HER2 vaccine composition

Abstract

The present invention relates to a HER2-ICD DNA vaccine composition. The vaccine composition according to the present invention can effectively suppress the growth of gastric cancer without serious side effects in an animal model transplanted with a human gastric cancer cell line expressing HER2, and therefore can be usefully used in the treatment of gastric cancer.

Description

[Technical Field]

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2021-0099411, filed on July 28, 2021, and all contents disclosed in the documents of this Korean patent application are incorporated herein by reference.

[0002] The present invention relates to a HER2 vaccine composition for the treatment of gastric cancer. [Background technology]

[0003] Gastric cancer essentially refers to all cancers that originate in the stomach. However, it can occur in a variety of forms, including adenocarcinoma, which arises in the glandular cells of the gastric mucosa, and gastric intes- tinal tumor, which arises in the interstitial cells of the stomach. Like other cancers, it can metastasize to other organs, including the esophagus, lungs, and liver. Gastric cancer is the fourth most frequently diagnosed cancer in the world, with 930,000 cases diagnosed in 2002. Its high incidence rate translates into a high mortality rate (approximately 800,000 cases per year), making it the second leading cause of cancer deaths in the world, after lung cancer. This cancer is more common in men than in women, and is more prevalent in Asian and developing countries.

[0004] Treatments for gastric cancer include surgery, chemotherapy, radiation therapy, and concurrent anticancer chemoradiotherapy. Surgery is the primary treatment for gastric cancer. The 5-year survival rate for surgical resection, the goal of treatment, is 30% to 50% for stage II patients and 10% to 25% for stage III patients. However, surgical resection and subsequent anticancer therapies, such as chemotherapy, can reduce patient quality of life, such as weight loss, so other treatment options are needed.

[0005] Vaccines help the body fight disease by training the immune system to recognize and destroy harmful components and diseased cells. Vaccines can be broadly divided into two types: preventive vaccines and therapeutic vaccines. Preventive vaccines are a form of traditional vaccine and are given to healthy individuals to prevent the development of specific diseases, while therapeutic vaccines, also known as immunotherapy, are given to individuals diagnosed with a disease to help interrupt the growth and spread of the disease or as a preventative measure. A typical form of therapeutic vaccine is a cancer vaccine.

[0006] Cancer vaccines are designed to activate cytotoxic T cells, which then recognize and act against cancer cells expressing specific antigens or induce the production of antibodies that bind to antigens present on the surface of cancer cells.The basic therapeutic goal of cancer vaccines is to reduce the size of existing cancer tissue in cancer patients or to prevent cancer recurrence after removal of cancer tissue by surgery or chemotherapy.

[0007] Over the past few years, extensive research has been conducted into vaccines aimed at treating or preventing cancer recurrence, but with the exception of the prostate cancer vaccine sipuleucel-T (Provenge™), no successful cases have been reported to date. The low success rate has been partially explained by the fact that, even if an antigen is specifically expressed in a particular type of tumor cell, each individual expresses the antigen at a low level, and in the case of metastatic tumors, the antigen profile changes between the initial tumor and the metastatic tumor, as well as the immune evasion mechanisms of tumor cells.

[0008] A desirable vaccine antigen must be expressed exclusively, or at least at elevated levels, in tumor cells. HER2 / neu (hereinafter referred to as "HER2") is a member of the EGFR (epidermal growth factor receptor) family (EGFR2). Overexpression of EGFR is observed in many carcinomas, and it is known that approximately 20% of all gastric cancer patients are diagnosed with HER2-positive advanced gastric cancer.

[0009] HER2 is a transmembrane protein having the amino acid sequence of SEQ ID NO: 1, and is composed of an intracellular domain (ICD (intracellular domain) (HER2 aa 676-1255)) with protein tyrosine kinase activity, a transmembrane domain (TM (HER2 aa 653-675)), and an extracellular domain (ECM (extracellular domain) (HER2 aa 22-652)).

[0010] HER2 vaccines have been extensively studied in breast cancer, and several immunogenic peptides have been identified. For example, a peptide consisting of amino acids 369-377 of the HER2 protein (nelipepimut-S [NeuVax™ (HER2 aa 369-377) (E75(NP-S))]) was tested as an immunotherapy by loading it into dendritic cells and then reinjecting them, but no clinically significant therapeutic or protective effects have been reported in women with advanced breast cancer. Furthermore, three large-scale clinical trials recently showed no difference in disease-free survival (DFS) compared to the placebo group, leading to the discontinuation of clinical trials.

[0011] Further examples of HER2 peptide vaccines include the HER2-ICD peptide vaccine AE37 (LRMK-GVGSPYVSRLLGICL (LRMK-HER2 aa 776-790)) and the HER2-TM peptide vaccine GP2 (HER2 aa 654-662), and some reports have shown that these vaccines increase survival rates. Furthermore, animal experiments have shown that a vaccine against a HER2-ICD fragment (hereinafter referred to as "HER2-ICD") reduces tumor size compared to a vaccine against a HER2-ECD fragment (hereinafter referred to as "HER2-ECD"), and there have been cases where a HER2-ICD DNA vaccine has been administered to breast cancer patients. However, no results have been reported on the administration of a HER2-ICD DNA vaccine to non-breast cancer gastric cancer.

[0012] Therefore, there is a need to explore the immunological effects and therapeutic potential of HER2-ICD DNA vaccines in gastric cancer patients to generate vaccines that provide reliable efficacy against the treatment or recurrence of gastric cancer. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] Korean Patent No. 10-1572474 (Publication date: 2015.11.30.) [Patent Document 2] US Patent Publication US2002 / 0193329 (Publication Date: 2002.12.19.) [Non-patent literature]

[0014] [Non-Patent Document 1] Breast Care 2016: 11, 116-121 [Non-patent document 2] Clin Cancer Res: 25 (14) July 15, 2019 Summary of the Invention [Problem to be solved by the invention]

[0015] As a result of research conducted by the inventors to solve the above-mentioned problems, they found that a DNA vaccine composition containing a plasmid containing a nucleic acid sequence encoding HER2-ICD can effectively suppress tumor growth in a mouse model transplanted with a human gastric cancer cell line, thereby completing the present invention.

[0016] An object of the present invention is to provide a vaccine composition comprising a plasmid containing a nucleic acid sequence encoding HER2-ICD (hereinafter referred to as "HER2-ICD DNA vaccine composition").

[0017] The present invention provides a kit for treating gastric cancer, which comprises the HER2-ICD DNA vaccine composition.

[0018] Another object of the present invention is to provide a method for suppressing the progression and / or recurrence of gastric cancer by administering the vaccine composition or kit to a subject.

[0019] The present invention provides the use of the composition or kit in the manufacture of a medicament for the treatment of gastric cancer. [Means for solving the problem]

[0020] To achieve the above object, the present invention provides a DNA vaccine composition comprising a plasmid containing a nucleic acid sequence encoding HER2-ICD.

[0021] According to one embodiment of the present invention, the vaccine composition can be used in gastric cancer patients.

[0022] According to one embodiment of the present invention, the gastric cancer patient group is also a patient group that expresses HER2 protein.

[0023] According to one embodiment of the present invention, the vaccine composition further comprises an adjuvant.

[0024] According to one embodiment of the present invention, the vaccine composition may be administered in combination with one or more anti-cancer agents selected from chemical anti-cancer agents, targeted anti-cancer agents, and immunological anti-cancer agents.

[0025] According to one embodiment of the present invention, the gastric cancer patient group is also a human patient group. [Effects of the Invention]

[0026] The vaccine composition according to the present invention is a vaccine composition comprising a plasmid containing a nucleic acid sequence encoding HER2-ICD, and can effectively suppress tumor growth in an animal model of gastric cancer, and can be useful for treating gastric cancer. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows the changes in body weight of each individual when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), and a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) were administered in a gastric cancer cell line xenograft mouse model. [Figure 2] This figure shows the tumor growth inhibitory effect when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), and a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) were administered in a mouse xenograft model implanted with a gastric cancer cell line. [Figure 3]This figure shows the tumor growth inhibitory effect when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), and a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) were administered in a mouse xenograft model implanted with a gastric cancer cell line. [Figure 4] This figure shows the tumor growth inhibitory effect when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), and a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) were administered in a mouse xenograft model implanted with a gastric cancer cell line. [Figure 5] FIG. 1 shows changes in the spleen in a gastric cancer cell line xenograft mouse model when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), or a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) was administered. [Figure 6] FIG. 1 shows changes in the spleen in a gastric cancer cell line xenograft mouse model when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), or a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) was administered. [Figure 7] FIG. 1 shows changes in the spleen in a gastric cancer cell line xenograft mouse model when a HER2-ICD DNA vaccine, an anti-HER2 therapeutic antibody (Herceptin), or a combination of the DNA vaccine and Herceptin (vaccine + Herceptin) was administered. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention will be described in further detail below.

[0029] In describing and claiming particular features of the present disclosure, the following terminology will be used in accordance with the definitions set forth below unless otherwise specified.

[0030] It should be understood that although an embodiment may be described herein with the term "comprising," other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0031] The term "treat" not only refers to the result or sign of the result of preventing or reducing tumor growth when a cancer patient is administered with the vaccine composition of the present invention and then examined by radiation or physical methods, but also refers to the result or sign of the result of preventing cancer recurrence / relapse when a cancer patient is administered with the vaccine composition of the present invention and then examined by radiation or physical methods, after cancer tissue has been removed from the patient by surgery or administration of an anticancer drug.

[0032] The term "anti-cancer effect" not only means the effect of preventing or reducing tumor growth when the vaccine composition of the present invention is administered to a cancer patient as a recipient, but also means the effect of preventing cancer recurrence or growth when the vaccine composition of the present invention is administered to the cancer patient after surgery or administration of an anti-cancer drug to remove cancer tissue.

[0033] The term "protective immunity" means that a subject mounts an immune response against the HER2-ICD antigen, allowing the subject's immune system to target and destroy cells expressing the same antigen, thereby reducing cancer progression or recurrence in the subject. In the present invention, this protective immunity is also expressed as an anti-cancer effect in which tumor growth is suppressed.

[0034] The term "anti-HER2 therapeutic antibody" means a monoclonal antibody that targets the HER2 receptor and can treat cancer by preventing the growth of HER2-expressing tumor cells, including, but not limited to, trastuzumab (Herceptin™) and its biosimilars.

[0035] The term "booster" refers to a dose of immunogen administered to a patient to enhance, prolong, or maintain protective immunity and overcome "down-regulation of T cell responses" mediated by regulatory T cells.

[0036] The term "pharmaceutically acceptable" means a substance that is acceptable to a patient from a pharmacological / toxicological standpoint in terms of composition, dosage form, safety, etc., and a "pharmaceutically acceptable carrier" refers to a medium that does not interfere with the effects of the biological activity of an active ingredient and is non-toxic to a subject when administered.

[0037] The term "patient" or "subject" refers to a living organism suffering from, being treated for, or susceptible to a condition that can be prevented or treated by administration of a vaccine composition of the present invention, such as gastric cancer, and includes both humans and animals. Such subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), preferably humans. Furthermore, "patient" or "subject" in the present invention includes, without distinction, gastric cancer patients and HER2-expressing gastric cancer patients.

[0038] The term "administering" refers to the introduction of a vaccine composition into a subject using any of a variety of methods and delivery systems known in the art. Exemplary administration routes include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes, such as injection or infusion. As used herein, "administration" generally refers to intradermal administration by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intraspinal, or intralymphatic injection and infusion. Other parenteral routes include topical, epithelial, or mucosal administration routes, such as intranasal, vaginal, rectal, or sublingual routes. As used herein, the terms "vaccine injection," "treatment," and "vaccination," which refer to the act of administering a vaccine to a subject, are used interchangeably.

[0039] In the present invention, the term "expressing HER2" means expressing the HER2 gene or protein in cancer tissue. The expression pattern of the HER2 gene or protein does not distinguish between high and low expression.

[0040] In the present invention, the term "immune anticancer agent" refers to a therapeutic agent that stimulates the immune system to induce immune cells to selectively attack only cancer cells, unlike existing anticancer agents that attack cancer itself, and that works by restoring or enhancing the immune system's ability to recognize or destroy tumors in order to overcome the immune suppression or immune evasion mechanisms acquired by cancer cells. The immune anticancer agent includes, but is not limited to, immune checkpoint inhibitors, immune cell therapy agents, and immune virus therapy agents.

[0041] In the present invention, the term "immune checkpoint inhibitor" refers to a type of immune anticancer agent that blocks the activation of immune checkpoint proteins involved in T cell inhibition when some cancer cells evade immunity by utilizing the immune checkpoint of T cells, which are immune cells in the body, thereby activating T cells to attack cancer cells. Examples include, but are not limited to, CTLA-4 inhibitors, PD-1 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, and VISTA inhibitors. Furthermore, the immune checkpoint inhibitor is an antagonist, and may be an antagonist antibody or a small molecule compound.

[0042] The compositions disclosed herein may be prepared into DNA vaccines. In some embodiments of the present invention, the DNA vaccine comprises a plasmid containing a promoter, appropriate transcriptional and translational control elements, and a nucleic acid sequence encoding one or more polypeptides of the present invention. In some embodiments, the plasmid may further comprise an enhancer sequence, e.g., a sequence that enhances expression levels, intracellular targeting, etc.

[0043] In some embodiments of the present invention, the DNA vaccine comprises a plasmid vector containing a nucleic acid sequence encoding the HER2-ICD of the present invention. In some embodiments, the plasmid is pUMVC3 (the conventional name is "pNGVL3," and the two names may be used interchangeably). The nucleic acid sequence encoding the HER2-ICD may have 100% homology to the nucleic acid sequence of SEQ ID NO: 3, or may have 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology. However, when the homology is limited to the specific value, the immunogenicity against HER2-ICD by vaccination is the same as or has an equivalent effect to that of a sequence with 100% homology.

[0044] In some examples of the present invention, NCI-N87 cell line, a human gastric cancer cell line expressing HER2 protein, was transplanted into athymic nude mice to create xenograft gastric cancer mouse models. The mice were then divided into four drug-treated groups: a control group, a HER2-ICD DNA vaccine-treated group (vaccine), an anti-HER2 therapeutic antibody-treated group (Herceptin), and a HER2-ICD DNA vaccine + Herceptin combination-treated group (vaccine + Herceptin).

[0045] In all three drug treatment groups, tumor growth was suppressed compared to the control group (up to 31 days after drug treatment), with the group receiving the HER2-ICD DNA vaccine alone showing the most effective tumor growth suppression effect. As a result, the results were even more superior than those of the test group receiving Herceptin alone, as well as the combined administration of Herceptin and the HER2-ICD DNA vaccine.

[0046] Considering that the common treatments for HER2-expressing advanced gastric cancer are HER2-direct targeting therapy using anti-HER2 therapeutic antibodies or combination therapy of anti-HER2 therapeutic antibodies with chemotherapy (5-fluorouracil (5-FU) + cisplatin or capecitabine + oxaliplatin), the fact that administration of the HER2-ICD DNA vaccine of the present invention alone shows a more superior tumor growth inhibitory effect than administration of Herceptin can be seen as offering new possibilities in the treatment of HER2-expressing gastric cancer.

[0047] In particular, the safety of the HER2-ICD DNA vaccine of the present invention has already been confirmed in the NCT00436254 phase 1 clinical study, and therefore the DNA vaccine therapy of the present invention may be applicable as a therapy that can reduce the burden on patients compared to existing therapies.

[0048] In some embodiments of the present invention, the DNA vaccines can further encode immunomodulatory molecules that modulate the generated immune response, such as enhancing vaccine efficacy, stimulating the immune system, or reducing immunosuppression.

[0049] In the present invention, since the vaccine composition is intended to induce and / or maintain a therapeutic effect against the growth or recurrence of gastric cancer, the vaccine composition of the present invention can be administered to a patient according to any schedule suitable for inducing and / or maintaining a cytotoxic T cell response against HER2-ICD. For example, a vaccine composition as described and exemplified herein can be administered to a patient according to a fixed schedule as a primary inoculation, followed by a booster to induce and / or maintain protective immunity.

[0050] In some embodiments of the present invention, the vaccine composition may be administered to a patient once, twice, or more times per month. Boosters may be administered one or more times at regular intervals, for example, monthly, after the completion of the vaccination schedule, and may be administered one or more times over a period of six months or more. In some embodiments of the present invention, the HER2-ICD vaccine may be administered three times, once per month, as the primary vaccination, followed by an additional booster six months or one year later.

[0051] In this case, the booster may have the same dose and composition as the vaccine composition used in the primary inoculation, or the dose and / or composition may be changed.

[0052] The vaccine composition described in the present specification may contain, in addition to the plasmid containing DNA encoding HER2-ICD, a pharmaceutically acceptable excipient, carrier, diluent, buffer, stabilizer, preservative, adjuvant, or other substances well known in the art to which the present invention pertains. Such substances are non-toxic and do not interfere with the pharmaceutical activity of the active ingredient. The pharmaceutical carrier or diluent may be, for example, an aqueous solution. The carrier or other substance included in the vaccine composition of the present invention may be selected differently depending on the administration route (e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intradermal, and intraperitoneal administration).

[0053] The vaccine compositions of the present invention also contain one or more "pharmaceutically acceptable carriers." These typically include proteins, saccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, and the like. Such carriers are well known in the art to which the present invention pertains. The vaccine compositions also contain diluents such as water, saline, and glycerol. Auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like may also be present. Sterile, pyrogen-free phosphate-buffered saline (PBS) and tromethamine (Tris) are typical carriers.

[0054] One aspect of the present invention relates to a vaccine composition or kit as described above, which contains one or more plasmid DNAs as an active ingredient. The kit of the present invention also includes the vaccine composition of the present invention and instructions for using the vaccine composition.

[0055] The vaccine composition or kit of the present invention is also for use in a method of inducing an immune response or providing treatment, vaccination, or immunotherapy in a subject, and the vaccine composition may be a vaccine or immunotherapeutic composition. Such treatment or vaccination comprises administering to the subject a vaccine composition of the present invention. The vaccine composition may be administered to the subject according to a set schedule.

[0056] The vaccine composition or kit of the present invention can be used to treat gastric cancer in a subject, which may be a human, and the gastric cancer may be gastric cancer that expresses the HER2 protein.

[0057] The vaccine compositions of the present invention may be lyophilized or in aqueous form, such as a solution or suspension. Such liquid formulations are ideal for injection, as the composition can be administered directly in its packaged form without the need for reconstitution in an aqueous medium. The vaccine compositions may be provided in vials or pre-filled syringes. The syringes may be supplied with or without needles. The syringes contain a single dose, while the vials may contain single or multiple doses. The vaccine compositions of the present invention may also be administered using microneedles.

[0058] The vaccine compositions of the present invention can also be formulated into slow-release vehicles or depot preparations. Such long-acting formulations can be administered by inoculation or implantation (e.g., subcutaneous or intramuscular), or by injection. Thus, for example, the vaccine compositions can be formulated with suitable polymeric materials, hydrophobic materials (e.g., emulsions in acceptable oils), or ion exchange resins, or as sparingly soluble derivatives (e.g., sparingly soluble salts). Liposomes and emulsions are well-known examples of suitable delivery vehicles for use as carriers.

[0059] The vaccine compositions of the present invention, when packaged in multiple dose formats, also contain an antimicrobial, which may be selected from 2-phenoxyethanol or parabens (methylparaben, ethylparaben, propylparaben), etc. Any preservatives are desirably present at low levels. The preservatives may be added exogenously and / or may be a component of the bulk antigens mixed to form the composition.

[0060] The vaccine composition of the present invention may be provided in the form of a kit including instructions and the like.

[0061] Furthermore, the kit of the present invention includes the vaccine composition of the present invention and instructions, but does not include an anti-HER2 therapeutic antibody.

[0062] The vaccine compositions of the present invention may also include one or more adjuvants to enhance the immunogenicity of the composition.

[0063] In the present invention, the composition may further comprise an anti-cancer adjuvant, and more preferably, the anti-cancer adjuvant is a STING (stimulator of interferon gene) agonist, but is not limited thereto.

[0064] Suitable adjuvants include aluminum salts such as aluminum hydroxide or aluminum phosphate, but may also be selected from, but are not limited to, calcium, iron, or zinc salts, insoluble suspensions of acylated tyrosine or acylated sugars, or cationically or anionically derivatized sugars, etc. The adjuvant may also include cytokines used as immunomodulators.

[0065] Cytokines suitable for use as the immunomodulator may be selected from, but are not limited to, interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), macrophage colony stimulating factor (M-CS), tumor necrosis factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), etc.

[0066] The vaccine composition of the present invention may be administered in combination with one or more anticancer agents selected from a chemical anticancer agent, a targeted anticancer agent, and an immunological anticancer agent. The one or more anticancer agents selected from a chemical anticancer agent, a targeted anticancer agent, and an immunological anticancer agent that can be administered in combination with the vaccine composition of the present invention may be administered simultaneously or sequentially with a time lag, and may be selected at an appropriate time and period.

[0067] In the present invention, the chemo-anticancer agent is also called a cytotoxic anticancer agent or a chemical drug anticancer agent. Examples of the chemo-anticancer agent include gemcitabine (gemcitabine injection), cytarabine, carboplatin (Paraplatin), cisplatin (Platinol, Platinol-AQ), crizotinib (Xalkori), cyclophosphamide (Siloxane, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), etoposide (Bepcid), 5-fluorouracil (5-FU), irinotecan (irinotecan), and the like. These include, but are not limited to, rinotecan (Camptosar), liposome-encapsulated doxorubicin (Doxil), methotrexate (Porex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), topotecan (Hycamtin), trabectedin (Yondelis), vincristine (Oncovin, Vincasar PFS), vinblastine (Velban), capecitabine, or oxaliplatin.

[0068] In the present invention, the targeted anticancer drug is trametinib, vemurafenib, alpelisib, dactolisib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenib, crizotinib, dabrafenib, trastuzumab. The therapeutic agent may be one or more targeted therapeutic agents selected from the group consisting of, but not limited to, rifacitinib, cetuximab, bevacizumab, panitumumab, ipilimumab, pertuzumab, tofacitinib, imatinib, bortezomib, ofatumumab, and alemtuzumab.

[0069] The targeted anti-cancer drugs include next-generation targeted anti-cancer drugs that are antibody-drug conjugates (ADCs).

[0070] In the present invention, the immunoanticancer agent may be an immune checkpoint inhibitor related to any one selected from the group consisting of CTLA-4 (cytotoxic tlymphocyte-associated protein 4), PD-1 (programmed cell death protein 1), PD-L1 (programmed death ligand 1), LAG3 (lymphocyte activation gene-3), TIM-3 (T-cell immunoglobulin and mucin-domain containing-3), TIGIT (T-cell immunoreceptor with IG and ITIM domain), and VISTA (V domain Ig suppressor of T cell activation), but is not limited thereto.

[0071] In some embodiments of the present invention, the HER2-ICD DNA vaccine composition may be administered concurrently with other cancer treatments, including, but not limited to, standard anticancer drugs such as fluorouracil, doxorubicin, and paclitaxel.

[0072] Unless otherwise indicated, all numbers used in the specification and claims, whether stated or not, should be understood to be modified in all instances by the term "about." It should also be understood that the precise numerical values ​​used in the specification and claims form further embodiments of the present disclosure. Efforts have been made to ensure the accuracy of the numerical values ​​disclosed in the examples. However, all measured numerical values ​​inherently contain certain error values ​​resulting from the standard deviations observed with their respective measuring techniques.

[0073] The present invention will be described in more detail below through examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples, according to the gist of the present invention. [Example]

[0074] The antitumor efficacy of the HER2-ICD DNA vaccine was measured when administered alone or in combination with Herceptin (an anti-HER2 antibody).A xenograft mouse model was used as an example of an animal model of gastric cancer in which athymic mice were implanted with NCI-N87 (ATCC CRL-2855), a human gastric cancer cell line expressing HER2 protein.

[0075] All animal studies were ethically conducted under the approval of the Laboratory Animal Steering Committee of the Experimental Animal Center of the Osong Advanced Medical Industry Promotion Foundation (KBIO-IACUC) (KBIO-IACUC-2020-120).

[0076] Xenograft gastric cancer mouse model construction Athymic nude mice (athymic-NCr-nu strain) were purchased from Coretech at 5 weeks of age and weighing 16-18 g, and were allowed to acclimate for a minimum of 1 week.

[0077] The human gastric cancer cell line, NCI-N87, was cultured at 5x10 cells per individual. 6 The cells were suspended in 200 μl of PBS and injected subcutaneously into the axillary region between the scapular region and the chest wall on the right side of the mice.

[0078] To analyze the antitumor effect of the HER2-ICD DNA vaccine, mice were implanted with NCI-N87 cells, and tumor size was measured at 11 days after cell implantation. 3 24 mice achieved the goal (average tumor size 150.5±16.4 mm3 ) were selected and assigned to each group with 6 animals.

[0079] vaccine preparations The DNA sequence (SEQ ID NO: 3) encoding HER2-ICD was inserted into the pNGVL3 (pUMVC3) plasmid vector and confirmed by DNA sequencing.

[0080] The plasmid DNA vaccine, pNGVL3-hICD, was amplified, quantified, and packaged in vials under GMP conditions. Each single-dose vial contains plasmid DNA (pNGVL3-HER2ICD) suspended in tromethamine / EDTA as a stabilizing buffer (1.2 ml). The vials were manufactured by VGXI (USA) using a master cell bank (MCB) in accordance with US Pharmacopoeia standards, after undergoing microbial, sterility, and stability testing. They were stored at -20°C.

[0081] The vaccine formulation was diluted in PBS and administered to each mouse at a final dose of 5 mg / kg (100 μg / 20 g / 150 μl).Herceptin (Roche) was administered in saline at a dose of 40 μg / 20 g / 100 μl.

[0082] Drug administration and administration schedule The HER2-ICD DNA vaccine was administered intradermally, and Herceptin was administered intraperitoneally.

[0083] The HER2-ICD DNA vaccine was administered intradermally three times on days 0, 7, and 14 to mice that had already been transplanted with NCI-N87 cells and had tumor development confirmed. Herceptin was administered intraperitoneally five times on days 0, 7, 14, 21, and 28.

[0084] As a control, PBS was administered.

[0085] Observation and inspection items All mice were observed once daily for general symptoms.

[0086] During the test period, body weight was measured a total of 14 times (days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, and 31), and changes in body weight up to day 31 were observed, based on the body weight on the day administration began (100%).

[0087] Tumor size and tumor growth inhibition rate were measured eight times (8th, 11th, 15th, 18th, 22nd, 25th, 30th, 32nd days) from day 8 to day 32 after cancer cell transplantation. Tumor size was calculated by measuring the tumor size in three directions using a Vernier caliper for each individual. On day 11 after cancer cell transplantation, the tumor size was an average of 150 mm. 3 Tumor size was calculated for each individual at 14 times (days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, and 31) from the time of tumor initiation (day 0) to day 31. The tumor growth rate was then calculated by comparing each test group with the control group.

[0088] Autopsy and examination items On the final day of the study, the mice were anesthetically induced by intramuscular injection of an anesthetic (a mixture of Zoletil 50 50 mg / kg and Rompun 10 mg / kg), and then exsanguinated. After that, the mice were euthanized, and the tumors and spleens were removed, weighed, and photographed.

[0089] Analysis of the antitumor effect of HER2-ICD DNA vaccine During this test period, no animals died in any test group, and no abnormal symptoms were observed in the single or combined administration of the test substances.

[0090] During the study, weight loss was observed in the Herceptin-treated group and the HER2-ICD DNA vaccine + Herceptin-treated group (hereafter referred to as the "vaccine + Herceptin-treated group") compared to the control group (PBS-treated group). From Day 5, all groups showed a gradual weight loss trend until the final day (Day 31), but this was not statistically significant. On the final day, baseline weights were 94.4±5.8% in the control group, 97.0±5.2% in the HER2 ICD DNA vaccine-treated group (hereafter referred to as the "vaccine-treated group"), 92.8±7.7% in the Herceptin-treated group, and 96.9±3.6% in the vaccine + Herceptin-treated group (Figure 1).

[0091] The rate of change in tumor size was measured, and tumor growth suppression was observed continuously from day 3 to the final day of the study in the vaccine-treated group, Herceptin-treated group, and vaccine + Herceptin-treated group compared to the control group. In particular, the vaccine-treated group showed a statistically significant tumor growth suppression effect in some periods (days 7, 12, 21, and 26) (p<0.05) (Figure 2). The tumors excised from each mouse are shown in Figure 3.

[0092] Therefore, it can be seen that the HER2-ICD vaccine induces a protective immune response and suppresses tumor growth in the xenograft gastric cancer mouse animal model.

[0093] When tumor weight was measured, a decrease in tumor weight was observed in the vaccine-treated group and the vaccine + Herceptin-treated group compared to the control group, but the decrease in tumor weight did not show statistical significance (Figure 4).

[0094] Macroscopic observation and weight measurement of the spleen revealed that the spleens were larger in the vaccine-treated group and the vaccine + Herceptin-treated group compared to the control group (Figure 5). Compared to the control group, the spleen weights were higher in both absolute and relative weights in the vaccine-treated group, Herceptin-treated group, and vaccine + Herceptin-treated group, and were particularly high in the vaccine + Herceptin-treated group, although statistical significance was not confirmed (Figures 6 and 7).

[0095] Taking all of these results together, no abnormal symptoms were observed in animals transplanted with NCI-N87 gastric cancer cells when the HER2-ICD DNA vaccine was administered alone or in combination with the HER2-ICD DNA vaccine and Herceptin.

[0096] Furthermore, in an antitumor effect analysis using animals transplanted with NCI-N87 gastric cancer cells, administration of the HER2-ICD DNA vaccine and the combined administration of the HER2-ICD DNA vaccine and Herceptin demonstrated antitumor effects that inhibited tumor growth more than administration of Herceptin alone. In particular, administration of the HER2-ICD DNA vaccine alone demonstrated a tendency for even greater antitumor effects than administration of Herceptin alone or the combined administration of the HER2-ICD DNA vaccine and Herceptin. Therefore, the present invention suggests that the use of the HER2-ICD DNA vaccine alone may be an effective treatment for HER2 protein-expressing gastric cancer. [Sequence List Free Text]

[0097] SEQ ID NO: 1 (full amino acid sequence of HER2 gene) TIFF0007780824000001.tif77170 SEQ ID NO: 2 (HER2-ICD amino acid sequence) TIFF0007780824000002.tif36170 SEQ ID NO: 3 (HER2-ICD nucleotide sequence) TIFF0007780824000003.tif103170

Claims

1. A vaccine composition for use in inducing an anti-cancer effect against HER2-expressing gastric cancer in a subject, the vaccine composition comprising a pharmaceutically acceptable carrier and a plasmid vector comprising a nucleotide sequence encoding the HER2-intracellular domain (ICD) region having SEQ ID NO: 2, the plasmid vector does not contain a nucleotide sequence encoding a HER2 extracellular domain and a HER2 transmembrane domain; The subject is not administered an anti-HER2 therapeutic antibody in combination with the vaccine composition.

2. The vaccine composition of claim 1, wherein the nucleotide sequence encoding the HER2-intracellular domain (ICD) region has SEQ ID NO:

3.

3. The vaccine composition of claim 1 or 2, wherein the subject is a gastric cancer patient that expresses HER2 protein.

4. The vaccine composition of claim 3 , wherein the subject is a human.

5. The vaccine composition of claim 1 or 2, further comprising an adjuvant.

6. The vaccine composition of claim 1 or 2, wherein the vaccine composition is administered by injection or inoculation.

7. The vaccine composition of claim 6, wherein the composition is administered by intradermal injection.

8. A kit for treating HER2-expressing gastric cancer, comprising the vaccine composition according to claim 1 or 2 and an instruction manual, The kit is a kit for treating gastric cancer expressing HER2, which does not include an anti-HER2 therapeutic antibody.

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