Anti-BCMA CAR antibodies, conjugates and methods of use

Abstract

The present invention provides improved methods for detecting anti-BCMA CAR expression on T cells. The present invention generally provides antibodies and antigen-binding fragments thereof, conjugates thereof, and methods of detecting, determining, or measuring CAR T cell and / or CAR expression in one or more T cells using the antibodies and antigen-binding fragments thereof and conjugates thereof. In various embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:7 and the variable heavy chain sequence set forth in SEQ ID NO:8.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 948,488, filed December 16, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing Description The sequence listing for this application has been submitted in text format in lieu of paper and is incorporated herein by reference. The name of the text file containing the sequence listing is BLUE-97PC2_ST25.txt. The text file is 26 KB, was created on December 10, 2020, and will be submitted electronically via EFS-Web simultaneously with the filing of this application.

[0003] The present invention relates to antibody compositions. More particularly, the present invention relates to antibodies and antigen-binding fragments thereof that bind to anti-BCMA CARs and conjugates thereof, and methods of using the same to detect anti-BCMA CAR T cells. [Background technology]

[0004] Description of related fields Genetic methods offer potential avenues for enhancing immune recognition and elimination of cancer cells. One promising strategy is to genetically engineer immune effector cells to express chimeric antigen receptors (CARs), which redirect cytotoxicity to tumor cells. However, accurate measurement of CAR expression and in vivo CAR T cell persistence has been difficult, confounding attempts to measure CAR T cell activity in one or more adoptively transferred CAR T cells. The most common approach to assessing CAR T cells is quantitative PCR using CAR-specific primers. However, this technique is insufficient and does not allow for multiparameter analysis of CAR T cells. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides a method for producing a CAR on one or more T cells. + Provided are antibodies and antigen-binding fragments thereof, conjugates thereof, and methods of using them to detect, determine, or measure T cell and / or CAR expression.

[0006] In various embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:7 and the variable heavy chain sequence set forth in SEQ ID NO:8.

[0007] In certain embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0008] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a Fab' fragment, a F(ab')2 fragment, a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), an Fv, a single-chain Fv protein ("scFv"), a bis-scFv, an (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0009] In certain embodiments, the antibody or antigen-binding fragment thereof is an scFv.

[0010] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain sequence set forth in SEQ ID NO:11 and the heavy chain sequence set forth in SEQ ID NO:12 or SEQ ID NO:13.

[0011] In certain embodiments, the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA CAR sequence set forth in SEQ ID NO:9.

[0012] In certain embodiments, the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA CAR sequence set forth in SEQ ID NO: 10. In certain embodiments, the antibody or antigen-binding fragment thereof is a murine antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody.

[0013] In certain embodiments, the antibody or antigen-binding fragment comprises an IgG1, IgG2, IgG3, or IgG4 constant domain. In various embodiments, the antibody or antigen-binding fragment comprises an IgG1 constant domain. In various embodiments, the antibody or antigen-binding fragment is of the murine IgG1 subtype.

[0014] In various embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof as contemplated herein and a detection means.

[0015] In various embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof as contemplated herein and a detection means.

[0016] In various embodiments, the conjugate comprises an antibody or antigen-binding fragment thereof as contemplated herein and a detectable label.

[0017] In some embodiments, the detectable label is selected from the group consisting of a hapten, a fluorescent dye, a fluorescent protein, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a luminescent compound, or an oligonucleotide.

[0018] In certain embodiments, the detectable label is a fluorescent dye selected from the group consisting of Oregon Green®, Pacific Blue™, Pacific Orange™, Pacific Green™, Cascade Blue™, Cascade Yellow™, Lucifer Yellow™, Marina Blue™, and Texas Red® (TxRed).

[0019] In certain embodiments, the detectable label is an AlexaFluor® (AF) dye selected from the group consisting of AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790 and AF800.

[0020] In some embodiments, the detectable label comprises the AlexaFluor® (AF) dye AF488.

[0021] In some embodiments, the detectable label is a QDot® selected from the group consisting of Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800.

[0022] In certain embodiments, the detectable label is a DyLight™ dye (DL) selected from the group consisting of DL549, DL649, DL680, and DL800.

[0023] In certain embodiments, the detectable label is a hapten selected from the group consisting of fluorescein or a derivative thereof, fluorescein isothiocyanate, carboxyfluorescein, dichlorotriazinylamine fluorescein, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), and biotin.

[0024] In certain embodiments, the detectable label is a Cy dye selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5.

[0025] In some embodiments, the detectable label is a fluorescent molecule selected from the group consisting of phycoerythrin (PE, R-phycoerythrin (RPE)), B-phycoerythrin (BPE), peridinin chlorophyll (PerCP), allophycocyanin (APC), and C-phycocyanin.

[0026] In certain embodiments, the detectable label is a fluorescent dye selected from the group consisting of Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740, Super Bright™ 436, Super Bright™ 600, Super Bright™ 645, Super Bright™ 702, Super Bright™ 780, Brilliant™ Violet 421, Brilliant™ Violet 480, Brilliant™ Violet 510, Brilliant™ Violet 605, Brilliant™ 650, Brilliant Violet™ 711, Brilliant Violet™ 786, Brilliant™ Ultraviolet 395 (BUV395), Brilliant™ Ultraviolet 496 (BUV496), Brilliant™ Ultraviolet 563 (BUV563), Brilliant™ Ultraviolet 661 (BUV661), Brilliant™ Ultraviolet 737 (BUV737), Brilliant™ Ultraviolet 805 (BUV805), Brilliant™ Blue 515 (BB515), Brilliant™ Blue 700 (BB700) and IR Dye 680, IR Dye680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR Dye 800RS and IR Dye 800CW.

[0027] In certain embodiments, the detectable label is a tandem fluorescent dye selected from the group consisting of RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-CF594, RPE-AlexaFluor® tandem conjugates, RPE-Alexa610, RPE-TxRed, APC-H7, APC-R700, APC-Alexa600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5, and APC-Cy7.

[0028] In certain embodiments, the detectable label is a fluorescent protein selected from the group consisting of GFP, eGFP, BFP, CFP, YFP, DsRed, DsRed2, mRFP, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mPlum, and mRaspberry.

[0029] In certain embodiments, the detectable label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase.

[0030] In certain embodiments, the detectable label is carbon (C), chromium (Cr), cobalt (Co), fluorine (F), gadolinium (Gd, Gd), germanium (Ge), holmium (Ho), indium (In, In, In, mIn), iodine (I, I, I), lanthanum (La), lutetium (Lu), manganese (Mn), molybdenum (Mo), palladium (Pd), phosphorus (P), praseodymium (I), or a combination thereof. The radionuclide comprises a radionuclide selected from the group consisting of 42Pr, promethium (149Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthenium (97Ru), samarium (153Sm), scandium (47Sc), selenium (75Se), (85Sr), sulfur (35S), technetium (99Tc), thallium (201Ti), tin (113Sn, 117Sn), tritium (3H), xenon (133Xe), ytterbium (169Yb, 175Yb) and yttrium (90Y).

[0031] In certain embodiments, the polynucleotide encodes an antibody or antigen-binding fragment thereof contemplated herein.

[0032] In certain embodiments, the host cell comprises an antibody or antigen-binding fragment thereof or a polynucleotide contemplated herein.

[0033] In some embodiments, the composition comprises an antibody or antigen-binding fragment thereof, a conjugate, a polynucleotide, or a host cell as contemplated herein.

[0034] In various embodiments, methods for producing an antibody or antigen-binding fragment thereof include expressing an antibody or antigen-binding fragment thereof contemplated herein in a host cell and recovering or isolating the antibody.

[0035] In various embodiments, the kit comprises an antibody or antigen-binding fragment or conjugate thereof contemplated herein and instructions for use.

[0036] In certain embodiments, the method of detecting expression of an anti-BCMA CAR on a T cell comprises contacting the T cell with an antibody or antigen-binding fragment thereof, or a conjugate contemplated herein, and detecting the formation of an antibody:anti-BCMA CAR complex.

[0037] In certain embodiments, the method of detecting expression of an anti-BCMA CAR in a T cell population comprises contacting a T cell with an antibody or antigen-binding fragment thereof, or a conjugate contemplated herein, and detecting the formation of an antibody:anti-BCMA CAR complex.

[0038] In certain embodiments, the method of determining expression of an anti-BCMA CAR on a T cell comprises contacting the T cell with an antibody or antigen-binding fragment thereof or a conjugate contemplated herein, detecting the formation of an antibody:anti-BCMA CAR complex, and measuring the amount of the complex to determine expression of the anti-BCMA CAR on the T cell.

[0039] In certain embodiments, the method of determining expression of an anti-BCMA CAR in a T cell population comprises contacting the T cell population with an antibody or antigen-binding fragment thereof or a conjugate contemplated herein, detecting the formation of an antibody:anti-BCMA CAR complex in one or more T cells, and measuring the amount of complex to determine expression of the anti-BCMA CAR on the one or more T cells.

[0040] In various embodiments, a method for determining the number of anti-BCMA CAR+ T cells in a T cell population comprises contacting the T cell population with an antibody or antigen-binding fragment thereof or a conjugate contemplated herein, detecting the formation of an antibody:anti-BCMA CAR complex in one or more T cells, and measuring the amount of complex to enumerate T cells expressing the anti-BCMA CAR.

[0041] In some embodiments, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS) are used to detect the formation of antibody:anti-BCMA CAR complexes.

[0042] In certain embodiments, the amount of antibody:anti-BCMA CAR complex is measured using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS).

[0043] In certain embodiments, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS) are used to enumerate T cells expressing the anti-BCMA CAR.

[0044] A brief explanation of sequence numbers SEQ ID NOs: 1-3 set forth the amino acid sequences of exemplary light chain CDR sequences of antibodies that bind to anti-BCMA CARs. SEQ ID NOs: 4-6 set forth the amino acid sequences of exemplary heavy chain CDR sequences of antibodies that bind to anti-BCMA CAR. SEQ ID NO: 7 sets forth the amino acid sequence of an exemplary variable light chain sequence of an antibody that binds to an anti-BCMA CAR. SEQ ID NO: 8 sets forth the amino acid sequence of an exemplary variable heavy chain sequence of an antibody that binds to an anti-BCMA CAR. SEQ ID NO: 9 specifies the amino acid sequence of an anti-BCMA CAR bound by antibodies comprising the sequences set forth in SEQ ID NOs: 1-8. SEQ ID NO: 10 specifies the amino acid sequence of an anti-BCMA CAR bound by antibodies comprising the sequences set forth in SEQ ID NOs: 1-8. SEQ ID NO: 11 sets forth the amino acid sequence of an exemplary light chain sequence of an antibody that binds to an anti-BCMA CAR. SEQ ID NO: 12 sets forth the amino acid sequence of an exemplary heavy chain sequence of an antibody that binds to an anti-BCMA CAR. SEQ ID NO: 13 sets forth the amino acid sequence of an exemplary heavy chain sequence of an antibody that binds to an anti-BCMA CAR. SEQ ID NOs: 14-24 specify the amino acid sequences of various linkers. DETAILED DESCRIPTION OF THE INVENTION

[0045] A. Overview The present invention relates generally to antibodies and antigen-binding fragments and conjugates thereof that bind to an anti-BCMA chimeric antigen receptor (CAR) and methods of using the same. In particularly preferred embodiments, the present invention relates to antibodies, fragments and conjugates that bind to an anti-BCMA CAR as set forth in SEQ ID NO: 9 or SEQ ID NO: 10, compositions and uses thereof.

[0046] BCMA is a member of the tumor necrosis factor receptor superfamily (see, e.g., Thompson et al., J. Exp. Medicine, 192(1):129-135, 2000, and Mackay et al., Annu. Rev. Immunol, 21:231-264, 2003). BCMA is a B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204:43-54, 2005). Among non-malignant cells, BCMA has been reported to be expressed predominantly on plasma cells and a subset of mature B cells (see, e.g., Laabi et al., EMBO J., 77(1):3897-3904, 1992; Laabi et al., Nucleic Acids Res., 22(7):1147-1154, 1994; Kalled et al., 2005; O'Connor et al., J. Exp. Medicine, 199(1):91-97, 2004; and Ng et al., J. Immunol., 73(2):807-817, 2004). Mice lacking BCMA are healthy and have normal numbers of B cells, but survival of long-lived plasma cells is impaired (see, e.g., O'Connor et al., 2004; Xu et al., Mol. Cell. Biol, 21(12):4067-4074, 2001; and Schiemann et al., Science, 293(5537):2 111-21 14, 2001). BCMA RNA is widely detected in cells of multiple myeloma and other lymphomas, and BCMA protein has been detected on the surface of plasma cells from patients with multiple myeloma by several investigators (see, e.g., Novak et al., Blood, 103(2):689-694, 2004; Neri et al., Clinical Cancer Research, 73(19):5903-5909, 2007; Bellucci et al., Blood, 105(10):3945-3950, 2005; and Moreaux et al., Blood, 703(8):3148-3157, 2004).

[0047] Anti-BCMA CARs offer a potentially curative, one-time therapy for subjects with multiple myeloma or B-cell lymphoma. Existing methods used to correlate anti-BCMA CAR expression and activity are suboptimal and require improvement. Using existing methods, it is difficult to accurately correlate anti-BCMA CAR expression and activity both ex vivo and in vivo in patients and to track the survival and persistence of anti-BCMA CAR T cells. The antibodies, fragments, and conjugates, compositions, and related methods of use contemplated herein that bind to anti-BCMA CARs provide solutions to these and other problems.

[0048] In various embodiments, an antibody or antigen-binding fragment thereof that binds to an anti-BCMA antibody or portion thereof is provided. In certain embodiments, the antibody or antigen-binding fragment thereof may further comprise one or more detectable labels.

[0049] Methods of using the antibodies and antigen-binding fragments thereof, conjugates thereof, and related compositions are also disclosed herein.

[0050] Recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays, tissue culture, transformation (e.g., electroporation, lipofection), enzymatic reactions, purification and related techniques and procedures may generally be performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology, cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach、Warren Strober 2001 John Wiley & Sons、NY、NY);Real-Time PCR:Current Technology and Applications、Edited by Julie Logan、Kirstin Edwards and Nick Saunders、2009、Caister Academic Press、Norfolk、UK;Anand、Techniques for the Analysis of Complex Genomes、(Academic Press、New York、1992);Guthrie and Fink、Guide to Yeast Genetics and Molecular Biology(Academic Press、New York、1991);Oligonucleotide Synthesis(N.Gait、Ed.、1984);Nucleic Acid The Hybridization(B.Hames & S.Higgins、Eds.、1985);Transcription and Translation(B.Hames & S.Higgins、Eds.、1984);Animal Cell Culture(R.Freshney、Ed.、1986);Perbal、A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz、2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park、Ed.、3rd Edition、2010 Humana Press);Immobilized Cells And Enzymes(IRL Press、1986);the treatise、Methods In Enzymology(Academic Press、Inc.、N.Y.);Gene Transfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Roitt, Essential Immunology, 6th Edition, (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (Q.E. Coligigan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and research articles in journals such as, for example, Advances in Immunology.

[0051] B. Definition Before describing this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

[0052] 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. Although methods and materials similar or equivalent to those described herein can be used to practice or test particular embodiments, preferred compositions, methods, and materials embodiments are disclosed herein. For purposes of this disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0053] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one, or to one or more) of the grammatical object of the article. By way of example, "an element" means one element or one or more elements.

[0054] The use of the alternative (eg, "or") should be understood to mean either one, both, or any combination of the alternatives.

[0055] The term "and / or" should be understood to mean either one or both of the alternatives.

[0056] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0057] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to imply the inclusion of the specified step or component or group of steps or components, but not the exclusion of any other step or component or group of steps or components. "Consisting of" means including and limited to everything that follows the word "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and that no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and any elements limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are necessary or mandatory, but that there are no other elements that materially affect the activity or function of the listed elements.

[0058] References throughout this specification to "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, mean that the particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, it should be understood that the affirmative recitation of a feature in one embodiment serves as grounds for the exclusion of that feature in certain embodiments.

[0059] C.Antibodies In certain embodiments, an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR is provided.

[0060] The term "antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region or fragment thereof that specifically recognizes and binds to an epitope of one or more antigens, such as peptides, lipids, polysaccharides, or antigenic determinant-containing nucleic acids, such as those recognized by immune cells.

[0061] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. Epitopes can be formed from both contiguous or noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, about nine, or about eight to ten amino acids in a unique spatial conformation.

[0062] An "isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from a component of its natural environment.

[0063] As used herein, the terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targets" describe the binding of an antibody or antigen-binding fragment thereof to an anti-BCMA CAR at a binding affinity higher than background binding. The antibody or antigen-binding fragment thereof may have a binding affinity of, for example, about 10 5 M -1 or greater affinity or K a (i.e., the equilibrium binding constant of the particular binding interaction, having units of 1 / M). In certain embodiments, the antibody or antigen-binding fragment thereof "specifically binds" to an anti-BCMA CAR if it binds to or associates with the anti-BCMA CAR with a binding constant of about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 109 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 More than K a A "high affinity" antibody or antigen-binding fragment thereof binds to an anti-BCMA CAR at a concentration of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 or higher K a It has.

[0064] Alternatively, affinity may be expressed in M ​​units (e.g., 10 -5 M~10 -13 The equilibrium dissociation constant (K d The affinity of an antibody or antigen-binding fragment thereof contemplated herein can be readily determined using conventional techniques, for example, competitive ELISA (enzyme-linked immunosorbent assay), or binding association or displacement assays using labeled ligands, or optical biosensor technologies such as a surface plasmon resonance instrument such as the Biacore T100, available from Biacore, Inc., Piscataway, NJ, or the EPIC system, available from Corning, or EnSpire, available from Perkin Elmer (see also, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Pat. Nos. 5,283,173; 5,468,614 or equivalents).

[0065] In one embodiment, the affinity of specific binding is about 2-fold greater than background binding, about 5-fold greater than background binding, about 10-fold greater than background binding, about 20-fold greater than background binding, about 50-fold greater than background binding, about 100-fold greater than background binding, or about 1000-fold greater than background binding, or more.

[0066] "Antigen (Ag)" refers to a compound, composition, or substance capable of stimulating antibody production or a T-cell response in an animal, including compositions (e.g., compositions comprising a cancer-specific protein) that are injected into or absorbed into an animal. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the antigens disclosed herein. In certain embodiments, the target antigen comprises an anti-BCMA CAR.

[0067] Antibodies include, for example, camelid Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single-domain antibodies (sdAb, nanobodies), as well as antigen-binding fragments thereof, such as the portion of a full-length antibody that is responsible for antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0068] As will be understood by those skilled in the art and as described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. Complete antibodies form a "Y" shape. The stem of the Y consists of the second and third constant regions of the two heavy chains (and for IgE and IgM, the fourth constant region) joined together, with disulfide bonds (interchain) formed at the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem Ig domains and a hinge region for additional flexibility, while heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and "CH3 domain," respectively. Each arm of the Y contains the variable and first constant regions of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0069] Light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." CDRs can be defined or identified by conventional methods, such as the sequences described in Kabat et al. (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84:2440-2443 (1987); (See Kabat et al., Sequences of Proteins of Immunological Interest, USDapartment of Health and Human Services, 1991, incorporated herein by reference) or the structures described in Chothia et al. (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4):901-917 (1987); Chothia, C. et al., Nature, 342:877-883 (1989)).

[0070] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 starts at about residue 24, is preceded by Cys, is about 10-17 residues, followed by Trp (typically Trp-Tyr-Gln, also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 starts about 16 residues after the end of CDR-L1, is generally preceded by Ile-Tyr, but also Val-Tyr, Ile-Lys, Ile-Phe, and is 7 residues; CDR-L3 starts about 33 residues after the end of CDR-L2, is preceded by Cys, is 7-11 residues, and is followed by Phe-Gly-XXX-Gly (XXX is any amino acid) (SEQ ID NO: 25).

[0071] Examples of rules for predicting heavy chain CDRs include: CDR-H1 begins at about residue 26, is preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 26), is 10-12 residues long, and is Trp (typically Trp-Val, also Trp-Ile, Trp-Ala); CDR-H2 begins about 15 residues after the end of CDR-H1, and is generally Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 27), or is preceded by some variations, is 16-19 residues long, and is followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala; CDR-H3 starts approximately 33 residues after the end of CDR-H2, is preceded by Cys-XXX-XXX (typically Cys-Ala-Arg), is 3-25 residues long, and is followed by Trp-Gly-XXX-Gly (SEQ ID NO: 28).

[0072] In one embodiment, the light chain and heavy chain CDRs are determined according to the Kabat method.

[0073] In one embodiment, the light chain CDRs and heavy chain CDR2 and CDR3 are determined according to the Kabat method, and the heavy chain CDR1 is determined according to the AbM method, which is a combination of the Kabat method and the Clothia method (see, for example, Whitelegg N & Rees AR, Protein Eng. 2000 Dec; 13(12): 819-24 and Methods Mol Biol. 2004; 248: 51-91). Programs for predicting CDRs are available, for example, at AbYsis (www.bioinf.org.uk / abysis / ).

[0074] The sequences of framework regions of different light or heavy chains are relatively conserved within species, such as humans. The framework region of an antibody is the combined framework region of the constituent light and heavy chains and serves to position and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to one or more epitopes of an antigen. The CDRs of each chain are commonly referred to as CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus, and generally identified by the chain in which the particular CDR is located. Thus, CDRs located in the variable domain of an antibody's heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while CDRs located in the variable domain of an antibody's light chain are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. Although it is the CDRs that differ from antibody to antibody, only a limited number of amino acid positions within the CDRs are directly involved in antigen binding. These positions within the CDRs are referred to as specificity-determining residues (SDRs). Illustrative examples of light chain CDRs include the CDR sequences set forth in SEQ ID NOs: 1 to 39. Illustrative examples of heavy chain CDRs include the CDR sequences set forth in SEQ ID NOs: 4 to 6.

[0075] References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein. An illustrative example of a light chain variable region includes the light chain variable region set forth in SEQ ID NO:7.

[0076] References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment disclosed herein. An illustrative example of a heavy chain variable region includes the heavy chain variable region set forth in SEQ ID NO:8.

[0077] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by a cell transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0078] A "chimeric antibody" has framework residues derived from one species, such as human, and CDRs (which generally confer antigen binding) derived from another species, such as mouse. In certain preferred embodiments, the antibody comprises an antigen-binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0079] In certain embodiments, the antibody is a human antibody (such as a human monoclonal antibody) or a fragment thereof. Human antibodies can be constructed by combining Fv clone variable domain sequence(s) selected from a human-derived phage display library with known human constant domain sequence(s) as described above. Alternatively, human monoclonal antibodies can be produced by hybridoma technology. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are described, for example, in Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991). Furthermore, transgenic animals (e.g., mice) can be used to produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Gene shuffling can also be used to derive human antibodies from non-human, e.g., rodent, antibodies, with similar affinities and specificities as the starting non-human antibody (see PCT WO93 / 06213, published April 1, 1993). Unlike traditional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies, which have no FR or CDR residues of non-human origin.

[0080] In one embodiment, the antibody is a "humanized" antibody. A humanized antibody is an immunoglobulin comprising a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDRs is referred to as the "donor," and the human immunoglobulin providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are derived from the donor immunoglobulin in the humanized immunoglobulin. Constant regions need not be present, but if present, should be substantially identical to human immunoglobulin constant regions, i.e., at least about 85-90%, e.g., about 95% or more. Thus, all portions of a humanized immunoglobulin are substantially identical to corresponding portions of native human immunoglobulin sequences, except potentially for the CDRs. Humanized or other monoclonal antibodies can have additional conservative amino acid substitutions, which do not substantially affect antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0081] In certain embodiments, antibodies or antigen-binding fragments thereof that bind to an anti-BCMA CAR include, but are not limited to, camel Ig (camel antibody (VHH)), Ig NAR, Fab fragment, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide stabilized Fv protein ("dsFv"), and single domain antibody (sdAb, nanobody).

[0082] As used herein, "Camel Ig" or "Camelidae VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte, et al., FASEB J., 21:3490-3498 (2007)). A "heavy chain antibody" or "camelidae antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079).

[0083] "IgNAR," for "immunoglobulin novel antigen receptor," refers to a class of antibodies from the shark immune repertoire consisting of a homodimer of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and possess highly stable and efficient binding properties. Their inherent stability can be attributed to both (i) the underlying Ig scaffold, which displays a significant number of charged and hydrophilic surface-exposed residues compared to traditional antibody VH and VL domains found in murine antibodies, and (ii) structural features in the complementarity-determining region (CDR) loops, including interloop disulfide bridges and stabilizing patterns of intraloop hydrogen bonds.

[0084] Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0085] An "Fv" is the minimum antibody fragment containing a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker, such that the light and heavy chains can associate in a "dimeric" structure similar to that of a two-chain Fv species. In this configuration, the three hypervariable regions (HVRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0086] Fab fragments contain heavy and light chain variable domains, as well as the light chain constant domain and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) in the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical linkages of antibody fragments are also known. A bispecific Fab dimer (Fab2) has two Fab' fragments, each binding to a different antigen. A trispecific Fab trimer (Fab3) has three Fab' fragments, each binding to a different antigen.

[0087] The term "diabody" refers to an antibody fragment having two antigen-binding sites, which fragment comprises a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to create two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097, WO 1993 / 01161, Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0088] A "single domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of an antibody heavy chain (VH domain) or the variable region of an antibody light chain (VL domain) (Holt, L. et al., Trends in Biotechnology, 21(11):484-490).

[0089] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, which domains are present in a single polypeptide chain and in any orientation (e.g., VL-VH or VH-VL). Generally, scFv polypeptides further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see, e.g., The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York, 1994), pp. 269-315.

[0090] In certain embodiments, the antigen-binding fragment is an scFv. In certain embodiments, the scFv is a mouse, human, or humanized scFv. Single-chain antibodies may be cloned from the V region genes of hybridomas specific to a desired target. The production of such hybridomas has become routine. Techniques that can be used to clone the variable heavy chain (VH) and variable light chain (VL) are described, for example, in Orlandi et al., PNAS, 1989;86:3833-3837.

[0091] In various embodiments, the antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs: 1-3 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs: 4-6.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO: 7 and / or a variable heavy chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in any one of SEQ ID NOs: 7, and / or a variable heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NO:8.

[0093] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain sequence set forth in SEQ ID NO: 11 and / or a heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 11 and / or a heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity to the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0094] In one embodiment, an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises the light chain CDR sequences set forth in SEQ ID NOs: 1-3. In specific embodiments, an anti-BCMA CAR antibody or antibody fragment, or an antibody or antigen-binding fragment thereof that binds to the anti-BCMA scFv portion of an anti-BCMA CAR, comprises a light chain CDR sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the light chain CDR sequences set forth in SEQ ID NOs: 1-3.

[0095] In one embodiment, the antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises a heavy chain CDR sequence set forth in SEQ ID NOs: 4-6. In specific embodiments, the antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises a heavy chain CDR sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity to the heavy chain CDR sequence set forth in SEQ ID NOs: 4-6.

[0096] In certain embodiments, an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises one or more light chain CDRs set forth in any one of SEQ ID NOs: 1-3, and / or one or more heavy chain CDR sequences set forth in any one of SEQ ID NOs: 4-6. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence set forth in SEQ ID NO: 7 and / or a variable heavy chain sequence set forth in SEQ ID NO: 8. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a light chain sequence set forth in SEQ ID NO: 11 and / or a heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0097] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence and / or a variable heavy chain sequence that binds to the anti-BCMA CAR set forth in SEQ ID NO:9 or SEQ ID NO:10.

[0098] D. Conjugates In various embodiments, a conjugate is provided comprising an antibody or antigen-binding fragment thereof and a label. In a preferred embodiment, the conjugate comprises an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR and a detectable label or a label that can produce a detectable signal. In a more preferred embodiment, the conjugate comprises an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR bound to a detectable label. In an even more preferred embodiment, the conjugate comprises an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR covalently or chemically bound to a detectable label.

[0099] As used herein, the term "label" refers to a detectable label or a label capable of producing a detectable signal. In certain embodiments, the label comprises a radionuclide, a nucleic acid, a small molecule, or a polypeptide. In some embodiments, the label is directly detectable. In some embodiments, the label is indirectly detectable.

[0100] Examples of detectable labels suitable for use in conjugates contemplated in certain embodiments include, but are not limited to, a hapten, a fluorescent molecule, a fluorescent dye, a fluorescent protein, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a radionuclide, a polypeptide, an enzyme, a luminescent compound, or an oligonucleotide.

[0101] Exemplary molecules suitable for use in certain embodiments include, but are not limited to: Oregon Green®; Pacific Blue™, Pacific Orange™, Pacific Green™; Cascade Blue™; Cascade Yellow™; Lucifer Yellow™; Marina Blue™, Texas Red® (TxRed); AlexaFluor® (AF) dyes, such as AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790, and AF800; QDot® nanocrystals, such as Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 700, and Qdot® 800. Qdot® 655, Qdot® 705, and Qdot® 800; DyLight™ dyes (DL), such as DL549, DL649, DL680, and DL800; fluorescein or its derivatives, such as fluorescein isothiocyanate, carboxyfluorescein, and dichlorotriazinylamine fluorescein; digoxigenin; dinitrophenol (DNP), trinitrophenol (TNP), biotin; Cy dyes, such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5;フィコエリスリン(PE、R-フィコエリスリン(RP E);B-フィコエリスリン(BPE)、ペリジニンクロロフAtto (registered trademark) dye, Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514 Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740; Super Bright (trademark) dyes, for example, Super Bright (trademark) 436, Super Bright (trademark) 600, Super Bright (trademark) 645, Super Bright (trademark) 702, Super Bright (trademark) 780; Brilliant (trademark) dyes, for example, Brilliant (trademark) Violet 421, Brilliant (trademark) Violet 480, Brilliant (trademark) Violet 510, Brilliant (trademark) Violet 605, Brilliant Violet 650, Brilliant Violet 711, Brilliant Violet trademark 786, Brilliant (trademark) UV 395 (BUV395), Brilliant (trademark) UV 496 (BUV496), Brilliant (trademark) UV 563 (BUV563), Brilliant (trademark) UV 661 (BUV661), Brilliant (trademark) UV 737 (BUV737), Brilliant (trademark) UV 805 (BUV805), Brilliant (trademark) Blue 515 (BB515), Brilliant (trademark) Blue 700 (BB700); IR Dye, for example, IR Dye 680, IR Dye 680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR Dye 800RS, IR Dye 800CW.

[0102] Illustrative examples of tandem fluorescent dye molecules suitable for use as detectable labels include, but are not limited to, the following: RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-CF594, RPE-AlexaFluor® tandem conjugates; RPE-Alexa610, RPE-TxRed, APC-H7, APC-R700, APC-Alexa600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5, and APC-Cy7.

[0103] Illustrative examples of fluorescent proteins suitable for use as detectable labels include, but are not limited to, GFP, eGFP, BFP, CFP, YFP, DsRed, DsRed2, mRFP, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mPlum, and mRaspberry.

[0104] Illustrative examples of enzymes suitable for use as detectable labels include, but are not limited to, alkaline phosphatase, horseradish peroxidase, luciferase, and B-galactosidase.

[0105] Illustrative examples of radionuclides suitable for use as detectable labels include, but are not limited to, carbon (C), chromium (Cr), cobalt (Co), fluorine (F), gadolinium (Gd, Gd), germanium (Ge), holmium (Ho), indium (In, In, In, In), iodine (I, I, I), lanthanum (La), lutetium (Lu), manganese (Mn), molybdenum (Mo), palladium (Pd), These include phosphorus (32P), praseodymium (142Pr), promethium (149Pm), rhenium (186Re, 188Re), rhodium (105Rh), ruthenium (97Ru), samarium (153Sm), scandium (47Sc), selenium (75Se), (85Sr), sulfur (35S), technetium (99Tc), thallium (201Ti), tin (113Sn, 117Sn), tritium (3H), xenon (133Xe), ytterbium (169Yb, 175Yb), and yttrium (90Y).

[0106] In certain embodiments, the conjugate comprises an antibody or antibody fragment conjugated, bound, or linked (e.g., covalently attached) to one or more labels. In certain embodiments, the label may be conjugated, bound, or linked to the antibody or fragment directly or indirectly (e.g., via a linker group). An antibody can be directly covalently attached to one or more labels when the antibody and label each have a substituent that can react with the other. For example, a nucleophilic group such as an amino group or sulfhydryl group can react on one side with a carbonyl-containing group such as an anhydride or acid halide, or on the other side with an alkyl group containing a good leaving group (e.g., a halide).

[0107] In certain embodiments, it may be desirable to couple, conjugate, or link an antibody or antibody fragment to one or more labels via a monovalent or polyvalent linker or spacer. A linker or spacer can be used to provide sufficient distance between the antibody and the label to avoid steric hindrance or interference with antibody binding capacity. It will be apparent to those skilled in the art that a variety of bifunctional or polyfunctional reagents, both homofunctional and heterofunctional (such as those listed in the catalog of Pierce Chemical Co., Rockford, IL), may be used as linker groups. Coupling may be performed, for example, via amino groups, carboxyl groups, sulfhydryl groups, or oxidized carbohydrate residues. Numerous references describe such methods, for example, U.S. Pat. No. 4,671,958 and Rodwell et al.

[0108] In certain embodiments, the linker has a total chain length of about 1 to 100 atoms, 1 to 80 atoms, 1 to 60 atoms, 1 to 40 atoms, 1 to 30 atoms, 1 to 20 atoms, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms, and the atoms in the chain include C, S, N, P, and O.

[0109] Examples of linkers or linkages useful in certain embodiments of the present invention include, but are not limited to, one or more of the following: -C(O)-, -NH-C(O)-, -C(O)-NH-, -C(O)-NH-(CH2). 2-6 -NH-C(O)-, -NH-(CH2) 2-6 -NH-C(O)-, -triazole-(CH2) 2-6 -NH-C(O)-, -S-(CH2) 2-6 -NH-C(O)-, -S-(CH2) 0-6 -CH(CONH2)-(CH2) 0-6 -NH-C(O)-, -S-(CH2) 0-6 -CH(CONH-PEG)-(CH2) 0-6 -NH-C(O)-, -SS-(CH2) 2-6 -NH-C(O)-, -SS-(CH2)0-6 -CH(CONH2)-(CH2) 0-6 -NH-C(O)-, -SS-(CH2) 0-6 -CH(CONH-PEG)-(CH2) 0-6 -NH-C(O)-, -NH-(CH2) 0-6 -CH(CONH-PEG)-(CH2) 0-6 -NH-C(O)-, -NH-(CH2) 0-6 -CH(CONH2)-(CH2) 0-6 -NH-C(O)--C=NO-(CH2) 2-6 -NH-C(O)-, -C=N-NH-(CO)-(CH2) 2-6 -NH-C(O)-, -succinimide-(CH2) 2-6 -NH-C(O)-, -diazodicarboxamido-(phenyl)-J-(CH2) 2-6 -NH-C(O)-, J is O, CH2, NH, S, NH(CO), (CO)NH, -NH-(CH2) 2-6 -, (CH2) 1-6 -NH-C(O)-NH-(CH2) 2-6 -, -C(S)-(CH2) 0-6 -,-(CH2) 1-6 -C(O)-NH-(CH2) 2-6 -, -(CH2) 1-6 -NH-C(O)-(CH2) 2-6 -, -(CH2) 1-6 -OC(O)-NH-(CH2) 2-6 -, -(CH2) 1-6 -NH-C(O)-O-(CH2) 2-6 -, (CH2) 1-6 -NH-(CH2) 2-6 , (CH2) 1-6 -C(O-(CH2) 2-6 -, branched or unbranched -C1-C16-alkyl, branched or unbranched -C1-C16-alkyl, one of the carbon atoms may be replaced by a heteroatom, R 2 -NH-(CH2) 2-6 -NH-C(O)-, R 2 -S-(CH2) 2-6 -NH-C(O)-, R 2 -triazole-(CH2)2-6 -NH-C(O)-, R 2 -NH-O-(CH2) 2-6 -NH-C(O)-, R 2 =N-NH-(CO)-(CH2) 2-6 -NH-C(O)-, R 2 is one to three bifunctional or trifunctional substituted bridging organic radicals selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl, and polyethylene glycol (PEG) [i.e., -(CH2CHO) 1-20 ].

[0110] In certain embodiments, the conjugate comprises an antibody or antibody fragment covalently attached to a polypeptide-based label, such as a fluorescent protein or enzyme, via a polypeptide linker as contemplated elsewhere herein.

[0111] In a preferred embodiment, the conjugate comprises an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR covalently attached to a PE label.

[0112] E. Polypeptides Various polypeptides, fusion polypeptides, and polypeptide variants are contemplated herein, including, but not limited to, antibodies and antigen-binding fragments thereof. In a preferred embodiment, the polypeptide comprises an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR.

[0113] "Polypeptide," "peptide," and "protein" are used interchangeably and are given their conventional meaning, i.e., as an amino acid sequence, unless specified to the contrary. A polypeptide is not limited to a particular length, e.g., a polypeptide may contain a full-length polypeptide or a polypeptide fragment, and a polypeptide may include one or more post-translational modifications of a polypeptide, such as glycosylation, acetylation, phosphorylation, and other modifications, both natural and non-natural, known in the art.

[0114] As used herein, "isolated protein," "isolated peptide," or "isolated polypeptide," etc., refers to the in vitro synthesis, isolation, and / or purification of a peptide or polypeptide molecule from its cellular environment and from association with other components of a cell, i.e., not significantly associated with substances in vivo.

[0115] Polypeptides include "polypeptide variants." Polypeptide variants can differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants can be natural or synthetically produced, for example, by modifying one or more of the above-described polypeptide sequences. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of an antibody or antigen-binding fragment thereof by introducing one or more amino acid substitutions, deletions, additions, and / or insertions. In certain embodiments, polypeptides include those having at least about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% amino acid identity to any of the reference sequences contemplated herein, and typically such variants retain at least one biological activity of the reference sequence.

[0116] Polypeptides include "polypeptide fragments." Polypeptide fragments may be monomeric or multimeric and refer to biologically active polypeptides having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions in naturally occurring or recombinantly produced polypeptides. Examples of biologically active polypeptide fragments include antibody fragments. As used herein, the term "biologically active fragment" or "minimal biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the activity of a naturally occurring polypeptide. In preferred embodiments, the biological activity is binding affinity for an epitope. In certain embodiments, a polypeptide fragment may contain an amino acid chain at least 5 to about 500 amino acids in length. In certain embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments contain functional domains, including antigen-binding domains or fragments of antibodies. In the case of antibodies, useful fragments include, but are not limited to, one or more CDR regions, the CDR3 region of a heavy or light chain, the variable region of a heavy or light chain, a portion of an antibody chain, or a variable region containing two CDRs.

[0117] As mentioned above, polypeptides may be modified in various ways, including amino acid substitution, deletion, truncation and insertion. Methods for such manipulation are generally known in the art. For example, amino acid sequence variants of reference polypeptides can be created by mutations in DNA. Methods for mutagenesis and nucleotide sequence modification are known in the art. For example, see Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al. (1987, Methods in Enzymol, 154: 367-382), U.S. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and the references cited therein. Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0118] In some embodiments, a polypeptide variant contains one or more conservative substitutions. A "conservative substitution" is one in which an amino acid is replaced with another amino acid having similar properties, and one skilled in the art of peptide chemistry would predict that such a substitution will not substantially alter the secondary structure and hydrophilic properties of the polypeptide. The amino acid changes in the protein variants disclosed herein are preferably conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids that are related by their side chains. Natural amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conservative substitutions of amino acids are known to those skilled in the art and can generally be made without changing the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not significantly change biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0119] Polypeptide variants also include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (for example, PEGylated molecules).Covalent variants can be prepared by linking functional groups to the groups present in amino acid chains or N-terminal or C-terminal residues, as known in the art.Variants also include allelic variants, species variants, and mutant proteins.Truncation or deletion of regions that do not affect the functional activity of protein is also considered a variant.

[0120] In certain embodiments, the polypeptides comprise a polypeptide linker. A "linker" refers to multiple amino acid residues added between two or more polypeptides for proper spacing, structure, and function of the molecules.

[0121] In certain embodiments, the polypeptide linker is a variable region binding sequence. H and V L The amino acid sequence connecting the two sub-binding domains provides a spacer function compatible with the interaction of the two sub-binding domains, and the resulting polypeptide retains the same specific binding affinity for the target molecule as an antibody containing the same light chain variable region and heavy chain variable region.

[0122] In certain embodiments, the conjugate comprises a polypeptide linker that covalently attaches the antibody or antibody fragment to one or more protein-based labels, such as fluorescent proteins or enzymes.

[0123] Exemplary linkers suitable for use in certain embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 14); TGEKP (SEQ ID NO: 15) (see, e.g., Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 16) (Pomerantz et al. 1995, supra); (GGGGS) n, where n=1, 2, 3, 4, or 5 (SEQ ID NO: 17) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 18) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 19) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 20); LQRDGERP (SEQ ID NO: 21); LRQKDGGGSERP (SEQ ID NO: 22); LRQKD(GGGS)2ERP (SEQ ID NO: 23). Alternatively, flexible linkers can be modeled using a computer program (Desjarlais & Berg, PNAS 93, 1156-1160 (1996)) that can model both the DNA binding site and the peptide itself. 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or rationally designed by phage display. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 24) (Cooper et al., Blood, 101(4):1637-1644 (2003)).

[0124] In preferred embodiments, the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 7 or 8. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain sequence set forth in SEQ ID NO: 11 and the heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0125] F. Polynucleotides In a preferred embodiment, a polynucleotide is provided that encodes an antibody or antigen-binding fragment thereof that binds to anti-BCMA CAR. As used herein, the term "polynucleotide" or "nucleic acid" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. In certain embodiments, polynucleotides include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity. In various exemplary embodiments, polynucleotides encoding the polypeptides contemplated herein include, but are not limited to, the polypeptide sequences set forth in SEQ ID NO: 7 or 8.

[0126] In certain embodiments, polynucleotides are provided that encode at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more consecutive amino acid residues of a polypeptide, as well as all intermediate lengths. In this context, "intermediate length" will be readily understood to mean any length between the recited values, e.g., 6, 7, 8, 9, etc., 101, 102, 103, etc., 151, 152, 153, etc., 201, 202, 203, etc.

[0127] In certain embodiments, a polynucleotide may be codon-optimized. As used herein, the term "codon optimization" refers to substituting codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or synthetically constructed bias tables; (ii) variation in the degree of codon bias within an organism, gene, or set of genes; (iii) systematic variation of codons with their context; (iv) variation of codons with their decoding tRNAs; (v) variation of codons with GC % either across triplets or at a single position in a triplicate; (vi) variation in similarity to a reference sequence, e.g., a natural sequence; (vii) variation in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence underlying the design of the codon substitution set; (x) synthetic variation of the codon set for each amino acid; and / or (xi) isolated removal of incorrect translation start positions.

[0128] As used herein, terms such as " polynucleotide variant " and " variant " refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence, or hybridizes with a reference sequence under stringent conditions as defined herein.These terms include polynucleotides in which one or more nucleotides are added or deleted or replaced with different nucleotides compared to a reference polynucleotide.In this regard, it is understood in the art that certain modifications, including mutations, additions, deletions and substitutions, can be made to a reference polynucleotide, and the modified polynucleotide can retain the biological function or biological activity of the reference polynucleotide.

[0129] Polynucleotide variants include polynucleotide fragments that encode biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a polynucleotide fragment that encodes a polypeptide that retains at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5%, of the activity of a naturally occurring polypeptide. It refers to polynucleotide fragments having a length of 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, a carboxy-terminal deletion, and / or an internal deletion or substitution of one or more amino acids of a naturally occurring or recombinantly produced polypeptide.

[0130] As used herein, "sequence identity" or phrases such as "a sequence 50% identical to" refer to the degree to which sequences are identical nucleotide-by-nucleotide or amino acid-by-amino acid over a comparison window. Thus, "percentage of sequence identity" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists in both sequences; calculating the number of matching positions; dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size); and multiplying the result by 100 to calculate the percentage of sequence identity. Included are nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein; typically, the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0131] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence," "comparison window," "sequence identity," "percentage of sequence identity," and "substantial identity." A "reference sequence" comprises nucleotides and amino acid residues that are at least 12 monomeric units in length, often 15-18 monomeric units, and often at least 25 monomeric units in length. Two polynucleotides may each contain (1) similar sequences between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) divergent sequences between the two polynucleotides. Sequence comparison between two (or more) polynucleotides is typically performed by comparing the sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least six contiguous positions, usually about 50 to about 100, more commonly about 100 to about 150, and a sequence is compared to the reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. The comparison window can contain about 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) for optimal alignment of two sequences. Optimal alignment of sequences for aligning the comparison window can be performed by computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA) in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, or by inspection and best alignment (i.e., resulting in the highest homology across the comparison window) generated by any of the various methods selected. For example, see the BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15, Unit 19.3.

[0132] As used herein, an "isolated polynucleotide" refers to a polynucleotide that has been purified from sequences that naturally flank it, e.g., a DNA fragment that has been removed from sequences that normally flank it. "Isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that are not found in nature and are made by the hand of man.

[0133] Furthermore, one of skill in the art will recognize that, as a result of the degeneracy of the genetic code, there are numerous nucleotide sequences that encode the polypeptides described herein, or variant fragments thereof. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any naturally occurring sequence. However, polynucleotides that vary due to differences in codon usage are contemplated, and in certain embodiments, polynucleotides that are optimized for, for example, human and / or primate codon preferences are contemplated.

[0134] Polynucleotides may be prepared, manipulated, and / or expressed using any of a variety of established techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding that polypeptide may be inserted into an appropriate vector.

[0135] G. Composition The compositions contemplated herein may comprise one or more antibodies or antibody fragments, as well as antigen-binding fragments thereof, conjugates, polypeptides, and polynucleotides. Compositions include, but are not limited to, pharmaceutical compositions. There is virtually no limit to the other components that may be included in the composition, provided that the additional agents do not adversely affect the function or activity of the active ingredients of the composition.

[0136] In certain embodiments, the compositions of the invention comprise an amount of an antibody or antigen-binding fragment thereof or conjugate thereof. As used herein, the term "amount" refers to an "effective amount" or "effective quantity" of an antibody that binds to a target polypeptide.

[0137] The composition may further comprise one or more carriers or excipients.

[0138] Examples of carriers suitable for incorporation into the compositions contemplated herein include, but are not limited to, buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, glucose, mannose, or other carbohydrates, including dextrins; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants, such as polyethylene glycol (PEG).

[0139] Acceptable carriers include, but are not limited to, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride; benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins , such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; other carbohydrates including monosaccharides, disaccharides, glucose, mannose or dextrins; chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0140] H.Kit In various embodiments, kits are provided that include one or more antibodies or antigen-binding fragments thereof that bind to an anti-BCMA CAR and instructions for using the antibodies to detect, determine and / or measure anti-BCMA CAR expression on one or more immune effector cells or anti-BCMA CAR T cells.

[0141] In certain embodiments, kits are provided that include one or more antibodies or antigen-binding fragments thereof that bind to an anti-BCMA CAR and instructions for using the antibodies to detect, determine, measure and / or enumerate anti-BCMA CAR expression on one or more immune effector cells or anti-BCMA CAR T cells.

[0142] In various embodiments, the antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs: 1-3 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs: 4-6.

[0143] In some embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO: 7 and / or the variable heavy chain sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody or antigen-binding fragment thereof comprises the light chain sequence set forth in SEQ ID NO: 11 and / or the variable heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0144] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence and / or a variable heavy chain sequence that binds to the anti-BCMA CAR set forth in SEQ ID NO:9 or SEQ ID NO:10.

[0145] In certain embodiments, the kit comprises an antibody or antibody fragment, a detectable label, and instructions for conjugation. In certain embodiments, the kit comprises an antibody or antibody fragment that is bound to a detectable label and instructions for use. In preferred embodiments, the detectable label is directly detectable. In more preferred embodiments, the detectable label is a fluorescent label. In even more preferred embodiments, the detectable label is RPE.

[0146] I. Method Anti-BCMA CAR T cell activity is related, in part, to anti-BCMA CAR expression on immune effector cells. The ability to accurately assess anti-BCMA CAR expression both ex vivo or in vitro and in vivo is necessary to estimate drug activity in patients and track the viability and persistence of anti-BCMA CAR T cells.

[0147] In certain embodiments, methods are provided that use one or more antibodies or antigen-binding fragments thereof. The antibodies are used to detect or measure expression of an anti-BCMA CAR. The antibodies, fragments, and conjugates contemplated herein can be used to detect or measure the presence or level of anti-BCMA CAR expression and / or the presence or level of anti-BCMA CAR in a population. + Used to bind, detect, determine, identify, measure, quantify, and / or enumerate the number of immune effector cells.

[0148] In various embodiments, the method comprises detecting anti-BCMA CAR expression on an immune effector cell. The immune effector cell, e.g., a T cell, NK cell, NKT cell, is contacted with an antibody or antigen-binding fragment thereof that binds to one or more epitopes of the anti-BCMA CAR in an amount effective and for a time sufficient for the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex.

[0149] In certain embodiments, the method comprises detecting anti-BCMA CAR expression on one or more immune effector cells in an immune effector cell population. The one or more immune effector cells, e.g., T cells, NK cells, NKT cells, are contacted with an antibody or antigen-binding fragment thereof that binds to one or more epitopes of the anti-BCMA CAR in an amount effective and for a time sufficient for the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. In certain embodiments, at least 25% of the immune effector cells express the anti-BCMA CAR. + In another embodiment, at least 50% of the immune effector cells are anti-BCMA CAR + In another embodiment, at least 75% of the immune effector cells are anti-BCMA CAR + is.

[0150] In certain embodiments, the method comprises determining the amount of expression of an anti-BCMA CAR on an immune effector cell. The immune effector cell, e.g., a T cell, NK cell, NKT cell, is contacted with an antibody or antigen-binding fragment thereof that binds to one or more epitopes of the anti-BCMA CAR in an amount effective and for a time sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. The amount of antibody:anti-BCMA CAR complex is measured and compared to a control.

[0151] In some embodiments, the method comprises determining the amount of expression of an anti-BCMA CAR on one or more immune effector cells in an immune effector cell population. The one or more immune effector cells, e.g., T cells, NK cells, NKT cells, are contacted with an antibody or antigen-binding fragment thereof that binds to one or more epitopes of the anti-BCMA CAR in an amount effective and for a time sufficient for the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. The amount of antibody:anti-BCMA CAR complex is measured and compared to a control.

[0152] In certain embodiments, the method comprises: + and determining the number of immune effector cells. One or more immune effector cells, e.g., T cells, NK cells, NKT cells, are contacted with an antibody or antigen-binding fragment thereof that binds to one or more epitopes of the anti-BCMA scFV portion in an amount effective and for a time sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. The immune effector cells containing the antibody:anti-BCMA CAR complex can then be enumerated.

[0153] In certain embodiments, anti-BCMA CAR expression or anti-BCMA CAR + The method for detecting, determining or enumerating T cells includes the use of anti-BCMA CAR + In certain embodiments, the sample comprises the isolation of a biological sample from a subject to which the T cells have been administered. +In a preferred embodiment, the sample is an apheresis sample, a leukapheresis sample, peripheral blood, bone marrow, lymph node tissue, spinal cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, or a tumor biopsy.

[0154] In certain embodiments, the antibodies and conjugates contemplated herein that bind to an anti-BCMA CAR are administered to a subject and / or a sample taken from the subject at one or more time points to study the pharmacokinetics, growth and / or persistence of the anti-BCMA CAR. + In certain embodiments, the sample is measured or assessed by detecting the amount of T cells. + The T cells are harvested, isolated and / or collected about 24 hours, about 48 hours, about 72 hours, about 4 days, about 5 days, about 6 days, about 7 days, about 10 days, about 14 days, about 21 days, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 3 months, about 4 months, about 6 months, about a year, or weekly, monthly or yearly after administration.

[0155] In various embodiments, the antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequences set forth in SEQ ID NOs: 1-3 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequences set forth in SEQ ID NOs: 4-6.

[0156] In some embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO: 7 and / or the variable heavy chain sequence set forth in SEQ ID NO: 8. In certain embodiments, the antibody or antigen-binding fragment thereof comprises the light chain sequence set forth in SEQ ID NO: 11 and / or the heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0157] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence and / or a variable heavy chain sequence that binds to the anti-BCMA CAR sequence set forth in SEQ ID NO:9 or SEQ ID NO:10.

[0158] In certain embodiments, anti-BCMA CAR expression or anti-BCMA CAR + The method for detecting, determining or counting T cell comprises using antibody or antibody fragment that is conjugated with detectable label.In a preferred embodiment, detectable label is directly detectable.In a more preferred embodiment, detectable label is fluorescent label.In an even more preferred embodiment, detectable label is RPE.

[0159] The presence of the antibody:anti-BCMA CAR complex can be detected, determined, and / or enumerated by one or more assays including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, fluorescence-activated cell sorting (FACS), or Western blot.

[0160] In a preferred embodiment, the presence of antibody:anti-BCMA CAR complexes is detected, determined, and / or enumerated using flow cytometry. In a preferred embodiment, the presence of antibody:anti-BCMA CAR complexes is detected, determined, and / or enumerated using FAC.

[0161] In certain embodiments, the level of background expression of the anti-BCMA CAR on one or more immune effector cells is determined and subtracted from the amount of antibody:anti-BCMA CAR complex measured on one or more immune effector cells, or used to normalize measurements.

[0162] All publications, patent applications, and issued patents cited herein are hereby incorporated by reference as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0163] Although the foregoing invention has been described in detail in the figures and examples for purposes of clarity and understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of the present invention, certain changes and modifications can be made without departing from the spirit or scope of the appended claims. The following examples are provided for purposes of illustration only, and not for purposes of limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to yield essentially similar results. [Example]

[0164] Example 1 Antibodies against anti-BCMA CAR Mouse monoclonal antibodies were generated against the anti-BCMA CAR sequence set forth in SEQ ID NO: 9. One of the antibodies was sequenced (GenScript).

[0165] Total RNA was isolated from hybridoma cells according to the TRIzol® Reagent technical manual. Total RNA was then reverse transcribed into cDNA using either an isotype-specific antisense primer or a universal primer according to the PrimeScript™ First Strand cDNA Synthesis Kit technical manual. Heavy and light chain antibody fragments were amplified according to the standard operating procedure (SOP) for rapid amplification of cDNA ends (RACE). The amplified antibody fragments were separately cloned into standard cloning vectors and sequenced.

[0166] In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the present disclosure. In certain embodiments, for example, the following items are provided: (Item 1) An antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO:7 and the variable heavy chain sequence set forth in SEQ ID NO:8. (Item 2) 2. The antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment. (Item 3) The antibody or antigen-binding fragment thereof may be a Fab' fragment, F(ab') 2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) 2 3. The antibody or antigen-binding fragment thereof according to item 1 or 2, wherein the antibody or antigen-binding fragment is selected from the group consisting of a minibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody). (Item 4) 4. The antibody or antigen-binding fragment thereof according to any one of items 1 to 3, wherein the antibody or antigen-binding fragment thereof is an scFv. (Item 5) 2. The antibody or antigen-binding fragment thereof according to item 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain sequence set forth in SEQ ID NO: 11 and a heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13. (Item 6) 6. The antibody or antigen-binding fragment thereof according to any one of items 1 to 5, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA CAR sequence set forth in SEQ ID NO: 9. (Item 7) 7. The antibody or antigen-binding fragment thereof according to any one of items 1 to 6, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA CAR sequence set forth in SEQ ID NO: 10. (Item 8) 8. The antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody or antigen-binding fragment is a mouse antibody. (Item 9) 8. The antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody or antigen-binding fragment is a humanized antibody. (Item 10) 8. The antibody or antigen-binding fragment thereof according to any one of items 1 to 7, wherein the antibody or antigen-binding fragment is a human antibody. (Item 11) 11. The antibody or antigen-binding fragment thereof according to any one of items 1 to 10, wherein the antibody or antigen-binding fragment comprises an IgG1, IgG2, IgG3, or IgG4 constant domain. (Item 12) 11. The antibody or antigen-binding fragment thereof according to any one of items 1 to 10, wherein the antibody or antigen-binding fragment comprises an IgG1 constant domain. (Item 13) A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 12, and a detection method thereof. (Item 14) A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 12, and a detection method thereof. (Item 15) 13. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of items 1 to 12, and a detectable label. (Item 16) 16. The conjugate of item 15, wherein the detectable label is selected from the group consisting of a hapten, a fluorescent dye, a fluorescent protein, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a luminescent compound or an oligonucleotide. (Item 17) 17. The conjugate of item 15 or item 16, wherein the detectable label is a fluorescent dye selected from the group consisting of Oregon Green®, Pacific Blue™, Pacific Orange™, Pacific Green™, Cascade Blue™, Cascade Yellow™, Lucifer Yellow™, Marina Blue™, and Texas Red® (TxRed). (Item 18) 17. The conjugate of item 15 or item 16, wherein the detectable label is an AlexaFluor® (AF) dye selected from the group consisting of AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790, and AF800. (Item 19) 19. The conjugate according to item 18, wherein the detectable label is the AlexaFluor® (AF) dye AF488. (Item 20) 17. The conjugate according to item 15 or item 16, wherein the detectable label is a QDot® selected from the group consisting of Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 655, Qdot® 705, and Qdot® 800. (Item 21) 17. The conjugate according to item 15 or 16, wherein the detectable label is a DyLight™ dye (DL) selected from the group consisting of DL549, DL649, DL680, and DL800. (Item 22) 17. The conjugate of claim 15 or 16, wherein the detectable label is a hapten selected from the group consisting of fluorescein or a derivative thereof, fluorescein isothiocyanate, carboxyfluorescein, dichlorotriazinylamine fluorescein, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), and biotin. (Item 23) 17. The conjugate of claim 15 or 16, wherein the detectable label is a Cy dye selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5. (Item 24) 17. The conjugate according to claim 15 or 16, wherein the detectable label is a fluorescent molecule selected from the group consisting of phycoerythrin (PE, R-phycoerythrin (RPE)), B-phycoerythrin (BPE), peridinin chlorophyll (PerCP), allophycocyanin (APC), and C-phycocyanin. (Item 25) The detectable label is Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740, Super Bright(TM) 436, Super Bright(TM) 600, Super Bright(TM) 645, Super Bright(TM) 702, Super Bright(TM) 780, Brilliant(TM) Violet 421, Brilliant(TM) Violet 480, Brilliant(trademark) Violet 510, Brilliant(TM) Violet 605, Brilliant Violet(TM) 650, Brilliant Violet(TM) 711, Brilliant Violet (trademark) 786, Brilliant (trademark) ultraviolet 395 (BUV395), Brilliant (trademark) ultraviolet 496 (BUV496), Brilliant (trademark) ultraviolet 563 (BUV563), B rilliant(TM) UV 661 (BUV661), Brilliant(TM) UV 737 (BUV737), Brilliant(TM) UV 805 (BUV805), Brilliant(TM) Blue 515(BB515), Brilliant(TM) Blue 700(BB700), and IR Dye 680, IR Dye680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR 17. The conjugate according to item 15 or 16, wherein the fluorescent dye is selected from the group consisting of IR Dye 800RS, and IR Dye 800CW. (Item 26) 17. The conjugate of claim 15 or 16, wherein the detectable label is a tandem fluorescent dye selected from the group consisting of RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-CF594, RPE-AlexaFluor® tandem conjugate; RPE-Alexa610, RPE-TxRed, APC-H7, APC-R700, APC-Alexa600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5, and APC-Cy7. (Item 27) 17. The conjugate of claim 15 or 16, wherein the detectable label is a fluorescent protein selected from the group consisting of GFP, eGFP, BFP, CFP, YFP, DsRed, DsRed2, mRFP, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mPlum, and mRaspberry. (Item 28) 17. The conjugate according to claim 15 or 16, wherein the detectable label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase. (Item 29) The detectable label may be carbon (C), chromium (Cr), cobalt (Co), fluorine (F), gadolinium (Gd, Gd), germanium (Ge), holmium (Ho), indium (In, In, In, In), iodine (I, I, I), lanthanum (La), lutetium (Lu), manganese (Mn), molybdenum (Mo), palladium (Pd), phosphorus (P), praseodymium (Pr), promethium ( 17. The conjugate of claim 15 or 16, comprising a radionuclide selected from the group consisting of Pm), rhenium (Re, Re), rhodium (Rh), ruthenium (Ru), samarium (Sm), scandium (Sc), selenium (Se), (Sr), sulfur (S), technetium (Tc), thallium (Ti), tin (Sn, Sn), tritium (H), xenon (Xe), ytterbium (Yb, Yb) and yttrium (Y). (Item 30) 13. A polynucleotide encoding the antibody or binding fragment thereof according to any one of items 1 to 12. (Item 31) A host cell comprising the antibody or binding fragment thereof according to any one of items 1 to 12 or the polynucleotide according to item 30. (Item 32) A composition comprising the antibody or binding fragment thereof according to any one of items 1 to 12, the conjugate according to any one of items 13 to 29, the polynucleotide according to item 30, or the host cell according to item 31. (Item 33) 13. A method for producing an antibody or antigen-binding fragment thereof, comprising expressing the antibody or antigen-binding fragment thereof according to any one of items 1 to 12 in a host cell and recovering or isolating the antibody. (Item 34) A kit comprising the antibody or antigen-binding fragment thereof according to any one of Items 1 to 12, or the conjugate according to any one of Items 13 to 29, and instructions for use. (Item 35) 30. A method for detecting expression of an anti-BCMA CAR on a T cell, the method comprising contacting the T cell with the antibody or antigen-binding fragment thereof of any one of items 1 to 12 or the conjugate of any one of items 13 to 29, and detecting the formation of an antibody:anti-BCMA CAR complex. (Item 36) 30. A method of detecting expression of an anti-BCMA CAR in a T cell population, the method comprising contacting a population of T cells with the antibody or antigen-binding fragment thereof of any one of items 1 to 12 or the conjugate of any one of items 13 to 29, and detecting formation of an antibody:anti-BCMA CAR complex in the one or more T cells. (Item 37) 30. A method for determining the expression of an anti-BCMA CAR on a T cell, the method comprising contacting a T cell with the antibody or antigen-binding fragment thereof of any one of items 1 to 12 or the conjugate of any one of items 13 to 29, detecting the formation of an antibody:anti-BCMA CAR complex, and measuring the amount of said complex to determine the expression of said anti-BCMA CAR on said T cell. (Item 38) 30. A method of determining expression of an anti-BCMA CAR in a T cell population, the method comprising contacting the T cell population with the antibody or antigen-binding fragment thereof of any one of items 1 to 12 or the conjugate of any one of items 13 to 29, detecting the formation of an antibody:anti-BCMA CAR complex in one or more T cells, and measuring the amount of said complex to determine the expression of said anti-BCMA CAR on said one or more T cells. (Item 39) Anti-BCMA CAR in T cell population+ 30. A method for determining the number of T cells, the method comprising contacting a population of T cells with the antibody or antigen-binding fragment thereof of any one of items 1 to 12 or the conjugate of any one of items 13 to 29, detecting the formation of an antibody:anti-BCMA CAR complex in one or more T cells, and enumerating the T cells that express the anti-BCMA CAR. (Item 40) 40. The method of any one of paragraphs 35-39, wherein formation of the antibody:anti-BCMA CAR complex is detected using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS). (Item 41) 39. The method of claim 37 or 38, wherein the amount of the antibody:anti-BCMA CAR complex is measured using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS). (Item 42) 40. The method of claim 39, wherein T cells expressing the anti-BCMA CAR are enumerated using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS).

Claims

1. An antibody or antigen-binding fragment thereof comprising the variable light chain sequence set forth in SEQ ID NO:7 and the variable heavy chain sequence set forth in SEQ ID NO:

8.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

3. The antibody or antigen-binding fragment thereof may be a Fab' fragment, F(ab') ２ fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single chain Fv proteins ("scFv"), bis-scFv, (scFv) ２ 3. The antibody or antigen-binding fragment thereof of claim 1 or claim 2, wherein the antibody or antigen-binding fragment is selected from the group consisting of a diabody, a triabody, a tetrabody, and a disulfide-stabilized Fv protein ("dsFv").

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is an scFv.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a light chain sequence set forth in SEQ ID NO: 11 and a heavy chain sequence set forth in SEQ ID NO: 12 or SEQ ID NO:

13.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA CAR sequence set forth in SEQ ID NO:

9.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA CAR sequence set forth in SEQ ID NO:

10.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment is a mouse antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment is a chimeric antibody.

10. The antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the antibody or antigen-binding fragment comprises an IgG1, IgG2, IgG3 or IgG4 constant domain.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment comprises an IgG1 constant domain.

12. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 and a detectable label.

13. 13. The conjugate of claim 12, wherein the detectable label is selected from the group consisting of a hapten, a fluorescent dye, a fluorescent protein, a chromophore, a metal ion, a gold particle, a silver particle, a magnetic particle, a polypeptide, an enzyme, a luminescent compound, or an oligonucleotide.

14. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is a fluorescent dye selected from the group consisting of Oregon Green®, Pacific Blue™, Pacific Orange™, Pacific Green™, Cascade Blue™, Cascade Yellow™, Lucifer Yellow™, Marina Blue™, and Texas Red® (TxRed).

15. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is an AlexaFluor® (AF) dye selected from the group consisting of AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790, and AF800.

16. 16. The conjugate of claim 15, wherein the detectable label is AlexaFluor® (AF) dye AF488.

17. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is a QDot® selected from the group consisting of Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 655, Qdot® 705, and Qdot® 800.

18. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is a DyLight™ dye (DL) selected from the group consisting of DL549, DL649, DL680, and DL800.

19. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is a hapten selected from the group consisting of fluorescein or a derivative thereof, fluorescein isothiocyanate, carboxyfluorescein, dichlorotriazinylamine fluorescein, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), and biotin.

20. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is a Cy dye selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.

5.

21. 14. The conjugate of claim 12 or 13, wherein the detectable label is a fluorescent molecule selected from the group consisting of phycoerythrin (PE, R-phycoerythrin (RPE)), B-phycoerythrin (BPE), peridinin chlorophyll (PerCP), allophycocyanin (APC), and C-phycocyanin.

22. The detectable label is Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, Atto 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740, Super Bright (trademark) 436, Super Bright (trademark) 600, Super Bright(TM) 645, Super Bright(TM) 702, Super Bright(TM) 780, Brilliant(TM) Violet 421, Brilliant(TM) Violet 480, Brilliant(TM) Violet 510, Brilliant(TM) Violet 605, Brilliant Violet(TM) 650, Brilliant Violet(TM) 711, Brilliant Violet™ 786, Brilliant™ Ultraviolet 395 (BUV395), Brilliant™ Ultraviolet 496 (BUV496), Brilliant™ Ultraviolet 563 (BUV563), B Brilliant™ Ultraviolet 661 (BUV661), Brilliant™ Ultraviolet 737 (BUV737), Brilliant™ Ultraviolet 805 (BUV805), Brilliant™ Blue 515 (BB515), Brilliant™ Blue 700 (BB700), and IR Dye 680, IR Dye680LT, IR Dye 700, IR Dye 14. The conjugate of claim 12 or claim 13, wherein the fluorescent dye is selected from the group consisting of IR Dye 700DX, IR Dye 800, IR Dye 800RS, and IR Dye 800CW.

23. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is a tandem fluorescent dye selected from the group consisting of RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-CF594, RPE-AlexaFluor® tandem conjugate; RPE-Alexa610, RPE-TxRed, APC-H7, APC-R700, APC-Alexa600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5, and APC-Cy7.

24. 14. The conjugate of claim 12 or 13, wherein the detectable label is a fluorescent protein selected from the group consisting of GFP, eGFP, BFP, CFP, YFP, DsRed, DsRed2, mRFP, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mPlum, and mRaspberry.

25. 14. The conjugate of claim 12 or claim 13, wherein the detectable label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase.

26. The detectable label may be carbon (C), chromium (Cr), cobalt (Co), fluorine (F), gadolinium (Gd, Gd), germanium (Ge), holmium (Ho), indium (In, In, In, In), iodine (I, I, I), lanthanum (La), lutetium (Lu), manganese (Mn), molybdenum (Mo), palladium (Pd), phosphorus (P), praseodymium (Pr), promethium (P 14. The conjugate of claim 12 or claim 13, comprising a radionuclide selected from the group consisting of rhenium (Re, Re), rhodium (Rh), ruthenium (Ru), samarium (Sm), scandium (Sc), selenium (Se), (Sr), sulfur (S), technetium (Tc), thallium (Ti), tin (Sn, Sn), tritium (H), xenon (Xe), ytterbium (Yb, Yb), and yttrium (Y).

27. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

28. A host cell comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 or the polynucleotide of claim 27.

29. A composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a conjugate according to any one of claims 12 to 26, a polynucleotide according to claim 27, or a host cell according to claim 28.

30. A method for producing an antibody or antigen-binding fragment thereof, comprising expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 in a host cell and recovering or isolating the antibody.

31. A kit comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a conjugate according to any one of claims 12 to 26, and instructions for use.

32. 27. A method for detecting expression of an anti-BCMA CAR on a T cell, the method comprising contacting the T cell with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the conjugate of any one of claims 12 to 26, and detecting formation of an antibody:anti-BCMA CAR complex.

33. 27. A method of detecting expression of an anti-BCMA CAR in a T cell population, the method comprising contacting the population of T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the conjugate of any one of claims 12 to 26, and detecting formation of an antibody:anti-BCMA CAR complex in the one or more T cells.

34. 27. A method of determining expression of an anti-BCMA CAR on a T cell, the method comprising contacting a T cell with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11, or the conjugate of any one of claims 12 to 26, detecting the formation of an antibody:anti-BCMA CAR complex, and measuring the amount of the complex to determine the expression of the anti-BCMA CAR on the T cell.

35. 27. A method of determining expression of an anti-BCMA CAR in a T cell population, the method comprising contacting the T cell population with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 or the conjugate of any one of claims 12 to 26, detecting formation of an antibody:anti-BCMA CAR complex in one or more T cells, and measuring the amount of the complex to determine the expression of the anti-BCMA CAR on the one or more T cells.

36. Anti-BCMA CAR in T cell populations ＋ 27. A method of determining the number of T cells, comprising contacting a population of T cells with the antibody or antigen-binding fragment thereof of any one of claims 1 to 11 or the conjugate of any one of claims 12 to 26, detecting formation of an antibody:anti-BCMA CAR complex in one or more T cells, and enumerating the T cells that express the anti-BCMA CAR.

37. 37. The method of any one of claims 32-36, wherein formation of the antibody:anti-BCMA CAR complex is detected using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore®), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS).

38. 36. The method of claim 34 or claim 35, wherein the amount of the antibody:anti-BCMA CAR complex is measured using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore®), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS).

39. 37. The method of claim 36, wherein T cells expressing the anti-BCMA CAR are enumerated using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore®), fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting (FACS).