Patent Description:
This application contains a Sequence Listing submitted electronically as a text file by EFS-Web. The text file, named "<NUM>-645P01US_SEQUENCE_LISTING. TXT", has a size in bytes of <NUM> bytes, and was recorded on July <NUM>, <NUM>. The information contained in the text file is incorporated herein by reference in its entirety pursuant to <NUM> CFR § <NUM>(e)(<NUM>).

Therapeutic heteromer protein complexes, including the interleukin-<NUM> (IL-<NUM>) superagonist N-<NUM>, have been found to modulate the immune response for treatment of various cancers. N-<NUM> is a multimeric protein complex including an IL-<NUM> mutant bound to an IL-<NUM> receptor α, which is in turn attached to an immunoglobulin G1 (IgG1) crystallizable fragment (Fc). N-<NUM> has been shown to display improved pharmacokinetic properties, longer persistence in lymphoid tissues and enhanced anti-tumor activity compared to native, non-complexed IL-<NUM>.

Continued evaluation of pharmacokinetic properties of therapeutic heteromer protein complexes such as N-<NUM> is critical for assessing clinical efficacy. Currently available pharmacokinetic assays do not distinguish between N-<NUM> and native IL-<NUM>, and may potentially measure IL-<NUM> levels in patient serum in addition to the target N-<NUM>. Thus, methods for specific detection of therapeutic heteromer protein complexes are needed. Provided herein, inter alia, are solutions to these and other problems in the art.

One embodiment relates to a composition including: (a) a first interleukin <NUM> receptor alpha Sushi domain (IL-15RαSu); (b) a second IL-15RαSu domain, wherein the first and second IL-15RαSu domains are covalently joined by a disulfide bond; (c) a first IL-<NUM> domain, bound by electrostatic interactions to the first IL-15RαSu domain to form a first IL-<NUM>/IL-15RαSu complex; (d) a second IL-<NUM> domain, bound by electrostatic interactions to the second IL-15RαSu domain to form a second IL-<NUM>/IL-15RαSu complex; (e) a first monoclonal antibody (mAb) bound to an epitope on the first IL-<NUM>/IL-15RαSu complex; and (f) a second mAb bound to the identical epitope on the second IL-<NUM>/IL-15RαSu complex, wherein the second mAb comprises a detection means selected from the group consisting of a fluorophore, a radioisotope, and an enzyme, and
wherein both the first mAb and the second mAb bind to the epitopes with equal affinity.

Another embodiment relates to a method for detecting a heterotetrameric IL-<NUM>/IL-15RαSu complex in a biological sample is provided, the method including: a) contacting the biological sample including the protein complex with a first mAb, wherein the mAb is conjugated to a polymeric surface, wherein the heterotetrameric complex includes two IL-<NUM> domains and two IL-15RαSu, wherein each IL-<NUM> domain is electrostatically bound to an IL-15RαSu domain, wherein the two IL-15RαSu domains are covalently bound to each other by a disulfide bond, and wherein the Fab portion of the mAb binds an epitope on the IL-<NUM>/IL-15RαSu complex with an affinity between <NUM> and <NUM> fM; b) contacting the biological sample with a second mAb under conditions such that the second antibody binds with the same affinity to the identical epitope on the second IL-<NUM>/IL-15RαSu complex, wherein the second mAb comprises a detection means selected from the group consisting of a fluorophore, a radioisotope, and an enzyme; c)
washing unbound complexes from the polymeric surface; and detecting binding of the second mAb.

In one aspect of any of the embodiments, at least one of the IL-<NUM> domains comprises an asparagine-to-aspartate mutation at amino acid position <NUM> (N72D).

In one aspect of any of the embodiments, the IL-15RαSu domains each further comprise an immunoglobulin crystalizable fragment (Fc) domain. In another aspect, the IL-15RαSu domains further comprise an scFv domain.

In one aspect of any of the embodiments, the first mAb is conjugated to a polymeric surface.

In one aspect of any of the embodiments, the second mAb comprises a detection means. In one aspect, the detection means is selected from the group consisting of a fluorophore, a radioisotope, and an enzyme.

In one aspect of any of the embodiments, the IL-<NUM> domains further comprise a single chain variable fragment (scFv) domain.

In one aspect of any of the embodiments, the polymeric surface is a polypropylene or polystyrene surface.

In one aspect of any of the embodiments, the first mAb and the second mAb have substantially the same amino acid sequence.

After reading this description it will become apparent to one skilled in the art how to implement the present disclosure in various alternative embodiments and alternative applications. However, all the various embodiments of the present invention will not be described herein. It will be understood that the embodiments presented here are presented by way of an example only, and not limitation. As such, this detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure as set forth herein.

Before the present technology is disclosed and described, it is to be understood that the aspects described below are not limited to specific compositions, methods of preparing such compositions, or uses thereof as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

The detailed description divided into various sections only for the reader's convenience and disclosure found in any section may be combined with that in another section. Titles or subtitles may be used in the specification for the convenience of a reader, which are not intended to influence the scope of the present disclosure.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:.

"Optional" or "optionally" means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "about" when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) <NUM>%, <NUM>%,<NUM>%, or any subrange or subvalue there between. Preferably, the term "about" when used with regard to an amount means that the amount may vary by +/- <NUM>%.

"Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others. "Consisting essentially of" when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of" shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.

"Antibody" refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Typically, the antigen-binding region of an antibody plays a significant role in determining the specificity and affinity of binding. In some embodiments, antibodies or fragments of antibodies may be derived from different organisms, including humans, mice, rats, hamsters, camels, etc. Antibodies of the invention may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate a desired function of the antibody (e.g. glycosylation, expression, antigen recognition, effector functions, antigen binding, specificity, etc.).

Antibodies are large, complex molecules (molecular weight of ~<NUM>,<NUM> or about <NUM> amino acids) with intricate internal structure. A natural antibody molecule contains two identical pairs of polypeptide chains, each pair having one light chain and one heavy chain. Each light chain and heavy chain in turn consists of two regions: a variable ("V") region involved in binding the target antigen, and a constant ("C") region that interacts with other components of the immune system. The light and heavy chain variable regions come together in <NUM>-dimensional space to form a variable region that binds the antigen (for example, a receptor on the surface of a cell). Within each light or heavy chain variable region, there are three short segments (averaging <NUM> amino acids in length) called the complementarity determining regions ("CDRs"). The six CDRs in an antibody variable domain (three from the light chain and three from the heavy chain) fold up together in <NUM>-dimensional space to form the actual antibody binding site which docks onto the target antigen. The position and length of the CDRs have been precisely defined by <NPL>. The part of a variable region not contained in the CDRs is called the framework ("FR"), which forms the environment for the CDRs.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about <NUM> kD) and one "heavy" chain (about <NUM>-<NUM> kD). The N-terminus of each chain defines a variable region of about <NUM> to <NUM> or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.

As used herein, the terms "Fc domain" or "fragment crystallizable domain" are used in accordance with their plain and ordinary meanings and refer to any of the recombinant or naturally-occurring forms of the "base" or tail-end region (C-terminal) of an antibody. The Fc domain is typically composed of two heavy chains that contribute two or three constant domains depending on the class of the antibody. The Fc region is comprised of two heavy chain constant Ig domains in the antibodies IgG, IgA, and IgD, and of three heavy chain constant Ig domains in the antibodies IgE and IgM.

The term "Fc" refers to a non-antigen-binding fragment of an antibody. Such an "Fc" can be in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins. In embodiments, the Fc is an IgG1 or IgG2 Fc. Native Fc's are made up of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from <NUM> to <NUM> depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see <NPL>). The term "Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

In embodiments, the term "Fc " refers to a molecule or sequence that is modified from a native Fc, but still comprises a binding site for a receptor. As with modified Fc and native Fc's, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by recombinant gene expression or by other means.

In embodiments, the Fc is attached, either directly or indirectly, to an IL-15Rα or an IL-<NUM> domain. In embodiments, the Fc is covalently and/or genetically fused with an IL-15Rα or an IL-<NUM> domain.

Antibodies exist, for example, as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'<NUM>, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)'<NUM> may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'<NUM> dimer into an Fab' monomer. The Fab' monomer is essentially the antigen binding portion with part of the hinge region (see <NPL>). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., <NPL>)).

A single-chain variable fragment (scFv) is typically a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short linker peptide of <NUM> to about <NUM> amino acids. The linker may usually be rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa.

The epitope of a mAb is the region of its antigen to which the mAb binds. Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1x, 5x, 10x, 20x or 100x excess of one antibody inhibits binding of the other by at least <NUM>% but preferably <NUM>%, <NUM>%, <NUM>% or even <NUM>% as measured in a competitive binding assay (see, e.g., <NPL>). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, often in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein at least two times the background and more typically more than <NUM> to <NUM> times background. Specific binding to an antibody under such conditions requires an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with the selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., <NPL>) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

A "protein complex" or "complex" as used herein refers to two or more polypeptides that assoicate simultaneously. The complexes may be constructed through binding between proteins and/or binding between receptors and ligands. The proteins may be associated through non-covalent protein-protein interactions, though certain polypeptides in the complex may also be covalently linked directly or indirectly through, for example, a chemical linker, a bond or another protein. For example, the heterotetrameric IL-<NUM>/IL-15RαSu complex includes two IL-<NUM> domains non-covalently bound to two IL-15RαSu domains, wherein the two IL-15RαSu domains are attached covalently by a disulfide bond.

A "detectable means" or "detectable moiety" is a composition, substance, element, or compound; or moiety thereof; detectable by appropriate means such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means.

For example, a detectable means includes <NUM>F, <NUM>P, <NUM>P, <NUM>Ti, <NUM>Sc, <NUM>Fe, <NUM>Fe, <NUM>Cu, <NUM>Cu, <NUM>CU, <NUM>Ga, <NUM>Ga, <NUM>As, <NUM>Y <NUM>Y. <NUM>Sr, <NUM>Zr, <NUM>Tc, <NUM>Tc, <NUM>Tc, <NUM>Mo, <NUM>Pd, <NUM>Rh, <NUM>Ag, <NUM>In, <NUM>I, <NUM>I, <NUM>I, <NUM>I, <NUM>Pr, <NUM>Pr, <NUM>Pm, <NUM>Sm, <NUM>-<NUM>Gd, <NUM>Tb, <NUM>Dy, <NUM>Ho, <NUM>Er, <NUM>Lu, <NUM>Lu, <NUM>Re, <NUM>Re, <NUM>Re, <NUM>Ir, <NUM>Au, <NUM>Au, <NUM>At, <NUM>Pb, <NUM>Bi, <NUM>Pb, <NUM>Bi, <NUM>Ra, <NUM>Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, <NUM>P, fluorophore (e.g. fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates, superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO nanoparticle aggregates, monochrystalline iron oxide nanoparticles, monochrystalline iron oxide, nanoparticle contrast agents, liposomes or other delivery vehicles containing Gadolinium chelate ("Gd-chelate") molecules, Gadolinium, radioisotopes, radionuclides (e.g. carbon-<NUM>, nitrogen-<NUM>, oxygen-<NUM>, fluorine-<NUM>, rubidium-<NUM>), fluorodeoxyglucose (e.g. fluorine-<NUM> labeled), any gamma ray emitting radionuclides, positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles (e.g. including microbubble shells including albumin, galactose, lipid, and/or polymers; microbubble gas core including air, heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexane lipid microsphere, perflutren, etc.), iodinated contrast agents (e.g. iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate), barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into a peptide or antibody specifically reactive with a target peptide. A detectable means may be a monovalent detectable agent or a detectable agent capable of forming a bond with another composition.

As used herein, the term "conjugate" refers to the association between atoms or molecules. The association can be direct or indirect. For example, a conjugate between an antigen binding domain and a peptide compound can be direct, e.g., by covalent bond (e.g., a disulfide bond), or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like). In embodiments, conjugates are formed using conjugate chemistry including, but are not limited to nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). These and other useful reactions are discussed in, for example,<NPL>; <NPL>; and <NPL>.

"Contacting" is used in accordance with its plain ordinary meaning and refers to the process of allowing at least two distinct species (e.g. chemical compounds including biomolecules or cells) to become sufficiently proximal to react, interact or physically touch. It should be appreciated, however, the resulting reaction product can be produced directly from a reaction between the added reagents or from an intermediate from one or more of the added reagents that can be produced in the reaction mixture.

The term "contacting" may include allowing two species to react, interact, or physically touch, wherein the two species may be an antibody and a fusion protein, biological sample, etc. as described herein.

A "control" sample or value refers to a sample that serves as a reference, usually a known reference, for comparison to a test sample. For example, a test sample can be taken from a test condition (e.g., in the presence of a test compound), and compared to samples from known conditions (e.g., in the absence of the test compound (negative control), or in the presence of a known compound (positive control)). A control can also represent an average value gathered from a number of tests or results. One of skill in the art will recognize that controls can be designed for assessment of any number of parameters. For example, a control can be devised to determine a background level of signal (negative control) or an expected level of signal (e.g., a standard curve or positive control). One of skill in the art will understand which controls are valuable in a given situation and be able to analyze data based on comparisons to control values. Controls are also valuable for determining the significance of data. For example, if values for a given parameter are widely variant in controls, variation in test samples will not be considered as significant.

"Biological sample" or "sample" refer to materials obtained from or derived from a subject or patient. A biological sample includes sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histological purposes. Such samples include bodily fluids such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like), sputum, tissue, cultured cells (e.g., primary cultures, explants, and transformed cells) stool, urine, synovial fluid, joint tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. A biological sample is typically obtained from a eukaryotic organism, such as a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

The term "polymeric" refers to a molecule including repeating subunits (e.g., polymerized monomers). For example, polymeric molecules may be based upon polypropylene (PP), polystyrene (PS), polyethylene glycol (PEG), poly[amino(<NUM>-oxo-<NUM>,<NUM>-hexanediyl)], poly(oxy-<NUM>,<NUM>-ethanediyloxycarbonyl-<NUM>,<NUM>-phenylenecarbonyl), tetraethylene glycol (TEG), polyvinylpyrrolidone (PVP), poly(xylene), or poly(p-xylylene). See, for example, "<NPL>; "<NPL>; "<NPL>, which are incorporated by reference in their entirety for all purposes.

An "interleukin-<NUM> protein" or "IL-<NUM>" as referred to herein includes any of the recombinant or naturally-occurring forms of the interleukin-<NUM> (IL-<NUM>) protein or variants or homologs thereof that maintain IL-<NUM> protein activity (e.g. within at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% activity compared to IL-<NUM> protein). In embodiments, the variants or homologs have at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a <NUM>, <NUM>, <NUM> or <NUM> continuous amino acid portion) compared to a naturally occurring IL-<NUM> protein. In embodiments, the IL-<NUM> protein is substantially identical to the protein identified by the UniProt reference number P40933 or a variant or homolog having substantial identity thereto.

An "interleukin-<NUM> receptor subunit alpha protein" or "IL-15Rα" as referred to herein includes any of the recombinant or naturally-occurring forms of the interleukin-<NUM> receptor subunit alpha (IL-15Rα) protein or variants or homologs thereof that maintain IL-15Rα protein activity (e.g. within at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% activity compared to IL-15Rα protein). In embodiments, the variants or homologs have at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a <NUM>, <NUM>, <NUM> or <NUM> continuous amino acid portion) compared to a naturally occurring IL-15Rα protein. In embodiments, the IL-15Rα protein is substantially identical to the protein identified by the UniProt reference number Q13261 or a variant or homolog having substantial identity thereto.

As used herein, "domain" refers to a conserved portion of a protein that functions and exists independently of the rest of the protein sequence. A domain may form a stable, three-dimensional structure that exists as a functional unit independent of the remaining protein. For example, the IL-15RαSu domain is the portion of IL-15Rα that retains the IL-<NUM> binding activity.

As used herein, "IL-<NUM> domain" refers to a polypeptide comprising at least a portion of a sequence of the IL-<NUM> protein. In embodiments, the IL-<NUM> domain comprises at least a portion of the sequence of the IL-<NUM> protein and includes one or more amino acid substitutions or deletions within the amino acid sequence of the IL-<NUM> protein. In embodiments, the IL-<NUM> domain is an IL-<NUM> variant that comprises a different a different amino acid sequence compared to the IL-<NUM> protein. In embodiments, the IL-<NUM> domain binds the IL-15Rα protein or a fragment thereof. In embodiments, the IL-<NUM> domain is bound to the IL-15Rα protein or a fragment thereof. In embodiments, the sequence of the IL-<NUM> domain has at least one amino acid change, e.g. substitution or deletion, compared to the IL-<NUM> protein. In embodiments, the amino acid substitutions/deletions are in the portions of IL-<NUM> that interact with IL-15Rβ and/or γC. In embodiments, the amino acid substitutions/deletions do not affect binding to the IL-15Rα polypeptide or the ability to produce the IL-<NUM> domain. In embodiments, amino acid substitutions can be conservative or non-conservative changes and insertions of additional amino acids compared to the IL-<NUM> protein. In embodiments, the IL-<NUM> domain comprises one or more than one amino acid substitutions/deletions at position <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, or <NUM> of the IL-<NUM> protein sequence. In embodiments, the IL-<NUM> domain comprises an N72D substitution of the IL-<NUM> protein sequence.

The term "sushi domain" as used herein refers to a common motif in proteins comprising a beta-sandwich arrangement. Sushi domains are common in protein-protein interactions, and typically include four cysteines forming two disulfide bonds in a <NUM>-<NUM> and <NUM>-<NUM> pattern. For example, the region of IL-15Rα that binds IL-<NUM> includes a sushi domain.

In embodiments, the IL-15Rα sushi domain includes the amino acid sequence comprising the sequence of SEQ ID NO: <NUM>. In embodiments, the variants or homologs have at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>% or <NUM>% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g. a <NUM>, <NUM>, <NUM> or <NUM> continuous amino acid portion) compared to the sequence of SEQ ID NO: <NUM>. In embodiments, the IL-15Rα sushi domain associates with the IL-<NUM> protein. In embodiments, the IL-15Rα sushi domain associates with the IL-<NUM> domain.

In an aspect is provided a composition including: (a) a first interleukin <NUM> receptor alpha Sushi domain (IL-15RαSu); (b) a second IL-15RαSu domain, wherein the first and second IL-15RαSu domains are covalently joined by a disulfide bond; (c) a first IL-<NUM> domain, bound by electrostatic interactions to the first IL-15RαSu domain to form a first IL-<NUM>/IL-15RαSu complex; (d) a second IL-<NUM> domain, bound by electrostatic interactions to the second IL-15RαSu domain to form a second IL-<NUM>/IL-15RαSu complex; (e) a first monoclonal antibody (mAb) bound to an epitope on the first IL-<NUM>/IL-15RαSu complex; and (f) a second mAb bound to an identical epitope on the second IL-<NUM>/IL-15RαSu complex, wherein the second mAb comprises a detection means selected from the group consisting of a fluorophore, a radioisotope, and an enzyme, and
wherein both the first mAb and the second mAb bind to the epitopes with equal affinity.

In embodiments, at least one of the IL-<NUM> domains includes an asparagine-to-aspartate mutation at amino acid position <NUM> (N72D). In embodiments, the IL-15RαSu domains each further include an immunoglobulin crystalizable fragment (Fc) domain. In embodiments, the first mAb is conjugated to a polymeric surface.

In embodiments, the second mAb includes a detection means. In embodiments, the detection means is selected from the group consisting of a fluorophore, a radioisotope, and an enzyme. In embodiments, the detection means is a fluorophore. In embodiments, the detection means is a radioisotope. In embodiments, the detection means is an enzyme.

In embodiments, the IL-<NUM> domains further include a single chain variable fragment (scFv) domain.

In an aspect is provided a method for detecting a heterotetrameric IL-<NUM>/IL-15RαSu complex in a biological sample, the method including: a) contacting the biological sample including the protein complex with a first mAb, wherein the mAb is conjugated to a polymeric surface, wherein the heterotetrameric complex comprises two IL-<NUM> domains and two IL-15RαSu, wherein each IL-<NUM> domain is electrostatically bound to an IL-15RαSu domain, wherein the two IL-15RαSu domains are covalently bound to each other by a disulfide bond, and wherein the Fab portion of the mAb binds an epitope on the IL-<NUM>/IL-15RαSu complex with an affinity between <NUM> and <NUM> fM; b) contacting the biological sample with a second mAb under conditions such that the second antibody binds with the same affinity to the identical epitope on the second IL-<NUM>/IL-15RαSu complex, wherein the second mAb comprises a detection means selected from the group consisting of a fluorophore, a radioisotope, and an enzyme; c) washing
unbound complexes from the polymeric surface; and d) detecting binding of the second mAb.

In embodiments, at least one of the IL-<NUM> domains includes an N72D mutation. In embodiments, both IL-<NUM> domains include an N72D mutation. In embodiments, the IL-15RαSu domains each further include an Fc domain.

In embodiments, the polymeric surface is a polypropylene or polystyrene surface. In embodiments, the polymeric surface is a polypropylene surface. In embodiments, the polymeric surface is a polystyrene surface.

In embodiments, the first mAb and the second mAb have substantially the same amino acid sequence.

In embodiments, the IL-<NUM> domains each further comprise an scFv domain. In embodiments, the IL-15RαSu domains each further comprise an scFv domain.

In embodiments, the heterotetrameric IL-<NUM>/IL-15RαSu complex is a fusion protein as described in <CIT>, <CIT>, <CIT>, and <CIT>, each of which is incorporated herein by reference in its entirety. In embodiments, the heterotetrameric IL-<NUM>/IL-15RαSu complex is N-<NUM> (also referred to as ALT-<NUM> or NANT-<NUM>). In embodiments, the complex is TxM. In embodiments, the complex is N-<NUM>.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the purview of this application and scope of the appended claims.

One skilled in the art would understand that descriptions of making and using the particles described herein is for the sole purpose of illustration, and that the present disclosure is not limited by this illustration.

The compositions and methods described herein including embodiments thereof allow for specific detection of heteromeric therapeutic protein complexes, including superagonist complexes N-<NUM>, TxM, and N-<NUM>.

Currently available assays are not capable of specifically detecting therapeutic protein complexes that include IL-<NUM>, and thus are unsuitable for assessing pharmacokinetic properties of the therapeutics in preclinical and clinical studies. As shown in <FIG>, existing detection systems that employ capture and detection antibodies that recognize different epitopes of IL-<NUM> bind non-specifically to both endogenous IL-<NUM> and N-<NUM>. This results in erroneously higher measurements of N-<NUM> concentration. Thus, various other approaches were tested for specific quantification and detection of various heteromeric protein complexes, as illustrated in the schematic of <FIG>.

First, an assay was tested where different antibodies were used for capture and detection of the N-<NUM> heteromeric protein. An anti-IL-<NUM> antibody was used for capture and an anti-human IgG Fc antibody for used for detection (<FIG>, left panel). However, serum antibodies interfered with detection of the N-<NUM> Fc domain, thus rendering this method unsuccessful. Another method was tested wherein an anti-IL-<NUM> antibody and an anti-IL-15Rα antibody were used for capture and detection, respectively (<FIG>, right panel). However, results indicated that this method is only accurate if all native IL-<NUM> in the sample is unbound and not complexed with IL-15Rα, which would result in non-specific detection of protein complexes.

An approach where the same monoclonal anti-IL-<NUM> antibody was employed for both capture and detection was tested (<FIG>, middle panel). Once the monoclonal anti-IL-<NUM> antibody bound to an IL-<NUM> molecule, the same IL-<NUM> molecule could not be recognized by a second antibody, for example the capture antibody, since the epitope is already bound. Since N-<NUM> comprises two IL-<NUM> molecules, a second anti-IL-<NUM> antibody was able to bind the second IL-<NUM> of the N-<NUM> heteromeric protein complex. This allows for binding of both the capture and detection antibody to a single N-<NUM> protein complex, without non-specific detection of native IL-<NUM>, as illustrated by <FIG>.

To optimize the assay, blocking buffers of various compositions were tested. N-<NUM> was used as a model for these experiments. As shown in <FIG>, the high background signal initially observed with <NUM>% BSA blocking solution was improved by exchanging the blocking solution with a solution comprising <NUM>% non-fat milk or <NUM>% mouse serum. Assays using blocking solution that included <NUM>% mouse serum blocking resulted in improved signal ranges and improved the lower limit of detection (LLOD) by approximately ten times. Blocking buffers with varying concentrations of mouse serum were then tested. Results illustrated in <FIG> show that buffers comprising either <NUM>% or <NUM>% mouse serum displayed similar blocking ability.

The concentrations of biotin-modified anti-IL15 capture antibody and SULFO-modified anti-IL15 detection antibody were optimized were then modified. The heteromeric N-<NUM> protein complex was used as an exemplary model for these experiments. Results are shown in Table <NUM> and <FIG>.

Matrix tolerance was tested using varying concentrations of serum, as listed in Table <NUM>. The highest signal to noise ratio was observed using samples in <NUM>% serum. The results indicated that the protein complex standards can be prepared in <NUM>% serum, and patient samples for pharmacokinetic analysis can be diluted at least <NUM> times with assay diluent.

Two-hundred microliter solutions comprising <NUM> ng/mL of IL-<NUM>, N-<NUM>, or N-<NUM> in <NUM>% serum were prepared. A <NUM> in <NUM> serial dilution in <NUM>% and <NUM>% serum, respectively, was completed as follows:.

For <NUM> ng/mL L-<NUM>, N-<NUM>, or N-<NUM> solutions, <NUM>µL of <NUM> ng/mL IL-<NUM>, N-<NUM>, or N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL solutions, <NUM>µL of <NUM> ng/mL IL-<NUM>, N-<NUM>, or N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL solutions, <NUM>µL of <NUM> ng/mL of <NUM> ng/mL IL-<NUM>, N-<NUM>, or N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL IL-<NUM>, N-<NUM>, or N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL solutions, <NUM>µL of <NUM> ng/mL IL-<NUM>, N-<NUM>, or N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL solutions, <NUM>µL of <NUM> ng/mL IL-<NUM>, N-<NUM>, or N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL, <NUM>µL of serum was used.

The Blocking Solution was comprised of <NUM>% (w/v) MSD Blocker A in PBS. For preparing the solution, <NUM> of Blocker A was dissolved in <NUM> PBS. The solution was stored at <NUM> for up to <NUM> days. Before using, the Blocking solution was allowed to equilibrate to ambient temperature.

The Assay Diluent was comprised of <NUM>% (w/v) Blocker A in PBS. The solution was prepared by adding <NUM> Blocking solution to <NUM> PBS. The solution was stored at <NUM>, and was allowed to equilibrate to ambient temperature before use.

The Read Buffer was comprised of <NUM> 2x Read Buffer T diluted in <NUM> H<NUM>O. The solution was mixed by inversion to avoid vortexing. The Read Buffer was stored at ambient temperature in a well-sealed bottle for a maximum of <NUM> days.

Biotin and SULFO-TAG conjugated anti-IL15 antibodies were prepared in Assay Buffer at final concentrations of <NUM>µg/mL.

First, a <NUM> solution of <NUM>µg/mL Biotin-anti-IL15 (R&D Systems, MAB247), a <NUM> solution of <NUM>µg/mL SULFO-TAG conjugated anti-IL15 (Thermo) and a <NUM> solution of <NUM> ug/mL SULFO-TAG conjugated anti-IL15 (R&D Systems MAB247) in Assay Buffer were prepared. Subsequently, <NUM> of the <NUM>µg/mL of Biotin-anti-IL15 and <NUM> of the <NUM>µg/mL SULFO-TAG conjugated anti-IL15 were mixed to make a <NUM> solution.

First, <NUM>µl Master Mix (comprising biotinylated and SULFO-TAG labeled antibody mixture) and <NUM>µl of either N-<NUM> or N-<NUM> was added to every other column of wells of a round-bottom <NUM>-well polypropylene (PP) plate. The plate was sealed and incubated for <NUM>-<NUM> hours at room temperature with moderate shaking or overnight at <NUM>.

During the Master Mix incubation, <NUM>µl of Blocking Buffer (<NUM>% Blocker A in PBS) was added per well to a Streptavidin GOLD (SA) plate. The plate was sealed and incubated at room temperature with shaking until sample incubation was completed (minimum <NUM>). Blocking Buffer was then removed from the SA plate. The plate was washed with Wash Buffer once (<NUM>µL/well). Fifty µl from each well of the PP plate was transferred to the SA plate. The SA plate was sealed and incubated with shaking at about <NUM> rpm for a minimum of <NUM> hour at room temperature. The plate was washed once with PBS-T (optional), and tapped dry. <NUM>µL of 2X Read Buffer T was added per well. The plate was then read. Results for this experiment are shown in <FIG>.

The Blocking Solution was comprised of either <NUM>% (w/v) BSA in PBS, <NUM>% non-fat milk in PBS, or <NUM>% mouse serum in PBS.

Serum used in this experiment was Millipore SI-<NUM> (MP Bromedical #<NUM> lot #S1449).

The Assay Diluent was comprised of PBS or PBS-T, and each blocking solution was diluted 5x in PBS or PBS-T.

The Read Buffer was comprised of <NUM> 4x Read Buffer T + <NUM> H<NUM>O. Mixing was completed by inversion, and the solution was stored at ambient temperature in a well-sealed bottle for no longer than <NUM> days.

The stock concentration of biotin conjugated anti-IL-<NUM> was <NUM>/mL (concentration was re-measured and confirmed by Bradford assay). The stock concentration of a new solution of SULFO-conjugated anti-IL-<NUM> antibody (25x excess) was <NUM>/mL (concentration was re-measured confirmed by Bradford assay).

A <NUM>µL solution of <NUM> ng/mL N-<NUM><NUM>% serum was prepared.

A <NUM> in <NUM> serial dilution of the N-<NUM> solution in <NUM>% serum or assay diluent, respectively, was performed as follows: For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum or assay diluent. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum or assay diluent. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum or assay diluent. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum or assay diluent. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum or assay diluent. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum or assay diluent. For a <NUM> ng/mL solution, <NUM>µL of <NUM>% serum was used.

An equimolar ratio of biotinylated and SULFO-TAG anti-IL15 at a concentration of <NUM>µg/mL of each antibody was used for this experiment. The Biotin and SULFO-TAG conjugated anti-IL15 (new) were prepared in the Assay Buffer at the final concentrations of <NUM>µg/mL. A total of <NUM> of <NUM>µg/mL of Biotin and SULFO-TAG conjugated antibody was prepared.

One-hundred µl Master Mix (containing biotinylated and SULFO-TAG labeled antibody mixture) and <NUM>µl of N-<NUM> solution was added to every other column of wells of a round-bottom <NUM>-well polypropylene (PP) plate. The plate was sealed and incubated overnight at <NUM> with moderate shaking.

During the Master Mix incubation, <NUM>µl of Blocking Buffer (<NUM>% BSA in PBS) was added per well to a small spot Streptavidin (SSA) plate. The plate was sealed and incubated at room temperature with shaking until sample incubation was finished (minimum <NUM>).

Blocking Buffer was removed from SSA plates, and the plates were tapped dry. From the PP plates, <NUM>µl from each well was transferred to the SSA plates. The SSA plates were sealed and incubated with shaking (<NUM> rpm) for minimum of <NUM> hour at room temperature. The plates were washed 3x with PBS-T, and subsequently were tapped dry. One hundred fifty µL of 2X Read Buffer T was added to each well, and the plate was read.

In this experiment, the plate was not washed after blocking, and washing was done three times prior to adding Read-T buffer. Subsequent experiments include a single wash after blocking and a single wash prior to adding Read-T buffer.

Results for this experiments are shown in <FIG>, and illustrate that the LLOD was improved by ten times using <NUM>% mouse serum.

The Blocking Solution comprised <NUM>% (v/v) Mouse Serum in PBS. The solution was prepared by diluting <NUM> mouse serum in <NUM> PBS. The solution was stored at <NUM> for a maximum of <NUM> days, and was allowed to eequilibrate to ambient temperature before use.

The Assay Diluent was comprised of <NUM>% (v/v) Mouse Serum in PBS. The solution was prepared by diluting <NUM> Blocking solution in <NUM> PBS. The Assay Diluent was stored at <NUM> and was allowed to equilibrate to ambient temperature before use.

The Read Buffer was used at a 2x concentration of Read Buffer T. Per plate, <NUM> of 4x Read Buffer T was diluted in <NUM> H<NUM>O. The solution was mixed by inversion, and vortexing was avoided. The solution was stored at ambient temperature in a well-sealed bottle for a maximum of <NUM> days.

Normal serum was used at <NUM>% and <NUM>% concentrations. Serum was diluted in assay diluent.

The following protocol required <NUM> each of <NUM>% and <NUM>% serum.

A <NUM>µl solution of <NUM> ng/mL N-<NUM> in either <NUM>% or <NUM>% serum was prepared, and was further diluted in a dilution series using <NUM>% and <NUM>% serum as diluent as follows.

A <NUM> in <NUM> serial dilution in <NUM>% and <NUM>% serum, respectively, was completed as follows: For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, either <NUM>% or <NUM>% serum was used.

Equimolar ratios of biotinylated and SULFO-TAG antibodies were used for this experiment. A <NUM>-fold serial dilution was prepared of the Biotin and SULFO-TAG conjugated anti-IL15 in the Assay Buffer with final concentrations of <NUM>, <NUM>, and <NUM>µg/mL.

A <NUM>µg/mL solution of Biotin and SULFO-TAG conjugated antibody in Assay Buffer was prepared. The protocol required <NUM> each of <NUM>µg/mL of Biotin and SULFO-TAG conjugated antibody in Assay Buffer.

The antibody solution was prepared by mixing <NUM> each of <NUM>µg/mL of Biotin and SULFO-TAG conjugated antibody. The resultant solution had <NUM>µg/mL of Biotin and SULFO-TAG conjugated antibody (final concentration).

From the above solution <NUM> was taken and diluted with <NUM> of Assay diluent to make a <NUM>µg/mL solution. From the <NUM>µg/mL solution <NUM> was taken and diluted with <NUM> of assay diluent to make a <NUM>µg/mL solution.

Results indicate that the heteromeric protein complex is detectable in <NUM>% serum samples using equimolar concentrations of capture and detection antibody.

The Blocking Solution comprised <NUM>% (v/v) Mouse Serum in PBS. The solution was prepared by diluting <NUM> mouse serum in <NUM> PBS. The solution was stored at <NUM> for up to <NUM> days and allowed to equilibrate to ambient temperature before use.

The Assay Diluent was comprised of <NUM>% (v/v) Mouse Serum in PBS, <NUM> Blocking solution, and <NUM> PBS. The solution was stored at <NUM> and equilibrated to ambient temperature before use.

The Read Buffer was comprised of 2x Read Buffer T. For each plate, <NUM> 4x Read Buffer T was added to <NUM> H<NUM>O. The solution was mixed by inversion, and vortexing was avoided. The solution was stored at RT in a sealed bottle for a maximum of <NUM> days.

Normal serum was diluted to <NUM>% for a total volume of <NUM>, and <NUM> of <NUM> ng/mL N-<NUM> in <NUM>% serum was prepared.

Two-fold serial dilutions of the Biotin and SULFO-TAG conjugated antibody in the Assay Buffer were prepared separately.

Two µg/mL of Biotin conjugated antibody and <NUM>µg/mL SULFO-TAG conjugated antibody were prepared. The study required <NUM> each of <NUM>µg/mL of Biotin and <NUM>µg/mL of SULFO-TAG conjugated antibody in Assay Buffer.

A <NUM>-fold serial dilution for Biotin conjugated antibody was completed as follows: For a <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution (above) was added to <NUM> of assay buffer. For a <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. For a <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. For a <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. Assay buffer only was used for <NUM>µg/mL.

Two-fold serial dilution for SULFO-TAG conjugated antibody was completed as follows: For <NUM>µg/mL, <NUM> of the above solution was used. For a <NUM>µg/mL solution, <NUM> of <NUM>µg/ml solution (above) was added to <NUM> of assay buffer. For <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. For <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. For <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. For <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer. Assay buffer only was used for <NUM>µg/mL.

To each well of a flat-bottom <NUM>-well polypropylene plate, <NUM>µl of biotinylated antibody and <NUM>µl of SULFO-TAG labeled antibody (total <NUM>µl/well of Master Mix) and <NUM>µl of N-<NUM> were added. The plate was sealed and incubated with moderate shaking (<NUM> rpm) overnight at <NUM>.

During the Master Mix incubation, <NUM>µl per well of Blocking Buffer (<NUM>% Mouse Serum in PBS) was added to a Small-spot Streptavidin GOLD (SSA) plate. The plate was sealed and incubate at room temperature with shaking until sample incubation was finished (minimum <NUM>).

The Blocking Buffer was removed from SA plate. The plate was washed with Wash Buffer once (<NUM>µL/well). From each well of the PP plate <NUM>µl of solution was transferred to the SSA plate. The SSA plate was sealed and incubated with shaking (<NUM> rpm) for a minimum <NUM> hour at room temperature. The plate was washed once with PBS-T and tapped dry. Per well, <NUM>µl of 2X Read Buffer T was added and the plate was read. Results are shown in Table <NUM>.

The effect of <NUM>% and <NUM>% mouse serum from two different vendors (Sigma vs MP Biomedicals) was compared in the below study.

The Blocking Solution comprised of either <NUM>% (v/v) Mouse Serum or <NUM>% (v/v) Mouse Serum in PBS. The solutions were made by diluting <NUM> mouse serum in <NUM> PBS, and were stored at <NUM> for a maximum of <NUM> days. The solutions were allowed to equilibrate to ambient temperature before use.

The Assay Diluent was comprised of <NUM>% (v/v) Mouse Serum in PBS. The Assay Diluent was prepared by adding <NUM> Blocking solution to <NUM> PBS. The solution was stored at <NUM>, was was allowed to equilibrate to ambient temperature before use.

The Read Buffer was comprised of 2x Read Buffer T. For each plate, <NUM> of 4x Read Buffer T was mixed with <NUM> H<NUM>O. The buffer was mixed by inversion, and vortexing was avoided. The solution was stored at ambient temperature in a well-sealed bottle for up to <NUM> days.

N-<NUM> was prepared at a concentration of <NUM> ng/mL at a volume of <NUM>µL. N-<NUM> samples were prepared in <NUM>% and <NUM>% serum.

One in five serial dilution in <NUM>% and <NUM>% serum were prepared as follows: For <NUM> ng/ mL N-<NUM>, <NUM>µL of <NUM> ng/mL was added to <NUM>µL serum. For <NUM> ng/mL N-<NUM>, <NUM>µL of <NUM> ng/mL was added to <NUM>µL serum. For <NUM> ng/mL N-<NUM>, <NUM>µL of <NUM> ng/mL was added to <NUM>µL serum. For <NUM> ng/mL N-<NUM>, <NUM>µL of <NUM> ng/mL was added to <NUM>µL serum. For <NUM> ng/mL N-<NUM>, <NUM>µL of <NUM> ng/mL was added to <NUM>µL serum. For <NUM> ng/mL N-<NUM>, <NUM>µL of <NUM> ng/mL was added to <NUM>µL serum. For <NUM> ng/mL N-<NUM>, <NUM> uL diluent was added to <NUM>µL of <NUM>% or <NUM>% serum.

<NUM> of <NUM>µg/mL biotin- and SULFO-anti-IL15 was prepared in either <NUM>% MS in PBS, <NUM>% MS (Sigma) in PBS, and <NUM>% MS (MB bioscience) in PBS.

<NUM>µl Master Mix (containing biotinylated and SULFO-TAG labeled antibody mixture) and <NUM>µl of N-<NUM> solution was added to every other column of a flat-bottom <NUM>-well polypropylene plate. The plate was sealed and incubated with moderate shaking (<NUM> rpm) overnight at <NUM>. The next day, <NUM>µl per well of Blocking Buffer (<NUM>% Mouse Serum in PBS) was added to a Small Spot Streptavidin GOLD (SSA) plate. The plate was sealed and incubated at room temperature with shaking for at minimum <NUM>.

The Blocking Buffer was removed from the SSA plate. The plate was washed with Wash Buffer once (<NUM>µL/well). From each well of the PP plate <NUM>µl was transferred to the SSA plate. The SSA plate was sealed and incubated with shaking (<NUM> rpm) for minimum <NUM> hour at room temperature.

The Blocking Buffer was removed from SSA plate. The plate was washed with Wash Buffer once (<NUM>µL/well). Then, <NUM>µl from each well of the PP plate was transferred to the SSA plate. The SSA plate was sealed and incubated with shaking (<NUM> rpm) for minimum <NUM> hour at room temperature. The source of mouse serum did not affect assay results.

The Blocking Solution comprised <NUM>% (v/v) Mouse Serum in PBS. The solution was prepared by adding <NUM> mouse serum to <NUM> PBS. The solution was stored at <NUM> for a maximum of <NUM> days, and was allowed to equilibrate to ambient temperature before use.

The Assay Diluent comprised <NUM>% (v/v) Mouse Serum in PBS. The solution was prepared by adding <NUM> Blocking solution to <NUM> PBS. The solution was stored at <NUM>, and was allowed to equilibrate to ambient temperature before use.

The Read Buffer was comprised of 2x Read Buffer T. For each plate, <NUM> of 4x Read Buffer T was added to <NUM> H<NUM>O. The solution was mixed by inversion, and vortexing was avoided. The solution was stored at ambient temperature in a sealed bottle for a maximum of <NUM> days.

<NUM>µL of <NUM> ng/mL N-<NUM> was first prepared in <NUM>% serum.

One in four serial dilutions in <NUM>% serum was prepared as follows: For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For a <NUM> ng/mL solution, <NUM>µL of <NUM> ng/mL N-<NUM> was added to <NUM>µL serum. For <NUM> ng/mL, <NUM>% serum was used.

A <NUM>-fold serial dilution for each antibody (biotin conjugated antibody) at a volume of <NUM> for each concentration was prepared.

First, <NUM> of <NUM>µg/mL antibody was prepared. For <NUM>µg/mL solution, <NUM> of <NUM>µg/mL solution was added to <NUM> of assay buffer.

A <NUM>-fold serial dilution for each antibody (SULFO-TAG conjugated antibody) at a volume of <NUM> for each concentration was prepared.

First <NUM> of <NUM>µg/mL antibody was prepared. For <NUM>µg/mL, <NUM> of <NUM>µg/mL solution was mixed with <NUM> of assay buffer. For <NUM>µg/mL, <NUM> of <NUM>µg/mL solution was mixed with <NUM> of assay buffer.

To every other column of a flat-bottom <NUM>-well polypropylene plate <NUM>µl of biotinylated antibody and <NUM>µl of SULFO-TAG labeled antibody (total <NUM>µl/well of Master Mix) and <NUM>µl of N-<NUM> solution was added. The plate was sealed and incubated with moderate shaking (<NUM> rpm) overnight at <NUM>. The next day, <NUM>µl of Blocking Buffer (<NUM>% Mouse Serum in PBS) was added to each well of a Small Spot Streptavidin GOLD (SSA) plate. The plate was sealed and incubated at room temperature with shaking for at minimum <NUM>. The Blocking Buffer was removed from the SSA plate. The plate was washed once with Wash Buffer (<NUM>µL/well). Subsequently, <NUM>µl was transferred from each well of the PP plate to the SSA plate. The SSA plate was sealed and incubated with shaking (<NUM> rpm) for minimum <NUM> hour at room temperature. The plate was washed once with PBS-T, and was tapped dry. Per well, <NUM>µL of 2X Read Buffer T was added, and the plate was read.

The Assay Diluent comprised <NUM>% (v/v) Mouse Serum in PBS. The solution was prepared by mixing <NUM> Blocking solution and <NUM> PBS. The solution was stored at <NUM>, and allowed to equilibrate to ambient temperature before use.

The Read Buffer was comprised of 2x Read Buffer T. For each plate, <NUM> 4x Read Buffer T was added to <NUM> H<NUM>O. The solution was mixed by inversion, and vortexing was avoided. The solution was stored at ambient temperature in a sealed bottle for a maximum of <NUM> days.

Solutions were prepared as follows: For <NUM>% serum, <NUM> of <NUM>% serum was used. For <NUM>% serum, <NUM> of <NUM>% serum (above) was added to <NUM> of assay diluent. For <NUM>% serum, <NUM> of <NUM>% serum was added to <NUM> of assay diluent. For <NUM>% serum, <NUM> of <NUM>% serum was added to <NUM> of assay diluent. For <NUM>% serum, <NUM> of <NUM>% serum was added to <NUM> of assay diluent. For <NUM>% serum, <NUM> of <NUM>% serum +<NUM> of assay diluent.

Next, <NUM> of <NUM> ng/mL of N-<NUM> in <NUM>% serum was prepared, and <NUM> of <NUM> ng/mL of N-<NUM> in assay diluent was prepared.

Mixtures were prepared as follows: For <NUM>% serum (<NUM> ng/mL N-<NUM>) sample, <NUM> of <NUM> ng/mL N-<NUM> in was prepared in <NUM>% serum. For <NUM>% serum (<NUM> ng/mL N-<NUM>) sample, <NUM> of <NUM> ng/mL N-<NUM> in <NUM>% serum was added to <NUM> of <NUM> ng/ mL N-<NUM> in assay diluent. For <NUM>% serum (<NUM> ng/mL N-<NUM>) sample, <NUM> of <NUM> ng/mL N-<NUM> in <NUM>% serum was added to <NUM> of <NUM> ng/mL N-<NUM> in assay diluent. For <NUM>% serum (<NUM> ng/mL N-<NUM>) sample, <NUM> of <NUM> ng/mL N-<NUM> in <NUM>% serum was added to <NUM> of <NUM> ng/mL N-<NUM> in assay diluent. For <NUM>% serum (<NUM> ng/mL N-<NUM>) sample, <NUM> of <NUM> ng/mL N-<NUM> in <NUM>% serum was added to <NUM> of <NUM> ng/mL N-<NUM> in assay diluent. For <NUM>% serum (<NUM> ng/mL N-<NUM>) sample, <NUM> of <NUM> ng/mL N-<NUM> in <NUM>% serum was added to <NUM> of <NUM>0ng/ mL N-<NUM> in assay diluent.

For testing <NUM>% serum, samples were prepared by subjecting the above samples to <NUM>-fold serial dilutions in <NUM>% serum.

For <NUM> ng/mL N-<NUM>, <NUM> of <NUM>% serum (<NUM> ng/mL N-<NUM>) was used. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL N-<NUM> and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL N-<NUM> solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/ mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM>µg/mL N-<NUM>, <NUM> <NUM>% serum was used.

For testing <NUM>% serum, the samples were prepared by subjecting the above samples to <NUM>-fold serial dilutions in <NUM>% serum.

For <NUM> ng/mL N-<NUM>, <NUM> of <NUM>% serum (<NUM> ng/mL N-<NUM>) was used. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL N-<NUM> and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/mL solution and <NUM> of <NUM>% serum were mixed. For <NUM> ng/mL N-<NUM>, <NUM> of <NUM> ng/ml solution and <NUM> of <NUM>% serum were mixed. For <NUM>µg/mL N-<NUM>, <NUM> of <NUM>% serum was used.

Dilutions were repeated as appropriate for <NUM>%, <NUM>%, <NUM>% and <NUM>% serum samples.

Biotin-anti-IL15 and SULFO-anti-IL15 at concentrations of <NUM> ug/mL were prepared in assay diluent at a final volumes of <NUM>. The solutions were combined and mixed well.

To every other well of a flat-bottom <NUM>-well polypropylene plate, <NUM>µl of Master Mix (containing biotinylated and SULFO-TAG labeled antibody mixture) and <NUM>µl of N-<NUM> solution were added. The plate was sealed and incubated with moderate shaking (<NUM> rpm) overnight at <NUM>.

During the Master Mix incubation, <NUM>µl was added per well of Blocking Buffer (<NUM>% Mouse Serum in PBS) to a Small-Spot Streptavidin GOLD (SSA) plate. The plate was sealed and incubated at room temperature with shaking until sample incubation was finished (minimum <NUM>).

Blocking Buffer was removed from the SSA plate. The plate was washed with <NUM>µL per well of Wash Buffer once. From the PP plate, <NUM>µl from each well was transferred to the SSA plate. The SSA plate was sealed and incubated with shaking (<NUM> rpm) at room temperature for <NUM> hours. The plate was washed once with PBS-T, and was tapped dry. To each well, <NUM>µL of 2X Read Buffer T was added and the plate was read.

Claim 1:
A composition comprising:
a) a first interleukin <NUM> receptor alpha Sushi domain (IL-15RαSu);
b) a second IL-15RαSu domain, wherein the first and second IL-15RαSu domains are covalently joined by a disulfide bond;
c) a first IL-<NUM> domain, bound by electrostatic interactions to the first IL-15RαSu domain to form a first IL-<NUM>/IL-15RαSu complex;
d) a second IL-<NUM> domain, bound by electrostatic interactions to the second IL-15RαSu domain to form a second IL-<NUM>/IL-15RαSu complex;
e) a first monoclonal antibody (mAb) bound to an epitope on the first IL-<NUM>/IL-15RαSu complex; and
f) a second mAb bound to the identical epitope on the second IL-<NUM>/IL-15RαSu complex, wherein the second mAb comprises a detection means selected from the group consisting of a fluorophore, a radioisotope, and an enzyme, and wherein both the first mAb and the second mAb bind to the epitopes with equal affinity.