Her2 vaccine composition and kit

Abstract

This invention relates to a HER2-ICD DNA vaccine composition and kit. The vaccine composition according to this invention can effectively inhibit gastric cancer growth in animal models transplanted with human gastric cancer cell lines expressing HER2 without serious side effects, and is therefore effective for the treatment of gastric cancer.

Description

Technical Field

[0001] This application claims the benefit of Korean Patent Application No. 10-2021-0099411, filed with the Korean Intellectual Property Office on July 28, 2021, the disclosure of which is incorporated herein by reference in its entirety.

[0002] This invention relates to a HER2 vaccine composition for the treatment of gastric cancer. Prior Technology

[0003] Stomach cancer, in principle, refers to all types of cancer that occur in the stomach and can take many forms, such as adenocarcinoma primarily found in the gastric mucosa, gastrointestinal tumors that occur in the stromal cells of the stomach, and similar forms. Like other cancers, stomach cancer can spread to other organs, including the esophagus, lungs, and liver. Stomach cancer is the fourth most frequently diagnosed cancer globally, with 930,000 cases diagnosed in 2002. Its high incidence leads to a high mortality rate (approximately 800,000 cases annually), and it is the second leading cause of cancer death worldwide after lung cancer. This cancer is more common in men than women and is more prevalent in Asia and developing countries.

[0004] Treatment options for gastric cancer include surgery, chemotherapy, and radiation therapy or concurrent chemoradiotherapy. Surgery is a first-line treatment for gastric cancer. For stage II patients, the 5-year survival rate for surgical resection for therapeutic purposes appears to be between approximately 30% and 50%, and for stage III patients, the survival rate appears to be between approximately 10% and 25%. However, because surgical resection and subsequent anticancer therapies such as chemotherapy can reduce patients' quality of life, such as causing weight loss, other treatment options are needed.

[0005] Vaccines help the body fight disease by training the immune system to recognize and destroy harmful substances and diseased cells. Vaccines can be broadly divided into two types: preventative vaccines and therapeutic vaccines. Preventative vaccines are the traditional form of vaccine provided to healthy individuals to prevent the development of certain diseases, while therapeutic vaccines (also known as immunotherapy) are provided or administered as a preventative measure to help stop the growth and spread of diseases in people diagnosed with those diseases. A representative form of therapeutic vaccine is the cancer vaccine.

[0006] Cancer vaccines can activate cytotoxic T cells, enabling these activated T cells to recognize and act on cancer cells expressing specific types of antigens. Alternatively, cancer vaccines can be designed to 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 cancerous tissue in cancer patients or to prevent cancer recurrence after cancerous tissue has been removed from cancer patients through surgery or chemotherapy.

[0007] Despite extensive research into vaccines for treating or preventing cancer recurrence over the past few years, no successful cases have been reported, except for Sipuleucel-T (Provenge®), a vaccine for prostate cancer. This low success rate is attributed to several factors: although the antigen is specifically expressed in certain types of tumor cells, it may be expressed at low levels depending on the individual; or in the case of metastatic tumors, the antigenic characteristics may change between the initial tumor and the metastatic tumor. Furthermore, immune evasion mechanisms of tumor cells are also explained as a contributing factor to the low success rate.

[0008] Ideally, the vaccine antigen should be expressed only in tumor cells or at least at elevated levels. HER2 / neu (hereinafter referred to as 'HER2') is a member of the epidermal growth factor receptor (EGFR) family (EGFR2), and EGFR overexpression has been observed in many cancers. In particular, 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 with the amino acid sequence SEQ ID NO: 1, and is composed of an intracellular domain (ICD, HER2 aa 676-1255), a transmembrane domain (TM, HER2 aa 653-675), and an extracellular domain (ECM, HER2 aa 22-652) with protein tyrosine kinase activity.

[0010] The HER2 vaccine has been extensively studied in breast cancer, and several immunogenic peptides have been identified. For example, in the case of the peptide nelipepimut-S (NeuVax; HER2 aa369-377; E75 (NP-S)) of the HER2 protein, immunotherapy has been attempted by loading the peptide into dendritic cells before injection. However, no clinically significant therapeutic or protective effects have been reported in women with advanced breast cancer. Furthermore, a recent large-scale phase III clinical trial was discontinued because there was no difference in disease-free survival (DFS) between the treatment and placebo groups.

[0011] Other examples of HER2 peptide vaccines include: AE37 (LRMK-GVGSPYVSRLLGICL:LRMK-HER2 aa 776-790), a HER2-ICD peptide vaccine; GP2 (HER2 aa 654-662), a HER2-TM peptide vaccine; and similar vaccines. There have been reports of vaccines increasing survival rates. Additionally, in animal studies, vaccines targeting the HER2-ICD fragment (hereinafter referred to as 'HER2-ICD') have been reported to reduce tumor size compared to vaccines targeting the HER2-ECD fragment (hereinafter referred to as 'HER2-ECD'). There are existing cases of HER2-ICD DNA vaccines being used in breast cancer patients. However, no results have been reported of HER2-ICD DNA vaccines being used in gastric cancer instead of breast cancer.

[0012] Therefore, in gastric cancer patients, there is a need to develop the immunomodulatory effects and therapeutic potential of HER2-ICD DNA vaccines to produce vaccines that will provide reliable treatment or prevention of gastric cancer recurrence. [Previous Technical Documents] [Patent Literature] KR 10-1572474 (Release date: November 30, 2015) US 2002 / 0193329 (Disclosure date: December 19, 2002) [Non-patent literature] Breast Care, 2016; 11:116-121 Clin Cancer Research; 25(14) July 15, 2019 Summary of the Invention

[0013] Technical issues

[0014] Therefore, the inventors conducted research to solve the above-mentioned problems, and as a result, the present invention was completed by demonstrating that a DNA vaccine composition comprising a plasmid encoding a nucleic acid sequence of HER2-ICD can effectively inhibit tumor growth in a mouse model of tumors transplanted with human gastric cancer cell lines.

[0015] The objective of this invention is to provide a vaccine composition (hereinafter referred to as "HER2-ICD DNA vaccine composition") comprising a plasmid including a nucleic acid sequence encoding HER2-ICD.

[0016] The objective of this invention is to provide a kit comprising a HER2-ICD DNA vaccine composition for the treatment of gastric cancer.

[0017] The objective of this invention is to provide a method for inhibiting the progression and / or recurrence of gastric cancer by administering a vaccine composition or kit to an individual.

[0018] The objective of this invention is to provide the use of components or kits in the manufacture of medical products for the treatment of gastric cancer.

[0019] Problem Solution

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

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

[0022] According to an embodiment of the present invention, the gastric cancer patient population may be a patient population expressing the HER2 protein.

[0023] According to one embodiment of the present invention, the vaccine composition may additionally include an adjuvant.

[0024] According to embodiments of the present invention, the vaccine composition may be administered in combination with one or more anticancer agents selected from chemoanticancer agents, targeted anticancer agents, and immunoanticancer agents.

[0025] According to an embodiment of the present invention, the gastric cancer patient population may be a human patient population.

[0026] The advantages of this disclosure

[0027] The vaccine composition according to the present invention comprises a plasmid containing a nucleic acid sequence encoding HER2-ICD, and can effectively inhibit tumor growth in animal models of gastric cancer, and is therefore effective for the treatment of gastric cancer. Simple Explanation of the Diagram

[0028] Figure 1 shows the weight changes of individuals in xenograft mouse models with transplanted gastric cancer cell lines when administered HER2-ICD DNA vaccine (Vaccine), anti-HER2 therapeutic antibody (Herceptin), and combinations thereof (Vaccine + Herceptin). Figures 2 to 4 show the inhibitory effect of administration of HER2-ICD DNA vaccine (vaccine), anti-HER2 therapeutic antibody (He'aiping), and combinations thereof (vaccine + He'aiping) on ​​tumor growth in xenograft mouse models with transplanted gastric cancer cell lines. Figures 5 to 7 show the spleen changes in xenograft mouse models with transplanted gastric cancer cell lines when administered HER2-ICD DNA vaccine (vaccine), anti-HER2 therapeutic antibody (He'aiping), and combinations thereof (vaccine + He'aiping). Implementation

[0029] The invention will be described in more detail below.

[0030] In describing and asserting certain features of this disclosure, unless otherwise indicated, the following terms will be used in accordance with the definitions set forth below.

[0031] It should be understood that although some states are described in this document using the term "comprising", other similar states described using the terms "consisting of" and / or "substantially consisting of" are also provided.

[0032] The term "treatment" refers to the result or indication of inhibition or reduction of tumor growth when the cancer patient is examined by radiation or physical methods after the cancer patient has been vaccinated with the vaccine composition of the present invention. Furthermore, the term also refers to the result or indication of prevention of cancer recurrence / relapse when the cancer patient is examined by radiation or physical methods after the cancerous tissue of the cancer patient has been removed by surgery or administration of anticancer drugs, followed by vaccination with the vaccine composition of the present invention.

[0033] The term "anti-cancer effect" refers to the effect of preventing or reducing tumor growth when administered to a cancer patient with the vaccine composition of the present invention. Furthermore, the term also refers to the effect of preventing cancer recurrence / relapse after removal of cancerous tissue from a cancer patient through surgery or administration of anti-cancer drugs, followed by vaccination with the vaccine composition of the present invention.

[0034] The term "protective immunity" refers to reducing cancer progression or recurrence / relapse in an individual by inducing an immune response against the HER2-ICD antigen, and by enabling the individual's immune system to target and destroy cells expressing the same antigen as the HER2-ICD antigen. In this invention, protective immunity manifests as an anti-cancer effect by inhibiting tumor growth.

[0035] The term "anti-HER2 therapeutic antibody" refers to a monoclonal antibody that can treat cancer by targeting the HER2 receptor and blocking the proliferation of tumor cells expressing HER2. Examples of anti-HER2 therapeutic antibodies include, but are not limited to, trastuzumab (Heyap®), its biosimilars, and similar analogues.

[0036] The term "enhancer" refers to a specific dose of immunogen administered to a patient to enhance, prolong, or maintain protective immunity and to overcome "downregulation of T cell response" mediated by regulatory T cells.

[0037] The term "medically acceptable" refers to a substance that is acceptable to a patient from a pharmacological / toxicological point of view, in terms of its composition, formulation, safety, and similar properties. The term "medically acceptable carrier" refers to a medium that does not interfere with the biological activity of the active ingredient and is non-toxic to the individual at the time of administration.

[0038] The terms "patient" or "individual" refer to a living organism in a situation where a disease can be prevented or treated by administration of the vaccine composition of the present invention, such as having gastric cancer, having been treated for gastric cancer, or being susceptible to gastric cancer, and may include both humans and animals. Examples of individuals include mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), but are not limited thereto, and individuals are preferably humans. Furthermore, the terms "patient" or "individual" in this invention include (but are not distinguished from) patients with gastric cancer or patients with gastric cancer expressing HER2.

[0039] The term "administration" refers to the introduction of a vaccine component into an individual using any of the various methods and delivery systems known in the art. Examples of administration routes include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other non-enteral administration routes, such as injection or infusion. As used herein, the term "administration" refers to intradermal administration typically performed by injection, and may include (but is not limited to) intravenous, intramuscular, intra-arterial, intrathecal, or intralymphatic injection and infusion. Other non-enteral routes may include local, epithelial, or transmucosal administration routes, such as intranasal, transvaginal, transrectal, or sublingual routes. In this invention, the terms "vaccine injection," "treatment," and "vaccination" are used indiscriminately with respect to the act of administering a vaccine to an individual.

[0040] In this invention, the term "HER2 expression" refers to the expression of the HER2 gene or protein in cancer tissue. The expression level of the HER2 gene or protein is not differentiated between high and low expression.

[0041] In this invention, the term "cancer immunotherapy drug" refers to a therapeutic drug that, unlike existing anticancer drugs that attack cancer itself, stimulates the immune system to induce immune cells to selectively attack only cancer cells. In other words, it is an anticancer drug with the following mechanism: it restores or enhances the immune system's ability to recognize or destroy tumors to overcome the immune suppression or immune evasion mechanisms acquired by cancer cells. Cancer immunotherapy drugs include, but are not limited to, immune checkpoint inhibitors, immune cell therapeutic agents, and immune viral therapeutic agents.

[0042] In this invention, the term "immune checkpoint inhibitor" refers to a class of cancer immunotherapy drugs that, when some cancer cells evade the immune system and utilize immune checkpoints of T cells (immune cells in the body), activate T cells to attack cancer cells by blocking the activation of immune checkpoint proteins involved in T cell suppression. Examples of immune checkpoint inhibitors 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, immune checkpoint inhibitors acting as antagonists can be antagonistic antibodies or small molecule compounds.

[0043] The components disclosed in this specification can be prepared as DNA vaccines. In some embodiments of the invention, the DNA vaccine may comprise a plasmid containing a promoter, suitable transcription and translation control factors, and a nucleic acid sequence encoding one or more polypeptides of the invention. In some embodiments, the plasmid may additionally comprise enhancer sequences, such as sequences that enhance expression levels, intracellular targeting, and the like.

[0044] In some embodiments of the present invention, the DNA vaccine may comprise a plasmid vector containing a nucleic acid sequence encoding the HER2-ICD of the present invention. In some embodiments, the plasmid may be a pUMVC3 plasmid (commonly known as 'pNGVL3', and therefore the two names are used interchangeably). The nucleic acid sequence encoding the HER2-ICD may have 100% homology with the nucleic acid sequence of SEQ ID NO: 3, or may have 99% or higher, 95% or higher, 90% or higher, 85% or higher, or 80% or higher homology with the nucleic acid sequence of SEQ ID NO: 3. However, when the homology is limited to the specific values ​​mentioned above, the immunogenicity against the HER2-ICD generated by vaccination may have the same or equivalent effect as that of a sequence with 100% homology.

[0045] In some embodiments of the present invention, the human gastric cancer NCI-N87 cell line expressing the HER2 protein can be transplanted into athymic nude mice to prepare a xenograft gastric cancer mouse model. The mouse model can then be divided into four groups and treated with the following drugs: control group; group treated with HER2-ICD DNA vaccine (vaccine); group treated with HER2-ICD DNA vaccine as an anti-HER2 antibody treatment group; and group treated with a combination of HER2-ICD DNA vaccine and HER2-ICD DNA vaccine (vaccine + HER2-ICD DNA vaccine).

[0046] Tumor growth was inhibited in all three drug treatment groups compared to the control group (up to day 31 after drug treatment). The group receiving the HER2-ICD DNA vaccine alone showed the greatest inhibitory effect on tumor growth, thus demonstrating excellent results compared to the test groups receiving Hepatopanol alone and the test groups receiving a combination of Hepatopanol and the HER2-ICD DNA vaccine.

[0047] Regarding HER2-expressing advanced gastric cancer, given that HER2-direct targeted therapy using anti-HER2 therapeutic antibodies or combinations of anti-HER2 therapeutic antibodies and chemotherapy (such as the combination of 5-fluorouracil and cisplatin or capecitabine and oxaliplatin) is a common treatment, the fact that administration of the HER2-ICD DNA vaccine of this invention alone has shown better tumor growth inhibition compared to administration of Hemodialyl alone can be regarded as proposing a new possibility for treating HER2-expressing gastric cancer.

[0048] In particular, the safety of the HER2-ICD DNA vaccine of the present invention has been confirmed in a phase 1 clinical trial (NCT00436254), and therefore the DNA vaccine treatment of the present invention can be used as a treatment that can reduce the burden on patients compared with existing treatments.

[0049] In some embodiments of the present invention, the DNA vaccine may additionally encode immunomodulatory molecules that regulate the resulting immune response, such as enhancing vaccine efficacy, stimulating the immune system, or reducing immunosuppression.

[0050] Because the vaccine composition of the present invention is designed to induce and / or maintain a therapeutic effect against the growth or recurrence of gastric cancer, it can be administered to patients at any timeframe suitable for inducing and / or maintaining a cytotoxic T-cell response to HER2-ICD. For example, after administering the vaccine composition as described and illustrated herein as a primary vaccination at the prescribed timeframe, a booster may be administered to induce and / or maintain protective immunity.

[0051] In some embodiments of the present invention, the vaccine components may be administered to the patient once a month, twice a month, or multiple times a month. After completion of the vaccination schedule, booster doses may be administered once or multiple times at regular intervals, such as once or multiple times at 1-month intervals, or once or multiple times at intervals of 6 months or more. In some embodiments of the present invention, the HER2-ICD vaccine may be administered three times a month as the initial vaccination, and additional booster doses may be administered after 6 months or 1 year.

[0052] Here, the booster can be used at the same dose and may have the same composition as the vaccine composition used for primary vaccination, or the dose and / or composition of the booster may vary.

[0053] In addition to plastids containing DNA encoding HER2-ICD, the vaccine composition described in this specification may also include pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, preservatives, adjuvants, and other substances well known in the art. These substances may be non-toxic and may not interfere with the pharmaceutical activity of the active ingredient. Pharmaceutical carriers or diluents may be, for example, aqueous solutions. The carriers or other substances that may be included in the vaccine composition of this invention may be selected depending on the route of administration (e.g., oral, intravenous, transdermal or subcutaneous, nasal, intramuscular, intradermal, and intraperitoneal administration).

[0054] The vaccine composition of this invention may contain one or more "medically acceptable carriers". Typical examples of pharmaceutically acceptable carriers include proteins, carbohydrates, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, sucrose, trehalose, and the like. Such carriers are well known in the art. The vaccine composition may also contain diluents such as water, saline, glycerol, and the like. In addition, excipients such as wetting agents, emulsifiers, pH buffers, and the like may be present in the vaccine composition. Sterile and pyrogen-free phosphate-buffered saline (PBS) and tris are representative carriers.

[0055] One aspect of this invention relates to a vaccine composition or kit as described above, comprising one or more plasmid DNAs as active ingredients. The kit of this invention may include the vaccine composition of this invention, instructions for using the vaccine composition, and the like.

[0056] The vaccine composition or kit of the present invention can be used in methods of inducing an immune response or providing treatment, vaccination, or immunotherapy in an individual, and the vaccine composition can be a vaccine or immunotherapy composition. Such treatment or vaccination may involve administering the vaccine composition of the present invention to an individual. The vaccine composition may be administered to the individual at fixed intervals.

[0057] The vaccine composition or kit of the present invention can be used to treat gastric cancer in an individual. The individual may be a human, and the gastric cancer may be gastric cancer expressing the HER2 protein.

[0058] The vaccine composition of this invention can be lyophilized or in aqueous solution form, such as an aqueous solution or suspension. This type of liquid formulation is ideal for injection because it can be administered directly in encapsulated form without reconstitution in an aqueous medium. The vaccine composition can be provided in vials or in pre-filled syringes. The syringes may or may not be equipped with needles. The syringes may contain a single dose, while the vials may contain a single dose or multiple doses. Additionally, the vaccine composition of this invention can be administered using microneedles.

[0059] Furthermore, the vaccine composition of the present invention can be formulated as a delayed-release medium or a reservoir-type formulation. Such long-acting formulations can be administered by inoculation or implantation (e.g., subcutaneous or intramuscular) or by injection. Therefore, for example, the vaccine composition can be formulated with suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or can be formulated as water-poorly soluble derivatives (e.g., water-poorly soluble salts). Liposomes and emulsions are well-known examples of suitable delivery media.

[0060] The vaccine composition of the present invention may contain an antimicrobial agent when packaged in a multi-dose form. The antimicrobial agent may be selected from 2-phenoxyethanol or parabens (e.g., methylparaben, ethylparaben, or propylparaben). Preservatives, if applicable, may preferably be present at low levels. Preservatives may be added exogenously and / or may be components of the main antigen of the composition after mixing.

[0061] The vaccine composition of the present invention may be provided in kit form including instructions and similar.

[0062] In addition, the kit of the present invention may include the vaccine composition of the present invention, the instruction manual and the like, but may not include anti-HER2 therapeutic antibodies.

[0063] The vaccine composition of the present invention may contain one or more adjuvants to enhance the immunogenicity of the composition.

[0064] In this invention, the composition may additionally include an anticancer adjuvant. More preferably, examples of anticancer adjuvants include, but are not limited to, an interferon gene stimulator (STING) agonist.

[0065] Suitable adjuvants may contain aluminum salts, such as aluminum hydroxide or aluminum phosphate, but may also be salts of calcium, iron, or zinc. Alternatively, suitable adjuvants may be insoluble suspensions of acetylated tyrosine or acetylated sugars, or may be selected from cationic or anionic inducible sugars and similar sources, but are not limited thereto. Additionally, adjuvants may contain cytokines used as immunomodulators.

[0066] Cytokines suitable as immunomodulators may be selected from interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF), and similar agents, but are not limited to these.

[0067] The vaccine composition of the present invention can be administered in combination with one or more anticancer agents selected from chemoanticancer agents, targeted anticancer agents, and immunoanticancer agents. The one or more anticancer agents selected from chemoanticancer agents, targeted anticancer agents, and immunoanticancer agents that can be administered in combination with the vaccine composition of the present invention can be administered simultaneously or sequentially at certain time intervals, and can be selected according to appropriate time and cycle.

[0068] In this invention, chemical anticancer agents can also be referred to as cytotoxic anticancer agents or chemical anticancer agents. Examples of chemical anticancer agents include gemcitabine, cytarabine, carboplatin (Paraplatin), cisplatin (Platinol, Pradinol-AQ), crizotinib (Zalkori), cyclophosphamide (Cytoxane, Neosar), docetaxel (Taxotere), doxorubicin (Adriamycin), and etoposide (BeP). esid), fluorouracil (5-fluorouracil, 5-FU), irinotecan (Camptosar), liposome-encapsulated cranberry (Doxyl), methotrexate (Polex, Mexate, Amethopterin), paclitaxel (Taxol, Abraxane), topotecan (Hycamtin), trabectedin (Yondelis), vincristine (Oncobin, Vincasar PFS), vinbrastine (Velban), capecitabine, oxaliplatin, and similar substances, but not limited to these.

[0069] In this invention, the targeted anticancer agent may be one or more targeted anticancer agents selected from the following group: trametinib, vemurafenib, alpelisib, dactolisib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenib, crizotinib, and others. Lafenib, trastuzumab, cetuximab, bevacizumab, panitumumab, ipilimumab, pertuzumab, tofacitinib, imatinib, bortezomib, ofatumumab, and alemtuzumab, but not limited to these.

[0070] Targeted anticancer agents may include antibody-drug conjugates (ADCs) as next-generation targeted anticancer agents.

[0071] In this invention, the immunomodulatory anticancer agent may be an immune checkpoint inhibitor selected from any of the following groups: cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), lymphocyte activation gene-3 (LAG3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), T-cell immunomunoreceptor with IG and ITIM domain (TIGIT), and V domain Ig suppressor of T cell activation (VISTA), but is not limited thereto.

[0072] In some embodiments of the present invention, the HER2-ICD DNA vaccine composition can be administered simultaneously with treatment with other cancer therapeutic agents. For the cancer therapeutic agents, standard substances such as fluorouracil, cranberry, paclitaxel, and similar substances can be used, but are not limited thereto.

[0073] Unless otherwise specified, all numbers used in this specification and the claims should be understood to be modified by the term "about" in all cases, whether or not they are stated. Furthermore, precise numbers used in this specification and the claims should be understood to form other embodiments of this disclosure. Efforts have been made to ensure the accuracy of the numerical values ​​disclosed in the following examples. However, all measurements may implicitly include some error values ​​resulting from the standard deviation measured by the respective measurement techniques. Disclosure mode

[0074] The invention will be described in more detail below by way of the following examples. These examples are merely for illustrating the invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention is not limited to these examples in accordance with the spirit of the invention. [Example]

[0075] The antitumor efficacy of the HER2-ICD DNA vaccine was measured by administering it alone or in combination with Helepin (an anti-HER2 antibody). One example is the use of athymic mice transplanted with NCI-N87 (ATCC CRL-2855) (a human-derived gastric cancer cell line expressing the HER2 protein) as an animal model for gastric cancer.

[0076] All animal testing was conducted ethically with the approval of the Institutional Animal Care and Use Committee (IACUC) of the laboratory animal facilities of the Osong Medical Innovation Foundation (KBIO) (KBIO-IACUC-2020-120). [Establishment of a xenograft mouse model for gastric cancer]

[0077] Purchase thymic nude mice (thymic-NCr-nu strain) weighing approximately 16 to 18 grams and aged approximately 5 weeks from KOATECH and acclimate them for at least 1 week.

[0078] Five × 10⁶ cells of the human-derived gastric cancer cell line NCI-N87 were suspended in 200 μL of PBS and then subcutaneously injected into the axillary region between the right scapula and chest wall of the mouse.

[0079] To analyze the antitumor effect of the HER2-ICD DNA vaccine, 24 mice with tumors reaching 150 cubic millimeters in size (mean tumor size 150.5 ± 16.4 cubic millimeters) on day 11 after transplantation of NCI-N87 cells were selected, and six mice were assigned to each group. [Vaccine formulation]

[0080] The DNA sequence encoding HER2-ICD (SEQ ID NO: 3) was inserted into the pNGVL3 (pUMVC3) plasmid vector and confirmed by DNA sequencing.

[0081] The plasmid DNA vaccine pNGVL3-hICD was amplified and quantified under GMP conditions and subsequently prepared in vials. Each single-dose vial contains plasmid DNA (pNGVL3-HER2 ICD) suspended in 1.2 mL of serotonin / EDTA as a stabilizing buffer. The vials were manufactured by VGXI Corporation in the United States and, after undergoing microbiological, sterility, and stability testing, were stored at -20°C in a master cell bank (MCB) according to US Pharmacopoeia standards.

[0082] For each mouse, the vaccine formulation was diluted with PBS and administered at a final dose of 5 mg / kg (100 μg / 20 g / 150 μL). Hexamethasone (Roche) was administered at a dose of 40 μg / 20 g / 100 μL after being dissolved in physiological saline. [Drug administration and administration schedule]

[0083] HER2-ICD DNA vaccine was administered intradermally, and He'aiping was administered intraperitoneally.

[0084] For mice that had been transplanted with NCI-N87 cells and confirmed to have developed tumors, the HER2-ICD DNA vaccine was administered intradermally three times on days 0, 7, and 14, and He'aiping was administered intraperitoneally five times on days 0, 7, 14, 21, and 28.

[0085] As a control group, mice were given PBS. [Observation Results and Inspection Items]

[0086] Observe the general symptoms of all mice once a day.

[0087] During the testing period, mice were weighed a total of 14 times (on days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28 and 31), and weight changes were observed up to day 31 based on the weight on the start date (100%).

[0088] From day 8 to day 32 after cancer cell transplantation, tumor size and tumor growth inhibition rate were measured in three directions using a vernier caliper for each individual, for a total of 8 times (days 8, 11, 15, 18, 22, 25, 30, and 32), and tumor size was calculated. Starting from day 11 after cancer cell transplantation when the tumor size reached 150 cubic millimeters (day 0), tumor size was calculated for each individual, for a total of 14 times (days 0, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28, and 31), and compared with the control group in each test group to calculate the rate of change in tumor growth. [Autopsy and Examination Items]

[0089] On the last day of the test, mice were injected intramuscularly with an anesthetic (a mixture of 50 mg / kg Zoltil 50 and 10 mg / kg Rompun) to induce euthanasia under anesthesia after scratching. Subsequently, the tumor and spleen were removed, and the mice were weighed and photographed. [HER2-ICD DNA] [Analysis of the anti-tumor effects of vaccines]

[0090] During the testing period, no animals died in any of the test groups, and no abnormal symptoms were observed when the test substance was administered alone or in combination.

[0091] The results of weight changes observed during the test period showed that, compared with the control group (PBS treatment group), the He'aoping treatment group and the group treated with He'aoping and HER2-ICD DNA vaccine (hereinafter referred to as the vaccine + He'aoping treatment group) showed a tendency for weight loss. In all test groups, there was a gradual tendency for weight loss from day 5 to the last day (day 31), but this was not statistically significant. The weights observed on the last day were: 94.4±5.8% in the control group, 97.0±5.2% in the HER2 ICD DNA vaccine treatment group (hereinafter referred to as the "vaccine treatment group"), 92.8±7.7% in the He'aoping treatment group, and 96.9±3.6% in the vaccine + He'aoping treatment group (Figure 1).

[0092] The results of measuring the rate of change in tumor size showed that, compared with the control group, tumor growth inhibition was continuously observed from day 3 to the last day in all vaccine treatment groups, Hecarbazine treatment groups, and vaccine + Hecarbazine treatment groups. In particular, statistically significant inhibitory effects on tumor growth were observed at several points in the vaccine treatment group (days 7, 12, 21, and 26) (p < 0.05) (Figure 2). Tumors extracted from each mouse are shown in Figure 3.

[0093] Therefore, it was confirmed that the HER2-ICD vaccine can inhibit tumor growth in xenograft gastric cancer mouse models by inducing a protective immune response.

[0094] The results of tumor weighing showed a reduction in tumor weight in both the vaccine treatment group and the vaccine + Hexamethasone treatment group compared to the control group. However, no statistical significance was observed in the reduction of tumor weight (Figure 4).

[0095] Macroscopic examination and weighing of the spleen revealed increased spleen size in both the vaccine treatment group and the vaccine + Hexamethasone treatment group compared to the control group (Figure 5). Furthermore, the spleen weight was significantly higher in both absolute and relative terms in these groups compared to the control group. Notably, the highest spleen weight was observed in the vaccine + Hexamethasone treatment group, but this was not statistically significant (Figures 6 and 7).

[0096] Overall, considering the above results, no abnormal symptoms were observed that were attributed to administration of HER2-ICD DNA vaccine alone or in combination with NCI-N87 gastric cancer cells to animal models.

[0097] Furthermore, analysis of the antitumor effects in animals transplanted with NCI-N87 gastric cancer cells revealed antitumor effects inhibiting tumor growth in both HER2-ICD DNA vaccine alone and in combination with HER2-ICD DNA vaccine and HER2-ICD DNA vaccine, but none were observed in HER2-ICD DNA vaccine alone. In particular, the antitumor effect was more significant in the case of HER2-ICD DNA vaccine alone compared to the case of HER2-ICD DNA vaccine alone or in combination with HER2-ICD DNA vaccine and HER2-ICD DNA vaccine. Therefore, this invention proposes the possibility that HER2-ICD DNA vaccine alone can effectively treat gastric cancer expressing HER2 protein.

[0098] none

[0099] TW202322855A_111128272_SEQL.xml

Claims

1. Use of a plasmonic carrier in the manufacture of a vaccine composition comprising a pharmaceutically acceptable carrier for inducing anticancer activity against gastric cancer expressing the HER2 protein in an individual without co-administration of an antiHER2 therapeutic antibody, wherein the plasmonic carrier comprises a nucleotide sequence encoding a HER2-ICD region having SEQ ID NO:2, wherein the vaccine composition exhibits enhanced anticancer activity compared to co-administration of the plasmonic carrier and the antiHER2 therapeutic antibody.

2. Use of the plasmid carrier as claimed in claim 1 in the manufacture of vaccine compositions, wherein the nucleotide sequence has SEQ ID NO:

3.

3. Use of the plasmid carrier as described in claim 1 or claim 2 in the manufacture of a vaccine composition, wherein the individual is a gastric cancer patient expressing the HER2 protein.

4. Use of the plasmid carrier as described in claim 3 in the manufacture of vaccine compositions, wherein the individual is a human.

5. Use of the plasmid carrier as described in claim 1 or claim 2 in the manufacture of a vaccine composition, wherein the anti-HER2 therapeutic antibody includes trastuzumab.

6. Use of the plasmid carrier as described in claim 1 or claim 2 in the manufacture of a vaccine composition, wherein the vaccine composition further includes an adjuvant.

7. Use of the plasmid carrier as described in claim 1 or claim 2 in the manufacture of a vaccine composition, wherein the vaccine composition is administered by injection or inoculation.

8. Use of the plasmid carrier as claimed in claim 7 in the manufacture of a vaccine composition, wherein the vaccine composition is administered by intradermal injection.

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