KR20220012809A - Agents for treating cancers - Google Patents

Abstract

Disclosed is an agent for treating cancer in combination with an immunotherapeutic agent, such as an immune checkpoint inhibitor. According to the present disclosure, a botulinum toxin or a composition comprising the same can be useful as a cancer treatment agent in combination with the immunotherapeutic agent, for example, the immune checkpoint inhibitor. A first object of the present disclosure is to provide a composition for treating cancer in combination with the immunotherapeutic agent, comprising botulinum toxin as an active ingredient.

Description

Cancer treatment {Agents for treating cancers}

The present disclosure relates to a cancer treatment agent, and more particularly, to a cancer treatment agent including botulinum toxin as an active ingredient, used in combination with an immunotherapeutic agent.

Botulinum toxin (Botulinum neurotoxin, BoNT) is a polypeptide product of the anaerobic bacterium Clostridium botulinum , and is a toxic substance that specifically acts on nerve cells. Botulinum toxin is a toxic substance that intrinsically causes lethality, but it is associated with a number of conditions including: It is used to treat urologic disease, migraine, and the like.

International Publication No. WO2010/062955 discloses that a therapeutically effective amount of botulinum toxin is administered in combination with an anticancer drug or anticancer therapy to a non-neoplastic area near a neoplasia, but a therapeutically effective amount of botulinum toxin penetrates the neoplasm ( A method for inhibiting the growth or metastasis of a neoplasm that does not penetrate) is disclosed. In addition, International Publication No. WO2006/094539 discloses a method for sensitizing cancer treatment by cytotoxic therapy, such as chemotherapy or radiation therapy, comprising administering a pharmaceutical composition comprising a botulinum toxin to cancer tissue or cells. and compositions therefor.

Anticancer immunotherapy is a treatment that activates the body's immune system to increase autoimmunity so that immune cells attack cancer cells. Immunotherapeutic agents can be largely classified into immune checkpoint inhibitors, immune cell therapy, therapeutic antibodies, and anticancer vaccines. Immune checkpoint inhibitors are drugs that attack cancer cells by activating T cells by blocking the activity of immune checkpoint proteins involved in T cell suppression, and typically recognizes CTLA-4, PD-1, and PD-L1. antibody is used. Immune cell therapy is a drug that strengthens cellular immunity against cancer cells by collecting, strengthening and transforming T cells from the patient's body, and injecting them back into the body. Therapeutic antibody is a drug that attacks cancer cells by releasing the drug when the antibody-drug conjugate binds to cancer cells, and an antibody-drug conjugate (ADC) is a representative example. Anticancer vaccines are to activate the immune system by administering to cancer patients a tumor-specific antigen or a protein or peptide molecule that can improve the overall immune response of cancer cells, thereby activating the immune function in the body to attack cancer cells.

Until now, there have been no known attempts to treat cancer by using botulinum toxin in combination with immunotherapeutic agents.

A first object of the present disclosure is to provide a composition for treating cancer in combination with an immunotherapeutic agent, comprising botulinum toxin as an active ingredient.

A second object of the present disclosure is to provide a method for treating cancer by administering to a subject in need thereof a therapeutically effective amount of botulinum toxin in combination with an immunotherapeutic agent.

A third object of the present disclosure is to provide a botulinum toxin for use as a cancer therapeutic agent in combination with an immunotherapeutic agent.

A fourth object of the present disclosure relates to the use of botulinum toxin as a cancer therapeutic agent in combination with an immunotherapeutic agent.

A first aspect of the present disclosure relates to a composition for treating cancer in combination with an immunotherapeutic agent, comprising botulinum toxin as an active ingredient.

A second aspect of the present disclosure relates to a method of treating cancer by administering to a subject in need thereof a therapeutically effective amount of a botulinum toxin in combination with an immunotherapeutic agent.

A third aspect of the present disclosure relates to a botulinum toxin for use as a cancer treatment in combination with an immunotherapeutic agent.

A fourth aspect of the present disclosure relates to the use of botulinum toxin as a cancer therapeutic agent in combination with an immunotherapeutic agent.

According to the present disclosure, a botulinum toxin or a composition comprising the same may be useful as a cancer treatment agent in combination with an immunotherapeutic agent, for example, an immune checkpoint inhibitor.

1 is a graph showing tumor growth over time after co-administration of botulinum toxin and an anti-PD-1 antibody in the mouse malignant melanoma transplant model described in Example 1. FIG.
2 shows the ratio of CD4+ T cells based on total viable cells infiltrating the tumor after co-administration of botulinum toxin and anti-PD-1 antibody in the mouse malignant melanoma transplant model described in Example 1 (1)), the ratio of CD8+ T cells (2)), a graph showing the actual number of CD4+ T cells in the tumor (3)) and the actual number of CD8+ T cells in the tumor (4)).
3 is a graph showing the number of exosomes in the blood of the vehicle group and each test group after co-administration of botulinum toxin and anti-PD-1 antibody in the mouse malignant melanoma transplant model described in Example 1. FIG.
4 is a graph showing tumor growth over time after co-administration of botulinum toxin and anti-PD-1 antibody by dose in the mouse malignant melanoma transplant model described in Example 2. FIG.
5 is a graph showing the survival rate of mice after co-administration of botulinum toxin and anti-PD-1 antibody by dose in the mouse malignant melanoma transplantation model described in Example 2.
6 is a graph showing tumor growth over time after co-administration of complex botulinum toxin or non-complex botulinum toxin and anti-PD-1 antibody in the mouse malignant melanoma transplant model described in Example 3. FIG.
7 is a graph showing tumor growth over time after co-administration of botulinum toxin and anti-CTLA4 antibody in the mouse lung tumor transplantation model described in Example 4.
8 is a graph showing tumor growth in the right and left sides over time after co-administration of botulinum toxin and anti-PD-1 antibody in the mouse malignant melanoma bilateral transplant model described in Example 5.
9 is a graph showing the survival rate of mice after co-administration of botulinum toxin and anti-PD-1 antibody in the mouse malignant melanoma bilateral transplant model described in Example 5.
FIG. 10 is a graph showing the ratio of viable cell-based CD11b+ cells and CD4+ and CD8a+ T cells infiltrating the left untreated tumor in the mouse malignant melanoma bilateral transplant model described in Example 5. FIG.
11 is a graph showing tumor growth over time after co-administration of botulinum toxin and anti-PD-1 antibody or propranolol and anti-PD-1 antibody co-administration in the mouse colorectal cancer transplantation model described in Example 6.
12 is a graph showing the survival rate of mice after co-administration of botulinum toxin and anti-PD-1 antibody or propranolol and anti-PD-1 antibody co-administration in the mouse colorectal cancer transplantation model described in Example 6.
13 is a graph showing tumor growth over time after co-administration of botulinum toxin and anti-CTLA4 antibody in the mouse colorectal cancer transplantation model described in Example 6.
14 is a graph showing the survival rate of mice after co-administration of botulinum toxin and anti-CTLA4 antibody in the mouse colorectal cancer transplantation model described in Example 6.

As used herein, the term 'botulinum toxin' refers to a botulinum toxin protein molecule produced by bacteria or recombinantly, including all serotypes and variants or fusion proteins thereof.

The Clostridium botulinum strain produces immunologically distinct neurotoxins (types A-G). The molecular masses of type A-G neurotoxin (NTX) are about 150 kDa (7S). In culture media and foods with acidic conditions, NTX combines with non-toxic components to form large complexes called progenitor toxins (PTX). PTX with molecular weights of 900 kDa (19S), 500 kDa (16S) and 300 kDa (12S) are found. 12S toxin consists of NTX and a nontoxic nonhemagglutinin (named NTNH) having no hemagglutinin activity, and 19S and 16S toxin consists of NTX, NTNH and hemagglutinin. Type A strains produce three types of toxins (19S, 16S and 12S). Type B, C and D strains produce 16S and 12S toxins. Under alkaline conditions, PTX dissociates into NTX and non-toxic components, 19S and 16S toxins dissociate into NTX and NTNH and non-toxic components (complex) of hemagglutinin, and 12S toxins dissociate into NTX and NTNH. In the present specification, the botulinum toxin may be of any of the above-described forms, for example, may have a molecular weight of 19S, 16S, 12S, or 7S.

Botulinum toxin includes botulinum toxin derivatives. The botulinum toxin derivative may be a part of a natural botulinum toxin having activity or a recombinant native botulinum toxin or a derivative comprising one or more chemical or functional modifications on a partial chain. For example, the botulinum toxin derivative may be a modified toxin having one or more amino acids deleted, modified or substituted. The botulinum toxin may be a recombinant peptide, a fusion protein, or a hybrid neurotoxin prepared, for example, from subunits or domains of different toxin serotypes. The botulinum toxin may also be a part of a whole molecule having a toxin activity, and may be used in combination or as part of a conjugated molecule, for example, a fusion protein.

In one aspect, the botulinum toxin is a botulinum toxin that does not comprise a chemical or functional modification, e.g., a natural botulinum toxin or a recombinant prototoxin or a portion thereof, e.g., a complexed botulinum toxin (eg 19S toxin) or a non- complexed botulinum toxin (eg, 7S toxin).

The term 'immunotherapeutic agent' refers to a compound, composition or treatment that indirectly or directly enhances, stimulates or increases the body's immune response to cancer cells and/or reduces the side effects of other anticancer therapies. Non-limiting examples of immunotherapeutic agents include cytokines, cancer vaccines, monoclonal antibodies, non-cytokine adjuvants, immune cells (T cells, NK cells, dendritic cells, B cells, etc.), immune checkpoint inhibitors, and the like.

The term 'combination' refers to any form of two or more different therapeutic agents such that a second therapeutic agent is administered while the previously administered therapeutic agent is still effective in the body. For example, two therapeutic agents are simultaneously effective in a subject, which may include a synergistic effect of the two therapeutic agents. The different therapeutic agents may be administered simultaneously or sequentially as a single agent or as separate agents.

'Administering' or 'administering' refers to a step of providing a pharmaceutical composition or an active ingredient to a subject. The pharmaceutical composition may be administered via a variety of suitable routes.

"Pharmaceutical composition" means a formulation containing an active ingredient. The term 'agent' means that in addition to the active ingredient (eg botulinum toxin), at least one additional ingredient, for example albumin (human serum albumin or recombinant human albumin) and/or sodium chloride, is present in the pharmaceutical composition. A pharmaceutical composition is a formulation suitable for administration to a subject, such as a human patient. The pharmaceutical composition may be in lyophilized form, for example a solution formed after reconstitution of the lyophilized pharmaceutical composition with saline or water, or in the form of a solution that does not require reconstitution. Pharmaceutical compositions may be liquid or solid. The pharmaceutical composition may be free of animal-derived proteins and/or albumin.

'therapeutically effective amount' means the level, amount or concentration of a pharmaceutical composition comprising an agent, e.g., botulinum toxin or botulinum toxin, required to treat a disease, disorder or condition without causing significant adverse or adverse side effects do.

'Treat', 'treating' or 'treatment' refers to, for example, healing of wounded or damaged tissue, or altering, altering, enhancing, ameliorating, ameliorating and improving an existing or perceived disease, disorder or condition. Alleviation or reduction (including partial reduction, significant reduction, near complete reduction and complete reduction), resolution or prevention (temporary or permanent) of a disease, disorder or condition, which results in the achievement of the desired therapeutic outcome by beautifying means As used herein, 'treatment' is a concept including prevention. By 'prevention' is meant delaying the onset of a disease, disorder or condition. Prevention may be considered complete if the onset of the disease, disorder or condition is delayed for a predetermined period.

In one aspect, 'treatment' refers to the treatment of a disease, disorder, or medical condition in a patient, such as a mammal (especially a human), comprising one or more of the following:

(a) preventing the occurrence of said disease, disorder, or medical condition, i.e., preventing recurrence of said disease or medical condition, or prophylactic treatment of a patient pre-disposed to said disease or medical condition;

(b) ameliorating said disease, disorder, or medical condition, i.e., eliminating or regressing said disease, disorder, or medical condition in a patient, including antagonizing the effect of another therapeutic agent;

(c) suppressing the disease, disorder, or medical condition, ie, slowing or arresting the development of the disease, disorder, or medical condition in a patient; or

(d) alleviating the symptoms of the disease, disorder, or medical condition in the patient.

The present disclosure is based on the discovery that a botulinum toxin or a composition comprising the same can be useful as a therapeutic agent for cancer in combination with an immunotherapeutic agent.

The present disclosure is based on the discovery that a botulinum toxin or a composition comprising the same can be useful as a therapeutic agent for metastatic cancer in combination with an immunotherapeutic agent.

In one embodiment, the botulinum toxin can be used without limitation of serotype, type A (BoNT/A), B (BoNT/B), C (BoNT/C), D (BoNT/D), E (BoNT/E) ), F(BoNT/F), G(BoNT/G), H(BoNT/H), X(BoNT/X), Enterococcus sp. botulinum toxin J (eBoNT/J) and mosaic botulinum toxin and/or variants thereof It may be selected from the group consisting of. Examples of mosaictoxins include BoNT/DC, BoNT/CD and BoNT/FA. For example, type A can be used.

In one aspect, the botulinum toxin is selected from various BoNT/A subtypes, eg, A1, A2, A3, A4, A5, A6, A7, A9, A10; BoNT/B subtypes such as B1, B2, B3, B4, B5, B6, B7, B8, Bnp and Bbv; BoNT/C subtypes such as C and CD; BoNT/D subtypes such as D and DC; BoNT/E subtypes such as E1, E2, E3, E4, E5, E6, E7, E8, E9; BoNT/F subtypes such as F1, F2, F3, F4, F5, F6, F7; and from the BoNT/G subtype, eg, subtype G. BoNT subtypes include chimeric BoNTs, such as BoNT/DC, BoNT/CD, BoNT/FA, and the like.

In one embodiment, the immunotherapeutic agent may be any substance or therapy known in the art, and may include cytokines, cancer vaccines, oncolytic viruses, monoclonal antibodies, non-cytokine adjuvants, immune cells (T cells, NK cells, dendritic cells, B cells, etc.), and immune checkpoint inhibitors. In an embodiment, the immunotherapeutic agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors include peptides, antibodies, nucleic acid molecules and small molecules. For example, an immune checkpoint inhibitor may be administered to enhance the proliferative, migratory, persistence and/or cytotoxic activity of CD8+ T cells in a subject, and particularly tumor invasiveness of CD8+ T cells in a subject. Typically, checkpoint inhibitors are activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PD-1), or the killer cell immunoglobulin-like receptor (KIR) family. antagonists that block immunosuppressive receptors expressed by NK cells, such as various members of It is a blocking antagonist. For example, an immune checkpoint inhibitor is an antibody or antigen-binding fragment thereof. Specifically, the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-IDO1 antibody. antibody, anti-TIGIT antibody, anti-B7H3 antibody, anti-B7H4 antibody, anti-BTLA antibody, and anti-B7H6 antibody, and antigen-binding fragment thereof. More specifically, immune checkpoint inhibitors include Ipilimumab (Yervoy®, BMS/Ono), Tremelimumab (AstraZeneca), atezolizumab (Tecentric®, Roche), and nivolumab (nivolumab) (Opdivo®, BMS/Ono), Pembrolizumab (Keytruda®, MSD), Avelumab (Bavencio®, Pfizer/Merck Germany), Durvalumab ( Imfinzi®, AstraZeneca/MedImmune), and antigen-binding fragments thereof may be at least one selected from, but not limited to.

In one aspect, the cancer may be exosome-mediated.

Exosomes are a type of extracellular vesicles, and have a size of approximately 100 nm in diameter. They are surrounded by a lipid bilayer and are secreted by most cells. Exosomes are generated as an intraluminal membrane vesicle (ILV) within the endocytic system and are secreted during the fusion of the cell membrane and the multivesicular body (MVB). It has been reported that exosomes contribute to maintaining tissue homeostasis by regulating cell-cell communication. Exosomes released from cancer cells are involved in cancer progression. Proliferative cancer cells (i) expand cancer tissue using angiogenesis, (ii) acquire the ability to migrate and invade, and (iii) acquire the ability to evade attack from immune cells, and ultimately metastatic lesion , and exosomes may be involved in each of these processes.

In one aspect, the cancer may be a solid tumor.

Non-limiting examples of solid cancer include breast cancer, lung cancer, head or neck cancer, colorectal cancer, esophageal cancer, laryngeal cancer, stomach cancer, liver cancer, pancreatic cancer, bone cancer, skin cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, perianal cancer, Colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, small intestine cancer, endocrine adenocarcinoma, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer , chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brain tumor, such as glioma (eg, astrocytoma) , glioblastoma, oligodendroglioma, ependymoma, germ cell tumor, meningioma, brainstem glioma, pituitary adenoma, schwannoma, congenital tumor, craniopharyngoma, metastatic brain tumor. For example, solid cancer may be at least one selected from the group consisting of melanoma, colon cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer, and stomach cancer, but is not limited thereto.

In one aspect, the cancer may be metastatic cancer.

In one aspect, the composition can be used in subjects with increased exosome secretion.

In one aspect, the composition comprising botulinum toxin may further comprise one or more pharmaceutically acceptable excipients or additives.

Pharmaceutically acceptable excipients or additives include stabilizers, ionic compounds, surfactants, buffers, lyophilization protectants, or combinations thereof, such as amino acids (eg methionine), salts (eg NaCl). ), buffers, nonionic surfactants (e.g. polysorbate, e.g. polysorbate 20), sugars (e.g. disaccharides such as sucrose), sugar alcohols (e.g. sorbitol), or their It can be a combination.

The composition may not contain albumin or animal-derived components or polysaccharides.

Said compositions include, for example, those disclosed in WO2009-008595 A1 or WO2012-134240 A2, the contents of which are incorporated herein by reference in their entirety.

The compositions include, without limitation, commercially available pharmaceutical compositions containing botulinum toxin, such as, for example, BOTOX® (botulinum toxin type A neurotoxin complex with human serum albumin and sodium chloride) (Allergan, Inc.) (Allergan, Inc.: Irvine, CA, USA)), DYSPORT® (a powder reconstituted with 0.9% sodium chloride prior to use, Clostridium Botulinum Type A Toxin hemaggluti with human serum albumin and lactose in the formulation. nin complex) (Ipsen Limited (Berkshire, UK)), MYOBLOC™ (injection solution containing botulinum toxin type B, human serum albumin, sodium succinate, and sodium chloride (approximately pH 5.6)) (Solstice Neuroscience) Seeds, Inc. (Solstice Neurosciences, Inc.: South San Francisco, CA)), Medytoxin® (a freeze-dried powder formulation containing Clostridium botulinum toxin type A, human serum albumin and sodium chloride) (Medytox, Korea) , Innotox® (Liquid formulation containing Clostridium botulinum toxin 　 type A, L-methionine, polysorbate 20 　, sodium chloride and water for injection) (Medytox, Korea), Coretox® (Clostridium botulinum toxin type A) (150 kD), polysorbate 20, lyophilized powder formulation containing sucrose and sodium chloride) (Medytox, Korea). In an embodiment, the composition may be Medytoxin®, Innotox®, Coretox®, or one or more combinations thereof, but is not limited thereto.

In one embodiment, the composition may be formulated in any form, such as a solid or liquid formulation, for example, a lyophilized powder, a liquid, or a pre-filled syringe formulation.

In one aspect, the composition may be a formulation for local administration. For example, topical administration may be intratumoral or precancerous tissue administration or peritumoral administration, and is typically accomplished by intratumoral injection or peritumoral injection of an aqueous botulinum toxin solution. can For example, the formulation can be administered subcutaneously or into a soft tissue, eg, intramuscularly. The composition can inhibit tumor growth or inhibit tumor development at a location away from the location by intratumoral or peripheral administration at a specific location, and the composition can exhibit systemic effects when administered locally.

In one aspect, a therapeutically effective amount of botulinum toxin is about 0.01 U/kg to about 100 U/kg, about 0.1 U/kg to about 100 U/kg, about 0.2 U/kg to about 100 U/kg, about 0.2 U/kg kg to about 50 U/kg, from about 0.2 U/kg to about 30 U/kg, from about 0.2 U/kg to about 10 U/kg, from about 0.2 U/kg to about 1 U/kg. In another aspect, based on an adult weighing 60 kg, from about 1 U to about 10,000 U, from about 1 U to about 5,000 U, from about 1 U to about 2,500 U, from about 1 U to about 1,000 U, from about 1 U to about 500 U, about 1 U to about 300 U, about 1 U to about 200 U, about 10 U to about 200 U, about 10 U to about 100 U, about 10 U to about 50 U.

As used herein, the term "unit", "unit(s)", or "U" refers to a dose of LD ₅₀ , defined as the amount of botulinum toxin that killed 50% of mice receiving a botulinum toxin injection. As such, it is used interchangeably within one product.

In one aspect, the botulinum toxin may be administered in single or multiple treatment sessions. Dosages may be administered as single or divided injections at the site of injection. In multiple treatment sessions, botulinum toxin may be administered at intervals of up to 6 months. In the multiple treatment session, the administration interval of the botulinum toxin includes the first treatment and the second treatment, and the dose of the second treatment may be less than, more than, or the same as the dose of the first treatment.

The dose, administration time, administration method, administration period or interval, etc. of the botulinum toxin may be appropriately selected and used by a person skilled in the art according to the patient's weight, age, sex, health condition, severity of disease, and the like.

In one aspect, the botulinum toxin may be administered concurrently or sequentially with the immunotherapeutic agent.

In one aspect, the botulinum toxin may be administered with the immunotherapeutic agent as a single agent or as separate agents.

In one embodiment, the immunotherapeutic agent is an oral formulation such as a solution, suspension, powder, granule, tablet, capsule, pill, extract, emulsion, syrup, aerosol, etc., according to a conventional method for each purpose of use, sterile injection It can be formulated and used in various forms such as parenteral formulations such as solution injections. The immunotherapeutic agent may be administered orally or parenterally via various routes including intravenous, intraperitoneal, subcutaneous, intradermal, intramuscular, spinal, spinal, or rectal administration or injection. The dosage may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, administration period or interval, excretion rate, constitutional specificity, properties of the preparation, severity of disease, etc. It may be appropriately selected by a person skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, but is not limited thereto, and may be administered in divided doses from once to several times a day.

In one aspect, a method of treating exosome-mediated cancer may comprise the steps of:

1) quantifying the exosomes secreted from the biological sample obtained from the subject; and,

2) administering a therapeutically effective amount of a botulinum toxin to a subject with increased exosome secretion in combination with an immunotherapeutic agent, either as a single agent or as a separate agent, simultaneously or sequentially.

The secreted exosomes can be separated and quantified from a biological sample of a subject, for example, blood according to a conventional method known in the art, and there is no particular limitation on the type of sample, separation and quantification method.

The subject to which the botulinum toxin or a composition comprising the same is administered may include, without limitation, a human or an animal, for example, a human, a pig, a dog, a cat, a cow, a horse, a rat, and the like.

Hereinafter, the present disclosure will be described in more detail with reference to examples, but this is only for explaining the present disclosure and is not intended to limit the scope in any way.

Example 1: Anticancer efficacy test of botulinum toxin and anti-PD-1 antibody against mouse malignant melanoma transplantation model

Male 6-week-old C57BL/6 (place of purchase: Orient Bio) mice were purchased, acclimatized and quarantined for 1 week, and 7-week-old mice were used for the experiment. For feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the period of acclimatization, quarantine, and experiment, the mouse was set at a temperature of 23±3 ℃, relative humidity of 55±15%, illumination time of 12 hours (8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination intensity of 150 to 300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

A B16-F10 mouse malignant melanoma cell line (KCLB No.: 80008) was obtained from the Korean Cell Line Bank (KCLB). B16-F10 cells were cultured in DMEM medium (CAT No.: 11965-092, Gibco) containing 10% FBS (CAT No.: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No.: 15140-122, Gibco). ) at 37 °C, 5% CO ₂ Conditions were incubated in an incubator. Using a syringe, 0.1 ml of 5×10 ⁵ cells were transplanted into the right flank of anesthetized C57BL/6 mice.

As a vehicle, sterile physiological saline (Lot No.: M8T7AF3, Daehan Pharmaceutical Co., Ltd.) used for botulinum toxin preparation was used, and 100 units Coretox (Lot No.: NSA19007A, Medytox) was used as the botulinum toxin. Anti-PD-1 antibody (Clone: RMP1-14, Cat No.: BE0146, Lot No.: 760220J1) was purchased from Bio X cell and used.

Each test substance was prepared and administered according to Table 1 below. To a 100 units vial of botulinum toxin, 6.667 ml of sterile physiological saline was added to prepare a concentration of 15 units/ml, and administered intratumorally at a dose of 1 ml/kg and 15 units/kg. The anti-PD-1 antibody was prepared at a final concentration of 1 mg/ml using sterile PBS (Cat No.: 10010, Gibco) on the day of administration, and administered intraperitoneally at a dose of 5 ml/kg, 5 mg/kg.

On the 8th day after transplantation of mouse malignant melanoma, the body weight and tumor size were measured, and the mice were randomly assigned to each group at an average size of about 33 mm 3 without any difference between the test groups. Vehicle and botulinum toxin were administered intratumorally according to Table 1 based on body weight on day 8 after tumor implantation. Anti-PD-1 antibody was administered intraperitoneally on days 8, 11, 14, and 17 of tumor implantation according to Table 1. Tumor size was measured twice a week after tumor transplantation. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the following equation using the measurements.

Tumor volume (mm3)=(W ² ×L)/2

W (mm) = width (short axis of tumor), L (mm) = length of tumor (long axis of tumor)

Tumor growth inhibition (TGI; tumor growth inhibition) (%) = (1- Mean tumor size _{test group} / Mean tumor size _vehicle ) × 100

army number of animals substance to be administered Volume route of administration Dosing frequency One 9 vehicle 1 ml/kg, 5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation)
2 times a week, 4 times in total 2 10 botulinum toxin 15 units/kg within the tumor Once (8 days after tumor transplantation) 3 10 anti-PD-1 antibody 5 mg/kg intraperitoneal 2 times a week, 4 times in total 4 9 botulinum toxin +
anti-PD-1 antibody 15 units/kg;
5 mg/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total

On day 19 after tumor implantation, all animals were euthanized before the tumor size reached 2000 mm 3 . After completion, the tumor tissue was excised and the weight was measured, and the number of T cells in the tumor tissue was measured using a flow cytometer (FACSVerse, BD). For T cell analysis in tumor tissue, the tumor tissue is separated into single cells using a gentle MACS C tube (Cat No.: 130-093-237, Miltenyi Biotec), and then LIVE/DEAD Fixable Aqua Dead Cell Stain (Cat No.: L34957, Invitrogen). ), Fc block (Cat No.: 553142, BD), CD45-PE-Cy7 (Cat No.: 25-0451-82, eBioscience), TCRbeta-FITC (Cat No.: 109206, Biolegend), CD4-pacific blue (Cat No.: 100428, Biolegend) and CD8a-PerCP-Cy5.5 (Cat No.: 100734, Biolegend) antibodies were stained and analyzed by flow cytometry. The number of CD4+ and CD8+ T cells in the tumor was calculated based on the tumor weight, and the ratio indicated the ratio of CD45 ⁺ TCRbeta ⁺ CD4 ⁺ and CD45 ⁺ TCRbeta ⁺ CD8a ⁺ T cells based on viable cells.

Exosomes in blood were isolated using Exoquick exosome precipitation solution (Cat No.: EXOQ20A-1, Lot No.: 200107-001, System Biosciences), and ExoELISA-ULTRA Complet Kit (Cat No.: EXEL-ULTRA-CD63- 1, Lot No.: 200226-002, System Biosciences).

Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) was used for statistical analysis. For parametric data, a two-tailed t -test was performed, and for non-parametric data, statistical analysis by Mann-Whitney test was performed at the significance level p = 0.05.

The measurement results of the tumor growth inhibition rate are shown in Table 2 and FIG. 1 .

army
substance to be administered Average tumor size ¹⁾
(average ㎣±SEM) average tumor weight ²⁾
(mean g±SEM) TGI (%) ¹⁾ One
vehicle 1684±284 2.02±0.38 0 2
botulinum toxin 1433±348 1.29±0.35 15 3
anti-PD-1 antibody 1415±121 1.83±0.18 16 4
botulinum toxin +
anti-PD-1 antibody 489±165 ^{**, †, ‡} 0.46±0.1 2 ^{**, †, ‡} 71

¹⁾ Results on day 18 after tumor transplantation, ²⁾ Results on day 19 after tumor transplantation, ^** p <0.01, vs. vehicle. ^† p < 0.05, vs. botulinum toxin, ^‡ p <0.001, vs. Anti-PD-1 antibody, two-tailed t -test.

As shown in Table 2 and FIG. 1 , on the 18th day after transplantation of mouse malignant melanoma, an average tumor size of 1433 mm 3 and a tumor growth inhibition rate of 15% were exhibited by intratumoral administration of botulinum toxin alone, and anti-PD-1 antibody showed an average of 1415 mm 3 and 16% tumor growth inhibition, but there was no statistical significance compared to the vehicle-administered group. In contrast, when the botulinum toxin and the anti-PD-1 antibody were administered in combination, the average tumor size was 489 mm 3 and the tumor growth inhibition rate was 71%. . In addition, even when tumors were excised and weighed on the 19th day after tumor transplantation, when the botulinum toxin and anti-PD-1 antibody were administered in combination, the vehicle-administered group, the botulinum toxin, and the anti-PD-1 antibody alone group administered the tumor significantly. weight was reduced.

The results of extracting the tumor tissue and analyzing the infiltrated CD4+ and CD8+ T cells in the tumor are shown in FIG. 2 . As shown in FIG. 2 , when botulinum toxin was administered alone, the number of CD8+ T cells in the tumor tends to increase compared to the vehicle-treated group. The ratio of CD4+ and CD8+ T cells was not significantly different from the vehicle-treated group when botulinum toxin and anti-PD-1 antibody were administered alone, but compared to the vehicle group in the group treated with botulinum toxin and anti-PD-1 antibody together It can be seen that a significant proportion of CD4+ and CD8+ T cells were infiltrated into the tumor. This was significantly increased compared to the anti-PD-1 antibody alone treatment group. In the group treated with botulinum toxin and anti-PD-1 antibody, the T cells in the tumor were significantly increased due to the synergy between the two substances, which could explain the significant decrease in tumor growth.

The results of measuring the number of exosomes in the blood are shown in FIG. 3 . As shown in FIG. 3 , the number of exosomes in the blood showed a tendency to decrease in the botulinum toxin administration group compared to the vehicle treatment group, and significantly decreased in the anti-PD-1 antibody and botulinum toxin + anti-PD-1 antibody administration group. became In addition, the number of exosomes in the blood was significantly reduced in the group administered with the botulinum toxin and the anti-PD-1 antibody in combination, compared to the group administered with the botulinum toxin and the anti-PD-1 alone.

Example 2: Anticancer efficacy test of botulinum toxin and anti-PD-1 antibody by dose in mouse malignant melanoma transplantation model

Male 6-week-old C57BL/6 (place of purchase: Orient Bio) mice were purchased, acclimatized and quarantined for 1 week, and 7-week-old mice were used for the experiment. For feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the acclimatization, quarantine and experimental period, the mouse was set at a temperature of 23±3 ℃, relative humidity of 55±15%, illumination time of 12 hours (8 am to 8 pm), ventilation frequency 15 times/hour, and illumination intensity of 150 to 300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

A B16-F10 mouse malignant melanoma cell line (KCLB No.: 80008) was obtained from the Korean Cell Line Bank (KCLB). B16-F10 cells were cultured in DMEM medium (CAT No.: 11965-092, Gibco) containing 10% FBS (CAT No.: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No.: 15140-122, Gibco). ) at 37 °C, 5% CO ₂ Conditions were incubated in an incubator. Using a syringe, 0.1 ml of 5×10 ⁵ cells were transplanted into the right flank of anesthetized C57BL/6 mice.

As a vehicle, only botulinum toxin was excluded from the botulinum toxin product, and the same placebo was used for the rest of the excipient ingredients, and 100 units of botulinum toxin Coretox (Lot No.: NSA19007A, Medytox) was used. Anti-PD-1 antibody (Clone: RMP1-14, Cat No.: BE0146, Lot No.: 780120J2) was purchased from Bio X cell and used. Each test substance was prepared and administered according to Table 3 below. Sterile physiological saline was added to 100 units vial of botulinum toxin to prepare concentrations of 1, 3, and 10 units/ml, and administered intratumorally at doses of 1 ml/kg, 1, 3, and 10 units/kg. The anti-PD-1 antibody was prepared at a final concentration of 1 mg/ml using sterile PBS (Cat No.: 10010, Gibco) on the day of administration, and administered intraperitoneally at a dose of 5 ml/kg, 5 mg/kg.

On the 8th day after transplantation of malignant melanoma in mice, tumor size was measured, and mice were randomly assigned to 12 mice according to the test group without any difference between the test groups. On the 8th day after tumor transplantation, vehicle and botulinum toxin were administered intratumorally according to the composition of the test group. Anti-PD-1 antibody was administered intraperitoneally twice a week for a total of 4 times according to Table 3. Tumor size was measured twice a week after tumor transplantation. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the equation shown in Example 1 using the measurements.

army number of animals substance to be administered Volume route of administration Dosing frequency One 12 vehicle 1 ml/kg within the tumor Once (8 days after tumor transplantation) 2 12 1 U/kg botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 3 12 3 U/kg botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 4 12 10 U/kg botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 5 12 1 U/kg botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total 6 12 3 U/kg botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total 7 12 10 U/kg botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total 8 12 vehicle +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total

After tumor transplantation, measurements were carried out until the tumor size of each mouse reached 2000 mm 3 , and subjects exceeding the euthanasia standard (2000 mm 3 ) were immediately euthanized and recorded as death.

Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. . A two-tailed t-test was performed for tumor size, and the survival rate was analyzed by Mantel-Cox log grading at significance level p=0.05.

The tumor size measurement results are shown in FIG. 4 (* p <0.05, ** p <0.01, *** p <0.001). As shown in FIG. 4 , in a mouse malignant melanoma model, when botulinum toxin was administered in a single tumor at a dose of 1, 3, or 10 U/kg, significant tumor growth inhibition was observed in the 10 U/kg administration group on the 14th day. On the 18th day after tumor transplantation, no significant tumor growth inhibitory effect was observed in the test group administered with botulinum toxin alone and the test group administered with vehicle + 5 mg/kg anti-PD1 antibody compared to the vehicle administration group. However, in the test group administered with 3 or 10 U/kg botulinum toxin and 5 mg/kg anti-PD1 antibody, a significant tumor growth inhibitory effect was observed compared to the vehicle-administered group.

The results of measuring the survival rate are shown in FIG. 5 . As shown in FIG. 5 , the mouse survival rate was significantly increased in the test group in which 3 or 10 U/kg botulinum toxin and 5 mg/kg anti-PD1 antibody were co-administered. A significant increase in survival was also observed in the vehicle + 5 mg/kg anti-PD1 administration group. From the above results, it was confirmed that when co-administered with an anti-PD1 antibody at a dose of 3 U/kg or more of botulinum toxin in a mouse melanoma model, there was a tumor growth inhibitory effect.

Example 3: Anti-PD-1 antibody co-administration anticancer efficacy test of complexed botulinum toxin and non-complexed botulinum toxin in mouse malignant melanoma transplantation model

Male 6-week-old C57BL/6 (place of purchase: Orient Bio) mice were purchased, acclimatized and quarantined for 1 week, and 7-week-old mice were used for the experiment. For feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the period of acclimatization, quarantine, and experiment, the mouse was set at a temperature of 23±3 ℃, relative humidity of 55±15%, illumination time of 12 hours (8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination intensity of 150 to 300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

A B16-F10 mouse malignant melanoma cell line (KCLB No.: 80008) was obtained from the Korean Cell Line Bank (KCLB). B16-F10 cells were cultured in DMEM medium (CAT No.: 11965-092, Gibco) containing 10% FBS (CAT No.: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No.: 15140-122, Gibco). ) at 37 °C, 5% CO ₂ Conditions were incubated in an incubator. Using a syringe, 0.1 ml of 5×10 ⁵ cells were transplanted into the right flank of anesthetized C57BL/6 mice.

As a vehicle, in the non-complexed botulinum toxin product, only the botulinum toxin was excluded and the remaining excipient ingredients were the same placebo. 100 units Coretoxin (Lot No.: NSA19007A, Medytox) was used as the non-complexed botulinum toxin, and 100 units Medytoxin (Lot No.: TFAA20024, Medytox) was used as the complexed botulinum toxin. Anti-PD-1 antibody (Clone: RMP1-14, Cat No.: BE0146, Lot No.: 780120J2) was purchased from Bio X cell and used. Each test substance was prepared and administered according to Table 4 below. Sterile physiological saline was added to a vial of 100 units of botulinum toxin to prepare a concentration of 15 units/ml, and administered intratumorally at a dose of 1 ml/kg and 15 units/kg. The anti-PD-1 antibody was prepared at a final concentration of 1 mg/ml using sterile PBS (Cat No.: 10010, Gibco) on the day of administration, and administered intraperitoneally at a dose of 5 ml/kg, 5 mg/kg.

On the 8th day after transplantation of malignant melanoma in mice, tumor size was measured, and mice were randomly assigned to 12 mice according to the test group without any difference between the test groups. On the 8th day after tumor transplantation, vehicle and botulinum toxin were administered intratumorally according to the composition of the test group. Anti-PD-1 antibody was administered intraperitoneally twice a week for a total of 4 times according to Table 4. Tumor size was measured twice a week after tumor transplantation. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the equation shown in Example 1 using the measurements.

army number of animals substance to be administered Volume route of administration Dosing frequency One 12 vehicle 1 ml/kg within the tumor Once (8 days after tumor transplantation) 2 12 15 U/kg non-complexed botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 3 12 15 U/kg complexed botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 4 12 15 U/kg non-complexed botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 ml/kg within the tumor Once (8 days after tumor transplantation) 5 12 15 U/kg complexed botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total 6 12 vehicle +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal Once (8 days after tumor transplantation),
2 times a week, 4 times in total

On day 19 after tumor implantation, all animals were euthanized and terminated before tumor size reached 2000 mm 3 .

Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. . Tumor size was assessed using a two-tailed t-test at significance level p = 0.05.

The results are shown in FIG. 6 (* p <0.05, ** p <0.01). As shown in FIG. 6 , on day 19 after transplantation of mouse malignant melanoma, 15 U/kg of non-complexed botulinum toxin and complexed botulinum toxin were administered in combination with 5 mg/kg anti-PD1 antibody. Tumor growth inhibition was observed. No significant difference was observed between the non-complexed botulinum toxin and the test group in which the complexed botulinum toxin was administered alone. Also, no significant difference was observed between the non-complexed botulinum toxin + anti-PD1 antibody administration group and the complexed botulinum toxin + anti-PD1 antibody and administration group. From the above results, it was confirmed that both the non-complexed botulinum toxin and the complexed botulinum toxin had a tumor growth inhibitory effect regardless of the type of botulinum toxin when administered in combination with the anti-PD1 antibody.

Example 4: Anticancer efficacy test of botulinum toxin and anti-CTLA4 antibody against mouse lung tumor transplantation model

Male 6-week-old C57BL/6 (place of purchase: Orient Bio) mice were purchased, acclimatized and quarantined for 1 week, and 7-week-old mice were used for the experiment. For feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the period of acclimatization, quarantine, and experiment, the mouse was set at a temperature of 23±3 ℃, relative humidity of 55±15%, illumination time of 12 hours (8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination intensity of 150 to 300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

LLC1 mouse lung cancer cell line (ATCC No.: CRL-1642) was obtained from ATCC. LLC1 cells were cultured in DMEM medium (CAT No.: LM001-05, Welgene) containing 10% FBS (CAT No.: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No.: 15140-122, Gibco). 37 °C, 5% CO ₂ Conditions were cultured in an incubator. Using a syringe, 0.1 ml of 5×10 ⁵ cells were transplanted into the right flank of anesthetized C57BL/6 mice.

As a vehicle, only botulinum toxin was excluded from the botulinum toxin product, and the same placebo was used for the rest of the excipient ingredients, and 100 units Coretox (Lot No.: NSA19007A, Medytox) was used as the botulinum toxin. Anti-CTLA4 antibody (Clone: 9H10, Cat No.: BE0131, Lot No.: 704019A2) was purchased from Bio X cell and used. Each test substance was prepared and administered according to Table 5 below. Sterile physiological saline was added to a vial of 100 units of botulinum toxin to prepare a concentration of 15 units/ml, and administered intratumorally at a dose of 1 ml/kg and 15 units/kg. The anti-CTLA4 antibody was prepared at a final concentration of 1 mg/ml using sterile PBS (Cat No.: 10010, Gibco) on the day of administration, and administered intraperitoneally at a dose of 5 ml/kg and 5 mg/kg.

The tumor size was measured on the 8th day after transplantation of the mouse LLC1 lung tumor cell line, and 10 mice were randomly assigned to each group according to the test group so that there was no difference between the test groups. On the 8th day after tumor transplantation, vehicle and botulinum toxin were administered intratumorally according to the composition of the test group. Anti-CTLA4 antibody was administered intraperitoneally twice a week for a total of 4 times according to Table 5. Tumor size was measured twice a week after tumor transplantation. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the equation shown in Example 1 using the measurements.

army number of animals substance to be administered Volume route of administration Dosing frequency One 10 vehicle 1 ml/kg within the tumor Once (8 days after tumor transplantation) 2 10 15 U/kg botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 3 10 15 U/kg botulinum toxin +
5 mg/kg anti-CTLA4 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal 1 time (8 days after tumor transplantation),
2 times a week, 4 times in total 4 10 vehicle +
5 mg/kg anti-CTLA4 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal 1 time (8 days after tumor transplantation),
2 times a week, 4 times in total

On day 21 after tumor implantation, all animals were euthanized and terminated before tumor size reached 2000 mm 3 .

Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. . Tumor size was assessed using a two-tailed t-test at significance level p = 0.05.

The results are shown in FIG. 7 (* p <0.05, ** p <0.01, *** p <0.001). As shown in FIG. 7 , on the 21st day after mouse lung tumor transplantation, a significant tumor growth inhibitory effect was not observed in the 15 U/kg botulinum toxin or vehicle + 5 mg/kg anti-CTLA4 antibody group compared to the vehicle test group, but 15 A significant tumor growth inhibitory effect was observed in the U/kg botulinum toxin + 5 mg/kg anti-CTLA4 combination group compared to the vehicle test group. In addition, the 15 U/kg botulinum toxin + 5 mg/kg anti-CTLA4 administration group was significantly compared to the test group administered with 15 U/kg botulinum toxin alone and the vehicle + 5 mg/kg anti-CTLA4 antibody alone administration group The tumor volume was reduced. From the above results, it was confirmed that the tumor growth inhibitory effect by the co-administration of botulinum toxin and anti-CTLA4 antibody in the mouse LLC1 lung tumor model.

Example 5: Anticancer efficacy test of botulinum toxin and anti-PD-1 antibody against both mouse malignant melanoma transplantation model

Male 6-week-old C57BL/6 (place of purchase: Orient Bio) mice were purchased, acclimatized and quarantined for 1 week, and 7-week-old mice were used for the experiment. For the feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the period of acclimatization, quarantine, and experiment, the mouse was set at a temperature of 23±3 ℃, relative humidity of 55±15%, illumination time of 12 hours (8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination intensity of 150 to 300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

A B16-F10 mouse malignant melanoma cell line (KCLB No.: 80008) was obtained from the Korean Cell Line Bank (KCLB). B16-F10 cells were cultured in DMEM medium (CAT No.: 11965-092, Gibco) containing 10% FBS (CAT No.: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No.: 15140-122, Gibco). ) at 37 °C, 5% CO ₂ Conditions were incubated in an incubator. 0.1 mL of 5 x 10 ⁵ cells were transplanted into the right flank of anesthetized C57BL/6 mice using a syringe, and 5 days later, 0.1 mL of 2 ~ 2.5 x 10 ⁵ cells were transplanted into the left flank of an anesthetized C57BL/6 mouse using a syringe.

As a vehicle, only botulinum toxin was excluded from the botulinum toxin product, and the same placebo was used for the rest of the excipient ingredients, and 100 units of botulinum toxin Coretox (Lot No.: NSA20015, Medytox) was used. Anti-PD-1 antibody (Clone: RMP1-14, Cat No.: BE0146, Lot No.: 780120J3 or 798921J1C) was purchased from Bio X cell and used. Each test substance was prepared and administered according to Table 6 below. Sterile physiological saline was added to a vial of 100 units of botulinum toxin to a concentration of 15 units/mL, and administered intratumorally at a dose of 1 mL/kg and 15 units/kg. The anti-PD-1 antibody was administered intraperitoneally at a dose of 5 mg/kg using PBS (Cat No.: LB004-02, Welgene).

The experiment was repeated twice with the same test group and method. The tumor size was measured on the 8th day after transplantation of malignant melanoma in the right flank, and 12 mice were randomly assigned to each test group without any difference between the test groups. On day 8 after tumor implantation in the right flank, vehicle and botulinum toxin were administered into the tumor of the right flank according to the composition of the test group. Anti-PD-1 antibody was intraperitoneally administered twice a week for a total of 4 times according to the composition of the test group. After tumor transplantation, the size of the transplanted tumor on the right and left sides was measured twice a week. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the equation shown in Example 1 using the measurements.

army number of animals substance to be administered Volume route of administration Dosing frequency One 12 vehicle 1 ml/kg within the tumor Once (8 days after tumor transplantation) 2 12 15 U/kg botulinum toxin 1 ml/kg within the tumor Once (8 days after tumor transplantation) 3 12 vehicle +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal 1 time (8 days after tumor transplantation),
2 times a week, 4 times in total 4 12 15 U/kg botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 ml/kg,
5 ml/kg within the tumor,
intraperitoneal 1 time (8 days after tumor transplantation),
2 times a week, 4 times in total

On day 21 after right tumor transplantation, all animals were euthanized to end the experiment. After completion, the presence or absence of transplanted tumor tissue in the left flank of all subjects was observed, and the left tumor was excised and immune cells in the tumor tissue were analyzed using a flow cytometer (FACSVerse, BD). Immune cell analysis in the tumor tissue is performed by separating the tumor tissue into single cells using a gentle MACS C tube (Cat No.: 130-093-237, Miltenyi Biotec) and then using LIVE/DEAD Fixable Aqua Dead Cell Stain (Cat No.: L34957, Invitrogen). ), Fc block (Cat No.: 553142, BD), CD45-PE-Cy7 (Cat No.: 25-0451-82, eBioscience), TCRbeta-APC-eFluor780 (Cat No.: 47-5961-82, Invitrogen ), CD4-pacific blue (Cat No.: 100428, Biolegend), CD8a-PerCP-Cy5.5 (Cat No.: 100734, Biolegend), CD11b-A647 (Cat No.: 101218, Biolegend) antibody staining and flow cytometry Analyzed with an analyzer. The ratio of CD4+ and CD8a+ T cells and CD11b+ cells in the tumor represents the ratio of CD45 ⁺ TCRbeta ⁺ CD4 ^{+ ,} CD45 ⁺ TCRbeta ⁺ CD8a ⁺ , and CD45 ⁺ CD11b ⁺ cells based on viable cells.

After tumor transplantation, when the sum of the right and left tumor sizes of each mouse reached 2000 mm ³ , the subject was recorded as dead.

Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. . A two-tailed t -test was performed for tumor size, a Pearson chi-square test was performed for the left tumor formation rate, and a Mantel-Cox log grading was performed for survival rate at significance level p = 0.05.

In FIG. 8 , tumor growth of the right and left sides over time was expressed as tumor volume (mm ³ ) (* p <0.05, ** p <0.01, *** p <0.001). As shown in FIG. 8 , in a malignant melanoma model transplanted to the right and left, when botulinum toxin was administered at a dose of 15 U/kg into the right tumor and 5 mg/kg anti-PD-1 antibody was concurrently administered intraperitoneally It was observed that the size of the right tumor was significantly reduced compared to the vehicle, and significantly reduced compared to the test group in which botulinum toxin and anti-PD-1 antibody were administered alone, respectively. The size of the left tumor was significantly reduced in the test group in which botulinum toxin and anti-PD-1 antibody were co-administered compared to the vehicle-administered group.

Table 7 shows the presence or absence of left tumor formation (* p <0.05 vs. G3 anti-PD-1 antibody).

army substance to be administered Left flank tumor formation
Observed (number of horses) Not observed (number of horses)
One vehicle 18 4 2 15 U/kg botulinum toxin 21 3 3 vehicle +
5 mg/kg anti-PD-1 antibody 18 2 4 15 U/kg botulinum toxin +
5 mg/kg anti-PD-1 antibody 14 8*

As shown in Table 7, the formation rate of the left tumor was significantly decreased in the group administered with the botulinum toxin and the anti-PD-1 antibody in combination compared to the group administered with the anti-PD-1 antibody alone.

Figure 9 shows the survival rate of mice (** p <0.01). It was confirmed that the survival rate was significantly improved in the test group in which the botulinum toxin and the anti-PD-1 antibody were co-administered.

FIG. 10 shows the ratio of standard CD11b+ cells and CD4+ and CD8a+ T cells infiltrating the left untreated tumor. The proportion of measured cells was calculated using a two-tailed t-test ( p <0.05, ** p <0.05). As a result of extracting the transplanted tumor tissue from the left side and analyzing the infiltrated CD4+ and CD8a+ T cells and CD11b+ cells, the CD11b+ cell ratio in the botulinum toxin and anti-PD1 antibody combination group was significantly decreased compared to the vehicle administration group, CD4+ T cells were significantly increased compared to the group administered with the anti-PD-1 antibody alone, and the ratio of CD8a+ T cells was significantly increased in the group administered with the botulinum toxin and anti-PD-1 antibody compared to the group administered with the vehicle and the botulinum toxin alone .

From the above results, in both mice melanoma transplantation model, when botulinum toxin and anti-PD-1 antibody were co-administered, significant improvement in survival rate and growth inhibition of tumors administered with botulinum toxin as well as formation and growth inhibition of non-administered tumors It was confirmed that it was effective.

Example 6: Co-administration anticancer efficacy test of botulinum toxin and immunotherapy (anti-PD-1 and anti-CTLA4) in a mouse colorectal cancer transplantation model

After purchasing 6-week-old female C57BL/6 (orient bio) mice, acclimatization and quarantine for 1 week, 7-week-old mice were used for the experiment. For the feed, sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and negative water was fed freely by autoclaving tap water at high temperature. During the acclimatization, quarantine and experimental period, the mouse was set at a temperature of 23±3 ℃, relative humidity of 55±15%, illumination time of 12 hours (8 am to 8 pm), ventilation frequency 15 times/hour, and illumination intensity of 150 to 300 Lux. Raised under specific-pathogen-free conditions. This experiment was conducted after review and approval (A-2020-004) by the Animal Experimental Ethics Committee of Medytox Co., Ltd.

The mouse colorectal cancer MC38 cell line (ENH204-FP) was obtained from Kerafast. The MC38 cell line was treated with 10% FBS (CAT No.: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No.: 15140-122, Gibco), 10 mM HEPES (CAT No.: 15630-080, Gibco). , DMEM medium (CAT No.: 11965-092, Gibco) containing 0.1 mM MEM NEAA (CAT No.: 11140-050, Gibco), 1 mM sodium pyruvate (CAT No.: 11360-070, Gibco) Incubated in an incubator at 37 ℃, 5% CO ₂ conditions. Using a syringe, 0.1 mL of 2.5x10 ⁵ cells were transplanted into the right flank of anesthetized C57BL/6 mice.

As a vehicle, only botulinum toxin was excluded from the botulinum toxin product, and the same placebo was used for the rest of the excipient ingredients, and 100 units of botulinum toxin (Lot No.: NSA20015, Medytox) was used. Anti-PD-1 antibody (Clone: RMP1-14, Cat No.: BE0146, Lot No.: 798921J1C) and anti-CTLA4 antibody (Clone: 9H10, Cat No.: BE0131, Lot No.: 755620A2) are Bio X Cell was purchased and used. Each test substance was prepared and administered according to Table 6 below. Sterile physiological saline was added to a vial of 100 units of botulinum toxin to a concentration of 15 units/mL, and administered intratumorally at a dose of 1 mL/kg and 15 units/kg. Anti-PD-1 and anti-CTLA4 antibodies were administered intraperitoneally at a dose of 5 mg/kg using PBS (Cat No.: LB004-02, Welgene). Propranolol (Cat No.: P0884, Sigma) was prepared at 2 mg/mL using PBS and administered intraperitoneally at a dose of 10 mg/kg.

The tumor size was measured on the 7th day after transplantation of the mouse colon cancer cell line, and the mice were randomly assigned to each test group by 10 mice without any difference between the test groups. On the 7th day after tumor transplantation, vehicle and botulinum toxin were administered intratumorally according to the composition of the test group. In the case of anti-PD-1 and anti-CTLA4 antibodies, according to Table 8, 2 times a week were administered intraperitoneally for a total of 4 times, and propranolol was intraperitoneally administered a total of 8 times between 7 and 16 days after tumor transplantation, and a week after tumor transplantation. Tumor size was measured twice. The tumor size was measured with a vernier caliper and the tumor volume was calculated using the equation shown in Example 1 using the measurements.

army number of animals substance to be administered Volume route of administration Dosing frequency One 10 vehicle 1 ml/kg within the tumor Once (7 days after tumor transplantation) 2 10 15 U/kg botulinum toxin 1 ml/kg within the tumor Once (7 days after tumor transplantation) 3 10 vehicle +
5 mg/kg anti-PD-1 antibody 1 mL/kg,
5 mL/kg within the tumor,
intraperitoneal 1 time (7 days after tumor transplantation),
2 times a week, 4 times in total 4 10 vehicle +
5 mg/kg anti-CTLA4 antibody 1 mL/kg,
5 mL/kg within the tumor,
intraperitoneal 1 time (7 days after tumor transplantation),
2 times a week, 4 times in total 5 10 15 U/kg botulinum toxin +
5 mg/kg anti-PD-1 antibody 1 mL/kg,
5 mL/kg within the tumor,
intraperitoneal 1 time (7 days after tumor transplantation),
2 times a week, 4 times in total 6 10 15 U/kg botulinum toxin +
5 mg/kg anti-CTLA4 antibody 1 mL/kg,
5 mL/kg within the tumor,
intraperitoneal 1 time (7 days after tumor transplantation),
2 times a week, 4 times in total 7 10 vehicle +
10 mg/kg Propranolol 1 mL/kg,
5 mL/kg within the tumor,
intraperitoneal 1 time (7 days after tumor transplantation)
A total of 8 times (7-16 days after tumor transplantation) 8 10 10 mg/kg Propranolol +
5 mg/kg anti-PD-1 antibody 10 mL/kg,
5 mL/kg within the tumor,
intraperitoneal A total of 8 times (7-16 days after tumor transplantation)
2 times a week, 4 times in total

After tumor transplantation, measurements were carried out until the tumor size of each mouse reached 2000 mm ³ , and subjects exceeding the euthanasia standard (2000 mm ³ ) were immediately euthanized and recorded as death.

Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software (version 25.0, SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. . A two-tailed t -test was performed for tumor size, and a Mantel-Cox log grading for survival was performed at significance level p = 0.05.

11 and 13 show tumor growth over time as tumor volume (mm ³ ) (FIG. 11: ** p <0.01 vs. vehicle, FIG. 13: ** p <0.01, *** p <0.001 vs. vehicle). vehicle). On the 20th day after mouse colon cancer transplantation, significant tumor growth inhibition was observed in the group administered with 15 U/kg botulinum toxin and 5 mg/kg anti-PD-1 antibody compared to the group administered with vehicle, botulinum toxin, and anti-PD-1 alone ( Fig. 11). In addition, on the 20th day after colon cancer transplantation, significant tumor growth inhibition was observed in the group administered with 5 mg/kg anti-CTLA4 alone and the group administered with botulinum toxin and anti-CTLA4 in combination compared to the vehicle administration group ( FIG. 13 ).

12 and 14 show the survival rates of mice (Fig. 12: * p <0.05, ** p < 0.01 vs vehicle, ^a p <0.05 vs. anti-PD-1, Fig. 14: (*** p < 0.001 vs vehicle, ^a p <0.05 vs. anti-CTLA4), the mouse survival rate was significantly increased in the test group administered with botulinum toxin and anti-PD1 antibody compared to the vehicle and anti-PD-1 antibody alone group, 10 In the test group in which mg/kg propranolol and anti-PD-1 antibody were administered in combination, the survival rate was significantly increased compared to the vehicle and anti-PD-1 antibody alone group. In the group administered with the botulinum toxin and anti-CTLA4 antibody, a significantly increased survival rate was observed compared to the group administered with the vehicle and the anti-CTLA4 antibody alone ( FIG. 14 ).

From the above results, it was confirmed that the synergistic effect and survival rate improvement effect of botulinum toxin and anti-PD-1 or anti-CTLA4 immunotherapy in mouse colorectal cancer model were co-administered. In addition, the synergistic effect of the beta-blocker (propranolol) was confirmed when administered in combination with the immune-cancer agent, and when the botulinum toxin and the immune-cancer agent were administered in combination, the effect was confirmed to be equal to or greater than that of the beta-blocker and the immune-cancer agent combination group.

Claims (23)

A composition for treating cancer in combination with an immunotherapeutic agent, comprising botulinum neurotoxin as an active ingredient. The composition of claim 1 , wherein the botulinum toxin is serotype A, B, C, D, E, F, G, H, or a combination thereof. The composition of claim 1 or 2, wherein the immunotherapeutic agent is an immune checkpoint inhibitor. The composition of claim 3 , wherein the checkpoint inhibitor is a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a VISTA antagonist, a BTLA antagonist, or a combination thereof. 5. The method of claim 4, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, an anti-TIM3 antibody, an anti-LAG3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti - At least one composition selected from the group consisting of a BTLA antibody or antigen-binding fragment thereof. The composition of claim 5, wherein the checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof. 7. The composition of any one of claims 1-6, wherein the cancer is exosome-mediated. The composition according to any one of claims 1 to 7, wherein the cancer is a solid tumor. The composition according to claim 8, wherein the solid cancer is at least one selected from the group consisting of melanoma, colorectal cancer, breast cancer, lung cancer, pancreatic cancer, prostate cancer, bladder cancer and stomach cancer. 10. The composition according to any one of claims 1 to 9, wherein the cancer is metastatic cancer. 11. A composition according to any one of claims 1 to 10 for use in subjects with increased exosome secretion. 12. The composition according to any one of claims 1 to 11, further comprising a pharmaceutically acceptable excipient or additive. The composition of claim 12, wherein the pharmaceutically acceptable excipient or additive is a stabilizer, an ionic compound, a surfactant, a buffer, a lyophilization protectant, or a combination thereof. The composition of claim 13, wherein the pharmaceutically acceptable excipient or additive is an amino acid, a salt, a buffer, a nonionic surfactant, a sugar, a sugar alcohol, or a combination thereof. 15. The composition according to any one of claims 12 to 14, wherein the composition is free of albumin or animal-derived components or polysaccharides. 16. The composition according to any one of claims 12 to 15, in the form of a lyophilized powder, liquid, or prefilled syringe formulation. 17. A composition according to any one of claims 12 to 16 for topical administration. 18. The composition of claim 17 for intratumoral or peritumoral administration. 19. The composition according to any one of claims 1 to 18, comprising 0.01 to 100 units/kg of botulinum toxin. 20. The composition of any one of claims 1-19, wherein the botulinum toxin is administered simultaneously or sequentially with the immunotherapeutic agent. 21. The composition of any one of claims 1-20, wherein the botulinum toxin is administered as a single agent or as a separate agent from the immunotherapeutic agent. 22. The composition of any one of claims 1-21, wherein the immunotherapeutic agent is administered parenterally. 23. The composition of claim 22, wherein the immunotherapeutic agent is administered or injected locally, intravenously, intraperitoneally, subcutaneously, intradermally, intramuscularly, spinally, intrathecally or intrarectally.

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