CN114685654A - Antibody for resisting FGF21 carboxyl terminal and application thereof - Google Patents

Abstract

The invention relates to an antibody for resisting the carboxyl terminal of FGF21 and application thereof, wherein the antigen binding site of the antibody is clear, and the antibody can be specifically bound with the complete carboxyl terminal of FGF 21; when FGF21 lacks the carboxy-terminal three amino acids (YAS), the antibody does not bind or binds very little thereto; the antibody specifically binds FGF21 or a fusion protein comprising FGF21 only if it is intact at the carboxy terminus (comprising YAS). Based on this, the present invention provides a biological sample analysis method that can distinguish between a carboxy-terminal intact molecule of FGF21 and a metabolite that has 3 or more amino acids missing from the carboxy-terminal.

Description

Antibody for resisting FGF21 carboxyl terminal and application thereof

Technical Field

The invention relates to the technical field of biology, and in particular relates to an antibody for resisting the carboxyl terminal of FGF21 and application thereof.

Background

Fibroblast growth factor 21(FGF21) consists of 181 amino acids, comprising a β clover-like core domain and random amino-and carboxy-terminal structures. In a series of animal models of insulin resistance, the use of recombinant FGF21 decreased plasma glucose and insulin levels, decreased triglyceride and cholesterol levels in the liver and circulation, and improved insulin sensitivity, energy expenditure, hepatic steatosis, and obesity (Xie T, Leung P S. fiber growth factor 21: a regulator of metabolic disease and health span [ J ]. American Journal of Physiology-Endocrinology and Metabolism,2017,313(3): E292-E302.). FGF21 has become the most potential therapeutic agent for the treatment of type 2 diabetes and related metabolic syndrome in humans.

FGF21 has been reported to have a decrease of about 90% in activity after 3 amino acids (YAS, leucine-alanine-serine) are lost at the carboxy terminus (Yi J, Hecht R, Patel J, et al, FGF21N-and C-tertiary plane differential roles in the interaction and activation [ J ] FEBS letters,2009,583(1): 19-24.). Therefore, establishing a biological sample analysis method that can distinguish between the intact molecule at the carboxy terminus of FGF21 and the metabolite that has 3 or more amino acids missing at the carboxy terminus is very important for accurately describing the pharmacokinetic profile of FGF21 or a fusion protein drug comprising FGF21 in vivo.

In the development of biomacromolecule drugs, it is challenging to establish a biological sample analysis method that can distinguish between intact protein/polypeptide molecules and their degraded metabolites. The protein/polypeptide biological sample analysis method is mainly based on immunoassay technology, and the essence of the method is the combination reaction of antigen and antibody, so the specificity of the antibody used in the analysis method determines the specificity of the method. Although there are a great many classes of antibodies currently commercially available, there is usually an undefined antigen binding site, such as the anti-FGF 21 antibody, and it is only known which amino acids bind FGF21, and the specific binding site is not clear which amino acids are bound. Therefore, it is necessary to develop a monoclonal antibody which binds to the carboxy-terminal 3 amino acids of FGF21 molecule and has a clear antigen binding site.

Disclosure of Invention

The invention aims to provide an antibody against the carboxyl terminal of FGF21 and application thereof, wherein the antibody has clear antigen binding sites and can be specifically combined with the carboxyl terminal of fibroblast growth factor 21; when FGF21 lacks the carboxy-terminal three amino acids (YAS), the antibodies of the invention do not bind or bind very little thereto; antibodies of the invention can specifically bind FGF21 or a fusion protein comprising FGF21 only if it is carboxy-terminally intact (comprising YAS).

To this end, in a first aspect, the present invention provides an anti-FGF 21 carboxy-terminal antibody comprising a light chain variable region comprising the amino acid sequences of LCDR1, LCDR2 and LCDR3 and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR 3; wherein the amino acid sequence of LCDR1 is RSSKSLLHSNGITYLY (SEQ ID NO: 1), the amino acid sequence of LCDR2 is QMSLAS (SEQ ID NO: 2) or QMSNLAS (SEQ ID NO: 3), the amino acid sequence of LCDR3 is AQTLELPT (SEQ ID NO: 4), the amino acid sequence of HCDR1 is GYTFTNY (SEQ ID NO: 5), the amino acid sequence of HCDR2 is NTYTGK (SEQ ID NO: 6), and the amino acid sequence of HCDR3 is NYYDYDVAY (SEQ ID NO: 7) or NYYDYDIAY (SEQ ID NO: 8).

Further, the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO: 9 or SEQ ID NO: shown at 10.

Further, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 11 or SEQ ID NO: shown at 12.

Further, the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO: 9, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 11 is shown in the figure; or the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO: 10, the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: shown at 12.

Further, the antibody is a full-length antibody, a Fab fragment, F (ab)₂A fragment, a two-chain Fv fragment or a single-chain Fv fragment (scFv).

Further, the antibody is a monoclonal antibody.

Further, the antibody further comprises a light chain constant region selected from a kappa or lambda subtype.

Further, the light chain constant region is of the kappa subtype.

Further, the light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 13 or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.

Further, the antibody also includes a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4 subtypes.

Further, the heavy chain constant region is of the IgG1 subtype.

Further, the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO: 15. SEQ ID NO: 16 or SEQ ID NO: 17.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding the anti-carboxy terminal antibody to FGF21, or an antigen-binding portion thereof, of the invention.

In a third aspect of the invention, there is provided a vector comprising a nucleic acid molecule according to the invention.

Further, the vector includes a plasmid, a bacteriophage, a plant cell virus, a mammalian cell virus, or a retrovirus.

In a fourth aspect of the invention, there is provided a host cell comprising a nucleic acid molecule according to the invention, or comprising a vector according to the invention.

Further, the host cell includes a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell.

Further, the host cell is a mammalian cell, such as a HEK293 cell.

In a fifth aspect of the invention, there is provided a detection kit comprising the antibody of the invention, the nucleic acid molecule of the invention, or the vector of the invention. In a sixth aspect of the invention, there is provided the use of an antibody according to the invention in an immunoassay for diagnostic or non-diagnostic purposes.

Further, the immunoassay comprises: the integrity of the carboxy terminus of FGF21 was examined.

Compared with the prior art, the technical scheme of the invention has the following advantages:

the antibody provided by the invention has clear antigen binding sites, and can be specifically bound with the complete carboxyl terminal of FGF 21; when FGF21 lacks the carboxy-terminal three amino acids (YAS), the antibodies of the invention do not bind or bind very little thereto; antibodies of the invention can specifically bind FGF21 or a fusion protein comprising FGF21 only if it is intact at the carboxy terminus (comprising YAS). Based on this, the present invention provides a biological sample analysis method that can distinguish between a carboxy-terminal intact molecule of FGF21 and a metabolite that has 3 or more amino acids missing from the carboxy-terminal.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

FIG. 1 shows the purified electrophoretogram of six monoclonal antibodies obtained by screening after the second subcloning; wherein, Lane M is molecular weight marker, Lane 1 is non-reducing electrophoresis, Lane 2 is reducing electrophoresis;

FIG. 2 shows the SDS-PAGE of MM21T antibody; wherein, Lane M is molecular weight marker, Lane 1 is reduction electrophoresis, Lane 2 is non-reduction electrophoresis;

FIG. 3 shows the SDS-PAGE of MM24T antibody; wherein, Lane M is the molecular weight Marker, Lane 1 is the reduction electrophoresis, and Lane 2 is the non-reduction electrophoresis.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

The examples herein employ, unless otherwise indicated, molecular biology, microbiology, cell biology, biochemistry, and immunology techniques which are conventional in the art.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

Definition of

The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. "antibody" as used herein includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, F (ab)₂Fv), variants thereof, fusion proteins comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylated variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies.

Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain contains a light chain variable region (VL) and a light chain constant region (CL). Full-length antibodies can be of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibodies need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further classified into subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2.

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen.

The term "antigen-binding fragment or antigen-binding portion" as used herein refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen. The antigen-binding portion may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2 and CDR3

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of an antibody. For the variable region amino acid sequence of a given antibody, the CDR amino acid sequence within the variable region amino acid sequence can be analyzed in a variety of ways, such as determined by Chothia herein using the online software, Abysis (http:// www.abysis.org /).

Example 1 animal immunization

4 polypeptides were designed and synthesized, 3 of which contained 3 amino acids from the carboxy terminus of FGF21 (YAS) and 1 did not contain YAS, the sequences of which are shown in Table 1.

TABLE 1 synthetic polypeptide sequences

The polypeptide A for immunization is respectively coupled for animal immunization.

Blood was taken after three immunizations to determine serum titers. The experimental groups for the immunized animals are shown in table 2.

TABLE 2 Immunity animal information

The indirect enzyme-linked immunoassay (indirect ELISA) method is used to screen animal serum with high titer for the polypeptide A for immunization and low titer for the polypeptide B for screening. The method comprises the following specific steps: the ELISA plates were coated with the polypeptide A for immunization, the polypeptide B for screening (SinoA8627), the protein A (SEQ ID NO: 22) and the protein B (SEQ ID NO: 23), respectively, and the serum titer was measured by an indirect ELISA method using a goat anti-mouse IgG Fc antibody (ICL, cat # GGPC-90P) labeled with HRP as a detection antibody. And (3) screening out animals which have strong reaction signals with the polypeptide A and the protein A for immunization and weak reaction signals with the polypeptide B and the protein B for screening. The results of the serum titer measurements are shown in tables 3 to 5 (S: signal value; S-B: signal value after background subtraction).

TABLE 3 ELISA test results for mouse sera one week after three immunizations

TABLE 4 ELISA test results for mouse sera one week after three immunizations

TABLE 5 ELISA test results for mouse sera one week after three immunizations

Example 2 fused hybridoma cells and screening

Knots according to tables 3-5Selecting a mouse with the number of SBI180064-2# C and injecting the polypeptide SinoA8624 to strengthen the immunity; after 3 days, the spleen was harvested for fusion, and the fused cells were suspended in HAT medium at 6X 10 per well⁴The individual cells were seeded in 96-well cell culture plates, i.e. 30 96-well cell culture plates were co-seeded. On each of the 6 th and 8 th days after the fusion, the medium in the plate was discarded, and fresh HAT medium was added, and hybridoma cell culture supernatants were collected on the 10 th day after the fusion and subjected to master clone screening (the results are shown in Table 6).

TABLE 6 Primary screening ELISA assay results for the Primary clones

The positive cells obtained by screening were counted and then seeded at 0.75 cells/well into 96-well cell culture plates, and 0.5 cell culture plates were seeded for each positive cell. After 7 days of culture, the cell state was observed under a microscope, wells containing only single cell masses were selected and labeled, culture supernatants were subjected to ELISA screening, and the screening results are shown in tables 7 and 8 (S: signal value; S-B: signal value after background subtraction). And carrying out secondary limiting dilution on the positive cells obtained by screening according to the steps until stable positive monoclonal cells are obtained. Through indirect ELISA screening, the supernatant of 6 hybridoma cells in Table 8 can be combined with SinoA8624 and protein A, and can not be combined with SinoA8627 and protein B.

TABLE 7 first subclone ELISA test results

TABLE 8 second subclone ELISA test results

EXAMPLE 3 monoclonal antibodies and Small-Scale culture and purification

Six hybridoma cells described in Table 8 were taken out, 1mL of each hybridoma cell was transferred to a 100mL culture flask, and a predetermined amount of medium was periodically added to the flask to amplify the cells, followed by culture for 10 days. Centrifuging and filtering the culture solution to obtain supernatant, and purifying by Protein A affinity chromatography, which comprises the following specific steps:

collecting culture solution, centrifuging at 6000rpm for 20min, filtering with filter, and collecting supernatant;

a. pretreatment of a chromatographic column: washing the Protein A affinity chromatographic column with ultrapure water, and then balancing with a balance buffer solution;

b. loading and balancing: loading the Protein A affinity chromatographic column with the feed liquid, and leaching with an equilibrium buffer solution until the baseline is stable after loading;

c. and (3) elution: eluting with Elution3.0, collecting elution peak, and neutralizing with 2M Tris;

d. regenerating a Protein A affinity chromatographic column;

e. desalting the purified sample to an appropriate buffer;

f. the purified samples were aseptically filtered and analyzed for purity by SDS-PAGE, and the results are shown in FIG. 1.

Example 4 detection of FGF21-Fc fusion proteins paired with HRP-labeled mouse anti-human IgG4 Fc antibodies

The monoclonal antibody obtained by purification in example 3 was used as a coating antibody, and an HRP-labeled mouse anti-human IgG4 Fc antibody (Southern Biotech, cat. No. 9200-05) was used as a detection antibody to detect protein A and protein B, respectively, by a double antibody sandwich ELISA, comprising the following steps:

(1) coating: coating the purified mouse monoclonal antibody at 2 mug/mL and 100 mug/hole overnight at 4 ℃;

(2) and (3) sealing: the liquid in the plate is thrown, cleaned and patted dry, 2% BSA (bovine serum albumin) is added into the plate at a concentration of 300 mu L/hole, and the plate is sealed and incubated for 1h at room temperature;

(3) washing the plate: washing the plate for 2 times by 300 mu L/hole washing solution, and finally drying by beating;

(4) sample preparation: respectively diluting protein A and protein B to 5ng/mL and 10ng/mL, adding 100 mu L/well into a pore plate, adding a sample diluent and 100 mu L/well into a blank control, uniformly mixing, and reacting at room temperature for 2 h;

(5) washing the plate: washing the plate for 3 times by using 300 mu L/hole washing solution, and drying by beating for the last time;

(6) adding a secondary antibody: HRP-labeled mouse anti-human IgG4 Fc monoclonal antibody (Southern biotech, cat No. 9200-05) was prepared as 1: diluting with 2000, 100 μ L/well, sealing, and incubating at room temperature for 1 h;

(7) washing the plate: the same step 5 is carried out;

(8) color development: mixing solution A and solution B at a ratio of 1:1, adding 200 μ L per well, and incubating at room temperature in dark for 20 min;

(9) and (4) terminating: 50. mu.L of stop buffer was added to each well, and the OD value was measured immediately at a wavelength of 450nm, and the results are shown in Table 9 (S: signal value; S-B: signal value after background subtraction).

ELISA detection result of double-antibody sandwich method of monoclonal antibody of table 96 strain and mouse anti-human IgG4 Fc antibody marked by HRP

According to table 9, MM21H, MM22H, MM23H, MM24H, MM25H, MM26H all paired with the detection antibody to detect protein a and were not interfered by protein B. MM21H and MM24H, which responded higher and were less background, were selected as candidate monoclonal antibody strains.

EXAMPLE 5 sequencing of monoclonal antibodies

The candidate monoclonal antibody strains MM21H and MM24H obtained by screening in example 4 were subjected to hybridoma cell line antibody subtype detection and sequencing by Sino Biological Inc., Beijing Yi Qian Shen science and technology Co. The supernatants of the respective MM21H and MM24H hybridomas were collected and subtype-tested using a subtype detection kit (Southern Biotech), and the MM21H and MM24H murine monoclonal antibodies were both IgG1/kappa according to the ELISA test results shown in Table 10.

TABLE 10 monoclonal antibody subtype detection results

Respectively centrifuging MM21H and MM24H hybridoma cells, discarding supernatant, extracting cell RNA by using a Tripure Isolation Reagent kit (Roche), carrying out reverse transcription to obtain cDNA, carrying out PrimeSTAR (TAKARA) high-fidelity PCR (polymerase chain reaction) by using cDNA as a template and specific primers (Sino Biological Inc.) according to a determined subtype, inserting the amplified gene fragment into an expression vector pcDNA series vector, and cloning and sequencing to obtain a gene sequence. The recombinant antibodies were named MM21T and MM24T, respectively, and the light chain variable region of MM21T contained the following three complementarity determining regions according to the sequencing results:

CDR-L1, the amino acid sequence is RSSKSLLHSNGITYLY (SEQ ID NO: 1), and the coding nucleotide sequence is SEQ ID NO: 24;

CDR-L2 having the amino acid sequence QMSLAS (SEQ ID NO: 2) and the coding nucleotide sequence SEQ ID NO: 25;

CDR-L3 having an amino acid sequence of AQTLELPT (SEQ ID NO: 4) and a coding nucleotide sequence of SEQ ID NO: 26.

according to the sequencing results, the amino acid sequence of the light chain of MM21T was SEQ ID NO: 27; wherein, the 1 st-19 th sites are signal peptides, the 20 th-130 th sites are light chain variable regions, and the 131 nd-237 th sites are light chain constant regions.

The nucleotide sequence encoding the light chain of MM21T was SEQ ID NO: 28.

the heavy chain variable region of MM21T comprises the following three complementarity determining regions:

CDR-H1, the amino acid sequence is GYTFTNY (SEQ ID NO: 5), the coding nucleotide sequence is SEQ ID NO: 29;

CDR-H2, the amino acid sequence thereof is NTYTGK (SEQ ID NO: 6), the coding nucleotide sequence thereof is SEQ ID NO: 30;

CDR-H3 having amino acid sequence NYYDYDVAY (SEQ ID NO: 7), and encoding nucleotide sequence SEQ ID NO: 31.

according to the sequencing results, the amino acid sequence of the heavy chain of MM21T is SEQ ID NO: 32, a first step of removing the first layer; wherein, the 1 st to 19 th sites are signal peptides, the 20 th to 137 th sites are heavy chain variable regions, and the 138 th and 461 th sites are heavy chain constant regions.

The nucleotide sequence encoding the heavy chain of MM21T is SEQ ID NO: 33.

the light chain variable region of MM24T comprises the following three complementarity determining regions:

CDR-L1', having an amino acid sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 1), and encoding nucleotide sequence of SEQ ID NO: 24;

CDR-L2', the amino acid sequence of which is QMSNLAS (SEQ ID NO: 3), the coding nucleotide sequence of which is SEQ ID NO: 34;

CDR-L3', having the amino acid sequence AQTLELPT (SEQ ID NO: 4), and the coding nucleotide sequence SEQ ID NO: 26.

according to the sequencing results, the amino acid sequence of the light chain of MM24T is SEQ ID NO: 35; wherein, the 1 st-19 th position is a signal peptide, the 20 th-130 th position is a light chain variable region, and the 131 nd-237 th position is a light chain constant region.

The nucleotide sequence encoding the light chain of MM24T was SEQ ID NO: 36.

the heavy chain variable region of MM24T comprises the following three complementarity determining regions:

CDR-H1', the amino acid sequence of which is GYTFTNY (SEQ ID NO: 5), the coding nucleotide sequence of which is SEQ ID NO: 29;

CDR-H2', the amino acid sequence of which is NTYTGK (SEQ ID NO: 6), the coding nucleotide sequence of which is SEQ ID NO: 37;

CDR-H3', having amino acid sequence NYYDYDIAY (SEQ ID NO: 8), encoding nucleotide sequence SEQ ID NO: 38.

according to the sequencing results, the amino acid sequence of the heavy chain of MM24T is SEQ ID NO: 39; wherein, the 1 st to 19 th sites are signal peptides, the 20 th to 137 th sites are heavy chain variable regions, and the 138 th and 461 th sites are heavy chain constant regions.

The nucleotide sequence encoding the heavy chain of MM24T is SEQ ID NO: 40.

example 6 expression and purification of recombinant antibodies MM21T and MM24T

HEK293 cells were subcultured in 293 serum-free CD medium (cat # SMM 293-TI), the expression plasmid containing the genes of the light and heavy chains of the target antibody constructed in example 5 was mixed with a transfection reagent TF1 and added to HEK293 cells, and 293 serum-free feed solution (cat # M293-SUPI-100) was added on days 1, 3, and 5 after transfection. Protein purification was performed 7 days after cell culture. After centrifugation of the HEK293 culture solution, the supernatant was collected by filtration through a filter, and then the collected cell culture solution was purified using a Protein a affinity chromatography column, and an absorption peak was collected. The protein expression process of the obtained protein sample is monitored by SDS-PAGE and UV OD280 methods, and QC detection is carried out on the product.

The results of SDS-PAGE are shown in FIGS. 2-3, and SDS-PAGE analysis revealed that the target antibody was purified from the HEK293 culture supernatant by Protein A affinity purification. Analyzing the molecular weight of the antibody in a range of 150-200 KDa by using non-reducing gel, and according with the characteristic of the molecular weight of the antibody. The bands below the main bands of the antibodies on the non-reducing gel may be due to differences in molecular weight and mode of movement due to varying degrees of glycosylation, while the band at-100 KDa on the non-reducing gel may be due to overexpression of the heavy chain of the antibody, but the overall percentage is < 5%.

Example 7 detection of Activity of MM24T recombinant antibody

The activity of the recombinant MM24T antibody was measured by ELISA method, protein A and protein B were coated at 0.1, 1 and 5. mu.g/mL, respectively, the spotting concentration of the antibody was 1. mu.g/mL, and it was confirmed that MM24T was able to bind to protein A and not to protein B, and was consistent with the binding ability of MM24H, and the measurement results are shown in Table 11 (two parallel experiments were set up, and the results of OD450 in the table were averaged).

TABLE 11 detection and verification of MM24T recombinant expression antibody activity

Example 8 detection of Activity of MM21T recombinant antibody

The activity of the MM21T recombinant antibody was measured by ELISA method, protein A and protein B were coated at 0.1, 1 and 5. mu.g/mL, respectively, the concentration of antibody spotting was 1. mu.g/mL, and it was confirmed that MM21T was able to bind to protein A and not to protein B, and the measurement results are shown in Table 12 (two parallel experiments were set up, and the results of OD450 in the table were averaged).

TABLE 12 detection and verification of MM21T recombinant expression antibody activity

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

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Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 14

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> CL

<400> 14

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

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

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 15

<211> 324

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> CH

<400> 15

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 16

<211> 325

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> CH

<400> 16

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

1 5 10 15

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

20 25 30

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

35 40 45

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

50 55 60

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

65 70 75 80

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

85 90 95

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

100 105 110

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

115 120 125

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

130 135 140

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

145 150 155 160

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

165 170 175

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

180 185 190

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

195 200 205

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

210 215 220

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

225 230 235 240

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

245 250 255

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

260 265 270

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

275 280 285

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

290 295 300

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

305 310 315 320

Leu Ser Pro Gly Lys

325

<210> 17

<211> 330

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> CH

<400> 17

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Val

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 18

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> SinoA8624

<400> 18

Cys Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser Tyr Ala

1 5 10 15

Ser

<210> 19

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> SinoA8625

<400> 19

Cys Gly Gly Gly Arg Ser Pro Ser Tyr Ala Ser

1 5 10

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> SinoA8626

<400> 20

Cys Gly Gly Ser Pro Ser Tyr Ala Ser

1 5

<210> 21

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> SinoA8627

<400> 21

Cys Leu Ser Met Val Gly Pro Ser Gln Gly Arg Ser Pro Ser

1 5 10

<210> 22

<211> 424

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> protein A

<400> 22

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly

245 250 255

Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr

260 265 270

Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala

275 280 285

Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly

290 295 300

Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg

305 310 315 320

Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys

325 330 335

Ser Phe Arg Glu Arg Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser

340 345 350

Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His

355 360 365

Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly

370 375 380

Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro

385 390 395 400

Pro Asp Val Gly Ser Ser Asp Pro Leu His Met Val Gly Ala Ser Gln

405 410 415

Gly Leu Ser Pro Ser Tyr Ala Ser

420

<210> 23

<211> 421

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> protein B

<400> 23

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala

1 5 10 15

Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

180 185 190

Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

225 230 235 240

Gly Gly Ser His Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly

245 250 255

Gly Gln Val Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr

260 265 270

Glu Ala His Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala

275 280 285

Asp Gln Ser Pro Glu Ser Leu Leu Gln Leu Lys Ala Leu Lys Pro Gly

290 295 300

Val Ile Gln Ile Leu Gly Val Lys Thr Ser Arg Phe Leu Cys Gln Arg

305 310 315 320

Pro Asp Gly Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys

325 330 335

Ser Phe Arg Glu Arg Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser

340 345 350

Glu Ala His Gly Leu Pro Leu His Leu Pro Gly Asn Lys Ser Pro His

355 360 365

Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly

370 375 380

Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro

385 390 395 400

Pro Asp Val Gly Ser Ser Asp Pro Leu His Met Val Gly Ala Ser Gln

405 410 415

Gly Leu Ser Pro Ser

420

<210> 24

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> LCDR1

<400> 24

aggtctagta agagtctcct acatagtaat ggcatcactt atttgtat 48

<210> 25

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> LCDR2

<400> 25

cagatgtcca gccttgcctc a 21

<210> 26

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> LCDR3

<400> 26

gctcaaactc tagaacttcc gacg 24

<210> 27

<211> 237

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val

20 25 30

Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu

35 40 45

Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Gln Val Leu Ile Tyr Gln Met Ser Ser Leu Ala Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

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

100 105 110

Ala Gln Thr Leu Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

130 135 140

Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn

145 150 155 160

Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser

165 170 175

Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu

195 200 205

Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser

210 215 220

Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 28

<211> 390

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

atgggctggt cctgtatcat cctgttcctg gtggctacag ccacaggagt gcatagtgac 60

attgtgatga cacaggctgc attctccaat ccagtcactc ttggaacatc agcttccatc 120

tcctgcaggt ctagtaagag tctcctacat agtaatggca tcacttattt gtattggttt 180

ctgcagaagc caggccagtc tcctcaggtc ctgatttatc agatgtccag ccttgcctca 240

ggagtcccag acaggttcag tagcagtggg tcaggaactg atttcacact gagaatcagc 300

agagtggagg ctgaggacgt gggtgtttat tactgtgctc aaactctaga acttccgacg 360

ttcggtggag gcaccaagct ggaaatcaaa 390

<210> 29

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> HCDR1

<400> 29

ggatatacct tcacaaacta t 21

<210> 30

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> HCDR2

<400> 30

aacacctaca ctggaaag 18

<210> 31

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> HCRD3

<400> 31

aactactatg attacgacgt tgcttac 27

<210> 32

<211> 461

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

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

20 25 30

Thr Gly Glu Thr Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Leu Met Gly Trp Ile Asn Thr Tyr Thr Gly Lys Pro Thr Tyr Ala

65 70 75 80

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

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Thr Asn Tyr Tyr Asp Tyr Asp Val Ala Tyr Trp Gly

115 120 125

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

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 33

<211> 1386

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtgc ccaagcacag 60

atccagttgg tgcagtctgg acctgagctg aagaagactg gagagacagt caagatgtcc 120

tgcaaggctt ctggatatac cttcacaaac tatggaatga actgggtgaa gcaggctcca 180

ggaaagggtt taaagttgat gggctggata aacacctaca ctggaaagcc aacatatgct 240

gatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300

cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgcaac aaactactat 360

gattacgacg ttgcttactg gggccaaggg actctggtca ctgtctctgc agccaaaacg 420

acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 480

accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 540

ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 600

ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 660

gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 720

tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 780

cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 840

agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 900

gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 960

cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 1020

gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1080

caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1140

tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1200

ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1260

tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 1320

gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 1380

aaataa 1386

<210> 34

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> LCDR2

<400> 34

cagatgtcca accttgcctc a 21

<210> 35

<211> 237

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Met Ala Pro Thr Gln Leu Leu Gly Leu Leu Val Leu Trp Ile Pro Gly

1 5 10 15

Ser Thr Ala Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro Val

20 25 30

Thr Leu Gly Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu

35 40 45

Leu His Ser Asn Gly Ile Thr Tyr Leu Tyr Trp Phe Leu Gln Lys Pro

50 55 60

Gly Gln Ser Pro Gln Val Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

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

100 105 110

Ala Gln Thr Leu Glu Leu Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu

115 120 125

Ile Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser

130 135 140

Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn

145 150 155 160

Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser

165 170 175

Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys

180 185 190

Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu

195 200 205

Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser

210 215 220

Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 36

<211> 714

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

atggccccaa ctcagctcct ggggctgctt gtgctctgga tccctggatc cactgcagat 60

attgtgatga cgcaggctgc attctccaat ccagtcactc ttggaacatc agcttccatc 120

tcctgcaggt ctagtaagag tctcctacat agtaatggca tcacttattt gtattggttt 180

ctgcagaagc caggccagtc tcctcaggtc ctgatttatc agatgtccaa ccttgcctca 240

ggagtcccag acaggttcag tagcagtggg tcaggaactg atttcacact gagaatcagc 300

agagtggagg ctgaggacgt gggtgtttat tactgtgctc aaactctaga acttccgacg 360

ttcggtggag gcaccaagct ggaaatcaaa cgggctgatg ctgcaccaac tgtatccatc 420

ttcccaccat ccagtgagca gttaacatct ggaggtgcct cagtcgtgtg cttcttgaac 480

aacttctacc ccaaagacat caatgtcaag tggaagattg atggcagtga acgacaaaat 540

ggcgtcctga acagttggac tgatcaggac agcaaagaca gcacctacag catgagcagc 600

accctcacgt tgaccaagga cgagtatgaa cgacataaca gctatacctg tgaggccact 660

cacaagacat caacttcacc cattgtcaag agcttcaaca ggaatgagtg ctaa 714

<210> 37

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> HCDR2

<400> 37

aacacctaca ctggaaaa 18

<210> 38

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221>

<222>

<223> HCDR3

<400> 38

aactactatg attacgacat tgcttac 27

<210> 39

<211> 461

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 39

Met Gly Trp Val Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

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

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Leu Met Gly Trp Ile Asn Thr Tyr Thr Gly Lys Pro Thr Tyr Ala

65 70 75 80

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

85 90 95

Thr Ala Tyr Leu Gln Leu Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Thr Asn Tyr Tyr Asp Tyr Asp Ile Ala Tyr Trp Gly

115 120 125

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

130 135 140

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

145 150 155 160

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro

195 200 205

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

210 215 220

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

225 230 235 240

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

245 250 255

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

260 265 270

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

275 280 285

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

290 295 300

Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu

305 310 315 320

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

325 330 335

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

340 345 350

Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro

355 360 365

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

370 375 380

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

385 390 395 400

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

405 410 415

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

420 425 430

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

435 440 445

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 40

<211> 1386

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atgggttggg tgtggaactt gctattcctg atggcagctg cccaaagtgc ccaagcacag 60

atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc 120

tgcaaggctt ctggatatac cttcacaaac tatggaatga actgggtgaa gcaggctcca 180

ggaaagggtt taaagttgat gggctggata aacacctaca ctggaaaacc aacatatgct 240

gatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300

cagctcaaca acctcaagaa tgaggacacg gctacatatt tctgtgcaac aaactactat 360

gattacgaca ttgcttactg gggccagggg actctggtcg ctgtctctgc agccaaaacg 420

acacccccat ctgtctatcc actggcccct ggatctgctg cccaaactaa ctccatggtg 480

accctgggat gcctggtcaa gggctatttc cctgagccag tgacagtgac ctggaactct 540

ggatccctgt ccagcggtgt gcacaccttc ccagctgtcc tgcagtctga cctctacact 600

ctgagcagct cagtgactgt cccctccagc acctggccca gcgagaccgt cacctgcaac 660

gttgcccacc cggccagcag caccaaggtg gacaagaaaa ttgtgcccag ggattgtggt 720

tgtaagcctt gcatatgtac agtcccagaa gtatcatctg tcttcatctt ccccccaaag 780

cccaaggatg tgctcaccat tactctgact cctaaggtca cgtgtgttgt ggtagacatc 840

agcaaggatg atcccgaggt ccagttcagc tggtttgtag atgatgtgga ggtgcacaca 900

gctcagacgc aaccccggga ggagcagttc aacagcactt tccgctcagt cagtgaactt 960

cccatcatgc accaggactg gctcaatggc aaggagttca aatgcagggt caacagtgca 1020

gctttccctg cccccatcga gaaaaccatc tccaaaacca aaggcagacc gaaggctcca 1080

caggtgtaca ccattccacc tcccaaggag cagatggcca aggataaagt cagtctgacc 1140

tgcatgataa cagacttctt ccctgaagac attactgtgg agtggcagtg gaatgggcag 1200

ccagcggaga actacaagaa cactcagccc atcatggaca cagatggctc ttacttcgtc 1260

tacagcaagc tcaatgtgca gaagagcaac tgggaggcag gaaatacttt cacctgctct 1320

gtgttacatg agggcctgca caaccaccat actgagaaga gcctctccca ctctcctggt 1380

aaataa 1386

Claims (12)

1. An anti-FGF 21 carboxy-terminal antibody comprising a light chain variable region comprising the amino acid sequences LCDR1, LCDR2 and LCDR3 and a heavy chain variable region comprising HCDR1, HCDR2 and HCDR 3; wherein the amino acid sequence of LCDR1 is SEQ ID NO: 1, the amino acid sequence of LCDR2 is SEQ ID NO: 2 or SEQ ID NO: 3, the amino acid sequence of the LCDR3 is SEQ ID NO: 4, the amino acid sequence of the HCDR1 is SEQ ID NO: 5, the amino acid sequence of HCDR2 is SEQ ID NO: 6, the amino acid sequence of HCDR3 is SEQ ID NO: 7 or SEQ ID NO: 8.

2. the antibody of claim 1, wherein the variable region of the light chain of said antibody has the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: shown at 10.

3. The antibody of claim 1, wherein the heavy chain variable region of said antibody has the amino acid sequence set forth in SEQ ID NO: 11 or SEQ ID NO: shown at 12.

4. The antibody of claim 1, wherein the variable region of the light chain of said antibody has the amino acid sequence of SEQ ID NO: 9, and the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: 11 is shown in the figure; or the amino acid sequence of the variable region of the light chain of the antibody is shown as SEQ ID NO: 10, and the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO: shown at 12.

5. The antibody of any one of claims 1-4, wherein said antibody is a full length antibody, a Fab fragment, F (ab)₂A fragment, a two-chain Fv fragment, or a single-chain Fv fragment; preferably, the antibody is a monoclonal antibody.

6. The antibody of any one of claims 1-4, further comprising a light chain constant region selected from the group consisting of a kappa or lambda subtype; preferably, the light chain constant region is of the kappa subtype; preferably, the light chain constant region comprises the amino acid sequence as set forth in SEQ ID NO: 13 or SEQ ID NO: 14, or a pharmaceutically acceptable salt thereof.

7. The antibody of any one of claims 1-4, further comprising a heavy chain constant region selected from the group consisting of IgG1, IgG2, IgG3, IgG4 subtypes; preferably, the heavy chain constant region is of the IgG1 subtype; preferably, the heavy chain constant region comprises the amino acid sequence as set forth in SEQ ID NO: 15. SEQ ID NO: 16 or SEQ ID NO: 17.

8. A nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-7.

9. A vector comprising the nucleic acid molecule of claim 8;

preferably, the vector comprises a plasmid, a bacteriophage, a plant cell virus, a mammalian cell virus or a retrovirus.

10. A host cell containing the nucleic acid molecule of claim 8, or comprising the vector of claim 9;

preferably, the host cell comprises a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell;

preferably, the host cell is a mammalian cell;

preferably, the host cell is a HEK293 cell.

11. A test kit comprising the antibody of any one of claims 1-7, the nucleic acid molecule of claim 8, or the vector of claim 9.

12. Use of an antibody according to any one of claims 1 to 7 for immunodetection for diagnostic or non-diagnostic purposes;

preferably, the immunoassay comprises: the integrity of the carboxy terminus of FGF21 was examined.

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