A monoclonal antibody and uses thereof

Abstract

The application utilizes tumor cells to immunize mice, and a monoclonal antibody is screened and prepared through a hybridoma fusion technology, the monoclonal antibody has obvious inhibiting effect on growth and migration of tumor cells, and has significant anti-tumor activity in animal tumor experiments.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, and more specifically, to a monoclonal antibody and its uses. Background Technology

[0002] Tumors develop when, under the influence of various factors, cells in a local tissue lose normal regulation of their growth at the gene level, leading to abnormal cell proliferation and the formation of new growths. Tumors are genetic diseases; their biological basis is gene abnormalities. The pathogenic factors are somatic cell gene mutations leading to the loss of normal genes and disordered gene expression, thereby affecting the biological and genetic activity of cells and forming tumor cells that differ from normal cells in morphology, metabolism, and function.

[0003] Targeted therapies are highly specific and have fewer side effects, demonstrating significant efficacy against various malignant tumors and have become the mainstream of new anti-tumor drugs in the past decade. In 1997, the FDA approved the first targeted cancer drug, rituximab, ushering in a new era of cancer treatment. Between 1997 and 2005, a few targeted small-molecule and large-molecule monoclonal antibodies were launched, indicating the rapid development of targeted therapies. From 2005 to 2017, the proportion of targeted drugs among the anti-tumor drugs approved by the FDA increased year by year. Of the 14 anti-tumor drugs approved in 2015, 12 were targeted therapies, and all approved in 2016 and 2017 were targeted therapies. Antibody-targeted cancer therapy is one of the most promising approaches.

[0004] Monoclonal antibodies (mAbs) are produced by B cells and specifically target antigens. The hybridoma technique introduced by Köhler and Milstein in 1975 made it possible to obtain large quantities of pure mAbs, greatly enhancing their potential for basic research and clinical application. Other technological advancements have also enabled the successful clinical application of mAbs. Globally, at least 570 therapeutic mAbs are being investigated in clinical trials by commercial companies, of which 79 have been approved by the U.S. Food and Drug Administration (FDA) and are currently on the market, including 30 antibodies used to treat cancer.

[0005] Although a large number of tumor antibody drugs have been successfully developed and clinically applied, there are still a large number of cancer patients who do not respond to existing antibody drugs. Summary of the Invention

[0006] In one aspect, the present invention provides a monoclonal antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3, wherein the sequence of CDR-L1 is selected from the amino acid sequence shown in SEQ ID NO: 4, the sequence of CDR-L2 is selected from the amino acid sequence shown in SEQ ID NO: 5, and the sequence of CDR-L3 is selected from the amino acid sequence shown in SEQ ID NO: 6;

[0007] The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3, wherein the sequence of CDR-H1 is selected from the amino acid sequence shown in SEQ ID NO: 1, the sequence of CDR-H2 is selected from the amino acid sequence shown in SEQ ID NO: 2, and the sequence of CDR-H3 is selected from the amino acid sequence shown in SEQ ID NO: 3.

[0008] In some embodiments, the light chain variable region further includes a leader sequence selected from the amino acid sequence shown in SEQ ID NO: 8;

[0009] The heavy chain variable region further includes a leader sequence selected from the amino acid sequence shown in SEQ ID NO: 7.

[0010] In some embodiments, it includes a light chain variable region selected from the amino acid sequence shown in SEQ ID NO: 10 and a heavy chain variable region selected from the amino acid sequence shown in SEQ ID NO: 9.

[0011] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0012] In another aspect, the present invention also provides a multispecific antibody comprising the monoclonal antibody described above.

[0013] In another aspect, the present invention also provides a vector comprising a nucleic acid molecule encoding the monoclonal antibody or multispecific antibody described above.

[0014] In another aspect, the present invention also provides a host cell that expresses the monoclonal antibody described above.

[0015] In another aspect, the present invention also provides a pharmaceutical composition comprising the monoclonal antibody described above and one or more pharmaceutically acceptable excipients.

[0016] In another aspect, the present invention also provides a combination drug comprising the monoclonal antibody described above, an anticancer drug, and one or more pharmaceutically acceptable excipients; said anticancer drug includes gefitinib and paclitaxel.

[0017] In another aspect, the use of the aforementioned monoclonal antibodies, multispecific antibodies, chimeric antigen receptors, and combination drugs in the preparation of medicaments for the treatment and / or prevention and / or diagnosis of tumor diseases.

[0018] In summary, this invention provides a method for immunizing mice with tumor cells and screening and preparing a monoclonal antibody using hybridoma fusion technology. This monoclonal antibody has a significant inhibitory effect on the growth and migration of tumor cells and exhibits significant anti-tumor activity in animal tumor experiments. Attached Figure Description

[0019] Figure 1 The results are from SDS-PAGE reduction electrophoresis of the 4A7 antibody.

[0020] Figure 2 The 4A7 antibody inhibits tumor cell migration induced by HRGβ1 molecules;

[0021] Figure 3 The combination of 4A7 antibody and gefitinib effectively inhibited the growth of triple-negative breast cancer cells;

[0022] Figure 4 The combination of 4A7 antibody and paclitaxel effectively inhibited the growth of triple-negative breast cancer cells;

[0023] Figure 5 The combination of 4A7 antibody and paclitaxel induces apoptosis;

[0024] Figure 6 The antitumor effect of 4A7 antibody in mice. Detailed Implementation

[0025] Unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0026] Typically, a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds, with each light chain connected to its respective heavy chain via disulfide bonds, forming a "Y"-shaped structure. Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain variable region contains three complementarity-determining regions (CDRs): CDR-H1, CDR-H2, and CDR-H3, and the heavy chain constant region contains three constant domains: CH1, CH2, and CH3. Each light chain contains a light chain variable region (VL) and a light chain constant region. The light chain variable region contains three CDRs: CDR-L1, CDR-L2, and CDR-L3, and the light chain constant region contains one constant domain CL. Within the heavy / light chain variable regions, the CDRs are separated by more conserved frame regions (FRs). The variable regions of the heavy / light chains are responsible for the recognition and binding of antigens, while the constant regions can mediate the binding of antibodies to host tissues or factors, including various cells of the immune system (such as effector cells) and the first component of the classical complement system.

[0027] The terms “complementarity-determining region” or “CDR” are well-known and interchangeable in the art and refer to a discontinuous amino acid sequence within the variable region of an antibody that confers antigen specificity and / or binding affinity. The terms “frame region” or “FR” are also known in the art and refer to the non-CDR portion within the variable region of an antibody, whose sequence is generally conserved.

[0028] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using many numbering schemes well-known in the art, including: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (“Kabat” numbering scheme); Al-Lazikani et al. (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745 (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily”. V-like domains,” Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272 (“AbM” numbering scheme).

[0029] Therefore, unless otherwise specified, it should be understood that the “CDR” of a given antibody or its region (such as its variable region) encompasses the CDRs defined by any of the above-described protocols or other known protocols. For example, in specifying that a particular CDR (e.g., CDR3) contains a given amino acid sequence, it should be understood that such a CDR may also have the sequence of the corresponding CDR (e.g., CDR3) as defined by any of the above-described protocols or other known protocols. Similarly, unless otherwise specified, it should be understood that the FR of a given antibody or its region (such as its variable region) encompasses the FRs defined by any of the above-described protocols or other known protocols.

[0030] As used herein, the term “sequence identity” refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147: 195-197), and Clustal W.

[0031] As used herein, the term "vector" is a medium nucleic acid molecule used to transfer (exogenous) genetic material into a host cell, in which the nucleic acid molecule may, for example, be replicated and / or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium for delivering isolated nucleic acids into the cell interior by, for example, homologous recombination or using a hybrid recombinase with a specific target site sequence. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences (e.g., those encoding the chimeric antigen receptor polypeptide of the present invention) in a suitable host cell and for the translation of their mRNA. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, the vectors of the present invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia virus, Raul's sarcoma virus (RSV, polyomavirus, and adeno-associated virus (AAV) etc.), bacteriophages, phage particles, granules, and artificial chromosomes (including BAC and YAC). The vector itself is typically a nucleic acid molecule, usually consisting of a DNA sequence containing an insert (transgenic) and a larger sequence serving as the vector "backbone." Engineered vectors typically also contain an origin of autonomous replication in the host cell (if stable expression of polynucleotides is required), a selection marker, and a restriction enzyme cleavage site (e.g., a multiple cloning site, MCS). The vector may additionally contain elements such as a promoter, polyA tail, 3'UTR, enhancer, terminator, insulator, operon, selection marker, reporter gene, target sequence, and / or protein purification tag.

[0032] As used herein, the term "pharmaceuticalally acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any undesirable local or systemic effects), which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adhesion agents, flow aids, antioxidants, flavoring agents, coloring agents, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delay agents, stabilizers, and tension modifiers. Those skilled in the art know how to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients used in the pharmaceutical compositions of the present invention include saline, buffered saline, glucose, and water. Generally, the selection of a suitable excipient depends in particular on the active agent used, the disease to be treated, and the desired dosage form of the pharmaceutical composition.

[0033] The pharmaceutical compositions according to the invention are suitable for administration via a variety of routes. Typically, administration is performed via parenteral delivery. Parenteral delivery methods include local, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0034] The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous, or lyophilized forms, particularly as ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for the desired method of administration. Processes known in this invention for producing pharmaceuticals may include, for example, conventional mixing, dissolving, granulation, coating, grinding, emulsification, encapsulation, embedding, or lyophilization processes.

[0035] Conventional antibody drugs involve immunizing animals with recombinant proteins, screening, and preparing antibodies. In this invention, mice were immunized using whole-cell tumor immunotherapy, and a monoclonal antibody with good anti-tumor activity was unexpectedly discovered, which exhibited good anti-tumor activity both in vitro and in vivo.

[0036] In one aspect, the present invention provides a monoclonal antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR-L1, CDR-L2, and CDR-L3, wherein CDR-L1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 4, CDR-L2 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 5, and CDR-L3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6;

[0037] The heavy chain variable region comprises CDR-H1, CDR-H2, and CDR-H3. CDR-H1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 1. CDR-H2 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 2. CDR-H3 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 3. Furthermore, the light chain variable region also includes a leader sequence, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 8; the heavy chain variable region also includes a leader sequence, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 7.

[0038] Preferably, the monoclonal antibody comprises a light chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the amino acid sequence shown in SEQ ID NO: 10, and a heavy chain variable region having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of the amino acid sequence shown in SEQ ID NO: 9.

[0039] The present invention also provides a nucleic acid molecule encoding the above-mentioned monoclonal antibody.

[0040] In another aspect, the present invention also provides a multispecific antibody comprising the monoclonal antibody described above.

[0041] In another aspect, the present invention also provides a vector comprising a nucleic acid molecule encoding the aforementioned monoclonal antibody or multispecific antibody.

[0042] In another aspect, the present invention also provides a host cell that expresses the aforementioned monoclonal antibody.

[0043] In another aspect, the present invention also provides a pharmaceutical composition comprising the above-described monoclonal antibody and one or more pharmaceutically acceptable excipients.

[0044] In another aspect, the present invention also provides a combination drug comprising the above-described monoclonal antibody, an anticancer drug, and one or more pharmaceutically acceptable excipients; the anticancer drug includes gefitinib and paclitaxel.

[0045] In another aspect, the use of the aforementioned monoclonal antibodies, the aforementioned multispecific antibodies, the aforementioned chimeric antigen receptors, and the aforementioned combination drugs in the preparation of medicaments for the treatment and / or prevention and / or diagnosis of tumor diseases. Specific Implementation

[0046] Example 1: Hybridoma Preparation

[0047] 1. Balb / C mice were selected for immunization, specifically by intraperitoneal immunization with SKBR3 cells overexpressing the HER3 molecule. The dose for each immunization was 1 × 10⁻⁶. 7 Cells / mice were immunized every two weeks for a total of four immunizations.

[0048] 2 Hybridoma fusion

[0049] 7-10 days before fusion, SP2 / 0 cells were thawed, and cell growth was observed daily. The medium was changed in half to ensure the cells were in the logarithmic growth phase. The viability of SP2 / 0 cells used for fusion was required to be above 95%. Peritoneal exudate cells from normal mice were seeded into 96-well plates at 100 μL / well one day before fusion. Three days after the last immunization, mice were bled, soaked in 75% ethanol for 3 min, and the abdomen was opened. The spleen was harvested, washed, and spleen cells were separated using a spleen cell separator, collecting the single-cell suspension. Viable myeloma cells (SP2 / 0) in the logarithmic growth phase were mixed with mouse spleen cells at a ratio of 1:4. Then, the cells were washed twice before fusion with PEG. After fusion, the cells were washed with DMEM and resuspended in cell growth medium supplemented with 10% FBS + HFCS + OPI + 1X HAT. The cell suspension was seeded at 200 μl per well into 96-well cell culture plates and incubated overnight in a CO2 incubator at 37°C and 10% humidity. After 7 days of cell incubation, the culture medium in the wells was aspirated and replaced with fresh growth medium. Hybridoma supernatant was screened 2-3 days after the medium change.

[0050] Example 2: Antibody Screening and Detection

[0051] 2.1 Post-fusion detection

[0052] Indirect ELISA: Coat plates with SKBR3 cells and incubate overnight at 4°C. Block with unrelated protein (BSA) at 37°C for 2 hours. Add the test sample and incubate at 37°C for 1 hour. Wash 5-6 times with 0.05% Tween 20-PBS. Add secondary antibody (e.g., HRP-GAM) and incubate at 37°C for another 2 hours. Wash as above. Develop color with substrate (OPD). Stop the reaction with stop solution (3M H2SO4) when the color reaches a certain level. Read the OD on a microplate reader. 492 .

[0053] Flow cytometry (FACS) detection: Take 2×10 5 Add 50 μL of supernatant to SKBR3 cells (approximately 50 μL in volume) (total volume approximately 50-100 μL), incubate at room temperature for 30 minutes, add 2 ml of PBS, mix well, centrifuge, and discard the supernatant. Add 20 μL of secondary antibody and mix well.

[0054] Incubate at room temperature, protected from light for 30 minutes, wash once as above, add 0.5 ml of PBS, mix well, and then analyze using an instrument to identify positive clones for further subcloning.

[0055] 2.2 Subcloning:

[0056] Stable hybridoma cells were collected, counted, and adjusted to a concentration of 10 / ml. 100 μL / well was seeded into 96-well plates pre-added with feeder cells and allowed to grow, ensuring a 100% monoclonal positivity rate. If this was not achieved, subcloning was performed again. The selected positive clones were expanded; a portion was cryopreserved for cell culture, and the remainder was used for ascites production.

[0057] 2.3 Cell cryopreservation:

[0058] The cell cryopreservation solution was 10% DMSO (prepared with 20% FBS-1640). Hybridoma cells with a viability of over 95% were collected, centrifuged, the supernatant was discarded, the cells were resuspended in the cryopreservation solution, transferred to cryovials, and immediately placed in a -80°C freezer. The next day, they were transferred to a liquid nitrogen tank.

[0059] Example 3: Antibody Sequencing

[0060] 2.4 Hybridoma Sequencing

[0061] The sequences of the light and heavy chain variable regions of the 4A7 antibody were obtained using polymerase chain reaction (PCR) amplification technology called 5'RACE (rapid amplification of cDNA ends). Total RNA from hybridoma cells producing the 4A7 antibody was isolated using Trizol (Invitrogen), and cDNA was synthesized using the Superscript first-strand synthesis system (Invitrogen) with Oligo (DT) 12-18 primers. The variable regions of the mouse IgG gene were cloned using PCR with a leader primer targeting the heavy chain variable region and a leader primer targeting the light chain variable region (NOVAGEN). The resulting antibody bands were cloned into the TOPO TA cloning vector, and the DNA from more than 10 clones was sequenced and analyzed using a sequencer.

[0062] Results: Through hybridoma screening, one superior hybridoma monoclonal strain was obtained and named 4A7. The antibody sequence is shown in Sequence Listing 1.

[0063] Table 1

[0064]

[0065] Example 4: Antibody Preparation and Identification

[0066] Ascites preparation: BALB / c mice were injected with 0.5 mL of Pristane. One week later, 5 to 10 million hybridoma cells were inoculated intraperitoneally. Ascites was collected repeatedly every 3 to 4 days, for a total of 2 to 4 times per mouse. The fluid was then centrifuged at 1000 x g for 20 min to remove cells and debris, and then rapidly frozen at -80°C.

[0067] Antibody purification: After thawing, centrifuge at 45,000 rpm for 20 min to remove additional fibrin and particulate matter. The antibody can be rapidly and reliably purified using protein A affinity chromatography according to the protein A protocol. Elute the antibody with a PBS equilibration column (binding buffer) and sodium citrate buffer (pH 3.6, elution buffer). The purified antibody is thoroughly dialyzed in PBS at 4°C and pH 7.4, aseptically filtered through a 0.22-Mm Millipore filter, and then lyophilized.

[0068] Example 5: Antibody in vitro activity test

[0069] 4A7 blocks HRGβ1-, but not SDF-1-, induced triple-negative breast cancer cell migration.

[0070] HCC1806 and MDA-MB-231 cells were pre-incubated with mitomycin C (2 μg / mL) and seeded in 96-well plates. After 24 h, the cell confluence reached 100%, covering the bottom of the 96-well plates. Cells were scratched using a scratcher. Different groups were established and treated with corresponding drugs, and cell migration was monitored using the IncuCyte system. The 4A7 antibody significantly inhibited HRGβ1-induced tumor cell migration (HRGβ1 (50 ng / ml); 4A7 (20 μg / ml), SDF-1 (200 ng / ml)).

[0071] The combination of 4A7 antibody and gefitinib can significantly inhibit the growth of tumor cells.

[0072] HCC1806 cells were seeded in 96-well cell culture plates, with different reagents added to the culture medium for treatment: in the presence of solvent control (DMSO), gefitinib (4 mM), or gefitinib (4 mM) + 4A7 (20 mg / ml). The cell culture plates were placed in an IncuCyte system and incubated for 64 h in a standard cell culture incubator. Cell growth curves were generated by scanning data obtained every 4 h using IncuCyte. The data showed results from three independent experiments, *, p < 0.05.

[0073] The combination of 4A7 antibody and paclitaxel can significantly inhibit the growth of tumor cells.

[0074] HCC1806 cells were seeded in 96-well plates. After 24 hours, the medium was replaced with fresh medium containing a specified concentration of paclitaxel (with or without 4A7 (20 μg / ml)) and incubated for another 72 hours. The percentage of surviving cells relative to the control group was determined by the MTS assay and defined as 100% survival.

[0075] The combination of 4A7 antibody and paclitaxel can significantly inhibit the growth of tumor cells.

[0076] HCC1806 cells seeded in 6-well plates were treated with either paclitaxel (2 nM) or a combination of 4A7 (20 g / ml) and paclitaxel (2 nM) for 48 h. Live / dead cell imaging was then performed. Green indicates live cells; red indicates dead cells. Dead cells were counted in three random fields.

[0077] Example 6: In vivo antitumor experiment of 4A7 antibody

[0078] Orthotopic tumor model: Athymic nu / nu female mice were purchased from Charles River Laboratories Inc. (Wilmington, MA) and fed according to procedures and guidelines approved by the Institutional Animal Care and Use Committee. Luciferase-labeled HCC1806 cells (5 x 10⁻⁶) were used. 5 The tumor was suspended in 100 μL PBS and mixed with Matrigel (BD Biosciences, Franklin Lakes, NJ) (1:1), then inoculated into the mammary fat pads of mice to establish in situ tumors. Mice were imaged weekly using a bioluminescent IVIS imaging system to monitor tumor growth. Tumor formation was also measured twice weekly using fine calipers. Tumor volume was calculated using the following formula: Volume = (Length × Width²) / 2, where length is the major axis and the width is measured perpendicular to the length. When the tumor volume reached ~80 mm², the tumor was considered complete. 3 Mice bearing tumors were intraperitoneally injected with PBS, 4A7 (20 mg / kg), and paclitaxel (6 mg / kg) (Selleck Chemicals LLC, Houston, TX) or a combination of 4A7 and paclitaxel (n=5). Tumor growth curves were plotted using the average tumor volume, followed by statistical analysis. At the end of the experiment (after 6 treatments), all mice were sacrificed. Breast tumors were dissected and imaged using a digital camera.

[0079] In a triple-negative breast cancer xenograft model, the combination with 4A7 significantly enhanced paclitaxel-mediated antitumor activity.

[0080] In summary, this invention utilizes tumor cells to immunize mice and screens and prepares a monoclonal antibody using hybridoma fusion technology. This monoclonal antibody has a significant inhibitory effect on the growth and migration of tumor cells and exhibits significant anti-tumor activity in animal tumor experiments.

[0081] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody comprising a light chain variable region and a heavy chain variable region, characterized in that, The light chain variable region includes CDR-L1, CDR-L2 and CDR-L3, wherein the sequence of CDR-L1 is selected from the amino acid sequence shown in SEQ ID NO: 4, the sequence of CDR-L2 is selected from the amino acid sequence shown in SEQ ID NO: 5, and the sequence of CDR-L3 is selected from the amino acid sequence shown in SEQ ID NO:

6. The heavy chain variable region includes CDR-H1, CDR-H2 and CDR-H3, wherein the sequence of CDR-H1 is selected from the amino acid sequence shown in SEQ ID NO: 1, the sequence of CDR-H2 is selected from the amino acid sequence shown in SEQ ID NO: 2, and the sequence of CDR-H3 is selected from the amino acid sequence shown in SEQ ID NO:

3. The antibody binds to SKBR3 cells that overexpress HER3 molecules.

2. The monoclonal antibody according to claim 1, characterized in that, The light chain variable region further includes a leader sequence, which is selected from the amino acid sequence shown in SEQ ID NO: 8; The heavy chain variable region further includes a leader sequence selected from the amino acid sequence shown in SEQ ID NO:

7.

3. The monoclonal antibody of claim 1, characterized in that, It comprises a light chain variable region selected from the amino acid sequence shown in SEQ ID NO: 10 and a heavy chain variable region selected from the amino acid sequence shown in SEQ ID NO:

9.

4. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody as described in any one of claims 1 to 3.

5. A multispecific antibody, characterized in that It comprises the monoclonal antibody as described in any one of claims 1 to 3.

6. A vector, characterized in that, It comprises encoding the monoclonal antibody as described in any one of claims 1 to 3.

7. A host cell, characterized in that, It expresses the monoclonal antibody as described in any one of claims 1 to 3.

8. A pharmaceutical composition, characterized by, It comprises the monoclonal antibody as described in any one of claims 1 to 3 and one or more pharmaceutically acceptable excipients.

9. A combination medicament, characterized in that It comprises a monoclonal antibody, an anticancer drug, and one or more pharmaceutically acceptable excipients as described in any one of claims 1 to 3; the anticancer drug includes gefitinib and paclitaxel.

10. Use of the monoclonal antibody according to any one of claims 1 to 3, the multispecific antibody according to claim 5, the pharmaceutical composition according to claim 8, or the combination of drugs according to claim 9 in the preparation of a medicament for treating triple-negative breast cancer.

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