CANCER THERAPEUTIC AGENT

Abstract

A cancer therapeutic agent used in combination with an immunotherapeutic agent, for example, an immune checkpoint inhibitor, is disclosed.

Claims

1-23. (canceled)

24. A method for treating cancer comprising administering a therapeutically effective amount of botulinum neurotoxin and an immunotherapeutic agent to a subject in need thereof.

25. The method of claim 24, wherein the immunotherapeutic agent is an immune checkpoint inhibitor.

26. The method of claim 25, wherein the immune checkpoint inhibitor is a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, an LAG3 antagonist, a TIGIT antagonist, a VISTA antagonist, a BTLA antagonist, or a combination thereof.

27. The method of claim 26, wherein the immune checkpoint inhibitor is at least one selected from the group consisting of 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-BTLA antibody, or an antigen-binding fragment thereof.

28. The method of claim 27, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA4 antibody, or an antigen-binding fragment thereof.

29. The method of claim 24, wherein cancer is exosome-mediated.

30. The method of claim 24, wherein cancer is a solid tumor.

31. The method of claim 30, wherein the solid tumor 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.

32. The method of claim 24, wherein cancer is metastatic cancer.

33. The method of claim 24, wherein the subject is a subject having increased secretion of exosomes.

34. The method of claim 24, a dosage form of botulinum neurotoxin comprises a pharmaceutically acceptable excipient or additive.

35. The method of claim 34, wherein the pharmaceutically acceptable excipient or additive is a stabilizer, an ionic compound, a surfactant, a buffer agent, a lyoprotectant, or a combination thereof.

36. The method of claim 35, wherein the pharmaceutically acceptable excipient or additive is an amino acid, a salt, a buffer solution, a non-ionic surfactant, a sugar, a sugar alcohol, or a combination thereof.

37. The method of claim 35, wherein the dosage form does not comprise albumin or an animal-derived ingredient or polysaccharide.

38. The method of claim 34, wherein the botulinum neurotoxin is locally administered.

39. The method of claim 38, wherein the botulinum neurotoxin is intratumorally or peritumorally administered.

40. The method of claim 24, wherein the botulinum neurotoxin in a range of about 0.01 units/kg to about 100 units/kg is administered.

41. The method of claim 24, wherein the botulinum neurotoxin is administered with the immunotherapeutic agent in the form of a single formulation or separate formulations.

42. The method of claim 24, wherein the immunotherapeutic agent is administered parenterally.

43. The method of claim 42, wherein the immunotherapeutic agent is locally administered or injected intravenously, intraperitoneally, subcutaneously, intradermally, intramuscularly, or into the spine, spinal cavity, or rectum.

Description

DESCRIPTION OF DRAWINGS

[0059] FIG. 1 is a graph showing the tumor growth over time after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma described in Example 1.

[0060] FIG. 2A is a graph showing the ratios of CD4+T cells based on total viable cells infiltrated in a tumor after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma described in Example 1.

[0061] FIG. 2B is a graph showing the ratios of CD8+T cells based on total viable cells infiltrated in a tumor after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma described in Example 1.

[0062] FIG. 2C is a graph showing the number of CD4+T cells in an actual tumor based on total viable cells infiltrated in a tumor after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma described in Example 1.

[0063] FIG. 2D is a graph showing the number of CD8+T cells in an actual tumor based on total viable cells infiltrated in a tumor after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma described in Example 1.

[0064] FIG. 3 is a graph showing the number of exosomes in blood of a vehicle group and each experimental group after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma described in Example 1.

[0065] FIG. 4 is a graph showing the tumor growth over time after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody by dose to a mouse model transplanted with malignant melanoma described in Example 2.

[0066] FIG. 5 is a graph showing the survival rate of a mouse after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody by dose to a mouse model transplanted with malignant melanoma described in Example 2.

[0067] FIG. 6 is a graph showing the tumor growth over time after an anti-PD-1 antibody is administered with complex botulinum neurotoxin or non-complex botulinum neurotoxin to a mouse model transplanted with malignant melanoma described in Example 3.

[0068] FIG. 7 is a graph showing the tumor growth over time after botulinum neurotoxin is administered in combination with an anti-CTLA4 antibody to a mouse model transplanted with pulmonary tumor described in Example 4.

[0069] FIG. 8A is a graph showing the tumor growth on the right over time after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma on both sides as described in Example 5.

[0070] FIG. 8B is a graph showing the tumor growth on the left over time after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma on both sides as described in Example 5.

[0071] FIG. 9 is a graph showing the survival rate of a mouse after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with malignant melanoma on both sides as described in Example 5.

[0072] FIG. 10A is a graph showing the ratio of CD11b+cells based on viable cells infiltrated on the left side of non-administered tumors in a mouse model transplanted with malignant melanoma on both sides as described in Example 5.

[0073] FIG. 10B is a graph showing the ratio of CD4+T cells based on viable cells infiltrated on the left side of non-administered tumors in a mouse model transplanted with malignant melanoma on both sides as described in Example 5.

[0074] FIG. 100 is a graph showing the ratio of CD8a+T cells based on viable cells infiltrated on the left side of non-administered tumors in a mouse model transplanted with malignant melanoma on both sides as described in Example 5.

[0075] FIG. 11 is a graph showing the tumor growth over time after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody or propranolol is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with colorectal cancer as described in Example 6.

[0076] FIG. 12 is a graph showing the survival rate of a mouse after botulinum neurotoxin is administered in combination with an anti-PD-1 antibody or propranolol is administered in combination with an anti-PD-1 antibody to a mouse model transplanted with colorectal cancer as described in Example 6.

[0077] FIG. 13 is a graph showing the tumor growth over time after botulinum neurotoxin is administered in combination with an anti-CTLA4 antibody to a mouse model transplanted with colorectal cancer as described in Example 6.

[0078] FIG. 14 is a graph showing the survival rate of a mouse after botulinum neurotoxin is administered in combination with an anti-CTLA4 antibody to a mouse model transplanted with colorectal cancer as described in Example 6.

BEST MODE

[0079] Hereinafter, the present disclosure will be described in more detail with Examples, but these are only for explaining the present disclosure and are not intended to limit the scope in any way.

Example 1: Anticancer Efficacy Test of Botulinum Neurotoxin and Anti-PD-1 Antibody on Mouse Model of Malignant Melanoma Transplantation

[0080] 6-week-old male C57BL/6 mice were purchased (Orient Bio), and after acclimation and quarantine for 1 week, 7-week-old mice were used in experiments. For a feed, a sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and for drinking water, tap water was autoclaved and freely fed. During the periods of acclimatization, quarantine, and experiments, the mice were bred under specific-pathogen-free conditions set at a temperature of 23±3° C., relative humidity of 55±15%, lighting time of 12 hours (from 8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination of 150 Lux to 300 Lux. This experiment was performed after review and approval (A-2020-004) by Institutional Animal Care and Use Committee of Medytox Inc.

[0081] 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 a DMEM medium (CAT No: 11965-092, Gibco) supplemented with 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco) in an incubator at 37° C. and 5% CO.sub.2 conditions. 0.1 ml of 5×10.sup.5 cells were transplanted into the right flank of anesthetized C57BL/6 mice by using a syringe.

[0082] Sterile physiological saline (Lot No: M8T7AF3, Daehan Pharmaceutical Co., Ltd.) used for preparing botulinum neurotoxin was used as a vehicle, and Coretox 100 unit (Lot No: NSA19007A, Medytox) was used as botulinum neurotoxin. Anti-PD-1 antibodies (Clone: RMP1-14, Cat No: BE0146, Lot No: 760220J1) were purchased from Bio X cell for use.

[0083] Each test material was prepared and administered according to Table 1 below. 6.667 ml of sterile physiological saline was added to a vial containing 100 units of botulinum neurotoxin to be prepared at a concentration of 15 units/ml, and then intratumorally administered at a dose of 1 ml/kg or 15 units/kg. An anti-PD-1 antibody was prepared at a final concentration of 1 mg/ml by using sterile PBS (Cat No: 10010, Gibco) on the day of administration, and then administered intraperitoneally at a dose of 5 ml/kg or 5 mg/kg.

[0084] On the 8th day after the mice were transplanted with malignant melanoma, the body weight and tumor size of the mice were measured, and when having the size of about 33 mm.sup.3 in average, 10 mice were randomly assigned per test group to have no difference among the test groups. On the 8th day after the tumor transplantation, a vehicle and botulinum neurotoxin were intratumorally administered according to Table 1 based on the body weight. An anti-PD-1 antibody was intraperitoneally administered on the 8th day, 11th day, 14th day, and 17th day after the tumor transplantation according to Table 1. The tumor size was measured twice a week after the tumor transplantation. The tumor size was measured with a vernier caliper, and the tumor volume was calculated by using the following equation with the measured values.

Tumor volume (mm.sup.3)=(W2×L)/2

[0085] W (mm)=width (short axis of tumor), L (mm)=length of tumor (long axis of tumor)

Tumor growth inhibition (TGI, %)=(1−average tumor size in test group/average tumor size in vehicle)×100

TABLE-US-00001 TABLE 1 Number of Material to be Administration Administration Group animal administered Dose route frequency 1 9* Vehicle 1 ml/kg, Intratumoral, Once (on 8th day 5 ml/kg intraperitoneal after tumor transplantation), twice a week, 4 times in total 2 10  Botulinum 15 Intratumoral Once (on 8th day neurotoxin units/kg after tumor transplantation) 3 10  Anti-PD-1 5 mg/kg Intraperitoneal Twice a week, 4 antibody times in total 4 9* Botulinum 15 Intratumoral, Once (on 8th day neurotoxin + units/kg, intraperitoneal after tumor anti-PD-1 5 mg/kg transplantation), antibody twice a week, 4 times in total *From each of Test Groups 1 and 4, one mouse was excluded due to a severe wound around the tumor caused by a fight between individuals.

[0086] On day 19th day after the tumor transplantation, all animals were euthanized before the tumor size reached 2,000 mm.sup.3. After completion of the euthanasia, the tumor tissue was excised and weighed, and the number of T cells in the tumor tissue was measured by using a flow cytometer (FACSVerse, BD). For the analysis of T cells in the tumor tissue, the tumor tissue was separated into single cells by using gentle MACS C tubes (Cat No: 130-093-237, Miltenyi Biotec), and then stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain (Cat No: L34957, Invitrogen), an Fc block (Cat No: 553142, BD), and CD45-PECy7 (Cat No: 25-0451-82, eBioscience), TCRbeta-FITC (Cat No: 109206, Biolegend), CD4-pacific blue (Cat No: 100428, Biolegend), CD8a-PerCPCy55 (Cat No: 100734, Biolegend) antibodies to be 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 was represented as a ratio of CD45+TCRbeta+CD4+ and CD45+TCRbeta+CD8a+ T cells based on viable cells.

[0087] Exosomes in blood were isolated by using an Exoquick exosome precipitation solution (Cat No: EXOQ20A-1, Lot No: 200107-001, System Biosciences), and then measured by using an ExoELISA-ULTRA Complete Kit (Cat No: EXEL-ULTRA-CD63-1, Lot No: 200226-002, System Biosciences).

[0088] A Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for the graph presentation, and an SPSS software (version 25.0, SPSS Inc., IL, USA) was used for the statistical analysis. A two-tailed t-test was performed with respect to parametric data, and Mann-Whitney test statistical analysis was performed with respect to non-parametric data at a significance level of p=0.05.

[0089] The results of measuring the tumor growth inhibition are shown in Table 2 and FIG. 1.

TABLE-US-00002 TABLE 2 Material to be Average tumor size.sup.1) Average tumor weight.sup.2) Group administered (mean mm.sup.3 ± SEM) (mean mm.sup.3 ± SEM) TGI (%).sup.1) 1 Vehicle 1684 ± 284 2.02 ± 038 0 2 Botulinum neurotoxin 1433 ± 348 1.29 ± 035 15 3 Anti-PD-1 antibody 1415 ± 121 1.83 ± 018 16 4 Botulinum neurotoxin +     .sup.  489 ± 165**, †, ‡    .sup. 0.46 ± 0.12**, †, ‡ 71 anti-PD-1 antibody .sup.1)Results at the 18th day after tumor transplantation, .sup.2)results at the 19th day after tumor transplantation, **p < 0.01, vs vehicle. †p < 0.05, vs botulinum neurotoxin, ‡p < 0.001, vs anti-PD-1 antibody, two-tailed t-test.

[0090] As shown in Table 2 and FIG. 1, on the 18th day after the mice were transplanted with malignant melanoma, the intratumoral administration of botulinum neurotoxin only showed an average tumor size of 1433 mm.sup.3 and a tumor growth inhibition rate of 15%, whereas the intratumoral administration of anti-PD-1 antibody only showed an average of 1415 mm.sup.3 and a tumor growth inhibition rate of 16%. However, there was no statistical significance compared to the vehicle-administered group. In contrast, when botulinum neurotoxin was administered in combination with anti-PD-1 antibody, an average tumor size was 489 mm.sup.3, and a tumor growth inhibition rate was 71%, confirming that the tumor growth was inhibited more significantly compared to the case where each of botulinum neurotoxin and an anti-PD-1 antibody was administered alone. In addition, even when the tumor was excised and weighed on the 19th day after the tumor transplantation, the case where botulinum neurotoxin was administered in combination with anti-PD-1 antibody showed a significant reduction in the tumor weight compared to the groups administered with vehicle only, botulinum neurotoxin only, and anti-PD-1 antibody only, respectively.

[0091] As a result of analyzing CD4+ and CD8+ T cells infiltrated into the tumor after the tumor tissue was excised, the case where only botulinum neurotoxin was administered showed a tendency to increase the number of CD8+ T cells in the tumor compared to the vehicle-treated group (FIGS. 2A and 2B). For the ratios of CD4+ and CD8+ T cells, there was no significant difference between the groups each administered with botulinum neurotoxin only and anti-PD-1 antibody only and the vehicle-treated group. However, in the group treated with botulinum neurotoxin in combination with anti-PD-1 antibody, a significantly high ratio of CD4+ and CD8+ T cells was confirmed to be infiltrated into the tumor compared to the vehicle-treated group (FIGS. 2C and 2D). That is, there was a significant increase compared to the group treated with anti-PD-1 antibody only. It can be explained that, in the group treated with botulinum neurotoxin and anti-PD-1 antibody together, T cells were significantly increased in the tumor due to a synergistic action between the two substances so that the tumor growth was significantly reduced.

[0092] The results of measuring the number of exosomes in blood are shown in FIG. 3. As shown in FIG. 3, the number of exosomes in the blood tended to decrease in the group administered with botulinum neurotoxin only compared to the vehicle-treated group, and was significantly decreased in the group administered with anti-PD-1 antibody only and the group administered with botulinum neurotoxin+anti-PD-1 antibody. In addition, the group administered with botulinum neurotoxin in combination with anti-PD-1 antibody showed a significant decrease in the number of exosomes in the blood compared to the groups each administered with botulinum neurotoxin only and anti-PD-1 only, respectively.

Example 2: Anticancer Efficacy Test of Botulinum Neurotoxin and Anti-PD-1 Antibody by Dose on Mouse Model of Malignant Melanoma Transplantation

[0093] 6-week-old male C57BL/6 mice were purchased (Orient Bio), and after acclimation and quarantine for 1 week, 7-week-old mice were used in experiments. For a feed, a sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and for drinking water, tap water was autoclaved and freely fed. During the periods of acclimatization, quarantine, and experiments, the mice were bred under specific-pathogen-free conditions set at a temperature of 23±3° C., relative humidity of 55±15%, lighting time of 12 hours (from 8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination of 150 Lux to 300 Lux. This experiment was performed after review and approval (A-2020-004) by Institutional Animal Care and Use Committee of Medytox Inc.

[0094] 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 a DMEM medium (CAT No: 11965-092, Gibco) supplemented with 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco) in an incubator at 37° C. and 5% CO.sub.2 conditions. 0.1 ml of 5×10.sup.5 cells were transplanted into the right flank of anesthetized C57BL/6 mice by using a syringe.

[0095] A placebo of a botulinum neurotoxin product containing the same excipient components as in the botulinum neurotoxin product, but only botulinum neurotoxin was excluded was used as a vehicle, and Coretox 100 unit (Lot No: NSA19007A, Medytox) was used as botulinum neurotoxin. An anti-PD-1 antibody (Clone: RMP1-14, Cat No: BE0146, Lot No: 780120J2) was purchased from Bio X cell for use. Each test material was prepared and administered according to Table 3 below. Sterile physiological saline was added to a vial containing 100 units of botulinum neurotoxin to be prepared at concentrations of 1 unit/ml, 3 units/ml, and 10 units/ml, and then intratumorally administered at a dose of 1 ml/kg or 1 unit/kg, 3 units/kg, or 10 units/kg. An anti-PD-1 antibody was prepared at a final concentration of 1 mg/ml by using sterile PBS (Cat No: 10010, Gibco) on the day of administration, and then administered intraperitoneally at a dose of 5 ml/kg or 5 mg/kg.

[0096] On the 8th day after the mice were transplanted with malignant melanoma, the tumor size was measured, and 12 mice were randomly assigned per test group to have no difference among the test groups. On the 8th day after the tumor transplantation, a vehicle and botulinum neurotoxin were intratumorally administered according to the compositions of the test groups. In the case of anti-PD-1 antibody, intraperitoneal administration thereof was performed twice a week for a total of 4 times according to Table 3. The tumor size was measured twice a week after the tumor transplantation. The tumor size was measured with a vernier caliper, and the tumor volume was calculated by using the equation described in Example 1.

TABLE-US-00003 TABLE 3 Number of Material to be Administration Administration Group animal administered Dose route frequency 1 12 Vehicle 1 ml/kg Intratumoral Once (on 8th day after tumor transplantation) 2 12 1 U/kg 1 ml/kg Intratumoral Once (on 8th day after botulinum tumor transplantation) neurotoxin 3 12 3 U/kg 1 ml/kg Intratumoral Once (on 8th day after botulinum tumor transplantation) neurotoxin 4 12 10 U/kg 1 ml/kg Intratumoral Once (on 8th day after botulinum tumor transplantation) neurotoxin 5 12 1 U/kg 1 ml/kg, Intratumoral, Once (on 8th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg anti- in total PD-1 antibody 6 12 3 U/kg 1 ml/kg, Intratumoral, Once (on 8th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg anti- in total PD-1 antibody 7 12 10 U/kg 1 ml/kg, Intratumoral, Once (on 8th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg anti- in total PD-1 antibody 8 12 Vehicle + 1 ml/kg, Intratumoral, Once (on 8th day after 5 mg/kganti- 5 ml/kg intraperitoneal tumor transplantation), PD-1 antibody twice a week, 4 times in total

[0097] After the tumor transplantation, measurements were performed until the tumor size in each mouse reached 2,000 mm.sup.3, and subjects having the tumor size exceeding the euthanasia standard (2,000 mm.sup.3) were immediately euthanized and recorded as dead.

[0098] A Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for the graph presentation, and an SPSS software (version 25.0, SPSS Inc., IL, USA) and an Excel (2013, MS, USA) were used for the statistical analysis. A two-tailed t-test was performed with respect to the tumor size, and Mantel-Cox log-rank analysis was performed with respect to the survival rates at a significance level of p=0.05.

[0099] Results of the tumor size measurement are shown in FIG. 4 (*p<0.05, **p<0.01, and ***p<0.001). As shown in FIG. 4, when botulinum neurotoxin was intratumorally administered in a single dose of 1 U/kg, 3 U/kg, or 10 U/kg to the mouse model of malignant melanoma, significant tumor growth inhibition was observed on the 14th day in in the 10 U/kg-administered group. On the 18th day after the tumor transplantation, no significant inhibitory effect on the tumor growth was observed in the test group administered with botulinum neurotoxin only and the test group administered with vehicle+5 mg/kg of anti-PD1 antibody, compared to the vehicle-administered group. However, a significant inhibitory effect on the tumor growth was observed in the test group administered with 3 U/kg or 10 U/kg of botulinum neurotoxin in combination with 5 mg/kg of anti-PD1 antibody, compared to the vehicle-administered group.

[0100] Results of the survival rate measurement are shown in FIG. 5. As shown in FIG. 5, the mouse survival rate was significantly increased in the test group administered with 3 U/kg or 10 U/kg of botulinum neurotoxin in combination with 5 mg/kg of anti-PD1 antibody. In addition, a significant increase in the survival rate was observed in the group administered with vehicle+5 mg/kg of anti-PD1. Based on the results above, it was confirmed that there is an inhibitory effect on tumor growth in the mouse model of melanoma when botulinum neurotoxin was administered at a dose of 3 U/kg or more in combination with anti-PD1 antibody.

Example 3: Anti-Cancer Efficacy Test of Complexed Botulinum Neurotoxin and Non-Complexed Botulinum Neurotoxin Administered in Combination with Anti-PD-1 Antibody on Mouse Model of Malignant Melanoma Transplantation

[0101] 6-week-old male C57BL/6 mice were purchased (Orient Bio), and after acclimation and quarantine for 1 week, 7-week-old mice were used in experiments. For a feed, a sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and for drinking water, tap water was autoclaved and freely fed. During the periods of acclimatization, quarantine, and experiments, the mice were bred under specific-pathogen-free conditions set at a temperature of 23±3° C., relative humidity of 55±15%, lighting time of 12 hours (from 8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination of 150 Lux to 300 Lux. This experiment was performed after review and approval (A-2020-004) by Institutional Animal Care and Use Committee of Medytox Inc.

[0102] 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 a DMEM medium (CAT No: 11965-092, Gibco) supplemented with 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco) in an incubator at 37° C. and 5% CO.sub.2 conditions. 0.1 ml of 5×10.sup.5 cells were transplanted into the right flank of anesthetized C57BL/6 mice by using a syringe.

[0103] A placebo of a non-complexed botulinum neurotoxin product containing the same excipient components as in the botulinum neurotoxin product, but only botulinum neurotoxin was excluded was used as a vehicle. Coretox 100 unit (Lot No: NSA19007A, Medytox) was used as non-complexed botulinum neurotoxin, Meditoxin 100 unit (Lot No: TFAA20024, Medytox) was used as complexed botulinum neurotoxin. An anti-PD-1 antibody (Clone: RMP1-14, Cat No: BE0146, Lot No: 780120J2) was purchased from Bio X cell for use. Each test material was prepared and administered according to Table 4 below. Sterile physiological saline was added to a vial containing 100 units of botulinum neurotoxin to be prepared at a concentration of 15 units/ml, and then intratumorally administered at a dose of 1 ml/kg or 15 units/kg. An anti-PD-1 antibody was prepared at a final concentration of 1 mg/ml by using sterile PBS (Cat No: 10010, Gibco) on the day of administration, and then administered intraperitoneally at a dose of 5 ml/kg or 5 mg/kg.

[0104] On the 8th day after the mice were transplanted with malignant melanoma, the tumor size was measured, and 12 mice were randomly assigned per test group to have no difference among the test groups. On the 8th day after the tumor transplantation, a vehicle and botulinum neurotoxin were intratumorally administered according to the compositions of the test groups. In the case of anti-PD-1 antibody, intraperitoneal administration thereof was performed twice a week for a total of 4 times according to Table 4. The tumor size was measured twice a week after the tumor transplantation. The tumor size was measured with a vernier caliper, and the tumor volume was calculated by using the equation described in Example 1.

TABLE-US-00004 TABLE 4 Number of Material to be Administration Administration Group animal administered Dose route frequency 1 12 Vehicle 1 ml/kg Intratumoral Once (on 8th day after tumor transplantation) 2 12 15 U/kg non- 1 ml/kg Intratumoral Once (on 8th day after complexed tumor transplantation) botulinum neurotoxin 3 12 15 U/kg 1 ml/kg Intratumoral Once (on 8th day after complexed tumor transplantation) botulinum neurotoxin 4 12 15 U/kg non- 1 ml/kg Intratumoral Once (on 8th day after complexed tumor transplantation) botulinum neurotoxin + 5 mg/kg anti- PD-1 antibody 5 12 15 U/kg 1 ml/kg, Intratumoral, Once (on 8th day after complexed 5 ml/kg intraperitoneal tumor transplantation), botulinum twice a week, 4 times neurotoxin + in total 5 mg/kg anti- PD-1 antibody 6 12 Vehicle + 1 ml/kg, Intratumoral, Once (on 8th day after 5 mg/kganti- 5 ml/kg intraperitoneal tumor transplantation), PD-1 antibody twice a week, 4 times in total

[0105] On day 19th day after the tumor transplantation, all animals were euthanized before the tumor size reached 2,000 mm.sup.3, and the experiment was terminated.

[0106] A Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for the graph presentation, and an SPSS software (version 25.0, SPSS Inc, IL, USA) and an Excel (2013, MS, USA) were used for the statistical analysis. A two-tailed t-test was performed with respect to the tumor size at a significance level of p=0.05.

[0107] The results are shown in FIG. 6 (*p<0.05, **p<0.01). As shown in FIG. 6, on the 19th day after the mice were transplanted with malignant melanoma, significant inhibition of the tumor growth was observed in the case where 15 U/kg of non-complexed botulinum neurotoxin and 15 U/kg of complexed botulinum neurotoxin were each administered in combination with 5 mg/kg of anti-PD1 antibody. Here, no significant difference was observed among the test groups administered with the non-complexed botulinum neurotoxin only and the test groups administered with complexed botulinum neurotoxin only. In addition, no significant difference was observed among the groups administered with non-complexed botulinum neurotoxin+anti-PD1 antibody and the groups administered with complexed botulinum neurotoxin+anti-PD1 antibody. Based on the results above, it was confirmed that, in both a case where non-complexed botulinum neurotoxin was administered in combination with anti-PD1 antibody and a case where complexed botulinum neurotoxin was administered in combination with anti-PD1 antibody, an inhibitory effect on tumor growth was exhibited regardless of the type of botulinum neurotoxin.

Example 4: Anticancer Efficacy Test of Botulinum Neurotoxin and Anti-CTLA4 Antibody on Mouse Model of Pulmonary Tumor Transplantation

[0108] 6-week-old male C57BL/6 mice were purchased (Orient Bio), and after acclimation and quarantine for 1 week, 7-week-old mice were used in experiments. For a feed, a sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and for drinking water, tap water was autoclaved and freely fed. During the periods of acclimatization, quarantine, and experiments, the mice were bred under specific-pathogen-free conditions set at a temperature of 23±3° C., relative humidity of 55±15%, lighting time of 12 hours (from 8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination of 150 Lux to 300 Lux. This experiment was performed after review and approval (A-2020-004) by Institutional Animal Care and Use Committee of Medytox Inc.

[0109] An LLC1 mouse lung cancer cell line (ATCC No: CRL-1642) was obtained from ATCC. LLC1 cells were cultured in a DMEM medium (CAT No: LM001-05, Welgene) supplemented with 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco) in an incubator at 37° C. and 5% CO.sub.2 conditions. 0.1 ml of 5×10.sup.5 cells were transplanted into the right flank of anesthetized C57BL/6 mice by using a syringe.

[0110] A placebo of a botulinum neurotoxin product containing the same excipient components as in the botulinum neurotoxin product, but only botulinum neurotoxin was excluded was used as a vehicle, and Coretox 100 unit (Lot No: NSA19007A, Medytox) was used as botulinum neurotoxin. Anti-CTLA4 antibody (Clone: 9H10, Cat No: 13E0131, Lot No: 704019A2) was purchased from Bio X cell for use. Each test material was prepared and administered according to Table 5 below. Sterile physiological saline was added to a vial containing 100 units of botulinum neurotoxin to be prepared at a concentration of 15 units/ml, and then intratumorally administered at a dose of 1 ml/kg or 15 units/kg. Anti-CTLA4 antibody was prepared at a final concentration of 1 mg/ml by using sterile PBS (Cat No: 10010, Gibco) on the day of administration, and then administered intraperitoneally at a dose of 5 ml/kg or 5 mg/kg.

[0111] On the 8th day after the mice were transplanted with LLC1 pulmonary tumor cell line, the tumor size was measured, and 10 mice were randomly assigned per test group to have no difference among the test groups. On the 8th day after the tumor transplantation, a vehicle and botulinum neurotoxin were intratumorally administered according to the compositions of the test groups. In the case of anti-CTLA4 antibody, intraperitoneal administration thereof was performed twice a week for a total of 4 times according to Table 5. The tumor size was measured twice a week after the tumor transplantation. The tumor size was measured with a vernier caliper, and the tumor volume was calculated by using the equation described in Example 1.

TABLE-US-00005 TABLE 5 Number of Material to be Administration Administration Group animal administered Dose route frequency 1 10 Vehicle 1 ml/kg Intratumoral Once (on 8th day after tumor transplantation) 2 10 15 U/kg 1 ml/kg Intratumoral Once (on 8th day after botulinum tumor transplantation) neurotoxin 3 10 15 U/kg 1 ml/kg, Intratumoral, Once (on 8th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg in total anti-CTLA4 antibody 4 10 Vehicle + 1 ml/kg, Intratumoral, Once (on 8th day after 5 mg/kg 5 ml/kg intraperitoneal tumor transplantation), anti-CTLA4 twice a week, 4 times antibody in total

[0112] On day 21st day after the tumor transplantation, all animals were euthanized before the tumor size reached 2,000 mm.sup.3, and the experiment was terminated.

[0113] A Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for the graph presentation, and an SPSS software (version 25.0, SPSS Inc, IL, USA) and an Excel (2013, MS, USA) were used for the statistical analysis. A two-tailed t-test was performed with respect to the tumor size at a significance level of p=0.05.

[0114] The results are shown in FIG. 7 (*p<0.05, **p<0.01, and ***p<0.001). As shown in FIG. 7, on the 21st day after the mice were transplanted with pulmonary tumor, no significant inhibitory effect on tumor growth was observed in the group administered with 15 U/kg of botulinum neurotoxin only or with vehicle+5 mg/kg of anti-CTLA4 antibody compared to the vehicle-treated test group, whereas a significant inhibitory effect on tumor growth was observed in the group administered with 15 U/kg of botulinum neurotoxin+5 mg/kg of anti-CTLA4 compared to the vehicle-treated test group. In addition, the tumor volume was significantly reduced in the group administered with 15 U/kg of botulinum neurotoxin+5 mg/kg of anti-CTLA4 compared to the test group administered with 15 U/kg of botulinum neurotoxin only and the group administered with vehicle+5 mg/kg of anti-CTLA4 antibody. Based on the results above, it was confirmed that the administration of botulinum neurotoxin in combination with anti-CTLA4 antibody showed an inhibitory effect on tumor growth in the mouse model of LLC1 pulmonary tumor.

Example 5: Anticancer Efficacy Test of Botulinum Neurotoxin and Anti-PD-1 Antibody on Mouse Model of Bilateral Malignant Melanoma Transplantation

[0115] 6-week-old male C57BL/6 mice were purchased (Orient Bio), and after acclimation and quarantine for 1 week, 7-week-old mice were used in experiments. For a feed, a sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and for drinking water, tap water was autoclaved and freely fed. During the periods of acclimatization, quarantine, and experiments, the mice were bred under specific-pathogen-free conditions set at a temperature of 23±3° C., relative humidity of 55±15%, lighting time of 12 hours (from 8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination of 150 Lux to 300 Lux. This experiment was performed after review and approval (A-2020-004) by Institutional Animal Care and Use Committee of Medytox Inc.

[0116] 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 a DMEM medium (CAT No: 11965-092, Gibco) supplemented with 10% FBS (CAT No: 10082-147, Gibco) and 1% penicillin-streptomycin (CAT No: 15140-122, Gibco) in an incubator at 37° C. and 5% CO.sub.2 conditions. 0.1 mL of 5×10.sup.5 cells were transplanted into the right flank of the anesthetized C57BL/6 mice by using a syringe, and after 5 days, 0.1 mL of 2×10.sup.5 cells to 2.5×10.sup.5 cells were transplanted into the left flank of the anesthetized C57BL/6 mice by using a syringe.

[0117] A placebo of a botulinum neurotoxin product containing the same excipient components as in the botulinum neurotoxin product, but only botulinum neurotoxin was excluded was used as a vehicle, and Coretox 100 unit (Lot No: NSA20015, Medytox) was used as botulinum neurotoxin. An anti-PD-1 antibody (Clone: RMP1-14, Cat No: BE0146, Lot No: 780120J2 or 798921J1C) was purchased from Bio X cell for use. Each test material was prepared and administered according to Table 6 below. Sterile physiological saline was added to a vial containing 100 units of botulinum neurotoxin to be prepared at a concentration of 15 units/m L, and then intratumorally administered at a dose of 1 mL/kg or 15 units/kg. An anti-PD-1 antibody was intraperitoneally administered at a dose of 5 mg/kg with PBS (Cat No: LB004-02, Welgene).

[0118] The experiment was repeated twice with the same test groups according to the same procedures. On the 8th day after the mice were transplanted with malignant melanoma on the right flank, the tumor size was measured, and 12 mice were randomly assigned per test group to have no difference among the test groups. On day 8 after the tumor transplantation on the right flank, a vehicle and botulinum neurotoxin were intratumorally administered on the right flank according to the compositions of the test groups. In the case of anti-PD-1 antibody, intraperitoneal administration thereof was performed twice a week for a total of 4 times according to the compositions of the test groups. After the tumor transplantation, the size of the tumors transplanted 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 by using the equation described in Example 1.

TABLE-US-00006 TABLE 6 Number of Material to be Administration Administration Group animal administered Dose route frequency 1 12 Vehicle 1 ml/kg Intratumoral Once (on 8th day after tumor transplantation) 2 12 15 U/kg 1 ml/kg Intratumoral Once (on 8th day after botulinum tumor transplantation) neurotoxin 3 12 Vehicle + 1 ml/kg, Intratumoral, Once (on 8th day after 5 mg/kg anti- 5 ml/kg intraperitoneal tumor transplantation), PD-1 antibody twice a week, 4 times in total 4 12 15 U/kg 1 ml/kg, Intratumoral, Once (on 8th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg anti- in total PD-1 antibody

[0119] On the 21st day after the tumor transplantation on the right side, all animals were euthanized to end the experiment. After completion of the experiment, the presence or absence of transplanted tumor tissue was observed in the left flank of all subjects. The tumor was excised from the left flank, and immune cells in the tumor tissue were analyzed by using a flow cytometer (FACSVerse, BD). For the analysis of immune cells in the tumor tissue, the tumor tissue was separated into single cells by using gentle MACS C tubes (Cat No: 130-093-237, Miltenyi Biotec), and then stained with a LIVE/DEAD Fixable Aqua Dead Cell Stain (Cat No: L34957, Invitrogen), an Fc block (Cat No: 553142, BD), and 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-Cy55 (Cat No: 100734, Biolegend), CD11b-A647 (Cat No: 101218, Biolegend) antibodies to be analyzed by flow cytometry. Ratios of CD4+ and CD8a+ T cells and CD11b+ cells in the tumor represent ratios of CD45+TCRbeta+CD4+, CD45+TCRbeta+CD8a+, and CD45+CD11b+ cells based on viable cells.

[0120] After the tumor transplantation, when the sum of the right and left tumor sizes of each mouse reached 2,000 mm.sup.3, the corresponding subjects were recorded as dead.

[0121] A Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for the graph presentation, and an SPSS software (version 25.0, SPSS Inc, IL, USA) and an Excel (2013, MS, USA) were used for the statistical analysis. A two-tailed t-test was performed with respect to the tumor size, a Pearson's Chi-Square test was performed with respect to the formation of left tumor, and Mantel-Cox log-rank analysis was performed with respect to the survival rates at a significance level of p=0.05.

[0122] The tumor growth on the right and left sides over time was shown in terms of tumor volume (mm.sup.3) as shown in FIGS. 8A and 8B (*p<0.05, **p<0.01, and ***p<0.001). When botulinum neurotoxin was intratumorally administered on the right side once at a dose of 15 U/kg and when 15 U/kg of botulinum neurotoxin was administered intraperitoneally in combination with 5 mg/kg of anti-PD-1 antibody, a significant reduction in the tumor size on the right side was observed in the mouse model transplanted with malignant melanoma on the right and left sides, compared to the test groups each administered with botulinum neurotoxin only and anti-PD-1 antibody only (FIG. 8A). Regarding the tumor size on the left side, the tumor size was significantly reduced in the test group administered with botulinum neurotoxin in combination with anti-PD-1 antibody, compared to the vehicle-administered group (FIG. 8B).

[0123] Table 7 shows the presence or absence of tumor formation on the left side (*p<0.05 vs G3 anti-PD-1 antibody).

TABLE-US-00007 TABLE 7 Formation of tumor on left flank Observed Not observed Material to be (number of (number of Group administered animals) animals) 1 Vehicle 18 4 2 15 U/kg 21 3 botulinum neurotoxin 3 Vehicle + 18 2 5 mg/kg anti- PD-1 antibody 4 15 U/kg 14  8* botulinum neurotoxin + 5 mg/kg anti- PD-1 antibody

[0124] As shown in Table 7, a formation rate of the tumor on the left side was significantly reduced in the group administered with botulinum neurotoxin in combination with anti-PD-1 antibody, compared to the group administered with the anti-PD-1 antibody only.

[0125] FIG. 9 shows the survival rate of the mice (**p<0.01), and it was confirmed that the survival rate was significantly improved in the test group administered with botulinum neurotoxin in combination with anti-PD-1 antibody.

[0126] The ratio of CD11b+ cells and the ratio of CD4+ and CD8a+ T cells were calculated by using a two-tailed t-test (*p<0.05) based on viable cells infiltrated in the tumor on the left non-administered side. As a result of excising the tumor tissue transplanted on the left side and analyzing the CD4+ and CD8a+ T cells and CD11b+ cells infiltrated in the tumor, in the group administered with botulinum neurotoxin in combination with anti-PD1 antibody, a significant decrease in the CD11b+ cell ratio was observed compared to the vehicle-administered group (FIG. 10A), the CD4+ T cell ratio was significantly increased compared to the group administered with anti-PD-1 antibody only (FIG. 10B), and the CD8a+ T cell ratio was significantly increased compared to the vehicle-administered group and the group administered botulinum neurotoxin only (FIG. 10C).

[0127] Based on the results above, the significant improvement in the survival rate was confirmed when the mouse model of bilateral melanoma transplantation was administered with botulinum neurotoxin in combination with anti-PD-1 antibody. It was also confirmed that there were effects on not only the growth of tumor to which botulinum neurotoxin was administered, but also the formation and growth of tumor to which botulinum neurotoxin was not administered were confirmed.

Example 6: Anti-Cancer Efficacy Test of Botulinum Neurotoxin Administered in Combination with Immuno-Anticancer Agent (Anti-PD-1 and Anti-CTLA4) on Mouse Model of Colorectal Cancer Transplantation

[0128] 6-week-old female C57BL/6 mice were purchased (Orient Bio), and after acclimation and quarantine for 1 week, 7-week-old mice were used in experiments. For a feed, a sterilized solid feed for laboratory animals (R40-10, SAFE, France) was freely fed, and for drinking water, tap water was autoclaved and freely fed. During the periods of acclimatization, quarantine, and experiments, the mice were bred under specific-pathogen-free conditions set at a temperature of 23±3° C., relative humidity of 55±15%, lighting time of 12 hours (from 8:00 am to 8:00 pm), ventilation frequency of 15 times/hour, and illumination of 150 Lux to 300 Lux. This experiment was performed after review and approval (A-2020-004) by Institutional Animal Care and Use Committee of Medytox Inc.

[0129] A mouse colorectal cancer MC38 cell line (ENH204-FP) was obtained from Kerafast. The MC38 cell line was cultured in a DMEM medium (CAT No: 11965-092, Gibco) supplemented with 10% FBS (CAT No: 10082-147, Gibco), 1% penicillin-streptomycin (CAT No: 15140-122, Gibco), 10 mM HEPES (CAT No: 15630-080, Gibco), 0.1 mM MEM NEAA (CAT No: 11140-050, Gibco), and 1 mM sodium pyruvate (CAT No.: 11360-070, Gibco) in an incubator at 37° C. and 5% CO.sub.2 conditions. 0.1 mL of 2.5×10.sup.5 cells were transplanted into the right flank of anesthetized C57BL/6 mice by using a syringe.

[0130] A placebo of a botulinum neurotoxin product containing the same excipient components as in the botulinum neurotoxin product, but only botulinum neurotoxin was excluded was used as a vehicle, and Coretox 100 unit (Lot No: NSA20015, Medytox) was used as botulinum neurotoxin. An anti-PD-1 antibody (Clone: RMP1-14, Cat No: BE0146, Lot No: 798921J1C) and an anti-CTLA4 antibody (Clone: 9H10, Cat No: BE0131, Lot No: 755620A2) were purchased from Bio X cell for use. Each test material was prepared and administered according to Table 6 below. Sterile physiological saline was added to a vial containing 100 units of botulinum neurotoxin to be prepared at a concentration of 15 units/mL, and then intratumorally administered at a dose of 1 mL/kg or 15 units/kg. Anti-PD-1 and anti-CTLA4 antibodies were intraperitoneally administered at a dose of 5 mg/kg with PBS (Cat No: LB004-02, Welgene). Propranolol (Cat No: P0884, Sigma) was prepared at a concentration of 2 mg/mL using PBS, and then intraperitoneally administered at a dose of 10 mg/kg.

[0131] On the 7th day after the mice were transplanted with colorectal cancer cell line, the tumor size was measured, and 10 mice were randomly assigned per test group to have no difference among the test groups. On the 7th day after the tumor transplantation, a vehicle and botulinum neurotoxin were intratumorally administered according to the compositions of the test groups. In the case of the anti-PD-1 and anti-CTLA4 antibodies, intraperitoneal administration thereof was performed twice a week for a total of 4 times according to Table 8. In the case of propranolol, intraperitoneal administration thereof was performed for a total of 8 times between the 7th day and the 16th day after the tumor transplantation, and the tumor size was measured twice a week after the tumor transplantation. The tumor size was measured with a vernier caliper, and the tumor volume was calculated by using the equation described in Example 1.

TABLE-US-00008 TABLE 8 Number of Material to be Administration Administration Group animal administered Dose route frequency 1 10 Vehicle 1 ml/kg Intratumoral Once (on 7th day after tumor transplantation) 2 10 15 U/kg 1 ml/kg Intratumoral Once (on 7th day after botulinum tumor transplantation) neurotoxin 3 10 Vehicle + 1 ml/kg, Intratumoral, Once (on 7th day after 5 mg/kg anti- 5 ml/kg intraperitoneal tumor transplantation), PD-1 antibody twice a week, 4 times in total 4 10 Vehicle + 1 ml/kg, Intratumoral, Once (on 7th day after 5 mg/kg 5 ml/kg intraperitoneal tumor transplantation), anti-CTLA4 twice a week, 4 times antibody in total 5 10 15 U/kg 1 ml/kg, Intratumoral, Once (on 7th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg anti- in total PD-1 antibody 6 10 15 U/kg 1 ml/kg, Intratumoral, Once (on 7th day after botulinum 5 ml/kg intraperitoneal tumor transplantation), neurotoxin + twice a week, 4 times 5 mg/kg in total anti-CTLA4 antibody 7 10 Vehicle + 1 ml/kg, Intratumoral, Once (on 7th day after 10 mg/kg 5 ml/kg intraperitoneal tumor transplantation), propranolol 8 times in total (between 7th day to 16th day after tumor transplantation) 8 10 10 mg/kg 10 ml/kg, Intratumoral, 8 times in total propranolol + 5 ml/kg intraperitoneal (between 7th day to 5 mg/kg 16th day after tumor anti-PD-1 transplantation), antibody twice a week, 4 times in total

[0132] After the tumor transplantation, measurements were performed until the tumor size in each mouse reached 2,000 mm.sup.3, and subjects having the tumor size exceeding the euthanasia standard (2,000 mm.sup.3) were immediately euthanized and recorded as dead.

[0133] A Graphpad Prism (version 7.05, GraphPad Software Inc., CA, USA) was used for the graph presentation, and an SPSS software (version 25.0, SPSS Inc., IL, USA) and an Excel (2013, MS, USA) were used for the statistical analysis. A two-tailed t-test was performed with respect to the tumor size, and Mantel-Cox log-rank analysis was performed with respect to the survival rates at a significance level of p=0.05.

[0134] FIGS. 11 and 13 show the tumor growth over time represented in tumor volume (mm.sup.3) (FIG. 11: **p<0.01 vs vehicle, and FIG. 13: **p<0.01 and ***p<0.001 vs vehicle). On the 20th day after the colorectal cancer transplantation on the mice, significant inhibition on the tumor growth was observed in the group administered with 15 U/kg of botulinum neurotoxin in combination with 5 mg/kg of anti-PD-1 antibody, compared to the vehicle-administered group, the group administered with botulinum neurotoxin only, and the group administered with anti-PD-1 only (FIG. 11). In addition, on the 20th day after the colorectal cancer transplantation, significant inhibition on the tumor growth was observed in the group administered with 5 mg/kg of anti-CTLA4 only and the group administered with botulinum neurotoxin in combination with anti-CTLA4, compared to the vehicle-administered group (FIG. 13).

[0135] FIGS. 12 and 14 show the survival rates of mice (FIG. 12: *p<0.05, **p<0.01 vs vehicle, and .sup.ap<0.05 vs anti-PD-1, and FIG. 14: ***p<0.001 vs vehicle and .sup.ap<0.05 vs anti-CTLA4). In the test group administered with botulinum neurotoxin in combination with the anti-PD1 antibody, the survival rate of mice was significantly increased compared to the vehicle-administered group and the group administered with the anti-PD-1 antibody only. Also, in the test group administered with 10 mg/kg of propranolol in combination with the anti-PD1 antibody, the survival rate of mice was significantly increased compared to the vehicle-administered group and the group administered with the anti-PD-1 antibody only. A significant increase in the survival rate of mice was observed also in the group administered with the anti-CTLA4 antibody only. Also, a significantly increased survival rate of the mice was observed in the group administered with botulinum neurotoxin in combination with the anti-CTLA4 antibody, compared to the vehicle-administered group and the group administered with the anti-CTLA4 antibody only (FIG. 14).

[0136] Based on the results above, the synergistic effect on the tumor growth inhibition and the survival rate improvement were observed in the mouse model of colorectal cancer due to the administration of botulinum neurotoxin in combination with the anti-PD-1 or anti-CTLA4 immuno-anticancer agent. In addition, the synergistic effect by the administration of a beta-blocker (propranolol) in combination with an immuno-anticancer agent was observed, and when botulinum neurotoxin was administered in combination with an immuno-anticancer agent, the equivalent or better effects were confirmed compared to the effects of the group administered with the beta-blocker in combination with the immuno-anticancer agent.