Materials, methods, and systems for biologic molecule occupancy determinations

Abstract

Materials, methods, means, steps and systems for determining occupancy of a multi-target agent at two or more targets in the same cell. The method generally includes use of multiple detection reagents to determine a bound condition of the multi-target agent to each of the multiple targets, respectively. Each detection reagent includes a detectable label and is designed to bind a specific region of the multi-target agent unique to the agent's ability to bind to one of the multiple targets on the cell surface, thus providing single bound signals. A total amount of the multi-target agent bound to the multiple targets is determined using a universal detection reagent designed to bind to a common region of the multi-target agent. Subtracting specific single bound signals from bound signals allows calculation of multiply bound signals and specific total signals. This approach allows for the measurement of target occupancy for each bound condition with each target.

Description

MATERIALS, METHODS, AND SYSTEMS FOR BIOLOGIC MOLECULE OCCUPANCY DETERMINATIONSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under 35 U.S.C. § 119(e) of prior U.S. Provisional patent Application Serial No. 63 / 709,612, filed October 21, 2024, the entire content of which is incorporated herein.FIELD

[0002] Materials, methods, and systems for determining occupancy of a multi-target agent at two or more targets in the same cell, such as cis binding of multi-specific antibodies to two or more receptors on the same cell.BACKGROUND

[0003] Receptor occupancy (RO) assays are frequently used to generate pharmacodynamic (PD) biomarker data which can be coupled to pharmacokinetic (PK) profiles to model PK / PD relationships. Such relationships provide an index for evaluating the association between drug concentration and drug efficacy, assessing the minimal biological effects of biological agents, and thus making appropriate dose and dosing regimen settings to ensure efficacy and safety.

[0004] The calculation of receptor occupancy is typically based on measurement of fluorescence from fluorescently labeled binding agents, e.g., the median fluorescence intensity (MFI) measured from cells in a flow cytometry assay. For agents that bind at a single site, calculation of the amount bound and free is generally straightforward.Development and application of double-target agents such as bispecific antibodies that target different antigens on the same cell, referred to as cis-binding, complicates these analyses as deconvolution of the various fluorescence signals is not possible.SUMMARY

[0005] The inventors of the present disclosure appreciated and have addressed the need for novel materials and methods useful for measurement of target occupancy of a multi-target agent at multiple targets on the same cell, such as cis-binding of a multi-specific antibodywith multiple receptors on the same cell, and accordingly, against this backdrop provide the following.

[0006] In a first aspect, a method for determining target occupancy of a multi-target agent is provided. In some embodiments, the method comprises adding the multi-target agent to a first set of cell samples at a standard concentration for incubation, wherein the multi-target agent comprises a first binding moiety targeted to a first target and a second binding moiety targeted to a second target.

[0007] In some embodiments, in a first sample of the first set of cell samples, a total amount of the multi-target agent bound to the first target at the standard concentration (Ti) is determined by adding a first detection reagent to the first sample, wherein the first detection reagent includes a detectable label and binds to the multi-target agent at a region unique to the second binding moiety, and detecting a signal intensity of the detectable label of the first detection reagent. In some embodiments, in a second sample of the first set of cell samples, a total amount of the multi-target agent bound to the second target at the standard concentration (T2) is determined by adding a second detection reagent to the second sample, wherein the second detection reagent includes the same detectable label and binds to the multi-target agent at a region unique to the first binding moiety, and detecting a signal intensity of the detectable label of the second detection reagent. In some embodiments, in a third sample of the first set of cell samples, a total amount of the multi-target agent bound to the first and second targets at the standard concentration (T3) is determined by adding a third detection reagent to the third sample, wherein the third detection reagent includes the same detectable label as the first and second detection reagents and binds to the multi-target agent at a region that is not the first or second binding moiety (i.e., common region), and detecting a signal intensity of the detectable label of the third detection reagent.

[0008] In some embodiments, a cis target occupancy of the multi-target agent at the standard concentration is calculated by subtracting the total amount of the multi-target agent bound to the first target in the first sample (Ti) and the total amount of the multi-target agent bound to the second target in the second sample (T2) from the total amount of the multi-target agent bound to the common region in the third sample (T3):Tcis-bound = T3 - (Tl + T2).

[0009] In some embodiments, the method comprises adding the multi-target agent to a second set of cell samples at a saturating concentration for incubation.

[0010] In some embodiments, in a first sample of the second set of cell samples, a total amount of the multi-target agent bound to the first target at the saturating concentration (T4) may be determined by adding the first detection reagent to the first sample and detecting a signal intensity of the detectable label of the first detection reagent. In some embodiments, in a second sample of the second set of cell samples, a total amount of the multi-target agent bound to the second target at the saturating concentration (T5) may be determined by adding the second detection reagent to the second cell sample and detecting a signal intensity of the detectable label of the second detection reagent. In some embodiments, in a third sample of the second set of cell samples, a total amount of the multi-target agent bound to the first and second targets at the saturating concentration (Te) may be determined by adding the third detection reagent to the third cell sample and detecting a signal intensity of the detectable label of the third detection reagent.

[0011] In some embodiments, the method comprises calculating a total target occupancy of the multi-target agent at the saturating concentration may for each target.

[0012] In some embodiments of the method, a total target 1 occupancy of the multi-target agent at the saturating concentration is calculated by subtracting the total amount of the multi-target agent bound to the first target in the first sample from the total amount of the multi-target agent bound to the common region in the third sample:Tsaturating-Targetl — T6 — T4.

[0013] In some embodiments of the method, a total target 2 occupancy of the multi-target agent at the saturating concentration is calculated by subtracting the total amount of the multi-target agent bound to the second target in the second sample from the total amount of the multi-target agent bound to the common region in the third sample:Tsaturating-Target2 — T6 — T5.

[0014] In some embodiments, the method further comprises adding the multi-target agent or single-target agents to a second set of cell samples at a saturating concentration for incubation.

[0015] In some embodiments, in a first sample of the second set of cell samples, an amount of the multi-target agent bound to the second target at the saturating concentration and to the first target at the standard concentration (T4*) is determined by adding (i) a first single-target agent configured to bind to the second target only and (ii) the third detection reagent, wherein the first single target agent includes a region recognized by the third detection reagent, anddetecting a signal intensity of the detectable label of the third detection reaction. In some embodiments, in a second sample of the second set of cell samples, an amount of the multitarget agent bound to the first target at the saturating concentration and to the second target at the standard concentration (Ts*) is determined by adding (i) a second single-target agent configured to bind to the first target only and (ii) the third detection reagent, wherein the second single target agent includes a region recognized by the third detection reagent, and detecting a signal intensity of the detectable label of the third detection agent. In some embodiments, in a third sample of the second set of cell samples, a total amount of the multitarget agent bound to the first and second targets at the saturating concentration (Te) may be determined by adding the third detection reagent to the third cell sample and detecting a signal intensity of the detectable label of the third detection reagent.

[0016] In some embodiments, a total target occupancy of the multi-target agent at the saturating concentration is calculated for each target. In some embodiments, a total target 1 occupancy of the multi-target agent at the saturating concentration is calculated by subtracting the multi-target agent that bound to the second target in the second sample of the first set of the cell samples from the total amount of the multi-target agent bound to the common region in the second sample of the second set of the cell samples:Tsaturating-Targetl — T5* — T2.

[0017] In some embodiments, a total target 2 occupancy of the multi-target agent at the saturating concentration is calculated by subtracting the multi-target agent bound to the first target in the first sample of the first set of the cell samples from the total amount of the multitarget agent bound to the common region in the first sample of the second set of the cell samples:Tsaturating-Target2 — 4* — Tl.

[0018] In some embodiments, a percent target occupancy at the standard concentration is determined by calculating a ratio of the total amount of the multi-target agent bound to the common region at the standard concentration to the total amount of the multi-target agent bound to the common region at the saturating concentration:%Ttot = (T3 / T6)X100.

[0019] In some embodiments, a percent cis-target occupancy at the standard concentration is determined by calculating a ratio of the cis target occupancy of the multi-target agent at thestandard concentration to the total amount of the multi-target agent bound to the first and second targets at the saturating concentration:%Tcis total = (Tcis-bound / T6)xl00.

[0020] In some embodiments, a percent cis-target occupancy at the standard concentration for the first target of the multi-target agent is determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the first target at the saturating concentration:%TcisTargetl — (Tcis-bound / (T6-4))xl00, Or%Tcis Targetl — ( cis-bound / ( s*-T2))xl00.

[0021] In some embodiments, a percent cis-target occupancy at the standard concentration for the second target of the multi-target agent may be determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the second target at the saturating concentration:%Tcis Target2 — (Tcis-bound / ( 6-Ts))xl00, OT%Tcis Target2 — (Tcis-bound / ( 4*-Tl))xl00.

[0022] In a second aspect, a system for determining target occupancy of a multi-target agent is provided. In some embodiments, the system comprises a multi-target agent comprising a first binding moiety targeted to a first target and a second binding moiety targeted to a second target; a first detection reagent having a detectable label and configured to bind to the multitarget agent at a region unique to the second binding moiety; a second detection reagent having the same detectable label and configured to bind to the multi-target agent at a region unique to the first binding moiety; and a third detection reagent having the same detectable label as the first and second detection reagents and configured to bind to the multi-target agent at a common region.

[0023] In some embodiments, the system further comprises first and second sets of cells.

[0024] In a third aspect, a use of the system to implement the method disclosed hereinabove is provided.

[0025] In some embodiments, the first target and the second target comprises proteins expressed on a surface of the same cell.

[0026] In some embodiments, the multi-target agent is a bispecific antibody.

[0027] In some embodiments of the methods disclosed herein, the methods are adapted for a multi-target agent having more than two binding moieties, such as a trispecific antibody.

[0028] In some embodiments, the standard concentration may be a non-saturating concentration.

[0029] In some embodiments, the multi-target agent is a therapeutic agent. In some embodiments, the standard concentration is a therapeutic concentration of the therapeutic agent.

[0030] In some embodiments, the cells of the first and second sets of cell samples are immune cells.

[0031] In some embodiments, the cells of the first and second sets of cell samples comprises B -cells, T-cells, NK-cells, Leukocytes, or monocytes.

[0032] In some embodiments, the cells of the first and second sets of cell samples comprises cells from a whole blood sample. In some embodiments, the whole blood sample are comprised from a mammal, such as a human. In some embodiments, the whole blood sample are from a non-human, such as a simian, llama, cow, rabbit, rat, or mouse.

[0033] In some embodiments, the first and second sets of cell samples are comprised from tissue samples. In some embodiments, the tissue samples are from a mammal, such as a human. In some embodiments, the tissue samples are comprised from a non-human, such as a simian, llama, cow, rat, or mouse.

[0034] In some embodiments, the first, second, and third detection reagents comprises antibodies.

[0035] In some embodiments, the detectable label comprises a fluorescent compound. In some embodiments, the step of detecting the signal intensity of the detectable label comprises flow cytometry or molecular imaging techniques.

[0036] In some embodiments, the detectable label may comprise a radiolabel, biotin, magnetic beads, or an enzymatic label.DESCRIPTION OF THE FIGURES

[0037] FIG. 1A illustrates a method for determining receptor occupancy using receptor binding agents and labeled detection agents at standard concentrations.

[0038] FIG. IB illustrates a method for determining percent receptor occupancy using receptor binding agents and labeled detection agents at saturating concentrations.

[0039] FIG. 1C illustrates a method for determining receptor occupancy using single arm receptor binding agents and labeled detection agents at saturating concentrations.

[0040] FIG. 2 illustrates various materials and method steps useful for measurement of target occupancy for the bound condition of a multi-target agent at standard concentrations with each target according to the present disclosure.

[0041] FIG. 3 illustrates various materials and method steps useful for measurement of target occupancy for the bound condition of a multi-target agent at saturating concentrations with each target according to the present disclosure.

[0042] FIG. 4 illustrates various materials and method steps useful for measurement of target occupancy for the bound condition of a multi-target agent at standard concentrations with each target according to the present disclosure.

[0043] FIG. 5 illustrates detection of target 1 and 2 expression levels in wild type and knockout cell lines according to the present disclosure.

[0044] FIGS. 6 A and 6B illustrate anti-multi-target agent binding for a range of multi-target agent concentrations in wild type and knockout cell lines (6A) and in human whole blood (6B).

[0045] FIGS. 7A and 7B illustrate detection of cis-target occupancy using the methods and materials of the present disclosure. Whole blood samples from three different donors were tested. The method was able to detect receptor occupancy (total RO) and cis-receptor occupancy (cisRO) (7 A), and occupancy of the bispecific antibody at each of target 1 (cisRO X) and target 2 (cisRO Y) (7B).DETAILED DESCRIPTIONDefinitions and Abbreviations

[0046] While the general inventive concepts are susceptible of embodiment in many forms, there are shown in the drawings, and will be described herein in detail, specific embodiments thereof with the understanding that the present disclosure is to be considered an exemplification of the principles of the general inventive concepts. Accordingly, the general inventive concepts are not intended to be limited to the specific embodiments illustrated herein.

[0047] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0048] The articles “a” and “an” are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a” detection reagent means one detection reagent or more than one detection reagent.

[0049] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±5%, preferably ±1%, and still more preferably ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

[0050] When a list is presented, unless stated otherwise, it is to be understood that each individual element of that list and every combination of that list is to be interpreted as a separate embodiment. For example, a list of embodiments presented as “A, B, or C” is to be interpreted as including the embodiments, “A,” “B,” “C,” “A or B,” “A or C,” “B or C,” or “A, B, or C.”

[0051] The terms “antibody” and "antibodies" as used herein are meant in a broad sense and include immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, human-adapted, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies, dimeric, tetrameric or multimeric antibodies, and single chain antibodies.

[0052] Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (k) and lambda (1), based on the amino acid sequences of their constant domains.

[0053] The term "antibody fragments" refers to a portion of an immunoglobulin molecule that retains the heavy chain and / or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragmentconsisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a domain antibody (dAb) fragment, which consists of a VH domain. VH and VL domains can be engineered and linked together via a synthetic linker to form various types of single chain antibody designs where the VH / VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in PCT Inti. Publ. Nos. W01998 / 44001, WO1988 / 01649, WO1994 / 13804, and WO 1992 / 01047. These antibody fragments are obtained using well known techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are full length antibodies.

[0054] The phrase "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody specifically binding CD20 is substantially free of antibodies that specifically bind antigens other than human CD20). An isolated antibody that specifically binds human CD20, however, can have cross-reactivity to other antigens, such as orthologs of human CD20, such as Baboon, chimpanzee, cynomolgus monkey, and Rhesus monkey CD20. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0055] "Humanized antibody" refers to an antibody in which the antigen binding sites are derived from non-human species and the variable region frameworks are derived from human immunoglobulin sequences. Humanized antibodies may include substitutions in the framework regions so that the framework may not be an exact copy of expressed human immunoglobulin or germline gene sequences.

[0056] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding sites are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin. A human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin wherein the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such systems include human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein. A human antibody may also contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction ofsubstitutions in the framework or antigen binding sites. Typically, a human antibody is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene.

[0057] Isolated humanized antibodies may be synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and / or synthetic frameworks, or can be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

[0058] The term "recombinant antibody" as used herein, includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody, antibodies isolated from a recombinant, combinatorial antibody library, and antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences, sequences, or antibodies that are generated in vitro using Fab arm exchange such as bispecific antibodies.

[0059] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope, or in a case of a bispecific monoclonal antibody, a dual binding specificity to two distinct epitopes. Monoclonal antibodies having higher order binding specificities display binding specificity to a greater number of epitopes, e.g., trispecific monoclonal antibodies display binding specificity to three epitopes.

[0060] The term "epitope" as used herein means a portion of an antigen to which an antibody specifically binds. Epitopes usually consist of chemically active (such as polar, non-polar or hydrophobic) surface groupings of moieties such as amino acids or polysaccharide side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope can be composed of contiguous and / or discontiguous amino acids that form a conformational spatial unit. For a discontiguous epitope, amino acidsfrom differing portions of the linear sequence of the antigen come in close proximity in 3-dimensional space through the folding of the protein molecule.

[0061] As used herein, the terms “determining,” “assessing,” “assaying,” “measuring,” and “detecting” refer to both quantitative and qualitative determinations, and as such, the term “determining” can be used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of binding and the like is used. Where a qualitative determination is intended, the phrase “determining a level” of binding or “detecting” binding is used.

[0062] The terms “binds” or “binding” refer to an interaction between molecules including, for example, to form a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site on an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio of dissociation rate (koff) to association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen (koff / kon) is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD for an antibody provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. The affinity at one binding site does not always reflect the true strength of the interaction between an antibody and an antigen. When complex antigens containing multiple, repeating antigenic determinants, such as a polyvalent antigen, come in contact with antibodies containing multiple binding sites, the interaction of antibody with antigen at one site will increase the probability of a reaction at a second site. The strength of such multiple interactions between a multivalent antibody and antigen is called the avidity.

[0063] An “antigen” is a structure to which an antibody can selectively bind. A target antigen may be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. The target antigen may be a polypeptide. An antigen may be associated with a cell, for example, is present on or in a cell.

[0064] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosed methods are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements. When ranges are given, any endpoints of those ranges and / or numbers within those ranges can be combined within the scope of the present disclosure. That is, a range is intended to comprise every integer or fraction or value within the range and endpoints of the range.

[0065] “Comprising,” “consisting essentially of,” and “consisting of’ are intended to connote their generally accepted meanings in the patent vernacular; that is, (i) “comprising,” which is synonymous with “including,” “containing,” “having,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; (ii) “consisting of’ excludes any element, step, or ingredient not specified in the claim; and (iii) “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristics” of the claimed invention. Embodiments described in terms of the phrase “comprising” (or its equivalents) also provide as embodiments those independently described in terms of “consisting of’ and “consisting essentially of.”

[0066] The conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning and therefore satisfy the requirement of the term “and / or.”METHODS

[0067] Receptor occupancy (RO) quantifies the binding of an agent, such as a therapeutic molecule(s), to a target(s), such as a receptor and or ligand in tissue, such as on a cell surface. RO provides valuable information related to the functional effects and safety of a dose of anagent. For example, RO may provide information that is useful in defining a starting dose of a therapeutic agent in pre-clinical and first-in-human clinical trials.

[0068] The calculation of RO is typically based on measurement of a detectable label. For example, detection of fluorescence from fluorescently labeled binding agents, e.g., the median fluorescence intensity (MFI) measured from cells in a flow cytometry assay, or autoradiography for tissue sections. For agents that bind at a single site, calculation of the amount bound and free is generally straight forward. There are two basic detection modes of RO, namely binding mode (Bound) and free mode (Free). The fraction of receptor sites on a cell surface occupied by a receptor binding agent may be determined by the ratio of (i) an amount of binding of a labeled non-competitive anti-binding agent to establish a bound level, e.g., labeled antibody against the receptor binding agent (FIG. 1A), to (ii) a total receptor level detected by adding excess receptor binding agent and labeled non-competitive antibinding agent to the sample (FIG. 1C at left). Alternatively, the total receptor level may be determined via an amount of binding of a labeled non-competitive binding agent that targets a different region of the receptor (e.g., different epitope; FIG. 1C at right). The free mode detects the level of receptor sites that are unoccupied by the receptor binding agent, such as via a labeled competitive binding agent (FIG. IB).

[0069] For therapeutic or diagnostic agents that are capable of binding to more than one molecular target, i.e., multi-target agents, conventional RO assays are not suitable. In such cases, the fluorescent or other reporter signals obtained from different target-binding events cannot be separated or accurately deconvoluted. As a result, it becomes impossible to determine which portion of the total signal corresponds to occupancy of a particular receptor or binding site. This limitation is especially evident when the multi-target agent binds to two or more distinct receptors or antigens that are expressed on the same cell surface, a situation often referred to as cis-binding. Under these conditions, the overlapping fluorescence spectra and signal interference prevent accurate quantification of receptor-specific binding events.

[0070] Given the increasing development and therapeutic relevance of multi-target agents (e.g., such as bispecific antibodies, bispecific fusion proteins, dual-target small molecules, and other engineered constructs capable of recognizing two or more targets simultaneously) there is a growing need for methods that can accurately determine RO for each individual target. The ability to assess target-specific binding and engagement of such agents is critical for understanding pharmacodynamics, optimizing dosage, evaluating efficacy, and ensuring safety during both preclinical and clinical development.

[0071] Accordingly, the development and implementation of novel receptor occupancy detection methods specifically designed for multi-target agents hold significant scientific and clinical importance. These methods would enable precise quantification of individual target occupancy even in the presence of cis-binding interactions, thereby overcoming the limitations of conventional single-target RO assays.

[0072] The present inventors have developed an assay method that allows measurement of target occupancy when using multi-target agents. Thus, provided in the embodiments herein, are methods for determining target occupancy of a multi-target agent.

[0073] The method generally comprises adding a multi-target agent to cell samples at a standard concentration or a saturating concentration for incubation. The standard concentration may be a non-saturating concentration. When the multi-target agent is a therapeutic agent, the standard concentration may be a therapeutic dose or a range of possible therapeutic doses. A saturating concentration is greater than the standard concentration, i.e., includes the standard concentration plus an added amount for consistency.

[0074] “Saturating amount” generally refers to an amount or concentration of a molecule (e.g., ligand, binding agent, antibody, receptor, or analyte) that is sufficient to occupy essentially all available binding sites of its corresponding binding partner under the given experimental or physiological conditions. In other words, a saturating amount is the amount at which further increases in the concentration of the molecule do not produce a measurable increase in binding, complex formation, or signal intensity. The saturating amount may correspond to a concentration that achieves at least about 90%, 95%, 98%, or 100% occupancy of the available binding sites. The exact concentration constituting a saturating amount may vary depending on the affinity of the interaction, temperature, ionic strength, and other environmental or assay conditions.

[0075] When the multi-target agent is bispecific, i.e., comprises a first binding moiety targeted to a first target and a second binding moiety targeted to a second target, the method includes use of two moiety specific detection reagents. Should the multi-target agent be trispecific, i.e., comprises a first binding moiety targeted to a first target, a second binding moiety targeted to a second target, and a third binding moiety targeted to a third site, the method would include use of three moiety specific detection reagents. Moreover, the methods of the present disclosure can be adapted to address multi-target agents having more than three binding moieties. Thus, while the methods of the present disclosure are described withrespect to multi-target agents that are bispecific, such as bispecific antibodies, they may be adapted to address multi-target agents having more than two binding moieties. In some embodiments of the methods herein, the multi-target agents have two of more binding moieties.

[0076] The multi-target agents disclosed herein may be multi-specific drugs, multi-specific antibodies, antibody-drug conjugates, antibody-cytokine fusions, proteolysis targeting chimeras, ribonuclease targeting chimeras, and the like. The multi-target agent may be a therapeutic agent. When the multi-target agent is a therapeutic agent, the standard concentration may be within a range of a therapeutic effect of the agent, such as a therapeutic concentration.

[0077] The targets of the multi-target agents may comprise proteins expressed on a surface of a cell, such as the same cell. For example, the proteins may be receptors, such as nuclear receptors, enzyme-linked receptors, G-protein coupled receptors, ligand-gated ion channels, and the like.

[0078] The cells useful in the disclosed methods may be from a tissue sample. In some embodiments, the cells comprise immune cells, such as B -cells, T-cells, NK-cells, Leukocytes, or monocytes. The cells may be cells from a whole blood sample. In some embodiments, the cells may be from a mammal, such as a human.

[0079] The moiety specific detection reagents, or “detection reagents”, may include any biomolecule capable of specific binding to one of the moiety regions of the multi-target agent only when that moiety region is free, i.e., not bound to a target. In an exemplary embodiment, the detection reagents include at least antibodies.

[0080] In the disclosed method, and with reference to FIG. 2, a multi-target agent is added to a first set of cell samples at a standard concentration and incubated for a time and at a temperature sufficient to provide binding of the multi-target agent to the target(s). A first detection reagent is added to a first sample of the first set of cell samples (Tube 1). The first detection reagent includes a detectable label and binds to the multi-target agent at a region unique to a second binding moiety of the multi-target agent. An amount of the multi-target agent bound to a first target at the standard concentration (Ti) is determined by the signal intensity from the detectable label of the first detection reagent (i.e., with standard controls known in the art).

[0081] Each of the detection reagents disclosed herein include a detectable label, such as a fluorescent compound. As such, detection of a signal from the detectable label may comprise flow cytometry or molecular imaging techniques. Alternatively, the detectable label may comprise a radiolabel, biotin, magnetic beads, or an enzymatic label.

[0082] A second detection reagent is added to a second sample of the first set of cell samples (Tube 2). The second detection reagent includes the same detectable label and binds to the multi-target agent at a region unique to a first binding moiety of the multi-target agent. An amount of the multi-target agent bound to a second target at the standard concentration (T2) is determined by the signal intensity from the detectable label of the second detection reagent (i.e., with standard controls known in the art).

[0083] A third detection reagent, which is configured to bind to the multi-target agent at a region that is not the first or second binding moiety (i.e., common region), is added to a sample of the first set of cell samples (Tube 3). The third detection reagent includes the same detectable label as the first and second detection reagents. A total amount of the multi-target agent bound to the first and second targets at the standard concentration (T3) is determined by the signal intensity from the detectable label of the third detection reagent (i.e., with standard controls known in the art).

[0084] A cis target occupancy of the multi-target agent at the standard concentration may be calculated by subtracting the total amount of the multi-target agent bound to the first target in the first sample and the total amount of the multi-target agent bound to the second target in the second sample from the total amount of the multi-target agent bound to the common region in the third sample:Tcis-bound = T3 - (T1 + T2).

[0085] The method may further include, and with reference to FIG. 3, measurement of the multi-target agent’s binding to the target(s) when included in a cell sample at a saturating concentration, i.e., the multi-target agent is added to a second set of cell samples at a concentration sufficient to saturate the target sites. The multi-target agent is incubated with the cells of the sell sample for a time and at a temperature sufficient to provide binding of the multi-target agent to the target(s).

[0086] In a first sample of the second set of cell samples (Tube 4), a total amount of the multi-target agent bound to the first target at the saturating concentration (T4) may be determined by adding the first detection reagent to the first sample and detecting a signalintensity of the detectable label. In a second sample of the second set of cell samples (Tube 5), a total amount of the multi-target agent bound to the second target at the saturating concentration (Ts) may be determined by adding the second detection reagent to the second cell sample and detecting a signal intensity of the detectable label. The third detection reagent is added to the sample of the second set of cell samples (Tube 6), and a total amount of the multi-target agent bound to the first and second targets at the saturating concentration (Te) is determined by the signal intensity from the detectable label of the third detection reagent.

[0087] A total target occupancy of the multi-target agent at the saturating concentration may be calculated for each target. A total target 1 occupancy of the multi-target agent at the saturating concentration may be calculated by subtracting the total amount of the multi-target agent bound to the first target in the first sample from the total amount of the multi-target agent bound to the common region in the third sample:Tsaturating-Targetl — T<> — T4.

[0088] A total target 2 occupancy of the multi-target agent at the saturating concentration may be calculated by subtracting the total amount of the multi-target agent bound to the second target in the second sample from the total amount of the multi-target agent bound to the common region in the third sample:Tsaturating-Target2 — T6 — T5.

[0089] A percent target occupancy at the standard concentration may be determined by calculating a ratio of the total amount of the multi-target agent bound to the common region at the standard concentration to the total amount of the multi-target agent bound to the common region at the saturating concentration:%Ttot = (T3 / T6)X100.

[0090] A percent cis-target occupancy at the standard concentration may be determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the first and second targets at the saturating concentration:%Tcis total = (Tcis-bound / Te)xl00.

[0091] A percent cis-target occupancy at the standard concentration for the first target of the multi-target agent may be determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the first target at the saturating concentration:%TcisTargetl — (Tcis-bound / (T6-T4))xl00.

[0092] A percent cis-target occupancy at the standard concentration for the second target of the multi-target agent may be determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the second target at the saturating concentration:%Tcis Target2 = (Tcis-bound / (T6-Ts))xl00.

[0093] The methods disclosed herein may include use of single-target agents for determination of the amount of the multi-target agent bound to the first and second targets. With reference to FIG. 4, the method may comprise adding the multi-target agent or singletarget agents to the second set of cell samples at the saturating concentration for incubation as described above. In a first sample of the second set of cell samples (Tube 4*), a first singletarget agent configured to bind to the second target only and the third detection reagent are added. The first single target agent includes a region recognized by the third detection reagent. The signal intensity of the detectable label of the third detection reagent provides an amount of the multi-target agent bound to the second target at the saturating concentration and to the first target at the standard concentration (T4*). Thus, a percent cis-target occupancy at the standard concentration for the second target of the multi-target agent may be determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the second target at the saturating concentration:%Tcis Target2 = (Tcis-bound / (T4*-Tl))xl00.

[0094] In a second sample of the second set of cell samples (Tube 5*), a second single-target agent configured to bind to the first target only and the third detection reagent are added. The second single target agent includes a region recognized by the third detection reagent. The signal intensity of the detectable label provides an amount of the multi-target agent bound to the first target at the saturating concentration and to the second target at the standard concentration (T5*). Thus, a percent cis-target occupancy at the standard concentration for the first target of the multi-target agent may be determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the first target at the saturating concentration:%Tcis Targetl — (Tcis-bound / (Ts*-T2))xl00.

[0095] Finally, in a third sample of the second set of cell samples (Tube 6), a total amount of the multi-target agent bound to the first and second targets at the saturating concentration (Te) may be determined by adding the third detection reagent to the third cell sample and detecting a signal intensity of the detectable label.SYSTEMS

[0096] Also provided in the present disclosure are embodiments directed to a system for determining target occupancy of a multi-target agent. The system generally comprises a multi-target agent comprising a first binding moiety targeted to a first target and a second binding moiety targeted to a second target; a first detection reagent having a detectable label and configured to bind to the multi-target agent at a region unique to the second binding moiety; a second detection reagent having the same detectable label and configured to bind to the multi-target agent at a region unique to the first binding moiety; and a third detection reagent having the same detectable label as the first and second detection reagents and configured to bind to the multi-target agent at a common region.

[0097] The system may further comprise first and second sets of cells (or a single set of cells into which different amounts of the multi-target agent and / or single-target agents are added).

[0098] The multi-target agent may be a bispecific or trispecific antibody, or a bispecific or trispecific drug. Each of the detection reagents may be an antibody configured to bind specifically to one of the first or second binding moieties of the multi-target agent. The detectable label may be a fluorescent label, such as useful in a flow cytometry assay to detect a signal strength. Other detectable labels, such as disclosed herein, are possible.KITS

[0099] In some embodiments, a kit is provided, comprising each of the components of the above noted system in a container. For example, the kit may comprise a container having separate tubes, such as a tube containing a multi-target agent; a tube containing a first detection reagent having a detectable label and configured to bind to the multi-target agent at a region unique to a binding moiety; a tube containing a second detection reagent having the detectable label and configured to bind to the multi-target agent at a region unique to a different binding moiety; and a tube containing a third detection reagent having the detectable label and configured to bind to the multi-target agent at a common region, such as a region not within the any of the binding moieties.

[0100] In some embodiments of the kit, cell samples may also be provided. Further, appropriate reagents for dilution, enhanced detection, and the like may be included. Further yet, various tubes or multi-well plates may be included to provide for formation of the various samples (Tubes 1-6) indicated in the above noted methods. Finally, an insert detailing the components of the kit and appropriate methods of use may be included in the kit.EXAMPLESExample 1: Preparing Samples

[0101] An exemplary method includes the following steps, certain of which may be performed in a different order.

[0102] A volume of appropriate cell samples is added to wells of a 2.2 mL / well preparation plate according to an experimental plate template. A multi-target agent is prepared according to the experimental plate layout (e.g., range of dilutions), and added to the appropriate wells of the 2.2 mL / well preparation plate according to the plate layout. The components in the each well of the preparation plate are mixed by pipetting up and down at least five times. The plate with cells is incubated for a minimum of 30 minutes in the dark at room temperature.

[0103] The cell samples are washed, such as by cycles of (i) centrifuge the samples at 400 X g for 5 minutes., (ii) aspirate the supernatant, (iii) add 1.8 mL of Stain Buffer (BSA) to each well containing sample, (vi) pipette up and down at least five times to mix, and (v) repeat.

[0104] An appropriate amount of the appropriate detection reagent(s) (e.g., 2.5 pg / sample) is added to the appropriate wells of the 2.2 mL / well plate according to the plate layout. The components in each well are mixed by pipetting up and down at least five times. The mixture(s) are the incubated for a minimum of 30 minutes in the dark in a refrigerator set to maintain 4°C.

[0105] The cell samples are washed, such as by cycles of (i) centrifuge the samples at 400 X g for 5 minutes., (ii) aspirate the supernatant, (iii) add 1.8 mL of Stain Buffer (BSA) to each well containing sample, (vi) pipette up and down at least five times to mix, (v) repeat.

[0106] Additional steps required to prepare the cell samples for label detection will depend on the label (e.g., fluorescent, radioactive, etc.) and are well known in the art.Example 2: Detection of two targets with a bispecific antibody

[0107] With reference to FIG. 5, a CD20+ cell line (Carnaval cell) was used to test detection of two targets. A wild type cell line, cell line absent target 1 (i.e., knockout for target 1), and acell line absent target 2 (i.e., knockout for target 2), were tested. The flow cytometry results shown in FIG. 5 demonstrate detection of signal for both targets in the wildtype, wherein each knockout shows no signal when probing for that absent target.

[0108] Of note, the third detection reagent in this assay is an anti-Human IgG antibody. Binding of the bispecific antibody to each of the targets in the various cells lines is shown in FIG. 6A (wildtype Carnaval CD20+ cells; and target 1 and 2 knockouts, each in Carnaval CD20+ cells). Binding of the bispecific antibody to both of the targets in whole blood is shown in FIG. 6B. Both illustrate a titration of the bispecific antibody to saturating concentrations.Example 3: Testing of donor blood samples

[0109] With reference to FIG. 7A and 7B, whole blood samples from three different donors were tested using the bispecific antibody of Example 2. As shown, variability in the assay is minimal, and the method is able to detect receptor occupancy (total RO), cis-receptor occupancy (cisRO), and occupancy of the bispecific antibody at each of target 1 (cisRO X) and target 2 (cisRO Y).

[0110] All publications and patents referred to herein are incorporated by reference. Various modifications and variations of the described subject matter will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to these embodiments. Indeed, various modifications for carrying out the invention are obvious to those skilled in the art and are intended to be within the scope of the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for determining target occupancy of a multi-target agent, the method comprising:adding the multi-target agent to a first set of cell samples at a standard concentration for incubation, wherein the multi-target agent comprises a first binding moiety targeted to a first target and a second binding moiety targeted to a second target;in a first sample of the first set of cell samples, determining a total amount of the multitarget agent bound to the first target at the standard concentration (Ti) by adding a first detection reagent to the first sample, wherein the first detection reagent includes a detectable label and binds to the multi-target agent at a region unique to the second binding moiety, and detecting a signal intensity of the detectable label;in a second sample of the first set of cell samples, determining a total amount of the multi-target agent bound to the second target at the standard concentration (T2) by adding a second detection reagent to the second sample, wherein the second detection reagent includes the detectable label and binds to the multi-target agent at a region unique to the first binding moiety, and detecting a signal intensity of the detectable label; andin a third sample of the first set of cell samples, determining a total amount of the multitarget agent bound to the first and second targets at the standard concentration (T3) by adding a third detection reagent to the third sample, wherein the third detection reagent includes the detectable label and binds to the multi-target agent at a common region or a region that is not the first or second binding moiety, and detecting a signal intensity of the detectable label.

2. The method according to claim 1 , wherein the standard concentration is a non-saturating concentration.

3. The method according to claim 1 or 2, comprising:calculating a cis target occupancy of the multi-target agent at the standard concentration by subtracting the total amount of the multi-target agent bound to the first target in thefirst sample and the total amount of the multi-target agent bound to the second target in the second sample from the total amount of the multi-target agent bound to the common region in the third sample:Tcis-bound = T3 - (T1 + T2).

4. The method according to any one of the preceding claims, comprising:adding the multi-target agent to a second set of cell samples at a saturating concentration for incubation;in a first sample of the second set of cell samples, determining a total amount of the multi-target agent bound to the first target at the saturating concentration (T4) by adding the first detection reagent to the first sample and detecting a signal intensity of the detectable label;in a second sample of the second set of cell samples, determining a total amount of the multi-target agent bound to the second target at the saturating concentration (T5) by adding the second detection reagent to the second cell sample and detecting a signal intensity of the detectable label; andin a third sample of the second set of cell samples, determining a total amount of the multi-target agent bound to the first and second targets at the saturating concentration (Te) by adding the third detection reagent to the third cell sample and detecting a signal intensity of the detectable label.

5. The method according to claim 4, comprising:calculating a total target 1 occupancy of the multi-target agent at the saturating concentration by subtracting the total amount of the multi-target agent bound to the first target in the first sample from the total amount of the multi-target agent bound to the common region in the third sample:Tsaturating-Targetl=Te — T4; andcalculating a total target 2 occupancy of the multi-target agent at the saturating concentration by subtracting the total amount of the multi-target agent bound to the second target in the second sample from the total amount of the multi-target agent bound to the common region in the third sample:Tsaturating-Target2 T6- T5.

6. The method according to any one of the preceding claims, comprising:adding the multi-target agent or single-target agents to a second set of cell samples at a saturating concentration for incubation;in a first sample of the second set of cell samples, determining an amount of the multitarget agent bound to the second target at the saturating concentration (T4*) by adding (i) a first single-target agent configured to bind to the second target only and (ii) the third detection reagent, wherein the first single target agent includes a region recognized by the third detection reagent, and detecting a signal intensity of the detectable label;in a second sample of the second set of cell samples, determining an amount of the multitarget agent bound to the first target at the saturating concentration (T5*) by adding (i) a second single-target agent configured to bind to the first target only and (ii) the third detection reagent, wherein the second single target agent includes a region recognized by the third detection reagent, and detecting a signal intensity of the detectable label; andin a third sample of the second set of cell samples, determining a total amount of the multi-target agent bound to the first and second targets at the saturating concentration (Te) by adding the third detection reagent to the third cell sample and detecting a signal intensity of the detectable label.

7. The method according to any one of the preceding claims, wherein a percent target occupancy at the standard concentration is determined by calculating a ratio of the total amount of the multi-target agent bound to the common region at the standard concentration to the total amount of the multi-target agent bound to the common region at the saturating concentration:%Ttot= (T3 / T6)X100.

8. The method according to any one of claims 3 to 7, wherein a percent cis-target occupancy at the standard concentration is determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the first and second targets at the saturating concentration:%Tcis total = (Tcis-bound / T6)xl00.

9. The method according to any one of claims 3 to 9, wherein a percent cis-target occupancy at the standard concentration for the first target of the multi-target agent is determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent:%T cis Targetl — ( cis-bound / (T6-T4))x 100, OT%Tcis Targetl=(Tcis-bound / (T5*-T2))x 100.

10. The method according to any one of claims 3 to 10, wherein a percent cis-target occupancy at the standard concentration for the second target of the multi-target agent is determined by calculating a ratio of the cis target occupancy of the multi-target agent at the standard concentration to the total amount of the multi-target agent bound to the second target at the saturating concentration:%T cis Target2 — ( cis-bound / (T6-T5))x 100, OT%T cis Target2 — (Tcis-bound / (T4*-Tl))xl00.

11. The method according to any one of the preceding claims, wherein the first target and the second target comprise proteins expressed on a surface of the same cell.

12. The method according to any one of the preceding claims, wherein the multi-target agent is a bispecific antibody.

13. The method according to any one of the preceding claims, adapted for a multi-target agent having more than two binding moieties.

14. The method according to claim 13, wherein the multi-target agent is a trispecific antibody.

15. The method according to any one of the preceding claims, wherein the multi-target agent is a therapeutic agent.

16. The method according to claim 15, wherein the standard concentration is a therapeutic concentration of the therapeutic agent.

17. The method according to any one of the preceding claims, wherein the cells of the first and second sets of cell samples are immune cells.

18. The method according to any one of the preceding claims, wherein the cells of the first and second sets of cell samples are B-cells, T-cells, NK-cells, Leukocytes, or monocytes.

19. The method according to any one of the preceding claims, wherein the cells of the first and second sets of cell samples are cells from a whole blood sample.

20. The method according to claim 19, wherein the whole blood sample is from a mammal.

21. The method of claim 20, wherein the mammal is a human.

22. The method of claim 20, wherein the mammal is a simian, llama, cow, rabbit, rat, or mouse.

23. The method according to any one of claims 1 to 19, wherein the first and second sets of cell samples are tissue samples.

24. The method according to claim 23, wherein the tissue samples are from a mammal.

25. The method of claim 23, wherein the mammal is a human.

26. The method of claim 23, wherein the mammal is a simian, llama, cow, rabbit, rat, or mouse.

27. The method according to any one of the preceding claims, wherein the first, second, and third detection reagents comprise antibodies.

28. The method according to any one of the preceding claims, wherein the detectable label comprises a fluorescent compound.

29. The method according to claim 28, wherein detecting the signal intensity of the detectable label comprises flow cytometry or molecular imaging techniques.

30. The method according to any one of the preceding claims, wherein the detectable label comprises a radiolabel, biotin, magnetic beads, or an enzymatic label.

31. A system for determining target occupancy of a multi-target agent, the system comprising:a multi-target agent comprising a first binding moiety targeted to a first target and a second binding moiety targeted to a second target;a first detection reagent having a detectable label and configured to bind to the multitarget agent at a region unique to the second binding moiety;a second detection reagent having the detectable label and configured to bind to the multi-target agent at a region unique to the first binding moiety; anda third detection reagent having the detectable label and configured to bind to the multitarget agent at a common region.

32. The system of claim 31, comprising first and second sets of cells.

33. The system of claim 31 or 32, wherein the multi-target agent is a bispecific antibody, wherein the first target and the second target comprise proteins expressed on a surface of a cell34. The system according to claim 32 or 33, wherein the cells are from a mammal.

35. The system according to claim 34, wherein the mammal is a human.

36. The system according to claim 34, wherein the mammal is a simian, llama, cow, rabbit, rat, or mouse.

37. The system according to any one of claims 31 to 36, wherein the cells of the first and second sets of cell samples are immune cells.

38. The system according to any one of claims 31 to 36, wherein the cells of the first and second sets of cell samples are B-cells, T-cells, NK-cells, Leukocytes, or monocytes.

39. The system according to any one of claims 31 to 36, wherein the cells of the first and second sets of cell samples are cells from a whole blood sample.

40. The system according to any one of claims 31 to 36, wherein the first and second sets of cell samples are tissue samples.

41. The system according to any one of claims 31 to 40, wherein the first, second, and third detection reagents comprise antibodies.

42. The system according to any one of claims 31 to 41, wherein the detectable label comprises a fluorescent compound.

43. The system according to claim 42, wherein detecting the signal intensity of the detectable label comprises flow cytometry or molecular imaging techniques.

44. The system according to any one of the claims 31 to 42, wherein the detectable label comprises a radiolabel, biotin, magnetic beads, or an enzymatic label.

45. The system of claim 31, adapted for a multi-target agent having more than two binding moieties.

46. The system of claim 45, wherein the multi-target agent is a trispecific antibody.

47. The system according to any one of claims 31 to 46, wherein the multi-target agent is a therapeutic agent.

48. The system according to claim 47, wherein the standard concentration is a therapeutic concentration of the therapeutic agent.

49. The system of claim 31 for use in a method according to any one of claims 1-30.

50. A method for determining target occupancy of a multi-target agent, the method comprising:adding the multi-target agent to a first set of cell samples at a standard concentration for incubation, wherein the multi-target agent comprises means for binding a first target and a means for binding a second target;in a first sample of the first set of cell samples, a means for determining a total amount of the multi-target agent bound to the first target at the standard concentration (Tl) by adding a first detection reagent to the first sample, wherein the first detection reagent includes a means for detecting the multi-target agent at a region unique to the second binding moiety, and a means for detecting a signal intensity of multi-target agent;in a second sample of the first set of cell samples, a means for determining a total amount of the multi-target agent bound to the second target at the standard concentration (T2) by adding a second detection reagent to the second sample, wherein the seconddetection reagent includes a means for detecting and binding to the multi-target agent at a region unique to the first binding moiety, and a means for detecting binding to the multitarget agent at the region unique to the first binding moiety; andin a third sample of the first set of cell samples, a means for determining a total amount of the multi-target agent bound to the first and second targets at the standard concentration (T3) by adding a third detection reagent to the third sample, wherein the third detection reagent includes a means for detecting and binding to the multi-target agent at a common region or a region that is not the first or second binding moiety, and a means for detecting the multi-target agent at a common region or a region that is not the first target or the second target.

5. Steps for determining target occupancy of a multi-target agent, the method comprising:a step for adding the multi-target agent to a first set of cell samples at a standard concentration for incubation, wherein the multi-target agent comprises a first binding moiety targeted to a first target and a second binding moiety targeted to a second target;in a first sample of the first set of cell samples, a step for determining a total amount of the multi-target agent bound to the first target at the standard concentration (Ti) by adding a first detection reagent to the first sample, wherein the first detection reagent includes a detectable label and binds to the multi-target agent at a region unique to the second binding moiety, and detecting a signal intensity of the detectable label;in a second sample of the first set of cell samples, a step for determining a total amount of the multi-target agent bound to the second target at the standard concentration (T2) by adding a second detection reagent to the second sample, wherein the second detection reagent includes the detectable label and binds to the multi-target agent at a region unique to the first binding moiety, and detecting a signal intensity of the detectable label; andin a third sample of the first set of cell samples, a step for determining a total amount of the multi-target agent bound to the first and second targets at the standard concentration (T3) by adding a third detection reagent to the third sample, wherein the third detection reagent includes the detectable label and binds to the multi-targetagent at a common region or a region that is not the first or second binding moiety, and detecting a signal intensity of the detectable label.

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