CN114790244B

CN114790244B - Use of DC tumor vaccine combined monoclonal antibody in preparation of pharmaceutical composition for treating cancer

Info

Publication number: CN114790244B
Application number: CN202210636789.4A
Authority: CN
Inventors: 史皓羽; 刘燕武
Original assignee: Zhongzhen Shenzhen Biomedical Research Co ltd; Ren Dianming
Current assignee: Ren Dianming; Zhongzhen Shenzhen Biomedical Research Co ltd
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2023-06-23
Anticipated expiration: 2042-06-07
Also published as: CN114790244A

Abstract

The invention relates to the use of DC tumor vaccine combined monoclonal antibodies in the preparation of pharmaceutical compositions for the treatment of cancer. The DC tumor vaccine for gastric cancer is specifically prepared, has better immune response stimulation, can effectively perform the function of killing tumor cells by being combined with a CLDN18.2 monoclonal antibody or being used independently, and has better application value.

Description

Use of DC tumor vaccine combined monoclonal antibody in preparation of pharmaceutical composition for treating cancer

Technical Field

The invention relates to the field of biology, in particular to application of a DC tumor vaccine combined monoclonal antibody in preparation of a pharmaceutical composition for treating cancers.

Background

The CLDJV18.2 gene is located at the 3q22 site of the human chromosome. CLDN18.2 consists of 261 amino acids, is a four-transmembrane structure, has a small relative molecular mass 277, and has amino and carboxyl ends both located inside the cell membrane. CLDN18.2 has 2 extracellular domains, extracellular loop 1 is a 4-fold inverted 3-lamellar structure consisting of 53 amino acids, in which 2 highly conserved cysteines exist between β3 and β4, and disulfide bonds formed between them contribute to the stabilization of the molecular structure; extracellular loop 2 is composed of 31 amino acids and is located between transmembrane region 3 and transmembrane region 4. CLDN18.2 is expressed restrictively on the surface of differentiated mature gastric mucosal epithelial cells and various tumor cells, and is not expressed in the stem cell area of stomach and other normal tissues.

Abnormal expression of CLDN18.2 is closely related to the development and progression of a variety of cancers. It has been shown that increased expression of CLDN18.2 is associated with bile duct tumor formation, and CLDN18.2 is involved in the processes of occurrence, proliferation and invasion of human intrabile duct adenocarcinoma cells, and is overexpressed on the surface of bile duct adenocarcinoma invading anterior cells. In addition, there is aberrant activation of CLDN18.2 in malignant transformation of non-small cell lung cancer. However, in the studies of Oshima et al, the decrease in the expression level of CLDN1&2 in the leading tissue of gastric cancer invasion revealed that proliferation and invasion of gastric cancer cells may be associated with down-regulation of CLDN18.2 expression. In pathological conditions, expression of claudin18.2 is significantly up-regulated in a variety of tumors, including 80% of gastrointestinal adenomas, 60% of pancreatic tumors. Furthermore, CLDN18.2 activation is also seen in esophageal, ovarian and lung adenocarcinomas and is therefore a hot target with potential for cancer treatment.

IMAB362 is a human murine chimeric mab raked to CLDN18.2 developed by Ganymed, germany, and is produced in Chinese hamster ovary cells using recombinant antibody expression techniques. IMAB362 specifically recognizes CLDM8.2 on the cell surface without binding to CLDN18.1, mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and has biological functions such as inhibition of cell proliferation and mediation of apoptosis. Studies in pancreatic cancer models have found that MAB362 is capable of specifically binding pancreatic cancer cells and mediating ADCC and CDC to apoptosis by cell lysis, the capacity of which is positively correlated with the expression level of cell surface CLDN 18.2. Furthermore, the chemotherapeutic agent gemcitabine can up-regulate expression of CLDN18.2 in human pancreatic cancer cells cultured in vitro, enhancing IMAB 362-induced ADCC activity. In mouse xenograft tumors derived from human pancreatic cancer cell lines (including gemcitabine-resistant lines), IMAB362 slows tumor growth, prolongs survival of mice, and reduces the potential for metastasis development.

Dendritic Cells (DCs) play a key role in initiating immune responses and are the most important, most functional professional antigen presenting cells in the body, and are therefore the best adjuvants for anti-tumor immunity. In recent years, many researches prove that DC loaded by different forms of tumor antigens can be made into vaccines, can induce the generation of specific killer T Cells (CTL) in vivo and in vitro, excite the effective specific anti-tumor immunity of human bodies, and achieve exciting curative effects in the trial treatment of malignant tumors. Currently, DC-mediated neoplasia is mainly DC stimulated by the following (1) antigenic peptides; (2) tumor extract stimulated DCs; (3) DC transfected with tumor antigen gene; (4) fusion cells of tumor cells and DCs; (5) the antigenic peptide stimulates a subcellular structure vaccine of DCs. Various load forms of tumor antigen can be used to payload DCs.

Studies of tumor models have also demonstrated that DC vaccines transduced with tumor antigen genes have therapeutic effects on established tumors. The therapeutic effect of the tumor antigen gene modified DC as a vaccine on B16/F10 melanoma expressing target tumor antigens was studied, and as a result, it was found that inoculation of MAGE21 gene transduced DC resulted in a reduction of the number of metastases in lung metastasis models, an extension of the survival time of animals, and a long-term cure of part of the subcutaneous tumor models. In addition, tumor mRNA isolated from tumor cell lines can be amplified in vitro without losing its biological function. mRNA transduced murine DCs amplified from the melanoma B16/F1019 cell line were able to stimulate CTL responses in mice, producing protective immune responses against post-operative tumor metastasis.

However, there are few studies on tumor DC vaccines, and particularly on the use of tumor DC vaccines with specific therapeutic antibodies, and further development is desired.

Disclosure of Invention

In one aspect, the invention provides a specific DC tumor.

The DC tumor vaccine is prepared by uniformly mixing DC mature cells with gastric cancer SNU601 cell strain mRNA, selecting voltage 700V, pulse time interval of 230 μs, shocking cells at 4deg.C, electrically transfecting for 30min, adding Alys-505 culture solution containing 10% fetal bovine serum, placing at 37deg.C and 5.0% CO ₂ Culturing in incubator for 48 hr.

In another aspect of the invention, there is provided the use of specific DC tumors Miao Zaizhi in the preparation of a pharmaceutical composition for the treatment of cancer.

Further, the pharmaceutical composition may further comprise other commonly used therapeutic agents, such as antibodies, small molecule compounds.

Further, in general, a therapeutically effective amount of an antibody, small molecule or other compound or composition described herein may range from 0.01mg/kg to 100mg/kg, and all values therebetween. For example, it may be 0.1mg/kg to 100mg/kg,0.1mg/kg to 50mg/kg,1mg/kg to 50mg/kg, etc.

Further, generally, a therapeutically effective amount of a DC tumor described herein may be at 1 x 10 ⁶ -1*10 ¹¹ In the range of/kg, and all values in between.

In another aspect, the invention also provides a monoclonal antibody specific for CLDN18.2, CLDN18.2-19, having an amino acid sequence of the light chain variable region as shown in SEQ ID No. 4:

DLVMTQTAPSVPVTPGESVSISCRSTWSPCVQPVWYQLYWFLQRPGQSPQLLIYIANNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCKLIFYEASMFGSGTKLEIK

the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 5:

VKPGGSLKLSCAASYPYKDISKMSWVRQTPDKRLEWVAIRSEMSAWRYYPDSVKGRFTISRDQDKQTLYLQMSSLKSEDTAMYYCMEEAFLDIMCYWGQGTTVTVS。

furthermore, the invention also provides application of the monoclonal antibody specific to CLDN18.2 in preparing a pharmaceutical composition for gastric cancer.

In some aspects, the invention relates to a pharmaceutical composition comprising at least one CLDN18.2 antibody as disclosed herein and a pharmaceutically acceptable carrier.

The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-CLDN 18.2 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers can include, for example, pharmaceutically acceptable liquid, gel or solid carriers, aqueous media, nonaqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispersing agents, chelating agents, diluents, adjuvants, excipients or non-toxic auxiliary substances, combinations of various components known in the art or more.

Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavouring agents, thickening agents, colouring agents, emulsifying agents or stabilizing agents such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, thioglycerol, thioglycolic acid, thiosorbitol, butylmethylanisole, butylated hydroxytoluene and/or propyl arsenate. As disclosed herein, the antibodies or antigen-binding fragments of the disclosed compositions may be oxidized in a solvent containing one or more antioxidants, such as methionine, that reduce the antibodies or antigen-binding fragments thereof. Redox can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Thus, in some embodiments, the invention provides compositions comprising one or more antibodies or antigen binding fragments thereof and one or more antioxidants, such as methionine. The invention further provides methods wherein the antibodies or antigen binding fragments thereof are admixed with one or more antioxidants such as methionine. Thus, the antibody or antigen binding fragment thereof may be protected from oxidation to extend its shelf life and/or increase activity.

For further illustration, pharmaceutically acceptable carriers may include, for example, aqueous carriers such as sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection or dextrose and lactate ringer's injection, non-aqueous carriers such as fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil, bacteriostats or antifungal concentrations of antimicrobial agents, isotonic agents such as sodium chloride or glucose, buffers such as phosphate or citrate buffers, antioxidants such as sodium bisulfate, local anesthetics such as procaine hydrochloride, suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone, emulsifying agents such as polysorbate 80 (TWEEN-80), chelating agents such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid), ethanol, polyethylene glycol, propylene glycol, sodium hydroxide, hydrochloric acid, citric acid or lactic acid. The antimicrobial agent used as a carrier may be added to a pharmaceutical composition in a multi-dose container containing phenol or cresol, a mercuric preparation, benzyl alcohol, chlorobutanol, methyl and propyl parahydroxybenzoates, thimerosal, benzalkonium chloride and benzethonium chloride. Suitable excipients may include, for example, water, saline, dextrose, glycerol or ethanol. Suitable non-toxic auxiliary substances may include, for example, wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or agents such as sodium acetate, sorbitan monolaurate, triethanolamine oleate, or cyclodextrins.

In another aspect, methods of treatment are provided for preventing or alleviating a disorder in a subject that would benefit from modulation of CLDN18 (particularly CLDN 18.2) activity comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof provided herein, and/or a pharmaceutical composition provided herein, and/or a chimeric antigen receptor provided herein. In certain embodiments, the disease, disorder or condition is a disease, disorder or condition associated with CLDN18 (particularly CLDN 18.2) as described above.

The therapeutically effective amount of an antibody or antigen binding fragment provided herein depends on various factors known in the art, such as weight, age, past medical history, current medications, the likelihood of health and cross-reactivity of the subject, allergies, sensitivity and adverse side effects, and the route and extent of administration of the disease progression. The dosage may be reduced or increased proportionally by those skilled in the art (e.g., a physician or veterinarian), as indicated by these and other circumstances or requirements.

In certain embodiments, the antibodies or antigen binding fragments provided herein can be administered at a therapeutically effective dose of about 0.01mg/kg to about 100 mg/kg. In certain embodiments, the dosage administered may vary during the course of treatment. For example, in certain embodiments, the initial dose may be higher than the subsequent doses. In certain embodiments, the dosage administered may vary throughout the course of treatment according to the subject's response.

The dosing regimen may be adjusted to provide the best desired response (e.g., therapeutic response). For example, a single dose may be administered, or several separate doses may be administered over time.

The antibodies or antigen-binding fragments thereof provided herein may be administered by any route known in the art, such as parenteral (e.g., subcutaneous, intraperitoneal, intravenous, including intravenous infusion, intramuscular or intradermal injection) or non-parenteral (e.g., oral, nasal, intraocular, sublingual, rectal or topical).

In some embodiments, an antibody or antigen binding fragment thereof provided herein can be administered alone or in combination with a therapeutically effective amount of a second therapeutic agent. For example, an antibody or antigen binding fragment thereof disclosed herein can be administered in combination with a second therapeutic agent, e.g., a chemotherapeutic agent, an anti-cancer drug, a radiation therapeutic agent, an immunotherapeutic agent, an anti-angiogenic agent, a targeted therapeutic agent, a cell therapeutic agent, a gene therapeutic agent, a hormonal therapeutic agent, an antiviral agent, an antibiotic, an analgesic, an antioxidant, a metal chelator, or a cytokine.

Furthermore, the invention also provides a monoclonal antibody specific to CLDN18.2 and application of the monoclonal antibody in preparing a pharmaceutical composition for gastric cancer by DC tumor Miao Zaizhi.

The DC cells are prepared by separating from peripheral blood and are subjected to induced maturation, and mRNA of gastric cancer SNU601 cells is introduced into the DC cells.

Advantageous effects

The DC tumor vaccine for gastric cancer is specifically prepared, has better immune response stimulation, can effectively perform the function of killing tumor cells by being combined with a CLDN18.2 monoclonal antibody or being used independently, and has better application value.

Drawings

FIG. 1 is a graph showing the results of flow cytometry detection of expression of surface molecules before and after transformation of DC cells

FIG. 2 is a graph showing the proliferation effect of T cell activation by DC vaccine

FIG. 3 graph of killing effect of DC vaccine and/or CLDN18.2 monoclonal antibody on tumor cells

Detailed Description

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

EXAMPLE 1 preparation of gastric cancer specific DC vaccine

Gastric cancer SNU601 cell strain is cultivated by adopting RPMI1640 culture medium (containing 10% of fetal calf serum), when the cells grow to 90% of culture dishes, the cells are digested by pancreatin, passage is carried out according to 1:3, and the cells with 3 rd generation in logarithmic growth phase are taken for mRNA extraction.

Extraction of mRNA of SNU601 gastric cancer cell line: the cells in the logarithmic growth phase were washed with PBS and total RNA was extracted by a standard Trizol-chloroform one-step method. Treatment with RNase-free DNaseI eliminates small amounts of contaminating DNA in the total RNA pool. RNA purification was performed according to the instructions for DNaseI reagent. The total RNA is quantified by using a micro-spectrophotometer, the result shows that the OD260/OD280 value is 1.86, the OD260/OD230 value is 1.95, the extracted RNA is relatively pure, no polysaccharide, protein, small molecular substances and other impurities exist, the concentration of the total RNA is measured to be 1054 mug/mL, the concentration is high, and the total RNA is stored at the temperature of minus 80 ℃ for standby.

Isolation, culture and loading of DC cells:

peripheral Blood Mononuclear Cells (PBMCs) were isolated from 50mL of peripheral blood, anticoagulated with heparin, and lymphocyte separation. The PBMC cells were resuspended in Alys-505 medium containing 10% autologous plasma and sub-packed in six well plates, 5X 10 ⁶ Every mL,2 mL/well, saturated humidity, 37 ℃, 5.0% CO ₂ Is cultured in an incubator for 2 hours. Washing and collecting adherent cells in six-well plate, wherein the adherent cells are DC cells, adding Alys-505 culture solution containing 100ng/mLGM-CSF, 20ng/mL IL-4 and 10% autologous plasma into six-well plate, placing in saturated humidity, 37deg.C, 5.0% CO ₂ DC cells were induced in the incubator. Half-amount of the culture was changed after 3 days and fresh cytokine was supplemented, maturation-inducing factor TNF-a (20 ng/mL) was added on day 5 of culture, and mature DCs were harvested on day 7.

0.5mL of DC mature cells was concentrated to 5X 10 ⁶ Uniformly mixing the single/mL with 200 mug gastric cancer SNU601 cell strain mRNA, selecting the voltage of 700V,pulse time course is 230 μs, cell is shocked at 4deg.C, alys-505 culture solution containing 10% foetal calf serum is added 30min after electrotransfection, and 37 deg.C and 5.0% CO are placed ₂ Culturing in incubator for 48 hr. Flow cytometry was used to detect expression of molecules on the surface of DCs, and the results are shown in figure 1.

FIG. 1 shows that DC tumor vaccine and DC are subjected to fluorescent labeling flow cytometry detection phenotype after electric transfection, CDla expression of DC vaccine is significantly reduced (P < 0.05), HLA-DR is not significantly changed (P > 0.05), and CD86 and CD40 expression is significantly up-regulated (P < 0.05) compared with mature DC, which also proves that DC tumor vaccine is successfully constructed.

Example 2 immune Effect validation of gastric cancer specific DC vaccine

Taking SD rat bone marrow-derived T lymphocytes, incubating with DC tumor vaccine prepared in example 1, and taking DC as a control, wherein the ratio of the cell density is 1: 10. 1:20, 1: 50. 1:100 (DC: T cells) were added to each of the 96-well plates, and after co-culturing for 96 hours, CCK8 lOul was added to each well, and after incubation for 2 hours, the optical density of each well was measured at 0D490 by a microplate reader, and the results are shown in FIG. 2.

As can be seen from the results of fig. 2, T cells can be activated with very small amounts of DC vaccine, and the ability to stimulate T cell proliferation increases with increasing proportion (P < o.05); at the same ratio, DC vaccines have a greater capacity to stimulate T cell proliferation than mature DCs (P < 0.05).

EXAMPLE 3 preparation of human CLDN18.2 monoclonal antibody

The epitope peptides of human CLDN18.1, human CLDN18.2 and mouse CLDN18.2 shown in SEQ ID NO. 1-3 are determined by searching dominant B cell epitopes and aiming at the results of computer analysis modeling and the dominant epitope sequences of specificity discrimination on the basis of the amino acid sequences of human and mouse CLDN18.1 and CLDN18.2, the epitope peptides are entrusted to the synthesis of Shenzhen Kagaku biological engineering Co., ltd, only human CLDN18.2 polypeptide is coupled with KLH, and three polypeptides are respectively coupled with BSA, wherein human CLDN18.2 polypeptide-KLH coupling complex is used for immunizing mice, and the polypeptide is coupled with BSA for ELISA detection of antibody titers of serum and hybridoma cell supernatants and ascites of immunized mice. The successful coupling of the polypeptide to KLH or BSA was identified by SDS-PAGE.

Establishment of hybridoma cell lines: 4 healthy 8-week-old female BALB/c mice were first immunized by mixing human CLDN18.2 polypeptide-KLH with an equal volume of complete adjuvant, and injected intraperitoneally, 150. Mu.L/mouse. Thereafter, an equal amount of injection was exchanged for incomplete adjuvant for a total of 3 times at interval 14 d. 3d prior to fusion, immunization was boosted directly intraperitoneally with 100 μl of immunogen without adjuvant. Mouse spleen cells were aseptically taken with Sp2/0 myeloma cells at 10:1, mixing evenly, fusing under the action of PEG4000, adding a 96-well plate after HAT culture medium is resuspended, culturing in a C2O incubator at 37 ℃, changing HT culture medium after 14d, and changing 100mL/L complete RPMI1640 culture medium after 20 d. Cell screening and positive cell cloning: screening by using an indirect ELISA method, detecting the coating of the original human CLDN18.2 polypeptide-KLH by 200 mug/L, sealing by using a 5Og/L skim milk powder solution, and detecting the culture supernatant of the hybridoma cells, wherein 53 positive holes are reserved in the primary screening. And (3) reversely screening by adopting a human CLDN18.1 polypeptide and a mouse CLDN18.2 polypeptide, removing non-specifically bound positive cells, continuously subcloning for 4 times by a limiting dilution method, finally screening to obtain positive and stably expressed 2 hybridoma cells which are CLDN18.2-5 and CLDN18.2-19 respectively, carrying out expanded culture on the positive hole cells, and establishing strains and freezing.

Preparation and purification of ascites by in vivo induction method, hybridoma cell strains CLDN18.2-5 and CLDN18.2-19,1 ×10 capable of stably secreting mAb against human CLDN18.2 polypeptide are selected ⁸ And (3) inoculating BALB/c mice injected with pristane, collecting ascites for 12 days, and purifying the ascites antibody by adopting an octanoic acid-ammonium sulfate method. The ascites after purification is tested for potency by indirect ELISA. The results are shown in Table 1.

TABLE 1 Indirect ELISA method for detecting 2 mAb titers

As can be seen from the results of Table 1, the titers of both CLDN18.2-5 and CLDN18.2-19 reached 1:10 ⁷ 。

The concentrations of the CLDN18.2-5 and the CLDN18.2-19 proteins were calculated by the Lowry-kalokar formula and were 7.82mg/mL and 7.59mg/mL, respectively, and stored at-70℃for further use.

Ig subclass detection: the hybridoma cell culture supernatant was assayed according to the mouse mAbIg subclass assay kit, and the subclasses to which the identified mAbs belong are shown in Table 2.

Table 2 indirect ELISA method to detect 2 mAb titers

As can be seen from the results in Table 2, the 2 monoclonal antibodies were of the IgG1 subtype.

Westernblot specificity identification: SDS-PAGE electrophoresis is carried out on artificial antigens of human CLDN18.2 polypeptide-BSA, human CLDN18.1 polypeptide-BSA, mouse CLDN18.2 polypeptide-BSA and BSA control, the artificial antigens are transferred to a nitrocellulose membrane, the nitrocellulose membrane is blocked overnight at 4 ℃ by 80g/L of skim milk powder, purified mAb is added as primary antibody, goat anti-mouse (1:10000) is marked by horseradish peroxidase, incubation is carried out for 2h at room temperature, TBST is washed for 3 times, and DAB color development is carried out. The results show that the CLDN18.2-5 and CLDN18.2-19 monoclonal antibodies only bind to human CLDN18.2 polypeptide-BSA, but not to other antigens or controls, which indicates that the monoclonal antibodies of the invention have better specificity.

EXAMPLE 4 characterization of human CLDN18.2 monoclonal antibody CLDN18.2-19

The affinity of antibodies CLDN18.2-19 to human CLDN18.2 antigen was measured using a surface plasmon resonance biosensor. Affinity analysis is carried out on an SPR (Biacore T200) instrument, an anti-human IgG Fc antibody is coupled on a CM5 chip in an amino coupling mode, the antibody to be detected flows in at a flow rate of 30 mu L/min, and the antibody to be detected is captured by the anti-human IgG Fc antibody coupled on the chip; after gradient dilution of the analyte (human CLDN 18.2) (100 nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 0 nM), the antibody to be tested was flowed in at a flow rate of 30 μl/min for a binding time of 120s with the analyte and a dissociation time of 1200s; HBS-EP was used as running buffer for the whole experiment, and 10mM glycine was used for the chipThe acid HCl, pH2.1 solution was regenerated by 60 second pulses. The assay data were fit to a 1:1 binding model to determine the equilibrium dissociation constant KD. The results showed that the affinity of monoclonal antibody CLDN18.2-19 was 2.5 x 10 ^-10 nM。

In addition, the sequence amplification and sequencing of the variable region of the light chain of the antibody are carried out by adopting an antibody light-heavy chain sequence identification kit, and the amino acid sequence of the variable region of the light chain is shown as SEQ ID NO.4 after sequence identification:

example 5 experiment of DC tumor in combination with CLDN18.2-19 mab in the treatment of gastric cancer

The killing effect experiment of the DC tumor vaccine and CLDN18.2-19 on tumor cells is as follows: (1) Culturing SNU601 gastric cancer cells, taking SNU601 gastric cancer cells in logarithmic growth phase, and regulating cell concentration to 2×10 ⁵ 96-well culture plates lcoul/well; (2) after 24 hours, adding in groups respectively: CLDN18.2-19 mab (50 ug/ml), DC vaccine + T cells (1:1 total 2 x 10 ⁶ Per well), CLDN18.2-19 mab (50 ug/ml) +dc vaccine+t cells (1:1 co 2 x 10) ⁶ Hole) and a negative control group without adding medicine, wherein the positive control group is Nibolumab (50 ug/ml), and each group is respectively provided with 3 compound holes; (3) After the culture is continued for 96 hours, the supernatant is sucked up by each group, after lOOul PBS is added into the holes, CCK8 lOul is added into the holes, and after 2 hours, the absorbance value is detected by an enzyme-labeling instrument; (4) And according to the formula, the tumor killing rate is = [ (control group 0D value-experimental group 0D value)/control group 0D value]The killing rate of each group on tumor cells was calculated separately. The results are shown in FIG. 3.

The experimental results show that: the single CLDN18.2-19 monoclonal antibodies and the single DC vaccine can effectively kill gastric cancer cells, and have obvious effect (P<0.05). In particular CLDN18.2-19 mab (50 ug/ml) +dc vaccine+t cells (1:1 co 2 x 10) ⁶ Hole) group has a synergistic killing-increasing effect, whichThe killing rate reaches (99.4+/-2.7)%, and is obviously higher than that of a single treatment group.

While the invention has been described and illustrated in detail as being sufficient to enable one skilled in the art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, are exemplary, and are not intended to limit the scope of the invention. Modifications and other uses thereof will occur to those skilled in the art. Such modifications are intended to be included within the spirit of the invention and are to be limited only by the scope of the appended claims.

Sequence listing

<110> Nanjing Haoyu biotechnology Co., ltd

Use of <120> DC tumor combined monoclonal antibody in preparation of pharmaceutical composition for cancer treatment

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<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu

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Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Pro Tyr Phe Thr Ile Leu

65 70

<210> 2

<211> 70

<212> PRT

<213> Artificial sequence (Artificial Sequence)

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

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu

65 70

<210> 3

<211> 69

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

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

1 5 10 15

Gly Phe Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu

65

<210> 4

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

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

1 5 10 15

Glu Ser Val Ser Ile Ser Cys Arg Ser Thr Trp Ser Pro Cys Val Gln

20 25 30

Pro Val Trp Tyr Gln Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser

35 40 45

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

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

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85 90 95

Phe Tyr Glu Ala Ser Met Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

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

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Arg Leu Glu Trp Val Ala Ile Arg Ser Glu Met Ser Ala Trp Arg Tyr

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100 105

Claims

1. A monoclonal antibody CLDN18.2-19 specific for CLDN18.2, characterized in that the amino acid sequence of the light chain variable region of said antibody is shown in SEQ ID No. 4:

DLVMTQTAPSVPVTPGESVSISCRSTWSPCVQPVWYQLYWFLQRPGQSPQLLIYIANNLASGVPDRFSGSGSGTA FTLRISRVEAEDVGVYYCKLIFYEASMFGSGTKLEIK

VKPGGSLKLSCAASYPYKDISKMSWVRQTPDKRLEWVAIRSEMSAWRYYPDSVKGRFTISRDQDKQTLYLQMSSLKSED TAMYYCMEEAFLDIMCYWGQGTTVTVS。

2. use of the monoclonal antibody of claim 1 in the preparation of a pharmaceutical composition for treating gastric cancer.

3. Use of a monoclonal antibody CLDN18.2-19 specific for CLDN18.2 in combination with a DC tumor vaccine for the manufacture of a pharmaceutical composition for the treatment of SNU601 cell-induced gastric cancer; wherein the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO. 4:

VKPGGSLKLSCAASYPYKDISKMSWVRQTPDKRLEWVAIRSEMSAWRYYPDSVKGRFTISRDQDKQTLYLQMSSL KSEDTAMYYCMEEAFLDIMCYWGQGTTVTVS；

the DC tumor vaccine is prepared by introducing DC cells into mRNA of gastric cancer SNU601 cells, and the DC cells are prepared by separating from peripheral blood and are subjected to induced maturation.

4. Use according to claim 2 or 3, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.