Anti-idiotype antibodies and uses thereof

Abstract

The invention relates to the technical field of immunology, in particular to an anti-idiotype antigen binding molecule and application thereof. According to the invention, the monoclonal antibody 8693-1G12 for the CEA-ScFv is obtained. Experimental results show that the anti-idiotypic antibody targeting CEA ScFv is high in specificity and affinity and has the capability of being combined with molecules containing CEA binding molecules.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and particularly to anti-idiotypic antibodies and their applications. Background Art

[0002] Engineered immune cells are cells obtained by modifying and transforming immune cells such as T cells, NK cells, macrophages, and various immune cells derived from iPSCs. For example, chimeric antigen receptor T cells (CAR-T cells) are formed by coupling the antigen-binding portion of an antibody that can recognize a certain tumor antigen with the intracellular portion of CD3-ζ chain or FcεRIγ in vitro to form a chimeric protein, and then transfecting the patient's T cells by gene transduction method to make them express chimeric antigen receptor (CAR). The detection of engineered immune cells mainly focuses on the modified parts. For example, for cells such as CAR-T and CAR-NK, the CAR expressed on the surface of T and NK cells is detected. Detecting CAR-positive cells of engineered immune cells is a key step in the quality control of CAR-T drugs, and is also an important link in the treatment dose control, process monitoring, and auxiliary diagnosis of clinical patients. Currently, the commonly used detection methods mainly have two types: one is to detect CAR gene positivity, and the other is to detect CAR protein positivity.

[0003] Detecting CAR gene positivity is to detect the CAR gene integrated on the genome of immune cells and the copy number of the CAR gene by qPCR. However, not all integrated genes can be normally expressed in immune cells. The unexpressed CAR gene will cause false positives in this detection method, reducing the accuracy of the qPCR method for detecting CAR positivity.

[0004] To detect CAR protein positivity, ProteinL is currently the most commonly used. ProteinL specifically binds to antibodies containing κ light chain. Although it has certain generality, it only binds to human κI, III, IV (not binding to VκII subtype and λ light chain antibodies) and the κI light chain subtype of mice, is not applicable to other subtypes, and has different affinities for each subtype. Therefore, although ProteinL can detect CARs with different targets, it is easily affected by residual immunoglobulins in serum or culture medium, and is prone to serious non-specific background in clinical sample detection. Moreover, different subtypes of antibodies used for CAR will lead to deviation in positive rates.

[0005] Therefore, there is an urgent need to develop a detection reagent and method for CAR-positive cells with high detection sensitivity and accuracy to meet the requirements of quality control of engineered immune cell drugs, clinical treatment, and auxiliary diagnosis. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide anti-idiotypic antibodies and their applications.

[0007] The antibody provided by the present invention is a monoclonal antibody against the ScFv targeting CEA. That is, the present invention provides a molecule that can bind to a molecule containing a CEA-binding molecule, which is an anti-antibody and an anti-idiotypic antibody against the ScFv targeting CEA.

[0008] The heavy chain of the antigen-binding molecule provided by the present invention contains three CDR regions, and the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO: 1, 2, or 3, or a sequence having at least 80% sequence homology therewith.

[0009] The light chain of the antigen-binding molecule provided by the present invention contains three CDR regions, and the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO: 4, 5, or 6, or a sequence having at least 80% sequence homology therewith.

[0010] In the present invention, the sequence having at least 80% sequence homology is an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids on the basis of the original sequence. The homology is 80%, 90%, 95%, 96%, 97%, 98%, or 99%. The plurality is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0011] In the present invention, the amino acid sequences of the three CDR regions of the heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 1, 2, and 3.

[0012] In the present invention, the amino acid sequences of the three CDR regions of the light chain respectively have the amino acid sequences shown in SEQ ID NO: 4, 5, and 6.

[0013] In some embodiments:

[0014] The amino acid sequence of CDR1 of the heavy chain is GFTFSNYW (SEQ ID NO: 1).

[0015] The amino acid sequence of CDR2 of the heavy chain is IRLKSNNYTT (SEQ ID NO: 2).

[0016] The amino acid sequence of CDR3 of the heavy chain is TRGPINNDYGGYGMDY (SEQ ID NO: 3).

[0017] The amino acid sequence of CDR1 of the light chain is ESVDNYGISF (SEQ ID NO: 4).

[0018] The amino acid sequence of CDR2 of the light chain is AAS (SEQ ID NO: 5).

[0019] The amino acid sequence of CDR3 of the light chain is QQNKEVPHT (SEQ ID NO: 6).

[0020] In some embodiments,

[0021] The amino acid sequence of FR1 of the heavy chain is EVKIQESGGGLVQPGGSMKLSCVTS (SEQ ID NO: 9).

[0022] The amino acid sequence of FR2 of the heavy chain is MNWVRQSPVKGLEWVAE (SEQ ID NO: 10).

[0023] The amino acid sequence of FR3 of the heavy chain is HYAESVKGRFTISRDDSKISVYLQMNNLRAEDTGIYYC (SEQ ID NO: 11).

[0024] The amino acid sequence of FR4 of the heavy chain is WGQGTSVTVSS (SEQ ID NO: 12).

[0025] In some embodiments,

[0026] The amino acid sequence of FR1 of the light chain is DIVMTQSPASLAVSLGQRATISCRAS (SEQ ID NO: 13).

[0027] The amino acid sequence of FR2 of the light chain is MNWFQQKPGQPPKLLIY (SEQ ID NO: 14).

[0028] The amino acid sequence of FR3 of the light chain is NQRSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYF C (SEQID NO: 15).

[0029] The amino acid sequence of FR4 of the light chain is FGGGTKLE (SEQ ID NO: 16).

[0030] In some specific embodiments, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, or a sequence having at least 80% sequence homology thereto.

[0031] In some specific embodiments, the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8, or a sequence having at least 80% sequence homology thereto.

[0032] More specifically, the amino acid sequence of the heavy chain variable region of the antigen-binding molecule of the present invention is as shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:8.

[0033] In some embodiments, the antigen-binding molecule of the present invention further comprises a constant region. The constant region of the heavy chain is any one of IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM or IgA; the constant region of the light chain is of the κ type or the λ type. In a specific embodiment, the constant region of the heavy chain of the antigen-binding molecule is IgG1, and the constant region of the light chain is of the κ type.

[0034] In other embodiments, the light chain variable region and the heavy chain variable region of the present invention are combined to form a single-chain antibody, which sequentially comprises, from the N-terminus to the C-terminus: light chain variable region - linker - heavy chain variable region or heavy chain variable region - linker - light chain variable region;

[0035] The linker is selected from: EGSTSGSGKPGSGEGSTKGE or (GGGGS)n, where n = 1 to 4. In some specific embodiments, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO:17.

[0036] In some embodiments, some single-chain antibodies may comprise the CDRs of SEQ ID NOs: 1-6 of the present invention. The single-chain antibody sequentially comprises, from the N-terminus to the C-terminus: light chain variable region - linker - heavy chain variable region or heavy chain variable region - linker - light chain variable region; the linker is selected from: EGSTSGSGKPGSGEGSTKGE or (GGGGS)n, where n = 1-4.

[0037] Through immunization, extensive screening and subcloning, the present invention obtained a monoclonal antibody 8693-1G12 against CEA-ScFv. The experimental results show that this monoclonal antibody targeting the CEA-specific ScFv has high specificity, strong affinity, and the ability to bind molecules containing the CEA-binding molecule.

[0038] Furthermore, the present invention also provides a nucleic acid encoding the antigen-binding molecule as described above.

[0039] The nucleic acid encoding the antigen-binding molecule of the present invention includes a nucleic acid encoding the light chain variable region, a nucleic acid encoding the heavy chain variable region, and also includes a nucleic acid encoding the single-chain antibody as described above.

[0040] Even further, the present invention also provides an expression vector comprising the nucleic acid encoding the antigen-binding molecule as described above.

[0041] Further, a host contains the expression vector as described above, or the nucleic acid as described above is integrated into its genome.

[0042] Further, the present invention also provides a method for preparing the antigen-binding molecule as described above, including: culturing the host cell as described above and inducing the expression of the antigen-binding molecule.

[0043] Further, the present invention also provides a conjugate, which is the antigen-binding molecule as described above linked with a chemical label or a biological label.

[0044] In the present invention, the chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope and / or a colloidal indicator. The biological label is biotin, avidin or an enzyme label.

[0045] In some embodiments, the enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, and β-galactosidase-anti-β-galactosidase conjugate.

[0046] In some embodiments, the fluorescent indicator is selected from one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red or PerCP dye.

[0047] In some embodiments, the chemiluminescent indicator is selected from one or more of acridinium ester, acridinium sulfonamide and its derivatives, luminol, isoluminol, isoluminol isothiocyanate and its derivatives, N-(4-aminobutyl)-N-ethylisoluminol, 4,5-diaminophthalohydrazide or aminobutylethylphthalohydrazide.

[0048] In some embodiments, the isotope is selected from one or more of 125I, 131I, 124I, 3H, 14C, 111In, 89Zr or 32P.

[0049] In some embodiments, the colloidal indicator is selected from one or more of colloidal gold, colloidal carbon or colloidal selenium.

[0050] Further, the present invention also provides a conjugate prepared by coupling the antigen-binding molecule as described above or the conjugate as described above with a medium.

[0051] The medium is an enzyme-linked immunosorbent assay (ELISA) plate, glass, magnetic beads, latex microspheres, or a polystyrene plate. In the present invention, the surface of the medium is modified with amino groups or carboxyl groups, and in the conjugate, the antigen-binding molecule or conjugate is linked to the medium through an amide bond.

[0052] The monoclonal antibody provided by the present invention is used as a detection reagent for the detection of CEAScFv by labeling with biotin, HRP, or various fluorescences. That is, the monoclonal antibody provided by the present invention can be used for the detection of CAR-positive cells during the in vitro preparation of engineered immune cells targeting CEA and during clinical detection, which has important guiding significance for the quality of the preparation of engineered immune cells and clinical applications. In some embodiments, the engineered immune cells are CAR-T cells, CAR-NK cells, etc. Furthermore, the present invention also provides the use of the antigen-binding molecule, the conjugate, and / or the conjugate as described above in the preparation of reagents for the quality control of engineered cell therapy drugs.

[0053] In some embodiments, the present invention also provides the use of the antigen-binding molecule, the conjugate, and / or the conjugate as described above in the preparation of reagents for the quality control of CAR-T drugs.

[0054] Furthermore, the present invention also provides a kit, which includes the antigen-binding molecule, the conjugate, and / or the conjugate as described above.

[0055] The kit of the present invention also includes a buffer; the buffer is selected from: PBS buffer, HEPES buffer, MES buffer, MOPS buffer, Tris buffer, citrate buffer, carbonate buffer, glycine buffer, or phosphate buffer.

[0056] The buffer of the present invention also includes 0-10% FBS.

[0057] In some embodiments, the kit of the present invention is applicable to immunological detection methods, including but not limited to: flow cytometry detection, enzyme-linked immunosorbent assay (ELISA) detection, immunofluorescence detection, and immunoelectrophoresis detection.

[0058] In some specific embodiments, the kit of the present invention includes the antigen-binding molecule, the conjugate, and / or the conjugate as described above, and also includes PBS buffer and PBS buffer containing 2% FBS.

[0059] Furthermore, the method for quality control of the engineered cell drug of the present invention includes: detecting the cells with the kit as described above to determine whether CAR is positive.

[0060] In some embodiments, the engineered cell drug quality control method described in the present invention refers to the CAR-T drug quality control method, which includes: detecting cells with the kit as described above to determine whether the CAR is positive.

[0061] The quality control method described in the present invention is used in the in vitro preparation process and clinical use process of engineered immune cells, so as to improve the effectiveness of clinical treatment. In some embodiments, the engineered immune cells are CAR-T cells.

[0062] The present invention also provides a method for judging the efficacy of an engineered immune cell drug targeting CEA, which detects a sample with the kit, the antibody or the conjugate, and determines the in vivo survival of the engineered immune cell targeting CEA according to whether the CAR is positive. The sample is blood or tumor tissue.

[0063] The present invention prepares an anti-idiotypic antibody targeting CEAScFv using the ScFv targeting CEA as an antigen. After immunization, extensive screening and subcloning, the monoclonal antibody 8693-1G12 against CEA-ScFv is obtained. Experimental results show that this monoclonal antibody targeting the ScFv with the target of CEA has high specificity, strong affinity, and the ability to bind molecules containing CEA-binding molecules at the same time. Description of the Drawings

[0064] Figure 1 Showing different monoclonal titers;

[0065] Figure 2 Showing the cell binding characteristics of the monoclonal antibody; where 2-A is the result of 1G12 recognizing the extracellular recognition region in CEACAR2; 2-B is the result of 1G12 recognizing the extracellular recognition region in CD19 CAR; 2-C is the result of 1G12 recognizing the extracellular recognition region in PSCACAR; 2-D is the result of 1G12 recognizing the extracellular recognition region in CD70 CAR; 2-E is the result of 1G12 recognizing control T cells; 2-F is the result of PL recognizing the extracellular recognition region in CEACAR2;

[0066] Figure 3 Showing the detection of the monoclonal antibody on the target tissue. Detailed Embodiments

[0067] The present invention provides anti-idiotypic antigen-binding molecules and their applications. Those skilled in the art can draw on the content of this article and appropriately improve process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate variations and combinations to the methods and applications herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0068] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as understood by those of ordinary skill in the art. Regarding the definitions and terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 common L-amino acids.

[0069] The term "antigen-binding molecule" refers to a molecule that specifically binds to an antigen. Exemplarily, antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics. An "antibody mimetic" refers to an organic compound or binding domain that can specifically bind to an antigen but is not related to the antibody structure. Exemplarily, antibody mimetics include, but are not limited to, affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers, or Kunitz-type domain peptides.

[0070] The term "antibody" refers to a polypeptide or polypeptide combination that contains sufficient sequences from the variable region of an immunoglobulin heavy chain and / or sufficient sequences from the variable region of an immunoglobulin light chain to be able to specifically bind to an antigen.

[0071] The term "antibody" includes antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins containing the antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be labeled and detected. For example, they can be labeled and detected by radioactive isotopes, enzymes that can produce detectable substances, fluorescent proteins, biotin, etc. Antibodies can also be bound to solid-phase carriers, including but not limited to polystyrene plates or beads, etc.

[0072] The "CDR region" or "CDR" refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy-chain CDRs and three light-chain CDRs. As the context requires, the terms CDR or CDRs as used herein are intended to indicate one of these regions, or several or even all of these regions, which regions contain most of the amino acid residues responsible for binding through the affinity of the antibody for the antigen or its epitope recognized.

[0073] The term "antibody" can be derived from any animal, including but not limited to humans and non-human animals, which non-human animals can be selected from primates, mammals, rodents, and vertebrates, such as camelids, llamas, rheas, alpacas, sheep, rabbits, mice, rats, or chondrichthyans (such as sharks).

[0074] The terms "antigen-binding fragment" and "antibody fragment" are used interchangeably herein and do not possess the entire structure of a full antibody, but only contain a portion or a variant of a portion of a full antibody that has the ability to bind an antigen. Exemplarily, the "antigen-binding fragment" or "antibody fragment" herein includes but is not limited to Fab, F(ab’)2, Fab’, Fab’-SH, Fd, Fv, scFv, diabody, and single-domain antibody.

[0075] The term "heavy-chain constant region" refers to the carboxyl-terminal portion of an antibody heavy chain that does not directly participate in the binding of the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, and has a more conserved amino acid sequence relative to the variable domain of the antibody. The "heavy-chain constant region" can be selected from: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. The "heavy-chain constant region" includes "full-length heavy-chain constant region" and "heavy-chain constant region fragment", the former having a structure substantially similar to the native antibody constant region, while the latter only includes "a part of the full-length heavy-chain constant region". Exemplarily, a typical "full-length antibody heavy-chain constant region" consists of CH1 domain - hinge region - CH2 domain - CH3 domain; when the antibody is IgE, it also includes the CH4 domain; when the antibody is a heavy-chain antibody, it does not include the CH1 domain. Exemplarily, a typical "heavy-chain constant region fragment" can be selected from Fc or CH3 domain.

[0076] The term "light chain constant region" refers to the carboxyl-terminal portion of an antibody light chain that does not directly participate in the binding of the antibody to an antigen, and the light chain constant region may be selected from a constant kappa domain or a constant lambda domain.

[0077] The term "identity" can be calculated as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. The molecule is identical at that position when the position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.

[0078] As used herein, the term "chimeric antigen receptor (CAR)" refers to an artificial cell surface receptor that has been engineered to be expressed on an immune effector cell and specifically binds an antigen, and that comprises at least (1) an extracellular antigen-binding domain, such as the variable heavy or light chain of an antibody, (2) a transmembrane domain that anchors the CAR into the immune effector cell, and (3) an intracellular signaling domain. The CAR is capable of redirecting T cells and other immune effector cells to a selected target, such as a cancer cell, in a non-MHC-restricted manner using the extracellular antigen-binding domain.

[0079] As used herein, the term "nucleic acid" includes any compound and / or substance that comprises a polymer containing nucleotides. Each nucleotide is composed of a base, in particular a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Generally, a nucleic acid molecule is described by the sequence of its bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually represented as 5' to 3'. As used herein, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including, for example, complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and hybrid polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. In addition, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Moreover, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleobases with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules that are suitable as vectors for the direct expression of the antibodies of the present invention in vitro and / or in vivo, e.g., in a host or patient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoded molecule, such that the mRNA can be injected into a subject to produce an antibody in vivo (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101 823B1). As used herein, an "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location different from its natural chromosomal location.

[0080] As used herein, the term "vector" refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors".

[0081] As used herein, the term "host cell" refers to a cell into which exogenous nucleic acid has been introduced, including progeny of such a cell. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical to the parental cell in terms of nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as selected or screened in the initially transformed cell are included herein.

[0082] As used herein, the term "engineered immune cell" refers to an immune cell that has been engineered, which can change cell characteristics through genetic modification / engineering, or express a target protein on the cell membrane, or secrete a target protein, etc. Immune cells modified by external forces can all be collectively referred to as "engineered immune cells". The "engineered immune cells" herein refer to immune cells such as T cells, NK cells, DC cells, macrophages, and iPSC-derived immune cells that express a fusion protein of ScFv targeting CEA, such as a chimeric antigen receptor (CAR).

[0083] As used herein, the term "anti-idiotypic antibody" refers to a specific antibody induced by using the idiotype of the variable region of an antibody as an epitope. The "anti-idiotypic antibody" as described herein refers to a specific antibody induced by using the idiotype of the variable region of an anti-CEA antibody (ScFv targeting CEA) as an epitope.

[0084] All the materials used in the present invention are common commercially available products and can be purchased in the market.

[0085] The antigen-binding molecule provided by the present invention is a monoclonal antibody against ScFv targeting CEA.

[0086] The variable region of the anti-CEA antibody (ScFv targeting CEA) has a sequence including:

[0087] CDR1 of the heavy chain variable region of ScFv targeting CEA: GFDFTTYW (SEQ ID NO:19)

[0088] CDR2 of the heavy chain variable region of ScFv targeting CEA: IHPDSSTI (SEQ ID NO:20)

[0089] CDR3 of the heavy chain variable region of ScFv targeting CEA: ASLYFGFPWFAY (SEQ ID NO:21)

[0090] CDR1 of the light chain variable region of ScFv targeting CEA: QDVGTS (SEQ ID NO:22)

[0091] CDR2 of the light chain variable region of ScFv targeting CEA: WTS (SEQ ID NO:23)

[0092] CDR3 of the variable region of the light chain of CEA-targeted ScFv: QQYSLYRS (SEQ ID NO:24)

[0093] Variable region of the light chain of CEA-targeted ScFv:

[0094] DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK (SEQ ID NO:25)

[0095] Variable region of the heavy chain of CEA-targeted ScFv:

[0096] EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO:26)

[0097] CEA-targeted ScFv

[0098] DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSS (SEQ ID NO:27)

[0099] Using the above-mentioned CEA-targeting ScFv as an antigen for screening anti-idiotypic antibodies, the obtained anti-idiotypic antibodies can recognize all anti-CEA antibodies containing heavy-chain CDRs: CDR1: GFDFTTYW (SEQ ID NO: 19), CDR2: IHPDSSTI (SEQ ID NO: 20), CDR3: ASLYFGFPWFAY (SEQ ID NO: 21) and light-chain CDRs: CDR1: QDVGTS (SEQ ID NO: 22), CDR2: WTS (SEQ ID NO: 23), CDR3: QQYSLYRS (SEQ ID NO: 24), including but not limited to the above SEQ ID NO: 27. Antibody CDR sequences have multiple coding rules, and common coding rules include: Kabat, IMGT, Chothia, etc. Antibodies containing CDRs under different coding rules are all applicable to the anti-idiotypic antibodies described in this article. In some embodiments, the CDRs of the anti-CEA antibodies may also be heavy-chain CDRs: TYWMS, EIHPDSSTINYAPSLKD, LYFGFPWF, and light-chain CDRs: KASQDVGTSVA, WTSTRHT, QQYSLYRS.

[0100] In some embodiments, the CEA-targeting ScFv used as an antigen may also be: containing the following heavy chain: EVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPS LKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFSASCFGPAYWGQGTPV TVSS, and the following light chain: DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKL LIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIK.

[0101] In some embodiments, the anti-CEA ScFv as an antigen may further be a ScFv comprising the following heavy chain: EVKLLESGGGLVQSGGSLKLSCAASGFDFTTYWMAWVRQAPGKGLEWIGEIHPDSSTINYAPS LKDKFIVSRDNAKNTLYLQMSKVRSEDTALYYCASLYFGFPWFSASCFGPAYWGQGTPV TVSA, and the following light chain: EIQLTQSHKMMSTSVGDRVSITCKASQDVGTSVAWYQQRPGQSPK LLIYWTSTRHTGVPSRFTGSVSGTDFTLTITNVQSEDLADYFCQQYSLYRSFGGGTKLEIK D.

[0102] The following examples are used to further illustrate the present invention, in which engineered immune cells are detected and verified using CAR-T cells as an example:

[0103] Example 1

[0104] I. Preparation method of anti-idiotypic antibody (8693-1G12) against scFv targeting CEA

[0105] 1. Preparation and purification of scFv targeting CEA:

[0106] CHO cells were infected with lentivirus of ScFv against CEA respectively and monoclonal cells were screened to obtain the target cell line. The CDR sequences of the ScFv targeting CEA (a molecule that can bind to CEA, used here as an antigen for screening anti-idiotypic antibody) are as follows: SEQ ID NO.19-24, and the ScFv sequence is as shown in SEQ ID NO.27. The target cell line was cultured in roller bottles and the culture supernatant was harvested. The target protein was preliminarily enriched by NI Sepharose excel (Cytiva), and the antigen molecule was purified.

[0107] 2. Immunize mice with recombinant antigen (ScFv targeting CEA)

[0108] According to the experimental requirements, an appropriate amount of recombinant antigen (ScFv targeting CEA) and adjuvant (QuickAntibody-Mouse5W) were quickly mixed at a ratio of 1:1, and then injected intramuscularly into the calf muscles of mice (50 μL was injected into each calf, and the converted dose was 20 μg / mouse). After 21 days, booster immunization was carried out in the same way. After 35 days, a small amount of tail blood was collected, and the titer was detected by ELISA. Detection method: The recombinant antigen (ScFv targeting CEA) was taken into the coating solution to prepare the working concentration (1 μg / ml), and 100 μl / well was added to a 96-well enzyme-linked immunosorbent assay (ELISA) plate and coated overnight at 2-8 °C. The ELISA plate was taken out and washed 3 times, then blocked with the blocking solution at room temperature for 1 hour. After washing 3 times, the test samples with different dilution gradients were added and incubated at room temperature for 1 hour. After washing 3 times, goat anti-mouse antibody was diluted at a ratio of 1:10000 and added, and incubated at room temperature for 1 hour. After washing 4 times, the substrate solution was added, incubated at room temperature in the dark for 10-30 minutes, and the reaction was terminated by adding the stop solution. The absorbance was measured with an ELISA reader.

[0109] Results: As Figure 1 shown in and Table 1, the highest titer of mouse antiserum could reach 1:102400, indicating that the recombinant antigen prepared in Embodiment 1 could efficiently produce specific antibodies against ScFv targeting CEA in mice, that is, anti-idiotypic antibodies against ScFv targeting CEA.

[0110] Table 1 Detection results of serum titer

[0111]

[0112] Figure 1 In the figure, the abscissa is the dilution factor of the tail blood of immunized mice from different clone sources, and the ordinate is the OD value of the enzyme-linked color development of the binding of ScFv targeting CEA and the target antibody (anti-idiotypic antibody) in the tail blood of immunized mice. It can be seen from the results that 8692 and 8693 have higher titers. Further combined with Table 1, 8693 was finally selected for the next monoclonal antibody screening.

[0113] 3. Screening of mouse B cell monoclonal antibodies

[0114] The spleens of immunized mice were removed and prepared into single-cell suspensions, which were fused with SP2 / 0. The fused cells were plated in a prepared feeder cell plate. After culturing for 7-10 days, the supernatant was taken for ELISA detection to screen out positive clones, and then subcloned more than 3 times to obtain the positive monoclonal cell line 8693-1G12.

[0115] 4. Preparation and purification of monoclonal antibody 8693-1G12

[0116] One week before injecting cells, sterilized paraffin was injected into the abdominal cavity, and 0.5 ml of sterilized paraffin was injected into each mouse for pre-stimulation. The obtained 8693-1G12 monoclonal cell line was expanded in culture, and 6e5 cells were injected into each mouse by intraperitoneal injection. After ascites was produced in the mice, the ascites was taken out for purification. The concentration of the 8693-1G12 finished product protein was measured by the method in the A205 range. After adding a lyoprotectant, it was sub-packed in ampoules and freeze-dried with a freeze dryer (Alpha) to obtain 8693-1G12 freeze-dried powder.

[0117] 5. Subtype identification of mouse 8693-1G12 monoclonal antibody

[0118] The antibodies corresponding to the antibody subtypes were coated on the ELISA plate. After blocking, the monoclonal antibody (cell culture supernatant or purified antibody) was added. Finally, the specific secondary antibody of HRP-labeled rabbit anti-was added respectively, and the substrate was added for color development. The monoclonal anti-subtype corresponded to the developed subtype. The results are shown in Table 2. 8693-1G12 had a higher OD value in the corresponding IgG1 and Igκ secondary antibodies, indicating that the subtype of 8693-1G12 was IgG1 and its light chain subtype was κ chain.

[0119] Table 2 Subtype identification results

[0120]

[0121]

[0122] 6. Labeling of 8693-1G12 monoclonal antibody:

[0123] The 8693-1G12 monoclonal antibody can be conjugated with a chemical label or a biological label to form a conjugate for binding to an scFv targeting CEA, a fusion protein containing the scFv targeting CEA, an engineered cell, etc.

[0124] Using a non-amino buffer, the protein concentration was adjusted within 1-10 mg / ml, and the concentration of AF647-NHS or Bio-NHS was adjusted to 5-10 mg / ml. The dissolved AF647-NHS or Bio-BHS was added to the protein solution to be labeled. A 40KMWCO desalting column was selected, and the solution from the previous step after the reaction was passed through the column to remove the unlabeled AF647-NHS or Bio-NHS. The labeled antibody can be applied to flow cytometry, ELISA, and immunohistochemistry.

[0125] The label can be one or more of the following labels: enzyme, biotin, fluorescent indicator, chemiluminescent indicator, isotope, colloid indicator, latex microsphere, magnetic bead indicator; wherein, the enzyme is selected from one or more of the following: horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase conjugate, alkaline phosphatase-anti-alkaline phosphatase conjugate, β-galactosidase-anti-β-galactosidase conjugate; the fluorescent indicator is selected from one or more of the following: AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red or PerCP dye; the chemiluminescent indicator is selected from one or more of the following: acridinium ester, acridinium sulfonamide and its derivatives, luminol (3-aminophthalhydrazide), isoluminol (4-aminophthalhydrazide), isoluminol isothiocyanate (ILITC) and its derivatives, N-(4-aminobutyl)-N-ethylisoluminol (ABEI), 4,5-diaminophthalhydrazide (DPH), aminobutylethyl phthalhydrazide (ABENH); the isotope is selected from one or more of the following: 125 I / 131 I / 124 I、 3 H、 14 C、 111 In、 89 Zr、 32 P; the colloid indicator is selected from one or more of the following: colloidal gold, colloidal carbon, colloidal selenium. 7. Characterization of the binding characteristics of 8693-1G12: Analyze the binding kinetics of 8693-1G12 to the scFv targeting CEA by Fortebio Octet. Detect the binding of 8693-1G12 to the CEA CAR on the cell surface containing the scFv targeting CEA by flow cytometry.

[0126] (1) The binding kinetics of 8693-1G12 to ScFv targeting CEA was analyzed by Fortebio Octet. The binding kinetic parameters of 8693-1G12 to ScFv targeting CEA were detected and analyzed using Fortebio Octet. The specific steps refer to the instruction manual. The HIS1K biosensor was equilibrated in PBST buffer and then the baseline was established by online equilibration (Baseline) for 60 seconds. The purified CEA scFv recombinant protein was diluted to 100 μg / mL with PBST and then loaded onto the sensor for immobilization (Loading) for 180 seconds. The sensor loaded with ScFv was then equilibrated (Baseline2) in PBST buffer for 120 seconds and then bound to different concentrations of 8693-1G12 mAb (Association) for 120 seconds. After that, the sensor was transferred to PBST for dissociation (Dissociation) for 180 seconds for dissociation rate measurement. The data was analyzed using Data Analysis 11.0.2.5 evaluation software, and the binding kinetic parameters were analyzed using a 1:2 binding model.

[0127] Table 3: Dissociation constant of 8693-1G12 analyzed by Fortebio Octet:

[0128] Serial number Loaded sample 8693-1G12 (nM) KD (M) 1 8693-1G12 400 5.210E-10 2 8693-1G12 200 5.210E-10 3 8693-1G12 100 5.210E-10 4 8693-1G12 25 5.210E-10

[0129] For the strength of affinity, the values of the association constant (Kon), dissociation constant (Kdis), and equilibrium dissociation constant (KD) are used as the evaluation criteria. The unit of Kon is 1 / Ms, which is used to represent the rate of antigen-antibody binding. The higher the Kon, the faster the antibody binds to the antigen to form a complex. The unit of Kdis is 1 / s, which is used to represent the dissociation rate of antigen-antibody. The higher the Kdis, the faster the antigen-antibody complex dissociates. KD is the ratio of Kdis to Kon, and the unit is M (mol / L). The smaller the KD value, the stronger the affinity. The equilibrium dissociation constant KD value of 8693-1G12Ab binding to ScFv targeting CEA reaches 5.210E-10 M, indicating that 8693-1G12 mAb binds to ScFv targeting CEA with high affinity.

[0130] (3) Flow cytometry was used to study the binding characteristics of 8693-1G12 to CAR-T cells with the antigen recognition domain targeting ScFv of CEA

[0131] T cells were infected with CEA-targeted CAR lentivirus, CD70 CAR lentivirus, PSCACAR lentivirus and CD19 CAR lentivirus (the sequences of patents 202111239047.X and 201710613317.6 were all incorporated into this patent) to obtain the corresponding CAR-T cells. Suspend 3×10 5 cells in 100 μl of PBS buffer containing 2% FBS, add AF647-NHS-labeled 8693-1G12-AF647 antibody at different concentrations, and incubate the mixture at 2 - 8 °C for 30 minutes, then wash the cells twice with cold PBS buffer. Use conventional PL (Protein-L, which can bind antibodies containing κ light chain) detection as a control. 8693-1G12-AF647 and PL were used to detect the recognition of CEA-targeted CAR: CEA CAR2 (the extracellular antigen recognition domain contains: ScFv with CDR sequences such as SEQ ID NO.19 - 24), CD19 CAR targeting CD19, PSCACAR targeting PSCA, CD70 CAR targeting CD70, and control T cells not expressing CAR. The results are as Figure 2 shown in Figure 2 and Table 4. In Figure 2 A is the result of 1G12 recognizing the extracellular recognition region in CEA CAR2; Figure 2 in Figure 2 B is the result of 1G12 recognizing the extracellular recognition region in CD19 CAR; Figure 2 in Figure 2 C is the result of 1G12 recognizing the extracellular recognition region in PSCA CAR;

[0132] Table 4: Recognition of 1G12 for CAR-T cells targeting different targets

[0133]

[0134] Note: " / " represents no experiment was conducted

[0135] The results showed that 8693-1G12 could specifically recognize CEACAR2, that is, it could specifically recognize the ScFv containing CDR sequences as shown in SEQ ID NOs. 19-24, and polypeptide structural molecules or fusion protein molecules containing the CDR combination of SEQ ID NOs. 19-24. 8693-1G12 did not recognize the CAR structures on the surfaces of CD19, CD70, and PSCACAR-T cells. The difference in each CAR structure lies in the scFv part. Therefore, 8693-1G12 specifically recognizes the ScFv targeting CEA and engineered immune cells such as CAR-T cells containing the ScFv targeting CEA. On the contrary, Protein L cannot distinguish CEACAR from other CARs, and the detection is not specific. 1G12 has the ability to bind to CAR (molecules containing CEA-binding molecules (CEA ScFv)). 8693-1G12 can be applied to the quality control detection method of engineered immune cells targeting CEA, such as CEACAR-T cell drugs, to judge the positive rate of CEACAR-T CAR.

[0136] 8. 8693-1G12 as a clinical detection reagent to detect engineered immune cells targeting CEA in the blood samples of subjects

[0137] Use the peripheral blood samples of 2 healthy people who have not received CAR-T cell infusion. Label the enrolled peripheral blood with 8693-1G12-AF647 to explore whether there is non-specific binding when the reagent labels peripheral blood samples. Prepare peripheral blood samples to be detected and cryopreserved CEACAR-T cells. After the CEA CAR-T cells are resuscitated, resuspend them with PBS containing 2% FBS. Take 80 μL of the blood sample to be detected, and mix 80 μL of the blood sample to be detected and 2E+6 cryopreserved and resuscitated CEA CAR-T cells. Add 5 μL of CD45-BV421 antibody, CD3-FITC antibody, and 2 μL of 8693-1G12-AF647 (151 μg / ml) antibody to the sample, incubate at 2-8°C in the dark for 30 minutes. After the incubation is completed, add 1 ml of red blood cell lysate, incubate at room temperature in the dark for 15 minutes, centrifuge at 400 g for 5 minutes, discard the supernatant, add 1 ml of PBS, wash by centrifuging at 400 g for 5 minutes, discard the supernatant, add 100 μL of PBS containing 2% FBS to resuspend, and load the sample on a flow cytometer; The experimental results are shown in Table 5:

[0138] Table 5: Detection of 8693-1G12 for peripheral blood samples of healthy people

[0139] Sample name 8693-1G12-AF647 measured value 4-71-PB 0.11％ 25-25-PB 0.00％

[0140] The results showed that the detection background value of 8693-1G12-AF647 in peripheral blood was low, and there was no non-specific recognition.

[0141] Furthermore, in the experiment of incorporating cryopreserved and resuscitated CEACAR2 cells into peripheral blood at high, medium, and low concentrations, according to the above detection steps, the proportion of CEA CAR-T was detected using 8693-1G12-AF647. The detection results are shown in Table 6:

[0142] Table 6: Detection of the proportion of CAR-T in peripheral blood samples with different proportions of CAR-T

[0143] Sample name Theoretical value Measured value Recovery rate 4-71-PB + CELL 48.22％ 48.91％ 101.43％ 25-25-PB + CELL 48.22％ 46.32％ 96.06％

[0144] The results showed that the CAR positive rate was between 90% and 110% for the high, medium, and low recoveries, meeting the quality standard requirements for clinical sample detection.

[0145] In summary, the 1G12 monoclonal antibody can detect engineered receptors or fusion proteins such as chimeric antigen receptors (CARs) in peripheral blood that contain a light chain with an antibody CDR region recognizing CEA as shown in SEQ ID NOs. 19 - 24, a variable region as shown in SEQ ID NO. 25, a heavy chain / ScFv structural region as shown in SEQ ID NO. 16, and a sequence as shown in SEQ ID NO. 26. 1G12 can be used for clinical detection, especially for the clinical detection of CEA CAR-T products.

[0146] CEA CAR

[0147] DIQLTQSPSSLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWTSTRHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYSLYRSFGQGTKVEIKGSTSGSGKPGSGEGSTKGEVQLVESGGGVVQPGRSLRLSCSASGFDFTTYWMSWVRQAPGKGLEWIGEIHPDSSTINYAPSLKDRFTISRDNAKNTLFLQMDSLRPEDTGVYFCASLYFGFPWFAYWGQGTPVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:18)

[0148] 1G12 can be applied to the in vivo monitoring of engineered immune cells targeting CEA. A kit containing 1G12 monoclonal antibody or single-chain antibody (ScFv) can be selected to detect engineered immune cells targeting CEA and determine the in vivo survival of the engineered immune cell drug targeting CEA. The 1G12 monoclonal antibody or single-chain antibody (ScFv) in the kit can be chemically labeled or biolabeled (the labeling protocol refers to part 6 of Example 1), or it can be an unlabeled monoclonal antibody. The kit also contains a buffer, and the buffer is selected from: PBS buffer, HEPES buffer, MES buffer, MOPS buffer, Tris buffer, citrate buffer, carbonate buffer, glycine buffer or phosphate buffer.

[0149] Antibody CDR sequences have multiple coding rules. Common coding rules include: Kabat, IMGT, Chothia, etc. Antibodies containing CDRs under different coding rules are all applicable to the anti-idiotypic antibodies described in this article. Therefore, 1G12 can also recognize ScFv, antibodies, fusion proteins containing heavy chain CDRs: TYWMS, EIHPDSSTINYAPSLKD, LYFGFPWF, and light chain CDRs: KASQDVGTSVA, WTSTRHT, QQYSLYRS, as well as engineered cells expressing the above ScFv, antibodies, and fusion proteins.

[0150] 9. The 8693-1G12 antibody was labeled with Bio-BHS. CAR-T cells targeting CEA CAR-T with a CAR positive rate of 92% were taken, and cells with a concentration of approximately 10×2^6 were resuspended with PBS (1×, pH 7.3). 30 μL was taken and evenly spread on a clean positively charged treated glass slide, and allowed to air dry naturally. The above glass slide was fixed with 4% PFA for 10 min, washed with PBS for 5 min × 3, and stained with 1G12-Bio 296 μg / ml. The results are as follows Figure 3 As shown: Analyzed using i-view32 software, the results showed that the detection rate of 8693-1G12 for CAR-T targeting CEA was ≥90%; there was no staining in the control T cells. 8693-1G12 is suitable for detecting CAR-T cells targeting CEA, and the extracellular recognition structure of CAR contains the CDR regions of the SEQ ID NO.19-244 sequence.

[0151] 10. Structural characterization (sequencing results) of 8693-1G12

[0152] The 8693-1G12 cell line was subcultured. Approximately 2×10^6 cells were taken, and total RNA was extracted by the Trizol method. The first-strand cDNA was obtained using a reverse transcription kit; murine VLVH primers were synthesized. Using cDNA as a template, the F and R primers were paired one by one, and a PCR reaction was carried out with high-fidelity DNA polymerase. The amplified fragments were detected by agarose gel electrophoresis, and the excised and recovered fragments were subjected to the reaction of adding an A tail with the r Taq DNA polymerase system, and then the product was purified immediately; the above products were respectively ligated to the pGEM-T vector, and each ligation product was transformed into Top10. The next day, single colonies were picked for sequencing identification, and the sequencing results were aligned and analyzed in NCBI and IMGT. The sequencing results were as follows

[0153] 8693-1G12 antibody heavy chain CDR1: GFTFSNYW (SEQ ID NO:1)

[0154] 8693-1G12 antibody heavy chain CDR2: IRLKSNNYTT (SEQ ID NO:2)

[0155] 8693-1G12 antibody heavy chain CDR3: TRGPINNDYGGYGMDY (SEQ ID NO:3)

[0156] 8693-1G12 antibody light chain CDR1: ESVDNYGISF (SEQ ID NO:4)

[0157] 8693-1G12 antibody light chain CDR2: AAS (SEQ ID NO:5)

[0158] 8693-1G12 antibody light chain CDR3: QQNKEVPHT (SEQ ID NO:6)

[0159] 8693-1G12 antibody heavy chain variable region (VH)

[0160] EVKIQESGGGLVQPGGSMKLSCVTSGFTFSNYWMNWVRQSPVKGLEWVAEIRLKSNNYTTHYAESVKGRFTISRDDSKISVYLQMNNLRAEDTGIYYCTRGPINNDYGGYGMDYWGQGTSVTVSS (SEQ ID NO:7)

[0161] 8693-1G12 antibody light chain variable region (VL)

[0162] DIVMTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQRSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQNKEVPHTFGGGTKLE (SEQ ID NO:8)

[0163] After obtaining the CDRs, light chain and heavy chain sequences of the above 1G12, in some embodiments, single-chain antibodies can be formed by combination. The single-chain antibody may comprise the CDRs of SEQ ID NO:1-6 of the present invention, or the heavy chain and light chain of SEQ ID NO:7 and SEQ ID NO:8. The single-chain antibody includes, in sequence from the N-terminus to the C-terminus: light chain variable region - linker - heavy chain variable region or heavy chain variable region - linker - light chain variable region; the linker is selected from: EGSTSGSGKPGSGEGSTKGE or (GGGGS)n, n = 1-4. In some embodiments, the light chain and heavy chain of the above antibody can also be combined to form a single-chain antibody (ScFv), which can be called 8693-1G12 antibody ScFv, and its amino acid sequence is as follows:

[0164] 8693-1G12 antibody ScFv

[0165] DIVMTQSPASLAVSLGQRATISCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNQRSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQNKEVPHTFGGGTKLEGSTSGSGKPGSGEGSTKGEVKIQESGGGLVQPGGSMKLSCVTSGFTFSNYWMNWVRQSPVKGLEWVAEIRLKSNNYTTHYAESVKGRFTISRDDSKISVYLQMNNLRAEDTGIYYCTRGPINNDYGGYGMDYWGQGTSVTVSS(SEQ ID NO:17)

[0166] In addition to the above light chain variable region-linker-heavy chain variable region connection mode, in some embodiments, the single-chain antibody (ScFv) can also be in the form of heavy chain variable region-linker-light chain variable region connection; the linker is selected from: EGSTSGSGKPGSGEGSTKGE or (GGGGS)n, where n = 1-4. In addition to the above applications, the 1G12 monoclonal antibody or single-chain antibody can also be conjugated to media such as enzyme-linked immunosorbent assay (ELISA) plates, glass, magnetic beads, latex microspheres, or polystyrene plates for the detection of the ScFv, antibody, fusion protein that recognizes CEA of the present invention, or engineered cells expressing the ScFv, antibody, fusion protein that recognizes CEA.

[0167] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. An antigen-binding molecule, the amino acid sequences of the three CDR regions of its heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 1, 2, and 3; the amino acid sequences of the three CDR regions of its light chain respectively have the amino acid sequences shown in SEQ ID NO: 4, 5, and 6.

2. The antigen-binding molecule according to claim 1, wherein, its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, or a sequence having at least 80% sequence homology therewith; its light chain variable region has the amino acid sequence shown in SEQ ID NO: 8, or a sequence having at least 80% sequence homology therewith.

3. The antigen-binding molecule according to claim 1 or 2, wherein, the constant region of its heavy chain is IgG1, and the constant region of its light chain is of the κ type.

4. The antigen-binding molecule according to claim 1 or 2, wherein, the combination of the light chain variable region and the heavy chain variable region forms a single-chain antibody, which sequentially includes from the N-terminus to the C-terminus: light chain variable region - linker - heavy chain variable region or heavy chain variable region - linker - light chain variable region; the linker is selected from: EGSTSGSGKPGSGEGSTKGE or (GGGGS)n, where n = 1 - 4.

5. A nucleic acid encoding the antigen-binding molecule according to any one of claims 1 to 4.

6. An expression vector, comprising the nucleic acid encoding the antigen-binding molecule according to any one of claims 1 to 4.

7. A host, which contains the expression vector according to claim 6, or the nucleic acid according to claim 5 is integrated into its genome.

8. A method for preparing the antigen-binding molecule according to any one of claims 1 to 4, comprising: culturing the host cell according to claim 7 and inducing the expression of the antigen-binding molecule.

9. A conjugate, which is the antigen-binding molecule according to any one of claims 1 to 4 linked with a chemical label or a biological label.

10. The conjugate according to claim 9, wherein, the chemical label is a fluorescent indicator, a chemiluminescent indicator, an isotope, and / or a colloidal indicator; the biological label is biotin, avidin, or an enzyme label.

11. The conjugate according to claim 10, wherein, the enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase - anti-peroxidase conjugate, alkaline phosphatase - anti-alkaline phosphatase conjugate, β-galactosidase - anti-β-galactosidase conjugate; the fluorescent indicator is selected from one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, Oregon Green 488, Pacific Blue dye, Pacific Orange dye, Texas Red, or PerCP dye; The chemiluminescent indicator is selected from one or more of acridinium esters, acridinium sulfonamides and their derivatives, luminol, isoluminol, isoluminol isothiocyanate and its derivatives, N-(4-aminobutyl)-N-ethylisoluminol, 4,5-diaminophthalhydrazide or aminobutylethyl phthaloylhydrazide; The isotope is selected from 125 I,[[]] 131 I,[[]] 124 I,[[]] 3 H,[[]] 14 C,[[]] 111 In,[[]] 89 Zr or 32 one or more of P; The colloidal indicator is selected from one or more of colloidal gold, colloidal carbon or colloidal selenium.

12. A conjugate prepared by conjugating the antigen-binding molecule according to any one of claims 1 to 4 or the conjugate according to any one of claims 9 to 11 with a medium.

13. The conjugate according to claim 12, wherein, the medium is an enzyme-linked immunosorbent assay (ELISA) plate, glass, magnetic beads, latex microspheres or a polystyrene plate.

14. Use of the antigen-binding molecule according to any one of claims 1 to 4, the conjugate according to any one of claims 9 to 11, and / or the conjugate according to claim 12 or 13 in the preparation of a reagent for quality control of engineered immune cell drugs targeting carcinoembryonic antigen (CEA).

15. A kit, wherein, it comprises the antigen-binding molecule according to any one of claims 1 to 4, the conjugate according to any one of claims 9 to 11, and / or the conjugate according to claim 12 or 13.

16. The kit according to claim 15, wherein, it further comprises a buffer; the buffer is selected from: phosphate-buffered saline (PBS) buffer, HEPES buffer, MES buffer, MOPS buffer, Tris buffer, citrate buffer, carbonate buffer, glycine buffer or phosphate buffer.

17. The kit according to claim 16, wherein, the buffer further comprises 0 to 10% fetal bovine serum (FBS).

18. A method for quality control of engineered immune cell drugs targeting CEA, wherein, it comprises: detecting cells with the kit according to any one of claims 15 to 17, the antibody according to any one of claims 1 to 4 or the conjugate according to any one of claims 9 to 11 to determine whether the chimeric antigen receptor (CAR) is positive.

19. A method for judging the efficacy of engineered immune cell drugs targeting CEA, wherein, detecting a sample with the kit according to any one of claims 15 to 17, the antibody according to any one of claims 1 to 4 or the conjugate according to any one of claims 9 to 11, and judging the in vivo survival of the engineered immune cells targeting CEA according to whether the CAR is positive; the sample is blood or tumor tissue.

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