KR20240038596A - A composition for preventing or treating cancer, having abscopal effect and a method of preventing or treating cancer using the same - Google Patents

Abstract

The present invention is intended to induce at least one immune stimulation, and is intended to treat primary cancer, cancer of cells scattered in the human body and its metastases, and in particular, the patient's own immune system to kill the cancer cells. It relates to a method and composition for inducing and promoting the establishment of memory to recognize, kill, and prevent recurrence of cancer disease.

Description

A composition for preventing or treating cancer, having an abscopal effect, and a method for preventing or treating cancer using the same {A composition for preventing or treating cancer, having abscopal effect and a method of preventing or treating cancer using the same}

The present invention relates to a composition for preventing or treating cancer, which has an abscopal effect, and a method for treating cancer using the same. Specifically, a method for increasing the abscopal effect for the treatment of cancer and tumors and inducing the abscopal effect. It relates to a composition for:

In general, it is known that radiation therapy worsens immune function. However, as more information is learned about the role of T cells in radiation therapy, new aspects of the immune function related to radiation therapy, such as immunomodulation, have become known. At the cellular level, the typical effects that appear after radiation are increased activity of natural killer cells (NK cells), increased infiltration of T cells (especially CD 8 T cells), increased antigen presentation by dendritic cells, There is an increase in the production of immunostimulatory cytokines.

The Abscopal effect is particularly well known as an immune control effect, and refers to an anticancer treatment effect that inhibits cancer growth not only in tumor tissue in the area where radiation was locally irradiated but also in metastatic tumors in areas where radiation was not irradiated. In other words, this is a case where radiation therapy sensitizes tumor cells and makes them more responsive to cancer treatment.

According to the Abscopal effect, radiation treatment affects the body's immune system to attack cancer cells distributed throughout the body as well as specific areas within the body. The immune effect makes it possible to eliminate cancer cells not only in the area exposed to radiation, but also in other distant parts of the body. Radiation therapy causes dendritic cells to send specific signals out of the cells, sends danger signals using HMGB1 or ATP, and promotes CD8 ⁺ T cells to induce an anticancer response. Therefore, during radiation treatment, not only the irradiated area but also cancer in other areas that were not irradiated are removed. Recently, it was revealed that high-dose ablative radiotherapy (20 Gy single fraction) increases the activity of CD8 ⁺ cytotoxic T cells and is effective in controlling not only cancer in the treated area but also cancer in untreated distant metastatic areas. Recently, there was a study that showed that when a vaccine (Flt3L) that inhibits TLR9 was administered to early-stage lymphoma (low grade lymphoma) and radiotherapy was performed on the cancer in a specific area, untreated cancer in distant areas also showed a reduction in response. In addition, when granulocy macrophage-colony stimulating factor (GM-CSF) is administered along with radiation therapy to patients with metastatic lung cancer, there are cases where the cancer in areas that were not treated with radiation therapy also showed a decreased response.

Meanwhile, MHC, which is an antigen presentation on cancer cells, causes CD8 ⁺ cytotoxic T lymphocytes to secrete interferon gamma, which promotes the production of PD-1 ligand in tumor cells. This PD-1 ligand binds to PD-1 on T cells and worsens T cell activity. Therefore, antibodies that can inhibit this (ex, anti-PD-1 antibody or bispecific T cell recruiting antibodies; CD 133 × CD3 Ab) can be used in cancer treatment.

Immune checkpoint inhibitors have been known to be effective in treating cancer through several research reports. Clinically, ipilimumab, which acts as an immune checkpoint inhibitor of cytotoxic T-Lymphocyte associated antigen-4 (CTLA-4), and nivolumab, which acts on programmed death-1 (PD-1), have lower survival rates in patients with advanced melanoma. It was found that it had the effect of increasing . Pembrolizumab, known as another anti-PD-1 Ab, has also been shown to be effective in stomach cancer, nasopharyngeal cancer, and lung cancer. However, the effectiveness of immunotherapy was limited to about 10-30% as monotherapy.

In summary, several studies are being conducted to discover ways to enhance the abscopal effect. However, no method has yet been proposed to effectively maximize the Abscopal effect.

Republic of Korea Patent Publication No. 10-2039520

The present invention was devised to solve the problems described above, by irradiating tumor tissue or cancer cells isolated from a cancer patient and treating them with an anti-cancer immunotherapy agent to inhibit the activity or induce death of the cancer cells, and then re-introducing them to the cancer patient. It was completed after confirming that when administered, it can activate the body's immune function against cancer, further enhance the effect of anticancer therapy, and suppress cancer growth, recurrence, and metastasis. In particular, it was confirmed that cancer cells whose activity was inhibited or whose death was induced as described above can exert abscopal effects when administered to cancer patients.

In addition, when cancer cells whose activity has been suppressed or killed as described above are combined with radiation therapy and/or immunotherapy, the anticancer effect is further maximized, anticancer immunity is enhanced, and a strong abscopal effect is achieved. It has been confirmed.

Therefore, the present invention is a pharmaceutical for the prevention or treatment of cancer, comprising as an active ingredient tumor tissue or cancer cells isolated from a cancer patient and subjected to one or more treatments selected from the group consisting of (a) and (b) below. The object is to provide a composition:

(a) Irradiation; and

(b) Immunoanticancer drugs.

Additionally, the present invention provides a kit for preventing or treating cancer comprising the composition.

Additionally, the present invention provides a method for producing the composition.

More specifically, the object to be achieved by the present invention is the invasive or non-invasive treatment of primary cancer and metastatic cancer thereof in patients suffering from cancer or tumors, including treatment using radiation therapy, anticancer agents, or immunostimulants. The aim is to provide a composition for the prevention or treatment of cancer that can enhance the anti-cancer effect by or exert the abscopal effect to prevent recurrence of cancer after treatment, and a method of preventing or treating cancer using the same.

Another object of the present invention is to provide a composition for preventing or treating cancer that can be used as a tumor vaccine, which can prevent immune evasion by cancer cells by allowing the immune system to more effectively recognize cancer cells, and a method for preventing or treating cancer using the same. is to provide. In particular, the composition according to the present invention can exert an abscopal effect to prevent recurrence of cancer in patients at risk of recurrence after cancer treatment, and is therefore very useful as a tumor vaccine.

Another object of the present invention is that, through the abscopal effect, it can provide memory and systemic immune response to cancer cells, especially to spreading cells of micrometastatic cancer, and can be used as a vaccine therapy, and after general cancer and tumor treatment. The aim is to provide a composition useful for preventing recurrence of cancer and a method for preventing or treating cancer using the same.

Another object of the present invention is to increase the effect of abscopal for the treatment of cancer and tumors in a subject, but the subject is subject to radiation, side effects and burden of the subject due to anticancer drugs, side effects of metabolites due to immunological agents, and The aim is to provide a composition that can suppress damage to the subject's immunity such as burden, local or systemic damage and burden, unwanted cancer and tumor mutation or increase in resistance, and a method of preventing or treating cancer using the same.

Furthermore, another object of the present invention is to reduce or delay the possibility of cancer occurrence by administering the composition according to the present invention to an individual who has not developed cancer, thereby enabling the immune function to learn and remember cancer and strengthening immunity against cancer. It's in what you're told to do. Therefore, the composition according to the present invention can achieve a cancer prevention effect in individuals who are genetically or environmentally likely to develop cancer.

Another object of the present invention is to achieve a more powerful anticancer effect by using the composition according to the present invention in combination with other anticancer treatments. For example, when the composition according to the present invention is used in combination with radiation therapy and/or immunotherapy, not only can the growth of primary tumors (or primary tumors) be more effectively inhibited, but also the growth of secondary tumors can be inhibited through the abscopal effect. Therefore, cancer metastasis can be suppressed more effectively. Furthermore, the above combination therapy can enhance anti-cancer immune function and achieve a synergistic anti-cancer effect.

For this purpose, the present invention extracts an appropriate amount of cancer and tumor tissue from the subject outside the body of the subject, and attenuates the extracted tissue or cancer cells by one or more physical, chemical, and medical methods, such as irradiation or immunotherapy, to the extracted tissue. Provides a method of inducing the effect of abscopal in the subject's body by inducing combustion or death and then re-administering it to the subject.

The present invention provides a pharmaceutical for the prevention or treatment of cancer, comprising as an active ingredient tumor tissue or cancer cells isolated from a cancer patient and subjected to one or more treatments selected from the group consisting of (a) and (b) below. Provided is a composition:

(a) Irradiation; and

(b) Immunoanticancer drugs.

Here, the cancer includes all primary cancer (primary tumor), secondary tumor, metastatic cancer, and recurrent cancer.

Additionally, the present invention provides a kit for preventing or treating cancer, comprising the composition.

Furthermore, the present invention provides a use of the composition for preventing or treating cancer.

In addition, the present invention provides a method for preventing or treating cancer, comprising administering the composition to an individual in need thereof. Preferably, the subject is an entity from which the tumor tissue or cancer cells are isolated, or an allogeneic entity of the subject. In one embodiment of the present invention, the method for preventing or treating cancer may further include performing anticancer therapy on the subject. The anti-cancer therapy may be two or more types of anti-cancer therapy (i.e., different types of anti-cancer therapy). Additionally, the anti-cancer therapy may be performed simultaneously or sequentially with administration of the composition. At this time, there is no limit to the execution order.

Furthermore, the present invention provides the use of the composition for the manufacture of a medicament for the treatment of cancer.

Furthermore, the present invention provides a combined use of the composition with anticancer therapy.

Furthermore, the present invention provides the use of the composition for preparing a combination preparation with anticancer therapy.

Furthermore, the present invention includes the steps of (i) administering the composition to an individual in need thereof; and (ii) applying a second anti-cancer therapy to the subject. The second anti-cancer therapy may be two or more types of anti-cancer therapy (i.e., different types of anti-cancer therapy). The steps (i) and (ii) may be performed simultaneously or sequentially, and there is no limitation on the order of execution. In addition, when performing two or more types of anticancer therapy, there is no limit to the order in which each anticancer therapy is performed, and each can be performed simultaneously with the administration of the composition, or can be performed sequentially.

In one embodiment of the present invention, the tumor tissue or the cancer cells may satisfy one or more characteristics selected from the group consisting of the following by the treatment, but are not limited thereto:

i) the activity of cancer cells is weakened;

ii) cancer cells become inactive; and

iii) Cancer cells die.

In another embodiment of the present invention, the composition contains autologous tumor tissue or cancer cells of the cancer patient and is administered to the cancer patient, or to a cancer patient allogeneic to the cancer patient. However, it is not limited to this.

In another embodiment of the present invention, the composition may be administered to an individual who does not have cancer for the purpose of preventing cancer, but the composition is not limited thereto. The individual may be an individual with a high risk of developing cancer genetically or environmentally.

In another embodiment of the present invention, when the treatment is (a) irradiation, the radiation may satisfy one or more characteristics selected from the group consisting of, but is not limited to:

i) the radiation is at least one selected from the group consisting of γ-rays, x-rays, ultraviolet rays, laser rays, and infrared rays; and

ii) The radiation dose is 1 to 500 Gy.

In another embodiment of the present invention, the immunotherapy agent may be one or more selected from the group consisting of immune checkpoint inhibitors, co-stimulatory molecule agents, cytokine treatments, CAR-T cell treatments, and autologous CD8 ⁺ T immune cell treatments. However, it is not limited to this.

In another embodiment of the present invention, the immune checkpoint inhibitor is a PD-L1 inhibitor, PD-1 inhibitor, CTLA-4 inhibitor, LAG3 inhibitor, TIM3 inhibitor, 4-1BB inhibitor, LAG-3 inhibitor, B7-H4 inhibitor, It may be one or more selected from the group consisting of HVEM inhibitor, TIM4 inhibitor, GAL9 inhibitor, VISTA inhibitor, KIR inhibitor, TIGIT inhibitor, and BTLA inhibitor, but is not limited thereto.

In another embodiment of the present invention, the composition may satisfy one or more characteristics selected from the group consisting of the following when administered to a cancer patient, but is not limited thereto:

i) exerts the abscopal effect;

ii) inhibits metastasis of cancer;

iii) reduces tumor load; and

iv) Inhibits the proliferation of cancer cells.

In another embodiment of the present invention, the composition may be for single administration or multiple administration, but is not limited thereto.

In another embodiment of the present invention, the composition contains two or more tumor tissues or cancer cells, and the two or more tumor tissues or cancer cells may have been subjected to the same treatment or different treatments, but are not limited thereto. .

In another embodiment of the present invention, the composition may be in the form of a mixture of the two or more tumor tissues or cancer cells, but is not limited thereto.

In another embodiment of the present invention, the composition may be in a form in which the two or more tumor tissues or cancer cells are each formulated and administered simultaneously, separately, or sequentially, but is not limited thereto.

In another embodiment of the present invention, the composition may be used in combination with anticancer therapy, but is not limited thereto.

In another embodiment of the present invention, the anticancer therapy may be one or more selected from the group consisting of radiation therapy, chemotherapy, targeted anticancer agent therapy, and immunotherapy, but is not limited thereto.

In another embodiment of the present invention, the composition may be used in combination with radiation therapy and immunotherapy, but is not limited thereto.

In another embodiment of the present invention, the composition may be administered simultaneously, separately, or sequentially with the anticancer therapy, but is not limited thereto.

In another embodiment of the present invention, the targeted anti-cancer agent may be one or more selected from the group consisting of tyrosine kinase inhibitors, PARP inhibitors, angiogenesis inhibitors, and CDK4/6 inhibitors, but is not limited thereto.

In another embodiment of the present invention, the immunotherapy agent may be one or more selected from the group consisting of immune checkpoint inhibitors, co-stimulatory molecule agents, cytokine treatments, CAR-T cell treatments, and autologous CD8 ⁺ T immune cell treatments. However, it is not limited to this.

In another embodiment of the present invention, the immune checkpoint inhibitor is a PD-L1 inhibitor, PD-1 inhibitor, CTLA-4 inhibitor, LAG3 inhibitor, TIM3 inhibitor, 4-1BB inhibitor, LAG-3 inhibitor, B7-H4 inhibitor, It may be one or more selected from the group consisting of HVEM inhibitor, TIM4 inhibitor, GAL9 inhibitor, VISTA inhibitor, KIR inhibitor, TIGIT inhibitor, and BTLA inhibitor, but is not limited thereto.

In another embodiment of the present invention, the tumor tissue may be pulverized and diluted or dissolved in a solution, but is not limited thereto.

In another embodiment of the present invention, the composition contains two or more tumor tissues, and the pulverized particle sizes of the two or more tumor tissues may be the same or different from each other, but the composition is not limited thereto.

In another embodiment of the present invention, the cancer may be one or more selected from the group consisting of blood cancer and solid cancer, but is not limited thereto.

In another embodiment of the present invention, the cancer is breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, skin melanoma, and intraocular melanoma. Tumor, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue Sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma, but is not limited thereto.

In addition, the present invention includes the steps of (S1) isolating tumor tissue or cancer cells from a cancer patient; and

(S2) It provides a method for producing the composition, comprising the step of applying one or more treatments selected from the group consisting of (a) and (b) below to the isolated tumor tissue or cancer cells:

(a) Irradiation; and

(b) Immunoanticancer drugs.

In one embodiment of the present invention, the method may further include, but is not limited to, pulverizing the tumor tissue and diluting or dissolving it in a solution after the step (S1).

In another embodiment of the present invention, the composition contains autologous tumor tissue or cancer cells of the cancer patient and is administered to the cancer patient, or to a cancer patient allogeneic to the cancer patient. It may be, but is not limited to this.

In another embodiment of the present invention, the composition may be administered to an individual who does not have cancer for the purpose of preventing cancer, but the composition is not limited thereto. The individual may be an individual with a high risk of developing cancer genetically or environmentally.

In addition, the present invention provides an anticancer vaccine composition or vaccine comprising, as an active ingredient, tumor tissue or cancer cells isolated from a cancer patient and subjected to one or more treatments selected from the group consisting of (a) and (b) below. to provide:

(a) Irradiation; and

(b) Immunoanticancer drugs.

In one embodiment of the present invention, the vaccine composition or vaccine may be administered to an individual who has not developed cancer or an individual whose cancer has been completely cured for the purpose of preventing cancer, but is not limited thereto.

In addition, the present invention provides a method for preventing cancer, comprising the step of administering tumor tissue or cancer cells to which the one or more treatments have been applied to an individual in need thereof.

In addition, the present invention provides the use of tumor tissue or cancer cells subjected to one or more of the above treatments for cancer prevention.

In addition, the present invention provides the use of tumor tissue or cancer cells to which one or more of the above treatments have been applied for the production of a cancer prevention drug (e.g., a cancer prevention vaccine).

When radiation is irradiated to a patient to induce the Abscopal effect, there is a problem that normal tissue damage and necrosis occur due to radiation exposure. There is a risk that radiation exposure to some cancer tissues may cause cancer mutations, resulting in the development of other types of cancer tissues that are resistant to radiation or are transformed. The present invention was developed to solve the above problems. It exerts an excellent abscopal effect to strengthen the body's immune function against tumors and further enhances the anticancer effect of anticancer therapy to prevent cancer growth, recurrence, and metastasis. It relates to a composition capable of inhibiting. The present invention is characterized by extracting cancer tissue or cancer cells from a cancer patient, inducing attenuation or death of the cancer cells through radiation or treatment with anti-cancer immunotherapy, and then administering the re-administered cancer cells or cancer cells to the cancer patient. Tumor tissue can enhance the sensitivity of the immune system to tumors, thus maximizing the anticancer effect of anticancer therapy through the abscopal effect and minimizing the risk of damage, necrosis, or mutation of normal tissue. In addition, the present invention reduces the minimal residual disease of cancer and tumor, increases the remission of the cancer or tumor, increases the remission period, lowers the cancer or tumor recurrence rate, prevents metastasis, or , it has the effect of lowering the transfer rate or achieving a combination of two or more thereof. Furthermore, when the composition according to the present invention is used in combination with other anticancer therapies, a more powerful tumor growth inhibition effect and abscopal effect can be achieved, and in particular, anticancer immune function is enhanced by increasing the levels of immune cells and interferons with anticancer activity. If this can be done, a synergistic anti-cancer effect can be obtained.

Figure 1 is a block diagram showing the order of application of the present invention and the expected positive effects.
Figures 2a and 2b show the results of observing the sensitization effect by radiation through MTT analysis after irradiating a high dose of radiation to cancer cells (Figure 2a, results of microscopic observation; Figure 2b, comparison results of cell viability) (C, control group; RT, Radiation treatment group).
Figure 3 shows the results of comparing the degree of growth of primary tumors after IVAM and radiation were used alone or in combination in a tumor animal model to confirm the anticancer effect of the combination of IVAM treatment and radiation therapy according to the present invention.
Figure 4 shows a secondary tumor (tumor on the left flank that was not irradiated with radiation) after IVAM and Radiation were used alone or in combination in a tumor animal model to confirm the abscopal effect of the combination of IVAM treatment and radiation therapy according to the present invention. This is the result of comparing the level of growth.
Figures 5a and 5b show the primary tumor (right hind limb) and secondary tumor through IVIS imaging after IVAM and radiation were used alone or in combination in a tumor animal model to confirm the anticancer effect of the combination of IVAM treatment and radiation therapy according to the present invention. This is the result of observing the tumor (left flank) (Figure 5a) and quantifying the degree of tumor growth (Figure 5b).
Figure 6 shows the results of measuring body weight changes over time in tumor animal models treated with IVAM and radiation alone or in combination to confirm the toxicity of IVAM treatment and radiation therapy according to the present invention.
Figure 7 is a schematic diagram of an animal experiment to confirm the combined effect of IVAM treatment, anticancer immunotherapy, and radiation therapy according to the present invention.
Figure 8 shows primary tumor (radiation) over time after administering IVAM therapy, immunotherapy (a-PD-L1), and radiation therapy (RT) according to the present invention to a tumor animal model, respectively, alone, in dual combination, or in triple combination. This is the result of observing the growth of the tumor on the irradiated right hind leg.
Figure 9 shows the secondary tumor (tumor on the left flank that was not irradiated) according to time after administering IVAM therapy, immunotherapy, and radiation therapy according to the present invention to a tumor animal model, respectively, in single, double, or triple combination. This is the result of observing the level of growth.
Figures 10a to 10c show the results of observing the degree of tumor growth through IVIS imaging after administering IVAM therapy, immunotherapy, and radiation therapy according to the present invention to a tumor animal model, respectively, alone, in dual combination, or in triple combination.
Figure 11 shows the results of observing the change in body weight of mice over time while administering IVAM therapy, anticancer immunotherapy, and radiation therapy according to the present invention to a tumor animal model, respectively, alone, in dual combination, or in triple combination.
Figures 12a and 12b show the degree of metastasis to the lungs by measuring the number of metastatic nodules in the lung tissue after administering IVAM therapy, immunotherapy, and radiation therapy according to the present invention to a tumor animal model, respectively, alone, in dual combination, or in triple combination. This is the result of comparison.
Figures 13a to 13c show the results of observing changes in the distribution of immune cells (CD8 ⁺ T cells, T _reg , and CD8 ⁺ effector memory T cells) within the tumor microenvironment of the primary tumor obtained from the tumor animal model. Figures 13a and 13b represent one view as a whole.
Figures 14a and 14b show the results of observing changes in various immune cell populations in the spleen obtained from the tumor animal model. Figures 14a and 14b represent one view as a whole.
Figure 15 shows the results of observing changes in interferon (IFN-β and IFN-γ) levels in serum obtained from the tumor animal model.
Figure 16 is a schematic diagram of an animal experiment to confirm the long-term survival rate and toxicity by the combined treatment of IVAM treatment, anticancer immunotherapy, and radiation therapy according to the present invention.
Figures 17a and 17b show the results of observing the long-term mouse survival rate (Figure 17a) and the change in body weight of mice over time (Figure 17b) due to the combined treatment of IVAM treatment, immunotherapy, and radiation therapy according to the present invention.
Figure 18 is a schematic diagram of an animal experiment to confirm the anticancer effect and toxicity of IVAM multiple administration according to the present invention.
Figure 19 shows the results of observing the degree of tumor growth caused by multiple administrations of IVAM according to the present invention.
Figure 20 shows the results of observing the degree of tumor growth due to multiple administration of IVAM according to the present invention through IVIS imaging.
Figure 21 shows the results of observing the change in body weight of mice over time upon multiple administration of IVAM according to the present invention.

The present invention relates to a composition that can strengthen the body's immune function against tumors through the abscopal effect and enhance the anti-cancer effect of anti-cancer therapy, by applying special treatment to tumor tissue or cancer cells isolated from cancer patients. When activity inhibition or death is induced and then re-administered to the cancer patient, the body's immune function against cancer is activated, and the anti-cancer effect of anti-cancer therapy is significantly increased, preventing cancer growth, recurrence, and metastasis. This was completed by confirming that this can be suppressed. In particular, it was confirmed that cancer cells whose activity was inhibited or whose death was induced as described above can exert abscopal effects when administered to cancer patients. Furthermore, when the composition according to the present invention is used in combination with other anticancer therapies, a more powerful tumor growth inhibition effect and abscopal effect are achieved, and in particular, the anticancer immune function is enhanced by increasing the levels of immune cells and interferons with anticancer activity. Confirmed. Therefore, a synergistic anticancer effect can be obtained through combined use of the composition of the present invention and other anticancer therapies.

Therefore, the main object of the present invention is a tumor tissue or cancer cell isolated from a cancer patient that has been subjected to one or more treatments selected from the group consisting of (a) and (b) below, comprising as an active ingredient, To provide a pharmaceutical composition for preventing or treating cancer:

(a) Irradiation; and

(b) Immunoanticancer drugs.

In the present specification, tumor tissue or cancer cells subjected to one or more of the above treatments; Alternatively, a pharmaceutical composition containing the same may be referred to as IVAM ( In vitro Abscopal Method, IVAM).

In addition, the composition not only achieves a cancer prevention or treatment effect by strengthening the body's immune function against cancer, but also can further enhance the anticancer effect of known anticancer therapy. Therefore, the composition of the present invention can be used not only for preventing or treating cancer, but also for suppressing the appearance or progression of metastatic cancer or recurrent cancer. For example, the composition according to the present invention can be used as a vaccine composition for preventing cancer, or can be used to manufacture a vaccine for preventing cancer.

In the past, when radiation was irradiated to a patient for the purpose of the abscopal effect, there was a problem that damage and necrosis of normal tissue due to radiation exposure could not be avoided. In particular, there is a risk that radiation exposure to some cancer tissues may cause cancer mutations, resulting in the development of other types of cancer tissues that are resistant to radiation or are transformed. However, the composition of the present invention extracts cancer tissue from a cancer patient and applies physical, chemical, and/or medical treatment, such as radiation, to the extracted cancer tissue from outside the body to attenuate or kill the cancer cells or tumor tissue. , it has the advantage of preventing problems such as damage to normal tissue, necrosis, and mutation. When tumor tissue or cancer cells that have been attenuated or killed through radiation irradiation are administered again to a cancer patient as described above, the body's immune system recognizes, attacks, and/or phagocytizes the cancer cells that have been killed or inactivated by heat treatment. Through the experience of attacking dead cancer cells, sensitivity to other cancer cells in the body increases, allowing them to be more sensitively recognized, attacked, and killed. In other words, the abscopal effect can be expected through irradiated tumor tissue or cancer cells according to the present invention. In particular, the present inventors found that when cancer cells irradiated in vitro as described above are re-administered to a tumor animal model and then combined with radiation therapy, not only does the anti-cancer effect of radiation therapy increase further, but metastasized cancer is also effectively suppressed. Confirmed.

In addition, the present invention is characterized in that cancer tissue extracted from a cancer patient is treated with an immuno-anticancer agent to induce death or attenuation of the cancer cells or tumor tissue in vitro, and then re-administered to the cancer patient. The cancer patient's immune system easily recognizes, attacks, and/or phagocytizes the re-administered tumor tissue or cancer cells, and through the experience of attacking dead cancer cells, sensitivity to other cancer cells in the body increases, making them more sensitive. It can recognize, attack and kill. In other words, the effect of abscopal can be expected through tumor tissue or cancer cells treated with an anti-cancer immunotherapy according to the present invention.

When tumor tissue or cancer cells that have been subjected to special treatment as described above are re-administered to the cancer patient from which they originated, they stimulate the immune system and cause the same effect as an immunostimulant. In other words, even if tumor tissue or cancer cells that were not previously recognized by the immune system are reintroduced into the body in an attenuated or killed state through special treatment, the immune system attacks them and kills previously unrecognized cancer cells and metastasis cells. A learning effect can be achieved. Therefore, the immune system of a subject with the ability to recognize cancer cells through learning can attack malignant cells throughout the body.

That is, since administration of the vaccinated tissue cells inducing abscopal of the present invention triggers an immune response and activates the immune system, administration of the vaccinated tissue cells of the present invention can be used to treat primary cancer and its metastatic cancer. It is useful in preventing recurrence of cancer disease in patients with a weak or suppressed immune system.

The order of applying the composition according to the present invention to a subject and the abscopal effect and anticancer effect thereof are schematically shown in Figure 1.

The term “cancer” used herein is characterized by uncontrolled cell growth. Due to this abnormal cell growth, a cell mass called a tumor is formed, which infiltrates surrounding tissues and, in severe cases, metastasizes to other organs of the body. It says that something can happen. Academically, it is also called a neoplasm. Cancer is an incurable chronic disease that cannot be fundamentally cured even when treated with surgery, radiation, and chemotherapy in many cases, causes pain to the patient, and ultimately leads to death. There are many causes of cancer, but internal factors are the cause. It is divided into and external factors. Although the exact mechanism by which normal cells are transformed into cancer cells has not been identified, it is known that a significant number of cancers are caused by external factors such as environmental factors. Internal factors include genetic factors and immunological factors, and external factors include chemicals, radiation, and viruses. Genes involved in the development of cancer include oncogenes and tumor suppressor genes, and cancer occurs when the balance between them is disrupted by the internal or external factors described above. In the present invention, cancer includes primary cancer, cancer to which radiation therapy has been applied, cancer to which no radiation therapy has been applied, metastatic cancer, and recurrent cancer. Additionally, the compositions according to the invention may be particularly suitable for the treatment of non-immunogenic tumors or cancer.

Additionally, in the present invention, cancer may include all types of solid cancer and all types of blood cancer. As a non-limiting example, the cancer of the present invention includes adenocarcinoma, choroidal melanoma, acute leukemia, acoustic neurinoma, ampullary carcinoma, and anal carcinoma. ), astrocytoma, basal cell carcinoma, pancreatic cancer, desmoid tumor, bladder cancer, bronchial carcinoma, non-small cell lung cancer (NSCLC) : non-small cell lung cancer, breast cancer, Burkitt's lymphoma, corpus cancer, carcinoma of unknown primary (CUP)-syndrome, colorectal cancer , small intestine cancer, small intestinal tumors, ovarian cancer, endometrial carcinoma, ependymoma, epithelial cancer types, Ewing tumor (Ewing's tumors), gastrointestinal tumors, gastric cancer, gallbladder cancer, gall bladder carcinomas, uterine cancer, cervical cancer, cervix ), glioblastomas, gynecologic tumors, ear, nose and throat tumors, hematologic neoplasias, hairy cell leukemia, urethral cancer ( urethral cancer, skin cancer, skin testis cancer, brain tumors (gliomas), brain metastases, testicle cancer, hypophysis tumor , carcinoids, Kaposi's sarcoma, laryngeal cancer, germ cell tumor, bone cancer, colorectal carcinoma, head and neck tumors: tumors of the nasopharynx area), colon carcinoma, craniopharyngiomas, oral cancer (cancer of the oral cavity area and lips), cancer of the central nervous system, liver cancer, liver metastases ), leukemia, eyelid tumor, lung cancer, lymph node cancer (Hodgkin's/NonHodgkin's), lymphomas, stomach cancer , malignant melanoma, malignant neoplasia, malignant tumors gastrointestinal tract, breast carcinoma, rectal cancer, medulloblastomas, melanoma ( melanoma, meningiomas, Hodgkin's disease, mycosis fungoides, nasal cancer, neurinoma, neuroblastoma, kidney cancer, kidney Renal cell carcinomas, non-Hodgkin's lymphomas, oligodendroglioma, esophageal carcinoma, osteolytic carcinomas and osteoplastic carcinomas, osteosarcoma (osteosarcomas), ovarian carcinoma, pancreatic carcinoma, penile cancer, plasmacytoma, squamous cell carcinoma of the head and neck (SCCHN), prostate Prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma, vaginal cancer, thyroid carcinoma, Schneeberger disease, esophageal cancer cancer), squamous cell carcinoma (spinalioms), T-cell lymphoma (mycosis fungoides), thymoma, tube carcinoma, eye tumors, urethral cancer , urologic tumors, urothelial carcinoma, vulva cancer, wart appearance, soft tissue tumors, soft tissue sarcoma, Wilms tumor (Wilm's tumor), cervical carcinoma, and tongue cancer. For example, it may be selected from the group consisting of astrocytomas, glioblastoma, and renal cell carcinoma.

The present invention can also be seen as inducing vaccination by applying radiation and/or immunological treatment to isolated tumor tissue or cancer cells of a cancer patient. Therefore, in one embodiment of the present invention, a vaccine composition for preventing cancer containing tumor tissue or cancer cells to which the treatment has been applied can be prepared, and the vaccine composition is prepared by the cancer patient's immune system judging it to be the patient's own body tissue. The function can be strengthened so that cancer cells that were not recognized as removal targets can be recognized as removal targets (i.e., increased sensitivity to cancer). In addition, the vaccine composition enables the immune system to recognize the metastatic cancer when metastatic cancer occurs and to create a memory of the cancer so that it can recognize new cancer cells that recur even after successful cancer treatment of the patient. The composition of the present invention exerts the above effects comprehensively, resulting in personalized vaccination against tumors and cancer in the subject's body. In other words, the composition of the present invention acts like an immunostimulant, allowing the subject's immune system to recognize and eliminate cancer cells, eradicate metastatic cancer, and recognize and eliminate new cancer cells that relapse after successful cancer treatment. Strengthen.

In one embodiment of the present invention, the tumor tissue or cancer cells may be tumor tissues or cancer cells isolated from an individual in need of cancer prevention or treatment. That is, the tumor tissue or cancer cells may be autologous tumor tissues or cancer cells of an individual in need of cancer prevention or treatment. In another embodiment of the present invention, the tumor tissue or cancer cells may be tumor tissues or cancer cells isolated from an individual different from the individual in need of cancer prevention or treatment. That is, the tumor tissue or cancer cells may be allogeneic tumor tissues or cancer cells from an individual in need of cancer prevention or treatment. Therefore, the pharmaceutical composition according to the present invention may be administered to a cancer patient from which tumor tissue or cancer cells contained as an active ingredient have been isolated (i.e., killed or attenuated autologous tumor tissue or cancer cells are re-administered), or The tumor tissue or cancer cells may be administered to an entity different from the cancer patient from which they were isolated (i.e., an allogeneic entity) (i.e., killed or attenuated allogeneic tumor tissue or cancer cells are administered).

In particular, the composition according to the present invention can be administered to individuals who do not develop cancer (i.e., normal individuals) for the purpose of preventing cancer. For example, the composition according to the present invention contains as an active ingredient tumor tissue or cancer cells isolated from a cancer patient and attenuated or killed by the treatment according to the present invention, and is administered to an allogeneic entity of the cancer patient. It may be characterized by, and in this case, the entity may be an entity in which cancer has not occurred. Therefore, even in individuals who are likely to develop cancer due to genetic or environmental reasons, the immune system experiences and learns about cancer by receiving allogeneic tumor tissue or cancer tissue that has been attenuated or killed by the treatment according to the present invention. It can strengthen the immune function against cancer and thus prevent or delay the occurrence of cancer.

The vaccinated tumor tissue or cancer cells of the present invention can act throughout the body through the abscopal effect or the like. In other words, the present invention enables systemic treatment only by collecting local cancer tissue. Since the composition according to the present invention can act on the entire body, it can discover tumor antigens that were not discovered even by existing anticancer drugs or radiation therapy, and treat cancer without artificial non-invasive or invasive measures that place a burden on the body. And even in weak patients, this immune-inducing function can be induced without any additional physical burden.

As used herein, the term “Abscopal effect” refers to a systemic anticancer effect that inhibits not only the local area directly irradiated with radiation, but also metastatic tumors in areas not irradiated with radiation. Accordingly, the abscopal effect causes shrinkage of tumors distributed elsewhere in the body beyond the scope of local tumor treatment. However, the abscopal effect cannot be said to be induced simply by radiation irradiation, and can also be induced by treatment with immunization agents.

In the present invention, substances that induce the abscopal effect, that is, substances that induce cancer cell-specific antibodies, such as tumor antigens, are considered as part of a vaccine in a broad sense. Therefore, the Abscopal effect-inducing substance can be expressed as ‘Abscopal effect-inducing vaccine.’

In particular, the present invention plays an important role as an immunostimulant useful in the treatment of primary cancer and its metastatic cancer in scattered cell states that cannot be detected by imaging methods, and prevents recurrence of cancer disease. It is useful for This immunostimulation method causes a systemic vaccine effect that activates the immune system, which is particularly useful in the treatment of metastatic and primary cancers that cannot be detected with prior art methods and prevents recurrence of successfully treated cancer. It is especially useful for

By the present invention, treatment of the following primary cancer forms can be achieved and the following cancer forms of metastatic cancer can be treated and also the recurrence of the following cancer forms can be prevented:

Adenocarcinoma, choroidal melanoma, acute leukemia, acoustic neuroma, ampullary carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma, pancreatic cancer, papillary tendonoma, bladder cancer, endobronchial carcinoma, non-small cell lung cancer (NSCLC), breast cancer, Burkitt's lymphoma, corpus Cancer, cancer of unknown primary site - syndrome, colon cancer, small intestine cancer, small intestine tumor, ovarian cancer, endometrial carcinoma, ependymoma, epithelial carcinoma, Ewing tumor, gastrointestinal stromal tumor, stomach cancer, cholangiocarcinoma, cholangiocarcinoma, uterine cancer, uterus Cervical cancer, uterine cervix, glioblastoma, gynecological tumor, otolaryngologic tumor, blood neoplasia, cytocytic leukemia, urethral cancer, skin cancer, skin testicular cancer, brain tumor (glioma), brain metastasis cancer, testicular cancer, pituitary tumor, carcinoid tumor. , Kaposi's sarcoma, laryngeal cancer, germ cell tumor, bone cancer, colorectal carcinoma, head and neck tumor (tumor of the ENT area), colon carcinoma, craniopharyngioma, oral cancer (cancer of the oral cavity area and lips), cancer of the central nervous system, liver cancer, liver metastasis. Ear cancer, leukemia, eyelid tumor, lung cancer, lymph node cancer (Hodgkin's/non-Hodgkin's), lymphoma, stomach cancer, malignant melanoma, malignant neoplasm, gastrointestinal malignancy, breast cancer, rectal cancer, medulloblastoma, melanoma, meningioma. , Hodgkin's disease, mycosis fungoides, nasal cancer, schwannoma, neuroblastoma, kidney cancer, renal cell carcinoma, non-Hodgkin's lymphoma, oligodendroglioma, esophageal carcinoma, osteolytic and osteoplastic carcinoma, osteosarcoma, ovarian carcinoma, pancreatic cancer. tumor, penile cancer, plasmacytoma, squamous cell carcinoma of the head and neck (SCCHN), prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma, vaginal cancer, thyroid carcinoma, Siniberg disease, esophageal cancer, squamous cell carcinoma, T-cell lymphoma (bacteria) common sarcoma), thymoma, fallopian tube carcinoma, ophthalmic tumor, urethral cancer, urological tumor, urothelial carcinoma, vulvar cancer, warty appearance, soft tissue tumor, soft tissue sarcoma, Wilms tumor, cervical carcinoma, and tongue cancer. For example, in the treatment of astrocytomas, glioblastoma, pancreatic cancer, bronchial cancer, breast cancer, colon cancer, ovarian cancer, stomach cancer, laryngeal cancer, malignant melanoma, esophageal cancer, cervical cancer, liver cancer, bladder cancer, and renal cell cancer. This may be particularly appropriate.

In other words, the composition according to the invention allows the patient's immune system to recognize that cancer cells of the above-mentioned cancer types are different from the subject's normal cells and kill them.

Furthermore, the composition according to the present invention is only performed by in vitro radiation irradiation or anticancer agent treatment on tumor tissue or cancer cells isolated from a cancer patient, and the above treatments are not directly applied to the cancer patient, so the above treatments There is no risk of serious side effects occurring. For example, tissues or cancer cells extracted from a patient are either completely killed or injected into the patient's body in an attenuated state that cannot grow or proliferate, so they cannot exist as another mutant cancer cell. Therefore, the application of the present invention is suitable for combination with conventional cancer treatment methods such as standard treatment and immunological treatment therapy involving one or more of the standard treatments developed so far (surgery, irradiation, cancer treatment). do.

In the present invention, the treatment may be radiation. In the present invention, the radiation may be selected from α-rays, β-rays, γ-rays,

When radiation is irradiated to tumor tissue or cancer cells extracted from a cancer patient, the following mechanism can be expected: Normal cancer cells hide factors that can be recognized by the immune system as much as possible, but when irradiated, various MHC antigens are expressed at high levels. It is revealed. Cancer cells killed by radiation produce large amounts of factors such as HMGB-1 or ATP, and these factors activate the innate immune system. For example, cancer cells irradiated with radiation have ICAM-1 expression on their surface, so immune cells can more easily recognize and attack them. In this way, cancer cells that die or die due to radiation not only present a clear marker (Fas receptor) so that immune cells can better recognize them (Calreticulin) and kill them, but also provide a combat signal to arm immune cells, that is, an activation signal (NKG2D). Send more. Additionally, cancer cells irradiated with radiation produce more Chemokine, which recruits immune cells. Therefore, when the composition containing irradiated tumor tissue or cancer cells according to the present invention is administered to a cancer patient, immune cells can gather around the administered tumor tissue or cancer cells, and thus the cancer cells are recognized by the immune cells. The possibility increases further. Therefore, the vaccine effect induced by the present invention contributes to the activation of the immune system in the subject's body. Furthermore, as described above, since the present invention irradiates radiation outside the body, unlike the prior art, it is possible to block the possibility of mutation of cancerous tissue in the subject's body ( in-vivo ) caused by radiation.

In the present invention, the radiation dose is 1 to 500 Gy, 1 to 400 Gy, 1 to 300 Gy, 1 to 200 Gy, 1 to 100 Gy, 1 to 80 Gy, 1 to 50 Gy, and 1 to 30 Gy. , 1 to 10 Gy, 5 to 10 Gy, 10 to 200 Gy, 10 to 100 Gy, 10 to 80 Gy, 20 to 60 Gy, 30 to 60 Gy, or 40 to 60 Gy, but is not limited thereto.

The radiation may be administered singly or in multiple doses. For example, rather than irradiating a single dose of radiation, multiple dose equivalents can be irradiated.

In the present invention, the treatment may be an anti-cancer immunotherapy agent.

As used herein, the term “anticancer agent” is used to collectively refer to substances used for the treatment of malignant tumors. Most anticancer drugs are drugs that mainly inhibit nucleic acid synthesis or exhibit anticancer activity by interfering with various metabolic pathways of cancer cells. Anticancer drugs currently used in cancer treatment include alkylating agents, antimetabolites, antibiotics, and mitotic inhibitors, depending on their biochemical mechanisms of action. , hormonal agents, and other six categories, but the anticancer agent according to the present invention may not be included in the above categories.

“Cancer immunotherapy” using immuno-anticancer drugs is a cancer treatment that activates the body's immune system to fight cancer cells. Immuno-anticancer drugs improve the specificity, memory, and adaptiveness of the immune system. It exhibits anti-cancer effects by enhancing . In other words, it uses the body's immune system to accurately attack only cancer cells, causing fewer side effects and utilizing the immune system's memory and adaptability, so patients who are responsive to immunotherapy can see sustained anticancer effects. Preferably, the immuno-anticancer agent may be one or more selected from the group consisting of immune checkpoint inhibitors, co-stimulatory molecule agents, cytokine therapeutic agents, CAR-T cell therapeutic agents, and autologous CD8 ⁺ T immune cell therapeutic agents, but is not limited thereto. No. Immune checkpoint inhibitors refer to agents that inhibit immune checkpoints involved in the immune evasion mechanism of cancer cells. Some cancer cells evade immunity by utilizing the immune checkpoints of immune cells. Immune checkpoint inhibitors block immune evasion signals by binding to the binding site of cancer cells and T cells, preventing the formation of an immunological synapse, thereby preventing immune evasion. Unhindered T cells have a mechanism to destroy cancer cells. The immune checkpoint inhibitors include PD-L1 inhibitor, PD-1 inhibitor, CTLA-4 inhibitor, LAG3 inhibitor, TIM3 inhibitor, 4-1BB inhibitor, LAG-3 inhibitor, B7-H4 inhibitor, HVEM inhibitor, TIM4 inhibitor, GAL9 inhibitor, It may be one or more selected from the group consisting of VISTA inhibitors, KIR inhibitors, TIGIT inhibitors, and BTLA inhibitors, but is not limited thereto. The inhibitor is not limited to a specific type, and includes without limitation as long as it can inhibit the function or expression of the target (immune checkpoint protein), but specific examples include antibodies or fragments thereof (Fab, Fab', F(ab') 2, scFv, (scFv) ₂ , Fv, dsFv, diabody, nanobody, Fd, and Fd', etc.), compounds, and other peptides. Examples of commercialized immune checkpoint inhibitors include pembrolizumab (Keytruda), ipilimumab (Yervoy), nivolumab (Opdivo), atezolizumab (Tecentriq), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), and tremelimumab. (Imjuno), Relatlimab, Nivolumab, etc., but are not limited to these.

Immunotherapy may include immune-cell therapy in addition to anticancer immunotherapy. Genetic modification of immune cells is well known as an immune-cell therapy for cancer. These immune-cell therapies are based on manipulating autologous or allogeneic immune cells and administering them to subjects in need. Immune-cell based therapies include natural killer cell therapy, dendritic cell therapy and naive T-cells, effector T-cells also known as T-helper cells, cytotoxic T-cells and regulatory T-cells (Tregs). ), including T-cell immunotherapy.

Genetically modified immune cells may be T-cells. In another embodiment, the T-cell is a naive T-cell. In another embodiment, the T-cell is a naive CD4+ T-cell. In another embodiment, the T-cell is a naive T-cell. In another embodiment, the T-cell is a naive CD8+ T-cell. In another embodiment, the genetically modified immune cells are natural killer (NK) cells. In another embodiment, the genetically modified immune cell is a dendritic cell. In another embodiment, the genetically modified T-cells are cytotoxic T lymphocytes (CTL cells). In another embodiment, the genetically modified T-cells are regulatory T-cells (Treg). In another embodiment, the genetically modified T-cell is a chimeric antigen receptor (CAR) T-cell. In another embodiment, the genetically modified T-cell is a genetically modified T-cell receptor (TCR) cell.

Immunotherapy involves cytokines. Cytokines are granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukins such as IL-2, and/or interferons such as IFN-alpha. Additional approaches to boost tumor-targeted immune responses include additional immune checkpoint inhibition. Immune checkpoint inhibition includes anti-CTLA4, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-TIM-3, anti-LAG-3, anti-A2aR or anti-KIR antibodies. Immunotherapy includes co-stimulatory receptor agonists such as anti-OX40 antibodies, anti-GITR antibodies, anti-CD137 antibodies, anti-CD40 antibodies and anti-CD27 antibodies. Immunotherapy involves inhibition of T regulatory cells (T-regulatory), myeloid derived suppressor cells (MDSC) and cancer associated fibroblasts (CAFs). Immunotherapy involves stimulation of innate immune cells such as natural killer (NK) cells, macrophages, and dendritic cells. Additional immunostimulatory treatments include IDO inhibitors, TGF-beta inhibitors, IL-10 inhibitors, stimulator of interferon gene (STING) agonists, toll-like receptor (TLR) agonists (e.g., TLR7, TLR8, or TLR9), tumor vaccines (e.g., whole tumor cellular vaccines, peptide and recombinant tumor-associated antigen vaccines), and adoptive cell therapy (ACT) (e.g., T cells, natural killer cells, TIL and LAK cells), and genetically engineered receptors (e.g., chimeric antigen receptor (CAR) and Includes ACT with T cell receptor (TCR).Combination of these agents can be used, such as combination of immune checkpoint inhibitors, checkpoint inhibition+action of T-cell co-stimulatory receptor, and checkpoint inhibition+TIL ACT. Additional anti-cancer treatments include combinations of immune checkpoint inhibitors (e.g., avelumab), 4-1BB (CD-137) agonists (e.g., utomirumab), and OX40 (TNFRS4) agonists.

The composition according to the present invention may contain only one type of tumor cell or cancer cell to which a specific treatment has been applied as an active ingredient, but may also contain two or more tumor tissues or cancer cells. At this time, the two or more tumor tissues or cancer cells may have been subjected to the same type of treatment, but may have been subjected to different types of treatment.

When the composition according to the present invention contains two or more tumor tissues or cancer cells, the composition may be in the form of a mixture in which the two or more tumor tissues or cancer cells are mixed. That is, at this time, the two or more tumor tissues or cancer cells can be administered simultaneously.

The composition may be formulated with two or more tumor tissues or cancer cells and administered simultaneously or sequentially. In this case, the composition may be a pharmaceutical composition for combined administration for simultaneous or sequential administration of the tumor tissue or cancer cells. At this time, in the case of sequential administration, the order of administration is not limited, and the administration regimen can be appropriately adjusted depending on the patient's condition, etc.

Additionally, the composition according to the present invention can be used in combination with other anticancer therapies. The composition according to the present invention can increase the sensitivity of an individual to anticancer therapy through the abscopal effect and maximize the anticancer effect of the anticancer agent by regulating the activity and level of immune cells and immunomodulators involved in the immune response. Therefore, the composition of the present invention can enhance the anticancer effect and reduce the side effects of combined anticancer therapy. In addition, it was confirmed that when the composition according to the present invention is used in combination with radiation therapy and anticancer immunotherapy, tumor growth is suppressed more effectively, and the abscopal effect caused by radiation therapy is further increased and cancer metastasis is reduced. In addition, it was confirmed that the excellent anti-cancer effect of the combination therapy was the result of an increase in anti-cancer active immune cells within the tumor and spleen and an increase in the level of anti-cancer interferon in the serum. In addition, it was confirmed that the composition according to the present invention maintains survival rate without causing side effects such as toxicity even for a long period of 50 days when used in combination with anticancer therapy.

According to one embodiment of the present invention, the composition according to the present invention does not exhibit toxicity and has a survival rate when administered once a day at intervals of 3 to 4 days for a total of 1 to 3 times, 1 to 2 times, or 2 to 3 times. It was confirmed that it was maintained. Accordingly, the composition according to the present invention can be administered 1 to 3 times, but is not limited thereto.

As used herein, the term “combination” can be achieved by administering (treating) the individual components of a treatment regimen simultaneously, sequentially, or separately. The combination treatment effect is obtained by performing two or more anticancer treatments simultaneously, sequentially, or alternately at regular or undetermined intervals. The combination treatment is not limited to this, but includes, for example, degree of response, Efficacy measured through response rate, time to disease progression, or survival period is therapeutically superior to and can provide a synergistic effect than the efficacy that can be obtained by administering one or the remaining components of the combination therapy at a typical dose. can be defined.

The composition according to the present invention may be used in combination with one or more selected from the group consisting of radiation therapy, chemotherapy, targeted anticancer drug therapy, and immunotherapy, but is not limited thereto.

In the present invention, “targeted anticancer agent” refers to an agent that exhibits an anticancer effect by targeting proteins or genes that are specifically changed in cancer cells or cancer tissues and interfering with the molecular activities involved in the growth and development of cancer. The targeted anticancer drugs include tyrosine kinase inhibitors (TKI), poly-ADP ribose polymerase inhibitors (PARP inhibitors), angiogenesis inhibitors, CDK4/6 inhibitors (cyclin-dependent kinases 4/6 inhibitor), and hormones. It may be one or more selected from the group consisting of hormone therapy drugs and antibody-drug conjugates, but is not limited thereto.

The term “chemical anticancer agent” used in this specification refers to a first generation anticancer agent, also called “cytotoxic anticancer agent” or “chemical anticancer agent.”

The composition according to the present invention may be used in combination with radiation therapy and immunotherapy. Detailed descriptions of immuno-anti-cancer drugs and immuno-anti-cancer therapy have been described above and will therefore be omitted.

The composition according to the present invention may be administered simultaneously, separately, or sequentially with the anticancer therapy. Additionally, each anticancer therapy can be performed repeatedly.

For example, the composition according to the present invention is used for 1 to 3 days, 1 to 5 days, 1 to 7 days, 1 to 10 days, 1 to 20 days, 1 to 30 days, and 5 days of anticancer therapy. It may be administered 15 days to 5 days, 5 to 20 days in advance, 10 minutes to 2 hours, 10 minutes to 1 hour, 10 minutes to 50 minutes, or 10 minutes to 30 minutes in advance, and may be administered simultaneously with the above-mentioned anticancer therapy. , 5 minutes to 10 minutes, 30 minutes to 1 hour, 6 hours to 12 hours, 12 hours to 24 hours, 1 day to 3 days, 1 day to 5 days, 1 day to 7 days, 1 day after performing the anticancer therapy. It may be administered after 1 to 10 days, 1 to 20 days, 1 to 30 days, 5 to 15 days, or 5 to 20 days. Additionally, the composition according to the present invention may be administered before or after the anticancer therapy.

There is no limitation to the anti-cancer therapy and may include all anti-cancer therapies known in the art. Non-limiting examples include surgical therapy, chemotherapy (e.g., administration of protein kinase inhibitors or EGFR-targeted therapeutics), embolization therapy, chemoembolization therapy, radiation therapy, cryotherapy, hyperthermia, phototherapy, and radioablation. therapy, hormonal therapy, immunotherapy, small molecule therapy, receptor kinase inhibitor therapy, anti-angiogenic therapy, cytokine therapy, or biological therapy, such as monoclonal antibodies, siRNA, miRNA, antisense oligonucleotides, ribozyme, or gene therapy. there is.

Treatment with anticancer drugs includes actinomycin D, aminoglutethimide, amsacrin, anastrozol, antagonists of purine and pyrimidine bases, anthracycline, Aromatase inhibitors, asparaginase, antiestrogens, bexaroten, bleomycin, buselerin, busulfan, camptothecin Derivatives (camptothecin derivates), capecitabine, carboplatin, carmustine, chlorambucil, cladribin, cyclophosphamide, cytarabine (cytarabin), cytosinarabinoside, alkylating cytostatics, dacarbacin, dactinomycin, daunorubicin, docetaxel, doxorubicin: Adriamycin), doxorubicin lipo (Lipodox), epirubicin, estramustine, etoposide, exemestan, fludarabine ), fluorouracil, folic acid antagonists, formestan, gemcitabine, glucocorticoids, goselerin, hormone antagonists, hycamtin ( hycamtin), hydroxy urea, idarubicin, ifosfamid, imatinib, irinotecan, letrozol, leuprorelin, Lomustin, melphalan, mercaptopurine, methotrexate, miltefosin, mitomycine, mitosis inhibitors, mitoxantron ), nimustine, oxaliplatin, paclitaxel, pentostatin, procarbacin, tamoxifen, temozolomid, teniposid, Testolactone, thiotepa, thioguanine, topoisomerase inhibitors, topotecan, treosulfan, tretinoin, triptorelin , trofosfamide, vinblastine, vincristine, vindesine, vinorelbine, or antibiotics with cytotoxic activity.

Treatment with immunological agents generally relies on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker present on the surface of tumor cells. Antibodies can function alone as effectors of therapy or can recruit other cells to actually affect cell death. Antibodies can also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) to function solely as targeting agents. Alternatively, the effector may be a lymphocyte that delivers surface molecules that directly or indirectly interact with the cellular target. Various effector cells include cytotoxic T cells and NK cells, as well as genetically engineered variants of these cell types that have been modified to express chimeric antigen receptors.

GM-CSF to increase the number of bone marrow-derived innate immune system cells, low dose cyclophosphamide or a PI3K inhibitor to eliminate T regulatory cells that suppress innate and adaptive immunity, and 5FU, PI3K, to eliminate bone marrow-derived suppressor cells. Other complementary immunotherapies, including inhibitors or histone deacetylase inhibitors, can be added to the above regimens to further enhance their efficacy.

Immunotherapy may also include administration of interleukins, such as IL-2, or interferons, such as INFα.

Examples of immunotherapies that can be combined with p53, ADP, and/or MDA-7 gene therapy and CD122/CD132 agonists include adjuvants (e.g., Mycobacterium tuberculosis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds), cytokines therapy, gene therapy and monoclonal antibodies. Therefore, it is contemplated that one or more anti-cancer therapies may be used in conjunction with the p53, ADP, and/or MDA-7 gene therapy described herein.

Other immunotherapies that may be combined with p53, ADP and/or MDA-7 gene therapy and CD122/CD132 agonists include immune checkpoint inhibitors, costimulatory receptor agonists, innate immune cell stimulators, or innate immune activators. In certain aspects, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, BTLA, B7H3, B7H4, TIM3, KIR, or A2aR. In certain aspects, the at least one immune checkpoint inhibitor is an anti-killer-cell immunoglobulin-like receptor (KIR) antibody.

In some aspects, the at least one immune checkpoint inhibitor is a human programmed cell death 1 (PD-1) axis binding antagonist. In certain aspects, the PD-1 axis binding antagonist is selected from the group consisting of PD-1 binding antagonists, PDL1 binding antagonists, and PDL2 binding antagonists. In some aspects, the PD-1 axis binding antagonist is a PD-1 binding antagonist. In certain aspects, a PD-1 binding antagonist inhibits the binding of PD-1 to PDL1 and/or PDL2. In particular, the PD-1 binding antagonist is a monoclonal antibody or antigen-binding fragment thereof.

The co-stimulatory receptor agonist may be an anti-OX40 antibody, an anti-GITR antibody, an anti-CD137 antibody, an anti-CD40 or an anti-CD27 antibody. Stimulators of innate immune cells include, but are not limited to, KIR monoclonal antibodies, inhibitors of cytotoxic-inhibiting receptors, and toll-like receptors. Activators of innate immune cells such as natural killer (NK) cells, macrophages, and dendritic cells include IDO inhibitors, TGF inhibitors, and IL-10 inhibitors. An exemplary activator of innate immunity is indoximod.

Immunotherapy may include inhibition of T regulatory cells, myeloid derived suppressor cells (MDSCs) and cancer associated fibroblasts (CAFs). In some embodiments, immunotherapy is a tumor vaccine (e.g., whole tumor cell vaccine, dendritic cell vaccine, DNA and/or RNA expression vaccine, peptide and recombinant tumor-associated antigen vaccine) or adoptive cell therapy (ACT) (e.g., T cells, natural killer cells, TIL and LAK cells). T cells and/or natural killer cells can be engineered with chimeric antigen receptors (CARs) or T cell receptors (TCRs) for specific tumor antigens. As used herein, chimeric antigen receptor (or CAR) may refer to any engineered receptor specific for an antigen of interest that, when expressed on T cells or natural killer cells, confers the specificity of the CAR to T cells or natural killer cells. there is. Once T cells or natural killer cells expressing chimeric antigen receptors are generated using standard molecular techniques, they can be introduced into patients, as in techniques such as adoptive cell transfer.

Immunotherapy comprises one or more cancer antigens, especially proteins or immunogenic fragments thereof, DNA or RNA encoding said cancer antigens, especially proteins or immunogenic fragments thereof, protein preparations obtained from cancer cell lysates and/or tumor cells. It could be a cancer vaccine. As used herein, cancer antigen is an antigenic substance present in cancer cells. In principle, proteins produced in cancer cells that are upregulated in cancer cells compared to normal cells or have abnormal structures due to mutations can act as cancer antigens. In principle, cancer antigens include products of mutated or overexpressed oncogenes and tumor suppressor genes, products of other mutated genes, overexpressed or abnormally expressed cellular proteins, cancer antigens produced by oncogenic viruses, tumor fetal antigens, transformations. These may be cell surface glycolipids and glycoproteins or cell type-specific differentiation antigens. Examples of cancer antigens include abnormal or overexpressed products of the ras and p53 genes. Other examples include tissue differentiation antigens, mutant protein antigens, oncogenic viral antigens, cancer-testis antigens, and vascular or matrix-specific antigens. Tissue differentiation antigens are antigens that are specific to a particular type of tissue. Mutant protein antigens are likely to be much more specific to cancer cells because normal cells should not contain these proteins. Normal cells display normal protein antigens on MHC molecules, while cancer cells display mutant forms. Some viral proteins are associated with cancer formation, and some viral antigens are also cancer antigens.

The immunotherapy may be part of an antibody preparation, such as a polyclonal antibody, or it may be a monoclonal antibody. Antibodies may be humanized antibodies, chimeric antibodies, antibody fragments, bispecific fragments, or single chain antibodies. Antibodies disclosed herein include antibody fragments, such as Fab, Fab' and F(ab')2, Fd, single chain Fv (scFv), single chain antibody, disulfide-linked Fv (sdfv) and VL or VH domains. This includes, but is not limited to, fragments. In some aspects, the antibody or fragment thereof is directed to epidermal growth factor receptor (EGFR1, Erb-B1), HER2/neu (Erb-B2), CD20, vascular endothelial growth factor (VEGF), insulin-like growth factor receptor (IGF-1R) , TRAIL-receptor, epithelial cell adhesion molecule, carcinoembryonic antigen, prostate-specific membrane antigen, mucin-1, CD30, CD33, or CD40.

Examples of monoclonal antibodies that can be used in combination with the compositions provided herein include, without limitation, trastuzumab (anti-HER2/neu antibody); Pertuzumab (anti-HER2 mAb); Cetuximab (chimeric monoclonal antibody against epidermal growth factor receptor EGFR); Panitumumab (anti-EGFR antibody); Nimotuzumab (anti-EGFR antibody); Zalutumumab (anti-EGFR mAb); necitumumab (anti-EGFR mAb); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-210 (humanized anti-HER-2 bispecific antibody); MDX-447 (humanized anti-EGF receptor bispecific antibody); Rituximab (chimeric murine/human anti-CD20 mAb); Obinutuzumab (anti-CD20 mAb); Ofatumumab (anti-CD20 mAb); Tositumumab-I131 (anti-CD20 mAb); Ibritumomab tiuxetan (anti-CD20 mAb); Bevacizumab (anti-VEGF mAb); Ramucirumab (anti-VEGFR2 mAb); ranibizumab (anti-VEGF mAb); aflibercept (extracellular domains of VEGFR1 and VEGFR2 fused to IgG1 Fc); AMG386 (angiopoietin-1 and -2 binding peptide fused to IgG1 Fc); dalotuzumab (anti-IGF-1R mAb); gemtuzumab ozogamicin (anti-CD33 mAb); Alemtuzumab (anti-campas-1/CD52 mAb); brentuximab vedotin (anti-CD30 mAb); Catumaxomab (dual specific mAb targeting epithelial cell adhesion molecule and CD3); Naptumomab (anti-5T4 mAb); Zirentuximab (anti-carbonic anhydrase ix); or parletuzumab (anti-folate receptor). Other examples include Panorex ^® (17-1A) (murine monoclonal antibody or chimeric murine monoclonal antibody); BEC2 (anti-idiotypic mAb, GD epitope mimic) (including BCG); Oncolym (Lym-1 monoclonal antibody); SMART M195 Ab, humanized 13'1 LYM-1 (Oncolym), Ovarex (B43.13, anti-idiotypic mouse mAb); 3622W94 mAb, which binds to the EGP40 (17-1A) pan-carcinoma antigen in adenocarcinoma; Zenapax (SMART anti-Tac (IL-2 receptor); SMART M195 Ab, humanized Ab, humanized); NovoMAb-G2 (pan-carcinoma specific Ab); TNT (chimeric mAb against histone antigen); TNT (chimeric mAb against histone antigen); Gliomab-H (monoclonal-humanized Abs); GNI-250Mab; EMD-72000 (chimeric-EGF antagonist); LymphoCide (humanized IL.L.2 antibody); and MDX-260 bispecific, targeting GD-2, ANA Ab, SMART IDIO Ab, SMART ABL 364 Ab or ImmuRAITCEA.

Other examples of antibodies include zanulimumab (anti-CD4 mAb), keliximab (anti-CD4 mAb); Ipilimumab (MDX-101; anti-CTLA-4 mAb); Tremilimumab (anti-CTLA-4 mAb); (daclizumab (anti-CD25/IL-2R mAb); basiliximab (anti-CD25/IL-2R mAb); MDX-1106 (anti-PD1 mAb); antibody against GITR; GC1008 (anti-TGF - Antibody); Metelimumab/CAT-192 (anti-TGF-antibody); Lerdelimumab/CAT-152 (anti-TGF-antibody); ID11 (anti-TGF-antibody); Denosumab (anti-RANKL mAb); BMS-663513 (humanized anti-4-1BB mAb); SGN-40 (humanized anti-CD40 mAb); CP870,893 (human anti-CD40 mAb); Infliximab (chimeric anti-TNF mAb; Dalimumab (human anti-TNF mAb); Certolizumab (humanized Fab anti-TNF); Golimumab (anti-TNF); Etanercept (extracellular domain of TNFR fused to IgG1 Fc); Belatacept (Fc (extracellular domain of CTLA-4 fused to); abatacept (extracellular domain of CTLA-4 fused to Fc); belimumab (anti-B lymphocyte stimulator); muromomab-CD3 (anti-CD3 mAb); Otel Rixizumab (anti-CD3 mAb); Neplizumab (anti-CD3 mAb); Tocilizumab (anti-IL6R mAb); REGN88 (anti-IL6R mAb); Ustekinumab (anti-IL-12/23 mAb); Briakinumab (anti-IL-12/23 mAb); Natalizumab (anti-α4 integrin); Vedolizumab (anti-α4 7 integrin mAb); T1 h (anti-CD6 mAb); Epratuzumab (anti-CD22 mAb); Efalizumab (anti-CD11a mAb); and Atacicept (the extracellular domain of the calcium-regulated ligand interactor and transmembrane activator fused to Fc).

When the composition according to the present invention includes tumor tissue, the tumor tissue may be pulverized and diluted or dissolved in a solution. Methods of grinding include physical impact, crushing, wet and dry grinding, disintegration, freezing, cryo-milling, and cutting. The solution may be water or a liquid containing water as a main ingredient, a buffer solution, isotonic solution, saline solution, etc., but is not limited to a particular type.

Additionally, when the composition includes two or more tumor tissues, the pulverized particle sizes of the two or more tumor tissues may be the same or different from each other.

Additionally, the present invention provides a kit for preventing or treating cancer comprising the composition. In addition to the above composition, the kit according to the present invention may include other components, compositions, solutions, devices, etc. commonly required for the prevention or treatment of cancer without limitation, and in particular provides instructions for appropriate use and storage of the composition according to the present invention. It may include manuals, etc.

The content of the tumor tissue or cancer cells in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the patient's condition, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 0.001% by weight, based on the total weight of the composition. It may be 50% by weight, but is not limited thereto. The content ratio is a value based on the dry amount with the solvent removed.

The pharmaceutical composition according to the present invention may further include appropriate carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

The pharmaceutical composition according to the present invention can be prepared as powder, granules, sustained-release granules, enteric-coated granules, solutions, eye drops, ellipsis, emulsions, suspensions, spirits, troches, perfumes, and limonadese according to conventional methods. , tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric-coated capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta preparations, sprays, inhalants, patches, sterilized injection solutions, or aerosols, and the external preparations include creams, gels, patches, sprays, ointments, and warning agents. , it may have a dosage form such as lotion, liniment, pasta, or cataplasma.

Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, and calcium. These include phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Additives to tablets, powders, granules, capsules, pills, and troches according to the present invention include corn starch, potato starch, wheat starch, lactose, white sugar, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, and phosphoric acid. Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl. Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin. , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. binders can be used, Hydroxypropyl methyl cellulose, corn starch, agar powder, methyl cellulose, bentonite, hydroxypropyl starch, sodium carboxymethyl cellulose, sodium alginate, calcium carboxymethyl cellulose, calcium citrate, sodium lauryl sulfate, silicic acid anhydride, 1-hydroxy Propylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen. Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, Macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, Lubricants such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.

Additives for the liquid according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

A solution of white sugar, other sugars, or sweeteners, etc. may be used in the syrup according to the present invention, and if necessary, flavoring agents, colorants, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc. may be used.

Purified water can be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as needed.

Suspensions according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. Topics may be used, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV solution, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV solution, ethanol, propylene glycol, non-volatile oil - sesame oil. , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristic acid, and benzene benzoate; Solubilizers such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, Tween, nicotinic acid amide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and buffering agents such as gums; Isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), and ethylenediaminetetraacetic acid; Sulfurizing agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; Analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; It may contain suspending agents such as CM sodium, sodium alginate, Tween 80, and aluminum monostearate.

Suppositories according to the present invention include cacao oil, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, lecithin, Lanet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75(S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Massaupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppositories type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), Tegestor triglyceride base (TG-95, MA, 57) and The same mechanism can be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include the extract with at least one excipient, such as starch, calcium carbonate, and sucrose. ) or prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.

Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and It can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field.

The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition of the present invention can be administered to an individual through various routes. All modes of administration are contemplated, including oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal injection, vaginal injection. It can be administered by internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, etc.

The pharmaceutical composition of the present invention is determined depending on the type of drug as the active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease. Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and is generally 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg per kg of body weight every day, every other day, or 3 days. It can be administered at intervals of 4 to 4 days or divided into 1 to 3 times per day. However, since it may increase or decrease depending on the route of administration, severity of disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

In the present invention, “individual” refers to a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cows, etc. refers to mammals of

In the present invention, “administration” means providing a given composition of the present invention to an individual by any appropriate method.

In the present invention, “prevention” refers to any action that suppresses or delays the onset of the desired disease, and “treatment” refers to the improvement or improvement of the desired disease and its associated metabolic abnormalities by administration of the pharmaceutical composition according to the present invention. It refers to all actions that are beneficially changed, and “improvement” refers to all actions that reduce parameters related to the target disease, such as the degree of symptoms, by administering the composition according to the present invention.

In addition, the present invention includes the steps of (S1) isolating tumor tissue or cancer cells from a cancer patient; and

(S2) It provides a method for producing a composition according to the present invention, comprising the step of applying one or more treatments selected from the group consisting of (a) and (b) below to the isolated tumor tissue or cancer cells:

(a) Irradiation; and

(b) Immunoanticancer drugs.

The tumor tissue or cancer cells can be obtained through a procedure to extract them, or tumor tissues or cancer cells extracted during the treatment or diagnosis process can be used. For example, specimens for biopsy to check for malignant tumors in the body can be used without discarding specimens after microscopic confirmation or other biopsy confirmation, thereby reducing the patient's pain accompanying the extraction of cancer tissue.

In addition, if the amount of extracted tissue or tissue reused after tissue examination is small, it can be cultured to increase the amount as needed. If culturing is not easy, a small amount of extracted tissue can be pulverized and diluted in water to increase the amount and then treated in vitro.

Therefore, the method may further include the step of pulverizing the tumor tissue and diluting or dissolving it in a solution after the step (S1).

The extracted tissue can be separated, labeled, and then treated in the quantity required to perform in vitro abscopal induction treatment.

Below, preferred embodiments are presented to aid understanding of the present invention. However, the following examples are provided only to make the present invention easier to understand, and the content of the present invention is not limited by the following examples.

[Example]

Example 1. Production of a composition having an abscopal effect using radiation irradiation

As a first means to achieve the effect of abscopal for cancer or tumor treatment, it was attempted to isolate tumor tissue from a subject, treat the extracted tissue with radiation, and then re-administer it. Specifically, when radiation is irradiated to an extracted tissue specimen, a single amount of radiation can be irradiated. The irradiation dose at this time can range from 1 Gy to 500 Gy, and is administered once or divided into more than one dose. Alternatively, instead of irradiating a single dose of radiation, multiple dose equivalents can be irradiated and mixed, or two or more total doses can be irradiated and mixed. In this case, the extracted tissue specimens are classified and irradiated with radiation, and then the radiation-treated tissues are mixed to prepare a composition for administration.

For example, in the case of a mixture of two (A, B) irradiation doses, it can be produced under the following conditions:

- Tissue A: 1~50 Gy/total irradiation dose

- Tissue B: 50~500 Gy/total irradiation dose

For a mixture of three (A, B, C) irradiation doses, it can be produced under the following conditions:

- Tissue A: 1~30 Gy/total irradiation dose

- Tissue B: 30~100 Gy/total irradiation dose

- Tissue C: 100~1000 Gy/total irradiation dose

The above examples present 2 to 3 total dose mixture compositions, but larger total dose mixture compositions can be made if necessary.

Example 2. Preparation of a composition with abscopal effect using immunological treatment

In this example, a composition that can exert an abscopal effect is prepared by isolating tumor tissue from a subject and then immunologically treating the extracted tissue. When performing immunological treatment on a tissue specimen extracted from a subject, the tissue specimen can be treated single-handedly with one agent, or the tissue specimen can be separated into two or more and treated with two or more immunological agents.

When immunologically treating a separated tissue specimen, the tissue cells in the separated tissue specimen are killed or attenuated so that they cannot grow or proliferate, and preferably, the immune cells can easily recognize the tissue cells of the immunologically treated composition. make it possible

Immunological treatments encompass the use of immune effector cells and molecules to target and destroy isolated cancer and tumor tissue cells.

The immune effector may be, for example, an antibody specific for some marker on the surface of tumor cells. Antibodies can be used alone as effectors of therapy, or they can recruit other cells to achieve actual cell death. Additionally, antibodies can be conjugated with drugs or toxins (radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and mainly used as targeting substances. As another example, the effector may be a lymphocyte that possesses surface molecules that interact directly or indirectly with the target tumor cell. Various effector cells include cytotoxic T cells and NK cells. It also encompasses the use of apoptotic cells in immunological treatments.

Immunological agents can be treated by mixing one or two or more of the conventionally developed agents.

As an example, when two (A, B) extracted specimens are treated with an immunological therapeutic agent, a composition is prepared by inducing death or attenuation of the cancer tissue of the extracted tissue specimen A through treatment with the immunological therapeutic agent. Treatment of the extracted tissue specimen B involves performing immunological treatments other than the immunological treatment on specimen A, thereby inducing death or attenuation of cancer cells in the tissue specimen B to create a composition. The compositions of treated specimens A and B are administered separately or mixed to the subject.

If there are three or more immunological methods to be treated, they are treated using the same division method as the two immunological treatment methods, and the prepared compositions are administered to the subject individually or in combination.

In another embodiment of the present invention, a composition made by individually or combining the composition of each tissue treated with one or more methods selected from the group consisting of irradiation treatment, immunological treatment, etc. on the extracted cancer tissue is applied to the subject's body. It can be administered. When necessary, a composition prepared from tissues treated by one or more methods, individually or by mixing two or more for convenience, is administered into the patient's body.

The timing of administration is not separately specified. When administered together with an immunotherapy agent, it can be administered 30 minutes to 2 hours before the immunotherapy agent, simultaneously with the immunotherapy agent, or 30 minutes to 1 week after the immunotherapy agent administration.

In the present invention, 'cancer' includes all types of solid cancer and all types of blood cancer.

In order to apply the present invention to blood cancer, the extracted blood was separated by centrifugation, mesh, filter, etc., and then only the cancer tissue was separated and treated in the same manner as the solid cancer tissue to be vaccinated with Abscopal.

Example 3. Confirmation of the anticancer effect of a composition with abscopal effect (IVAM) prepared using radiation treatment.

The In vitro Abscopal Method (IVAM) of the present invention is expected to activate the individual's anti-tumor immunity and induce an abscopal effect by administering irradiated tumor cells. Accordingly, the present inventors confirmed the effect of the present invention using a breast cancer animal model with a low immunogenicity and immune-suppressive tumor microenvironment.

3-1. Experiment method

Specifically, the in vitro experiment was conducted as follows: 4T1 murine triple negative breast cancer cells were seeded with 2×10 ⁵ cells in a 60mm culture dish and 1×10 ⁴ cells in a 24 well plate, followed by irradiation the next day ( 50Gy) was administered. At 2, 4, and 7 days after irradiation, cell morphology was observed through a microscope, and MTT was performed to measure cell viability.

In vivo experiments were conducted in compliance with animal ethics rules in accordance with the IACUC animal experiment protocol (BA-2112-333-009-01) of Seoul National University Bundang Hospital. First, to create a tumor animal model, 4T1 murine triple negative breast cancer cells (6×10 ⁵ cells at right hindlimb or 1×10 ⁵ cells at left flank) were placed on the hind limbs and flanks of a 6-week-old immune-competent BALB/c mouse. was injected. In the present invention, the 4T1 cell can be injected with 4T1-Luc cell expressing luciferase (the same example below), and when luciferin is injected into a mouse, the expressed luciferase (enzyme) uses it to emit light. This light can be detected using in vivo bioluminescence imaging to monitor the size and location of cancer in mice. Seven days after injection, it was confirmed that the tumor had grown steadily, and experimental groups were assigned. The experimental group was divided into a total of 4 groups, and 5 mice were assigned to each group: control group; IVAM administration group; Radiation treatment group; and IVAM+Radiation combination treatment group. Specifically, for IVAM administration, 4T1 cells irradiated with 50Gy were cultured for a week, then the cell supernatant (with dead cells) was obtained, and 200 μl of this was administered once (Day 10) through IV injection. Additionally, during radiation treatment, 8Gy was administered with a 6MeV electron beam three times a week (Days 10, 12 and 14), for a total of 24Gy.

Each experimental group measured and recorded the size and weight of the tumor every Monday, Wednesday, and Friday. On the 4th and 28th days after tumor implantation, IVIS imaging was used to determine the tumor growth inhibition effect and the abscopal effect by radiation. was compared and analyzed.

3-2. High-dose radiation sensitization effect in 4T1 murine triple negative breast cancer cell line

A cytotoxicity test (MTT analysis) was performed to measure the cell viability of 4T1 cells when irradiated with high-dose radiation (50Gy), and changes in cell shape were observed under a microscope.

As a result, as shown in Figures 2a and 2b, compared to the control group, cancer cells irradiated with radiation were observed to have damaged cell membranes, and the fraction of viable cells was also significantly lower than the control group, confirming the sensitization effect by radiation. There was (p<0.0001).

3-3. Confirmation of synergistic anti-cancer effect and abscopal effect by combination of IVAM and Radiation

As a result of observing tumor growth for 28 days after tumor inoculation, it was confirmed that tumor growth was significantly (p<0.05) delayed in the group administered IVAM alone compared to the control group. In addition, when radiation and IVAM were combined, a synergistic effect was confirmed in which tumor growth was delayed more significantly (p<0.01) compared to the control group (Figure 3).

In addition, it was confirmed that the growth of the tumor (secondary tumor) on the left side that did not receive radiation was significantly (p>0.05, p<0.01) suppressed in the radiation treatment alone or radiation+IVAM combination treatment group compared to the control group. did. In the group administered IVAM alone, the tumor in the left flank slightly decreased, but this was not statistically significant (Figure 4).

In addition, IVIS imaging using the Luciferase system was used to compare and analyze the 4th day and the 28th day after tumor implantation. As a result of directly measuring the size of the tumor, similar to the previously confirmed results, it was confirmed that a synergistic anti-cancer effect was exerted when IVAM and radiation were used together. Even in secondary tumors that were not irradiated with radiation, the combination of IVAM and radiation showed tumor growth. As it was confirmed that the inhibitory effect was exerted synergistically, the results showing the abscopal effect were confirmed (Figures 5a and 5b).

The above results show that when the IVAM of the present invention is used in combination with Radiation, the anti-cancer effect is significantly increased compared to each monotherapy, and a better anti-cancer effect can be achieved through the abscopal effect.

3-4. Confirmation of toxicity of combined use of IVAM and Radiation

When the mouse's body weight was measured for 28 days, it was observed that there was no significant weight loss due to IVAM and radiation compared to the control group (FIG. 6). The above results suggest that the combined use of IVAM and Radiation of the present invention can only exert excellent anticancer and abscopal effects without causing side effects such as toxicity to the subject.

Example 4. Confirmation of the combined effect of IVAM, radiation therapy, and immunotherapy

In this example, the combined effect of IVAM according to the present invention and other anticancer treatments was confirmed. Specifically, the anticancer effects of IVAM combined with radiation therapy and/or immunotherapy (immune checkpoint inhibitor) were compared.

4-1. Experiment method

The animal experiment schedule according to this example is shown in Figure 7. All animal experiments were conducted in compliance with animal ethics rules and in accordance with the IACUC animal experiment protocol (BA-2112-333-009-01) of Seoul National University Bundang Hospital.

Specifically, the experiment was conducted as follows: 4T1 murine triple negative breast cancer cells were inoculated into the hind limbs and flanks of 6-week-old immune-competent BALB/c mice (6×10 ⁵ cells in the right hind limb or 1×10 ⁵ cells in the left flank). was injected. 7 days after injection, it was confirmed that the tumor grew consistently, and the experimental group was divided into 8 groups, with 5 mice assigned to each group: ① Control group, ② Radiation (RT) treatment group, ③ IVAM administration group, ④ IVAM administration group + radiation combination treatment group, ⑤ a-PD-L1 administration group, ⑥ a-PD-L1 + radiation combination treatment group, ⑦ IVAM administration group + a-PD-L1 administration group, and ⑧ IVAM administration group + a-PD-L1 administration group + radiation triple combination treatment group. (Triple combination). Here, each anticancer therapy was performed as follows:

1) IVAM: ① After culturing 4T1 cells irradiated with 5, 25, 50, and 100 Gy for a week, the cell supernatant (with dead cells 1x10 ⁵ cells/injection) was injected at a ratio of 1:1:1:1. It was collected and administered intravenously (IV injection) in a total of 200 μl once (Day 10).

2) Radiation therapy (RT): Using Precision X-ray's Radiation was applied only to the right hind limb of the mouse.

3) a-PD-L1 (anti-Programmed Death-Ligand 1 antibody): Administered intraperitoneally at a concentration of 5mg/kg, 100 μl a total of 2 times (Day 11, 18).

The tumor size and body weight of mice in each group were measured and recorded every Monday, Wednesday, and Friday. On the 7th and 28th days after tumor implantation, IVIS imaging was used to compare and analyze the tumor growth inhibition effect of each therapy and the presence or absence of abscopal effect by radiation. On the 19th day after tumor implantation, orbital blood was collected from the mouse, and interferon-γ (IFN-γ) and interferon-β (IFN-β) levels were measured through multiplex immunoassay using serum separated through a centrifuge. All mice were euthanized on the 31st day after tumor implantation. At this time, tumor tissue, spleen, and tumor-draining lymph nodes (dLN) in the mouse's hind limbs and flanks, respectively, primary (tumor in the irradiated right hind limb) and secondary (secondary (tumor in the left flank that was not irradiated)). , lung tissue was extracted. A portion of each tumor tissue and lung tissue were fixed with 4% paraformaldehyde immediately after extraction, and other tumors and spleen tissue were extracted and then used for flow cytometry analysis (FACS) through a single cell isolation process. The number of metastatic lung nodules observed in the fixed lung tissue was counted through a dissecting microscope. Statistical analysis used one-way ANOVA (Tukey’s multiple comparison tests) and unpaired two-tailed Student’s t-tests in PRISM statistical analysis and graphing software (GraphPad 8).

4-2. Observation of tumor growth inhibition effect following IVAM, a-PD-L1, and Radiation combination treatment

The results of observing tumor growth in the irradiated right hind limb (primary tumor) are shown in Figure 8. Tumor growth was observed for 28 days after tumor inoculation in mice. First, it was confirmed that tumor growth in the triple combination (IVAM + a-PD-L1 + radiation) group in the irradiated right hind limb (primary tumor) was significantly (p<0.0001) delayed compared to the control group. In addition, the tumor size was smaller than that of the RT alone group (p<0.001), IVAM + RT group, IVAM + a-PD-L1 group, and a-PD-L1 + RT group (p<0.0001 for all three groups), compared to the triple combination group. Although it is somewhat large, it was confirmed that tumor growth was significantly suppressed compared to the control group. Compared to the control group, a slight tumor growth delay was observed in the IVAM alone group, but no significant differences were observed.

When compared to the IVAM alone group, RT alone group (p≤<0.01), IVAM + RT group, IVAM + a-PD-L1 group, a-PD-L1 + RT group (p<0.001 for all three groups), and It was confirmed that tumor growth in the triple combination group (p<0.0001) was significantly delayed.

When comparing the a-PD-L1 alone group and the IVAM + a-PD-L1 group, there was no significant difference (p=0.6380), but the tumor size tended to be smaller when combined treatment was used. However, there was no difference in tumor size between the RT alone group and the IVAM + RT group.

As a result, as a result of comparing the effects of IVAM, a-PD-L1, and RT alone, double combination, and triple combination, it was confirmed that the anticancer treatment effect was the strongest in the triple combination group in which all three treatments were used together. .

The results of observing the growth of secondary tumors are shown in Figure 9. As shown in the figure, as a result of comparing the growth of the secondary tumor on the left side that was not irradiated with the control group, tumor growth was significantly delayed in the triple combination group of IVAM + a-PD-L1 + RT. As shown, it was confirmed that the abscopal effect appeared (p<0.05). There was no significant difference in tumor size between different treatment groups.

In addition, IVIS imaging using the Luciferase system was used to compare and analyze tumors on the 4th day and tumors on the 28th day after tumor implantation. As a result, a similar tumor growth inhibition effect pattern as the previously confirmed tumor growth inhibition effect (FIGS. 8 and 9) was confirmed (FIGS. 10a to 10c). In other words, tumor growth was shown to be suppressed in all groups compared to the control group, and in particular, the triple combination of IVAM + a-PD-L1 + RT was confirmed to have the strongest anticancer effect.

Summarizing the above results, in the case of tumor growth of the primary tumor, a decrease in tumor size was observed in the IVAM-only group compared to the control group, and the tumor size was larger in the IVAM + a-PD-L1 combination group compared to the a-PD-L1-only group. A decreasing trend was observed. In particular, it was confirmed that tumor growth was most strongly suppressed in the triple combination group of IVAM + a-PD-L1 + Radiation. In addition, in the case of secondary tumors that did not receive direct radiation, the abscopal effect was observed in the triple combination group. These results show that the combination therapy of IVAM, radiation therapy, and immunotherapy according to the present invention not only achieves a synergistic anticancer effect, but also exerts a more powerful anticancer effect through the abscopal effect. In addition, as a result of measuring changes in the immune cell population in animal models, the primary tumor growth inhibition effect of the triple combination was anti-tumor due to an increase in CD8 ⁺ T cells and CD8 ⁺ effector memory T cells in the tumor microenvironment and a decrease in T _regs . It was confirmed that this was due to an increase in the dual immune effect, and in particular, an increase in Ki67 ⁺ CD8 + T cells and Ki67 ⁺ CD4 + T cells was confirmed in the spleen due to the triple combination. Furthermore, an increase in IFN-γ, which contributes to the anti-tumor immune effect, was observed in mice to which the triple combination was applied, which is an important clue to the effect of Abscopal in the triple combination.

4-3. Confirmation of toxicity according to IVAM, a-PD-L1, and Radiation combination treatment

In order to confirm the presence or absence of side effects due to the combination of IVAM therapy, a-PD-L1, and Radiation according to the present invention, the change in body weight of mice over time during the treatment process was confirmed. As a result of measuring the body weight of the mice for 31 days, it was confirmed that there was no significant weight loss in the groups treated with IVAM, a-PD-L1, and/or Radiation compared to the control group (FIG. 11). The above results suggest that the combined use of IVAM, immuno-anticancer agent, and radiation of the present invention can only exert excellent anti-cancer and abscopal effects without causing side effects such as toxicity to the subject.

4-4. Confirmation of lung metastasis inhibition effect of IVAM, a-PD-L1, and Radiation combination treatment

In order to confirm the effect of suppressing metastasis by the triple combination of IVAM, immunotherapy, and radiation according to the present invention, lung tissue was extracted from the euthanized mouse on the 31st day after inoculating the tumor in the mouse, and observed on the tissue surface. Metastatic lung nodules were measured. As a result, a very high number of nodules were observed in the control group, confirming that lung metastasis occurred, whereas in the a-PD-L1 alone group, a-PD-L1 + RT double combination group, IVAM + a-PD-L1 A significant reduction in metastasis was confirmed in the double combination group and triple combination group (all p<0.0001). Metastasis was significantly reduced in the IVAM + RT dual combination group (p<0.001) and the RT alone group (p<0.01), but there was no significant difference from the IVAM alone group (FIGS. 12a and 12b).

Compared to the IVAM alone group, the greatest difference was observed in the triple combination group (p<0.0001), followed by the a-PD-L1 + RT double combination group and the IVAM + a-PD-L1 double combination group (p<0.0001 for both groups). 0.01), and differences were also found in the a-PD-L1 group (p<0.05). Through this, even if only two or more of the three therapies of IVAM, RT, and a-PD-L1 of the present invention are used in combination, the lung metastasis inhibitory effect is significantly increased. In particular, when all of the above therapies are used together, a synergistic metastasis inhibitory effect appears. Confirmed.

4-5. Confirmation of changes in the distribution of immune cells within the primary tumor following combination treatment of IVAM, a-PD-L1, and Radiation

The results of analyzing the population changes of three types of immune cells (CD8 ⁺ T cell, T _reg , and CD8 ⁺ effector memory T cell) in the tumor microenvironment of the primary tumor (hind limb tumor) using flow cytometry are shown in Figures 13A to 13C.

First, the number of CD8 ⁺ T cells attacking tumor cells significantly increased in the IVAM + a-PD-L1 + Radiation triple combination group compared to the control group (p<0.0001), RT alone group (p<0.05), and IVAM + RT dual combination group, a-PD-L1 + RT dual combination group (p<0.01 for both groups), and IVAM + a-PD-L1 dual combination group (p<0.001) also showed a significant increase in CD8 ⁺ T cells. Confirmed.

In the case of immunosuppressive regulatory T cells (T _reg ), which suppress the cytotoxic activity of CD8 ⁺ T cells, previous studies have shown that they increase due to radiation. Consistent with this, the number of T _regs increased in the RT-only group compared to the control group, and the number of T _regs increased in the IVAM + RT group compared to the IVAM-only group. On the other hand, a decrease in T _reg was observed in the triple combination group compared to the single group that did not receive RT (IVAM +a-PD-L1 group).

In addition, in the case of CD8+ effector memory T cells, in which memory T cells with memory for a specific antigen can attack tumor cells with the same antigen, the RT alone group and the IVAM + a-PD-L1 group compared to the control group. A significant increase was observed in all the double combination group (p<0.01), IVAM + RT double combination group, a-PD-L1 + RT double combination group, and triple combination group (p<0.001).

The above results show that the triple combination of IVAM + anticancer immunotherapy + radiation increases the number of CD8 ⁺ T cells with anticancer activity and decreases the number of T _regs that suppress the activity of CD8 ⁺ T cells, resulting in enhanced anticancer immune effect. It shows that it is done.

In summary, it can be seen that in the triple combination group, the number of CD8+ T cells and CD8+ effector memory T cells increased and the number of T _regs decreased, enhancing the antitumor immune effect.

4-6. Confirmation of changes in distribution of immune cells in the spleen following combination treatment of IVAM, a-PD-L1, and Radiation

The spleen contains a variety of immune cells, including lymphocytes such as T cells, as well as monocytes that later differentiate into macrophages and dendritic cells, and thus plays an important role in regulating the immune system throughout the body. Accordingly, the effect of the combined use of IVAM, anticancer immunotherapy, and/or radiation according to the present invention on the distribution of immune cells in the spleen was confirmed.

The results are shown in Figures 14a and 14b. First, as a result of observing population changes in CD8 ⁺ and CD4 ⁺ T cells, no significant differences were observed in all groups. However, the population of Ki67 ⁺ CD8 ⁺ T cells and Ki67 ⁺ CD4 ⁺ T cells expressing Ki67, a cell proliferation marker, was found to significantly increase in the triple combination group compared to the control group. The above results suggest that the abscopal effect seen in the triple combination of IVAM, anticancer immunotherapy, and Radiation according to the present invention is related to changes in immune cell distribution in the spleen.

In the case of T _reg , RT alone group compared to the control group, similar to the aspects shown in Figures 13a to 13a; IVAM + RT combination group compared to IVAM alone group; The number of T _regs increased in both the a-PD-L1 + RT group compared to the a-PD-L1 single group, but the number of T _regs slightly decreased in the triple combination group compared to the IVAM + a-PD-L1 double combination group. pattern was observed.

4-7. Confirmation of changes in cytokines in serum following combination treatment of IVAM, a-PD-L1, and Radiation

The effect of the combined use of IVAM, anticancer immunotherapy, and/or radiation according to the present invention on the level of cytokines in serum was confirmed. For this purpose, serum was obtained by collecting orbital blood from mice 19 days after tumor inoculation, and the levels of two types of interferons, IFN-β and IFN-γ, were measured in the serum.

As a result, as shown in Figure 15, a significant increase in IFN-γ was observed in the triple combination group (IVAM + a-PD-L1 + Radiation) compared to the control group (p<0.05), and IFN-β was observed in the group that received RT. In all cases, an increasing trend was observed in the control group.

The above results suggest that radiation therapy increases the level of interferon, which is consistent with the immune-enhancing and interferon-increasing effects of RT known through previous studies. In particular, in the case of IFN-γ, the increase was greater in the triple combination group where IVAM and a-PD-L1 were used together with RT treatment. Furthermore, the overall increase in IFN-γ in the body following triple therapy is considered the basis for explaining the growth delay effect of primary tumor and the abscopal effect caused by triple therapy.

Example 5. Confirmation of long-term survival experiment results by combination treatment of IVAM, radiation therapy, and immunotherapy

In this example, the combined effect of IVAM according to the present invention and other anticancer therapies was confirmed for a long period of time. Specifically, the anticancer effects of IVAM combined with radiation therapy and/or immunotherapy (immune checkpoint inhibitor) were compared.

5-1. Experiment method

The animal test schedule for the long-term survival rate and toxicity test according to this example is shown in Figure 16. All animal experiments were conducted in compliance with animal ethics rules and in accordance with the IACUC animal experiment protocol (BA-2112-333-009-01) of Seoul National University Bundang Hospital.

Specifically, the experiment was conducted as follows: 4T1 murine triple negative breast cancer cells (6 × 10 ⁵ cells in the right hind limb or 1 × 10 ⁵ cells in the left flank) were injected into the hind limbs and flanks of 6-week-old immune-competent BALB/c mice. Injected. 7 days after injection, it was confirmed that the tumor grew consistently, and the experimental group was divided into 7 groups, with 5 mice assigned to each group: ① Control group, ② Radiation (RT) treatment group, ③ IVAM administration group, ④ IVAM administration group + Radiation combination treatment group, ⑤ a-PD-L1 administration group, ⑥ IVAM administration group + a-PD-L1 administration group, ⑦ IVAM administration group + a-PD-L1 administration group + radiation triple combination group. Here, each anticancer therapy was performed as follows:

1) IVAM: ① After culturing 4T1 cells irradiated with 5, 20, 50, and 100 Gy for a week, the cell supernatant (with dead cells 1x10 ⁵ cells/injection) was injected at a ratio of 1:1:1:1. It was collected and administered intravenously (IV injection) in a total of 200 μl once (Day 10).

2) Radiation therapy (RT): Using Precision X-ray's X-RAD320 equipment, 8 Gy was administered twice (Day 11 and 12) for a total of 16 Gy. Radiation was applied only to the right hind limb of the mouse.

3) a-PD-L1: 100 μl was administered intraperitoneally at a concentration of 5 mg/kg a total of 4 times (Days 11, 14, 17, and 20).

The body weight of mice in each group was measured and recorded every Monday, Wednesday, and Friday. All mice were euthanized on the 50th day after tumor implantation, and the survival rate of mice in each group for 50 days was measured. Statistical analysis used one-way ANOVA (Tukey’s multiple comparison tests) and unpaired two-tailed Student’s t-tests in PRISM statistical analysis and graphing software (GraphPad 8).

5-2. Confirmation of long-term survival rate and toxicity following IVAM, a-PD-L1, and Radiation combination treatment

In order to confirm the long-term survival rate and presence of side effects due to the combination of IVAM therapy, a-PD-L1, and Radiation according to the present invention, the survival rate and body weight changes of mice over time during the treatment process were confirmed.

As a result of measuring the survival rate of mice for 50 days, it was confirmed that in the control group, the survival rate decreased to 0% after 40 days, whereas in the combination treatment of IVAM, a-PD-L1, and Radiation, the survival rate was maintained at 100% even after 50 days. (p<0.001), and it was found that the survival rate was maintained above 80% after 50 days even in the radiation-only treatment group (p<0.05) (FIG. 17a).

In addition, as a result of measuring the change in body weight of mice for 50 days, it was confirmed that there was no significant weight loss in the groups treated with IVAM, a-PD-L1, and/or Radiation compared to the control group (FIG. 17b).

The above results suggest that the combined use of IVAM, immuno-anticancer agent, and radiation of the present invention can only exert excellent anti-cancer and abscopal effects without causing side effects such as toxicity to the subject even for a long period of time.

Example 6. Confirmation of anticancer effect and toxicity by IVAM multiple administration

In this example, the anticancer effect of IVAM multiple administration was confirmed through tumor size and IVIS imaging.

6-1. Experiment method

The animal test schedule for testing to confirm the anticancer effect and toxicity of IVAM multiple administration according to this example is shown in Figure 18. This study was conducted in compliance with animal ethics rules in accordance with the IACUC animal experiment protocol (BA-2112-333-009-01) at Seoul National University Bundang Hospital. 4T1 murine triple negative breast cancer cells (6×10 ⁵ cells in the right hind limb or 1×10 ⁵ cells in the left flank) were injected into the hind limbs and flanks of 6-week-old immune-competent BALB/c mice. Seven days after injection, it was confirmed that the tumor had grown steadily, and experimental groups were assigned. The experimental group was divided into a total of 4 groups, and 5 mice were assigned to each group: control group; IVAM single dose group (IVAM-1); IVAM 2-dose group (IVAM-2); and IVAM 3-dose group (IVAM-3).

Specifically, IVAM administration involves seeding 4T1 cells at 2.5x10 ⁵ cells/dish on a 60 pi dish, irradiating 5, 20, 50, and 100 Gy the next day, culturing for a week, and then dissolving the cell supernatant (with Dead cells (1.5x10 ⁵ cells/injection) were collected at a ratio of 1:1:1:1 and administered IV injection at 200 μl a total of 3 times (Days 10, 13, and 17).

The body weight of mice in each group was measured and recorded every Monday, Wednesday, and Friday. On the 7th and 21st days after tumor implantation, the tumor growth inhibition effect and the effect of abscopal by radiation were compared and analyzed using IVIS imaging. All mice were euthanized on the 31st day after tumor implantation, and statistical analysis was performed using one-way ANOVA (Tukey's multiple comparison tests) and unpaired two-tailed Student's t-tests in PRISM statistical analysis and graphing software (GraphPad 8). did.

6-2. Confirmation of tumor growth inhibition effect and toxicity following IVAM multiple administration

As a result of observing tumor growth for 24 days after tumor inoculation, the growth of the tumor on the irradiated right hind limb (primary tumor) and the tumor on the left flank without radiation (secondary tumor) was significantly higher in the IVAM group compared to the control group. It was confirmed that there was a delay in the 3-time IVAM administration group compared to the 1- and 2-time IVAM administration group, but it was not statistically significant (Figure 19).

In addition, IVIS imaging using the Luciferase system was used to compare and analyze the 7th and 21st days after tumor implantation. As a result of directly measuring the size of the tumor, similar to the previously confirmed results, a tumor suppressive effect was observed in the IVAM administration group compared to the control group in the primary tumor of the irradiated right hind limb, and compared to the 1 and 2 IVAM administration groups. It was confirmed that a higher inhibitory effect was observed in the 3-time administration group (FIG. 20).

In addition, as a result of measuring the change in body weight of mice for 24 days, it was confirmed that there was no significant weight loss in the groups treated with IVAM once, twice, and three times compared to the control group (FIG. 21).

The above results suggest that the anticancer effect and abscopal effect may be achieved without causing side effects such as toxicity to the subject even when IVAM of the present invention is administered three times.

Through the above examples, the present inventors applied radiation and/or immunological treatment to tumor tissue or cancer cells isolated from cancer patients to suppress or kill the activity of the cancer cells, and then administered them to cancer patients to prevent cancer in the body's immune system. It was confirmed that it not only effectively suppresses the progression or growth of primary cancer by activating the immune function, but also exerts a tumor killing function on other tumors in the body. In particular, when radiation therapy is combined with re-administration of tumor cells whose activity has been suppressed or killed by irradiation (IVAM), the anticancer effect on the primary tumor is further enhanced and the abscopal effect also inhibits the growth of secondary tumors. It was confirmed that this was in effect. Furthermore, it is believed that the triple combination therapy of IVAM along with radiation therapy and immune checkpoint inhibitor treatment therapy can exert significant anticancer effects, and the activation function of immune cells is also expected to be achieved. Therefore, the composition and treatment method according to the present invention are expected to be used in the treatment of various cancers because they can enhance the body's immune function against tumors and further improve the anticancer effect of other anticancer therapies through relatively simple treatment.

Other objects, features and advantages of the present invention will become apparent from the detailed description below. However, it should be understood that the detailed descriptions are provided for illustrative purposes only, and that various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the following detailed description.

Claims (30)

A pharmaceutical composition for the prevention or treatment of cancer, comprising as an active ingredient tumor tissue or cancer cells isolated from a cancer patient and subjected to one or more treatments selected from the group consisting of (a) and (b) below:
(a) Irradiation; and
(b) Immunoanticancer drugs.
According to paragraph 1,
A pharmaceutical composition wherein the tumor tissue or the cancer cells satisfy one or more characteristics selected from the group consisting of the following by the treatment:
i) the activity of cancer cells is weakened;
ii) cancer cells become inactive; and
iii) Cancer cells die.
According to paragraph 1,
The composition is a pharmaceutical composition comprising autologous tumor tissue or cancer cells of the cancer patient and administered to the cancer patient, or to a cancer patient allogeneic to the cancer patient. .
According to paragraph 1,
A pharmaceutical composition, characterized in that the composition is administered to an individual who does not have cancer for the purpose of preventing cancer.
According to paragraph 1,
When the treatment is (a) radiation, the radiation satisfies one or more characteristics selected from the group consisting of:
i) the radiation is at least one selected from the group consisting of γ-rays, x-rays, ultraviolet rays, laser rays, and infrared rays; and
ii) The radiation dose is 1 to 500 Gy.
According to paragraph 1,
A pharmaceutical composition, characterized in that the immuno-anticancer agent is at least one selected from the group consisting of immune checkpoint inhibitors, co-stimulatory molecule agents, cytokine therapeutic agents, CAR-T cell therapeutic agents, and autologous CD8 ⁺ T immune cell therapeutic agents.
According to clause 6,
The immune checkpoint inhibitors include PD-L1 inhibitor, PD-1 inhibitor, CTLA-4 inhibitor, LAG3 inhibitor, TIM3 inhibitor, 4-1BB inhibitor, LAG-3 inhibitor, B7-H4 inhibitor, HVEM inhibitor, TIM4 inhibitor, GAL9 inhibitor, A pharmaceutical composition comprising at least one selected from the group consisting of VISTA inhibitors, KIR inhibitors, TIGIT inhibitors, and BTLA inhibitors.
According to paragraph 1,
The composition is a pharmaceutical composition that satisfies one or more characteristics selected from the group consisting of the following when administered to a cancer patient:
i) exerts the abscopal effect;
ii) inhibits metastasis of cancer;
iii) reduces tumor load; and
iv) Inhibits the proliferation of cancer cells.
According to paragraph 1,
A pharmaceutical composition, wherein the composition is for single administration or multiple administration.
According to paragraph 1,
The composition includes two or more tumor tissues or cancer cells, and the two or more tumor tissues or cancer cells are each subjected to the same treatment or different treatments.
According to clause 10,
A pharmaceutical composition, characterized in that the composition is in the form of a mixture of the two or more tumor tissues or cancer cells.
According to clause 10,
The composition is a pharmaceutical composition, characterized in that the two or more tumor tissues or cancer cells are each formulated and administered simultaneously, separately, or sequentially.
According to paragraph 1,
A pharmaceutical composition, characterized in that the composition is used in combination with anticancer therapy.
According to clause 13,
A pharmaceutical composition, characterized in that the anticancer therapy is at least one selected from the group consisting of radiation therapy, chemotherapy, targeted anticancer drug therapy, and immunotherapy.
According to clause 13,
A pharmaceutical composition, characterized in that the composition is used in combination with radiation therapy and immunotherapy.
According to clause 13,
A pharmaceutical composition, characterized in that the composition is administered simultaneously, separately, or sequentially with the anticancer therapy.
According to clause 14,
A pharmaceutical composition, wherein the targeted anticancer agent is at least one selected from the group consisting of tyrosine kinase inhibitors, PARP inhibitors, angiogenesis inhibitors, and CDK4/6 inhibitors.
According to clause 14,
A pharmaceutical composition, characterized in that the immuno-anticancer agent is at least one selected from the group consisting of immune checkpoint inhibitors, co-stimulatory molecule agents, cytokine therapeutic agents, CAR-T cell therapeutic agents, and autologous CD8 ⁺ T immune cell therapeutic agents.
According to clause 18,
The immune checkpoint inhibitors include PD-L1 inhibitor, PD-1 inhibitor, CTLA-4 inhibitor, LAG3 inhibitor, TIM3 inhibitor, 4-1BB inhibitor, LAG-3 inhibitor, B7-H4 inhibitor, HVEM inhibitor, TIM4 inhibitor, GAL9 inhibitor, A pharmaceutical composition comprising at least one selected from the group consisting of VISTA inhibitors, KIR inhibitors, TIGIT inhibitors, and BTLA inhibitors.
According to paragraph 1,
A pharmaceutical composition, characterized in that the tumor tissue is pulverized and diluted or dissolved in a solution.
According to clause 20,
The composition is a pharmaceutical composition comprising two or more tumor tissues, wherein the two or more tumor tissues have the same or different crushed particle sizes.
According to paragraph 1,
A pharmaceutical composition, wherein the cancer is at least one selected from the group consisting of hematological cancer and solid cancer.
According to paragraph 1,
The above cancers include breast cancer, colon cancer, lung cancer, head and neck cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, cervical cancer, skin melanoma, intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, Anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer One selected from the group consisting of cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney cancer, ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. Lee Sang-in, pharmaceutical composition.
A kit for preventing or treating cancer, comprising the pharmaceutical composition of claim 1.
(S1) separating tumor tissue or cancer cells from a cancer patient; and
(S2) A method for producing the composition of claim 1, comprising the step of applying one or more treatments selected from the group consisting of (a) and (b) below to the isolated tumor tissue or cancer cells:
(a) Irradiation; and
(b) Immunoanticancer drugs.
According to clause 25,
The method further includes the step of pulverizing the tumor tissue and diluting or dissolving it in a solution after the step (S1).
According to clause 25,
A manufacturing method, wherein the composition contains autologous tumor tissue or cancer cells of the cancer patient and is administered to the cancer patient, or to a cancer patient allogeneic to the cancer patient.
According to clause 25,
A manufacturing method, characterized in that the composition is administered to an individual who does not have cancer for the purpose of preventing cancer.
An anticancer vaccine composition comprising, as an active ingredient, tumor tissue or cancer cells isolated from a cancer patient and subjected to one or more treatments selected from the group consisting of (a) and (b) below:
(a) Irradiation; and
(b) Immunoanticancer drugs.
According to clause 29,
The vaccine composition is characterized in that it is administered to an individual who has not developed cancer or an individual whose cancer has been completely cured for the purpose of preventing cancer.