Method for detecting expression or clustering of cell surface moieties

Abstract

To provide a robust method of demonstrating the capacity of two or more antigens to cluster in the presence of a multispecific agent.SOLUTION: The present disclosure relates to a method for detecting and / or quantifying expression of at least a first cell surface moiety and of a second cell surface moiety in a patient sample, and to a method for detecting and / or quantifying clustering of at least a first cell surface moiety with a second cell surface moiety in a sample. The present disclosure further relates to a method for predicting the responsiveness of a subject to such a binding molecule, a method for determining the effectiveness of such a binding molecule, a method for confirming the mode of action of such a binding molecule, a method for treating a subject, and a method 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates preferably to methods for detecting and / or quantifying the expression or clustering of at least a first and a second cell surface moiety in a patient tumor sample, hi certain embodiments, the methods of the present disclosure can be used to predict whether a patient is likely to benefit from treatment with a binding agent that binds to both cell surface moieties, or to ascertain the mode of action of such a binding agent. [Background technology]

[0002] The development of therapeutic antibodies for the treatment of cancer has increased rapidly in the last few years. For various cancer types, diagnostic assays have been used to assess whether a particular patient will benefit from treatment with a particular drug, so that it can be predicted that it is likely to be safe and / or effective. One category of diagnostic assays that has been used with biologics or large molecule therapeutics involves testing the expression level of the antigen targeted by the biologic in a patient's tissue sample. For example, a tissue biopsy can be taken from the patient's tumor and subjected to a quantitative assay. Examples of such quantitative assays include immunohistochemistry (IHC), double in situ hybridization assays, chromogenic in situ hybridization (CISH) assays, and fluorescent in situ hybridization (FISH) assays.

[0003] IHC has been the standard test method for the assessment of, for example, HER2 expression, for example in breast cancer. IHC tests utilize specific monoclonal or polyclonal antibodies that bind to the HER2 protein on the cell surface. The addition of a secondary tagged antibody with a reporter function, followed by an enzymatic reaction, results in a signal that is 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 in situ hybridization assays use single or dual probe technology to quantify gene copy number per cell. FISH has become a widely accepted platform for HER2 testing, for example. However, FISH assays are expensive, labor intensive, and require fluorescence microscopy and advanced training. Bright-field in situ hybridization assays, such as CISH and silver-enhanced in situ hybridization, do not require fluorescence microscopy and are less expensive.

[0005] Another development is the validation of assays as methods for measuring total HER2, HER2 homodimer, or p95HER2 expression in, for example, breast cancer, which are proximity-based assays designed to quantify protein expression, dimerization, and protein-protein interactions (Diagn Mol Pathol 2009;18:11-21; described in detail in Shi et al.). Summary of the Invention [Problem to be solved by the invention]

[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 a multispecific agent, where such antigens do not normally associate under somatic cell conditions. Here, we demonstrate such an assay, and an exemplary application of its use. [Means for solving the problem]

[0007] The inventors have developed an assay to detect and quantify the expression levels of CD137, a cell surface moiety expressed on T cells, and PD-L1, a cell surface moiety expressed on tumor cells. This allows prediction of whether a particular patient is likely to respond to and benefit from a treatment that binds these two cell surface moieties. In certain embodiments, other applicable methods based on measuring a signal produced when two cell surface moieties are present in the same sample, such as the assay used herein, may be used. In this context, the signal may be the presence or absence of a signal. In certain embodiments, both such readouts provide information regarding the expression level of the cell surface moiety. This method may also be used to detect and / or quantify the expression level of any two or more cell surface moieties that may be bound by a particular drug, such as a multispecific agent, such as a bispecific or trispecific antibody.

[0008] The present inventors have further developed assays used to detect and quantify CD137 and its clustering with PD-L1. CD137 and PD-L1 do not form a cognate receptor-ligand pair, and they do not naturally cluster or form direct interactions between proteins. However, when targeted by a drug that simultaneously binds to both cell surface moieties, such as a multispecific agent, such as a bispecific or trispecific antibody, these two cell surface moieties are brought into close proximity to each other, thereby producing a signal that can be detected. Similarly, when targeted by a multivalent drug that binds to at least two of the same cell surface moieties, such as a monospecific bivalent agent, such as a monospecific antibody, or a multispecific agent, such as a bispecific or trispecific antibody, at least two cell surface moieties are brought into close proximity to each other, thereby producing a signal that can be detected.

[0009] In certain embodiments, the present 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 tumor cells and / or cells from the immune system. The multispecific agents thereby induce clustering of the two or more target antigens. Thus, clustering of the 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 with which the patient is being treated actually binds to two cell surface moieties simultaneously and exhibits its predicted mode of action. In certain embodiments, other applicable methods based on measuring the signal produced when two cell surface moieties are in close proximity, such as the assay used herein, can be used. In this context, the signal can be the presence or absence of a reporter or feature of the assay. In certain embodiments, both such readouts (presence or absence of reporters) provide information about the proximity of the cell surface moieties. This method can also be used to detect and / or quantify the clustering of any two or more cell surface moieties that can be simultaneously bound to a particular drug, such as, for example, a multispecific agent, such as a bispecific or trispecific antibody.

[0011] In certain embodiments, the present disclosure provides a method for detecting and / or quantifying 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 a first and a second cell surface moiety have been exposed to an agent having binding specificity for at least the first and the 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, where 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 the molecular tag to detect the presence of clustering of the first cell surface moiety and the second cell surface moiety in the sample.

[0012] The present disclosure also provides a method for detecting and / or quantifying expression of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, in a sample, 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 a first cell surface moiety and at least one binding molecule that detects a second cell surface moiety comprise a molecular tag; and detecting and / or quantifying the presence or absence of molecular tags to detect expression of a first cell surface moiety and a second cell surface moiety in a sample.

[0013] The disclosure further provides 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, the method comprising: - detecting and / or quantifying the expression levels of a first cell surface moiety and a second cell surface moiety in a biological sample from a subject; - determining whether the expression levels of a first cell surface moiety and a second cell surface moiety in the subject's sample are above or below a threshold level; and - 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 a threshold level.

[0014] The present disclosure further provides a method for treating a subject in need of treatment having cancer, the method comprising: - predicting the responsiveness of a subject, in particular a subject with cancer, to an agent or agents that bind to the first and second cell surface moieties described herein; and administering to a subject likely to respond an agent or agents that bind to a first cell surface moiety and a second cell surface moiety.

[0015] The present disclosure further relates to a method for determining the effectiveness of a drug, the drug comprising a binding molecule comprising 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 and / or 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, as described herein.

[0016] The disclosure further relates to a method for ascertaining a mode of action of an agent, the agent comprising a binding molecule comprising 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 and / or 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 agent, as described herein.

[0017] The present disclosure further provides a method for treating a subject in need of treatment, particularly a subject having cancer, the method comprising: - treating a subject in need of treatment with an agent that binds to a first cell surface moiety and a second cell surface moiety; - Analysing the efficacy of a drug or the mode of action of a drug as described herein.

[0018] The disclosure further provides a method for screening one or more test agents for an 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, contacting, wherein the test cell culture comprises cells expressing a first cell surface moiety and cells expressing a second cell surface moiety; detecting a level of clustering of the first and second cell surface moieties as described herein; and comparing the level of clustering to the level of clustering detected in a control cell culture not contacted with the test agent or not contacted with a reference agent; The method relates to a method in which the control cell culture comprises a first cell surface moiety and a second cell surface moiety. [Brief description of the drawings]

[0019] [Figure 1] 1 is a schematic diagram of the VeraTag® assay concept used in accordance with certain embodiments of the present disclosure. The VeraTag® assay format in this figure utilizes two primary antibodies that bind to one or two specific antigens, one of which contains a tag and the other contains a cleavage-inducing moiety (scissors symbol). Upon photoactivation, the cleavage-inducing moiety is released from one of the antibodies, which 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. [Diagram 2]Schematic diagram of an example of a format of a VeraTag® assay using a primary antibody. In this figure, the first and second cell surface moieties are different cell surface moieties. However, the present disclosure also includes embodiments in which the first and second cell surface moieties are the same. A) A first and second binding molecule that specifically binds to a first moiety expressed on a cell surface, and a third and fourth binding molecule that specifically binds to a second moiety expressed on a cell surface, where the first binding molecule comprises a first molecular tag attached to the first binding molecule via a cleavable linker, the second binding molecule comprises a cleavage-inducing moiety (scissors symbol), the third binding molecule comprises a second molecular tag attached to the third binding molecule via a cleavable linker, and the fourth binding molecule comprises a cleavage-inducing moiety (scissors symbol). B) A first and second binding molecule that specifically bind to a first moiety expressed on the cell surface, and a third and fourth binding molecule that specifically bind to a second moiety expressed on the cell surface, where the second binding molecule comprises a first molecular tag attached to the second binding molecule via a cleavable linker, the first binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fourth binding molecule comprises a second molecular tag attached to the fourth binding molecule via a cleavable linker, and the third binding molecule comprises a cleavage-inducing moiety (scissors symbol). C) first and second binding molecules that specifically bind to a first moiety expressed on the cell surface, and third and fourth binding molecules that specifically bind to a second moiety expressed on the cell surface, where the first binding molecule comprises a first molecular tag attached to the first binding molecule via a cleavable linker, the second binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fourth binding molecule comprises a second molecular tag attached to the fourth binding molecule via a cleavable linker, and the third binding molecule comprises a cleavage-inducing moiety (scissors symbol).D) A first and second binding molecule that specifically binds to a first portion expressed on the cell surface, and a third and fourth binding molecule that specifically binds to a second portion expressed on the cell surface, where the second binding molecule comprises a first molecular tag attached to the second binding molecule via a cleavable linker, where the first binding molecule comprises a cleavage-inducing moiety (scissors symbol), where the third binding molecule comprises a second molecular tag attached to the third binding molecule via a cleavable linker, and where the fourth binding molecule comprises a cleavage-inducing moiety (scissors symbol). [Diagram 3]1 is a schematic diagram of an example of a VeraTag® assay format using two primary and secondary antibodies for each target. In this diagram, the first and second cell surface moieties are different cell surface moieties. However, the present disclosure also includes embodiments in which the first and second cell surface moieties are the same. A) A first and second binding molecule that specifically binds to a first moiety expressed on the cell surface, a third and fourth binding molecule that specifically binds to a second moiety expressed on the cell surface, a fifth binding molecule, and a sixth binding molecule, where the first and third binding molecules comprise a first and second molecular tag, respectively, attached thereto via a cleavable linker, the fifth binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the fourth binding molecule and comprises a cleavage-inducing moiety (scissors symbol). B) a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 comprise a first and a second molecular tag, respectively, attached thereto via a cleavable linker, the fifth binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the third binding molecule and comprises a cleavage-inducing moiety (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 comprise a cleavage-inducing moiety (scissors symbol), the fifth binding molecule is bound to the second binding molecule and comprises a first molecular tag bound to the fifth binding molecule via a cleavable linker, and the sixth binding molecule is bound to the fourth binding molecule and comprises a second molecular tag bound to the sixth binding molecule via a cleavable linker.D) a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 comprise a cleavage-inducing moiety (scissors symbol), the fifth binding molecule comprises a first molecular tag bound to the first binding molecule and bound to the fifth binding molecule via a cleavable linker, and the sixth binding molecule comprises a second molecular tag bound to the third binding molecule and 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, where the first binding molecule comprises a first molecular tag attached thereto via a cleavable linker, the fourth binding molecule comprises a second molecular tag attached thereto via a cleavable linker, the fifth binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the third binding molecule and comprises a cleavage-inducing moiety (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 comprises a first molecular tag attached thereto via a cleavable linker, the third binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fifth binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the fourth binding molecule and comprises a second molecular tag attached 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 comprises a first molecular tag attached thereto via a cleavable linker, the fourth binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fifth binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the third binding molecule and comprises a second molecular tag attached thereto via a cleavable linker. H) A first and second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and fourth binding molecule 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 comprises a first molecular tag attached thereto via a cleavable linker, the third binding molecule comprises a second molecular tag attached thereto via a cleavable linker, the fifth binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the fourth binding molecule and comprises a cleavage-inducing moiety (scissors symbol). I) a first and second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and fourth binding molecule 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 comprises a first molecular tag attached thereto via a cleavable linker, the third binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fifth binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker.J) A first and second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and fourth binding molecule 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 comprises a first molecular tag attached thereto via a cleavable linker, the fourth binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fifth binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the sixth binding molecule binds to the third binding molecule and comprises a second molecular tag attached thereto via a cleavable linker. K) A first and second binding molecule that specifically bind to a first portion expressed on a cell surface, a third and fourth binding molecule 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 comprises a cleavage-inducing moiety (scissors symbol), the third binding molecule comprises a second molecular tag attached thereto via a cleavable linker, the fifth binding molecule binds to the second binding molecule and comprises the first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the fourth binding molecule and comprises the cleavage-inducing moiety (scissors symbol). 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 comprises a cleavage-inducing moiety (scissors symbol), the fourth binding molecule comprises a second molecular tag attached thereto via a cleavable linker, the fifth binding molecule binds to the second binding molecule and comprises the first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the third binding molecule and comprises the cleavage-inducing moiety (scissors symbol).M) a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 and the fourth binding molecule comprise a cleavage-inducing moiety (scissors symbol), the fifth binding molecule binds to the second binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the third binding molecule and comprises a second molecular tag attached thereto via a cleavable linker. N) a first and second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and fourth binding molecule 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 comprises a cleavage-inducing moiety (scissors symbol), the third binding molecule comprises a second molecular tag attached thereto via a cleavable linker, the fifth binding molecule binds to the first binding molecule and comprises the first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the fourth binding molecule and comprises the cleavage-inducing moiety (scissors symbol). O) a first and a second binding molecule that specifically bind to a first portion expressed on a cell surface, a third and a fourth binding molecule that specifically bind to a second portion expressed on a cell surface, a fifth binding molecule, and a sixth binding molecule, wherein the second binding molecule comprises a cleavage-inducing moiety (scissors symbol), the fourth binding molecule comprises a second molecular tag attached thereto via a cleavable linker, the fifth binding molecule binds to the first binding molecule and comprises the first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the third binding molecule and comprises the cleavage-inducing moiety (scissors symbol). 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 comprise a cleavage-inducing moiety (scissors symbol), the fifth binding molecule binds to the first binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker. [Figure 4]1 is a schematic diagram of an example of a VeraTag® assay format using two primary antibodies and two secondary antibodies for each target. In this diagram, the first and second cell surface moieties are different cell surface moieties. However, the present disclosure also includes embodiments in which the first and second cell surface moieties are the same. A) A first and second binding molecule that specifically binds to a first moiety expressed on the cell surface, a third and fourth binding molecule that specifically binds to a second moiety expressed on the cell surface, a fifth binding molecule, a sixth binding molecule, a seventh binding molecule, and an eighth binding molecule, where the fifth binding molecule binds to the first binding molecule and includes a first molecular tag attached thereto via a cleavable linker, the seventh binding molecule binds to the second binding molecule and includes a cleavage-inducing moiety (scissors symbol), the sixth binding molecule binds to the third binding molecule and includes a second molecular tag attached thereto via a cleavable linker, and the eighth binding molecule binds to the fourth binding molecule and includes a cleavage-inducing moiety (scissors symbol). B) a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 comprises a cleavage-inducing moiety (scissors symbol), the seventh binding molecule binds to the second binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, the sixth binding molecule binds to the third binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the eighth binding molecule binds to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker.C) a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 comprises a first molecular tag attached thereto via a cleavable linker, the seventh binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol), the sixth binding molecule binds to the third binding molecule and comprises a cleavage-inducing moiety (scissors symbol), and the eighth binding molecule binds to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker. D) a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 comprises a cleavage-inducing moiety (scissors symbol), the seventh binding molecule binds to the second binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, the sixth binding molecule binds to the third binding molecule and comprises a second molecular tag attached thereto via a cleavable linker, and the eighth binding molecule binds to the fourth binding molecule and comprises a cleavage-inducing moiety (scissors symbol). [Diagram 5]1 is a schematic diagram of an example of a format of the VeraTag® assay 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 figure, the first and second cell surface moieties are different cell surface moieties. However, the present disclosure also includes embodiments in which the first and second cell surface moieties are the same. A) A first binding molecule that specifically binds to a first moiety expressed on the cell surface, a second binding molecule that specifically binds to a second moiety expressed on the cell surface, and a third binding molecule that binds to the first binding molecule, wherein the second binding molecule comprises the first molecular tag attached thereto via a cleavable linker, and the third binding molecule comprises the second molecular tag attached thereto 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 comprises a first molecular tag attached thereto via a cleavable linker and the third binding molecule comprises a second molecular tag attached thereto via a cleavable linker. [Figure 6]FIG. 1 is a schematic diagram of an example of a VeraTag® assay format using one primary antibody for each target to detect and / or quantify target clustering. In this diagram, the first and second cell surface moieties are different cell surface moieties expressed on different cells. However, the present disclosure also includes embodiments in which the first and second cell surface moieties are present on the same cell. Also in this diagram, the molecule having binding specificity for the first and second cell surface moieties is a bivalent bispecific antibody. However, the present disclosure also encompasses the situation in which the molecule having binding specificity for the cell surface moiety is a multivalent bispecific or multispecific antibody, such as a trispecific or tetraspecific antibody. A) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, the first binding molecule comprising a molecular tag attached thereto via a cleavable linker, and the second binding molecule comprising a cleavage-inducing moiety (scissors symbol). B) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, the second binding molecule comprising a molecular tag attached thereto via a cleavable linker, and the first binding molecule comprising a cleavage-inducing moiety. [Figure 7]FIG. 1 is a schematic diagram of an example of a VeraTag® assay format that uses one primary antibody for each target and a secondary antibody against one of the primary antibodies to detect and / or quantify clustering of targets. In this diagram, the first and second cell surface moieties are different cell surface moieties expressed on different cells. However, the present disclosure also includes embodiments in which the first and second cell surface moieties are present on the same cell. Also in this diagram, the molecule having binding specificity for the first and second cell surface moieties is a bivalent bispecific antibody. However, the present disclosure also encompasses the situation in which the molecule having binding specificity for the cell surface moiety is a multivalent bispecific or multispecific antibody, such as a trispecific or tetraspecific antibody. A) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, where the first binding molecule comprises a molecular tag attached to it via a cleavable linker and the third binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol). B) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, where the second binding molecule comprises a molecular tag attached to it via a cleavable linker and the third binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety (scissors symbol). C) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, where the first binding molecule comprises a cleavage-inducing moiety (scissors symbol) and the third binding molecule binds to the second binding molecule and comprises a molecular tag attached thereto via a cleavable linker.D) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, where the second binding molecule comprises a cleavage-inducing moiety (scissors symbol) and the third binding molecule binds to the first binding molecule and comprises a molecular tag attached to it via a cleavable linker. [Figure 8]Schematic diagram of a VeraTag® assay format using one primary antibody for each target and two secondary antibodies against the primary antibodies to detect and / or quantify target clustering. In this diagram, the first and second cell surface moieties are different cell surface moieties expressed on different cells. However, the disclosure extends to where the first and second cell surface moieties are present on the same cell. Also in this diagram, the molecule having binding specificity for the first and second cell surface moieties is a bivalent bispecific antibody. However, the disclosure also encompasses the situation where the molecule having binding specificity for the cell surface moiety is a multivalent bispecific or multispecific antibody, for example a trispecific or tetraspecific antibody. A) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, thereby bringing the first and second cell surface moieties into close proximity to one another, 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, where the third binding molecule binds to the first binding molecule and comprises a molecular tag attached to it via a cleavable linker, and the fourth binding molecule binds to the second binding molecule and comprises a cleavage-inducing moiety (scissors symbol). B) A bispecific antibody (100) that binds to a first cell surface moiety and a second cell surface moiety, whereby the first and second cell surface moieties are in close proximity to one another, 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, where the third binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety (scissors symbol) and the fourth binding molecule binds to the second binding molecule and comprises a molecular tag attached thereto via a cleavable linker. [Figure 9]Figure 2 shows PD-L1 expression levels 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 CD137 and PD-L1, Sample C: cell pellet prepared by incubation with a negative control antibody that binds RSV. [Figure 10] Figure 1 shows CD137 expression levels in relative peak area (RPA) 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 an anti-CD137 positive control antibody, Sample B: cell pellet prepared by incubation with a bispecific antibody that binds CD137 and PD-L1, Sample C: cell pellet prepared by incubation with a negative control antibody that binds RSV. [Figure 11] Graph showing CD137 clustering in relative peak area (RPA) 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 RSV, Sample B: cell pellet prepared by incubation with a bispecific antibody that binds CD137 and PD-L1, Sample C: cell pellet prepared by incubation with an anti-CD137 positive control antibody. [Figure 12]Figure 1 shows 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 RSV, Sample B: cell pellet prepared by incubation with a bispecific antibody that binds 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 control experiment (ITC). [Figure 13] A - an assay described herein for detecting expression of a receptor (antigen 1) present on the cell membrane, B - a proximity assay described herein for detecting clustering of receptors (antigen 1) on the same cell, and C - a proximity assay described herein for detecting clustering of a receptor (antigen 1) on one cell with a receptor (antigen 2) on another cell, thereby forming an immunological synapse. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In one aspect, the disclosure provides a method for detecting and / or quantifying the expression of at least a first and a second cell surface moiety in a sample, the method comprising detecting a signal generated when the first and second cell surface moieties are expressed in the same sample. In certain embodiments, preferably, two different signals are generated so that it is possible to quantify the expression of each cell surface moiety. Alternatively, in certain embodiments, the signal generating moieties are selected such that the combined signal provides information regarding the expression level of each cell surface moiety.

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

[0022] Methods according to the present disclosure can be carried out in different formats. In one format, the present disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: a) contacting a sample 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 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, the binding molecule comprising a molecular tag, and optionally, the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; and b) detecting the presence or absence or measuring the amount of the molecular tag to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0023] In another format, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: a) contacting a sample with at least two binding molecules that detect a first cell surface moiety and at least two binding molecules that detect a second cell surface moiety; contacting, wherein one of the binding molecules that detect the first cell surface moiety and one of the binding molecules that detect the second cell surface moiety preferably comprises a molecular tag attached thereto via a cleavable linker, and optionally another of the binding molecules that detect the first cell surface moiety and another of the binding molecules that detect the second cell surface moiety comprises a cleavage-inducing moiety; b) optionally inducing cleavage of the molecular tag; and c) detecting the presence or absence or measuring the amount of the molecular tag to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0024] In certain embodiments, the method according to the present disclosure can be used to measure the co-expression of at least two different cell surface moieties in a single sample, preferably a patient sample.Therefore, this is particularly useful for predicting the response of a patient, preferably a cancer patient, to treatment with an agent or agents that bind to at least two different cell surface moieties.An example of such an agent is, for example, a multispecific antibody.

[0025] In certain embodiments, the methods used in the present disclosure include the VeraTag® assay, which is well known in the art and 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 formats: One format is a proximity assay that uses two primary binding molecules for each target moiety, one of which contains a molecular tag and the other of which contains a cleavage-inducing moiety.

[0027] Thus, in one embodiment, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: 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, contacting a first binding molecule comprising a first molecular tag attached thereto via a cleavable linker, a second binding molecule comprising a cleavage-inducing moiety, a third binding molecule comprising a second molecular tag attached thereto via a cleavable linker, and a fourth binding molecule comprising a cleavage-inducing moiety; 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 comprising a first molecular tag attached thereto via a cleavable linker, the first binding molecule comprising a cleavage-inducing moiety, a fourth binding molecule comprising a second molecular tag attached thereto via a cleavable linker, and a third binding molecule comprising a cleavage-inducing moiety; 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, contacting a first binding molecule comprising a first molecular tag attached thereto via a cleavable linker, a second binding molecule comprising a cleavage-inducing moiety, a fourth binding molecule comprising a second molecular tag attached thereto via a cleavable linker, and a third binding molecule comprising a cleavage-inducing moiety; or a4) contacting the sample with first and second binding molecules that specifically bind to a first portion expressed on the cell surface, and with third and fourth binding molecules that specifically bind to a second portion expressed on the cell surface, a second binding molecule comprising a first molecular tag attached thereto via a cleavable linker, the first binding molecule comprising a cleavage-inducing moiety, a third binding molecule comprising a second molecular tag attached thereto via a cleavable linker, and a fourth binding molecule comprising a cleavage-inducing moiety; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the first and second molecular tags; and and c) detecting the presence or absence of the released first and second molecular tags to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0028] Another format is a proximity assay, which uses two primary binding molecules for each target moiety, and a secondary binding molecule directed against one of the primary binding molecules.

[0029] Thus, in one embodiment, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: 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; a first and a third binding molecule comprising a first and a second molecular tag, respectively, attached thereto via a cleavable linker, a fifth binding molecule attached to the second binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule attached to the fourth binding molecule and comprising a cleavage-inducing moiety; 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; a second and a fourth binding molecule comprising the first and second molecular tags, respectively, attached thereto via a cleavable linker, a fifth binding molecule attached to the first binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule attached to the third binding molecule and comprising a cleavage-inducing moiety; 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, wherein the first and third binding molecules comprise a cleavage-inducing moiety, the fifth binding molecule is bound to the second binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule is bound to the fourth binding molecule and comprises 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, and a sixth binding molecule; contacting, wherein the second and fourth binding molecules comprise a cleavage-inducing moiety, the fifth binding molecule binds to the first binding molecule and a first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the third binding molecule and comprises a second molecular tag attached thereto 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 comprising a first molecular tag attached thereto via a cleavable linker, a fourth binding molecule comprising a second molecular tag attached thereto via a cleavable linker, a fifth binding molecule attached to the second binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule attached to the third binding molecule and comprising a cleavage-inducing moiety; 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; a first binding molecule comprising a first molecular tag attached thereto via a cleavable linker, a third binding molecule comprising a cleavage-inducing moiety, a fifth binding molecule bound to a second binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule bound to a fourth binding molecule and comprising a second molecular tag attached 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; a first binding molecule comprising a first molecular tag attached thereto via a cleavable linker, a fourth binding molecule comprising a cleavage-inducing moiety, a fifth binding molecule bound to a second binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule bound to a third binding molecule and comprising a second molecular tag attached 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 comprising a first molecular tag attached thereto via a cleavable linker, a third binding molecule comprising a second molecular tag attached thereto via a cleavable linker, a fifth binding molecule attached to the first binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule attached to the fourth binding molecule and comprising a cleavage-inducing moiety; 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; a second binding molecule comprising a first molecular tag attached thereto via a cleavable linker, a third binding molecule comprising a cleavage-inducing moiety, a fifth binding molecule bound to the first binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule bound to the fourth binding molecule and comprising a second molecular tag attached thereto via a cleavable linker; 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; a second binding molecule comprising a first molecular tag attached thereto via a cleavable linker, a fourth binding molecule comprising a cleavage-inducing moiety, a fifth binding molecule bound to the first binding molecule and comprising a cleavage-inducing moiety, and a sixth binding molecule bound to the third binding molecule and comprising a second molecular tag attached thereto 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 comprising a cleavage-inducing moiety, a third binding molecule comprising a second molecular tag attached thereto via a cleavable linker, a fifth binding molecule bound to the second binding molecule and comprising the first molecular tag attached thereto via a cleavable linker, and a sixth binding molecule bound to the fourth binding molecule and comprising the cleavage-inducing moiety; 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 comprising a cleavage-inducing moiety, a fourth binding molecule comprising a second molecular tag attached thereto via a cleavable linker, a fifth binding molecule bound to the second binding molecule and comprising the first molecular tag attached thereto via a cleavable linker, and a sixth binding molecule bound to the third binding molecule and comprising the cleavage-inducing moiety; 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 a first binding molecule and a fourth binding molecule comprising a cleavage-inducing moiety, a fifth binding molecule bound to a second binding molecule and comprising a first molecular tag attached thereto via a cleavable linker, and a sixth binding molecule bound to a third binding molecule and comprising a second molecular tag attached thereto via a cleavable linker; 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 comprising a cleavage-inducing moiety, a third binding molecule comprising a second molecular tag attached thereto via a cleavable linker, a fifth binding molecule bound to the first binding molecule and comprising the first molecular tag attached thereto via a cleavable linker, and a sixth binding molecule bound to the fourth binding molecule and comprising the cleavage-inducing moiety; 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 comprising a cleavage-inducing moiety, a fourth binding molecule comprising a second molecular tag attached thereto via a cleavable linker, a fifth binding molecule bound to the first binding molecule and comprising the first molecular tag attached thereto via a cleavable linker, and a sixth binding molecule bound to the third binding molecule and comprising the cleavage-inducing moiety; 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 comprise a cleavage-inducing moiety, the fifth binding molecule binds to the first binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, and the sixth binding molecule binds to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the first and second molecular tags; and and c) detecting the presence or absence of the released first and second molecular tags to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0030] Another format is a proximity assay that uses two primary binding molecules for each target moiety and a secondary binding molecule for each of the primary binding molecules, one of the secondary binding molecules for each target moiety containing a molecular tag and the other containing a cleavage-inducing moiety.

[0031] Thus, in one embodiment, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: 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 comprises a first molecular tag attached thereto via a cleavable linker, a seventh binding molecule is bound to the second binding molecule and comprises a cleavage-inducing moiety, a sixth binding molecule is bound to the third binding molecule and comprises a second molecular tag attached thereto via a cleavable linker, and an eighth binding molecule is bound to the fourth binding molecule and comprises a cleavage-inducing moiety; 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; a fifth binding molecule is bound to the first binding molecule and comprises a cleavage-inducing moiety, a seventh binding molecule is bound to the second binding molecule and comprises a first molecular tag attached thereto via a cleavable linker, a sixth binding molecule is bound to the third binding molecule and comprises a cleavage-inducing moiety, and an eighth binding molecule is bound to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker; or a3) contacting the sample with a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 comprises a first molecular tag attached thereto via a cleavable linker, a seventh binding molecule is bound to the second binding molecule and comprises a cleavage-inducing moiety, a sixth binding molecule is bound to the third binding molecule and comprises a cleavage-inducing moiety, and an eighth binding molecule is bound to the fourth binding molecule and comprises a second molecular tag attached thereto via a cleavable linker; or a4) contacting the sample with a first and a second binding molecule that specifically bind to a first portion expressed on the cell surface, a third and a fourth binding molecule 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 binds to the first binding molecule and comprises a cleavage-inducing moiety; a seventh binding molecule binds to the second binding molecule and comprises a first molecular tag attached thereto via a cleavable linker; a sixth binding molecule binds to the third binding molecule and comprises a second molecular tag attached thereto via a cleavable linker; and an eighth binding molecule binds to the fourth binding molecule and comprises a cleavage-inducing moiety; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the first and second molecular tags; and and c) detecting the presence or absence of the released first and second molecular tags to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0032] Another format is DTT-mediated release, wherein the method for detecting and / or quantifying expression of at least a first cell surface moiety and a second cell surface moiety in a sample comprises: a) contacting a sample 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; 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 comprise a molecular tag attached thereto via a cleavable linker; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the molecular tag; and c) detecting the presence or absence of the released molecular tag to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0033] In one format, the DTT-mediated release assay uses a primary binding molecule for each target moiety that contains a molecular tag.

[0034] Thus, in one embodiment, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: a) contacting a 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 comprises a first molecular tag attached thereto via a cleavable linker; the second binding molecule comprises a second molecular tag attached thereto via a cleavable linker; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the first and second molecular tags; and c) detecting the presence or absence of the released molecular tag to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0035] Another format is a DTT-mediated release assay that uses a primary binding molecule and a secondary binding molecule for each target moiety, where the secondary binding molecule comprises a molecular tag.

[0036] Thus, in one embodiment, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: a) contacting a 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; the third binding molecule comprises the first molecular tag attached thereto via a cleavable linker; the fourth binding molecule comprises a second molecular tag attached thereto via a cleavable linker; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the first and second molecular tags; and c) detecting the presence or absence of the released molecular tag to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

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

[0038] Thus, in one embodiment, the disclosure provides a method for detecting and / or quantifying expression in a sample of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, the method comprising: 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 comprises the first molecular tag attached thereto via a cleavable linker; a third binding molecule comprising a second molecular tag attached thereto 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 comprises a first molecular tag attached thereto via a cleavable linker; the third binding molecule comprises a second molecular tag attached thereto via a cleavable linker; contacting, wherein the molecular tag attached to the binding molecule that detects the first cell surface moiety is different from the molecular tag attached to the binding molecule that detects the second cell surface moiety; b) inducing cleavage of the first and second molecular tags; and c) detecting the presence or absence of the released molecular tag to detect and / or quantify expression of the first cell surface moiety and the second cell surface moiety in the sample.

[0039] In each of the above exemplified formats in which a primary binding molecule and a secondary binding molecule are used, one or more binding molecules may be present between the primary binding molecule and the secondary binding molecule.

[0040] Methods according to the present disclosure may include a combination of a proximity assay and a DTT-mediated release assay, where a first cell surface moiety is detected using a proximity assay and a second cell surface moiety is detected using a DTT-mediated release assay.

[0041] Methods according to the present disclosure can be used to measure the co-expression of at least two different cell surface moieties in a single sample, and the knowledge gained therefrom can be used to predict the responsiveness of a patient, particularly a cancer patient, to an agent or agents that bind to the two different cell surface moieties.

[0042] Accordingly, the present disclosure provides a method for predicting the responsiveness of a patient, particularly a cancer patient, to an agent or agents that bind to at least 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, the method comprising: a) detecting the expression levels of a first cell surface moiety and a second cell surface member in a biological sample from a subject, in particular from a tumor of the subject, using a method according to the present disclosure; b) 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; and c) 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 a threshold level. The agent or agents include those further defined herein. This method is also referred to herein as a "prediction method."

[0043] The present disclosure also provides a method for treating a subject in need of treatment, particularly a subject having cancer, the method comprising: a) 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 prediction method described above; and b) administering to a subject likely to respond according to the prediction an agent or agents that bind to the first cell surface moiety and the second cell surface moiety, The agent or agents include agents as further defined herein.

[0044] The present disclosure provides an agent or agents that bind to a first cell surface moiety and a second cell surface moiety for the treatment of a subject, particularly a subject having cancer, the treatment comprising: a) 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 prediction method described above; and b) administering to a subject likely to respond according to the prediction an agent or agents that bind to the first cell surface moiety and the second cell surface moiety, The agent or agents include agents as further defined herein.

[0045] The present disclosure further provides a method for detecting and / or quantifying the presence of clustering of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, in a sample, the method comprising detecting the presence or absence of a signal, which is not detected unless the first and second cell surface moieties are in close proximity to each other in a sample in which the first and second cell surface moieties are exposed to an agent having binding specificity for at least the first and second cell surface moieties. This method can be used to indicate whether two or more cell surface moieties are in close proximity to each other. When two or more cell surface moieties are in close proximity to each other, they are considered to cluster if they generate a signal as described herein. The proximity of the at least two cell surface moieties is caused or induced by an agent having binding specificity for at least two cell surface moieties, such as, for example, a multispecific antibody. Thus, in certain embodiments, the method can also be defined as comprising detecting a signal produced when the first and second cell surface moieties are simultaneously bound by an agent having binding specificity for at least two cell surface moieties.

[0046] The method according to the present disclosure can be carried out in different ways, including different formats of proximity assay.One way of carrying out this method involves quenching the signal from the fluorophore attached to the binding molecule that detects one of the cell surface moieties, and the quenching is caused by the quencher attached to the binding molecule that detects another part of the cell surface moieties.Another way of carrying out this method involves the signal produced by the interference of the fluorophore attached to the binding molecule that detects one of the cell surface moieties with another different fluorophore attached to the binding molecule that detects another part of the cell surface moieties.

[0047] In one format of a proximity assay, two primary binding molecules are used, one for each target moiety, where one of the primary binding molecules contains a molecular tag and the other primary binding molecule contains a cleavage-inducing moiety.

[0048] Thus, in one embodiment, the present disclosure provides a method for detecting and / or quantifying 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: a1) contacting a sample in which a first and a second cell surface moiety have been exposed to an agent having binding specificity for at least the first and the second cell surface moiety 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 a first binding molecule comprises a molecular tag attached thereto via a cleavable linker and a second binding molecule comprises a cleavage-inducing moiety; or a2) 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, contacting a second binding molecule comprising a molecular tag attached thereto via a cleavable linker, and a first binding molecule comprising a cleavage-inducing moiety; and b) inducing cleavage of the molecular tag; and c) detecting the presence or absence of the released molecular tag to detect and / or quantify clustering of the first cell surface moiety with a second cell surface moiety in the sample.

[0049] In another format, a primary binding molecule is used for each target moiety and a secondary binding molecule is used for one of the primary binding molecules, and the primary binding molecule not bound by the secondary binding molecule comprises a molecular tag and the secondary binding molecule comprises a cleavage-inducing moiety, or the primary binding molecule not bound by the secondary binding molecule comprises a cleavage-inducing moiety and the secondary binding molecule comprises a molecular tag.

[0050] Thus, in one embodiment, the present disclosure provides a method for detecting and / or quantifying the presence of clustering of at least two cell surface moieties in a sample, comprising a first cell surface moiety with a second cell surface moiety, the method comprising: a1) contacting a sample in which a first and a second cell surface moiety have been exposed to an agent having binding specificity for at least the first and the second cell surface moiety 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 a first binding molecule comprising a molecular tag attached thereto via a cleavable linker, and a third binding molecule attached to the second binding molecule and comprising a cleavage-inducing moiety; or a2) 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, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule, contacting a second binding molecule comprising a molecular tag attached thereto via a cleavable linker, and a third binding molecule attached to the first binding molecule and comprising a cleavage-inducing moiety; or a3) 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, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule, contacting a first binding molecule comprising a cleavage-inducing moiety and a third binding molecule binding to the second binding molecule and comprising a molecular tag attached thereto via a cleavable linker; or a4) 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, a second binding molecule that specifically binds to the second cell surface moiety, and a third binding molecule, contacting, wherein a second binding molecule comprises a cleavage-inducing moiety and a third binding molecule binds to the first binding molecule and comprises a molecular tag attached thereto via a cleavable linker; b) inducing cleavage of the molecular tag; and c) detecting the presence or absence of the released molecular tag to detect and / or quantify clustering of the first cell surface moiety with a second cell surface moiety in the sample.

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

[0052] Thus, in one embodiment, the present disclosure provides a method for detecting and / or quantifying 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: a1) contacting a sample in which a first and a second cell surface moiety have been exposed to an agent having binding specificity for at least the first and the second cell surface moiety 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 a third binding molecule that binds to the first binding molecule and comprises a molecular tag attached thereto via a cleavable linker, and a fourth binding molecule that binds to the second binding molecule and comprises a cleavage-inducing moiety; or a2) 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, a second binding molecule that specifically binds to the second cell surface moiety, a third binding molecule, and a fourth binding molecule; contacting, wherein a third binding molecule is bound to the first binding molecule and comprises a cleavage-inducing moiety, and a fourth binding molecule is bound to the second binding molecule and comprises a molecular tag attached thereto via a cleavable linker; b) inducing cleavage of the molecular tag; and and c) detecting the released molecular tag to detect and / or quantify clustering of the first cell surface moiety with a second cell surface moiety in the sample.

[0053] In each of the above exemplified formats in which a primary binding molecule and a secondary binding molecule are used, one or more binding molecules may be present between the primary binding molecule and the secondary binding molecule.

[0054] The present disclosure provides a method for detecting and / or 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 a first and a third cell surface moiety have been exposed to an agent having binding specificity for at least the first and the third cell surface moiety, 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; Further provided is a method comprising detecting the presence or absence of the molecular tag to detect the presence of clustering of the first cell surface moiety and the second cell surface moiety in the sample.

[0055] In this context, at least the first and third cell surface moieties are preferably different cell surface moieties. In certain embodiments, the first cell surface moiety is CD137 or another costimulatory molecule, and the third cell surface moiety is a moiety on another cell, such as a tumor-associated moiety or an immune checkpoint moiety, preferably PD-L1. The first and second cell surface moieties are preferably the same cell surface moiety. In certain embodiments, the first and second cell surface moieties are CD137.

[0056] The methods according to the present disclosure can be used to measure the clustering of two or more different cell surface moieties in a single sample, particularly where the clustering is induced by an agent having binding specificity for two or more different cell surface moieties, and can be used according to the present disclosure. In certain embodiments, knowledge gained therefrom can be used to determine whether treatment with an agent having binding specificity for two or more different cell surface moieties is effective, which determination is based on confirmation of simultaneous binding of an agent having binding specificity for two different cell surface moieties to those moieties.

[0057] The agent can be any single moiety that can bind to at least two cell surface moieties simultaneously. In certain embodiments, the agent 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. For example, suitable agents include binding molecules such as antibodies, including multispecific antibodies, such as bispecific and trispecific antibodies, antibody fragments, molecules that contain antibody-derived domains, and fusion proteins. The agent can also be referred to as a drug.

[0058] Thus, the present disclosure provides a method for determining the effectiveness of a drug, the drug comprising 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 and / or 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 a method according to the present disclosure. The drug is an agent as further defined herein. This method is also referred to herein as a "monitoring method".

[0059] The present disclosure also provides a method for confirming the mode of action of an agent, the agent comprising at least a binding domain that specifically binds at least a first cell surface moiety and a binding domain that specifically binds a second cell surface moiety, the method comprising detecting and / or 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 agent by using a method according to the present disclosure. The agent is an agent as further defined herein. The mode of action is, for example, simultaneous binding of the agent to the first and second cell surface moieties. In this context, the first and second cell surface moieties are preferably different cell surface moieties and the agent is a multispecific antibody. Another mode of action is, for example, clustering of two or more cell surface moieties. In this context, in one embodiment, at least the first and second cell surface moieties are the same and the agent is a monospecific antibody, and in another embodiment, at least the first and second cell surface moieties are different cell surface moieties and the agent is a multispecific antibody. This method is also referred to herein as a "confirmation method". For example, it can be determined whether an agent, preferably a multispecific antibody having specificity for a cell surface moiety expressed on an immune effector cell, preferably CD137 or any other immune effector cell costimulatory moiety, and a cell surface moiety expressed on a tumor cell, preferably PD-L1 or any other tumor associated moiety or immune checkpoint, induces clustering of two or more of the cell surface moieties expressed on an immune effector cell, preferably one or more CD137, or any other immune effector cell costimulatory proteins.

[0060] The present disclosure provides a method for treating a subject in need of treatment, particularly a subject having cancer, the method comprising: a) treating a subject with an agent that binds to a first cell surface moiety and a second cell surface moiety; b) analyzing the efficacy of the agent using the monitoring methods described herein or confirming the mode of action of the agent using the confirmation methods described herein. The agent is an agent as further defined herein. In certain embodiments, the method may further include continuing or adapting the treatment based on the results of the analysis or confirmation, where adapting includes, but is not limited to, increasing or decreasing the dosage and / or increasing or decreasing the frequency of administration of the agent, or terminating the treatment.

[0061] The present disclosure provides an agent that binds to a first cell surface moiety and a second cell surface moiety for use in treating a subject, particularly a subject having cancer, the treatment comprising: a) treating a subject with an agent that binds to a first cell surface moiety and a second cell surface moiety; b) analyzing the effectiveness of the agent using the monitoring methods described herein or confirming the mode of action of the agent using the confirmation methods described herein. The agent is an agent as further defined herein. In certain embodiments, the treatment may further include continuing or adapting the treatment based on the results of the analysis or confirmation, where adapting includes, but is not limited to, increasing or decreasing the dosage and / or increasing or decreasing the frequency of administration of the agent, or terminating the treatment.

[0062] Methods according to the present disclosure can further be used to screen one or more test agents for their ability to induce clustering of at least a first cell surface moiety with a second cell surface moiety.

[0063] Accordingly, the present disclosure provides a method for screening one or more test agents for an ability to induce clustering of at least a first cell surface moiety with a second cell surface moiety, the method comprising: a) contacting one or more test cell cultures with a test agent, contacting, wherein the test cell culture comprises at least a cell expressing a first cell surface moiety and a second cell expressing a second cell surface moiety; b) detecting a level of clustering of the first and second cell surface moieties using a method according to the present disclosure; and c) comparing the level of clustering detected in step b) with the level of clustering detected in a control cell culture not contacted with the test agent or not contacted with a reference agent; Further provided is a method, wherein the control cell culture comprises a first cell surface moiety and a second cell surface moiety, hi certain embodiments, the method may further comprise selecting a test agent that induces a level of clustering equal to or greater than the level of clustering in the control cell culture.

[0064] This method can be used to identify novel, further or alternative binding molecules that have binding specificity for two or more different cell surface moieties in addition to those already known. For example, this method can be used to identify further or alternative binding molecules that have binding specificity for CD137, or any other immune effector cell co-stimulatory moiety, and PD-L1, or any other tumor-associated or immune checkpoint moiety.

[0065] The present disclosure further provides a kit of parts. In certain embodiments, the kit comprises a binding molecule that specifically binds to at least a first and a second cell surface moiety according to the method described herein. In one embodiment, the kit of parts comprises at least two binding molecules that specifically bind to a first and a second cell surface moiety, where optionally one of the binding molecules comprises a molecular tag attached thereto via a cleavable linker, and the other binding molecule comprises a cleavage-inducing moiety, and instructions for contacting a patient sample with the at least two binding molecules, optionally inducing cleavage of the molecular tag, and measuring the signal induced by contacting the patient sample with the at least two binding molecules.

[0066] The following is a further description of the features of the methods described herein: Although for clarity and concise description, features are described herein as part of the same or separate embodiments, it will be understood that the scope of the disclosure may include embodiments having all or any combination of the described features.

[0067] The method according to the present disclosure can be used to detect the expression of a first cell surface moiety and a second cell surface moiety. The present disclosure further provides a method that can be used to indicate whether two cell surface moieties are in close proximity to each other. The detection method used, the release of a molecular tag from a binding molecule bound to (one of) the cell surface moieties, also allows quantification of the expression of (a complex of) the first and second cell surface moieties.

[0068] In certain embodiments, the method according to the present disclosure allows for the detection and / or quantification of a first and a second cell surface moiety in a single sample. Thus, the molecular tag attached to the antibody that binds to the first cell surface moiety is different from the molecular tag attached to the antibody that binds to the second cell surface moiety. In certain embodiments, the method can be used to determine whether the first and second cell moieties are co-expressed in a particular sample.

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

[0070] In certain embodiments, the first cell surface moiety and the second cell surface moiety are preferably different moieties and may be expressed on the same cell type, e.g., a tumor cell or an immune cell, on the same cell, or on different cells. In certain embodiments, the first cell surface moiety and the second cell surface moiety are preferably expressed on different cell types.

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

[0072] In certain embodiments, one of the at least two cell surface moieties is preferably expressed on 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, preferably a T cell.

[0073] In certain embodiments, one of the at least two cell surface moieties is expressed on a cell, which may be from a tumor or immune cell origin, such as, for example, but not limited to, a tumor cell, a B cell, a myeloid cell, a dendritic cell, or a neutrophil.

[0074] In certain embodiments, one of the at least two cell surface moieties is expressed on 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, preferably a T cell, and the other of the at least two cell surface moieties is expressed on a cell that may be from a tumor or immune cell origin, such as, for example, but not limited to, a tumor cell, a B cell, a myeloid cell, a dendritic cell, or a neutrophil.

[0075] In certain embodiments, the immune effector cell may be a NK cell, a T cell, a B cell, a monocyte, a macrophage, a dendritic cell, or a neutrophil granulocyte, preferably a T cell.

[0076] In certain embodiments, the immune effector 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 associated 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 moieties can be any cell surface moieties that cluster in response to an agent that brings both cell surface moieties into close proximity with each other. In certain embodiments, the first and second cell surface moieties can be the same, and the method is used to detect and / or quantify the clustering, e.g., dimerization or trimerization, of these cell surface moieties in response to an agent. This is exemplified herein, for example, for the clustering of at least two CD137 molecules.

[0079] In certain embodiments, the first cell surface moiety is CD137 and the second cell surface moiety 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, PD-1-PD-L1 complexes are not part of this disclosure.

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

[0082] The present disclosure also provides methods for detecting and / or quantifying the presence of clustering of at least two cell surface moieties, including a first cell surface moiety and a second cell surface moiety, in a sample, as described herein, such that the first and second cell surface moieties 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 disclosed methods, the sample is contacted with binding molecules that bind to the first and second cell surface moieties, also referred to as assay binding molecules.

[0084] In certain formats of the method according to the present disclosure, two binding molecules are used for each moiety. The present disclosure refers to the two binding molecules that bind to the first cell surface moiety as the first binding molecule and the second binding molecule. The two binding molecules that bind to the second cell surface moiety are referred to herein as the third binding molecule and the fourth binding molecule. The numbers used in referring to these binding molecules indicate that the binding molecules differ from each other in terms of binding specificity and / or to facilitate visualization of examples of useful assay formats. The numbers do not refer to any particular order or required presence of one or more of the binding molecules. Also, in formats 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 moiety, and the secondary binding molecule, i.e., the binding molecule that includes a molecular tag or a cleavage-inducing moiety.

[0085] In a particular form of the method according to the present disclosure, the two binding molecules that bind to the first cell surface moiety are binding molecules that bind to different epitopes and are selected so that they do not interfere with each other's binding to the first cell surface moiety. Similarly, the two binding molecules that bind to the second cell surface moiety are binding molecules that bind to different epitopes and are selected so that they do not interfere with each other's binding to the second cell surface moiety. The first, second, third and / or fourth binding molecules can bind to the extracellular domain of the cell surface moiety, but can also bind to the intracellular domain of the cell surface moiety. Combinations of these are also possible. For example, two binding molecules that bind to a first cell surface moiety may be directed to its extracellular domain while two binding molecules that bind to a second cell surface moiety may bind to its intracellular domain, or one of the binding molecules that bind to a first cell surface moiety may bind to the extracellular domain and the other may bind to its intracellular domain and the same for a second cell surface moiety, or one of the binding molecules that bind to a first cell surface moiety may bind to the extracellular domain and the other may bind to its intracellular domain while both binding molecules that bind to a second cell surface moiety may bind to the extracellular or intracellular domain, or vice versa.

[0086] In certain embodiments, one primary binding molecule is used for each moiety. The present disclosure refers to two primary binding molecules that bind to a first cell surface moiety and a second cell surface moiety as a first binding molecule and a second binding molecule. The numbers used in referring to these binding molecules indicate that the binding molecules differ from each other in terms of binding specificity and / or to facilitate visualization of examples of useful assay formats. The numbers do not refer to any particular order or necessary 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 the present disclosure. Also, in formats 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 moiety, and the secondary binding molecule, i.e., the binding molecule that includes a molecular tag or a cleavage-inducing moiety.

[0087] In certain embodiments, the two binding molecules that bind to the first and second cell surface moieties are binding molecules that bind to different cell surface moieties. The first and second binding molecules can bind to the extracellular domain of the cell surface moiety, but can also bind to the intracellular domain of the cell surface moiety. Combinations of these are also possible. For example, the binding molecule that binds to the first cell surface moiety may bind to its extracellular domain, while the binding molecule that binds to the second cell surface moiety may bind to its intracellular domain, or vice versa.

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

[0089] In a particular form of the method according to the present disclosure, one of the primary binding molecules for each cell surface moiety comprises a molecular tag or a cleavage-inducing moiety. In a particular form of the method according to the present disclosure, one of the primary binding molecules comprises a molecular tag, and the other comprises a cleavage-inducing moiety. Such molecular tags or cleavage-inducing moieties can be attached to primary binding molecules, particularly antibodies, using standard techniques in the art. In some cases, it may be difficult to attach molecular tags or cleavage-inducing moieties to certain binding molecules. In that case, a secondary binding molecule, usually an antibody, to which the molecular tag or cleavage-inducing moiety is attached can be used. Such secondary binding molecules, including molecular tags or cleavage-inducing moieties, are typically directed to the Fc region of primary binding molecules and are generally commercially available.

[0090] A molecular tag can be any molecular moiety, e.g., a molecule, that can be detected. In certain cases, upon release, the molecular moiety provides a measurable signal. A molecular tag may be selected based on one or more of its properties that distinguish it from other moieties, 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 the molecular tag in an assay that measures multiple cell surface moieties in a single sample. In certain embodiments, the molecular tag includes a detection moiety, such as, but not limited to, a fluorescent label, a chromogenic label, a radioactive label, or an electrochemical label. Exemplary fluorescent dyes include water-soluble rhodamine dyes, fluorescein, 4,7-dichlorofluorescein, benzoxanthene dyes, and energy transfer dyes, as disclosed in the following references: Handbook of Molecular Probes and Research Reagents, 8:111-115, 1999; Handbook of Molecular Probes and Research Reagents, 8:111-115, 1999; thed. (2002), Molecular Probes, Eugene, Oreg., WO 2001 / 32783, US Patent Application Publication Nos. 2002-0081616, 2002-0086985, and Lee et al., 1997, Nucleic Acids Research 25:2816-2822. Examples of suitable molecular tags include, but are not limited to, VeraTag® reporter molecules. VeraTag® reporter molecules are well known in the art. Preferred molecular tags are, for example, VeraTag® reporter molecules Pro11 and Pro125. Pro11 is an example of a photoreleasable tag, and Pro125 is an example of a DTT-releasable tag. Other VeraTag® molecules are described, for example, in U.S. Patent Application Publication Nos. 2004-0166529, 2004-0126818, 2003-0013126, 2005-0079565, and 2011-0180408, each of which and the references cited therein are incorporated herein in their entirety.

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

[0092] In certain assay formats, molecular tags are linked to binding molecules via cleavable linkers. The cleavable linker can be any cleavable linker, including but not limited to linkers that can be cleaved by singlet oxygen or hydrogen peroxide, or DTT. The linker that can be cleaved by DTT is a SS linker. The cleavable bond can also include bonds that are unstable to agents acting on the entire reaction mixture, such as base-labile bonds, photocleavable bonds, reductively cleavable bonds, oxidatively cleaved bonds, acid-labile bonds, and peptide bonds that can be cleaved by certain proteases. References that describe many such bonds include Greene and Wuts, 1991, Protective Groups in Organic Synthesis, Second Edition, John Wiley & Sons, New York; Henanson, 1996, Bioconjugate Techniques, Academic Press, New York; and US Patent Publication No. 2003-0119059.

[0093] The cleavage of molecular tag from binding molecule can be induced by methods known in the art, including but not limited to the use of light induction and DTT-mediated release.Light induction induces the activation of light-absorbing molecules that convert molecular oxygen into singlet oxygen when activated by light.Inducing cleavage by using DTT involves DTT-induced cleavage of the disulfide linker that is cleavable by reduction and connects the molecular tag to the binding molecule.

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

[0095] In the context of the present disclosure, "proximity" means that the binding molecule containing the bound molecular tag and the binding molecule containing the cleavage-inducing moiety are within a distance that allows cleavage of the molecular tag induced by the cleavage-inducing moiety. In the VeraTag® assay, this is about 1000 nm, preferably within about 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 easily determined by the skilled artisan, information provided by suppliers of commercially available tags, quenchers and reporter moieties. The information necessary for the skilled artisan to apply a particular proximity assay in the present disclosure is available in the art. For example, Nathan P. 2020, Assay Guidance Manual, Compound-Mediated Assay Interferences in Homogenous Proximity Assays, incorporated herein by reference in its entirety, provides information regarding, among other things, 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, e.g., Tables 2 and 3).

[0096] Using the method according to the present disclosure, the co-expression of two different cell surface moieties in a single sample can be measured. The knowledge gained therefrom can be used to determine a treatment plan for the patient. For example, if two cell surface moieties are co-expressed in a patient's tissue or blood sample, it can be decided to treat the patient with a drug or drugs that target these two cell surface moieties. One example of such a situation is when a tumor biopsy sample of a cancer patient shows co-expression of two tumor-associated antigens on the 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 pathways of the tumor-associated antigens, and / or induce T cell-mediated tumor cell killing. If the patient's tumor sample does not show co-expression of such tumor-associated antigens, it can be determined by the treating physician that the patient is unlikely to benefit from such treatment. Another situation is, for example, when a tumor biopsy shows co-expression of a tumor-associated antigen on the tumor cells and an antigen expressed on immune effector cells. This indicates that immune effector cells, such as, for example, T cells and / or NK cells, are present in the tumor microenvironment. Such patients may benefit from treatment with agents that bring immune cells into close proximity to tumor cells and / or activate immune effector cells so that tumor cells are selectively killed.Thus, in certain embodiments, the methods of the present disclosure may be used to predict the response of a patient, preferably a cancer patient, to treatment with an agent or agents that bind to two different cell surface moieties.

[0097] An example of an agent that binds to two different cell surface moieties is, for example, a multispecific antibody. Such a multispecific antibody can be a bispecific or trispecific antibody, or an antigen-binding fragment thereof, that binds simultaneously to both cell surface moieties. Such a multispecific antibody can exhibit monovalent binding to both cell surface moieties, such that the multispecific antibody contains a single antigen-binding fragment for each cell surface moiety. The cell surface moieties can be any of the first and second cell surface moieties as disclosed herein.

[0098] A specific example of a multispecific antibody that binds to two different cell surface moieties and in relation to which the methods of the disclosure are useful is a multispecific antibody that binds to PD-L1 on tumor cells and CD137 on T cells. Such a multispecific antibody may comprise a CD137-binding domain comprising a heavy chain CDR3 (HCDR3) having an 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, in which one, two, or three amino acid substitutions may be tolerated. In certain embodiments, the CD137-binding domain comprises a heavy chain CDR3 (HCDR3) having an 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. The CD137 binding domain may further comprise 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, allowing for one, two, or three amino acid substitutions therein, 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, allowing for one, two, or three amino acid substitutions therein. In certain embodiments, the CD137 binding domain may comprise 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, 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. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred CD137 binding domains comprise a combination of HCDR1, HCDR2 and HCDR3 of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16; SEQ ID NO:18, SEQ ID NO:3, and SEQ ID NO:19; SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; SEQ ID NO:10, SEQ ID NO:29, and SEQ ID NO:30; SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34; SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42; SEQ ID NO:44, SEQ ID NO:41, and SEQ ID NO:45; SEQ ID NO:2, SEQ ID NO:47, and SEQ ID NO:48; or SEQ ID NO:50, SEQ ID NO:52, and SEQ ID NO:52. The CD137 binding domain of such a multispecific antibody may comprise a heavy chain variable region having any one of SEQ ID NO: 1, 5, 9, 13, 17, 20, 24, 28, 31, 35, 39, 43, 46, or 49, or having at least 80%, 85%, 90%, 95%, or 99%, preferably 95%, sequence identity to their framework regions. In certain embodiments, the CD137 binding domain of such a multispecific antibody also comprises a CH1 domain. Any CH1 domain may be used. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

[0099] The multispecific antibody may further comprise a PD-L1 binding domain comprising 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, allowing for one, two, or three amino acid substitutions therein. In certain embodiments, the PD-L1 binding domain comprises 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. The PD-L1 binding domain may further comprise 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 allows for one, two, or three amino acid substitutions therein, and / or may further comprise 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 allows for one, two, or three amino acid substitutions therein. In certain embodiments, the PD-L1 binding domain 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, 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. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred PD-L1 binding domains are those set forth in 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, SEQ ID NO: SEQ ID NO:83, and SEQ ID NO:84; SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88; SEQ ID NO:90, SEQ ID NO:79, and SEQ ID NO:91; SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95; SEQ ID NO:68, SEQ ID NO:55, and SEQ ID NO:56; SEQ ID NO:70, SEQ ID NO:98, and SEQ ID NO:99; SEQ ID NO:93, SEQ ID NO:101, and SEQ ID NO:102; SEQ ID NO:74, SEQ ID NO:105, and SEQ ID NO:106; or a combination of HCDR1, HCDR2, and HCDR3 of SEQ ID NO:86, SEQ ID NO:108, and SEQ ID NO:88. The PD-L1 binding domain of such a multispecific antibody may comprise 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 having at least 80%, 85%, 90%, 95%, 99%, preferably 95% sequence identity to their framework regions. In certain embodiments, the PD-L1 binding domain of such a multispecific antibody also comprises a CH1 domain. Any CH1 domain may be used. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

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

[0101] In certain embodiments, the multispecific antibody may further comprise an optional light chain. An example of a suitable light chain comprises a light chain variable region comprising a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113, allowing one, two or three amino acid substitutions therein. In certain embodiments, the light chain variable region comprises a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113. The light chain variable region may further comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, allowing one, two or three amino acid substitutions therein, and / or may further comprise a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, allowing one, two or three amino acid substitutions therein. In certain embodiments, the light chain variable region comprises a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, and / or a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112. The light chain variable region of the multispecific antibody may comprise a light chain variable region having SEQ ID NO: 110 or having at least 80%, 85%, 90%, 95%, 99% sequence identity to the framework regions thereof. In certain embodiments, the light chain of such a multispecific antibody also comprises a CL domain. Any CL domain may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 115.

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

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

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

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

[0106] The multispecific antibody may further comprise a PD-L1 binding domain comprising 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, allowing for one, two, or three amino acid substitutions therein. In certain embodiments, the PD-L1 binding domain comprises 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. The PD-L1 binding domain may further comprise 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 allows for one, two, or three amino acid substitutions therein, and / or may further comprise 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 allows for one, two, or three amino acid substitutions therein. In certain embodiments, the PD-L1 binding domain 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, 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. Any combination of HCDR1, HCDR2, and HCDR3 is possible.Preferred PD-L1 binding domains are those set forth in 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, SEQ ID NO: SEQ ID NO:83, and SEQ ID NO:84; SEQ ID NO:86, SEQ ID NO:87, and SEQ ID NO:88; SEQ ID NO:90, SEQ ID NO:79, and SEQ ID NO:91; SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95; SEQ ID NO:68, SEQ ID NO:55, and SEQ ID NO:56; SEQ ID NO:70, SEQ ID NO:98, and SEQ ID NO:99; SEQ ID NO:93, SEQ ID NO:101, and SEQ ID NO:102; SEQ ID NO:74, SEQ ID NO:105, and SEQ ID NO:106; or a combination of HCDR1, HCDR2, and HCDR3 of SEQ ID NO:86, SEQ ID NO:108, and SEQ ID NO:88. The PD-L1 binding domain of such a multispecific antibody may comprise 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 having at least 80%, 85%, 90%, 95%, 99%, preferably 95% sequence identity to their framework regions. In certain embodiments, the PD-L1 binding domain of such a multispecific antibody also comprises a CH1 domain. Any CH1 domain may be used. An example of a suitable CH1 domain is provided by the amino acid sequence provided as SEQ ID NO: 116.

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

[0108] In certain embodiments, the multispecific antibody may further comprise an optional light chain. An example of a suitable light chain comprises a light chain variable region comprising a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113, allowing one, two or three amino acid substitutions therein. In certain embodiments, the light chain variable region comprises a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113. The light chain variable region may further comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, allowing one, two or three amino acid substitutions therein, and / or may further comprise a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, allowing one, two or three amino acid substitutions therein. In certain embodiments, the light chain variable region comprises a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, and / or a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112. The light chain variable region of the multispecific antibody may comprise a light chain variable region having SEQ ID NO: 110 or having at least 80%, 85%, 90%, 95%, 99% sequence identity to the framework regions thereof. In certain embodiments, the light chain of such a multispecific antibody also comprises a CL domain. Any CL domain may be used. An example of a suitable CL domain is provided by the amino acid sequence provided as SEQ ID NO: 115.

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

[0110] As used herein, "comprises" and its conjugations are used in their open-ended sense to mean that the items preceding the word are included, but not excluding items not specifically mentioned.

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

[0112] The reference in this specification to a patent document or other matter is not to be considered an admission that the document or matter was known or that the information contained in it was part of the common general knowledge at the priority date of any of the claims.

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

[0114] The present disclosure is illustrated in the following examples, which are not intended to limit the scope of the disclosure. EXAMPLES

[0115] Example 1 - Preparation of cell pellets for the VeraTag® assay T75 flasks were coated overnight with 2 μg / mL anti-CD3 (clone OKT3, eBioscience, cat. no. 16-0037-85) in PBS. Jurkat T cells expressing CD137 (Jurkat_CD137K) were then cultured at 1.8 × 10 in 50 mL of medium (9% FBS-HI RPMI 1640, 2 mM L-glutamine). 6 Jurkat cells were then added at a concentration of 0.45 × 10 cells / mL in 50 mL of medium and incubated at 37 °C for 4 hours. 6The cells were co-cultured (Jurkat to CHO) with CHO-K1 cells expressing PD-L1 (CHO-PD-L1) at a concentration of 10000 cells / mL. The cells were allowed to interact for 4 hours, and then a bispecific antibody binding CD137 and PD-L1, an anti-CD137 positive control antibody, or a negative control antibody binding RSV (10 μg / mL) was added for an additional 2 hours. The cells were then harvested from the flasks by resuspension and scraping, and fixed as follows: after centrifugation at 1,200 rpm (125×g) for 10 minutes at 4°C, the medium was poured off, the cell pellet was loosened, and resuspended in ice-cold PBS. The centrifugation was repeated twice, whereby the PBS was poured off 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, Cat. No. 5701, Thermo Fisher Scientific) and gently rotated overnight at 4°C. After centrifugation at 1,500 rpm for 10 min at 4°C, the formalin was removed and the cell pellet was diluted with 25 × 10 6 cells / mL and resuspended in 80% ethanol, stored at 4°C, and then processed as previously described ( 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. 5 Cells were placed onto positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology as briefly described below.

[0118] Antigen retrieval was performed by heating using a pressure cooker (Biocare Medical). After antigen retrieval, antibody pairs were added and DTT-mediated released fluorescent VeraTags were detected by capillary electrophoresis. Released VeraTags were normalized to sample buffer volume and were calculated to be 4.5 × 10 5Relative fluorescence units per cell were obtained.

[0119] The antibodies used were anti-PD-L1 rabbit mAb E1L3N (Cell Signaling Technology cat#13684) and a pepsin digest of 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 means for detecting the expression levels of PD-L1. 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. 5 Cells were placed onto positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology as briefly described below.

[0122] Antigen retrieval was performed by heating using a pressure cooker (Biocare Medical). After antigen retrieval, antibody pairs were added and DTT-mediated released fluorescent VeraTags were detected by capillary electrophoresis. Released VeraTags were normalized to sample buffer volume and were calculated to be 4.5 × 10 5 Relative fluorescence units per cell were 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 a VeraTag (Jackson Immuno Research cat#115-005-146) was paired with the primary antibody. In isotype control experiments, the CD137 antibody is 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 expression levels of CD137. Similar amounts of CD137 were measured in all three samples for both primary assay antibodies.

[0125] Example 4 - CD137 Clustered VeraTag® Assay The cell pellet prepared in Example 1 was used in this assay. 5 Cells were placed onto 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, antibody pairs were added and released fluorescent VeraTags were detected by capillary electrophoresis. Released VeraTags were normalized to the sample buffer volume and were calculated to be 4.5 × 10 5 Relative fluorescence units per cell were obtained.

[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). Equal concentrations of anti-CD137 antibodies were labeled with either the fluorescent VeraTag reporter or biotin.

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

[0129] Example 5 - CD137-PD-L1 Proximity VeraTag® Assay The cell pellet prepared in Example 1 was used in this assay. 5 Cells were placed onto positively charged glass slides (Fisher Scientific) and analyzed using VeraTag® technology as briefly described below.

[0130] Antigen retrieval was performed by heating using a pressure cooker (Biocare Medical). After antigen retrieval, antibody pairs were added and released fluorescent VeraTags were detected by capillary electrophoresis. Released VeraTags were normalized to the sample buffer volume and were calculated to be 4.5 × 10 5 Relative fluorescence units per cell were obtained.

[0131] CD137-PD-L1 proximity was measured by proximity-dependent release of the VeraTag reporter from mouse mAb BBK2 (ThermoFisher cat#MS-621, ectodomain) paired with anti-CD137 mouse mAb M127 (BD Pharmingen cat#552532, ectodomain), or anti-PD-L1 rabbit mAb E1L3N (Cell Signaling Technology cat#13684, c-terminus) and a 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 tool for detecting CD137-PD-L1 complexes. Sample B shows the strongest VeraTag signal for both primary assay antibodies. This indicates that the CD137xPD-L1 bispecific antibody binds simultaneously to CD137 and PD-L1, clustering these antigens and thus cells expressing these antigens.

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

Claims

1. 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; whereby the first cell surface moiety is CD137 or another immune effector cell costimulatory molecule and the second cell surface moiety is PD-L1 or another tumor-associated moiety or immune checkpoint moiety; the agent is a multispecific antibody, e.g., a bispecific or trispecific 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; The binding domain of the bispecific antibody that binds to CD137 comprises: allowing one, two, or three amino acid substitutions in each of heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3); 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; 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; and 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; and a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113; The method comprises:

1. A method for detecting 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, wherein the first and second cell surface moieties have been exposed to an agent, 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; and detecting the presence or absence of the molecular tag to detect the presence of clustering of the first cell surface moiety and the second cell surface moiety in the sample. detecting clustering of a first cell surface moiety with a second cell surface moiety in a biological sample of a subject being treated with said agent.

2. 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; whereby the first cell surface moiety is CD137 or another immune effector cell costimulatory molecule and the second cell surface moiety is PD-L1 or another tumor-associated moiety or immune checkpoint moiety; the agent is a multispecific antibody, e.g., a bispecific or trispecific 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; The binding domain of the bispecific antibody that binds to CD137 comprises: allowing one, two, or three amino acid substitutions in each of heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3); 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; 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; and 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; and a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113; The method comprises: contacting a sample in which the first and second cell surface moieties have been exposed to an agent 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; and measuring the amount of the molecular tag to quantify the presence of clustering of the first cell surface moiety and the second cell surface moiety in the sample. quantitating clustering of a first cell surface moiety with a second cell surface moiety in a biological sample of a subject being treated with said agent.

3. The method of claim 1, wherein the binding domain of the multispecific antibody that binds to CD137 comprises a heavy chain binding domain having HCDR1, HCDR2, and HCDR3 of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12; SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:25; SEQ ID NO:18, SEQ ID NO:3, and SEQ ID NO:19; SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; SEQ ID NO:10, SEQ ID NO:29, and SEQ ID NO:30; SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34; SEQ ID NO:36, SEQ ID NO:37, and SEQ ID NO:38; SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42; SEQ ID NO:44, SEQ ID NO:41, and SEQ ID NO:45; SEQ ID NO:2, SEQ ID NO:47, SEQ ID NO:30, and SEQ ID NO:48; or SEQ ID NO:50, SEQ ID NO:52, and SEQ ID NO:

52.

4. A method described in any one of claims 1 to 3, wherein the binding domain of the multispecific 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.

5. The binding domain of a multispecific antibody that binds to PD-L1 comprises: allowing one, two, or three amino acid substitutions in each of heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3); a heavy chain variable region having 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; 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; and 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; a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO: 113; 5. The method according to any one of claims 1 to 4.

6. A multispecific antibody that binds to PD-L1, the binding domains of which 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, SEQ ID NO:83, and SEQ ID NO:

84.

6. The method of claim 1 , wherein the heavy chain binding domain has a combination of HCDR1, HCDR2, and HCDR3 of SEQ ID NO: 86, SEQ ID NO: 87, and SEQ ID NO: 88; SEQ ID NO: 90, SEQ ID NO: 79, and SEQ ID NO: 91; SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95; SEQ ID NO: 68, SEQ ID NO: 55, and SEQ ID NO: 56; SEQ ID NO: 70, SEQ ID NO: 98, and SEQ ID NO: 99; SEQ ID NO: 93, SEQ ID NO: 101, and SEQ ID NO: 102; SEQ ID NO: 74, SEQ ID NO: 105, and SEQ ID NO: 106; or SEQ ID NO: 86, SEQ ID NO: 108, and SEQ ID NO:

88.

7. The method of claim 1, wherein the binding domain of the multispecific 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 having at least 80%, 85%, 90%, 95%, or 99% sequence identity thereto.

8. The method of claim 1, wherein the light chain variable region of the binding domain that binds to CD137 and the light chain variable region of the binding domain that binds to PD-L1 comprise a light chain variable region having SEQ ID NO: 110 or having at least 80%, 85%, 90%, 95%, or 99% sequence identity to its framework region.

9. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO:50, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO:51, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO:52; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 68, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 56; 9. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113. Claim 10: A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 32, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 33, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 34; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 68, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 56; 9. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

11. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO:25, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO:26, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO:26; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 68, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 55, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 56; 9. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113. Claim 12: A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 50, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 51, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 52; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 74, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 105, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 106; 9. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

13. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 32, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 33, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 34; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 74, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 105, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 106; 9. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

14. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO:25, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO:26, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO:26; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain CDR1 (HCDR1) having the amino acid sequence set forth in SEQ ID NO: 74, a heavy chain CDR2 (HCDR2) having the amino acid sequence set forth in SEQ ID NO: 105, and a heavy chain CDR3 (HCDR3) having the amino acid sequence set forth in SEQ ID NO: 106; 9. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

15. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:49; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:67; 10. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

16. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:49; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 96; 10. The method of claim 1, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

17. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:67; 11. The method of any one of claims 1 to 8 and 10, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

18. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 96; 11. The method of any one of claims 1 to 8 and 10, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

19. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:67; 12. The method of any one of claims 1 to 8 and 11, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

20. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 96; 12. The method of any one of claims 1 to 8 and 11, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

21. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:49; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 104; 13. The method of any one of claims 1 to 8 and 12, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

22. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 31; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 104; 14. The method of any one of claims 1 to 8 and 13, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

23. A multispecific antibody that binds to CD137, wherein the binding domain comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:24; and the binding domain of the multispecific antibody that binds to PD-L1 comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 104; 15. The method of any one of claims 1 to 8 and 14, wherein both binding domains comprise a light chain CDR1 (LCDR1) having the amino acid sequence set forth in SEQ ID NO: 111, a light chain CDR2 (LCDR2) having the amino acid sequence set forth in SEQ ID NO: 112, and a light chain CDR3 (LCDR3) having the amino acid sequence set forth in SEQ ID NO:

113.

24. The method of any one of claims 9 to 23, wherein both binding domains comprise a light chain variable region having the amino acid sequence set forth in SEQ ID NO:

110.

25. A method for detecting a cell surface region comprising contacting a sample with a first binding molecule that specifically binds to a first cell surface portion and a second binding molecule that specifically binds to a second cell surface portion, contacting the first binding molecule with a molecular tag attached thereto via a cleavable linker and the second binding molecule with a cleavage-inducing moiety; or a2) contacting the sample with a first binding molecule that specifically binds to a first cell surface moiety and a second binding molecule that specifically binds to a second cell surface moiety; contacting, wherein the second binding molecule comprises a molecular tag attached thereto via a cleavable linker, and the first binding molecule comprises a cleavage-inducing moiety; b) inducing cleavage of the molecular tag; and 25. The method of any one of claims 1 to 24, comprising: c) detecting the presence or absence or measuring the amount of released molecular tags to detect and / or quantify clustering of the first cell surface moiety with the second cell surface moiety in the sample.

26. A method for detecting a cell surface region comprising contacting a sample with a first binding molecule that specifically binds to a first cell surface portion, a second binding molecule that specifically binds to a second cell surface portion, and a third binding molecule, contacting the first binding molecule with a molecular tag attached thereto via a cleavable linker, and the third binding molecule with the second binding molecule and with a cleavage-inducing moiety; or a2) contacting the sample with a first binding molecule that specifically binds to a first cell surface moiety, a second binding molecule that specifically binds to a second cell surface moiety, and a third binding molecule; contacting the second binding molecule, wherein the second binding molecule comprises a molecular tag attached thereto via a cleavable linker, and the third binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety; or a3) contacting the sample with a first binding molecule that specifically binds to a first cell surface moiety, a second binding molecule that specifically binds to a 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 binds to the second binding molecule and comprises a molecular tag attached thereto via a cleavable linker; or a4) contacting the sample with a first binding molecule that specifically binds to a first cell surface moiety, a second binding molecule that specifically binds to a 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 binds to the first binding molecule and comprises a molecular tag attached thereto via a cleavable linker; b) inducing cleavage of the molecular tag; and 26. The method of any one of claims 1 to 25, comprising: c) detecting the presence or absence or measuring the amount of released molecular tags to detect or quantify clustering of the first cell surface moiety with the second cell surface moiety in the sample.

27. A method of detecting a cell surface region comprising contacting a sample with a first binding molecule that specifically binds to a first cell surface portion, a second binding molecule that specifically binds to a second cell surface portion, 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 thereto via a cleavable linker, and the fourth binding molecule with the second binding molecule and comprising a cleavage-inducing moiety; or a2) contacting the sample with a first binding molecule that specifically binds to a first cell surface moiety, a second binding molecule that specifically binds to a second cell surface moiety, a third binding molecule, and a fourth binding molecule; contacting, wherein the third binding molecule binds to the first binding molecule and comprises a cleavage-inducing moiety, and the fourth binding molecule binds to the second binding molecule and comprises a molecular tag attached thereto via a cleavable linker; b) inducing cleavage of the molecular tag; and 27. The method of any one of claims 1 to 26, comprising: c) detecting the presence or absence or measuring the amount of released molecular tags to detect and quantify clustering of the first cell surface moiety with the second cell surface moiety in the sample.

28. A method described in any one of claims 1 to 27, wherein the sample is a tissue sample, a blood sample, or a cultured cell.

29. The method of claim 28, wherein the sample is a fresh sample or a fixed sample.

30. The method of any one of claims 1 to 29, wherein the sample is a tumor biopsy sample from a subject with cancer.

Images