A method for enhanced BCMA immunohistochemical detection in human and monkey tissues

Abstract

Provided herein are methods for detecting the presence of a molecule in a sample, such as a bodily fluid or tissue, from a patient.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Patent Application No. 63 / 272,600, filed October 27, 2021, the contents of which are incorporated by reference herein in their entirety.

[0002] (Field) Provided herein are methods for detecting the presence of a molecule in a sample, such as a bodily fluid or tissue, from a patient.

[0003] (overview) In one aspect, provided herein is a method of detecting molecules of B-cell maturation antigen ("BCMA") in a formalin-fixed paraffin-embedded ("FFPE") sample, comprising: sectioning and mounting the FFPE sample; deparaffinizing the sample; heat-mediated antigen retrieval of the sample; pre-treating the sample with a blocking agent; contacting the sample with a first detection agent, which optionally comprises an antibody or antigen-binding fragment thereof that binds to at least one BCMA molecule in the sample; optionally removing unbound sample; contacting the sample bound to the first detection agent with a second detection agent, which optionally is an antibody or fragment thereof; optionally removing unbound second detection agent; and detecting the presence of the second detection agent bound to the sample; wherein detection of an amount of the second detection agent bound to the sample above background indicates the presence of at least one BCMA molecule in the sample.

[0004] In some embodiments, heat-mediated antigen retrieval is performed using an EDTA-based solution.

[0005] In some embodiments, the EDTA-based solution has a pH of 8.5 to 9.5, In some embodiments, the EDTA-based solution has a pH of 9.0.

[0006] In some embodiments, the step of heat-mediated antigen retrieval is carried out for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, or 60 minutes.

[0007] In some embodiments, the step of heat-mediated antigen retrieval is carried out at a temperature between 85° C. and 100° C. In some embodiments, the step of heat-mediated antigen retrieval is carried out at 100° C.

[0008] In some embodiments, the blocking agent blocks endogenous peroxidase, hi some embodiments, the blocking agent is a peroxide blocker.

[0009] In some embodiments, the peroxide block pretreatment step is carried out for 2 minutes, 5 minutes, 8 minutes, 10 minutes, or 15 minutes.

[0010] In some embodiments, the blocking agent blocks non-specific antibody binding. In some embodiments, the blocking agent is Dako / Agilent Protein Block.

[0011] In some embodiments, the pretreatment step with Dako / Agilent Protein Block is carried out for 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes.

[0012] In some embodiments, the first detection agent is an antibody or an antigen-binding fragment thereof.

[0013] In some embodiments, the sample is contacted with the first detection agent at room temperature or at 37°C.

[0014] In some embodiments, the sample is contacted with the first detection agent for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 95 minutes, or 120 minutes.

[0015] In some embodiments, the second detection agent is an antibody or an antigen-binding fragment thereof.

[0016] In some embodiments, the sample bound to the first detection agent is contacted with the second detection agent for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

[0017] In some embodiments, the sample comprises cells from a body fluid or tissue. In some embodiments, the tissue is brain tissue. In other embodiments, the tissue is from the striatum, thalamus, midbrain, or medullary regions of the brain.

[0018] In some embodiments, the tissue is a tumor tissue.

[0019] In some embodiments, the sample is from a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a cynomolgus monkey.

[0020] In one aspect, provided herein are kits for carrying out the methods disclosed herein.

[0021] In one aspect, provided herein is a method of treating a subject with a BCMA targeting agent, the method comprising detecting BCMA in a sample from the subject.

[0022] In some embodiments, detecting BCMA in the sample comprises a method disclosed herein.

[0023] In some embodiments, the targeting agent is a chimeric antigen receptor (CAR) T cell. In some embodiments, the targeting agent is a T cell redirecting antibody.

[0024] In some embodiments, the subject has cancer, hi some embodiments, the cancer is multiple myeloma. [Brief description of the drawings]

[0025] The foregoing Summary of the Invention, as well as the following Detailed Description of certain embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1-1] Detection of B-cell maturation antigen ("BCMA") protein by immunohistochemistry ("IHC") on formalin-fixed, paraffin-embedded (FFPE) cell pellets and tissue controls, and detection of BCMA RNA by in situ hybridization ("ISH"). Figure 1A shows BCMA expression in H929 cells. Figure 1B shows BCMA expression in MM1R cells. Figure 1C shows BCMA expression in Jeko-1 cells. Dark chevrons show BCMA detection in the perinuclear region. Figure 1D shows BCMA expression in Raji cells. Dark chevrons show BCMA detection in the perinuclear region. Figure 1E shows BCMA expression in K562 cells. Figure 1F shows BCMA expression in U-937 cells. Figure 1G shows BCMA expression in HEK293 cells. Figures 1L-1R show demonstrative ISH on H929, MM1R, Jeko-1, Raji, K562, U-937, and HEK293 cells, respectively. Light chevrons indicate detection of BCMA RNA in Figures 1N and 1O. Figure 1H shows BCMA IHC in a human colon FFPE sample. BCMA expression is membranous and perinuclear in putative resident plasma cells within the lamina propria. The inset in Figure 1H shows a higher magnification of the putative plasma cells. Figure 1I shows ISH of BCMA expression in a human colon FFPE with positive putative plasma cells. The inset in Figure 1I shows a higher magnification of the putative plasma cells. [Figure 1-2]Detection of B-cell maturation antigen ("BCMA") protein by immunohistochemistry ("IHC") on formalin-fixed, paraffin-embedded (FFPE) cell pellets and tissue controls, and detection of BCMA RNA by in situ hybridization ("ISH"). Figure 1A shows BCMA expression in H929 cells. Figure 1B shows BCMA expression in MM1R cells. Figure 1C shows BCMA expression in Jeko-1 cells. Dark chevrons show BCMA detection in the perinuclear region. Figure 1D shows BCMA expression in Raji cells. Dark chevrons show BCMA detection in the perinuclear region. Figure 1E shows BCMA expression in K562 cells. Figure 1F shows BCMA expression in U-937 cells. Figure 1G shows BCMA expression in HEK293 cells. Figures 1L-1R show demonstrative ISH on H929, MM1R, Jeko-1, Raji, K562, U-937, and HEK293 cells, respectively. Light chevrons indicate detection of BCMA RNA in Figures 1N and 1O. Figure 1H shows BCMA IHC in a human colon FFPE sample. BCMA expression is membranous and perinuclear in putative resident plasma cells within the lamina propria. The inset in Figure 1H shows a higher magnification of the putative plasma cells. Figure 1I shows ISH of BCMA expression in a human colon FFPE with positive putative plasma cells. The inset in Figure 1I shows a higher magnification of the putative plasma cells. [Figure 1-3] Detection of B-cell maturation antigen ("BCMA") protein by immunohistochemistry ("IHC") on formalin-fixed, paraffin-embedded (FFPE) cell pellets and tissue controls, and detection of BCMA RNA by in situ hybridization ("ISH") are shown. Figures 1J and 1K show IHC of BCMA expression on BCMA-negative cells transfected with BAFFR (Figure 1J) or TACI (Figure 1K). Insets in Figures 1J and 1K show that despite successful transfection of the cell lines as shown by anti-tag immunoreactivity (inset), no immunoreactivity is detected. [Figure 2-1]The results of IHC assays performed on brain samples using various BCMA antibodies are shown. Figure 2A shows BCMA immunoreactivity with the Cell Signaling E6D7B clone (tag) in the putamen. Figures 2B-2C show BCMA immunoreactivity with the Cell Signaling E6D7B clone (tag) in the medulla. [Figure 2-2] The results of IHC assays performed on brain samples using various BCMA antibodies are shown. Figures 2D and 2G show BCMA immunoreactivity with Santa Cruz Biotech clone D6(tag) in the putamen. Figures 2E-2F and 2H-2I show BCMA immunoreactivity with Santa Cruz Biotech clone D6(tag) in the medulla. [Figure 2-3] The results of IHC assays performed on brain samples using various BCMA antibodies are shown. Figures 2D and 2G show BCMA immunoreactivity with Santa Cruz Biotech clone D6(tag) in the putamen. Figures 2E-2F and 2H-2I show BCMA immunoreactivity with Santa Cruz Biotech clone D6(tag) in the medulla. [Figure 3-1] Figures 3A-3D show the co-localization of BCMA with trans- and cis-Golgi markers. Figures 3A-3D show the results of immunofluorescence staining in H929 cells with various antibodies. Figure 3A shows DAPI staining. Figure 3B shows GOLM1 staining. Figure 3C shows E6D7B staining. Figure 3D shows the co-localization of GOLM1 and E6D7B. Arrows indicate the co-localization of GOLM1 and E6D7B. [Figure 3-2] Figures 3E-3H show the results of immunofluorescence staining in the medulla with various antibodies. Figure 3E shows DAPI staining. Figure 3F shows GOLM1 staining. Figure 3G shows E6D7B staining. Figure 3H shows the colocalization of GOLM1, E6D7B and BCMA. The chevrons indicate autofluorescence in scattered neurons. [Figure 4-1]Figure 4 shows BCMA RNA expression in various brain regions. Figure 4A shows RNA sequencing results in GTEx, Allen BrainSpan, and aggregate striatal data for all brain regions. [Figure 4-2] Figure 4B shows BCMA RNA expression in various brain regions. Figure 4B shows Allen BrainSpan bulk RNA-seq data for all brain regions plotted by developmental stage. [Figure 4-3] Figure 4C shows aggregate BCMA RNA expression data for the striatum and components of the striatum (GTEx: caudate and putamen) from Allen BrainSpan plotted by donor age. Abbreviations: TPM, transcripts per million. [Figure 5-1] Assay validation of the Santa Cruz D6 clone is shown in Figure 5A, which shows BCMA-expressing multiple myeloma cells in bone marrow using the D6 clone, and Figure 5B, which shows BCMA-expressing putative plasma cells in the lamina propria of the colon using the D6 clone. [Figure 5-2] Assay validation of the Santa Cruz D6 clone is shown in Figures 5C-5I and Figures 5J-5P show BCMA expression in cell pellets using the D6 clone. [Figure 6] Comparison of E6D7B immunoreactivity with protein aggregation markers is shown. Figures 6A-6C show BCMA immunoreactivity observed in neurons of human brain using E6D7B clones. Figure 6D shows phosphorylated tau protein (pTau) immunoreactivity (peaks) in the medulla. Figures 6E-6F show Bielschowsky silver staining in the medulla.

[0026] (Detailed description) In the "Background" section and throughout the specification, various publications, articles and patents are cited or described, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, operations, materials, devices, articles and the like which is included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0027] Unless otherwise defined, 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 invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0028] The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using conventional techniques, such as the commonly used techniques described in Sambrook et al: A Laboratory Manual (3d ed.2001), Current Protocols in Molecular Biology (Ausubel et al. eds., 2003), Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed.2009), Monoclonal Antibodies: Methods and Protocols (Albitar ed.2010), and Antibody Engineering Vols 1 and 2 (Kontermann and Dubel eds., 2d ed.2010). Unless otherwise defined herein, technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. For the purposes of interpreting this specification, the following explanations of terms apply, and where appropriate, terms used in the singular also include the plural and vice versa. In the event that any explanation of a term provided herein conflicts with any document incorporated by reference, the explanation of the term provided below shall prevail.

[0029] The following references are incorporated by reference in their entireties: Bingham et al., Oncotarget 2017;8(55):93392-403; Bolon et al., Toxicol Pathol. 2013;41(7):1028-48; Gras et al., 1995;7(7):1093-106; Uchihara et al., Neuropathology. 2014;34(6):571-7; Schuh et al., J Immunol. 2017;198(8):3081-8; and Shah et al., Leukemia. 2020;34(4):985-1005.

[0030] definition It should be noted that as used herein in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0031] In the event that there are a plurality of definitions for terms herein, those in this section prevail unless stated otherwise.

[0032] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" refers to within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" or "approximately" refers to within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

[0033] Unless otherwise indicated, the term "at least" preceding a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0034] It will be understood that as used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are intended to be inclusive of the stated element or elements, but not to the exclusion of other elements or elements, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or device that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or that are inherent to such composition, mixture, process, method, article, or device. Further, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0035] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined options. For example, when two elements are connected by "and / or," the first option refers to the first element being applicable without the second element. The second option refers to the second element being applicable without the first element. The third option refers to the first and second elements being applicable together. Any one of these options is understood to be within the meaning and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the options is also understood to be within the meaning and thus meets the requirements of the term "and / or."

[0036] As used herein, the term "consists of," or variations such as "consist of" or "consisting of," as used throughout the specification and claims, includes any listed element or elements, but indicates that no additional element or elements are added to the specified method, structure, or composition.

[0037] As used herein, the term "consists essentially of," or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, indicates the inclusion of any recited element or group of elements, optionally including any recited element or group of elements that do not materially change the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0038] As used herein, "administering" or "administration" refers to the act of injecting or otherwise physically delivering an exogenous substance to a patient, such as by oral, mucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When a disease or a symptom thereof is being treated, administration of the substance typically occurs after the onset of the disease or a symptom thereof. When a disease or a symptom thereof is being prevented, administration of the substance typically occurs before the onset of the disease or a symptom thereof.

[0039] As used herein, the term "polynucleotide" is also referred to interchangeably as "nucleic acid molecule," "nucleotide," or "nucleic acid," and refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules containing DNA and RNA that may be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to triple-stranded regions that include RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA that contain one or more modified bases, and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. A variety of modifications can be made to DNA and RNA. Thus, "polynucleotide" includes chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as chemical forms having characteristics of viral and cellular DNA and RNA. "Polynucleotide" also includes relatively short nucleic acid strands, often referred to as oligonucleotides.

[0040] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody may be present in the cytoplasm of a host cell, in an extracellular environment such as the growth medium of a cell culture, or may be anchored to the cell membrane.

[0041] As used herein, the term "peptide", "polypeptide", or "protein" can refer to a molecule composed of amino acids and can be recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide", and "protein" can be used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that are naturally modified or modified by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0042] The peptide sequences described herein are written according to the usual convention with the N-terminal region of the peptide on the left and the C-terminal region on the right, although isomeric forms of amino acids are known, unless expressly indicated otherwise, it is the L-form of the amino acid that is shown.

[0043] The terms "antibody", "immunoglobulin", or "Ig" are used interchangeably herein and are used in the broadest sense, specifically including, for example, monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), antibody compositions with polyepitopic or monoepitopic specificity, polyclonal or monovalent antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single chain antibodies, single domain antibodies (e.g., VHH), and fragments thereof (e.g., domain antibodies). Antibodies may be human, humanized, chimeric, and / or affinity matured, and may also be antibodies from other species, e.g., mouse, rabbit, llama, etc. The term "antibody" is intended to include polypeptide products of B cells within the immunoglobulin class of polypeptides capable of binding to a specific molecular antigen and composed of two identical paired polypeptide chains, each pair having one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), the respective amino-terminal portions of each chain containing a variable region of about 100 to about 130 or more amino acids, and the respective carboxy-terminal portions of each chain containing a constant region. See, e.g., Antibody Engineering (Borrebaeck ed., 2d ed. 1995); and Kuby, Immunology (3d ed. 1997). Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, single domain antibodies such as those derived from Camelidae species (e.g., llamas and alpacas) or humanized variants thereof, intracellular antibodies, anti-idiotypic (anti-Id) antibodies, and functional fragments of any of the above (e.g., antigen-binding fragments), and refer to portions of antibody heavy or light chain polypeptides that retain some or all of the binding activity of the antibody from which the fragment is derived.Non-limiting examples of functional fragments (e.g., antigen-binding fragments) include single chain Fvs (scFv) (including, e.g., monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (dsFv), Fd fragments, Fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen-binding domains or molecules that contain an antigen-binding site that binds an antigen (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane, Antibodies: A Laboratory Manual (1989), Mol. Biology and Biotechnology: A Comprehensive Desk Reference (Myers ed., 1995), Huston et al., 1993, Cell Biophysics 22:189-224, Pluckthun and Skerra, 1989, Meth. Enzymol. 178:497-515, and Day, Advanced Immunochemistry (2d ed. 1990). The antibodies provided herein can be of any class (e.g., IgG, IgE, IgM, IgD, and IgA) or any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) of immunoglobulin molecules. The antibodies can be agonist or antagonist antibodies. The antibody may be neither an agonist nor an antagonist.

[0044] An "antigen" is a structure to which an antibody can selectively bind. A target antigen can be a polypeptide, carbohydrate, nucleic acid, lipid, hapten, or other naturally occurring or synthetic compound. In some embodiments, the target antigen is a polypeptide. In certain embodiments, the antigen is associated with a cell, e.g., present on or within a cell.

[0045] As used herein, the term "BCMA" refers to B cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), a protein encoded by the TNFRSF17 gene in humans. BCMA is a cell surface receptor of the TNF receptor superfamily that recognizes B cell activating factors. BCMA is preferentially expressed in mature B lymphocytes. The term "BCMA" includes any BCMA variants, isoforms, and interspecies homologs that can be naturally expressed by cells (including B cells) or expressed on cells transfected with a gene or cDNA encoding the polypeptide. Unless otherwise stated, preferably, the BCMA is human BCMA. An exemplary human BCMA nucleotide sequence is provided by GenBank Accession No. BC058291. There are four major haplotypes of the BCMA gene in the human genome, and in this disclosure, the term "BCMA" is meant to encompass all four (Kawasaki et al., Genes Immun. 2:276-9, 2001).

[0046] "Antigen-binding domain" or "antigen-binding fragment" or "domain that binds to antigen" refers to a portion of a molecule that specifically binds to an antigen. An antigen-binding domain may comprise a portion of an immunoglobulin that binds to an antigen, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAbs) consisting of one VH or one VL, shark variable IgNAR domains, camelized VH domains, VHH, a minimal recognition unit consisting of amino acid residues that mimic the CDRs of an antibody such as a FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, and LCDR1, LCDR2, and / or LCDR3, and a non-antibody scaffold that binds to an antigen.

[0047] As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which a binding molecule (e.g., an antibody comprising a single-chain antibody sequence) can specifically bind. An epitope can be a linear or conformational epitope, a non-linear epitope, or a discontinuous epitope. In the case of a polypeptide antigen, for example, an epitope can be consecutive amino acids of a polypeptide (a "linear" epitope), or an epitope can include amino acids from two or more non-contiguous regions of a polypeptide (a "conformational", "non-linear", or "discontinuous" epitope). In general, it will be understood by those skilled in the art that a linear epitope may or may not depend on secondary, tertiary, or quaternary structure. For example, in some embodiments, a binding molecule binds to a group of amino acids, regardless of whether the amino acids are folded into a native three-dimensional protein structure. In other embodiments, the binding molecule requires that the amino acid residues that make up the epitope exhibit a particular conformation (eg, a bend, twist, turn, or fold) in order to recognize and bind to the epitope.

[0048] An "intact" antibody is one that contains an antigen-binding site as well as a CL and at least a heavy chain constant region, CH1, CH2, CH3. The constant region may include a human constant region or an amino acid sequence variant thereof. In certain embodiments, an intact antibody has one or more effector functions.

[0049] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising a VH antibody domain and a VL antibody domain connected in a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding. For a description of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0050] As used herein, a "single domain antibody" or "sdAb" refers to a single monomeric variable antibody domain capable of antigen binding. Single domain antibodies include VHH domains as described herein. Examples of single domain antibodies include, but are not limited to, antibodies naturally lacking light chains, such as those from Camelidae species (e.g., llamas), single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be derived from any species, including, but not limited to, mouse, human, camel, llama, goat, rabbit, and cow. For example, single domain antibodies may be derived from antibodies produced in Camelidae species, such as camel, llama, dromedary, alpaca, and guanaco, as described herein. Other species outside of Camelidae may also produce heavy chain antibodies that do not naturally have light chains. VHHs derived from such other species are within the scope of this disclosure. In some embodiments, the single domain antibodies (e.g., VHH) provided herein have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Single domain antibodies may be genetically fused or chemically conjugated to another molecule (e.g., a drug) as described herein. Single domain antibodies may be part of a larger binding molecule (e.g., a multispecific antibody or a functional exogenous receptor).

[0051] The term "binding" or "binding" refers to interactions between molecules, including, for example, forming a complex. The interactions can be non-covalent interactions, including, for example, hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single antigen-binding site of an antibody and a single epitope of a target molecule, such as an antigen, is the affinity of the antibody or functional fragment for that epitope. The ratio (koff / kon) of the dissociation rate (koff) and the association rate (kon) of a binding molecule (e.g., an antibody) to a monovalent antigen is the dissociation constant KD, which is inversely related to affinity. The lower the KD value, the higher the affinity of the antibody. The value of KD varies for different complexes of antibody and antigen and depends on both kon and koff. The dissociation constant KD of the antibodies provided herein can be determined using any method provided herein or any other method known to one of skill in the art. Affinity at one binding site does not necessarily reflect the true strength of the interaction between the antibody and the antigen. When a complex antigen containing multiple repeating antigenic determinants, such as a multivalent antigen, comes into contact with an antibody containing multiple binding sites, the interaction of the antibody with the antigen at one site will increase the probability of reaction at a second site. The strength of multiple interactions between such a multivalent antibody and the antigen is called avidity.

[0052] As used herein, the term "body fluid" or "bodily fluid" refers to a fluid obtained from a patient, such as a mammalian (e.g., human) patient. For example, the body fluid can be blood, cerebral spinal fluid (CSF), breast milk, or urine. The body fluid can also be blood that has been fractionated to remove cells (i.e., plasma) or blood that has been fractionated to remove cells and clotting factors (i.e., serum).

[0053] As used herein, the term "capture moiety" or "first antibody" refers to a composition that can be specifically bound by another composition immobilized, e.g., bound, or otherwise linked to a solid support. Many of the detection moieties provided herein can also be used as capture moieties, so long as a binding event is involved. For example, useful capture moieties include affinity labels for which specific and selective ligands are available (e.g., biotin and avidin, glutathione and GST), haptens and proteins for which antisera or monoclonal antibodies are available (e.g., c-Myc), nucleic acid molecules with sequences complementary to the target, and peptides for which specific and selective ligands are available (e.g., histidine tags and Ni). Molecules that affect the binding properties to a chromatographic resin are also contemplated. The solid support can be, for example, a filter, a plate, a membrane, a chromatographic resin, or a bead.

[0054] As used herein, the term "cut-point factor" or "threshold" generally refers to a value used to mathematically manipulate the signal from a naive pool matrix (e.g., serum or plasma) to set the minimum signal from a sample required to be considered positive.

[0055] The term "derivative," as used in reference to antibody agents and polypeptides used in the methods provided herein, refers to polypeptides that have been chemically modified by techniques including, but not limited to, ubiquitination, conjugation to therapeutic or diagnostic agents, labeling (e.g., with radionuclides or various enzymes), covalent polymer attachment such as pegylation (i.e., derivatization with polyethylene glycol), and chemically synthesized insertion or substitution of amino acids such as ornithine that do not normally occur in human proteins. Derivatives can retain the binding properties of the non-derivatized molecule.

[0056] As used herein, the terms "detectable moiety," "detection moiety," or "label" refer to a composition (e.g., a polypeptide or antibody) that is detectable by means including, but not limited to, spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful detectable moieties or labels include ruthenium (Ru)-based catalysts, europium, 32 P, 35 Detectable moieties or labels include, for example, fluorescent dyes, electron-dense reagents, enzymes (such as those commonly used in ELISA), biotin-streptavidin, dioxygenin, haptens and proteins for which antisera or monoclonal antibodies are available, and nucleic acid molecules having a sequence complementary to a target. Detectable moieties or labels often generate a measurable signal, such as a radioactive, colorimetric, luminescent, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety or label in a sample.

[0057] Examples of labels that may be used in the present invention include fluorophores, chromophores, electrochemiluminescent labels, bioluminescent labels, polymers, polymeric particles, beads or other solid surfaces, gold or other metal particles or heavy atoms, spin labels, radioisotopes, enzyme substrates, haptens, antigens, quantum dots, aminohexyl, pyrene, nucleic acids or nucleic acid analogs, or proteins such as, for example, receptors, peptide ligands or substrates, enzymes, and antibodies (including antibody fragments).

[0058] Some labels according to the present invention include "color labels" in which a target is detected by the presence or absence of color or a color change in a sample. Examples of "color labels" are chromophores, fluorophores, chemiluminescent compounds, electrochemiluminescent labels, bioluminescent labels, and enzymes that catalyze a color change in a substrate. In some embodiments, more than one type of color may be used, for example, by attaching distinguishable color labels to a single detection unit, or by using two or more detection units, each carrying a different distinguishable color label.

[0059] A "fluorophore" as described herein is a molecule that emits detectable electromagnetic radiation when excited with electromagnetic radiation of one or more wavelengths. A wide variety of fluorophores are known in the art and have been developed by chemists for use as labels and can be conjugated to the linkers of the present invention. Examples include fluorescein or its derivatives, such as fluorescein-5-isothiocyanate (FITC), 5-(and 6)-carboxyfluorescein, 5- or 6-earboxyfluorescein, 6-(fluorescein)-5-(and 6)-carboxamidohexanoic acid, fluorescein isothiocyanate, rhodamine or its derivatives, such as tetramethylrhodamine and tetramethylrhodamine-5-(and -6)-isothiocyanate (TRITC).Other exemplary fluorophores that may be conjugated to the linkers of the invention include coumarin dyes such as (diethyl-amino)coumarin or 7-amino-4-methylcoumarin-3-acetic acid, succinimidyl ester (AMCA); sulforhodamine 101 sulfonyl chloride (TexasRed™ or TexasRed™ sulfonyl chloride; 5-(and-6)-carboxy-X-rhodamine, also known as succinimidyl ester (CXR); Lissamine or Lissamine derivatives such as 5-(and-6)-carboxyrhodamine 101, succinimidyl ester, Lissamine rhodamine B sulfonyl chloride (LiSR); 5-(and-6)-carboxyfluorescein succinimidyl ester; ester (CFI); fluorescein-5-isothiocyanate (FITC); 7-diethylaminocoumarin-3-carboxylic acid, succinimidyl ester (DECCA); 5-(and-6)-carboxytetramethylrhodamine, succinimidyl ester (CTMR); 7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester (HCCA); 6-(fluorescein-5-(and-6)-carboxamidohexanoic acid (FCHA); N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid, succinimidyl ester; also known as 5,7-dimethylBODIPY™ propionic acid succinimidyl ester (DMBP), Molecular "Activated fluorescein derivative" (FAP) available from Probes, Inc.; eosin-5-isothiocyanate (EITC); erythrosine-5-isothiocyanate (ErITC); and Cascade™ Blue acetyl azide (CBAA) (the 0-acetyl azide derivative of 1-hydroxy-3,6,8-pyrenetrisulfonic acid).Further potential fluorophores useful in the present invention include fluorescent proteins such as green fluorescent protein and analogs or derivatives thereof, fluorescent amino acids such as tyrosine and tryptophan and their analogs, fluorescent nucleosides, and other fluorescent molecules such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, IR dyes, Dyomics dyes, phycoerythrin, Oregon Green 488, Pacific Blue, Rhodamine Green, and Alexa dyes. Further examples of fluorescent labels that may be used in the present invention include inorganic fluorescent labels such as conjugates of R-phycoerythrin or aliiophycoerythrin, particles based on semiconducting materials such as coated CdSe nanocrystals.

[0060] Many of the above fluorophores, as well as others, are commercially available from companies such as Molecular Probes, Inc. (Eugene, OR), Pierce Chemical Co. (Rockford, IL), or Sigma-Aldrich Co. (St. Louis, MO).

[0061] Examples of polymer particle labels which may be used in the present invention include polystyrene, PMMA or silica microparticles, beads or latex particles which can be embedded with fluorescent dyes, or polymer micelles or capsules containing dyes, enzymes or substrates.

[0062] Examples of metal particles that may be used in the present invention include gold particles and coated gold particles, which can be transformed by silver staining.

[0063] Examples of haptens that may be conjugated in some embodiments are fluorophores, myc, nitrotyrosine, biotin, avidin, streptavidin, 2,4-dinitrophenyl, digoxigenin, bromodeoxyuridine, sulfonates, acetylaminofluorene, mercury trinitrophenol, and estradiol.

[0064] Examples of enzymes that may be used in the present invention include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (GAL), glucose-6-phosphate dehydrogenase, β-N-acetylglucosaminidase, β-glucuronidase, invertase, xanthine oxidase, firefly luciferase and glucose oxidase (GO).

[0065] Examples of commonly used substrates for horseradish peroxidase (HRP) include 3,3'-diaminobenzidine (DAB), diaminobenzidine with nickel enhancement, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC), Hanker-Yates reagent (HYR), indophane blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), α-naphtholpyronine (α -NP), o-dianisidine (OD), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), nitro blue tetrazolium (NBT), 2-(p-iodophenyl)-3-p-nitrophenyl-5-phenyl tetrazolium chloride (INT), tetranitro blue tetrazolium (TNBT), 5-bromo-4-chloro-3-indoxyl-β-D-galactoside / ferro-ferricyanide (BCIG / FF).

[0066] Examples of commonly used substrates for alkaline phosphatase include naphthol-AS-B1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / new fuchsin (NABP / NF), bromochloroindolyl phosphate / nitro blue tetrazolium (BCIP / NBT), 5-bromo-4-chloro-3-indolyl-b(beta)-d(delta)-galactopyranoside (BCIG).

[0067] Examples of luminescent labels that may be used in the present invention include luminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines. Examples of electrochemiluminescent labels include ruthenium derivatives.

[0068] Examples of radiolabels that may be used in the present invention include radioactive isotopes of iodide, cobalt, selenium, hydrogen, carbon, sulfur and phosphorus.

[0069] As used herein, the term "detectable antibody" refers to any antibody that can be detected. In some embodiments, the antibody is directly labeled with a detectable moiety. In certain embodiments, the antibody is a detectable anti-Ig antibody. As used herein, the term "detectable anti-Ig antibody" refers to an anti-Ig antibody that can be detected. In some embodiments, the anti-Ig antibody is directly labeled with a detectable moiety in addition to its inherent binding to an Ig molecule. The Ig antibody can be, for example, of the IgG, IgE, IgM, IgD, IgA, or IgY isotype.

[0070] As used herein, the term "primary antibody" refers to an antibody that directly binds to an antigen of interest. As used herein, the term "secondary antibody" refers to an antibody that is conjugated to a detection label. In some embodiments, the secondary antibodies provided herein directly bind to the primary antibody. In other embodiments, the secondary antibodies provided herein indirectly bind to the primary antibody, for example, by binding to another antibody that recognizes the primary antibody.

[0071] As used herein in the context of a peptide or polypeptide, the term "fragment" refers to a peptide or polypeptide that comprises less than the full-length amino acid sequence. Such fragments can result, for example, from truncations at the amino terminus, truncations at the carboxy terminus, and / or internal deletion of a residue(s) from the amino acid sequence. Fragments can result, for example, from alternative RNA splicing or in vivo protease activity. In certain embodiments, a fragment comprises a polypeptide comprising an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, at least 200 contiguous amino acid residues, or at least 250 contiguous amino acid residues of an amino acid sequence of an antibody that immunospecifically binds to a target antigen. In certain embodiments, an antibody fragment that immunospecifically binds to a target antigen retains at least one, at least two, or at least three functions of an antibody.

[0072] The term "identical" or percent "identity" in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same nucleotide or amino acid residues when compared and aligned for maximum correspondence, as determined using a sequence comparison algorithm or by visual inspection.

[0073] The term "antibody that immunospecifically binds" to a target antigen and similar terms are used interchangeably herein and refer to antibodies and fragments thereof that specifically bind only to a target antigen or epitope. In yet other embodiments, the antibodies provided herein immunospecifically bind to Ig, such as IgG, IgE, IgM, IgD, IgA isotypes.

[0074] As used herein, the term "interference" generally refers to the presence of a substance in a bodily fluid (e.g., serum or plasma) sample that prevents accurate detection and measurement of a target analyte. As used herein, interference generally refers to the effect of free drug or the effect of the matrix (e.g., serum or plasma) on the concentration-response relationship. For example, interference from the matrix may be evaluated relative to a sample without potential interference to target a range of relative accuracy of 75-125%.

[0075] The term "in vivo" in the context of a sample refers to a biological sample, e.g., a sample obtained from a subject, e.g., a patient, e.g., a human patient, including a biological or bodily fluid, e.g., blood, plasma, serum, bone marrow, cerebrospinal fluid, cerebral fluid, or tissue, e.g., lymphatic tissue, thin layer cytological samples, fresh frozen tissue samples, or tumor tissue. The term "in vivo" should be distinguished from the term "in vitro," which includes cells or cell lines cultured or grown outside a living organism, or biomolecular components of cells.

[0076] The terms "limit of detection," "LOD," or "sensitivity," as used herein, generally refer to the lowest analyte concentration in a bodily fluid (e.g., serum or plasma) sample that can be detected but not necessarily quantified as an exact value. For example, the LOD can be defined as the analyte concentration that consistently produces a signal greater than the measured mean response of the pooled naive matrix plus the cutpoint coefficient.

[0077] As used herein, the term "matrix" or "matrices" generally refers to the biological background in which an antibody is measured. Examples of matrices include, for example, body fluids and tissues.

[0078] The term "monoclonal antibody" refers to an antibody obtained from a homogeneous or substantially homogeneous population of antibodies, with each monoclonal antibody typically recognizing a single epitope on an antigen. In certain embodiments, a "monoclonal antibody" as used herein is an antibody produced by a single hybridoma or other cell. The term "monoclonal" is not limited to a particular method for making the antibody. For example, the monoclonal antibodies used in the methods provided herein can be made by the hybridoma method described in Kohler et al.; Nature, 256:495 (1975) or isolated from a phage library using techniques known in the art. Other methods for the preparation of clonal cell lines and the monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

[0079] As used herein, "polyclonal antibody" refers to an antibody population generated in an immunogenic response to a protein with many epitopes, and thus includes a variety of different antibodies directed to the same and different epitopes within the protein. Methods for producing polyclonal antibodies are known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York).

[0080] As used herein, the term "precision" generally refers to the variability of the signal between analysts and days. For example, precision can be assessed as the coefficient of variation, range of values, or using ANOVA statistics.

[0081] As used herein, the terms "prevent," "preventing," and "prevention" refer to the total or partial inhibition of the occurrence, recurrence, development, or spread of a disease and / or its associated symptoms (e.g., a disease or its associated symptoms associated with elevated phenylalanine levels, such as PKU or cancer in a patient) resulting from the administration of a therapy or combination of therapies provided herein.

[0082] As used herein, the term "reagent stability" generally refers to the robustness of a reagent preparation and storage stability. For example, reagent stability may be established by conditions that still allow values ​​to be measured within 75-125% accuracy for a freshly prepared reagent.

[0083] As used herein, the term "robustness" generally refers to the ability of an assay to remain unaffected by small variations in method parameters and indicates the reliability of the assay during normal running conditions. For example, robustness can be assessed as the percent change in reagent concentration, reagent volume, or incubation time that still produces a signal within 75-125% accuracy relative to nominal conditions.

[0084] As used herein, the term "sample" generally refers to a test fluid or tissue, e.g., taken from a patient, that can be used in the methods provided herein. In some embodiments, the sample is an in vivo sample, e.g., a bodily fluid (or biological fluid) from a subject, e.g., a patient, e.g., a human patient. Non-limiting examples of such bodily fluids include blood (e.g., human peripheral blood (HPB)), blood lysate, serum, plasma, fine needle aspirate, ductal lavage, cerebrospinal fluid, brain fluid, bone marrow, ascites, or any combination thereof. In other embodiments, the sample is taken from a biopsy tissue, such as a tumor tissue from a subject, or a thin layer cytological sample of other body tissues or organs. In certain embodiments, the sample comprises a peripheral blood sample, a tumor tissue or suspected tumor tissue, a thin film cytology sample, a fine needle aspirate sample, a bone marrow sample, a lymph node sample, a urine sample, a peritoneal fluid sample, a lavage sample, an esophageal brushing sample, a bladder or lung lavage sample, a cerebrospinal fluid sample, a brain fluid sample, a ductal aspirate sample, a breast secretion sample, a pleural effusion sample, a fresh frozen tissue sample, a paraffin-embedded tissue sample, In other embodiments, the sample is an extract from or a processed sample produced from any of a peripheral blood sample, a tumor tissue or suspected tumor tissue, a thin film cytology sample, a fine needle aspirate sample, a bone marrow sample, a urine sample, a peritoneal fluid sample, a lavage sample, an esophageal brushing sample, a bladder or lung lavage sample, a cerebrospinal fluid sample, a brain fluid sample, a ductal aspirate sample, a breast secretion sample, a pleural effusion sample, a fresh frozen tissue sample, or a paraffin-embedded tissue sample.

[0085] As used herein, the term "specificity" generally refers to the ability of an assay to detect an antibody that reacts with a specific protein.For example, specificity can refer to a proportional detection response with a specific analyte, while the response to non-specific proteins should be less than LOD.Proportional response can be evaluated against a correlation coefficient R value of 0.98 or more.When used in connection with the method provided herein for detecting a target antigen, specificity refers to the ability to detect an antigen that reacts with a specific protein.

[0086] As used herein, the terms "subject" and "patient" are used interchangeably. As used herein, a subject is preferably a mammal, such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans), most preferably a human. In one embodiment, the subject is a mammal, preferably a human. In some embodiments of the methods and kits provided herein, the patient has a disease or condition, or cancer. In other embodiments of the methods and kits provided herein, the patient is a patient undergoing cancer therapy. In yet other embodiments of the methods and kits provided herein, the patient is a pregnant woman or an infant (e.g., 0 to about 36 months of age).

[0087] As used herein, the terms "tag" and "label" are used interchangeably and refer to any type of moiety attached to an antibody or antigen-binding fragment thereof, or other polypeptide used in the methods provided herein. The term "detectable" or "detection" with respect to an antibody or tag refers to any antibody or tag that can be visualized, or the presence of the antibody or tag can be otherwise determined and / or measured (e.g., by quantification). Non-limiting examples of detectable tags include fluorescent or other chemiluminescent tags, and tags that can be amplified and quantified using PCR. In certain embodiments, the secondary antibody used in the methods provided herein is a biotinylated secondary antibody used in combination with labeled streptavidin.

[0088] As used herein, the term "therapy" refers to any protocol, method, and / or agent that may be used in the prevention, management, treatment, and / or amelioration of a disease (or symptoms associated therewith) or cancer. In certain embodiments, the terms "therapies" and "therapy" refer to biologic, supportive, and / or other therapies useful in the prevention, management, treatment, and / or amelioration of a disease or cancer known to one of skill in the art, such as a medical practitioner.

[0089] As used herein, the term "tissue" refers to tissue obtained from a mammal, such as a human. For example, the tissue may be from a biopsy sample, surgically removed tissue, or a post-mortem collection. Additionally, the tissue may be homogenized and extracted to isolate enzymes or antibodies from the tissue.

[0090] As used herein, the terms "treat," "treatment," and "treating" refer to the reduction or amelioration of the progression, severity, and / or duration of a disease (or its associated symptoms) or cancer resulting from the administration of one or more therapies.

[0091] As used herein, the term "variant" refers to a polypeptide sequence that contains at least one amino acid substitution, deletion, or insertion in the coding region relative to the original polypeptide coding domain. Variants retain the biological activity of the naturally occurring polypeptide.

[0092] As used herein, the term "in situ hybridization" or "ISH" refers to a technique for localizing and visualizing specific target nucleic acids while preserving the morphology of the source sample.

[0093] As used herein, the term "immunohistochemistry" or "IHC" refers to a technique for detecting a protein of interest in a source sample utilizing antibodies while preserving the morphology of the source sample. Immunofluorescence (IF) refers to fluorescent labeling and is therefore also encompassed by the term IHC.

[0094] As used herein, the term "crosslinking" refers to the process of linking two or more molecules together. A "crosslinker" or equivalent refers to an agent that contains two or more chemically reactive termini that attach themselves to functional groups found on proteins and other molecules. Specifically, when the crosslinker is formaldehyde or its equivalent, the nucleophilic group on an amino acid or nucleic acid base forms a covalent bond with the formaldehyde, which is often stabilized in a second step with another functional group on another molecule, resulting in the formation of a methylene bridge. When the crosslinker is an oxidizing agent, it can react with the side chains of proteins and other biomolecules, allowing the formation of crosslinks that stabilize tissue structures.

[0095] As used herein, the term "fixation" or "fixing", when performed with respect to fixation of a sample in an IHC process, refers to a procedure to protect the sample from decay, for example by autolysis or putrefaction, which may terminate any ongoing biochemical reactions and also increase the mechanical strength or stability of the processed tissue.

[0096] As used herein, the term "detecting" generally refers to any form of measurement, including determining whether an element is present or not. The term includes quantitative and / or qualitative determinations.

[0097] IHC methods for detecting BCMA Immunohistochemistry (IHC) on formalin-fixed paraffin-embedded (FFPE) tissues is a critical step in R&D therapeutic campaigns by identifying cells expressing target proteins of interest and predicting potential toxicity. A robust IHC assay relies on a suitable primary antibody that reliably recognizes the target with optimal specificity and sensitivity. FFPE tissues often present conformationally altered overfixed proteins, which makes repurposing of antibodies validated in non-IHC assays highly uncertain. If crosslinking agents such as formalin are used during the tissue preparation and preservation process, formalin fixation may mask epitopes and result in reduced immunoreactivity (see Arnold et al., Biotech Histochem 71:224-230 (1996)). Formalin fixation is a time-dependent process in which increasing fixation times result in continued binding of formaldehyde groups to proteins up to an equilibrium point (see Fox et al., J Histochem Cytochem 33:845-853 (1985)). Studies have shown that formalin fixation, especially if prolonged, reduces antigenicity (see Battifora and Kopinski, J Histochem Cytochem 34:1095-1100 (1986)), limiting the use of formalin-fixed tissues for diagnostic IHC (see Ramos-Vara, Vet Pathol 42:405-426 (2005); Webster et al., J Histochem Cytochem. 57(8):753-761 (2009)). The generation of new IHC antibodies requires screening many candidates against relevant controls when suitable reagents are not commercially available.

[0098] In one aspect, provided herein is a method of detecting BCMA in a formalin-fixed paraffin-embedded ("FFPE") sample. In some embodiments, the method includes (1) sectioning and mounting the FFPE sample, (2) deparaffinizing the sample, (3) heating the sample to mediate antigen retrieval, (4) pretreating the sample with a blocking agent, (5) contacting the sample with a first detection agent, (6) contacting the sample bound to the first detection agent with a second detection agent, and (7) detecting the presence of unbound second detection agent bound to the sample.

[0099] In another embodiment, the method includes (1) sectioning and mounting an FFPE sample, (2) deparaffinizing the sample, (3) heating the sample to mediate antigen retrieval, (4) pretreating the sample with a blocking agent, (5) contacting the sample with a first detection agent, (6) removing unbound sample, (7) contacting the sample bound to the first detection agent with a second detection agent, (8) removing unbound second detection agent, and (9) detecting the presence of unbound second detection agent bound to the sample.

[0100] In some embodiments, BCMA detected by the methods of the present disclosure includes any BCMA variants, isoforms, and interspecies homologs that can be naturally expressed by cells (including B cells) or expressed on cells transfected with a gene or cDNA encoding the polypeptide. Unless otherwise stated, preferably, the BCMA is human BCMA.

[0101] In some embodiments, the sample used in the methods of the present disclosure comprises cells collected from bodily fluids or tissues. In some embodiments, the sample may comprise any tissue found in an organism. In some embodiments, the organism comprises a vertebrate or an invertebrate. In some embodiments, the vertebrate comprises a human or a non-human primate. In some embodiments, the monkey comprises a cynomolgus monkey (Macaca fascicularis), a rhesus monkey (Macaca mulatta), a marmoset (Callithrix jacchus), a chimpanzee (Pan troglodytes), a bonobo (Pan paniscus), a Bornean orangutan (Pongo pygmaeus), a Sumatran orangutan (Pongo abelii), a Tapanuliensis, or any other known primate species. In some embodiments, the tissue comprises a tumor tissue. In some embodiments, the tumor tissue comprises a benign tumor. In another embodiment, the tumor tissue comprises a pre-malignant tumor. In another embodiment, the tumor tissue comprises a malignant tumor. A non-exhaustive list of exemplary tumor tissues contemplated by the present disclosure includes sarcoma, carcinoma, adenocarcinoma, lymphoma, breast tumor, prostate tumor, head and neck tumor, brain tumor, pituitary tumor, glioblastoma, medulloblastoma, atypical teratoma / rhabdoid tumor, bladder tumor, pancreatic tumor, pancreatic islet tumor, liver tumor, ovarian tumor, colorectal tumor, lung tumor, bronchial tumor, tracheobronchial tumor, skin tumor, lymphatic tumor, and gastrointestinal tumor.

[0102] In further embodiments, the tissue may include brain tissue. The brain tissue may further include the striatal, thalamic, midbrain, or medulla regions of the brain.

[0103] In some embodiments, the samples used in the methods of the present disclosure are obtained from a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human or a non-primate human. In other embodiments, the mammal is a cynomolgus monkey (Macaca fascicularis). Samples may be collected by any method known in the art. Those skilled in the art will appreciate that collection procedures will vary depending on the type of sample and the intended analysis. For example, blood samples may be collected by an intravenous route using a vacuum tube system. Brain tissue may be collected by making an incision in the scalp, drilling a hole in the skull, and inserting a needle into the brain to obtain brain tissue.

[0104] In some embodiments of the present disclosure, tissue or cell samples may be fixed or embedded. Fixatives may be required, for example, to preserve cells and tissues in a reproducible and life-like manner. Fixatives may also stabilize cells and tissues, thereby protecting them from the harsh conditions of processing and staining techniques. For example, samples including tissue blocks, sections, or smears may be immersed in a fixative or, in the case of smears, dried.

[0105] Many methods of fixing and embedding tissue specimens are known, such as formalin fixation followed by paraffin embedding (FFPE). Any suitable fixative may be used. Examples include ethanol, acetic acid, picric acid, 2-propanol, 3,3'-diaminobenzidine tetrahydrochloride dihydrate, acetoin (mixture of monomers) and dimers, acrolein, crotonaldehyde (cis+trans), formaldehyde, glutaraldehyde, glyoxal, potassium dichromate, potassium permanganate, osmium tetroxide, paraformaldehyde, mercuric chloride, tolylene-2,4-diisocyanate, trichloroacetic acid, tungstic acid. Other examples include formalin (aqueous formaldehyde) and neutral buffered formalin, glutaraldehyde, carbodiimides, imidates, benzoequinone, osmic acid, and osmium tetroxide. Fresh biopsy specimens, cytological preparations (including touch preparations and blood smears), frozen sections, and tissues for IHC analysis may be fixed in organic solvents including ethanol, acetic acid, methanol, and / or acetone samples.

[0106] In some embodiments of the present disclosure, the method includes a pretreatment step to increase the reactivity or accessibility of target molecules in the sample while reducing non-specific interactions. This process is called antigen retrieval, also known as target retrieval, epitope retrieval, target unmasking, or antigen unmasking. See, for example, Shi et al., J Histochem Cytochem, 45(3):327 (1997). Antigen retrieval encompasses a variety of methods, including enzymatic digestion with proteolytic enzymes, such as proteinase, pronase, pepsin, papain, trypsin, or neuraminidase. In some embodiments, heat may be used, such as "heat-mediated antigen retrieval." Heating may include microwave irradiation, or a water bath, steamer, conventional oven, autoclave, or pressure cooker in a buffer stabilized to an appropriate pH. In some embodiments, heat-mediated antigen retrieval is performed using an EDTA-based solution.

[0107] In some embodiments, the EDTA-based solution has a pH of 8.0-9.0, 8.5-9.5, 9.0-10.0, 9.5-10.5, 10-11, 10.5-11.5, 12.0-13.0, 12.5-13.5, or 13.0-14.0. In other embodiments, the EDTA-based solution has a pH of 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, or 14.0. Additional pH stabilizing buffers may include EGTA, Tris-HCl, citric acid, urea, glycine-HCl, or boric acid.

[0108] In some embodiments, the heat-mediated antigen retrieval step is performed for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes. In other embodiments, the heat-mediated antigen retrieval step is performed at a temperature of 80°C to 120°C. In some embodiments, the heat-mediated antigen retrieval step is performed at a temperature of 80°C to 95°C, 85°C to 100°C, 90°C to 105°C, 95°C to 110°C, 100°C to 115°C, or 105°C to 120°C. In other embodiments, the heat-mediated antigen retrieval method is performed at 98°C, 99°C, 100°C, or 120°C. In certain embodiments, the heat-mediated antigen retrieval method is performed at 100°C. In some embodiments, any combination of the above antigen retrieval methods may be used.

[0109] In some embodiments of the present disclosure, the method includes blocking the sample with a blocking agent. Blocking agents known in the art include serum (bovine serum albumin), casein, gelatin, salmon sperm DNA, avidin / biotin, among others. In some embodiments, the blocking agent blocks endogenous peroxidase. Thus, the blocking agent may include a peroxide block. Blocking may be performed for a duration. In some embodiments, blocking with a peroxide block may be performed for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, or 20 minutes. In other embodiments, the blocking agent blocks non-specific antibody binding. Thus, the blocking agent may include Dako / Agilent Protein Block. In some embodiments, blocking with Dako / Agilent Protein Block may be performed for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, or 120 minutes. In some embodiments, blocking with Dako / Agilent Protein block may be performed for 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. It is understood that additional blocking agents may be used. Those skilled in the art will understand that the blocking agent and the time for blocking will depend on the tissue being treated.

[0110] The present disclosure is compatible with many known detection formats and their associated samples. For example, the present invention may be used in conjunction with immunoassays, protein detection assays, or nucleic acid hybridization assays, such as immunohistochemistry (IHC), immunocytochemistry (ICC), and in situ hybridization (ISH), among others, labeling on a surface or array. All of these detection assays are useful not only in research, but also in the detection and diagnosis of, for example, various diseases and conditions.

[0111] For example, IHC specifically provides a method to detect targets in a sample or tissue specimen in situ (see Mokry 1996, ACTA MEDICA 39:129). IHC maintains the overall cellular integrity of the sample, allowing detection of both the presence and location of targets of interest. Typically, samples are fixed in formalin, embedded in paraffin, and cut into sections for staining and subsequent examination by light microscopy. Current methods of IHC use either direct labeling or secondary antibody-based or hapten-based labeling. Examples of known IHC systems include, for example, EnVision™ (DakoCytomation), Powervision™ (Immunovision, Springdale, AZ), NBA™ kit (Zymed Laboratories Inc., South San Francisco, CA), HistoFine™ (Nichirei Corp, Tokyo, Japan). The present invention may allow for enhanced signal or increased flexibility in IHC detection platforms.

[0112] IHC, ISH and pathology techniques may be performed in matrices of tissues, cells and proteins that may be partially crosslinked and highly heterogeneous in nature. The rate of diffusion increases with increasing concentration and temperature, but decreases with molecular weight and size. Therefore, the physical size of the components is very important. For example, large molecules can be excluded from diffusing into parts of the sample, while components of small size may easily diffuse in and out of different compartments of the sample. In some embodiments, the units of the invention may be designed to be small in size, e.g., smaller than an antibody or a biotin-streptavidin complex, to improve target recognition and detection.

[0113] In some embodiments of the present disclosure, the method includes contacting the sample with a detection agent. As used herein, the term "detection agent" refers to a binding agent that can specifically bind to a target. Such binding agents include, for example, antibodies and ligands. Antibodies include full-length antibodies and functional fragments such as those exemplified above. Ligands include full-length polypeptides and functional binding fragments thereof such as those exemplified above. Ligands also include non-polypeptide ligands as exemplified above. When referring to specific binding to a target, the detection agent of the present disclosure can directly bind to the target or can be specific to the target by indirect means. In some embodiments, the sample is contacted with a first detection agent and a second detection agent. In some embodiments, the detection agent comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the sample is contacted with the first detection agent at room temperature. In some embodiments, the sample is contacted with the first detection agent at 4°C or 37°C. In some embodiments, the sample is contacted with the first detection agent for 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, 115 minutes, or 120 minutes. In some embodiments, the sample is contacted with the first detection agent for 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours. In some embodiments, the sample is contacted with the second detection agent at room temperature. In some embodiments, the sample is contacted with the second detection agent at 37° C. In other embodiments, the sample is contacted with the second detection agent at 4° C. In some embodiments, the sample is contacted with the second detection agent for 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, or 60 minutes.

[0114] In some embodiments, detection of above background an amount of second detection agent bound to the sample indicates the presence of at least one molecule of BCMA in the sample.

[0115] In some embodiments, a method for detecting BCMA" in FFPE samples comprises sectioning and mounting FFPE samples of colon, spleen, and lymph nodes from human and cynomolgus monkeys, deparaffinizing the samples; heating the samples at 100°C for 20 minutes in an EDTA-based solution at pH 9.0; pretreating the samples with endogenous peroxidase solution for 10 minutes and then pretreating the samples with Dako serum-free protein block for 10 minutes; contacting the samples with a rabbit monoclonal anti-BCMA antibody at a concentration of 7.8 μg / mL in antibody diluent for 30 minutes; washing the samples extensively; and contacting the samples bound to the first detection agent with a second detection agent comprising DAB.

[0116] ISH ISH involves contacting a sample (e.g., a slide-mounted cell or tissue sample) containing a target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) with a probe (i.e., a target nucleic acid probe as described above) that is specifically hybridizable or specific to the target nucleic acid sequence (e.g., a genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation. The slide is optionally pretreated, for example, to remove paraffin or other substances that may interfere with uniform hybridization. Both the chromosome sample and the probe are treated, for example, by heating to denature double-stranded nucleic acids. The probe (formulated in an appropriate hybridization buffer) and the sample are combined under conditions and for a sufficient time to allow hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess target nucleic acid probe, and detection of the specific label of the chromosomal target is performed. The probes and probe systems of the present disclosure can be used for nucleic acid detection, such as in situ hybridization procedures (e.g., fluorescent in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)). Hybridization between complementary nucleic acid molecules is mediated through hydrogen bonds, including Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonds, between complementary nucleotide units. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. If a nucleotide unit at a particular position of the probe of the present disclosure can hydrogen bond with a nucleotide unit at the same position of a DNA or RNA molecule (e.g., a target nucleic acid sequence), the oligonucleotides are complementary to each other at that position. A probe and a DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotide units that can hydrogen bond with each other and thus produce a detectable bond. A probe need not be 100% complementary to its target nucleic acid sequence (eg, a genomic target nucleic acid sequence) to be specifically hybridizable.However, sufficient complementarity is required so that the probe will bind, double-strand or hybridize only, or substantially only, to the target nucleic acid sequence when that sequence is present in a complex mixture (e.g., total cellular DNA or RNA).

[0117] In some embodiments of the present disclosure, detection is facilitated by hybridization of a detection agent to the target nucleic acid probe. The detection agent may be detected by direct detection or indirect detection. For example, in some direct detection embodiments, the detection agent is labeled with one or more fluorescent compounds, and the sample is analyzed by fluorescence microscopy or imaging. In some indirect detection embodiments, the detection agent comprises a plurality of detectable moieties, including a first member of a binding pair (i.e., a hapten or biotin), and the first member is detected by contacting the sample with a compound that includes a second member of the binding pair (i.e., an anti-hapten antibody or avidin) conjugated to a detectable moiety (i.e., a fluorescent dye or a quantum dot). For a general description of in situ hybridization procedures, see, for example, U.S. Pat. No. 4,888,278. Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Patent Nos. 5,447,841, 5,472,842, 5,427,932, and, for example, Pinkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988, and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described, for example, in Tanneret al., Am. J. Pathol. 157:1467-1472, 2000, and U.S. Patent No. 6,942,970. Further detection methods are provided in U.S. Patent No. 6,280,929. Exemplary procedures for detecting viruses by in situ hybridization can be found in Poddighe et al., J. Clin. Pathol. 49:M340-M344, 1996.

[0118] Numerous reagents and detection schemes can be used in combination with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As described above, detection agents labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS™) can be directly optically detected when performing FISH. Alternatively, detection agents can be labeled with non-fluorescent molecules such as haptens (including, but not limited to, the following: biotin, digoxigenin, DNP, and various oxazoles, pyrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenone, coumarins, coumarin-based compounds, podophyllotoxins, podophyllotoxin-based compounds, and combinations thereof), ligands, or other indirectly detectable moieties. Such non-fluorescent molecule-labeled detection agents (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., a cell or tissue sample to which the probes are bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for a selected hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT™) or another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., a secondary antibody or specific antibody) that can be labeled with a fluorophore. Optionally, the detectable label is directly attached to the antibody, receptor (or other specific binding agent). Alternatively, the detectable label is attached to the binding agent via a linker, such as a hydrazide thiol linker, a polyethylene glycol linker, or any other flexible binding moiety with comparable reactivity. For example, specific binding agents such as antibodies, receptors (or other antiligands), avidin, etc., can be covalently modified with a fluorophore (or other label) via a heterobifunctional polyalkylene glycol linker, such as a heterobifunctional polyethylene glycol (PEG) linker.Heterobifunctional linkers combine two different reactive groups, e.g., selected from a carbonyl-reactive group, an amine-reactive group, a thiol-reactive group, and a photoreactive group, the first of which binds to a label and the second of which binds to a specific binding agent.

[0119] In other examples, the detection agent or specific binding agent (e.g., an antibody such as a primary antibody, a receptor or other binding agent) comprises an enzyme capable of converting a fluorogenic or chromogenic composition into a detectable fluorescence, color, or other detectable signal (e.g., like the deposition of detectable metal particles in SISH). As noted above, the enzyme can be directly or indirectly attached to the associated probe or detection reagent via a linker. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linkers and binding chemistries) are described in U.S. Patent Application Publication Nos. 2006 / 0246524, 2006 / 0246523, and 2007011715.

[0120] It will be appreciated by those skilled in the art that by appropriate selection of labeled detection agents and / or labeled binding pairs, multiplex detection schemes can be created to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample, or on more than one cell or tissue sample). For example, a first detection agent corresponding to a first target nucleic acid probe can be labeled with a first hapten, such as biotin, and a second detection agent corresponding to a second target nucleic acid sequence can be labeled with a second hapten, such as DNP. After exposing the sample to the probe set, the bound probes can be detected by contacting the sample with a first specific binding agent (in this case avidin labeled with a first fluorophore, e.g., a first spectrally distinct QUANTUM DOT™ that emits at 585 nm) and a second specific binding agent (in this case an anti-DNP antibody or antibody fragment labeled with a second fluorophore, e.g., a second spectrally distinct QUANTUM DOT™ that emits at 705 nm). Additional probe / binding agent pairs can be added in a multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations, direct and indirect (one step, two step or more) can be envisioned, all of which are suitable in the context of the disclosed probes and assays.

[0121] Standard fluorescence microscopes are inexpensive tools for detecting reagents and probes that incorporate fluorescent compounds, such as quantum dot bioconjugates. Because quantum dot conjugates are substantially photostable, the microscope can be used over time to find an area of ​​interest and to focus well on the sample. Quantum dot conjugates are useful whenever bright, photostable emission is required, and are particularly useful in multicolor applications where only one excitation source / filter is available and minimal crosstalk between colors is required.

[0122] Treatment method In one aspect, provided herein is a method of treating a subject with a BCMA targeting agent, comprising detecting BCMA in a sample from the subject. In some embodiments, the method of treating a subject with a BCMA targeting agent comprises first detecting BCMA. Any of the methods disclosed herein may be used in the method of detecting BCMA.

[0123] In some embodiments, the targeting agent includes any targeting agent known in the art. As used herein, "targeting agent" refers to any molecule capable of interacting with a target of interest. Thus, in some embodiments, the targeting agent includes a chimeric antigen receptor (CAR) T cell or a T cell redirecting antibody. In other embodiments, the targeting agent includes an antibody or an antigenic fragment thereof. The antibody or an antigenic fragment thereof may include an anti-BCMA antibody or an antigenic fragment thereof. In further embodiments, the antibody may include a bispecific or multispecific antibody. The bispecific or multispecific antibody may include an anti-BCMA bispecific or multispecific antibody. In other embodiments, the targeting agent may include a small molecule capable of binding to BCMA. In other embodiments, the targeting agent may include a BCMA polypeptide.

[0124] In some embodiments, the target of interest is a soluble polypeptide. In other embodiments, the target of interest is a membrane-bound polypeptide that comprises a receptor. In some embodiments, the receptor comprises BCMA.

[0125] In some embodiments, the subject of the treatment methods of the invention has a disease, disorder, condition, or syndrome. In some embodiments, the disease, disorder, condition, or syndrome may be any human disease, disorder, condition, or syndrome known in the art. In some embodiments, the targeting agents of the present disclosure may also be used to treat diseases that express BCMA.

[0126] In some embodiments, the subject of the treatment methods of the invention has a cancer or tumor. In other embodiments, the cancer may include anal cancer, bile duct cancer, bladder cancer, bone cancer, breast cancer, cervical cancer, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, fallopian tube cancer, gallbladder cancer, gastric (stomach) cancer, head and neck cancer, liver cancer, hepatocellular carcinoma, Hodgkin's lymphoma, laryngeal cancer, leukemia, lung cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, melanoma, Merkel cell carcinoma (skin cancer), mesothelioma, malignant, paranasal sinus and nasal cavity cancer (head and neck cancer), parathyroid cancer, penile cancer, pharyngeal cancer (head and neck cancer), prostate cancer, rectal cancer, renal cell carcinoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, and vulvar cancer.

[0127] In further embodiments, examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, intraductal carcinoma in situ, and lobular carcinoma in situ. Examples of respiratory tract cancer include, but are not limited to, small cell lung cancer, non-small cell lung cancer, bronchial adenoma, and pleuropulmonary blastoma. Examples of brain cancer include, but are not limited to, brain stem and hypothalamic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumor. Tumors of the male reproductive system include, but are not limited to, prostate cancer and testicular cancer. Tumors of the female reproductive system include, but are not limited to, endometrial cancer, cervical cancer, ovarian cancer, vaginal cancer, vulvar cancer, and hysteroma. Gastrointestinal tumors include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, gastric cancer, pancreatic cancer, rectal cancer, small intestine cancer, and salivary gland cancer. Urethral tumors include, but are not limited to, bladder cancer, penile cancer, renal cell carcinoma, renal pelvis cancer, ureter cancer, and urethral cancer. Eye cancers include, but are not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancers include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without fibrolamellar variation), cholangiocarcinoma (intrahepatic cholangiocarcinoma), and mixed hepatocellular carcinoma-cholangiocarcinoma. Skin cancers include, but are not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell carcinoma, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, laryngeal / hypopharyngeal / nasopharyngeal / oropharyngeal cancer, and lip and oral cavity cancer. Lymphomas include, but are not limited to, AIDS-related lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma.

[0128] In some embodiments, the cancer may include a hematological cancer. A hematological cancer is a cancer of the blood or bone marrow. In some embodiments, a hematological (or hematologic) cancer of the present disclosure includes leukemia (acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous ... leukemia), myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (chronic myeloid leukemia, chronic granulocytic leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade forms), multiple myeloma, Waldenstrom's hypergammaglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, B-cell lymphoma, plasma cell leukemia, myelodysplasia, secondary leukemia, myeloproliferative syndrome (MPS) or Burkitt's lymphoma lymphoma) (endemic Burkitt lymphoma or sporadic Burkitt lymphoma), malignant plasma cell neoplasms, BCMA+ high-grade lymphoma, Kahler's disease and myelomatosis, plasma cell leukemia; plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, follicular lymphoma (including follicular non-Hodgkin's lymphoma types), marginal zone lymphoma (mucosa-associated lymphoid tissue: MALT 1 lymphoma; large cell lymphoma (diffuse large cell, diffuse mixed cell, immunoblastic lymphoma, primary mediastinal B cell lymphoma, hemocentric lymphoma pulmonary B cell); small lymphocytic lymphoma (SLL), precursor B lymphoblastic lymphoma; subacute myeloid leukemia, myeloid sarcoma, chloroma, granulocytic sarcoma, acute promyelocytic leukemia, acute myelomonocytic leukemia) or other B cell leukemias or lymphomas.

[0129] In some embodiments, the subject of the present disclosure has a plasma cell disorder, such as heavy chain disease, primary or immune cell-associated amyloidosis, and monoclonal gammopathy of undetermined significance (MGUS).

[0130] In some embodiments, the subject of the treatment method of the present invention has a disease or disorder. In some embodiments, the disease or disorder may include acquired immune deficiency syndrome (AIDS), brain disease, acute flaccid myelitis (AFM), amyotrophic lateral sclerosis (ALS), Alzheimer's disease, amyotrophic lateral sclerosis, arthritis, bone disease, inflammatory disease, osteoarthritis (OA), rheumatoid arthritis (RA), asthma, blood disorder, brain disease, dementia, diabetes, intestinal disease, liver disease, kidney disease, lung disease, skin disease, gastrointestinal disease, ulcerative colitis, inflammatory bowel disease, hypertension, or cardiovascular disorder. In other embodiments, the disease or condition may include a disease or disorder of the breast, airway, brain, reproductive organs, digestive tract, urethrae, eye, liver, skin, head and neck, thyroid and parathyroid.

[0131] In some embodiments, the subject of the treatment methods of the invention has an immunological disorder, particularly an autoimmune disorder, which in some embodiments includes systemic lupus erythematosus, myasthenia gravis, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, antiphospholipid syndrome, Chagas disease, Graves disease, Wegener's granulomatosis, polyarteritis nodosa, rapidly progressive glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus (SLE), type I diabetes, asthma, atopic dermatitis, allergic rhinitis, thrombocytopenic purpura, multiple sclerosis, psoriasis, Sjögren's syndrome, and the like. These conditions include, but are not limited to, Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis, Wegener's granulomatosis, tuberculosis, and graft-versus-host disease, immune-mediated thrombocytopenia, hemolytic anemia, bullous pemphigoid, myasthenia gravis, Graves' disease, Addison's disease, pemphigus foliaceus, psoriasis, psoriatic arthritis, and ankylosing spondylitis.

[0132] In some embodiments, the subject of the treatment method of the present invention has an inflammatory disease. In some embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, psoriasis, allergies, asthma, Crohn's disease, IBD, fibromyalgia, mastocytosis, autoimmune diseases such as celiac disease, and any combination thereof. Additionally, the method of the present invention may be useful for treating diabetes, particularly type 1 diabetes.

[0133] Embodiment The present invention provides the following non-limiting embodiments.

[0134] In one set of embodiments, the following is provided: 1. A method for detecting B-cell maturation antigen ("BCMA") molecules in a formalin-fixed paraffin-embedded ("FFPE") sample, comprising: a. Sectioning and mounting FFPE samples; b. Deparaffinizing the sample; c. Heat-mediated antigen retrieval of the sample; d. Pretreatment of the sample with a blocking agent; e. contacting the sample with a first detection agent, the first detection agent optionally comprising an antibody or antigen-binding fragment thereof that binds to at least one BCMA molecule in the sample; f. optionally, removing unbound sample; g. contacting the sample bound to the first detection agent with a second detection agent, which is optionally an antibody or fragment thereof; h. optionally, removing unbound second detection agent; i. detecting the presence of a second detection agent bound to the sample; Methods are provided wherein detection of an above background amount of the second detection agent bound to the sample indicates the presence of at least one BCMA molecule in the sample. 2. The method of embodiment 1, wherein heat-mediated antigen retrieval is carried out using an EDTA-based solution. 3. The method of embodiment 2, wherein the EDTA-based solution has a pH of 8.5 to 9.5. 4. The method of embodiment 3, wherein the EDTA-based solution has a pH of 9.0. 5. The method according to any one of the preceding embodiments, wherein the step of heat-mediated antigen retrieval is carried out for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or 60 minutes. 6. The method according to any one of the preceding embodiments, wherein the step of heat-mediated antigen retrieval is carried out at a temperature between 85°C and 100°C. 7. The method of embodiment 6, wherein the step of heat-mediated antigen retrieval is carried out at 100°C. 8. The method of any one of embodiments 1 to 7, wherein the blocking agent blocks endogenous peroxidase. 9. The method of embodiment 8, wherein the blocking agent is a peroxide blocker. 10. The method of embodiment 9, wherein the step of pretreating with a peroxide block is carried out for 2 minutes, 5 minutes, 8 minutes, 10 minutes or 15 minutes. 11. The method of any one of embodiments 1 to 10, wherein the blocking agent blocks non-specific antibody binding. 12. The method of embodiment 11, wherein the blocking agent is Dako / Agilent Protein Block. 13. The method of embodiment 12, wherein the step of pretreating with Dako / Agilent Protein Block is performed for 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes. 14. The method of any one of embodiments 1-13, wherein the first detection agent is an antibody or an antigen-binding fragment thereof. 15. The method of any one of embodiments 1 to 14, wherein the sample is contacted with the first detection agent at room temperature or at 37°C. 16. The method of any one of the preceding embodiments, wherein the sample is contacted with the first detection agent for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 95 minutes, or 120 minutes. 17. The method of any one of embodiments 1-16, wherein the second detection agent is an antibody or an antigen-binding fragment thereof. 18. The method of any one of the preceding embodiments, wherein the sample bound to the first detection agent is contacted with the second detection agent for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes. 19. The method of any one of embodiments 1 to 18, wherein the sample comprises cells from a body fluid or tissue. 20. The method of embodiment 19, wherein the tissue is brain tissue. 21. The method of embodiment 20, wherein the tissue is derived from the striatum, thalamus, midbrain, or medullary regions of the brain. 22. The method of embodiment 19, wherein the tissue is a tumor tissue. 23. The method of any one of embodiments 1 to 22, wherein the sample is derived from a subject. 24. The method of embodiment 23, wherein the subject is a mammal. 25. The method of embodiment 24, wherein the mammal is a human. 26. The method of embodiment 24, wherein the mammal is a cynomolgus monkey. 27. A kit for carrying out the method according to any one of embodiments 1 to 26. 28. A method of treating a subject with a BCMA targeting agent, comprising detecting BCMA in a sample from the subject. 29. The method of embodiment 28, wherein the step of detecting BCMA in the sample comprises a method according to any one of embodiments 1 to 22 or 24 to 26. 30. The method of embodiment 28 or 29, wherein the targeting agent is a chimeric antigen receptor (CAR) T cell. 31. The method of embodiment 28 or 29, wherein the targeting agent is a T cell redirecting antibody. 32. The method of any one of embodiments 28-31, wherein the subject has cancer. 33. The method of embodiment 32, wherein the cancer is multiple myeloma.

[0135] Specific embodiments of the present invention are described herein. Upon reading the foregoing description, it is expected that variations of the disclosed embodiments may become apparent to those skilled in the art, and such variations may be adopted as necessary. It is therefore intended that the present invention be practiced otherwise than as specifically described herein, and that the present invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto, as permitted by applicable law. Moreover, any combination of the above elements in all possible these variations is encompassed herein, unless otherwise noted herein or the context clearly contradicts. Many embodiments of the present invention have been described. However, it will be understood that various modifications may be made without departing from the spirit and scope of the present invention. Thus, the description in the Examples section is intended to illustrate, rather than limit, the scope of the invention as recited in the claims. EXAMPLES

[0136] The following is a description of various methods and materials used in the testing, which are set forth to provide those skilled in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure, nor are they intended to represent all of the experiments that have been performed and can be performed. It should be understood that the exemplary descriptions written in the present tense have not necessarily been performed, but rather the descriptions are those that can be performed to generate data and the like relevant to the teachings of the present disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental error and deviation should be taken into account.

[0137] Example 1: Development of immunochemical methods The antibodies screened are shown in Table 1.

[0138] [Table 1]

[0139] Generation of assay controls. Seven cell lines were selected according to their endogenous BCMA RNA expression levels based on RNA-seq data from publicly available CCLE (https: / / sites.broadinstitute.org / ccle / ) and Genentech (https: / / ega-archive.org / dacs / EGAC00001000055) datasets. The selected cell lines served as qualified reagent controls with negative, low, medium, and high levels of BCMA expression (Table 1). Because BCMA is part of a larger family of tumor necrosis factor receptors that includes BAFFR and TACI, which share ligands with BCMA, further studies were performed to confirm the lack of antibody cross-reactivity to potentially conserved epitopes. Engineered cell lines expressing TACI or BAFFR were generated by transfecting HEK293 parental cells with lentiviruses carrying human TACI or human BAFFR genes linked to a tag epitope. Cell lines were grown to 100% confluence, non-enzymatically harvested, and pelleted by centrifugation. Cell pellets were fixed in 10% neutral buffered formalin for 24 hours, routinely paraffinized, embedded and arrayed in a single paraffin block.

[0140] FFPE samples of colon, spleen, and lymph nodes were obtained as endogenous tissue controls for the presence of resident BCMA-expressing plasma cells. The same tissues from cynomolgus monkeys were used to confirm antibody cross-reactivity with this species. All tissues were quality controlled (QCed) for anatomical location, lack of histopathology, and suitability for IHC and ISH, and only samples that met the QC criteria were used in the experiments. To be considered suitable for IHC, samples had to show the immunolabeling pattern expected by synaptophysin IHC. Suitability for ISH was confirmed by abundant positive signal for the mRNA housekeeping gene, peptidyl prolyl isomerase B (PPIB), and lack of DapB (negative control probe) signal.

[0141] Screening of candidate IHC reagents. IHC assays were developed on a Leica Bond Rx automated stainer (Leica Biosystems, Buffalo Grove, IL). Table 1 summarizes the IHC candidate reagents considered for developing the final BCMA IHC assay. IHC reagents were tested with cell pellet reagent controls to assess specificity and sensitivity for BCMA protein. Of all reagents tested, only rabbit monoclonal anti-BCMA antibody clone [E6D7B] demonstrated acceptable IHC binding specificity and sensitivity. Therefore, anti-BCMA antibody clone [E6D7B] was used in the purification assay as the primary antibody for detection of endogenous expression of BCMA protein on tissue sections.

[0142] Assay conditions. The final IHC method used rabbit monoclonal anti-BCMA antibody clone [E6D7B] (Cell Signaling Technology #88183). FFPE blocks were sectioned at 4 μm and samples were mounted on SuperFrost Plus glass slides (VWR, Cat. No. 48311-703). Briefly, unfired glass slides were loaded into an autostainer and deparaffinized according to the general Leica deparaffinization protocol. Heat-mediated antigen retrieval with EDTA-based solution (pH approx. 9.0) was performed at 100°C for 20 min. Slides were then pretreated with endogenous peroxidase solution for 10 min. Dako serum-free protein block (Agilent catalog number X0909) was applied to the samples for 10 min immediately prior to a 30 min incubation with a 1 / 200 dilution of rabbit monoclonal anti-BCMA antibody clone [E6D7B] (final concentration 7.8 μg / mL in antibody diluent [Diagnostic Biosystem #K0004]). After extensive washing steps, bound primary antibodies were detected by a chromogenic Leica refine DAB detection kit (Leica catalog number DS9800) according to the manufacturer's recommendations. Slides were cover-slip mounted and examined under a brightfield microscope.

[0143] Study of human brain tissue for IHC and ISH. FFPE human normal brain tissue was commercially sourced from various vendors. Each brain tissue sample was checked for positional accuracy by H&E and IHC suitability by synaptophysin staining (rabbit monoclonal anti-synaptophysin antibody 1 / 8000 [Abcam#32127]). A total of 107 brain samples covering a wide range of brain loci were used in this study. Samples were derived from 63 different donors. A subset of samples was further evaluated for ISH suitability using PPIB (positive control probe).

[0144] FFPE cynomolgus monkey normal brain tissue from two sexually immature males and two sexually immature females was evaluated from coronal sections following a species-specific brain trimming scheme. Cynomolgus monkey tissue was included because this species is routinely studied in the preclinical safety of several biotherapeutics due to the lack of biotherapeutic cross-reactivity and pharmacological activity in other species. Standardized tissue sampling and processing protocols were followed to reduce preanalytical variables and maximize tissue sample quality.

[0145] Colocalization studies. Colocalization studies between IHC immunoreactivity observed in E6D7B and the Golgi apparatus and neurofibrillary tangle markers were performed by immunofluorescence. Evaluation of staining was completed using a Zeiss confocal microscope (Zeiss, White Plains, NY). The following antibody probes were included in the colocalization experiments: cis and trans Golgi markers, mouse monoclonal anti-TGN46 antibody 1 / 500 (LSBio #LS-C133654-100), mouse polyclonal anti-GOLGA2 antibody 1 / 100 (Abnova #H00002801-B01P), mouse monoclonal anti-GOLGA5 antibody 1 / 300 (NovusBio #NBP2-66875), and mouse monoclonal anti-GOLM1 antibody 1 / 100 (Sino Biological #13066-MM12). Each specific antibody was detected by the following secondary binders: goat polyclonal Alexa Fluor® 488 AffiniPure F(ab')2 fragment anti-mouse IgG(H+L) (Jackson ImmunoResearch Laboratories #115-546-146), and goat polyclonal Alexa Fluor® 594 AffiniPure F(ab')2 fragment anti-rabbit IgG(H+L) (Jackson ImmunoResearch Laboratories #111-586-144).

[0146] Birchowsky silver staining. To highlight neurofibrillary tangles, vital tissue samples were stained with Birchowsky silver staining (Abcam ab245877). The method was performed according to the manufacturer's recommendations. Briefly, 4 μm tissue sections were deparaffinized and hydrated, then incubated in silver nitrate solution at 40°C for 15 min, followed by ammoniacal silver solution for 10 min. The silver stain was developed in developer with agitation until desired coloration. Precipitated silver was fixed with 5% sodium thiosulfate for 2 min, and slides were dehydrated and mounted with Permount (Fisher Scientific, SP15-100) before digital capture. Detection of phosphorylated tau protein was also performed using a tangle marker probe: mouse monoclonal anti-pTau 1 / 100 (RnD Systems #MAB34941-100).

[0147] Example 2: Development of an RNA IN SITU Hybridization Assay Controls. Cell pellet arrays and human FFPE colon samples served as reagent controls as described above in Example 1. For each ISH staining assay, FFPE blocks were sectioned at 4 μm and samples were mounted on SuperFrost Plus glass slides.

[0148] Reagents. ISH assays were developed on a Leica Bond Rx autostainer (Leica Biosystems, Buffalo Grove, IL) using the following key reagents: mRNA detection probes including human BCMA specific probe (Hs-TNFRSF17, ACDBio Catalog No. 585791), human positive tissue PPIB control probe (Hs-PPIB, ACDBio Catalog No. 313908), and negative control probe DapB (ACDBio Catalog No. 312038). Hybridized probes were detected using RNAscope® 2.5 LSx Reagent Kit-Red (ACDBio Catalog No. 322750).

[0149] Assay conditions. The BCMA ISH assay was performed on a Leica Bond Rx autostainer. Briefly, slides were loaded into the autostainer, baked at 60°C for 30 min, and deparaffinized according to the general Leica deparaffinization protocol. Heat-mediated antigen retrieval with an EDTA-based solution (pH approx. 9.0) was performed at 85°C for cell pellet reagent controls or at 95°C for tissue samples for 15 min. Then, non-specific enzymatic digestion with proteinase K (provided in the ACDBio RNAscope® 2.5 LSx kit) was applied to each histological sample for 15 min at 40°C. Hybridization of specific probes was performed at 42°C for 120 min. After extensive washing, specifically bound probes were detected by a series of signal amplification steps. Finally, alkaline phosphatase enzymatic activity reacted with the chromogen, resulting in a red precipitate signal visible under a bright-field microscope.

[0150] ISH staining signals result in intracellular dot-like patterns. The number of positive dots per cell generally correlates with the amount of detectable mRNA transcripts present. The size of each dot preferentially depends on the overall probe set design. Evaluation of ISH signals was performed visually according to the manufacturer's guidelines. The minimum number of dots to consider a cell positive and its relationship to the correlative protein expression depends on each target of interest. Since there can be differences between mRNA and protein levels, the contrast characterization between ISH signal levels and IHC immunoreactivity intensity provides an acceptable indication of the ISH signal threshold to be considered relative to protein expression.

[0151] Preanalytical variables in tissue collection and processing, such as extended periods in ethanol, may affect the quality of mRNA that can be detected with the ISH method. PPIB is a housekeeping gene, and its ISH signal was used to assess the overall quality of the mRNA present in the FFPE samples (i.e., a QC check). Each cell pellet included in the control array showed an ISH PPIB signal well above the manufacturer's minimum recommended threshold of 3-4 dots per cell.

[0152] GTEx RNA-seq data. RNA-seq data from GTEx Analysis Release V8 were obtained from the dbGaP database (https: / / www.ncbi.nlm.nih.gov / projects / gap / cgi-bin / study.cgi?study_id=phs000424.v8.p2) and processed using Omicsoft Array Suite tools (https: / / www.arrayserver.com / wiki / ). Human genome organization GRCh38 and gene model GENCODE Release 33 (https: / / www.gencodegenes.org / human / ) were employed to map the RNA-seq sequences to the genome and quantify gene expression. A total of 2641 samples from 13 brain regions, including 246 caudate nucleus samples and 204 putamen samples, were selected for profiling BCMA expression in human brain. Transcripts per million ("TPM") was adopted as the unit of gene expression measurement.

[0153] Allen BrainSpan RNA-seq data. All gene expression data were downloaded from the Allen BrainSpan website (https: / / www.brainspan.org / ) using the link https: / / www.brainspan.org / api / v2 / well_known_file_download / 267666525. Data were converted from reads per million kilobases to TPM to allow for compatibility with GTEx data. BCMA expression was reported with samples grouped by brain region and developmental stage.

[0154] Aggregated RNA-seq data. To determine how expression of the BCMA gene in the human striatum changes with developmental stage, we extracted BCMA gene expression data in striatal samples from the Allen BrainSpan dataset and in caudate and putamen samples from the GTEx dataset. The combined data were plotted together and split by donor age group.

[0155] Example 3: IHC and ISH assay results IHC Assays. Cell pellet reagent control array staining results are summarized in Table 1 and Figures 1A-1R. Mouse monoclonal anti-BCMA antibody clone [1004023] (RnD Systems, Catalog No. MAB1931), mouse monoclonal anti-BCMA antibody clone [clone 19F2] (BioLegend, Catalog No. 357502), and rat monoclonal anti-BCMA antibody clone [Vicky-1] (Novus Biologicals, Catalog No. NBP1-97637SS) did not produce acceptable specificity and sensitivity for expressed BCMA protein or with control cell pellets (data not shown). Rabbit monoclonal anti-BCMA antibody clone [E6D7B] produced the expected pattern of BCMA-specific immunoreactivity (i.e., membrane and / or Golgi-like pattern) in cell pellet controls (Figures 1A-1R). H929 and MM1.R cells showed prominent positive membrane staining and prominent Golgi-like staining patterns in their cytoplasm. JEKO-1 and Raji cells showed low to very low intensity staining observable in Golgi-like structures mostly present in the cytoplasm of the cells. As expected, no immunoreactivity was observed with E6D7B in BCMA-negative cell lines (K562, U937 and HEK293). BCMA immunoreactivity produced by clone [E6D7B] was orthogonally confirmed using an ISH assay specific for detecting BCMA mRNA. HEK293-BAFFr and HEK293-TACI cell lines overexpressing the closely related targets BAFFR and transmembrane activator and interactor of CAML (TACI) did not show any BCMA immunoreactivity, further demonstrating the specificity of E6D7B for BCMA.

[0156] In tissue sections, specific positive labeling was identified in cells with morphology consistent with plasma cells in human lymph node, spleen, and colon samples. Similar to the BCMA positive cell pellet reagent control, plasma cells displayed strong membrane staining and Golgi-like positive structures when present in the sectioned sections. The relative intensity of positive cells in lymph node and spleen was less than that observed in colon tissue samples. Evidence of cross-reactivity with cynomolgus monkey tissue samples was identified in similar anatomical locations. The overall intensity of positive BCMA IHC cells was less in cynomolgus monkeys than that observed in humans. Cynomolgus monkey colon showed a higher density of BCMA positive cells than human colon. The exact peptide immunogen sequence used to generate the rabbit monoclonal anti-BCMA antibody clone [E6D7B] is proprietary to Cell Signaling Technology (CST). CST has published that the monoclonal antibody was produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu115 of the human TNFRSF17 / BCMA protein. Bioinformatic alignment of the cytoplasmic portions (amino acid residues 78-184) of the human and cynomolgus BCMA proteins showed 92.5% sequence identity between the cynomolgus and human proteins (BLAST-P, NIH).

[0157] ISH assay. BCMA-specific ISH staining was identified in cell lines displaying a range of endogenous expression, from low to high BCMA mRNA fragments per kilobases per million (FPKM) scores (Table 1, Figures 1A-1R). Cell lines with high BCMA FPKM scores (H929 and MM1.R) showed 15 or more dots per cell. In cells with lower FPKM scores (JEKO-1 and Raji), 5 or 1 dot per cell were observed, respectively. As expected, the BCMA ISH assay failed to generate a specific signal in cell lines (K562, U937, and HEK293) displaying extremely low or 0 FPKM scores.

[0158] A direct correlation between BCMA ISH signal and BCMA IHC immunoreactivity in the same control cell pellets was visually estimated (Table 1, Figures 1A-1R). In FFPE cell pellets, an acceptable agreement between the intensity of BCMA IHC immunoreactivity and BCMA ISH signal was observed. Not all Raji cells showed BCMA ISH positivity in the sectioned sections, with positive cells showing mainly one dot and rarely two dots. For that cell line, the overall density of positive BCMA ISH signal was consistent with that observed in the BCMA IHC assay and reached the threshold of positivity as determined by the scoring recommended by the manufacturer (https: / / www.indicalab.com / wp-content / uploads / 2018 / 04 / MK_51_103_RNAScope_data_analysis_guide_RevB.pdf). Although ISH signals were occasionally present in the negative control cell lines (K562, U-937, and HEK293), the signals were rare and did not reach the threshold of one dot per 10 cells.

[0159] In human normal colon tissue sections (positive control), specific positive BCMA ISH labeling was identified in cells with morphology consistent with plasma cells. Most of these cells showed low levels of BCMA ISH signal (1 dot / cell and occasionally 2-3 dots / cell). Groups of plasma cells showing a single BCMA ISH dot signal appeared to stain intensely by IHC assay in subsequent adjacent sections, indicating that, similar to Raji control cells, IHC-detectable BCMA protein expression in tissues may be expected for cells showing persistent ISH levels as low as 1 dot / cell.

[0160] The presence of an observable positive signal produced by the BCMA ISH assay required a certain overall mRNA quality threshold (≥4 dots / cell), as assessed by the PPIB QC ISH assay. Plasma cells that presented a specific positive BCMA ISH signal generally had a minimum of 4 dots per cell present in the QC PPIB ISH signal. In contrast, in areas where plasma cells had a low PPIB ISH signal, below 4 dots / cell, no corresponding BCMA ISH signal was detected, despite a strong and specific positive signal at the protein level (IHC on directly adjacent sections).

[0161] BCMA IHC in human FFPE normal brain samples. Immunoreactivity with rabbit monoclonal anti-BCMA antibody clone [E6D7B] (CST) was observed in a small subset of neurons in specific anatomical regions of several normal human brain samples. The immunoreactive pattern was seen in the cell body (soma) and axonal processes of certain neurons (Figure 2A-I), and was manifested as cytoplasmic fibril or needle-like structures rarely seen in glial cell processes. Neurons with IHC immunoreactivity were observed mainly in the dorsal striatum, thalamus, midbrain, and medulla oblongata. Among all neurons present on tissue sections, the density of cells with immunoreactivity varied depending on the location examined and was generally low. Similar regional patterns of immunoreactivity were consistently reproduced across brain tissues and donors, regardless of the demographics or commercial origin of the samples. Due to the relatively small number of samples evaluated in this study, no clear parallels were found between the IHC immunoreactivity with E6D7B and the available demographic data associated with the tissue samples.

[0162] The unique fibrillar or needle-like intracellular, nonmembranous, nonGolgi-like pattern of immunoreactivity in human normal brain was unexpected because it was significantly different from the characteristic pattern in cells known to express this protein (e.g., plasma cells). The unexpected immunoreactivity necessitated further characterization. To that end, orthogonal experiments were performed on representative human brain samples in which IHC immunoreactivity was observed.

[0163] To further evaluate BCMA expression in human brain samples, an external molecular pathology service provider (CRO) was contracted to develop an independent BCMA IHC assay at least as sensitive as E6D7B for application in detecting low levels of BCMA protein. The two BCMA IHC assays developed at Reveal Biosciences (San Diego, CA) and Hematogenix (Tinley Park, IL) were based on mouse monoclonal anti-BCMA clone D6 from Santa Cruz Biotechnology. These two assays produced acceptable and comparable specificity and sensitivity when targeting BCMA on the same cell pellet reagents and colon tissue controls used to develop the internal BCMA IHC assay. The same set of brain samples that showed immunoreactivity using the E6D7B clone were sent to each CRO for staining in a blinded fashion. In the control samples, the BCMA IHC based on the Santa Cruz anti-BCMA antibody clone D6 showed a very similar staining pattern to the internal BCMA IHC (Figures 5A-5P). In contrast, neither BCMA staining nor fibril-like immunoreactivity was seen in brain samples. Thus, BCMA IHC assays based on the Santa Cruz mouse monoclonal anti-BCMA antibody clone D6, performed independently in two contract laboratories, did not reproduce the immunoreactivity initially observed in brain samples using the rabbit monoclonal anti-BCMA antibody clone [E6D7B].

[0164] BCMA ISH in human FFPE normal brain samples. Specific detection of mRNA transcripts from the gene of the protein of interest in the same cellular and anatomical regions could increase confidence in the specificity of the observed immunoreactivity. Therefore, BCMA ISH was used to corroborate the immunoreactivity observed when using the E6D7B clone, as it reveals gene expression with anatomical and cellular resolution. A total of 49 brain samples randomly selected without regard to E6D7B-mediated immunoreactivity were stained with the mRNA QC probe PPIB. Of these 49 samples, 25 showed acceptable PPIB control signals (4 dots or more per cell) and were used for subsequent experiments. A BCMA-specific probe was applied to these 25 samples, and only 4 of these samples yielded a very low (1-2 dots / cell) positive signal spotted in 1 or 2 cells out of the several thousand present in the section. A similar signal could be detected in the negative control cell pellet sample (HEK293 cells), but this signal was below the threshold of what was considered positive based on the assay. Immunoreactivity generated by the E6D7B clone was seen occasionally in neurons throughout the brain sections, but ISH signals were very rare.

[0165] Colocalization assays. To further characterize the immunoreactivity resulting from BCMA IHC assays performed on normal human brain tissue, immunofluorescence colocalization experiments with Golgi apparatus markers were performed. In plasma cells, which endogenously express BCMA protein, BCMA can be found on the plasma membrane or within the Golgi apparatus. The Golgi apparatus in neurons is significantly different in shape, size, and distribution when compared to plasma cells, and therefore it was deemed appropriate to determine whether BCMA immunoreactivity colocalizes with known Golgi proteins within neuronal cell bodies. To cover the span of the Golgi apparatus, cis (GOLGA2 and GOLM1, PMID:18953438) and trans (TGN46, PMID:29311477) Golgi IHC markers were examined. In tissue-resident plasma cells and reagent control cell pellets, BCMA IHC assays located the protein at the plasma membrane and consistently in Golgi-like structures in the cytoplasm. As expected, confocal observations performed between Golgi markers and BCMA antibodies showed clear colocalization in cell pellet controls, whereas no colocalization could be observed between cis- and trans-Golgi markers and immunoreactivity resulting from E6D7B in neurons of normal human brain samples (Figures 3A-3H).

[0166] Furthermore, we also investigated the colocalization of pTau (the most common aggregated protein seen preclinically and clinically in Alzheimer's disease) with BCMA, since immunoreactive fibrils are morphologically reminiscent of neurofibrillary tangles seen in neurodegenerative diseases such as Alzheimer's disease. Colocalization was assessed by confocal microscopy. pTau IHC staining showed no changes in the tissue samples evaluated, and thus the immunoreactivity observed with E6D7B did not show colocalization with that marker (Figure 6A-6F). pTau colocalization studies did not reveal any relationship to the immunoreactivity seen in the tissue samples. Since protein aggregates can be formed by a wide variety of proteins other than pTau, Bielschowsky silver staining was used to identify most changes independent of the protein(s) involved. Selected neurons showed punctate intraneuronal argyrophilic aggregates, and the majority of the cells showed clear protoplasm. In the medulla, the distribution and morphology of argyrophilic aggregates did not resemble the pattern produced by E6D7B IHC, suggesting that the immunoreactivity most likely did not originate from aggregated proteins (Figures 6A-6F).

[0167] Comparative BCMA IHC in Cynomolgus monkey brain samples. Cynomolgus monkey normal brain locations corresponding to those considered in humans were stained with BCMA IHC assay using E6D7B clone. No BCMA immunoreactivity was observed in the Cynomolgus monkey tissue samples evaluated, including the same anatomical locations studied in humans, such as the caudate nucleus, putamen, and thalamus.

[0168] GTEx RNA-Seq analysis. Across brain samples, BCMA RNA detection is generally negligible (0-0.21 TPM). In the caudate and putamen (components of the striatum), there is a small subset of samples with slightly higher TPM values ​​(1-3 TPM).

[0169] Allen BrainSpan RNA-seq analysis. BCMA (TNFRSF17) RNA expression (Figures 4A-4C) is detectable in the striatum in samples from fetuses (mean TPM=5.2), infants (mean TPM=15.1), juveniles (mean TPM=9.5), adolescents (one donor, TPM=2.2), and young adults (19 and 20 year olds, mean TPM=4.6). BCMA RNA levels are negligible (mean TPM less than 0.4) in all other brain regions examined across multiple developmental stages.

[0170] Aggregated BCMA RNA-seq expression data: striatum. For GTEx and Allen BrainSpan BCMA RNA expression levels in the striatum and striatal components (caudate and putamen), samples (n=478) show a clear decrease in expression with increasing age (Figures 4A-4C). BCMA RNA expression is generally negligible in the striatum of donors over 30 years of age.

[0171] Those skilled in the art will appreciate that changes could be made to the embodiments described above without departing from the broad inventive concept. It is understood therefore that the invention is not limited to the particular embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined herein.

Claims

1. 1. A method for detecting B-cell maturation antigen ("BCMA") molecules in a formalin-fixed, paraffin-embedded ("FFPE") sample, comprising: a. sectioning and mounting FFPE samples; b. Deparaffinizing the sample; c. Heat-mediated antigen retrieval of the sample; d. Pre-treating the sample with a blocking agent; e. contacting the sample with a first detection agent, optionally comprising an antibody or antigen-binding fragment thereof that binds to at least one BCMA molecule in the sample; f. Optionally, removing unbound sample; g. contacting the sample bound to the first detection agent with a second detection agent, which is optionally an antibody or fragment thereof; h. Optionally, removing unbound second detection agent; and i. detecting the presence of the second detection agent bound to the sample; wherein detecting an above background amount of said second detection agent bound to said sample indicates the presence of at least one BCMA molecule in said sample.

2. 10. The method of claim 1, wherein the heat-mediated antigen retrieval is performed using an EDTA-based solution.

3. 3. The method of claim 2, wherein the EDTA-based solution has a pH of 8.5 to 9.

5.

4. 4. The method of claim 3, wherein the EDTA-based solution has a pH of 9.

0.

5. 5. The method of any one of claims 1 to 4, wherein the heat-mediated antigen retrieval step is carried out for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes or 60 minutes.

6. The method of any of claims 1 to 4, wherein the heat-mediated antigen retrieval step is carried out at a temperature of 85°C to 100°C.

7. 7. The method of claim 6, wherein the heat-mediated antigen retrieval step is performed at 100°C.

8. The method of any one of claims 1 to 4, wherein the blocking agent blocks endogenous peroxidase.

9. The method of claim 8 wherein the blocking agent is a peroxide blocker.

10. 10. The method of claim 9, wherein the step of pretreating with a peroxide block is carried out for 2 minutes, 5 minutes, 8 minutes, 10 minutes, or 15 minutes.

11. The method of any one of claims 1 to 4, wherein the blocking agent blocks non-specific antibody binding.

12. 12. The method of claim 11, wherein the blocking agent is Dako / Agilent Protein Block.

13. 13. The method of claim 12, wherein the step of pretreating with Dako / Agilent Protein Block is performed for 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes.

14. The method of any one of claims 1 to 4, wherein the first detection agent is an antibody or an antigen-binding fragment thereof.

15. The method of any one of claims 1 to 4, wherein the sample is contacted with the first detection agent at room temperature or 37°C.

16. 5. The method of any one of claims 1 to 4, wherein the sample is contacted with the first detection agent for 15 minutes, 30 minutes, 45 minutes, 60 minutes, 95 minutes, or 120 minutes.

17. The method of any one of claims 1 to 4, wherein the second detection agent is an antibody or an antigen-binding fragment thereof.

18. 5. The method of any one of claims 1 to 4, wherein the sample bound to the first detection agent is contacted with the second detection agent for 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes.

19. The method of any one of claims 1 to 4, wherein the sample comprises cells derived from a body fluid or tissue.

20. 20. The method of claim 19, wherein the tissue is brain tissue.

21. 21. The method of claim 20, wherein the tissue is derived from the striatum, thalamus, midbrain, or medullary region of the brain.

22. 20. The method of claim 19, wherein the tissue is a tumor tissue.

23. The method of any one of claims 1 to 4, wherein the sample is derived from a subject.

24. 24. The method of claim 23, wherein the subject is a mammal.

25. 25. The method of claim 24, wherein the mammal is a human.

26. 25. The method of claim 24, wherein the mammal is a cynomolgus monkey.

27. A kit for carrying out the method according to any one of claims 1 to 4.

28. A method of treating a subject with a BCMA targeting agent, comprising detecting BCMA in a sample from the subject.

29. 29. The method of claim 28, wherein the step of detecting BCMA in the sample comprises the method of any one of claims 1 to 4.

30. 29. The method of claim 28, wherein the targeting agent is a chimeric antigen receptor (CAR) T cell.

31. 29. The method of claim 28, wherein the targeting agent is a T cell redirecting antibody.

32. 29. The method of claim 28, wherein the subject has cancer.

33. 33. The method of claim 32, wherein the cancer is multiple myeloma.

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