knowledge obtained therefrom can be used to determine whether the patient is benefiting from treatment with a drug having binding specificity to two different cell surface regions, and therefore whether to continue, adapt, or discontinue the treatment. For example, if a therapeutic agent is administered but no clustering is observed, or if it is below a certain threshold level, it can be decided to discontinue the treatment. Or, if some clustering is observed but not at a desired level, it can be decided to increase the therapeutic dose and / or treatment interval. Thus, in certain embodiments, the method according to this disclosure can be considered a method for monitoring a patient's response to a particular treatment.

[0104] An example of a drug having binding specificity to two different cell surface regions is, for example, a multispecific antibody. Such a multispecific antibody may be a bispecific or triplicate antibody, or its antigen-binding fragment, that binds simultaneously to both cell surface regions. Such a multispecific antibody may exhibit monovalent binding to both cell surface regions, such that the multispecific antibody contains a single antigen-binding fragment for each cell surface region. The method of this disclosure can be used in any situation in which two or more cell surface regions are clustered by a drug having binding specificity to those two or more cell surface regions. Thus, a drug having binding specificity to two different cell surface regions can bind to any cell surface region, such as those disclosed herein, but is not limited to these.

[0105] Specific examples of multispecific antibodies that bind to two different cell surface regions and are relevant to the usefulness of the methods disclosed herein are multispecific antibodies that bind to PD-L1 on tumor cells and CD137 on T cells. Such multispecific antibodies may include a CD137-binding domain comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 12, 16, 19, 23, 27, 30, 34, 38, 42, 45, 48, or 52, allowing for one, two, or three amino acid substitutions therein. In certain embodiments, the CD137-binding domain comprises a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 12, 16, 19, 23, 27, 30, 34, 38, 42, 45, 48, or 52. The CD137 binding domain may further comprise heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 21, 25, 32, 36, 40, 44, or 50, allowing for one, two, or three amino acid substitutions, and / or further comprise heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 11, 15, 22, 26, 29, 33, 37, 41, 47, or 51, allowing for one, two, or three amino acid substitutions. In certain embodiments, the CD137-binding domain includes a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 21, 25, 32, 36, 40, 44, or 50, and / or a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 11, 15, 22, 26, 29, 33, 37, 41, 47, or 51. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred CD137-binding domains include combinations of HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 2, 3, and 4; 6, 7, and 8; 10, 11, and 12; 14, 15, and 16; 18, 3, and 19; 21, 22, and 23; 25, 26, and 27; 10, 29, and 30; 32, 33, and 34; 36, 37, and 38; 40, 41, and 42; 44, 41, and 45; 2, 47, and 48; or 50, 52, and 52. The CD137-binding domain of such a multispecific antibody may have one of SEQ ID NOs: 1, 5, 9, 13, 17, 20, 24, 28, 31, 35, 39, 43, 46, or 49, or may include a heavy chain variable region having at least 80%, 85%, 90%, 95%, or 99%, preferably 95%, sequence identity with respect to those framework regions. In certain embodiments, the CD137-binding domain of such a multispecific antibody also includes a CH1 domain. Any CH1 domain may be used. An example of a preferred CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

[0106] The multispecific antibody may further include a PD-L1 binding domain comprising a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs. 56, 58, 61, 72, 76, 80, 84, 88, 91, 95, 99, 102, or 106, allowing for one, two, or three amino acid substitutions therein. In a particular embodiment, the PD-L1 binding domain comprises a heavy chain CDR3 (HCDR3) having the amino acid sequence shown in any one of SEQ ID NOs. 56, 58, 61, 72, 76, 80, 84, 88, 91, 95, 99, 102, or 106. The PD-L1 binding domain may further comprise a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs. 54, 60, 65, 68, 70, 74, 78, 82, 86, 90, or 93, allowing for one, two, or three amino acid substitutions, and / or further comprise a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs. 55, 3, 63, 66, 71, 75, 79, 83, 87, 94, 98, 101, 105, or 108, allowing for one, two, or three amino acid substitutions. In certain embodiments, the PD-L1 binding domain includes a heavy chain CDR1 (HCDR1) having the amino acid sequence shown in any one of SEQ ID NOs: 54, 60, 65, 68, 70, 74, 78, 82, 86, 90, or 93, and / or a heavy chain CDR2 (HCDR2) having the amino acid sequence shown in any one of SEQ ID NOs: 55, 3, 63, 66, 71, 75, 79, 83, 87, 94, 98, 101, 105, or 108. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred PD-L1 binding domains are: SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56; SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 58; SEQ ID NO: 60, SEQ ID NO: 3, and SEQ ID NO: 61; SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 56; SEQ ID NO: 65, SEQ ID NO: 66, and SEQ ID NO: 56; SEQ ID NO: 68, SEQ ID NO: 55, and SEQ ID NO: 56; SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 72; SEQ ID NO: 74, SEQ ID NO: 75, and SEQ ID NO: 76; SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; SEQ ID NO: 82, sequence number Includes sequences 83 and 84; 86, 87, and 88; 90, 79, and 91; 93, 94, and 95; 68, 55, and 56; 70, 98, and 99; 93, 101, and 102; 74, 105, and 106; or combinations of HCDR1, HCDR2, and HCDR3 of sequences 86, 108, and 88. The PD-L1 binding domain of such a multispecific antibody may have one of SEQ ID NOs: 53, 57, 59, 62, 64, 67, 69, 73, 77, 81, 85, 89, 92, 96, 97, 100, 103, 104, or 107, or may include a heavy chain variable region having at least 80%, 85%, 90%, 95%, 99%, preferably 95%, sequence identity with respect to those framework regions. In certain embodiments, the PD-L1 binding domain of such a multispecific antibody also includes a CH1 domain. Any CH1 domain may be used. An example of a preferred CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

[0107] In certain embodiments, the multispecific antibody may include any combination of CD137 and PD-L1 binding domains disclosed herein, see, for example, Table 1. One such multispecific antibody is MCLA-145.

[0108] In certain embodiments, the multispecific antibody may further include any light chains. A preferred example of a light chain is a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 113, and including a light chain variable region that allows for one, two, or three amino acid substitutions. In certain embodiments, the light chain variable region includes a light chain CDR3 (LCDR3) having the amino acid sequence shown in SEQ ID NO: 113. The light chain variable region may further include a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 111, and / or a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 112, and / or including a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 112. In certain embodiments, the light chain variable region includes a light chain CDR1 (LCDR1) having the amino acid sequence shown in SEQ ID NO: 111, and / or a light chain CDR2 (LCDR2) having the amino acid sequence shown in SEQ ID NO: 112. The light chain variable region of a multispecific antibody may include a light chain variable region having SEQ ID NO: 110 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity with respect to its framework region. In certain embodiments, the light chain of such a multispecific antibody also includes a CL domain. Any CL domain may be used. An example of a preferred CL domain is provided by the amino acid sequence provided as SEQ ID NO: 115.

[0109] In certain embodiments, the multispecific antibody may further include an Fc region, or a portion thereof. Such an Fc region may include any modifications known in the art, such as, but are not limited to, modifications to eliminate or reduce Fc effector function, and / or modifications to promote heterodimerization of different CH3 domains. Any Fc region may be used. Examples of preferred Fc regions are provided by amino acid sequences provided as SEQ ID NOs: 116-120. [Table 1-1] [Table 1-2]

[0110] As used herein, “includes” and its conjugations are used in their non-restrictive sense, meaning that the item preceding the word is included, but items not specifically mentioned are not excluded.

[0111] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical objects of the article. For example, "element" means one element or more elements.

[0112] References to patent documents or other matters in this specification shall not be deemed to acknowledge that such documents or matters were known, or that the information contained herein was part of the common general knowledge as of the priority date of any of the claims.

[0113] All patent and document references cited herein are incorporated herein by reference in their entirety.

[0114] This disclosure is illustrated by the following embodiments, which do not limit the scope of this disclosure. [Examples]

[0115] Example 1 - Preparation of cell pellets for the VeraTag® assay T75 flasks were coated overnight with 2 μg / mL of anti-CD3 (clone OKT3, eBioscience, catalog number 16-0037-85) in PBS. Next, Jurkat T cells expressing CD137 (Jurkat_CD137K) were placed in 50 mL of medium (9% FBS-HI RPMI 1640, 2 mM L-glutamine) at a rate of 1.8 × 10⁶ 6 The solution was added at a concentration of cells / mL and incubated at 37°C for 4 hours. Then, Jurkat cells were added to 50 mL of culture medium at a concentration of 0.45 × 10⁶. 6CHO-K1 cells expressing PD-L1 at a concentration of cells / mL (CHO-PD-L1) were co-cultured (Jurkat to CHO). The cells were interacted for 4 hours, and then bispecific antibodies binding to CD137 and PD-L1, anti-CD137 positive control antibodies, or negative control antibodies binding to RSV (10 μg / mL) were added for a further 2 hours. The cells were then collected from the flask by resuspending and scraping, and fixed as follows: centrifuged at 1,200 rpm (125 × g) at 4°C for 10 minutes, then the medium was poured out, the cell pellet was loosened, and resuspended in ice-cold PBS. The centrifugation was repeated twice, the PBS was poured out, and the cell pellet was loosened by vortexing. After the second wash, the pellet was resuspended in 30 mL of 10% neutral buffered formalin (10% NBF, catalog no. 5701, Thermo Fisher Scientific) and gently swirled overnight at 4°C. After centrifugation at 1,500 rpm and 4°C for 10 minutes, remove the formalin and divide the cell pellet into 25 × 10 6 The cells were resuspended in 80% ethanol at a concentration of cells / mL, stored at 4°C, and then treated as described above (Shi et al, 2009).

[0116] Suitable bispecific antibodies that bind to CD137 and PD-L1 are, for example, those specifically disclosed herein.

[0117] Example 2 - PD-L1 Expression VeraTag® Assay The cell pellet prepared in Example 1 was used in this assay. 4.5 × 10 5 Individual cells were placed on positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology, as briefly described below.

[0118] Antigen recovery was performed by heating using a pressure cooker (Biocare Medical). After antigen recovery, antibody pairs were added, and the DTT-mediated released fluorescent VeraTags were detected by capillary electrophoresis. The released VeraTags were normalized to the sample buffer volume and measured 4.5 × 10⁶. 5The unit of relative fluorescence per individual cell was obtained.

[0119] The antibodies used were pepsin digests of anti-PD-L1 rabbit mAb E1L3N (Cell Signaling Technology cat#13684) and goat anti-rabbit IgG (H+L) (Southern Biotech cat#4052-01), labeled with a fluorescent VeraTag® reporter via a disulfide bond. In isotype control experiments, the PD-L1 antibody was replaced with rabbit IgG (Cell Signaling Technology cat#3900).

[0120] The results are shown in Figure 9. The VeraTag® assay appears to be a suitable method for detecting PD-L1 expression levels. Similar amounts of PD-L1 were measured in all three samples.

[0121] Example 3 - CD137 Expression VeraTag® Assay The cell pellet prepared in Example 1 was used in this assay. 4.5 × 10 5 Individual cells were placed on positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology, as briefly described below.

[0122] Antigen recovery was performed by heating using a pressure cooker (Biocare Medical). After antigen recovery, antibody pairs were added, and the DTT-mediated released fluorescent VeraTags were detected by capillary electrophoresis. The released VeraTags were normalized to the sample buffer volume and measured 4.5 × 10⁶. 5 The unit of relative fluorescence per individual cell was obtained.

[0123] Two different primary antibodies were evaluated: anti-CD137 mouse mAb M127 (BD Pharmingen cat#552532) and anti-CD137 mouse mAb BBK2 (ThermoFisher cat#MS-621). A goat anti-mouse IgG secondary antibody conjugated to VeraTag (Jackson Immuno Research cat#115-005-146) was paired with the primary antibody. In the isotype control experiment, the CD137 antibody was replaced by mouse IgG (BD Pharmingen cat#554121).

[0124] The results are shown in Figure 10. The VeraTag® assay appears to be a suitable means for detecting the expression level of CD137. Similar amounts of CD137 were measured in all three samples for both primary assay antibodies.

[0125] Example 4 - CD137 Clustering VeraTag® Assay The cell pellet prepared in Example 1 was used for this assay. 4.5×10 5 cells were placed on positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology as briefly described below.

[0126] Antigen retrieval was performed by heating using a pressure cooker (Biocare Medical). After antigen retrieval, the antibody pair was added and the released fluorescent VeraTags were detected by capillary electrophoresis. The released VeraTags were normalized to the sample buffer volume to obtain relative fluorescence units per 4.5×10 5 cells.

[0127] Two different primary antibodies were evaluated: anti-CD137 mouse mAb M127 (BD Pharmingen cat#552532) and anti-CD137 mouse mAb BBK2 (ThermoFisher cat#MS-621). Anti-CD137 antibodies at equal concentrations were labeled with either a fluorescent VeraTag reporter or biotin.

[0128] The results are shown in Figure 11. The VeraTag® assay appears to be a suitable method for detecting CD137 clustering. The VeraTag® signal is more potent when BBK antibody is used as the primary assay antibody compared to M127 antibody.

[0129] Example 5 - CD137-PD-L1 proximity VeraTag® assay The cell pellet prepared in Example 1 was used in this assay. 4.5 × 10 5 Individual cells were placed on positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology, as briefly described below.

[0130] Antigen recovery was performed by heating using a pressure cooker (Biocare Medical). After antigen recovery, antibody pairs were added, and the released fluorescent VeraTags were detected by capillary electrophoresis. The released VeraTags were normalized to the sample buffer volume and measured 4.5 × 10⁶. 5 The unit of relative fluorescence per individual cell was obtained.

[0131] CD137-PD-L1 proximity was measured by proximity-dependent release of a VeraTag reporter from mouse mAb BBK2 (ThermoFisher cat#MS-621, external domain) paired with anti-CD137 mouse mAb M127 (BD Pharmingen cat#552532, external domain) or anti-PD-L1 rabbit mAb E1L3N (Cell Signaling Technology cat#13684, c-terminus) and biotinylated goat anti-rabbit IgG secondary antibody (Rockland Immunochemicals cat#611-101-122). In isotype control experiments, the PD-L1 antibody was replaced with rabbit IgG (Cell Signaling Technology cat#3900). The results are shown in Figure 12. The VeraTag® assay appears to be a suitable means for detecting the CD137-PD-L1 complex. Sample B shows the strongest VeraTag signal for both primary assay antibodies. This indicates that the CD137 × PD-L1 bispecific antibody simultaneously binds to CD137 and PD-L1, clustering these antigens and, consequently, cells expressing these antigens.

[0132] array Sequence ID 1: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRRAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWGVIGGHYMDVWGKGTTVTVSS Sequence ID: 2: HCDR1 by KABAT from Sequence ID: 1 NFAMN Sequence ID 3: HCDR2 by KABAT from Sequence ID 1 WINTNTGNPTYAQGFTG Sequence ID 4: HCDR3 by KABAT from Sequence ID 1 DWGVIGGHYMDV Sequence ID 5: Heavy Chain Variable Region QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDSDGYGPKAFDYWGQGTLVTVSS Sequence ID 6: HCDR1 by KABAT from Sequence ID 5 SYGIS Sequence ID 7: HCDR2 by KABAT from Sequence ID 5 WISAYNGNTNYAQKLQG Sequence ID 8: HCDR3 by KABAT from Sequence ID 5 DSDGYGPKAFDY Sequence ID 9: Heavy Chain Variable Region EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPDDSDTRYSPSFQGQVTISADKSSSTAYLQWSSLKASDTAMYYCASFYTGIVGATGAFDVWGQGTTVTVSS Sequence ID 10: HCDR1 by KABAT from Sequence ID 9 SYWIG Sequence ID 11: HCDR2 by KABAT from Sequence ID 9 IIYPDDSDTRYSPSFQG Sequence ID 12: HCDR3 by KABAT from Sequence ID 9 FYTGIVGATGAFDV Sequence ID 13: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSDAISWVRQAPGQGLEWMGGMIPILGTANYAQKFQGRVTITADRSSTSTAYMELSSLRSEDTAVYYCVRGATYYYGSGTYYSINWFDPWGQGTLVTVSS Sequence ID 14: HCDR1 by KABAT from Sequence ID 13 SDAIS Sequence ID 15: HCDR2 by KABAT from Sequence ID 13 GMIPILGTANYAQKFQG Sequence ID 16: HCDR3 by KABAT from Sequence ID 13 GATYYYGSGTYYSINWFDP Sequence ID 17: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCRASGYTFTNFAMTWVRQAPGQGPEYMGWINTNTGNPTYAQGFTGRFVFSLDTSVNTAYLQISSLKAEDTAVYYCARDWASVMVRGDLDYWGQGTLVTVSS Sequence ID 18: HCDR1 by KABAT from Sequence ID 17 NFAMT Sequence ID 19: HCDR3 by KABAT from Sequence ID 17 DWASVMVRGDLDY Sequence ID 20: Heavy Chain Variable Region QVQLVQSGAEVKKPGASVKVSCKVSGYTLSELSIHWVRQAPGKGVEWMGGFYPEDVEPIYARKFQGRVTMTEDTSTDTAYMELNSLRSEDTAVYYCAAEGFDNYGSGIRGNWFDPWGQGTLVTVSS Sequence ID 21: HCDR1 by KABAT from Sequence ID 20 ELSIH Sequence ID 22: HCDR2 by KABAT from Sequence ID 20 GFYPEDVEPIYARKFQG Sequence ID 23: HCDR3 by KABAT from Sequence ID 20 EGFDNYGSGIRGNWFDP Sequence ID 24: Heavy Chain Variable Region EVQLVQSGAEVKKPGASVKVSCKVSGYTLTELSMHWVRQSPGKGLEWMGSFYPEDGETIYAQKFQGRITMTEDTSADTAYMELSSLRSEDTAVYYCATEGVGVIRGNWFDPWGQGTLVTVSS Sequence ID 25: HCDR1 by KABAT from Sequence ID 24 ELSMH Sequence ID 26: HCDR2 by KABAT from Sequence ID 24 SFYPEDGETIYAQKFQG Sequence ID 27: HCDR3 by KABAT from Sequence ID 24 EGVGVIRGNWFDP Sequence ID: 28: Heavy Chain Variable Region EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIFPDDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKPSDTAMYYCVRLGGYSGYAEDFVDFWGQGTLVTVSS Sequence ID 29: HCDR2 by KABAT from Sequence ID 28 IIFPDDSDTRYSPSFQG Sequence ID 30: HCDR3 by KABAT from Sequence ID 28 LGGYSGYAEDFVDF Sequence ID 31: Heavy Chain Variable Region EVQLVQSGAEVKKPGASVKVSCKVSGYTLTKLSMHWVRQAPGKGLEWMGGFEPEDGETINAQKFQGRVTMTEDTSTDTAYMELSSLRSEDTAVYYCATDLRLGASYYYSYMDVWGRGTMVTVSS Sequence ID 32: HCDR1 by KABAT from Sequence ID 31 KLSMH Sequence ID 33: HCDR2 by KABAT from Sequence ID 31 GFEPEDGETINAQKFQG Sequence ID 34: HCDR3 by KABAT from Sequence ID 31 DLRLGASYYYSYMDV Sequence ID 35: Heavy Chain Variable Region QITLKESGPTLVKPTQTLTLSCTFSGFSLSTSGMSVGWIRQPPGKALEWLALIYWNDDKYFSPSLKSRLTITKDTSKNQVVLTLTNMDPVDTATYYCAHTLWGSDDVFDVWGQGTMVTVSS Sequence ID 36: HCDR1 by KABAT from Sequence ID 35 TSGMSVG Sequence ID 37: HCDR2 by KABAT from Sequence ID 35 LIYWNDDKYFSPSLKS Sequence ID 38: HCDR3 by KABAT from Sequence ID 35 TLWGSDDVFDV Sequence ID 39: Heavy Chain Variable Region EVQLVQSGAEVKKPGESLKISCKVSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWHTLKASDTAMYYCARHQGYSFSGSHIDDYWGQGTLVTVSS Sequence ID 40: HCDR1 by KABAT from Sequence ID 39 NYWIG Sequence ID 41: HCDR2 by KABAT from Sequence ID 39 IIYPGDSDTRYSPSFQG Sequence ID 42: HCDR3 by KABAT from Sequence ID 39 HQGYSFSGSHIDDY Sequence ID 43: Heavy Chain Variable Region EVQLVQSGAEVRKPGESLKISCKGSGYSFTTYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTVYLQWSSLKASDTAMYYCARHAGFIITSQNIDDYWGQGTLVTVSS Sequence ID 44: HCDR1 by KABAT from Sequence ID 43 TYWIG Sequence ID 45: HCDR3 by KABAT from Sequence ID 43 HAGFIITSQNIDDY Sequence ID 46: Heavy Chain Variable Region EVQLVQSGSELKKPGASVKVSCKASGYTFTNFAMNWVRQAPGQGLEWMGWINTNTGNPTYAQDFTGRFVFSLDTSGNTAYLQISSLKAEDTAVYYCARDWGLVAIGYFDYWGQGTLVTVSS Sequence ID 47: HCDR2 by KABAT from Sequence ID 46 WINTNTGNPTYAQDFTG Sequence ID 48: HCDR3 by KABAT from Sequence ID 46 DWGLVAIGYFDY Sequence ID 49: Heavy Chain Variable Region QITLKESGPTLVKPTQTLTLTCTFSGFSLSTTGVGVNWIRQPPGEALEWLALIYWNDDTYYSPSLKSRLTITKDTSKNQVVLTMTNMPDPVDTATYYCAHEGIIGFLGGNWFDPWGQGTLVTVSS Sequence ID 50: HCDR1 by KABAT from Sequence ID 49 TTGVGVN Sequence ID 51: HCDR2 by KABAT from Sequence ID 49 LIYWNDDTYYSPSLKS Sequence ID 52: HCDR3 by KABAT from Sequence ID 49 EGIIGFLGGNWFDP Sequence ID 53: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGQGTLVTVSS Sequence ID 54: HCDR1 by KABAT from Sequence ID 53 SHAMN Sequence ID 55: HCDR2 by KABAT from Sequence ID 53 WINPNTGNPTYAQGFTG Sequence ID 56: HCDR3 by KABAT from Sequence ID 53 DRKYVTNWVFAEDFQH Sequence ID 57: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAIDRGYMSNWVFAEYFPHWGQGTLVTVSS Sequence ID 58: HCDR3 by KABAT from Sequence ID 57 DRGYMSNWVFAEYFPH Sequence ID 59: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSYAMNWVRQAPGQGLEWMGWINTNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCATDRGYISSWVFAEDFQHWGQGTLVTVSS Sequence ID 60: HCDR1 by KABAT from Sequence ID 59 SYAMN Sequence ID 61: HCDR3 by KABAT from Sequence ID 59 DRGYISSWVFAEDFQH Sequence ID 62: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCTASGYTFTSYAMNWVRQAPGQRLEWMACVNPNTGSPTYAQGSTGRFVVSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGHGTLVTVSS Sequence ID: 63: HCDR2 by KABAT from Sequence ID: 62 CVNPNTGSPTYAQGSTG Sequence ID 64: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAMNWVRQAPGQGLEWMGWMNPNTGNPTYAQGSTGRFVVSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGRGTLVTVSS Sequence ID 65: HCDR1 by KABAT from Sequence ID 64 NYAMN Sequence ID 66: HCDR2 by KABAT from Sequence ID 64 WMNPNTGNPTYAQGSTG Sequence ID 67: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAINWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGRGTLVTVSS Sequence ID 68: HCDR1 by KABAT from Sequence ID 67 NYAIN Sequence ID 69: Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVKVSCKASGDTFNTYSITWVRQAPGQGLEWMGSIVPIFGTINNAQKFQGRVTITADKSANTAYMELSSLRSEDTAVYYCARDNTMVRGVDYYYMDVWGKGTMVTVSS Sequence ID 70: HCDR1 by KABAT from Sequence ID 69 TYSIT Sequence ID 71: HCDR2 by KABAT from Sequence ID 69 SIVPIFGTINNAQKFQG Sequence ID 72: HCDR3 by KABAT from Sequence ID 69 DNTMVRGVDYYYMDV Sequence ID 73: Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVKVSCKASGGIFSTYAISWVRQAPGQGLEWMGGIIPIFDTPNYAQKFQGRVTITADKSTSTAYMDLSSLRSEDTAVYYCAKNVRGYSAYDLDYWGQGTLVTVSS Sequence ID 74: HCDR1 by KABAT from Sequence ID 73 TYAIS Sequence ID 75: HCDR2 by KABAT from Sequence ID 73 GIIPIFDTPNYAQKFQG Sequence ID 76: HCDR3 by KABAT from Sequence ID 73 NVRGYSAYDLDY Sequence ID 77: Heavy Chain Variable Region EVQLVQSGAEVKNPGSSVKVSCKATGGTFNTYGTNWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTTTAYMEVSSLRSEDTAVYYCARGGADMGTLDYWGQGTLVTVSS Sequence ID 78: HCDR1 by KABAT from Sequence ID 77 TYGTN Sequence ID 79: HCDR2 by KABAT from Sequence ID 77 GIIPIFGTANYAQKFQG Sequence ID 80: HCDR3 by KABAT from Sequence ID 77 GGADMGTLDY Sequence ID 81: Heavy Chain Variable Region EVQLVQSGAEVMRPGSSVKVSCKASGGIFTYTIIWVRQAPGQGLEWMGGIIPIFDTPNFAQKFQGRLTITADKSTNTAYMELTSLRSEDTAVYYCAREGCNHGVCYPYWGQGTLVTVSS Sequence ID 82: HCDR1 by KABAT from Sequence ID 81 TYTII Sequence ID 83: HCDR2 by KABAT from Sequence ID 81 GIIPIFDTPNFAQKFQG Sequence ID 84: HCDR3 by KABAT from Sequence ID 81 EGCNHGVCYPY Sequence ID 85: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGDTFRSYGITWVRQAPGQGLEWMGGIIPIFGTTNYAQKFQGRVTITADKSTSTVYMELSSLRSEDTAVYYCARRRGYSNPHWLDPWGQGTLVTVSS Sequence ID 86: HCDR1 by KABAT from Sequence ID 85 SYGIT Sequence ID 87: HCDR2 by KABAT from Sequence ID 85 GIIPIFGTTNYAQKFQG Sequence ID 88: HCDR3 by KABAT from Sequence ID 85 RRGYSNPHWLDP Sequence ID 89: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYGILWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADISTSTAYMELSSLRSEDTAVYYCARGGGNYYEFVYWGQGTLVTVSS Sequence ID 90: HCDR1 by KABAT from Sequence ID 89 TYGIL Sequence ID 91: HCDR3 by KABAT from Sequence ID 89 GGGNYYEFVY Sequence ID 92: Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVRVSCKASGGTFNTYAINWVRQAPGQGLEWVGRIIPIFDTANYAQKFQGRVTISADKSTTTAYMELSSLRSEDTAVFYCAKDETGYSSSNFQHWGQGTLVTVSS Sequence ID 93: HCDR1 by KABAT from Sequence ID 92 TYAIN Sequence ID 94: HCDR2 by KABAT from Sequence ID 92 RIIPIFDTANYAQKFQG Sequence ID 95: HCDR3 by KABAT from Sequence ID 92 DETGYSSSNFQH Sequence ID 96: Heavy chain variable region QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAINWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDRKYVTNWVFAEDFQHWGQGTLVTVSS Sequence ID 97: Heavy Chain Variable Region QVQLVQSGAEVKRPGSSVKVSCKASGGTFNTYSITWVRQAPGQGLEWMGIIPVFGTSKYAQKFQDRVTITADKSTNTAYMELSSLRSEDTAVYYCARDPSFSSSSGWFDPWGQGTLVTVSS Sequence ID 98: HCDR2 by KABAT from Sequence ID 97 GIIPVFGTSKYAQKFQD Sequence ID 99: HCDR3 by KABAT from Sequence ID 97 DPSFSSSSGWFDP Sequence ID 100: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGGTFNTYAINWVRQAPGQGLEWMGGIIPIFDTANYAQRFQGRVTITADKSTSTAYMELSSLRSEDTAVYFCAKDQTGYSSTLFDYWGQGTLVTVSS Sequence ID 101: HCDR2 by KABAT from Sequence ID 100 GIIPIFDTANYAQRFQG Sequence ID 102: HCDR3 by KABAT from Sequence ID 100 DQTGYSSTLFDY Sequence ID 103: Heavy Chain Variable Region QVQLVQSGSELKKPGASVKVSCKASGYTFTSHAMNWVRQAPGQGLEWMGWINPNTGNPTYAQGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCAIDRGYMSNWVFAEYFPHWGQGTLVTVSS Sequence ID 104: Heavy Chain Variable Region EVQLVQSGAEVKKPGSSVKVSCKASGGTFSTYAISWVRQAPGQGLEWMGWIIPIFDTGNYAQKIQGRVTITADKSTSTAYMELTSLRSEDTAVYYCARHDYTNTVDAFDIWGQGTMVTVSS Sequence ID 105: HCDR2 by KABAT from Sequence ID 104 WIIPIFDTGNYAQKIQG Sequence ID 106: HCDR3 by KABAT from Sequence ID 104 HDYTNTVDAFDI Sequence ID 107: Heavy Chain Variable Region QVQLVQSGAEVKKPGSSVKVSCKASGDTFRSYGITWVRQAPGQGLEWMGGIIPVFGTTNYAQKFQGRVTITADKSTSTVFMELNSLRSEDTAVYYCARRRGYSNPHWLDPWGQGTLVTVSS Sequence ID 108: HCDR2 by KABAT from Sequence ID 107 GIIPVFGTTNYAQKFQG Sequence ID 109: Amino acid sequence of the human common light chain IGKV1-39 / jk1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 110: Amino acid sequence of the common light chain variable domain DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK Sequence ID 111: LCDR1 by KABAT from Sequence ID 110 RASQSISSYLN Sequence ID 112: LCDR2 by KABAT from Sequence ID 110 AASSLQS Sequence ID 113: LCDR3 by KABAT from Sequence ID 110 QQSYSTPPT Sequence ID 114: Amino acid sequence of the common light chain constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 115: Amino acid sequence of the light chain constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 116: Amino acid sequence of CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV Sequence ID 117: Amino acid sequence of the hinge EPKSCDKTHTCPPCP SEQ ID NO: 118: Amino acid sequence of CH2 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK Sequence ID 119: Amino acid sequence of CH3 with KK mutation GQPREPQVYTKPPSREEMTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 120: Amino acid sequence of CH3 with DE mutation GQPREPQVYTDPPSREEMTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

1. 1. A method for detecting the presence of clustering of at least two cell surface moieties in a sample, comprising a first cell surface moiety and a second cell surface moiety, the method comprising: contacting a sample in which the first and second cell surface moieties have been exposed to an agent having binding specificity for at least the first and second cell surface moieties with a first binding molecule that specifically binds to the first cell surface moiety and a second binding molecule that specifically binds to the second cell surface moiety, wherein at least one of the first binding molecule and the second binding molecule comprises a molecular tag that is not detectable unless the first and second cell surface moieties are in close proximity to one another; detecting the presence or absence of said molecular tag to detect said presence of clustering of said first cell surface moiety and said second cell surface moiety in said sample.

2. 1. A method for quantifying the presence of clustering of at least two cell surface moieties in a sample, the clustering comprising a first cell surface moiety and a second cell surface moiety, the method comprising: contacting a sample in which the first and second cell surface moieties have been exposed to an agent having binding specificity for at least the first and second cell surface moieties with a first binding molecule that specifically binds to the first cell surface moiety and a second binding molecule that specifically binds to the second cell surface moiety, wherein at least one of the first binding molecule and the second binding molecule comprises a molecular tag that is not detectable unless the first and second cell surface moieties are in close proximity to one another; measuring the amount of said molecular tag to quantify said presence of clustering of said first cell surface moiety and said second cell surface moiety in said sample.

3. The method comprises: - a) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety and a second binding molecule that specifically binds to the second cell surface moiety; contacting the first binding molecule with a molecular tag attached to the first binding molecule via a cleavable linker and the second binding molecule with a cleavage-inducing moiety; or b) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety and a second binding molecule that specifically binds to the second cell surface moiety; contacting, wherein the second binding molecule comprises a molecular tag attached to the second binding molecule via a cleavable linker, and the first binding molecule comprises a cleavage-inducing moiety; - inducing cleavage of said molecular tag; - detecting or quantifying clustering of the first cell surface moiety with the second cell surface moiety in the sample by detecting the presence or absence or measuring the amount of released molecular tags.

4. The method comprises: - a) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule; contacting the first binding molecule with a molecular tag attached to the first binding molecule via a cleavable linker, and the third binding molecule attached to the second binding molecule and comprising a cleavage-inducing moiety; or b) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule; contacting the second binding molecule, wherein the second binding molecule comprises a molecular tag attached to the second binding molecule via a cleavable linker, and the third binding molecule is attached to the first binding molecule and comprises a cleavage-inducing moiety; or c) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule; contacting, wherein the first binding molecule comprises a cleavage-inducing moiety and the third binding molecule is bound to the second binding molecule and comprises a molecular tag attached to the third binding molecule via a cleavable linker; or d) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule; contacting, wherein the second binding molecule comprises a cleavage-inducing moiety and the third binding molecule comprises a molecular tag bound to the first binding molecule and attached to the third binding molecule via a cleavable linker; - inducing cleavage of said molecular tag; - detecting or quantifying clustering of the first cell surface moiety with the second cell surface moiety in the sample by detecting the presence or absence or measuring the amount of released molecular tags.

5. The method comprises: - a) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety, a second binding molecule that specifically binds to the second cell surface moiety, a third binding molecule, and a fourth binding molecule; contacting the third binding molecule with the first binding molecule and comprising a molecular tag attached to the third binding molecule via a cleavable linker, and the fourth binding molecule with the second binding molecule and comprising a cleavage-inducing moiety; or b) contacting the sample with a first binding molecule that specifically binds to the first cell surface moiety, a second binding molecule that specifically binds to the second cell surface moiety, a third binding molecule, and a fourth binding molecule; contacting, wherein the third binding molecule is bound to the first binding molecule and comprises a cleavage-inducing moiety, and the fourth binding molecule is bound to the second binding molecule and comprises a molecular tag attached to the fourth binding molecule via a cleavable linker; - inducing cleavage of said molecular tag; - detecting or quantifying clustering of the first cell surface moiety with the second cell surface moiety in the sample by detecting the presence or measuring the amount of absence of released molecular tags.

6. The method according to any one of claims 1 to 5, wherein the sample is preferably a tissue sample, a blood sample or cultured cells from a subject or patient.

7. The method of claim 6, wherein the sample is a fresh sample or a fixed sample.

8. The method of any one of claims 1 to 7, wherein the sample is a tumor biopsy sample from a subject with cancer.

9. 1. A method for detecting expression of at least two cell surface moieties in a sample, comprising a first cell surface moiety and a second cell surface moiety, the method comprising: contacting a tumor biopsy sample from a subject with cancer with at least one binding molecule that detects a first cell surface moiety and at least one binding molecule that detects a second cell surface moiety; contacting, wherein at least one binding molecule that detects the first cell surface moiety and at least one binding molecule that detects the second cell surface moiety comprise a molecular tag; detecting the presence or absence of said molecular tag to detect said expression of said first cell surface moiety and said second cell surface moiety in said sample.

10. 1. A method for quantifying expression of at least two cell surface moieties in a sample, comprising a first cell surface moiety and a second cell surface moiety, the method comprising: contacting a tumor biopsy sample from a subject with cancer with at least one binding molecule that detects a first cell surface moiety and at least one binding molecule that detects a second cell surface moiety; contacting, wherein at least one binding molecule that detects the first cell surface moiety and at least one binding molecule that detects the second cell surface moiety comprise a molecular tag; measuring the amount of said molecular tag to quantify said expression of said first cell surface moiety and said second cell surface moiety in said sample.

11. The method of any one of claims 1 to 10, wherein the first and second cell surface moieties are expressed by the same cell or cell of the same type.

12. The method of any one of claims 1 to 10, wherein the first and second cell surface moieties are expressed by different cells or different types of cells.

13. 11. The method of any one of claims 1 to 10, wherein the first cell surface moiety is expressed by an immune effector cell, in particular an NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell or a neutrophil granulocyte, and the second cell surface moiety is expressed by a tumor cell or an immune cell.

14. The method of any one of claims 1 to 13, wherein at least one cell surface moiety is CD137 or another immune effector cell co-stimulatory moiety.

15. 15. The method of any one of claims 1 to 14, wherein at least one cell surface moiety is PD-L1 or another tumor-associated moiety or immune checkpoint moiety.

16. 16. The method of any one of claims 1 to 15, wherein at least one cell surface moiety is CD137 or another immune effector cell co-stimulatory moiety and at least one cell surface moiety is PD-L1 or another tumor-associated moiety or immune checkpoint moiety.

17. 17. The method of any one of claims 1 to 8 and 11 to 16, wherein the agent having binding specificity for the cell surface moiety is a multispecific antibody, such as a bispecific or trispecific antibody.

18. 18. The method of claim 17, wherein the multispecific antibody is a bispecific antibody that binds to CD137 or another immune effector cell costimulatory molecule and to PD-L1 or another tumor-associated moiety or immune checkpoint moiety.

19. 19. The method of claim 18, wherein the binding domain of the bispecific antibody that binds to CD137 comprises a heavy chain variable region having a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in any one of SEQ ID NOs: 4, 8, 12, 16, 19, 23, 27, 30, 34, 38, 42, 45, 48, or 52, which allows for 1, 2, or 3 amino acid substitutions, preferably 1 amino acid substitution.

20. 20. The method of claim 18 or 19, wherein the binding domain of the bispecific antibody that binds to CD137 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in any one of SEQ ID NOs: 2, 6, 10, 14, 18, 21, 25, 32, 36, 40, 44, or 50, which tolerates 1, 2, or 3 amino acid substitutions, preferably 1 amino acid substitution, and / or a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in any one of SEQ ID NOs: 3, 7, 11, 15, 22, 26, 29, 33, 37, 41, 47, or 51, which tolerates 1, 2, or 3 amino acid substitutions, preferably 1 amino acid substitution.

21. 21. The method of any one of claims 18 to 20, wherein the binding domain of the bispecific antibody that binds to CD137 comprises a heavy chain variable region having any one of SEQ ID NOs: 1, 5, 9, 13, 17, 20, 24, 28, 31, 35, 39, 43, 46, or 49, or at least 80%, 85%, 90%, 95%, 99% sequence identity thereto, in particular at least 80%, 85%, 90%, 95%, 99% sequence identity thereto, for the framework regions thereof.

22. 22. The method of any one of claims 18 to 21, wherein the binding domain of the bispecific antibody that binds to PD-L1 comprises a heavy chain variable region having a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in any one of SEQ ID NOs: 56, 58, 61, 72, 76, 80, 84, 88, 91, 95, 99, 102, or 106, which tolerates one, two, or three amino acid substitutions, preferably one amino acid substitution.

23. 23. The method of any one of claims 18 to 22, wherein the binding domain of the bispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in any one of SEQ ID NOs: 54, 60, 65, 68, 70, 74, 78, 82, 86, 90, or 93, which tolerates one, two, or three amino acid substitutions, preferably one amino acid substitution, and / or a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in any one of SEQ ID NOs: 55, 3, 63, 66, 71, 75, 79, 83, 87, 94, 98, 101, 105, or 108, which tolerates one, two, or three amino acid substitutions, preferably one amino acid substitution.

24. 24. The method of any one of claims 18 to 23, wherein the binding domain of the bispecific antibody that binds to PD-L1 comprises a heavy chain variable region having any one of SEQ ID NOs: 53, 57, 59, 62, 64, 67, 69, 73, 77, 81, 85, 89, 92, 96, 97, 100, 103, 104, or 107, or at least 80%, 85%, 90%, 95%, 99% sequence identity thereto, and in particular at least 80%, 85%, 90%, 95%, 99% sequence identity thereto to the framework regions thereof.

25. 1. A method for predicting the responsiveness of a subject, particularly a cancer patient, to an agent or agents that bind to a first cell surface moiety and a second cell surface moiety, particularly a moiety expressed on an immune effector cell and a moiety expressed on a tumor cell or an immune cell, the method comprising: - detecting the expression levels of a first cell surface moiety and a second cell surface member in a biological sample from a subject; - determining whether the expression levels of the first cell surface moiety and the second cell surface moiety in the subject's sample are above or below a threshold level; - predicting that the subject is likely to respond to an agent or agents that bind to the first cell surface moiety and the second cell surface moiety if the expression levels of the first cell surface moiety and the second cell surface moiety in the subject's sample are equal to or greater than the threshold level.

26. 26. The method of claim 25, wherein the expression levels of a first cell surface moiety and a second cell surface member are measured using a method according to any one of claims 9 to 16.

27. 1. A method for treating a subject in need of treatment, particularly a subject with cancer, said method comprising: - predicting the responsiveness of a subject to an agent or agents that bind to a first cell surface moiety and a second cell surface moiety using the method of claim 25 or 26; - administering to a subject likely to respond an agent or agents that bind to said first cell surface moiety and said second cell surface moiety.

28. 28. The method of any one of claims 25 to 27, wherein at least one cell surface moiety is CD137 or another immune effector cell co-stimulatory moiety.

29. 29. The method of any one of claims 25 to 28, wherein at least one cell surface moiety is PD-L1 or another tumor-associated moiety or immune checkpoint moiety.

30. 30. The method of any one of claims 25 to 29, wherein at least one cell surface moiety is CD137 and at least one cell surface moiety is PD-L1.

31. 31. The method of any one of claims 25 to 30, wherein the agent that binds to the cell surface moiety is a multispecific antibody, such as a bispecific or trispecific antibody, in particular a bispecific antibody, that specifically binds to CD137 or another immune effector cell co-stimulatory moiety and PD-L1 or another tumor-associated moiety or immune checkpoint moiety according to any one of claims 17 to 24.

32. 25. A method for determining the effectiveness of a drug, wherein the drug comprises at least a binding domain that specifically binds to a first cell surface moiety and a binding domain that specifically binds to a second cell surface moiety, the method comprising detecting clustering of the first cell surface moiety with the second cell surface moiety in a biological sample of a subject being treated with the drug by using the method of any one of claims 1 to 24.

33. 25. A method for determining the effectiveness of a drug, wherein the drug comprises at least a binding domain that specifically binds to a first cell surface moiety and a binding domain that specifically binds to a second cell surface moiety, the method comprising quantifying clustering of the first cell surface moiety with the second cell surface moiety in a biological sample of a subject being treated with the drug by using the method of any one of claims 1 to 24.

34. 25. A method for ascertaining the mode of action of a drug, wherein the drug comprises at least a binding domain that specifically binds to a first cell surface moiety and a binding domain that specifically binds to a second cell surface moiety, the method comprising detecting clustering of the first cell surface moiety with the second cell surface moiety in a biological sample of a subject being treated with the drug by using the method of any one of claims 1 to 24.

35. 25. A method for ascertaining the mode of action of a drug, wherein the drug comprises at least a binding domain that specifically binds to a first cell surface moiety and a binding domain that specifically binds to a second cell surface moiety, the method comprising quantifying clustering of the first cell surface moiety with the second cell surface moiety in a biological sample of a subject being treated with the drug by using the method of any one of claims 1 to 24.

36. 36. The method of claim 34 or 35, wherein the mode of action is simultaneous binding of the agent to the first and second cell surface moieties.

37. 36. The method of claim 34 or 35, wherein the mode of action is clustering of two or more cell surface moieties.

38. 38. The method of claim 37, wherein the clustering is of two or more cell surface moieties expressed on immune effector cells, in particular the clustering of two or more CD137 proteins.

39. 1. A method for treating a subject in need of treatment, particularly a subject with cancer, said method comprising: - treating the subject with an agent that binds to the first cell surface moiety and the second cell surface moiety; - Analysing the efficacy of the drug or the mode of action of the drug using a method according to any one of claims 32 to 38.

40. 40. The method of claim 39, further comprising continuing or adapting the treatment based on the results of the analysis or confirmation.

41. 1. A method for screening one or more test agents for the ability to induce clustering of at least a first cell surface moiety with a second cell surface moiety, said method comprising: contacting one or more test cell cultures with a test agent, contacting the test cell culture, wherein the test cell culture comprises cells expressing a first cell surface moiety and cells expressing a second cell surface moiety; - detecting or quantifying the level of clustering of said first and second cell surface moieties using a method according to any one of claims 1 to 24; - comparing said level of clustering to the level of clustering detected for said clustering in a control cell culture not contacted with said test agent or not contacted with a reference agent, The method, wherein the control cell culture comprises the first cell surface moiety and the second cell surface moiety.

42. 42. The method of claim 41, further comprising selecting a test agent that induces a level of clustering that is equal to or greater than the level of clustering in the control cell culture.

43. 43. The method of claim 41 or 42, wherein at least one cell surface moiety is CD137 or another immune effector costimulatory moiety.

44. 44. The method of any one of claims 41 to 43, wherein at least one cell surface moiety is PD-L1 or another tumor-associated moiety or immune checkpoint molecule.

45. 45. The method of any one of claims 41 to 44, wherein at least one cell surface moiety is CD137 and at least one cell surface moiety is PD-L1.

46. The method of any one of claims 41 to 45, wherein the reference agent is a multispecific antibody that specifically binds to CD137 or another immune effector co-stimulatory moiety and PD-L1 or another tumor-associated moiety or immune checkpoint moiety according to any one of claims 17 to 24.

47. 1. A kit of parts comprising: at least two binding molecules that specifically bind to first and second cell surface moieties, optionally one of said binding molecules comprising a molecular tag attached to said one via a cleavable linker and the other binding molecule comprising a cleavage-inducing moiety; and instructions for contacting a patient sample with said at least two binding molecules, optionally inducing cleavage of said molecular tag, and detecting the presence or absence of a signal induced by contacting said patient sample with said at least two binding molecules.

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