Anti-idiotype antibody against anti-GPRC5D antibody

Abstract

In some aspects, the present disclosure relates to anti-idiotype antibodies and antigen-binding portions thereof that specifically bind to GP5B83-containing proteins, such as antibodies or antigen-binding portions thereof. In some aspects, the anti-idiotype antibodies and antigen-binding portions thereof of the present disclosure can be used in methods for detecting and quantifying cells that express a chimeric antigen receptor comprising GP5B83.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 053,286, filed on 17 July 2020. The entire contents of the aforementioned application are incorporated herein by reference.

[0002] (Sequence Listing) This application includes a sequence listing, which has been electronically filed in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 7, 2021, is named JBI6352WOPCT1_SL.txt, and has a size of 37,057 bytes.

[0003] (Field of invention) This invention relates to GP5B83-containing proteins, such as antibodies or anti-idiotype antibodies that specifically bind to the antigen-binding portion thereof, and the antigen-binding portion thereof. A method for detecting and quantifying cells expressing chimeric antigen receptors containing GP5B83 is also provided. [Background technology]

[0004] Recent advances in understanding the delivery and integration of genomic materials into target genomes have great potential to adapt standard treatments to a variety of diseases. T-cell therapy utilizes genetically modified and isolated T cells to enhance specificity to particular tumor-associated antigens. Genetic modification, involving the expression of chimeric antigen receptors (CARs) or exogenous T cell receptors, can provide novel antigen specificity to T cells. T cells expressing chimeric antigen receptors (CAR-T cells) can induce tumor immunoreactivity.

[0005] One specific CAR target of interest is G protein-coupled receptor family C group 5 member D (GPRC5D). GPRC5D has been identified as a potential target for immunotherapy in multiple myeloma and potentially other cancers. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, CAR-T cell therapy is needed to treat cancer. Anti-idiotype antibodies against such CARs are also needed to detect, purify, or select proteins and cells that express CARs. [Means for solving the problem]

[0007] This disclosure provides GP5B83-containing proteins, such as anti-idiotype antibodies and their antigen-binding moieties that specifically bind to antibodies or their antigen-binding moieties. This disclosure also provides nucleic acids encoding anti-idiotype antibodies and their antigen-binding moieties, methods for producing anti-idiotype antibodies and their antigen-binding moieties, methods for detecting GP5B83 using anti-idiotype antibodies and their antigen-binding moieties, and kits comprising anti-idiotype antibodies and their antigen-binding moieties.

[0008] In one embodiment, the disclosure provides an anti-idiotype antibody or an antigen-binding moiety thereof that specifically binds to an anti-GPRC5D antibody, such as a target antibody containing GP5B83. In some embodiments, the target antibody or antigen-binding moiety includes a VH domain having the amino acid sequence of SEQ ID NO: 41 and a VL domain having the amino acid sequence of SEQ ID NO: 42.

[0009] In other embodiments, the anti-idiotype antibody or antigen-binding moiety is for use in the detection of GP5B83 in a biological sample, which includes (a) providing the biological sample, (b) contacting the biological sample with the anti-idiotype antibody or antigen-binding moiety, and (c) detecting the anti-idiotype antibody or antigen-binding moiety.

[0010] In another embodiment, the present disclosure provides an anti-idiotype antibody or antigen-binding moiety that specifically binds to GP5B83, comprising a heavy chain variable (VH) domain including VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-7, VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14, and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20; and further comprising a light chain variable (VL) domain including VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-26, VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-30, and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34.

[0011] In some embodiments, the anti-idiotype antibody or its antigen-binding moiety includes a VH domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45, and the VL domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety includes a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37, and further includes a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 39. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety includes a VH domain having an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 45. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety includes a VL domain having an amino acid sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 46.

[0012] In some embodiments, the anti-idiotype antibody or its antigen-binding moiety includes a VH domain having the amino acid sequence of SEQ ID NO: 45 and a VL domain having the amino acid sequence of SEQ ID NO: 46. In some other embodiments, the anti-idiotype antibody or its antigen-binding moiety includes a heavy chain having the amino acid sequence of SEQ ID NO: 37 and further includes a light chain having the amino acid sequence of SEQ ID NO: 39.

[0013] In some embodiments, the antigen-binding moiety is selected from Fab, F(ab')2, or scFv. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the chimeric antibody comprises a mouse IgG2a framework. In some other embodiments, the antibody is a fully human antibody. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is specific to GP5B83, where GP5B83 is located within the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). In some embodiments, GP5B83 is scFv, and the anti-idiotype antibody or antigen-binding moiety specifically binds to an epitope in the scFv of the CAR. In some embodiments, GP5B83 specifically binds to GPRC5D. In some embodiments, the antibody or antigen-binding moiety does not cross-react with other GPRC5D antibodies or other GPRC5D-binding CARs. In some embodiments, the CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs. 43-44.

[0014] In some embodiments, the present disclosure provides nucleic acids encoding the heavy chain, light chain, or both of an anti-idiotype antibody or antigen-binding moiety.

[0015] In another embodiment, the disclosure provides nucleic acids encoding an anti-idiotype antibody that specifically binds to GP5B83, or the heavy chain, light chain, or both of the antigen-binding portion thereof, comprising the nucleotide sequence of SEQ ID NO: 38, the nucleotide sequence of SEQ ID NO: 40, or both. In another embodiment, the disclosure provides a vector comprising a nucleic acid sequence. In some embodiments, the vector is an expression vector. In another embodiment, the disclosure provides a host cell comprising the vector. In some embodiments, the host cell is a mammalian cell.

[0016] In another aspect, the present disclosure provides a method for producing an anti-idiotype antibody or an antigen-binding portion thereof that specifically binds to GP5B83, the method comprising culturing a host cell comprising nucleotide sequences encoding the heavy and light chains of the antibody or antigen-binding portion under conditions that allow for the expression of the antibody or antigen-binding portion, and isolating the antibody or antigen-binding portion from the culture. In some embodiments, the host cell encodes a vector comprising a nucleic acid encoding the anti-idiotype antibody or an antigen-binding portion thereof.

[0017] In another aspect, the present disclosure provides a method for detecting GP5B83 in a biological sample, the method comprising: (a) providing a biological sample; (b) contacting the biological sample with an anti-idiotype antibody or an antigen-binding portion thereof; and (c) detecting the anti-idiotype antibody or an antigen-binding portion thereof.

[0018] In another aspect, the present disclosure provides a method for detecting the expression of a chimeric antigen receptor (CAR) comprising GP5B83 in a biological sample, the method comprising: (a) providing a biological sample; (b) contacting the biological sample with an anti-idiotype antibody or an antigen-binding portion thereof; and (c) detecting the anti-idiotype antibody or an antigen-binding portion thereof, thereby detecting the expression of the CAR.

[0019] In some embodiments, the antibody comprises a detectable label. In some embodiments, the method further comprises contacting the anti-idiotype antibody or an antigen-binding portion thereof with a detectable label prior to detecting the anti-idiotype antibody or an antigen-binding portion thereof. In some embodiments, the biological sample is blood, serum, or urine.

[0020] In some aspects, the present disclosure provides a kit for detecting GP5B83 in a biological sample, the kit comprising: (a) an anti-idiotype antibody or an antigen-binding portion thereof; and (b) instructions for detecting the anti-idiotype antibody or an antigen-binding portion thereof.

[0021] In other embodiments, the Disclosure provides a method for purifying GP5B83 from a sample, comprising: (a) providing a biological sample containing GP5B83; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety; and (c) capturing the anti-idiotype antibody or antigen-binding moiety to thereby purify GP5B83.

[0022] In other embodiments, the Disclosure provides a method for selecting CAR-T cells from a cell population, comprising: (a) providing a biological sample containing CAR-T cells; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety; and (c) capturing the anti-idiotype antibody or antigen-binding moiety, thereby selecting CAR-T cells. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is specific to GP5B83.

[0023] This disclosure intends to include all combinations of any of the aforementioned aspects and embodiments, as well as any combination of any of the embodiments described in the Detailed Description and Examples. [Brief explanation of the drawing]

[0024] For the purpose of illustrating the present invention, certain embodiments of this disclosure are illustrated in the drawings. However, this disclosure is not limited to the precise arrangements and means of the embodiments illustrated in the drawings. [Figure 1] This figure shows a graph of GCDB332-specific binding enrichment after four rounds of panning detected by polyclonal ELISA. [Figure 2-1] This figure shows a graph of the results of monoclonal Fab binding screening from a GCDB332 target binding assay compared to an HRP-conjugated streptavidin counter screening reagent binding assay. [Figure 2-2] (As stated above.) [Figure 3] This figure shows a graph illustrating the dose-dependent binding of GP5B318 to GP5DB83-LH SupT1 cells. [Figure 4] This figure shows a graph illustrating the dose-dependent blocking of PE-P5B318 binding to GP5DB83-HL SupT1 cells by the GPDB83-HL scFv-Fc fusion protein. [Modes for carrying out the invention]

[0025] Overview This disclosure provides GP5B83-containing proteins, such as anti-idiotype antibodies and their antigen-binding moieties that specifically bind to antibodies or their antigen-binding moieties. The anti-idiotype antibodies and antigen-binding moieties of this disclosure can be used in methods for detecting and quantifying cells expressing CARs containing GP5B83. Such methods may enable researchers to determine whether a given batch of CAR-T cells produced in vitro expresses a desired CAR, and therefore whether the cells are therapeutically useful for targeting a desired protein. In this disclosure, the anti-idiotype antibodies and antigen-binding moieties target GP5B83, which itself targets GPRC5D, a protein associated with cancer, including multiple myeloma.

[0026] definition As used herein and in the attached Claims, the singular forms "a," "an," and "the" encompass multiple references unless otherwise explicitly indicated. For example, the reference "a cell" includes combinations of two or more cells.

[0027] The transitional phrases “comprising,” “consisting essentially of,” and “consisting” are intended to imply the generally accepted meanings in patent terminology, namely, (i) “comprising” is synonymous with “containing,” “containing,” or “characterizing,” and is comprehensive or non-restrictive, not excluding other unlisted elements or process steps; (ii) “consisting of” excludes any elements, processes, or components not specified in the claims; and (iii) “consisting essentially of” limits the scope of the claims to specified materials or processes, and those that “do not substantially affect the basic and novel features” of the claimed invention. Embodiments described with the phrase “comprising” (or its equivalent) are also provided as embodiments described independently with “consisting” and “consisting essentially of.”

[0028] "Activation," "stimulation," "activated," or "stimulated" refers to the induction of a change in the biological state of a cell that results in the expression of activation markers, cytokine production, or mediation of target cell proliferation or cytotoxicity. Cells can be activated by a primary stimulatory signal. Co-stimulatory signals can amplify the magnitude of the primary signal and suppress cell death after initial stimulation, resulting in a more durable activated state and, consequently, higher cytotoxicity. A "co-stimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, induces the proliferation of T cells and / or NK cells, and / or the upregulation or downregulation of key molecules.

[0029] An "anti-idiotype antibody" or "anti-idiotypic antibody" refers to an antibody that specifically binds to the variable region of another antibody. In the case of GPRC5D, the anti-idiotype antibody specifically binds to the anti-GPRC5D2 antibody.

[0030] An "antigen-binding portion," "antigen-binding fragment," or "antigen-binding domain" refers to a portion of a protein that binds to an antigen. Antigen-binding fragments may be synthetic polypeptides, enzymatically available polypeptides, or genetically engineered polypeptides, and include portions of immunoglobulins that bind to antigens, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAb) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelid VH domains, VHH domains, minimal recognition units consisting of amino acid residues that mimic the CDR of an antibody, such as the FR3-CDR3-FR4 portion, HCDR1, HCDR2, and / or HCDR3, as well as LCDR1, LCDR2, and / or LCDR3, alternative scaffolds that bind to antigens, and multispecific proteins including antigen-binding fragments. Antigen-binding fragments (such as VH and VL) can be linked to each other via synthetic linkers to form various types of single-chain antibody designs, where the VH / VL domains can pair intramolecularly or intermolecularly to form a monovalent antigen-binding domain, such as a single-chain Fv (scFv) or diabody, when the VH and VL domains are expressed as separate single-chain units. Antigen-binding fragments may also be conjugated to other antibodies, proteins, antigen-binding fragments, or alternative scaffolds, which may be monospecific or multispecific, for genetic engineering of bispecific and multispecific proteins.

[0031] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and growth can lead to the formation of malignant tumors that invade adjacent tissues and can metastasize to distal parts of the body via the lymphatic system or bloodstream. "Cancer" or "cancer tissue" may include tumors.

[0032] A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC), which are interconnected by disulfide bonds, as well as a polymer of these (e.g., IgM). Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, the heavy chain constant domain consisting of subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). The VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed with framework regions (FRs). Each VH and VL consists of three CDRs and four FR segments arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0033] Complementarity determining regions (CDRs) are antigen-binding sites in antibodies. CDRs can be defined using various terms: (i) Three complementarity determining regions (CDRs) in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence diversity (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) The "hypervariable regions," "HVR," or "HV," consisting of three (H1, H2, H3) within the VH and three (L1, L2, L3) within the VL, refer to regions of antibody variable domains that are structurally hypervariable, as defined by Chothia and Lesk (Chothia and Lesk Mol Biol 196:901-17, 1987). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standard numbers and definitions for antigen-binding sites. The correspondence between CDR, HV, and IMGT summaries is described in Lefranc et al., Dev Comparat Immunol 27:55-77, 2003. As used herein, the terms “CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2,” and “LCDR3” include CDRs as defined by any of the methods described above by Kabat, Chothia, or IMGT, unless otherwise specified herein.

[0034] "Human antibody" means an antibody optimized to produce a minimal immune response when administered to a human subject. The variable region of a human antibody is derived from a human immunoglobulin sequence. If a human antibody contains a constant region or a portion of a constant region, that constant region is also derived from a human immunoglobulin sequence. If the variable region of a human antibody is obtained from a system using human germline immunoglobulin or a rearranged immunoglobulin gene, the human antibody includes heavy-chain and light-chain variable regions "derived" from a human sequence. Such exemplary systems include human immunoglobulin gene libraries displayed on phages, and transgenic non-human animals possessing human immunoglobulin loci, such as mice or rats. "Human antibody" typically contains amino acid differences when compared to immunoglobulin expressed in humans, due to differences in the human antibody and the system used to obtain the human immunoglobulin locus, intentional introduction of somatic mutations or substitutions into the framework or CDR, or both. Typically, a “human antibody” is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin or rearranged immunoglobulin gene. In some cases, a “human antibody” may contain, for example, a consensus framework sequence obtained from human framework sequence analysis described in Knappik et al., (2000) J Mol Biol 296:57-86, or synthetic HCDR3 incorporated into a human immunoglobulin gene library presented on phages, for example, Shi et al., (2010) J Mol Biol 397:385-96 and International Publication No. 2009 / 085462. Antibodies in which at least one CDR originates from a non-human species are not included in the definition of "human antibodies."

[0035] A "humanized antibody" means an antibody in which at least one CDR is derived from a non-human species and at least one framework is derived from a human immunoglobulin sequence. Because humanized antibodies can contain substitutions in their framework, the framework may not be an exact copy of the expressed human immunoglobulin or human immunoglobulin germline gene sequence.

[0036] "Isolated" refers to a homogeneous collection of molecules (e.g., synthetic polynucleotides or polypeptides) that have been substantially separated and / or purified from other components of a system in which molecules are produced, such as in recombinant cells, as well as proteins subjected to at least one purification or isolation step. "Isolated" refers to molecules that are substantially free from other cellular materials and / or chemicals, and includes molecules isolated to a higher purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0037] "Modifying" refers to either an enhanced or reduced ability of a test molecule to mediate a greater or lesser response (i.e., a downstream effect) compared to the response mediated by the control or vehicle.

[0038] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. NK cells are CD16 + CD56 + and / or CD57 + TCR - NK cells refer to differentiated lymphocytes that exhibit a specific phenotype. NK cells are characterized by their ability to bind to and kill cells that cannot express "self" MHC / HLA antigens through the activation of specific cytolytic enzymes, their ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and their ability to release protein molecules called cytokines that stimulate or inhibit the immune response.

[0039] "Specifically binds", "specific binding", "specifically bound", or "binds" refers to a proteinaceous molecule binding to an antigen or an epitope within the antigen with a higher affinity than to other antigens. Typically, the proteinaceous molecule binds to the antigen or epitope within the antigen with an equilibrium dissociation constant (K -7 M or less, for example about 5×10 -8 M or less, about 1×10 -8 M or less, about 1×10 -9 M or less, about 1×10 -10 M or less, about 1×10 -11 M or less, or about 1×10 -12 M, 1×10 -13 M, 1×10 -14 M, 1×10 -15 M or less, and typically K D is at least 100-fold smaller than K D for binding to non-specific antigens (e.g., BSA, casein). In the context of the prostate neoantigens described herein, "specific binding" refers to a proteinaceous molecule binding to a prostate neoantigen without detectably binding to the wild-type protein of which the prostate neoantigen is a variant.

[0040] "Tumor cell" or "cancer cell" refers to a cancerous, pre-cancerous, or transformed cell having a naturally occurring or introduced phenotypic change, either in vivo, ex vivo, or in tissue culture. These changes do not necessarily involve the uptake of new genetic material. Transformation can also be induced by infection with a transforming virus and integration of new genomic nucleic acid, uptake of exogenous nucleic acid, occur spontaneously, or occur after exposure to a carcinogen, resulting in mutation of endogenous genes in some cases. Transformation / cancer is exemplified by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, regulation of tumor-specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, etc., in vitro, in vivo, and ex vivo. <00001​​​​The terms “chimeric antigen receptor” or “CAR,” as used herein, are defined as cell surface receptors comprising an extracellular target-binding domain, a transmembrane domain, and an intracellular signaling domain, all of which are combinations not found together in a single protein in nature. This includes, in particular, receptors in which the extracellular domain and intracellular signaling domain are not found together in a single receptor protein in nature. The chimeric antigen receptors of the present invention are primarily intended for use in lymphocytes such as T cells and natural killer (NK) cells.

[0042] The terms “T cell” and “T lymphocyte” are interchangeable and are used synonymously herein. As used herein, T cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cell may be a T helper (Th) cell, e.g., a T helper 1 (Th1) or T helper 2 (Th2) cell. T cell may be a helper T cell (HTL, CD4+ T cell), a CD4+ T cell, a cytotoxic T cell (CTL; ​​CD8+ T cell), a tumor infiltrating cytotoxic T cell (TIL; CD8+ T cell), a CD4+CD8+ T cell, or any other subset of T cell. Other exemplary populations of T cells suitable for use in particular embodiments include naive T cells and memory T cells. Furthermore, "NKT cells" are also included, which refer to a special population of T cells that not only express semi-invariant αβ T cell receptors but also various molecular markers typically associated with NK cells, such as NK1.1. Examples of NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+, and CD8 cells. The TCR of NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or detrimental effects due to their ability to produce cytokines that promote either inflammation or immune tolerance. Also included are "gamma delta T cells (γδ T cells)." Gamma delta T cells refer to a special population of a small subset of T cells that have different TCRs on their surface, unlike most T cells whose TCR consists of two glycoprotein chains denoted as α and β-TCR chains. The TCR in γδ T cells consists of a γ chain and a δ chain. γδ T cells have been found to play a role in immune surveillance and immunomodulation, to be an important source of IL-17, and to induce active CD8+ cytotoxic T cell responses. This also includes "regulatory T cells" or "Tregs," which are T cells that suppress abnormal or excessive immune responses and play a role in immune tolerance.Tregs are typically Foxp3-positive CD4+ T cells, but may also include Foxp3-negative regulatory T cells, which are IL-10-producing CD4+ T cells.

[0043] As used herein, the term “antigen” refers to any agent molecule (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, part thereof, or combination thereof) to which a T cell receptor can bind. Antigens can also evoke an immune response. Examples of immune responses, though not limited to, may include antibody production, activation of specific immunocompetent cells, or both. Those skilled in the art will understand that antigens do not necessarily have to be encoded by a “gene.” It is readily apparent that antigens may be synthesized, derived from biological samples, or be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells, or fluids containing lysates.

[0044] The term “antibody” refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fv(scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fv(sdFv), intrabodies, minibodies, diabodies, and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against antigen-specific TCRs), and any of the epitope-binding fragments described above. The terms “antibody” and “antibody” also refer to covalent diabodies, such as those described in U.S. Patent Application Publication 2007 / 0004909, and Ig-DARTS, such as those disclosed in U.S. Patent Application Publication 2009 / 0060910. Antibodies useful as TCR-binding molecules include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules containing antigen-binding sites. The immunoglobulin molecule may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2), or a subclass.

[0045] The term "host cell" means any cell containing heterologous nucleic acids. Heterologous nucleic acids can be vectors (e.g., expression vectors). For example, a host cell may be a cell of any organism that is selected, modified, transformed, grown, used, or manipulated in any way for the production of substances by the cell, such as the expression of genes, DNA or RNA sequences, proteins, or enzymes by the cell. A suitable host can be determined. For example, a host cell may be selected based on the vector backbone and the desired outcome. As an example, plasmids or cosmids can be introduced into prokaryotic host cells to replicate several types of vectors. Bacterial cells, SURE® competent cells, and SOLOPACK Gold cells, such as DH5α, JM109, and KCB, as well as others, can be used as host cells for vector replication and / or expression. Furthermore, bacterial cells such as E. coli LE392 can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for vector replication and / or expression include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12.

[0046] The terms "express" and "expression" mean enabling or causing the generation of information from a gene or DNA sequence, for example, producing a protein by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence is expressed within or by a cell to form "expression products," such as proteins. The expression product itself, for example, the resulting protein, can also be said to have been "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0047] The term “transfection” means introducing “foreign” (i.e., exogenous or extracellular) nucleic acids into a cell using recombinant DNA technology. The term “genetic modification” means introducing “foreign” (i.e., exogenous or extracellular) genes, DNA, or RNA sequences into a host cell so that the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence and may include regulatory or control sequences that are manipulably ligated to a polynucleotide encoding a chimeric antigen receptor, such as start, stop, promoter, signal, secretion, or other sequences used by the cell’s genetic mechanisms. The gene or sequence may include non-functional sequences that do not have a known function. A host cell that receives and expresses introduced DNA or RNA is “genetically modified.” The DNA or RNA introduced into a host cell may be of any origin, including cells of the same genus or species as the host cell, or from a different genus or species.

[0048] The term "transduction" refers to the introduction of foreign nucleic acids into cells using a viral vector.

[0049] The term “regulatory element” refers to any cis-acting genetic element that controls several aspects of nucleic acid sequence expression. In some embodiments, the term “promoter” essentially includes the minimum sequence necessary to initiate transcription. In some embodiments, the term “promoter” includes the sequence for initiating transcription, plus sequences that can upregulate or downregulate transcription, which are commonly referred to as “enhancer elements” and “repressor elements,” respectively.

[0050] As used herein, “operably linked” and similar phrases, when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequences arranged in a functional relationship with one another, respectively. For example, operablely linked promoters, enhancer elements, open reading frames, 5' and 3' UTRs, and terminator sequences result in the precise production of nucleic acid molecules (e.g., RNA). In some embodiments, operablely linked nucleic acid elements result in the transcription of an open reading frame and ultimately the production of a polypeptide (i.e., expression of the open reading frame). As another example, operablely linked peptides refer to those in which functional domains are arranged at appropriate distances from one another to confer the intended function of each domain.

[0051] "Enhancement" or "promotion," or "increase," "expansion," or "improvement," generally refers to the ability of the composition intended herein to produce, induce, or cause (i.e., downstream effect) a greater physiological response compared to the response caused by either the vehicle or the control molecule / composition. Measurable physiological responses may include, as will be evident from the understanding of the art and the description herein, an increase in T cell expansion, activation, effector function, persistence, and / or cancer cell killing ability. In certain embodiments, the "increased" or "enhanced" amount may be a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) of the response produced by the vehicle or the control composition (including all integers and decimals, such as 1.5, 1.6, 1.7, 1.8, etc.).

[0052] "Decrease," "lower," "less," "reduce," or "weaken" generally refers to the ability of the composition intended herein to produce, induce, or cause a less physiological response (i.e., downstream effect) compared to the response caused by either the vehicle or the control molecule / composition. In certain embodiments, the amount "decreased" or "reduced" may be a "statistically significant" amount and may include a 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times or more (e.g., 500, 1000 times) of the response produced by the vehicle, the control composition (reference response), or the response in a particular cell lineage (intermediate and greater than 1, e.g., 1.5, 1.6, 1.7, 1.8, etc.).

[0053] The term "effective" as applied to dosage or quantity refers to the amount of compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject requiring it. When administering a combination of active ingredients, it should be noted that the effective dose of the combination may or may not include the amount of each individual ingredient that would be effective if administered individually. The exact required dose will vary depending on the subject's species, age, and overall health, the severity of the condition being treated, the specific drug used, and the mode of administration.

[0054] As used herein in reference to the compositions, the term “pharmaceutically acceptable” refers to molecular entities and other components of such compositions that are physiologically acceptable and do not typically produce adverse reactions when administered to mammals (e.g., humans). Preferably, the term “pharmaceutically acceptable” means that the substance is approved for use in mammals, more specifically in humans, by a federal or state regulatory agency, or is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0055] When used herein, the term "protein" encompasses all types of modified proteins, including but not limited to natural and synthetic proteins, fusion proteins, and glycoproteins, including protein fragments of all lengths, as well as all other types of modified proteins (e.g., proteins obtained from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0056] The terms “nucleic acid,” “nucleotide,” and “polynucleotide” encompass both DNA and RNA unless otherwise specified. “Nucleic acid sequence” or “nucleotide sequence” means a nucleic acid sequence that codes for amino acids, and these terms may also refer to a nucleic acid sequence that includes any amino acid coding by a linker, or any amino acid coding portion that is added as a cloning artifact.

[0057] The term "carrier" refers to a diluent, auxiliary, excipient, or vehicle to which a compound is administered together. Such pharmaceutical carriers may be sterile liquids such as water and oil, including those of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water or aqueous solutions, physiological saline, and aqueous solutions of dextrose and glycerin are particularly preferred as carriers for injectable solutions. Alternatively, the carrier may be a solid dosage form carrier containing, but not limited to, one or more of the following: a binder (for compressed pills), a flow enhancer, a encapsulant, a flavoring agent, and a coloring agent. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.

[0058] The terms “about” or “approximately” include being within a statistically meaningful range of values. Such a range may be within one order of magnitude of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the terms “about” or “approximately” depends on the specific system under study and can be readily understood by those skilled in the art.

[0059] "GPRC5D" refers to a known protein, member D of group C5 of the G protein-coupled receptor family. The amino acid sequence of the full-length human GPRC5D is shown in SEQ ID NO: 47.

[0060] The term "GP5B83" refers to any antibody, its antigen-binding portion, or any other protein containing variable regions derived from GP5B83-VH-N23S-N30S VH (SEQ ID NO: 41) and GP5B83 VL (SEQ ID NO: 42), including CARs. "GP5B83" may be used interchangeably with "GP5DB83". In certain embodiments, the anti-idiotype antibody of this disclosure specifically binds to a protein containing the VH domain shown in SEQ ID NO: 41 and / or the VL domain shown in SEQ ID NO: 42. In certain embodiments, the anti-idiotype antibody of this disclosure specifically binds to a protein containing three CDRs of the VH domain shown in SEQ ID NO: 41 and three CDRs of the VL domain shown in SEQ ID NO: 42.

[0061] The term "GCDB332" refers to the scFv fusion protein derived from GP5B83, specifically GP5B83-VH-N23S-N30S-LH-scFv.

[0062] The term "GP5B318" refers to a chimeric mAb with human VH / VL that targets scFv GCDB332 derived from GP5B83 and mouse IgG2a / k.

[0063] The terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting. Where used herein, the articles "a," "an," and "the" should be understood to include multiple referents unless otherwise clearly indicated by the context.

[0064] This disclosure further provides variants of the antibodies, nucleic acids, polypeptides, and proteins described herein, such as functional variants. “Variant” means a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as substitution, insertion, or deletion. As used herein, the term “functional variant” means an antibody, polypeptide, or protein having substantial or significant sequence identity or similarity to a parent antibody, polypeptide, or protein, and which retains the biological activity of the variant antibody, polypeptide, or protein. Functional variants include, for example, variants of the antibodies, polypeptides, or proteins (parent antibodies, polypeptides, or proteins) described herein, and which retain the ability to recognize target cells to the same degree, the same degree, or a higher degree than the parent antibody, polypeptide, or protein. With respect to the parent antibody, polypeptide, or protein, the functional variant may have an amino acid sequence that is identical to, for example, the parent antibody, polypeptide, or protein by at least about 30%, about 40%, about 50%, about 60%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more.

[0065] In this specification, the structure of a polypeptide is defined based on its % sequence identity with an enumerated reference sequence (having a given sequence number). In this regard, the % sequence identity between two amino acid sequences can be determined by comparing these two sequences that have been optimally aligned, and the amino acid sequences being compared may contain additions or deletions to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions where amino acid residues are identical between the two sequences, dividing this number of identical positions by the total number of positions in the comparison window, and multiplying the result obtained to obtain the percentage of identity between these two sequences by 100. Typically, the comparison window corresponds to the total length of the sequences being compared. For example, the BLAST program, "BLAST 2 Sequences" (Tatusova et al, "Blast 2 Sequences - A Novel Tool for Comparing Protein and Nucleotide Sequences," FEMS Microbiol Lett. 174:247-250), available at the site http: / / www.ncbi.nlm.nih.gov / gorf / bl2.html, can be used, and the parameters used are those specified by default (specifically, the parameters "open gap penalty": 5 and "extension gap penalty": 2; the selected matrix is, for example, the matrix "BLOSUM 62" suggested by the program), and the percentage of identity between the two sequences being compared is calculated directly by the program. Determining the sequence identity of a query sequence to a reference sequence is within the capabilities of those skilled in the art and can be performed using commercially available analytical software such as BLAST®.

[0066] A functional variant may include, for example, an amino acid sequence of a parent antibody, polypeptide, or protein having at least one conserved amino acid substitution. In another embodiment, a functional variant may include an amino acid sequence of a parent antibody, polypeptide, or protein having at least one non-conserved amino acid substitution. In this case, the non-conserved amino acid substitution cannot suppress or inhibit the biological activity of the functional variant. The non-conserved amino acid substitution can enhance the biological activity of the functional variant, resulting in increased biological activity compared to the parent antibody, polypeptide, or protein.

[0067] The amino acid substitutions in the antibodies of the present invention may be conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having a particular physical and / or chemical property is replaced with another amino acid having the same or similar chemical or physical property. For example, conservative amino acid substitutions may include acidic amino acids substituted with another acidic amino acid (e.g., Asp or Glu), amino acids with nonpolar side chains substituted with another amino acid having a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), basic amino acids substituted with another basic amino acid (e.g., Lys, Arg), and amino acids with polar side chains substituted with another amino acid having a polar side chain (e.g., Asn, Cys, Gln, Ser, Thr, Tyr, etc.).

[0068] The antibodies, polypeptides, and proteins (including the functional portions and functional variants of the present invention) of embodiments of the present invention may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β- Examples include hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, N'-benzyl-N'-methyllysine, N',N'-dibenzyllysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, and α-tert-butylglycine.

[0069] The antibodies, polypeptides, and proteins (including functional moieties and functional variants) of embodiments of the present invention can undergo post-translational modifications. These can be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, esterified, cyclized via disulfide crosslinking, for example, or converted to acid addition salts. In some embodiments, they are dimerized, polymerized, or conjugated.

[0070] The antibodies, polypeptides, and / or proteins (including functional portions and functional variants thereof) of embodiments of the present invention can be obtained by methods known in the art. Preferred methods for the de novo synthesis of polypeptides and proteins are described in reference to Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; and Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Polypeptides and proteins can also be produced by recombination using standard recombination methods and nucleic acids as described herein. For example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Furthermore, some of the antibodies, polypeptides, and proteins (including functional portions and their functional variants) of the present invention can be isolated and / or purified from sources such as plants, bacteria, insects, and mammals. Methods for isolation and purification are known in the art. Alternatively, the antibodies, polypeptides, and / or proteins (including functional portions and their functional variants) described herein can be commercially synthesized. In this regard, antibodies, polypeptides, and proteins can be synthesized, recombinant, isolated, and / or purified.

[0071] Methods and Uses of This Disclosure This disclosure provides GP5B83-containing proteins, such as anti-idiotype antibodies and their antigen-binding moieties, which specifically bind to antibodies or their antigen-binding moieties. For example, the anti-idiotype antibody may contain an amino acid sequence complementary to a portion of the GP5B83 antibody to facilitate specific binding. This disclosure also provides nucleic acids encoding the anti-idiotype antibody and its antigen-binding moiety, methods for producing the anti-idiotype antibody and its antigen-binding moiety, methods for detecting GP5B83 using the anti-idiotype antibody and its antigen-binding moiety, and kits containing the anti-idiotype antibody and its antigen-binding moiety. For example, the anti-idiotype antibody may be included in a kit containing other reagents and may be used, for example, to determine whether a given biological sample contains a GP5B83 / GCDB332 antibody or fragment thereof expressed on the surface of T cells in a CAR.

[0072] Methods for testing antibodies for their ability to bind to any functional portion of GP5B83 are known in the art and include any antibody-antigen binding assay, such as radioimmunoassay (RIA), Western blotting, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, and competitive inhibition assay.

[0073] Suitable methods for producing antibodies are known in the art. For example, the standard hybridoma method is described, for instance, in Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods are known in the art, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression systems (see, for example, Huse et al., Science, 246, 1275-81 (1989)). Furthermore, methods for producing antibodies in non-human animals are described, for example, in U.S. Patents No. 5,545,806, No. 5,569,825, and No. 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266 (A1)).

[0074] Antibodies can also be generated using phage display. In this regard, phage libraries encoding the antigen-binding variable (V) domain of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al. (above) and Ausubel et al. (above)). Phages encoding variable regions with desired specificity are selected for specific binding to the desired antigen, and complete or partial antibodies containing the selected variable domains are reconstituted. The nucleic acid sequences encoding the reconstituted antibodies are introduced into suitable cell lines, such as myeloma cells used for hybridoma production, resulting in the secretion of antibodies with monoclonal antibody characteristics by the cells (see, e.g., Janeway et al. (above), Huse et al. (above), and U.S. Patent No. 6,265,150).

[0075] In one embodiment, the disclosure provides an anti-idiotype antibody or antigen-binding moiety thereof that specifically binds to a target antibody or CAR containing GP5B83. For example, the anti-idiotype antibody or antigen-binding moiety can specifically bind to one or more domains of a fragment antigen-binding region (Fab) including VH and VL. In some embodiments, the anti-idiotype antibody or antigen-binding moiety includes a VH domain having the amino acid sequence of SEQ ID NO: 45 and a VL domain having the amino acid sequence of SEQ ID NO: 46.

[0076] In other embodiments, the anti-idiotype antibody or antigen-binding moiety is for use in the detection of GP5B83 in a biological sample, comprising (a) providing the biological sample, (b) contacting the biological sample with the anti-idiotype antibody or antigen-binding moiety, and (c) detecting the anti-idiotype antibody or antigen-binding moiety. For example, the anti-idiotype antibody may be added to any biological sample, including tissue samples, tumor samples, cells or fluids containing other biological components, organisms, protein / antigen subunits, killed or inactivated whole cells or lysates. The anti-idiotype antibody may be contained in a solution containing pharmaceutically acceptable reagents, including but not limited to buffers, stabilizers, and / or polymers. The anti-idiotype antibody can be contacted with the biological sample by pipetting and / or mixing. The anti-idiotype antibody can then specifically bind to GP5B83-containing proteins in the biological sample, such as the antibody or its antigen-binding moiety. For example, whether or not an anti-idiotype antibody has bound to GP5B83 can be determined by washing away the unbound anti-idiotype antibody, leaving only the anti-idiotype antibody that has formed a complex. In this example, the anti-idiotype antibody may also contain a fluorophore that can be irradiated to emit a signal proportional to the amount of GP5B83 in the biological sample. Detection of the binding complex of the anti-idiotype antibody to GP5B83 is further described below.

[0077] In another embodiment, the present disclosure provides an anti-idiotype antibody or antigen-binding moiety that specifically binds to an anti-GPRC5D2 antibody, comprising a heavy chain variable (VH) domain including a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4-7, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19-20; and further comprising a light chain variable (VL) domain including a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25-26, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-30, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33-34. The six CDRs may be selected according to any known method. VH and VL CDRs determined according to Kabat, AbM, Chothia, and contact methods are shown in Table 3.

[0078] In certain embodiments, the present disclosure provides an anti-idiotype antibody or antigen-binding moiety that specifically binds to an anti-GPRC5D2 antibody, comprising a heavy chain variable (VH) domain including VH CDR1 having the amino acid sequence of SEQ ID NO: 4, VH CDR2 having the amino acid sequence of SEQ ID NO: 11, and VH CDR3 having the amino acid sequence of SEQ ID NO: 19, and further comprising a light chain variable (VL) domain including VL CDR1 having the amino acid sequence of SEQ ID NO: 25, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 33.

[0079] In certain embodiments, the present disclosure provides an anti-idiotype antibody or antigen-binding moiety that specifically binds to an anti-GPRC5D2 antibody, comprising a heavy chain variable (VH) domain including VH CDR1 having the amino acid sequence of SEQ ID NO: 5, VH CDR2 having the amino acid sequence of SEQ ID NO: 12, and VH CDR3 having the amino acid sequence of SEQ ID NO: 19, and further comprising a light chain variable (VL) domain including VL CDR1 having the amino acid sequence of SEQ ID NO: 25, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 33.

[0080] In certain embodiments, the present disclosure provides an anti-idiotype antibody or antigen-binding moiety that specifically binds to an anti-GPRC5D2 antibody, comprising a heavy chain variable (VH) domain including VH CDR1 having the amino acid sequence of SEQ ID NO: 6, VH CDR2 having the amino acid sequence of SEQ ID NO: 13, and VH CDR3 having the amino acid sequence of SEQ ID NO: 19, and further comprising a light chain variable (VL) domain including VL CDR1 having the amino acid sequence of SEQ ID NO: 25, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 33.

[0081] In certain embodiments, the present disclosure provides an anti-idiotype antibody or antigen-binding moiety that specifically binds to an anti-GPRC5D2 antibody, comprising a heavy chain variable (VH) domain including VH CDR1 having the amino acid sequence of SEQ ID NO: 7, VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and VH CDR3 having the amino acid sequence of SEQ ID NO: 20, and further comprising a light chain variable (VL) domain including VL CDR1 having the amino acid sequence of SEQ ID NO: 26, VL CDR2 having the amino acid sequence of SEQ ID NO: 30, and VL CDR3 having the amino acid sequence of SEQ ID NO: 34.

[0082] In some embodiments, the anti-idiotype antibody or its antigen-binding portion that specifically binds to the anti-GPRC5D2 antibody includes a VH domain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45, and the VL domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46. In some embodiments, the anti-idiotype antibody or its antigen-binding portion that specifically binds to the anti-GPRC5D2 antibody includes a heavy chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37, and further includes a light chain having an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 39.

[0083] In some embodiments, the anti-idiotype antibody or its antigen-binding moiety that specifically binds to the anti-GPRC5D2 antibody includes a VH domain having the amino acid sequence of SEQ ID NO: 45 and a VL domain having the amino acid sequence of SEQ ID NO: 46. In some other embodiments, the anti-idiotype antibody or its antigen-binding moiety that specifically binds to the anti-GPRC5D2 antibody includes a heavy chain having the amino acid sequence of SEQ ID NO: 37 and further includes a light chain having the amino acid sequence of SEQ ID NO: 39.

[0084] In some embodiments, the antigen-binding moiety is selected from Fab, F(ab')2, or scFv. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the chimeric antibody comprises a mouse IgG2a framework. In certain embodiments, the mouse IgG2a framework may comprise a mouse Ig heavy chain signal peptide derived from Mix FVB / N, C57BL / 6J containing the sequence MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 48).

[0085] In some other embodiments, the antibody is a fully human antibody. For example, a fully human antibody may be IgG, IgM, IgA, IgE, or IgD. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is specific to GP5B83, where GP5B83 is located within the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). For example, GP5B83 encoding a nucleic acid can be introduced into T cells in vitro, and at least a portion of it is then expressed in the extracellular portion of the CAR. The anti-idiotype antibody can then specifically bind to the extracellular portion of the CAR. In some embodiments, GP5B83 is an scFv, and the anti-idiotype antibody or antigen-binding moiety specifically binds to an epitope in the scFv of the CAR. In some embodiments, GP5B83 specifically binds to GPRC5D. In some embodiments, the antibody or antigen-binding moiety does not cross-react with other GPRC5D antibodies or other GPRC5D-binding CARs. For example, in an assay to determine whether GP5B83 is expressed on the extracellular portion of a CAR, to prevent false positives, the anti-idiotype antibody may be specific to GP5B83 and may not have recognizable binding to its target GPRC5D or other GPRC5D-targeting ligands other than GP5B83. In some embodiments, the CAR contains an amino acid sequence selected from the group consisting of SEQ ID NOs. 43-44.

[0086] In some embodiments, the disclosure provides nucleic acids encoding the heavy chain, light chain, or both of an anti-idiotype antibody or antigen-binding moiety. For example, the nucleic acid may be DNA, RNA, or any chemical modifications thereof (e.g., nucleoside modifications).

[0087] In another embodiment, the disclosure provides nucleic acids encoding an anti-idiotype antibody that specifically binds to GP5B83, or the heavy chain, light chain, or both of the antigen-binding portion thereof, comprising the nucleotide sequence of SEQ ID NO: 38, the nucleotide sequence of SEQ ID NO: 40, or both. In another embodiment, the disclosure provides a vector comprising a nucleic acid sequence. For example, the vector may be a self-replicating nucleic acid structure or may be incorporated into the genome of a host cell into which it is introduced. In some embodiments, the vector is an expression vector. In another embodiment, the disclosure provides a host cell comprising the vector. In some embodiments, the host cell is a mammalian cell.

[0088] In another embodiment, the disclosure provides a method for producing an anti-idiotype antibody or its antigen-binding moiety that specifically binds to GP5B83, comprising culturing host cells containing nucleotide sequences encoding the heavy and light chains of the antibody or antigen-binding moiety under conditions that enable the expression of the antibody or antigen-binding moiety, and isolating the antibody or antigen-binding moiety from the culture. For example, anti-idiotype antibodies can be produced by homogeneous suspension culture in deep-tank agitated fermenters, perfusion tank systems, air-lift reactors, and continuous culture systems. Anti-idiotype antibodies can be isolated from the reaction mixture and / or growth mixture by physical or chemical separation procedures, including affinity separation using protein A or G, size exclusion chromatography, and charge separation. In some embodiments, the host cells encode a vector containing nucleic acids encoding the anti-idiotype antibody or its antigen-binding moiety.

[0089] In another aspect, the Disclosure provides a method for detecting GP5B83 in a biological sample, comprising (a) providing the biological sample, (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety, and (c) detecting the anti-idiotype antibody or antigen-binding moiety. For example, an anti-idiotype antibody may bind to GP5B83 expressed in the biological sample. The binding complex can be detected by any detection method, including both chemical and physical detection methods. For example, this detection method may be used to identify the mere presence of the antibody of interest in the biological sample, or to test whether the antibody of interest is present in the sample at a detectable level, or to quantify the amount of the antibody of interest in the sample and further to compare the antibody level with that of different samples. For example, the detection method may be one or more of immunoprecipitation, immunocytochemistry, immunoblotting, and immunoadsorption assays. As a specific example, an immunoadsorption assay may be an ELISA or ELISA-type assay, which includes a bound anti-idiotype antibody or fragment thereof on which the biological sample is washed.

[0090] In another aspect, the present disclosure provides a method for detecting the expression of a chimeric antigen receptor (CAR) comprising GP5B83 in a biological sample, the method comprising (a) providing a biological sample, (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety, and (c) detecting the anti-idiotype antibody or antigen-binding moiety, thereby detecting the expression of a CAR.

[0091] In some embodiments, the antibody includes a detectable label. In some embodiments, the method further comprises contacting the anti-idiotype antibody or antigen-binding moiety with the detectable label before detecting the anti-idiotype antibody or antigen-binding moiety. For example, the detectable label may be any chemical tag or component that is integrated with the anti-idiotype antibody, conjugates to the anti-idiotype antibody, or otherwise forms a complex with the anti-idiotype antibody and emits or otherwise provides a unique, identifiable signal. For example, the detectable label may be an isotope marker, a colorimetric biosensor, a photochromic compound, a fluorescent label, a fluorescence-generating label, or an electrochemical sensor. As a specific example, the fluorescent label may be a green fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, fluorescein, rhodamine, coumarin, cyanine, phycoerythrin, or derivatives thereof.

[0092] In some embodiments, the biological sample is blood, serum, or urine. For example, the biological sample may be whole blood, serum, plasma, urine, feces, cerebrospinal fluid, ascites, etc. In some embodiments, the biological sample is fresh biological material, such as biological material obtained at a given time for the purpose of this analysis. The biological sample may also be biological material obtained using patient material obtained at another point in time during the treatment of the patient for this purpose or other purposes, or archived patient material. The biological sample may be newly obtained or previously obtained, and if previously obtained, may be stored (e.g., at room temperature, refrigerated, or frozen) before use.

[0093] In some embodiments, the Disclosure provides a kit for detecting GP5B83 in a biological sample, comprising (a) an anti-idiotype antibody or antigen-binding moiety, and (b) instructions for detecting the anti-idiotype antibody or antigen-binding moiety. For example, the kit may contain the anti-idiotype antibody or antigen-binding moiety as a solid powder, lyophilized powder, liquid solution, or liquid component for mixing to form a solution, or conjugated to a solid support. The kit may also include further reagents, including stabilizers, buffers, and other pharmaceutically acceptable excipients necessary to facilitate the use of the kit in the assay of the biological sample. The kit may also include written instructions for the user on how to perform the assay.

[0094] In other embodiments, the Disclosure provides a method for purifying GP5B83 from a sample, comprising: (a) providing a biological sample containing GP5B83; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety of the Disclosure; and (c) capturing an anti-idiotype antibody or antigen-binding moiety containing a CAR or other protein containing GP5B83, thereby purifying GP5B83. For example, the anti-idiotype antibody can be captured using any separation method, including physical and chemical methods. Specifically, GP5B83 can be captured and isolated from cell culture media, host cells, or both using art-known techniques for purifying proteins, including ion-exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification, using antibodies specific to a particular epitope of the anti-idiotype antibody. In some embodiments, the anti-idiotype antibody is a fusion protein containing a domain that facilitates its purification. In certain embodiments, the purified GP5B83 composition is substantially isolated from a protein that does not contain GP5B83. In some embodiments, the purified GP5B83 composition is 100% pure, 99% pure, 98% pure, 97% pure, 96% pure, 95% pure, or 90% pure or higher.

[0095] In other embodiments, the Disclosure provides a method for selecting CAR-T cells from a cell population, comprising: (a) providing a biological sample containing CAR-T cells; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety; and (c) capturing the anti-idiotype antibody or antigen-binding moiety, thereby selecting CAR-T cells. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is specific to GP5B83.

[0096] Embodiment Specific non-limiting embodiments of the present invention are described in the following numbered paragraphs. 1. An anti-idiotype antibody or its antigen-binding moiety that specifically binds to a target antibody containing GP5B83. 2. The target antibody or antigen-binding moiety thereof comprises a VH domain having the amino acid sequence of SEQ ID NO: 41 and a VL domain having the amino acid sequence of SEQ ID NO: 42, as described in paragraph 1. 3. An anti-idiotype antibody or antigen-binding moiety that specifically binds to GP5B83, comprising a heavy chain variable (VH) domain including VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 7, VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 14, and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 20, and further comprising a light chain variable (VL) domain including VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 26, VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 30, and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 to 34. 4. An anti-idiotype antibody or antigen-binding moiety according to any one of paragraphs 1 to 3, wherein the VH domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45, and the VL domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46. 5. The anti-idiotype antibody or antigen-binding moiety according to any one of paragraphs 1 to 3, comprising a heavy chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37, and further comprising a light chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 39. 6. An anti-idiotype antibody or antigen-binding moiety described in any one of paragraphs 1 to 3, wherein the VH domain has the amino acid sequence of SEQ ID NO: 45 and the VL domain has the amino acid sequence of SEQ ID NO: 46. 7. The anti-idiotype antibody or antigen-binding moiety according to any one of paragraphs 1 to 3, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 37, and further comprising a light chain containing the amino acid sequence of SEQ ID NO: 39. 8. The antigen-binding moiety is an anti-idiotype antibody or antigen-binding moiety selected from Fab, F(ab')2, or scFv, as described in any one of paragraphs 1-3. 9. The antibody is a monoclonal antibody, an anti-idiotype antibody or antigen-binding moiety as described in any one of paragraphs 1-3. 10. The antibody is a chimeric antibody, an anti-idiotype antibody or antigen-binding moiety as described in any one of paragraphs 1-3. 11. The antibody is an anti-idiotype antibody or antigen-binding moiety as described in paragraph 10, comprising a mouse IgG2a framework. 12. The antibody is a fully human antibody, an anti-idiotype antibody or antigen-binding moiety as described in any one of paragraphs 1-3. 13. A nucleic acid encoding the heavy chain, light chain, or both of the heavy chain or antigen-binding moiety of an anti-idiotype antibody or antigen-binding moiety as described in any one of paragraphs 1 to 3. 14. An anti-idiotype antibody that specifically binds to GP5B83, or a nucleic acid that encodes the heavy chain, light chain, or both of the antigen-binding portion thereof, a) Nucleotide sequence of Sequence ID No. 38, b) Nucleotide sequence of sequence number 40, c) both a) and b) Nucleic acids, including 15. A vector containing the nucleic acid described in paragraph 14. 16. The vector is an expression vector, as described in paragraph 15. 17. Host cells containing the vector described in paragraph 16. 18. The cells are mammalian cells, the host cells described in paragraph 17. 19. A method for producing an anti-idiotype antibody or an antigen-binding moiety thereof that specifically binds to GP5B83, comprising culturing a host cell as described in paragraph 17, which comprises a nucleotide sequence encoding the heavy and light chains of the antibody or antigen-binding moiety, under conditions that enable the expression of the antibody or antigen-binding moiety, and isolating the antibody or antigen-binding moiety from the culture. 20. A method for detecting GP5B83 in a biological sample, comprising: (a) providing a biological sample; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any one of paragraphs 1 to 3; and (c) detecting the anti-idiotype antibody or antigen-binding moiety. 21. A method for detecting the expression of a chimeric antigen receptor (CAR) containing GP5B83 in a biological sample, comprising: (a) providing a biological sample; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any one of paragraphs 1 to 3; and (c) detecting the anti-idiotype antibody or antigen-binding moiety, thereby detecting the expression of a CAR. 22. The method according to paragraph 20, wherein the antibody includes a detectable label. 23. The method according to paragraph 20, further comprising contacting the anti-idiotype antibody or antigen-binding moiety with a detectable label before detecting the anti-idiotype antibody or antigen-binding moiety. 24. The method according to paragraph 20, wherein the biological sample is blood, serum, or urine. 25. GP5B83 is an anti-idiotype antibody or antigen-binding moiety located within the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR), as described in any one of paragraphs 1-3. 26.GP5B83 is an scFv, and the anti-idiotype antibody or antigen-binding moiety specifically binds to an epitope in the scFv of the CAR, as described in paragraph 25. 27. GP5B83 is an anti-idiotype antibody or antigen-binding moiety described in paragraph 25, which specifically binds to GPRC5d. 28. The anti-idiotype antibody or antigen-binding moiety described in paragraph 25, wherein the antibody or antigen-binding moiety does not cross-react with other GPRC5d antibodies or other GPRC5d-binding CARs. 29.CAR is an anti-idiotype antibody or antigen-binding moiety as described in paragraph 25, having an amino acid sequence selected from the group consisting of SEQ ID NOs. 43-44. 30. A kit for detecting GP5B83 in a biological sample, comprising (a) an anti-idiotype antibody or antigen-binding moiety described in any one of paragraphs 1 to 3, and (b) instructions for detecting the anti-idiotype antibody or antigen-binding moiety. 31. An anti-idiotype antibody or antigen-binding moiety according to any one of paragraphs 1 to 3, for use in the detection of GP5B83 in a biological sample, wherein the detection comprises (a) providing a biological sample, (b) contacting the biological sample with the anti-idiotype antibody or antigen-binding moiety, and (c) detecting the anti-idiotype antibody or antigen-binding moiety. 32. A method for purifying GP5B83 from a sample, comprising: (a) providing a biological sample containing GP5B83; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any one of paragraphs 1 to 3; and (c) capturing the anti-idiotype antibody or antigen-binding moiety to purify GP5B83. 33. A method for selecting CAR-T cells from a cell population, comprising: (a) providing a biological sample containing CAR-T cells; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any one of paragraphs 1 to 3; and (c) capturing the anti-idiotype antibody or antigen-binding moiety, thereby selecting CAR-T cells. [Examples]

[0097] Example 1: Determination of GCDB332-bound Fab GP5B83-derived scFv fusion protein GCDB332-bound Fab was selected from two sets of de novo Fab-pIX phage display libraries, as described in Shi et al., J Mol Biol 397:385-96, 2010; International Publication No. 2009 / 085462; U.S. Patent Application Publication No. 2010 / 0021477).

[0098] In phage selection using purified recombinant antigens, biotinylated GCDB332 was used as a "bait" to capture and immobilize phage binders. After several selection rounds, polyclonal phage ELISA using purified antigens was performed to detect specific enrichment from individual panning experiments. Phages collected from these panning experiments demonstrating binder enrichment against GCDB332 were expressed in E. coli for primary screening. Monoclonal Fab lysates were prepared from the enriched Fab library and screened by ELISA for binding to GCDB332, which does not bind to the background control. Selected Fabs were sequenced to identify unique Fab clones, and their V-region genes were isolated. The unique Fab V-regions were cloned into mammalian expression vectors to express chimeric mAbs containing the mouse IgG2a / mouse κ constant region. We evaluated the specific binding of chimeric mAbs to scFv expressed on SupT1 cells (GP5B83) corresponding to the scFv in the GCDB332 scFv fusion protein, and measured the binding dynamics using SPR.

[0099] Phagepanning Six individual panning experiments using individual V3.0 and V5.0 de novo Fab phage libraries were performed against biotinylated GCDB332 according to a standard protocol (Cheadle, EJet al. Antibody Engineering. 907:645-666 (2012)). Briefly, the phage libraries and paramagnetic streptavidin (SA) beads were panned using 50% Chemiblocker (Millipore catalog no. 2170) / 50% Blocking was performed in 1xTBST (Teknova catalog number T0310) for 1 hour. The library was added to SA beads to adsorb clones that nonspecifically bind to the beads. The SA beads were discarded, and the adsorbed library was added to biotinylated GCDB332. Bead / antigen / phage complexes were formed by adding SA beads, and the binder was recovered by washing these complexes in 1xTBST. After the final wash, phages were rescued by infection with log-phase TG1 E. coli cells (OD600nm=0.4~0.6). Phage-infected TG1 cells were spread on three 150 mm LB / agar plates containing 75 μg / mL carbenicillin and 1% glucose, and then grown overnight at 37°C. Phages were prepared and subjected to further panning. To increase the selective pressure, the antigen concentration was reduced and the incubation time was extended for each subsequent round: R1 100 nM, 1 hour; R2 10 nM, 1 hour; R3 10nM for 16 hours.

[0100] Polyclonal phage OLED Binder enrichment was measured by panning experiments using polyclonal phage ELISA. Briefly, 100 μL of 20 nM unbiotinized GCDB332 diluted in 1× TBS (Teknova catalog number T9530) was captured on an NA-coated plate (Thermo catalog number 15217). After incubation at 37°C for 1 hour, the plate was washed three times with 300 μL of 1× TBST. 300 μL of blocking buffer (50% Chemiblocker / 50% 1× TBST) was added to each well of the plate and incubated at room temperature for 1 hour. After blocking, the plate was washed three times with 300 μL of 1× TBST. 100 μL of polyclonal phage excretion from each panning round, diluted 1 / 100 with assay buffer (10% Chemiblocker / 90% TBST), was added to an ELISA plate and incubated at room temperature for 1 hour to conjugate the Fab displayed on the phage particles to the immobilized GCDB332. After incubation, the plate was washed three times with 1×TBST. 100 μL of HRP-conjugated anti-M13(pVIII) antibody (GE Healthcare catalog no. 27942101), diluted 1:2500 with assay buffer, was added to the plate and incubated at room temperature. After 1 hour of incubation, the plate was washed six times with 300 μL of 1×TBST. 100 μL of the prepared BM chemiluminescent ELISA substrate (Roche catalog no. 11582950001) was added to the plate. Chemiluminescence or relative light units (RLU) were measured using an Envision plate reader. As shown in Figure 1, all six panning experiments showed GCDB332-specific binding enrichment after three rounds of panning. GP5B318 originated from the XP3 panning experiment.

[0101] Fab production Plasmid DNA was isolated and purified from glycerol stocks of specific rounds of phage panning experiments identified to demonstrate binder enrichment to GCDB332, transformed into TG-1 E. coli cells, and grown overnight on LB / agar plates. Overnight cultures were used for (i) colony PCR and V-region sequencing, and (ii) initiation of culture for Fab production. For Fab production, overnight cultures were diluted 10-100-fold with fresh medium and grown at 37°C for 5-6 hours. Fab production was induced by adding fresh medium containing IPTG, and cultures were grown overnight at 30°C. The cultures were spun down, and the bacterial pellet was lysed using BugBuster® (Millipore) to release soluble Fab protein. The cell lysates were spun down, and the supernatant was used for Fab ELISA.

[0102] Primary screening Phages collected from panning experiments demonstrating binder enrichment for GCDB332 were expressed in E. coli for primary screening. Soluble Fab proteins were captured on plates using polyclonal sheep anti-human Fd(CH1) antibody (The Binding Site). After appropriate washing and blocking, biotinylated GCDB332 was added at a concentration of 50 nM. This biotinylated GCDB332 was detected using HRP-conjugated streptavidin and ELISA Pico Chemiluminescent Substrate (Thermo catalog number 37069), and then read with a plate reader. Furthermore, HRP-conjugated streptavidin was used for counterscreening. Fab clones with a binding signal to GCDB332 that was more than four times stronger than that of the negative control Fab, and a binding signal to streptavidin-HRP that was less than or equal to that of the negative control Fab, were selected and sequenced.

[0103] Figure 2 shows the results of the primary screening. Monoclonal Fabs were screened by ELISA for binding to GCDB332. Clones with a 4x binder signal relative to the background were selected and sequenced. The two clones identified by the asterisk produced VH and VL at GP5B318.

[0104] Example 2: Preparation of a monoclonal antibody against GP5B83 (GP5B318) Fab selection Fabs selected from the primary screening were sequenced to determine the V region sequence and identify unique clones. The unique Fab V region was cloned into a mammalian expression vector and expressed as a chimeric mAb containing the mouse IgG2a / mouse κ constant region.

[0105] The variable regions of GP5B318 were identified by phage display using the human Fab-pIX de novo library against the soluble scFv-Fc fusion protein GCDB332. These V regions did not undergo affinity maturation. DNA sequences were obtained from the de novo Fab library without codon optimization.

[0106] V H and V L Cloning Two pcDNA3.1-derived mammalian expression vectors (vDR000368 and vDR000961) were used to construct single-gene constructs encoding the heavy chain (HC) or light chain (LC) of a chimeric mAb. Each vector contains a human cytomegalovirus (hCMV) promoter to drive HC and LC expression, and both contain an ampicillin resistance gene (Amp(R)) to facilitate cloning. vDR000368 also contains unique HindIII and DraIII restriction enzyme sites for cloning, as well as a mouse IgG2a constant region. vDR000961 also contains unique HindIII and Tth111I restriction enzyme sites for cloning, as well as a mouse κ constant region.

[0107] DNA fragments containing the variable regions of HC(VH) or LC(VL) were synthesized by IDT and ligated into the HC vector vDR000368 and the LC vector vDR000961. The HC synthesized fragment contained a HindIII restriction enzyme site, a Kosack sequence, a DNA sequence encoding a signal peptide, parts of VH and CH1, and a DraIII cloning site. The LC synthesized fragment contained a HindIII restriction enzyme site, a Kosack sequence, a DNA sequence encoding a signal peptide, parts of VL and the κ constant region, and a Tth111I restriction cloning site. The final HC construct was PBD000094819, and the final LC construct was PBD000094818. The two constructs were simultaneously transfected into the mammalian cell line HEK293 Expi or CHO to produce GP5B318.

[0108] Protein expression Prior to transfection, the HC construct PBD000094819 and LC construct PBD000094818 were sequenced. HEK Expi293® cells (Thermo catalog number A14527) were grown in Expi293® expression medium (Thermo catalog number A1435101). Cells were grown at 37°C with shaking at 125 RPM under 8% CO2. Cells were expressed in 2.5 × 10⁶ cells using the Expi293® expression kit (Thermo catalog number A14524). 6Cells were transfected at a rate of cells / mL. For every liter of transfected cells, 1 mg of total DNA was diluted in 25 mL of Opti-MEM (Thermo catalog number 319850620), and 2.6 mL of Expi293® reagent was diluted in 25 mL of Opti-MEM. The mixture was incubated at room temperature for 5 minutes. The diluted DNA and diluted Expi293 reagent were added together and incubated at room temperature for 20 minutes. This DNA complex was then added to the cells. The cells were placed in a shaking incubator overnight. The day after transfection, 5 mL of enhancer 1 was diluted in 50 mL of enhancer 2, and the entire contents of both enhancers were added to the cells. The transfected cells were returned to the incubator for another 4 days before harvesting. The cells were removed by centrifugation at 4,500 g for 35 minutes, and then filtered through a 0.2 μm filter before checking the expression levels.

[0109] Expression was quantified using Octet. Mouse IgG2 (Sigma catalog number M9144) was used as the standard. A Protein A biosensor was used. Samples and standards were diluted in used Expi293 medium. The standard curve was started at 100 μg / mL at a 2-fold dilution. Samples were diluted 1:10. The standard curve was a linear point curve. Calculations were performed using Forte Biosystems software.

[0110] Example 3: Binding assay of anti-idiotype antibody GP5B318 Protein binding assay using soluble proteins A binding assay was performed using the ProteOn XPR36 system (BioRad). ProteOn GLC chips (BioRad, catalog number 176-5011) were coated with anti-mouse Fc antibody. Library antibody (mouse IgG2a) was diluted to 0.25 ug / mL to 1 ug / mL to reach approximately 100-200 RL. The antigen (scFv Fc fusion) was associated with 50 nM to 0.2 nM at a 1:4 dilution for 3 minutes. Dissociation was performed for 30 minutes. The assay results (Table 1 below) show the picomolar affinity of GP5B318 to GP5B305. GP5B305 is HC1 N-terminal CD3B376-Fab / C-terminal BCMB516-LH-scFv * and HC2 N-terminus GP5B83 N23T,N30S-LH-scFv * It is a bispecific antibody containing [specific compound].

[0111] [Table 1]

[0112] Cell binding assay scFv-transfected SupT1-GP5B680 HL, SupT1-GP5B680 LH, or GP5B83 cells were cultured in RMPI 1640, 10% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 10 mM HEPES, and 0.1% bicarbonate. Cell culture media and supplements were ThermoFisher Scientific Gibco products. mAbs were diluted to either 1.0 or 0.1 micrograms / mL in staining buffer (BSA) (BD Pharmingen catalog no. 554657). 50,000 cells / well of scFv-expressing SupT1 cells were added to 384-well V-bottom polypropylene plates (Greiner Bio-One #781280 or #781281). The plates containing the cells were centrifuged at 450 × g for 2 minutes, and the supernatant was aspirated. Diluted mAb samples were added to the cell pellet, mixed, and incubated on ice for 1 hour. The plate containing the cells was centrifuged at 450 × g for 2 minutes, and the supernatant was aspirated. Anti-mouse IgG heavy and light chain-PE conjugated secondary antibody (JacksonImmunoResearch, catalog no. 115-116-146), diluted 1:200 in Stain Buffer (BSA), was added to the cell pellet, mixed, and incubated on ice for 30 minutes. The plate containing the cells was centrifuged at 450 × g for 2 minutes, the supernatant was aspirated, the cell pellet was resuspended in Stain Buffer (BSA), mixed, and analyzed using an LSR II flow cytometer with an HTS autosampler (Beckton Dickenson). Data were analyzed using Flowjo software (Treestar) for the mean fluorescence intensity of the gated viable population. All liquid handling was performed using an Agilent Bravo system, and 384-well plates were aspirated using a BioTek 405 Select plate washer.

[0113] As shown in Table 2, GP5B318 showed specific binding to GP5B83-expressing cell lines, but not to the negative controls SupT1-GP5B680-HL and SupT1-GP5B680-LH.

[0114] [Table 2]

[0115] Example 4: Characterization of detection antibodies against GPRC5D CAR GP5B83-LH on CAR+SupT1 cells Antibodies for detecting GPRC5D CAR (GP5DB83-HL) expressed on NK cells and T cells were identified from a panel of proteins derived from phage display screening. As discussed in previous examples, the proteins were first tested for binding to recombinant CAR proteins, and potential binders were scaled up. The proteins were purified, and dose-dependent binding to GP5DB83-HL-expressing SupT1 cells was tested by flow cytometry. Binding was confirmed to be CAR-specific by competitive binding experiments using Fc-GP5DB83-HL fusion proteins and by the absence of binding to parental SupT1 cells. After selecting the best binder, the antibodies were directly conjugated to recombinant phycoerythrin ("PE") for use as CAR detection reagents. The antibodies were purified in a 1:1 PE:antibody ratio to enable receptor counting (number of CARs expressed on the cell surface).

[0116] purification Cell culture supernatant was loaded onto a MabSelect column and eluted with a low pH buffer such as 100 mM sodium acetate pH 3.0. The buffer was then replaced with 1×SSC, 8.5% sucrose pH 7.0 using a Sephadex G-25 column. The protein-containing fraction was collected. After purification, the protein was quality-controlled using SDS-PAGE, SEC-HPLC, and LC-MS.

[0117] Phycoerythrin labeling For every 10 μL of antibody to be labeled, 1 μL of Modifier reagent was added and gently mixed. The antibody sample (with Modifier reagent added) was pipetted directly onto lyophilized PE (Expedeon, catalog no. 703-0015), then gently resuspended, and incubated in the dark at room temperature (20-25°C) for 1 hour. 1 μL of Quencher reagent was added for every 10 μL of antibody used, and incubated for 30 minutes.

[0118] After labeling, the PE-antibody conjugate was purified using a size exclusion chromatography column. The fractions were collected and analyzed by SEC-HPLC. Fractions containing only one antibody with one PE were pooled together and concentrated as needed. The final product was analyzed by SEC-HPLC.

[0119] Characterization of GPRC5D CAR (GP5DB83-HL)CAR anti-idiotype antibody GP5B318 SupT1 cells expressing GP5DB83-HL were compared with parental CAR-SupT1 cells. Both GP5DB83-HL-expressing SupT1 cells and parental CAR-SupT1 cells were stained with near-IR LIVE / DEAD fluorescent dye on ice for 20 minutes. After incubation, the cells were washed and resuspended in BSA staining buffer. Cells (100,000 cells / well) were seeded into 96-well plates and incubated on ice for 30 minutes with gradually increasing concentrations of GP5B318 in and without 10 μg / mL of the CAR-Fc fusion protein, GP5B83_N24T_N315-HL-hu Fc-scFv (GP5B30.002). After incubation, the samples were washed with BSA staining buffer and stained with PE-goat anti-mouse IgG polyclonal to detect bound antibodies on viable SupT1 cells. After incubation, washing, and fixation, samples were acquired using a 10-color FACSCanto II flow cytometer. Analysis was performed using FlowJo, and the median PE fluorescence intensity of viable SupT1 cells was plotted in Figure 3. Figure 3 shows the dose-dependent binding of GP5B318 to GP5DB83-LH SupT1 cells. No binding was detected in CAR-SupT1 parent cells.

[0120] GP5B318 was conjugated to R-PE as described above, and its binding to GP5DB83-HL SupT1 was tested in the presence or absence of 10 μg / mL of GP5DB83-scFv Fc fusion protein to evaluate the specificity of GP5B318 binding to GP5DB83-HL CARs and confirm that CAR detection was not affected by PE labeling. GP5DB83-HL SupT1 cells were stained with near-IR LIVE / DEAD fluorescent dye for 20 minutes in a 50 mL tube on ice. After incubation, the cells were washed and 2 × 10⁶ solution was added to BSA staining buffer. 6 The cells were resuspended to the specified cell / mL. 100,000 cells / well were seeded into 96-well plates and incubated on ice for 45 minutes with increasing concentrations of PE-GP5B318 and 10 μg / mL of GP5DB83-scFv Fc. After incubation, washing, and fixation, samples were acquired using a 10-color FACSCanto II flow cytometer. Analysis was performed using FlowJo analysis software, and the median PE fluorescence intensity of viable SupT1 cells was plotted in Figure 4. As shown in Figure 4, the dose-dependent binding of PE-GP5B318 to GP5DB83-HL SupT1 cells is specific to GP5DB83-HL CAR and can be blocked by 10 μg / mL of GPDB83-HL scFv Fc.

[0121] array

[0122] [Table 3]

[0123] [Table 4]

[0124] Sequence ID 37: Heavy chain (amino acids) of GP5B318 containing muIgG2a EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGWDYGTGEFDYWGQ GTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIK PCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER TISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[0125] Sequence ID 38: Heavy chain (DNA) of GP5B318 containing muIgG2a

[0126] Sequence ID 39: Light chain (amino acid) of GP5B318 containing muKappa EIVLTQSPGTLSLSPGERATLSCRASQSIGNWLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQSLSFPITFGQGTKVEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[0127] Sequence ID 40: Light strand (DNA) of GP5B318 containing muKappa GAAATTGTGCTGACCCAGAGCCCGGGCACCCTGAGCCTGAGCCCGGGCGAACGCGCGACCCTGAGCTGCCGCGCGAGCCAGAGCATCGGTAACTGGCTGAACTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATTACGCGAGCAGCCTGCAGAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGGTGTATTATTGCCAGCAGTCCCTTTCCTTTCCGATTACATTTGGCCAGGGCACCAAAGTGGAAATTAAACGGGCTGATGCTGCACCGACTGTGTCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT

[0128] Accession No. 41: GP5B83 N23T / N30S VH QLQLQESGPGLVKPSETLSLTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRGTLVTVSS

[0129] Accession No. 42: GP5B83 VL EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIK

[0130] Sequence ID 43-CAR 1 (GP5B83-HL) QLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFWYFDLWGRG TLVTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRS NWPPTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0131] Sequence ID 44-CAR 2 (GP5B83-LH) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPTFGQGTKVEIKGGSEGKSSGSG SESKSTGGSQLQLQESGPGLVKPSETLSLTTCTVSGGSLSSSSYWWGWTRQPPGRGLEWIGTMYYSGNIYYNPSLQSRATISVDTSKNQFSLKLSSVTAADTAVYYCARHVGYSYGRRFW YFDLWGRGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE EEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0132] Sequence ID 45: Heavy chain variable domain (amino acid) of GP5B318 EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGWDYGTGEFDYWGQGTLVTVSS

[0133] Sequence ID 46: Light chain variable domain (amino acid) of GP5B318 EIVLTQSPGTLSLSPGERATLSCRASQSIGNWLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFAVYYCQQSLSFPITFGQGTKVEIK

[0134] Sequence ID 47 - Human GPRC5D sequence (amino acids) MYKDCIESTGDYFLLCDAEGPWGIILESLAILGIVVTILLLLAFLFLMRKIQDCSQWNVLPTQLLFLLSVLGLFGLAFAFIIELNQQTAPVRYFLFGVLFALCFSCLLAHASNLVKLVRGCVSFSWTTILCIAIGCSLLQIIIATEYVTLIMTRGMMFVNMTPCQLNVDFVV LLVYVLFLMALTFFVSKATFCGPCENWKQHGRLIFITVLFSIIIWVVWISMLLRGNPQFQRQPQWDDPVVCIALVTNAWVFLLLYIVPELCILYRSCRQECPLQGNACPVTAYQHSFQVENQELSRARDSDGAEEDVALTSYGTPIQPQTVDPTQECFIPQAKLSPQQDAGGV

[0135] Embedding by reference All publications and patent applications referenced herein are incorporated herein by reference to the same extent as each individual publication or patent application is specifically and individually indicated as being incorporated by reference.

[0136] While specific embodiments of the subject matter disclosure have been discussed, the above specification is illustrative and not limiting. Many variations of this disclosure will become apparent to those skilled in the art upon consideration of this specification and the following claims. The full scope of this disclosure should be determined by referring to the claims together with the full scope of the equivalents, and the specification together with such variations. Various embodiments of the present invention are shown below. 1. An anti-idiotype antibody or its antigen-binding moiety that specifically binds to a target antibody containing GP5B83. 2. The target antibody or antigen-binding moiety thereof comprises a VH domain having the amino acid sequence of SEQ ID NO: 41 and a VL domain having the amino acid sequence of SEQ ID NO: 42, as described in 1 above. 3. An anti-idiotype antibody or antigen-binding moiety that specifically binds to GP5B83, comprising a heavy chain variable (VH) domain including VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 7, VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 14, and VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 20, and further comprising a light chain variable (VL) domain including VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 26, VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 30, and VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 to 34. 4. The anti-idiotype antibody or antigen-binding moiety described in 1 above, wherein the VH domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45, and the VL domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46. 5. The anti-idiotype antibody or antigen-binding moiety according to 1 above, wherein the anti-idiotype antibody or antigen-binding moiety comprises a heavy chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37, and further comprises a light chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 39. 6. The anti-idiotype antibody or antigen binding moiety described in 1 above, wherein the VH domain has the amino acid sequence of SEQ ID NO: 45 and the VL domain has the amino acid sequence of SEQ ID NO: 46. 7. The anti-idiotype antibody or antigen-binding moiety according to item 1 above, wherein the anti-idiotype antibody or antigen-binding moiety comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 37, and further comprises a light chain containing the amino acid sequence of SEQ ID NO: 39. 8. The antigen-binding portion is Fab, F(ab') 2 An anti-idiotype antibody or antigen-binding moiety as described in 1 above, selected from , or scFv. 9. The antibody is a monoclonal antibody, or the anti-idiotype antibody or antigen-binding moiety described in 1 above. 10. The antibody is a chimeric antibody, or the anti-idiotype antibody or antigen-binding moiety described in 1 above. 11. The antibody is an anti-idiotype antibody or antigen-binding moiety as described in 10 above, comprising a mouse IgG2a framework. 12. The antibody is a fully human antibody, the anti-idiotype antibody or antigen-binding moiety described in 1 above. 13. A nucleic acid encoding the heavy chain, light chain, or both of the anti-idiotype antibody or antigen-binding moiety described in item 1 above. 14. An anti-idiotype antibody that specifically binds to GP5B83, or a nucleic acid that encodes the heavy chain, light chain, or both of the antigen-binding portion thereof, a) Nucleotide sequence of Sequence ID No. 38, b) Nucleotide sequence of sequence number 40, c) both a) and b) Nucleic acids, including 15. A vector containing the nucleic acid described in item 14 above. 16. The vector described in 15 above is an expression vector. 17. A host cell containing the vector described in item 16 above. 18. The host cell described in 17 above, wherein the cell is a mammalian cell. 19. A method for producing an anti-idiotype antibody or an antigen-binding moiety thereof that specifically binds to GP5B83, comprising culturing the host cells described in 17 above, which include nucleotide sequences encoding the heavy and light chains of the antibody or antigen-binding moiety, under conditions that enable the expression of the antibody or antigen-binding moiety, and isolating the antibody or antigen-binding moiety from the culture. 20. A method for detecting GP5B83 in a biological sample, comprising: (a) providing a biological sample; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any of items 1 to 3 above; and (c) detecting the anti-idiotype antibody or antigen-binding moiety. 21. A method for detecting the expression of a chimeric antigen receptor (CAR) containing GP5B83 in a biological sample, comprising: (a) providing a biological sample; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any of items 1 to 3 above; and (c) detecting the anti-idiotype antibody or antigen-binding moiety, thereby detecting the expression of the CAR. 22. The method according to 20 above, wherein the antibody includes a detectable label. 23. The method according to 20, further comprising contacting the anti-idiotype antibody or antigen-binding moiety with a detectable label before detecting the anti-idiotype antibody or antigen-binding moiety. 24. The method according to 20 above, wherein the biological sample is blood, serum, or urine. 25. GP5B83 is an anti-idiotype antibody or antigen-binding moiety described in item 1 above, located within the antigen-binding domain of the extracellular portion of a chimeric antigen receptor (CAR). 26. The anti-idiotype antibody or antigen-binding moiety described in 25 above, wherein GP5B83 is an scFv, and the anti-idiotype antibody or antigen-binding moiety specifically binds to an epitope in the scFv of the CAR. 27. GP5B83 is an anti-idiotype antibody or antigen-binding moiety described in 25 above, which specifically binds to GPRC5d. 28. The anti-idiotype antibody or antigen-binding moiety described in 25 above, wherein the antibody or antigen-binding moiety does not cross-react with other GPRC5d antibodies or other GPRC5d-binding CARs. 29. The CAR is an anti-idiotype antibody or antigen-binding moiety according to 25, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 43 to 44. 30. A kit for detecting GP5B83 in a biological sample, comprising (a) an anti-idiotype antibody or antigen-binding moiety as described in 1 above, and (b) instructions for detecting the anti-idiotype antibody or antigen-binding moiety. 31. An anti-idiotype antibody or antigen-binding moiety as described in 1 above, for use in the detection of GP5B83 in a biological sample, wherein the detection comprises (a) providing a biological sample, (b) contacting the biological sample with the anti-idiotype antibody or antigen-binding moiety, and (c) detecting the anti-idiotype antibody or antigen-binding moiety. 32. A method for purifying GP5B83 from a sample, comprising: (a) providing a biological sample containing GP5B83; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any of items 1 to 3 above; and (c) capturing the anti-idiotype antibody or antigen-binding moiety to purify GP5B83. 33. A method for selecting CAR-T cells from a cell population, comprising: (a) providing a biological sample containing CAR-T cells; (b) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in 1 above; and (c) capturing the anti-idiotype antibody or antigen-binding moiety, thereby selecting CAR-T cells.

Claims

1. An anti-idiotype antibody or its antigen-binding moiety that specifically binds to a target antibody, The target antibody is an anti-idiotype antibody or its antigen-binding moiety, comprising a VH domain having the amino acid sequence of SEQ ID NO: 41 and a VL domain having the amino acid sequence of SEQ ID NO:

42.

2. The anti-idiotype antibody or antigen-binding moiety according to claim 1, a) A heavy chain variable (VH) domain comprising VH CDR1 having the amino acid sequence of SEQ ID NO: 4, VH CDR2 having the amino acid sequence of SEQ ID NO: 11, and VH CDR3 having the amino acid sequence of SEQ ID NO: 19, and further comprising a light chain variable (VL) domain comprising VL CDR1 having the amino acid sequence of SEQ ID NO: 25, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 33, b) A heavy chain variable (VH) domain comprising VH CDR1 having the amino acid sequence of SEQ ID NO: 5, VH CDR2 having the amino acid sequence of SEQ ID NO: 12, and VH CDR3 having the amino acid sequence of SEQ ID NO: 19, and further comprising a light chain variable (VL) domain comprising VL CDR1 having the amino acid sequence of SEQ ID NO: 25, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 33, c) A heavy chain variable (VH) domain comprising VH CDR1 having the amino acid sequence of SEQ ID NO: 6, VH CDR2 having the amino acid sequence of SEQ ID NO: 13, and VH CDR3 having the amino acid sequence of SEQ ID NO: 19, further comprising a light chain variable (VL) domain comprising VL CDR1 having the amino acid sequence of SEQ ID NO: 25, VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and VL CDR3 having the amino acid sequence of SEQ ID NO: 33, or d) A heavy chain variable (VH) domain comprising VH CDR1 having the amino acid sequence of SEQ ID NO: 7, VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and VH CDR3 having the amino acid sequence of SEQ ID NO: 20, and further comprising a light chain variable (VL) domain comprising VL CDR1 having the amino acid sequence of SEQ ID NO: 26, VL CDR2 having the amino acid sequence of SEQ ID NO: 30, and VL CDR3 having the amino acid sequence of SEQ ID NO: 34, Anti-idiotype antibody or antigen-binding moiety.

3. a) The VH domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 45, and the VL domain has an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 46, and / or b) The anti-idiotype antibody or antigen-binding moiety comprises a heavy chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37, and further comprises a light chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

39. The anti-idiotype antibody or antigen-binding moiety according to claim 2.

4. The anti-idiotype antibody or antigen binding moiety according to claim 3, wherein the VH domain has the amino acid sequence of SEQ ID NO: 45, and the VL domain has the amino acid sequence of SEQ ID NO:

46.

5. The anti-idiotype antibody or antigen-binding moiety according to claim 3 or 4, wherein the anti-idiotype antibody or antigen-binding moiety comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 37, and further comprises a light chain containing the amino acid sequence of SEQ ID NO:

39.

6. a) The antigen-binding portion is Fab, F(ab') ２ , or selected from scFv, b) The antibody is a monoclonal antibody, and / or c) The VH domain described in Sequence ID No. 41 and the VL domain described in Sequence ID No. 42 are located within the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). The anti-idiotype antibody or antigen-binding moiety according to any one of claims 1 to 3.

7. a) The antigen-binding portion is selected from Fab, F(ab')2, or scFv b) The antibody is a monoclonal antibody, and / or c) The VH domain described in Sequence ID No. 41 and the VL domain described in Sequence ID No. 42 are located within the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). The anti-idiotype antibody or antigen-binding moiety according to any one of claims 4 to 5.

8. The anti-idiotype antibody or antigen-binding moiety according to claim 6, wherein the antibody is a chimeric antibody or a fully human antibody.

9. The anti-idiotype antibody or antigen-binding moiety according to claim 8, comprising a mouse IgG2a framework.

10. i) The VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42 are scFv, and the anti-idiotype antibody or antigen-binding portion specifically binds to the epitope in the scFv of the CAR. ii) The VH domain described in Sequence ID No. 41 and the VL domain described in Sequence ID No. 42 specifically bind to GPRC5d, and / or iii) The CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs: 43 to 44. The anti-idiotype antibody or antigen-binding moiety according to any one of claims 6 to 9.

11. A nucleic acid encoding the heavy chain and light chain of an anti-idiotype antibody or antigen-binding moiety according to any one of claims 1 to 10.

12. a) The nucleotide sequence of Sequence ID No. 38, and / or b) Nucleotide sequence of sequence number 40, The nucleic acid according to claim 11, comprising:

13. A vector comprising the nucleic acid according to claim 11 or 12.

14. The vector according to claim 13, which is an expression vector.

15. A host cell comprising the vector according to claim 13 or 14.

16. The host cell according to claim 15, wherein the cell is a mammalian cell.

17. A method for producing an anti-idiotype antibody or an antigen-binding moiety thereof that specifically binds to a protein comprising the VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42, comprising culturing a host cell according to claim 15 or 16, which comprises a nucleotide sequence encoding the heavy chain and light chain of the antibody or antigen-binding moiety, under conditions that enable the expression of the antibody or antigen-binding moiety, and isolating the antibody or antigen-binding moiety from the culture.

18. A method for detecting a protein in a biological sample that includes the VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42, comprising: (a) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any one of claims 1 to 10; and (b) detecting the anti-idiotype antibody or antigen-binding moiety.

19. A method for detecting the expression of a chimeric antigen receptor (CAR) in a biological sample, comprising a protein containing the VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42, the method comprising: (a) contacting the biological sample with an anti-idiotype antibody or antigen-binding moiety described in any one of claims 1 to 10; and (b) detecting the anti-idiotype antibody or antigen-binding moiety, thereby detecting the expression of the CAR.

20. a) The antibody contains a detectable label, b) The method further comprises contacting the anti-idiotype antibody or antigen-binding moiety with a detectable label before detecting the anti-idiotype antibody or antigen-binding moiety, or c) The method according to claim 18 or 19, wherein the biological sample is blood, serum, or urine.

21. A kit for detecting a protein in a biological sample that includes the VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42, comprising: (a) an anti-idiotype antibody or antigen-binding moiety described in any one of claims 1 to 10; and (b) instructions for detecting the anti-idiotype antibody or antigen-binding moiety.

22. An anti-idiotype antibody or antigen-binding moiety according to any one of claims 1 to 10, for use in detecting a protein comprising the VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42 in a biological sample, wherein the detection comprises (a) contacting the biological sample with the anti-idiotype antibody or antigen-binding moiety, and (b) detecting the anti-idiotype antibody or antigen-binding moiety.

23. A method for purifying a protein from a sample comprising the VH domain described in SEQ ID NO: 41 and the VL domain described in SEQ ID NO: 42, comprising: (a) contacting a biological sample containing the protein with an anti-idiotype antibody or antigen-binding moiety described in any one of claims 1 to 10; and (b) capturing the anti-idiotype antibody or antigen-binding moiety and thereby purifying the protein.

24. A method for selecting CAR-T cells from a cell population, comprising: (a) contacting a biological sample containing the CAR-T cells with an anti-idiotype antibody or antigen-binding moiety described in any one of claims 1 to 10; and (b) capturing the anti-idiotype antibody or antigen-binding moiety to select the CAR-T cells.

Images