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.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 685,043, filed on June 14, 2018, which is incorporated herein by reference in its entirety.

[0003] Statement regarding sequence listing

[0004] The sequence table associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence table is BLBD_099_01WO_ST25.txt. The text file is 19KB, created on June 14, 2019 and submitted electronically through EFS-Web at the same time as submitting this specification. Technical Field

[0005] The present invention relates to antibody compositions. More specifically, the present invention relates to antibodies and antigen-binding fragments that bind to anti-BCMA CAR and conjugates thereof, and methods for detecting anti-BCMA CAR T cells using the same. Background Art

[0006] Genetic approaches offer a potential means of enhancing immune recognition and elimination of cancer cells. One promising strategy is to genetically engineer immune effector cells to express chimeric antigen receptors (CARs) that redirect cytotoxicity toward tumor cells. However, it is difficult to accurately measure CAR expression on one or more adoptively transferred CAR T cells and the persistence of CAR T cells in vivo, which confounds attempts to measure CAR T cell activity. The most commonly used method to evaluate CAR T cells is quantitative PCR using CAR-specific primers. However, this technique has shortcomings and does not allow for multi-parameter analysis of CAR T cells. Summary of the invention

[0007] The present invention generally provides antibodies and antigen-binding fragments thereof, conjugates thereof, and use thereof to detect, determine or measure CAR on one or more T cells. + Methods of T cell and / or CAR expression.

[0008] In various embodiments, an antibody or antigen-binding fragment thereof comprises one or more CDRs that bind an anti-BCMA chimeric antigen receptor (CAR).

[0009] In certain embodiments, said one or more CDRs bind to an anti-BCMA scFv domain of said anti-BCMA CAR.

[0010] In some embodiments, three light chain CDRs bind to the anti-BCMA scFv domain of the anti-BCMA CAR.

[0011] In certain embodiments, three heavy chain CDRs bind to the anti-BCMA scFv domain of the anti-BCMA CAR.

[0012] In certain embodiments, three light chain CDRs and three heavy chain CDRs bind to the anti-BCMA scFv domain of the anti-BCMA CAR.

[0013] In certain embodiments, the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA scFv sequence shown in SEQ ID NO:25.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof is human.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof is humanized.

[0016] In certain embodiments, the antibody or antigen-binding fragment thereof is murine.

[0017] In certain embodiments, the antibody or antigen-binding fragment thereof is chimeric.

[0018] In certain embodiments, the antibody or antigen-binding fragment thereof is monoclonal.

[0019] In certain embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0020] In certain 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), Fv, a single-chain Fv protein ("scFv"), a bis-scFv, (scFv)2, a miniantibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0021] In some embodiments, the antibody or antigen-binding fragment thereof is a scFv.

[0022] In various embodiments, an antibody or antigen-binding fragment thereof comprises a variable light chain comprising the CDRL1-CDRL3 sequence shown in SEQ ID NOs: 1-3 and a variable heavy chain comprising the CDRH1-CDRH3 sequence shown in SEQ ID NOs: 4-6.

[0023] In further embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:7.

[0024] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the variable heavy chain sequence set forth in SEQ ID NO:8.

[0025] In further 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.

[0026] In various embodiments, an antibody or antigen-binding fragment thereof comprises a variable light chain comprising the CDRL1-CDRL3 sequence shown in SEQ ID NOs: 9-11 and a variable heavy chain comprising the CDRH1-CDRH3 sequence shown in SEQ ID NOs: 12-14.

[0027] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:15.

[0028] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the variable heavy chain sequence set forth in SEQ ID NO:16.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:15 and the variable heavy chain sequence shown in SEQ ID NO:16.

[0030] In various embodiments, an antibody or antigen-binding fragment thereof comprises a variable light chain comprising the CDRL1-CDRL3 sequence set forth in SEQ ID NOs: 17-19 and a variable heavy chain comprising the CDRH1-CDRH3 sequence set forth in SEQ ID NOs: 20-22.

[0031] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence set forth in SEQ ID NO:23.

[0032] In certain embodiments, the antibody or antigen-binding fragment thereof comprises the variable heavy chain sequence shown in SEQ ID NO:24.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises the variable light chain sequence shown in SEQ ID NO:23 and the variable heavy chain sequence shown in SEQ ID NO:24.

[0034] In certain embodiments, the antibody or antigen-binding fragment thereof is human.

[0035] In certain embodiments, the antibodies or antigen-binding fragments thereof are humanized.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof is murine.

[0037] In certain embodiments, the antibodies or antigen-binding fragments thereof are chimeric.

[0038] In certain embodiments, the antibody or antigen-binding fragment thereof is monoclonal.

[0039] In certain embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment.

[0040] 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), Fv, a single-chain Fv protein ("scFv"), a bis-scFv, (scFv)2, a miniantibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody).

[0041] In certain embodiments, the antibody or antigen-binding fragment thereof is a scFv.

[0042] In certain embodiments, the antibody or antigen-binding fragment thereof binds to one or more epitopes of the anti-BCMA scFv sequence shown in SEQ ID NO:25.

[0043] In various embodiments, a conjugate includes an antibody or antigen-binding fragment thereof contemplated herein and a means for detection.

[0044] In various embodiments, a conjugate includes an antibody or antigen-binding fragment thereof contemplated herein and a detection device.

[0045] In various embodiments, a conjugate includes an antibody or antigen-binding fragment thereof contemplated herein and a detectable label.

[0046] 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.

[0047] In certain embodiments, the detectable label is a fluorescent dye selected from the group consisting of: Oregon Pacific Blue TM 、Pacific Orange TM 、Pacific Green TMCascade Blue TM CascadeYellow TM Lucifer Yellow TM 、Marina Blue TM and Texas (TxRed).

[0048] In certain embodiments, the detectable label is selected from the group consisting of (AF) Dyes: AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790 and AF800.

[0049] In some embodiments, the detectable label is selected from the group consisting of 525, 565, 585, 605, 655, 705 and 800.

[0050] In certain embodiments, the detectable label is a DyLight TM Dyes (DL): DL549, DL649, DL680 and DL800.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 514Atto 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 TM 436.Super Bright TM 600、SuperBright 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、BrilliantViolet TM 650、Brilliant Violet TM 711、Brilliant Violet TM 786、Brilliant TM Ultraviolet395(BUV395)、Brilliant TM Ultraviolet 496(BUV496)、Brilliant TM Ultraviolet 563(BUV563)、Brilliant TM Ultraviolet 661(BUV661)、Brilliant TM Ultraviolet 737(BUV737)、Brilliant TM Ultraviolet 805(BUV805)、Brilliant TM Blue 515(BB515), Brilliant TMBlue 700 (BB700), as well as IR Dye 680, IR Dye 680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR Dye 800RS and IR Dye 800CW.

[0055] 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, Tandem conjugates; RPE-Alexa610, RPE-TxRed, APC-H7, APC-R700, APC-Alexa600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5 and APC-Cy7.

[0056] 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.

[0057] In particular embodiments, the detectable label is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase.

[0058] In certain embodiments, the detectable label is a radionuclide selected from the group consisting of carbon (14C), chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd, 159Gd), germanium (68Ge), holmium (166Ho), indium (115In, 113In, 112In, mIn), iodine (125I, 123I, 121I), lanthanum (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium (154Mn), iodine (155In, ... (103Pd), 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).

[0059] In various embodiments, a polynucleotide encodes a contemplated antibody or antigen-binding fragment thereof.

[0060] In certain embodiments, a host cell comprises an antibody or antigen-binding fragment thereof or a polynucleotide contemplated herein.

[0061] In some embodiments, a composition includes an antibody or antigen-binding fragment thereof, conjugate, polynucleotide, or host cell contemplated herein.

[0062] In various embodiments, a method of producing an antibody or antigen-binding fragment thereof comprises: expressing the antibody or antigen-binding fragment thereof contemplated herein in a host cell; and recovering or isolating the antibody.

[0063] In certain embodiments, a kit includes an antibody or antigen-binding fragment thereof or conjugate contemplated herein and instructions for use.

[0064] In certain embodiments, a 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 conjugate thereof, as contemplated herein; and detecting formation of an antibody:anti-BCMA CAR complex.

[0065] In various embodiments, a method of detecting 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 conjugate thereof, as contemplated herein; and detecting formation of an antibody:anti-BCMA CAR complex on said one or more T cells.

[0066] In certain embodiments, a 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 conjugate thereof, contemplated herein; detecting 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.

[0067] In a specific embodiment, a method of determining the expression of an anti-BCMA CAR in a T cell population comprises: contacting a T cell population with an antibody or antigen-binding fragment thereof, or conjugate thereof, contemplated herein; detecting the formation of an antibody:anti-BCMA CAR complex on 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.

[0068] In various embodiments, a method of 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, conjugate contemplated herein; detecting the formation of an antibody: anti-BCMA CAR complex on one or more T cells; and enumerating the T cells expressing the anti-BCMA CAR.

[0069] In some embodiments, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy or flow cytometry, fluorescence activated cell sorting (FACS) are used to detect the formation of the antibody: anti-BCMA CAR complex.

[0070] In certain embodiments, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy or flow cytometry, fluorescence activated cell sorting (FACS) are used to measure the amount of the antibody: anti-BCMA CAR complex.

[0071] In specific embodiments, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy or flow cytometry, fluorescence activated cell sorting (FACS) are used to enumerate the T cells expressing the anti-BCMA CAR. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Representative FACs graphs of anti-BCMA CAR antibodies binding to a T cell population comprising an anti-BCMA CAR (SEQ ID NO: 25) are shown.

[0073] A brief description of sequence identifiers

[0074] SEQ ID NOs: 1-3 show the amino acid sequences of exemplary light chain CDR sequences of antibodies that bind to anti-BCMA CAR.

[0075] SEQ ID NOs: 4-6 show the amino acid sequences of exemplary heavy chain CDR sequences of antibodies that bind to anti-BCMA CAR.

[0076] SEQ ID NO: 7 shows the amino acid sequence of an exemplary light chain sequence of an antibody that binds to anti-BCMA CAR.

[0077] SEQ ID NO: 8 shows the amino acid sequence of an exemplary heavy chain sequence of an antibody that binds to anti-BCMA CAR.

[0078] SEQ ID NOs:9-11 show the amino acid sequences of exemplary light chain CDR sequences of antibodies that bind anti-BCMA CAR.

[0079] SEQ ID NOs: 12-14 show the amino acid sequences of exemplary heavy chain CDR sequences of antibodies that bind to anti-BCMA CAR.

[0080] SEQ ID NO: 15 shows the amino acid sequence of an exemplary light chain sequence of an antibody that binds to anti-BCMA CAR.

[0081] SEQ ID NO: 16 shows the amino acid sequence of an exemplary heavy chain sequence of an antibody that binds to anti-BCMA CAR.

[0082] SEQ ID NOs: 17-19 show the amino acid sequences of exemplary light chain CDR sequences of antibodies that bind anti-BCMA CAR.

[0083] SEQ ID NOs: 20-22 show the amino acid sequences of exemplary heavy chain CDR sequences of antibodies that bind anti-BCMA CAR.

[0084] SEQ ID NO: 23 shows the amino acid sequence of an exemplary light chain sequence of an antibody that binds to anti-BCMA CAR.

[0085] SEQ ID NO: 24 shows the amino acid sequence of an exemplary heavy chain sequence of an antibody that binds to anti-BCMA CAR.

[0086] SEQ ID NO:25 shows the amino acid sequence of anti-BCMACAR bound by the antibody including the sequence shown in SEQ ID NO:1-24.

[0087] SEQ ID NOs: 26-36 show the amino acid sequences of various linkers. DETAILED DESCRIPTION

[0088] A. Overview

[0089] The present invention generally relates to antibodies and antigen-binding fragments and conjugates thereof, compositions and methods of using the same that bind to anti-BCMA chimeric antigen receptors (CARs). In particularly preferred embodiments, the present invention relates to antibodies, fragments and conjugates, compositions and uses thereof that bind to anti-BCMA CARs shown in SEQ ID NO: 25.

[0090] 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 binds to 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). In non-malignant cells, BCMA is reported to be expressed mostly in plasma cells and subsets of mature B cells (see, e.g., Laabi et al., EMBO J., 77(1):3897-3904, 1992; Laabi et al., Nucleic Acids Res., 1993). Res., 22(7):1147-1154, 1994; Kalled et al., 2005; O'Connor et al., Journal of Exp. Med., 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 the 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):2111-2114, 2001). BCMA has been commonly detected in multiple myeloma cells and other lymphomas. BCMA RNA is expressed in the blood, and BCMA protein has been detected on the surface of plasma cells of 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).

[0091] Anti-BCMA CAR provides a potentially curative one-time therapy for subjects with multiple myeloma or B-cell lymphoma. Existing methods for associating anti-BCMA CAR expression and activity are suboptimal and need to be improved. Using existing methods, it is difficult to accurately associate both anti-BCMA CAR expression and activity in vivo and in vitro and track anti-BCMA CAR T cell survival and persistence in patients. Antibodies, fragments and conjugates, compositions and related methods of use contemplated herein in combination with anti-BCMA CAR provide solutions to these and other problems.

[0092] In various embodiments, antibodies or antigen-binding fragments thereof that bind to an anti-BCMA antibody or portion thereof are provided. In certain embodiments, the antibodies or antigen-binding fragments thereof may further include one or more detectable labels.

[0093] Also disclosed herein are methods of using the antibodies and antigen-binding fragments thereof, conjugates thereof, and related compositions.

[0094] Techniques of 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 can be generally performed as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Current Protocols in Molecular Biology (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, Volumes I and II (IRL Press, Oxford University Press, USA). USA, 1985); Current Protocols in Immunology (by John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober, ed., 2001); 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 (edited by N. Gait, 1984); Nucleic Acid Hybridization, ed., 1996; Hybridization (B.Hames and S.Higgins, 1985); Transcription and Translation (B.Hames and S.Higgins, 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 ed., 2010, Humana Press); Immobilized Cells and Enzymes (IRL Press, 1986); Methods in Enzymology (Academic Press, Inc., New York); Gene Transfer Vectors for Mammalian Cells (Gene Transfer Vectors for Mammalian Cells). Cells (JH Miller and MP Calos, eds., 1987, Cold Spring Harbor Laboratory Press); Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998); Immunochemical Methods in Cell and Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook of Experimental Immunology, Vols. I to IV (DM Weir and CC Blackwell, eds., 1986); Roitt, Essential Immunology, 6th ed. (Blackwell Scientific Publications, Oxford, 1988); Contemporary Protocols in Immunology (QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober, eds., 1991); Annual Review of Immunology (eds., 1993); Immunology; and monographs in journals such as Advances in Immunology.

[0095] B. Definition

[0096] Before setting forth the present disclosure in greater detail, an understanding of the invention may be helpful to provide definitions of certain terms used herein.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art to which the invention belongs. Although any method and material similar to or equivalent to the method and material described herein can be used to practice or test a specific embodiment, preferred embodiments of compositions, methods and materials are described herein. For purposes of this disclosure, the following terms are defined as follows. Additional definitions are set forth throughout this disclosure.

[0098] The articles "a", "an" and "the" are used herein to refer to one or more than one (i.e., at least one or one or more) of the grammatical object of the article. For example, "element" means one element or one or more elements.

[0099] The use of alternatives (eg, "or") should be understood to mean one, two, or any combination of the alternatives.

[0100] The term "and / or" should be understood to mean one or both of the alternatives.

[0101] 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% compared 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 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%. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0102] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the stated steps or elements or groups of steps or elements, but not the exclusion of any other steps or elements or groups of steps or elements. "Consisting of" means including and limited to, no matter what follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or facilitate the activities or actions specified for the listed elements in the present disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but no other elements are present that substantially affect the activities or actions of the listed elements.

[0103] References throughout this specification to "one embodiment," "an embodiment," "a specific 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 combination with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the above phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, in one or more embodiments, particular features, structures, or characteristics may be combined in any suitable manner. It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for excluding the feature in a specific embodiment.

[0104] C. Antibodies

[0105] In certain embodiments, antibodies or antigen-binding fragments thereof that bind to an anti-BCMA CAR are provided.

[0106] The term "antibody" refers to a binding agent that is a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region or fragment thereof that specifically recognizes and binds one or more epitopes of an antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing antigenic determinants, such as those recognized by immune cells.

[0107] "Epitope" or "antigenic determinant" refers to the region of an antigen to which a binding agent binds. An epitope can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein. Epitopes formed by contiguous amino acids are generally retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are generally lost upon treatment with denaturing solvents. An epitope generally comprises at least 3, and more commonly at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation.

[0108] An "isolated antibody or antigen-binding fragment thereof" is an antibody or antigen-binding fragment thereof that has been identified and separated and / or recovered from a component of its natural environment.

[0109] As used herein, the term "specific binding affinity" or "specifically binds" or "specifically binds" or "specifically binds" or "specifically targets" describes an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR with a binding affinity greater than background binding. 5 M -1 Affinity or K a The antibody or antigen-binding fragment thereof "specifically binds" to the anti-BCMA CAR if the antibody or antigen-binding fragment thereof binds or associates with the anti-BCMA CAR at an equilibrium association constant of greater than or equal to about 10 (i.e., the equilibrium association constant for a specific binding interaction in units of 1 / M). In certain embodiments, the antibody or antigen-binding fragment thereof binds or associates with the anti-BCMA CAR at an equilibrium association constant of greater than or equal to about 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 or 10 13 M -1 K a Binding to anti-BCMA CAR. K of a “high affinity” antibody or antigen-binding fragment thereof a For 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 larger.

[0110] Alternatively, affinity can be defined as the equilibrium dissociation constant (K) in M ​​for a specific binding interaction. d )(For example, 10 -5 M to 10-13 M or less). The affinity of the antibodies or antigen-binding fragments thereof contemplated herein can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or displacement assay using a labeled ligand, or using a surface plasmon resonance apparatus such as the Biacore T100 available from Biacore, Inc., Piscataway, NJ, or optical biosensor technology such as the EPIC system or EnSpire available from Corning and Perkin Elmer, respectively (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).

[0111] In one embodiment, the affinity of specific binding is about 2 times greater than background binding, about 5 times greater than background binding, about 10 times greater than background binding, about 20 times greater than background binding, about 50 times greater than background binding, about 100 times greater than background binding, or about 1000 times greater than background binding or more.

[0112] "Antigen (Ag)" refers to a compound, composition or substance that can stimulate antibody production or T cell response in an animal, including a composition injected or absorbed into an animal (such as a composition comprising a cancer-specific protein). Antigens react with products having specific humoral or cellular immunity, including products induced by heterologous antigens such as disclosed antigens. In a specific embodiment, the target antigen includes an anti-BCMA CAR.

[0113] Antibodies include antigen-binding fragments thereof, such as 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"), bi-scFv, (scFv)2, miniantibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv") and single domain antibodies (sdAb, nanobodies) as well as the portion of the full-length antibody responsible for antigen binding. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heterologous conjugate 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., WH Freeman & Co., New York, 1997.

[0114] As 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 a first, second and third constant region, 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. Immunoglobins including α, δ, ε, γ and μ heavy chains are classified as immunoglobin (Ig) A, IgD, IgE, IgG and IgM. Complete antibodies form a "Y" shape. The stem of the Y consists of the second constant region and the third constant region (and for IgE and IgM, the fourth constant region) of the two heavy chains combined together, and a (interchain) disulfide bond is formed in the hinge. Heavy chains γ, α and δ have a constant region consisting of three series (on a line) Ig domains and a hinge region for increasing flexibility; heavy chains μ and ε have a constant region consisting of four immunoglobin domains. The second constant region and the third constant region are called "CH2 domain" and "CH3 domain", respectively. Each arm of the Y contains the variable region of a single heavy chain combined with the variable region and constant region of a single light chain and the first constant region. The variable regions of the light and heavy chains are responsible for antigen binding.

[0115] The light and heavy chain variable regions comprise 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 by sequence according to 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, USD Department of Health and Human Services, 1991, which is incorporated herein by reference) or by structure according to 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)).

[0116] An illustrative example of a rule for predicting a light chain CDR includes: CDR-L1 starts at about residue 24, after Cys, is about 10-17 residues and is followed by Trp (usually Trp-Tyr-Gln, but can also be Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 starts at about 16 residues after the end of CDR-L1, usually after Ile-Tyr and also Val-Tyr, Ile-Lys, Ile-Phe and is 7 residues; and CDR-L3 starts at about 33 residues after the end of CDR-L2, after Cys, is 7-11 residues and is followed by Phe-Gly-XXX-Gly (SEQ ID NO: 37) (XXX is any amino acid).

[0117] An illustrative example of a rule for predicting heavy chain CDRs includes: CDR-H1 starts at about residue 26, after Cys-XXX-XXX-XXX (SEQ ID NO: 38), is 10-12 residues and is followed by a Trp (usually Trp-Val, but can also be Trp-Ile, Trp-Ala); CDR-H2 starts about 15 residues after the end of CDR-H1, usually at Leu-Glu-Trp-Ile-Gly (SEQ ID NO: NO:39) or multiple variations, followed by 16-19 residues and followed by Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala; and CDR-H3 begins approximately 33 residues after the end of CDR-H2, after Cys-XXX-XXX (usually Cys-Ala-Arg), followed by 3 to 25 residues and followed by Trp-Gly-XXX-Gly (SEQ ID NO:40).

[0118] In one embodiment, the light chain CDRs and heavy chain CDRs are determined according to the Kabat method.

[0119] In one embodiment, the light chain CDR 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 compromise between the Kabat method and the Clothia method, see, e.g., Whitelegg N. and Rees AR, Protein Eng, December 2000, 13(12):819-24 and Methods Mol Biol. 2004; 248:51-91. Programs for predicting CDRs are publicly available, e.g., AbYsis (www.bioinf.org.uk / abysis / ).

[0120] The sequences of the framework regions of different light or heavy chains are relatively conservative in species such as humans. The framework regions of the combined framework regions of the constitutive light and heavy chains of antibodies are used to locate and arrange 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 generally referred to as CDR1, CDR2, and CDR3, which are numbered sequentially from the N-terminus, and are generally also identified by the chain in which a specific CDR is located. Therefore, the CDRs located in the variable domains of the heavy chain of an antibody are referred to as CDRH1, CDRH2, and CDRH3, while the CDRs located in the variable domains of the light chain of an antibody are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., having different combination 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 in the CDRs are directly involved in antigen binding. These positions in the CDRs are referred to as specificity determining residues (SDRs). Illustrative examples of light chain CDRs include the CDR sequences shown in SEQ ID NOs: 1-3, 9-11, and 17-19. Illustrative examples of heavy chain CDRs include the CDR sequences shown in SEQ ID NOs: 4-6, 12-14, and 20-22.

[0121] As disclosed herein, reference to "VL" or "VL" refers to the variable region of an immunoglobin light chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab or other antibody fragment. Illustrative examples of light chain variable regions include the light chain variable region sequences shown in SEQ ID NOs: 7, 15 and 23.

[0122] As disclosed herein, reference to "VH" or "VH" refers to the variable region of an immunoglobin heavy chain, including the variable region of an antibody, Fv, scFv, dsFv, Fab or other antibody fragment. Illustrative examples of heavy chain variable regions include the heavy chain variable region sequences shown in SEQ ID NOs: 8, 16 and 24.

[0123] "Monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells into which the light and heavy chain genes of a single antibody have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by making hybrid antibody-forming cells based on the fusion of myeloma cells with immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0124] "Chimeric antibodies" have framework residues from one species, such as human, and CDRs (which typically confer antigen binding) from another species, such as mouse. In certain preferred embodiments, the antibody includes an antigen-specific binding domain that is a chimeric antibody or antigen-binding fragment thereof.

[0125] In a particular embodiment, the antibody is a human antibody (such as a human monoclonal antibody) or a fragment thereof. As described above, human antibodies can be constructed by combining one or more Fv clone variable domain sequences selected from a human phage display library with one or more known human constant domain sequences. Alternatively, human monoclonal antibodies can be prepared by a hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described, for example, by: Kozbor, Journal of Immunology, 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., Journal of Immunology, 147: 86 (1991). In addition, transgenic animals (e.g., mice) can be used to produce a complete human antibody library in the absence of endogenous immunoglobulin production. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. 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 obtain human antibodies from non-human, e.g., rodent antibodies, wherein the human antibodies have similar affinity and specificity to the starting non-human antibodies. (See PCT WO 93 / 06213 published April 1, 1993). Unlike conventional humanization of non-human antibodies by CDR grafting, this technique provides fully human antibodies that do not have non-human FR or CDR residues.

[0126] 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 CDR is referred to as a "donor", and the human immunoglobulin providing the framework is referred to as an "acceptor". In one embodiment, all CDRs are from the donor immunoglobulin in the humanized immunoglobulin. It is not necessary to have a constant region, but if present, the constant region must be substantially the same as the human immunoglobulin constant region, that is, at least about 85-90% such as about 95% or more identical. Therefore, except for possible CDRs, all parts of the humanized immunoglobulin are substantially the same as the corresponding parts of the natural human immunoglobulin sequence. Humanization or other monoclonal antibodies may have additional conservative amino acid substitutions, which have substantially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed by genetic engineering (see, e.g., U.S. Patent No. 5,585,089).

[0127] In certain embodiments, antibodies or antigen-binding fragments thereof that bind to anti-BCMA CAR include, but are not limited to, camelid Ig (camelid antibodies (VHH)), Ig NARs, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv antibodies ("scFv"), bis-scFv, (scFv)2, miniantibodies, diabodies, triabodies, tetrabodies, disulfide-stabilized Fv proteins ("dsFv"), and single domain antibodies (sdAb, nanobodies).

[0128] As used herein, "camel Ig" or "camelid VHH" refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-Nolte et al., FASEB J., 21:3490-3498 (2007)). "Heavy chain antibody" or "camelid antibody" refers to an antibody comprising two VH domains and no light chain (Riechmann L. et al., J. Immunol. Methods 231:25-38 (1999); WO 94 / 04678; WO 94 / 25591; U.S. Pat. No. 6,005,079).

[0129] "IgNAR" for "immunoglobin new antigen receptor" refers to a class of antibodies from the shark immune repertoire that consists of a homodimer of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains. IgNARs represent some of the smallest immunoglobin-based protein scaffolds known and are highly stable and have efficient binding properties. The inherent stability can be attributed to both: (i) the underlying Ig scaffold, which presents a considerable number of charged and hydrophilic surface-exposed residues compared to conventional antibody VH and VL domains found in murine antibodies; and (ii) stabilizing structural features in the complementarity determining region (CDR) loops, which contain inter-loop disulfide bonds and intra-loop hydrogen bonding patterns.

[0130] 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, whose name reflects its ability to crystallize readily. Pepsin treatment yields a F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0131] "Fv" is the smallest antibody fragment containing a complete antigen binding site. In one embodiment, a double-chain Fv species is composed of a dimer of a heavy chain variable domain and a light chain variable domain in a tight non-covalent association. In a single-chain Fv (scFv) species, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker so that the light chain and the heavy chain can associate into a "dimer" structure similar to the dimer structure in the double-chain Fv species. It is in this structure that the three hypervariable regions (HVR) of each variable domain interact to define the antigen binding site on the surface of the VH-VL dimer. Six HVRs jointly confer antigen binding specificity to antibodies. However, even a single variable domain (or only half of an Fv including three HVRs specific for an antigen) has the ability to recognize and bind antigens, although with a lower affinity than a complete binding site.

[0132] The Fab fragment comprises a heavy chain variable domain and a light chain variable domain and also comprises a constant domain of a light chain and a first constant domain (CH1) of a heavy chain. The difference between a Fab' fragment and a Fab fragment is that several residues are added to the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the hinge region of the antibody. Fab'-SH is the nomenclature for Fab' herein, wherein one or more cysteine ​​residues of the constant domain carry a free thiol group. The F(ab')2 antibody fragment was originally produced as a Fab' fragment pair with hinge cysteine ​​therebetween. Other chemical couplings of antibody fragments are also known. A bispecific Fab dimer (Fab2) has two Fab' fragments, each of which binds to different antigens. A trispecific Fab trimer (Fab3) has three Fab' fragments, each of which binds to different antigens.

[0133] The term "diabody" refers to an antibody fragment with two antigen binding sites, the fragment comprising 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 the complementary domains of another chain and produce two antigen binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med., 9:129-134 (2003); and Hollinger et al., Proceedings of the National Academy of Sciences of the United States of America 90:6444-6448 (1993). Tribodies and tetrabodies are also described in Hudson et al., Nat. Med., 9:129-134 (2003).

[0134] "Single domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment consisting of the variable region of the antibody heavy chain (VH domain) or the variable region of the antibody light chain (VL domain) (Holt, L. et al., Trends in Biotechnology, 21(11):484-490).

[0135] "Single-chain Fv" or "scFv" antibody fragments include the VH domain and VL domain of an antibody, wherein these domains are present in a single polypeptide chain and in any orientation (e.g., VL-VH or VH-VL). Typically, the scFv polypeptide further includes a polypeptide linker between the VH domain and the VL domain that enables the scFv to form a desired structure for antigen binding. For a review of scFv, see Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds., (Springer Verlag, New York, 1994), pp. 269-315.

[0136] In a particular embodiment, the antigen binding fragment is a scFv. In a particular embodiment, the scFv is a murine, human or humanized scFv. Single-chain antibodies can be cloned from the V region genes of a hybridoma that is specific for a desired target. The production of such hybridomas has become routine. Techniques that can be used to clone variable region heavy chains (VH) and variable region light chains (VL) have been described, for example, in Orlandi et al., Proceedings of the National Academy of Sciences of the United States of America, 1989; 86: 3833-3837.

[0137] In various embodiments, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO: 1-3, 9-11, or 17-19 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO: 4-6, 12-14, or 20-22.

[0138] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence as set forth in any one of SEQ ID NOs: 7, 15, or 23 and / or a variable heavy chain sequence as set forth in any one of SEQ ID NOs: 8, 16, or 24. 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, 15, or 23 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 any one of SEQ ID NOs: 8, 16, or 24.

[0139] In one embodiment, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises a light chain CDR sequence shown in SEQ ID NO: 1-3, 9-11 or 17-19. In a specific embodiment, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA antibody or antibody fragment or anti-BCMA scFv portion of the anti-BCMA CAR comprises a light chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the light chain CDR sequence shown in SEQ ID NO: 1-3, 9-11 or 17-19.

[0140] In one embodiment, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises a heavy chain CDR sequence shown in SEQ ID NO: 4-6, 12-14 or 20-22. In a specific embodiment, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises a heavy chain CDR sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid identity with the heavy chain CDR sequence shown in SEQ ID NO: 4-6, 12-14 or 20-22.

[0141] In certain embodiments, the antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR comprises one or more light chain CDRs as set forth in any one of SEQ ID NOs: 1-3 and / or one or more heavy chain CDRs as 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 as set forth in SEQ ID NO: 7 and / or a variable heavy chain sequence as set forth in SEQ ID NO: 8.

[0142] In some embodiments, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises one or more light chain CDRs as shown in any one of SEQ ID NOs: 9-11 and / or one or more heavy chain CDRs as shown in any one of SEQ ID NOs: 12-14. In further embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 15 and / or a variable heavy chain sequence as shown in SEQ ID NO: 16.

[0143] In additional embodiments, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises one or more light chain CDRs as shown in any one of SEQ ID NOs: 17-19 and / or one or more heavy chain CDRs as shown in any one of SEQ ID NOs: 20-22. In specific embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO: 23 and / or a variable heavy chain sequence as shown in SEQ ID NO: 24.

[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 the anti-BCMA CAR shown in SEQ ID NO:25.

[0145] D. Conjugate

[0146] In various embodiments, a conjugate is provided, comprising an antibody or an antigen binding fragment thereof and a label. In a preferred embodiment, a conjugate comprises an antibody or an 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, a conjugate comprises an antibody or an antigen binding fragment thereof that binds to an anti-BCMA CAR and is coupled to a detectable label. In an even more preferred embodiment, a conjugate comprises an antibody or an antigen binding fragment thereof that binds to an anti-BCMA CAR and is covalently bound to a detectable label or chemically coupled to a detectable label.

[0147] As used herein, the term "label" refers to a detectable marker or a label that can produce a detectable signal. In certain embodiments, the label includes 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.

[0148] Illustrative examples of detectable labels suitable for use in the conjugates contemplated in particular 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.

[0149] Illustrative examples of molecules suitable for use as detectable labels in certain embodiments include, but are not limited to: Oregon Pacific Blue TM ;Pacific Orange TM ;Pacific Green TM Cascade Blue TM Cascade Yellow TM;Lucifer Yellow TM ; Marina Blue TM ;Texas (TxRed); (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; Nanocrystals, e.g. 525, 565, 585, 605, 655, 705 and 800; DyLight TM Dyes (DL), such as DL549, DL649, DL680 and DL800; fluorescein or its derivatives, such as fluorescein isothiocyanate, carboxyfluorescein and dichlorotriazinylamine fluorescein; digoxin; dinitrophenol (DNP); trinitrophenol (TNP); biotin; Cy dyes, such as Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5; phycoerythrin (PE, R-phycoerythrin (RPE)); B-phycoerythrin (BPE); peridinin chlorophyll (PerCP); allophycocyanin (APC); and C-phycocyanin; Dyes such as Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514, Atto520, Atto532, 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 and Atto 740; Super Bright TM Dyes, such as Super Bright TM 436.Super Bright TM 600、Super Bright TM 645、Super Bright TM 702 and Super Bright TM 780; Brilliant TMDyes, such as Brilliant TM Violet 421, Brilliant TM Violet480, Brilliant TM Violet 510, Brilliant TM Violet 605, Brilliant Violet TM 650、BrilliantViolet TM 711、Brilliant Violet TM 786、Brilliant TM Ultraviolet 395(BUV395)、Brilliant TM Ultraviolet 496(BUV496)、Brilliant TM Ultraviolet 563(BUV563)、Brilliant TM Ultraviolet 661(BUV661)、Brilliant TM Ultraviolet 737(BUV737)、Brilliant TM Ultraviolet 805(BUV805)、Brilliant TM Blue 515 (BB515) and Brilliant TM Blue700 (BB700); and IR dyes such as IR dye 680, IR dye 680LT, IR dye 700, IR dye 700DX, IR dye 800, IR dye 800RS, and IR dye 800CW.

[0150] Illustrative examples of tandem fluorescent dye molecules suitable for use as detectable labels include, but are not limited to: RPE-Cy5, RPE-Cy5.5, RPE-Cy7, RPE-CF594, Tandem conjugates; RPE-Alexa610, RPE-TxRed, APC-H7, APC-R700, APC-Alexa600, APC-Alexa610, APC-Alexa750, APC-Cy5, APC-Cy5.5 and APC-Cy7.

[0151] 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.

[0152] Illustrative examples of enzymes suitable for use as detectable labels include, but are not limited to, alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase.

[0153] Illustrative examples of radionuclides suitable for use as detectable labels include, but are not limited to, carbon (14C), chromium (51Cr), cobalt (57Co), fluorine (18F), gadolinium (153Gd, 159Gd), germanium (68Ge), holmium (166Ho), indium (115In, 113In, 112In, mIn), iodine (125I, 123I, 121I), lanthanum (140La), lutetium (177Lu), manganese (54Mn), molybdenum (99Mo), palladium ( 103Pd), 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).

[0154] In certain embodiments, a conjugate includes an antibody or antibody fragment conjugated, coupled or connected (e.g., covalently bound) to one or more labels. In certain embodiments, the label can be conjugated, coupled or connected to the antibody or fragment directly or indirectly (e.g., through a linker group). When the antibody and label each have a substituent that can react with each other, the antibody can be directly covalently bound to one or more labels. For example, a nucleophilic group (e.g., an amino group or a sulfhydryl group) on a polypeptide can react with a carbonyl-containing group (e.g., anhydride or acyl halide) on another polypeptide or an alkyl group (e.g., a halide) containing a good leaving group.

[0155] In a particular embodiment, it is desirable that the antibody or antibody fragment be coupled, conjugated or connected to one or more labels by a monovalent linker or a multivalent linker or a spacer. A linker or a spacer can be used to provide sufficient distance between the antibody and the label to avoid steric hindrance or interfere with the antibody binding ability. It will be apparent to those skilled in the art that a variety of bifunctional or multifunctional reagents can be used, including homofunctional and heterofunctional reagents (such as reagents described in the catalog of Pierce Chemical Co., Rockford, IL, Rockford, IL) as linker groups. Coupling can be affected, for example, by amino, carboxyl, sulfhydryl or oxidized carbohydrate residues. There are many references describing this method, for example, U.S. Patent No. 4,671,958 to Rodwell et al.

[0156] In certain embodiments, the linker can have 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, wherein the atoms in the chain include C, S, N, P, and O.

[0157] Illustrative examples of linkers or connections that can be used in specific 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)-, -diazodicarbonamide-(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 of which may be optionally 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 2is one to three difunctional or trifunctional substituted cross-linking organic groups selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl, polyethylene glycol (PEG) [i.e., -(CH2CH2O) 1-20 ].

[0158] In certain embodiments, a conjugate includes an antibody or antibody fragment covalently bound to a polypeptide-based label (eg, a fluorescent protein or an enzyme) via a polypeptide linker as contemplated elsewhere herein below.

[0159] In a preferred embodiment, a conjugate comprises an antibody or antigen-binding fragment thereof conjugated to an anti-BCMA CAR and covalently bound to a PE marker.

[0160] E. Peptide

[0161] 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, a polypeptide comprises an antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR.

[0162] Unless specified to the contrary, "polypeptide", "peptide" and "protein" are used interchangeably and are used according to the conventional meaning (i.e., as an amino acid sequence). Polypeptides are not limited to a specific length, for example, a polypeptide can include a full-length polypeptide or a polypeptide fragment, and can contain one or more post-translational modifications of the polypeptide (e.g., glycosylation, acetylation, phosphorylation, etc.) as well as other naturally occurring and non-naturally occurring modifications known in the art.

[0163] As used herein, "isolated protein", "isolated peptide" or "isolated polypeptide" etc. refers to a peptide or polypeptide molecule that is synthesized, separated and / or purified in vitro from a cellular environment and from the association with other components of the cell, i.e., has no significant association with in vivo substances.

[0164] Polypeptides include "polypeptide variants". Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring or may be produced synthetically, e.g., 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 specific embodiments, the polypeptide comprises a polypeptide 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, typically wherein the variant maintains at least one biological activity of the reference sequence.

[0165] Polypeptides include "polypeptide fragments". Polypeptide fragments refer to polypeptides that can be monomeric or multimeric, with amino-terminal deletions, carboxyl-terminal deletions and / or internal deletions or substitutions of naturally occurring or recombinantly produced polypeptides. Illustrative examples of biologically active polypeptide fragments include antibody fragments. As used herein, the term "biologically active fragment" or "minimum 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 a preferred embodiment, the biological activity is binding affinity to an epitope. In certain embodiments, a polypeptide fragment may include an amino acid chain having a length of at least 5 to about 500 amino acids. It will be appreciated that in certain embodiments, the fragment length is 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 6, 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. Particularly useful polypeptide fragments include functional domains, which include antigen binding domains or antibody fragments. In the case of antibodies, useful fragments include but are not limited to: one or more CDR regions, CDR3 regions of a heavy chain or light chain; a variable region of a heavy chain or light chain; a portion of an antibody chain or a variable region comprising two CDRs; etc.

[0166] As noted above, polypeptides can be altered in various ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are well known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutation of the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA 82:488-492); Kunkel et al., (1987, Methods in Enzymol, 154:367-382); U.S. Pat. No. 4,873,192; Watson, JD et al., (Molecular Biology of the Gene, 4th ed., Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0167] In certain embodiments, polypeptide variants include one or more conservative substitutions. "Conservative substitutions" are substitutions in which an amino acid is substituted by another amino acid with similar properties so that the secondary structure and hydrophilicity of the polypeptide are expected to be substantially unchanged by the technician in the field of peptide chemistry. Preferably, the amino acid changes of the protein variants disclosed herein are conservative amino acid changes, i.e., the substitution of amino acids with similar charges or uncharged amino acids. Conservative amino acid changes involve the substitution of an amino acid in a family of amino acids related in terms of side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and polar uncharged 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, appropriate conservative amino acid substitutions are known to those skilled in the art, and can generally be carried out without changing the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter the biological activity (see, e.g., Watson et al., Molecular Biology of the Gene, 4th ed., 1987, Benjamin / Cummings Publishing Company, p. 224).

[0168] Polypeptide variants further include glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). As known in the art, covalent variants can be prepared by linking the function to a group found in an amino acid chain or at the N-terminal or C-terminal residue. Variants also include allelic variants, species variants, and mutant proteins. Regions that do not affect the functional activity of the protein are also variants, such as truncations or deletions.

[0169] In certain embodiments, the polypeptide includes a polypeptide linker. "Linker" refers to a plurality of amino acid residues between two or more polypeptide domains added for proper spacing, conformation, and function of the molecule.

[0170] In certain embodiments, the polypeptide linker is a variable region linker sequence. A "variable region linker sequence" is an amino acid sequence that links V H Domain and V L domains and provides a spacer function that is compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region.

[0171] In certain embodiments, the conjugate includes a polypeptide linker that covalently binds the antibody or antibody fragment to one or more protein-based labels (eg, a fluorescent protein or an enzyme).

[0172] Illustrative examples of linkers suitable for use in the specific embodiments contemplated herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO: 26); TGEKP (SEQ ID NO: 27) (see, e.g., Liu et al., Proc. Natl. Acad. Sci. USA 5525-5530 (1997)); GGRR (SEQ ID NO: 28) (Pomerantz et al., 1995, supra); (GGGGS)n, where n = 1, 2, 3, 4 or 5 (SEQ ID NO: 29) (Kim et al., Proc. Natl. Acad. Sci. USA 93, 1156-1160 (1996)); EGKSSGSGSESKVD (SEQ ID NO: 30) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 31) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); NO:31) (Bird et al., 1988, Science 242:423-426); GGRRGGGS (SEQ ID NO:32); LRQRDGERP (SEQ ID NO:33); LRQKDGGGSERP (SEQ ID NO:34); LRQKD(GGGS)2ERP (SEQ ID NO:35). Alternatively, flexible linkers can be rationally designed using computer programs that can model both the DNA binding site and the peptide itself (Desjarlais and Berg, Proc. Natl. Acad. Sci. USA 90:2256-2260 (1993), Proc. Natl. Acad. Sci. USA 91:11099-11103 (1994)) or by phage display methods. In one embodiment, the linker comprises the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 36) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).

[0173] In a preferred embodiment, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1-24.

[0174] F. Polynucleotides

[0175] In a preferred embodiment, a polynucleotide encoding an antibody or antigen-binding fragment thereof binding to an anti-BCMA CAR is provided. As used herein, the term "polynucleotide" or "nucleic acid" refers to messenger RNA (mRNA), RNA, genomic DNA (gDNA), complementary DNA (cDNA) or recombinant DNA. Polynucleotides include single-stranded polynucleotides and double-stranded polynucleotides. In particular embodiments, the 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 to any of the reference sequences contemplated herein. In various illustrative embodiments, the polynucleotides encoding the polypeptides contemplated herein include, but are not limited to, SEQ ID NOs: 1-24.

[0176] In a specific embodiment, polynucleotides encoding at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750 or 2000 or more and all intermediate lengths of consecutive amino acid residues of a polypeptide are provided. It is readily understood that in this context, "intermediate length" means any length between the cited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0177] In a particular embodiment, the polynucleotide may be codon optimized. As used herein, the term "codon optimized" refers to replacing codons in a polynucleotide encoding a polypeptide to increase the expression, stability and / or activity of the polypeptide. Factors affecting codon optimization include, but are not limited to, one or more of the following: (i) changes in codon bias or synthetically constructed bias tables between two or more organisms or genes; (ii) changes in codon bias within an organism, gene, or group of genes; (iii) systematic changes in codons including background; (iv) changes in codons according to their decoding tRNA; (v) changes in codons according to GC% in general or at one position in a triplet; (vi) changes in similarity to a reference sequence, such as a naturally occurring sequence; (vii) changes in codon frequency cutoffs; (viii) structural properties of mRNA transcribed from a DNA sequence; (ix) prior knowledge of the function of the DNA sequence on which the codon substitution set is designed; (x) systematic changes in the codon set for each amino acid; and / or (xi) removal of isolated spurious translation start sites.

[0178] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity to a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides, compared to a reference polynucleotide. In this regard, it is well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, may be made to a reference polynucleotide, whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

[0179] 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 of 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, 106, 107, 108, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 300, 311, 321, 33 In the present invention, the present invention relates to a polynucleotide fragment of 10, 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 that encodes a polypeptide variant 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 the naturally occurring polypeptide. A polynucleotide fragment refers to a polynucleotide that encodes a polypeptide having an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution of one or more amino acids of a naturally occurring or recombinantly produced polypeptide.

[0180] As used herein, the statement "sequence identity" or, for example, including "a sequence that is 50% identical to ... " refers to the degree to which the sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis within a comparison window. Therefore, the "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences within the comparison window, determining the number of positions at which 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) appears in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity. %, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity to any of the reference sequences described herein, typically wherein the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0181] 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" is at least 12, but typically 15 to 18 and often at least 25 monomer units in length, comprising nucleotides and amino acid residues. Because two polynucleotides can each include (1) a sequence that is similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence) and (2) a sequence that differs between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides within a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of at least 6 consecutive positions, typically about 50 to about 100, more typically about 100 to about 150, in which a sequence is compared to a reference sequence having the same number of consecutive positions after the two sequences are optimally aligned. The comparison window can include about 20% or less addition or deletion (that is, gap) compared with the reference sequence (the reference sequence does not include addition or deletion) for the best comparison of the two sequences. The best comparison for comparing the sequence of the comparison window can be by the computerized implementation scheme of the algorithm (GAP, BESTFIT, FASTA and TFASTA of Wisconsin Genetics Software Package 7.0 version (WisconsinGenetics Software Package Release 7.0) of No. 575 Science Avenue, Madison, Wisconsin, USA, Genetics Computer Group (Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by checking and by any one of the selected various methods The best comparison generated (that is, resulting in the highest percentage homology in the comparison window) is carried out. It can also be referenced as, for example, by Altschul et al., 1997, " Nucleic Acids Research " 25: BLAST program family disclosed in 3389. A detailed discussion of sequence analysis can be found in Ausubel et al., Current Molecular Biology Laboratory Manual, John Wiley & Sons, Inc., 1994-1998, Chapter 15, Unit 19.3.

[0182] As used herein, "isolated polynucleotide" refers to a polynucleotide that has been purified from its naturally occurring sequences on both sides, for example, a DNA fragment that has been removed from sequences that are normally adjacent to it. "Isolated polynucleotide" also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature and have been made by human hands.

[0183] In addition, it will be appreciated by those of ordinary skill in the art that, due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides or variant fragments thereof, as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native sequence. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon selection, are specifically contemplated in certain embodiments.

[0184] Various established techniques well known and available in the art can be used to prepare, manipulate and / or express polynucleotides. In order to express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector.

[0185] G. Composition

[0186] Compositions contemplated herein may include one or more antibodies or antibody fragments and antigen-binding fragments thereof, conjugates, polypeptides and polynucleotides. Compositions include but are not limited to pharmaceutical compositions. There is virtually no limitation on other components that may also be included in the composition, provided that the additional agent does not adversely affect the function or activity of the active ingredient of the composition.

[0187] In certain embodiments, the compositions of the present invention include a certain amount of an antibody or antigen-binding fragment or conjugate thereof. As used herein, the term "amount" refers to an "effective amount" of an antibody that binds to a target polypeptide.

[0188] The composition may further include one or more carriers or excipients.

[0189] Illustrative examples of carriers suitable for incorporation into the compositions contemplated herein include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl paraben 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 and other carbohydrates including 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 nonionic surfactants such as polyethylene glycol (PEG).

[0190] Acceptable carriers also include, but are not limited to: buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben 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; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; 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 nonionic surfactants such as polyethylene glycol (PEG).

[0191] H. Kit

[0192] In various embodiments, a kit is provided that includes one or more antibodies or antigen-binding fragments thereof that bind to 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.

[0193] In certain embodiments, a kit is provided that includes one or more antibodies or antigen-binding fragments thereof that bind to 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.

[0194] In various embodiments, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO: 1-3, 9-11, or 17-19 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO: 4-6, 12-14, or 20-22.

[0195] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in any one of SEQ ID NOs: 7, 15 or 23 and / or a variable heavy chain sequence as shown in any one of SEQ ID NOs: 8, 16 or 24.

[0196] 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 scFv fragment shown in SEQ ID NO:25.

[0197] In a specific embodiment, the kit includes an antibody or antibody fragment and a detectable label and a conjugation description. In a specific embodiment, the kit includes an antibody or antibody fragment coupled with a detectable label and instructions for use. In a preferred embodiment, the detectable label is directly detectable. In a more preferred embodiment, the detectable label is a fluorescent label. In an even more preferred embodiment, the detectable label is RPE.

[0198] I. Methods

[0199] Anti-BCMA CAR T cell activity is partially related to anti-BCMA CAR expression on immune effector cells. The ability to accurately assess anti-BCMA CAR expression ex vivo or in vitro and in vivo is needed to estimate drug product activity and track the survival and persistence of anti-BCMA CAR T cells in patients.

[0200] In certain embodiments, methods using one or more antibodies or antigen-binding fragments thereof are provided. The antibodies are used to detect or measure the expression of anti-BCMA CAR. The antibodies, fragments and conjugates contemplated herein are used to bind, detect, determine, identify, measure, quantify and / or enumerate the presence or level of anti-BCMA CAR expression and / or anti-BCMA CAR in a population. + The number of immune effector cells.

[0201] In various embodiments, a method includes detecting anti-BCMA CAR expression on immune effector cells. Immune effector cells (e.g., T cells, NK cells, NKT cells) are contacted with antibodies or their antigen-binding fragments, which are sufficient to bind to anti-BCMA CAR and form antibodies: Anti-BCMA CAR complexes in an effective amount and time to bind to one or more epitopes of anti-BCMA CAR.

[0202] In a particular embodiment, a method includes detecting anti-BCMA CAR expression on one or more immune effector cells in an immune effector cell population. One or more immune effector cells (e.g., T cells, NK cells, NKT cells) are contacted with an antibody or its antigen-binding fragment, which is sufficient to bind the antibody to anti-BCMA CAR and form an antibody: an effective amount and time for an anti-BCMA CAR complex to bind to one or more epitopes of the anti-BCMA CAR. In a particular embodiment, at least 25% of the immune effector cells are 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 + .

[0203] In certain embodiments, a method includes determining the amount of expression of anti-BCMA CAR on immune effector cells.Immune effector cells (e.g., T cells, NK cells, NKT cells) are contacted with antibodies or their antigen-binding fragments, which are sufficient to bind to anti-BCMA CAR and form antibodies: effective amounts of anti-BCMA CAR complexes and time to bind one or more epitopes of anti-BCMA CAR.Antibodies are measured: the amount of anti-BCMA CAR complexes and compared with controls.

[0204] In certain embodiments, a method includes determining the amount of expression of anti-BCMA CAR on one or more immune effector cells in immune effector cell colonies.One or more immune effector cells (e.g., T cells, NK cells, NKT cells) are contacted with an antibody or its Fab, and the antibody or its Fab is enough to make antibody binding anti-BCMA CAR and form an antibody: the effective amount of anti-BCMA CAR complexes and time combine one or more epitopes of anti-BCMA CAR.Antibody is measured: the amount of anti-BCMA CAR complexes and compared with a control.

[0205] In certain embodiments, a method comprises determining an anti-BCMA CAR in a population of immune effector cells. + 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 its antigen-binding fragment, which is sufficient to bind the antibody to anti-BCMA CAR and form an antibody: an effective amount of an anti-BCMA CAR complex and time to bind one or more epitopes of the anti-BCMA CAR scFv part of CAR. Then, it is possible to enumerate the immune effector cells including antibodies: anti-BCMA CAR complexes.

[0206] In certain embodiments, detecting, determining, or enumerating anti-BCMA CAR expression or anti-BCMA CAR + T cell approaches include administration of anti-BCMA CAR + In certain embodiments, the sample contains or is suspected of containing anti-BCMA CAR +T cells. In preferred embodiments, the sample is an apheresis sample, a leukapheresis sample, peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, or a tumor biopsy.

[0207] In certain embodiments, the antibodies and conjugates contemplated herein that bind to anti-BCMA CARs are used to study anti-BCMA CARs by detecting anti-BCMA CARs in subjects and / or samples collected from subjects at one or more time points. + The pharmacokinetics, expansion and / or persistence of T cells measured or assessed. In a specific embodiment, after administering an anti-BCMA CAR to a subject + The samples are collected, isolated and / or taken from the subject at 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 one year, or every week, every month, or every year after the T cells are taken.

[0208] In various embodiments, the antibody or antigen-binding fragment thereof that binds to the anti-BCMA CAR comprises a variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO: 1-3, 9-11, or 17-19 and / or a variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO: 4-6, 12-14, or 20-22.

[0209] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in any one of SEQ ID NOs: 7, 15 or 23 and / or a variable heavy chain sequence as shown in any one of SEQ ID NOs: 8, 16 or 24.

[0210] 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 the anti-BCMACAR shown in SEQ ID NO:25.

[0211] In a particular embodiment, the method of detecting, determining or enumerating anti-BCMA CAR expression or anti-BCMA CAR+T cells includes the use of an antibody or antibody fragment conjugated to a detectable marker. In a preferred embodiment, the detectable marker is directly detectable. In a more preferred embodiment, the detectable marker is a fluorescent marker. In an even more preferred embodiment, the detectable marker is RPE.

[0212] In particular embodiments, the presence of the antibody: anti-BCMA CAR complex can be detected, determined, and / or enumerated using one or more of the following assays: immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance (SPR)-based biosensor (e.g., BIAcore), fluorescence microscopy, flow cytometry, fluorescence activated cell sorting (FACS), or western blot.

[0213] In a preferred embodiment, flow cytometry is used to detect, determine and / or enumerate the presence of antibody: anti-BCMA CAR complexes. In a more preferred embodiment, FACs are used to detect, determine and / or enumerate the presence of antibody: anti-BCMA CAR complexes.

[0214] In specific embodiments, a background expression level of 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 the one or more immune effector cells or used to normalize the measurement results.

[0215] All publications, patent applications, and issued patents cited in this specification are herein incorporated by reference to the same extent as if each individual publication, patent application, or issued patent was specifically and individually indicated to be incorporated by reference.

[0216] Although the above invention has been described in some detail by way of illustration and example for the purpose of clear understanding, it should be readily apparent to those of ordinary skill in the art, inspired by the teachings of the present invention, that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize various non-critical parameters that may be changed or modified to produce substantially similar results.

[0217] Examples

[0218] Example 1

[0219] Evaluation of Antibodies Targeting Anti-BCMA CAR

[0220] A mouse monoclonal antibody was generated against the anti-BCMA CAR sequence shown in SEQ ID NO:25.

[0221] Peripheral blood mononuclear cells (PBMCs) were cultured in static flasks in medium containing IL-2 (CellGenix) and antibodies specific for CD3 and CD28 (Miltenyi Biotec). One day after the start of culture, 2×10 lentivirus encoding anti-BCMA CAR was added. 8 Transduction units. Anti-BCMA CAR T cells were maintained in the logarithmic phase by adding fresh medium containing IL-2 for a total of ten days of culture. T cells were transduced with anti-BCMA CAR (BCMA-02), humanized anti-BCMA CAR (BCMA-03) or anti-EGFR CAR and expanded for 10 days. CAR expression of CAR T cells was evaluated at the end of culture using three antibodies against the anti-BCMA scFv sequence shown in SEQ ID NO: 25.

[0222] Antibody clones 7G2.A1.B2, 11E6.G2.B5 and 11E6.G7.F2 were conjugated to phycoerythrin (PE). 1×10 6 Anti-BCMA CAR T cells or untransduced control T cells were incubated with 1 μg of antibody conjugate in a total volume of 100 μL for 20 minutes and then washed and fixed in 1% paraformaldehyde. Samples were subjected to FACS analysis to detect, identify, and enumerate anti-BCMA CAR + T cells. All antibody conjugates detected anti-BCMA CAR expression on approximately 70% of cells, Figure 1 .

[0223] In summary, 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 range of equivalents to which such claims are entitled. Therefore, the claims are not limited by the present disclosure. Sequence Listing <110> bluebird bio, Inc. Friedman, Kevin Perkins, Molly Reed <120> Anti-BCMA CAR antibodies, conjugates, and methods of use <130> BLBD-099 / 01WO 315698-2946 <150> US 62 / 685,043 <151> 2018-06-04 <160> 40 <170> PatentIn Version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR1 <400> 1 Lys Ala Ser Gln Asp Val Asp Thr Thr Val Ala 1 5 10 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR2 <400> 2 Trp Ala Ser Thr Arg His Thr 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR3 <400> 3 Gln Gln Tyr Thr Phe Tyr Pro Tyr Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR1 <400> 4 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 5 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR2 <400> 5 Glu Ile Asn Pro Arg Asn Gly Arg Ser Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR6 <400> 6 Glu Val His Tyr Tyr Gly Ser Asp Tyr Asp Ala Met Asp Phe 1 5 10 <210> 7 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Lab-made variable light chain <400> 7 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Thr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Thr Phe Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 8 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - variable heavy chain <400> 8 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ser Cys 85 90 95 Ala Arg Glu Val His Tyr Tyr Gly Ser Asp Tyr Asp Ala Met Asp Phe 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 9 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Light chain CDR1 <400> 9 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR2 <400> 10 Trp Ala Ser Thr Arg His Thr 1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR3 <400> 11 Gln Gln Tyr Asn Thr Tyr Pro Tyr Thr 1 5 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR1 <400> 12 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> 13 <211> 17 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR2 <400> 13 Glu Ile Asn Pro Arg Asn Gly Arg Ser Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR6 <400> 14 Glu Val His Tyr Tyr Gly Ser Asp Tyr Asp Ala Met Asp Tyr 1 5 10 <210> 15 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Lab-made variable light chain <400> 15 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Thr Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - variable heavy chain <400> 16 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ser Cys 85 90 95 Ala Arg Glu Val His Tyr Tyr Gly Ser Asp Tyr Asp Ala Met Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 17 <211> 11 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR1 <400> 17 Lys Ala Ser Gln Asp Val Asp Thr Thr Val Ala 1 5 10 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR2 <400> 18 Trp Ala Ser Thr Arg His Thr 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - light chain CDR3 <400> 19 Gln Gln Tyr Thr Phe Tyr Pro Tyr Thr 1 5 <210> 20 <211> 10 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR1 <400> 20 Gly Tyr Thr Phe Thr Ser Tyr Trp Met His 1 5 10 <210> twenty one <211> 17 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR2 <400> twenty one Glu Ile Asn Pro Arg Asn Gly Arg Ser Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> twenty two <211> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - Heavy chain CDR6 <400> twenty two Glu Val His Tyr Tyr Gly Ser Asp Tyr Asp Ala Met Asp Phe 1 5 10 <210> 23 <211> 107 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - variable light chain <400> 23 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Asp Thr Thr 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Thr Phe Tyr Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 24 <211> 123 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Prepared in the laboratory - variable heavy chain <400> 24 Glu Val Gln Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Arg Asn Gly Arg Ser Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Ser Cys 85 90 95 Ala Arg Glu Val His Tyr Tyr Gly Ser Asp Tyr Asp Ala Met Asp Phe 100 105 110 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Pro 115 120 <210> 25 <211> 493 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Laboratory preparation - BCMA CAR <400> 25 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu 20 25 30 Ala Met Ser Leu Gly Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu 35 40 45 Ser Val Thr Ile Leu Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys 50 55 60 Pro Gly Gln Pro Pro Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln 65 70 75 80 Thr Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe 85 90 95 Thr Leu Thr Ile Asp Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr 100 105 110 Cys Leu Gln Ser Arg Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys 115 120 125 Leu Glu Ile Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly 130 135 140 Glu Gly Ser Thr Lys Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu 145 150 155 160 Leu Lys Lys Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly 165 170 175 Tyr Thr Phe Thr Asp Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly 180 185 190 Lys Gly Leu Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro 195 200 205 Ala Tyr Ala Tyr Asp Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr 210 215 220 Ser Ala Ser Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp 225 230 235 240 Thr Ala Thr Tyr Phe Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr 245 250 255 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Thr Thr 260 265 270 Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln 275 280 285 Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala 290 295 300 Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 305 310 315 320 Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr 325 330 335 Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 340 345 350 Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser 355 360 365 Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys 370 375 380 Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln 385 390 395 400 Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu 405 410 415 Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg 420 425 430 Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met 435 440 445 Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly 450 455 460 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 465 470 475 480 Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 485 490 <210> 26 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 26 Asp Gly Gly Gly Ser 1 5 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 27 Thr Gly Glu Lys Pro 1 5 <210> 28 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 28 Gly Gly Arg Arg 1 <210> 29 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 29 Gly Gly Gly Gly Ser 1 5 <210> 30 <211> 14 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 30 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> 31 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 31 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 32 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 32 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 33 Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> 34 <211> 12 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 34 Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10 <210> 35 <211> 16 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 35 Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro 1 5 10 15 <210> 36 <211> 18 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary linker sequences <400> 36 Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 37 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Rules for Determining Light Chain CDR-L3 Motifs <220> <221> SITE <222> (3) <223> Xaa is any amino acid <400> 37 Phe Gly Xaa Gly 1 <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Rules for Determining Heavy Chain CDR-H1 Motifs <220> <221> SITE <222> (2)..(4) <223> Xaa is any amino acid <400> 38 Cys Xaa Xaa Xaa 1 <210> 39 <211> 5 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Rules for Determining Heavy Chain CDR-H2 Motifs <400> 39 Leu Glu Trp Ile Gly 1 5 <210> 40 <211> 4 <212> PRT <213> Artificial Sequence <220> <221> <222> <223> Exemplary Rules for Determining Heavy Chain CDR-H3 Motifs <220> <221> SITE <222> (3) <223> Xaa is any amino acid <400> 40 Trp Gly Xaa Gly 1

Claims

1. An antibody or antigen-binding fragment thereof that binds to an anti-BCMA chimeric antigen receptor (CAR), the antibody or antigen-binding fragment thereof comprising: a) a variable light chain comprising a CDRL1-CDRL3 sequence as shown in SEQ ID NOs: 1-3, and a variable heavy chain comprising a CDRH1-CDRH3 sequence as shown in SEQ ID NOs: 4-6; or b) a variable light chain comprising the CDRL1-CDRL3 sequence as shown in SEQ ID NOs: 9, 2 and 11, and a variable heavy chain comprising the CDRH1-CDRH3 sequence as shown in SEQ ID NOs: 4-5 and 14, Wherein the anti-BCMA CAR comprises the scFv sequence shown in SEQ ID NO:

25.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is human, humanized, murine or chimeric.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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), Fv, a single-chain Fv protein ("scFv"), a bis-scFv, (scFv)2, a miniantibody, a diabody, a triabody, a tetrabody, a disulfide-stabilized Fv protein ("dsFv"), and a single domain antibody (sdAb, nanobody). The antibody or antigen-binding fragment thereof according to claim 3 , wherein the antibody or antigen-binding fragment thereof is a scFv.

5. An antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR, comprising a variable light chain comprising a CDRL1-CDRL3 sequence as shown in SEQ ID NOs: 1-3 and a variable heavy chain comprising a CDRH1-CDRH3 sequence as shown in SEQ ID NOs: 4-6, wherein the anti-BCMA CAR comprises the scFv sequence shown in SEQ ID NO:

25.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein the variable light chain comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO:

7.

7. The antibody or antigen-binding fragment thereof according to claim 5, wherein the variable light chain comprises an amino acid sequence that is at least 95% identical to the amino acids shown in SEQ ID NO:

7.

8. The antibody or antigen-binding fragment thereof of claim 5, wherein the variable light chain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

7.

9. The antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, wherein the antibody or antigen-binding fragment thereof is a scFv.

10. The antibody or antigen-binding fragment thereof according to any one of claims 5 to 8, wherein the variable heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO:

8.

11. The antibody or antigen-binding fragment thereof according to any one of claims 5-8, wherein the variable heavy chain comprises an amino acid sequence that is at least 95% identical to the amino acids shown in SEQ ID NO:

8.

12. The antibody or antigen-binding fragment thereof according to any one of claims 5-8, wherein the variable heavy chain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

8.

13. The antibody or antigen-binding fragment thereof according to claim 10, wherein the antibody or antigen-binding fragment thereof is a scFv.

14. The antibody or antigen-binding fragment thereof according to claim 5, wherein the variable light chain sequence comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO: 7, and the variable heavy chain sequence comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO:

8.

15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the antibody or antigen-binding fragment thereof is a scFv.

16. The antibody or antigen-binding fragment thereof according to claim 5, comprising the variable light chain sequence shown in SEQ ID NO:

7.

17. The antibody or antigen-binding fragment thereof according to claim 5 or claim 16, comprising the variable heavy chain sequence shown in SEQ ID NO:

8.

18. The antibody or antigen-binding fragment thereof according to claim 5, comprising the variable light chain sequence shown in SEQ ID NO: 7 and the variable heavy chain sequence shown in SEQ ID NO:

8.

19. An antibody or antigen-binding fragment thereof that binds to an anti-BCMA CAR, comprising a variable light chain comprising a CDRL1-CDRL3 sequence as shown in SEQ ID NOs: 9, 2 and 11 and a variable heavy chain comprising a CDRH1-CDRH3 sequence as shown in SEQ ID NOs: 4-5 and 14, wherein the anti-BCMA CAR comprises the scFv sequence shown in SEQ ID NO:

25.

20. The antibody or antigen-binding fragment thereof of claim 19, wherein the variable light chain comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO:

15.

21. The antibody or antigen-binding fragment thereof of claim 19, wherein the variable light chain comprises an amino acid sequence that is at least 95% identical to the amino acids shown in SEQ ID NO:

15.

22. The antibody or antigen-binding fragment thereof of claim 19, wherein the variable light chain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

15.

23. The antibody or antigen-binding fragment thereof according to any one of claims 19-22, wherein the antibody or antigen-binding fragment thereof is a scFv.

24. The antibody or antigen-binding fragment thereof according to any one of claims 19-22, wherein the variable heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO:

16.

25. The antibody or antigen-binding fragment thereof according to any one of claims 19-22, wherein the variable heavy chain comprises an amino acid sequence that is at least 95% identical to the amino acids shown in SEQ ID NO:

16.

26. The antibody or antigen-binding fragment thereof according to any one of claims 19-22, wherein the variable heavy chain comprises an amino acid sequence that is at least 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

16.

27. The antibody or antigen-binding fragment thereof of claim 19, wherein the variable light chain comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO: 15, and the variable heavy chain comprises an amino acid sequence that is at least 90% identical to the amino acids shown in SEQ ID NO:

16.

28. The antibody or antigen-binding fragment thereof according to claim 27, wherein the antibody or antigen-binding fragment thereof is a scFv.

29. The antibody or antigen-binding fragment thereof of claim 19, comprising the variable light chain sequence shown in SEQ ID NO:

15.

30. The antibody or antigen-binding fragment thereof of claim 19 or claim 29, comprising the variable heavy chain sequence shown in SEQ ID NO:

16.

31. The antibody or antigen-binding fragment thereof of claim 19, comprising the variable light chain sequence shown in SEQ ID NO: 15 and the variable heavy chain sequence shown in SEQ ID NO:

16.

32. The antibody or antigen-binding fragment thereof of claim 5, comprising the variable heavy chain sequence shown in SEQ ID NO:

24.

33. The antibody or antigen-binding fragment thereof of claim 5, comprising the variable light chain sequence shown in SEQ ID NO: 7 and the variable heavy chain sequence shown in SEQ ID NO:

24.

34. A conjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33; and a detectable label.

35. The conjugate of claim 34, wherein the detectable label is selected from the group consisting of: a) haptens, fluorescent dyes, fluorescent proteins, chromophores, metal ions, gold particles, silver particles, magnetic particles, peptides, enzymes, luminescent compounds or oligonucleotides; b) 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; c) QDot® selected from the group consisting of Qdot® 525, Qdot® 565, Qdot® 585, Qdot® 605, Qdot® 655, Qdot® 705 and Qdot® 800; d) a hapten selected from the group consisting of fluorescein or its derivatives, fluorescein isothiocyanate, carboxyfluorescein, dichlorotriazinylamine fluorescein, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP) and biotin; e) a Cy dye selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 and Cy7.5; f) 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; g) a fluorescent dye selected from the group consisting of: 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, Atto 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740 and IR Dye 680, IR Dye 680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR Dye 800RS and IR Dye 800CW; h) 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; i) 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; j) an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase and β-galactosidase; and k) a radionuclide selected from the group consisting of carbon, chromium, cobalt, fluorine, gadolinium, germanium, holmium, indium, iodine, lanthanum, lutetium, manganese, molybdenum, palladium, phosphorus, praseodymium, promethium, rhenium, rhodium, ruthenium, samarium, scandium, selenium, sulfur, technetium, thallium, tin, tritium, xenon, ytterbium and yttrium.

36. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33.

37. A host cell comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or the polynucleotide according to claim 36, wherein the host cell is of a non-animal or plant species.

38. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33, the conjugate according to claim 34 or 35, the polynucleotide according to claim 36, or the host cell according to claim 37.

39. A method of producing an antibody or an antigen-binding fragment thereof, the method comprising expressing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 in a host cell and recovering or isolating the antibody.

40. A kit comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or the conjugate according to claim 34 or 35 and instructions for use.

41. A method for detecting the expression of anti-BCMA CAR on T cells for non-disease diagnosis and treatment purposes, the method comprising: contacting T cells with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or the conjugate according to claim 34 or 35; and detecting formation of an antibody: anti-BCMA CAR complex.

42. A method for detecting the expression of anti-BCMA CAR in a T cell population for non-disease diagnosis and treatment purposes, the method comprising: contacting a population of T cells with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or a conjugate according to claim 34 or 35; and detecting the formation of an antibody:anti-BCMACAR complex on the one or more T cells.

43. A method for determining expression of an anti-BCMA CAR on a T cell for non-disease diagnostic and therapeutic purposes, the method comprising: Contacting T cells with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or the conjugate according to claim 34 or 35; 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 cells.

44. A method for determining the expression of an anti-BCMA CAR in a T cell population for non-disease diagnostic and therapeutic purposes, the method comprising: Contacting a population of T cells with the antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or the conjugate according to claim 34 or 35; detecting formation of an antibody:anti-BCMA CAR complex on one or more T cells; and measuring the amount of the complex to determine expression of the anti-BCMA CAR on the one or more T cells.

45. A method for identifying anti-BCMA CAR in a T cell population + A method for non-disease diagnosis and treatment of T cell population, the method comprising: Contacting a population of T cells with an antibody or antigen-binding fragment thereof according to any one of claims 1 to 33 or a conjugate according to claim 34 or 35; detecting the formation of an antibody: anti-BCMA CAR complex on one or more T cells; and enumerating the T cells expressing the anti-BCMA CAR.

46. ​​The method of any one of claims 41 to 45, wherein immunohistochemistry, enzyme-linked immunosorbent assay, immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance-based biosensor, fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting is used to detect the formation of the antibody:anti-BCMA CAR complex.

47. The method of claim 43 or claim 44, wherein immunohistochemistry, enzyme-linked immunosorbent assay, immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance-based biosensor, fluorescence microscopy, flow cytometry, or fluorescence-activated cell sorting is used to measure the amount of the antibody: anti-BCMA CAR complex.

48. The method of claim 45, wherein immunohistochemistry, enzyme-linked immunosorbent assay, immunosorbent assay, chemiluminescent assay, electrochemiluminescent assay, surface plasmon resonance-based biosensor, fluorescence microscopy or flow cytometry, fluorescence activated cell sorting is used to enumerate the T cells expressing the anti-BCMA CAR.

Images