B-cell maturation antigen (BCMA) anti-idiotypic antibodies

Abstract

Provided herein are anti-idiotypic antibodies that specifically recognize anti-BCMA antibody moieties, particularly anti-BCMA antibody moieties that are present in recombinant receptors, including chimeric antigen receptors (CARs). The disclosure also relates to the use of anti-idiotypic antibodies for the specific identification and / or selection of cells expressing such recombinant receptors, such as anti-BCMA CAR T cells. The disclosure also relates to the use of anti-idiotypic antibodies for specific activation of such cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 376,156, filed on September 19, 2022, the contents of which are hereby incorporated by reference in their entirety.

[0003] Reference to a sequence listing

[0004] This application contains a sequence listing, which is electronically submitted in XML file format and is hereby incorporated by reference in its entirety. The XML copy was created on August 31, 2023, named AT-036_02WO_SL.xml, and is 44,155 bytes in size. Technical Field

[0005] The present disclosure relates in some aspects to anti-idiotype (anti-id) antibodies, which specifically recognize anti-BCMA antibody parts, particularly anti-BCMA antibody parts present in recombinant receptors (including chimeric antigen receptors (CAR)). The present disclosure also relates to the use of anti-idiotype antibodies, such as for specific identification, quantification or selection of cells expressing such recombinant receptors, such as anti-BCMA CART cells. The present disclosure also relates to the use of anti-idiotype antibodies for specific activation of such cells. Background Art

[0006] Anti-idiotypic antibodies are a subset of antibodies produced against immune antibodies. These anti-idiotypic antibodies demonstrate specific binding to the idiotype (unique antigenic determinant on the antibody surface) of the immune antibody. Anti-idiotypic antibodies can generally be classified into three different groups: (1) antibodies that recognize idiotypes different from the antigen binding site (ABS) on the immune antibody; (2) antibodies that recognize epitopes within the ABS and mimic the structure of the nominal antigen; and (3) antibodies that recognize epitopes within the ABS without the structure of the nominal antigen (see, e.g., Pan et al., (1995) FASEB J 9: 43-49).

[0007] Conventional methods for detecting CAR expression (such as anti-BCMA CAR expression) (e.g., using soluble human BCMA-Fc fusion protein) may lack specificity, have batch-to-batch variability, produce low-intensity signals in analytical methods (e.g., flow cytometry), or be difficult to prepare in sufficient quantities, thereby potentially underestimating the true number of CAR-expressing cells.

[0008] Therefore, there is a need for robust reagents to accurately detect anti-BCMACAR expression on engineered T cells or in in vitro assays. Provided herein are methods and compositions that address this and other needs. Summary of the invention

[0009] Provided herein are reagents that specifically bind to antibodies (including antibody fragments, such as single-chain variable fragments (scFv)) and chimeric molecules containing them (such as chimeric antigen receptors). Embodiments provided herein also include the purposes and methods of use of such reagents, including for detecting, using, manipulating and / or stimulating cells or therapies containing or suspected of containing the antibody or chimeric molecule, such as in the detection, stimulation or use of CAR-expressing cells.

[0010] In some aspects, the antibody is or includes an anti-idiotype (anti-id) antibody or an antigen-binding fragment thereof, which specifically binds to a target antibody, the target antibody being or containing the variable region of an antibody named C29, and / or specifically binds to a chimeric molecule containing such an antibody fragment, such as a CAR having a binding domain, the binding domain containing an antibody variable region derived from C29 or a portion thereof, such as in the form of an scFv. In some aspects, the antibody is or includes an anti-idiotype antibody or an antigen-binding fragment thereof, which specifically binds to a target antibody, the target antibody being or containing the variable region of an antibody named P5A2, which exhibits a high degree of sequence identity with C29 in the heavy and light chain variable regions, and / or specifically binds to a chimeric molecule containing such an antibody fragment, such as a CAR having a binding domain, the binding domain containing an antibody variable region derived from P5A2 or a portion thereof, such as in the form of an scFv.

[0011] In some embodiments, the anti-BCMA scFv derived from C29 comprises an amino acid sequence having at least 80%, at least 90%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the anti-BCMA scFv derived from P5A2 comprises an amino acid sequence having at least 80%, at least 90%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the isolated antibody does not bind to the framework regions of C29 or P5A2. In some embodiments, the isolated antibody binds to an anti-BCMA scFv derived from C29 or P5A2 with a KD of no more than 10 nM, no more than 5 nM, no more than 1 nM, no more than 100 pM, no more than 90 pM, no more than 80 pM, no more than 70 pM, no more than 60 pM, no more than 50 pM, no more than 40 pM, no more than 30 pM, or no more than 20 pM, as determined by Biacore analysis at 25°C. In various embodiments, the antigen binding molecule is selected from the group consisting of: antibody; scFv; Fab; Fab'; Fv, F(ab')2; dAb; human antibody; humanized antibody; chimeric antibody; monoclonal antibody; polyclonal antibody; recombinant antibody; IgE antibody; IgD antibody; IgM antibody; IgG1 antibody; IgG1 antibody having at least one mutation in the hinge region; IgG2 antibody; IgG2 antibody having at least one mutation in the hinge region; IgG3 antibody; IgG3 antibody having at least one mutation in the hinge region; IgG4 antibody; IgG4 antibody having at least one mutation in the hinge region; antibodies comprising at least one non-naturally occurring amino acid, and any combination thereof.

[0012] In further embodiments, the isolated anti-idiotypic antibody comprises a heavy chain (HC), and in specific embodiments, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 2. In further specific embodiments of the antibodies provided herein, a heavy chain CDR1 selected from SEQ ID NO: 6, 7 and 8 is included. In additional specific embodiments of the antibodies provided herein, a heavy chain CDR2 selected from SEQ ID NO: 9 and 10 is included. In still other embodiments of the antibodies provided herein, a heavy chain CDR3 selected from SEQ ID NO: 11 is included. In still further embodiments, the heavy chain of the antibodies provided herein comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, each CDR comprising the amino acid sequence shown in Table 1c.

[0013] In some embodiments, the anti-idiotypic antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen binding molecule provided herein.

[0014] In some embodiments, the isolated anti-idiotypic antibodies provided herein comprise a light chain (LC), and in various embodiments, the LC comprises a light chain variable region (VL) sequence of SEQ ID NO: 4. In some embodiments, the light chain variable region (VL) of the antibodies provided herein comprises one or more of (a) CDR1, (b) CDR2, and (c) CDR3. In further specific embodiments, the light chain CDR1 of the antibodies provided herein may comprise SEQ ID NO: 12. In other embodiments, the light chain CDR2 of the antibodies provided herein may comprise SEQ ID NO: 13. In still further embodiments, the light chain CDR3 of the antibodies provided herein may comprise SEQ ID NO: 14. In still further embodiments, the light chain of the antibodies provided herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, each CDR comprising the amino acid sequence in Table 1d.

[0015] In some embodiments, provided herein is a VL amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VL of the antigen binding molecule.

[0016] In some embodiments, the anti-idiotypic antibodies provided herein comprise (a) a VH comprising the amino acid sequence of SEQ ID NO: 2; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 4. In another embodiment, the antibodies provided herein comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 6, 7 or 8; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 9 or 10; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 12; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 13; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 14.

[0017] In further embodiments, the isolated anti-idiotypic antibody comprises a heavy chain (HC), and in specific embodiments, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 20. In further specific embodiments of the antibodies provided herein, a heavy chain CDR1 is selected from SEQ ID NOs: 24, 25, and 26. In additional specific embodiments of the antibodies provided herein, a heavy chain CDR2 is selected from SEQ ID NOs: 27 and 28. In still other embodiments of the antibodies provided herein, a heavy chain CDR3 is selected from SEQ ID NO: 29. In still further embodiments, the heavy chain of the antibodies provided herein comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, each CDR comprising the amino acid sequence shown in Table 1c.

[0018] In some embodiments, the anti-idiotypic antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen binding molecule provided herein.

[0019] In some embodiments, the isolated anti-idiotypic antibodies provided herein comprise a light chain (LC), and in various embodiments, the LC comprises a light chain variable region (VL) sequence of SEQ ID NO: 22. In some embodiments, the light chain variable region (VL) of the antibodies provided herein comprises one or more of (a) CDR1, (b) CDR2, and (c) CDR3. In further specific embodiments, the light chain CDR1 of the antibodies provided herein may comprise SEQ ID NO: 30. In other embodiments, the light chain CDR2 of the antibodies provided herein may comprise SEQ ID NO: 31. In still further embodiments, the light chain CDR3 of the antibodies provided herein may comprise SEQ ID NO: 32. In still further embodiments, the light chain of the antibodies provided herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, each CDR comprising the amino acid sequence in Table 1d.

[0020] In some embodiments, provided herein is a VL amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VL of the antigen binding molecule.

[0021] In some embodiments, the anti-idiotypic antibodies provided herein comprise (a) a VH comprising the amino acid sequence of SEQ ID NO: 20; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 22. In another embodiment, the antibodies provided herein comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 24, 25, or 26; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 27 or 28; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 29; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 30; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 31; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 32.

[0022] In further embodiments, the isolated anti-idiotypic antibody comprises a heavy chain (HC), and in specific embodiments, the HC comprises the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 33. In further specific embodiments of the antibodies provided herein, a heavy chain CDR1 selected from SEQ ID NO: 37, 38 and 39 is included. In additional specific embodiments of the antibodies provided herein, a heavy chain CDR2 selected from SEQ ID NO: 40 and 41 is included. In still other embodiments of the antibodies provided herein, a heavy chain CDR3 selected from SEQ ID NO: 42 is included. In still further embodiments, the heavy chain of the antibodies provided herein comprises a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3, each CDR comprising the amino acid sequence shown in Table 1c.

[0023] In some embodiments, the anti-idiotypic antibody comprises a VH amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VH of an antigen binding molecule provided herein.

[0024] In some embodiments, the isolated anti-idiotypic antibodies provided herein comprise a light chain (LC), and in various embodiments, the LC comprises a light chain variable region (VL) sequence of SEQ ID NO: 35. In some embodiments, the light chain variable region (VL) of the antibodies provided herein comprises one or more of (a) CDR1, (b) CDR2, and (c) CDR3. In further specific embodiments, the light chain CDR1 of the antibodies provided herein may comprise SEQ ID NO: 43. In other embodiments, the light chain CDR2 of the antibodies provided herein may comprise SEQ ID NO: 44. In still further embodiments, the light chain CDR3 of the antibodies provided herein may comprise SEQ ID NO: 45. In still further embodiments, the light chain of the antibodies provided herein comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, each CDR comprising the amino acid sequence in Table 1d.

[0025] In some embodiments, provided herein is a VL amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the VL of the antigen binding molecule.

[0026] In some embodiments, the anti-idiotypic antibodies provided herein comprise (a) a VH comprising the amino acid sequence of SEQ ID NO: 33; and (b) a VL comprising the amino acid sequence of SEQ ID NO: 35. In another embodiment, the antibodies provided herein comprise (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, 38 or 39; (b) a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 40 or 41; (c) a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42; (d) a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 43; (e) a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 44; and (f) a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 45.

[0027] In some of any such embodiments, the target antibody or antigen-binding fragment is a single chain fragment. In some aspects, the fragment contains an antibody variable region joined by a flexible linker. In some of any such embodiments, the fragment contains a scFv.

[0028] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope of a target antibody or antigen-binding fragment thereof that is the same as or overlaps with the epitope specifically bound by the anti-id antibody or antigen-binding fragment according to any of the embodiments described herein.

[0029] In some of any such embodiments, the target antibody or antigen binding fragment is within the antigen binding domain of the extracellular portion of the chimeric antigen receptor or is included in the antigen binding domain of the extracellular portion of the chimeric antigen receptor; and / or the anti-id antibody or antigen binding fragment specifically binds to the target antibody or antigen binding fragment contained in the antigen binding domain of the extracellular portion of the CAR or is included in the antigen binding domain of the extracellular portion of the CAR. In some embodiments, the target antibody or antigen binding fragment is a scFv, and the anti-id antibody or antigen binding fragment specifically binds to an epitope in the scFv of the CAR.

[0030] In some of any such embodiments, the antibody or fragment specifically binds to a scFv derived from antibody C29 contained in the extracellular portion of a chimeric antigen receptor, optionally wherein the scFv derived from antibody C29 contains the heavy chain variable region shown in SEQ ID NO: 15 and / or the light chain variable region shown in SEQ ID NO: 16 and / or contains the amino acid sequence shown in SEQ ID NO: 1. In some of any such embodiments, the antibody or fragment specifically binds to a scFv derived from antibody P5A2 contained in the extracellular portion of a chimeric antigen receptor, optionally wherein the scFv derived from antibody P5A2 contains the heavy chain variable region shown in SEQ ID NO: 17 and / or the light chain variable region shown in SEQ ID NO: 18 and / or contains the amino acid sequence shown in SEQ ID NO: 19.

[0031] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope within or included in all or a portion of a complementarity determining region (CDR) of a target antibody or antigen-binding fragment.

[0032] In some of any such embodiments, the anti-idiotypic antibody or fragment is an antagonist antibody of a CAR containing a target antibody or antigen binding fragment. In some of any such embodiments, the antibody or fragment is an antagonist of a CAR containing a target antibody or antigen binding fragment.

[0033] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is humanized. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is recombinant. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is monoclonal.

[0034] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a Fab' fragment, a Fv fragment, a single-chain variable fragment (scFv), or a single domain antibody.

[0035] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof contains at least a portion of an immunoglobulin constant region. In some embodiments, at least a portion of an immunoglobulin constant region contains an Fc region or a portion of an Fc containing CH2 and CH3 domains. In some aspects, the constant region is derived from human IgG. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is a complete antibody or a full-length antibody.

[0036] Provided herein is a vector containing a nucleic acid molecule according to any one of the embodiments described herein. Also provided herein is a cell containing an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of the embodiments described herein or a nucleic acid molecule according to any one of the embodiments described herein.

[0037] Provided herein is a method for producing an anti-idiotypic antibody or an antigen-binding fragment thereof, the method comprising expressing in a suitable host cell a

[0038] Nucleic acid molecules according to any one of the embodiments described herein or heavy and / or light chains encoded by a vector according to any one of the embodiments described herein and recovering or separating antibodies. In certain embodiments, the method for producing anti-idiotypic antibodies or antigen binding fragments includes culturing cells according to any one of the embodiments described herein under conditions of expressing heavy and / or light chains and recovering or separating antibodies. Anti-idiotypic antibodies or antigen binding fragments thereof produced by methods according to any one of the embodiments described herein are also provided herein.

[0039] Also provided are methods for detecting using any provided reagents (such as anti-idiotypic antibodies). In some embodiments, a method for detecting a target antibody or an antigen-binding fragment thereof (such as a CAR containing it) is provided, the method comprising (a) contacting a composition containing a target antibody (such as those having a variable region derived from antibody C29 or derived from antibody P5A2 or derived from an antigen-binding fragment of any of such antibodies) with an anti-idiotypic antibody or an antigen-binding fragment thereof; and (b) detecting an anti-idiotypic antibody bound to a target antibody or antigen-binding fragment and / or detecting the presence or absence of a target antibody or reagent.

[0040] In some embodiments, the method provided relates to detecting a CAR containing a target antibody or its antigen binding fragment according to any one of the embodiments, such as a CAR containing a variable domain derived from C29 or P5A2. In some aspects, the method includes (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody for antibody C29 or its antigen binding fragment with an anti-idiotype antibody or its antigen binding fragment according to any one of the described embodiments or a conjugate according to any one of the described embodiments, the anti-idiotype antibody or its antigen binding fragment or the conjugate specifically binds to a target antibody for antibody C29 or its antigen binding fragment; and (b) detecting cells bound to anti-idiotype antibodies. In some embodiments, the method includes (a) contacting a cell expressing a chimeric antigen receptor (CAR) containing a target antibody that is antibody P5A2 or an antigen binding fragment thereof with an anti-idiotypic antibody or an antigen binding fragment thereof of any one of the described embodiments or a conjugate of any one of the described embodiments, wherein the anti-idiotypic antibody or an antigen binding fragment thereof or the conjugate specifically binds to a target antibody that is antibody P5A2 or an antigen binding fragment thereof; and (b) detecting cells bound to the anti-idiotypic antibody. In some of any such embodiments, the anti-idiotypic antibody or its antigen binding fragment is directly or indirectly labeled for detection.

[0041] In some embodiments, a method for selecting cells from a cell population is provided, the method comprising (a) contacting a cell population expressing a chimeric antigen receptor (CAR) containing a target antibody or a cell bound to a target antibody with an anti-idiotypic antibody or its antigen-binding fragment according to any one of the embodiments described herein or a conjugate according to any one of the described embodiments, wherein the anti-idiotypic antibody or its antigen-binding fragment or the conjugate specifically binds to a target antibody that is antibody C29 or its antigen-binding fragment, wherein the target antibody is antibody C29 or its antigen-binding fragment; and (b) selecting cells bound to the anti-idiotypic antibody. In some embodiments, the method comprises (a) contacting a cell population expressing a chimeric antigen receptor (CAR) containing a target antibody or a cell bound to a target antibody with an anti-idiotypic antibody or its antigen-binding fragment according to any one of the described embodiments or a conjugate according to any one of the described embodiments, wherein the anti-idiotypic antibody or its antigen-binding fragment or the conjugate specifically binds to a target antibody that is antibody P5A2 or its antigen-binding fragment, wherein the target antibody is antibody P5A2 or its antigen-binding fragment; and (b) selecting cells bound to the anti-idiotypic antibody.

[0042] In some cases, cells that bind to the anti-idiotypic antibody are selected by affinity-based separation. In some aspects, affinity-based separation is immunoaffinity-based separation. In some of any such embodiments, affinity-based separation is performed by flow cytometry.

[0043] The provided methods also include methods of stimulating cells with reagents, such as stimulating cells containing molecules such as CARs, which are or contain target antibodies recognized by anti-idiotypic antibodies. In some aspects, the method involves incubating an input composition containing a chimeric antigen receptor (CAR) containing a target antibody for antibody C29 or its antigen binding fragment with an anti-idiotypic antibody or its antigen binding fragment according to any one of the described embodiments or a conjugate of any one of the described embodiments, wherein the anti-idiotypic antibody or its antigen binding fragment or the conjugate specifically binds to a target antibody for antibody C29 or its antigen binding fragment, thereby generating an output composition containing stimulated cells. In some embodiments, the method includes incubating an input composition containing a chimeric antigen receptor (CAR) containing a target antibody for antibody P5A2 or its antigen binding fragment with an anti-idiotypic antibody or its antigen binding fragment of any one of the described embodiments or a conjugate of any one of the described embodiments, wherein the anti-idiotypic antibody or its antigen binding fragment or the conjugate specifically binds to a target antibody for antibody P5A2 or its antigen binding fragment, thereby generating an output composition containing stimulated cells.

[0044] In some embodiments, the methods result in proliferation, activation, stimulation, cytokine release, or other functional outcome, such as upregulation of activation markers or cytokine release or production, of cells expressing a chimeric receptor, such as a CAR recognized by an anti-ID antibody.

[0045] In some aspects, CAR contains a target antibody that specifically binds to BCMA. In some embodiments, the target antibody is antibody C29 or an antigen-binding fragment thereof. In some of any such embodiments, the anti-idiotypic antibody or its antigen-binding fragment is an anti-idiotypic antibody or its antigen-binding fragment specifically bound to a target antibody that is antibody C29 or its antigen-binding fragment according to any one of the described embodiments. In some cases, the target antibody is antibody P5A2 or its antigen-binding fragment. In some of any such embodiments, the anti-idiotypic antibody or its antigen-binding fragment specifically binds to a target antibody, and the target antibody is antibody P5A2 according to any one of the described embodiments, which specifically binds to a target antibody that is antibody P5A2 or its antigen-binding fragment.

[0046] In some of any such embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO: 15 and / or the light chain variable region set forth in SEQ ID NO: 16. In some of any such embodiments, the target antibody or antigen-binding fragment contains the heavy chain variable region set forth in SEQ ID NO: 17 and / or the light chain variable region set forth in SEQ ID NO: 18.

[0047] In some embodiments, a method for purifying an antibody or antigen-binding fragment thereof is provided, the method comprising (a) contacting a composition or sample containing a target antibody that is antibody C29 or an antigen-binding fragment thereof with an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of the embodiments described herein or a conjugate according to any one of the described embodiments, wherein the anti-idiotypic antibody or antigen-binding fragment thereof or the conjugate specifically binds to the target antibody that is antibody C29 or an antigen-binding fragment thereof; and (b) isolating a complex containing the anti-idiotypic antibody. In some embodiments, the method comprises (a) contacting a composition or sample containing a target antibody that is antibody P5A2 or an antigen-binding fragment thereof with an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of the described embodiments or a conjugate according to any one of the described embodiments, wherein the anti-idiotypic antibody or antigen-binding fragment thereof or the conjugate specifically binds to the target antibody that is antibody P5A2 or an antigen-binding fragment thereof; and (b) isolating a complex containing the anti-idiotypic antibody.

[0048] In some of any such embodiments, the antigen binding fragment contains the variable heavy chain region and / or variable light chain region of the target antibody. In some embodiments, the antigen binding fragment is a single chain fragment. In some aspects, the antigen binding fragment is a scFv. In some of any such embodiments, the antigen binding fragment is within the antigen binding domain of the extracellular portion of the chimeric antigen receptor (CAR) or is included in the antigen binding domain of the extracellular portion of the chimeric antigen receptor (CAR).

[0049] In some of any such embodiments, the target antibody binds to BCMA. In some embodiments, the antigen-binding fragment of the target antibody is derived from antibody C29, optionally wherein the antigen-binding fragment of the target antibody contains the heavy chain variable region shown in SEQ ID NO: 15 and / or the light chain variable region shown in SEQ ID NO: 16. In some of any such embodiments, the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from antibody C29, optionally wherein the scFv contains the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the antigen-binding fragment of the target antibody is derived from antibody P5A2, optionally wherein the antigen-binding fragment of the target antibody contains the heavy chain variable region shown in SEQ ID NO: 17 and / or the light chain variable region shown in SEQ ID NO: 18. In some embodiments, the antigen-binding fragment of the target antibody is a single chain variable fragment (scFv) derived from antibody P5A2, optionally wherein the scFv contains the amino acid sequence shown in SEQ ID NO: 19.

[0050] In some embodiments, a method of depleting cells is provided, the method comprising administering to a subject a composition comprising an anti-idiotype antibody or its antigen-binding fragment according to any one of the embodiments described herein or a conjugate according to any one of the described embodiments, the anti-idiotype antibody or its antigen-binding fragment or the conjugate specifically binds to a target antibody that is antibody C29 or its antigen-binding fragment, wherein the subject has been administered a cell expressing a chimeric antigen receptor (CAR) comprising a target antibody that is antibody C29 or its antigen-binding fragment. In some embodiments, the method comprises administering to a subject a composition comprising an anti-idiotype antibody or its antigen-binding fragment or a conjugate of any one of the embodiments described herein or any one of the described embodiments, the anti-idiotype antibody or its antigen-binding fragment or the conjugate specifically binds to a target antibody that is antibody P5A2 or its antigen-binding fragment, wherein the subject has been administered a cell expressing a chimeric antigen receptor (CAR) containing a target antibody that is antibody P5A2 or its antigen-binding fragment. In some embodiments, depletion occurs via antibody-dependent cell-mediated cytotoxicity (ADCC).

[0051] In various embodiments, the antibodies provided herein further comprise a detectable label, and the detectable label can be selected from fluorescent labels, photochromic compounds, protein fluorescent labels, magnetic labels, radioactive labels, and haptens. When a fluorescent label is needed, the fluorescent label can be selected from Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Fluorescent Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhodamine B, B), Texas Red, Allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire Blue, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midorishi Blue, Wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Bright Green, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Sapphire Yellow, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), strawberry fluorescent protein (mStrawberry), TurboFP602, AsRed2, mRFP1, J-Red (J-Red), R-phycoerythrin (RPE), B-phycoerythrin (BPE), cherry fluorescent protein (mCherry), HcRed1, Katusha, P3, peridinin-chlorophyll (PerCP), mKate (TagFP635), TurboFP635, plum fluorescent protein (mPlum), and raspberry fluorescent protein (mRaspberry).In specific embodiments, the fluorescent label may be R-phycoerythrin (PE) or allophycocyanin (APC).

[0052] Also provided herein is a composition comprising an antibody provided herein and optionally a pharmaceutically acceptable carrier or vehicle.

[0053] Provided herein are polynucleotides encoding the heavy chain of the separated antibodies of the antibodies provided herein. In addition, polynucleotides encoding the light chain of the separated antibodies of the antibodies provided herein are also provided. Also provided are vectors, which contain polynucleotides encoding the heavy chain of the separated antibodies of the antibodies provided herein and the light chain of the separated antibodies of the antibodies provided herein. Cells comprising such vectors are also provided, and in various embodiments, the cells include cells selected from: CHO cells, Sp2 / 0 cells, rabbit cells, and Escherichia coli (E.coli) cells.

[0054] Methods of making the isolated antibodies provided herein are also provided and can comprise culturing the cells provided herein under suitable conditions.

[0055] A method for determining the number of cells expressing C29 or P5A2 derived scFv is provided, and can include contacting a sample with an isolated antibody that specifically binds to a C29 or P5A2 derived scFv conjugated to a detectable label, and determining the number of cells expressing C29 or P5A2 derived scFv in the sample. In some embodiments, the isolated antibody that specifically binds to a C29 or P5A2 derived scFv is an antibody provided herein or a humanized form thereof.

[0056] Also provided is a method for determining the number of cells presenting a polypeptide comprising an anti-BCMA scFv derived from C29 or P5A2, wherein the method comprises: (a) providing a sample comprising cells known or suspected of presenting a polypeptide comprising an anti-BCMA scFv derived from C29 or P5A2; (b) contacting the sample with an isolated antigen-binding molecule provided herein or a humanized form thereof under conditions that allow binding of the polypeptide to the antigen-binding molecule; and (c) determining the number of cells presenting the polypeptide in the sample.

[0057] Provided herein is a method for determining the presence or absence of a polypeptide comprising an anti-BCMA scFv derived from C29 or P5A2, wherein the method comprises: (a) providing a sample known or suspected to contain a polypeptide comprising an anti-BCMA scFv derived from C29 or P5A2; (b) contacting the sample with an isolated antigen binding molecule or a humanized form thereof provided herein under conditions that allow the polypeptide to bind to the antigen binding molecule; and (c) detecting the presence or absence of a polypeptide: antigen binding molecule complex. In one embodiment of the method, the sample is a formalin fixed sample. In another embodiment, C29 or P5A2 derived scFv is a component of a chimeric antigen receptor (CAR), and in a further embodiment, the cell expressing C29 or P5A2 derived scFv CAR is an immune cell selected from the following: CD8+T cells, CD4+T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR expressing cells, dendritic cells and NK-T cells. In some embodiments, the isolated antigen binding molecules are detectably labeled, and the detectable label can be selected from a fluorescent label, a photochromic compound, a protein fluorescent label, a magnetic label, a radioactive label, and a hapten.When the detectable label is a fluorescent label, the fluorescent label can be selected from Atto dyes, Alexafluor dyes, quantum dots, hydroxycoumarins, aminocoumarins, methoxycoumarins, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midoris hi Cyan, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), bright green, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Ruby Yellow, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 ("RFP"), strawberry fluorescent protein (mStrawberry), TurboFP602, AsRed2, mRFP1, J-red (J-Red), R-phycoerythrin (RPE), B-phycoerythrin (BPE), cherry fluorescent protein (mCherry), HcRed1, Katusha, P3, polydinoxanthin-chlorophyll (PerCP), mKate (TagFP635), TurboFP635, plum fluorescent protein (mPlum) and raspberry fluorescent protein (mRaspberry). In a specific embodiment, the fluorescent marker is R-phycoerythrin (PE) or allophycocyanin (APC). In some embodiments, the cell expressing C29 or P5A2 derived scFv CAR is an immune cell, wherein the immune cell is a T cell, and it can be placed in vitro or in vivo. In some embodiments, T cells are placed in blood, extracted tissue, ex vivo grown tissue, or cell culture medium. In one embodiment, T cells are autologous T cells.In another embodiment, the T cells are allogeneic T cells.

[0058] In a further aspect, methods for preparing and using anti-idiotypic antibodies are provided herein. In a related aspect, provided herein is a method for quantifying or determining the number of cells expressing anti-BCMA antibodies, the anti-BCMA antibodies comprising the amino acid sequences of SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18, the method comprising contacting cells with the anti-idiotypic antibodies disclosed herein and determining the number of cells expressing anti-BCMA antibodies in the cells. In another aspect, the disclosure provides a method for determining the presence or absence of cells expressing anti-BCMA antibodies, the anti-BCMA antibodies comprising the amino acid sequences of SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18, the method comprising contacting cells with isolated antibodies and determining the presence or absence of cells expressing anti-BCMA antibodies in the cells. In yet another aspect, the present disclosure provides a method for purifying an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the anti-BMCA antibody comprises an amino acid sequence of SEQ ID NO: 15 and 16 or SEQ ID NO: 17 and 18, the method comprising (a) contacting a sample comprising an anti-BCMA antibody with an anti-idiotypic antibody disclosed herein to form a complex; and (b) isolating or purifying the complex comprising the anti-BCMA antibody and the separated antibody from the sample. In some embodiments, the anti-BCMA antibody is a scFv comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 19. In some embodiments, the cell expresses an anti-BCMA chimeric antigen receptor (CAR) comprising an amino acid sequence of SEQ ID NO: 1 or 19. In some embodiments, the cell is a CAR T cell.

[0059] Also provide the method for the cell of selecting to express anti-BCMACAR, described anti-BCMACAR comprises the aminoacid sequence of SEQ ID NO:1 or 19, described method comprises making cell contact with anti-idiotype antibody described herein and selecting the cell that is combined with anti-idiotype antibody.In certain embodiments, anti-idiotype antibody also comprises detectable label.In certain embodiments, by separation based on affinity, select the cell that is combined with anti-idiotype antibody.

[0060] Also provided is a method for stimulating anti-BCMA CAR T cells, comprising contacting anti-BCMA CAR T cells with antibodies described herein. In certain embodiments, antibodies are conjugated to beads or solid surfaces. In certain embodiments, anti-BCMA CAR T cells are present in a drug substance or drug product. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 A sequence alignment of the VH and VL sequences of anti-BCMA antibody clones C29 and P5A2 is shown. The VH sequences of clones C29 and P5A2 share 95% identity, and the VL sequences share 99% identity. The CDR names according to Kabat are underlined.

[0062] Figure 2A-2B Shown are the results of flow cytometry analysis showing the binding specificity of anti-BCMA anti-id antibody H6 to anti-BCMA CART clone P5A2 and no binding to anti-Flt3 CAR, anti-CD19 CAR or anti-CD70 CAR used as scFv or framework controls. NT: non-transduced. DETAILED DESCRIPTION

[0063] Provided herein are reagents for specifically recognizing anti-BCMA antibody portions (such as anti-BCMA antibody portions present in recombinant receptors (including chimeric antigen receptors)), such as anti-idiotype (anti-id) antibodies and antigen-binding fragments (such as Fv, Fab or single-chain fragments, including scFv). Also provided are its uses and methods of use, as well as compositions and products comprising such reagents, including for specific identification, quantification, selection, separation, purification and / or stimulation and / or activation of cells expressing or including target antibodies or fragments, such as anti-BCMACAR T cells. In some embodiments, the antibodies provided can be used for specific identification, quantification and / or selection of various anti-BCMACARs, such as CARs that are bound to or expressed on the cell surface, and can also be used to specifically activate cells expressing target CARs, such as CAR T cells. In some embodiments, there are provided antibodies specific to anti-BCMA antibodies named C29 or P5A2 or antibody fragments derived therefrom, including antibodies and CARs containing variable regions derived from such antibodies, and / or antibodies containing unique positions contained therein.

[0064] In some aspects, the anti-idiotype antibodies provided are superior to conventional reagents in detecting, identifying, manipulating and / or influencing and / or engineering cells expressing CAR (particularly CAR containing an anti-BCMA antibody scFv extracellular domain or CAR containing a recognized idiotype). In some available methods, the presence or absence or amount of CAR or CAR-expressing cells in a sample is detected (and / or the stimulation or manipulation of CAR) by assessing the presence or absence or amount of alternative molecules (such as those included in the construct encoding CAR and thus acting as indirect or alternative markers of its expression).

[0065] The anti-idiotypic antibodies and antigen-binding fragments provided in some embodiments overcome the challenges of low binding affinity associated with target antibody ligands and non-specific binding associated with antibody reagents directed to the constant region of the target antibody, thereby providing reagents with high affinity and specificity for its target antibody or its antigen-binding fragment. In some embodiments, compared with BCMA-Fc and other reagents currently available for detecting or identifying CARs containing anti-BCMA antibodies, the antibodies provided exhibit higher specificity and binding affinity for their target antibodies or antigen-binding fragments (such as anti-BCMA antibodies named C29 or P5A2).

[0066] In certain embodiments, anti-idiotypic antibodies are multispecific.Multispecific binding molecules include multispecific antibodies, such as bispecific antibodies.Multispecific binding partners, such as antibodies, have binding specificity to at least two different sites, and the at least two different sites can be in the same or different antigens.In certain embodiments, one of the binding specificities is for anti-BCMA antibody part, and the other is for another antigen.In certain embodiments, bispecific antibodies can be combined with two different epitopes of anti-BCMA antibody part.Bispecific antibodies can also be used to locate cytotoxic agents to cells expressing anti-BCMA antibody parts on their surfaces, such as anti-BCMA CART cells.Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.Bispecific antibodies include multispecific single-chain antibodies, such as diabodies, triabodies and tetrabodies, tandem two-scFv and tandem three-scFv.

[0067] In addition, in certain embodiments, anti-idiotypic antibodies and antigen-binding fragments can be selected as agonists or antagonists of chimeric receptors (comprising their target antibodies or antigen-binding fragments), thereby allowing selective detection, separation, ablation and / or depletion (e.g., via antibody-dependent cell-mediated cytotoxicity (ADCC) killing) and / or stimulation or activation of cells with such chimeric receptors bound to or expressed on their surfaces. In some embodiments, anti-idiotypic antibodies can be humanized or fully human antibodies. Anti-idiotypic antibody agonists are provided herein, which exhibit stimulation (such as activation) of CAR containing an extracellular binding domain of an anti-BCMA antibody derived from C29 or P5A2. In some aspects, such antibodies can be used in methods for stimulating and amplifying cells expressing specific CARs, the methods being included in the process for generating and preparing CAR-expressing cells. In some embodiments, the method of stimulation and amplification can be in an in vitro method. In some embodiments, the method of stimulation and amplification can be incorporated into the CAR T manufacturing process. In some embodiments, the method of stimulation and amplification can be in an in vivo method. In some embodiments, anti-idiotypic antibodies can be humanized or fully human antibodies.

[0068] Also provided herein are nucleic acids encoding the anti-idiotype antibodies and fragments provided herein, as well as cells, such as recombinant cells, expressing and used to produce these anti-idiotype antibodies and fragments. Also provided are methods of making and using the anti-idiotype antibodies and fragments, as well as cells expressing or containing the anti-idiotype antibodies and fragments.

[0069] The scFv part of some chimeric antigen receptors (CAR) is derived from fully human antibody clones C29 or P5A2 with high affinity for BCMA. The present disclosure provides a reagent for detecting anti-BCMACAR comprising scFv parts derived from antibody clones C29 or P5A2. Anti-idiotype antibodies specifically bound to anti-BCMA clones C29 or P5A2 and anti-BCMA molecules derived from C29 or P5A2 are disclosed herein. The anti-id antibodies from C29 derived molecules disclosed herein exhibit specific high affinity binding to chimeric antigen receptors (CAR) comprising C29 derived scFv. A non-limiting example of C29 derived scFv includes SEQ ID NO:1 amino acid sequence, which is also found to cross-react with antibody clone P5A2 and recognize the antibody clone. A non-limiting example of P5A2 derived scFv includes SEQ ID NO:19 amino acid sequence.

[0070] When conjugated with bright fluorescent dye, anti-id antibodies disclosed herein stain cells expressing chimeric antigen receptors (CAR) comprising C29 or P5A2 derived scFv with high MFI and low background. Antibodies described herein can be used in the method for detecting anti-BCMACAR expression. These antibodies can be used for identification by both flow cytometry and immunohistochemistry. These antibodies can also be used in the context of, for example, non-clinical studies, for the manufacture of immune cells comprising C29 or P5A2 derived scFv, as pharmacokinetic reagents based on clinical flows, and for clinical immunogenicity studies.

[0071] C29 is a BCMA monoclonal antibody that recognizes human BCMA. The single-chain variable fragment (scFv) formed by C29 includes some targeting components of chimeric antigen receptors (CAR). In some embodiments, the scFv derived from BCMA monoclonal antibody C29 includes a portion of BCMA monoclonal antibody C29 immunoglobulin γ1 heavy chain connected together by a flexible linker (SEQ ID NO: 15) and a portion of anti-BCMA monoclonal antibody C29 immunoglobulin κ light chain (SEQ ID NO: 16). In some embodiments, scFv includes variable fragments of anti-BCMA monoclonal antibody C29 immunoglobulin γ1 heavy chains and anti-BCMA monoclonal antibody C29 immunoglobulin κ light chains connected together by a flexible linker.

[0072] P5A2 is also a BCMA monoclonal antibody that recognizes BCMA. The single-chain variable fragment formed by P5A2 contains some targeting components of chimeric antigen receptors. In some embodiments, the scFv derived from the BCMA monoclonal antibody P5A2 comprises a portion of the BCMA monoclonal antibody P5A2 immunoglobulin γ1 heavy chain (SEQ ID NO: 17) and a portion of the anti-BCMA monoclonal antibody P5A2 immunoglobulin κ light chain (SEQ ID NO: 18) connected together by a flexible linker. In some embodiments, the scFv comprises a variable fragment of the anti-BCMA monoclonal antibody P5A2 immunoglobulin γ1 heavy chain and a variable fragment of the anti-BCMA monoclonal antibody P5A2 immunoglobulin κ light chain connected together by a flexible linker.

[0073] The anti-BCMA monoclonal antibody C29 immunoglobulin gamma 1 heavy chain variable domain comprises the amino acid sequence:

[0074] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLE WVSAIGGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CARYWPMDIWGQGTLVTVSS(SEQ ID NO:15)

[0075] The anti-BCMA monoclonal antibody C29 immunoglobulin kappa light chain variable domain comprises the amino acid sequence:

[0076] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL MYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTF GQGTKVEIK(SEQ ID NO:16)

[0077] The anti-BCMA monoclonal antibody P5A2 immunoglobulin gamma 1 heavy chain variable domain comprises the amino acid sequence:

[0078] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLE WVSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYY CARYWPMDIWGQGTLVTVSS(SEQ ID NO:17)

[0079] The anti-BCMA monoclonal antibody P5A2 immunoglobulin kappa light chain variable domain comprises the amino acid sequence:

[0080] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLL MYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSWPLTF GQGTKVEIK(SEQ ID NO:18)

[0081] An exemplary C29-derived scFv comprises the amino acid sequence:

[0082] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYPMSWVRQAPGKGLEWVSAIGGSGGSLPYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYQSWPLTFGQGTKVEIK(SEQ ID NO:1)

[0083] An exemplary P5A2-derived scFv comprises the amino acid sequence:

[0084] EVQLLESGGGVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISDSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYWPMDIWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLMYDASIRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSWPLTFGQGTKVEIK(SEQ ID NO:19)

[0085] In some embodiments, the scFv comprises at least a portion of the amino acid sequence of SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, the scFv comprises at least a portion of the amino acid sequence of SEQ ID NO: 15 and / or SEQ ID NO: 16, with or without a signal sequence. In some embodiments, the scFv comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15 and / or SEQ ID NO: 16. In some embodiments, the scFv comprises at least a portion of the amino acid sequence of SEQ ID NO: 17 and / or SEQ ID NO: 18. In some embodiments, the scFv comprises at least a portion of the amino acid sequence of SEQ ID NO: 17 and / or SEQ ID NO: 18, with or without a signal sequence. In some embodiments, the scFv comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence identity to the variable region of the amino acid sequence of SEQ ID NO: 17 and / or SEQ ID NO: 18. In some embodiments, the scFv comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the scFv comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 19. Disclosed herein are antigen binding molecules, including antibodies, that specifically bind to anti-BCMA scFv derived from C29 or P5A2, as well as molecules comprising these sequences and cells presenting such molecules. Humanized forms of the antigen binding molecules are also formed as aspects of the present disclosure. Applications and uses of these antigen binding molecules are also disclosed.

[0086] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0087] I. Antigen-binding molecules and polynucleotides encoding the same

[0088] As used herein, "antigen binding domain" means any polypeptide that binds to a specified target antigen, for example, the specified target antigen may be a BCMA protein or a fragment thereof (interchangeably referred to herein as a "BCMA antigen," "BCMA target antigen," or "BCMA target"). In the context of the anti-idiotypic antibodies of the present disclosure, the target antigen is an antigen binding molecule that specifically binds to BCMA (e.g., antibody clones C29 or P5A2 and antigen binding molecules derived from or related to C29 or P5A2, including scFv).

[0089] In some embodiments, the antigen binding domain binds to a BCMA antigen on a tumor cell. In some embodiments, the antigen binding domain binds to a BCMA antigen on a cell implicated in a hyperproliferative disease or to a viral or bacterial antigen.

[0090] Antigen binding domains include but are not limited to antibody binding regions as immune function fragments.The term "immune function fragment" (or "fragment") of antigen binding domains is the species of antigen binding domains, and the species of the antigen binding domains includes at least some of the amino acid present in the lack of full-length chain, but still can be a part of the antibody specifically bound to the target antigen (obtained or synthesized in any case). Such fragments are biologically active because they are bound to the target antigen, and can be bound to a given epi-position including complete antibody competition with other antigen binding domains. In certain embodiments, fragments are neutralizing fragments. In certain embodiments, fragments can block or reduce the activity (for example, blocking effect) of anti-BCMACAR. In certain embodiments, fragments can antagonize the activity of anti-BCMACAR.

[0091] In specific embodiments, the anti-id antibodies of the present disclosure are those identified herein as clone H6, and comprise heavy and light chain amino acid, variable region, CDR sequences, and nucleotide sequences encoding such sequences, as provided and labeled herein.

[0092] Immunologically functional immunoglobulin fragments include, but are not limited to, scFv fragments, Fab fragments (Fab', F(ab')2, etc.), one or more complementarity determining regions ("CDRs"), diabodies (heavy chain variable domains and light chain variable domains are located on the same polypeptide, connected by a short peptide linker that is too short to allow pairing between the two domains on the same chain), domain antibodies, bivalent antigen binding domains (comprising two antigen binding sites), multispecific antigen binding domains, and single-chain antibodies. These fragments may be derived from any mammalian source, including, but not limited to, humans, mice, rats, camels, or rabbits. As will be appreciated by those skilled in the art, antigen binding domains may include non-protein components.

[0093] The variable region usually exhibits the same general structure of a relatively conservative framework region (FR) joined by three hypervariable regions (CDRs). The CDRs from the two chains of each pair are usually aligned by a framework region that can bind to a specific epitope. From the N-terminus to the C-terminus, both the light chain variable region and the heavy chain variable region usually include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. By convention, the CDR regions in the heavy chain are usually referred to as HC CDR1, CDR2, and CDR3. The CDR regions in the light chain are usually referred to as LC CDR1, CDR2, and CDR3.

[0094] In some embodiments, the antigen binding domain includes one or more complementary binding regions (CDRs) present in the full-length light or heavy chains of an antibody, and in some embodiments includes a single heavy and / or light chain or a portion thereof. These fragments may be produced by recombinant DNA techniques or may be produced by enzyme or chemical cleavage of the antigen binding domain (including intact antibodies).

[0095] In some embodiments, the antigen binding domain is an antibody of a fragment thereof, including one or more of its complementary determining regions (CDRs). In some embodiments, the antigen binding domain is a single chain variable fragment (scFv), comprising light chain CDRs CDR1, CDR2, and CDR3, and heavy chain CDRs CDR1, CDR2, and CDR3.

[0096] In general, the assignment of amino acids to each of the framework, CDRs, and variable domains generally conforms to the numbering scheme of Kabat numbering (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIH Publication No. 91-3242, Bethesda Md. 1991), Chothia numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226), contact numbering (see, e.g., Chothia & Lesk, (1987), J Mol Biol 196:901-917; Al-Lazikani et al., (1997) J Mol Biol 273:927-948; Chothia et al., (1992) J Mol Biol 227:799-817; Tramontano et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226), numbering) or AbM protocol (Antibody Modeling program, Oxford Molecular).

[0097] Thus, in some embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the Kabat numbering scheme. In other embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the Chothia numbering scheme. In other embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the contact numbering scheme. In other embodiments, the CDRs of the anti-idiotypic antibodies presented herein are numbered according to the AbM numbering scheme.

[0098] Humanized antibodies are described herein and can be prepared by known techniques. In certain embodiments, humanized monoclonal antibodies include the variable domains of anti-ID antibodies (or all or part of their antigen binding sites) and constant domains derived from human antibodies. Alternatively, humanized antibody fragments may include the antigen binding sites of murine or rabbit monoclonal antibodies and variable domain fragments (lacking antigen binding sites) derived from human antibodies. Procedures for producing engineered monoclonal antibodies include, for example, Riechmann et al., (1988) Nature 332:323, Liu et al., (1987) Proc. Nat. Acad. Sci. USA 84:3439, Larrick et al., (1989) Bio / Technology 7:934, and Winter et al., (1993) TIPS 14:139 described in those. In certain embodiments, chimeric antibodies are CDR-transplanted antibodies. Techniques for humanizing antibodies are discussed in, for example, U.S. Pat. Nos. 5,869,619; 5,225,539; 5,821,337; 5,859,205; 6,881,557; Padlan et al., (1995) FASEB J. 9:133-39; Tamura et al., (2000) J. Immunol. 164:1432-41; Zhang et al., (2005) Mol. Immunol. 42(12):1445-1451; Hwang et al., Methods. (2005) 36(1):35-42; Dall'Acqua et al., (2005) Methods 36(1):43-60; and Clark, (2000) Immunology Today. 21(8):397-402.

[0099] Variants of anti-idiotypic antibodies are also within the scope of the present disclosure, such as variable light chains and / or variable heavy chains, each of which has at least 70-80%, 80-85%, 85-90%, 90-95%, 95-97%, 97-99% or more than 99% identity to the amino acid sequence of the antigen binding domain sequence described herein. In some embodiments, the anti-idiotypic antibodies have at least about 75%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identity to the heavy chain variable region sequences provided in Table 1a and / or the light chain variable sequences provided in Table 1b.

[0100] In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant form contains two variable light chains and two variable heavy chains (or sub-portions thereof). One skilled in the art will be able to determine suitable variants of anti-idiotypic antibodies as described herein using well-known techniques. In certain embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without destroying activity by targeting regions not considered important for activity.

[0101] The anti-ID antibodies disclosed herein can also be fully human monoclonal antibodies. Fully human monoclonal antibodies can be produced by any number of techniques that will be familiar to those of ordinary skill in the art. Such methods include, but are not limited to, transformation of human peripheral blood cells (e.g., containing B lymphocytes) by Epstein Barr Virus (EBV), in vitro immunization of human B-cells, fusion of spleen cells from immune transgenic mice carrying inserted human immunoglobulin genes, separation from human immunoglobulin V region phage libraries, or other procedures known in the art and based on the disclosure of this text.

[0102] Anti-id antibodies that specifically bind to anti-BCMA clones C29 or P5A2 and anti-BCMA molecules derived from C29 or P5A2 are described as "selective" when they bind one target more tightly than they bind a second target.

[0103] When the dissociation constant (Kd) is ~1nM, anti-id antibodies that specifically bind to anti-BCMA clones C29 or P5A2 and anti-BCMA molecules derived from C29 or P5A2 are described as "specifically binding" to their target antigens (e.g., mouse C29 or P5A2 and molecules derived from C29 or P5A2). When the Kd is 1-5nM, the antigen binding domain specifically binds to the antigen with "high affinity", and when the Kd is 0.1-0.5nM, the antigen binding domain specifically binds to the antigen with "very high affinity". In one embodiment, the Kd of the antigen binding domain is about 1nM. In one embodiment, the dissociation rate is <1×10 -5 In other embodiments, the antigen binding domain will be present at about 1×10 -7 M to 1×10 -12 The antigen binding domain will bind to mouse C29 or P5A2 and molecules derived from C29 or P5A2 with a Kd between about 1×10 -5 Up to 1×10 -12 Kd binding between.

[0104] As provided herein, the anti-ID antibodies of the present disclosure specifically bind to mouse C29 or P5A2 and molecules derived from C29 or P5A2 (e.g., murine C29 or P5A2, humanized C29 or P5A2, C29 or P5A2 derived CAR). In certain embodiments, the anti-ID antibodies of the present disclosure are expressed in an amount of less than 1×10 -6 M, less than 1×10 -7 M, less than 1×10 -8 M or less than 1×10 -9 In a particular embodiment, the anti-ID antibody binds to mouse C29 or P5A2 and molecules derived from C29 or P5A2 with a KD of less than 1×10 -7 In another embodiment, the anti-ID antibody binds to mouse C29 or P5A2 and molecules derived from C29 or P5A2 with a KD of less than 1×10 -8 The KD of M binds to mouse C29 or P5A2 and molecules derived from C29 or P5A2. In some embodiments, the anti-ID antibody is at about 1×10 -7 M, about 2×10 -7 M, about 3×10 -7 M, about 4×10 -7 M, about 5×10 -7 M, about 6×10 -7 M, about 7×10 -7 M, about 8×10 -7 M, about 9×10 -7 M, about 1×10 -8M, about 2×10 -8 M, about 3×10 -8 M, about 4×10 -8 M, 5×10 - 8 M, about 6×10 -8 M, about 7×10 -8 M, about 8×10 -8 M, about 9×10 -8 M, about 1×10 -9 M, about 2×10 -9 M, about 3×10 -9 M, about 4×10 -9 M, about 5×10 -9 M, about 6×10 -9 M, about 7×10 -9 M, about 8×10 -9 M, about 9×10 -9 M, about 1×10 -10 M or about 5×10 -10 The Kd of M binds to mouse C29 or P5A2 and molecules derived from C29 or P5A2. In certain embodiments, Kd is calculated as K off / K on The quotient of K on and K off The assay is performed using monovalent antibodies such as Fab fragments, as determined by e.g. In other embodiments, Kd is calculated as K off / K on The quotient of K on and K off Using bivalent antibodies such as Fab fragments, as determined by e.g. Measured by surface plasmon resonance technique.

[0105] In some embodiments, the anti-ID antibody is present in an amount less than 1×10 -4 M -1 s -1 , less than 2×10 -4 M -1 s -1 , less than 3×10 - 4 M -1 s -1 , less than 4×10 -4 M -1 s -1 , less than 5×10 -4 M -1 s -1 , less than 7×10 -4 M -1 s-1 , less than 8×10 -4 M -1 s -1 , less than 9×10 -4 M -1 s -1 , less than 1×10 -5 M -1 s -1 , less than 2×10 -5 M -1 s -1 , less than 3×10 -5 M -1 s -1 , less than 4×10 -5 M -1 s -1 , less than 5×10 -5 M -1 s -1 , less than 6×10 -5 M -1 s -1 , less than 7×10 -5 M -1 s -1 , less than 8×10 -5 M -1 s -1 , less than 9×10 -5 M -1 s -1 , less than 1×10 -6 M -1 s -1 , less than 2×10 -6 M -1 s -1 , less than 3×10 -6 M -1 s -1 , less than 4×10 -6 M -1 s -1 , less than 5×10 -6 M - 1 s -1 , less than 6×10 -6 M -1 s -1 , less than 7×10 -6 M -1 s -1 , less than 8×10 -6 M -1 s -1 , less than 9×10 -6 M -1 s -1 or less than 1×10-7 M -1 s -1 The association rate (k on ) binds to mouse C29 or P5A2 and molecules derived from C29 or P5A2. In certain embodiments, k on The assay is performed using monovalent antibodies such as Fab fragments, as determined by e.g. In other embodiments, k on Using bivalent antibodies to determine, e.g. Measured by surface plasmon resonance technique.

[0106] In some embodiments, the anti-ID antibody is present in an amount less than 1×10 -2 s -1 , less than 2×10 -2 s -1 , less than 3×10 -2 s -1 , less than 4×10 -2 s -1 , less than 5×10 -2 s -1 , less than 6×10 -2 s -1 , less than 7×10 -2 s -1 , less than 8×10 -2 s -1 , less than 9×10 - 2 s -1 , less than 1×10 -3 s -1 , less than 2×10 -3 s -1 , less than 3×10 -3 s -1 , less than 4×10 -3 s -1 , less than 5×10 -3 s -1 , less than 6×10 -3 s -1 , less than 7×10 -3 s -1 , less than 8×10 -3 s -1 , less than 9×10 -3 s -1 , less than 1×10 -4 s -1 , less than 2×10 -4 s -1 , less than 3×10 -4 s -1 , less than 4×10-4 s -1 , less than 5×10 -4 s -1 , less than 6×10 -4 s -1 , less than 7×10 -4 s -1 , less than 8×10 -4 s -1 , less than 9×10 -4 s -1 , less than 1×10 -5 s -1 or less than 5×10 -5 s -1 The dissociation rate (k off ) binds to mouse C29 or P5A2 and molecules derived from C29 or P5A2. In certain embodiments, k off The assay is performed using monovalent antibodies such as Fab fragments, as determined by e.g. In other embodiments, k off Using bivalent antibodies to determine, e.g. Measured by surface plasmon resonance technique.

[0107] Provided herein are anti-idiotypic antibodies that specifically bind to anti-BCMA clones C29 or P5A2 and antigen-binding molecules derived from C29 or P5A2, comprising a variable heavy chain (VH), wherein the amino acid sequence or polynucleotide sequence of VH is selected from the VH sequences presented in Table 1a.

[0108] Table 1a: Heavy chain variable region (VH)

[0109]

[0110]

[0111]

[0112] Provided herein are anti-idiotypic antibodies that specifically bind to anti-BCMA clone C29 or P5A2, comprising a variable light chain (VL), wherein the amino acid sequence or polynucleotide sequence of VL is selected from the VL sequences presented in Table 1b.

[0113] Table 1b: Light chain variable region

[0114]

[0115]

[0116] Provided herein are anti-idiotypic antibodies that specifically bind to anti-BCMA clones C29 or P5A2 and antigen-binding molecules derived from C29 or P5A2, wherein the anti-idiotypic antibodies comprise a variable heavy chain (VH) and a variable light chain (VL), wherein the amino acid sequence or polynucleotide sequence of VH is selected from the VH sequences presented in Table 1a; and wherein the amino acid sequence or polynucleotide sequence of VL is selected from the VL sequences presented in Table 1b.

[0117] In some embodiments, the anti-idiotypic antibodies that specifically bind to the anti-BCMA clones C29 or P5A2 and antigen binding molecules derived from C29 or P5A2 comprise VH CDR 1, CDR2 and CDR3 of the VH sequences presented in Table 1a. In some embodiments, VH CDR1, CDR2 and CDR3 are selected from the CDR sequences presented in Table 1c.

[0118] Table 1c: Heavy Chain CDRs

[0119]

[0120] In some embodiments, the anti-idiotypic antibodies that specifically bind to the anti-BCMA clones C29 or P5A2 and antigen binding molecules derived from C29 or P5A2 comprise VL CDR 1, CDR2 and CDR3 of the VL sequences presented in Table 1b. In some embodiments, VH CDR1, CDR2 and CDR3 are selected from the CDR sequences presented in Table 1d.

[0121] Table 1d: Light chain CDRs

[0122]

[0123] In some of any such embodiments, the target antibody or antigen binding fragment is within the antigen binding domain of the extracellular portion of the chimeric antigen receptor or is included in the antigen binding domain of the extracellular portion of the chimeric antigen receptor; and / or the anti-id antibody or antigen binding fragment specifically binds to the target antibody or antigen binding fragment contained in the antigen binding domain of the extracellular portion of the CAR or is included in the antigen binding domain of the extracellular portion of the CAR. In some embodiments, the target antibody or antigen binding fragment is a scFv, and the anti-id antibody or antigen binding fragment specifically binds to an epitope in the scFv of the CAR.

[0124] In some of any such embodiments, the antibody or fragment specifically binds to a scFv derived from antibody C29 contained in the extracellular portion of a chimeric antigen receptor, optionally wherein the scFv derived from antibody C29 contains the heavy chain variable region shown in SEQ ID NO: 15 and / or the light chain variable region shown in SEQ ID NO: 16 and / or contains the amino acid sequence shown in SEQ ID NO: 1. In some of any such embodiments, the antibody or fragment specifically binds to a scFv derived from antibody P5A2 contained in the extracellular portion of a chimeric antigen receptor, optionally wherein the scFv derived from antibody P5A2 contains the heavy chain variable region shown in SEQ ID NO: 17 and / or the light chain variable region shown in SEQ ID NO: 18 and / or contains the amino acid sequence shown in SEQ ID NO: 19.

[0125] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment specifically binds to an epitope within or included in all or a portion of a complementarity determining region (CDR) of a target antibody or antigen-binding fragment.

[0126] In some of any such embodiments, the anti-idiotypic antibody or fragment is an antagonist antibody of a CAR containing a target antibody or antigen binding fragment. In some of any such embodiments, the antibody or fragment is an antagonist of a CAR containing a target antibody or antigen binding fragment.

[0127] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is humanized. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is recombinant. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is monoclonal.

[0128] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some aspects, the antigen-binding fragment is selected from a fragment antigen-binding (Fab) fragment, a F(ab')2 fragment, a Fab' fragment, a Fv fragment, a single-chain variable fragment (scFv), or a single domain antibody.

[0129] In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof contains at least a portion of an immunoglobulin constant region. In some embodiments, at least a portion of an immunoglobulin constant region contains an Fc region or a portion of an Fc containing CH2 and CH3 domains. In some aspects, the constant region is derived from human IgG. In some of any such embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof is a complete antibody or a full-length antibody.

[0130] Provided herein is a vector containing a nucleic acid molecule according to any one of the embodiments described herein. Also provided herein is a cell containing an anti-idiotypic antibody or antigen-binding fragment thereof according to any one of the embodiments described herein or a nucleic acid molecule according to any one of the embodiments described herein.

[0131] The present disclosure encompasses modifications of anti-ID antibodies comprising the sequences shown in Tables 1a to 1d, including functionally equivalent anti-ID antibodies with modifications that do not significantly affect their properties and variants with enhanced or reduced activity and / or affinity. For example, the amino acid sequence can be mutated to obtain anti-BCMA clones C29 or P5A2 and antigen-binding molecules derived from C29 or P5A2 with the desired binding affinity for anti-ID antibodies. The modification of polypeptides is a routine practice in the art and therefore does not need to be described in detail herein. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly adversely change the functional activity or maturation (enhancement) of the polypeptide to its ligand, or the use of chemical analogs.

[0132] Amino acid sequence insertions include amino and / or carboxyl terminal fusions within the length range of a polypeptide from one residue to one hundred or more residues, and single or multiple amino acid residues inserted within the sequence. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions of enzymes or polypeptides that extend the half-life of the antibody in the blood circulation to the N-terminus or C-terminus of the antibody.

[0133] Substitution variants remove at least one amino acid residue in the antigen binding domains and insert different residues into their positions. In certain embodiments, the relevant sites of substitution mutations include hypervariable regions / CDRs, but also encompass FR changes. Conservative substitutions are shown in Table 2 under the heading of "conservative substitutions". If such substitutions cause changes in biological activity, more substantial changes and screening products named "exemplary substitutions" or as further described below about amino acid classes may be introduced into Table 2.

[0134] Table 2: Amino Acid Substitutions

[0135]

[0136] II. Methods for preparing anti-ID antibodies

[0137] In order to clone a polynucleotide, a vector can be introduced into a host cell (isolated host cell) to allow replication of the vector itself, and thereby amplify copies of the polynucleotide contained therein. The cloning vector may contain sequence components, generally including but not limited to a replication origin, a promoter sequence, a transcription initiation sequence, an enhancer sequence, and a selectable marker. These elements may be selected as appropriate by a person of ordinary skill in the art. For example, a replication origin may be selected to promote autonomous replication of the vector in a host cell.

[0138] In certain embodiments, the present disclosure provides an isolated host cell containing a vector provided herein. The host cell containing the vector can be used for expression or cloning of the polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells, such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, true bacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae, such as Escherichia, such as Escherichia coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, such as Salmonella typhimurium; Serratia, such as Serratia marcescens. marcescens); and Shigella, as well as Bacilli, such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces.

[0139] The vector can be introduced into the host cell using any suitable method known in the art, including but not limited to DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipid mediated delivery, liposome mediated transfection, electroporation, gene gun method, receptor mediated gene delivery, delivery mediated by polylysine, histone, chitosan and peptide. Standard methods for transfecting and transforming cells for expressing the vector of interest are well known in the art. In another embodiment, a mixture of different expression vectors can be used for a donor population of genetically modified immune effector cells, wherein each vector encodes a different CAR as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, wherein a certain proportion of the engineered cells express more than one different CAR.

[0140] In one embodiment, the present disclosure provides a method for evaluating a genetically engineered cell expressing a CAR targeting BCMA. In some embodiments, the engineered cell is evaluated after thawing the cryopreserved immune cells.

[0141] In some embodiments, cells are formulated by first collecting the cells from their culture medium, and then washing and concentrating the cells in a culture medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration in a therapeutically effective amount. Suitable infusion media can be any isotonic medium formulation, typically saline Normosol TM R (Abbott) or Plasma-Lyte TM A (Baxter), but 5% dextrose in water or Ringer's lactate may also be used. The infusion medium may be supplemented with human serum albumin.

[0142] In some aspects, the anti-ID antibodies disclosed herein are used to quantify the therapeutic amount of cells required in an engineered T cell composition, the engineered T cell composition comprising a C29 or P5A2 derived CAR, for example, an anti-BCMACAR (such as a CAR comprising a C29 or P5A2 derived scFv) or a fragment thereof. In some embodiments, the required therapeutic amount is typically at least 2 cells (e.g., at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell subset), or more typically greater than 10 2 cells, and up to 10 6 up to and including 10 8 or 10 9 cells, and can be greater than 10 10 The number of cells will depend on the intended use of the composition and the type of cells included therein. The desired cell density is generally greater than 10 6 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 Cells / ml or greater. Clinically relevant numbers of immune cells can be assigned to cells that accumulate to or exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12 In some aspects of the present disclosure, particularly since all infused cells will be redirected to a specific target antigen (BCMA), 10 6 / kg(10 6 -1011 The CAR therapy may be administered multiple times at doses within these ranges. The cells may be autologous, allogeneic, or xenogeneic to the patient being treated.

[0143] The CAR expressing cell colonies disclosed herein can be administered alone or in the form of a pharmaceutical composition in combination with a diluent and / or with other components (such as IL-2 or other cytokines or cell colonies). The pharmaceutical composition disclosed herein may include CAR expression or TCR expression cell colonies, such as T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions disclosed herein can be formulated for intravenous administration. Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents, such as water for injection, saline solutions (such as physiological saline), Ringer's solution, isotonic sodium chloride; fixed oils, such as synthetic mono- or diglycerides, polyethylene glycol, glycerol, propylene glycol or other solvents that can serve as solvents or suspension media; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral formulations may be sealed in ampoules, disposable syringes or multiple-dose vials made of glass or plastic. Injectable pharmaceutical compositions may be sterile.

[0144] III. Method for Determining the Number of Cells Expressing Anti-BCMACAR

[0145] The present disclosure provides a method for determining the number of cells of anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragments present in a sample.For example, it may be necessary to determine the number of immune cells expressing anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragments present in a sample obtained from a subject. Alternatively, it may be necessary to determine the number of cells transfected and expressing anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragments, which can be used as a measure of transfection efficiency level. The disclosed method can be used in these and other applications in which it is necessary to determine the number of cells of the expression molecules of interest such as anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragments present in a sample.

[0146] Thus, provided is a method of determining the number of cells presenting a molecule in a sample, wherein the molecule comprises a polypeptide comprising an anti-BCMACAR (eg, a CAR comprising a C29- or P5A2-derived scFv) or a fragment thereof.

[0147] In some embodiments, a sample comprising cells known or suspected to express a molecule of interest is provided, comprising a polypeptide comprising an anti-BC MA CAR (e.g., a CAR comprising a C29- or P5A2-derived scFv) or a fragment thereof.

[0148] Then the sample is contacted with the antigen binding molecules of the molecule of specific binding concern under the conditions of allowing to form a binding complex comprising cells and antigen binding molecules present in the sample.Antigen binding molecules can be antigen binding molecules disclosed herein (or its fragment), such as in the diagram, sequence table or example part of the present disclosure.Any antigen binding molecules of the polypeptide comprising anti-BCMACAR (for example, comprising C29- or P5A2-derived scFv CAR) or its fragment can be used in the disclosed method.Multiple examples of suitable antigen binding molecules are provided herein, for example, with one or more of those in CDR shown in Tables 1c and 1d and described herein.

[0149] The cell can be of any type, and can be human or non-human (e.g., mouse, rat, rabbit, hamster, etc.). In some embodiments, the cell is an immune cell. The immune cell of the method can be any type of immune cell (e.g., B lymphocyte, monocyte, dendritic cell, Langerhans cell (Langerhans cells), keratinocyte, endothelial cell, astrocyte, fibroblast and oligodendrocyte). In some embodiments, the immune cell is a T cell, including T cell toxicity, T helper cell and Treg cell. In a specific embodiment, the cell is a T cell, which can be obtained as described herein and by methods known in the art. In this embodiment of the disclosed method, any type of immune cell can be used, and the cell can be a human or non-human cell (including both prokaryotic and eukaryotic cells). Exemplary cells include, but are not limited to, immune cells, such as T cells, tumor infiltrating lymphocytes (TIL), NK cells, TCR expressing cells, dendritic cells and NK-T cells. T cells can be autologous, allogeneic or heterologous. In additional embodiments, the cell is a T cell presenting CAR. The T cells can be CD4+T cells or CD8+T cells. When T cells are used in the disclosed methods, the T cells can be in vivo T cells or in vitro T cells. In addition, the cells can be placed in or separated from any environment capable of maintaining the cells in a viable form, such as blood, tissue or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, suitable buffer, etc.

[0150] In some embodiments, a sample comprising anti-BCMACAR (e.g., CAR comprising C29- or P5A2-derived scFv) or a fragment thereof is contacted with an anti-ID antibody specifically binding to BCMA derived binding molecules disclosed herein. In some embodiments, the anti-ID antibody comprises a detectable label. In some embodiments, a detectable label is conjugated to an anti-ID antibody and a sample expressing anti-BCMACAR (e.g., CAR comprising C29- or P5A2-derived scFv) is contacted under conditions allowing formation of a binding complex comprising cells present in the sample with anti-ID antibodies. Any anti-ID antibody specifically binding to anti-BCMACAR (e.g., CAR comprising C29- or P5A2-derived scFv) can be used in the disclosed method. Multiple examples of suitable anti-ID antibodies are provided herein, for example, those with one or more of the CDRs shown in Table 1c or 1d.

[0151] Any detectable label can be employed in the methods described herein, and a suitable label can be selected using a desired set of criteria. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPYFL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, and Texas Red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Sciences). Science). Suitable optical dyes, including fluorophores, are described in Johnson, Molecular Probes Handbook:A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition , Life Technologies, (2010), hereby expressly incorporated by reference, radiolabeled (e.g., isotopically labeled, e.g., 3 H. 11 C. 14 C. 15 N. 18 F. 35 S. 64 CU, 90 Y. 99 Tc, 111 In, 124 I. 125 I. 131I), photochromic compounds, halogen tags, Atto dyes, Tracy dyes, protein fluorescent markers (e.g., protein fluorescent markers also include (but are not limited to) green fluorescent protein, including Renilla, Ptilosarcus or aequorin species of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP (Clontech Labs., Inc., GenBank Accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (Clontech Labs., Inc), luciferase (Ichiki et al., (1993) J. Immunol. 150: 5408-5417), magnetic labels (e.g., DYNABEADS), etc. Strategies for labeling proteins are well known in the art and can be used in the disclosed methods. See, e.g., Obermaier et al., (2015) Methods Mol Biol 1295:153-65;Srack(2016)NatureMethods 13:33; Site-Specific Protein Labeling:Methods and Protocols , (Gautier and Hinner eds.) 2015, Springer. In some embodiments, the detectable label is a phycoerythrin (PE) or allophycocyanin (APC) fluorescent probe.

[0152] The label may be associated with the anti-ID antibody at any position in the molecule, but it may be desirable to associate the label with the antibody at a position (or positions, if multiple labels are employed) at a point such that the binding properties of the molecule are not altered (unless such altered binding activity is desired). Any antigen binding molecule that specifically binds to a BCMA-binding molecule (or fragment thereof) may be employed, such as those disclosed herein, e.g., those having one or more of the CDRs shown in Table 1c or 1d.

[0153] The antigen binding molecules can be placed on any surface, or not placed on a surface at all. For example, the antigen binding molecules can be present in a buffer and the buffer-antigen binding molecules can be contacted with the sample. Alternatively, the antigen binding molecules can be associated with a surface. Suitable surfaces include agarose beads, magnetic beads (such as ) or plastic, glass or ceramic plates (such as perforated plates), bags (such as cell culture bags), etc. The surface can itself be arranged in another structure (such as a column).

[0154] The conditions that allow for formation of the binding complex will depend on a variety of factors, however generally an aqueous buffer at physiological pH and ionic strength (such as in phosphate buffered saline (PBS)) will facilitate formation of the binding complex and is desirable in the disclosed methods.

[0155] The number of cells present in the binding complex in the sample is determined. The specific method used to determine the number of cells present in the binding complex will depend on the nature of the label selected. For example, when a fluorescent label is selected, FACS can be used; when an isotopic label is selected, mass spectrometry, NMR or other techniques can be used; when a magnetic label is selected, magnetic-based cell sorting can be used; microscopy can also be used. The output of these detection methods can be in the form of a plurality of units, or the output can be in a form that allows the number of units to be calculated based on the output.

[0156] IV. Methods for Determining the Presence or Absence of C29 or P5A2-Derived Anti-BCMACARs

[0157] In certain embodiments, it is sufficient information to know whether or not a sample contains anti-BCMACAR (e.g., CARs comprising C29- or P5A2-derived scFv) or molecules of its fragments. For example, regardless of expression level, it may be advantageous to know that such molecules are expressed. In other cases, it may be necessary to know whether the purification process or steps designed to remove such molecules are effective. Therefore, the presence or absence of anti-BCMACAR (e.g., CARs comprising C29- or P5A2-derived scFv) or its fragments can be used in a variety of applications.

[0158] In some embodiments, a method of determining the presence or absence of a polypeptide comprising an anti-BCMA CAR (e.g., a CAR comprising a C29- or P5A2-derived scFv) or a fragment thereof in a sample is provided.

[0159] In some embodiments, the method comprises providing a sample known or suspected to contain a polypeptide comprising an anti-BC MA CAR (e.g., a CAR comprising a C29- or P5A2-derived scFv) or a fragment thereof.

[0160] The present disclosure provides a kind of specific binding comprising anti-BCMACAR (for example, comprising C29- or P5A2-derived scFv CAR) or the antigen binding molecule of the polypeptide of its fragment, it includes detectable label.Suitable label can be selected using required criterion set. Examples of types of detectable labels include fluorescent labels (e.g., fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, fluorescein yellow, Cascade Blue, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes), FITC, rhodamine, and Texas Red (Pierce), Cy5, Cy5.5, Cy7 (Amersham Life Sciences). Science). Suitable optical dyes, including fluorophores, are described in Johnson, Molecular Probes Handbook:A Guide to Fluorescent Probes and Labeling Techniques, 11th Edition , Life Technologies, (2010), hereby expressly incorporated by reference, radiolabeled (e.g., isotopically labeled, e.g., 3 H. 11 C. 14 C. 15 N. 18 F. 35 S. 64 CU, 90 Y. 99 Tc, 111 In, 124 I. 125 I. 131I). Photochromic compounds, Halo-tags, Atto dyes, Tracy dyes, protein fluorescent labels (e.g., protein fluorescent labels also include, but are not limited to, green fluorescent protein, including Renilla, Ptilosarcus, or aequorin species of GFP (Chalfie et al., (1994) Science 263:802-805), EGFP (Clontech Labs, Inc., GenBank Accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc.; Stauber, (1998) Biotechniques 24:462-471; Heim et al., (1996) Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (Clontech Labs, Inc.), luciferase (Ichiki et al., (1993) J. Immunol. 150:5408-5417), magnetic labels (e.g., ) etc. The strategy for labeling proteins is well known in the art and can be used in the disclosed method. The label can be associated with the antigen binding molecule at any position in the molecule, but it may be necessary to associate the label with the molecule at a point where the binding properties of the molecule are not changed (unless the binding activity of such changes is required) in one position (or multiple positions, if multiple labels are used). Any antigen binding molecule of a polypeptide comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment can be used, such as those disclosed herein, for example, those with one or more of the CDRs described in Tables 1c and 1d.

[0161] Next, the sample is contacted with the antigen-binding molecule under conditions that allow the formation of a binding complex comprising the cells present in the sample and the antigen-binding molecule.

[0162] Sample and antigen binding molecules are allowed to contact under the conditions of forming binding complex between the polypeptide and antigen binding molecules comprising anti-BCMACAR (for example, comprising C29- or P5A2-derived scFv CAR) or its fragment. Allowing the formation of binding complex conditions will depend on multiple factors. Because the component parts of the binding complex can be placed on the surface as described herein, the formed binding complex can also be placed on the surface.

[0163] At this stage, binding complex may not yet be formed, or multiple binding complexes may have been formed, and the binding complex comprises one or more antigen binding molecules combined with the polypeptide comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment. It is also possible that there is an unbound molecule and / or uncombined antigen binding molecule comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment in the local environment of any formed binding complex.

[0164] Then any molecule that is not a part of the binding complex is separated from any formed binding complex. The method of removal will depend on the structure and / or local environment of the binding complex. For example, if the antigen binding molecules are placed on beads, plates or bags, the unbound components of the reaction mixture can be washed off using a solution that makes the formed binding complex complete. In certain embodiments, it is not necessary to separate the binding complex for detection.

[0165] The solution used to induce the formation of the binding complex can be used, for example, as a washing solution to remove unbound components. Any suitable buffer or solution that does not destroy the formed binding complex can also be used. Generally, when performing this step of the method, buffers with high salt concentrations, non-physiological pH containing chaotropic agents or denaturants should be avoided.

[0166] The presence or absence of binding complex can be detected, and the binding complex will include polypeptides and antigen binding molecules comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment.The specific method for detecting the presence or absence of binding complex will generally depend on the property of the selected mark.In certain embodiments, detection method is by colorimetric analysis.The result of the method is qualitative assessment of the presence or absence of antigen binding molecules comprising detectable labeling, and therefore qualitative assessment of the presence or absence of its binding partner--comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment polypeptide.

[0167] As in the case of the disclosed method, the polypeptide comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment can be placed in any environment. In certain embodiments, the polypeptide comprising anti-BCMACAR (for example, CAR comprising C29- or P5A2-derived scFv) or its fragment is expressed on the cell surface. In this embodiment, the cell can be any type, and can be human or non-human (such as mouse, rat, rabbit, hamster, etc.). In certain embodiments, the cell is an immune cell. The immune cell of the method can be any type of immune cell (such as B lymphocyte, monocyte, dendritic cell, Langerhans cell, keratinocyte, endothelial cell, astrocyte, fibroblast and oligodendrocyte). T cell (including T cell toxicity, T helper cell and Treg cell) is particularly suitable. In a specific embodiment, the cell is a T cell, which can be as described herein and obtained by methods known in the art. In this embodiment of the disclosed method, any type of immune cell can be used, and the cell can be human or non-human cell. Exemplary cells include, but are not limited to, immune cells, such as T cells, tumor infiltrating lymphocytes (TIL), NK cells, dendritic cells, and NK-T cells. T cells can be autologous, allogeneic, or heterologous. In additional embodiments, the cells are T cells presenting TCR. T cells can be CD4+T cells or CD8+T cells. When T cells are used in the disclosed methods, T cells can be in vivo T cells or in vitro T cells. In addition, cells can be derived from stem cells, such as iPSC cells, cord blood cells, or mesenchymal stem cells.

[0168] In some embodiments, the cells can be placed in or isolated from any environment capable of maintaining the cells in a viable form, such as blood, tissue, or any other sample obtained from a subject, cell culture medium, ex vivo grown tissue, a suitable buffer, etc. In some embodiments, the cells are in a formalin fixed sample. In some embodiments, the sample is formalin fixed paraffin embedded tissue (FFPE).

[0169] V. Methods and Uses of Antibodies

[0170] In some embodiments, provided herein are methods involving the use of one or more anti-idiotypic antibodies. In some aspects, provided herein are methods for measuring or detecting target antibodies such as CAR or cells expressing CAR, and methods for changing the activity of target antibodies such as CAR or cells expressing CAR. In certain embodiments, the one or more anti-idiotypic antibodies bind, detect, identify and / or quantify CAR and / or cells expressing CAR. In some embodiments, provided herein are methods for contacting and / or incubating together one or more steps of one or more anti-idiotypic antibodies with cells expressing chimeric antigen receptors (CAR) or containing or believed to contain cells expressing chimeric antigen receptors. In some embodiments, the composition or sample treatment, incubation and / or contact with anti-idiotypic antibodies are used under conditions allowing the formation of a complex between anti-idiotypic antibodies and target antibodies (e.g., CAR). In some aspects, the complex can be used for the purpose of detecting, separating and / or measuring CAR. In some embodiments, the formation of the complex changes the activity of the target antibody (e.g., CAR), such as by stimulating receptor signaling activity, or in some embodiments, by preventing the association of CAR with antigens and antagonizing the activity of the target antibody (e.g., CAR).

[0171] A. Detection / Separation Methods

[0172] In some embodiments, there is provided a method involving the use of one or more anti-idiotypic antibodies and / or molecules containing one or more such anti-idiotypic antibodies (such as conjugates and complexes) to detect, bind and / or separate antibodies (e.g., target antibodies). In certain embodiments, the method provides one or more steps of contacting one or more anti-idiotypic antibodies with a sample and / or composition, incubating with a sample and / or composition, and / or exposing to a sample and / or composition. In some embodiments, the sample and / or composition has, may have and / or is suspected of having a target antibody and / or its antigen-binding fragment bound and / or recognized by one or more anti-idiotypic antibodies. In certain embodiments, the antibody or its antigen-binding fragment bound or recognized by one or more anti-idiotypic antibodies contains one or more fusion domains and / or is a fusion protein. In certain embodiments, the target antibody and / or its antigen-binding fragment is CAR. In certain embodiments, the anti-idiotypic antibody binds and / or recognizes an anti-BCMA antibody (e.g., antibody C29 or P5A2) or an antigen-binding fragment thereof, including a chimeric molecule or a conjugate, which includes a CAR containing such an anti-BCMA antibody (e.g., an antibody fragment).

[0173] The method in some embodiments comprises incubating a sample and / or composition containing or suspected of containing a target antibody with an anti-idiotypic antibody, treating a sample and / or composition containing or suspected of containing a target antibody with an anti-idiotypic antibody, and / or contacting a sample and / or composition containing or suspected of containing a target antibody with an anti-idiotypic antibody. In certain embodiments, incubation is performed under conditions that allow the anti-idiotypic antibody to bind to the target antibody present in the composition, for example to form a complex containing the anti-idiotypic antibody and the target antibody.

[0174] In some embodiments, the sample and / or composition contains or is suspected of containing a target antibody, such as CAR. In certain embodiments, the sample and / or composition contains or is suspected of containing cells expressing a target antibody (e.g., CAR). In certain embodiments, the sample is a biological sample. In a particular embodiment, the sample is a serum sample or a blood sample. In some embodiments, the biological sample contains one or more immune cells. In some embodiments, the biological sample is or is derived from a tissue, such as connective tissue, muscle tissue, neural tissue, or epithelial tissue. In a particular embodiment, the biological sample is taken from a human subject, collected from a human subject, and / or obtained. In certain embodiments, the sample contains living and / or intact cells. In some embodiments, the sample is or contains a homogenate and / or cell that has been destroyed and / or lysed. In some embodiments, the biological sample contains proteins and / or antibodies separated from blood, serum, and / or tissue.

[0175] In a particular embodiment, the anti-idiotypic antibody forms a complex with a target antibody, such as CAR, or can form a complex with a target antibody, such as CAR. In a particular embodiment, the complex is detected, measured, quantified and / or evaluated, for example to allow detection, identification, measurement and / or quantification of the target antibody, for example in a composition or sample. In certain embodiments, the method includes detecting whether a complex is formed between the anti-idiotypic antibody and the target antibody in the sample, and / or detecting the presence or absence or level of such binding. In some embodiments, the complex contains a detectable marker. In a particular embodiment, the anti-idiotypic antibody is an immunoconjugate containing a detectable marker. In certain embodiments, the anti-idiotypic antibody contains a detectable marker, is conjugated to a detectable marker, is combined with a detectable marker and / or is attached to a detectable marker. In some embodiments, the complex contains an antibody that binds and / or recognizes an anti-idiotypic antibody, such as a secondary antibody, which is conjugated, combined and / or attached to a detectable marker.

[0176] In some embodiments, methods for detecting, quantifying, detecting and / or evaluating a target antibody in, for example, a sample or composition include detecting a complex of a target antibody and an anti-idiotypic antibody. In some embodiments, the complex contains a detectable label. In certain embodiments, the complex is detected and / or contacted with a detectable label. In some embodiments, the complex is detected by any suitable method or means, such as, but not limited to, flow cytometry, immunocytochemistry, immunohistochemistry, Western blot analysis, and ELISA.

[0177] In some embodiments, the target antibody or antigen binding fragment is bound to a cell or expressed on the surface of a cell. In a particular embodiment, the target antibody (e.g., CAR) is not bound to a cell or contained in a cell, for example, in some embodiments, the target antibody is secreted. In certain embodiments, the antibody has been separated, removed and / or cracked from the surface of a cell.

[0178] In some embodiments, the target antibody is an anti-BCMA antibody. In some embodiments, the target antibody is or is derived from antibody C29 or an antigen-binding fragment thereof. In some embodiments, the target antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 15 and / or the light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody is or is derived from antibody P5A2 or its antigen-binding fragment. In some embodiments, the target antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 17 and / or the light chain variable region shown in SEQ ID NO: 19.

[0179] In some embodiments, a method for detecting a target antibody (such as antibody C29) or an antigen-binding fragment thereof (and / or a chimeric molecule comprising such an antibody (e.g., an antibody fragment), such as a CAR) is provided, the method comprising contacting a composition comprising the target antibody or antigen-binding fragment with an anti-idiotypic antibody or antigen-binding fragment thereof or an anti-idiotypic antibody immunoconjugate described herein, and detecting the anti-idiotypic antibody bound to the target antibody or antigen-binding fragment. In some embodiments, the method further comprises detecting whether a complex is formed between the anti-idiotypic antibody and the target antibody in the composition, such as detecting the presence or absence or level of such binding. In some embodiments, the target antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell, and the detection comprises detecting a cell bound to the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody or its antigen-binding fragment is directly or indirectly labeled for detection. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 17 and / or a light chain variable region shown in SEQ ID NO: 18.

[0180] In some embodiments, a method of separating a target antibody (such as antibody P5A2) or an antigen-binding fragment thereof (and / or a chimeric molecule comprising such an antibody (e.g., an antibody fragment), such as a CAR) is provided, the method comprising contacting a composition and / or a sample containing or suspected of containing the target antibody or antigen-binding fragment with an anti-idiotypic antibody or antigen-binding fragment thereof or an anti-idiotypic antibody immunoconjugate described herein, and separating a complex comprising the anti-idiotypic antibody bound to the target antibody or antigen-binding fragment. In certain embodiments, the target antibody is separated together with the anti-idiotypic antibody described in Section I. In some embodiments, the target antibody or antigen-binding fragment is bound to a cell or expressed on the surface of a cell, and the separation comprises separating cells bound to the anti-idiotypic antibody. In some embodiments, the complex comprising the anti-idiotypic antibody is separated by affinity-based separation. In some embodiments, affinity-based separation is selected from immunoaffinity-based separation, magnetic-based separation, and affinity chromatography. In some embodiments, the target antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO:17 and / or the light chain variable region set forth in SEQ ID NO:18.

[0181] In certain embodiments, the target antibody in the subject is evaluated using the method for detecting the target antibody with an anti-idiotypic antibody described herein.For example, in some embodiments, provided herein is a method for evaluating, measuring and / or quantifying the in vivo pharmacokinetics, amplification and / or persistence of the CAR-expressing cells of the therapeutic cell composition using an anti-idiotypic antibody.In some embodiments, the in vivo pharmacokinetics, amplification and / or persistence of the cell can be measured with the anti-idiotypic antibody provided herein in the method provided herein, and / or changes in cell phenotype or cell function activity, such as CAR-expressing cells applied to immunotherapy (e.g., CAR-T cell therapy).In some embodiments, the presence and / or amount of cells expressing CAR in the subject and / or in the sample obtained from the subject after the therapeutic cell composition is administered is detected during and / or after the therapy of the anti-idiotypic antibody provided herein is measured, and the pharmacokinetics, amplification and / or persistence of the CAR-expressing cells are evaluated.

[0182] In some aspects, anti-idiotypic antibodies are used together with flow cytometry to assess the amount of cells expressing recombinant receptors (e.g., CAR expressing cells for T cell-based therapy) in blood or serum or organ or tissue samples (e.g., disease sites, such as tumor samples) of the subject. In some aspects, persistence is quantified as the number of CAR expressing cells in each microliter sample (e.g., blood or serum sample), or the total number of peripheral blood mononuclear cells (PBMC) or leukocytes or T cells in each microliter sample. In certain aspects, amplification is quantified as the number of CAR expressing cells between each microliter sample (e.g., blood or serum sample) or the total number of peripheral blood mononuclear cells (PBMC) or leukocytes or T cells in each microliter sample over time. In certain embodiments, pharmacokinetics, amplification and / or persistence are measured or assessed by detecting the amount of CAR expressing cells in a subject and / or in a sample collected from a subject at multiple time points.

[0183] In some embodiments, a method for selecting cells expressing a CAR comprising a target antibody (such as antibody C29 or P5A2) or an antigen-binding fragment thereof is provided, the method comprising contacting a cell population comprising cells expressing CAR with an anti-idiotypic antibody or its antigen-binding fragment described herein, and selecting cells bound to the anti-idiotypic antibody. In some embodiments, cells bound to anti-idiotypic antibodies are selected by affinity-based separation. In some embodiments, affinity-based separation is selected from immunoaffinity-based separation, flow cytometry, magnetic-based separation, and affinity chromatography. In some embodiments, anti-idiotypic antibodies or their antigen-binding fragments or anti-idiotypic antibody immunoconjugates are reversibly bound or fixed to a support or fixed phase. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 17 and / or a light chain variable region shown in SEQ ID NO: 18.

[0184] In some embodiments, a method for validating a CAR comprising a target antibody (such as antibody C29 or P5A2) or an antigen-binding fragment thereof is provided, the method comprising a) incubating a sample comprising T cells transduced with CAR with an anti-idiotypic antibody or an antigen-binding fragment thereof targeting CAR; b) determining the percentage of cells bound to the anti-idiotypic antibody or its antigen-binding fragment; and c) validating the CAR based on the percentage of T cells bound to the anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody is labeled, and the T cells bound to the anti-idiotypic antibody are determined by flow cytometry. In some embodiments, the target antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 15 and / or the light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or its antigen-binding fragment comprises the heavy chain variable region shown in SEQ ID NO: 17 and / or the light chain variable region shown in SEQ ID NO: 18.

[0185] Also provided is a method for providing treatment decision information in an individual using a provided anti-idiotypic antibody and a molecule (such as a conjugate and a complex) containing one or more of such anti-idiotypic antibodies, such as by detecting a CAR identified by an anti-idiotypic antibody, such as a CAR comprising a target antibody (such as an anti-BCMA antibody (e.g., antibody C29 or P5A2)) or its antigen-binding fragment. In some embodiments, the method is used to provide treatment decision information in an individual, and these treatment decisions are related to the therapy for administration including CART cells (such as anti-BCMA CART cells). The method in some embodiments includes incubating a biological sample with an anti-idiotypic antibody and / or detecting the biological sample with an anti-idiotypic antibody and / or administering an anti-idiotypic antibody to an individual. In certain embodiments, the biological sample includes a cell or tissue or a part thereof, such as a tumor or cancer tissue or a biopsy or section thereof. In certain embodiments, incubation is performed under conditions that allow the anti-idiotypic antibody to bind to the CAR present in the sample. In some embodiments, the method also includes detecting whether a complex is formed between the anti-idiotypic antibody and the CAR in the sample, such as detecting the presence or absence or level of such a combination. Such a method can be in vitro or in vivo methods.

[0186] B. Use in cell stimulation

[0187] In some embodiments, the anti-idiotypic antibody or its antigen-binding fragment provided is an agonist and / or exhibits specific activity of stimulating cells expressing target antibodies, and the target antibodies include conjugates or chimeric receptors containing them, such as anti-BCMA antibodies (e.g., antibody C29 or P5A2) or their antigen-binding fragments. In some embodiments, there is provided a method involving the use of provided anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies to stimulate or activate cells expressing CAR or cells expressing other chimeric receptors (such as T cells). In some aspects, CAR or other receptors include target antibodies, such as anti-BCMA antibodies (e.g., antibody C29 or P5A2) or their antigen-binding fragments.

[0188] In some embodiments, the method can be used in combination with a method for preparing genetically engineered T cells, such as for amplifying genetically engineered T cells or other cells, wherein the cell has been introduced, for example, by a non-viral method (such as a transposon-based method) of transfection, transduction or nucleic acid transfer, encoding a chimeric receptor (such as CAR) comprising a target antibody Nucleic acid molecule. In some aspects, the target antibody is an anti-BCMA antibody (for example, antibody C29 or P5A2) or its antigen-binding fragment. In a particular embodiment, the target antibody is or contains CAR, such as anti-BCMA CAR. In a particular embodiment, anti-BCMACAR contains scFv, and the scFv is from and / or derived from anti-BCMA antibodies, such as antibody C29 or P5A2.

[0189] The method in some embodiments includes incubating a sample containing T cells transduced with CAR with an anti-idiotypic antibody. In certain embodiments, the method further includes detecting whether the CAR T cells are activated or stimulated, such as by assessing the activity, proliferation and / or expression of activation markers in the CAR T cells. In some embodiments, the target antibody is an anti-BCMA antibody. In some embodiments, the target antibody is or is derived from antibody C29 or P5A2 or an antigen-binding fragment thereof. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 17 and / or a light chain variable region shown in SEQ ID NO: 18.

[0190] In some embodiments, a method of simulating cells is provided, comprising incubating an input composition comprising cells expressing CAR (comprising a target antibody, such as antibody C29 or P5A2 or an antigen-binding fragment thereof) with an anti-idiotypic antibody or its antigen-binding fragment described herein, thereby generating an output composition comprising stimulated cells. In some embodiments, incubation is performed under conditions where the anti-idiotypic antibody or its antigen-binding fragment is combined with CAR, thereby inducing or regulating a signal in one or more cells of the input composition. In some embodiments, the cell comprises a T cell. In some embodiments, the T cell comprises CD4+ and / or CD8+T cells.

[0191] In certain embodiments, an anti-idiotypic antibody is administered to a subject, such as a subject to whom a therapeutic cell composition containing CAR-expressing cells has been previously administered. In some embodiments, administration of an anti-idiotypic antibody to a subject promotes the re-expansion of CAR-expressing cells in the subject, and in some cases, the CAR-expressing cells may reach or exceed the initial peak amplification level before the administration of the anti-idiotypic antibody. In some embodiments, when the level of CAR-expressing cells decreases or is undetectable, anti-idiotypic antibodies are administered to regulate the amplification and / or persistence of CAR-expressing cells. In some embodiments, for example, compared to the efficacy before the administration of the anti-idiotypic antibody, the CAR-expressing cells re-expanded by the anti-idiotypic antibody show increased efficacy in the subject in which it is administered.

[0192] In some embodiments, a method for producing a cell composition is provided, the method comprising introducing a nucleic acid molecule encoding CAR into a cell, thereby generating an input composition, and incubating the input composition with an anti-idiotypic antibody or its antigen-binding fragment specific to the antigen-binding domain of CAR, thereby producing a cell composition. In some embodiments, CAR comprises a target antibody or its antigen-binding fragment that specifically binds to BCMA. In some embodiments, the target antibody is antibody C29 or P5A2 or its antigen-binding fragment. In some embodiments, the anti-idiotypic antibody or its antigen-binding fragment is an anti-idiotypic antibody or its antigen-binding fragment described herein. In some embodiments, the anti-idiotypic antibody or its antigen-binding fragment is an agonist of CAR. In some embodiments, the introduction includes introducing nucleic acid molecules into cells by viral transduction, transposition, electroporation or chemical transfection. In some embodiments, the introduction includes transduction with a retroviral vector comprising nucleic acid molecules, transduction with a lentiviral vector comprising nucleic acid molecules, transposition with a transposon comprising nucleic acid molecules, or electroporation or transfection of a vector comprising nucleic acid molecules to introduce the nucleic acid molecules into cells.

[0193] C. Use in Cell Inactivation / Depletion

[0194] In some embodiments, the anti-idiotypic antibody or antigen-binding fragment thereof provided is an antagonist and / or exhibits specific activity of inhibiting, ablating and / or depleting (e.g., killing via antibody-dependent cell-mediated cytotoxicity (ADCC)) cells expressing a target antibody, such as an anti-BCMA antibody (e.g., antibody C29 or P5A2) or an antigen-binding fragment thereof. Also provided are methods involving the use of the provided anti-idiotypic antibodies and molecules (such as conjugates and complexes) containing one or more of such anti-idiotypic antibodies for inactivation, ablation and / or depletion of CAR T cells, wherein the CAR comprises a target antibody, such as an anti-BCMA antibody (e.g., antibody C29 or P5A2) or an antigen-binding fragment thereof.

[0195] The method in some embodiments includes treating, contacting and / or incubating a composition and / or sample comprising T cells transduced with CAR with an anti-idiotypic antibody. In certain embodiments, the method also includes detecting whether the CAR T cells are inactivated, such as by assessing the viability, proliferation and / or expression of activation markers in CART cells. In some embodiments, the method is associated with a therapy comprising administering CAR T cells. The method in some embodiments includes administering an anti-idiotypic antibody to an individual. In one embodiment, an anti-idiotypic antibody or conjugate is used to ablate and / or deplete (such as kill) CART cells in an individual. In some embodiments, the target antibody is an anti-BCMA antibody. In some embodiments, the target antibody is or is derived from antibody C29 or P5A2 or an antigen-binding fragment thereof. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 17 and / or a light chain variable region shown in SEQ ID NO: 18.

[0196] In some embodiments, anti-idiotypic antibodies are administered to deplete, reduce and / or reduce the number of CAR expressing cells in a subject. In a particular embodiment, anti-idiotypic antibodies are administered to deplete, reduce and / or reduce the amount of CAR expressing cells (e.g., circulating CAR-T cells) by at least 25%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, at least 99.9%, 100% or about 100%. In certain embodiments, depletion, reduction and / or reduction are related to the amount of CAR expressing cells in a subject before the administration of anti-idiotypic antibodies. In a particular embodiment, depletion, reduction and / or reduction are related to the amount of CAR expressing cells in a subject to which anti-idiotypic antibodies are not administered. In some embodiments, after the administration of anti-idiotypic antibodies, CAR expressing cells are not detected in a subject. In a particular embodiment, anti-idiotypic antibodies are human antibodies or humanized antibodies.

[0197] In some embodiments, a method for inactivating CAR T cells is provided, wherein the CAR comprises a target antibody, such as antibody C29 or P5A2 or an antigen-binding fragment thereof, the method comprising incubating a sample comprising CART cells with an antagonistic anti-idiotypic antibody or an antigen-binding fragment thereof targeting CAR, thereby inactivating the CAR T cells in the sample. In some embodiments, anti-idiotypic antibodies are used in an amount sufficient to attenuate the activation of CAR T cells in the sample. In some embodiments, anti-idiotypic antibodies are used in an amount sufficient to substantially inactivate CAR T cells in the sample. In some embodiments, incubation with anti-idiotypic antibodies results in ablation and / or depletion of CAR T cells in the sample. In some embodiments, anti-idiotypic antibodies are used in an amount sufficient to cause clearance of CAR T cells in the sample. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or its antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 17 and / or a light chain variable region shown in SEQ ID NO: 18.

[0198] In some embodiments, a method for adjusting CAR T cell therapy in an individual is provided, wherein the CAR comprises a target antibody, such as antibody C29 or P5A2 or an antigen binding fragment thereof, the method comprising administering to an individual an anti-idiotypic antibody immunoconjugate targeting CAR, wherein the anti-idiotypic antibody immunoconjugate comprises a cytotoxic agent. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to attenuate CAR T cell therapy in an individual. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to substantially stop CAR T cell therapy in an individual. In some embodiments, the anti-idiotypic antibody immunoconjugate is administered in an amount sufficient to cause CAR T cell clearance in an individual. In some embodiments, the cytotoxic agent is selected from a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a protein toxin of bacterial, fungal, plant or animal origin, an enzymatically active toxin or a fragment thereof) and a radioactive isotope. In some embodiments, the target antibody or its antigen binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 15 and / or a light chain variable region shown in SEQ ID NO: 16. In some embodiments, the target antibody or antigen-binding fragment thereof comprises the heavy chain variable region set forth in SEQ ID NO:17 and / or the light chain variable region set forth in SEQ ID NO:18.

[0199] D. Use in Binding Assays or Methods

[0200] Provided herein is a method for assessing the presence or absence of a molecule in a sample, the molecule is bound to a chimeric antigen receptor (CAR) such as an extracellular domain of a CAR or a portion thereof containing an antigen binding domain. In some embodiments, the method may be used to assess the presence or absence of a humoral response or antibody response to a cell therapy comprising a chimeric antigen receptor (CAR) administered in a subject. In some embodiments, the chimeric antigen receptor comprises a target antibody, which is an antibody P5A2 or an antigen binding fragment thereof. In some embodiments, the chimeric antigen receptor comprises a target antibody, which is an antibody C29 or an antigen binding fragment thereof. In some embodiments, an anti-idiotypic antibody or an antigen binding fragment thereof specific to the extracellular domain of CAR, such as any described herein, can be used as a positive control in the method.

[0201] In some embodiments, there is provided a method involving contacting or incubating a binding agent with a sample from a subject, wherein the subject has been administered a cell therapy comprising cells engineered with a chimeric antigen receptor, wherein the binding agent is a protein comprising the extracellular domain of CAR or a portion thereof (containing a target antibody or its antigen binding fragment). In some embodiments, the method also includes detecting whether a complex is formed between a molecule (e.g., a binding molecule, such as an antibody) present in a binding agent and a sample, and / or detecting the presence or absence or level of such binding. In certain embodiments, contact or incubation is performed under conditions that allow binding agents to bind to molecules present in a sample from a subject. In some aspects, the method can also be performed on a positive control sample, which contains an anti-idiotypic antibody or its antigen binding fragment specific to any CAR as described. In some embodiments, determining the presence, absence or level of binding of the molecule to the binding agent may include comparing the binding or detection with the binding or detection of the positive control sample with the binding agent.

[0202] In some embodiments, the method includes detecting whether a complex is formed between a binding agent and a molecule (e.g., a binding molecule, such as an antibody) present in a sample, and / or detecting the presence or absence or level of such a combination. In certain embodiments, contact or incubation is performed under conditions that allow the binding agent to bind to a molecule present in a sample from a subject. In some aspects, the complex is detected by an immunoassay, optionally a sandwich assay or a bridging assay. For example, an immunoassay is an enzyme-linked immunosorbent assay (ELISA), chemiluminescence, electrochemiluminescence, a biosensor (e.g., BIAcore), flow cytometry, or protein blotting based on surface plasmon resonance (SPR). In some embodiments, an immunoassay is or includes mesoscale discovery.

[0203] In some aspects, the immunoassay is a sandwich assay or a bridging assay. In a sandwich or bridging assay, the binding reagent is a first binding reagent, and the presence or absence of a molecule or a complex comprising a molecule is detected by contacting the complex formed between the first binding reagent and the molecule with a second binding reagent, wherein the second binding reagent is a reagent capable of binding to the same or similar molecule as the first binding reagent. In some embodiments, the second binding reagent comprises an extracellular domain of a CAR or a portion thereof. In some aspects, the extracellular domain of a CAR or a portion thereof of a first binding reagent and a second binding reagent is the same or substantially the same.

[0204] Incorporated by Reference

[0205] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. However, citation of references herein should not be construed as an admission that such references are prior art to the present disclosure. To the extent that any of the definitions or terms provided in the references incorporated by reference differ from the terms and discussions provided herein, the terms and definitions herein shall prevail.

[0206] The foregoing written description is considered sufficient to enable those skilled in the art to practice the present disclosure. The foregoing description and examples describe in detail certain embodiments of the present disclosure and describe the best mode considered by the inventor. However, it should be understood that no matter how detailed the foregoing content is in the text, the present disclosure can be practiced in many ways, and the present disclosure should be interpreted according to the attached claims and any equivalents thereof.

[0207] Examples

[0208] Example 1: Generation of anti-idiotypic antibodies against human BCMA antibody clone C29

[0209] Balb / c mice were immunized with the anti-BCMA antibody clone C29-human Fab-8xHis (HHHHHHHH-SEQ ID NO: 46) fusion protein (Combo29DI_Fab). Hybridomas were generated, cloned, and screened for binding specificity of the secreted antibodies to C29. Antibody clones were identified and selected based on their ability to specifically bind to C29-derived scFv. Anti-id clone E2008 was isolated and several E2008-derived subclones were purified for further analysis. Since the anti-BCMA antibody C29 shares a high degree of sequence identity with the anti-BCMA antibody P5A2 (see Figure 1), so anti-ID antibody clones were also screened for specific binding to P5A2. Three anti-ID clones, E2008.51H1.H6 (H6), 54E4.F3 (F3), and 87B12.E10 (E10), showed promising activity and were further sequenced. Antibodies produced by hybridoma cell lines or recombinantly produced antibodies were tested. Anti-ID clones E2008.51H1.H6 (H6) and 87B12.E10 (E10) were analyzed below.

[0210] Example 2: Anti-idiotypic Antibodies H6 and E10 Against Anti-Human BCMA Antibody Clone C29 or P5A2

[0211] E10 purified from the supernatant of the hybridoma cell line was analyzed by Biacore assay against the antigen anti-BCMA clone C29 Fab. For example, the Biacore assay was run at 25°C in 10 mM HEPES buffer with 150 mM NaCl and 0.05% Tween 20 at pH 7.4 and 1 mg / mL BSA. E10 did not bind to the Flt3 Fab used as a framework control (data not shown).

[0212] Similarly, clone H6 purified from hybridoma cell lines or recombinantly produced was analyzed by Biacore with the anti-BCMA scFv P5A2 (which shares high sequence identity with C29, see Figure 1 The results are shown in the table below.

[0213] Table 3. Binding specificity of anti-BCMA anti-id antibodies E10 and H6

[0214]

[0215] Example 3: Flow cytometry analysis of anti-BCMA anti-id antibody H6

[0216] The specificity of clone H6 was tested in this experiment by flow cytometry. Figure 2A The data in Figure 4 show that the anti-BCMA anti-ID antibody clone H6 detected the anti-BCMA CAR clone P5A2 expressed on the cell surface. Clone H6 did not bind to the negative control anti-CD19 CAR even at high concentrations ( Figure 2A upper inset). Figure 2B The data in show that clone H6 specifically binds to the anti-BCMACAR clone P5A2 but not to other CARs used as negative controls.

Claims

1. An isolated antibody that specifically binds to a molecule comprising an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the anti-BCMA antibody comprises the amino acid sequence of SEQ ID NO: 1 or 19.

2. The isolated antibody according to claim 1, wherein the isolated antibody comprises (a) a heavy chain variable (VH) region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable (VL) region comprising the amino acid sequence of SEQ ID NO: 4; (b) a VH region comprising the amino acid sequence of SEQ ID NO: 20 and a VL region comprising the amino acid sequence of SEQ ID NO: 22; or (c) a VH region comprising the amino acid sequence of SEQ ID NO:33 and a VL region comprising the amino acid sequence of SEQ ID NO:

35.

3. The separated antibody according to claim 1 or 2, wherein the separated antibody comprises a VH region, wherein the VH region comprises (a) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, 7 or 8, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 9 or 10, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11; (b) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 24, 25 or 26, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 or 28, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 29; or (c) a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 37, 38 or 39, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 40 or 41, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:

42.

4. according to the separation antibody described in any one of claims 1-3, wherein said separation antibody also comprises a VL region, and said VL region comprises (a) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; (b) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 32; or (c) a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:43, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:44, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

45.

5. The isolated antibody of claim 4, wherein the VH region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 2, and the VL region comprises an amino acid sequence at least 95% identical to SEQ ID NO:

4.

6. The isolated antibody of claim 5, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 2, and the VL region comprises the amino acid sequence of SEQ ID NO:

4.

7. The isolated antibody of claim 4, wherein the VH region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 20, and the VL region comprises an amino acid sequence at least 95% identical to SEQ ID NO:

22.

8. The isolated antibody of claim 7, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 20, and the VL region comprises the amino acid sequence of SEQ ID NO:

22.

9. The isolated antibody of claim 4, wherein the VH region comprises an amino acid sequence at least 95% identical to SEQ ID NO: 33, and the VL region comprises an amino acid sequence at least 95% identical to SEQ ID NO:

35.

10. The isolated antibody of claim 9, wherein the VH region comprises the amino acid sequence of SEQ ID NO: 33, and the VL region comprises the amino acid sequence of SEQ ID NO:

35.

11. The isolated antibody of claim 1, wherein the isolated antibody comprises: a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9 and SEQ ID NO: 10, and the VH CDR3 comprises the amino acid sequence SEQ ID NO: 11, and wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 12, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 13, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

14.

12. The isolated antibody of claim 1, wherein the isolated antibody comprises: a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 27 and SEQ ID NO: 28, and the VH CDR3 comprises the amino acid sequence SEQ ID NO: 29, and wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 30, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 31, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

32.

13. The isolated antibody of claim 1, wherein the isolated antibody comprises: a heavy chain variable region comprising VH CDR1, VH CDR2 and VH CDR3 and a light chain variable region comprising VL CDR1, VL CDR2 and VL CDR3, wherein the VH CDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 37, SEQ ID NO: 38 and SEQ ID NO: 39, the VH CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40 and SEQ ID NO: 41, and the VH CDR3 comprises the amino acid sequence SEQ ID NO: 42, and wherein the VL CDR1 comprises the amino acid sequence of SEQ ID NO: 43, the VL CDR2 comprises the amino acid sequence of SEQ ID NO: 44, and the VL CDR3 comprises the amino acid sequence of SEQ ID NO:

45.

14. The isolated antibody of any one of the preceding claims, wherein the isolated antibody further comprises a detectable label.

15. The isolated antibody of claim 14, wherein the detectable label is selected from the group consisting of a fluorescent label, a photochromic compound, a magnetic label, a radioactive label, and a hapten.

16. The isolated antibody according to claim 15, wherein the fluorescent label is selected from Atto dye, Alexafluor dye, quantum dot, hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R-phycoerythrin (PE), PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-rhodamine, Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 conjugate, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire Blue, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Mid orishi blue, wild-type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), bright green, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Ruby Yellow, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed The proteins included mCysteine, DsRed2 ("RFP"), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinocyanin-chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry.

17. A polynucleotide encoding the isolated antibody according to any one of claims 1-13. A vector comprising the polynucleotide according to claim 17 .

19. A cell comprising the polynucleotide according to claim 17 or the vector according to claim 18.

20. A method for preparing an isolated antibody that specifically binds to a molecule comprising an anti-BCMA scFv comprising the amino acid sequence of SEQ ID NO: 1 or 19, the method comprising growing or culturing the cell of claim 19 under suitable conditions.

21. A method for determining the number of cells expressing an anti-BCMA antibody, the anti-BCMA antibody comprising the amino acid sequence of SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18, the method comprising contacting the cell with an isolated antibody according to any one of claims 1 to 16 and determining the number of cells expressing the anti-BCMA antibody in the cell.

22. A method for determining the presence or absence of cells expressing an anti-BCMA antibody, the anti-BCMA antibody comprising the amino acid sequence of SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18, the method comprising contacting the cell with the isolated antibody according to any one of claims 1-16 and determining the presence or absence of cells expressing the anti-BCMA antibody in the cell.

23. The method of claim 21 or 22, wherein the anti-BCMA antibody is a scFv comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

19.

24. The method of claim 23, wherein the cell expresses an anti-BCMA chimeric antigen receptor (CAR) comprising the amino acid sequence of SEQ ID NO: 1 or 19.

25. The method of claim 24, wherein the cell is a CAR T cell.

26. A method for selecting cells expressing anti-BCMACAR, wherein the anti-BCMACAR comprises the amino acid sequence of SEQ ID NO: 1 or 19, the method comprising (a) contacting the cell with an isolated antibody according to any one of claims 1 to 16; and (b) selecting the cell that binds to the antibody, Wherein the isolated antibody further comprises a detectable label.

27. The method of claim 25, wherein the cells that bind to the isolated antibody are selected by affinity-based separation.

28. The method of claim 25, wherein the cells that bind to the isolated antibody are selected by flow cytometry.

29. A method for purifying an anti-BCMA antibody or an antigen-binding fragment thereof, wherein the anti-BMCA antibody comprises the amino acid sequence of SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18: (a) contacting a sample comprising the anti-BCMA antibody with an isolated antibody according to any one of claims 1-16 to form a complex; and (b) purifying the complex comprising the anti-BCMA antibody and the isolated antibody from the sample.

30. A method for stimulating cells expressing anti-BCMA chimeric antigen receptors (anti-BCMA CART cells), the method comprising incubating the anti-BCMA CART cells with an isolated antibody according to any one of claims 1-13, wherein the anti-BCMA CART cells express the anti-BCMA antibody, and the anti-BCMA antibody comprises the amino acid sequence of SEQ ID NOs: 15 and 16 or SEQ ID NOs: 17 and 18.

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