Anti-bcma car antibodies, conjugates, and methods of use

Abstract

The present invention provides improved methods for detecting anti-BCMA CAR expression on T cells. The present invention generally provides antibodies and antigen-binding fragments thereof, conjugates thereof, and methods of using the same to detect, determine, or measure CAR T cell and / or CAR expression on one or more T cells.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 948,488, filed December 16, 2019, pursuant to 35 U.S. SC §119(e), which is incorporated herein by reference in its entirety.

[0003] Declaration of sequence list

[0004] The sequence list relating to this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The text file containing the sequence list is named BLUE-97PC2_ST25.txt. The text file is 26KB in size, created on December 10, 2020, and submitted electronically via EFS-Web at the same time as this specification is filed. Background Technology Technical Field

[0006] This invention relates to antibody compositions. More specifically, this invention relates to antibodies and antigen-binding fragments that bind to anti-BCMA CAR, and conjugates thereof, as well as methods for detecting anti-BCMA CAR T cells using said antibodies and antigen-binding fragments.

[0007] Existing technology

[0008] Genetic approaches offer potential means to enhance immune recognition and eliminate cancer cells. One promising strategy is to genetically engineer immune effector cells to express chimeric antigen receptors (CARs), which redirect cytotoxicity to tumor cells. However, the difficulty in accurately measuring CAR expression on one or more adoptive CAR T cells and the persistence of CAR T cells in vivo confounds attempts to measure CAR T cell activity. The most common method for evaluating CAR T cells is quantitative PCR using CAR-specific primers. However, this technique is insufficient and does not allow for multiparameter analysis of CAR T cells. Summary of the Invention

[0009] This invention generally provides antibodies and their antigen-binding fragments, conjugates thereof, and methods for using them to detect, determine, or measure CAR. + Methods for CAR expression on T cells and / or one or more T cells.

[0010] In various embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence as shown in SEQ ID NO:7 and a variable heavy chain sequence as shown in SEQ ID NO:8.

[0011] In some implementations, the antibody or its antigen-binding fragment is an antigen-binding fragment.

[0012] In some implementations, the antibody or its antigen-binding fragment is selected from the group consisting of: Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv protein (“scFv”), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein (“dsFv”), and single-domain antibody (sdAb, nanobody).

[0013] In some implementations, the antibody or its antigen-binding fragment is scFv.

[0014] In some embodiments, the antibody or its antigen-binding fragment comprises the light chain sequence shown in SEQ ID NO:11 and the heavy chain sequence shown in SEQ ID NO:12 or SEQ ID NO:13.

[0015] In some embodiments, the antibody or its antigen-binding fragment binds to one or more epitopes of anti-BCMACAR as shown in SEQ ID NO:9.

[0016] In some embodiments, the antibody or its antigen-binding fragment binds to one or more epitopes of anti-BCMACAR as shown in SEQ ID NO:10. In some embodiments, the antibody or antigen-binding fragment is a mouse antibody. In some embodiments, the antibody or antigen-binding fragment is a humanized antibody. In some embodiments, the antibody or antigen-binding fragment is a human antibody.

[0017] In some embodiments, the antibody or antigen-binding fragment includes a constant IgG1, IgG2, IgG3, or IgG4 domain. In various embodiments, the antibody or antigen-binding fragment thereof includes a constant IgG1 domain. In various embodiments, the antibody or antigen-binding fragment thereof is a mouse IgG1 subtype.

[0018] In various implementations, the conjugate comprises an antibody or its antigen-binding fragment covered herein and a means for detection.

[0019] In various implementation schemes, the conjugate comprises an antibody or its antigen-binding fragment covered herein and a detection method.

[0020] In various embodiments, the conjugate comprises an antibody or its antigen-binding fragment as covered herein and a detectable label.

[0021] In some implementations, the detectable markers are selected from the group consisting of: haptens, fluorescent dyes, fluorescent proteins, chromophores, metal ions, gold particles, silver particles, magnetic particles, peptides, enzymes, luminescent compounds, or oligonucleotides.

[0022] In a particular implementation, the detectable marker is selected from the following group: Oregon PacificBlue TM Pacific Orange TM Pacific Green TM Cascade Blue TM Cascade Yellow TM LuciferYellow TM Marina Blue TM and Texas (TxRed).

[0023] In some implementations, the detectable marker is selected from the group consisting of the following: (AF) Dyes: AF350, AF405, AF488, AF500, AF514, AF532, AF546, AF555, AF568, AF594, AF610, AF633, AF635, AF647, AF680, AF700, AF710, AF750, AF790 and AF800.

[0024] In some implementations, the detectable marker contains (AF) dye AF488.

[0025] In some implementations, the detectable marker is selected from a group consisting of the following: and

[0026] In a particular implementation, the detectable marker is DyLight selected from the group consisting of the following. TM Dyes (DL): DL549, DL649, DL680 and DL800.

[0027] In some embodiments, the detectable marker is a hapten selected from the group consisting of fluorescein or its derivatives, fluorescein isothiocyanate, carboxyfluorescein, dichloroimide fluorescein, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), and biotin.

[0028] In a particular embodiment, the detectable marker is a Cy dye selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.5.

[0029] In some implementations, the detectable marker is a fluorescent molecule selected from the group consisting of: phycoerythrin (PE, R-fish phycoerythrin (RPE)), β-phycoerythrin (BPE), polydinophytin (PerCP), allophycocyanin (APC), and C-phycocyanin.

[0030] In a specific embodiment, the detectable label is selected from the group consisting of: Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, Atto 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto700, Atto 725, Atto 740, Super Bright TM 436, Super Bright TM 600, Super Bright TM 645, Super Bright TM 702, Super Bright TM 780, Brilliant TM Violet 421, Brilliant TM Violet480, Brilliant TM Violet 510, Brilliant TM Violet 605, Brilliant Violet TM 650, Brilliant Violet TM 711. Brilliant Violet TM 786, Brilliant TM Ultraviolet 395(BUV395)、Brilliant TM Ultraviolet 496(BUV496)、Brilliant TM Ultraviolet 563(BUV563)、Brilliant TMUltraviolet 661 (BUV661), Brilliant TM Ultraviolet 737(BUV737)、Brilliant TM Ultraviolet 805 (BUV805), Brilliant TM Blue 515 (BB515), Brilliant TM Blue 700 (BB700) and IR Dye 680, IR Dye 680LT, IR Dye 700, IR Dye700DX, IR Dye 800, IR Dye 800RS and IR Dye 800CW.

[0031] In some implementations, the detectable marker 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.

[0032] In some implementations, the detectable marker 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.

[0033] In a particular implementation, the detectable marker is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase.

[0034] In some implementations, radionuclides labeled with a subset of the following can be detected: 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).

[0035] In various implementations, the polynucleotide encodes the covered antibody or its antigen-binding fragment.

[0036] In some implementations, the host cell contains an antibody or its antigen-binding fragment or polynucleotide as covered herein.

[0037] In some embodiments, the composition comprises an antibody or an antigen-binding fragment thereof, a conjugate, a polynucleotide, or a host cell as covered herein.

[0038] In various embodiments, methods for generating antibodies or antigen-binding fragments thereof include expressing the antibodies or antigen-binding fragments covered herein in host cells and recovering or separating the antibodies.

[0039] In some implementations, the kit contains an antibody or its antigen-binding fragment or conjugate covered herein, along with instructions for use.

[0040] In a particular embodiment, the method for detecting the expression of anti-BCMA CAR on T cells includes contacting T cells with an antibody or its antigen-binding fragment or conjugate as covered herein, and detecting the formation of an antibody:anti-BCMA CAR complex.

[0041] In various embodiments, methods for detecting the expression of anti-BCMA CAR in a T cell population include contacting the T cell population with an antibody or its antigen-binding fragment or conjugate as covered herein, and detecting the formation of one or more antibody:anti-BCMA CAR complexes on T cells.

[0042] In some embodiments, the method for determining the expression of anti-BCMA CAR on T cells includes contacting T cells with an antibody or its antigen-binding fragment or conjugate as covered herein, detecting the formation of an antibody:anti-BCMA CAR complex, and measuring the amount of the complex to determine the expression of anti-BCMA CAR on T cells.

[0043] In a particular embodiment, a method for determining the expression of anti-BCMA CAR in a T cell population includes contacting the T cell population with an antibody or its antigen-binding fragment or conjugate as covered 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 anti-BCMA CAR on one or more T cells.

[0044] In various implementations, methods for determining the expression of anti-BCMA CAR+ T cells in a T cell population include contacting the T cell population with an antibody or its antigen-binding fragment or conjugate as covered herein, detecting the formation of one or more antibody-anti-BCMA CAR complexes on T cells, and listing T cells expressing anti-BCMA CAR.

[0045] In some implementations, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR) based biosensors (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence activated cell sorting (FACS) are used to detect the formation of antibody:anti-BCMA CAR complex.

[0046] In some implementations, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR) based biosensors (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence activated cell sorting (FACS) are used to measure the amount of antibody:anti-BCMA CAR complex.

[0047] In certain implementations, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR) based biosensors (e.g., BIAcore), fluorescence microscopy, flow cytometry, or fluorescence activated cell sorting (FACS) are used to enumerate T cells expressing anti-BCMA CAR.

[0048] Brief description of sequence identifiers

[0049] SEQ ID NO:1-3 shows the amino acid sequence of an exemplary light chain CDR sequence of an antibody that binds to anti-BCMA CAR.

[0050] SEQ ID NO:4-6 shows the amino acid sequences of exemplary heavy chain CDR sequences of antibodies that bind to anti-BCMA CAR.

[0051] SEQ ID NO:7 shows an exemplary variable light chain sequence of an antibody that binds to anti-BCMA CAR, representing an amino acid sequence.

[0052] SEQ ID NO:8 shows an exemplary variable heavy chain sequence of an antibody that binds to anti-BCMA CAR, containing an amino acid sequence.

[0053] SEQ ID NO:9 shows the amino acid sequence of anti-BCMACAR bound by an antibody containing the sequences shown in SEQ ID NO:1-8.

[0054] SEQ ID NO:10 shows the amino acid sequence of anti-BCMACAR bound by an antibody containing the sequences shown in SEQ ID NO:1-8.

[0055] SEQ ID NO:11 shows an exemplary light chain sequence of an antibody that binds to anti-BCMA CAR, comprising an amino acid sequence.

[0056] SEQ ID NO:12 shows an exemplary heavy chain sequence of an antibody that binds to anti-BCMA CAR, containing an amino acid sequence.

[0057] SEQ ID NO:13 shows an exemplary heavy chain sequence of an antibody that binds to anti-BCMA CAR, containing an amino acid sequence.

[0058] SEQ ID NO:14-24 shows the amino acid sequences of various linkers. Detailed Implementation

[0059] A. Overview

[0060] This invention generally relates to antibodies and antigen-binding fragments that bind to anti-BCMA chimeric antigen receptor (CAR), conjugates thereof, compositions thereof, and methods of use thereof. In a particular preferred embodiment, the invention relates to antibodies, fragments and conjugates that bind to the anti-BCMA CAR shown in SEQ ID NO:9 or SEQ ID NO:10, and their uses therein.

[0061] BCMA is a member of the tumor necrosis factor receptor superfamily (see, for example, 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, for example, Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204:43-54, 2005). In non-malignant cells, BCMA has been reported to be mainly expressed in plasma cells and mature B cell subsets (see, for example, Laabi et al., EMBO J., 77(1):3897-3904, 1992; Laabi et al., Nucleic Acids Res., 22(7):1147-1154, 1994; Kalled et al., 2005; O'Connor et al., J. Exp. Medicine, 199(1):91-97, 2004; and Ng et al., J. Immunol., 73(2):807-817, 2004). Mice lacking BCMA are healthy and have a normal number of B cells, but the survival of long-lived plasma cells is impaired (see, for example, O'Connor et al., 2004; Xu et al., Mol. Cell. Biol, 21(12):4067-4074, 2001; and Schiemann et al., Science, 293(5537):2 111-21 14, 2001). BCMA RNA has been widely detected in multiple myeloma cells and other lymphomas, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several researchers (see, for example, 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).

[0062] Anti-BCMA CARs offer a potentially curative, one-time therapy for patients with multiple myeloma or B-cell lymphoma. Existing methods for correlating anti-BCMA CAR expression and activity are suboptimal and require improvement. Using existing methods, it is difficult to accurately correlate anti-BCMA CAR expression and activity in vitro and in vivo, and to track the survival and persistence of anti-BCMA CAR cells in patients. The antibodies, fragments and conjugates, compositions, and related methods of use for binding anti-BCMA CARs covered in this article provide solutions to these and other problems.

[0063] In various embodiments, antibodies or antigen-binding fragments thereof that bind to anti-BCMA antibodies or portions thereof are provided. In certain embodiments, the antibody or antigen-binding fragment thereof may also comprise one or more detectable markers.

[0064] This article also discloses methods for using antibodies and their antigen-binding fragments, their conjugates, and related compositions.

[0065] Techniques, immunoassays, tissue cultures, transformations (e.g., electroporation, liposome transfection), enzymatic reactions, purification, and related techniques and procedures used for the synthesis of recombinant (i.e., engineered) DNA, peptides, and oligonucleotides can generally be performed as described in the various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (updated July 2008 by John Wiley and Sons); 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 Univ. Press USA, 1985); Current Protocols in Immunology (Editors: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications, edited by Julie Logan, Kirstin Edwards and Nick Saunders, 2009, Caister Academic Press, Norfolk, UK; Anand, Techniques for the Analysis of Complex Genomes (Academic Press, New York, 1992); Guthrie and Fink, Guide to Yeast Genetics and Molecular Biology (Academic Press, New York, 1991); Oligonucleotide Synthesis (edited by N. Gait, 1984); Nucleic Acid Hybridization (edited by B. Hames & S. Higgins, 1985); Transcription and Translation (edited by B. Hames & S. Higgins, 1984); Animal Cell Culture (edited by R. Freshney, 1986); Perbal, A Practical Guide to Molecular Cloning (1984); Next-Generation Genome Sequencing (Janitz, 2008 Wiley-VCH); PCR Protocols (Methods in Molecular Biology) (edited by Park, 3rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P.Calos (ed., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Immunochemical Methods in Cell and Molecular Biology (eds., Mayer and Walker, Academic Press, London, 1987); Handbook of Experimental Immunology, Volumes I-IV (eds., D.M. Weir and C.C. Blackwell, 1986); Roitt, Essential Immunology, 6th Edition (Blackwell Scientific Publications, Oxford, 1988); Current Protocols in Immunology (eds., Q.E. Coligan, A.M. Kruisbeek, H.D. Margulies, E.M. Shevach, and W. Strober, 1991); Annual Review of Immunology; and feature articles in journals such as Advances in Immunology.

[0066] B. Definition

[0067] Before elaborating on this disclosure in more detail, providing definitions of certain terms used herein may help in understanding this disclosure.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While particular embodiments may be practiced or tested using any methods and materials similar to or equivalent to those described herein, preferred embodiments of the compositions, methods, and materials described herein are preferred. For the purposes of this disclosure, the following terms are defined. Other definitions are set forth throughout this disclosure.

[0069] The article “a / an” as used in this article refers to one or more grammatical objects of the article described (i.e., at least one or more). As an example, “an element” means one element or one or more elements.

[0070] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0071] The term “and / or” should be understood to mean one or both of the alternative options.

[0072] As used herein, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, dimensions, quantity, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length. In one embodiment, the term "about" or "approximately" means a quantity, level, value, number, frequency, percentage, size, quantity, weight, or length that is approximately ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length.

[0073] Throughout this specification, unless the context otherwise requires, the word "comprise / comprises / comprising" should be understood to imply that it includes the stated step or element or group of steps or elements, but does not exclude any other step or element or group of steps or elements. "Constitutes of" means to include and is limited to what follows the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the listed element is essential or necessary, and no other element can exist. "Substantially constitutes of" is intended to include any element listed following the phrase, and is limited to other elements that do not interfere with or affect the activity or function described in the disclosure regarding the listed element. Therefore, the phrase "substantially constitutes of" indicates that the listed element is essential or necessary, and no other element substantially affects the activity or function of the listed element.

[0074] Throughout this specification, the terms "an embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "a particular embodiment," "an additional embodiment," or "another embodiment," or combinations thereof, mean that a specific feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Therefore, the foregoing phrases appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, a specific feature, structure, or characteristic can be combined in one or more embodiments in any suitable manner. It should also be understood that a positive description of a feature in one embodiment serves as the basis for excluding said feature in a particular embodiment.

[0075] C. Antibody

[0076] In a particular implementation, an antibody or antigen-binding fragment thereof that binds to anti-BCMA CAR is provided.

[0077] The term "antibody" refers to a binding agent comprising at least a light chain or heavy chain immunoglobulin variable region or fragment thereof, which specifically recognizes and binds to one or more epitopes of an antigen, such as peptides, lipids, polysaccharides, or nucleic acids containing antigenic determinants, such as those recognized by immune cells.

[0078] An epitope, or antigenic determinant, is a region of an antigen that binds to a binding agent. Epitopes can be formed from consecutive amino acids or from non-consecutive amino acids linked in parallel through the ternary folding of a protein. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed through ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least three, and more usually at least five, about nine, or about eight to ten amino acids in a distinctive spatial conformation.

[0079] "Isolated antibody or antigen-binding fragment thereof" refers to an antibody or antigen-binding fragment thereof that has been identified, isolated and / or recovered from components of its natural environment.

[0080] As used herein, the terms “specific binding affinity,” “specifically bound,” “specific binding,” or “specific targeting” describe antibodies or antigen-binding fragments thereof binding to anti-BCMA CARs with a binding affinity greater than background binding. If the antibody or antigen-binding fragment binds with an affinity greater than or equal to about 10, for example… 5 M -1 Affinity or K a When an antibody or its antigen-binding fragment binds to or associates with an anti-BCMA CAR (i.e., the equilibrium association constant of a specific binding interaction in units of 1 / M), then the antibody or its antigen-binding fragment "specifically binds" to the anti-BCMA CAR. In some embodiments, the antibody or its antigen-binding fragment binds at a ratio 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 aBinding to anti-BCMA CAR. The K+ of a "high-affinity" antibody or its antigen-binding fragment. 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.

[0081] Alternatively, affinity can be defined as the equilibrium dissociation constant (K) of a specific binding interaction. d And in units of M (e.g., 10) -5 M to 10 -13 M, or lower). The affinity of the antibody or its antigen-binding fragment as anticipated herein can be readily determined using conventional techniques, such as by competitive ELISA (enzyme-linked immunosorbent assay), or by binding association, or by displacement assay using labeled ligands, or by surface plasmon resonance devices such as the Biacore T100 (available from Biacore, Inc., Piscataway, NJ), or optical biosensor technologies such as the EPIC system or EnSpire (available from Corning and Perkin Elmer, respectively) (see also, for example, Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Patent Nos. 5,283,173; 5,468,614 or equivalents).

[0082] In one embodiment, the specific binding affinity is approximately 2 times, 5 times, 10 times, 20 times, 50 times, 100 times, or 1000 times or more than the background binding affinity.

[0083] "Antigen (Ag)" means a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, including compositions injected or absorbed into an animal (such as compositions containing cancer-specific proteins). Antigens react with products that induce specific humoral or cellular immunity, including those induced by heterologous antigens such as disclosed antigens. In a particular embodiment, the target antigen comprises an anti-BCMA CAR.

[0084] Antibodies include their antigen-binding fragments, such as camel Ig, Ig NAR, Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins (“scFv”), bis-scFv, (scFv)2, microantibodies, biantibodies, triantibodies, tetraantibodies, disulfide-stabilized Fv proteins (“dsFv”), and single-domain antibodies (sdAb, nanobodies), as well as portions of full-length antibodies responsible for antigen binding. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and their antigen-binding fragments. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd ed., WH Freeman & Co., New York, 1997.

[0085] As those skilled in the art will understand 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. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a “Y” shape. The stem of the Y consists of the second and third constant regions (and, for IgE and IgM, a fourth constant region) of the two heavy chains linked together, and disulfide bonds (interchain) are formed in the hinge. Heavy chains γ, α, and δ have constant regions consisting of three tandem (row) Ig domains, and hinge domains for added flexibility; heavy chains μ and δ have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and the "CH3 domain," respectively. Each arm of the Y chain includes a variable region of a single heavy chain and a first constant region, which bind to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0086] The light chain variable region and the heavy chain variable region contain “framework” regions, also known as “complementarity-determining regions” or “CDRs”, which are interrupted by three hypervariable regions. CDRs can be defined or identified by conventional methods, such as by the 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, USDapartment of Health and Human Services, 1991, which is incorporated by reference), or by the structure according to Chothia et al. (Chothia, C. or Lesk, AM, J Mol. Biol., 196(4):901-917 (1987); Chothia, C. et al., Nature, 342:877-883 (1989)).

[0087] Illustrative examples of rules for predicting light chain CDRs include: CDR-L1 begins at approximately residue 24, preceded by Cys for approximately 10-17 residues, and followed by Trp (usually Trp-Tyr-Gln, but also Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu); CDR-L2 begins at approximately 16 residues after the end of CDR-L1, usually preceded by Ile-Tyr, but also preceded by Val-Tyr, Ile-Lys, Ile-Phe, and consists of 7 residues; and CDR-L3 begins at approximately 33 residues after the end of CDR-L2, preceded by Cys for 7-11 residues, and followed by Phe-Gly-XXX-Gly (XXX is any amino acid) (SEQ ID NO:25).

[0088] Illustrative examples of rules for predicting heavy chain CDRs include: CDR-H1 begins at approximately residue 26, preceded by Cys-XXX-XXX-XXX (SEQ ID NO: 26), consisting of 10-12 residues, followed by Trp (usually Trp-Val, but also Trp-Ile, Trp-Ala); CDR-H2 begins approximately 15 residues after the end of CDR-H1, usually preceded by Leu-Glu-Trp-Ile-Gly (SEQ ID NO: 27), or many variations, consisting of 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, preceded by Cys-XXX-XXX (usually Cys-Ala-Arg), consisting of 3 to 25 residues. The residue is followed by Trp-Gly-XXX-Gly (SEQ ID NO:28).

[0089] In one implementation, the light chain CDR and heavy chain CDR are determined according to the Kabat method.

[0090] In one implementation, 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 combination of the Kabat and Clothia methods, see, for example, Whitelegg N & Rees AR, Protein Eng. Dec 2000; 13(12):819-24 and Methods Mol Biol. 2004; 248:51-91. Procedures for predicting CDRs are publicly available, for example, AbYsis (www.bioinf.org.uk / abysis / ).

[0091] The sequences of the structural regions of different light or heavy chains are relatively conserved within species such as humans. The structural regions of an antibody (i.e., the combined structural regions that make up the light and heavy chains) are used to locate and align CDRs in three-dimensional space. CDRs are primarily responsible for binding one or more epitopes of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are often also identified by the chain in which the specific CDR resides. Thus, CDRs located in the variable domains of the antibody's heavy chain are referred to as CDRH1, CDRH2, and CDRH3, while CDRs located in the variable domains of the antibody's light chain are referred to as CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combination sites for different antigens) have different CDRs. Although CDRs vary from antibody to antibody, only a limited number of amino acid positions within a CDR are directly involved in antigen binding. These positions within a CDR are called assay residues (SDRs). Illustrative examples of light chain CDRs include the CDR sequences shown in SEQ ID NO:1-39. Illustrative examples of heavy chain CDRs include the CDR sequences shown in SEQ ID NO:4-6.

[0092] The term "VL" or "VL" refers to the variable region of an immunoglobulin light chain, including variable regions of antibodies, Fv, scFv, dsFv, Fab, or other antibody fragments as disclosed herein. Illustrative examples of light chain variable regions include the light chain variable region sequence shown in SEQ ID NO:7.

[0093] Mentioning "V" H "VH" or "VH" refers to the variable region of the immunoglobulin heavy chain, including variable regions of antibodies, Fv, scFv, dsFv, Fab, or other antibody fragments as disclosed herein. Illustrative examples of heavy chain variable regions include the heavy chain variable region sequence shown in SEQ ID NO:8.

[0094] "Monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, such as by preparing hybrid antibody-forming cells from a fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0095] A "chimeric antibody" has a framework residue from one species (such as a human) and a CDR (which typically confers antigen binding) from another species (such as a mouse). In a particular preferred embodiment, the antibody comprises an antigen-specific binding domain as a chimeric antibody or an antigen-binding fragment thereof.

[0096] In certain embodiments, the antibody is a human antibody (such as a human monoclonal antibody) or a fragment thereof. Human antibodies can be constructed by combining an Fv clone variable domain sequence selected from a human-derived phage display library with a known human constant domain sequence as described above. Alternatively, human monoclonal antibodies can be prepared using hybridoma methods. Human myeloma and mouse-human allogeneic myeloma cell lines used to generate human monoclonal antibodies have been described, for example, by Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991). Additionally, transgenic animals (e.g., mice) can be used to generate a complete human antibody library in the absence of endogenous immunoglobulin production. See, for example, Jakobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993). Genetic transection can also be used to derive human antibodies from non-humans, such as rodent antibodies, where the human antibody has similar affinity and specificity to the original non-human antibody. (See PCT WO 93 / 06213, published April 1, 1993). Unlike traditional humanization of non-human antibodies via CDR transplantation, this technique provides fully human antibodies that do not contain FR or CDR residues from non-human sources.

[0097] In one embodiment, the antibody is a “humanized” antibody. A humanized antibody is an immunoglobulin comprising a human framework region and one or more core receptors (CDRs) derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin providing the CDR is referred to as the “donor”, ​​and the human immunoglobulin providing the framework is referred to as the “recipient.” In one embodiment, all CDRs are derived from the donor immunoglobulin within the humanized immunoglobulin. The presence of a constant region is not required, but if present, it must be substantially identical to the constant region of the human immunoglobulin, i.e., at least about 85-90%, such as about 95% or more. Thus, apart from the possible CDRs, all portions of the humanized immunoglobulin are substantially identical to the corresponding portions of the native human immunoglobulin sequence. Humanized or other monoclonal antibodies may have additional conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions. Humanized antibodies can be constructed through genetic engineering (see, for example, U.S. Patent No. 5,585,089).

[0098] In certain implementations, antibodies or antigen-binding fragments of the anti-BCMA CAR include, but are not limited to: camel Ig (camel antibody (VHH)), Ig NAR, Fab fragment, Fab' fragment, F(ab')2 fragment, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), Fv, single-chain Fv antibody (“scFv”), bis-scFv, (scFv)2, microantibody, biantibody, triantibody, tetraantibody, disulfide-stabilized Fv protein (“dsFv”), and single-domain antibody (sdAb, nanobody).

[0099] As used herein, “camel Ig” or “camel family VHH” refers to the smallest known antigen-binding unit of a heavy chain antibody (och-Nolte et al., FASEB J., 21:3490-3498 (2007)). “Heavy chain antibody” or “camel family antibody” refers to an antibody containing two VH domains and no light chain (Riechmann L. et al., J. Immunol. Methods 231:25–38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079).

[0100] The term "IgNAR" in "immunoglobulin neoantigen receptor" refers to a class of antibodies derived from a shark immune repertoire, consisting of homodimers of a variable neoantigen receptor (VNAR) domain and five constant neoantigen receptor (CNAR) domains. IgNAR represents some of the smallest known immunoglobulin-based protein scaffolds and is highly stable with efficient binding characteristics. This inherent stability can be attributed to (i) the basic Ig scaffold, which exhibits a large number of charged and hydrophilic surface-exposed residues compared to the VH and VL domains of conventional antibodies found in murine antibodies; and (ii) structural features in the stable complementarity-determining region (CDR) loop, including patterns of intercyclic disulfide bonds and intracyclic hydrogen bonds.

[0101] 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 tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still able to cross-link the antigen.

[0102] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, a double-stranded Fv species consists of a dimer of a tightly, non-covalently bound heavy-chain variable domain and a light-chain variable domain. In a single-stranded Fv (scFv) species, a heavy-chain variable domain and a light-chain variable domain are covalently linked by a flexible peptide linker, allowing the light and heavy chains to associate in a "dimeric" structure similar to that in the double-stranded Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind antigens, although its affinity is lower than that of the entire binding site.

[0103] The Fab fragment contains variable domains of both the heavy and light chains, and also includes a constant domain of the light chain and a first constant domain (CH1) of the heavy chain. The Fab′ fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteine ​​residues from the antibody hinge region. Fab′-SH is referred to herein as Fab′, where the cysteine ​​residues of the constant domain are accompanied by free thiol groups. The F(ab′)2 antibody fragment was initially generated as a pair of Fab′ fragments with a hinge cysteine ​​residue between them. Other chemical conjugations of antibody fragments are also known. The bispecific Fab dimer (Fab2) has two Fab′ fragments, each binding to a different antigen. The trispecific Fab trimer (Fab3) has three Fab′ fragments, each binding to a different antigen.

[0104] The term "biantibody" refers to an antibody fragment having two antigen-binding sites, the fragment comprising a light chain variable domain (VH) linked to a heavy chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to pair between the two domains on the same chain, the domain is forced to pair with a complementary domain of the other chain, creating two antigen-binding sites. Biantibodies can be bivalent or bispecific. Biantibodies are described more comprehensively in, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90:6444-6448 (1993). Triantibodies and tetraantibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0105] "Single-domain antibody" or "sdAb" or "nanobody" refers to an antibody fragment composed of the variable region (VH domain) of the antibody heavy chain or the variable region (VL domain) of the antibody light chain (Holt, L., et al., Trends in Biotechnology, 21(11):484-490).

[0106] A "single-chain Fv" or "scFv" antibody fragment contains the antibody's VH and VL domains, which are located within a single polypeptide chain and are oriented in either direction (e.g., VL-VH or VH-VL). Typically, scFv polypeptides also contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the structure required for antigen binding. For a review of scFv, see, for example, Pluckthün, in *The Pharmacology of Monoclonal Antibodies*, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York, 1994), pp. 269-315.

[0107] In a particular embodiment, the antigen-binding fragment is an scFv. In a particular embodiment, the scFv is a murine, human, or humanized scFv. Single-chain antibodies can be cloned to form V region genes of hybridomas specific to the desired target. The generation 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., PNAS, 1989; 86:3833-3837.

[0108] In various embodiments, the antibody binding to the anti-BCMA CAR or its antigen-binding fragment comprises a variable light chain sequence and / or a variable heavy chain sequence, the variable light chain sequence comprising the CDRL1-CDRL3 sequence as shown in SEQ ID NO:1-3, and the variable heavy chain sequence comprising the CDRH1-CDRH3 sequence as shown in SEQ ID NO:4-6.

[0109] In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence as shown in SEQ ID NO:7 and / or a variable heavy chain sequence as shown in SEQ ID NO:8. In some embodiments, the antibody or its antigen-binding fragment 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 with the amino acid sequence shown in any of SEQ ID NO:7 and / or a variable heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with the amino acid sequence shown in any of SEQ ID NO:8.

[0110] In some embodiments, the antibody or its antigen-binding fragment comprises a light chain sequence as shown in SEQ ID NO:11 and / or a heavy chain sequence as shown in SEQ ID NO:12 or SEQ ID NO:13. In some embodiments, the antibody or its antigen-binding fragment comprises a light chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with the amino acid sequence shown in SEQ ID NO:11 and / or a heavy chain sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid identity with the amino acid sequence shown in SEQ ID NO:12 or SEQ ID NO:13.

[0111] In one embodiment, the antibody binding to the anti-BCMA CAR or its antigen-binding fragment comprises the light chain CDR sequence shown in SEQ ID NO:1-3. In a particular embodiment, the anti-BCMA antibody or antibody fragment binding to the anti-BCMA CAR or the anti-BCMA scFv portion or its antigen-binding fragment 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.

[0112] In one embodiment, the antibody binding to anti-BCMA CAR or its antigen-binding fragment comprises the heavy chain CDR sequence shown in SEQ ID NO:4-6. In a particular embodiment, the antibody binding to anti-BCMA CAR or its antigen-binding fragment 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.

[0113] In certain 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 of SEQ ID NO:1-3 and / or one or more heavy chain CDRs as shown in any of SEQ ID NO:4-6. In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable light chain sequence as shown in SEQ ID NO:7 and / or a variable heavy chain sequence as shown in SEQ ID NO:8. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain sequence as shown in SEQ ID NO:11 and / or a heavy chain sequence as shown in SEQ ID NO:12 or SEQ ID NO:13.

[0114] In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence and / or a variable heavy chain sequence that binds to the anti-BCMA CAR shown in SEQ ID NO:9 or SEQ ID NO:10.

[0115] D. Conjugate

[0116] In various embodiments, conjugates comprising an antibody or an antigen-binding fragment thereof and a label are provided. In a preferred embodiment, the conjugate comprises an antibody or an antigen-binding fragment thereof binding to an anti-BCMA CAR, and a detectable label or marker capable of generating a detectable signal. In a more preferred embodiment, the conjugate comprises an antibody or an antigen-binding fragment thereof binding to an anti-BCMA CAR conjugated to a detectable label. In even more preferred embodiments, the conjugate comprises an antibody or an antigen-binding fragment thereof binding to an anti-BCMA CAR covalently or chemically conjugated to a detectable label.

[0117] As used herein, the term "tag" refers to a detectable tag or label capable of generating a detectable signal. In certain embodiments, the tag comprises a radionuclide, nucleic acid, small molecule, or peptide. In some embodiments, the tag is directly detectable. In some embodiments, the tag is indirectly detectable.

[0118] Illustrative examples suitable for detectable labeling in conjugates covered in a particular embodiment include, but are not limited to: haptens, fluorescent molecules, fluorescent dyes, fluorescent proteins, chromophores, metal ions, gold particles, silver particles, magnetic particles, radionuclides, peptides, enzymes, luminescent compounds, or oligonucleotides.

[0119] Illustrative examples of molecules suitable for use as detectable labels in specific implementation schemes include, but are not limited to: Pacific Blue TM Pacific Orange TM Pacific Green TM CascadeBlue TM Cascade Yellow TM Lucifer Yellow TM Marina Blue TM ; (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, for example and DyLight TM Dyes (DL), such as DL549, DL649, DL680 and DL800; fluorescein or its derivatives, such as fluorescein isothiocyanate, carboxyfluorescein and dichloroimide fluorescein; digoxigenin; and 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)); β-phycoerythrin (BPE); perCP; allophycocyanin (APC); and C-phycocyanin; dyes, such as Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, Atto 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725 and Atto 740; SuperBright TM dyes, such as Super Bright TM 436, Super Bright TM 600, Super Bright TM 645, SuperBright TM 702 and Super Bright TM 780; Brilliant TM dyes, such as 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 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 Blue515 (BB515) and Brilliant TMBlue 700 (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.

[0120] 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.

[0121] 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.

[0122] Illustrative examples of enzymes suitable for use as detectable labels include, but are not limited to: alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosase.

[0123] Illustrative examples of radionuclides suitable for detection labeling 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).

[0124] In certain embodiments, the conjugate comprises an antibody or antibody fragment conjugated, coupled, or linked (e.g., covalently bound) to one or more tags. In some embodiments, the tag may be conjugated, coupled, or linked to the antibody or fragment directly or indirectly (e.g., via a linker group). When the antibody and tag each have substituents capable of reacting with each other, the antibody may directly covalently bind one or more tags. For example, a nucleophilic group (such as an amino or mercapto group) may be capable of reacting with a carbonyl-containing group (such as an anhydride or acid halide) or with an alkyl group containing a good leaving group (e.g., a halide) on another group.

[0125] In certain embodiments, it may be necessary to conjugate, conjugate, or link an antibody or antibody fragment to one or more tags via monovalent or multivalent linkers or spacers. Linkers or spacers can be used to provide sufficient distance between the antibody and the tag to avoid steric hindrance or interference with antibody binding ability. It will be apparent to those skilled in the art that a variety of bifunctional or multifunctional reagents (both purely functional and heterofunctional reagents, such as those described in the Pierce Chemical Co., Rockford, IL catalogue) can be used as linker groups. Conjugation can be achieved, for example, via amino, carboxyl, thiol, or oxidized carbohydrate residues. Numerous references describe such methods, such as U.S. Patent No. 4,671,958 to Rodwell et al.

[0126] In some embodiments, the linker has about 1-100 atoms, 1-80 atoms, 1-60 atoms, 1-40 atoms, 1-30 atoms, 1-20 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.

[0127] Illustrative examples of connectors or bonds 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)—, —S—S—(CH2) 2-6 —NH—C(O)—, —S—S-(CH2) 0-6 — CH(CONH2)—(CH2) 0-6 —NH—C(O)—, —S—S-(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=N—O— (CH2) 2-6 —NH—C(O)—, —C=N—NH—(CO)—(CH2) 2-6 —NH—C(O)—、—succinimide—(CH2) 2-6 —NH—C(O)—、—diazodicarboxamide—(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 — O—C(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 straight-chain —C1-C16—alkyl, branched or straight-chain —C1-C16—alkyl, wherein one of the carbon atoms may optionally be substituted by: heteroatom, R 2 —NH—(CH2) 2-6 —NH—C(O)—、R2 —S—(CH2) 2-6 —NH—C(O)—、 R 2 —triazole—(CH2) 2-6 —NH—C(O)—、R 2 —NH—O—(CH2) 2-6 —NH—C(O)—、 R 2 =N—NH—(CO)—(CH2) 2-6 —NH—C(O)—、R 2 It is one to three bifunctional or trifunctional substituted crosslinked organic groups selected from the group consisting of alkyl groups: alkyl, substituted alkyl, cycloalkyl, aryl, heteroaryl, polyethylene glycol (PEG) [i.e., -(CH2CH2O)]. 1-20 ].

[0128] In a particular embodiment, the conjugate comprises a polypeptide-based labeled antibody or antibody fragment, such as a fluorescent protein or enzyme, covalently bound via a polypeptide linker as covered herein, as described below.

[0129] In a preferred embodiment, the conjugate comprises an antibody or antigen-binding fragment thereof that is covalently bound to the PE marker and binds to the anti-BCMA CAR.

[0130] E. polypeptide

[0131] This document covers a variety of peptides, fusion peptides, and peptide variants, including but not limited to antibodies and their antigen-binding fragments. In a preferred embodiment, the peptide comprises an antibody or its antigen-binding fragment that binds to an anti-BCMA CAR.

[0132] Unless otherwise stated, “polypeptide,” “peptide,” and “protein” are used interchangeably and, by their conventional meaning, are defined as an amino acid sequence. A polypeptide is not limited to a specific length; for example, it may comprise a full-length polypeptide or a polypeptide fragment, and may include one or more post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

[0133] As used herein, "isolated protein," "isolated peptide," or "isolated polypeptide," etc., refers to peptide or polypeptide molecules that are synthesized, isolated, and / or purified in vitro from the cellular environment and from association with other components of the cell, i.e., the peptide or polypeptide molecules do not significantly associate with substances in vivo.

[0134] Peptides include "peptide variants." A peptide variant differs from a naturally occurring peptide in that it may involve one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be generated synthetically, for example, by modifying one or more of the aforementioned peptide sequences. For example, in a particular embodiment, it may be necessary to improve the binding affinity and / or other biological properties of an antibody or its antigen-binding fragment by introducing one or more amino acid substitutions, deletions, additions, and / or insertions. In a particular embodiment, 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 acids identical to any of the reference sequences covered herein, typically wherein the variant maintains at least one biological activity of the reference sequence.

[0135] Peptides include “peptide fragments.” A peptide fragment refers to a peptide, which may be a monomer or polymer, having an N-terminal deletion, a C-terminal deletion, and / or internal deletion or substitution of a naturally occurring or recombinant peptide. Illustrative examples of bioactive peptide fragments include antibody fragments. As used herein, the term “bioactive fragment” or “minimum bioactive fragment” refers to a peptide 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 bioactivity of a naturally occurring peptide. In a preferred embodiment, bioactivity refers to the binding affinity to an epitope. In some embodiments, the peptide fragment may comprise an amino acid chain of at least 5 to about 500 amino acids in length. It should be understood that in some implementations, 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, 55, 60, 65, 70, 75, 80, 85, or 90. 1, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids. Particularly useful peptide fragments include functional domains, including 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 the heavy or light chain; variable regions of the heavy or light chain; antibody chains comprising two CDRs or portions of variable regions; and so on.

[0136] As described above, peptides can be altered in various ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such operations are generally known in the art. For example, amino acid sequence variants of a reference peptide can be prepared by mutation in DNA. Methods for mutagenesis and nucleotide sequence alteration 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. Patent No. 4,873,192, Watson, JD et al. (Molecular Biology of the Gene, 4th Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and references cited therein. Guidelines on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0137] In some embodiments, peptide variants include one or more conserved substitutions. A “conservative substitution” is the replacement of an amino acid with another amino acid having similar properties, such that those skilled in peptide chemistry can expect the secondary structure and hydrophilic properties of the peptide to remain substantially unchanged. Preferably, the amino acid changes in the protein variants disclosed herein are conserved amino acid changes, i.e., substitutions of amino acids with similar or no charge. Conservative amino acid changes involve the substitution of one of a group of amino acids associated with the side chain. Naturally occurring amino acids are generally classified into four groups: acidic amino acids (aspartic acid, glutamic acid), basic amino acids (lysine, arginine, histidine), nonpolar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and nonpolar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids. In peptides or proteins, suitable conserved amino acid substitutions are known to those skilled in the art and can generally be performed without altering the biological activity of the resulting molecule. Those skilled in the art will recognize that, in general, a single amino acid substitution in the non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th ed., 1987, The Benjamin / Cummings Pub. Co., p. 224).

[0138] Peptide variants also include glycosylated forms, aggregate conjugates with other molecules, and covalent conjugates with unrelated chemical motifs (e.g., PEGylated molecules). As is known in the art, covalent variants can be prepared by attaching functional groups to groups found in the amino acid chain or N-terminal or C-terminal residues. Variants also include allelic variants, species variants, and mutant proteins. Truncation or deletion of regions that do not affect protein function or activity also constitutes a variant.

[0139] In a particular embodiment, the polypeptide includes a polypeptide linker. A "linker" is a plurality of amino acid residues added between two or more polypeptide domains for proper spacing, conformation, and function of the molecule.

[0140] In a particular embodiment, the peptide linker is a variable region linker sequence. The "variable region linker sequence" is an amino acid sequence that links to V... H Domain and V L The domain also provides a spacer function compatible with the interaction of the two sub-binding domains, such that the resulting peptide retains specific binding affinity to the same target molecules as antibodies that include the same light chain variable region and heavy chain variable region.

[0141] In a particular embodiment, the conjugate comprises a covalently bound antibody or antibody fragment to one or more protein-based tags, such as fluorescent proteins or peptide linkers of enzymes.

[0142] Illustrative examples of linkers suitable for use in the specific embodiments covered herein include, but are not limited to, the following amino acid sequences: GGG; DGGGS (SEQ ID NO:14); TGEKP (SEQ ID NO:15) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO:16) (Pomerantz et al. 1995, ibid.); (GGGGS) nWhere n = 1, 2, 3, 4, or 5 (SEQ ID NO: 17) (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 18) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 19) (Bird et al., 1988, Science 242: 423-426); GGRRGGGS (SEQ ID NO: 20); LRQRDGERP (SEQ ID NO: 21); LRQKDGGGSERP (SEQ ID NO: 22); LRQKD(GGGS)2ERP (SEQ ID NO: 23). Alternatively, a computer program capable of modeling DNA binding sites and the peptide itself can be used (Desjarlais & Berg, PNAS). 90:2256-2260(1993), PNAS 91:11099-11103(1994)) or by rationally designing flexible adapters through phage display methods. In one embodiment, the adapter comprises the following amino acid sequence: GSTGSGSGKPGSGEGSTKG (SEQ ID NO:24) (Cooper et al., Blood, 101(4):1637-1644(2003)).

[0143] In a preferred embodiment, the polypeptide comprises the amino acid sequence shown in SEQ ID NO:7 or 8. In some embodiments, the antibody or its antigen-binding fragment comprises the light chain sequence shown in SEQ ID NO:11 and the heavy chain sequence shown in SEQ ID NO:12 or SEQ ID NO:13.

[0144] F. Polynucleotides

[0145] In a preferred embodiment, a polynucleotide is provided encoding an antibody or an antigen-binding fragment thereof that binds to an anti-BCMA CAR. As used herein, the terms "polynucleotide" or "nucleic acid" refer to messenger RNA (mRNA), RNA, genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. In certain embodiments, the polynucleotide includes a polynucleotide or variant thereof 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 with any of the reference sequences covered herein. In various exemplary embodiments, the polynucleotide encoding the polypeptides covered herein includes, but is not limited to, the polypeptide sequences shown in SEQ ID NO: 7 or 8.

[0146] In certain embodiments, at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more adjacent amino acid residues encoding a polypeptide, as well as all intermediate lengths of polynucleotides, 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.

[0147] In certain embodiments, polynucleotides may undergo codon optimization. As used herein, the term "codon-optimized" refers to the substitution of codons in a polynucleotide encoding a polypeptide to increase the expression, stability, and / or activity of said polypeptide. Factors influencing codon optimization include, but are not limited to, one or more of the following: (i) changes in codon preferences between two or more organisms or genes or synthetically constructed preference tables; (ii) changes in the degree of codon preference within an organism, gene, or gene set; (iii) systematic changes in codons, including those related to the environment; (iv) codon changes derived from decoding tRNA; (v) codon changes derived from the GC% at a position in a total or triplet; (vi) changes in the degree of similarity to a reference sequence, such as a naturally occurring sequence; (vii) changes in codon frequency cutoff values; (viii) structural characteristics of mRNA transcribed from a DNA sequence; (ix) prior knowledge about the function of the DNA sequence on which the design of codon substitution sets is based; (x) systematic changes in the codon set for each amino acid; and / or (xi) the separation and removal of pseudo-translation initiation sites.

[0148] As used herein, the terms "polynucleotide variant" and "variant," etc., refer to a polynucleotide that exhibits considerable sequence identity with a reference polynucleotide sequence or a polynucleotide that hybridizes to a reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides have been added to or deleted from, or substituted with, different nucleotides compared to the reference polynucleotide. In this regard, it will be well understood in the art that certain changes, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide, thereby allowing the altered polynucleotide to retain the biological function or activity of the reference polynucleotide.

[0149] Polynucleotide variants include polynucleotide fragments encoding biologically active polypeptide fragments or variants. As used herein, the term "polynucleotide fragment" refers to a fragment with a length 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, or 95. Polynucleotide fragments of 1, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700 or more nucleotides encoding polypeptide variants that retain 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 naturally occurring polypeptide activity. A polynucleotide fragment is a polynucleotide that encodes a polypeptide with an amino-terminal deletion, a carboxyl-terminal deletion, and / or an internal deletion or substitution, which is either naturally occurring or recombinantly generated.

[0150] As used herein, the description of “sequence identity” or, for example, “a sequence that is 50% identical to…” refers to the degree to which sequences are identical within a comparison window on a nucleotide-to-nucleotide or amino acid-to-amino acid basis. Therefore, the “sequence identity percentage” can be calculated as follows: comparing two optimally aligned sequences within a comparison window, determining the number of positions in which identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to obtain 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 obtain the sequence identity percentage. It comprises nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 86%, 97%, 98%, or 99% sequence identity with any of the reference sequences described herein, wherein the polypeptide variant typically maintains at least one biological activity of the reference polypeptide.

[0151] 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 units long, but often 15 to 18, and typically at least 25 monomeric units, including nucleotide and amino acid residues. Because two polynucleotides can each contain (1) sequences similar between the two polynucleotides (i.e., only a portion of the complete polynucleotide sequence); and (2) sequences dissimilar 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 the similarity of local regions of the sequence. A “comparison window” is a conceptual segment having at least 6, typically about 50 to about 100, more typically about 100 to about 150 consecutive positions, wherein the two sequences are compared after optimally aligning one sequence with a reference sequence having the same number of consecutive positions. To achieve optimal alignment of two sequences, the comparison window can contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (excluding added or missing sequences). Optimal sequence alignment for the comparison window can be performed using computer-implemented algorithms (GAP, BESTFIT, FASTA, and TFASTA, WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575 ScienceDrive Madison, WI, USA) or through checks and optimal alignments (i.e., those producing the highest percentage of homology within the comparison window) generated by any of the chosen methods. Reference can also be made to the family of BLAST programs, such as those disclosed, for example, Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994–1998, Chapter 15, Unit 19.3.

[0152] As used herein, “isolated polynucleotide” refers to a polynucleotide that has been purified from the naturally occurring sequences flanking it, for example, a DNA fragment that has been removed from the sequence 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 artificially created.

[0153] Furthermore, those skilled in the art will understand 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 natural sequence. Nevertheless, polynucleotides that vary due to differences in codon usage, such as polynucleotides optimized for human and / or primate codon selection, are specifically covered in particular embodiments.

[0154] Polynucleotides can be prepared, manipulated, and / or expressed using any of a variety of recognized techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector.

[0155] G. Composition

[0156] The compositions covered herein may comprise one or more antibodies or antibody fragments and their antigen-binding fragments, conjugates, peptides, and polynucleotides. Compositions include, but are not limited to, pharmaceutical compositions. There are practically no limitations on other components that may be included in the composition, provided that additional reagents do not adversely affect the function or activity of the active ingredient in the composition.

[0157] In certain embodiments, the compositions of the present invention comprise an amount of an antibody or an antigen-binding fragment or conjugate thereof. As used herein, the term “amount” refers to an “effective amount” of the antibody binding to the target polypeptide.

[0158] The composition may also contain one or more carriers or excipients.

[0159] Illustrative examples of carriers suitable for use in compositions incorporated herein include, but are not limited to: buffers, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzyl chloride); phenol, butanol or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0160] Acceptable carriers also include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexachlorocyclohexane quaternary ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) peptides; 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 dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0161] H. Reagent kit

[0162] In various embodiments, a kit is provided comprising one or more antibodies or antigen-binding fragments thereof that bind to anti-BCMA CAR, and instructions for using said antibodies to detect, determine and / or measure anti-BCMA CAR expression on one or more immune effector cells or anti-BCMA CAR T cells.

[0163] In a particular embodiment, a kit is provided comprising one or more antibodies or antigen-binding fragments thereof that bind to anti-BCMA CAR, and instructions for using said 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.

[0164] In various embodiments, the antibody binding to the anti-BCMA CAR or its antigen-binding fragment comprises a variable light chain sequence and / or a variable heavy chain sequence, the variable light chain sequence comprising the CDRL1-CDRL3 sequence shown in SEQ ID NO:1-3, and the variable heavy chain sequence comprising the CDRH1-CDRH3 sequence shown in SEQ ID NO:4-6.

[0165] In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence as shown in SEQ ID NO:7 and / or a variable heavy chain sequence as shown in SEQ ID NO:8. In some embodiments, the antibody or its antigen-binding fragment comprises a light chain sequence as shown in SEQ ID NO:11 and / or a variable heavy chain sequence as shown in SEQ ID NO:12 or SEQ ID NO:13.

[0166] In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence and / or a variable heavy chain sequence that binds to the anti-BCMA CAR shown in SEQ ID NO:9 or SEQ ID NO:10.

[0167] In a particular embodiment, the kit comprises an antibody or antibody fragment and a detectable label, along with instructions for conjugation. In a particular embodiment, the kit comprises an antibody or antibody fragment conjugated to the 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 an RPE (Reactive Protein Equivalent).

[0168] I. Method

[0169] Anti-BCMA CAR T cell activity is partly associated with anti-BCMA CAR expression on immune effector cells. The ability to accurately assess anti-BCMA CAR expression in vitro, in vitro, and in vivo is needed to estimate drug product activity and track anti-BCMA CAR T cell survival and persistence in patients.

[0170] In certain embodiments, methods are provided for using one or more antibodies or antigen-binding fragments thereof. The antibodies are used to detect or measure anti-BCMA CAR expression. The antibodies, fragments, and conjugates covered herein are used for binding, detecting, determining, identifying, measuring, quantifying, and / or enumerating the presence or level of anti-BCMA CAR expression in populations and / or anti-BCMA CAR. + The number of immune effector cells.

[0171] In various embodiments, the 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 an antibody or an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding to one or more epitopes of the anti-BCMA CAR in an effective amount and for a duration sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex.

[0172] In a particular embodiment, the 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 an antigen-binding fragment thereof, said antibody or antigen-binding fragment binding to one or more epitopes of the anti-BCMA CAR in an effective amount and for a duration sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. In one 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 CARs. + In another embodiment, at least 75% of the immune effector cells are anti-BCMA CARs. + .

[0173] In some embodiments, the method includes determining the expression level of anti-BCMA CAR on immune effector cells. Immune effector cells (e.g., T cells, NK cells, NKT cells) are contacted with an antibody or its antigen-binding fragment, said antibody or antigen-binding fragment binding to one or more epitopes of the anti-BCMA CAR in an effective amount for a duration sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. The amount of the antibody:anti-BCMA CAR complex is measured and compared to a control.

[0174] In some embodiments, the method includes determining the expression level of anti-BCMA CAR 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, said antibody or antigen-binding fragment binding to one or more epitopes of the anti-BCMA CAR in an effective amount and for a duration sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. The amount of the antibody:anti-BCMA CAR complex is measured and compared to a control.

[0175] In a particular implementation, the method includes measuring anti-BCMA CAR in a population of immune effector cells. + 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, said antibody or its antigen-binding fragment binding to one or more epitopes of the anti-BCMA scFv portion of the CAR in an effective amount and for a duration sufficient to allow the antibody to bind to the anti-BCMA CAR and form an antibody:anti-BCMA CAR complex. Immune effector cells containing the antibody:anti-BCMA CAR complex can then be enumerated.

[0176] In a specific implementation, the expression of anti-BCMA CAR or anti-BCMA CAR is detected, measured, or enumerated. + T-cell methods include administration of anti-BCMA CARs. + T-cell-containing biological samples isolated from subjects. In a particular embodiment, the samples contain or are suspected of containing anti-BCMA CARs. + T cells. In a preferred embodiment, the cells are single samples, leukocyte single samples, peripheral blood, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, or tumor biopsy.

[0177] In a particular implementation, the antibodies and conjugates that bind to anti-BCMA CAR as described herein are used to detect anti-BCMA CAR in subjects and / or in samples collected from subjects at one or more time points. + The number of T cells is used to measure or assess pharmacokinetics, amplification, and / or persistence. In a specific implementation, this is done after administering anti-BCMA CAR to the subject. +Samples are collected, isolated, and / or harvested from subjects approximately 24 hours, 48 ​​hours, 72 hours, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 6 months, 1 year, or weekly, monthly, or annually.

[0178] In various embodiments, the antibody binding to the anti-BCMA CAR or its antigen-binding fragment comprises a variable light chain sequence and / or a variable heavy chain sequence, the variable light chain sequence comprising the CDRL1-CDRL3 sequence as shown in SEQ ID NO:1-3, and the variable heavy chain sequence comprising the CDRH1-CDRH3 sequence as shown in SEQ ID NO:4-6.

[0179] In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence as shown in SEQ ID NO:7 and / or a variable heavy chain sequence as shown in SEQ ID NO:8. In some embodiments, the antibody or its antigen-binding fragment comprises a light chain sequence as shown in SEQ ID NO:11 and / or a heavy chain sequence as shown in SEQ ID NO:12 or SEQ ID NO:13.

[0180] In some embodiments, the antibody or its antigen-binding fragment comprises a variable light chain sequence and / or a variable heavy chain sequence that binds to the anti-BCMA CAR shown in SEQ ID NO:9 or SEQ ID NO:10.

[0181] In a specific implementation, the expression of anti-BCMA CAR or anti-BCMA CAR is detected, measured, or enumerated. + T-cell methods include using antibodies or antibody fragments 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 a reactive ionomer (RPE).

[0182] In a particular implementation, one or more of the following assays may be used to detect, measure, and / or enumerate the presence of the anti-BCMA CAR complex: immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR) based biosensors (e.g., BIAcore), fluorescence microscopy, flow cytometry or fluorescence activated cell sorting (FACS) or Western blot.

[0183] In a preferred embodiment, flow cytometry is used to detect, determine, and / or enumerate the presence of the antibody:anti-BCMA CAR complex. In a more preferred embodiment, FAC is used to detect, determine, and / or enumerate the presence of the antibody:anti-BCMA CAR complex.

[0184] In a particular implementation, the background expression level of anti-BCMA CAR on one or more immune effector cells is determined, and the amount of antibody:anti-BCMA CAR complex measured on one or more immune effector cells is subtracted from or used for normalization of the measurement.

[0185] All publications, patent applications and granted patents cited in this specification are incorporated herein by reference as if each individual publication, patent application or granted patent were specifically and individually incorporated by reference.

[0186] While the foregoing invention has been described in considerable detail by way of illustration and example for the purpose of clarity, it will be apparent to those skilled in the art, based on the teachings of the invention, that various changes and modifications may be made therein without departing from the spirit or scope of the appended claims. The following embodiments are provided for illustrative purposes only and are not intended to be limiting. Those skilled in the art will readily recognize that many non-critical parameters can be altered or modified to produce substantially similar results.

[0187] Example

[0188] Example 1

[0189] Antibodies against BCMA CAR

[0190] Mouse monoclonal antibodies were generated against the anti-BCMA CAR sequence shown in SEQ ID NO:9. One of the antibodies was sequenced (GenScript).

[0191] follow The reagent's technical manual describes the isolation of total RNA from hybridoma cells. Then, following PrimeScript... TM The technical manual for the first-strand cDNA synthesis kit guides you through reverse transcription of total RNA into cDNA using isotype-specific antisense primers or universal primers. Amplify heavy and light chain antibody fragments according to the standard operating procedure (SOP) for rapid cDNA end amplification (RACE). Clone the amplified antibody fragments individually into standard cloning vectors and sequence them.

[0192] Generally, the terminology used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but rather as encompassing the full scope of all possible embodiments together with the equivalents claimed by these claims. Therefore, the claims are not limited by this disclosure. sequence list <110> Bluebird Bio, Inc. Kevin Friedman; Molly Reed Perkins <120> Anti-BCMA CAR antibody, conjugate and usage <130> BLUE-97PC2 <150> US 62 / 948,488 <151> 2019-12-16 <160> 28 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made - 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> <223> Laboratory-made - Light chain CDR2 <400> 2 Trp Ala Ser Thr Arg His Thr 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-manufactured - 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> <223> Laboratory-made – 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> <223> Laboratory-made – 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> <223> Laboratory-made – 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> <223> Laboratory-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 Thr Leu Glu Ile Lys 100 105 <210> 8 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Laboratory manufacturing - Variable weight chain <400> 8 Gln Val Gln Leu Gln Gln Pro 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> 493 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-made – anti-BCMA CAR <400> 9 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> 10 <211> 472 <212> PRT <213> Artificial Sequence <220> <223> Lab-made – Anti-BCMA CAR Construct <400> 10 Asp Ile Val Leu Thr Gln Ser Pro Pro Ser Leu Ala Met Ser Leu Gly 1 5 10 15 Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Thr Ile Leu 20 25 30 Gly Ser His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Thr Leu Leu Ile Gln Leu Ala Ser Asn Val Gln Thr Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Arg Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Glu Asp Asp Val Ala Val Tyr Tyr Cys Leu Gln Ser Arg 85 90 95 Thr Ile Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly 100 105 110 Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys 115 120 125 Gly Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly 130 135 140 Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp 145 150 155 160 Tyr Ser Ile Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp 165 170 175 Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp 180 185 190 Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala 195 200 205 Tyr Leu Gln Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe 210 215 220 Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Ser Ala Ala Ala Thr Thr Thr Pro Ala Pro Arg 245 250 255 Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg 260 265 270 Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly 275 280 285 Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr 290 295 300 Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg 305 310 315 320 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 325 330 335 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 340 345 350 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 405 410 415 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 420 425 430 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 435 440 445 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 450 455 460 His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 11 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-manufactured light chain sequence of antibody binding to anti-BCMA CAR <400> 11 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 Thr Leu Glu Ile Lys Arg Ala Asp Ala Ala 100 105 110 Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly 115 120 125 Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile 130 135 140 Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu 145 150 155 160 Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser 165 170 175 Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr 180 185 190 Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser 195 200 205 Phe Asn Arg Asn Glu Cys 210 <210> 12 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-manufactured heavy chain sequence of antibody binding to anti-BCMA CAR <400> 12 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr 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 Ala Lys Thr Thr Pro 115 120 125 Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser 130 135 140 Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr 180 185 190 Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala 195 200 205 His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp 210 215 220 Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr 245 250 255 Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu 260 265 270 Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln 275 280 285 Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser 290 295 300 Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys 305 310 315 320 Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro 340 345 350 Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met 355 360 365 Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn 370 375 380 Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr 385 390 395 400 Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn 405 410 415 Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu 420 425 430 His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys 435 440 445 <210> 13 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> Laboratory-manufactured heavy chain sequence of antibody binding to anti-BCMA CAR <400> 13 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr 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 Ala Lys Thr Thr Pro 115 120 125 Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser 130 135 140 Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr 180 185 190 Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala 195 200 205 His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp 210 215 220 Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val 225 230 235 240 Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr 245 250 255 Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu 260 265 270 Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln 275 280 285 Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser 290 295 300 Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys 305 310 315 320 Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro 340 345 350 Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met 355 360 365 Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn 370 375 380 Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr 385 390 395 400 Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn 405 410 415 Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu 420 425 430 His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly 435 440 445 <210> 14 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 14 Asp Gly Gly Gly Ser 1 5 <210> 15 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 15 Thr Gly Glu Lys Pro 1 5 <210> 16 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 16 Gly Gly Arg Arg 1 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 17 Gly Gly Gly Gly Ser 1 5 <210> 18 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 18 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp 1 5 10 <210> 19 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 19 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 20 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> 20 Gly Gly Arg Arg Gly Gly Gly Ser 1 5 <210> twenty one <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> twenty one Leu Arg Gln Arg Asp Gly Glu Arg Pro 1 5 <210> twenty two <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> twenty two Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro 1 5 10 <210> twenty three <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> twenty three Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro 1 5 10 15 <210> twenty four <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Exemplary connector sequence <400> twenty four Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr 1 5 10 15 Lys Gly <210> 25 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Exemplary rules for determining light chain CDR-L3 motifs <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is any amino acid <400> 25 Phe Gly Xaa Gly 1 <210> 26 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Exemplary rules for determining heavy chain CDR-H1 motifs <220> <221> MISC_FEATURE <222> (2)..(4) <223> Xaa is any amino acid <400> 26 Cys Xaa Xaa Xaa 1 <210> 27 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Exemplary rules for determining heavy chain CDR-H2 motifs <400> 27 Leu Glu Trp Ile Gly 1 5 <210> 28 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Exemplary rules for determining heavy chain CDR-H3 motifs <220> <221> MISC_FEATURE <222> (3)..(3) <223> Xaa is any amino acid <400> 28 Trp Gly Xaa Gly 1

Claims

1. An antibody or an antigen-binding fragment thereof comprising the variable light chain sequence shown in SEQ ID NO: 7 and the variable heavy chain sequence shown in SEQ ID NO: 8, wherein the antibody or the antigen-binding fragment thereof binds the anti-BCMA CAR sequence shown in SEQ ID NO: 9 or SEQ ID NO:

10.

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

3. The antibody or antigen-binding fragment thereof as described in claim 1, wherein the antibody or antigen-binding fragment thereof is selected from: Fab' fragment, F(ab')2 fragment, Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, microantibody, biantibody, triantibody, tetraantibody, disulfide bond-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, nanobody).

4. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof is scFv.

5. The antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the antibody or antigen-binding fragment thereof comprises the light chain amino acid sequence shown in SEQ ID NO: 11 and the heavy chain amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO:

13.

6. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a mouse antibody.

7. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a humanized antibody.

8. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof is a human antibody.

9. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof comprises a constant IgG1, IgG2, IgG3 or IgG4 domain.

10. The antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 5, wherein the antibody or antigen-binding fragment comprises an IgG1 constant domain.

11. A conjugate comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10; and a detectable label.

12. The conjugate of claim 11, wherein the detectable marker is selected from the group consisting of: metal ions, gold particles, silver particles, magnetic particles, polypeptides, luminescent compounds, or oligonucleotides.

13. The conjugate of claim 11, wherein the detectable marker is selected from the group consisting of: fluorescent dyes, fluorescent proteins, or enzymes.

14. The conjugate of claim 11, wherein the detectable marker is selected from the group consisting of haptens and chromophores.

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

16. The conjugate of claim 13, wherein the detectable marker is an Alexa Fluor® 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.

17. The conjugate of claim 16, wherein the detectable marker is Alexa Fluor® dye AF488.

18. The conjugate of claim 13, wherein the detectable marker is QDot® selected from the group consisting of: Qdot®525, Qdot®565, Qdot®585, Qdot®605, Qdot®655, Qdot®705 and Qdot®800.

19. The conjugate of claim 13, wherein the detectable marker is a DyLight™ dye selected from the group consisting of DL549, DL649, DL680 and DL800.

20. The conjugate of claim 11 or claim 12, wherein the detectable marker is a hapten selected from the group consisting of: fluorescein or a derivative thereof, digoxigenin, dinitrophenol (DNP), trinitrophenol (TNP), and biotin.

21. The conjugate of claim 20, wherein the fluorescein or its derivative is fluorescein isothiocyanate, carboxyfluorescein, or dichloroimide fluorescein.

22. The conjugate of claim 13, wherein the detectable marker is a Cy dye selected from the group consisting of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, and Cy7.

5.

23. The conjugate of claim 11, wherein the detectable marker is a fluorescent molecule selected from the group consisting of: phycoerythrin, polydinophytic chlorophyll (PerCP), allophycocyanin (APC), and C-phycocyanin.

24. The conjugate of claim 23, wherein the phycoerythrin is selected from R-fish phycoerythrin and B-phycoerythrin.

25. The conjugate of claim 13, wherein the detectable marker is a fluorescent dye selected from the group consisting of: Atto 390, Atto 425, Atto 465, Atto 488, Atto 495, Atto 514, Atto 520, Atto 532, Atto 550, Atto 565, Atto 590, Atto 594, Atto 610, Atto 620, Atto 633, Atto 647, Atto 655, Atto 665, Atto 680, Atto 700, Atto 725, Atto 740, Super Bright™ 436, Super Bright™ 600, Super Bright™ 645, Super Bright™ 702, Super Bright™ 780, Brilliant™ Violet 421, Brilliant™ Violet 480, Brilliant™ Violet 510, Brilliant™ Violet 605, Brilliant Violet™ 650, Brilliant Violet™ 711, Brilliant Violet™ 786, Brilliant™ Ultraviolet 395 (BUV395), Brilliant™ Ultraviolet 496 (BUV496), Brilliant™ Ultraviolet 563 (BUV563), Brilliant™ Ultraviolet 661 (BUV661), Brilliant™ Ultraviolet 737 (BUV737), Brilliant™ Ultraviolet 805 (BUV805), Brilliant™ Blue 515 (BB515), Brilliant™ Blue 700 (BB700) and IR Dye 680, IR Dye 680LT, IR Dye 700, IR Dye 700DX, IR Dye 800, IR Dye800RS and IR Dye 800CW.

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

27. The conjugate of claim 13, wherein the detectable marker 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.

28. The conjugate of claim 13, wherein the detectable marker is an enzyme selected from the group consisting of alkaline phosphatase, horseradish peroxidase, luciferase, and β-galactosidase.

29. The conjugate of claim 12, wherein the detectable marker comprises 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, 85Sr, sulfur, technetium, thallium, tin, tritium, xenon, ytterbium, and yttrium.

30. A polynucleotide encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10.

31. A host cell comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10, or a polynucleotide as described in claim 30.

32. A composition comprising an antibody or an antigen-binding fragment thereof as claimed in any one of claims 1 to 10, a conjugate as claimed in any one of claims 11 to 29, a polynucleotide as claimed in claim 30, or a host cell as claimed in claim 31.

33. A method for producing an antibody or an antigen-binding fragment thereof, comprising expressing the antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 10 in a host cell, and recovering or separating the antibody.

34. A kit comprising an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 10 or a conjugate as claimed in any one of claims 11 to 29, and instructions for use.

35. A method for detecting the expression of anti-BCMA CAR on T cells for non-diagnostic and non-therapeutic purposes, comprising: Contact T cells with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10 or a conjugate as described in any one of claims 11 to 29; and detect the formation of an antibody:anti-BCMA CAR complex.

36. A method for non-diagnostic and non-therapeutic purposes of detecting the expression of anti-BCMA CAR in a T cell population, comprising: Contact a population of T cells with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10 or a conjugate as described in any one of claims 11 to 29; and detect the formation of an antibody-anti-BCMA CAR complex on one or more T cells.

37. A method for non-diagnostic and non-therapeutic purposes of determining the expression of anti-BCMA CAR on T cells, comprising: The T cells are contacted with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10 or a conjugate as described in any one of claims 11 to 29; the formation of an antibody:anti-BCMA CAR complex is detected; and the amount of the complex is measured to determine the expression of the anti-BCMA CAR on the T cells.

38. A method for non-diagnostic and non-therapeutic purposes of determining the expression of anti-BCMA CAR in a T cell population, comprising: Contact a population of T cells with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 10 or a conjugate as described in any one of claims 11 to 29; detect the formation of an antibody:anti-BCMA CAR complex on one or more T cells; and measure the amount of the complex to determine the expression of the anti-BCMA CAR on the one or more T cells.

39. A method for determining anti-BCMA CAR in T cell populations + Methods for expressing T cells for non-diagnostic and non-therapeutic purposes include: Contact a population of T cells with an antibody or antigen-binding fragment thereof as claimed in any one of claims 1 to 10 or a conjugate as claimed in any one of claims 11 to 29; detect the formation of an antibody-anti-BCMA CAR complex on one or more T cells; and enumerate the T cells expressing the anti-BCMA CAR.

40. The method of any one of claims 35 to 39, wherein immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR) based biosensor, fluorescence microscopy, flow cytometry, or fluorescence activated cell sorting (FACS) is used to detect the formation of the antibody:anti-BCMA CAR complex.

41. The method of claim 37 or 38, wherein immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR) based biosensor, fluorescence microscopy, flow cytometry, or fluorescence activated cell sorting (FACS) is used to measure the amount of the antibody:anti-BCMA CAR complex.

42. The method of claim 39, wherein immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), immunosorbent assay, chemiluminescence assay, electrochemiluminescence assay, surface plasmon resonance (SPR)-based biosensor, fluorescence microscopy, flow cytometry, or fluorescence activated cell sorting (FACS) is used to enumerate the T cells expressing the anti-BCMA CAR.