Monoclonal antibody and application thereof, nucleotide molecule, vector or recombinant vector, and transformant

Abstract

The invention relates to a monoclonal antibody, the monoclonal antibody is specifically bound with epithelial cell adhesion molecules, and the monoclonal antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.4; or, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.6, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.8. The monoclonal antibody disclosed by the invention has high affinity and high specificity aiming at epithelial cell adhesion molecules; the monoclonal antibody fused with the interleukin-2 shows excellent uniformity, stability and high activity, and shows remarkable anti-tumor effects of targeting, long half-life period, immune activation and the like on colorectal cancer.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a monoclonal antibody and application thereof, a nucleotide molecule, a vector or a recombinant vector, and a transformant. Background Art

[0002] Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein with a molecular weight of approximately 40 kilodaltons (kDa). EpCAM is a well-known antigen in cancer research because it is overexpressed in a variety of human cancers, including tumors of epithelial origin such as pancreatic cancer, colorectal cancer, and prostate cancer, as well as circulating tumor cells and cancer stem cells.

[0003] Due to its high expression level and antigenicity, EpCAM has become a potential target for monoclonal antibody therapy. Although a variety of monoclonal antibodies against EpCAM have been developed and tested, some early candidate drugs have shown limited clinical benefits and there are concerns about their safety in cancer patients. For example, Edrecolomab (or MAb17-1A) is a murine monoclonal antibody against EpCAM that kills tumor cells through antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). However, a phase III clinical study showed that Edrecolomab did not significantly improve the overall survival (OS) of patients with colon cancer.

[0004] To improve efficacy, researchers have developed fully human recombinant IgG1 antibodies, such as adecatumumab (MT201), which have a stronger ability to bind to Fcγ receptors of effector cells and peripheral blood mononuclear cells (PBMCs), thereby exhibiting higher ADCC activity.

[0005] Recently, therapeutic strategies based on bispecific EpCAM / CD3 antibodies, antibody-drug conjugates (ADCs), and antibody-cytokine fusion proteins (immunocytokines) have shown potential as EpCAM-targeted cancer therapies. These therapeutic approaches offer improved efficacy and safety by delivering drugs or cytokines directly to tumor sites and activating immune cells in the tumor microenvironment. For example, Tucotuzumab celmoleukin (huKS-IL2), an immunocytokine that fuses interleukin-2 (IL-2) with an anti-EpCAM monoclonal antibody, has demonstrated safety and immunological activity in cancer patients. In addition, the combination of Tucotuzumab celmoleukin with radiofrequency ablation enhanced antitumor effects and immune memory in a mouse colon cancer model.

[0006] In summary, the present invention provides a monoclonal antibody and its application, a nucleotide molecule, a vector or a recombinant vector, and a transformant for EpCAM, an antigen marker overexpressed in epithelial tumor cells. Summary of the invention

[0007] The object of the present invention is to provide a monoclonal antibody and its application, nucleotide molecule, vector or recombinant vector, and transformant in view of the deficiencies in the prior art.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] The first aspect of the present invention is to provide a monoclonal antibody, which specifically binds to an epithelial cell adhesion molecule, and the monoclonal antibody comprises: a light chain variable region and a heavy chain variable region; wherein,

[0010] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2;

[0011] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4.

[0012] Preferably, the monoclonal antibody comprises: a light chain, a first heavy chain, and a second heavy chain;

[0013] Wherein, the light chain comprises: the light chain variable region and the light chain constant region;

[0014] Wherein, at least one of the first heavy chain and the second heavy chain comprises: the heavy chain variable region;

[0015] Wherein at most one of the first heavy chain and the second heavy chain comprises:

[0016] Interleukin-2, or

[0017] A polypeptide derived from interleukin-2 in which interleukin-2 is substituted, deleted, or added with one or more amino acids and has interleukin-2 activity.

[0018] Preferably, in the monoclonal antibody,

[0019] The amino acid sequence of the light chain is shown in SEQ ID NO.3;

[0020] The amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.5, or the amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.10.

[0021] Preferably, in the monoclonal antibody,

[0022] The amino acid sequence of the light chain is shown in SEQ ID NO.3;

[0023] The amino acid sequence of the first heavy chain is shown in SEQ ID NO.12;

[0024] The amino acid sequence of the second heavy chain is shown in SEQ ID NO.13.

[0025] Preferably, in the monoclonal antibody,

[0026] The amino acid sequence of the light chain is shown in SEQ ID NO.3;

[0027] The amino acid sequence of the first heavy chain is shown in SEQ ID NO.14;

[0028] The amino acid sequence of the second heavy chain is shown in SEQ ID NO.15.

[0029] The second aspect of the present invention is to provide a monoclonal antibody, which specifically binds to an epithelial cell adhesion molecule, and the monoclonal antibody comprises: a light chain variable region and a heavy chain variable region; wherein,

[0030] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.6;

[0031] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8.

[0032] Preferably, the monoclonal antibody comprises: a light chain, a first heavy chain, and a second heavy chain;

[0033] Wherein, the light chain comprises: the light chain variable region and the light chain constant region;

[0034] Wherein, at least one of the first heavy chain and the second heavy chain comprises: the heavy chain variable region;

[0035] Wherein at most one of the first heavy chain and the second heavy chain comprises:

[0036] Interleukin-2, or

[0037] A polypeptide derived from interleukin-2 in which interleukin-2 is substituted, deleted, or added with one or more amino acids and has interleukin-2 activity.

[0038] Preferably, in the monoclonal antibody,

[0039] The amino acid sequence of the light chain is shown in SEQ ID NO.7;

[0040] The amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.9, or the amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.11.

[0041] Preferably, in the monoclonal antibody,

[0042] The amino acid sequence of the light chain is shown in SEQ ID NO.7;

[0043] The amino acid sequence of the first heavy chain is shown in SEQ ID NO.16;

[0044] The amino acid sequence of the second heavy chain is shown in SEQ ID NO.17.

[0045] Preferably, in the monoclonal antibody,

[0046] The amino acid sequence of the light chain is shown in SEQ ID NO.7;

[0047] The amino acid sequence of the first heavy chain is shown in SEQ ID NO.18;

[0048] The amino acid sequence of the second heavy chain is shown in SEQ ID NO.19.

[0049] The third aspect of the present invention is to provide a nucleotide molecule encoding the monoclonal antibody as described above.

[0050] The fourth aspect of the present invention is to provide a vector or a recombinant vector, comprising: the nucleotide molecule as described above.

[0051] The fifth aspect of the present invention is to provide a transformant, the transformant comprising: the vector or recombinant vector as described above, or

[0052] The nucleotide molecules described above are integrated into the genes of the transformants.

[0053] The sixth aspect of the present invention is to provide a use of the monoclonal antibody as described above in the preparation of a drug for treating colorectal cancer.

[0054] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0055] The monoclonal antibody of the present invention has high affinity and high specificity for epithelial cell adhesion molecules; the monoclonal antibody fused with interleukin-2 exhibits excellent uniformity, stability, and high activity, and shows significant anti-tumor effects such as targeting, long half-life, and immune activation against colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Schematic diagram of the screening strategy for fully human monoclonal antibodies against epithelial cell adhesion molecules;

[0057] Figure 2 Enrichment of polyclonal phages for antibodies targeting epithelial cell adhesion molecules;

[0058] Figure 3 is the binding activity of purified Fab-E3 and Fab-D11 to EpCAM, respectively;

[0059] Figure 4 are the binding activities of IgG1-E3 and IgG1-D11 with EpCAM and irrelevant proteins, respectively;

[0060] Figure 5 The cell binding activity of IgG1-E3 and IgG1-D11 and the human colon cancer cell line HCT116;

[0061] Figure 6 Schematic diagram of the structures of IgG1-E3 (m801), m801.2, and m801.3;

[0062] Figure 7 The purification test results of m801, m801.2, and m801.3 are shown;

[0063] Figure 8 HPLC test results of m801, m801.2, and m801.3;

[0064] Fig. 9 DLS detection results for m801, m801.2, and m801.3;

[0065] Fig.10 CD detection results for m801, m801.2, and m801.3;

[0066] Fig.11 The binding kinetics of m801, m801.2, and m801.3 to human EpCAM, respectively;

[0067] Fig.12 The binding kinetics of m801, m801.2, and m801.3 to mouse EpCAM, respectively;

[0068] Fig.13 The binding of m801, m801.2, and m801.3 to SW480 cells, respectively;

[0069] Fig.14 are the binding kinetics of m801, m801.2, and m801.3 to the IL-2Rβ subunit, respectively;

[0070] Fig.15 are the binding kinetics of m801, m801.2, and m801.3 to the IL-2Rα subunit, respectively;

[0071] Fig.16 The effects of m801, m801.2, and m801.3 on the expression level of CD69 in effector cells, respectively;

[0072] Fig.17 The stimulation of IFN-γ secretion level by m801, m801.2, and m801.3, respectively;

[0073] Fig.18 Schematic diagram of the experimental flow for in vivo antitumor activity evaluation;

[0074] Fig.19 The changes of tumor volume after treatment of NSG tumor-bearing mice;

[0075] Fig. 20 The effect of the tumor after the mouse was peeled off was shown in the picture;

[0076] Fig.21 Comparison of tumor weight changes after tumor removal in mice. DETAILED DESCRIPTION

[0077] The specific embodiments of the present invention will be described in detail below.

[0078] Unless otherwise defined, technical or scientific terms used in the claims and the specification shall have the common meanings understood by persons having ordinary skills in the technical field to which the present invention belongs.

[0079] The words "include" or similar words used in the patent application specification and claims of the present invention mean that the items appearing before "include" include the items listed after "include" or their equivalents, and do not exclude other items.

[0080] The numerical values ​​mentioned in the present invention include all numerical values ​​that increase from low to high by one unit, assuming that there are at least two units between any lower value and the higher value. For example, if a component or a physical quantity is said to be from 1 to 100, 10 to 90 is more preferred, and 20 to 80 is the most preferred, it is intended to express that values ​​such as 5 to 95, 14 to 76, 23 to 67, 32 to 58, 41 to 49 are clearly listed in this specification; for values ​​less than 1, 0.0001, 0.001, 0.01 or 0.1 are considered to be more suitable as a unit. The above examples are for illustrative purposes only. In fact, all numerical combinations between the lowest value and the highest value listed are deemed to be clearly listed in this specification in a similar manner.

[0081] The biological materials used in the embodiments of the present invention, such as proteins or cells, etc., if not specified, those skilled in the art can use any commercially available products without affecting the final experimental results.

[0082] Example

[0083] like Figure 1 As shown in Figure 1, a high-affinity monoclonal antibody specifically targeting epithelial cell adhesion molecule (EpCAM, SEQ ID NO. 1) was screened from a large-capacity fully human antibody phage display library constructed in the early stage of the laboratory (Cell Host Microbe 2017, 22 (4): 471-483.e5.); after 4 rounds of "adsorption-elution-amplification" enrichment screening, the phage library obtained in each round of screening was subjected to polyclonal phage enzyme-linked immunosorbent assay (ELISA) with EpCAM, that is, after each round of enrichment screening, 1×10 11 The phages screened were incubated with the antigen EpCAM, and then detected using the anti-phage secondary antibody. The data were displayed as the light absorption value at OD405nm. The results are as follows: Figure 2 As shown, anti-EpCAM specific antibodies began to be significantly enriched in the third round; the enriched anti-EpCAM antibodies obtained in the third and fourth rounds of screening were selected, 200 monoclonal colonies were randomly selected, and they were induced to produce antibodies, and the binding ability of these antibodies to the EpCAM antigen was determined using monoclonal ELISA. The results showed that more than 90% of the clones expressed antibodies that had the ability to bind to the EpCAM antigen.

[0084] By sequencing and comparing the cloned antibody sequences, two types of antibodies with different sequences were obtained, which were named Fab-E3 (the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2, the amino acid sequence of the light chain is shown in SEQ ID NO.3, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.4, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.5) and Fab-D11 (the amino acid sequence of the light chain variable region is shown in SEQ ID NO.6, the amino acid sequence of the light chain is shown in SEQ ID NO.7, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8, and the amino acid sequence of the heavy chain is shown in SEQ ID NO.9); these two antibody strains were induced to express and purified to obtain a large amount of high-purity Fab antibodies ( Figure 3A), then the gradient diluted Fab-E3 and Fab-D11 were incubated with EpCAM at 37°C for 1.5 hours, and detected using anti-Fab HRP secondary antibody. The data were displayed as OD405nm light absorption value. The results are as follows Figure 3 As shown in B, both Fab-E3 and Fab-D11 have strong binding activity with the antigen EpCAM protein. The EC50 value of Fab-E3 is 14.65 nM, which is better than the EC50 value of Fab-D11 (37.17 nM).

[0085] Since the immune antibodies produced by the human body are generally present in the form of IgG1, the screened antibodies Fab-E3 and Fab-D11 were transformed into IgG1-E3 (the amino acid sequence of the light chain is still as shown in SEQ ID NO.3, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.10) and IgG1-D11 (the amino acid sequence of the light chain is still as shown in SEQ ID NO.7, and the amino acid sequence of the heavy chain is as shown in SEQ ID NO.11), respectively. Then, the gradient diluted IgG1-E3 and IgG1-D11 were incubated with EpCAM and irrelevant protein (ZIKV protein) at 37°C for 1.5 hours, and detected with anti-human Fc HRP secondary antibody. The data are shown as OD405nm light absorption value. The results are as follows Figure 4 As shown in the figure, compared with the Fab form of the antibody, the binding activity of the IgG1 form of the antibody to the antigen EpCAM protein was significantly improved, and similar to the results of the Fab form, the binding activity of IgG1-E3 to the antigen EpCAM protein was stronger (the EC50 value of IgG1-E3 was 1.092nM, and the EC50 value of IgG1-D11 was 1.530nM, left figure), and it had no binding activity with other proteins (right figure); in order to further verify whether IgG1-E3 and IgG1-D11 can bind to cells expressing EpCAM, the human colon cancer cell line HCT116 was selected for the antibody cell binding experiment, and IgG1-E3 and IgG1-D11 were incubated with the human colon cancer cell line HCT116 at concentrations of 100nM and 10nM, respectively, for 1 hour, and then FITC-labeled anti-human IgG fluorescent secondary antibody was added, and the flow cytometer was used to detect it and the Flowjo software was used for data analysis. The results are shown in the figure. Figure 5 As shown, IgG1-E3 (left figure) and IgG1-D11 (right figure) have strong binding activity to HCT116 cells and are concentration-dependent. However, there is no significant difference in cell binding activity between IgG1-E3 and IgG1-D11.

[0086] like Figure 6As shown in the figure, IgG1-E3 (hereinafter referred to as m801, left figure) was fused with interleukin-2 mutant (IL-2v) by genetic engineering technology. Since the form and structure of the fusion have a great influence on the functions of the antibody m801 and IL-2v, two different forms of bifunctional antibodies were constructed: one bifunctional antibody is a monovalent bifunctional antibody m801.2 formed by connecting IL-2v to the C-terminus of the Knob after the Fc domain of m801 is mutated into a "Knob into Hole" (the amino acid sequence of the light chain is still as shown in SEQ ID NO.3, the amino acid sequence of the first heavy chain knob is as shown in SEQ ID NO.12, and the amino acid sequence of the second heavy chain hole is as shown in SEQ ID NO.13, middle figure); the other bifunctional antibody is a monovalent bifunctional antibody m801.3 formed by connecting IL-2v to the N-terminus of the Knob after the Fc domain of m801 is mutated into a "Knob into Hole" (the amino acid sequence of the light chain is still as shown in SEQ ID NO.1 NO.3, the amino acid sequence of the first heavy chain knob is shown in SEQ ID NO.14, and the amino acid sequence of the second heavy chain hole is shown in SEQ ID NO.15 (right figure);

[0087] Similarly, after the Fc domain of IgG1-D11 is subjected to the "Knob into Hole" mutation, IL-2v is connected to the C-terminus of the Knob to form a monovalent bifunctional antibody, and the amino acid sequence of its light chain is still as shown in SEQ ID NO.7, the amino acid sequence of the first heavy chain knob is shown in SEQ ID NO.16, and the amino acid sequence of the second heavy chain hole is shown in SEQ ID NO.17; and after the Fc domain of IgG1-D11 is subjected to the "Knob into Hole" mutation, IL-2v is connected to the N-terminus of the Knob to form a monovalent bifunctional antibody, the amino acid sequence of its light chain is still as shown in SEQ ID NO.7, the amino acid sequence of the first heavy chain knob is shown in SEQ ID NO.18, and the amino acid sequence of the second heavy chain hole is shown in SEQ ID NO.19.

[0088] Since each bifunctional antibody contains an Fc fragment, the correctly constructed m801.2 and m801.3 plasmids were transiently transfected into Expi392 cells. The expressed antibodies were purified using a Protein G affinity column. The purified antibodies were tested by SDS-PAGE to determine whether the antibodies were correctly expressed. The results are shown in Figure 2. Figure 7 As shown, the purified monoclonal antibody m801, bifunctional antibody m801.2, and bifunctional antibody m801.3 all had correct expression bands, and the purity was more than 95%.

[0089] Next, the physicochemical properties of the antibodies were analyzed:

[0090] The purity and aggregation state of the bifunctional antibody were evaluated by high performance liquid chromatography (HPLC) (the rate of passing the monoclonal antibody m801 and the bifunctional antibody m801.3 through the chromatographic column was 0.5 mL / min; the rate of passing the bifunctional antibody m801.2 through the chromatographic column was 0.25 mL / min). Figure 8 As shown, monoclonal antibody m801 (left), bifunctional antibody m801.2 (middle), and bifunctional antibody m801.3 (right) all have only one sharp peak, indicating that the protein is single, highly pure, and very stable, without the generation of polymers.

[0091] The particle size of the bifunctional antibody was further measured using a nanomolecular particle size analyzer (Dynamic Light Scattering, DLS) to evaluate whether the homogeneity was good (the loading volume of monoclonal antibody m801, bifunctional antibody m801.2, and bifunctional antibody m801.3 was 100 μL). The results are as follows: Fig. 9 As shown, the particle sizes of monoclonal antibody m801 (left), bifunctional antibody m801.2 (middle), and bifunctional antibody m801.3 (right) are evenly dispersed, with diameters of about 10 nm, showing good uniformity.

[0092] The thermodynamic stability of the bifunctional antibody was further evaluated by circular dichroism (CD) spectroscopy (the loading amount of monoclonal antibody m801, bifunctional antibody m801.2, and bifunctional antibody m801.3 was 300 μL, and the curves were fitted by the Boltzmann sigmoidal equation). The results are as follows: Fig.10 As shown, the Tm value of monoclonal antibody m801 is (86.68±0.21)℃ (left figure), the Tm value of bifunctional antibody m801.2 is [(86.32±0.30)℃] (middle figure) and the Tm value of bifunctional antibody m801.3 is [(86.29±0.23)℃] (right figure), which is consistent with the Tm value of monoclonal antibody m801, indicating that monoclonal antibody m801, bifunctional antibody m801.2, and bifunctional antibody m801.3 all have good thermal stability.

[0093] Explore the biological activity and mechanism of action of bifunctional antibodies:

[0094] In order to further detect the binding kinetics of the bifunctional antibody to human EpCAM, the biolayer interferometry (BLI) technique was used to measure the binding kinetics of the human EpCAM protein to the Ni-NTA biosensor. The bifunctional antibody diluted 3 times the concentration gradient was used to bind and dissociate with human EpCAM. The obtained data were fitted and analyzed according to the 1:1 binding mode by using ForteBio Data analysis 8.1 software, and the kinetic curve was drawn. The results are shown in the figure. Fig.11 As shown in the figure, the binding affinity of the bifunctional antibody m801.2 (middle figure) and the bifunctional antibody m801.3 (right figure) are both higher than that of the monoclonal antibody m801 (left figure). The bifunctional antibody m801.2 has the highest binding affinity, and its K D is 0.60 nM, because the binding rate (k on , 9.90×10 4 M -1 s -1 ) is very fast, while the dissociation rate (k off , 5.94×10 -5 s -1 ) is very slow, while the binding affinity of the bifunctional antibody m801.3 is 2.33nM. Therefore, the bifunctional antibody m801.2 and the bifunctional antibody m801.3 have the characteristics of fast binding and slow dissociation, and are suitable as targeted anti-tumor drugs;

[0095] Similarly, the binding kinetics of the bifunctional antibody to mouse EpCAM was determined by BLI. The mouse EpCAM protein was immobilized on the Ni-NTA biosensor, and the bifunctional antibody diluted 3 times the concentration gradient was used to bind and dissociate with mouse EpCAM. The obtained data was fitted and analyzed according to the 1:1 binding mode by using ForteBio Data analysis 8.1 software, and the kinetic curve was drawn. The results are shown in Fig.12 As shown in the figure, the binding pattern of the bifunctional antibody m801.2 (middle figure) and the bifunctional antibody m801.3 (right figure) to mouse EpCAM is very similar to that of human EpCAM. The binding affinity of the bifunctional antibody m801.2 (middle figure) and the bifunctional antibody m801.3 (right figure) to mouse EpCAM is also higher than that of the monoclonal antibody m801 (left figure). The bifunctional antibody m801.2 still has the highest binding affinity, and its K D is 3.76 nM, and the binding rate (k on ) is 2.91×10 4 M -1 s -1 , dissociation rate (k off ) is 1.10×10 -4 s -1, while the binding affinity of the bifunctional antibody m801.3 was 10.30 nM;

[0096] To further verify whether the bifunctional antibody can bind to EpCAM on the cell surface and thus exert its targeting function, a human colon cancer cell line SW480 with high expression of EpCAM was selected, and the bifunctional antibody diluted 3 times with concentrations of 1.23nM, 11.11nM and 100nM was evenly divided into 1×10 5 The SW480 cells of each cell were co-cultured and analyzed by flow cytometry. The flow data were analyzed using FlowJo V10 software. The results are as follows: Fig.13 As shown, monoclonal antibody m801 (left) and bifunctional antibody m801.2 (middle) have similar affinities, while bifunctional antibody m801.3 (right) has an intermediate affinity due to its monovalent EpCAM binding arm, indicating that the binding affinity of bifunctional antibody m801.2 is relatively better;

[0097] Afterwards, the kinetics of different bifunctional antibodies and IL-2Rβ subunits were measured by flow cytometry. The IL-2Rβ subunit protein was immobilized on the Ni-NTA biosensor, and the bifunctional antibody diluted 3 times the concentration gradient was combined with the IL-2Rβ subunit protein. The obtained data were fitted and analyzed according to the 1:1 binding mode by ForteBio Data analysis 8.1 software. The results are shown as follows Fig.14 As shown in Figure 2, both the bifunctional antibody m801.2 (middle) and the bifunctional antibody m801.3 (right) have high binding affinity to the IL-2Rβ subunit. The equilibrium constant (K D ) was 27.3 nM, and the binding rate was 1.51×10 5 M -1 s -1 , the dissociation rate is 4.12×10 -3 s -1 , higher than the bifunctional antibody m801.3, while the monoclonal antibody m801 (left figure) could not bind to the IL-2Rβ subunit;

[0098] Similarly, the IL-2Rα subunit protein was immobilized on the Ni-NTA biosensor, and the bifunctional antibody diluted 3 times in concentration gradient was bound to the IL-2Rα subunit protein. The obtained data was fitted and analyzed according to the 1:1 binding mode using ForteBio Data analysis 8.1 software. The results are as follows: Fig.15As shown, monoclonal antibody m801 (left), bifunctional antibody m801.2 (middle), and bifunctional antibody m801.3 (right) all have no binding ability to IL-2Rα subunit, indicating that bifunctional antibody m801.2 and bifunctional antibody m801.3 both have the ability to activate effector cells;

[0099] In order to further explore the activation level of bifunctional antibodies on effector cells such as T cells and NK cells, the expression level of cell surface CD69 was detected. After co-incubation of effector cells PBMC, target cells SW480 and bifunctional antibodies, the expression of cell CD69 was detected by flow cytometry. GraphPad Prism data processing, the Y axis is the percentage of cells expressing CD69 (%), the results are shown as follows Fig.16 As shown, monoclonal antibody m801 and irrelevant control antibody did not detect the expression level of CD69, indicating that monoclonal antibody m801 cannot activate T cells, while bifunctional antibody m801.2 and bifunctional antibody m801.3 can both activate T cells well and make them express CD69 on their surface, and at the same concentration, bifunctional antibody m801.2 can activate CD69 with higher expression, that is, bifunctional antibody m801.2 can activate T cells with higher activity;

[0100] When T cells or NK cells are activated, they will secrete IFN-γ cytokines, and their secretion level reflects the activation level of immune cells. Therefore, after the effector cells PBMC are co-incubated with the bifunctional antibody, the IFN-γ content in the cell supernatant is detected using the IFN-γ detection kit. The results are as follows: Fig.17 As shown, both the bifunctional antibody m801.2 and the bifunctional antibody m801.3 can stimulate immune cells to secrete a large amount of IFN-γ. The EC50 value of the bifunctional antibody m801.2 is 0.7nM, while the EC50 value of the bifunctional antibody m801.3 is 1.3nM. The secretion level of IFN-γ stimulated by the monoclonal antibody m801 is consistent with that of the negative control group, and it cannot effectively activate T cells or NK cells to produce secretion.

[0101] like Fig.18 To further evaluate the anti-tumor activity of the bifunctional antibody in vivo, 6-week-old NOD-PrkdcscidIl2rgem1 / Smoc (NSG) mice were subcutaneously inoculated with SW480 (5×10 7 / mL cells, 100 μL / mouse) and PBMC (5×10 7 / mL cells, 100μL / cell) in a 1:1 ratio of cell mixture, a total of 1×10 7 SW480 xenograft tumor mouse model was established, and 5×106 PBMC cells were used to establish humanized mice with PBMC immune system reconstruction, so that the mice had a complete human immune system. On the 22nd day of inoculation, the average size of the subcutaneous tumors of the mice reached 100 mm 3 -200mm 3 At about 14 days, the mice were randomly divided into groups: negative control group (CR3022 antibody, a fully human monoclonal antibody targeting the new coronavirus), IL-2v administration group, monoclonal antibody m801 administration group, monoclonal antibody m801 and IL-2v combined administration group, bifunctional antibody m801.2 administration group, and bifunctional antibody m801.3 administration group, and then intraperitoneal administration (concentration of 1 mg / mL) was performed at a dose of 4 mg / kg, once every 3 days, for a total of 5 times, and PBMC cells were injected into the tail vein once a week. When the medication course was completed, the PBMC injection was stopped, and the longest and shortest sides of the mouse tumors were measured and recorded on the 23rd, 26th, 29th, 32nd, 35th, 38th, 41st, 44th, 47th, and 50th days. The tumor volume calculation formula is tumor volume (mm 3 ) = longest side × shortest side × shortest side ÷ 2, and draw a tumor growth curve. The result is as follows Fig.19 As shown in the figure, the tumor growth curves of the negative control group and the monoclonal antibody m801 administration group are similar, indicating that its anti-tumor growth inhibitory activity is very weak. The IL-2v administration group can inhibit tumor growth, and the monoclonal antibody m801 and IL-2v combined administration group has no significant enhancement in inhibitory effect compared with it. It is worth noting that the bifunctional antibody m801.2 administration group and the bifunctional antibody m801.3 administration group both have tumor growth inhibitory activity. Since the molecular weight of the bifunctional antibody m801.2 is 165kDa, the molecular weight of the bifunctional antibody m801.3 is 115kDa, and the molecular weight of IL-2v is 15kDa, so at the same 4mg / kg dose The molar number of IL-2v in the bifunctional antibody m801.2 administration group was almost one-tenth of that in the IL-2v administration group, and the molar number of IL-2v in the bifunctional antibody m801.3 administration group was almost one-seventh of that in the IL-2v administration group. Therefore, from the tumor growth curve, the bifunctional antibody m801.2 had the strongest tumor growth inhibition activity, and was stronger than the IL-2v administration group or the monoclonal antibody m801 and IL-2v combined administration group. In order to more intuitively observe the therapeutic effect of the bifunctional antibody on tumors, the mice were euthanized, and the corresponding subcutaneous tumors of each group of mice were peeled off. The peeled tumors were placed in groups and photographed. The results are shown in Figure 2. Fig. 20As shown in the figure, the anti-tumor effect of the bifunctional antibody m801.2 group was the most prominent, and the tumor of each mouse was significantly reduced. What was exciting was that the tumor of one mouse completely disappeared. The tumor tissues peeled off from each group of mice were weighed, and the data were expressed as mean±sem. For comparisons between different groups, ** indicates p<0.01, and *** indicates p<0.005. The results are shown in the figure. Fig.21 As shown, the anti-tumor activity of the bifunctional antibody m801.2 was the best (p=0.0023), while the tumor weight of the negative control group did not change significantly. It can be seen that the combination of monoclonal antibody m801 and cytokine IL-2v does not have a synergistic effect in inhibiting tumor growth, but the anti-tumor effect of the constructed bifunctional antibody is significantly enhanced.

[0102] Similarly, the bifunctional antibody constructed by fusing monoclonal antibody IgG1-D11 with cytokine IL-2v also has high binding affinity for specific targeting of EpCAM and significantly enhanced anti-tumor effect compared to cytokine IL-2v.

[0103] All nucleotide or amino acid sequences involved in the embodiments of the present invention are as follows:

[0104] Epithelial cell adhesion molecule, EpCAM, AA24-265, SEQ ID NO.1:

[0105] QEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWI IIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLK

[0106] Fab-E3, light chain, SEQ ID NO.3:

[0107] DIQMTQSPSSSLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGTGT DFTLTISSLQPEDFATYYCQQANSFPLTFGGGTKVDIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0108] (The underlined part is the light chain variable region, SEQ ID NO.2)

[0109] Fab-E3, heavy chain, SEQ ID NO.5:

[0110] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFT ISRDNSKNTLYLQMNSLRSEDTAVYYCARGLPARAFDIWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAG

[0111] (The underlined part is the heavy chain variable region, SEQ ID NO.4)

[0112] Fab-D11, light chain, SEQ ID NO.7:

[0113] SYVLTQPPSVSVSPGQTARITCSGDALPKQYAYWYQQKPGQAPVLVIYKDSERPSGIPERFSGSSSGTT VTLTISGVQAEDEADYYCQSADSSGTYVVFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

[0114] (The underlined part is the light chain variable region, SEQ ID NO.6)

[0115] Fab-D11, heavy chain, SEQ ID NO.9:

[0116] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCASASGTYHANYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTSGQAG

[0117] (The underlined part is the heavy chain variable region, SEQ ID NO.8)

[0118] IgG1-E3, m801, heavy chain, SEQ ID NO.10:

[0119] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARGLPARAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0120] IgG1-D11, heavy chain, SEQ ID NO. 11:

[0121] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASASGTYHANYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0122] m801.2, first heavy chain, SEQ ID NO.12:

[0123] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARGLPARAFDIWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEE VLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

[0124] (The underlined part is interleukin-2 mutant, IL-2v, the same below)

[0125] m801.2, second heavy chain, SEQ ID NO.13:

[0126] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARGLPARAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0127] m801.3, the first heavy chain, SEQ ID NO.14:

[0128] APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEV LNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0129] m801.3, the second heavy chain, SEQ ID NO.15:

[0130] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYGMHWVRQAPGKGLEWVAVIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRSEDTAVYYCARGLPARAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0131] IgG1-D11-C-IL-2v, first heavy chain, SEQ ID NO.16:

[0132] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASASGTYHANYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGS APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEE VLNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT

[0133] IgG1-D11-C-IL-2v, second heavy chain, SEQ ID NO.17:

[0134] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASASGTYHANYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0135] IL-2v-N-IgG1-D11, first heavy chain, SEQ ID NO.18:

[0136] APASSSTKKTQLQLEHLLDLQMILNGINNYKNPKLTRMLTAKFAMPKKATELKHLQCLEEELKPLEEV LNGAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT GGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0137] IL-2v-N-IgG1-D11, second heavy chain, SEQ ID NO.19:

[0138] EVQLVESGGGVVQPGRSLRLSCAASGFTFSGYAMHWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASASGTYHANYWGQGT LVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0139] In summary, the monoclonal antibodies of the present invention have high affinity and high specificity for epithelial cell adhesion molecules; the monoclonal antibodies fused with interleukin-2 exhibit excellent uniformity, stability, and high activity, and show significant anti-tumor effects such as targeting, long half-life, and immune activation against colorectal cancer.

[0140] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A monoclonal antibody that specifically binds to an epithelial cell adhesion molecule, the monoclonal antibody comprising: A light chain variable region and a heavy chain variable region; characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

2. The monoclonal antibody according to claim 1, characterized in that The monoclonal antibody comprises: a light chain, a first heavy chain, and a second heavy chain; Wherein, the light chain comprises: the light chain variable region and the light chain constant region; Wherein, at least one of the first heavy chain and the second heavy chain comprises: the heavy chain variable region; Wherein at most one of the first heavy chain and the second heavy chain comprises: Interleukin-2, or A polypeptide derived from interleukin-2 in which interleukin-2 is substituted, deleted, or added with one or more amino acids and has interleukin-2 activity.

3. The monoclonal antibody according to claim 2, characterized in that In the monoclonal antibody, The amino acid sequence of the light chain is shown in SEQ ID NO.3; The amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.5, or the amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.

10.

4. The monoclonal antibody according to claim 2, characterized in that In the monoclonal antibody, The amino acid sequence of the light chain is shown in SEQ ID NO.3; The amino acid sequence of the first heavy chain is shown in SEQ ID NO.12; The amino acid sequence of the second heavy chain is shown in SEQ ID NO.

13.

5. The monoclonal antibody according to claim 2, characterized in that In the monoclonal antibody, The amino acid sequence of the light chain is shown in SEQ ID NO.3; The amino acid sequence of the first heavy chain is shown in SEQ ID NO.14; The amino acid sequence of the second heavy chain is shown in SEQ ID NO.

15.

6. A monoclonal antibody that specifically binds to an epithelial cell adhesion molecule, the monoclonal antibody comprising: A light chain variable region and a heavy chain variable region; characterized in that, The amino acid sequence of the light chain variable region is shown in SEQ ID NO.6; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

8.

7. The monoclonal antibody according to claim 6, characterized in that The monoclonal antibody comprises: a light chain, a first heavy chain, and a second heavy chain; Wherein, the light chain comprises: the light chain variable region and the light chain constant region; Wherein, at least one of the first heavy chain and the second heavy chain comprises: the heavy chain variable region; Wherein at most one of the first heavy chain and the second heavy chain comprises: Interleukin-2, or A polypeptide derived from interleukin-2 in which interleukin-2 is substituted, deleted, or added with one or more amino acids and has interleukin-2 activity.

8. The monoclonal antibody according to claim 7, characterized in that In the monoclonal antibody, The amino acid sequence of the light chain is shown in SEQ ID NO.7; The amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.9, or the amino acid sequences of the first heavy chain and the second heavy chain are both shown as SEQ ID NO.

11.

9. The monoclonal antibody according to claim 7, characterized in that In the monoclonal antibody, The amino acid sequence of the light chain is shown in SEQ ID NO.7; The amino acid sequence of the first heavy chain is shown in SEQ ID NO.16; The amino acid sequence of the second heavy chain is shown in SEQ ID NO.

17.

10. The monoclonal antibody according to claim 7, characterized in that In the monoclonal antibody, The amino acid sequence of the light chain is shown in SEQ ID NO.7; The amino acid sequence of the first heavy chain is shown in SEQ ID NO.18; The amino acid sequence of the second heavy chain is shown in SEQ ID NO.

19.

11. A nucleotide molecule, characterized in that: Encodes the monoclonal antibody according to any one of claims 1 to 10.

12. A vector or a recombinant vector, characterized in that: include: The nucleotide molecule according to claim 11.

13. A transformant, characterized in that: The transformant comprises: the vector or recombinant vector as claimed in claim 12, or the nucleotide molecule as claimed in claim 11 is integrated into the gene of the transformant.

14. Use of the monoclonal antibody according to any one of claims 4, 5, 9, or 10 in the preparation of a drug for treating colorectal cancer.

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