CN117247453A - Monoclonal antibody of anti-CD19scFv FMC63 sequence, preparation and application thereof - Google Patents

Abstract

The invention provides an anti-CD19scFv FMC63 sequence monoclonal antibody, a preparation method and application thereof. Specifically, the monoclonal antibody of the anti-CD19scFv FMC63 sequence provided by the invention has the advantages of high stability, high specificity, high sensitivity, high affinity and the like, and can be used for effectively detecting the expression positive rate of CD19scFv (FMC 63).

Description

Monoclonal antibody of anti-CD19scFv FMC63 sequence, preparation and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a monoclonal antibody of an anti-CD19scFv FMC63 sequence, and preparation and application thereof.

Background

Traditional tumor treatment modes comprise operation treatment, chemotherapy, radiation treatment and the like, and although certain results are achieved, the survival rate of patients is low, and tumor recurrence and metastasis are still difficult problems to be overcome. Unlike traditional anti-cancer therapies, tumor immunotherapy targets are not directed against tumor cells and tumor tissue, but rather against the human immune system itself. Among them, the method of combining high affinity of antigen-antibody and killing effect of T cells and NK cells to construct T cells or NK cell immunotherapy (CAR-T, CAR-NK) based on Chimeric Antigen Receptor (CAR) with tumor specificity has obvious advantages.

CAR-T, collectively Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy, introduces chimeric antigen receptors for specific targets and co-stimulatory molecules into T cells by gene editing techniques, thereby binding tumor cells expressing the target antigen under non-Major Histocompatibility Complex (MHC) -dependent conditions, and killing by activating T cells with co-stimulatory molecules. Has remarkable curative effect on acute leukemia and non-Hodgkin lymphoma, and is considered as one of the most promising tumor treatment modes. The CAR-T, CAR-NK cells can specifically kill tumor cells, have small damage to normal cells, have no toxic or side effect of radiotherapy and chemotherapy, and have good tolerance. Meanwhile, the CAR-T can reconstruct and enhance the specific anti-tumor immunity of patients, and can resist tumors for a long time.

In recent years, as CAR structures continue to be optimized, CAR-T has also been moved to clinical trials by the laboratory. Many malignant tumors, B cell tumors, express tumor-specific or tumor-associated antigens on their cell surfaces, such as CD19, glypican 3, and Mesothelin, etc., and can be used as targets recognized by CAR-T, CAR-NK cells to mediate killing of tumor cells.

The CAR-T is based on cancer immunotherapy in which T cells are modified in vitro to express a receptor fragment specifically recognizing tumor surface antigens on the surface, combines the high affinity of antigen antibodies with the killing effect of T lymphocytes, and enables the T lymphocytes to express the chimeric antigen receptor through gene transduction by constructing a specific chimeric antigen receptor, and specifically recognizes target antigens so as to kill the target cells. Through the gene modification technology, the targeting, killing activity and durability of the effector T cells are higher than those of immune cells which are conventionally applied, and the tumor local immunosuppression microenvironment can be overcome and the host immune tolerance state can be broken. According to this principle, the CAR structure comprises a region specifically recognizing an antigen, the structure of the region is derived from a single chain variable region (ScFv) of a monoclonal antibody, and CD3 and a co-stimulatory factor (CD 28/41 BB) capable of signaling transmit an activation signal, so that T cells are continuously activated and proliferated, and cytokines are continuously secreted, thereby enhancing the effect of killing tumor cells. A representative CAR structure is shown in figure 1.

An important loop of quality control of CAR-T, CAR-NK therapies is CAR positive expression rate detection, a common detection scheme is cell detection by using anti-idiotype (anti-idiotype) antibodies, protein L or target proteins in combination with flow cytometry. The target protein combined flow cytometry has the defects of poor stability and high cost; the detection scheme of the anti-idiotype antibody combined flow cytometry is an optimal tool for CAR-T quality control supervision and dynamic tracking of CAR-T cells after being input into a human body due to the advantages of strong specificity, no reaction with non-CAR-T cells in the body and the like.

However, satisfactory quality control techniques are currently lacking for CAR-T cells and CAR-NK cells developed based on targeting CD19scFv FMC63, in particular based on anti-idiotype monoclonal antibodies specific for the scFv sequence of the chimeric antigen receptor.

Disclosure of Invention

The invention aims to provide a monoclonal antibody of a specific anti-CD19scFv FMC63 sequence, a preparation method and application thereof.

It is another object of the invention to provide a method of quality control for CAR-T cells and CAR-NK cells developed based on targeting CD19scFv FMC 63.

In a first aspect of the invention there is provided a heavy chain variable region of an anti-FMC 63 antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO. 3,

CDR2 as shown in SEQ ID NO. 4, and

CDR3 shown in SEQ ID No. 5.

In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which is capable of retaining the binding affinity of FMC63 of the CD19 scFv.

In another preferred embodiment, the heavy chain variable region further comprises an FR region of human origin or an FR region of murine origin.

In another preferred embodiment, the heavy chain variable region has the amino acid sequence shown in SEQ ID NO. 2.

In a second aspect of the invention there is provided a heavy chain of an anti-FMC 63 antibody, said heavy chain having a heavy chain variable region according to the first aspect of the invention.

In another preferred embodiment, the heavy chain of the antibody further comprises a heavy chain constant region.

In another preferred embodiment, the heavy chain constant region is of human or murine origin.

In a third aspect of the invention there is provided a light chain variable region of an anti-FMC 63 antibody, said light chain variable region comprising the following three complementarity determining region CDRs:

CDR1' shown in SEQ ID NO. 8,

CDR2' shown in SEQ ID NO 9, and

CDR3' shown in SEQ ID NO 10.

In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which is capable of retaining the binding affinity of FMC63 of the CD19 scFv.

In another preferred embodiment, the light chain variable region further comprises an FR region of human origin or an FR region of murine origin. )

In another preferred embodiment, the light chain variable region has the amino acid sequence shown in SEQ ID NO. 7.

In a fourth aspect of the invention there is provided an antibody light chain having a light chain variable region according to the third aspect of the invention.

In another preferred embodiment, the light chain of the antibody further comprises a light chain constant region.

In another preferred embodiment, the light chain constant region is of human or murine origin.

In a fifth aspect of the invention, there is provided an anti-FMC 63 antibody having:

(1) A heavy chain variable region according to the first aspect of the invention; and/or

(2) A light chain variable region according to the third aspect of the invention;

alternatively, the antibody has: a heavy chain according to the second aspect of the invention; and/or a light chain according to the fourth aspect of the invention.

In another preferred embodiment, the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

In another preferred embodiment, the antibody is a double-chain antibody or a single-chain antibody.

In another preferred embodiment, the antibody is a monoclonal antibody.

In another preferred embodiment, the antibody is a partially or fully humanized monoclonal antibody.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO. 2; and/or the light chain variable region sequence of the antibody is shown as SEQ ID NO. 7.

In another preferred embodiment, the heavy chain variable region sequence of the antibody is shown in SEQ ID NO. 2; and the light chain variable region sequence of the antibody is shown as SEQ ID NO. 7.

In a sixth aspect of the present invention, there is provided a recombinant protein having:

(i) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; and

(ii) Optionally a tag sequence to assist expression and/or purification.

In another preferred embodiment, the tag sequence comprises a 6His tag.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In a seventh aspect of the invention, there is provided a polynucleotide encoding a polypeptide selected from the group consisting of:

(1) A heavy chain variable region according to the first aspect of the invention, a heavy chain according to the second aspect of the invention, a light chain variable region according to the third aspect of the invention, a light chain according to the fourth aspect of the invention, or an antibody according to the fifth aspect of the invention; or (b)

(2) The recombinant protein according to the sixth aspect of the invention.

In another preferred embodiment, the polynucleotide comprises DNA, RNA, cDNA or a combination thereof.

In an eighth aspect of the invention there is provided a vector comprising a polynucleotide according to the seventh aspect of the invention.

In another preferred embodiment, the carrier comprises: bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

In another preferred embodiment, the vector comprises an expression vector.

In a ninth aspect of the invention there is provided a genetically engineered host cell comprising a vector or genome according to the eighth aspect of the invention incorporating a polynucleotide according to the seventh aspect of the invention.

In a tenth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: the heavy chain variable region according to the first aspect of the invention, the heavy chain according to the second aspect of the invention, the light chain variable region according to the third aspect of the invention, the light chain according to the fourth aspect of the invention, or the antibody according to the fifth aspect of the invention, the recombinant protein according to the sixth aspect of the invention, or a combination thereof, is used for (a) preparing a detection reagent or kit.

In another preferred embodiment, the assay or assay kit is used for quality control detection of a CAR-T or CAR-NK product of a scFV comprising FMC 63.

In another preferred embodiment, the detection reagent or kit is used to detect the proportion of CAR-T or CAR-NK positive cells in the product that express the chimeric antigen receptor of FMC63 scFV.

In another preferred embodiment, the assay or assay kit is used for quality control detection of a CAR-T or CAR-NK product of a scFV comprising FMC 63.

In an eleventh aspect of the invention, there is provided a method of detecting CAR-T or CAR-NK positive cells expressing a chimeric antigen receptor of FMC63scFV in vitro (including non-diagnostic) in a sample, the method comprising the steps of:

(1) Contacting the sample with an antibody according to the fifth aspect of the invention in vitro;

(2) Detecting whether a FMC63 scFV-antibody complex is formed, wherein the formation of the complex indicates the presence of a CAR-T or CAR-NK positive cell expressing a chimeric antigen receptor of FMC63scFV in the sample.

In another preferred embodiment, the method is a qualitative or quantitative method.

In a twelfth aspect of the present invention, there is provided a kit comprising:

(1) A first container comprising an antibody according to the fifth aspect of the invention; and/or

(2) A second container comprising a second antibody against an antibody according to the fifth aspect of the invention.

In a thirteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, the method comprising:

(a) Culturing a host cell according to the ninth aspect of the invention under conditions suitable for expression;

(b) Isolating the recombinant polypeptide from the culture, said recombinant polypeptide being an antibody according to the fifth aspect of the invention or a recombinant protein according to the sixth aspect of the invention.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows a schematic representation of the CAR (chimeric antigen receptor) structure.

FIG. 2 shows that purified CD19scFv (FMC 63) recognizes OD450 absorbance of human CD19-Fc recombinant protein.

FIG. 3 shows flow cytometry analysis of GFP-FMC63 transfected Jurkat cells with antibodies of the invention.

Detailed Description

The present inventors have made extensive and intensive studies to obtain an anti-CD19scFv FMC63 sequence monoclonal antibody having extremely excellent affinity and specificity, and to obtain nucleic acid and amino acid sequences of the antibody variable region. The antibody provided by the invention can be combined with the CD19scFv FMC63 sequence antigen on the surface of the CAR-T cell or the CAR-NK cell with high specificity, and has high affinity. The present invention has been completed on the basis of this finding.

Terminology

As used herein, the term "conjugate" refers to an antibody or fragment or analog thereof that is capable of binding to the scFv FMC63 sequence of anti-CD 19. The term "FMC63 conjugate" as used herein refers to an antibody or fragment or analog thereof that specifically recognizes FMC63 and binds to FMC 63.

In order that the invention may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specifically recited value, the term "about" means that the value can vary no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that there may be, but need not be, 1, 2, or 3 antibody heavy chain variable regions of a particular sequence.

Antibodies to

As used herein, the term "antibody" refers to an immunoglobulin that is a tetrapeptide chain structure formed from two identical heavy chains and two identical light chains joined by an interchain disulfide bond. The immunoglobulin heavy chain constant region differs in amino acid composition and sequence, and thus, in antigenicity. Accordingly, immunoglobulins can be assigned to five classes, or different types of immunoglobulins, i.e., igM, igD, igG, igA and IgE, and the heavy chain constant regions corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. IgG represents the most important class of immunoglobulins, which can be divided into 4 subclasses again due to differences in chemical structure and biological function: igG1, igG2, igG3 and IgG4. Light chains are classified as either kappa or lambda chains by the difference in constant regions. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known to those skilled in the art.

The sequences of the heavy and light chains of the antibody near the N-terminus vary widely, being the variable region (V region); the remaining amino acid sequence near the C-terminus is relatively stable and is a constant region (C-region). The variable region includes 3 hypervariable regions (HVRs) and 4 FR Regions (FR) that are relatively conserved in sequence. The amino acid sequences of the 4 FRs are relatively conserved and do not directly participate in the binding reaction. The 3 hypervariable regions determine the specificity of the antibody, also known as Complementarity Determining Regions (CDRs). Each of the Light Chain Variable Region (LCVR) and Heavy Chain Variable Region (HCVR) consists of 3 CDR regions and 4 FR regions, arranged in sequence from amino-to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain, namely the light chain hypervariable region (LCDR), refer to LCDR1, LCDR2 and LCDR3; the 3 CDR regions of the heavy chain, namely heavy chain hypervariable regions (HCDR), refer to HCDR1, HCDR2 and HCDR3. The CDR amino acid residues of the LCVR and HCVR regions of the antibodies or antigen-binding fragments of the invention are in numbers and positions that meet the known Kabat numbering convention (LCDR 1-3, HCDR 2-3), or that meet the numbering convention of Kabat and chothia (HCDR 1). The four FR regions in the natural heavy and light chain variable regions are generally in a β -sheet configuration, connected by three CDRs forming the connecting loops, which in some cases may form part of the β -sheet structure. The CDRs in each chain are held closely together by the FR regions and form together with the CDRs of the other chain an antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody. The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

As used herein, the term "antigen binding fragment" refers to a Fab fragment, fab 'fragment, F (ab') 2 fragment, or single Fv fragment having antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding.

As used herein, the term "epitope" refers to a discrete, three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the invention.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared by techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human portions, can be prepared using DNA recombination techniques well known in the art.

As used herein, the term "monoclonal antibody" refers to an antibody secreted from a clone derived from a single cell source. Monoclonal antibodies are highly specific, being directed against a single epitope. The cells may be eukaryotic, prokaryotic or phage clonal cell lines.

As used herein, the term "chimeric antibody" is an antibody molecule expressed by a host cell by splicing the V region gene of a murine antibody to the C region gene of a human antibody into a chimeric gene, followed by insertion into a vector. The high specificity and affinity of the parent mouse antibody are maintained, and the human Fc segment of the parent mouse antibody can effectively mediate biological effect functions.

As used herein, the term "humanized antibody" is an engineered version of murine antibodies having CDR regions derived (or substantially derived) from a non-human antibody (preferably a mouse monoclonal antibody), and FR regions and constant regions substantially derived from human antibody sequences; i.e., grafting murine-resistant CDR region sequences onto different types of human germline antibody framework sequences. Because CDR sequences are responsible for most of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular naturally occurring antibody can be expressed by constructing expression vectors.

In the present invention, antibodies may be monospecific, bispecific, trispecific, or more multispecific.

In the present invention, the antibodies of the invention also include conservative variants thereof, meaning that up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are replaced by amino acids of similar or similar nature to the amino acid sequence of the antibodies of the invention to form a polypeptide. These conservatively mutated polypeptides are preferably produced by amino acid substitution according to Table 1.

TABLE 1

anti-CD19scFv FMC63 antibodies

As used herein, the term "CD19 scFv FMC63" refers generally to natural or recombinant human CD19scFv FMC63, as well as non-human homologs of human CD19scFv FMC 63. The molar concentration of CD19scFv FMC63 was calculated using the molecular weight of the homodimer of CD19scFv FMC63 (e.g. 30KDa for human CD19scFv FMC 63), unless otherwise indicated.

As used herein, the terms "anti-CD 19scFv FMC63 antibody", "anti-FMC 63 antibody", "antibody of the invention" are used interchangeably, all referring to antibodies of the invention (as described in the fifth aspect) that are highly specific and high affinity for CD19scFv FMC 63.

Typically, the high specificity and high affinity antibodies of the invention against CD19scFv FMC63 comprise a heavy chain comprising a heavy chain variable region (VH) amino acid sequence and a light chain comprising a light chain variable region (VL) amino acid sequence.

Preferably, the respective CDRs of the heavy chain variable region (VH) amino acid sequence and the light chain variable region (VL) amino acid sequence are selected from the group consisting of:

a1 SEQ ID No. 3 (heavy chain CDR 1);

a2 SEQ ID No. 4 (heavy chain CDR 2);

a3 SEQ ID No. 5 (heavy chain CDR 3);

a4 SEQ ID No. 8 (light chain CDR 1');

a5 SEQ ID No. 9 (light chain CDR 2');

a6 SEQ ID No. 10 (light chain CDR 3');

a7 Any one of the above amino acid sequences is added, deleted, modified and/or substituted for at least one (e.g., 1-5, 1-3, preferably 1-2, more preferably 1) amino acid sequence having the binding affinity of a CD19scFv FMC 63.

In another preferred embodiment, the sequence formed by adding, deleting, modifying and/or substituting at least one amino acid sequence is preferably an amino acid sequence having a homology of at least 80%, preferably at least 85%, more preferably at least 90%, most preferably at least 95%.

The antibody of the present invention may be a double-chain or single-chain antibody, and may be selected from animal-derived antibodies, chimeric antibodies, humanized antibodies, more preferably humanized antibodies, human-animal chimeric antibodies.

The antibody derivatives of the invention may be single chain antibodies, and/or antibody fragments, such as: fab, fab ', (Fab') ₂ Or other antibody derivatives known in the art, and IgA, igD, igE, igG and any one or more of IgM antibodies or other subclasses of antibodies.

Wherein the animal is preferably a mammal, such as a mouse.

The antibodies of the invention may be murine, chimeric, humanized, CDR grafted and/or modified antibodies that target human CD19scFv FMC 63.

In a preferred embodiment of the invention, any one or more of the sequences of SEQ ID Nos 3, 4 and 5 described above, or a sequence thereof having binding affinity to FMC63 of CD19scFv, which is added, deleted, modified and/or substituted with at least one amino acid, is located in the CDR region of the heavy chain variable region (VH).

In a preferred embodiment of the invention, any one or several of the sequences of SEQ ID Nos. 8, 9 and 10 described above, or a sequence thereof having binding affinity to FMC63 of CD19scFv, which is added, deleted, modified and/or substituted with at least one amino acid, is located in the CDR region of the light chain variable region (VL).

In a more preferred embodiment of the invention, the VH CDR1, CDR2, CDR3 are each independently selected from any one or several of SEQ ID No. 3, 4 and 5, or a sequence with binding affinity of the CD19scFv FMC63 with addition, deletion, modification and/or substitution of at least one amino acid; VL CDR1, CDR2, CDR3 are each independently selected from any one or several sequences of SEQ ID No. 8, 9 and 10, or a sequence with binding affinity of CD19scFv FMC63 by adding, deleting, modifying and/or substituting at least one amino acid.

In the above-described aspect of the present invention, the number of amino acids added, deleted, modified and/or substituted is preferably not more than 40%, more preferably not more than 35%, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, more preferably 15 to 20% of the total amino acids in the original amino acid sequence.

In the present invention, the number of the added, deleted, modified and/or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, most preferably 1.

Preparation of antibodies

Any suitable method for producing monoclonal antibodies may be used to produce the anti-CD19scFv FMC63 sequence monoclonal antibodies of the invention. For example, animals may be immunized with linked or naturally occurring CD19scFv FMC63 homodimers or fragments thereof. Suitable immunization methods may be used, including adjuvants, immunostimulants, repeated booster immunizations, and one or more routes may be used.

Any suitable form of CD19scFv FMC63 can be used as an immunogen (antigen) for generating non-human antibodies specific for CD19scFv FMC63, screening the antibodies for biological activity. The priming immunogen may be full length mature human CD19scFv FMC63, including natural homodimers, or a peptide containing single/multiple epitopes. The immunogens may be used alone or in combination with one or more immunogenicity enhancing agents known in the art. The immunogen may be purified from a natural source or produced in genetically modified cells. The DNA encoding the immunogen may be genomic or non-genomic (e.g., cDNA) in origin. DNA encoding the immunogen may be expressed using suitable genetic vectors including, but not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, prime and non-viral vectors.

An exemplary method of producing an anti-human CD19scFv FMC63 antibody of the invention is described in example 1.

The humanized antibody may be selected from any class of immunoglobulins, including IgM, igD, igG, igA and IgE. In the present invention, the antibody is an IgG antibody, and an IgG1 subtype is used. Optimization of the necessary constant domain sequences to produce the desired biological activity is readily achieved by screening antibodies using the biological assays described in the examples below.

Also, any type of light chain may be used in the compounds and methods herein. In particular, kappa, lambda chains or variants thereof are useful in the compounds and methods of the present invention.

The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained by a conventional technique such as amplification by PCR or screening of a genomic library. In addition, the coding sequences for the light and heavy chains may be fused together to form a single chain antibody.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Preferred animal cells include (but are not limited to): CHO-S, CHO-K1, HEK-293 cells.

The steps described herein for transforming a host cell with recombinant DNA may be performed using techniques well known in the art. The transformant obtained can be cultured by a conventional method, and the transformant expresses the polypeptide encoded by the gene of the present invention. Depending on the host cell used, it is cultivated in conventional medium under suitable conditions.

Typically, the transformed host cell is cultured under conditions suitable for expression of the antibodies of the invention. The antibodies of the invention are then purified by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography or affinity chromatography, using conventional separation and purification means well known to those skilled in the art.

The resulting monoclonal antibodies can be identified by conventional means. For example, the binding specificity of a monoclonal antibody can be determined using immunoprecipitation or in vitro binding assays, such as Radioimmunoassays (RIA) or enzyme-linked immunosorbent assays (ELISA).

Application of

The invention provides uses of the antibodies of the invention, for example for quality control of CAR-T or CAR-NK expressing a chimeric antigen receptor of FMC63 scFV.

Preferably, the antibodies of the invention are useful for qualitative detection, thereby obtaining the proportion of CAR-T or CAR-NK positive cells in the cell product that express the chimeric antigen receptor of FMC63 scFV.

Detection application and kit

The antibodies of the invention may be used in detection applications, for example for detecting samples, thereby providing quality control information.

In the present invention, the sample (specimen) used includes a cell sample, for example, a cell sample during or after the preparation of CAR-T cells or CAR-NK cells.

The invention also provides a kit comprising an antibody (or fragment thereof) of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, etc. In a preferred embodiment, the antibody of the present invention may be immobilized on a detection plate.

The main advantages of the invention include:

(a) The antibody has excellent bioactivity and specificity and high binding force. In addition, it has good binding affinity for the specific conformational CD19scFv FMC63 sequence on the cell surface and can be used as a quality control antibody targeting the CD19scFv FMC63 sequence.

(b) The antibody of the invention has high stability, is derived from stable monoclonal antibody secreting cells, can be produced in an infinitely amplified manner, has mature antibody production and quality control methods, and has small batch-to-batch difference.

(c) The antibody of the invention has low cost, can be produced in large scale engineering, and has mature various labeling and detection methods.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 preparation of hybridoma cell lines

1.1 Synthesis and preparation of antigens for immunization

The following are the Anti-CD19Scfv (FMC 63) nucleic acid sequences and amino acid sequences.

Anti-CD19Scfv (FMC 63) nucleic acid sequence:

gacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaaccagatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaagaagatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggaggggggactaagttggaaataacaggctccacctctggatccggcaagcccggatctggcgagggatccaccaagggcgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtccgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagcctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccacatactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaagttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacattattactacggtggtagctatgctatggactactggggtcaaggaacctcagtcaccgtctcctcagcggccgca(SEQ ID No:11)

Anti-CD19Scfv (FMC 63) amino acid sequence:

MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAA(SEQ ID No:12)

the CD19scfv (FMC 63) mammalian cell expression vector was total gene synthesized by Shanghai switzerland, constructed into an invitrogen pcdna3.1 plasmid, in which the C-terminal was fused with a6 xhis tag. Recombinant expression was performed using transiently transfected ExpiCHO-S CHO cells. One day before transfection, seed-plated ExpiCHO-S ^TM Cells with a final density of 4X 10 ⁶ Each living cell/mL. The next day, cells were counted and diluted to a final density of 6×10 with pre-warmed fresh medium ⁶ The flask was gently shaken to mix the cells at each viable cell/mL. Using precooled OptiPRO ^TM Preparing an Expifectamine by a culture medium ^TM CHO/plasmid complex.

The total plasmid DNA transfected was 0.6. Mu.g/ml culture volume. Plasmid and Expifectamine ^TM CHO ratio 1:5.3. using precooled OptiPRO ^TM Dilution of plasmid DNA and Expifectamine respectively ^TM CHO transfection reagent, diluted Expifectamine ^TM The CHO reagent was added to the diluted DNA and mixed upside down. The mixture is placed at room temperature for 1-5 minutes, and the mixture is slowly transferred into a culture flask to be transfected and is evenly mixed. The cells were incubated on an orbital shaker at 37℃in an incubator with humidified air containing 8% CO 2. The following day after transfection, the ExpiFectamine was added in the required ratio as indicated ^TM CHO enhancers and adjuvants. Culturing for 7-10 days. The optimal collection time for a protein depends on the nature and stability of the protein expression. The cells were collected by centrifugation at 4000rpm at 4℃for 30 minutes, followed by verification and purification.

Ni column affinity chromatography purified the his-tagged recombinant CD19Scfv (FMC 63) protein. The method comprises the following steps:

(1) The Ni Sepharose High Performance column was washed with 5-10 column volumes of 1 XPBS buffer, and the column interior was thoroughly washed with the protective solution at a wash rate of 2ml/min.

(2) The sample was injected into Ni Sepharose High Performance column, 1ml/min, and the effluent was collected.

(3) The second step was repeated and 1 XPBS buffer was injected into Ni Sepharose High Performance column until the eluate OD280 was <0.08. Imidazole was used for elution, imidazole was competitively bound to nickel sulfate, and the permeate containing the target protein was collected at this time and dialyzed against 1 XPBS buffer.

For the purified and prepared CD19scfv (FMC 63) protein, ELISA method was used to test the binding performance of the protein with CD19-Fc recombinant protein.

As a result, the purified CD19scfv (FMC 63) can recognize a human CD19-Fc recombinant protein as shown in FIG. 2.

1.2 immunization of animals

SPF-class 6 week old female Balb/c strain mice (Shanghai, southern mode) were prepared and 5 mice were immunized with CD19scfv (FMC 63). The immunization method is as follows:

1) Antigen 0.1mg was mixed with Freund's complete adjuvant at a volume ratio of 2:1, emulsified and injected subcutaneously and intradermally at 5 points (100. Mu.l) for immunization. The dose of antigen was 0.1 mg/dose.

2) After 3 weeks, the antigen was emulsified 0.1mg with incomplete Freund's adjuvant (emulsification method same as first), and the subcutaneous and intradermal multiple injections were re-immunized. The dose of antigen was 0.1 mg/dose.

3) Three immunizations were performed 3 weeks after the second immunization, and re-immunization was performed by subcutaneous and intradermal multipoint injections after emulsification of 0.1mg of antigen with incomplete Freund's adjuvant (emulsification method same as first). The dose of antigen was 0.1 mg/dose.

4) 7-10 days after the third immunization, blood was taken and placed at 4 ℃.

5) The next day, 10000rpm, serum was separated and stored at-20 ℃.

6) Serum titers were determined by ELISA. The mice were boosted once 3 days before cell fusion and the immunization method was intraperitoneal injection.

The following immunization protocol was used to immunize mice:

(a) Primary immunization (antigen CD19scfv (FMC 63) 100 μg/d, subcutaneous multipoint injection with complete adjuvant of foci);

(b) After 3 weeks, a second immunization (antigen CD19scfv (FMC 63) 100. Mu.g/dose, subcutaneous multipoint injection with incomplete Freund's adjuvant)

(c) After 3 weeks, a third immunization (antigen CD19scfv (FMC 63) 100. Mu.g/dose, subcutaneous multipoint injection with incomplete Freund's adjuvant)

(d) After 1 week, blood was drawn and serum antibody titers were measured.

Table 2: results of CD19scfv (FMC 63) immunized mice

ELISA determination of mouse antiserum antibody titers after third immunization with CD19scfv (FMC 63)

1.3 hybridoma cell lines

1.3.1 cell preparation

Preparation of myeloma cells: myeloma cells SP2/0 selected by 8-azaguanine (Invitrogen) were collected, observed for cell activity under a microscope, and cells in a logarithmic growth phase with good activity were counted after washing and suspended in DMEM (Invitrogen) medium for use.

Preparation of feeder cells: the day before fusion, 5ml DMEM culture solution is injected into the abdominal cavity of the mice, the abdominal cavity solution is pumped out after slight shaking, the centrifugation and the counting are carried out, and the cell concentration is regulated to be 1 multiplied by 10 ⁵ Per ml, inoculated into 96-well plates, 50 μl/well.

Preparation of spleen cells: the #3 mice were selected as immune spleen cell donors for cell fusion. Dissecting the mice, taking spleens, dispersing spleen cells by a mechanical method, filtering by a filter screen to obtain spleen cell suspension, washing by DMEM culture solution, and counting.

1.3.2 cell fusion

Taking 1×10 ⁷ SP2/0 cells and 5X 10 ⁷ Spleen cells (1:5 ratio) were mixed in a 50ml centrifuge tube, DMEM serum-free medium was added, centrifuged at 1500rpm for 3min,the supernatant was discarded. The cells were pelleted by shaking, 50% (v/v) PEG (molecular weight 1500) 1ml was added dropwise, and shaking was performed while adding the solution, and the addition was completed within 1 min. Let stand for 90 seconds, allow PEG to continue to act. Then 10ml of serum-free DMEM culture solution preheated at 37 ℃ is added dropwise within 2.5min, and the mixture is left stand for 5min to terminate the action of PEG. After fusion, the cell suspension was centrifuged at 1000rpm for 3min. Removing supernatant, gently stirring the pellet, adding 25ml of complete culture solution (DMEM+10% FBS), inoculating into 96-well plate with feeder cells, 50 μl/well, placing at 37deg.C, 5% CO ₂ Culturing in an incubator. The next day, 100 μl/well of complete medium containing 2 XHAT (Sigma-Aldrich) was added to kill unfused myeloma cells.

The results of the fusion rate and antibody positive rate of the spleen cells of the mice and the myeloma cells of the mice are shown in Table 3.

TABLE 3 fusion Rate of spleen cells of mice and cells of mouse myeloma lines and antibody Positive Rate

Construction of anti-CD19scfv (FMC 63) hybridomas

1.3.3 hybridoma cell screening

1.3.3.1 hybridoma cell cloning

The hybridoma cells in the wells, which are positive for antibodies and have a high OD, are cloned by limiting dilution, typically to 0.8 cells/well. When the cells are cultured to 20% of the plate bottom area, the cell culture supernatant is sucked and the antibody positive holes are screened again by ELISA method. If 3 clones were continued, the cloning efficiency was less than 2/3 each time and the positive rate was 100%, the cells thus obtained were monoclonal. 1X 10 ⁶ After cells were cultured in 10ml of culture supernatant for 3 days, the supernatant was taken for ELISA detection

The results of cloning of hybridoma clones are shown in Table 4.

TABLE 4 results of hybridoma clones

Cryopreservation of 1.3.3.2 hybridoma cells

Cell cryopreservation solution: 50% calf serum, 40% RPMI 1640 solution, 10% dimethyl sulfoxide.

Centrifuging hybridoma cells, and re-suspending the cells in pre-chilled frozen stock solution with concentration of 10 ⁶ -10 ⁷ Per ml, transferred to a cryopreservation flask 1ml. Placed in a refrigerator at-70 deg.c and transferred into liquid nitrogen the next day.

EXAMPLE 2 monoclonal antibody preparation and purification

In the invention, the scheme for preparing a large amount of monoclonal antibodies is a mouse abdominal water method, and the method comprises the following steps:

2.1 preparation of ascites

Mice of F1 generation were given a warp beam and 0.5ml of pristane (prine) was intraperitoneally injected. After 7-10 days, 0.5ml of 1X 10 is again injected intraperitoneally ⁶ A hybridoma cell. The growth condition of the mice is observed every day, abdominal bulge is visible about 7 days, and ascites is collected in time.

2.2 monoclonal antibody purification

The monoclonal antibody ascites obtained above is purified by Protein G affinity chromatography, pre-packed with Protein G-sepharosose 4B (GE) column and equilibrated with about 50ml PB; filtering 2ml of ascites with 0.45 μm filter membrane, diluting with 20mM PB buffer solution, loading onto column, eluting protein not bound to column with 20mM PB buffer solution with pH of 7.0, and measuring protein concentration until no protein exists in the eluate; eluting the antibody bound on the column by 100mM pH 2.7glycine, and neutralizing the eluted liquid by using 1M Tris-HCl with the pH value of 9.0 to make the pH value neutral; collecting the eluent, and measuring the protein concentration. The solution is put into a dialysis bag, and is dialyzed by PBS buffer solution with the pH of 7.4 and 10mM, the solution is changed once for 4 hours, the solution is changed for 3 times, and the dialyzed monoclonal antibody is subpackaged and stored in a refrigerator with the temperature of minus 70 ℃.

EXAMPLE 3 identification of purified CD19scfv (FMC 63) monoclonal antibody

To evaluate the specificity of monoclonal antibodies to recognize antigens, a series of antibodies screened were tested for ELISA using different antigens.

The results are shown in Table 5.

Table 5 ELISA assay of anti-CD19scfv (FMC 63) monoclonal antibody titers in mouse IgG

Example 4 use of a cd19scfv (FMC 63) monoclonal antibody for detection of FMC63 CAR at the cell surface

Human T lymphocyte leukemia cell cells (Jurkat) stably transfected with FMC63 CAR were used, which simultaneously expressed Green Fluorescent Protein (GFP). Detection was performed using a flow cytometer.

The detection results are shown in FIG. 3. Clone S-541-9 can best recognize the FMC63 CAR-expressing Jurkat cells. This suggests that the clone S-541-9 antibody can well detect cell-expressed CD19scfv (FMC 63) with the correct specific conformation. This suggests that the antibodies of the invention can be used to detect FMC63 scFV-based CAR-T cells or CAR-NK cells.

Example 5 sequencing of hybridoma antibodies

5.1 obtaining variable region genes of monoclonal antibodies

5.1.1 RNA extraction of hybridoma cell lines

Resuscitation and culture was performed after using RDF medium, and RNA extraction was performed using the Rneasy MiNi Kit of Qiagen after cell culture to an appropriate number.

5.1.2 RT-PCR and purification of cDNA

5.1.2.1 RT-PCR

Reverse kit is utilizedqPCR RT Kit, the specific steps are as follows:

5.1.2.1.1 Denaturation of RNA: RNA was denatured at 65℃for 5min, immediately after which it was cooled on ice.

5.1.2.1.2 the reaction system is as follows:

5.1.2.1.3 The procedure for the RT-PCR reaction was as follows:

37℃,30min；98℃,5min；4℃,∞。

5.1.2.2 purification of cDNA

To ensure the quality of the subsequent PCR, a method of usingSV Gel and PCR Clean-Up System kit cDNA was purified.

5.1.3 Variable region genes of monoclonal antibodies obtained by PCR

5.1.3.1 primer Synthesis

The key to this step is the design of a murine antibody variable region Primer library, which we refer to the Novagen Ig-Primer Sets Primer library, which synthesizes the desired primers.

5.1.3.2 PCR amplification of cDNA Using KOD Fx Neo Polymerase

The annealing temperature for the primers of the A and B Sets was 50℃and the annealing temperature for the primers of the C-G Sets was 60℃according to the Novagen Ig-Primer Sets Primer specification. Two PCR sets were set, and the primers of the A-B set were mixed and the primers of the C-G set were mixed, and PCR was performed separately. The reaction system is as follows:

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5.1.3.3 adding A tail

To the PCR product was added 1. Mu.L of Taq Ex Polymerase at 72℃for 30min.

5.1.3.4 gel electrophoresis

Recovered using a rubber cutting recovery kit, 50. Mu.L dd H ₂ And (3) eluting O.

5.1.3.5 T vector ligation overnight at 16 ℃

The connection system is as follows:

5.1.3.6 transformation and blue and white spot screening

When plating each plate was previously plated with a mixture of 20. Mu. L X-Gal and 5. Mu.L IPTG, white plaques were positive for T-carriers. Sequencing the positive clone to obtain the variable region gene sequence of the antibody in the monoclonal. And (5) preserving the glycerol bacteria.

Antibody sequences

Heavy chain variable region VH of antibody S-541-9

Nucleic acid sequence: (SEQ ID No: 1)

ATGGAATGTAACTGGATACTTCCTTTTATTCTGTCAGTAACTTCAGGTGTCTACTCACAGGTTCAGCTCCAGCAGTCTGGGGCTGAGTTGGCAAGACCTGGGGCTTCAGTCAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTAC TAACTTCTGGCTGCAGTGGGTCAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTCATCCTGGAAATG GTGATACTAGATACACTCAGAACTTCACGGGCAAGGCCACATTGACTACAGATACATCTTTTAGTACAGCCTACATGCAACTCAGCAGCTTGGCATCTGAAGACTCTGCGGTCTATTACTGTGCCAGGGGATGGGACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA

Amino acid sequence: (SEQ ID No: 2)

QVQLQQSGAELARPGASVKLSCKASGYTFTNFWLQWVKQRPGQGLEWIGAIHPGNGDTRYTQNFTGKATLTTDTSFSTAYMQLSSLASEDSAVYYCARGWDWGQGTLVTVSA

H-CDR1：GYTFTNFW(SEQ ID No:3)

H-CDR2：IHPGNGDT(SEQ ID No:4)

H-CDR3：ARGWD(SEQ ID No:5)

Light chain variable region VL of antibody S-541-9

Nucleic acid sequence: (SEQ ID No: 6)

ATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGGAGTGATGTTGTGTTGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGAATTGT ACATAGTAATGGAGACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAG TTTCCACCCGATTTTCTGGGGTCCCAGACAGGTTCAGTGCCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTACTGCTTTCAAGCTTCTCATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAACA

Amino acid sequence: (SEQ ID No: 7)

DVVLTQTPLSLPVSLGDQASISCRSSQRIVHSNGDTYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSASGSGTDFTLKISRVEAEDLGIYYCFQASHVPYTFGGGTKLEI

L-CDR1：QRIVHSNGDTY(SEQ ID No:8)

L-CDR2：KVS(SEQ ID No:9)

L-CDR3：FQASHVPYT(SEQ ID No:10)

EXAMPLE 6 recombinant expression of antibodies

The heavy chain coding sequence (containing the VH and heavy chain constant regions) and the light chain coding sequence (containing the VL and light chain constant regions) of antibody S-541-9 were cloned by PCR. The 5 'and 3' ends of the mouse cDNA sequence are modified with PCR primers containing added leader sequences for the heavy and light chains and added restriction sites that allow cloning into conventional antibody expression vectors. And (3) after enzyme digestion, connecting the PCR products to corresponding restriction sites on the antibody expression vector to obtain corresponding recombinant expression plasmids respectively.

The host cell used for protein expression was a CHO-K1 cell (Cat#CCL-61) purchased from ATCC. And transferring the constructed recombinant expression plasmids of the light chain and the heavy chain into cells by an electrotransformation method. Culturing in incubator for 3-5 days. The concentration of antibodies from CHO-K1 transfection supernatants was measured by indirect ELISA.

The results show that the recombinantly expressed antibody S-541-9 can specifically bind to CD19scFv FMC 63.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Inbath Biotechnology (Shanghai) Co., ltd

<120> monoclonal antibody of anti-CD19scFv FMC63 sequence, preparation and application thereof

<130> P2021-3438

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<212> DNA

<213> mice (Mus musculus)

<400> 1

atggaatgta actggatact tccttttatt ctgtcagtaa cttcaggtgt ctactcacag 60

gttcagctcc agcagtctgg ggctgagttg gcaagacctg gggcttcagt caagttgtcc 120

tgcaaggctt ctggctacac ctttactaac ttctggctgc agtgggtcaa acagaggcct 180

ggacagggtc tggaatggat tggggctatt catcctggaa atggtgatac tagatacact 240

cagaacttca cgggcaaggc cacattgact acagatacat cttttagtac agcctacatg 300

caactcagca gcttggcatc tgaagactct gcggtctatt actgtgccag gggatgggac 360

tggggccaag ggactctggt cactgtctct gca 393

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<211> 112

<212> PRT

<213> mice (Mus musculus)

<400> 2

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Phe

20 25 30

Trp Leu Gln Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile His Pro Gly Asn Gly Asp Thr Arg Tyr Thr Gln Asn Phe

50 55 60

Thr Gly Lys Ala Thr Leu Thr Thr Asp Thr Ser Phe Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Trp Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

100 105 110

<210> 3

<211> 8

<212> PRT

<213> mice (Mus musculus)

<400> 3

Gly Tyr Thr Phe Thr Asn Phe Trp

1 5

<210> 4

<211> 8

<212> PRT

<213> mice (Mus musculus)

<400> 4

Ile His Pro Gly Asn Gly Asp Thr

1 5

<210> 5

<211> 5

<212> PRT

<213> mice (Mus musculus)

<400> 5

Ala Arg Gly Trp Asp

1 5

<210> 6

<211> 393

<212> DNA

<213> mice (Mus musculus)

<400> 6

atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgcttc caggagtgat 60

gttgtgttga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120

tcttgcagat ctagtcagag aattgtacat agtaatggag acacctattt agaatggtac 180

ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccac ccgattttct 240

ggggtcccag acaggttcag tgccagtgga tcagggacag atttcacact caagatcagc 300

agagtggagg ctgaggatct gggaatttat tactgctttc aagcttctca tgttccgtac 360

acgttcggag gggggaccaa gctggaaata aca 393

<210> 7

<211> 111

<212> PRT

<213> mice (Mus musculus)

<400> 7

Asp Val Val Leu Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Arg Ile Val His Ser

20 25 30

Asn Gly Asp Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr Tyr Cys Phe Gln Ala

85 90 95

Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

<210> 8

<211> 11

<212> PRT

<213> mice (Mus musculus)

<400> 8

Gln Arg Ile Val His Ser Asn Gly Asp Thr Tyr

1 5 10

<210> 9

<211> 3

<212> PRT

<213> mice (Mus musculus)

<400> 9

Lys Val Ser

1

<210> 10

<211> 9

<212> PRT

<213> mice (Mus musculus)

<400> 10

Phe Gln Ala Ser His Val Pro Tyr Thr

1 5

<210> 11

<211> 744

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggactaagt tggaaataac aggctccacc tctggatccg gcaagcccgg atctggcgag 360

ggatccacca agggcgaggt gaaactgcag gagtcaggac ctggcctggt ggcgccctca 420

cagagcctgt ccgtcacatg cactgtctca ggggtctcat tacccgacta tggtgtaagc 480

tggattcgcc agcctccacg aaagggtctg gagtggctgg gagtaatatg gggtagtgaa 540

accacatact ataattcagc tctcaaatcc agactgacca tcatcaagga caactccaag 600

agccaagttt tcttaaaaat gaacagtctg caaactgatg acacagccat ttactactgt 660

gccaaacatt attactacgg tggtagctat gctatggact actggggtca aggaacctca 720

gtcaccgtct cctcagcggc cgca 744

<210> 12

<211> 270

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly

50 55 60

Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln

100 105 110

Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

115 120 125

Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser

130 135 140

Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala

145 150 155 160

Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu

165 170 175

Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu

180 185 190

Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser

195 200 205

Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln

210 215 220

Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr

225 230 235 240

Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr

245 250 255

Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala

260 265 270

Claims (10)

1. A heavy chain variable region of an anti-FMC 63 antibody, said heavy chain variable region comprising the following three complementarity determining region CDRs:

CDR1 shown in SEQ ID NO. 3,

CDR2 as shown in SEQ ID NO. 4, and

CDR3 shown in SEQ ID No. 5.

2. A heavy chain of an anti-FMC 63 antibody, wherein said heavy chain has a heavy chain variable region of claim 1.

3. A light chain variable region of an anti-FMC 63 antibody, said light chain variable region comprising the following three complementarity determining region CDRs:

CDR1' shown in SEQ ID NO. 8,

CDR2' shown in SEQ ID NO 9, and

CDR3' shown in SEQ ID NO 10.

4. A light chain of an anti-FMC 63 antibody, wherein the light chain has the light chain variable region of claim 3.

5. An anti-FMC 63 antibody, comprising:

(1) The heavy chain variable region of claim 1; and/or

(2) A light chain variable region according to claim 3;

alternatively, the antibody has: the heavy chain of claim 2; and/or the light chain of claim 4.

6. The antibody of claim 5, wherein the antibody is selected from the group consisting of: an animal-derived antibody, a chimeric antibody, a humanized antibody, or a combination thereof.

7. The antibody of claim 5, wherein the heavy chain variable region sequence of the antibody is set forth in SEQ ID No. 2; and/or

The light chain variable region sequence of the antibody is shown as SEQ ID NO. 7.

8. A recombinant protein, said recombinant protein comprising:

(i) A heavy chain variable region according to claim 1, a heavy chain according to claim 2, a light chain variable region according to claim 3, a light chain according to claim 4, or an antibody according to claim 5; and

(ii) Optionally a tag sequence to assist expression and/or purification.

9. A polynucleotide encoding a polypeptide selected from the group consisting of:

(1) A heavy chain variable region according to claim 1, a heavy chain according to claim 2, a light chain variable region according to claim 3, a light chain according to claim 4, an antibody according to claim 5; or (b)

(2) The recombinant protein of claim 8.

10. Use of an active ingredient selected from the group consisting of: the heavy chain variable region of claim 1, the heavy chain of claim 2, the light chain variable region of claim 3, the light chain of claim 4, the antibody of claim 5, the recombinant protein of claim 8, or a combination thereof, wherein the active ingredient is used in (a) the preparation of a detection reagent or kit.