Her2 vaccine composition

Abstract

To provide a method for predicting the acquisition of immunogenicity against a DNA vaccine and the therapeutic efficacy thereof through a method of examining immunogenicity against a vaccine antigen (HER2-ICD antigen) before DNA vaccination.SOLUTION: The present disclosure relates to a method for predicting the reactivity of a HER2-ICD DNA vaccine composition, that is, the acquisition of immunogenicity and the therapeutic efficacy thereof, by measuring immunogenicity against a HER2-ICD antigen before vaccination. Additionally, by using the method for predicting the reactivity, a DNA vaccination target may be selected.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of priority to U.S. patent application Ser. No. 63 / 183,869, filed May 4, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to HER2 vaccine compositions. [Background technology]

[0003] Breast cancer is not only the most frequently diagnosed cancer and the most common cause of cancer death in women, but it also shows a trend toward increasing incidence and a lower age range for onset.

[0004] Over the past 20 years, various targeted therapies and diagnostic methods have significantly improved disease-free survival (DFC) in breast cancer treatment compared to other cancers. For example, In addition to traditional cancer treatments such as surgery and chemotherapy, such as paclitaxel, or radiation therapy, patients with estrogen receptor (ER)-positive breast cancer may be treated with estrogen therapy. A variety of targeted therapies are used to treat or slow the progression of breast cancer, such as ER-targeted therapies such as tamoxifen, which inhibits activation of the genotype receptor, and HER2-targeted antibodies such as trastuzumab, which are used for HER2-positive patients.

[0005] However, in the treatment of breast cancer, despite the progress of treatment methods, a considerable number of patients die due to metastasis or recurrence. Therefore, it is necessary to reduce the recurrence of breast cancer and increase the overall survival (OS) or disease-free survival (DFS) rate. Attempts have been made to do so.

[0006] In an attempt to prevent the metastasis or recurrence of breast cancer, we are researching methods to discover biomarkers for breast cancer stem cells and to administer targeted therapy to them. Both are being investigated as immunotherapies that utilize vaccines against breast cancer-specific antigens. Cancer vaccines are designed to activate cytotoxic T cells that recognize and act against specific types of cancer cells or induce the production of antibodies that bind to antigens present on the surface of cancer cells.

[0007] In the past few years, extensive research has been conducted into vaccines for cancer treatment or prevention of cancer recurrence, but with the exception of the prostate cancer vaccine sipuleucel-T (Provenge™), there have been no successful cases to date. The low success rate is partly 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, or in the case of metastatic tumors, the antigen profile changes between the initial tumor and the metastatic tumor, and the immune evasion mechanism of tumor cells.

[0008] A desirable vaccine antigen must be expressed exclusively or at least at an elevated level in tumor cells. HER2 / neu (hereinafter referred to as "HER2") is a member of the EGFR (epidermal growth factor receptor) family (EGFR2), and overexpression of EGFR is observed in many carcinomas. In particular, HER2 overexpression is observed in 15-30% of all breast cancer patients, and HER2 expression is known to be associated with recurrence and poor prognosis in breast cancer patients.

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

[0010] HER2 in breast cancer has been studied quite extensively, and several immunogenic peptides have been identified. For example, a peptide of amino acids 369-377 of the HER2 protein (nelipepimut-S [NeuVax; HER2 aa 369-377; E75 (NP-S)]) was found to be loaded onto dendritic cells. Immunotherapy, such as injection and reinfusion, has been attempted, but no clinically significant therapeutic or protective effects have been reported in women with advanced breast cancer. As a result, clinical trials have recently been halted in three large clinical trials, with no difference in disease-free survival (DFS) between the placebo group and the control group.

[0011] Other examples of HER2 peptide vaccines include the HER2-ICD peptide vaccine AE37 (LRMK-GVGSPYVSRLLGICL: LRMK-HER2 aa 776-790), the HER2-TM peptide There are vaccines such as GP2 (HER2 aa 654-662), which is a type of vaccine that can There have been some reports of an effect of increasing survival rates. Also, animal experiments have shown that a vaccine against HER2-ICD fragments (hereinafter referred to as "HER2-ICD") reduces tumor size compared to a vaccine against HER2-ECD fragments (hereinafter referred to as "HER2-ECD"). However, there are no reports of a direct administration of the HER2-ICD DNA vaccine to breast cancer patients. The results of this application were not reported.

[0012] By their very nature, vaccines for disease treatment are often administered twice or more repeatedly at a specific interval, and long-term immunogenicity must be generated using the subject's immune system, so a long period of time is required to confirm the effectiveness of the vaccine.

[0013] However, when considering the characteristics of the cancer disease and the mechanism of action of the drug, if it is confirmed after long-term observation that a cancer vaccine is ineffective, it is often the case that the disease has already worsened and no further treatment options are available. Therefore, it is most important to preselect patients who will respond to the vaccine and administer the drug to them.

[0014] However, so far, nothing is known about selecting recipients for breast cancer therapeutic vaccination. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] Republic of Korea Patent No. 10-1572474 (Announced: 2015.11.30.) [Patent Document 2] US Patent Publication US2002 / 0193329 (Publication date: 2002.12.19.) [Non-patent literature]

[0016] [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]

[0017] Therefore, the present inventors conducted research to solve the above-mentioned problems, and found that HER2-I The present invention was completed by discovering a patient group that exhibited long-term immunogenicity to the vaccine composition, in which a DNA vaccine composition containing a plasmid containing a nucleic acid sequence encoding CD was used to stably enhance immunogenicity against the HER2-ICD antigen in breast cancer patients without side effects.

[0018] The present invention aims to provide a method for predicting the acquisition of immunogenicity and therapeutic efficacy of a DNA vaccine through a method for testing the immunogenicity of a vaccine antigen (HER2-ICD antigen) prior to DNA vaccination.

[0019] Another object of the present invention is to provide a method for enriching subjects for DNA vaccination through the method of predicting the acquisition of immunogenicity and therapeutic efficacy of a DNA vaccine.

[0020] Furthermore, the present invention aims to provide a method of providing information for selecting patients who will have acquired immunogenicity and therapeutic effect against a DNA vaccine, via the method for selecting subjects for DNA vaccination. [Means for solving the problem]

[0021] To achieve the above objective, the present invention provides a method for predicting the acquisition of immunogenicity and therapeutic efficacy against a DNA vaccine through a method for testing the immunogenicity against a vaccine antigen (HER2-ICD antigen) before DNA vaccination.

[0022] Using the above-mentioned method for predicting reactivity to a DNA vaccine, subjects (patient groups) to be vaccinated with the DNA vaccine can be selected. Effect of the Invention

[0023] Acquisition of immunogenicity of the DNA vaccine (HER2-ICD DNA vaccine) according to the present invention This method for predicting immunogenicity and therapeutic efficacy not only predicts the acquisition of immunogenicity and therapeutic efficacy against a DNA vaccine before DNA vaccination without placing a heavy burden on the patient, but also has the advantage of being able to select vaccine recipients (patient groups) who are capable of exhibiting immunogenicity and therapeutic efficacy against a DNA vaccine. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 shows baseline pre-DNA vaccine immunogenicity against HER2-ICD antigen based on exposure to HER2-directed therapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present invention will now be described in further detail.

[0026] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027] 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.

[0028] In this application, even if an embodiment is described with the term "comprising," it should be understood that other similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0029] The term "prevent" refers to success, or evidence of success, in preventing the recurrence / relapse of breast cancer when a patient in clinical remission is examined, such as by radiological or physical methods.

[0030] The term "recurrence free" refers to a state in which a patient in clinical remission is free of recurrence of breast cancer, or cancer at other sites, when examined, such as by radiological or physical methods.

[0031] Thus, the terms "recurrence, relapse" or "resurgence" refer to Used interchangeably herein, the terms "breast cancer" and "cancerous" refer to signs or symptoms of breast cancer, or the development of cancer at other sites, following a period of improvement, palliation, or remission from breast cancer treatment. do.

[0032] The terms "free of breast cancer," "disease free," "Complete remission," or "no evidence of disease (NED)," means that a patient is currently in clinical remission with standard medical therapy, where the clinical signs, radiological signs, and symptoms of breast cancer have been significantly reduced or completely disappeared, based on clinical diagnosis, even though cancer cells may still be present in the body. Thus, remission is meant to include partial and complete remission.

[0033] The term "immunity" refers to the ability of a specific substance, such as an antigen, to induce an immune response in a subject. In this specification, it refers to the ability to induce an immune response against the HER2-ICD antigen, and the immune response can be measured using an IFN-γ ELISPOT assay (using peripheral blood mononuclear cells isolated from a subject). (PBMC: peripheral blood mononuclear cells) were stimulated with HER2-ICD antigen. Measurement shall be performed at, but not limited to, the following conditions:

[0034] In one embodiment of the present specification, the amount of IFN-γ produced by peripheral blood mononuclear cells (PBMCs) stimulated by HER2-ICD was measured by ELISpot (enzyme-linked immuno-spot assay), and compared to baseline, If HER2-ICD antigen-specific IFN-γ production is increased by more than 2-fold, it is considered that immunogenicity has been generated after vaccination due to an increase in HER2-ICD antigen-specific T cell response.

[0035] The term "immune response" refers to the physiological reaction of a subject's immune system to an antigen. The term "immunotherapy" as used herein refers to, but is not limited to, protective immunity against the HER2-ICD antigen as a result of such an immune response.

[0036] In this specification, the above-mentioned "protective immunity" or "protective immune response" means that a subject mounts an immune response against the HER2-ICD antigen, and the subject's immune system can target and destroy cells expressing the same antigen, thereby reducing cancer recurrence or progression in the subject. In the present invention, the protective immunity is provided by, but is not limited to, T lymphocytes.

[0037] In this specification, when the amount of IFN-γ produced by a subject's peripheral blood mononuclear cells (PBMCs) in response to stimulation by HER2-ICD was measured by ELISpot assay before (baseline) and after vaccine administration, a HER2-ICD antigen-specific T cell response was considered to have increased if HER2-ICD antigen-specific IFN-γ production was more than twice as high as baseline.

[0038] The term "booster" refers to a compound that enhances, prolongs or maintains protective immunity and is mediated by regulatory T cells. The term refers to a dose of an immunogen administered to a patient to overcome downregulation of T cell responses that may result from immunotherapy.

[0039] The term "pharmaceutical 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 "pharmaceutical acceptable carrier" means a medium that does not interfere with the effect of the biological activity of an active ingredient and is non-toxic to a subject when administered.

[0040] The term "about" when referring to a measurable value, e.g., weight, duration, etc., can mean, but is not limited to, a variation of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, including variations suitable for carrying out the described method, and meaning a range that does not affect reactivity.

[0041] The term "patient" or "subject" refers to a living organism suffering from, or susceptible to, the condition to be prevented or treated by administration of the vaccine composition of the invention, e.g., breast cancer, and includes both humans and animals. Subjects include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), preferably humans. Additionally, the term "patient" or "subject" in the present invention is not limited to any particular species and includes breast cancer patients or breast cancer patients expressing HER2.

[0042] The term "administering" refers to introducing a vaccine composition into a subject using any of a variety of methods and delivery systems known in the art to which the present invention pertains. Exemplary routes of administration include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion. As used herein, "administration" generally refers to intradermal administration by injection, and may also refer to intravenous, intramuscular, or intradermal administration. Parenteral routes include, but are not limited to, intraarterial, intrathecal or intralymphatic injection and infusion. Other parenteral routes include topical, epidermal or mucosal routes of administration, such as intranasal, vaginal, rectal or sublingual. As used herein, the terms "vaccine injection," "treatment," and "vaccination" refer to a subject. " are used without distinction.

[0043] In the present invention, the term "immune anticancer agent" refers to a therapeutic drug that induces immune cells to selectively attack only cancer cells by injecting artificial immune proteins into the body and stimulating the immune system, unlike existing anticancer agents that attack cancer itself, and is an anticancer agent that restores or strengthens the immune system's tumor recognition or destruction ability to overcome the immune suppression or immune evasion mechanism acquired by cancer cells. The immune anticancer agent includes immune checkpoint inhibitors, immune cell therapeutic agents, and immune virus therapeutic agents, It is not limited to these.

[0044] In the present invention, the term "immune checkpoint inhibitor" refers to a drug that blocks the activation of immune checkpoint proteins involved in suppressing T cells, which are immune cells in the body, when some cancer cells evade the immune system by utilizing the immune checkpoint of T cells. It is a type of immune anticancer agent that acts to block the immune checkpoints, activate T cells, and attack cancer cells, and includes, but is not limited to, CTLA-4 inhibitors, PD-1 inhibitors, LAG-3 inhibitors, TIM-3 inhibitors, TIGIT inhibitors, and VISTA inhibitors. The immune checkpoint inhibitors are antagonists, and may be antagonist antibodies or small molecule compounds.

[0045] The term "HER2-targeted therapy" refers to a therapy that relies on a mechanism that inhibits the activity of the HER2 protein, and refers to a therapy that uses a drug that blocks the active domain of HER2. Specific examples of such drugs include therapeutic antibodies that act against HER2, Small molecules, antibody-drug conjugates (ADCs) conjugated with drugs, etc. A representative example of an antagonist antibody is trastuzumab, but is not limited thereto. In this specification, the terms "HER2-targeted therapy" and "HER2-targeted therapy" are used interchangeably.

[0046] The compositions disclosed herein may be prepared into DNA vaccines. In some embodiments of the present invention, the DNA vaccine comprises a plasmid having 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. For example, they may also include sequences that enhance expression levels, intracellular targeting, and the like.

[0047] The antigen of the vaccine composition of the present invention is a polypeptide of the HER2 gene, which is a region between aa676 and a region between 1255 and 1300. The HER2-ICD polypeptide sequence may have 100% homology with the amino acid sequence of SEQ ID NO: 2, 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 the HER2-ICD antigen by vaccination has the same or equivalent effect as a sequence having 100% homology.

[0048] In some embodiments of the present invention, the DNA vaccine comprises a plasmid vector comprising a nucleic acid sequence encoding the HER2-ICD of the present invention. In some embodiments, the plasmid is a pUMVC3 (the conventional name is "pNGVL3", so 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 the HER2-ICD antigen by vaccination has the same or equivalent effect as a sequence having 100% homology.

[0049] In some embodiments of the present invention, the DNA vaccines can be encoded with additional immunomodulatory molecules that modulate the immune response generated, such as enhancing the efficacy of the vaccine, stimulating the immune system, or reducing immunosuppression.

[0050] In some embodiments of the present invention, a HER2-ICD DNA vaccine composition is administered. The patients were stage 3 or 4 breast cancer patients who expressed HER2. After completing standard breast cancer treatment, each patient was in complete remission or, in the case of stage 4 breast cancer patients, in stable bone only disease (SBD). It is acceptable.

[0051] In the present invention, since the vaccine composition is intended to induce and / or maintain protective immunity against breast cancer recurrence, 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 the HER2-ICD antigen. For example, a vaccine composition as described and exemplified herein can be administered to a patient according to a fixed schedule as a primary vaccination, followed by a booster to induce and / or maintain protective immunity.

[0052] In some embodiments of the present invention, the vaccine composition may be administered to a patient once, twice or more times per month. The booster may be administered once or multiple times at regular intervals, for example, monthly intervals, after the completion of the vaccination schedule, and may be administered once or multiple times for a period of six months or more. In some embodiments of the present invention, the HER2-ICD vaccine is administered three times, once per month, for the primary vaccination, and then again six months or a year later. A booster vaccination can be administered.

[0053] In this case, the booster may have the same dose and composition as the vaccine composition used in the primary vaccination, or the dose and / or composition may be changed.

[0054] The term "dose" refers to the total amount of the active ingredient (HER2-ICD plasmid DNA) of the vaccine composition administered once to a subject. In some embodiments of the present invention, the HER2-ICD DNA vaccine composition was administered at a dose of 10 μg, 100 μg, and 500 μg per arm (group of 22 patients) in three arms. It was observed that the administration of the vaccine in each arm increased immunogenicity against the HER2-ICD antigen, and the side effects related to the vaccine that occurred in each arm were all graded. ) 1,2 (very low grade), and no serious side effects of grade 3 to 5 were found. There were no statistically significant differences between the arms in terms of side effects.

[0055] Notably, the immunogenicity against HER2-ECD was increased in the patient group administered with the vaccine composition of the present invention. The DNA vaccine composition of the present invention is a vaccine that uses HER2-ICD as an antigen, and although the sequence is different from that of HER2-ECD or a peptide, the immune reactivity against HER2-ECD was also increased in the patient group administered with the vaccine composition of the present invention, particularly in the arm 2 and arm 3 patient groups.

[0056] Considering such effects, it has been confirmed that the vaccine composition of the present invention maintains immune reactivity against HER2-ICD for a long period of time even after vaccine administration has ended, and has excellent effects in preventing recurrence or metastasis of breast cancer in patients in remission during breast cancer treatment.

[0057] In the vaccine composition of the present invention, the total amount of HER2-ICD plasmid DNA contained in the vaccine administered once is 10 μg to 1,000 μg, 20 μg to 1,000 μg, 30 μg to 1,000 μg, 40 μg to 1,000 μg, 50 μg to 1,000 μg, 60 μg to 1,000 μg, or 70 μg to 1,000 μg, and is also 10 μg to 900 μg, 20 μg to 900 μg, 30 μg to 900 μg, 40 μg to 900 μg, 50 μg to 900 μg, 60 μg to 900 μg, 70 μg to 1,000 μg. 00μg, 10μg to 800μg, 20μg to 800μg, 30μg to 800μg, 40μg to 800μg, 50μg to 800μg, 60μg to 800μg, 70μg to 800μg, 10μg to 700μg, 20μg to 700μg, 30μg to 700μg, 40μg to 700μg, 50μg to 700μg, 60μg to 700μg, 70μg to 700μg, 10μg to 600μg, 20μg to 600μg, 30μg to 600μg, 40μg to 600μg , 50μg to 600μg, 60μg to 600μg, 70μg to 600μg, also 10μg to 500μg, 20μg to 500μg, 30μg to 500μg, 40μg to 500μg, 50μg to 500μg, 60μg to 500μg, 70μg to 500μg; also 10μg to 900μg, 10μg to 800μg, 10μg to 700μg, 10μg to 600μg, 10μg to 500μg, 10μg to 400μg, 10μg to 300μg, 20μg to 900μg, 20 μg to 800 μg, 20 μg to 700 μg, 20 μg to 600 μg, 20 μg to 500 μg, 20 μg to 400 μg, 20 μg to 300 μg, also 30 μg to 900 μg, 30 μg to 800 μg, 30 μg to 700 μg, 30 μg to 600 μg, 30 μg to 500 μg, 30 μg to 400 μg, 30 μg to 300 μg, also 40 μg to 900 μg, 40 μg to 800 μg, 40 μg to 700 μg, 40 μg to 600 μg, 40 μg to 500 μg, 40 μg to 400 μg, 40 μg to 300 μg, 50 μg to 900 μg, 50 μg to 800 μg, 50 μg to 700 μg, 50 μg to 600 μg, 50 μg to 500 μg, 50 μg to 400 μg, and 50 μg to 300 μg.

[0058] If the total amount of HER2-ICD plasmid DNA contained in a single dose of vaccine is less than 1 μg, the desired immune response will not occur, but if it exceeds 1,000 μg, injection site hypersensitivity reactions and immune tolerance will occur, and immunity will actually be reduced.

[0059] In vaccination, predicting immune reactogenicity is of utmost importance because vaccines generally require repeated administration two or more times, and the purpose of vaccines is to acquire long-term immunogenicity, so a long period of time is required to confirm the efficacy of the vaccine.

[0060] In particular, for therapeutic vaccines, the acquisition of immunogenicity against the antigen contained in the vaccine and the associated persistence (long-term immunogenicity) are of utmost importance, because long-term immunogenicity is directly linked to the therapeutic efficacy.

[0061] Specifically, in the case of cancer vaccines for treating cancer patients or preventing recurrence, if it is confirmed after vaccination that the vaccine is ineffective, there is often not enough time to apply other treatment methods, so it is important to pre-select patients who can be expected to benefit from the therapeutic effects of the vaccine, i.e., patients who are likely to acquire immunogenicity and have long-term immunogenicity (therapeutic effect) against the antigen contained in the vaccine, and then administer the vaccine to them.

[0062] In the present invention, before HER2-ICD DNA vaccination, peripheral blood mononuclear cells (PBM C) In the case where there is almost no immunogenicity (immunity) against the HER2-ICD antigen, i.e., in the patient group where there is almost no immune response in the immune test against the HER2-ICD antigen at baseline, after vaccination, the immunogenicity against the HER2-ICD antigen increases (2-fold or more), and the increased immunogenicity continues until 12 months after completion of vaccination (LTFU: long-term follow-up). However, in patients who already have immunogenicity against the HER2-ICD antigen at baseline, not only do immunogenicity against the HER2-ICD antigen not increase by more than two-fold after vaccination compared to the immunogenicity (baseline) before vaccination in many cases (it cannot be determined that it has been increased), but even if immunogenicity is increased, the increased immunogenicity is often not maintained for a long period of time.

[0063] There may be various mechanisms by which immunogenicity against the HER2-ICD antigen can be acquired before vaccination, but exposure to a therapy targeting HER2 can also lead to immunogenicity against the HER2-ICD antigen. In one embodiment of the present invention, in a patient group (naive group) that has not received a therapy targeting HER2, Although the present study showed little immunogenicity against the HER2-ICD antigen in the first line, the HER2-direct therapy (in the present invention, trastuzumab is prescribed) was completed (prior treatment) or during the vaccination period. We found that patients who had been exposed to HER2-targeted therapy, such as those receiving concurrent treatment, had increased immunogenicity against the HER2-ICD antigen at baseline.

[0064] Therefore, subjects for HER2-ICD DNA vaccination were: It is desirable to select a patient group that shows little immunogenicity against HER2-ICD. Many of these patients have not been exposed to HER2-targeting therapy, and even for patients who have experience with HER2-targeting therapy, it is desirable to select a patient group that has low or almost no immunogenicity against the HER2-ICD antigen at baseline.

[0065] Such results are also related to the persistence of vaccine DNA at the site of vaccination, and in DNA vaccines, DNA (plasmid DNA) acts as a continuous source of transfection of cells at the site of vaccination to produce the target antigen. Therefore, if DNA is present at the site of vaccination for a long period of time, it can continuously produce antigens, which is thought to be useful for maintaining antigen-specific immunity. However, if plasmid DNA, which is a DNA carrier of a foreign substance, persists in the patient's body for a long period of time, it will cause an immune response against the plasmid DNA itself in the subject.

[0066] In one embodiment of the present invention, one month after completion of vaccination, skin tissue was isolated through a biopsy from the vaccine administration site, and vector DNA was amplified through PCR. As a result, vector DNA was detected in 31 patients, and not detected in 21 patients. As a result of analyzing the immunogenicity against HER2-ICD antigen in peripheral blood mononuclear cells (PBMCs) from each patient, it was confirmed that the group of 21 patients in which vector DNA was not detected showed a tendency for increased immune reactivity against HER2-ICD antigen compared to patients in which vector DNA was detected, and in particular, immune reactivity at LTFU (12 months after completion of vaccination) showed a significant difference.

[0067] Notably, more than half (61%) of patients with detectable vaccine DNA had received HER2-targeted therapy during the vaccination period, whereas 76% of patients without detectable vaccine DNA had not received HER2-targeted therapy, and the difference between the two was statistically significant.

[0068] These results indicate that the patient group in which the vaccine DNA persisted for a long time at the injection site showed the same distribution as the patient group that was exposed to a therapy targeting HER2, which shows high immunogenicity against the HER2-ICD antigen before vaccination. This can be seen as supporting the result mentioned above that the more little immunogenicity there is against the HER2-ICD antigen before vaccination, the greater the immunogenicity due to vaccination, which is advantageous for ensuring long-term immunogenicity.

[0069] The vaccine composition described in the present specification does not contain any pharma- ceutical acceptable excipients, carriers, diluents, or vaccinia agents other than the plasmid containing the DNA encoding HER2-ICD. The vaccine composition of the present invention may include a buffer, stabilizer, preservative, adjuvant, or other substances well known in the art to which the present invention belongs. 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. Carriers or other substances that may be included in the vaccine composition of the present invention may be selected differently depending on the route of administration (e.g., oral administration, intravenous administration, cutaneous or subcutaneous administration, nasal administration, intramuscular administration, intradermal administration, and intraperitoneal administration).

[0070] The vaccine composition of the present invention also comprises one or more "pharmaceutical 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 composition also comprises a diluent, such as water, saline, or glycerol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffer substances, may be present. Sterile, pyrogen-free Phosphate buffered saline, tromethamine (Tris) are typical carriers.

[0071] One aspect of the present invention relates to vaccine compositions or kits as described above, which contain, as an active ingredient, one or more plasmid DNAs, for inducing an immune response in a subject or for providing a therapeutic, vaccinating or immunotherapeutic treatment. The vaccine composition may also be a vaccine or immunotherapeutic composition for use in the method. Such treatment or vaccination comprises administering to a subject a vaccine composition of the invention. The vaccine composition may be administered to the subject on a defined schedule.

[0072] The vaccine composition of the present invention may be lyophilized or may be in aqueous form, for example as an aqueous solution or suspension. Such types of liquid formulations are suitable for use in the preparation of vaccines in which the composition is reconstituted in an aqueous medium. Vaccine compositions are ideal for injection because they can be administered directly in packaged form without the need for injection. Vaccine compositions can be provided in vials or prefilled. The vaccine composition of the present invention may be provided in a syringe with a needle. The syringe may be supplied with or without a needle. The syringe may contain a single dose, while the vial may contain single or multiple doses. The vaccine composition of the present invention may also be administered using a microneedle.

[0073] The vaccine compositions of the present invention may also be formulated in a delayed release vehicle or depot formulation. Such long acting formulations may be administered by inoculation or implantation (e.g., subcutaneously or intramuscularly) or by injection. Thus, for example, the vaccine compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., an emulsion in an acceptable oil), or ion exchange resins, or as sparingly soluble derivatives (e.g., a sparingly soluble salt). Liposomes and emulsions are well known examples of delivery vehicles suitable for use as carriers.

[0074] The vaccine compositions of the present invention, when packaged in multiple dose formats, also include an antimicrobial agent. The antimicrobial agent may be 2-phenylindole, ... The optional preservatives may be selected from among, for example, 2-phenoxyethanol or the parabens (methylparaben, ethylparaben, propylparaben). Any preservatives are desirably present at low levels. The preservatives may be added exogenously and / or may also be components of the bulk antigens that are mixed to form the composition.

[0075] The vaccine composition of the present invention may be provided in the form of a kit including a manual and the like.

[0076] Vaccine compositions of the invention may also include one or more adjuvants to enhance the immunogenicity of the composition.

[0077] 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 cationic or anionic derivatized sugars, etc. The adjuvant may also include cytokines used as immunomodulators.

[0078] Suitable cytokines for use as the immunomodulatory agent include interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-2 L-12, etc.), macrophage colony stimulating factor (M-CSF: macrophage colony stimulating factor ing factor), tumor necrosis factor (TNF), granulocyte macrophage colony-stimulating factor (GC-CSF), The immunosuppressant may be selected from, but is not limited to, granulocyte-macrophage colony stimulating factor (GM-CSF) and the like.

[0079] In one embodiment of the invention, the adjuvant also includes granulocyte macrophage colony stimulating factor (GM-CSF).

[0080] The adjuvant may be added in an amount of 0.075 mg to 0.2 mg, or 0.08 mg to 0.12 mg per dose.

[0081] The vaccine composition of the present invention can be administered in combination with one or more anticancer agents selected from chemical anticancer agents, targeted anticancer agents, and immune anticancer agents. The one or more anticancer agents selected from chemical anticancer agents, targeted anticancer agents, and immune anticancer agents that can be administered in combination with the vaccine composition of the present invention can be administered simultaneously or sequentially with a time lag, and can be selected according to an appropriate time and period.

[0082] In the present invention, the chemical anticancer agent is also called a cytotoxic anticancer agent or a chemical drug anticancer agent. The chemical anticancer agent is, for example, gemcitabine (gemcitabine injection), cytarabine (Cytarabine), Carboplatin (Paraplatin), Cisplatin (Platinol, Platinol-AQ), Crizotinib (Xalkori), Cyclophosphamide (Cytoxan, Neosar), Docetaxel (Taxotere), Doxorubicin (Adriamycin), Etoposide (Bepcid), 5-fluorouracil (5-FU), Irinotecan (Camptosar), Liposome-encapsulated Doxorubicin (Doxorubicin), Sorubicin (Doxil), Methotrexate (Porex, Mexate, Amethopterin), Paclitaxel (Taxol, Abraxane), Topotecan (Hycamtin), Trabectedin (Yondelis), Vincristine (Oncovin, Vincasar PFS) or Vinbu Examples include, but are not limited to, Vinbrastine (Velban) .

[0083] In the present invention, the targeted anticancer drug is trametinib, vemurafenib, alpelisib, dactolisib, gefitinib, erlotinib, lapatinib, sunitinib, sorafenib, crizotinib, dabrafenib. Dabrafenib, Trastuzumab, Cetuximab, Bevacizumab, Panitumumab, Ipilimumab, Pertuzumab, Tofacitinib, Imatinib ), Bortezomib, Ofatumumab, and Alemtuzumab, but also one or more targeted therapeutic agents selected from the group consisting of However, the present invention is not limited to these.

[0084] In the present invention, the immunoanticancer agent is selected from the group consisting of CTLA-4 (cytotoxic T-lymphocyte-associated protein 4), PD-1 (programmed cell death protein 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). The present invention is also directed to, but not limited to, any one of the immune checkpoint inhibitors listed above.

[0085] In the present invention, the composition may further include an anti-cancer adjuvant, and more preferably, the anti-cancer adjuvant is a STING (stimulator of interferon gene) agonist. However, it is not limited to this.

[0086] In some embodiments of the present invention, administration of the HER2-ICD DNA vaccine composition comprises: Treatment with other cancer therapeutic agents may be performed simultaneously. The cancer therapeutic agents include anticancer drugs such as fluorouracil, doxorubicin, and paclitaxel that are standardly used in breast cancer treatment, trastuzumab (Herceptin), which is an antibody therapeutic agent against HER2, and ER (Estrogen Tamoxifen or bisphosphonates, which are vasopressin receptor targeted therapeutic agents, may be used, but are not limited thereto.

[0087] Unless otherwise indicated, all numbers used in the specification and claims, whether or not mentioned, should be understood to be modified in all cases 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 ​​generated from the standard deviations observed with their respective measuring techniques.

[0088] The present invention will be described in more detail below with reference to 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.

[0089] Clinical trial (NCT00436254) design and results patient group Patients enrolled in the study were stage 3 or 4 breast cancer patients who met the following criteria: (1) Only patients who had completed standard therapy and were in complete remission and, in stage 4 patients, had stable bone only disease; (2) tumors found to overexpress the HER2 gene or protein, either in the primary tumor or at the metastatic stage, via immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH); (3) ECOG performance score is 0 (SCOG Performance Score O); (4) must have white blood cell (WBC) ≥ 3,000 / mL, platelet count ≥ 100,000 / mL, hematocrit ≥ 30, and normal renal and hepatic function; (5) not have received chemotherapy or other immunosuppressant drug treatments, such as steroids, within 30 days prior to clinical trial enrollment; (6) Left ventricular ejection fraction (LVEF) must be within the normal range.

[0090] During the vaccination period, standard therapies such as bisphosphonates, anti-estrogen therapy, and anti-trastuzumab therapy were permitted. All patients were treated according to institutional regulations and and provided written informed consent as required by federal regulations. Sixty-six patients were enrolled from May 2004 through August 2007, with 64 patients completing all three doses of the vaccine. Two patients enrolled in arm 1 experienced disease progression after receiving one dose of the vaccine and discontinued the study. As required by federal regulations, patients were followed for 10 years for long-term toxicity.

[0091] Study design and treatment schedule Patients enrolled in this study received three pNGVL3-HER2- They were sequentially assigned to one of the ICD groups (arm 1: 10 μg, arm 2: 100 μg, arm 3: 500 μg). All vaccines were administered intradermally (id). Patients were given trastuzumab in 2 doses, but in the same draining lymph node site, once a month for a total of three vaccines, split into two injection sites per vaccine dose. Patients received a tetanus toxoid vaccine as a positive control before the first vaccination. Patients who received trastuzumab also underwent multi-gate (MUGA) scans at baseline and one month after the last vaccine. Toxicity assessments were performed monthly, and at 3, 6, and 12 months after completion of the vaccine series, and included physical exams, autoimmune serology, CBC, and complete metabolic panel. Toxicity was assessed using the CTCAE v3.0 Vaccine-related toxicities were graded by the WHO, and vaccine-related toxicities were defined as definitely, probably, or possibly adverse effects, regardless of severity. Long-term side effects were assessed via annual medical record review for surviving patients. Peripheral blood mononuclear cells (PBMCs) were collected at baseline, and after the third vaccine dose (12 weeks), and at 1, 3, 6, and 12 months after the final vaccination (corresponding to 16, 24, 36, and 60 weeks after enrollment, respectively) to evaluate induced immune responses against HER2-ICD and HER2-ECD.

[0092] To assess plasmid DNA persistence, skin biopsies were obtained from the vaccine injection site 1 and 3 months after the last vaccine dose. Peripheral blood mononuclear cells (PBMCs) were assessed for the presence of plasmid DNA at baseline and 1 month after the last vaccine dose.

[0093] Vaccine preparations The HER2-ICD sequence (SEQ ID NO:2) was inserted into pNGVL3 and confirmed by DNA sequencing. The plasmid DNA vaccine pNGVL3-HER2-ICD was amplified, quantified, and manufactured in vials under GMP conditions. Single-dose vials contain 10 μg, 100 μg, or 500 μg of plasmid DNA (pNGVL3-HER2-ICD) suspended in tromethamine / EDTA as a stabilizing buffer (1.2 ml), and commonly 100 μg of granulocyte macrophage colony-stimulating factor (GM-CSF). The vials underwent microbial, sterility, and stability testing and were stored between -25°C and 10°C in the Investigational Drug Services Pharmacy at the University of Washington in accordance with US Pharmacopoeia standards.

[0094] Assessment of antigen-specific T cell responses Peripheral blood mononuclear cell (PBMC) samples from all patients were processed and cryopreserved using a standardized method validated to preserve lymphocyte function. All samples from each patient were thawed and analyzed simultaneously to reduce experimental variability. (J Clin Oncol. 2009 Oct 1; 27 (28): 4685-92) specific response to HER2 was measured via IFN-γ ELISPOT assay stimulated with the antigen (stimulant) described below. HER2-ICD antigen or HER2-ECD antigen (stimulant) was used as an antigen, a 15 amino acid peptide pool (overlapping peptide pool (1 mg / mL)) with 11 amino acids overlapping.

[0095] Patients were considered to have augmented HER2-ICD antigen-specific T cell responses if, after vaccination, they demonstrated a two-fold increase in HER2-ICD antigen-specific IFN-γ production from baseline in peripheral blood mononuclear cells (PBMCs) measured by ELISpot assay.

[0096] Assessment of plasmid DNA persistence in skin and lymphocytes After vaccination, the vaccine was administered between the two tattoo marks to serve as a consistent marker for skin biopsies. (skin) was biopsied. The isolated skin tissue was stored at 70°C. DNA from tissue and peripheral blood mononuclear cells (PBMCs) was isolated using a DNA isolation kit, and vector DNA was amplified using the primer couple PRDM36 primer [GACTAACAGACTGTTCCTTTCCATGG: coding primer] and PRDM52 primer [GCCAGAAGTCAGATGCTCAA: complementary primer].

[0097] Patient group analysis and adverse effect analysis by arm Arm 1, Arm 2, and Arm 3 each consisted of 22 patients, and each received a vaccine composition containing 10 μg, 100 μg, and 500 μg of HER2-ICD plasmid DNA, respectively, and 100 μg of granulocyte-macrophage colony-stimulating factor (GM-CSF) intradermally. Patient characteristics for each arm are listed in Table 1. It has been done.

[0098] [Table 1]

[0099] A total of 990 adverse effects were observed across all arms, with 517 were considered to be vaccine-related (Table 2). The most common vaccine-related adverse events included injection site reactions (23%), fatigue (7%), and myalgia (7%). All vaccine-related toxicities were mostly grade 1 (89%) or grade 2 (11%), with no significant differences between arms.

[0100] The safety of DNA vaccines is attributable to the administration of exogenous DNA to the subject, which may result in the occurrence of autoimmune responses and / or the insertion of exogenous DNA into the subject's chromosomal DNA. There is a very low risk of infection. In this study, 16 abnormal serological reactions related to autoimmunity were found in all arms (Table 2). In 11 patients, ANA positivity was observed during the vaccination period, and in 5 patients, a temporary decrease in C3 was observed.

[0101] All autoimmune-related toxicities were grade 1, anti-ds-DNA antibodies were not found in any patients, and no signs or development of autoimmune disease was found. The DNA vaccine was Not detected.

[0102] Therefore, the side effects of administering the vaccine composition of the present invention are extremely low and not of concern when administered to a subject, and no signs or symptoms of autoimmune disease were observed. In addition, since no insertion into the genome of a subject was observed, the vaccine composition of the present invention is considered to be safe in clinical application.

[0103] [Table 2]

[0104] Immunogenicity analysis of HER2-ICD before and after vaccination To analyze the baseline immunogenicity of each patient against the HER2-ICD antigen, immune reactivity against the HER2-ICD antigen was analyzed in peripheral blood mononuclear cells (PBMCs) before vaccination using interferon-gamma ELISPOT assay, and this value was used as a baseline to calculate the change in immune reactivity against the HER2-ICD antigen after vaccination.

[0105] Before vaccination, patients were divided into three groups based on their experience with HER2-targeted therapy: the naive group, who had no experience with HER2-targeted therapy (HER2-direct therapy; in this study, trastuzumab was prescribed), the prior group, who had completed HER2-targeted therapy before vaccination, and the concurrent group, who were undergoing HER2-targeted therapy at the time of vaccination.

[0106] The immunogenicity of HER2-ICD antigen by ELISpot in each group is shown in Figure 1. In the naive group, the immunogenicity was 1:1,000,000 (1:10,870 to 1:1, 000,000), and in the prior group, it was 1:9,707 (1:498~1:100, The mean values ​​of 1:1,000,000, 1:9,707, and 1:12,840 in the naive group were the mean values ​​of the ELISPOT measurements. The values ​​of the group showed a statistical difference from the values ​​of the prior group and the concurrent group, but there was no statistical difference between the values ​​of the prior group and the concurrent group.

[0107] Therefore, patients with experience of HER2-targeted therapy can be known to have immunogenicity against the HER2-ICD antigen before vaccination, specifically, to have immunogenicity that is approximately 10-fold increased compared to patients without experience of HER2-targeted therapy.

[0108] After that, the subjects were assessed at 1 month and 12 months (LTFU: long-term follow-up) based on the time of vaccination completion. In peripheral blood mononuclear cells (PBMCs) isolated from patients with HER2-ICD As a result of analyzing immune reactivity against antigens, it was noted that not only did the immune response against HER2-ICD antigen increase to a high level (2-fold or more) in patients who did not show immunogenicity against HER2-ICD antigen at baseline, but the immune response level was maintained until LTFU. Moreover, the patients who did not show immunogenicity against HER2-ICD antigen at baseline included most of the naive patient group, and also included some of the patients who had received HER2 target therapy.

[0109] Specifically, by the ELISPOT method of the present specification, 6 If reactivity to the HER2-ICD antigen is measured at baseline (at least 1:20,000) in 50 or more peripheral blood mononuclear cells (PBMCs), then the patient is deemed to be immunogenic to the HER2-ICD antigen and should be vaccinated with the HER2-ICD DNA vaccine. Even after multiple revaccination, very few patients showed a 2-fold or greater increase in immunogenicity and / or long-term maintenance.

[0110] Therefore, it is highly likely that patients with no or low immunogenicity to the HER2-ICD antigen will be able to acquire and maintain long-term immunogenicity induced by the HER2-ICD DNA vaccine. Therefore, for effective treatment, the patient group to be vaccinated can be enriched in advance based on specification based on clinical criteria, and can be determined as a group of patients who have no experience of HER2-targeted therapy, or who have received HER2-targeted therapy but have little immunogenicity against the HER2-ICD antigen at baseline before vaccination.

[0111] Analysis of plasmid DNA persistence at vaccination sites Sixteen weeks after the start of vaccination (one month after the last vaccine administration), the presence of DNA vaccine in skin biopsies was analyzed by PCR amplification of vector DNA into which HER2-ICD had been cloned.

[0112] Skin biopsies were obtained from 52 patients overall, and vector DNA was detected in 31 (60%) of them: 61% in arm 1, 50% in arm 2, and 73% in arm 3.

[0113] As a result of following up patients in whom the DNA was detected and those in whom it was not detected (Table 3), it was confirmed that patients in whom the DNA was detected not only had lower immunogenicity against the HER2-ICD antigen on average over time, but also had significantly lower immune reactivity against the HER2-ICD antigen at 60 weeks after vaccination (12 months after vaccination) (p=0.02).

[0114] [Table 3]

[0115] Of note, patients with detectable vaccine DNA at week 16 were more likely to have been exposed to HER2-targeted therapy than those without detectable vaccine DNA: 19 of 31 patients with detectable vaccine DNA had received HER2-targeted therapy (61%), whereas 1 of 21 patients without detectable DNA had been previously exposed to HER2-targeted therapy (24%) (p=0.008).

[0116] These results indicate that the patient group in which the vaccine DNA persisted for a long time at the injection site showed the same distribution as the patient group that was exposed to a therapy targeting HER2, which shows high immunogenicity against the HER2-ICD antigen before vaccination. This can be seen as consistent with the result mentioned above, which shows that the less immunogenicity there is against the HER2-ICD antigen before vaccination, the more advantageous it is for increasing immunogenicity and ensuring long-term immunogenicity through vaccination. [Sequence List Free Text]

[0117] Sequence information SEQ ID NO: 1 (HER2 full length sequence) YLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPE YVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:2 (HER2-ICD amino acid sequence) MKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGS QDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSE CRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPL QRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERAKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO:3 (HER2-ICD nucleotide sequence) GGGACCTGGTGGATGCTGAGGAGTATCTGGTACCCCAGCAGGGCTTCTTCTGTCCAGACCCTGCCCCGGGCGCTGGGGGCATGGTCCACCACAGGCACCGCAGCTCATCTACCAGGAGTGGCGGTGGGGACCTGACACTAGGGCTGGAGCCCTCTGAAGAGGAGGCCCCCAGGTCTCCACTGGCACCCTCCGAAGGGGCTGGCTCCGATGTATTTGATGGTGACCTGGGAATGGGGGCAGCCAAGGGGCTGCAAAGCCTCCCCACACATGACCCCAGCCCTCTACAGCGGTACAGTGAGGACCCCACAGTACCCCTGCCCTCTGAGACTGATGGCTACGTTGCCCCCCTGACCTGCAGCCCCCAGCCTGAATATGTGAACCAGCCAGATGTTCGGCCCCAGCCCCCTTCGCCCCGAGAGGGCCCTCTGCCTGCTGCCCGACCTGCTGGTGCCACTCTGGAAAGGGCCAAGACTCTCTCCCCAGGGAAGAATGGGGTCGTCAAAGACGTTTTTGCCTTTGGGGGTGCCGTGGAGAACCCCGAGTACTTGACACCCCAGGGA GGAGCTGCCCCTCAGCCCCACCCTCCTCCTGCCTTCAGCCCAGCCTTCGACAACCTCTATTACTGGGACCAGGACCCACCAGAGCGGGGGGCTCCACCCAGCACCTTCAAAGGGACACCTACGGCAGAGAACCCAGAGTACCTGGGTCTGGACGTGCCAGTGTAA

Claims

1. A method for predicting the reactivity of a HER2-ICD (intracellular domain) DNA vaccine, including a method for testing immunogenicity against a HER2-ICD antigen in peripheral blood mononuclear cells (PBMCs) of patients receiving the HER2-ICD DNA vaccine prior to vaccination. How to do it.

2. The patient receiving the HER2-ICD DNA vaccine is a patient with a cancer that expresses HER2. The method of claim 1 ,

3. The patient to be vaccinated with the HER2-ICD DNA vaccine has HER2 ICD prior to vaccination. The method of claim 2, wherein the patient is bi-targeted therapy naive.

4. The method of claim 3 , wherein the HER2 targeted therapy is by a pharmaceutical composition comprising a therapeutic antibody against HER2.

5. 5. The method of claim 4, wherein the therapeutic antibody against HER2 is from a pharmaceutical composition comprising trastuzumab.

6. HER2-ICD DNA is isolated by the method according to any one of claims 1 to 5. How to select patients for vaccination.

7. The HER2-ICD DNA vaccine comprises a sequence identical to SEQ ID NO: 2 or a sequence having 80% or more similar thereto. The method of any one of claims 1 to 5, comprising a plasmid vector comprising a nucleotide sequence encoding a homologous HER2-ICD region.

8. The method of claim 7, wherein the nucleotide sequence is SEQ ID NO: 3 or has 80% or more sequence identity thereto.

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