METHOD FOR THE CANCER TREATMENT AND PREVENTION OF METASTATIC DISEASE

Abstract

The disclosure provides a method of new use of an immunomodulatory drug comprising a water-soluble acidic peptidoglycan from sprouts of the plant Solanum tuberosum (WSPG) for prevention and treatment of metastatic tumor growth, comprising an application of a therapeutically effective dose of a said immunomodulatory drug or a pharmaceutical composition of a said immunomodulatory drug with a pharmaceutically acceptable carrier or excipient, or a pharmaceutically effective amount of mammalian immune cells treated with said immunomodulatory drug WSPG in vitro/ex vivo to a patient in need thereof.

Claims

1. A method of new use of the compound water-soluble acidic peptidoglycan from sprouts of the plant Solanum tuberosum (WSPG) for treating cancerous tumors and preventing, treating, inhibiting and/or controlling the formation or establishment of metastases at one or more sites distinct from primary tumor or cancer.

2. The method according to claim 1 wherein a pharmaceutically effective amount of the compound WSPG and a pharmaceutically acceptable carrier, excipient and/or diluent are administered to a patient in need thereof.

3. The method according to claim 1 wherein a pharmaceutically effective amount of mammalian immune cells treated with compound WSPG or with similarly acting other TLR4-agonist in vitro/ex vivo are administered to a patient in need thereof.

4. The method according to claim 1 wherein said WSPG is a compound according to the patent RU 2195308.

5. The method according to claim 1 wherein said WSPG is a compound according to the patent application RU 2013151824.

6. The method according to claim 1 wherein said WSPG is a pharmacological compound registered in Russian Federation under No. 001919/02-2002

7. The method according to claim 1 wherein said compound WSPG is an immunostimulator.

8. The method according to claim 1 wherein said compound WSPG activates dendritic cells and macrophages transforming them into killer cells and/or tumor cell growth inhibitors.

9. The method according to claim 1 wherein said compound WSPG activates NK cells, increasing their antitumor activity.

10. The method according to claim 3, wherein said mammalian cells are autologous peripheral blood mononuclear cells or dendritic cells or macrophages.

11. The method according to claim 3, wherein said mammalian cells activated with WSPG and administered to a patient in need thereof inhibit the growth of tumor and the spread of metastases.

12. The method of claim 1, for use in treating cancer selected from the group consisting of melanoma, colon, breast, prostate, bladder, lung, kidney, liver, stomach, pancreas, ovary, uterus, head and neck, brain, bone marrow and a hematological malignancy, as well as sarcoma, glandular tissue, skin tumor, mucosal and other epithelia.

13. An immune cell activated with compound WSPG endowed with tumoricidal activity, said cell selected from a group of cells comprising of: a) T cells; b) monocytes; c) dendritic cells; d) macrophages; e) NK cells; f) peripheral blood mononuclear cells; g) NKT cells; h) lymphokine activated killers; i) NK-92 cells; j) DC-CIK; k) combination of dendritic cells and NK cells; l) dendritic cells and T cells; and m) CD8 T cells cultured with antigen presenting cells.

14. The immune cell of claim 13, wherein said immune cell is pretreated with a source of tumor antigen.

15. The immune cell of claim 13, wherein said tumoricidal activity is determined by ability to reduce viability of a malignant cell.

16. A pharmaceutical kit for treatment of cancer comprising an antibody derived therapeutic whose activity is mediated in part by antibody dependent cytotoxicity (ADCC) and a therapeutically sufficient amount of WSPG capable of augmenting activity in vivo of cells mediating ADCC.

17. The pharmaceutical kit of claim 16, wherein said antibody derived therapeutic whose activity is mediated in part by ADCC is selected from a group of antibodies comprising of: a) Herceptin; b) rituximab; and c) cetuximab.

18. A pharmaceutical kit for treatment of cancer comprising a checkpoint inhibitor and a therapeutically sufficient amount of WSPG capable of augmenting in vivo activation of innate immune cells.

19. The pharmaceutical kit of claim 18, wherein said checkpoint inhibitor is an inhibitor of immune associated proteins selected from the group comprising of: a) PD-1; b) PD-1 ligand; c) CTLA-4; d) TIM-1; and d) LAG-3.

20. The method of treating cancer with immune cell of claim 13, wherein said immune cells are autologous or allogeneic.

21. A method of treating cancer comprising administration of a therapeutic amount of WSPG and an intervention selected from a group of interventions comprising of: a) chemotherapy; b) radiotherapy; c) hormone therapy; and d) anti-angiogenic therapy.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] FIGS. 1 A and B show killing of 4T1 cell line as well as metastatic 4T1 cells by mouse splenocytes in vitro activated with WSPG (A) shows that naive splenocytes and WSPG alone did not affect the growth of 4T1 cells in vitro, whereas incubation of splenocyte/4T1 co-culture with WSPG led to complete inhibition of 4T1 cell growth. Photographs of the respective wells are presented. (B) shows that activation of splenocytes isolated from 4T1 tumor bearing mice with WSPG inhibited the growth of splenic metastatic tumor cells.

[0027] FIG. 2 shows that WSPG enhances the antitumor activity of mouse macrophages against 4T1 cells in vitro. Peritoneal macrophages from Balb/c mice were co-cultured in the wells of 96-well plate in different ratios with 4T1 tumor cells (50 cells) for 6 days in a medium (white squares) or medium, supplemented with WSPG (black dots). All samples were analyzed in triplets. The color intensity of the 4T1 tumor cell colonies is normalized to the rate in the control 4T1 cell monoculture (mean and standard deviation). P<0.01.

[0028] FIGS. 3 A and B show that normal macrophages and tumor associated macrophages, acquire the capacity to kill 4T1 tumor cells as a result of activation by WSPG. Peritoneal macrophages from tumor-free mice (low-dotted bars) and mice inoculated in the peritoneal cavity with 150000 4T1 cells (high-dotted bars), were washed out of the peritoneum cavity, and then sorted as F4/80 positive cells using FACS Aria II (BD Biosciences) in triplets. A. Sorted macrophages were seeded in the wells of 96-well plate at a density of 1×10.sup.5 cells/well, and cultured for 48 hours in complete medium supplemented with 10 μg/ml WSPG (2) or without the addition of WSPG (1), and then the supernatant was collected and the concentration of NO was evaluated as the stable oxidation product of NO into nitrite in Griess reaction. B. Sorted macrophages were seeded in the wells of 96-well plate at a density of 6×10.sup.3 cells/well and co-cultured with 200 4T1-GFP cells for 4 days in the presence of 10 μg/ml WSPG (2) or without it (1). The number of live 4T1-GFP cells was determined using FACS Aria II after staining with propidium iodide (2 μg/ml). White bar—monoculture of 4T1-GFP cells without the addition of macrophages, black bars—co-cultures of 4T1-GFP cells with macrophages from tumor-free mice, gray bars—co-cultures of 4T1-GFP cells with macrophages, associated with the tumor. Mean values for the three experiments are presented. Significance of differences * p<0.05, ** p<0.01.

[0029] FIG. 4A-C show killing of tumor cells by highly purified BMDC activated with WSPG. A and B show the purity of NK cells isolated from naive mouse spleen (A) and purity of bone marrow derived dendritic cells (BMDC) (B). (C) Antitumor effect of highly purified NK cells and BMDC activated by WSPG during co-cultivation with 4T1 tumor cells in vitro. 4T1 cells (200 cells per well in 96-well Plate) were co-cultured either alone or with purified NK cells (50,000 cells per well), BMDC (25,000 cells per well) or NK/BMDC in the presence of 10 μg/ml WSPG (low-dotted bars) or without it (high-dotted bars). * P<0.05.

[0030] FIG. 5A-E show the activation and proliferation of NK cells by Dendritic cells pre-activated with WSPG. (A, B, C)—analysis by flow cytometer FACS Aria II (BD Biosciences, USA). Allocation strategy of NK cells as negative for CD4 and CD8 (A) and negative for CD19, but positive for DX5 (B). Activated and proliferating NK cells were determined by increased expression of CD69 and a decrease in fluorescence CellTraceViolet (C). (D). Murine BMDC were activated with or without WSPG [incubation for 18 hours in the absence of WSPG (black squares) or in the presence of 10 μg/ml WSPG, white squares], and added in different proportions to murine splenocytes stained with CellTraceViolet (CTV). After 4 days incubation flow cytometry was performed, the percentage of activated CD69.sup.+, DX5.sup.+, CD4.sup.−, CD8.sup.−, CD19.sup.− and proliferating (reduced content CellTraceViolet) NK cells was calculated. Mean values and standard deviations from three independent experiments are presented. Significance of difference (*) P<0.05. (E) Highly purified dendritic cells pre-activated with WSPG, activate the highly purified NK cells. High-dotted bars—monoculture of 5×10.sup.4 highly purified NK cells, low-dotted bars—the co-culture of highly purified NK cells (5×10.sup.4) and dendritic cells (2.5×10.sup.4). Mean values and standard deviations from two independent experiments are presented. Significance of difference (*) P<0.05.

[0031] FIG. 6 shows the activation of NK cells in mice injected with WSPG intravenously. BALB/c mice (female) were administered with WSPG (10 μg per mouse) through orbital sinus. After 18 hours blood mononuclear cells were taken. YAC-1 target cell line was stained with CellTraceViolet™ Proliferation Kit (Invitrogen). Target cells (T) and effector cells (E) were incubated at various ratios of E:T in round bottom 96-well plates for 4 hours. Cells were stained with propidium iodide and the percentage of killed target cells was evaluated by flow cytometer FACS AriaII. Black squares—mice received an intravenous injection of saline (control group), white squares—mice treated with intravenous injection of WSPG. Mean values with standard deviations from 4 independent experiments are presented. * P<0.05

[0032] FIGS. 7A and B show that administration of WSPG inhibits metastatic disease and prolongs the survival of 4T1 tumor-resected mice. 15×10.sup.3 4 T1 tumor cells were injected subcutaneously in the mammary gland of female BALB/c mice. Upon reaching a diameter of 3.5-5 mm tumors were surgically removed. WSPG (10 μg in 100 μl) was injected intravenously via the retro-orbital sinus when tumor became palpable and weekly after tumor removal. (A) Three injections of WSPG significantly decreased the number of clonogenic tumor cells in the lungs of mice. Number of clonogenic 4T1 cells in the lungs of control mice (black circles, n=8) and mice treated by intravenous injection of a solution of WSPG (white triangles, n=9) was determined at day 20 post-surgery, after three injections of WSPG. Significance of differences between groups is defined by an unpaired t-test (*P<0.05). (B) Continued weekly injections with WSPG until the 69.sup.th day significantly prolonged the survival of mice (***P<0.0001). Thirty one percent of mice from this group were completely cured from the disease. Survival curves were compared using the log rank test with GraphPad Prism. Black circles—control group (n=14), isotonic NaCl solution without WSPG, white triangles—experimental group (n=15), WSPG 10 ng/100 μl per mouse weekly. Significance of differences between the groups (***), defined in the test Log—rank (Mantel-Cox) is 0.001.

[0033] FIGS. 8A and B show that WSPG increases the frequency of activated NK (A) and CD8.sup.+ (B) cells in spleens of 4T1 tumor-resected mice. 15×10.sup.3 4 T1 tumor cells were injected subcutaneously in the mammary gland of female BALB/c mice. Upon reaching the diameter of 3.5-5 mm the tumors were surgically removed. WSPG (10 μg in 100 μl) was injected intravenously via the retro-orbital sinus when tumor became palpable and on days 6 and 13 post-resection. On day 20 post-resection the animals were sacrificed and the activation state of NK cells and CD8.sup.+ T cells in the spleen was evaluated by flow cytometry. Significant differences * P<0.01.

[0034] FIGS. 9A and B show that administration of BMDC pre-activated ex vivo with WSPG inhibited tumor growth (A) and decreased the number of clonogenic tumor cells in lungs (B) of tumor bearing mice in 4T1 metastatic breast cancer model. 7×10.sup.3 4 T1 tumor cells were injected subcutaneously into the mammary gland of female BALB/c mice. 5×10.sup.5 BMDC pre-activated ex vivo with 30 ng WSPG were administered subcutaneously in experimental mice (n=8) three times with weekly intervals. Mice in control group (n=8) were administered with vehicle only. On day 19 mice were sacrificed and the number of clonogenic 4T1 tumor cells in lungs were quantified. (A) The volume of the tumor in cm.sup.3. Black circles—mice treated with BMDC/WSPG injection, white squares—control group. (B) The number of clonogenic metastatic cells in the lungs of mice. Low-dotted bars—mice injected of BM-DC combined with WSPG, high-dotted bars—control group. * P<0.05

[0035] FIG. 10 shows the ability of WSPG to enhance the cytolytic activity of NK cells against human tumor cells K526. Mononuclear cells from peripheral blood of healthy donors were incubated for 18 hours in the presence of WSPG (10 ng/ml). K562 target cells were stained with a set CellTraceViolet™ Proliferation Kit (Invitrogen). Effector cells (E) and target cells (T) were incubated at various ratios of E:T in round bottom 96-well plates for 4 hours. Cells were stained with propidium iodide and the percentage of killed target cells was counted on a flow cytometer FACS Aria II. Mean values and standard deviations of the results of experiments with blood cells from three donors are presented. * P<0.05

EXAMPLES

Example 1

[0036] Mouse splenocytes activated with WSPG in vitro are capable of killing 4T1 cell line as well as metastatic 4T1 cells

[0037] This example shows that after being activated with WSPG splenocytes from tumor-free mice as well as mice inoculated with 4T1 tumor (breast cancer) can kill 4T1 cells in vitro.

[0038] 4T1 cells were seeded in the wells of 96-well plate at a density of 50 cells/well in 200 μl of complete DMEM and cultured in quadruplicates either alone or in the presence of 5×10.sup.4 splenocytes from naive BALB/c mice, and in the presence or absence of WSPG (10 μg/ml). Cultures were incubated for 7 days at 37° C. and 5% CO.sub.2. For visualization and quantification, 4T1-colonies were fixed using 1% paraformaldehide, stained using 0.5% methylene blue in 50% ethanol. Digital images of each well were taken, then the integrated color density (blue channel) was calculated using ImageJ software (NIH, USA).

[0039] As shown in FIG. 1A adding WSPG to monoculture of 4T1 cells does not alter their viability or the rate of reproduction—the number and size of 4T1 colonies in the presence of WSPG does not differ from the number and size of colonies in monoculture of 4T1 cells without WSPG after 6-7 days of culture.

[0040] Co-culturing of 4T1 cells with 5×10.sup.4 tumor-free mouse splenocytes also had no effect on tumor cell growth. However, adding WSPG (10 ng/ml) into co-cultures of 4T1 cells/splenocytes, dramatically inhibited tumor cell growth.

[0041] According to the literature it is known that the development of 4T1 tumors induces large number of myeloid derived suppressor cells (MDSC) that inhibits the function of the immune effector cells (Gabrilovich et al. Nat Rev Immunol 12:253-68, 2012). Indeed, in the spleens of mice inoculated with 4T1 tumor, we observed the increasing content of the MDSC (CD11b.sup.+ Gr1.sup.+ cells). 25-30 days after subcutaneous inoculation of 7×10.sup.3 4 T1 cells, when the primary tumor reached the size of 1 cm.sup.3, the level of MDSC in the spleens of tumor-bearing mice reached to 60-70%. It was of great interest to determine whether WSPG can activate antitumor properties of splenocytes when it is massively infiltrated by suppressor cells. FIG. 1B shows that splenocytes from tumor-bearing mice almost completely inhibited the growth of 4T1 cells in the presence of WSPG in vitro despite the presence of 70% of MDSC.

Example 2

WSPG Enhances Antitumor Properties of Mouse Macrophages In Vitro

[0042] In Example 1, we demonstrated that mouse splenocytes activated by WSPG inhibited the growth of 4T1 breast cancer cell line in vitro. To clarify the nature of the cells that have anti-tumor effect when activated by WSPG, we investigated isolated populations of various cell subsets of the immune system.

[0043] Tissue macrophages were obtained from the peritoneal cavity swabs of female BALB/c mice. 5 ml of PBS supplemented with 1% glucose and 1 mg/ml bovine serum albumin (BSA) was administered into the peritoneal cavity of mice (mPBS, pH 7,4). After several minutes of gentle massage frontal abdominal wall was dissected, peritoneal washings were collected with plastic Pasteur pipette, further the peritoneal cavity was washed three more times, each time introducing 5 ml of PBS. All portions of washes were combined in the centrifuge tube, cells were pelleted by centrifugation at 1200 rpm/min for 10 min, resuspended in complete culture medium at the concentration of 1×10.sup.6 cells/ml (RMPI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 mM β-mercaptoethanol and 10 μg/ml of gentamicine). Cells were incubated overnight at 37° C. and 5% CO.sub.2. Next day, non-adherent cells were washed with warm PBS, pH 7.4, Versene solution was added to adherent macrophages and cells were kept in the refrigerator for 60 min at 4° C., and then washed off with a jet of Versene solution. Macrophages were pelleted by centrifugation at 1200 rpm/min for 10 min and resuspended in CM. Various amounts of macrophages were incubated in wells of 96-well plate (in a volume of 200 μl per well in triplets) with the 4T1 tumor cells (50 cells/well) in the presence of WSPG (10 μg/ml) or without it. After 6 days of incubation at 37° C. and 5% CO.sub.2, The number of 4T1 colonies was quantified as described in Example 1.

[0044] FIG. 2 shows quantitative analysis of the color intensity. It is evident that the macrophages themselves are capable of killing tumor cells at a sufficiently high amount of 45×10.sup.3 cells per well. Adding WSPG greatly enhances the antitumor activity of macrophages. 11×10.sup.3 macrophages per well activated by WSPG are sufficient for complete inhibition of the tumor growth. Thus, WSPG increased the antitumor activity of macrophages more than 4 fold.

Example 3

WSPG Converts Tumor-Associated Pro-Tumoral Macrophages, into Killer Macrophages

[0045] To obtain macrophages, associated with a tumor, 1.5×10.sup.4 4 T1 tumor cells were injected into the peritoneal cavity of BALB/c mice. The tumor growth rate was determined by detection of MDSC in blood of injected mice (Espagnolle et al. Cancers (Basel), 6:472-90, 2014). After 7-14 days, when MDSC level in blood was above 10.sup.6 cells/ml the mice were sacrificed and the peritoneal macrophages were eluted as described in Example 2. In order to achieve a greater purity of the population, macrophages were stained with APC-conjugated anti-F4/80 antibodies and by FACS Aria II (BD Biocseinces).

[0046] To determine the NO producing ability of WSPG-activated macrophages, 1×10.sup.5 cells were seeded per well and cultured in complete medium for 48 hours with or without addition of WSPG. Then the supernatant was taken and NO production was estimated from determining the concentrations of nitrite and nitrate end products of NO oxidation using calorimetric method based on Griess reaction (Green et al. Anal Biochem 126:131-8, 1982).

[0047] To determine the antitumor activity of macrophages sorted cells (6×10.sup.3 macrophages/well) were co-cultured with 4T1-GFP cells in complete culture medium for 4 days. 4T1-GFP cells were harvested and the number of live 4T1-GFP cells was determined by flow cytometry on FACS Aria II (BD Biosciences) after staining the cells with propidium iodide.

[0048] As shown in FIG. 3A the level of NO production by tumor-associated macrophages is significantly (p<0.05) reduced compared with macrophages isolated from tumor-free mice indicating their pro-tumoral properties. Importantly, incubation in the presence of WSPG significantly increased the level of NO production in both populations: in macrophages from tumor-free mice and in tumor-associated macrophages, rising it to almost the same level.

[0049] Data presented in FIG. 3B shows that macrophages isolated from tumor-free mice significantly inhibited growth of 4T1 tumor cells while the tumor-associated macrophages did not affect the growth of cells. Adding WSPG to the co-culture led to activation of antitumor properties of both populations and inhibition of the growth of 4T1 tumor cells by macrophages isolated from tumor-free mice as well as tumor-associated macrophages.

Example 4

Various Preparations Based on Acidic Peptidoglycan from Sprouts of Solanum tuberosum Stimulate Antitumor Activity of Mouse BMDC

[0050] We compared the antitumour properties of several preparations of the same type of water-soluble acidic peptidoglycans from sprouts of Solanum tuberosum, in particular WSPG-1 [RU 2195308], similar thereto WSPG-2 [RU 2013151824] and the pharmaceutical formulation, WSPG-3 (R No. 001919 Identification Number/02).

[0051] Mouse BMDC (4×10.sup.4/well) were co-cultured with 4T1-GFP tumor cells (200 cells/well) in the presence of WSPG-1, WSPG-2 or WSPG-3 (10 μg/ml). After 4 days numbers of live 4T1-GFP tumor cells were counted in the wells by flow cytometery after staining with propidium iodide. The data in Table 1 shows that stimulation of antitumor activity of BMDC is a common feature for all the investigated formulations of acidic peptidoglycan from sprouts of Solanum tuberosum.

TABLE-US-00001 TABLE 1 The inhibition of 4T1 tumor cells by mouse BMDC activated by different WSPG drugs Number of live 4T1 cells/well Standard Percent Sample Mean deviation inhibition Significance 4T1 tumor cells 5667 317 4T1 tumor cells + 239 68 96% 0.001 *** BMDC + WSPG-1 4T1 tumor cells + 66 2 99% 0.001 *** BMDC + WSPG-2 4T1 tumor cells + 64 20 99% 0.001 *** BMDC + WSPG-3

Example 5

Sorted Dendritic Cells Stimulated with WSPG Independently Kill Tumor Cells as Well as Activate Antitumor Activity of NK Cells

[0052] In this example, we have shown a direct activating effect of WSPG on pure population of BMDC as well as NK cells, which are widely recognized as the killers of tumor cells.

[0053] Mouse BMDC cells have been purified by cell sorter FACS Aria II to a purity of 99% by the selection of cells positive for CD11c and IA/E (FIG. 4B). Highly purified NK cells were prepared from a suspension of naive mouse splenocytes in two stages. First NK cells were enriched to a purity of 70-80% using EasySep™ Mouse NK Cell Enrichment Kit (STEMCELL Technologies Inc. Canada) according to the manufacturer's protocol, and then 98-99% purity was reached by cell sorting on BD FACS Aria II, as cells positive for DX5.sup.+ and negative for I-A/E (FIG. 4A).

[0054] 4T1-GFP Tumor cells were cultured (at density 200 cells/well) in monoculture or in combination with highly purified NK cells and/or highly purified BMDC. As shown in FIG. 4C, highly purified NK or highly purified BMDCs individually were unable to inhibit 4T1-GFP growth. However co-cultures of these purified populations of cells exerted significant anti-4T1-GFP tumor effect (**P<0.01). Importantly, although WSPG-2 itself had no effect on 4T1-GFP growth, the addition of WSPG-2 either to the BMDCs or NK/BMDC co-cultures dramatically improved the inhibition of 4T1-GFP growth (***P<0.001). No effect was observed when WSPG-2 was added to the NK cells (FIG. 4C).

Example 6

Activation of NK Cell by WSPG is Mediated by Mouse Dendritic Cells

[0055] Example 5 shows that WSPG-2 does not directly activate purified splenic NK cells, but does significantly increase the capacity of BMDC to activate NK cells. Murine BMDC were pre-activated with WSPG-2 (incubation for 18 hours in the presence of 10 μg/ml) and added in different proportions to murine splenocytes stained with CellTraceViolet (CTV). After 4 days the percentage of activated (CD69.sup.+ DX5.sup.+ CD4.sup.−CD8.sup.−CD19.sup.−) and proliferating (reducing CTV) NK cells was determined by flow cytometry (FIG. 5 A, B, C). Data show that significantly higher (P<0.05) number of activated NK cells was detected when splenocytes were co-cultured with 2.5-10% of purified BMDC pre-activated with WSPG (FIG. 5 D).

[0056] Direct activating effect of dendritic cells previously stimulated with WSPG was confirmed using highly purified populations of NK and BMDC cells (FIG. 5E). Highly purified NK cells (5×10.sup.4/well) and BMDC (2.5×10.sup.4/well) were co-cultured for 18 hours without (control) or with WSPG and percentage of activated NK cells (CD69.sup.+ cells) was determined. BMDC themselves activated considerably NK cells. However, the addition of WSPG to the NK/BMDC co-cultures dramatically increased the percentage of activated NK cells and led to a 2-fold increase in the level of expression of the CD69 on the NK cells (FIG. 5E).

Example 7

WSPG Injected Intravenously into Mice, Enhances the Cytolytic Activity of NK Cells

[0057] Example 6 shows the effect of WSPG on activation of mouse NK cells in vivo. BALB/c mice (n=12/group) were administered with 10 μg WSPG-2 through orbital sinus. Mice in the control group were administered with vehicle only. Peripheral blood mononuclear cells (PBMC) collected from mice 18-20 hrs after injection of WSPG have significantly higher cytotoxic activity (about 2-fold, P<0.05) against YAC-1, a classical mouse NK-target tumor cell line, as compared to PBMC from vehicle only injected control mice (FIG. 6).

Example 8

Anti-Tumor Activity of WSPG in the 4T1 Post-Resection Metastatic Breast Cancer Model

[0058] Example 8 shows the effect of WSPG on survival, the number of clonogenic metastatic cells in the lungs, as well as immune suppression and/or activation in a model of 4T1 metastatic breast cancer after surgical removal of the tumor. The 4T1 mouse model is the most clinically relevant and stringent breast cancer model yielding metastatic disease to the lungs, brain and bone, even after primary tumor resection, remarkably similar to human breast cancer (Pulaski et al. Current protocols in immunology 4:20.2.1, 2001; Heppner et al. Breast Cancer Res 2:331-4, 2000).

[0059] Resection of tumor in 4T1 breast cancer mouse model creates a “window of opportunity” with decreased tumor-associated immune suppression and increased frequency of CD4 and CD8 positive activated T cells, which exists for approximately 10 days (from the fourth to the thirteenth day) after resection (Ghochikyan et al. J Transl Med 12:322, 2014). Administration of WSPG was initiated prior to tumor resection, when tumors were just palpable and was continued during the “window of opportunity” (on 6th and 13th days after tumor resection). One half of the mice were terminated on day 20 post-surgery and clonogenic tumor cells from metastatic disease in the lungs were analyzed, while the other half of mice continued to be injected with WSPG every week until day 69 with survival of mice monitored to day 140. Treatment with WSPG significantly reduced the number of clonogenic tumor cells in lungs of mice detected on day 20 post-resection compared with control animals injected with PBS. Further injections of mice with WSPG resulted in a statistically significant prolongation of survival of mice and complete cure in 31% of the mice (FIG. 7).

[0060] The frequencies of activated NK, CD4.sup.+ and CD8.sup.+ cells as well as myeloid-derived suppressor cells (MDSC) and Treg suppressor cells were evaluated in spleens of WSPG treated mice on day 20 post-resection. Treatment with WSPG significantly increased frequency of activated NK, CD4.sup.+ and CD8.sup.+ cells compared with vehicle injected control mice. The frequency of MDSC was slightly but significantly decreased in the spleens (7.86±1.26% vs 12.43±1.84%, *P<0.05) of mice treated with WSPG relative to control animals. No differences were observed in frequency of Treg cells in spleens of treated and control mice.

Example 9

BMDC Pre-Activated with WSPG Reduces Tumor Growth and Number of Metastasis in 4T1 Tumor-Bearing Mice

[0061] 7×10.sup.3 4 T1 tumor cells were injected subcutaneously into the mammary gland of female BALB/c mice. 5×10.sup.5 BMDC pre-activated ex vivo with 30 μg WSPG were administered subcutaneously in experimental mice (n=8) three times with weekly intervals. Mice in control group (n=8) were administered with vehicle only. On day 19 tumor size was measured, mice were sacrificed and the number of clonogenic 4T1 tumor cells in lungs were quantified.

[0062] As shown in FIG. 9A mean tumor volume in BMDC/WSPG injected mice was significantly (P<0.05) smaller than in control group.

[0063] FIG. 9B shows that 2 mice (out of 12) treated with BMDC/WSPG were free of lung metastases. 6 mice had less than 25 metastases and only one mouse had >25 metastases, while 100% of animals from control group had greater than 5 clonogenic metastatic cells in the lungs. Average numbers of clonogenic metastatic cells were 9.5 and 37.7 in mice vaccinated with BMDC/WSPG and control animals, respectively.

Example 10

Mononuclear Cells of Human Peripheral Blood Activated with WSPG Effectively Kill

[0064] This example shows the ability of WSPG to activate human antitumor killer cells.

[0065] PBMC from healthy donors were incubated (18 hours, 37° C., 5% CO.sub.2) in the absence or presence of WSPG-2 (10 μg/ml). The target K562-cells were labelled by the CellTrace™ Violet. Co-cultures of PBMC and K562 at different effector:target ratios were incubated for 4 hrs at 37° C. and 5% CO.sub.2. Cells were stained with propidium iodide and the percentage of killed target cells was counted on a flow cytometer FACS Aria II. The experiment was repeated using PBMC from three donors.

[0066] The data in FIG. 10 shows, that WSPG significantly (P<0.05) enhances the cytotoxic activity of human blood mononuclear cells at all tested ratios of effector cells to target cells (E:T).

Example 11

WSPG Activates Antitumor Properties of Killer Dendritic Cells of Humans

[0067] Dendritic cells were prepared by differentiation of human peripheral blood monocytes from healthy donors as described (Nair et al. Curr Protoc Immunol, Chapter 7:32, 2012). 12.5×10.sup.3 moDC cells were co-cultured with 200 4T1-GFP tumor cells for 4 days without or with WSPG-2. The numbers of live 4T1-GFP per well were determined using a flow cytometer BD FACS Aria II as previously described. Data in Table 2 show that the activation of human dendritic cells by WSPG-2 significantly enhances their ability to inhibit the growth of 4T1-GFP cells.

TABLE-US-00002 TABLE 2 Inhibition of the growth of tumor cells by human dendritic cells (MoDC) affected by WSPG. Number of live 4T1 per well Standard Percent The Sample Mean deviation inhibition accuracy 4T1 tumor cells + 11603 717 MoDC 4T1 tumor cells + 4692 86 60% 0.05 * MoDC + WSPG-2, 10 μg/ml

Example 12

Injection of Pharmaceutical Composition of WSPG Activates Antitumor Properties of Human Immune Cells

[0068] This example shows the ability of WSPG to activate anti-tumor properties of the immune cells directly in the human body after injection of a pharmaceutical-grade WSPG. Studies have been conducted on human volunteers with the pharmaceutical preparation of WSPG designated as “Immunomax” (LLC <<Immafarma” Moscow, P No. 001919/02-2002). “Immunomax” is available for injection in vials. The content of the vial (200 IU) were dissolved in 1 ml of water and administered intravenously.

[0069] Blood was collected into BD Vacutainer tubes before the intravenous administration and 1 hour, 3 hours, 7 hours, 24 hours after the intravenous administration for detection of cytokines and analyses of cellular composition of blood. The effect of WSPG was evaluated by characterization of blood lymphocyte populations: detection of activation markers, determination of cytolytic properties (presence of perforin), measuring of cytokines concentration.

[0070] Table 3 presents data, showing increasing concentration of immunostimulating cytokines in peripheral blood after intravenous administration of “Immunomax”. After 1 hour the level of TNF-α was increased up to 40 pg/ml and almost returned to the baseline level after 2 hours (Table 3).

TABLE-US-00003 TABLE 3 Changes in the content of TNF-α in the blood of volunteers after the intravenous administration of Immunomax Time after The content of TNF-α in blood, pg/mL Significant injection of Standard differences Immunomax (h) Mean deviation (P) 0 3.0 2.7 — 1 42 42   0.05 * 2 6.5 4.3 0.1 3 4.2 3.9  0.51 4 2.2 1.2 0.5 5 3.2 4.1 0.9

[0071] Tables 4-8 presents the changes in the composition of the immune cells in the peripheral blood of volunteers after intravenous administration of Immunomax.

[0072] 1-3 hours after the administration of Immunomax, monocytes in the peripheral blood decreased about 2-fold of the baseline, and in 7 hours returned to the original level, and during the next day were increased 3-fold relative to the baseline (Table 4).

TABLE-US-00004 TABLE 4 Changes in the content of monocytes in the peripheral blood of volunteers after intravenous administration of Immunomax Time after Monocytes in 1 ml of blood Significant injection Standard differences Immunomax (h) Mean deviation (P) 0 539 157 0.02 * 1 245 257 0.02 * 3 218 143  0.001 * 7 671 251 0.2   24 1025 381  0.008 *

[0073] Around the same period of time, changes in the expression of CD49d molecules on the surface of monocytes (Table 5) were observed. CD49d is the integrin (very late antigen-4, VLA-4) that is responsible for the adhesion of monocytes to endothelial cells via vascular cell adhesion molecule-1 (VCAM-1). Expression of VCAM-1 is usually increased at sites of inflammation. Thus, monocytes overexpressing CD49d have greater capacity for adhesion and migration to sites of inflammation, such as in a tumor or metastases. Thus, WSPG may contribute to the body's ability to combat tumor growth by increasing the number, activity and the ability of monocytes to migrate to the center of the tumor.

TABLE-US-00005 TABLE 5 Changes in the expression of CD49 on monocytes in the peripheral blood of volunteers following intravenous administration of Immunomax. Expression of CD49 molecules on monocytes, Time after normalized to the initial value Significant injection of Standard differences Immunomax (h) Mean deviation (P) 1 0.99 0.29 3 0.58 0.30  0.02 * 7 0.57 0.20 20.00 * 24 1.67 0.36 20.00 *

[0074] Table 6 shows that after 24 hours there is an increase in the expression of HLA-DR molecules on monocytes. HLA-DR molecules on monocytes/macrophages are necessary for antigen presentation to T cells and for the effective development of protective T cell responses against the tumor. The ability of WSPG to increase

TABLE-US-00006 TABLE 6 Change in the expression of HLA - DR on monocytes in peripheral blood of volunteers after intravenous administration of Immunomax Expression of the HLA - DR molecule on monocytes, Time after normalized to the initial value Significant injection of Standard differences Immunomax (h) Mean deviation (P) 1 1.2 0.4 0.2.sup.  3 0.5 0.1 10.00 * 7 0.6 0.2 10.00 * 24 1.2 0.6 0.36 
the expression level of the antigen-presenting HLA-DR molecules can enhance the specific reaction of the immune system against a tumor.

[0075] Injection of “Immunomax” (WSPG) increases the number of cytolytic CD8.sup.+ T cells containing perforin in the peripheral blood (Table 7). In the first hours after administration of the drug content of CD8.sup.+ T-cells decreases, but within 1 day, it increased to 1.5 times compared with the level of these cells prior to administration of “Immunomax”. Decrease in the amount of circulating blood cells in the first few hours after the drug administration may indicate their massive migration to the tumor site or to the tissue with inflammation. Later, after 1 day, the drug stimulates the proliferation of cells and the maximum number of CD8.sup.+ T cells is detected on the third day after the administration of Immunomax. Thus, WSPG promotes specific defense reaction of the immune system against the tumor.

TABLE-US-00007 TABLE 7 Changes in the perform content of cytolytic CD8.sup.+ T cells in the blood of volunteers after intravenous administration of Immunomax Content of perform in cytolytic CD8.sup.+ T Time after cells in 1 ml of blood Significant injection of Standard differences Immunomax (h) Mean deviation (P) 0 101 70 1 57 46 <0.05*  3 27 19 <0.001* 7 30 25 <0.001* 24 155 101 0.2 

[0076] Cytolytic NK cells are generally considered to be a component of the first line of defense against cancer (Lauzon et al. Adv Exp Med Biol 598:1-11, 2007) and play an important role in dealing with the neoplastic cells in the body. As shown in Table 8, even at 1 hour after intravenous injection Immunomax significantly enhanced activation state of NK cells.

TABLE-US-00008 TABLE 8 Activation of NK cells (upregulation of CD69) in volunteers after intravenous administration of Immunomax The content of activated CD 69+ NK cells in the blood, normalized to the Time after initial value (Before administration) Significant injection of Standard differences Immunomax (h) Mean deviation (P) 1 2.10 0.13 <0.05 * 3 5.87 0.32 <0.05 * 7 5.23 0.64 <0.05 * 24 1.29 0.85 .

[0077] Overall, the data presented in Example 12 indicate that Immunomax activates protective immune cells (monocytes, CD8.sup.+ T cells and NK cells) and directs them to the pathologically altered tissues. This effect of the drug may provide the increased protection against cancer.