CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 62/966,946, filed January 28, 2020, which is incorporated by reference herein.

TECHNICAL FIELD

[0002] This disclosure relates to a new bioluminescent reporter assay technology that can enable real time kinetic capacity and high sensitivity for imaging and quantification of uptake of biomolecules inside the cytosolic compartment of the cell. Since most of the biological targets are located inside the cells and require entry in the cytosol, this assay represents a valuable tool for drug discovery of novel therapeutics and understanding of basic mechanism of cell entry of biologically active biomolecules. The biomolecules suitable for this assay include, but not limit, to the following classes of biomolecules: small molecule drugs, vitamins, metabolites (lipids, glucose, etc.), peptides, antibodies, antibody drug conjugates, proteins, nanoparticles, viruses, cells, and bacteria. The assay is suitable for both in vitro (ex. live cells) and in vivo (live animals) applications enabling important aspect of drug discovery. This assay is termed in this disclosure as Bioluminescence Imaging of Cellular Uptake (BL-ICU).

BACKGROUND

[0003] Biologically active compounds such as small molecules, peptides, antibodies and antibodies drug conjugates constitute a dynamic and rapidly expanding class of biotherapeutics capable of targeting multiple human disease indications including cardiovascular, respiratory, autoimmune and oncology disorders.

[0004] For example, small molecules represent the most common class of pharmaceuticals. The majority of small molecule drug targets are inside the cells and therefore efficiency to cross cellular barriers is an important parameter for their biological activity.

[0005] Peptide therapeutics is another important class of drugs that play a central role in medical practice. Over 60 peptide drugs have been approved in the United States and other major markets in the last few years. They have many advantages over small-molecule drugs such as low toxicity and fast clearance. However, the major obstacle for the peptidic drug development is their high polarity that is the main reason for their low levels of cell permeability.

[0006] Small molecules metabolites and vitamins play an important role in drug discovery. One of the examples is the development of inhibitors of tumor metabolism that are normally given in combination with existing drugs to increase their overall efficacy. Understanding of intracellular internalization properties of various metabolites and vitamins is important for screening of such drugs. [0007] Nanoparticle drug delivery is a novel technologies that utilizes nanoparticles for the targeted delivery followed by controlled release of drugs. The main advantages of nanoparticles is their high drug load capacity, targeted delivery to the tissues of interest (ex. cancer) and their ability to facilitate intracellular uptake. Despite these advantages, there is a strong need in understanding their biodistribution and accumulation, as well as intracelluar drug delivery. Due to the lack of existing tools for quantification of intracellular drug release, the development of a nanoparticle drug delivery system remains to be a long and expensive process.

[0008] The last decade has seen a strong year-on-year increase in the number of therapeutic Abs, with around 59 antibody or antibody-derived products awarded market approval by the United States Food and Drug Administration (FDA) and European Medicines Agency (EMEA). Indeed, several mAbs have achieved extraordinary success as so-called blockbusters (e.g. rituximab, adalimumab, trastuzumab) while others occupy niches for orphan indications (dinutximab, obiltoxaximab). Several Abs are now listed as WHO’s essential medicines (e.g. trastuzumab, rituximab and bevacizumab).

[0009] This rapid growth and success within the Ab field has led to the recent emergence of a class of specialist cancer bio-therapeutics termed Antibody Drug Conjugates (ADCs). These ADCs seek to combine - through a physical linkage - the exquisite specificity and disease targeting potential of mAbs with the potent, but indiscriminate, cell killing activity of various well-characterized natural and synthetic toxin molecules (e.g. aurostatins, maytansinoids). It is widely considered that specific targeting of such chemotherapeutic ‘payloads’ to sites of oncological disease, may offer significant advantages with regard to treatment efficacy, side-effect avoidance and general patient outcomes. [0010] Notable advances in mAh production technologies and in the development of linker properties, toxin cargo, and conjugation strategies have fueled a substantial increase in new ADC investigational new drug (IND) submissions over recent years resulting in a strong clinical pipeline of some 53 ADC molecules with approximately one-third of these in Phase II/III of development. This dynamic pipeline also includes an impressive array of molecules (about 60) in early discovery/pre-clinical research. With about 10 new ADC commercial launches predicted over the coming decade, the overall ADC market is expected to be worth an estimated USD 10 billion annually by 2025. Therefore, there is a strong ongoing interest for the development of novel ADCs (www.reporterlinker.com/p02280919-summary/Antibody -Drug-Conjugates-Market-Edition.html). [0011] Despite the compelling early successes of the clinical ADC pioneers outlined above, the wider field faces significant technical and conceptual challenges. Over the last several years it has become increasingly apparent that the therapeutic performance of a given ADC depends on several empirically-determined variables which include not only the chemical design features of the conjugated ‘payload’ toxin, but also on the affinity and epitope-recognition properties of the targeting antibody component itself. Hence, current ADC opinion leaders now acknowledge that effective ADC performance can be dependent on a complex interplay between the antibody, internalization properties of the cell surface target antigen and the chemistry of the conjugated toxin moiety. The important implication of the above is that simply re-purposing existing off-the-shelf antibodies that were developed specifically for classical tumor cell binding or ligand/receptor blockade applications may not yield optimal ADCs and may ultimately limit the potential of this drug class. Therefore, typical ADC development platforms could be substantially improved through integration of de novo discovery and selection strategies early in the process that will speed up and streamline the identification of high-quality ADC-compatible antibodies that are optimally fit-for- purpose as payload carriers.

[0012] The selection of appropriate mAbs for ADC development ideally requires knowledge of: a) the affinity and selectivity of mAb binding to a target cell antigen (more specifically, an epitope), b) the extent to which the mAb-antigen complex is internalized (endocytosed), c) the extent to which the mAb is then degraded within the lytic compartments of the cell, and/or d) the extent to which the resultant lytic fragments (containing the toxin ‘payload‘) are trafficked into the cytosolic compartment of the cell (where the ‘payload‘ will exert its cytotoxicity). [0013] A means to comprehensively monitor or observe these sequentially linked processes would be considered a highly desirable and enabling ADC platform capability.

[0014] At present, however, in vitro assay systems employed to assess and rank the kinetics of mAb cellular internalization and intracellular toxin release all suffer from significant and important limitations. In brief, the existing methodologies all rely on so-called ‘non-homogeneous‘ assay formats that require multiple "invasive" mechanical manipulation steps (typically cell washing, lysis etc.) and yield only end-point read-outs. Therefore, these methods are not capable of assessing or ranking antibodies or their conjugates with regard to the real-time kinetics of internalization and processing in cells. Moreover, ADC uptake by cells is a dynamic and metabolic process which could be significantly perturbed by mechanical interference/intervention leading to artificial or misleading observations. Consequently, typical ADC lead discovery and early development platforms currently lack robust and informative screening tools to guide the selection of those antibodies or antibody variants that are i) internalized efficiently by target cells, and/or ii) are processed appropriately to deliver an active toxin payload into the cytosolic compartment of the cell.

[0015] This application discloses an enabling, scalable reporter assay technology that is developed by combining chemical and biochemical innovation. This reporter assay can provide real time kinetic data for the uptake and processing of prospective ADC candidate Abs as well as other therapeutic molecules such as small-molecule drugs, peptides, metabolites, vitamins, nanoparticles and viruses that rely on cellular internalization. The assay can, in addition, yield information on the internalization behavior of different cell-surface antigens. This assay, named Bioluminescence Imaging of Cellular Uptake (BL-ICU), relies on labeling biomolecules through a linker such as a disulfide bond with a novel luciferin molecule (ex. Thiol Tag Luciferin (TT-Luc), FIG. 1). The labeling can also be done via chemical conjugation to amines, that relies on the same reported molecule (ex. Amino Tag Luciferin (AT-Luc), FIG. 1). Upon delivery into the reducing cytosolic environment the linker such as the disulfide bond is cleaved to release the luciferin derivative, a process that can be monitored in cells expressing luciferase enzyme.

[0016] BL-ICU assay provides significant advantages over regular caging strategy. For example, Amino Tag Luciferin (AT-Luc) and Thiol Tag Luciferin (TT-Luc) produce bright light upon reaction with luciferase enzyme and possess unprecedented stability producing no background in biological environment. They can be attached to a wide variety of biomolecules using easy-to-perform chemistry resulting in stable conjugates. That makes them excellent tags for studies of biological uptake. No similar molecules are known in the literature as the general “believe” that luciferin has to be free to produce light (the tag compounds provided herein have luciferin tagged with special non-releasable cytosol-environment sensitive linker).

[0017] BL-ICU assay is highly flexible, easy to apply and can be readily adapted to early drug discovery platforms.

BRIEF SUMMARY

[0018] In one aspect, a compound of Formula I or a salt thereof is provided:

Formula I wherein L is a bioluminescent reporter or a pro-luminescent moiety.

[0019] In another aspect, provided is a conjugate of Formula II or a salt thereof:

Formula II wherein B comprises a biomolecule and L is a bioluminescent reporter or a pro-luminescent moiety. [0020] In another aspect, provided is a compound of Formula III or a salt thereof,

Formula III

[0021] In another aspect, provided is a conjugate of Formula IV or a salt thereof:

Formula IV wherein B comprises a biomolecule.

[0022] In each of Formulae I-IV, X is an alkylene.

[0023] In each of Formulae I and III, Z is an optionally substituted alkyl, an optionally substituted

L _g _ I aryl, an optionally substituted heteroaryl, , or 1 ; wherein Y is an optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted arylene, or polyethylene glycol PEGn, where n=l-50; and A is selected from the group consisting of , wherein R is optionally substituted alkyl.

[0024] In each of Formulae I

[0025] In another aspect, provided is a compound of Formula V or a salt thereof:

Formula V

[0026] wherein X is an alkylene and L is a bioluminescent reporter or a pro-luminescent moiety. [0027] In another aspect, provided is a compound of Formula VI or a salt thereof: Formula VI, wherein X is an alkylene

[0028] In another aspect, a method for preparing a conjugate of Formula II or IV is provided, comprising incubating a biomolecule with a compound of Formula I or III, respectviely. [0029] In another aspect, a method for detecting cellular uptake and processing of a biomolecule is provided, wherein the method comprises contacting a sample with a conjugate of Formula II or IV. [0030] In some embodiments, the sample comprises a cell.

[0031] In some embodiments, the method comprises measuring light emission, wherein detection of light emission indicates that the biomolecule has been processed by a lysosome.

[0032] In another aspect, a kit is provided, comprising a compound of Formula I or III, and a biomolecule.

[0033] In some embodiments, the kit comprises a conjugate of Formula II or IV.

BRIEF DESCRIPTION OF DRAWINGS [0034] FIG. 1A illustrates Probe/ Assay design.

[0035] FIG. IB describes a mechanism whereby conjugates of the present disclosure allow monitoring of internalization of biomolecules of interest.

[0036] FIG. 2 illustrates a dose dependent bioluminescence from thioluciferin and thioluciferin- labeled octa-arginine peptide (r8-SS-luc).

[0037] FIG. 3 shows a general principle of the antibody uptake assay design.

[0038] FIG. 4A shows bioluminescent signal from a cellular uptake of Trastuzumab-thioluciferin (Trastuzumab-SS-Luc) and IgG-thioluciferin (IgG-SS-Luc) antibodies in Her2 positive SK-BR-3 luciferase expressing cell lines at different time points.

[0039] FIG. 4B shows the bioluminescent signal from Trastuzumab-thioluciferin normalized to that from IgG-thioluciferin at each time points over the course of 48 hrs.

[0040] FIG. 5A shows bioluminescent signal from a cellular uptake of Trastuzumab- thioluciferin and IgG-thioluciferin antibodies in Her2 negative 4T1 luciferase expressing cell lines over the course of 48 hrs.

[0041] FIG. 5B shows the bioluminescent signal from Trastuzumab-thioluciferin normalized to that from IgG- thioluciferin at each time points.

[0042] FIGS. 6A-6D shows comparison of Trastuzumab-thioluciferin uptake normalized to IgG- thioluciferin uptake in two Her2 positive (SK-BR-3 and BT474 in FIG. 6A and FIG. 6B, respectively) and two Her2 negative luciferase expressing cell lines (4T1 and MDA-MB231 in FIG. 6C and FIG. 6D, respectively).

[0043] FIG. 7 provides data of cold antibody competition assay with Trastuzumab-thioluciferin and Trastuzumab along in BT474 Her2 positive cell line.

[0044] FIG. 8 provides signal from in vivo uptake of Trasuzumab-thioluciferin and control IgG- thioluciferin in BT474-Luc tumor xenograft mouse model. [0045] FIGS. 9A-9B illustrate data from bioluminescence from 4T1 RLR transfected with DNA- SS-luc (DNA-thioluciferin) using lipofectamine (FIG. 9A) and polyethyleneimine (FIG. 9B) transfection reagents.

DETAILED DESCRIPTION

I. Definitions

[0046] As used in this specification, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a "polypeptide" includes a single polypeptide as well as two or more of the same or different polypeptides, reference to an "agent" includes a single agent as well as two or more of the same or different agents, and the like.

[0047] Where a range of values is provided, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 pm to 8 pm is stated, it is intended that 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, and 7 pm are also explicitly disclosed, as well as the range of values greater than or equal to 1 pm and the range of values less than or equal to 8 pm. As used herein, the term “about” can mean ±2%, ±3%, ±5%, ±10%, or ±20% of the value being modified. [0048] “Luciferase,” as used herein refers to an enzyme that oxidizes a corresponding luciferin, thereby causing bioluminescence. Luciferase enzymes can be found in bacteria, fireflies, fish, squid, dinoflagellates, and other organisms capable of bioluminescence. Luciferase, as used herein, can include prokaryotic and eukaryotic luciferases, as well as variants possessing varied or altered optical properties.

[0049] “Luciferin,” as used herein refers to a substrate for a luciferase enzyme. Luciferins typically undergo an enzyme-catalyzed oxidation and the resulting excited state intermediate emits light (photons) upon decaying to its ground state. Luciferin as used herein can refer to any one or more of the following non-exhaustive list: a luciferin derivative or analog, a preluciferin or analog, coelenterazine or a coelenterazine derivative or analog, pro-luciferin, aminoluciferin, quionolyl- luciferin, napthyl luciferin, chloroluciferin, coelenterazine, furimazine, coelenterazine-n, coelenterazine-f, coelenterazine -h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxycoelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl coelenterazine.

[0050] The term "alkyl," as used herein is intended to include a monovalent branched and straight- chain saturated aliphatic hydrocarbon groups having one to twenty number of carbon atoms. Ci-Cio, as in "Ci-Cio alkyl" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement. For example, "Ci-Cio alkyl" specifically includes methyl, ethyl, n- propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, and so on. [0051] The term “alkylene,” as used herein is intended to include a bivalent, branched or straight- chain saturated aliphatic hydrocarbon groups having one to twenty number of carbon atoms. For example, Ci-io, as in "Ci-io alkylene" is defined to include groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbons in a linear or branched arrangement. For example, C1-3 alkylene includes methylene, ethylene, and propylene (-CH2CH2CH2- or -CFECHiOFE)-).

[0052] The term "alkoxy" as used herein refers to -O-alkyl wherein alkyl is defined as above. [0053] The term "cycloalkyl" means a monovalent monocyclic, bicyclic or spirocyclic saturated aliphatic hydrocarbon group having three to twenty number of carbon atoms. The cycloalkyl can be bridged (i.e., forming a bicyclic moiety), for example with a methylene, ethylene or propylene bridge. The cycloalkyl may be fused with an aryl group such as phenyl, and it is understood that the cycloalkyl substituent is attached via the cycloalkyl group. For example, "cycloalkyl" includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and so on. "cycloalkyl" also includes cycloalkyl rings as described above wherein =CH2 replaces two available hydrogens on the same ring carbon atom.

[0054] The term "cycloalkylene" means a bivalent monocyclic, bicyclic or spirocyclic saturated aliphatic hydrocarbon group having three to twenty number of carbon atoms. The ring structure of cycloalkylene is the same as that of cycloalkyl.

[0055] "Aryl" is intended to mean any stable monovalent monocyclic, bicyclic or tricyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. The aryl can have six to twenty carbon atoms. Examples of such aryl elements include phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. In cases where the aryl substituent is bicyclic or tricyclic and one ring is non aromatic, it is understood that attachment is via the aromatic ring.

[0056] In one embodiment, "aryl" is an aromatic ring of 6 to 14 carbon atoms, and includes a carbocyclic aromatic group fused with a 5- or 6-membered cycloalkyl group such as indan. Examples of carbocyclic aromatic groups include, but are not limited to, phenyl, naphthyl, e.g. 1-naphthyl and 2-naphthyl; anthracenyl, e.g. 1-anthracenyl, 2-anthracenyl; phenanthrenyl; fluorenonyl, e.g. 9- fluorenonyl, indanyl and the like.

[0057] “Arylene” is intended to mean any stable bivalent monocyclic, bicyclic or tricyclic carbon ring of up to 7 atoms in each ring, wherein at least one ring is aromatic. The bovalent attachment point can be on a saturated or unsaturated carbon. The ring structure of arylene is the same as that of aryl.

[0058] The term “heteroaryl,” as used herein, represents a stable monovalent monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and contains carbon and from 1 to 4 heteroatoms selected from the group consisting of O, N and S. In another embodiment, the term heteroaryl refers to a monocyclic, bicyclic or tricyclic aromatic ring of 5- to

14-ring atoms of carbon and from one to four heteroatoms selected from O, N, or S. As with the definition of heterocycle below, "heteroaryl" is also understood to include the N-oxide derivative of any nitrogen-containing heteroaryl. In cases where the heteroaryl substituent is bicyclic or tricyclic and one ring is non-aromatic or contains no heteroatoms, in one embodiment, the attachment is via the heteroatom containing aromatic ring, respectively.

[0059] The term “optionally substituted” as used herein such as in “optionally substituted alkyl”, refers to the moiety following “optionally substituted” (such as alkyl) is not substituted or substituted. When substituted, the moiety can be substituted with one, two, or three groups independently selected from alkyl, alkoxy, halogen, CN, cycloalkyl, aryl and heteroaryl.

[0060] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (11). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region. The heavy chain constant region typically is comprised of three domains, CHI, CH2, and CH3. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region. The light chain constant region typically is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and/or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Typically, the numbering of amino acid residues in this region is performed by the method described in Rabat et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Rabat) after residue 52 of V.sub.H CDR2 and inserted residues (for instance residues 82a, 82b, and 82c, etc. according to Rabat) after heavy chain FR residue 82. The Rabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Rabat numbered sequence.

[0061] The term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a half-life of significant periods of time, such as at least about 30 minutes, at least about 45 minutes, at least about one hour, at least about two hours, at least about four hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally-defined period (such as a time sufficient to induce, promote, enhance, and/or modulate a physiological response associated with antibody binding to the antigen and/or time sufficient for the antibody to recruit an Fc-mediated effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. The constant regions of the antibodies (Abs) may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as Clq, the first component in the classical pathway of complement activation. As indicated above, the term antibody herein, unless otherwise stated or clearly contradicted by context, includes fragments of an antibody that comprise a mutated or wildtype core hinge region and retain the ability to specifically bind to the antigen. [0062] It has been shown that the antigen-binding function of an antibody may be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antibody" include, e.g. F(ab')2 fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region. Although such fragments are generally included within the meaning of antibody, they collectively and each independently are unique features of the present invention, exhibiting different biological properties and utility. It also should be understood that the term antibody, unless specified otherwise, also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and antibody fragments retaining the ability to specifically bind to the antigen (antigen-binding fragments) provided by any known technique, such as enzymatic cleavage, peptide synthesis, and recombinant techniques. An antibody as generated can possess any isotype.

[0063] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer 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. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be generated by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal nonhuman animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

[0064] As used herein, the term "binding" in the context of the binding of an antibody to a predetermined antigen typically is a binding with an affinity corresponding to a KD of about 10 ⁷ M or less, such as about 10 ⁸ M or less, such as about 10-9 M or less, about 10 ¹⁰ M or less, or about

10 ¹¹ M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a KD that is at least ten-fold lower, such as at least 100 fold lower, for instance at least 1000 fold lower, such as at least 10,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely -related antigen. The amount with which the affinity is lower is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.

[0065] The term "kd" (sec ¹ ), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the k _0ff value.

[0066] The term "k _a " (M l x sec ¹ ), as used herein, refers to the association rate constant of a particular antibody-antigen interaction.

[0067] The term "KD" (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0068] The term "KA" (M ¹ ), as used herein, refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the k _a by the k _d .

[0069] The term “therapeutic polypeptide” as used herein refers to a polypeptide which provides therapeutic efficacy when administered to a subject suffering from a disease or disorder. A therapeutic polypeptide includes but is not limited to a therapeutic antibody, fragment or derivative thereof.

[0070] The term "reducing agent" refers to a compound which reduces molecules in its environment, i.e., which changes molecules in its environment to become more reduced and more reducing. A reducing agent acts by donating electrons, thereby becoming itself oxidized after having reduced a substrate. Thus, a reducing agent is an agent which donates electrons. Examples of reducing agents include but are not limited to dithiothreitol (DTT), mercaptoethanol, cysteine, thioglycolate, cysteamine, glutathione, and sodium borohydride. In one embodiment, the reducing agent does not comprise an enzyme.

II. Bioluminescent Compositions for Monitoring Internalization

[0071] Antibody Drug Conjugates (ADCs) bind to an extracellular domain of their target receptor, leading to receptor internalization via endocytosis followed by lysosomal degradation of the Ab- receptor complex and cytosolic release of the toxin. Success of an ADC is closely correlated with endosomal sorting and lysosomal degradation kinetics of the antibody-receptor complex. Currently, there is no method which can directly measure the rate of such antibody internalization or cytosolic release of the cargo (e.g. drug conjugate) in real-time. The existing methodologies rely on imaging fluorophore- or radio-labelled antibodies using invasive techniques, which do not directly reveal cytosolic delivery of the cargo and are not in real-time.

[0072] In order to address the need for a homogeneous assay to real-time monitor antibody internalization and cytosolic drug release, a novel bioluminescence based assay termed as Bioluminescence Imaging of Cellular Uptake (BL-ICU) has been developed. BL-ICU is unique in that the generation of a read-out signal is strictly conditional on the end-point trafficking of a surrogate reporter ‘payload probe’ to the cytosolic compartment. The measurements are kinetic (real-time data sampling) and high-resolution, and reflect only the desired bio-active productive processing of a candidate ADC and other therapeutic molecules i.e. the unproductive association of an ADC at the cell surface or its residency in non-bioactive cellular compartments yields no read out.

[0073] The assay is based on the well-characterized chemical energetic transition that occurs when the Firefly luciferase enzyme catalyzes the oxidation of D-luciferin to oxyluciferin. The resulting electronically excited state, emits a yellow-green photon which can be detected via photomultiplier instruments. Such bioluminescence is a highly sensitive imaging modality due to the extremely low auto-luminescence present in mammalian cells and tissues, and it can easily be adapted to high- throughput screening systems.

[0074] The overall BL-ICUP in vitro assay principle is summarized in FIG. 3. Relevant target cells (pre-engineered to constitutively express Firefly luciferase in the cytosol) are treated with an antibody conjugated with luciferin (Ab-SS-Luciferin). If the specific cognate receptor antigen is present, a bound surface complex is formed. This complex can either reversibly dissociate, remain largely at the cell surface, or become internalised into the endosomal vesicular network. In the latter scenario, early endosomal vesicles can either recycle the bound complex back to the cell surface or direct the contents for lysosomal degradation. In this case, strong proteolytic processes hydrolyse the receptor-antibody components into peptidic fragments. These subsequently enter the reducing environment of the cytosol where disulfide-bonded peptide-luciferin fragments are efficiently reduced, liberating free luciferin which can be oxidized to generate a light signal that can be kinetically monitored and quantitated in real time.

[0075] While examples of the present disclosure are directed to an antibody conjugate comprising an antibody or fragment thereof conjugated to a thioluciferin derivative, it is understood that the luciferin compounds or moieties described herein, e.g., Formulae I or III, can be conjugated to a biomolecule such as polypeptide or protein to form a conjugate of Formula II or IV. A polypeptide according to the present disclosure, alternatively referred to herein as a protein, can be a monomeric, dimeric or multimeric protein. Similarly, it is understood that the luciferin compounds or moieties described herein, e.g., Formulae I or III, can be conjugated to other biomolecules, such as molecules with a specific binding activity. Such biomolecules can further include small molecules, nucleotides, oligonucleotides and siRNA. It can also be conjugated to therapeutic viral particles.

[0076] Provided herein is a compound of Formula I or a salt thereof: Formula I, wherein

L is a bioluminescent reporter or a pro-luminescent moiety,

X is an alkylene,

Z is an optionally substituted alkyl, an optionally substituted aryl, an optionally substituted

L _Y _ j heteroaryl, or 1 ; wherein Y is an optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted arylene, or polyethylene glycol PEGn, where n=l-50; and A is selected from the group consisting of

O and O , wherein R is optionally substituted alkyl.

[0077] In some embodiments, the optionally substituted alkyl is an alkyl optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. [0078] In some embodiments, the optionally substituted alkylene is an alkylene optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiments, the optionally substituted alkylene is a C1-C7 alkylene, such as Ci, C2, C3, C4, C5, Ce, or C7 alkylene, optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiment, the optionally substituted alkylene is a unsubstituted C1-C7 alkylene, such as unsubstituted Ci, C2, C3, C4, C5, Ce, or C7 alkylene. [0079] In some embodiments, the optionally substituted aryl is an aryl optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. [0080] In some embodiments, the optionally substituted arylene is an arylene optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl.

[0081] In some embodiments, the optionally substituted cycloalkylene is a cycloalkene optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl.

[0082] In some embodiments, Z is wherein Y is C1-C6 alkylene and A is as defined above. Z

L _g _ 1 L -U _ I is 1 wherein Y is C1-C6 alkylene some embodiments, Z is * wherein Y is C2 or Ce alkylene and A is as defined above. In some embodiments, Z is wherein

Y is C2 or Ce alkylene

[0083] In some embodiments, Z is an aryl optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiments, Z is an unsubstituted aryl such as phenyl.

[0084] In some embodiments, Z is a heteroaryl optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiments, Z is an unsubstituted heteroaryl such as pyridine.

[0085] In some embodiments, Z is an alkyl optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiments, Z is C1-C4 alkyl optionally substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiments, Z is a methyl substituted with one, two, or three groups selected from alkyl, alkoxy, CN, halogen, aryl, cycloalkyl, and heteroaryl. In some embodiments, Z is a methyl substituted with three aryl groups such as phenyl groups.

[0086] In some embodiments, [0087] In some embodiments, X is a Ci-Ce alkylene. In some embodiments, X is a C1-C4 alkylene. In some embodiments, X is C2, C3 or C4 alkylene.

[0088] In some embodiments, L is a luciferin, a luciferin derivative or analog, a preluciferin or analog, coelenterazine or a coelenterazine derivative or analog thereof. In other embodiments, L is selected from the group consisting of aminoluciferin, quionolyl-luciferin, napthyl luciferin, chloroluciferin, fluoroluciferin, coelenterazine, furimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxy coelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine- icp, coelenterazine-v, and 2-methyl coelenterazine.

[0089] In some embodiments, a compound of Formula I is a compound of Formula III or a salt thereof , and X are the same as defined for Formula I including each embodiments thereof.

[0090] In some embodiments, a compound of Formula III is a compound selected from yanobenzothiazoles) [0091] Also provided is a compound of Formula V or a salt thereof:

Formula V wherein X is an alkylene and L is a bioluminescent reporter or a pro-luminescent moiety.

[0092] In some embodiments, L is a luciferin, a luciferin derivative or analog, a preluciferin or analog, coelenterazine or a coelenterazine derivative or analog thereof. In other embodiments, L is selected from the group consisting of aminoluciferin, quionolyl-luciferin, napthyl luciferin, chloroluciferin, fluoroluciferin, coelenterazine, furimazine, coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, bis-deoxy coelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl coelenterazine.

[0093] In some embodiments, X is a Ci-Ce alkylene. In some embodiments, X is a C1-C4 alkylene. In some embodiments, X is C2, C3 or C4 alkylene.

[0094] In some embodiments, a compound of Formula V is a compound of Formula VI or a salt thereof: r -CN, and X is as defined in Formula V including each embodiment thereof.

[0095] In some embodiments, the compound of Formula VI is In the application, the moiety of this compound without H attached to the sulfur is referred to -S-Luc. This compound or the moiety of this compound without H attached to the sulfur is also termed thioluciferin.

III. Conjugates

[0096] The compound described above can be conjugated to any polypeptide (protein) or derivative thereof that comprises a free amine. The free amine containing protein can be directly incubated in the presence of a composition comprising a compound of Formula I or III. In preferred embodiments, the compound incubated with a free amine containing (lysine, arginine, or N-terminus) protein is AT-Luc or AT-CBT. In other preferred embodiments, the protein is an antibody or fragment thereof. [0097] The compound described above, such as TT-Luc or TT-CBT compound, can be conjugated to any polypeptide (protein) or derivative thereof that comprises a sulfur group such as the -SH group of a cysteine residue, wherein the sulfur group can form a disulfide bond. The sulfur- containing residue can be present in a disulfide bond within the native polypeptide. The disulfide bond can be intramolecular (between two residues on a single polypeptide) or inter molecular (between two residues, wherein each residue is on a separate polypeptide in a dimeric or multimeric protein). Upon partial reduction of disulfide bonds between polypeptides, addition of the compound of Formula I or III to the partially reduced protein composition results in disulfide linkage of the compound of Formula I or III to the protein to generate the conjugate of Formula II or IV. Accordingly, any exposure of the protein conjugate to a reducing environment will result in cleavage of the luciferin thereby rendering the luciferin available as a substrate to a luciferase.

[0098] Conjugating a compound with a protein containing a disulfide bond can be achieved by first partial disulfide bond reduction, wherein no more than 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the disulfide bonds in the protein on average are reduced. The partially reduced protein is then incubated in the presence of a composition comprising a compound of Formula I or III. In preferred embodiments, the compound incubated with the partially reduced protein is TT-Luc. In other preferred embodiments, the protein is an antibody or fragment thereof.

[0099] In some embodiments, the polypeptide of interest is site specifically engineered to include a free Cysteine, Lysine or Arginine residue, which could be directly conjugated to the imaging reagent without reducing the disulfide bonds.

[0100] In preferred embodiments, the compound of Formula III is conjugated to free amino groups or cysteine residues of an antibody or fragment or derivative thereof via amide or disulfide bond to form a conjugate referred to herein as “Ab-SS -Luciferin”.

[0101] The compound of Formula I or III may also be conjugated to a peptide, such as a peptide having a length ranging from 10-20, 15-30, 20-40, 10 to 50 or 25 to 50 amino acids in length, wherein the peptide comprises 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4 or 5 cysteines. Such peptides can be treated to allow disulfide bond formation between two peptides, then the disulfide-bonded peptides are incubated under reducing conditions to conjugate a TT-Luc compound to one or both peptides. [0102] The compound of Formula I or III may also be conjugated to a peptide, such as a peptide having a length ranging from 2-8, 5-15, 10-20, 15-30, 20-40, 10 to 50 or 25 to 50 amino acids in length. When there is a free Cysteine residue, such peptides can be directly treated with TT-Luc to conjugate the luciferin molecule.

[0103] The luciferin derivative such TT-luc and AT-luc can be conjugates to any small molecule drugs, metabolites (ex. lipids), vitamins, nanoparticles, and viruses using the same strategy described for peptides and antibodies above.

[0104] Provided is a conjugate of Formula II or a salt thereof:

Formula II

[0105] wherein B comprises a biomolecule, X and L are the same as defined for Formula I including each embodiment thereof. [0106] In some embodiments, the biomoleculeis a protein, peptide or derivatives as described above. [0107] In some embodiments, the biomolecule is a polypeptide or peptide.

[0108] In some embodiments, the biomolecule is a protein containing viral particle.

[0109] In some embodiments, the polypeptide is an antibody or fragment or derivative thereof. In other embodiments, the antibody or fragment or derivative thereof is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, an F(ab)2, a diabody or a probody. In yet other embodiments, the antibody is a bispecific antibody, a multispecific antibody, an scFv, a F(ab)2, a bis-scFv, a diabody, a triabody or a tetrabody.

[0110] In some embodiments, the antibody or fragment or derivative thereof is therapeutic.

[0111] In some embodiments, the biomoleculeis a nucleotide or an oligonucleotide. In other embodiments, the biomolecule is siRNA, shRNA or any other silencing RNA molecule.

[0112] In some embodiments, the polypeptide is an antibody, fragment or derivative thereof which specifically binds an antigen, wherein the binding activity of the conjugate is not reduced by more than 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60% or 75% as compared to the binding activity of the antibody, fragment or derivative thereof which is not conjugated.

[0113] In some embodiments, the polypeptide is an antibody or fragment or derivative thereof comprising a disulfide bond, wherein no more than 10%, 20%, 30%, 40%, 50% or 60% of the disulfide bonds in the polypeptide are reduced for the formation of the conjugate.

[0114] In some embodiments, the biomolecules are selected from, for example, small molecule drugs, sugars, vitamin, peptides (oligo or poly), lipids, necleotides, oligosaccharides, anitbodies, antibody drug conjugates, DNA, siRNA, RNA, nanoparticles, and virus. In some embodiments, the biomolecules comprise, for example, an amino, hydroxyl, or thiol group.

[0115] In some embodiments, a conjugate of Formula II is a conjugate of Formula IV or a salt thereof: or-CN, B comprises a biomolecule, and X is the same as defined for

Formula I including each embodiment thereof.

IV. Methods for Use

[0116] Provided herein are methods for monitoring the binding, uptake and processing by a cell of a conjugate according to Formula II or IV. The method can comprise incubating a conjugate of Formula II or IV with an enzyme such as luciferase wherein the enzyme such as luciferase can act on the bioluminescent such as luciferin moiety of Formula II or IV after the disulfide bond of Formula II or IV is cleaved to generate light. In some embodiments, the luciferase is expressed by a cell which is contacted with the conjugate for a period of time, e.g., 1-10 min, 10 to 20 min, 10-30 min, 15 to 60 min, 1-2 h, 1-5 h, 1-10 h, 1-20 h, or 1-30 h. The luciferase expressed by the cell can be an endogenous, naturally occurring luciferase or a recombinant luciferase wherein the cell was engineered to express the luciferase.

[0117] When W in Formula IV is -CN, the “full”-luciferin moiety capable of bioluminescent light emission, e.g., the moiety comprising , can be formed inside the cell or outside cell by reacting with a D-Cystein. This “split luciferin” approached is illustrated in the scheme below:

[0118] In the above split luciferin approach, a biomolecule of interest, such as a protein, can be labelled by cyanobenzothiazole (CBT) derivative via a free thiol or amino group to form protein- SS-CBT conjugate. This can be converted into corresponding luciferin conjugate (protein-SS-Luc) by addition of D-cysteine or the protein-SS-CBT can be used directly to probe cell uptake process to form active luciferin upon reaction with intracellular D-cysteine. This approach similarly does limit the biomolecules to proteins. Neither does this approach limit the compound of Formula IV to AT-CBT and TT-CBT. The biomolecules disclosed for Formula II or IV and the compounds of Formula IV when W is CN are all suitable for this approach.

[0119] Cells for use in the methods can be eukaryotic or prokaryotic. Eukaryotic cells include but are not limited to yeast, avian, plant, insect and mammalian cells.

[0120] In an alternative embodiment, the conjugate is incubated with a cell which does not express endogenous or recombinant luciferase. In this embodiment, the cell and conjugate are incubated with a composition comprising the luciferase.

[0121] In a preferred use, the method comprises incubating a luciferase-expressing cell with a conjugate of Formula IV wherein B is an antibody. Upon incubating with or exposing a cell to the Ab-SS-Luciferin, an effective antibody that binds to a receptor or protein on the surface of the cell will be endocytosed by the cell. Once the Ab-SS-Luciferin is shuttled to the lysosome, it is degraded to yield luciferin fragments with the disulfide bond. Then, these fragments are delivered into the reducing cytosolic environment where the disulfide bond is cleaved, releasing the luciferin. Luciferase expressed by the cell is then able to use the free luciferin as a substrate, thereby generating light. It is understood that the antibody (Ab) in this embodiment can be a fragment or derivative of an antibody as described herein or may be any protein or polypeptide which can bind to a receptor or protein on the surface of the cell.

[0122] FIG.1A describes examples of conjugates of Formula IV. The biomolecule comprises a free amino or thiol group. The biomolecules can be small molecule drugs, sugars, vitamin, peptides, lipids, nucleotides, oligosaccharides, antibodies, antibody drug conjugates, DNA, siRNA, RNA, nanoparticles, and virus. The biomolecule of interest bearing free amino or thiol group or modified with linker bearing such groups can be tagged with Amino Tag Luciferin (AT-Luc) or Thiol Tag Luciferin (TT-Luc) respectively to afford a SS-Luc tagged biomolecule.

[0123] FIG. IB uses an exemplary conjugate, i.e., a SS-Luc tagged biomolecule, illustrating the uptake probe mechanism: SS-Luc tagged molecule is taken up by a luciferase-expressing cell; upon internalization, the probe is reduced by ubiquitous glutathione liberating reporter molecule mercaptopropylaminoluciferin capable of enzymatic oxidation with firefly luciferase emitting light. Amount of detected light is proportional to intensity of cellular uptake of the probe.

[0124] Example 1 describes synthetic schemes and procedures for compounds AT-Luc and TT-Luc (precursors for thioluciferin labeling).

[0125] Examples 2, 4, 6, 7, 8, and 9 describe labeling procedures for various biological molecules with thioluciferin either with AT-Luc or TT-Luc molecules, including labeling of peptides, antibodies, small molecules such as a free fatty acids and triglycerides, and DNA.

[0126] Examples 3, 5, and 10 below describes cell uptake of peptide or antibody labeled with thioluciferin or transfection with DNA labeled with thioluciferin.

[0127] The study in Example 3 is a dose dependent uptake study of a peptide conjugate D- Octaarginine-thioluciferin (abbreviated as r8-SS-Luc or 8-SS-Luc) in cells. The conjugate r8-SS- Luc was prepared according to Example 2. FIG. 2 displays integrated luminescence over 3 h from cells incubated with r8-SS-luc in HBSS and 25 mM glucose.

[0128] FIG. 3 shows general antibody-thioluciferin uptake assay design. The thioluciferin labeled antibody undergoes endocytosis with subsequent release of thioluciferin molecule. Upon cleavage of thioluciferin by luciferase, the light is emitted proportionally to the amount of endocytosed antibody.

[0129] Example 5 shows the successful use of the antibody conjugates of Formula II or IV to monitor cellular uptake and processing of a protein upon binding to a cell surface receptor both in vitro and in vivo. Incubation of luciferase-expressing cells with a protein conjugate (trastuzumab-thioluciferin antibody conjugate, also abbreviated as Trastuzumab-SS-luc, prepare according to Example 4) which binds specifically to a receptor (Her2) on the cell shows increasing luminescence over time, demonstrating the uptake and processing of the conjugate by the cells to release the luciferin moiety from the conjugate. A control conjugate, (IgG antibody -thioluciferin conjugate, also abbreviated as IgG-SS-luc prepared according to Example 4) which did not contain a protein which internalizes inside the cells resulted in the production of only background signal. As expected, an increase in luminescence was observed only when the conjugate contains a protein which binds specifically to the cell surface receptor and gets internalized and processed via lysosome in the subsequent step of the assay. If no internalization occurs upon binding to the receptor, no light is produced from the conjugates of interest. That makes the assay very specific for sensitive monitoring of internalization of biomolecules inside the cells. The process can be extended to monitoring of intracellular internalization in live cells and living animals. This conclusion is further supported by competition assays showing the specificity of the assay. Importantly, studies were done in which luciferase- expressing cells were pretreated with a competitive inhibitor. Such cells generate lower levels of light when incubated with a protein conjugate that specifically binds to the cell surface. Without pretreatment with the competitive inhibitor, the cells generated light as expected with incubated with the protein conjugate.

[0130] Longitudinal imaging of luminescence by luc-expressing cells treated with protein conjugates demonstrate that the methods described herein are able to measure protein uptake and processing over extended periods of time, such as up to 48 hours.

The studies with conjugate of trastuzumab-thioluciferin antibody described in Example 4 were performed with cell lines that were positive and negative for Her2 expression, further validating specificity of the assay.

[0131] FIGS. 4A-4B show uptake of trastuzumab-thioluciferin and IgG-thioluciferin antibodies in Her2 positive SK-BR-3 luciferase expressing cell lines as a function of time. FIG. 4A shows the bioluminescent signal measured at different time points for the positive cells incubated with trastuzumab-thioluciferin and IgG-thioluciferin antibodies. FIG. 4B is a chart showing signal from Trastuzumab-thioluciferin normalized to the negative IgG-thioluciferin control signal at each time point. The peak ratio of over 40 folds was achieved at 24hrs time point. These data suggest that the luciferin labeled antibody can be used to determine the efficiency and kinetics of antibody uptake and processing inside the cells.

[0132] Example 5 also treated Her2 negative 4T1 luciferase expressing cell lines with incubated trastuzumab-thioluciferin and IgG-thioluciferin antibodies. No increase of the signal from the Trastuzumab treated cells was observed. FIG. 5A shows bioluminescent signal measured at different time points for the negative cells incubated with trastuzumab-thioluciferin and IgG- thioluciferin antibodies. FIG. 5B is a chart showing signal from Trastuzumab-thioluciferin normalized to the negative IgG-thioluciferin control signal at each time point. The ratio of Trastuzumab signal over the IgG signal remained close to 1 in this experiment.

[0133] In Example 5, experiments were repeated with one additional Her 2 positive cells (BT474) and one additional Her 2 negative cells (MDA-MB231). FIGS. 6A-6D show high normalized signal ratios of Trastuzumab-thioluciferin over IgG-thioluciferin for Her 2 positive cells (FIG. 6A and FIG. 6B) and low normalized signal ratios for Her 2 negative cells (FIG. 6C and FIG. 6D).

[0134] Example 5 further conducted a competition assay. In this assay, BT474 Her 2 positive cells were incubated with either 2ng of trastuzumab-thioluciferin antibody alone or with 2 ng trastuzumab-thioluciferin plus high amounts of cold unlabeled trastuzumab antibody (lOng or lOOng). FIG. 7 shows signals from the cells treated with labeled antibody plus non labelled cold antibody was dramatically lower than the signal obtained with labeled antibody alone, further validating the specificity of the assay.

[0135] Example 5 additionally conducted an in vivo study on the uptake of Trasuzumab- thioluciferin and control IgG-thioluciferin in BT474-Luc tumor xenograft mouse model. Mice were injected with equal amounts of either Trastuzumab-thioluciferin or IgG-thioluciferin and imaged at different time points using IVIS Spectrum instrument. FIG. 8 displays normalized photon flux for Trastuzumab-thioluciferin and IgG-thioluciferin at different time points. The kinetics and strength of the signal from trastuzumab injected animals was significantly higher than the signal from the IgG injected animals, suggesting that the BL-ICUP signal can be used in vivo as well.

[0136] The cellular uptake assay described herein can also be used to assess the activity of extracellular enzyme (such as lipase) in situations where internalization of a conjugate occurs after the biomolecule moiety of the conjugate is modified by an extracellular enzyme wherein the conjugate/biomolecule is a substrate or potential substrate to the enzyme.

[0137] For example, Example 8 demonstrates applications of thioluciferin as a bioluminescent reporter in enzymatic and cell-based internalization assays simultaneously. Only in case if both are present/active the light production will occur. Example 8 specifically demonstrates that only if the lipase is active and fatty acid is cleaved from triglyceride backbone as the result of lipase activity, the cellular internalization of thioluciferin-labeled fatty acid can occur. That would result in light production that can be imaged and quantified by the bioluminescence assay disclosed herein. Here the biomolecule (Triglyceride-SS-Luc) is a substrate for lipase, after cleavage a part of the conjugate (palmitic acid - SS- Luc) is internalized and quantified by BL-ICU assay.

[0138] Example 10 reports a transfection study with a DNA-thioluciferin (also abbreviated DNA- SS-Luc), which was prepared according to Example 9. In this study, DNA-SS-Luc plus lipofectamine or polyethylene imine at different concentration were incubated with cells for a period of time. The bioluminescence was measured in the IVIS spectrometer, and the results were shown in FIG. 9A and FIG. 9B. FIG. 9A shows bioluminescence from 4T1 RLR cells after 6 h incubation with bpofectamine (TA) and DNA-SS-luc, wherein +++TA corresponds to 0.6 pi/ 96 well, ++TA to 0.3 mΐ/ 96 well, +TA to 0.15 mΐ/ 96 well. FIG. 9B shows integrated bioluminescence from 4T1 RLR cells during 80 min incubation with polyethyleneimine (TA) and DNA-SS-luc, wherein +++TA corresponds to 1.0 pg/ 96 well, +TA to 0.25 pg/ 96 well.

V. The BL-ICU Assay

[0139] The flexible nature of the BL-ICU assay can allow sensitive and real-time screening of cellular internalization of a number of biomolecules such as small molecule drugs, vitamins, metabolites (lipids, glucose, etc.), peptides, antibodies, antibody drug conjugates, proteins, nanoparticles, viruses, cells, and bacteria in the same assay as well as in various cell lines including suspension cells.

[0140] The assay developed here does not involve any invasive steps, which will readily allow longitudinal imaging both in vitro and in vivo. Additionally, knowing the real-time kinetics of the molecule uptake in a target organ can be highly valuable for dose treatment and pharmacokinetic (PK) studies, therefore, this approach could be useful for many biomolecules including therapeutic antibody development and biomedical applications.

[0141] BL-ICUP assay can directly be translated to in vivo applications. In addition to real-time uptake in the target organ, cellular internalization of the antibody in healthy tissues could be detected, which would give prior information about the potential side effects.

[0142] Moreover, the in vivo imaging techniques available for biomolecules, give information mainly about the tissue distribution of the biomolecules rather than cellular internalization kinetics in vivo. For example, they do not allow monitoring in real time degradation of antibodies and cytosolic release of the drug cargo from ADC.

[0143] Furthermore, this BL-ICU assay is not limited to the biomolecules provided in the examples and could readily be extended to other proteins, peptides, small molecules, viruses, nanoparticles and oligonucleotides such as siRNA to monitor cellular uptake both in vitro and in vivo.

[0144] The Examples below are illustrative in nature and are in no way intended to be limiting.

Example 1

Synthesis of Novel Luciferin Labeling Molecules

[0145] Reaction conditions: i) TEA, CH2CI2, room temperature, overnight; ii) NaBH(OAc)3, AcOH, THF, room temperature, overnight; iii) THF/water (2:1), room temperature, 1 h; iv) hydrazine monohydrate, N2, 0°C to room temperature, 30 min; v) TEA, DMF, N2, room temperature, 2 h.

[0146] S-(3-oxopropyl) ethanethioate (3)

[0147] To an ice-cold solution of acrolein 2 (0.5g, 6.6 mmol) and TEA (0.31 mL) in DCM (lOmL), thioacetic (1) acid (0.68g, 12.1 mmol) in DCM (3 mL) was added dropwise over 10 min at 0 ^° C. Then the reaction mixture was stirred at room temperature overnight before it was concentrated at room temperature (60 mbar). The remaining dark yellow liquid was distilled at 55 ^° C at 2 mbar. The colorless liquid (0.67 g) was collected. 77 % yield.

[0148] S-(3-((2-cyanobenzo[r/]thiazol-6-yl)amino)propyl) ethanethioate (5)

[0149] To a solution of aldehyde 3 (0.2 g, 1.5 mmol) and aromatic amine 4 (0.13 g, 0.75 mmol) in THF (5 mL), NaBH(OAc)3 (0.24 g, 1.13 mmol) and glacial acetic acid (0.09 ml, 1.5 mmol) was added, and the reaction mixture was stirred at room temperature overnight. Then, the reaction was quenched with saturated aqueous NaHCCh, diluted with ethyl acetate and the product was extracted with ethyl acetate. The remaining crude product was purified over column chromatography using n- hexane:EtAc (3:1) yielding 0.15 g (0.5 mmol) yellow solid. 67 % yield. MS (ESI): m/z calc for [M + H] ⁺ 292.06, found 292.14 . ¾ NMR (400 MHz, CDCb) d : 7.91 (d, 1H, J=8.9Hz), 6.93 (d, 1H, J=2.3Hz), 6.90 (dd, 1H, J=2.3Hz, J=8.9Hz), 4.52 (brs, 1H), 3.27 (t, 2H, J=6.6Hz), 3.00 (t, 2H, J=6.9Hz), 2.37 (s, 3H), 1.99-1.91 (m, 2H). ¹³ C NMR (100 MHz, CDCB) d: 196.53, 148.90, 144.85, 138.91, 129.92, 125.75, 117.20, 113.98, 99.88, 42.06, 30.85, 29.00, 26.41.

[0150] (A)-2-(6-((3-mercaptopropyl)amino)benzo[d]thiazol-2-yl)-4,5- dihydrothiazole-4- carboxylic acid (8) Thioluciferin

[0151] To a mixture of intermediate 5 (0.15 g, 0.5 mmol), D-cysteine 6 (73 mg, 0.6 mmol), and THF (2 mL) was added water (1 mL) at room temperature. The reaction mixture was stirred for 1 h at the same temperature. Completion of the reaction was monitored with UPLC (Waters, Cl 8 BEH column). The reaction mixture was placed on an ice bath, and hydrazine monohydrate (50 uL, 1.0 mmol) was added under nitrogen gas flow. After stirring at room temperature for another 30 min (HPLC monitoring) the pH was adjusted to pH 4 with 0.5 M HC1 solution, and the product was purified by preparative HPLC (Cl 8 RP column, acetonitrile- water gradient over 10 min), and lyophilized to afford 8 (88 mg, 50 %) as a dark yellow solid. MS (ESI): m/z calc for [M + H] ⁺ 354.04, found 354.05.

[0152] (A)-2-(6-((3-((3-((2,5-dioxopyrrolidin-l-yl)oxy)-3-oxopropyl )disulfaneyl) propyl)amino)benzo[r/]thiazol-2-yl)-4,5-dihydrothiazole-4-ca rboxylic acid (AT-Luc) -Luc

[0153] To a mixture of 2,5-dioxopyrrolidin-l-yl 3-(pyridin-2-yldisulfaneyl)propanoate 9 (34 mg, 0.11 mmol), 8 (35 mg, 0.1 mmol), and DMF (1 mL) was added TEA (27 uL, 0.2 mmol) under nitrogen gas flow at room temperature, and the resulting mixture was stirred at room temperature for 2 h. Then, the pH was adjusted to pH 4 with 0.5 M HC1 solution, and the product was purified by preparative HPLC (Cl 8 RP column, acetonitrile-water gradient over 10 min), and lyophilized to afford AT-Luc (23 mg, 42 %) as a dark orange solid. MS (ESI) m/z calc for [M+H] ⁺ 555.04, found

555.05. [0154] (5)-2-(6-((3-(pyridin-2-yldisulfanyl)propyl)amino)benzo[cf]t hiazol-2-yl)-4,5- dihydrothiazole-4-carboxylic acid (TT-Luc)

[0155] To a mixture of 2,2'-dithiopyridine 10 (24 mg, 0.11 mmol), 8 (35 mg, 0.1 mmol), and DMF (lmL) was added TEA (27 uL, 0.2 mmol) under nitrogen gas flow at room temperature, and the resulting mixture was stirred at room temperature for 2 h. Then, the pH was adjusted to pH 4 with 0.5 M HC1 solution, and the product was purified by preparative HPLC (Cl 8 RP column, acetonitrile-water gradient over 10 min), and lyophilized to afford TT-Luc (17 mg, 37 %) as a dark orange solid. HRMS (ESI): m/z calc for [M + H] ⁺ 463.0385, found 463.0383.

Example 2

Labelling a peptide with thioluciferin

[0156] Octa-arginine peptide Z-r8C-OH (150 mg, 0.1 mmol) was resuspended in DMF (1 mL) and formic acid (9 mg, 0.2 mmol) and added to TT-Luc (46 mg, 0.1 mmol) at room temperature. MeOH (1 mL) was added to solubilize the ingredients. The yellow solution was stirred for 30 min and direct injection into LC/MS revealed that starting material was consumed. Thus, the solvent was removed by rotary evaporation and the remaining resuspended in water. The suspension was centrifuged and the following filtration was difficult because filter was blocked by starting material and thiopyridine. It was purified on a argilent zarbax-C8 column starting with 0-1% MeCN in 4 min followed by a 40 min gradient up to 50 % MeCN. The product eluted at around 17 % MeCN. Direct injection reveals the +2H to +5H ( 928, 619, 464, 372) ions in the mass spectrum. The solvents were removed and the product dissolved in 1 milli Q water. It was desalted on an ammonium solid phase carbonate column and eluted with MeCN and water. The solution was lyophilized over night to give r8-SS-Luc 13 (82 mg, 44%).

Example 3

Dose dependent uptake of thioluciferin 8 and r8-SS-Luc 13 in 4T1-RLR cells [0157] 4T1-RLR cells were plated in 96 well plates in DMEM medium supplemented with FBS

(10%). After 2 days, at ca 90 % confluency, the medium was removed and the cells washed with HBSS buffer (25 mM glucose). Then 8 or 13 dissolved in HBSS at various concentrations were added to cells. The luminescence was measured for 60 min. FIG. 2 is a chart showing luminescence at different doses of 8 (A) and 13 (B).

Example 4

Labeling a Protein via acylation of free amino groups with thioluciferin via AT-Luc derivatization

[0158] To a solution of purified antibody (100 pL, 1.0 mg/mL) in 0.1 M NaHC03 buffer (pH 8.23) was added solution of AT-Luc (0.7 pL of 10 mM stock soln in DMSO). After mixing the reaction mixture was left at +4°C for 4 hours. Then, the mixture was purified through Zeba Spin Desalting Column (7K) using PBS (pH 7.4) as elution buffer. The absorbance of the conjugate was measured at 280 nm and 380 nm (Amax). Protein Concentration and Degree of labeling were calculated using standard equations

^280 ^” (^max ^x ^ f )

Protein Concentration (M) = - x dilution factor

^ Protein . and

Degree of Labeling (DOL) = x dilution factor

Si L _n u _H ci _f ere _r ni _n n ^x Protein concentration and , were fround . for Trastuzumab-SS-Luc 0.58 mg/mL (protein concentration) and 5.16 (degree of labeling) and for

IgG-SS-Luc 0.77 mg/mL (protein concentration) and 4.11 (degree of labeling).

Example 5

Antibody uptake assay

[0159] To determine if the antibody conjugate can be used for monitoring uptake and processing of the antibody in vitro, luciferase expressing cells were seeded in 24 well plates at approximately 50% confluency. Thioluciferin labeled antibody (2 ng) were then added directly into the cell culture media, the cells were incubated with antibody for different time intervals and the bioluminescent signal was acquired using IVIS imaging instrument (Perkin Elmer). Fig. 4A and FIG. 4B show the results of the experiment where SK-BR-3 Her2 positive cells were incubated with either thioluciferin labeled trastuzumab antibody (trastuzumab-SS-Luc or Trastuzumab-thioluciferin) which specifically binds Her2 receptor and efficiently internalizes inside the cells, or with nonspecific thioluciferin labeled IgG antibody (IgG-SS-Luc) which does not bind any cell surface antigens. As shown in Fig. 4A, the signal from Trastuzumab-SS-Luc treated cells significantly increases during the first 48 hours of incubation, while the signal from negative control IgG-SS-Luc antibody remains at the background level for the duration of experiment. Fig 4B shows the ratio of trastuzumab- thioluciferin signal over the control IgG signal at different time points.

[0160] Essentially the same experiment using Her2 negative 4T1 breast cancer cells was conducted. Cells again were treated with 2 ng of luciferin labeled antibodies. In this case no increase of the signal from the Trastuzumab-thioluciferin treated cells was observed, further proving the specificity of the assay. See FIG. 5A and 5B.

[0161] To confirm these results, essentially the same experiments with 1 additional Her2 positive (BT474) and Her2 negative cell lines (MDA-MB231) were conducted. See FIG. 6A-D, which summarize the results with high ratios of Trastuzumab-thioluciferin over IgG for Her2 positive cell lines, and low ratios for Her2 negative cell lines.

[0162] To further validate the specificity of the assay, BT474 Her 2 positive cells were incubated either with 2ng of trastuzumab-thioluciferin antibody alone, or with 2 ng of trastuzumab- thioluciferin plus high amounts of cold unlabeled trastuzumab antibody (lOng, lOOng, competition assay). The signal from the cells treated with labeled antibody plus non-labelled cold antibody was dramatically lower than the signal obtained with labeled antibody alone, further validating the specificity of the assay. See FIG. 7.

[0163] Finally, the antibody uptake assay was investigated in vivo in subcutaneous xenograft tumors. 5M luciferase labeled BT474 cells were injected subcutaneously into the right flank of nude mice. The tumors were allowed to grow until approximately 0.5 cm3 in size. The mice were then injected with 50ng of trastuzumab-thioluciferin or IgG-thioluciferin antibodies, respectively. The bioluminescent signal was acquired at different time intervals using IVIS Spectrum imaging instrument (Perkin Elmer). See FIG. 8. The kinetics and strength of the signal from trastuzumab injected animals was significantly higher than the signal from the IgG injected animals, suggesting that the BL-ICUP assay can be used in vivo as well.

Example 6

Labeling a Reagent Protein with thioluciferin via TT-Luc (partial reduction)

[0164] A solution of monoclonal antibody (10 pL of 20 mg/mL in PBS pH 7.4) was added to reduction buffer (90 pi of 0.025 M sodium borate pH 8, 0.025 M NaCl, 1 mM DTP A). To this mixture 0.5-4 equivalent of TCEP was added to partially reduce the interchain disulfide bonds at 37 °C for 1 hour. Then, the mixture was purified through Zeba Spin Desalting Column (7K) using PBS (1 mM DTP A) as elution buffer. Then to this elute TT-Luc (1.6 mΐ 10 mM in DMSO) was added and shaken at room temperature for 2 hours before purifying through ZebaSpin desalting Column (7K) using PBS as elution buffer. The absorbance of the conjugate was measured at 280 nm and 380 nm (Amax), and degree of labeling was calculated as

A280 (A _max ^x CF)

Protein Concentration (M) = - x dilution factor

^ Protein . and

Degree of Labeling (DOL) = - x dilution factor sLuciferin ^x Protein concentration Example 7

Labeling a Long chain fatty acid molecule with thioluciferin via TT-Luc

[0165] Reaction conditions: i) TT-Luc, DIPEA, THF-DMF, RT, 12 h.

[0166] (A)-2-(6-((3-((15-carboxypentadecyl)disulfaneyl)propyl)amino )benzo[d]thiazol-2-yl)- 4,5-dihydro-thiazole-4-carboxylic acid (15) To a solution of 16-mercaptohexadecanoic acid 14 (29 mg, 0.1 mmol) in THF (1 mL) was added a solution of the TT-Luc (46 mg, 0.1 mmol) in DMF (1 mL) and DIPEA (53 pL, 0.3 mmol). The mixture was stirred at RT for 12 h and then was concentrated in vacuo to dryness. The residue was purified by preparative HPLC on a C18 column (MeCN/water gradient + 0.1% formic acid) to give the product 15 as an orange solid (42 mg, 65% yield). HRMS: calculated for C30H46N3O4S4 [M+H] ⁺ 640.2371, found 640.2369.

Example 8

Labeling a Triglyceride molecule with thioluciferin [0167] Thioluciferin (SS-Luc) labelled glycerol tripalmitate molecule was designed for measuring of lipase enzyme activity. Prior to labeling with TT-Luc agent, a terminal thiol group was introduced to the structure triglyceride molecule according to next synthetic scheme

[0168] Reaction conditions: i) 2,2'-dithiopyridine, DIPEA, THF, rt, 3h; ii) EDCI, DMAP, DCM, 0°C to RT, 16 h; iii) 8, DIPEA, DCM, rt, 16 h.

[0169] 16-(pyridin-2-yldisulfaneyl)hexadecanoic acid (17) A mixture of 16- mercaptohexadecanoic acid 16 (289 mg, 1.0 mmol), 2,2'-dithiopyridine (231 mg, 1.05 mmol), and THF (6 mL) stirred at RT for 3 h under nitrogen atmosphere. Then the reaction mixture was diluted with DCM (20 mL), washed with water (3x20 mL), dried over Na2S04, and evaporated to dryness to give 17 as a yellowish solid (near quantitative yield).

[0170] 3-((16-(pyridin-2-yldisulfaneyl)hexadecanoyl)oxy)propane-l,2 -diyl dipalmitate (19) To a mixture of 3-hydroxypropane-l,2-diyl dipalmitate 18 (60 mg, 0.105 mmol), 17 (50 mg, 0.127 mmol), DMAP (3 mg, 0.02 mmol), and DCM (5 mL) was added EDCI (60 mg, 0.316 mmol) with stirring on an ice bath. After stirring for 16 h the reaction mixture was concentrated to 1/3 volume and flash chromatographed through a silica gel column with EtAc-Hex 1 :2 mixture, and evaporated to dryness to afford 19 as a white powder (80 mg, 80%).

[0171] (A)-2-(6-((3-((16-(/?A-2,3-bis(palmitoyloxy)propoxy)-16- oxohexadecyl)disulfaneyl)propyl)-amino)-benzo[</]thiazol- 2-yl)-4,5-dihydrothiazole-4- carboxylic acid (14) A mixture of 19 (40 mg, 0.042 mmol), 8 (15 mg, 0.042 mmol), DIPEA (20 uL), and DCM (2 mL) was stirred at ambient temperature for 16 h. The resulting mixture was concentrated to 1/3 volume and chromatographed through a silica gel column eluting with DCM- MeOH 40: 1 + HO Ac (2% vv), and evaporated to dryness to afford 20 as a terracotta powder (27 mg, 54%). HRMS (ESI/QTOF) m/z: [M + H]+ Calcd for CesHmNiOsSA 1190.7327; Found 1190.7335.

Example 9 DNA labelling

[0172] 0.751 mg DNA was dissolved in 751 mΐ of TE buffer (pH=7.4). Dilutions were measured on Nanodrop to confirm a concentration of 0.98 g/L. Then the mixture is aliquoted into 4 and one aliquot is purified from glutathion/ mercaptoethanol on a centripure Zetadex column (NAP 25). The column was opened on both sides and the storage buffer run through. It was equilibrated with 25 ml of TE buffer and 188 mΐ of DNA were put on the column. 2- 2-5 ml of buffer were run through the column and the next 2-4 ml of eluate were collected in 1.5 ml fractions. The first fraction contained almost all the DNA. The nanodrop dilution series indicated 0.260 mg in this fraction which was much more than the expected amount. 9.7 mΐ (97 pg, 210 nmol, 6 eq.) of TT-Luc solution in DMF is added to the DNA in buffer and the mixture was stirred at room temperature for 4 h. The solution was concentrated with viva spin columns and transferred to 2 mL Eppendorf tubes. NaOAc was added (to 0.3 M end concentration) and pure EtOH (to 75 % end concentration). The solution was cooled to -80 °C over night (30 min are usually sufficient) and centrifuged at 4 °C and 10000 g for 20 min. The precipitation worked fine, but washing with 1 mL 0.25 NEEOAc in 70 % EtOH, dissolved the pellet partially. The supernatant was removed, the wet pellet was lyophilized. The oligonucleotide was purified on an Inert Sustain C18 column with 3 pm diameter (4.6 x 150 mm). The LC- system was Agilent infinity 1260. Solvent A was 95 % 0.1 M TEAA, pH=7 and 5 % MeCN, solvent B was

100 % MeCN. A linear gradient from 15 % to 55 % B was used as the compound seemed to be quite hydrophobic. The flow rate was 1 ml/min. The mass was confirmed on ESI-QTOF by the -5 and -6 ions (1527.7 and 1272.). Calculated mass is 7642.5. Found mass is 7643.5.

Example 10

Transfection with DNA-SS-Luc (DNA-thioluciferin)

[0173] lxlO ⁴ cells per well in RPMI were plated in 96 well plates and incubated for two days. For 200 nM final DNA concentration a 40 mM solution of DNA was prepared by measuring the concentration on Nanodrop with an absorption coefficient of 289282 L/(cm*mol) at 650 nm. The absorption coefficient of the DNA strand is 220000 L/(cm*mol) at 260 nm, but less exact. 4T1RLR cells were seeded in RPMI medium with 10 ⁴ cells/96 well two days before the experiment.

[0174] The medium was removed and the cells were washed three times with 100 pL optimem medium. The transfecting mixtures in medium are added. Different concentrations of DNA, and transfecting agent were used. +TA means 0.15 pl/well, ++TA means 0.3 pl/well. +++TA means 0.6 pl/well for lipofectamine transfection and +TA means 0.12 pg/well, ++TA means 0.25 pg/well, +++TA means 0.5 pg/well polyethylene imine.

[0175] The linear polyethylene imine had an average molecular weight of 13 kDa and a maximum molecular weight of 25 kDa.

[0176] For PEI transfection in three 96 wells at 200 nM DNA concentration and a N/P ratio of 25, a 40 pM solution of DNA (306 ng/pl) was prepared. 2 pi DNA (0.6 pg) were diluted with 38 pi 150 mM NaCl. 2 pi of PEI (1 g/L, 2 pg) were diluted with 36 pi of 150 mM NaCl. DNA and PEI solutions were mixed, incubated for 50 minutes at room temperature and vortexed. 80 pi transfection solution was diluted with 320 pi optimem and 100 pi added to each well.

[0177] For lipofectamine transfection in three 96 wells at 200 nM DNA concentration a 40 pM solution of DNA (306 ng/pl) was prepared. 2 pi DNA (0.6 pg) were diluted with 38 pi Optimem. 2.4 pi of lipofectamine 3000 were diluted with 37.6 pi of 150 Optimem. DNA and PEI solutions were mixed and incubated for 10 minutes at room temperature. 80 pi transfection solution was diluted with 320 pi optimem and 100 pi added to each well.

[0178] The bioluminescence was measured in the IVIS spectrometer with an exposure time of 3 minutes for 1.5 hours. Then, the plate was put in the incubator for 5 hours and the bioluminescence measured again for 15 min. The same was repeated after 8 h and 22 h. The medium is removed and the cells were washed three times with 100 pL optimem medium. 100 pi Optimem were added and at the fluorescence was measured at 650/ 670 nm on Tecan plate reader. The average background fluorescence was subtracted from the signal.

[0179] The results of our biological experiments clearly demonstrate that the novel method could be used for determination of kinetics and quantification of uptake of various classes of biomolecules and the bioluminescent signal produced is proportional to their cytosolic internalization.