CN101701039B - Variable regions of light chains and heavy chains of FMU-EPCAM-2A9 monoclonal antibodies - Google Patents

Abstract

The present invention discloses the variable regions of the light chain and the heavy chain of the FMU-EPCAM-2A9 monoclonal antibody. The amino acid sequence of the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the variable region of the heavy chain is shown in Shown in SEQ ID NO.2. By immunizing BALB/c mice with recombinant human EPCAM, a group of mouse anti-human EPCAM monoclonal antibodies were prepared, a hybridoma cell line that could stably secrete high-affinity anti-human EPCAM monoclonal antibodies was screened, and ascites was prepared to obtain high-affinity anti-human EPCAM antibodies. Human EPCAM monoclonal antibody. Confirm the uniqueness of the gene sequence and corresponding protein sequence and its CDR sequence; provide support for anti-human EPCAM chimeric or humanized genetic engineering antibodies.

Description

Light and heavy chain variable regions of FMU-EPCAM-2A9 monoclonal antibody

technical field

The invention belongs to the field of biomedical technology, and relates to a monoclonal antibody, in particular to a light chain and heavy chain variable region of an anti-human EPCAM FMU-EPCAM-2A9 monoclonal antibody, including its amino acid sequence and nucleotide sequence .

Background technique

Epithelial cell adhesion molecule (EPCAM) was originally discovered as a dominant epitope in colon cancer, and it was considered to be a reliable binding site for cell adhesion and therapeutic antibodies. Current studies believe that EPCAM is a glycosylated type I transmembrane glycoprotein with a molecular weight of 30 to 40kD. Its structure includes an epidermal growth factor-like and a thyroglobulin-like extracellular region, a transmembrane region and a Cytoplasmic domain of 26 amino acids. In normal cells, EPCAM is mainly located in the tight junction of the epithelial cell space. As shown by the intensity and frequency of EPCAM expression, it is highly expressed in substantially all human adenocarcinomas, some squamous cell carcinomas, retinoblastomas and hepatocellular carcinomas.

The research on the function of EPCAM before 2007 found that EPCAM plays an important role in cell proliferation, migration and signal transduction. For example: enhanced expression of EPCAM in human and rodent cells enhanced cell anchorage and serum growth factor-independent proliferation and increased expression of various target genes such as c-myc and cyclins; in breast cancer cells Line, EPCAM signal through RNA interference, can cause the reduction of cell proliferation, migration and invasion ability.

Recent studies have confirmed that: EPCAM can be used as one of the surface markers of some solid tumor stem cells, and can also be used as a component of the surface markers of normal stem cells. At the same time, the research results also explained the reason for the high expression of EPCAM in tumor cells, and found that it was related to the poor prognosis and low survival rate of some tumor patients; and further explained the relationship between EPCAM and tumors. In addition, mouse embryonic stem cell experiments have confirmed that the high expression of EPCAM on tumor stem cells and normal stem cells and the upregulation of its own positive feedback are very important to ensure the maintenance of the proliferation of these normal stem cells and malignant stem cells.

The study of anti-EPCAM antibody for tumor therapy has always been a hotspot in the study of antibody therapy. The mouse-derived anti-EPCAM antibody 17-1A developed as early as 1979 was used in the clinical treatment of tumors in 1982. However, when the mouse-derived antibody drug is used in the human body, due to its immunogenicity, it will cause an immune response in the human body, resulting in the clearance of the antibody drug or hypersensitivity mediated by immune complexes. Genetic modification of murine antibodies can partially solve the problem of immunogenicity, such as constructing chimeric antibodies or humanized antibodies.

The antibody monomer molecule is a tetrapeptide chain structure formed by two identical heavy chains (H chains) and two identical light chains (L chains) connected by interchain disulfide bonds. The H chain and the L chain include the amino (N) terminal and the carboxyl (C) terminal, and the variable region (V region) near the N-terminal is composed of a hypervariable region/complementarity determining region (HVR/CDR) and a framework region (FR); The C-terminus is the constant region (C region). The protein fold formed by the variable region of the heavy chain (VH) and the variable region of the light chain (VL) is the antigen binding site, and the CDR/HVR is the site where the antibody and the epitope are complementary to each other, and the C region triggers the recognition of the antigen and the antibody. reaction. Antibodies can be divided into human-derived and mouse-derived antibodies according to the FR/C region. Mouse-derived antibodies are immunogenic when used in the human body and are likely to cause immune responses in the human body. These immune reactions can cause the removal of mouse-derived antibodies and Immune complex-mediated hypersensitivity. In order to overcome the defects of murine antibodies, it is necessary to construct high-affinity specific chimeric antibodies, single-chain antibodies or humanized antibodies.

In the transformation process, the most important thing is to first obtain the murine parent antibody with good specificity and affinity, clone its light chain and heavy chain variable region genes, and then transform these two genes to construct the recombinant antibody gene. In response to these problems, the research and development of anti-EPCAM therapeutic antibodies continue to make progress, anti-EPCAM rat and mouse bispecific antibody Catumaxomab, single-chain antibody Proxinium, humanized antibody ING-1, humanized fusion antibody EMD and The fully humanized antibody Adecatumumab (MT201) has entered clinical research on tumor treatment. Currently in clinical trials, the modified antibody Adecatumumab has achieved good results in the treatment of breast cancer. Therefore, screening out high-affinity mouse-derived anti-human EPCAM monoclonal antibodies, and cloning the light and heavy chain variable region genes from them, will further improve the preparation of drugs targeting EPCAM and the treatment of tumors. .

Contents of the invention

The problem solved by the present invention is to provide a light chain and heavy chain variable region of an anti-human EPCAM FMU-EPCAM-2A9 monoclonal antibody, including its amino acid and nucleotide sequences, for the construction of high-affinity anti-EPCAM chimeric or Humanized genetically engineered antibodies provide support.

The present invention is achieved through the following technical solutions:

The light chain and heavy chain variable regions of the FMU-EPCAM-2A9 monoclonal antibody, the three complementarity determining region (CDR) sequences of the light chain variable region are respectively:

CDR1: Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-Thr-Tyr;

CDR2: Val-Val-Ser-Lys-Leu-Asp-Ser;

CDR3: Trp-Gln-Gly-Thr-His-Phe-Pro-Trp-Thr;

The three complementarity determining region (CDR) sequences of the heavy chain variable region are:

CDR1: Gly-Tyr-Ser-Phe-Thr-Ser-Tyr-Trp;

CDR2: Ile-Tyr-Pro-Gly-Asn-Ser-Ala;

CDR3: Ile-Arg-Gly-Gly-Asn-Tyr.

The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

The gene sequence encoding the variable region of the light chain of the monoclonal antibody is shown in SEQ ID NO.3, and the sequence of the gene encoding the variable region of the heavy chain of the monoclonal antibody is shown in SEQ ID NO.4.

The monoclonal antibody is applied to the construction of genetically engineered antibodies or diagnostic reagents targeting human EPCAM.

Compared with the prior art, the present invention has the following beneficial technical effects:

1. The FMU-EPCAM-2A9 monoclonal antibody provided by the present invention is a high-affinity monoclonal antibody against human EPCAM, which is confirmed by indirect ELISA detection, flow cytometry detection and pathological section immunohistochemical detection Can specifically bind human EPCAM;

The monoclonal antibody light chain, heavy chain variable region genes and amino acid sequences were cloned, and sequence analysis confirmed the uniqueness of the antibody sequence.

2. Analyze the CDR regions of the light chain and heavy chain variable regions, and provide support for the construction of high-affinity anti-human EPCAM chimeric or humanized genetically engineered antibodies on this basis.

Description of drawings

Fig. 1 is the result figure of the flow cytometry detection of the FMU-EPCAM-2A9 monoclonal antibody against human EPCAM combined with the EPCAM expressed on the surface of COLO205 and SKBr-3 cell lines; 1b is a graph of the detection results of the combination with the SKBr-3 cell line; wherein, FITC: fluorescein isothiocyanate, the horizontal axis is the fluorescence intensity, the vertical axis is the relative cell count, and M1 represents the range of positive cells;

Figure 2 is the results of immunohistochemical staining of the combination of FMU-EPCAM-2A9 monoclonal antibody against human EPCAM and EPCAM expressed in colon cancer specimens; ;

Fig. 3 is the FMU-EPCAM-2A9 anti-human EPCAM monoclonal antibody light chain variable region gene homology sequence detection result figure;

Fig. 4 is the FMU-EPCAM-2A9 monoclonal antibody heavy chain variable region gene homology sequence detection result figure of anti-human EPCAM;

Figure 5 is a diagram showing the detection results of amino acid sequence homology in the light chain variable region of the FMU-EPCAM-2A9 monoclonal antibody against human EPCAM;

Fig. 6 is a diagram showing the detection results of the amino acid sequence homology of the heavy chain variable region of the anti-human EPCAM FMU-EPCAM-2A9 monoclonal antibody.

Detailed ways

The applicant immunized BALB/c mice with recombinant human EPCAM, prepared a group of mouse anti-human EPCAM monoclonal antibodies, and screened a hybridoma cell line that can stably secrete FMU-EPCAM-2A9 monoclonal antibody with high affinity against human EPCAM , Prepare ascites fluid to obtain high-affinity anti-human EPCAM monoclonal antibody. Confirm the uniqueness of the gene sequence and corresponding protein sequence and its CDR sequence; provide support for anti-human EPCAM chimeric or humanized genetic engineering antibodies.

The present invention will be described in detail below in conjunction with the accompanying drawings, which are explanations of the present invention rather than limitations.

The present invention is specifically implemented according to the following steps:

1 Preparation of mouse anti-human EPCAM high-affinity antibody

1.1 Preparation and purification of monoclonal antibodies

Design primers with reference to the sequence of GENBANK NM_002354, and amplify the gene encoding the extramembrane protein of human EPCAM; then clone this gene into the pGEX-4T-3 vector, construct a prokaryotic expression vector and transfect host cells; and then induce by IPTG, The soluble protein in the extracellular region of recombinant human EPCAM was obtained by freeze-thawing and ultrasonic lysis.

According to the monoclonal antibody preparation method (Cellular and Molecular Immunology Experimental Technology First Edition, P9-P17), BALB/c mice (purchased from the Experimental Animal Center of Fourth Military Medical University) were immunized with recombinant human EPCAM extracellular domain protein; Freund's complete adjuvant was used for the initial immunization, and Freund's incomplete adjuvant was used for the subsequent immunizations, with an interval of 3 weeks between each time, subcutaneous injections at multiple points, and a total of 4 immunizations. Blood was collected 7-10 days after the last immunization to measure its potency and detect the immune effect. After an interval of 2-3 weeks, the antigen was injected intravenously to boost the immunization, and the animals were sacrificed 3 days later to obtain the spleen for cell fusion.

The logarithmic growth of mouse myeloma cells SP2/0 was counted, and the immune spleen cell suspension was prepared at the same time. Myeloma cells and spleen cells were mixed at a ratio of 1:10 for cell fusion. After fusion, the cell suspension was added to a 96-well plate containing feeder cells, and cultured in a 37°C, 5% CO ₂ incubator. After the clones appeared, they were tested by indirect ELISA and positive clones were selected. Cells containing positive cloning wells were cloned by the limiting dilution method until a hybridoma cell line capable of stably secreting antibodies was obtained (in vitro continuous culture for more than 6 months), and routine karyotype identification and Ig subclass determination were performed on them ( The result is IgG2a subclass).

After obtaining a hybridoma cell line capable of stably secreting antibodies, prepare ascites fluid containing monoclonal antibodies according to the method for preparing mouse ascites (Experimental Techniques of Cellular and Molecular Immunology First Edition, P9-P17). Ascitic fluid was precipitated with 40% saturated ammonium sulfate and purified by QFF anion exchange chromatography. The purity of the purified antibody was identified by SDS-PAGE, and the purity of the purified monoclonal antibody FMU-EPCAM-2A9 reached 95%.

1.2 Titer determination of anti-human EPCAM monoclonal antibody

The relative affinities of the ascites fluid before and after purification were determined by indirect ELISA. The coating antigen is the soluble protein in the extracellular region of recombinant human EPCAM, the sample to be tested is serially diluted ascitic fluid and purified mAb, the detection antibody is goat anti-mouse-HRP enzyme-labeled antibody, and ABTS is used as the substrate. The high-affinity FMU-EPCAM-2A9mAb screened out has a titer of 1×10 ^-7 in ascites, and a titer of 0.5 ng/ml after purification. Generally, indirect ELISA is used to detect antibodies with a titer of more than 1×10 ^-5 in ascites Ready to use.

1.3 Fluorescent staining by flow cytometry to detect the activity of anti-human EPCAM monoclonal antibody FMU-EPCAM-2A9 binding to EPCAM on the cell surface

Using SEDmAb as a negative antibody control, flow cytometry was used to detect the binding of FMU-EPCAM-2A9mAb to EPCAM expressed on the surface of COLO205 and SKBr-3 cell lines;

COLO205 and SKBr-3 cell lines were cultured with 10% FCS-RPMI1640 medium to the logarithmic growth phase, and the cell concentration was adjusted to 5×10 ⁶ ～1×10 ⁷ /ml; take 50 μl of cell suspension and add specific FMU-EPCAM -2A9mAb 1 μl of ascites fluid, plus 1 μl of inactivated normal rabbit serum, incubated at 4°C for 30 minutes; the same method was used to prepare a negative control group.

Wash the cells twice with washing solution (5% FCS-PBS-4% NaN ₃ ), add 2 ml of washing solution each time, centrifuge at 1000 rpm for 5 min and discard the supernatant; add 100 μl of working concentration of goat anti-mouse fluorescently labeled antibody, shake well Shake, incubate at 4°C for 30 minutes;

Wash twice with washing solution, add 2ml each time, discard the supernatant after centrifuging at 1000rpm×5min; add a certain amount of fixative, and then perform flow cytometry analysis of the binding of FMU-EPCAM-2A9mAb to EPCAM on the cell surface;

The results are shown in Figure 1, in the COLO205 cell line: compared with the negative control, the COLO205 cells combined with FMU-EPCAM-2A9mAb and falling into the range of M1 positive cells reached 46%;

In the SKBr-3 cell line: Compared with the negative control, 24% of SKBr-3 cells that fall into the range of M1 positive cells combined with FMU-EPCAM-2A9 mAb;

The above flow cytometry fluorescence staining detection shows that FMU-EPCAM-2A9mAb can recognize and bind to the natural EPCAM expressed on the surface of COLO205 and SKBr-3 cells.

1.4 Immunohistochemical staining to detect the binding of FMU-EPCAM-2A9mAb to EPCAM in colon cancer specimens

Colon cancer paraffin sections (provided by the Department of Pathology, Fourth Military Medical University) were dewaxed to water by passing through xylene I, II, 100% alcohol I, II, 95%, 90%, 80% and 70% alcohol for 10 min each; 0.3% methanol-H ₂ O ₂ for 30 minutes to inactivate endogenous peroxidase; 0.01M PBS (pH7.3) rinse 10 minutes, rinse 3 times; 95 ° C antigen retrieval solution (purchased from Fuzhou Maixin Biotechnology Development Co., Ltd.) for 20 minutes; block for 30 minutes (blocking solution is 1% normal rabbit serum or 5% normal sheep serum);

Add primary antibody (FMU-EPCAM-2A9mAb ascites diluted 1:100), incubate at 4°C in a wet box for more than 18 h; rinse with 0.01M PBS (pH7.3) for 10 min×3 times; add HRP-labeled goat anti-mouse secondary antibody, and store at room temperature Incubate in a wet box for 2h to 3h;

Rinse with 0.01M PBS (pH7.3) for 10min x 3 times; stop with PBS after 7-8min of DAB color development.

Observed under a light microscope, counterstained with hematoxylin for 10-20 seconds; the results are shown in Figure 2, the part of the colon cancer specimen bound to FMU-EPCAM-2A9 mAb turned brown, indicating that the EPCAM expressed in the colon cancer specimen could be identified.

Through the above three kinds of FMU-EPCAM-2A9mAb binding experiments, the identification results at different levels show that FMU-EPCAM-2A9mAb can recognize both recombinantly expressed EPCAM (indirect ELISA detection), and can recognize EPCAM naturally expressed on the surface of cell lines and tissues (Immunohistochemical staining), indicating that FMU-EPCAM-2A9mAb has good specificity and affinity in combination with the corresponding antigen EPCAM.

2 Cloning of light chain and heavy chain variable region genes of anti-human EPCAM FMU-EPCAM-2A9 monoclonal antibody

2.1 Culture of FMU-EPCAM-2A9 hybridoma cells

Resuscitate (cell culture, first edition, P88) FMU-EPCAM-2A9 hybridoma cells according to conventional methods, culture with RPMI 1640 containing 10% calf serum, and cultivate to logarithmic growth in an incubator at 37°C with 5% CO ₂ Expect.

2.2 Extraction of total RNA and synthesis of cDNA first strand

Total RNA was extracted with TRIZOL Reagent (purchased from GIBCO, USA) according to the instructions.

2.3 RT-PCR method to amplify the VL and VH genes of FMU-EPCAM-2A9mb

The one-step RT-PCR amplification kit was purchased from TakaRa Company, and the VL of FMU-EPCAM-2A9mAb and the VH gene of FMU-EPCAM-2A9mAb were amplified according to the kit instructions;

Using two pairs of primers VL F (upstream primer) and VL B (downstream primer) and VHF and VH B, RT-PCR was carried out using total RNA as a template;

The reaction volume is 50μl, and the reaction conditions are: 94°C for 5min; 95°C for 30s, 58°C for 1min, 72°C for 1min, cycle 35 times; 72°C for 7min; the primer sequence is:

VL F: gttagatctc cagcttggtc cc 22;

VL B: gacattcagc tgacccagtc tcca 24;

VH F: tgaggagacg gtgaccgtgg tcccttggcc ccag 34;

VH B: aggtsmarct gcagsagtcw gg 22;

(IUB standard merger base code: s:c/g; m:a/c; r:a/g; w:a/t)

2.4 Cloning and screening of PCR amplification products

The PCR product was subjected to 1.5% agarose gel electrophoresis, and the PCR amplified fragment was recovered with a small gel recovery kit (purchased from Shanghai Huashun Bioengineering Co., Ltd.), and the fragment was reclaimed with a DNA ligation kit (purchased from TakaRa Company). According to the instructions, insert the A tail into the pMD-T18 vector (purchased from TakaRa Company), and transform the linker into E.coli (purchased from China General Microorganism Culture Collection Center, CGMCC, Beijing), and culture the plate on Amp-resistant LB agar. Incubate overnight at 37°C.

Pick the clones from the LB agar culture plate, shake the bacteria in the Amp-resistant LB medium at 37°C overnight, use 1 μl of the bacterial liquid as a template, and use the PCR method to screen for recombinants through the above-mentioned primers designed for the variable regions of the light chain and heavy chain. Positive E.coli clone;

Shake the obtained recombinant positive E.coli clone, and send the bacterial solution to Shanghai Bioengineering Technology Service Co., Ltd. to complete the gene sequence determination. The gene sequence of the light chain variable region is shown in SEQ ID NO.3, and the heavy chain variable region The gene sequence is shown in SEQ ID NO.4.

Nucleotide sequence and homology analysis of the light chain and heavy chain variable regions of 3FMU-EPCAM-2A9 mAb

3.1 After confirming that the sequencing is correct, perform nucleotide sequence homology analysis (Blastn) in the GenBank+EMBL+DDBJ+PDB database.

The FMU-EPCAM-2A9mAb light chain variable region gene has the highest homology with the mouse Ig light chain variable region gene numbered GeneID: 243420, reaching 292/299 (97%), as shown in Figure 3;

The heavy chain variable region gene of FMU-EPCAM-2A9 mAb has the highest homology with the mouse Ig heavy chain variable region gene numbered GeneID: 668469, which is 262/288 (90%), as shown in FIG. 4 .

Homology analysis shows that although the nucleotide sequence of the light and heavy chain variable regions of coding FMU-EPCAM-2A9mAb has certain homology with other gene sequences, no gene sequence completely identical with the present invention has been found, indicating that The invention has uniqueness in gene sequence.

3.2 Translate the variable region gene into amino acid sequence and analyze the amino acid sequence

The amino acid sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2.

Amino acid sequence homology analysis (Blastp) was performed in the non-redundant Genbank CDS translations+PDB+SwissProt+PIR+PRF protein database.

The analysis results showed that the light chain amino acid sequence of FMU-EPCAM-2A9mAb had the highest homology with the mouse Igκ chain numbered ABR32168.1GI: 149799217, reaching 106/111 (95%), as shown in Figure 5;

The heavy chain amino acid sequence of FMU-EPCAM-2A9 mAb has the highest homology with the mouse Ig heavy chain protein numbered S55541 GI: 1363159, which is 98/117 (83%), as shown in Figure 6.

Homology analysis shows that although the amino acid sequences of FMU-EPCAM-2A9mAb light and heavy chain variable regions have certain homology with other protein amino acid sequences, no amino acid sequences completely identical to those of the present invention have been found, indicating that the present invention is in Amino acid sequence is also unique.

3.3 Use IMGT/V-QUEST to analyze the variable region structure and determine the CDR region.

The light chain and heavy chain variable region sequences of the FMU-EPCAM-2A9 mAb antibody obtained by sequencing were analyzed on the IMGT/V-QUEST website (http://imgt.cines.fr/IMGT_vquest/vquest) to obtain the CDR region.

The 3 complementarity determining region (CDR) sequences of the light chain variable region, as shown in the underlined part of SEQ ID NO.1, are specifically:

CDR1: Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-Thr-Tyr;

CDR2: Val-Val-Ser-Lys-Leu-Asp-Ser;

CDR3: Trp-Gln-Gly-Thr-His-Phe-Pro-Trp-Thr;

The 3 complementarity determining regions (CDR) sequences of the heavy chain variable region are shown in the underlined part of SEQ ID NO.2, specifically:

CDR1: Gly-Tyr-Ser-Phe-Thr-Ser-Tyr-Trp;

CDR2: Ile-Tyr-Pro-Gly-Asn-Ser-Ala;

CDR3: Ile-Arg-Gly-Gly-Asn-Tyr.

4. Genetic engineering antibody design

Based on the expression, purification and sequence analysis of the anti-human EPCAM monoclonal antibody FMU-EPCAM-2A9, the following biological products were designed and constructed

1) Construction of a single-chain antibody: the FMU-EPCAM-2A9mAb light and heavy chain variable region genes of the present invention can be connected through a linker, inserted into a prokaryotic or eukaryotic expression vector, transformed into a host bacterium or transfected into a eukaryotic cell for use in Preparation of single-chain antibodies that can treat tumors such as breast cancer, colon cancer, and pancreatic cancer.

2) Construction of human-mouse anti-EPCAM chimeric antibody: FMU-EPCAM-2A9mAb light and heavy chain variable region genes of the present invention can be inserted into a general-purpose chimeric antibody expression vector to obtain vector transfection containing the chimeric gene Eukaryotic cells are used to prepare chimeric antibodies that are expected to have therapeutic effects on tumors such as breast cancer, colon cancer, and pancreatic cancer.

3) Construction of humanized antibody: The CDR regions of the FMU-EPCAM-2A9mAb light and heavy chain variable region genes of the present invention can be grafted into the framework region (FR) of the variable region of the human antibody to form complementarity determining regions (CDR) grafted antibody (CDR-grafted antibody), also known as reshaped antibody (reshaping antibody) or humanized antibody (humanized antibody).

Using CDR transplantation technology to modify antibodies, we can obtain new antibodies that not only maintain the specificity of mouse-derived parental mAbs, but also are closer to human antibodies, and are used to prepare humanized antibodies that can treat tumors such as breast cancer, colon cancer, and pancreatic cancer. Antibody.

4) Other biological products targeting human EPCAM functional epitopes can be prepared according to the gene sequence of the present invention and its encoded amino acid sequence.

5) The FMU-EPCAM-2A9 specific monoclonal antibody against human EPCAM of the present invention can be used as a tool for distinguishing epithelial and non-epithelial-derived tissues to diagnose some confusing diseases. By staining with the FMU-EPCAM-2A9 specific monoclonal antibody against human EPCAM of the present invention, the expression intensity of EPCAM in tissues can be observed, which may be used as a marker for judging the prognosis and degree of differentiation of some epithelial-derived tumors.

6) The high-affinity FMU-EPCAM-2A9mAb of the present invention can be used to prepare an ELISA kit for measuring the EPCAM extracellular domain protein in body fluids, which is expected to have extensive application value in the clinical diagnosis of tumor occurrence, development and prognosis.

List of Amino Acid and Nucleotide Sequences

<110> Fourth Military Medical University of Chinese People's Liberation Army

<120> Light chain and heavy chain variable regions of FMU-EPCAM-2A9 monoclonal antibody

<160>4

<210>1

<211>111

<212>PRT

<213> Synthetic

<400>1

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

1 5 10 15 20

Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn Trp

25 30 35 40

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

45 50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro

85 90 95 100

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

105 110

<210>2

<211>113

<212>PRT

<213> Synthetic

<400>2

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

1 5 10 15 20

Ser Cys Arg Ala Ser Gly Tyr Ser Phe Tyr Ser Tyr Trp Leu His Trp Ile Lys Gln Arg

25 30 35 40

Pro Gly Gln Gly Leu Glu Trp Val Gly Gly Ile Tyr Pro Gly Asn Ser Ala Thr Ser Tyr

45 50 55 60

Lys Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Ala Val Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Ile Arg Gly Gly

85 90 95 100

Asn Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

105 110

<210>3

<211>333

<212>DNA

<213> Synthetic

<400>3

gatgttgtgc tgacccagtc tccactcact ttgtcggtta ccattggaca accagcctcc 60

atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120

ttgttccaga ggccaggcca gtctccaaag cgcctgatct atgtggtgtc taaactggac 180

tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240

agtagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acactttccg 300

tggacgttcg gtggagggac caagctggag atc 333

<210>4

<211>339

<212>DNA

<213> Synthetic

<400>4

gaggtgaagc tgcaggagtc tgggactgtg ctggcaaggc ctgggacttc cgtgaagatg 60

tcctgcaggg cttctggcta cagttttacc agctactggt tgcactggat aaaacagagg 120

cctggacagg gtctagaatg ggttggtggt atctatcctg gaaatagtgc tactagttac 180

aaacagaagt ttaaggacaa ggccacactg actgcagtca catccgccag tactgcctac 240

atggaactca gtagcctgac aaatgaggac tctgcggtct attackgcat aagagggggg 300

aactactggg gccaagggac cacggtcacc gtctcctca 339

Claims (4)

1.FMU-EPCAM-2A9 monoclonal antibody comprises light chain and heavy chain, it is characterized in that, 3 complementary determining regions (CDR) sequence of the variable region of described light chain is:

CDR1：Gln-Ser-Leu-Leu-Asp-Ser-Asp-Gly-Lys-Thr-Tyr；

CDR2：Val-Val-Ser-Lys-Leu-Asp-Ser；

CDR3：Trp-Gln-Gly-Thr-His-Phe-Pro-Trp-Thr；

3 complementary determining regions (CDR) sequence of the variable region of described heavy chain is:

CDR1：Gly-Tyr-Ser-Phe-Thr-Ser-Tyr-Trp；

CDR2：Ile-Tyr-Pro-Gly-Asn-Ser-Ala；

CDR3：Ile-Arg-Gly-Gly-Asn-Tyr。

2. FMU-EPCAM-2A9 monoclonal antibody as claimed in claim 1 is characterized in that, the aminoacid sequence of described monoclonal antibody variable region of light chain such as SEQ ID NO.1, and the aminoacid sequence of variable region of heavy chain is shown in SEQ ID NO.2.

3. FMU-EPCAM-2A9 monoclonal antibody as claimed in claim 1 is characterized in that, the gene order of coding monoclonal antibody variable region of light chain is shown in SEQ ID NO.3, and the gene order of coding monoclonal antibody variable region of heavy chain is shown in SEQ ID NO.4.

4. to be applied to make up with people EPCAM be the preparation of the genetic engineering antibody or the diagnostic reagent of target spot to the described FMU-EPCAM-2A9 monoclonal antibody of claim 1.

Images