Anti-idiotype antibodies against anti-KLK2 antibodies

Abstract

This invention provides anti-idiotype antibodies for detecting, purifying, or selecting CAR-T cells for the treatment of prostate cancer. [Solution] The following are provided: an anti-idiotype antibody or its antigen-binding portion that specifically binds to a KL2B413-containing protein; a nucleic acid encoding the anti-idiotype antibody and its antigen-binding portion; a method for producing the anti-idiotype antibody and its antigen-binding portion; a method for detecting KL2B413 (or cells expressing KL2B413) using the anti-idiotype antibody and its antigen-binding portion; and a kit containing the anti-idiotype antibody and its antigen-binding portion.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application is U.S. Provisional Patent Application No. 63 / 053,275, filed on 17 July 2020. This claims priority. The entire contents of the aforementioned application are referred to herein by reference. It will be incorporated into it.

[0002] (Sequence Listing) This application has been submitted electronically in ASCII format, and the entire application is by reference. This specification includes sequence listings incorporated herein. The above ASCII copy was made on July 7, 2021. A file was created, named JBI6351WOPCT1_SL.txt, with a size of 36,1 It is 87 bytes.

[0003] (Field of invention) The present invention relates to an anti-idiotype antibody that specifically binds to KL2B413-containing proteins, and This relates to the antigen-binding portion of an antibody or its antigen-binding portion, including KL2B413. A method for detecting and quantifying cells that express chimeric antigen receptors is also provided. [Background technology]

[0004] Recent advances in understanding the delivery and integration of genomic materials to target genomes are proving to be a major factor in standard treatment. It has great potential to be adapted to various diseases. In T-cell therapy, specific tumors Genetically modified and isolated T cells are used to enhance specificity to related antigens. Genetic modification involves the expression of chimeric antigen receptors (CARs) or exogenous T cell receptors, and T cells This can provide new antigen specificity. T cells expressing chimeric antigen receptors (CAR-T cells) These cells can induce tumor immunoreactivity.

[0005] One of the specific CAR targets of interest is kallikrein-related peptidase 2 (hK2, H This is K2). This is driven by androgen receptors (ARs) and the prostate tissue and prostate hK2 is a trypsin-like enzyme that is specifically expressed in cancer cells. It is activated by TMPRSS2 and secreted into the prostatic ducts, where it is present in the semen. A cascade that enhances sperm motility by cleaving semenogerin, which is part of the extracellular matrix. To initiate. hK2 expression is limited to prostate and prostate cancer tissue, but recently, steroids have been found to be In breast cancer cell lines and primary patient samples after the AR pathway has been appropriately activated by the hormone, It was proven that K2 was detectable (U.S. Patent Application Publication No. 2018 / 032610). (2) When the highly structured tissue of the prostate is damaged during hypertrophy or malignant transformation. This results in the retrograde release of catalytically inactive hK2 into the blood. [Overview of the Initiative] [Problems that the invention aims to solve]

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

[0007] This disclosure relates to an anti-idiotype antibody that specifically binds to KL2B413-containing proteins and The present disclosure provides an antigen-binding portion thereof, for example, an antibody or its antigen-binding portion. The present disclosure also provides an anti Idiotype antibodies and nucleic acids encoding their antigen-binding moieties, anti-idiotype antibodies and Methods for producing the antigen-binding portion, anti-idiotype antibodies and their antigen-binding portions are used to detect KL2B413 (or cells expressing KL2B413), and also provide a kit containing anti-idiotype antibodies and their antigen-binding portions.

[0008] In one aspect, the present disclosure provides an anti-idiotype antibody or an antigen-binding portion thereof that specifically binds to an anti-hK2 target antibody such as a target antibody containing KL2B413. In some embodiments, the target antibody or antigen-binding portion has a VH domain having an amino acid sequence containing SEQ ID NO: 41 and a VL domain having an amino acid sequence containing SEQ ID NO: 42.

[0009] In other embodiments, the anti-idiotype antibody or antigen-binding portion is for use in detecting KL2B413 in a biological sample, and the detection includes (a) preparing the biological sample, (b) contacting the biological sample with the anti-idiotype antibody or antigen-binding portion, and (c) detecting the anti-idiotype antibody or antigen-binding portion.

[0010] In another aspect, the present disclosure includes a heavy chain variable (VH) domain having a VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 7, a VH CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 11 to 14, and a VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 20, and a light chain variable (VL) domain having a VL CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 26, a VL CDR2 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 30, and a VL CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 33 to 34. ​ Furthermore, it includes an anti-idiotype antibody or antigen-binding portion that specifically binds to KL2B413. provide.

[0011] In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is SEQ ID NO: 4 A VH domain having an amino acid sequence with at least 90% sequence identity to 5 The VL domain contains amino acids that have at least 90% sequence identity with SEQ ID NO: 46. It has a sequence. In some embodiments, the anti-idiotype antibody or its antigen-binding portion , a heavy chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 37 It includes an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 39. It further comprises a light chain. In some embodiments, an anti-idiotype antibody or its antigen-binding portion. The minutes represent at least approximately 80%, 81%, 82%, 83%, 84%, and 8% of sequence number 45. 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 V has an amino acid sequence with 5%, 96%, 97%, 98%, or 99% sequence identity. Includes an H domain. In some embodiments, an anti-idiotype antibody or its antigen-binding portion. The minutes represent at least approximately 80%, 81%, 82%, 83%, 84%, and 8% of sequence number 46. 5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 9 V has an amino acid sequence with 5%, 96%, 97%, 98%, or 99% sequence identity. Includes L domains.

[0012] In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is SEQ ID NO: 4 A VH domain having the amino acid sequence of 5, and a VL domain having the amino acid sequence of SEQ ID NO: 46. Includes the main component. In some other embodiments, an anti-idiotype antibody or its antigen binding. The portion contains a heavy chain containing the amino acid sequence of SEQ ID NO: 37, and the amino acid sequence of SEQ ID NO: 39 It further contains light chains.

[0013] In some embodiments, the antigen-binding moiety is Fab, F(ab')2, or scFv. They are selected. In some embodiments, the antibody is a monoclonal antibody. In this embodiment, the antibody is a chimeric antibody. In some embodiments, the chimeric antibody is Includes a mouse IgG2a framework. In some other embodiments, the antibody is fully functional. It is an antibody. In some embodiments, the anti-idiotype antibody or its antigen-binding portion is It is specific to KL2B413, and KL2B413 is a chimeric antigen receptor (CAR) cell. It is located within the antigen-binding domain of the external portion. In some embodiments, KL2B413 is scF v is an anti-idiotype antibody or antigen-binding moiety, and the scFv of the CAR is an epitope within the scFv. It binds specifically to KLK2. In some embodiments, KL2B413 specifically binds to KLK2. It binds. In some embodiments, the antibody or antigen binding portion is another KLK2 antibody or other It does not cross-react with the KLK2-bound CAR. In some embodiments, CAR is SEQ ID NO: It has an amino acid sequence selected from the group consisting of 43 to 44.

[0014] In some embodiments, the present disclosure relates to the heavy chain of an anti-idiotype antibody or antigen-binding moiety. The present invention provides nucleic acids that encode a light chain, or both.

[0015] In another aspect, the present disclosure provides an anti-idiotype antibody that specifically binds to KL2B413 or This is a nucleic acid that codes for the heavy chain, light chain, or both of the antigen-binding portion, and SEQ ID NO: 3 A nucleic acid containing the nucleotide sequence of sequence number 8, the nucleotide sequence of sequence number 40, or both. To provide. In another aspect, the Disclosure provides a vector comprising a nucleic acid sequence. Several practical In one embodiment, the vector is an expression vector. In another embodiment, the disclosure includes a vector. A host cell is provided. In some embodiments, the host cell is a mammalian cell.

[0016] In another aspect, the present disclosure provides an anti-idiotype antibody that specifically binds to KL2B413 or A method for producing the antigen-binding portion, wherein the antibody or the antigen-binding portion is expressed Under certain conditions, the nucleotide sequence includes the heavy and light chains of the antibody or antigen-binding portion. The process involves culturing host cells and isolating the antibody or antigen-binding portion from the culture. The present invention provides a method that includes the use of anti-idiotype antibodies in host cells. In some embodiments, host cells are subjected to anti-idiotype antibodies. Alternatively, it encodes a vector containing a nucleic acid that codes for the antigen-binding portion.

[0017] In another embodiment, the present disclosure provides a method for detecting KL2B413 in a biological sample. This involves (a) preparing a biological sample, and (b) treating the biological sample with an anti-idiotype antibody or anti- (c) Contact with the proto-binding site and (c) detection of the anti-idiotype antibody or antigen-binding site. To provide a method that includes doing so.

[0018] In another aspect, the disclosure relates to a chimeric antigen receptor (CA) containing KL2B413 in a biological sample. A method for detecting the expression of R), comprising (a) preparing a biological sample and (b) (c) Contacting the body sample with an anti-idiotype antibody or antigen-binding site, and (c) anti-idio This includes detecting type antibodies or antigen-binding moieties, thereby detecting CAR expression. Hmm, I will provide a method.

[0019] In some embodiments, the antibody includes a detectable label. In some embodiments, This method involves detecting the anti-idiotype antibody or antigen-binding portion before detecting the anti-idiotype antibody. The method further includes contacting the body or antigen-binding portion with a detectable label. Therefore, biological samples are blood, serum, or urine.

[0020] In some aspects, the present disclosure provides a kit for detecting KL2B413 in biological samples. (a) an anti-idiotype antibody or antigen-binding moiety, and (b) an anti-idiotype antibody A kit is provided that includes instructions for detecting the body or antigen-binding site.

[0021] In other embodiments, the present disclosure relates to a method for purifying KL2B413 from a sample, (a) K (b) Prepare a biological sample containing L2B413 and (b) apply the biological sample to the anti-idiota of the present disclosure. (c) Contacting the ip antibody or antigen binding portion, and (c) anti-idiotype antibody or antigen binding portion The present invention provides a method that includes capturing the combined portion and thereby purifying KL2B413. .

[0022] In other embodiments, the present disclosure is a method for selecting CAR-T cells from a cell population, (a (b) Prepare a biological sample containing CAR-T cells, and (b) apply an anti-idiotype to the biological sample. (c) Contact with the antibody or antigen binding portion, and the anti-idiotype antibody or antigen binding portion The present invention provides a method that includes capturing cells and thereby selecting CAR-T cells. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is KL2B413 It is specific to.

[0023] This disclosure covers all combinations of the aforementioned aspects and embodiments, as well as details. This is intended to be combined with any of the embodiments described in the description and examples. [Brief explanation of the drawing]

[0024] For the purpose of illustrating the present invention, certain embodiments of this disclosure are shown in the drawings. However, However, this disclosure is not limited to the precise arrangement and means of the embodiments shown in the drawings. do not have. [Figure 1] This graph shows the specific binding and enrichment of KL2B513 or KL2B610 after four rounds of panning detected by polyclonal ELISA. [Figure 2] This graph shows the results of monoclonal Fab binding screening from KL2B513 or KL2B610 target binding assays compared with the GCDB734 counter screening reagent binding assay. [Figure 3] This is a graphical representation of a cell-based binding assay in which monoclonal antibodies were screened for binding to scFv-transfected SupT1 cells. [Figure 4] This graph shows the dose-dependent binding of A002B39 to KL2B413-LH SupT1 cells. [Figure 5] This graph shows the dose-dependent blocking of PE-A002B39 binding to KL2B413-LH SupT1 cells by the KL2B413-LH scFv-Fc fusion protein. [Figure 6] This graph shows the detection of PE-A002B39 in KL2B413-LH-SupT1 cells spiked in whole blood. [Figure 7] This is a graph showing two different lots of PE-A002B39 that have a similar binding profile to KL2B413-LH-SupT1. [Modes for carrying out the invention]

[0025] Overview This disclosure relates to an anti-idiotype antibody that specifically binds to KL2B413-containing proteins and The present disclosure provides an antigen-binding portion thereof, for example, an antibody or its antigen-binding portion. The otype antibody and antigen-binding moiety detect cells expressing CARs containing KL2B413. It can be used in methods for quantitative analysis. By such methods, researchers can A given batch of CAR-T cells generated in vitro expresses the desired CAR. Therefore, whether the cells are therapeutically useful in targeting the desired protein. It may be possible to determine whether or not. In this disclosure, anti-idiotype antibody and antigen binding site The target is KL2B413, which is itself a protein associated with prostate tissue. It targets a specific KLK2.

[0026] definition As used herein and in the attached "Claims", "a", "an", And the singular form "the" implies plural unless otherwise explicitly indicated. It encompasses the referent. Therefore, for example, the reference "a cell" can refer to two things. This includes combinations of three or more cells, etc.

[0027] Transition phrases: "comprising," "consisting essential" "ly of" and "consisting of" are generally accepted in patent terminology. It is intended to imply the meaning of "to be equipped / include (comp "Rising" is synonymous with "includes," "contains," or "characterizes," and is comprehensive or This is not restrictive and does not exclude other elements or methods not listed. (ii) "consisting of" is not specified in the claims. (iii) "Exclude any element, process or component that is not present, and (iii) "essentially from "The specified materials or processes, as well as the "basic and novel features" of the claimed invention" The scope of the patent claims is limited to "things that do not substantially affect" the term "equipment / includes" (and Embodiments described with the equivalent terms are also "consisting of" and "essentially from" It is provided as an embodiment that is described independently with the phrase "ru".

[0028] "Activation," "stimulation," "activated," or "stimulated" refers to the activation marker. This can lead to the expression of - cytokines, cytokine production, or mediation of target cell proliferation or cytotoxicity. This refers to the induction of a change in the biological state of a cell. Cells are activated by a primary stimulus signal. This can happen. Co-stimulatory signals amplify the magnitude of the primary signal and suppress cell death after initial stimulation. This allows for a more durable activation state, and consequently, higher cytotoxicity. This results in "co-stimulatory signals" such as primary signals like TCR / CD3 ligation. In combination with this, the proliferation of T cells and / or NK cells and / or the enhancement of key molecules This refers to a signal that causes preregulation or downregulation.

[0029] An "anti-idiotype antibody" or "anti-idiotype antibody" is a variable in the variable region of another antibody. This refers to antibodies that specifically bind to KLK2. In the case of KLK2, anti-idiotype antibodies are anti-KLK2 antibodies. It binds specifically to the body.

[0030] The "antigen-binding portion," "antigen-binding fragment," or "antigen-binding domain" is a part that binds to the antigen. This refers to the protein portion. The antigen-binding portion is a synthetic polypeptide or an enzymatically available polypeptide. This may be a peptide or a genetically modified polypeptide, which binds to an antigen. Some immunoglobulins, e.g., VH, VL, VH and VL, Fab, Fab', F(a b')2, Fd and Fv fragments, one VH domain or one VL domain In antibody (dAb), shark variable IgNAR domain, camelid VH domain, VHH domain It consists of amino acid residues that mimic the CDR of antibodies, such as the FR3-CDR3-FR4 region. The minimum recognition unit, HCDR1, HCDR2, and / or HCDR3, and LCDR 1. LCDR2 and / or LCDR3, alternative scaffolds that bind to the antigen. Examples include multispecific proteins containing antigen-binding moieties. Antigen-binding moieties (VH and The VH and VL domains (and VL, etc.) are linked to each other via a synthetic linker, so that the VH and VL domains are separate. When expressed as a single strand, the VH / VL domains pair intramolecularly or intermolecularly to form a monovalent form. It can form an antigen-binding domain, such as a single-stranded Fv (scFv) or a diabody. It is possible to form various types of single-chain antibody designs. The antigen-binding portion is also double-chain. To genetically engineer specific and multispecific proteins, single-specific or multispecific proteins are used. Possible other antibodies, proteins, antigen-binding sites, or alternative scaffolds It may be denjuted.

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

[0032] A "full-length antibody" consists of two heavy chains (HC) linked together by disulfide bonds. It is composed of two light chains (LCs) and their polymers (e.g., IgM). The chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, and the heavy chain constant domain is It consists of subdomains CH1, hinge, CH2, and CH3. Each light chain is a light chain variable It consists of a main (VL) and a light chain constant domain (CL). VH and VL are frames Work areas (FRs) are scattered, complementarity determining regions (FRs) It can be further subdivided into a region of hypervariability called gions (CDR). From the amino terminus to the carboxyl terminus, the order is as follows: FR1, CDR1, FR2, CD Three CDRs and four FR segments are arranged in R2, FR3, CDR3, and FR4. It is composed of elements.

[0033] The "complementarity-determining region (CDR)" is the antigen-binding site in an antibody. CDRs vary. It can be defined using the following terms: (i) Three within VH (HCDR1, HCDR2, HCD R3) and within VL there are three complementarity determination regions (C DR is based on sequence diversity (Wu and Kabat, J Exp Med 132:211-50,1970;Kabat et al.,Sequences of Proteins of Immunological Interest,5t h Ed.Public Health Service,National Inst. Itutes of Health, Bethesda, Md., 1991). (ii) Three "super-variable" elements within VH (H1, H2, H3) and three within VL (L1, L2, L3) "Region", "HVR", or "HV" refers to Chothia and Lesk (Chothia Defined by and Lesk Mol Biol 196:901-17, 1987) As described above, it refers to the antibody variable domain region, which is highly variable in structure. International ImmunoGeneTics (IMGT) Database (http http: / / www_imgt_org provides standard numbers and definitions for antigen-binding sites. Provided. CDR, HV, and IMGT summary descriptions are available from Lefranc et al. ., described in Dev Comparat Immunol 27:55-77, 2003. The terms "CDR", "HCDR1", "HCDR2", and "as used herein" are used in this specification. The terms "HCDR3", "LCDR1", "LCDR2", and "LCDR3" are used in this context. Unless otherwise stated in the specification, the above-mentioned Kabat, Chothia, or IMGT Includes a CD-R as defined by any of the methods described.

[0034] Human antibodies are optimized to produce the minimum possible immune response when administered to human subjects. This refers to antibodies that have been modified. The variable region of human antibodies is derived from the human immunoglobulin sequence. If the antibody contains a constant region or a portion of a constant region, that constant region is also human immunoglobulin Derived from the row. Human antibodies are derived from human germline immunoglobulins or rearranged in the variable region of human antibodies. When obtained from a system using the immunoglobulin gene, the sequence is of human origin. This includes a heavy chain variable region and a light chain variable region. Such exemplary systems are used to phages. A sprayed human immunoglobulin gene library and human immunoglobulin gene loci. This includes transgenic non-human animals possessing "human antibodies," such as mice or rats. " is typically a system used to obtain human antibodies and human immunoglobulin gene loci. The difference lies in the intentional introduction of somatic mutations into the framework or CDR. By means of, or both of these, the amino acid content when compared to immunoglobulins expressed in humans It contains differences. Typically, "human antibodies" are human germline immunoglobulins or reorganized immunoglobulins. For the amino acid sequence encoded by the immunoglobulin gene, The sequence is at least approximately 80%, 81%, 82%, 83%, 84%, 85%, 86%, and 87%. 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% They are 98% or 99% identical. In some cases, "human antibodies" are, for example, Knappi k et al., (2000) J Mol Biol 296:57-86 Consensus framework sequences obtained from human framework sequence analysis, or for example Shi et al., (2010) J Mol Biol 397:385-96 and The human immune phages presented in International Publication No. 2009 / 085462 It may contain synthetic HCDR3 incorporated into a globulin gene library. Antibodies in which one of the CDRs originates from a non-human species are not included in the definition of "human antibodies."

[0035] "Humanized antibodies" are those in which at least one CDR is derived from a non-human species and at least one F The framework refers to antibodies derived from human immunoglobulin sequences. Humanized antibodies are frameworks. Because the framework can include substitutions, the framework expresses human immunoglobulins. It may not be an exact copy of the germline gene sequence of phosphorus or human immunoglobulin. .

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

[0037] "To regulate" means to have a greater response compared to a response mediated by a control or vehicle. Enhanced or reduced test molecules that mediate a response of or less (i.e., downstream effects) It refers to one of the abilities that have been described.

[0038] "Natural killer cells" and "NK cells" are used interchangeably and synonymously in this specification. NK cells are CD16 + CD56 + and / or CD57 + TCR - phenotype Refers to activated differentiated lymphocytes. NK cells are "self" by activation of specific cytolytic enzymes Cells that cannot express MHC / HLA antigens, the ability to bind and kill, tumor cells or NK activity The ability to kill other diseased cells that express ligands of activating receptors, and the ability to stimulate or inhibit Able to release protein molecules called cytokines that are harmful.

[0039] "Specifically binds", "specific binding", "specifically bound", or "binds " refers to a proteinaceous molecule binding to an antigen or an epitope within an antigen with a higher affinity than to other antigens. Typically, the proteinaceous molecule is about 1×10 M or less, for example about 5×10 -7 M or less, about 1×10 -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, 1×1 0 -13 M, 1×10 -14 M, 1×10 -15 M or less of the equilibrium dissociation constant (K D ) binds to an antigen or is an epitope within an antigen, and typically, K D is at least 100-fold smaller than K for binding to non-specific antigens (e.g., BSA, casein). As described herein D In the context of prostate neoantigens, "specific binding" means that a proteinaceous molecule binds to a prostate neo antigen without detectably binding to the wild-type protein of which the prostate neo antigen is a variant. Refers to binding to prostate neoantigens.

[0040] "Tumor cells" or "cancer cells" refer to in vivo, ex vivo, or tissue culture In any of the following cases, cancer or precancerous conditions exhibiting spontaneously occurring or induced phenotypic changes. This refers to sex-changed or transformed cells. These changes do not necessarily involve the uptake of new genetic material. Transformation is not the result of infection with a transformed virus and the incorporation of a new genomic nucleic acid. It can also be generated by the incorporation of exogenous nucleic acids, spontaneously or through exposure to carcinogens. It can develop after exposure, and in some cases, this can lead to mutations in endogenous genes. Transformation / cancer This involves morphological changes in vitro, in vivo, and ex vivo. Cell immortalization, control of abnormal proliferation, lesion formation, proliferation, malignant lesions, tumor-specific marker levels By controlling the growth rate, invasiveness, and tumor growth in suitable animal hosts such as nude mice, Examples are given.

[0041] The terms "chimeric antigen receptor" or "CAR" are used herein in reference to all naturally occurring substances. A combination not found together in a single protein, extracellular target binding domain It is defined as a cell surface receptor containing a transmembrane domain and an intracellular signaling domain. This is understood to mean that, in particular, the extracellular domain and intracellular signaling domain are naturally The present invention includes receptors that are not found together in a single receptor protein. Chimeric antigen receptors are mainly found in lymphocytes such as T cells and natural killer (NK) cells. It is intended for use.

[0042] The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. When used herein, T cells refer to thymocytes, naive T lymphocytes, and immature T cells. Includes lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells are, T helper (Th) cells, for example, T helper 1 (Th1) or T helper 2 (Th2) cells. T cells include helper T cells (HTL, CD4+ T cells), C D4+ T cells, cytotoxic T cells (CTLs, CD8+ T cells), tumors Infiltrating cytotoxic T cells (TILs, CD8+ T cells), CD4+CD8+ T cells, or T cells It may be any other subset of cells. T cells suitable for use in a particular embodiment. Other exemplary populations include naive T cells and memory T cells. NKT cells are also included, and these not only express the semi-invariant αβ T cell receptor, but also NK1 T cells also express various molecular markers typically associated with NK cells, such as .1. This refers to a specific group of cells. NKT cells include NK1.1+ and NK1.1-, as well as CD4+ cells. Examples include CD4-, CD8+, and CD8 cells. The TCR of NKT cells is MHC I It is unique in that it recognizes glycolipid antigens presented by the similar molecule CD Id. T cells have the ability to produce cytokines that promote either inflammation or immune tolerance. It may have either a protective effect or a harmful effect. Also, "gamma delta T cells (γδT cells) ) also includes a small subset of T cells that have different TCRs on their surface. This refers to a special group of individuals, where TCR is represented as two glycoprotein chains, α and β-TCR chains. Unlike most T cells, which are composed of the gamma and delta chains, the TCR in γδ T cells is different. It is composed of the following. γδT cells can play a role in immune surveillance and immunomodulation. It is an important source of IL-17 and induces an active CD8+ cytotoxic T cell response. It was found that it suppresses abnormal or excessive immune responses and plays a role in immune tolerance. This also includes "regulatory T cells" or "Tregs," which refer to T cells that perform this function. Tregs are typical... Specifically, these are Foxp3-positive CD4+ T cells, and also IL-10-producing CD4+ cells. This may also include Foxp3-negative regulatory T cells, which are a type of T cell.

[0043] As used herein, the term “antigen” means an antigen to which a T cell receptor can bind. Ingredients (for example, proteins, peptides, polysaccharides, glycoproteins, glycolipids, nucleic acids, etc.) It refers to a molecule (part of or a combination thereof). Antigens can also trigger an immune response. Examples of immune responses include, but are not limited to, antibody production or specific immunological conditions. This may involve activation of pitent cells, or both. Those skilled in the art will know that the antigen never becomes a "gene". Therefore, you will understand that it does not need to be coded. Antigens are produced by synthesis. It may also be derived from a biological sample, or it may be a macromolecule other than a polypeptide. It is readily apparent that this is acceptable. Such biological samples include tissue samples and tumor samples. Cerebral samples, cells, or other biological components, organisms, protein / antigen subunits, killing or Alternatively, fluids containing inactivated whole cells or lysates can be cited, but these Not limited to this.

[0044] The term "antibody" refers to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, and more. Mela antibody, single-chain Fv(scFv), single-chain antibody, Fab fragment, F(ab') fragment, dys Rufid linkage Fv (sdFv), intrabody, minibody, diamondbody, and anti- Diotype (anti-Id) antibodies (e.g., anti-Id antibodies against antigen-specific TCRs), This refers to either the epitope-binding site or any of the above. The terms "antibody" and "antibody" are also used. Covalent dies, such as those described in U.S. Patent Application Publication No. 2007 / 0004909. Abody, and those disclosed in U.S. Patent Application Publication No. 2009 / 0060910 Which Ig-DARTS are you referring to? Antibodies useful as TCR-binding molecules include immunoglobulins. It contains immunologically active fragments of immunoglobulin molecules and immunoglobulin molecules, i.e., antigen-binding sites. Examples of such molecules include immunoglobulin molecules of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG) 3. IgG4, IgM1, IgM2, IgA1, and IgA2), or subclasses That's fine.

[0045] The term "host cell" refers to any cell containing heterologous nucleic acids. Heterologous nucleic acids are vectors. - (For example, an expression vector). For example, a host cell produces a substance by the cell, For example, the expression of genes, DNA or RNA sequences, proteins, or enzymes by cells. Any biological material that is selected, modified, transformed, grown, used, or manipulated in any manner for the purpose of It can be a native cell. A suitable host can be determined. For example, the host cell is a vector The ter skeleton and the desired result can be selected. For example, plasmids or cosmids Several types of vectors can be introduced into prokaryotic host cells to replicate them. DH Bacterial cells such as 5α, JM109, and KCB, but not limited to these, SURE (Registered Trademark) Competent cells, as well as SOLOPACK Gold cells, are vector-compounded. It can be used as a host cell for production and / or expression. Furthermore, Escherichia coli LE39 Bacterial cells such as those listed in 2 can be used as host cells for phage viruses. Eukaryotic cells that can be used as cells include yeast (e.g., YPH499, YPH500 and YPH499). This includes, but is not limited to, PH501), insects, and mammals. Vector replication Examples of mammalian eukaryotic host cells for expression include HeLa, NIH3T3, J Examples include urkat, 293, COS, CHO, Saos, and PC12, but these Not limited to this.

[0046] The terms "express" and "expression" refer to the generation of information about a gene or DNA sequence. To enable or generate, for example, the transcription and translation of a corresponding gene or DNA sequence. This means generating proteins by activating cellular functions involved in D NA sequences are expressed within or by cells to form "expression products" such as proteins. The expression product itself, for example, the resulting protein, is said to have been "expressed" by the cell. It is also possible to characterize the expression product as intracellular, extracellular, or transmembrane. ru.

[0047] The term "transfection" refers to the use of recombinant DNA technology to "external" ( In other words, it means introducing exogenous or extracellular nucleic acids into cells. This is called "genetic modification." The term refers to a host cell expressing an introduced gene or sequence to produce a desired substance, usually introduced. To produce a protein or enzyme encoded by the gene or sequence, "externally The introduction of (i.e., exogenous or extracellular) genes, DNA, or RNA sequences into host cells. This means that the introduced gene or sequence is also "cloned" or "foreign." They may also be called genes or sequences, and are starters, terminaters, promoters, signals, secretions, or The genes that encode chimeric antigen receptors, such as other sequences used by the cellular genetic mechanism. It may contain a regulatory or control sequence that is manipulably linked to a renucleotide. The sequence may include non-functional sequences that do not have known functions. The introduced DNA or The host cells that receive and express RNA are "genetically engineered." The DNA or RNA obtained may originate from any source, including cells of the same genus or species as the host cell, or It may originate from a different genus or species.

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

[0049] The term "regulatory element" refers to any systolic function that controls several aspects of nucleic acid sequence expression. It refers to a functional genetic element. In some embodiments, the term "promoter" essentially means a transforming genetic element. Includes the minimum sequence required to start the copy. In some embodiments, the term "promoter" is used. The "-" contains a sequence to initiate transcription, and in addition, each is generally referred to as an "enhancer". Upregulate or This also includes sequences that can be downregulated.

[0050] When used herein, “connected in an operable manner” and similar phrases refer to the core When used in relation to acids or amino acids, the nuclei are arranged in a functional relationship with each other. This refers to a manipulable linkage of acid sequences or amino acid sequences. For example, a manipulable linkage. A connected promoter, enhancer element, and open reading frame. The 5' and 3' UTRs, as well as the terminator sequence, are the positive terminals of a nucleic acid molecule (e.g., RNA). This ensures reliable production. In some embodiments, nucleic acid ligated to be operable is used. Rement is responsible for the transcription of open reading frames and ultimately the generation of polypeptides. That is, it results in the emergence of an open reading frame. Another example is the operable The linked peptides are such that their functional domains are positioned at appropriate distances from each other. This refers to something that provides the intended functionality of a domain.

[0051] "To strengthen," "to promote," "to increase," "to expand," or "to improve" In general, the response and ratio are determined by either the vehicle or the control molecule / composition. In comparison, it produces, induces, or causes a larger physiological response (that is, (Downstream effects) refer to the capabilities of the compositions intended herein. Measurable physiological responses are In particular, as is evident from the understanding of the relevant technical field and the description herein, T cell expansion, activation This may include enhancement, increased effector function, increased persistence, and / or increased cancer cell killing ability. In certain embodiments, the “increased” or “enhanced” amount is a “statistically significant” amount. The response produced by the vehicle or control composition is 1.1, 1.2, 1.5, 2, 3. 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more (for example, 500, (1000 times) (All integers and decimals between them, and greater than 1, e.g., 1.5, 1.6, 1.7) This may include increases (including 1.8, etc.).

[0052] "Decrease" or "Lower" or "Make less" or "Reduce" or "Weaken" "Responding" generally refers to a response caused by either the vehicle or the control molecule / composition. Compared to that, it produces or induces fewer physiological responses (i.e., downstream effects), This refers to the ability of the composition intended herein to cause or result in. In certain embodiments, "reduce" The amount "reduced" or "decreased" may be a "statistically significant" amount, and the vehicle, control composition Responses caused by a substance (reference response), or responses in a specific cell lineage, 1.1, 1.2 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 times, or more. (For example, 500, 1000 times) (All integers and decimals between them, and greater than 1, for example, 1. This may include decreases in 5, 1.6, 1.7, 1.8, etc.

[0053] The term “effective” when applied to a dose or amount refers to the time of administration to a subject who requires it. This refers to the amount of a compound or pharmaceutical composition sufficient to produce the desired activity. The combination of active ingredients... When administering a combination of drugs, the effective dose of that combination would likely be the same as if administered individually. Please note that the amount of each component may or may not be present. The required amount depends on the species, age, and overall health, the severity of the condition being treated, and the specific medication used. The appropriate dosage varies depending on the substance, method of administration, and the target individual.

[0054] The phrase "pharmaceutically acceptable" as used in relation to the compositions described herein means Physiologically tolerable and usually harmful when administered to mammals (e.g., humans). This refers to molecular entities that do not undergo a reaction and other components of such compositions. Preferably, the term "medical "Pharmacologically acceptable" means that for use in mammals, more specifically in humans, it has been approved by the federal government. Alternatively, it may be authorized by a state government regulatory agency, or it may be regulated by the United States Pharmacopeia or other general regulations. It means that it is listed in the officially recognized pharmacopoeia.

[0055] The term "protein," as used herein, includes protein fragments of all lengths. In addition to natural and synthetic proteins, fusion proteins, and glycoproteins, all other Types of modified proteins (e.g., phosphorylation, acetylation, myristoylation, palmitoylation) Formulation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosyl Modifications (including, but not limited to, proteins obtained from pegylation, biotinylation, etc.) It encompasses all types of proteins.

[0056] Unless otherwise specified, the terms "nucleic acid," "nucleotide," and "polynucleotide" are used. It includes both DNA and RNA. "Nucleic acid sequence" or "nucleotide sequence" refers to a It refers to the nucleic acid sequence that codes for amino acids, and these terms are also coded by linkers. A cloning artifact that includes any amino acid being cloned. It can also refer to a nucleic acid sequence that contains the portion coding for a specific amino acid.

[0057] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle to which a compound is administered together. This refers to petroleum, animal, and plant-based medicinal carriers such as peanut oil, soybean oil, mineral oil, and sesame oil. Sterilizing liquids such as water and oil, including substances or synthetic materials. Water or aqueous solution, Physiological saline, as well as aqueous solutions of dextrose and glycerin, are used as carriers, especially for injection solutions. It is preferably used as a carrier. Alternatively, the carrier may be a binder (for compressed pills), a flow promoter, or a sealant. A solid dosage form carrier containing, but not limited to, one or more of the following: fillers, flavoring agents, and coloring agents. This may also be the case. An example of a suitable pharmaceutical carrier is "Reming" by EW Martin. It is listed in "ton's Pharmaceutical Sciences".

[0058] The terms "approximately" or "about" include the fact that the value falls within a statistically meaningful range. Such a range is within one decimal place of a given value or range, preferably within 50%, more preferably within 2. It may be within 0%, more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the words "approximately" or "about" is a specific system under study. It depends on the element and can be easily understood by those skilled in the art.

[0059] "Kallikrein-related peptidase 2", "hK2", "KLK2", or "klk2" This refers to a known protein also called kallikrein-2, granular kallikrein 2, or HK2. It refers to the protein. hK2 is produced as a preproprotein and is cleaved during proteolysis. It then produces an active protease. All hK2 isoforms and mutants are "hK2 It is included in ". The amino acid sequences of various isoforms are GenBank accessions Numbers NP_005542.1, NP_001002231.1, and NP_00124 Searchable from 3009. The amino acid sequence of the full-length human hK2 in SEQ ID NO: 47 The sequence is shown. The sequence consists of a signal peptide (residues 1-18) and a propeptide region (residues 19-2 Includes 4).

[0060] The term "KL2B413" includes CAR, KL2B413 VH (Sequence ID 4) 1) Any variable region containing the variable region derived from KL2B413 VL (SEQ ID NO: 42) This refers to an antibody, its antigen-binding portion, or any other protein. In certain embodiments, this opening The anti-idiotype antibody shown contains the VH domain and / or SEQ ID NO: 41. It specifically binds to proteins containing the VL domain shown in 42. In certain embodiments, The anti-idiotype antibody of this disclosure has three CDs in the VH domain as shown in SEQ ID NO: 41 Specifically for proteins containing three CDRs in the VL domain as shown in R and SEQ ID NO: 42. Combine.

[0061] The term "KL2B513" refers to KL2B413, which has a variable region in the VL-VH direction. This refers to the derived scFv fusion protein. KL2B513 is KL2B413-LH-sc It is sometimes called Fv for compatibility.

[0062] The term "KL2B610" refers to the KL2B413 having a variable region in the VH-VL direction. This refers to the derived scFv fusion protein. KL2B610 is KL2B413-HL-sc It is sometimes called Fv for compatibility.

[0063] The term "A002B39" refers to human VH / VL and macules that target KL2B413. This refers to a chimeric mAb containing Us IgG2a / k. A002B39 is a chimeric mAb containing A002M39. It may be called interchangeably.

[0064] The terms used herein are for the sole purpose of describing specific embodiments. This is not intended to be an exhaustive limitation. When used herein, it is clearly indicated otherwise depending on the context. Unless otherwise specified, the articles "a," "an," and "the" are understood to refer to multiple things. I want to be treated that way.

[0065] This disclosure includes variants of antibodies, nucleic acids, polypeptides, and proteins described herein, e.g. For example, it provides functional variants. A "variant" is, for example, a substitution, insertion, or deletion. One or more modifications result in a different polypeptide from the reference polypeptide or reference polynucleotide. Refers to a nucleotide or polynucleotide. The term “functional variant” as used herein , having substantial or significant sequence identity or similarity with the parent antibody, polypeptide, or protein This refers to an antibody, polypeptide, or protein that is a variant of an antibody. It preserves the biological activity of the body, polypeptide, or protein. Functional variants are parental antibodies, polypeptides, etc. Recognizes target cells to the same degree, the same degree, or a higher degree than lipeptides or proteins. For example, antibodies, polypeptides, or proteins described herein that retain the ability to do so. This includes variants of parent antibodies, polypeptides, or proteins. Or, in the case of proteins, functional variants are, for example, parental antibodies, polypeptides, or proteins. For the protein, the amino acid sequence is at least approximately 30%, approximately 40%, approximately 50%, approximately 60%, and approximately 75%, approximately 80%, approximately 85%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately They may be 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or more identical.

[0066] In this specification, the structure of a polypeptide is defined as having an enumerated reference sequence (a given sequence number). It is located in a place defined based on % sequence identity with ). In relation to this, two amino acids The % sequence identity between sequences is calculated by comparing these two sequences that have been aligned in the most optimal way. This can be determined by comparing these two amino acid sequences. This may involve additions or deletions to the reference sequence for optimal alignment between the sequences. The percentage of uniformity is determined by finding the number of identical amino acid residues at the same position between two sequences. Divide the number of identical positions by the total number of positions in the comparison window, and multiply the resulting result by 100. This is calculated by obtaining the percentage of identity between these two sequences. Typically, the comparison window corresponds to the total length of the sequences being compared. For example, BLAST Program, "BLAST 2 Sequence" (Tatusova et al, "Blast 2 sequences-a new tool for comparing pro tein and nucleotide sequences”,FEMS Micr It is possible to use obiol Lett.174:247-250) and site http: / / www.ncbi.nlm.nih.gov / gorf / bl2.htm It is available in l, and the parameter used is the default (specifically, the parameter " Open gap penalty: 5, and extension gap penalty: 2 The selected matrix is, for example, the matrix "B" proposed by the program. The parameters given in LOSUM 62 are between the two arrays being compared. The percentage of identity is calculated directly by the program. Determining sequence identity is within the scope of the skills of those skilled in the art, and commercially available methods such as BLAST (trademark) are available. This can be done using analysis software.

[0067] Functional variants are, for example, parent antibodies having at least one conserved amino acid substitution, poly It may include the amino acid sequence of a peptide or protein. In another embodiment, a functional variant. This refers to a parent antibody, polypeptide, or protein having at least one non-conservative amino acid substitution. It may contain a non-conservative amino acid sequence. In this case, non-conservative amino acid substitutions result in the creation of functional mutants. It is not possible to suppress or inhibit the physical activity. Functional mutants are created by non-conservative amino acid substitutions. The biological activity of the parent antibody can be improved, and as a result, the biological activity of the functional mutant is improved, Enhances compared to lipeptides or proteins.

[0068] The amino acid substitutions in the antibody of the present invention may be conservative amino acid substitutions. This is one known in the art and having specific physical and / or chemical properties. The amino acid is replaced with another amino acid having the same or similar chemical or physical properties. This includes amino acid substitutions. For example, a conservative amino acid substitution is an acidic amino acid substitution with another acidic amino acid. An amino acid (e.g., Asp or Glu), substituted with another amino acid having a nonpolar side chain. Amino acids with nonpolar side chains (e.g., Ala, Gly, Val, Ile, Leu, Me) Basic amino acids (such as t, Phe, Pro, Trp, Val), which are substituted with other basic amino acids. amino acids (Lys, Arg, etc.), polar side chains substituted with other amino acids that have polar side chains It may contain amino acids such as Asn, Cys, Gln, Ser, Thr, Tyr, etc. .

[0069] Antibodies, polypeptides, and proteins of embodiments of the present invention (functional parts and mechanisms of the present invention) Potential mutants (including activating mutants) contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, for example, , aminocyclohexanecarboxylic acid, norleucine, α-aminon-decanoic acid, homoceryl S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydro Xyproline, 4-aminophenylalanine, 4-nitrophenylalanine, α-(2- (Amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminobutyric acid Ropionic acid, homophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine Nylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine α-Naphthylalanine, Cyclohexylalanine, Cyclohexylglycine, N'-B N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine N, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid 1,2, 3,4-Tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid Examples include monoamides and α-tert-butylglycine.

[0070] Antibodies, polypeptides, and proteins (functional portions and functional mutations) according to embodiments of the present invention (including the body) can undergo post-translational modifications. These include glycosylation and esterification. N-acylation, amidation, carboxylation, phosphorylation, esterification, for example, disulfide It can be cyclized via cyclobridged crosslinking, or converted into an acid addition salt. In this embodiment, these are dimerized, polymerized, or conjugated. ru.

[0071] Antibodies, polypeptides, and / or proteins (functional portions and their respective embodiments) of the present invention Functional variants (including functional variants) can be obtained by methods known in the art. A suitable method for the de novo synthesis of polypeptides and proteins is described by Chan et al. al.,Fmoc Solid Phase Peptide Synthesis,O xford University Press,Oxford,United Kin gdom,2000, Peptide and Protein Drug Analysis sis, ed. Reid, R., Marcel Dekker, Inc., 2000, and and Epitope Mapping, ed.Westwood et al.,Oxfo rd University Press,Oxford,United Kingdom This is described in references such as m,2001. Furthermore, polypeptides and proteins are, Using standard recombination methods, nucleic acids described herein can be produced by recombination. It is possible. For example, Sambrook et al., Molecular Cloni ng:A Laboratory Manual,3rd ed.,Cold Spri ng Harbor Press, Cold Spring Harbor, NY2 001, and Ausubel et al.,Current Protocols i n Molecular Biology, Greene Publishing As See Sociates and John Wiley & Sons, NY, 1994. Please refer to the following. Furthermore, the antibody, polypeptide, and protein (functional portion and its Some of these (including functional variants) were isolated from sources such as plants, bacteria, insects, and mammals. / or it can be purified. Methods of isolation and purification are known in the art. Alternatively, antibodies, polypeptides, and / or proteins (functional portions) as described herein. (and its functional variants) can be commercially synthesized. In this regard, Body, polypeptides, and proteins are synthesized, recombinant, isolated, and / or purified. It is possible.

[0072] Methods and Uses of This Disclosure This disclosure relates to an anti-idiotype antibody that specifically binds to KL2B413-containing proteins and The antigen-binding portion thereof, for example, an antibody or its antigen-binding portion, is provided. For example, an antibody Otype antibodies contain an amino acid complementary to the KL2B413 antibody to promote specific binding. It may contain an acid sequence. This disclosure also describes anti-idiotype antibodies and their antigen-binding portions. Nucleic acids, anti-idiotype antibodies and methods for producing their antigen-binding moieties, anti-idiota A method for detecting KL2B413 using an anti-Ide antibody and its antigen-binding moiety, and an anti-Ide antibody. The kit provides an iotype antibody and its antigen-binding moiety. For example, an anti-iotype antibody. The antibody can be included in a kit containing other reagents, and a given biological sample can be, for example, CAR Determine whether the T cells inside contain the KL2B413 antibody or a fragment thereof, which is expressed on the surface of the T cells. It can be used for that purpose.

[0073] A method for testing an antibody for its ability to bind to any functional part of KL2B413 is as follows: Known in the art, any antibody-antigen binding assay, for example, radioimmuno Assay (radioimmunoassay, RIA), Western blot, enzyme-linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA), immunoprecipitation, and competitive inhibition assays Examples include Sei.

[0074] Suitable methods for producing antibodies are known in the art. For example, standard methods The ibridomas method is, for example, described by Kohler and Milstein, Eur.JI. mmunol.,5,511-519(1976),Harlow and Lane( eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CAJaneway et al. (eds.), Immunobiology,5th Ed.,Garland Publishing It is described in , New York, NY (2001). Alternatively, by other means, For example, the EBV-hybridoma method (Haskard and Archer, J.Im munol.Methods,74(2),361-67(1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986 )), and bacteriophage vector expression systems (e.g., Huse et al.,Sc See ience, 246, 1275-81 (1989), etc., in the relevant technical field. This is known in [location]. Furthermore, methods for producing antibodies in non-human animals are known, for example, in the United States. Licenses No. 5,545,806, No. 5,569,825, and No. 5,714,352, (and as described in U.S. Patent Application Publication No. 2002 / 0197266(A1)).

[0075] It is also possible to generate antibodies using phage display. In this regard, antibodies Phage libraries encoding antigen-binding variable (V) domains are standard molecular biology. and can be produced using recombinant DNA technology (e.g., Sambrook See et al. (above) and Ausubel et al. (above). Phages encoding a variable region with desired specificity are capable of specific binding to the desired antigen. A complete or partial antibody containing the selected variable domain is reconstituted. The nucleic acid sequence encoding the constructed antibody is used in myeloma cells for hybridoma production. In which suitable cell line is introduced, and as a result, an antibody having the characteristics of a monoclonal antibody is introduced into the cell It is secreted by (e.g., Janeway et al. (above), Huse et al.) See also al. (above) and U.S. Patent No. 6,265,150).

[0076] In one embodiment, the disclosure specifically binds to a target antibody or CAR containing KL2B413. The present invention provides an anti-idiotype antibody or its antigen-binding moiety. For example, an anti-idiotype antibody Alternatively, the antigen-binding portion is one or more of the fragment antigen-binding regions (Fab) including VH and VL. It can specifically bind to the ion. In some embodiments, it binds to an anti-idiotype antibody or antigen. The combined portion consists of a VH domain having the amino acid sequence of SEQ ID NO: 45 and the amino acid sequence of SEQ ID NO: 46. It contains a VL domain having an acid sequence.

[0077] In other embodiments, the anti-idiotype antibody or antigen-binding moiety is KL2B in the biological sample. This is intended for use in the detection of 413, and the detection involves (a) preparing a biological sample. (b) Contacting a biological sample with an anti-idiotype antibody or antigen-binding site, c) Includes detecting an anti-idiotype antibody or antigen-binding moiety. For example, anti-idiotype Otype antibodies are found in tissue samples, tumor samples, liquids containing cells or other biological components, and other biological materials. Any protein / antigen subunits, dead or inactivated whole cells or lysates It can be added to biological samples. Anti-idiotype antibodies can be used as buffers, stabilizers, and / or This is contained in a solution containing pharmaceutically acceptable reagents, including but not limited to polymers. Anti-idiotype antibodies can be obtained by pipetting and / or mixing with biological samples. It can be brought into contact with KL2B41 in the biological sample. 3. It can specifically bind to proteins, such as antibodies or their antigen-binding sites. Therefore, whether or not the anti-idiotype antibody bound to KL2B413 can be determined by the presence or absence of the anti-idiotype antibody. By washing the diotype antibody and leaving only the complexed anti-idiotype antibody, It can be determined. Continuing with this example, the anti-idiotype antibody is present in the KL in the biological sample. It may contain a fluorophore that can be irradiated to give a signal proportional to the amount of 2B413. The detection of the binding complex of the anti-idiotype antibody against KL2B413 is described below. It will be published.

[0078] In another aspect, the present disclosure includes an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 7. VH CDR1 has an amino acid sequence selected from the group consisting of SEQ ID NOs: 11-14. It has an amino acid sequence selected from the group consisting of VH CDR2 and SEQ ID NOs: 19-20. It contains a heavy chain variable (VH) domain including VH CDR3, and sequence numbers 25-26 VL CDR1, consisting of sequence numbers 29-30, having an amino acid sequence selected from the group. VL CDR2 having an amino acid sequence selected from the group, and from SEQ ID NOs: 33-34 A light chain variable (VL) domain containing a VL CDR3 having an amino acid sequence selected from the group. An anti-idiotype antibody or antigen binding site that further contains an anti-KLK2 antibody that specifically binds to the anti-KLK2 antibody. Provides minutes. Six CDRs can be selected according to any known method. Kabat method VH and VL CDRs determined according to the AbM method, Chothia method, and contact method. This is shown in Table 3.

[0079] In certain embodiments, the present disclosure relates to 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 the amino acid sequence of SEQ ID NO: 19 It contains a heavy chain variable (VH) domain including VH CDR3, and the amino of SEQ ID NO: 25 VL CDR1 has an acid sequence, VL CDR2 has the amino acid sequence of SEQ ID NO: 29, and VL Domains containing VL CDR3 having the amino acid sequence of SEQ ID NO: 33 An anti-idiotype antibody or antigen binding site that further contains an anti-KLK2 antibody that specifically binds to the anti-KLK2 antibody. Provide minutes.

[0080] In certain embodiments, the present disclosure relates to 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 the amino acid sequence of SEQ ID NO: 19 It contains a heavy chain variable (VH) domain including VH CDR3, and the amino of SEQ ID NO: 25 VL CDR1 has an acid sequence, VL CDR2 has the amino acid sequence of SEQ ID NO: 29, and VL Domains containing VL CDR3 having the amino acid sequence of SEQ ID NO: 33 An anti-idiotype antibody or antigen-binding portion that further comprises and specifically binds to an anti-KLK2 antibody. is provided.

[0081] In certain embodiments, the disclosure includes a heavy chain variable (VH) domain comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 6, a VH CDR2 having the amino acid sequence of SEQ ID NO: 13, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 19, and further comprises a light chain variable (VL) domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 25, a VL CDR2 having the amino acid sequence of SEQ ID NO: 29, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 33, and provides an anti-idiotype antibody or antigen-binding portion that specifically binds to an anti-KLK2 antibody. An anti-idiotype antibody or antigen-binding portion that further comprises and specifically binds to an anti-KLK2 antibody. is provided.

[0082] In certain embodiments, the disclosure includes a heavy chain variable (VH) domain comprising a VH CDR1 having the amino acid sequence of SEQ ID NO: 7, a VH CDR2 having the amino acid sequence of SEQ ID NO: 14, and a VH CDR3 having the amino acid sequence of SEQ ID NO: 20, and further comprises a light chain variable (VL) domain comprising a VL CDR1 having the amino acid sequence of SEQ ID NO: 26, a VL CDR2 having the amino acid sequence of SEQ ID NO: 30, and a VL CDR3 having the amino acid sequence of SEQ ID NO: 34, and provides an anti-idiotype antibody or antigen-binding portion that specifically binds to an anti-KLK2 antibody. An anti-idiotype antibody or antigen-binding portion that further comprises and specifically binds to an anti-KLK2 antibody. is provided.

[0083] In some embodiments, the anti-idiotype antibody or its antigen-binding portion that specifically binds to an anti-KLK2 antibody has a VH domain having an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 45, and the VL domain has at least an amino acid sequence with at least 90% sequence identity to SEQ ID NO: 46. It has an amino acid sequence having at least 90% sequence identity. In some embodiments, anti-K An anti-idiotype antibody or an antigen-binding portion thereof that specifically binds to the LK2 antibody has a heavy chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 37 and further comprises a light chain comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 39. In some embodiments, an anti-idiotype antibody or an antigen-binding portion thereof that specifically binds to the anti-KLK2 antibody has 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:

[0084] 46. In some other embodiments, an anti-idiotype antibody or an antigen-binding portion thereof that specifically binds to the anti-KLK2 antibody has a heavy chain comprising the amino acid sequence of SEQ ID NO: 37 and further comprises a light chain comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antigen-binding portion 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 comprises the hybrid FVB / N, C57BL / 6J-derived mouse Ig heavy chain signal peptide having the sequence MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 48). In some other embodiments, the antibody is a fully human antibody. For example, the fully human antibody can be

[0085] IgG, IgM, IgA, IgE, or IgD. In some embodiments, 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 comprises the hybrid FVB / N, C57BL / 6J-derived mouse Ig heavy chain signal peptide having the sequence MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 48). In some other embodiments, the antibody is a fully human antibody. For example, the fully human antibody can be IgG, IgM, IgA, IgE, or IgD. In some embodiments, the mouse IgG2a framework comprises the sequence MAWVWTLLFLMAAAQSIQA (SEQ ID NO: 48) and may comprise a mouse Ig heavy chain signal peptide derived from FVB / N, C57BL / 6J. In some other embodiments, the antibody is a fully human antibody. For example, the fully human antibody can be

[0086] In some other embodiments, the antibody is a fully human antibody. For example, the fully human antibody can be IgG, IgM, IgA, IgE, or IgD. In some embodiments, The anti-idiotype antibody or its antigen-binding moiety is specific to KL2B413, and KL2 B413 is located within the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). For example, it is possible to introduce the nucleic acid encoding KL2B413 into T cells in vitro. Then, at least a portion of it is expressed in the extracellular portion of CAR. Subsequently, anti-Idi Otype antibodies can specifically bind to the extracellular portion of CARs. In some embodiments, KL2B413 is an scFv, and the anti-idiotype antibody or antigen-binding moiety is CAR It specifically binds to epitopes in scFv. In some embodiments, KL2B413 It specifically binds to KLK2. In some embodiments, the antibody or antigen binding moiety is It does not cross-react with other KLK2 antibodies or other KLK2-binding CARs. For example, KL2B41. In an assay to determine whether 3 is expressed in the extracellular portion of CAR, a false positive was observed. To prevent sexual activity, anti-idiotype antibodies may be specific to KL2B413 and target its target Recognizable binding to KLK2-targeting ligands other than KLK2 or KL2B413 It may not have. In some embodiments, CAR consists of SEQ ID NOs: 43-44 It has an amino acid sequence selected from the group.

[0087] In some embodiments, the present disclosure relates to the heavy chain of an anti-idiotype antibody or antigen-binding moiety. The present invention provides nucleic acids that encode light chains, or both. For example, nucleic acids include DNA, RNA, and These can be any chemical modifications (e.g., nucleoside modifications).

[0088] In another aspect, the present disclosure provides an anti-idiotype antibody that specifically binds to KL2B413 or It is a nucleic acid encoding the heavy chain, light chain, or both of the antigen-binding portion thereof, and SEQ ID NO: 3 A nucleic acid containing the nucleotide sequence of 8, the nucleotide sequence of SEQ ID NO: 40, or both is provided. In another aspect, the present disclosure provides a vector containing a nucleic acid sequence. For example, the vector may be a self-replicating nucleic acid structure, or it may be integrated into the genome of the host cell into which it is introduced . In some embodiments, the vector is an expression vector. In another aspect , the present disclosure provides a host cell containing the vector. In some embodiments, the host cell is a mammalian cell.

[0089] In another aspect, the present disclosure is a method for producing an anti-idiotype antibody that specifically binds to KL2B413 or its antigen-binding portion, comprising culturing a host cell containing a nucleotide sequence encoding the heavy and light chains of the antibody or antigen-binding portion under conditions that allow expression of the antibody or antigen-binding portion, and isolating the antibody or antigen-binding portion from the culture . For example, the anti-idiotype antibody can be produced by homogeneous suspension culture in a deep tank stirred fermenter, perfusion tank system, air-lift reactor, and continuous culture system . The anti-idiotype antibody 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 cell encodes a vector containing a nucleic acid encoding the anti-idiotype antibody or its antigen-binding portion . . In some embodiments, the host cell encodes a vector containing a nucleic acid encoding the anti-idiotype antibody or its antigen-binding portion . .

[0090] In another aspect, the present disclosure is a method for detecting KL2B413 in a biological sample This involves (a) preparing a biological sample, and (b) treating the biological sample with an anti-idiotype antibody or anti- (c) Contact with the proto-binding site and (c) detection of the anti-idiotype antibody or antigen-binding site. This includes the following: For example, an anti-idiotype antibody is a KL2 expressed in a biological sample. It can bind to B413. The bound complex can be detected by both chemical and physical methods. It can be detected by any detection method, including the detection method in a biological sample. It can be used to identify the mere presence of the antibody of interest, or to detect the antibody of interest in the sample. It can be used to test whether it is present at an acceptable level or the target in the sample. It can be used to quantify the amount of antibodies and to compare antibody levels from different samples. For example, detection methods include immunoprecipitation, immunocytochemistry, immunoblotting, and immunoadsorption. It can be one or more of the following. For example, in immunoadsorption assays, the biological sample is washed ELISA or ELISA-type ELISA containing a conjugated anti-idiotype antibody or a fragment thereof It could be true.

[0091] In another aspect, the disclosure relates to a chimeric antigen receptor (CA) containing KL2B413 in a biological sample. A method for detecting the expression of R), comprising (a) preparing a biological sample and (b) (c) Contacting the body sample with an anti-idiotype antibody or antigen-binding site, and (c) anti-idio This includes detecting type antibodies or antigen-binding moieties, thereby detecting CAR expression. Hmm, I will provide a method.

[0092] In some embodiments, the antibody includes a detectable label. In some embodiments, This method involves detecting the anti-idiotype antibody or antigen-binding portion before detecting the anti-idiotype antibody. The further step involves contacting the body or antigen-binding site with a detectable label. For example, a detectable label The label either integrates with the anti-idiotype antibody, or binds to the anti-idiotype antibody, Otherwise, it forms a complex with an anti-idiotype antibody and produces a unique, identifiable signal. It may be any chemical tag or component that emits or otherwise provides a signal. For example, detectable labels include isotope markers, colorimetric biosensors, and photochromic markers. It may be a compound, a fluorescent label, a fluorescence-generating label, or an electrochemical sensor. For example, Fluorescent labels include green fluorescent protein, yellow fluorescent protein, blue fluorescent protein, and fluorescein. Cein, rhodamine, coumarin, cyanine, phycoerythrin, and their derivatives It is possible.

[0093] In some embodiments, the biological sample is blood, serum, or urine. For example, the biological sample is This could be whole blood, serum, plasma, urine, feces, cerebrospinal fluid, ascites, etc. In some embodiments, The biological sample is a fresh biological material such as a biological material collected at a given time for this analysis. Therefore, biological samples may also be collected at another point in the patient's treatment for this purpose or any other purpose. This may be biomaterial obtained from biomaterials or biomaterials made from archived patient materials. The sample may be newly obtained or previously obtained. Items that have been stored before use may be kept in storage (for example, at room temperature, refrigerated, or frozen).

[0094] In some aspects, the present disclosure provides a kit for detecting KL2B413 in biological samples. (a) an anti-idiotype antibody or antigen-binding moiety, and (b) an anti-idiotype antibody A kit is provided that includes instructions for detecting a body or antigen-binding site. The component may be a solid powder, freeze-dried powder, liquid solution, or a liquid component that can be mixed to form a solution. Alternatively, it may contain an anti-idiotype antibody or antigen-binding moiety conjugated to a solid support. The kit contains stabilizers necessary to facilitate the use of the kit in assays of biological samples. The kit may include additional reagents, including buffers and other pharmaceutically acceptable excipients. It may also include instructions for the user on how to perform the assay.

[0095] In other embodiments, the present disclosure relates to a method for purifying KL2B413 from a sample, (a) K (b) Prepare a biological sample containing L2B413 and (b) apply the biological sample to the anti-idiota of the present disclosure. Contacting the ip antibody or antigen binding portion, and (c) CAR containing KL2B413 Alternatively, it captures an anti-idiotype antibody or antigen-binding site containing other proteins, thereby The present invention provides a method that includes purifying KL2B413, for example, physically and chemically. Anti-idiotype antibodies can be captured using any separation method, including the method described above. Specifically, ion exchange chromatography, gel filtration chromatography, ultrafiltration, and electrochemistry. Electrophoresis and immunoaffinity analysis using antibodies specific to particular epitopes of anti-idiotype antibodies. Using techniques known in the art for purifying proteins, including purification, cell culture KL2B413 can be captured and isolated from soil, host cells, or both. In some embodiments, the anti-idiotype antibody contains a domain that facilitates its purification. It is a fusion protein. In a particular embodiment, the purified KL2B413 composition is K It is substantially isolated from proteins that do not contain L2B413. In some embodiments, The purified KL2B413 composition is available in 100% pure, 99% pure, 98% pure, and 97% pure. It is pure, 96% pure, 95% pure, or 90% pure or higher.

[0096] In other embodiments, the present disclosure is a method for selecting CAR-T cells from a cell population, (a (b) Prepare a biological sample containing CAR-T cells, and (b) apply an anti-idiotype to the biological sample. (c) Contact with the antibody or antigen binding portion, and the anti-idiotype antibody or antigen binding portion The present invention provides a method that includes capturing cells and thereby selecting CAR-T cells. In some embodiments, the anti-idiotype antibody or its antigen-binding moiety is KL2B413 It is specific to.

[0097] Embodiment Specific non-limiting embodiments of the present invention are described in the following numbered sections. 1. An anti-idiotype antibody that specifically binds to a target antibody containing KL2B413, or the same Antigen-binding portion. 2. The target antibody or its antigen-binding moiety has the amino acid sequence of SEQ ID NO: 41. The anti-in described in item 1, comprising a VL domain having the amino acid sequence of SEQ ID NO: 42. Diotype antibody or antigen-binding portion. 3. An anti-idiotype antibody that specifically binds to KL2B413 or its antigen-binding portion 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 VH CDR3 having an amino acid sequence selected from the group consisting of SEQ ID NOs. 19-20 A heavy chain variable (VH) domain is included and selected from the group consisting of SEQ ID NOs. 25-26. Amino acids selected from the group consisting of VL CDR1 and SEQ ID NOs: 29-30, which have a mino acid sequence. Amino acids selected from the group consisting of VL CDR2 and SEQ ID NOs: 33-34, which have an acid sequence. Further comprising a light chain variable (VL) domain containing a VL CDR3 having an acid sequence, anti-idiolytic Otype antibody or its antigen-binding portion. 4. The VH domain has at least 90% sequence identity with SEQ ID NO: 45. It has a mino acid sequence, and the VL domain is at least 90% sequence-identical to SEQ ID NO: 46. An anti-idiotype anti- The body or antigen-binding portion. 5. Contains an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 37. An amino acid sequence containing a heavy chain and having at least 90% sequence identity with SEQ ID NO: 39. An anti-idiotype antibody or antigen according to any one of claims 1 to 3, further comprising a light chain containing joining part. 6. 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: An anti-idiotype antibody having a sequence of 46 amino acids, as described in any one of items 1 to 3, or This is the antigen-binding site. 7. Contains a heavy chain containing the amino acid sequence of SEQ ID NO: 37, and contains the amino acid sequence of SEQ ID NO: 39. An anti-idiotype antibody or antigen binding according to any one of items 1 to 3, further comprising a light chain. portion. 8. The antigen-binding site is selected from Fab, F(ab')2, or scFv, item 1~ An anti-idiotype antibody or antigen-binding moiety as described in any one of item 3. 9. The antibody is a monoclonal antibody, and is an anti-idiom as described in any one of items 1 to 3. Type antibody or antigen-binding portion. 10. The antibody is a chimeric antibody, according to any one of the anti-idiotypes described in items 1 to 3. The antibody or antigen-binding portion. 11. Antibodies comprising the mouse IgG2a framework, as described in item 10. IP antibody or antigen binding portion. 12. The antibody is a fully human antibody, as described in any one of items 1 to 3. The antibody or antigen-binding portion. 13. The heavy chain of the anti-idiotype antibody or antigen-binding moiety described in any one of items 1 to 3. A nucleic acid that codes for a light chain, or both. 14. Anti-idiotype antibody or its antigen-binding moiety that specifically binds to KL2B413. A nucleic acid that codes for the heavy chain, light chain, or both, a) Nucleotide sequence of Sequence ID No. 38, b) Nucleotide sequence of sequence number 40, c) Nucleic acids containing both a) and b). A vector containing the nucleic acid described in Section 15, Paragraph 14. 16. The vector is an expression vector, as described in item 15. 17. Host cells containing the vector described in Section 16. 18. A host cell as described in item 17, wherein the cell is a mammalian cell. 19. Anti-idiotype antibody or its antigen-binding moiety that specifically binds to KL2B413 A method for producing the antibody or the antigen-binding portion, wherein the antibody or The accommodation described in item 17 includes nucleotide sequences encoding the heavy and light chains of the antigen-binding region. The process involves culturing the principal cells and isolating the antibody or antigen-binding portion from the culture. Includes, method. 20. A method for detecting KL2B413 in a biological sample, wherein (a) the biological sample (b) Prepare the biological sample and (b) apply the anti-idiotype described in any one of items 1 to 3. (c) Contact with the antibody or antigen binding portion, and the anti-idiotype antibody or antigen binding portion A method including detecting minutes. 21. Detection of the expression of chimeric antigen receptors (CARs) including KL2B413 in biological samples. A method for doing so, comprising (a) preparing a biological sample and (b) placing the biological sample in items 1-3 Contacting the anti-idiotype antibody or antigen-binding moiety described in any one of the items, c) Detect anti-idiotype antibodies or antigen-binding moieties to detect CAR expression. A method that includes doing something. 22. The method according to item 20, wherein the antibody includes a detectable label. 23. Before detecting anti-idiotype antibodies or antigen-binding moieties, The method according to claim 20, further comprising contacting the antigen-binding portion with a detectable label. 24. The method according to item 20, wherein the biological sample is blood, serum, or urine. 25. KL2B413 is the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). An anti-idiotype antibody or antigen-binding portion contained within the . 26. KL2B413 is scFv, and the anti-idiotype antibody or antigen-binding portion is C The anti-idiotype anti-idiotype described in item 25, which specifically binds to the epitope in scFv of AR. The body or antigen-binding portion. 27. KL2B413 is an anti-idiotype antibody described in item 25 that binds to KLK2. Or the antigen-binding portion. 28. The antibody or antigen binding site cross-links with other KLK2 antibodies or other KLK2-binding CARs. An anti-idiotype antibody or antigen-binding moiety as described in item 25 that does not react. 29.CAR has an amino acid sequence selected from the group consisting of SEQ ID NOs. 43-44. , the anti-idiotype antibody or antigen-binding moiety described in item 25. 30. A kit for detecting KL2B413 in biological samples, wherein (a) items 1-3 (b) an anti-idiotype antibody or antigen-binding moiety as described in any one of the items, and (b) an anti-idiota A kit including instructions for detecting IP antibodies or antigen-binding sites. 31. Anti-idiotype for use in the detection of KL2B413 in biological samples. An antibody or antigen-binding portion, the detection involves (a) preparing a biological sample and (b) the biological (c) Contacting the sample with an anti-idiotype antibody or antigen-binding site, and The anti- Idiotype antibody or antigen-binding moiety. 32. A method for purifying KL2B413 from a sample, comprising (a) a sample containing KL2B413 (b) Prepare a biological sample and (b) apply the biological sample to any one of the anti-idiolytic agents described in items 1 to 3. (c) Contacting with an iotype antibody or antigen-binding portion, and (c) an anti-iotype antibody or A method comprising capturing an antigen-binding site and thereby purifying KL2B413. 33. A method for selecting CAR-T cells from a cell population, wherein (a) CAR-T cells (b) Prepare a biological sample containing the antimicrobial agent described in any one of items 1 to 3. (c) Contacting with an idiotype antibody or antigen-binding moiety, and (c) anti-idiotype antibody A method comprising capturing an antigen-binding site and thereby selecting CAR-T cells. . [Examples]

[0098] Example 1: Determination of KL2B513 or KL2B610 bound Fab Shi et al., J Mol Biol 397:385-96, 2010, country (International Publication No. 2009 / 085462, U.S. Patent Application Publication No. 2010 / 0021477) As described, the KL2B413-derived scFv fusion protein-binding Fab is 2 Selected from the set of de novo Fab-PIX phage display libraries . Two scFv fusion proteins derived from KL2B413, namely KL2B513(V There are L-VH and KL2B610(VH-VL), and within scFv, VH and VL are... The directions are different.

[0099] Phage selection using purified recombinant antigens is performed using biotinylated KL2B513 or KL2 B610 was used as "bait" to capture and fix the phage binder. Several times After the selection round, polyclonal phage ELISA using purified antigens is performed to identify individual cells. Specific enrichment was detected in the panning experiment for KL2B513 or KL2B6. Phages collected from these panning experiments, which showed binder enrichment relative to 10 For primary screening, the expression was produced in E. coli. Monochromator was extracted from the enriched Fab library. Prepare a solution of Fluorine Fab and test its binding to KL2B513 or KL2B610. Screening was performed with ISA, but a similar negative control scFv fusion protein GC5B734 was found. Alternatively, binding to GCDB332 was not screened. The selected Fab was The genes were sequenced to identify unique Fab clones, and their V-region genes were isolated. The Fab V region was cloned into a mammalian expression vector, and mouse IgG2a / mau Chimeric mAbs with a scupper constant region were expressed. (KL2B513 or KL2B61) 0 KL expressed on SupT1 cells corresponding to scFv in scFv fusion protein We evaluated chimeric mAbs for specific binding to scFv derived from 2B413, using SPR. The binding dynamics were then measured.

[0100] Phagepanning Using individual V3.0 and V5.0 de novo Fab phage libraries Six individual panning experiments were performed according to the standard protocol using biotinylated KL2B513 or K (Cheadle, EJet al. Antibody) Engineering.907,645-666(2012). In short, Phage library and paramagnetic streptavidin (SA) beads, 50% Chemib locker (Millipore catalog number 2170) / 50% 1 × TBST (Te Blocked for 1 hour with knova catalog number T0310. Library SA B It was added to the beads, and clones that nonspecifically bind to the beads were adsorbed. The SA beads were discarded. Biotinylated KL2B513 or KL2B610 in the presence of 100 nM GP5B305 A pre-adsorbed library was added. SA beads were added to create a bead / antigen / phage complex. The binder was recovered by forming a composite and washing it with 1x TBST. After the final wash... Phages are introduced by infection with log-phase TG1 E. coli cells (OD600nm=0.4~0.6). Rescued phage-infected TG1 cells with 75 μg / mL carbenicillin and 1% Spread the mixture onto three 150mm LB / agar plates containing glucose and incubate overnight at 37°C. Phages were generated and subjected to additional panning. To increase the selective pressure, subsequent lau The antigen concentration was reduced at room temperature for each step: R1 100nM, 1 hour; R2 50nM, 1 hour The intervals were set as R3 10nM for 1 hour and R4 5nM for 1 hour.

[0101] Polyclonal phage OLED Binder enrichment was determined from each panning experiment using polyclonal phage ELISA. To put it simply, dilute with 1x TBS (Teknova catalog number T9530). 100 μL of 20 nM non-biotinized KL2B513 or KL2B610, NA Co Captured on a heating plate (Thermo catalog number 15217). 37°C for 1 hour. After incubation, the plate was washed three times with 300 μL of 1×TBST. 30 0 μL of blocking buffer (50% Chemiblocker / 50% 1×TBST) The mixture was added to each well of the plate and incubated at room temperature for 1 hour. After blocking, 30 The plate was washed three times with 0 μL of 1×TBST. Assay buffer, 10% Chemib Polychloride from each panning round diluted to 1 / 100 with locker / 90% TBST Add 100 μL of ronal phage output to the ELISA plate and leave at room temperature for 1 hour. Incubate and immobilize the Fab presented on the phage particle with KL2B513 or K It was coupled to L2B610. After incubation, the plate was washed three times with 1×TBST. 100 μL of HRP conjugate anti-M was diluted 1:2500 with assay buffer. 13(pVIII) antibody (GE Healthcare catalog number 27942101) The mixture was added to the plate and incubated at room temperature. After 1 hour of incubation, 300 The plate was washed six times with 1 μL of 1×TBST. 100 μL of prepared BM chemiluminescent EL ISA substrate (Roche catalog number 11582950001) was added to the plate. Chemiluminescence or relative luminous units (RLU) are measured using an Envision plate reader. Ta.

[0102] Figure 1 shows the results of polyclonal phage ELISA. From 12 panning experiments... The panning output for rounds 1-4 is KL2B513 or KL2B61 In binding to 0 and the negative control GCDB332, the NA coated plate was screened. Leaned. Panning output with specific enrichment, monoclonal Fab production It was selected for this purpose. A002B39 originated from the APD316XP_19 panning experiment. .

[0103] Fab production The plasmid DNA is enriched with a binder for KL2B513 or KL2B610. To demonstrate, the glycerol stocks of a specific round of phage panning experiments were identified. The cells were isolated and purified, transformed into TG-1 E. coli cells, and cultured overnight on LB / agar plates. The cultures were subjected to (i) colony PCR and V region sequencing, and (ii) Used for initial culture for Fab production. For Fab production, the overnight culture was transferred to fresh medium. The solution was diluted 10 to 100 times and grown at 37°C for 5 to 6 hours. Fab production contained IPTG. The culture was induced by adding fresh culture medium and grown overnight at 30°C. Spin down the nutrients and the bacterial pellets with BugBuster(trademark)(Millipor The cells were lysed using (e) to release the soluble Fab protein. The lysates were then spun down. The supernatant was then used in Fab ELISA.

[0104] Primary screening Panning experiment showing binder enrichment for KL2B513 or KL2B610? The collected phages were expressed in E. coli for primary screening. MSD 3 84-well streptavidin plate (Meso Scale #L21SA-5) , 50 μL / well SuperBlock T20 (TBS) blocking buffer (T The area was blocked with hermo #37536 at room temperature for 30 minutes, and then aspirated. 2.5 nM biotinylated ScFv-Fc fusion antigen, target antigen KL2B513 or KL2B6 10, or the counter screening reagent GCDB734, blocked 384U The plates were placed in a ELL MSD plate and incubated on a shaker at room temperature for 30 minutes. 80 μL / d with 1x phosphate-buffered saline ("1x PBST") containing Tween detergent. Wash twice with a lint roller, and take the crude Fab lysate from the E. coli expression in the 96-well plate. Double-stamp the mixture onto an 84-well assay plate and incubate at room temperature on a shaker for 1 hour. The assay plate was washed once with 80 μL / well of 1×PBST, then 6 nM SULFO-TAG anti-human / NHP kappa antibody (Meso Scale #D20TF) -6) Add 20 μL / well and incubate the plate on a shaker at room temperature for 1 hour. The plate was washed once with 80 μL / well 1× PBST, and then with 35 μL / well 1× Each MSD Read Buffer T (Meso Scale #R92TC-1) In addition to the wells, the plate was analyzed using an MSD Sector S600 plate reader. All liquid processing is performed using the Agilent Bravo system, and the 384-well plate is processed. The plates were cleaned using a BioTek 405 Select plate washer.

[0105] Figure 2 shows the results of the primary screening. The results indicate a connection to KL2B513 or KL2B610. For the combination, monoclonal Fab was screened on an MSD plate. KL2B Standard deviation of mean background signal in 513 or KL2B610 target binding assays It has a larger signal than when three times the amount is added, and the GCDB734 counter reagent binding is increased. Signals smaller than the average background signal plus three times the standard deviation are considered to be signals in the same category. Clones possessing the specified trait were selected for sequencing. Clones identified by the asterisk are selected. This is the parent clone of VH and VL of A002B39.

[0106] Example 2: Monochrome for KL2B413-LH / HL-scFv(A002B39) Production of ronal antibodies Fab selection The Fab selected from the primary screening is sequenced to determine the V region sequence, and unique We identified the clone. We cloned the unique Fab V region into a mammalian expression vector. It was then expressed as a chimeric mAb containing the mouse IgG2a / mouse kappa constant region.

[0107] The variable region of A002B39 is controlled by the soluble scFv-Fc fusion protein KL2B513 or This is a method for treating KL2B610 using the human Fab-pIX de novo library. These V regions were identified by a page display. These V regions did not undergo any affinity maturation. The DNA sequence was obtained from the de novo Fab library without codon optimization. It was done.

[0108] V H and V L Cloning Two mammalian expression vectors derived from pcDNA3.1 (vDR000368 and vDR0 Using 00961), a single code encoding the heavy chain (HC) or light chain (LC) of a chimeric mAb. Genetic constructs were generated. Each vector drives the expression of human cytoplasmic megadoses of HC and LC. It contains a hCMV (H4CV) promoter, and both facilitate cloning. It contains the picillin resistance gene (Amp(R)). vDR000368 is cloned. Unique HindIII and DraIII restriction enzyme sites for mouse IgG2a It also has a steady-state region. vDR000961 is a unique HindIII for cloning. It also contains a Tth111I restriction enzyme site and a constant region of mouse kappa.

[0109] HC(V H ) or LC(V L A DNA fragment containing the variable region of ) was synthesized, and the HC vector v Ligation was performed on DR000368 and LC vector vDR000961. HC synthesis. The fragment consists of a HindIII restriction enzyme site, a Kozak sequence, a signal peptide, and V H and C H1 It contained a DNA sequence that encoded part of the DraIII cloning site. The LC synthesis fragment contains the Hind III restriction enzyme site, Kozak sequence, signal peptide, V L , and DNA sequences encoding a portion of the kappa constant region, and Tth111I restriction It contained a ronning site. The final HC construct was PBD000105920. The LC construct is PBD000105921. The two constructs are expressed in the mammalian cell line HE. A002B39 was prepared by co-transfecting with K293 Expi or CHO.

[0110] Protein expression HC structure PBD000105920 and LC structure PBD000105921, Sequence validation was performed before lance infection. Expi293(trademark) expression medium (Thermo HEK Expi293(trademark) cells (T) grown using catalog number A1435101 (Hermo catalog number A14527). Shake the cells at 125 RPM under 8% CO2. It was grown at 37°C. Expi293(trademark) expression kit (Thermo catalog number) Using (No. A14524), cells are divided into 2.5 × 10⁶ cells per 1 mL. 6 Transfect individually For every 1 liter of transfected cells, 1 mg of total DNA was added to 25 mL of Opti-M Dilute with EM (Thermo catalog number 319850620) and add 2.6 mL of Expiry. Dilute reagent 293 (trademark) in 25 mL of Opti-MEM and incubate at room temperature for 5 minutes. The diluted DNA and diluted Expi293 reagent were added together and incubated at room temperature for 20 minutes. The cells were incubated. Next, this DNA complex was added to the cells. The cells were then shaken in an incubator. - It was left overnight. The day after transfection, 5 mL of Enhancer 1 was added to 50 mL. The total amount of the two enhancers was added to the cells after dilution with Enhancer2. The septic cells were returned to the incubator for another four days before being harvested. 4,500 The cells were removed by centrifugation at 35 minutes, and then filtered through a 0.2 μm filter. The expression levels were then checked.

[0111] Expression was quantified using an Octet instrument. Mouse IgG2 (Sigma catalog number M 9144) was used as the standard. A protein A biosensor was used. Sample and label The standard solution was diluted with used Expi293 medium. The standard curve is 100 μg / mL at a 2-fold dilution. The process began with the sample being diluted 1:10. The standard curve was a linear point curve. The calculation was performed using F. This was done using Orte Biosystems software.

[0112] Example 3: Binding assay of anti-idiotype antibody A002B39 Biacore-based binding assay using soluble proteins Anti-mouse Fc antibody directly immobilized on the M5 sensor chip. Reaching approximately 20-50 RU. Library antibody (mouse IgG2a) diluted to 1 μg / mL. Antigen (scFV Associate the Fc fusions in a 500 nM to 0.8 nM solution at a 1:5 dilution for 3 minutes. Dissolve for 30 minutes. Separate them. The assay results (Table 2 below) are the same as those obtained using ProteOn. This shows the binding affinity to KLB513.

[0113] Binding assays using soluble proteins by Proteon and Biacore The binding assay was performed using the ProteOn XPR36 system (BioRad). ProteOn GLC chip (BioRad, catalog number 176-5011) This was coated with an anti-mouse Fc antibody. The library antibody (mouse IgG2a) was 0 Diluted to 0.25 μg / mL to 1 μg / mL, resulting in approximately 100-200 RL. Antigen (sc The Fv-Fc fusion was associated with a 1000nM to 0.4nM solution at a 1:4 dilution for 3 minutes, followed by The samples were dissociated for 30 minutes. The assay results (Table 1 below) are for KLB513 and KLB610. A002B39 exhibits similar binding affinity to both.

[0114] [Table 1]

[0115] Anti-mouse Fc antibody directly immobilized on M5 sensor chip. Library antibody (mouse I The antigen (scFV Fc) was diluted to 1 μg / mL to approximately 40-80 RU. Associate the fused molecules at a 500nM to 0.8nM concentration, diluted 1:5, for 3 minutes. Dissociate them for 30 minutes. The assay results (Table 2 below) show the binding affinity of A002B39 to KLB513. show.

[0116] [Table 2]

[0117] Cell binding assay ScFv Transfect SupT1-KL2B413 HL, SupT1-KL2B 413 LH, or SupT1-CD9B337-HL cells, RMPI 1640, 1 0% FBS, 1% non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamate Cell culture supplements were cultured in 10 mM HEPES and 0.1% bicarbonate. It was a ThermoFisher Scientific Gibco product. mA b in staining buffer (BSA) (BD Pharmingen catalog number 554657) Diluted to 6 micrograms / mL. Liv was used to fix scFv-expressing SupT1 cells. Labeled with e / Dead stain (Molecular Probes #L34974), 3 84-well V-bottom microplate (Greiner Bio-One #781281) It was added at a concentration of 50,000 cells / well.

[0118] Gently mix the normalized mAb sample into the cell suspension at a volume of 20 μL / well. The cells were then incubated on ice for 30 minutes. The cell suspension was chilled on ice in 70 μL / wells. Dilute with staining buffer (BSA), pellet the cells at 400×g at 4°C for 5 minutes, and remove the supernatant. The cells were aspirated. The cell pellet was then immersed again in 70 μL / well of ice-cold staining buffer (BSA). Washed. 3 μg / mL AF488 anti-mouse IgG(H+L) specific goat F(ab') 2 (Jackson ImmunoResearch #115-546-062) Add 40 μL / well to the cell pellet while gently mixing, and store the cells on ice in the dark for 30 days. Incubate for 1 minute. Wash the cells as described above and add 40 μL / well of BD Cyt Fixation on ice for 20 minutes using ofix (BD Pharmingen #554655) The fixed cells were washed as described above, and the cell pellet was re-suspended in 20 μL of staining buffer. The cells were turbid and analyzed using an iQue PLUS VBR flow cytometer. And it gates to the singlet population and inhibits the BL1-H (AF488) channel. Body bonding was analyzed. All liquid processing was performed using the Agilent Bravo system. , 384-well plate aspiration using BioTek 405 Select plate washer went.

[0119] As shown in Figure 3, in the binding of scFv-transfected SupT1 cells, numerous Candidate mAbs were screened. A002B39 (labeled as A002M39) is Su Specificity for both pT1-KL2B413 HL and SupT1-KL2B413 LH cells. It showed binding, but did not show specific binding to the negative control SupT1-CD9B337-HL. It was.

[0120] Example 4: KLK2 CAR KL2B413-LH(T) on CAR+SupT1 cells Characterization of detection antibodies against protein ID#A002B39 Detection of KLK2 CAR (KL2B413-LH) expressed on NK cells and T cells. Identify antibodies from a panel of proteins derived from phage display screening. As discussed in the above example, regarding the binding to recombinant CAR proteins first... We tested the protein and scaled up the potential binder. We purified the protein, SupT1 cells expressing KL2B413-LH as determined by flow cytometry. Dose-dependent binding to the Fc-KL2B413-LH fusion protein was tested. Competitive binding experiments and the lack of binding to parental SupT1 cells indicate that binding is specific to CAR. It was found that, after selecting the best binder, it was used as a CAR detection reagent. The antibody was recombinant and directly conjugated to phycoerythrin ("PE"). The antibody was then purified. The PE:antibody ratio was set to 1:1, enabling receptor counting tests (expressed on the cell surface). (The number of CARs).

[0121] purification Load the cell culture supernatant into a MabSelect column and set the pH to 100 mM sodium acetate. Elute with a low pH buffer such as 3.0, followed by elution using a Sephadex G-25 column. The buffer was then replaced with 1×SSC, 8.5% sucrose, pH 7.0. (The sample contains protein.) The fraction was collected. After purification, it was analyzed by SDS-PAGE, SEC-HPLC, and LC-MS. We performed protein quality control using [this method].

[0122] Phycoerythrin labeling Add 1 μL of Modifier reagent for every 10 μL of antibody to be labeled, and mix gently. The antibody sample (with modifier reagent added) was freeze-dried into PE (Expede). Transfer directly to (catalog number 703-0015) using a pipette, gently resuspend, and leave at room temperature. Incubated in the dark at 20-25°C for 1 hour. Add 1 μL of Quencher reagent. Add 10 μL of the antibody used to each sample and incubate for 30 minutes.

[0123] After labeling, the PE antibody conjugate was purified on a size exclusion chromatography column. The sample was collected and analyzed by SEC-HPLC. It contained only one antibody with one PE molecule. The fractions were pooled together and concentrated as needed. The final product was analyzed by SEC-HPLC. did.

[0124] Characteristics of KLK2 CAR (KL2B413-LH) anti-idiotype antibody A002B39 Sexual evaluation SupT1 cells expressing KL2B413LH were compared to parental CAR-SupT1 cells. Cells (100,000 cells / well) LIVE / DEAD Fixable Nea Stained with r-IR viability dye (Life Technologies L10119), The A002B39 was incubated on ice for 30 minutes while increasing its concentration. Afterward, the sample was stained with BSA staining buffer (BD Biosciences #554657). Wash and use PE goat anti-mouse IgG polyclonal antibody (Biolegend #4053) Staining was performed with 07) to detect bound antibodies on live CAR+SupT1 cells. Incubation After cleaning, washing, and fixing (Cytofix, BD Biosciences), the sample is prepared. 10-color FACSCanto II (BD Biosciences) Flow Cytometry The data was obtained using a ter. Analysis was performed using FlowJo to determine the PE central fluorescence of surviving SupT1 cells. The intensity is plotted in Figure 4. As shown in Figure 4, KL2B413-LH SupT1 cells Dose-dependent binding of A002B39 to was observed. Binding occurred in CAR-SupT1 parent cells. It was not detected.

[0125] A002B39 was conjugated to R-PE as described above, and 10 μg / mL of KL2 KL2B413 in the presence or absence of the B413-LH scFv Fc fusion protein - Tested for binding to LH SupT1, and A002B to KL2B413 CAR The specificity of the 39-binding was evaluated. KL2B413-LH SupT1 cells (100,000 Cells / wells) LIVE / DEAD Fixable Near-IR survival dye (L Stained with ife Technologies L10119, and PE-A002B39 and The concentration of 10 μg / mL KL2B413-LH scFv Fc was increased on ice for 3 Incubated for 0 minutes. After incubation, washing, and fixation, the sample was colored 10 colors. Acquired on a FACSCanto II flow cytometer. FlowJo analysis software. Using WEAR, the analysis was performed, and the PE central fluorescence intensity of surviving SupT1 cells is plotted in Figure 5. As shown in Figure 5, PE-A002B3 is injected into KL2B413-LH SupT1 cells. The dose-dependent binding of 9 is specific to the KL2B413-LH CAR, at 10 μg / mL It can be blocked by KL2B413-LH scFv-Fc.

[0126] To reflect the detection of CAR cells in clinical samples, KL2B413LH SupT1 cells The cells were spiked into whole blood. As shown in Figure 3 below, KL2 by PE-A002B39 B413-LH CAR detection was not inhibited in the presence of whole blood. KL2B413L H SupT1 cells LIVE / DEAD Fixable Near-IR viability dye Stain with [a specific solution], wash, suspend in staining buffer or fresh whole blood (n=2), and measure 100,000 cells. KL2B413LH SupT1 cells were seeded at 50 μL / well, and PE-A002B The solution was incubated on ice for 30 minutes while gradually increasing the concentration of solution 39. Incubation, washing. After fixation, the sample is acquired using a 10-color FACSCanto II flow cytometer. The analysis was performed using FlowJo software, and the central PE of viable SupT1 cells was analyzed. The fluorescence intensity is plotted in Figure 6. As shown in Figure 6, KL2B4 spiked in whole blood. Detection of PE-A002B39 in 13-LH-SupT1 cells.

[0127] By comparing multiple lots of PE-A002B39, we can distinguish between batches of KL2B413LH-S The difference in binding to upT1 cells was evaluated. KL2B413-LH SupT1 cells (200 ,000 cells / well) LIVE / DEAD Fixable Near-IR viability Stain with dye and incubate on ice for 30 minutes while increasing the concentration of PE-A002B39. After incubation, washing, and fixation, the sample was processed using 10-color FACSCanto. Data was acquired on a flow cytometer. Analysis was performed using FlowJo analysis software. The PE central fluorescence intensity of surviving SupT1 cells is plotted in Figure 7. Two different lots of PE-A002B39 are KL2B413-LH-SupT1. In contrast, it has a similar coupling profile.

[0128] array

[0129] [Table 3-1]

[0130] [Table 3-2]

[0131] Sequence ID 37: Heavy chain (amino acids) of A002B39 containing muIgG2a EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCARVQWGLDYWGQGTLVTVSSAKT TAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNS GSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN VAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSV FIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVN NVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFK CKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTK KQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDS DGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKS FSRTPGK

[0132] Heavy chain (DNA) of A002B39 having SEQ ID NO: 38: muIgG2a GAAGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAA CCGGGCAGCAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCG GCACCTTTAGCAGCTATGCGATTAGCTGGGTGCGCCAGGC GCCGGGCCAGGGCCTGGAATGGATGGGCGGCATTATTCCG ATTTTTGGCACCGCGAACTATGCGCAGAAATTTCAGGGCC GCGTGACCATTACCGCGGATGAAAGCACCAGCACCGCGTA TATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTG TATTATTGCGCGCGCGTGCAGTGGGGCTTGGACTATTGGG GCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAAAACAAC AGCACCAAGTGTCTATCCACTGGCCCCTGTGTGTGGAGAT ACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGG GTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGG ATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTG CAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAA CCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGT GGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATT GAGCCCAGAGGGCCCACAATCAAGCCCTGTCCTCCATGCA AATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTT CATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCC CTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCG AGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAA CGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAG GATTACAACAGTACTCTCCGGGTGGTCAGTGCCCTCCCCA TCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATG CAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGA ACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGG TATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAA ACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCT GAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAG AGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGA TGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAG AAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGG TCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTT CTCCCGGACTCCGGGTAAA

[0133] SEQ ID NO: 39: Light chain (amino acid) of A002B39 with muKappa EIVLTQSPATLSLSPGERATLSCRASQSVDSALAWYQQK PGQAPRLLIYGASNRATGIPARFSGSGSGTDFTLTISSLE PEDFAVYYCQQRFNWPITFGQGTKVEIKRADAAPTVSIFP PSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGV LNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHK TSTSPIVKSFNRNEC

[0134] SEQ ID NO: 40: Light chain (DNA) of A002B39 having muKappa GAAATTGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTG AGCCCGGGCGAACGCGCGACCCTTAGCTGCCGTGCAAGTC AGAGTGTGGACAGCGCGCTGGCGTGGTATCAGCAGAAACC GGGCCAGGCGCCGCGCCTGCTGATTTATGGTGCGAGCAAC CGCGCGACCGGCATTCCGGCGCGCTTTAGCGGCAGCGGCA GCGGCACCGATTTTACCCTGACCATTAGCAGCCTGGAACC GGAAGATTTTGCGGTGTATTATTGCCAGCAGCGTTTCAAC TGGCCGATCACCTTTGGCCAGGGCACCAAAGTGGAAATTA AACGGGCTGATGCTGCACCGACTGTGTCCATCTTCCCACC ATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTG TGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCA AGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCT GAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTAC AGCATGAGCAGCACCCTACAGGTTGACCAAGGACGAGTATG AACGACATAACAGCTATAACCTGTGAGGCCACTCACAAGAC ATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAG TGT

[0135] Sequence ID 41: KL2B413 VH EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQ APGKGLEWVANIKQDGSERYYVDSVKGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARDQNYDILTGHYGMDVWGQGTT VTVSS

[0136] Sequence ID 42: KL2B413 VL EIVLTQSPSFLSASVGDRVTITCRASQGISSYLSWYQQK PGKAPKLLIYATSTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQLNSYPRTFGQGTKVEIK

[0137] Sequence ID 43-CAR1(KL2B413_HL;pBD000091628) EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMTWVRQ APGKGLEWVANIKQDGSERYYVDSVKGRFTISRDNAKNSL YLQMNSLRAEDTAVYYCARDQNYDILTGHYGMDVWGQGTT VTVSSGGSEGKSSGSGSESKSTGGSEIVLTQSPSFLSASV GDRVTITCRASQGISSYLSWYQQKPGKAPKLLIYATSTLQ SGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP RTFGQGTKVEIKTSTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0138] Sequence ID 44-CAR2(KL2B413_LH;pBD000091623) EIVLTQSPSFLSASVGDRVTITCRASQGISSYLSWYQQK PGKAPKLLIYATSTLQSGVPSRFSGSGSGTEFTLTISSLQ PEDFATYYCQQLNSYPRTFGQGTKVEIKGGSEGKSSGSGS ESKSTGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSY WMTWVRQAPGKGLEWVANIKQDGSERYYVDSVKGRFTISR DNAKNSLYLQMNSLRAEDTAVYYCARDQNYDILTGHYGMD VWGQGTTVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLY CKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG CELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0139] Heavy chain variable domain (amino acid) of SEQ ID NO: 45:A002B39 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQ APGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTA YMELSSLRSEDTAVYYCARVQWGLDYWGQGTLVTVSS

[0140] Light chain variable domain (amino acid) of sequence number 46:A002B39 EIVLTQSPATLSLSPGERATLSCRASQSVDSALAWYQQK PGQAPRLLIYGASNRATGIPARFSGSGSGTDFTLTISSLE PEDFAVYYCQQRFNWPITFGQGTKVEIK

[0141] Sequence ID 47-Human kallikrein-2 sequence (signal sequence: amino acids 1-18) MWDLVLSIALSVGCTGAVPLIQSRIVGGWECEKHSQPWQ VAVYSHGWAHCGGVLVHPQWVLTAAHCLKKNSQVWLGRHN LFEPEDTGQRVPVSHSFPHPLYNMSLLKHQSLRPDEDSSH DLMLLRLSEPAKITDVVKVLGLPTQEPALGTTCYASGWGS IEPEEFLRPRSLQCVSLHLLSNDMCARAYSEKVTEFMLCA GLWTGGKDTCGGDSGGPLVCNGVLQGITSWGPEPCALPEK PAVYTKVVHYRKWIKDTIAANP

[0142] Built-in by reference All publications and patent applications referred to herein are subject to change without notice. To the same extent as when it is specifically and individually indicated that the request is incorporated by reference, The entirety of this is incorporated herein.

[0143] While we have discussed specific embodiments of the disclosure of the subject matter, the above specification is illustrative and not limited. Not applicable. Considering this specification and the following claims, many modifications of this disclosure are applicable. This will become clear to the industry. The claims, along with the full scope of the equivalent, and such The full scope of this disclosure should be determined by referring to the specification along with the modifications.

Claims

1. An anti-idiotype antibody or its antigen that specifically binds to a target antibody containing KL2B413. joining part.

2. The target antibody or its antigen-binding portion has the amino acid sequence of SEQ ID NO:

41. The claim according to claim 1, comprising an in and a VL domain having the amino acid sequence of SEQ ID NO:

42. Anti-idiotype antibody or antigen-binding moiety.

3. An anti-idiotype antibody that specifically binds to KL2B413, or its antigen-binding portion. VH CDR1 having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 7, VH CDR2 and a compound having an amino acid sequence selected from the group consisting of row numbers 11 to 14. VH CDR3 containing an amino acid sequence selected from the group consisting of row numbers 19 to 20 An amino acid containing a heavy chain variable (VH) domain, selected from the group consisting of SEQ ID NOs: 25-26. Amino acids selected from the group consisting of VL CDR1 and SEQ ID NOs: 29-30, which have an acid sequence. amino acids selected from the group consisting of VL CDR2 having a sequence and SEQ ID NOs: 33-34 The anti-idiot further comprises a light chain variable (VL) domain containing a VL CDR3 sequence, Ip antibody or its antigen-binding portion.

4. The VH domain has at least 90% sequence identity with respect to sequence number 45. It has a mino acid sequence, and the VL domain has at least 90% of the sequence relative to SEQ ID NO:

46. An anti-idiotype antibody or antigen according to claim 1, having an identical amino acid sequence. joining part.

5. A heavy chain containing an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

37. It includes 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 1, further comprising a light chain.

6. The VH domain has the amino acid sequence of Sequence ID No. 45, and the VL domain has the sequence The anti-idiotype antibody or antigen bindance according to claim 1, having amino acid sequence number 46. portion.

7. It contains a heavy chain containing the amino acid sequence of SEQ ID NO: 37, and a light chain containing the amino acid sequence of SEQ ID NO:

39. The anti-idiotype antibody or antigen-binding moiety according to claim 1, further comprising a chain.

8. The antigen-binding portion is Fab, F(ab') ２ A claim selected from, or scFv The anti-idiotype antibody or antigen-binding moiety described in 1.

9. The anti-idiotype antibody according to claim 1, wherein the antibody is a monoclonal antibody or Antigen-binding portion.

10. The anti-idiotype antibody or antigen binding according to claim 1, wherein the antibody is a chimeric antibody. portion.

11. The antibody comprises a mouse IgG2a framework, according to claim 10. Type antibody or antigen-binding portion.

12. The anti-idiotype antibody or antigen antibody according to claim 1, wherein the antibody is a fully human antibody. Joint part.

13. The heavy chain, the light chain, or the anti-idiotype antibody or antigen-binding moiety described in claim 1. Nucleic acids that encode both.

14. An anti-idiotype antibody that specifically binds to KL2B413, or the antigen-binding portion thereof. A nucleic acid encoding a heavy chain, the light chain, or both, a) Nucleotide sequence of Sequence ID No. 38, b) Nucleotide sequence of sequence number 40, c) A nucleic acid comprising both a) and b).

15. A vector comprising the nucleic acid described in claim 14.

16. The vector according to claim 15, wherein the vector is an expression vector.

17. A host cell comprising the vector according to claim 16.

18. The host cell according to claim 17, wherein the cell is a mammalian cell.

19. It produces an anti-idiotype antibody or its antigen-binding moiety that specifically binds to KL2B413. A method comprising expressing the antibody or the antigen-binding portion under conditions that cause the antibody or The claim includes a nucleotide sequence encoding the heavy chain and the light chain of the antigen-binding portion. Culturing the host cells described in 17, and obtaining the antibody or antigen-binding portion from the culture. A method comprising isolating the substance.

20. A method for detecting KL2B413 in a biological sample, wherein (a) a biological sample is prepared (b) the biological sample is subjected to the anti-idiotitis described in any one of claims 1 to 3. (c) Contacting the antibody or antigen binding portion, and the anti-idiotype antibody or the A method comprising detecting an antigen-binding site.

21. To detect the expression of chimeric antigen receptors (CARs) including KL2B413 in biological samples. A method comprising (a) preparing a biological sample, and (b) the biological sample, according to claim 1 to Contacting the anti-idiotype antibody or antigen-binding portion described in any one of item 3, (c) The anti-idiotype antibody or the antigen-binding portion is detected, thereby the CA A method comprising detecting the expression of R.

22. The method according to claim 20, wherein the antibody includes a detectable label.

23. Before detecting the anti-idiotype antibody or the antigen-binding moiety, Claim 20 further comprises contacting the antibody or the antigen-binding portion with a detectable label. Methods used.

24. The method according to claim 20, wherein the biological sample is blood, serum, or urine.

25. KL2B413 is located within the antigen-binding domain of the extracellular portion of the chimeric antigen receptor (CAR). A certain anti-idiotype antibody or antigen-binding moiety according to claim 1.

26. KL2B413 is scFv, and the anti-idiotype antibody or the antigen-binding portion The anti-I according to claim 25, which specifically binds to the epitope in the scFv of the CAR. Diotype antibody or antigen-binding portion.

27. The anti-idiotype antibody according to claim 25, wherein KL2B413 binds to KLK2 or This is the antigen-binding site.

28. The antibody or antigen-binding portion interferes with other KLK2 antibodies or other KLK2-binding CARs. The anti-idiotype antibody or antigen-binding moiety according to claim 25, which does not react with differential reactions.

29. 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 claim 25.

30. A kit for detecting KL2B413 in a biological sample, wherein (a) the description in claim 1 (b) an anti-idiotype antibody or antigen-binding moiety of the above, and (b) the anti-idiotype antibody or the above A kit including instructions for detecting antigen-binding sites.

31. Anti-idiotype antibodies or for use in the detection of KL2B413 in biological samples The antigen-binding portion is the antigen-binding portion, and the detection involves (a) preparing a biological sample and (b) the biological (c) Contacting the body sample with the anti-idiotype antibody or the antigen-binding portion, and The method described in claim 1, comprising detecting the anti-idiotype antibody or the antigen-binding portion. The anti-idiotype antibody or antigen-binding portion.

32. A method for purifying KL2B413 from a sample, wherein (a) a biological sample containing KL2B413 (b) Prepare the material and (b) the biological sample with the anti-idiotype antibody described in claim 1 or (c) bringing the antigen-binding portion into contact with the antigen-binding portion, and (c) the anti-idiotype antibody or the antigen binding portion. A method comprising capturing a portion and thereby purifying KL2B413.

33. A method for selecting CAR-T cells from a cell population, wherein (a) a living cell population containing CAR-T cells (b) Prepare a biological sample, and (b) the biological sample is subjected to the anti-idiotype anti- (c) bringing the body or antigen-binding portion into contact with the body, and (c) the anti-idiotype antibody or the antigen A method comprising capturing a binding site and thereby selecting CAR-T cells.

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