Methods and compositions for treating cancer and enhancing therapeutic immunity by selectively reducing immunomodulatory M2 monocytes.

Abstract

To provide methods and compositions for treating cancers and enhancing therapeutic immunity by selectively reducing immunomodulatory M2 monocytes.SOLUTION: A pharmaceutical composition comprises an effective amount of an Insulin-like Growth Factor 1 Receptor antisense oligodeoxynucleotide (IGF-1R AS ODN). Administering the pharmaceutical composition to a subject having M2 cells in their circulation, M2 cells in a tumor microenvironment, or a serum that polarizes undifferentiated monocytes towards M2 cells reduces the number of M2 cells in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This disclosure relates to methods and compositions for treating cancer and enhancing therapeutic immunity by selectively reducing M2 cells by targeting them with antisense nucleic acids specific to the insulin-like growth factor 1 receptor (IGF-1R). [Background technology]

[0002] Monocytes are a type of white blood cell that originates from myeloid progenitor cells in the bone marrow. Monocytes enter the peripheral bloodstream from the bone marrow and later migrate to tissues. Within the tissues, after exposure to local growth factors, pro-inflammatory cytokines, and microbial compounds, monocytes differentiate into macrophages and dendritic cells. Macrophages derived from monocyte progenitor cells undergo specific differentiation into classically polarized (M1) macrophages and non-classically activated (M2) macrophages. Macrophages typically perform three main functions in the immune system: phagocytosis, antigen presentation, and cytokine presentation. Furthermore, certain types of cancer (e.g., breast cancer, astrocytoma, head and neck squamous cell carcinoma, papillary renal cell carcinoma type II, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, glioma, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer) exhibit elevated levels of M2-like macrophages within the tumor and similar M2 monocytes circulating in the periphery. [Overview of the project] [Problems that the invention aims to solve]

[0003] Despite advances in cancer treatment, the prognosis for these cancers remains poor. Attempts to treat them with conventional therapies such as chemotherapy, external beam radiation, and brachytherapy have yielded only marginal improvements in progression-free survival and overall survival. Therefore, there is a need for new and improved treatments for these cancers in this field. [Means for solving the problem]

[0004] In some embodiments, the Disclosure provides a pharmaceutical composition comprising an effective amount of insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN) to which the number of M2 cells in the subject is reduced by administering the pharmaceutical composition to a subject having circulating M2 cells, M2 cells in the tumor microenvironment, or serum that polarizes undifferentiated monocytes into M2 cells.

[0005] In other embodiments, the present disclosure provides a method for selective removal of M2 cells in a subject, comprising the step of systemically administering a pharmaceutical composition to the subject in an effective amount. In other embodiments, the present disclosure provides a method for treating cancer by reducing the number of M2 cells, comprising the step of systemically administering an effective amount of a pharmaceutical composition to a subject suffering from cancer.

[0006] In further embodiments, the present disclosure provides a method for enhancing an immune response in a subject, comprising the step of systemically administering a pharmaceutical composition to the subject in an effective amount. [Brief explanation of the drawing]

[0007] [Figure 1] Figure showing CD163+ cell expression in the periphery of patients with glioma. This subset of monocytes is initiated by the presence of tumor, and this subpopulation supports tumor growth and invasion due to its angiogenic and immunosuppressive nature. The grade of glioma is associated with the accumulation and activity of cells with the M2 monocyte marker. The presence of M2-like CD163+ macrophages within the tumor and similar M2 monocytes in the circulating periphery of this population also negates the strategies of any pro-inflammatory antitumor vaccine. a. Flow cytometry is shown reflecting the increase in CD14+ cells within WHO grade III astrocytoma. b. Graph showing comparison of CD163+ cell levels according to WHO grade. Grade III and grade IV tumors show significantly different % monocytes in PMBC compared to either a normal control or a WHO grade II astrocytoma. [Figure 2a] This figure illustrates the uptake of insulin-like growth factor 1 receptor-specific labeled antisense nucleic acid (IFG-1R AS ODN) by cell type. Macrophages (CD14+) derived from tumors and corresponding blood samples in glioma patients greedily take up insulin-like growth factor 1 receptor-specific antisense (IGF-1R AS ODN). [Figure 2b] This figure illustrates the uptake of insulin-like growth factor 1 receptor-specific labeled antisense nucleic acid (IFG-1R AS ODN) by immunotypes. Macrophages (CD14+) derived from tumors and corresponding blood samples in glioma patients greedily take up insulin-like growth factor 1 receptor-specific antisense (IGF-1R AS ODN). [Figure 3a] This figure shows flow cytometry of cells expressing insulin-like growth factor 1 receptor (IFG-1R). Normal peripheral monocytes, which are polarized to M2 cells in vitro, overexpress IGF-1R compared to macrophages, which are induced to M1 polarization. Furthermore, IGF-1R AS ODN selectively induces cell death in the M2 subpopulation in a dose-dependent manner. Figure 3a details how IGF-1R AS ODN selectively targets the removal of M2 macrophages so that their protumor-promoting effects can be eliminated under conditions where these cells are dominant, and therapeutic Th1 immunity can be rescued. White circles represent differentiated, unstimulated cells, and black circles represent differentiated, stimulated cells. [Figure 3b] This figure shows flow cytometry of cells expressing insulin-like growth factor 1 receptor (IFG-1R). Normal peripheral monocytes, which are polarized to M2 cells in vitro, overexpress IGF-1R compared to macrophages, which are induced to M1 polarization. Furthermore, IGF-1R AS ODN selectively induces cell death in the M2 subpopulation in a dose-dependent manner. Figure 3b shows the difference in monocyte subset distribution after treatment with IGF-1R AS ODN due to macrophage polarization. [Figure 4]Figure 4 shows the quantification of tumor-associated CD163+ cells in patients throughout the course of treatment. The mean and standard deviation of the 5400x field of view were measured by Aperio quantification. Aperio CD163+ cells are provided on the y-axis at four time points: first surgery, first recurrence, second recurrence, and autopsy. Figure 4 shows that the method disclosed herein is effective in reducing CD163+ cells in patients who have not responded to standard treatment. [Figure 5] This figure shows immunohistochemistry for IGF-1R in six consecutive glioblastoma multiforme specimens. All tumors showed immunoreactivity to IGF-1R. IGF-1R immunoreactivity indicates the presence of one or more IGF-1R-expressing cells in the tumor microenvironment, and identifies IGF-1R as a target in cancer therapy. [Figure 6] Figure showing mass spectrometry of two different sequence lots of IGF-1R AS ODN. ac: Avecia lot product of the DWA sequence; df: Girindus lot product of the NOBEL sequence; a,d: Stability of AS ODN in lyophilized powder form; b,e: Formulation in sterile saline; c,f: Formulation in sterile saline. The stability results for IMV118 lot number GAI-08-060-S3-B1 show that the minimum degradation product is approximately 300 Da, and therefore the measured mass spectrum meets the requirement of 5709 ± 300 Da and the acceptable stability in storage from lot release to the present. The Avecia sequence (DWA) shows stability for 9 years. [Figure 7]A figure showing that circulating CD68+CD163+ cells decrease in animals at least 14 days after systemic (intraperitoneal) administration of one dose of NOBEL (SEQ ID NO. 1) following transplantation into the central nervous system of GL261. NOBEL is an 18-mer phosphorothioate oligodeoxynucleotide IGF1-R antisense oligodeoxynucleotide (AS ODN) beginning with six nucleotides downstream from the starting methionine codon. NOBEL is prepared by solid-phase organic synthesis using a well-established methodology in a synthesizer equipped with a closed chemical column reactor using flow-through technology. Each synthetic cycle sequence on a solid support consists of multiple steps, which are carried out sequentially until the full-length oligonucleotide is established. NOBEL is then lyophilized, packaged in HDPE containers with screw caps, and then vacuum heat-sealed inside 5 mL Mylar pouches for storage at -80°C. Before use, the lyophilized powder is dissolved in physiological saline until a 100 mg / mL solution is obtained. The resulting solution is sterile filtered through a 0.22 μm membrane filter. Before storage at -80°C, 1 mL aliquots are filled into USP Type 1 glass vials and sealed with appropriate rubber stoppers and aluminum caps. In this experiment, leukocytes were stained with biotinylated anti-mouse CD163 (Biorbyt), washed, and secondary streptavidin-APC was added. After two washes and fixation, the cells were permeabilized, intracellularly stained with anti-mouse CD68-PE, and subsequently washed twice with palm buffer to close the membrane, followed by a final PBS wash. The samples flowed on a Millipore Guava flow cytometer and analyzed using FlowJo. Samples taken at the time of slaughter show significant changes in the WBC population after intraperitoneal administration of Nobel. a. PBS intraperitoneal injection control; b. Nobel intraperitoneal injection. [Figure 8]This figure shows that administration of NOBEL (SEQ ID NO: 1) alone before tumor formation is effective in delaying the initiation of GL261 cell growth. While 60% and 100% of C57 and Tbet knockout animals, respectively, developed tumors after intraperitoneal administration of the solvent (PBS), 20% and 50% of C57 and Tbet knockout mice, respectively, developed tumors after intraperitoneal administration of NOBEL. Significance was assessed using the log-rank test (*=p<0.05). [Figure 9] A diagram showing that cytosine-phosphorothioate-guanosine-DNA activates TLR9 expressed on B cells and plasmacytoid dendritic cells (DCs). [Figure 10] Figure showing that antigen-presenting cells take up AS ODN, express increased co-stimulatory molecules, and express various levels of CD80 / 83 / 86 in PBMCs before and after AS ODN treatment (mDC, bone marrow dendritic cells; pDC, plasmacytoid dendritic cells). [Figure 11] This figure shows that NOBEL (SEQ ID NO. 1) activates monocyte-derived dendritic cells (DCs), as determined by a decrease in median fluorescence intensity. Immature DCs phagocytose large amounts of fluorescent protein, resulting in higher fluorescence intensity (indicated by larger bars). Mature (activated) DCs downregulate endocytosis, resulting in reduced uptake of fluorescent protein and lower fluorescence intensity (indicated by smaller bars). Treatment of monocyte-derived dendritic cells with IGF-1R AS ODN shows a significant dose-dependent maturation response. [Figure 12]Figure showing that CpG motifs, 5'G*G motifs, and phosphorothioate binding all provide further maturation stimuli to dendritic cells. a: Immature DCs exhibit highly enhanced endocytosis, phagocytosing large amounts of fluorescent protein (upper panel), resulting in higher fluorescence intensity. b: Monocyte-derived DCs were incubated for 24 hours in the presence of various IGF-1R / AS ODNs (1 μg / ml). LPS-treated DCs (1 μg / ml) served as a positive control in maturation. Immature DCs phagocytosed large amounts of fluorescent protein, resulting in higher fluorescence intensity (indicated by larger bars). (Activated) mature DCs downregulated endocytosis, resulting in reduced uptake of fluorescent protein and lower fluorescence intensity (indicated by smaller bars). The CpG motif contained in IGF-1R / AS ODN also provides maturation stimuli to DCs (see control and LNA DCs). Phosphothioate binding provides further maturation stimuli to DCs (NOBEL DCs). The 5'G*G motif provides a third maturation stimulus to DCs (see DWA PT DC). The oligomers tested included SEQ ID NOBEL, SEQ ID NO. 11 (IDT1220 phosphorothioate AS ODN (IDT1220)), SEQ ID NO. 15 (DWA phosphorothioate AS ODN (DWA PT)), SEQ ID NO. 16 (DWA immobilized nucleic acid AS ODN (LNA)), and SEQ ID NO. 17 (DWA phosphodiester AS ODN (DWA control)). [Figure 13] a. The DWA sequence maintains a 5' hairpin loop secondary structure (shaded inset) at 37°C, which may influence base pairing with targeted mRNA sequences. b. The NOBEL (SEQ ID NO. 1) sequence does not have a 5' hairpin loop (shaded inset) of the CpG motif at 37°C for two alternating secondary structures with MPs at 18°C, allowing for increased targeted base pairing and thus CpG potential. [Figure 14]Figure showing the Nobel (SEQ ID NO: 1) adaptation in GL261. Cells were seeded at 20k / well in a 96-well plate with growth medium and incubated for 4 hours (37 °C, 5% CO2 humidified); the growth medium was removed and serum-free Opti-MEM (100 μL) with the desired AS ODN concentration was added to each well. The cells were returned to the culture medium for a further 24 hours. a. Effect of Nobel adaptation on the expression of IGF-1R in GL261 cells. Final mg / well (or mg / 20k cells) in the microtiter plate versus IGF-1R copy number. Cells were seeded. Significance was determined using analysis of variance (* = P < 0.05; ** = P < 0.001). b. Cells were collected, stained with an antibody specific for mouse IGF-1R, and analyzed by flow cytometry. Median fluorescence intensity was plotted against the final AS ODN concentration (mg / 20k cells). Expression of IGF-1R was significantly decreased in GL261 cells treated with 1 mg of Nobel AS ODN / well (P < 0.001), and in cells treated with 0.1 mg of Nobel AS ODN / well (P < 0.05). [Figure 15] Figure showing the results of quantitative RT-PCR to evaluate downregulation downstream of hexokinase isoform 2 mRNA. Expression of the L13, IGF-1R and HexII genes is linearly correlated in cells of the human glioma line U118 treated with Nobel (SEQ ID NO: 1). mRNA copy numbers specific for the housekeeping gene L13 (▼) and hexokinase 2 [HEX] (■) are shown, plotted against the IGF-1R copy number detected under individual culture with treatment with Nobel at different concentrations. The solid line represents the best-fit linear regression line between L13 and IGF-1R, and the dotted line represents the best-fit linear regression line between Hex-II and IGF-1R, where r2 represents the degree of linearity (from 1.0) and P represents the significance of the gradient. [Figure 16]Figure showing cumulative tumor growth in C57 / B6 mice injected with 106 GL261 cells 2 weeks after AS ODN treatment. In all mice in the AS ODN group, GL261 (overnight AS ODN treatment, 20 mg / 5×106 GL261) treated with 106 NOBEL (SEQ ID NO: 1) was injected once into the flank, and 2 weeks after treatment, WT GL261 was loaded on the opposite flank; in mice in the AS ODN / GL261 mixed group, NOBEL (20 mg / 5×106 GL261) mixed with untreated GL261 cells was injected once into the flank immediately before injection, and 2 weeks after treatment, WT GL261 was loaded on the opposite flank. Tumors developed from the post-treatment load (WT GL261). [Figure 17] Figure showing that the combination of GL261 cells and NOBEL (SEQ ID NO: 1) at the administration site inhibits tumor formation in a subcutaneous model. [Figure 18] Figure showing that NOBEL (SEQ ID NO: 1) induces radiosensitization. [Figure 19] Figure showing the safety evaluation test. a. Overall survival of patients in the trial; b. Overall survival against the interval between surgeries; c. Survival protocol in two survival cohorts. Nine patients died due to disease progression, while 1 died of intracerebral hemorrhage and 2 died of sepsis. The overall survival protocols were 48.2 weeks and 9.2 weeks for the longer-term (N = 4) and shorter-term (N = 8) survival cohorts, respectively (log-rank = 0.0025). c. Excluding deaths not due to disease progression, the median survival was 48.2 weeks and 10 weeks for the longer-term cohort (N = 4) and shorter-term cohort (N = 5), respectively (log-rank = 0.0049); d. Excluding one outlier (longer-term cohort), linear regression showed a high correlation between the survival protocol and the lymphocyte count at registration (R2 = 0.8, p = 0.0028). [Figure 20a]This figure shows the X-ray response of an anatomical tumor, an example from a short-term survival cohort. T12:AD; TJ10:EH; A,E: Preoperative T1-gadolinium-enhanced axial image; G: T1-gadolinium-enhanced coronal image; C: Preoperative axial FLAIR image; B,D,F,H: Images taken 3 months postoperatively for each individual. [Figure 20b] This figure shows the X-ray response of anatomical tumors, an example from a longer-term survival cohort. TJ06:AD; TJ09:EH. A,E: Preoperative T1 gadolinium-enhanced axial image; C,F: Preoperative axial FLAIR image; B,D,F,H: Images 3 months postoperatively for each surgery. [Figure 20c] This figure shows the X-ray response of anatomical tumors, specifically the relationship between the apparent diffusion coefficient and the relative cerebral blood volume within the tumor in a short-term survival cohort. [Figure 20d] This figure shows the X-ray response of anatomical tumors in a longer-term survival cohort. There is a high correlation between the apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) (R²=0.96, p=0.0005). [Figure 20e] This figure shows the X-ray response of anatomical tumors and provides an overview of cytokine responses in a longer-term survival cohort (N=3). [Figure 20f] A figure showing an example of the decrease in CD163+ cells associated with rCBV and ADC over time in patient TJ06. Furthermore, an assay for activated nitric oxide synthetase (Greiss assay), a drug of hyperemia reflected as serum nitrate levels, when associated with rCBV. [Figure 21a] This figure shows the examination of explanted chambers and pathological specimens, with composite optical micrographs of chambers explanted from TJ09. Left column: PBS chamber; Right column: Vaccine chamber; Top row: H&E staining of the extramembrane surface; Bottom row: CD163+ immunostaining of the extramembrane surface. [Figure 21b]This figure shows the examination of the explanted chamber and pathological specimen, with immunofluorescence staining (red) for CD163. a. CD163+TAM in the tumor of patient TJ14 at the time of initial resection; b. CD163+TAM in the tumor of patient TJ14 at the time of recurrence before vaccination; CD163+TAM was increased; c. CD163+TAM in the tumor of patient TJ14 at the time of second recurrence; a decrease in TAM was observed in the tumor microenvironment, and CD163 TAM was found to be associated only with blood vessels; d, e, f. Each at increased magnification. [Figure 21c] Figure showing examination of explanted chambers and pathological specimens. Aperio immunohistochemical quantification of CD163+TAM according to treatment stage (left two panels); levels were similar in both Phase I trials, but significantly lower levels were observed in undiagnosed, untreated patients who underwent autopsy (right panel). [Figure 22] Figures showing continuous measurements of immunoeffector cell changes and cytokine / chemokine changes after vaccination during the post-treatment period; longer survival cohorts (patients TJ03, TJ14, TJ06, TJ09); examples of short-term survival cohorts (see patient TJ13, Figure 25 for all other short-term survival cohorts). Rows: a. Absolute number of CD4 and CD8 compared to the relative amount of WBC in PBMCs; b. Levels of CCL21 and CXCL12; c. Relationship between relative T cell number and macrophage number; d. Relationship between the relative proportion of CD14+CD16-macrophages and CCR2 and MCP-1 (CCL2); e. Estimated Th-1 cytokine response after vaccination. [Figure 23] A summary of cytokine levels (pg / ml) at day 14 in a. estimated Th-1 cytokines and b. Th-2-related cytokines after PMA / ionomycin stimulation in the survival cohort. Comparison of mean (Chuke) and independent t-test. Significance at p<.05. TJ03 was consistently excluded as an outlier with values ​​outside the 95% CI. [Figure 24]Figure showing the radiographic response of anatomical tumors. a and c: Axial gadolinium-enhanced T-1W images; panels a and b: patient TJ06, panels 4c and d: patient TJ07; b and d: Delayed PET / CT images with same axial registration. In panel b, note the absence of normal hypermetabolism in the left temporal lobe cortical ribbon compared to the right temporal lobe; photopenia including a small portion of hypermetabolism in the anterolateral temporal lobe. The majority of the enhancement in (panel a) is interpreted as inflammation. In d, note the clear correlation between hypermetabolism and the corresponding volume of enhancement in panel c, which is interpreted as disease progression. [Figure 25] The figure shows a comparison of mean cytokine levels (pg / ml) by source (Cp, PBS chamber; Cv, vaccine chamber; serum; SN, autologous tumor cell supernatant). CCL21 was significantly elevated in the vaccine chamber compared to both Cp and serum. CCL20 was significantly elevated in Cv and Cp compared to serum, and CCL19 was significantly elevated in Cv compared to Cp or serum. HSP-70 was significantly elevated compared to serum, CCL2 was significantly elevated compared to serum, and CXCL12 was the only cytokine that was significantly elevated in serum compared to Cp (*p<0.035, **p<0.025, ***p<0.015, †p<0.004, ††p<0.0002, †††p<0.0001). [Figure 26a] This figure shows the examination of explanted chambers and pathological specimens. Left panel: Mean comparison of the number of immunopositive cells / 400x field and CD163 TAMs at initial diagnosis and at relapse before vaccination; Right panel: Mean difference in matched pair comparisons -19.2% increase, p<0.0001. [Figure 26b] This figure shows the examination of explanted chambers and pathological specimens. Left panel: Mean comparison of the number of CD163 TAMs at relapse before vaccination and at autopsy; Right panel: Mean difference in matched pair comparisons -26.35% decrease, p<0.0001. [Figure 26c]This figure shows the examination of explanted chambers and pathological specimens, retrospectively comparing CD163 TAM in paraffin samples from initial and current studies with that in six autopsy specimens obtained from undiagnosed and untreated glioblastomas. [Figure 26d] This figure shows the examination of explanted chambers and pathological specimens, and the evaluation of IGF-1R+ cells in paraffin sections obtained at initial diagnosis, relapse before vaccination, and autopsy. [Figure 27a] Comparison of mean numbers of immunopositive cells / 400x field-of-view detected CD163 cells in the survival cohort; left panel: at diagnosis, long-term vs. short-term, p<0.0002; right panel: at tumor resection before vaccination induction, long-term vs. short-term, p<0.0127. [Figure 27b] Linear regression of the relationship between peripheral and tumor-associated macrophages (R²=0.96, p=0.004). [Figure 28-1] Figure showing sequential measurements of immunoeffector cell changes and cytokine / chemokine changes after vaccination during the post-treatment period in short-term survival cohorts (patients TJ01, TJ02, TJ07, TJ08, TJ10, TJ11, and TJ12, respectively). Rows: a. Absolute number of CD4 and CD8 compared to the relative amount of WBCs in PBMCs; b. Levels of CCL21 and CXCL12; c. Relationship between relative T cell count and macrophage count. [Figure 28-2] Figure showing sequential measurements of immunoeffector cell changes and cytokine / chemokine changes after vaccination during the post-treatment period in short-term survival cohorts (patients TJ01, TJ02, TJ07, TJ08, TJ10, TJ11, and TJ12, respectively). Rows: d. Relationship between the relative proportions of CD14+CD16-macrophages and CCR2 and MCP-1 (CCL2); e. Estimated Th-1 cytokine response after vaccination. [Figure 29A] A figure showing that the majority of IGF-1R AS ODN uptake occurs in monocytes and neutrophils. [Figure 29B]Despite similar IGF-1R AS ODN uptake in M1 and M2 cells, the figure shows that increasing the concentration of IGF-1R AS ODN targets selective elimination of only M2 CD163+ cells, accompanied by upregulation of IGF-1R. [Figure 29C] A figure showing that the rate of apoptotic cell death in CD163+ cells is directly related to the concentration of IGF-1R AS ODN. [Figure 30] Figure showing the polarization of monocytes into M2 cells by incubation of normal monocytes in the serum of cancer patients. a. Mean comparison of CD163+ macrophages treated with IGF-1R AS ODN (NOBEL, 250 μg) against PBS controls; b. Matched pair analysis shows a highly significant decrease in the M2 cell population. [Figure 31] Figure showing that monocytes polarized to the M2 CD163+ phenotype by treatment with serum from patients with different cancers exhibit upregulation of both CD163 and PDL-1, and that treatment with AS ODN knocks down both CD163 and PDL-1 by selectively targeting this cell population in both cases. a. Mean comparison of CD163+ macrophages expressing PDL-1 in PBS control versus IGF-1R AS ODN (NOBEL, 250 μg); b. Matched-pair analysis shows a highly significant decrease in this cell population, reflected as a significant decrease in PDL-1. [Figure 32] This figure shows that monocytes polarized to M2 by IL-10 treatment produce significantly more glutamine (gln) than monocytes polarized to M1 by LPS / IFNγ treatment, and are therefore more likely to promote tumor cell growth. Normal human monocytes were polarized to M1 in vitro by LPS / IFNγ treatment and to M2 in vitro by MCSF or IL-10 treatment. a. shows glutamine levels accumulated in the culture medium at various time points; b. shows intracellular glutamine levels assessed after 24 hours of culture. [Figure 33]This figure shows the differences in circulating CD163+ monocytes between a normal individual and an astrocytoma patient. Panel A: A normal individual with intermediate levels of CD163 along with approximately 6% CD14+ monocytes in circulation. In cancer patients, two changes are observed: an increase in the number of monocytes and higher levels of CD163 in the monocytes. Other cells (red boxes) have no CD163 at all. Panel B: A normal individual may have a wide range of monocytes due to infection, etc. (Panel B, CD11b+CD14 positive cells), but these are elevated in patients with malignant astrocytoma. The histogram in Panel C shows that monocytes from cancer patients have higher levels of CD163 on their CD14 monocytes than control cells (red histogram). [Figure 34-1] This figure shows that gadolinium enhancement on MRI in tumor-infiltrating M2 monocytes, wild-type IDH1 status, and undifferentiated astrocytoma patients clearly identifies more invasive tumors associated with poor prognosis. Formalin-fixed paraffin-embedded tissue was stained for IDHR1 mutations R132H (A) and CD163 (B). [Figure 34-2] Representative images in FLAIR (C and D, left panel) and gadolinium-enhanced T1-weighted axial MRI (C and D, right panel) show an AIII (IDH1 R132H mutant grade III) tumor in unenhanced form (C) and an AIII-G (IDH1 wild-type grade III with glioblastoma features) tumor in enhanced form (D). [Figure 34-3]This figure shows that gadolinium enhancement by MRI in patients with tumor-infiltrating M2 monocytes, wild-type IDH1 status, and undifferentiated astrocytocytes distinguishes more invasive tumors associated with poor prognosis. Patients were divided into multiple groups (E, F, and G) based on these three parameters (AD), particularly AIII and AIII-G, which are more similar to the more invasive GBM. Panel E shows the results for the presence (R132H+) or absence (R132H-) of IDH1 mutations in 38 randomly selected AA patients on enhanced and unenhanced MRI (where nd indicates no detection). Panel F presents the CD163+ cell content in resected tumor specimens for AA specimens separated by enhancement, after counting using an automated cell counting system. Box plots show the 75th, 50th, and 25th percentiles, while maximum and minimum data values ​​are represented by upper and lower whiskers. The statistical significance of the differences between groups was assessed using the Mann-Whitney test (***, p<0.001). [Figure 34-4] This figure shows that gadolinium enhancement on MRI in patients with tumor-infiltrating M2 monocytes, wild-type IDH1 status, and undifferentiated astrocytoma distinguishes more invasive tumors associated with poor prognosis. Panel G presents Kaplan-Meier survival curves for patients, segregated based on the invasiveness of their tumors. Statistically significant survival differences between groups (**) were determined by log-rank (p=0.0019) and Wilcoxon test (p=0.0088). The results indicate that IDH R132H mutant grade III astrocytomas are rarely enhanced with gadolinium, and that accumulation of CD163+ M2 cells within tumor tissue is associated with loss of vascular integrity. [Figure 35]This figure shows elevated circulating monocyte counts and elevated levels of M2 marker CD163 expression in AIII and AIII-G patients. PBMCs obtained from 18 randomly selected anaplastic astrocytoma (AA) patients (i.e., patients with astrocytomas characterized morphologically as Grade III according to WHO histological criteria) and 24 normal donors were stained with antibodies specific to CD11b, CD14, and CD163 and evaluated by flow cytometry. Forward scatter (FSC) and side scatter (SSC) characteristics were used to establish a live gate, and monocytes were defined as living cells expressing CD11b and CD14 (Panel A). Representative contour plots in the live gate and analysis of CD11b and CD14 positivity in PBMCs from normal and AA donors are shown in Panel A, where axes are on a log-scale and numbers indicate the frequency of gated cells. Panel B is a summary chart showing the frequency of CD11b+CD14+ monocytes in PBMCs obtained by flow cytometry from 12 patients with AIII, 6 patients with AIII-G, and 24 normal individuals. The statistical significance of the difference in cell percentage between normal individuals and the AA patient subset was assessed by Student's t-test (**, p<0.01). The median fluorescence intensity (MFI) of gated CD11b+CD14+ monocytes stained with CD163 is overlaid in Panel C from representative histogram plots of AIII, AIII-G, and normal blood samples. The axes are shown as logarithmic scales. Panel D shows the MFI of gated monocyte subsets stained with CD163 in PBMC samples obtained from different donor groups. [Figure 36]This figure shows that antibodies present in the serum of AIII and AIII-G patients that bind to a common antigen on astrocytoma exosomes have different isotype characteristics. Exosomes isolated from primary tumor cell lines of three astrocytoma patients were coated onto 96-well plates and incubated with patient serum (13AIII, 8AIII-G) and normal control serum (4) collected before initial surgery. Binding antibodies were detected with fluorescently conjugated whole IgG (Panel A) or IgG isotype-specific secondary antibodies (Panel B), and the degree of antibody binding was measured as MFI. [Figure 37] A figure showing that soluble factors generally associated with Th1 and Th2 immunity are elevated in the serum of patients with AIII and AIII-G, respectively. [Figure 38] This figure shows that the expression levels of genes encoding leukocyte phenotypic markers, cytokines and chemokine receptors, and their ligands differ between AIII and AIII-G patients in PBMCs. The copy numbers of genes for monocyte phenotypic markers (A), interleukins (B), interleukin receptors (C), CC chemokines (D) and receptors (E), and CXC chemokines (F) and receptors (G) in PBMCs obtained from 17 randomly selected AA patients were evaluated by high-throughput quantitative RT-PCR and normalized to the copy number of the housekeeping gene L13a present in each sample. [Figure 39] This figure shows that AIII and AIII-G patient subsets can be accurately distinguished by the expression of selected immunologically relevant genes in PBMCs. First, discriminant analysis was used to identify gene expression data that best differentiate PBMCs from AIII and AIII-G patients (Panel A). Next, principal component analysis was used to determine which of these genes—CCL3, CCR4, CCR5, CCR7, CXCL7, IL-15, IL-32, IL-15R, IL-21R, IL-23R, IL-31RA, and CD163—was most effective in distinguishing the two patient cohorts (Panel B). [Modes for carrying out the invention]

[0008] This disclosure is the first to demonstrate that a crucial difference between M1 and M2 cells is that the M2 subpopulation produces higher levels of insulin-like growth factor type 1 receptor (IGF-1R) than the M1 subpopulation. This indicates that IGF-1R plays a vital role in the polarization and survival of M2 cells. Naturally, this disclosure shows that both M1 and M2 cells greedily take up IGF-1R-specific antisense nucleic acid (IGF-1R AS ODN), but that IGF-1R AS ODN induces a dose-dependent selective reduction in M2 cells derived from cancer patients compared to M1 cells. More importantly, this disclosure shows that the selective reduction of M2 cells leads to cancer regression in these patients. Thus, this specification provides for the first time a viable and efficient mechanism for treating specific cancers by selectively reducing the number of M2 cells in patients through systemic administration of IGF-1R AS ODN.

[0009] In addition, in patients with certain types of cancer, including but not limited to glioma, astrocytoma, breast cancer, head and neck squamous cell carcinoma, papillary renal cell carcinoma type II, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer, M2 cells make the tumor environment immunomodulatory to type 2 immunity. This suppresses type 1 immunity and repels immunotherapeutic measures. As a result, M2 cells attenuate the induction of therapeutic antitumor immunity. Consequently, therapies that aim to improve Th1 immunity are ineffective or have reduced efficacy in terms of the M2 cells present in these patients. This disclosure is the first to show that reducing the M2 subpopulation also promotes type 1 immunity in cancer patients. This disclosure shows that by targeting and neutralizing the M2 cell population, the ability to generate protective type 1 antitumor immunity in cancer patients is restored, thereby promoting the use of immunotherapeutic measures.

[0010] More importantly, this disclosure demonstrates that selective reduction of M2 cells by administration of IGF-1R AS ODN leads to a mechanism for delaying cancer development or even preventing cancer in a target population. Therefore, using IGF-1R AS ODN to selectively knock down M2 cells provides a novel and important immunotherapeutic approach for the treatment and prevention of cancer in cancer patients, as well as for enhancing therapeutic immunity. Thus, this disclosure provides new information about the immune system and supports therapeutic interventions, including the removal of targeted M2 cells associated with poor prognosis in patients with various cancers.

[0011] Accordingly, this disclosure provides a pharmaceutical composition comprising a nucleic acid capable of targeting the expression of IGF-1R in M2 cells. This disclosure also provides a method for selective reduction of M2 cells by targeting the expression of IGF-1R in these cells. This disclosure further provides a method for treating cancer by targeting the expression of IGF-1R in M2 cells in a patient. Importantly, the pharmaceutical compositions of the present invention are effective when administered systemically to a target that requires them. The ease of administration of the pharmaceutical compositions facilitates treatment and enhances patient compliance.

[0012] The term "selective," as used herein, refers to an effect that affects M2 cells but not M1 cells. Alternatively, it may refer to an effect that affects M2 cells more broadly than M1 cells. For example, a selective reduction in the number of M2 cells may have no effect on the number of M1 cells, or it may result in a greater reduction in the number of M2 cells compared to a reduction in the number of M1 cells.

[0013] The term "targeting IGF-1R expression" or "targeting IGF-1R expression," as used herein, refers to administering a nucleic acid having a sequence designed to bind to IGF-1R.

[0014] When used herein, the term “M2 cells” includes M2 macrophages present within the tumor of interest and / or M2 monocytes circulating in the periphery of the subject. As used herein, the term "M1 cells" includes M1 macrophages present within the tissue of interest and / or M1 monocytes circulating in the periphery of the subject.

[0015] As used herein, terms such as "a," "an," and "the" include singular and plural references unless otherwise specified by the context. As used herein, the term "about" when preceding a number indicates a value within a 10% range, either added or subtracted. For example, "about 100" includes 90 and 110.

[0016] In some embodiments, the disclosure provides a pharmaceutical composition comprising an effective amount of nucleic acid that targets the expression of IGF-1R in M2 cells, wherein administration of the pharmaceutical composition to a patient with cancer induces a reduction in M2 cells.

[0017] In certain embodiments, the Disclosure provides a pharmaceutical composition comprising an effective amount of a nucleic acid capable of targeting the expression of IGF-1R in M2 cells, wherein administration of the pharmaceutical composition to a patient with cancer induces downregulation of IGF-1R expression in M2 cells. In other embodiments, the Disclosure provides a pharmaceutical composition comprising an effective amount of a nucleic acid capable of targeting the expression of IGF-1R in M2 cells, wherein administration of the pharmaceutical composition to a patient with cancer induces downregulation of the expression of genes other than IGF-1R in M2 cells.

[0018] In some embodiments, nucleic acids downregulate the expression of genes downstream of the IGF-1R pathway within the cell. In certain embodiments, the downstream gene is hexokinase (HexII). In some embodiments, nucleic acids downregulate the expression of housekeeping genes within the cell. In some embodiments, the housekeeping gene is L13.

[0019] In some embodiments, the nucleic acid is a naturally occurring nucleic acid. In other embodiments, the nucleic acid is a non-naturally occurring nucleic acid. In certain embodiments, the nucleic acid is recombinantly produced. In some embodiments, the nucleic acid is recombinantly produced in microorganisms. In some embodiments, the nucleic acid is recombinantly produced in bacteria. In other embodiments, the nucleic acid is recombinantly produced in mammalian cell lines. In yet another embodiment, the nucleic acid is recombinantly produced in insect cell lines.

[0020] In certain embodiments, nucleic acids are chemically synthesized. In certain embodiments, nucleic acids are prepared by solid-phase organic synthesis. In some embodiments, nucleic acid synthesis is carried out in a synthesizer equipped with a closed chemical column reactor using flow-through technology. In some embodiments, each synthesis cycle sequence on a solid support consists of multiple steps, which are carried out sequentially until a full-length nucleic acid is obtained. In certain embodiments, nucleic acids are stored in liquid form. In other embodiments, nucleic acids are lyophilized prior to storage. In some embodiments, lyophilized nucleic acids are dissolved in water before use. In other embodiments, lyophilized nucleic acids are dissolved in an organic solvent before use. In yet another embodiment, lyophilized nucleic acids are formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a liquid pharmaceutical composition. In other embodiments, the pharmaceutical composition is a solid pharmaceutical composition.

[0021] In some embodiments, the nucleic acid is RNA. In other embodiments, the nucleic acid is DNA. In yet another embodiment, the nucleic acid is an RNAi molecule. In yet another embodiment, the nucleic acid is an oligonucleotide.

[0022] In certain embodiments, the nucleic acid is an antisense oligomer. In some embodiments, the nucleic acid is an antisense oligodeoxynucleotide (AS ODN). Antisense oligomers function at the molecular level by binding to the complementary sequence of mRNA targeted by the Watson-Crick base pairing rule. Translation of the target mRNA is inhibited by active and / or passive mechanisms when hybridization occurs between the complementary helices. In the passive mechanism, hybridization between the mRNA and the exogenous nucleotide sequence results in double-strand formation, preventing the ribosome complex from reading the message. In the active mechanism, hybridization facilitates the binding of ribonuclease H, disrupting the RNA but leaving the AS ODN intact to hybridize with another complementary mRNA target. One or both mechanisms inhibit the translation of proteins that contribute to or persist a malignant phenotype. As therapeutic agents, they are far more selective and, as a result, more effective and less toxic than conventional drugs. The presence of one or more phosphorothioate modifications stabilizes the oligomer by conferring nuclease resistance, thereby extending its half-life.

[0023] In some embodiments, the nucleic acid contains a modified phosphate backbone. In certain embodiments, the phosphate backbone modification makes the nucleic acid more resistant to nuclease degradation. In certain embodiments, the modification is immobilized nucleic acid modification. In other embodiments, the modification is a phosphorothioate bond. In certain embodiments, the nucleic acid contains one or more phosphorothioate bonds. In certain embodiments, the phosphorothioate bond makes the nucleic acid more resistant to nuclease cleavage. In some embodiments, the nucleic acid may be partially phosphorothioate-bonded. For example, up to about 1%, up to about 3%, up to about 5%, up to about 10%, up to about 20%, up to about 30%, up to about 40%, up to about 50%, up to about 60%, up to about 70%, up to about 80%, up to about 90%, up to about 95%, or up to about 99% of the nucleic acid may be phosphorothioate-bonded. In some embodiments, the nucleic acid is completely phosphorothioate-bonded. In other embodiments, the phosphorothioate bond may be replaced by a phosphodiester bond. In certain embodiments, the nucleic acid has at least one terminal phosphorothioate monophosphate.

[0024] In some embodiments, the nucleic acid contains one or more CpG motifs. In other embodiments, the nucleic acid does not contain CpG motifs. In certain embodiments, one or more CpG motifs are methylated. In other embodiments, one or more CpG motifs are not methylated. In certain embodiments, when the nucleic acid is administered to a subject, one or more unmethylated CpG motifs induce an innate immune response. In some embodiments, the innate immune response is mediated by the binding of the unmethylated CpG-containing nucleic acid to a Toll-like receptor (TLR). In some embodiments, the TLR is TLR9. In other embodiments, the binding of the TLR to the unmethylated CpG-containing nucleic acid causes activation of TLR9. In certain embodiments, the activated TLR9 is expressed on B cells. In other embodiments, the activated TLR is expressed on plasmacytoid dendritic cells. In certain embodiments, TLR9 activation may be measured by the secretion of cytokines by B cells. In one embodiment, the cytokine is IL-6. In another embodiment, the cytokine is IL-10. In other embodiments, TLR9 activation may be measured by cytokine secretion by plasmacytoid dendritic cells. In one embodiment, the cytokine is IFNα. In another embodiment, the cytokine is IFNβ. In yet another embodiment, the cytokine is TNFα.

[0025] In certain embodiments, the nucleic acid includes at least one terminal modification, i.e., a “cap.” The cap may be a 5' and / or 3' cap structure. The term “cap” or “end cap” includes a chemical modification at either end of an oligonucleotide (relative to a terminal ribonucleotide) and includes a modification in the binding between the last two nucleotides on the 5' end and the last two nucleotides on the 3' end. The cap structure may enhance the resistance of the nucleic acid to exonucleases without impairing molecular interactions with target sequences or cellular mechanisms. Such modifications may be selected based on their enhanced potency in vitro or in vivo. The cap may be present at the 5' end (5' cap) or the 3' end (3' cap), or at both ends. In certain embodiments, the 5' cap and / or 3' cap are independently selected from phosphorothioate monophosphate, debasic residue(s), phosphorothioate bond, 4'-thionucleotide, carbocyclic nucleotide, phosphorodithioate bond, inverted nucleotide or inverted debasic moiety(2'-3' or 3'-3'), phosphorodithioate monophosphate, and methylphosphonic acid moiety. If the phosphorothioate or phosphorodithioate bond(s) are part of the cap structure, they are generally located between the two terminal nucleotides on the 5' end and the two terminal nucleotides on the 3' end.

[0026] In certain embodiments, nucleic acids target the expression of specific genes within cells. In some embodiments, nucleic acids target the expression of one or more growth factors within cells. In some embodiments, the growth factor is the insulin-like growth factor 1 receptor (IGF-1R). IGF-1R is a tyrosine kinase cell surface receptor that shares 70% homology with the insulin receptor. When activated by its ligands (IGF-I, IGF-II, and insulin), IGF-1R regulates a wide range of cellular functions, including proliferation, transformation, and cell survival. While IGF-1R is not an absolute requirement for normal proliferation, it is essential for growth under anchorage-independent conditions that can occur in malignant tissues. A review of the role of IGF-IR in tumors is provided in Baserga et al., "Vitamins and Hormones," 1997, 53, pp. 65–98 (the entire article is incorporated herein by reference).

[0027] In certain embodiments, the nucleic acid is a growth factor or growth factor receptor, such as an oligonucleotide specific to DNA or RNA, like IGF-IR. In certain embodiments, the cells are mammalian cells. In other embodiments, the cells are immune system cells, including, but not limited to, monocytes, neutrophils, eosinophils, basophils, leukocytes, natural killer (NK) cells, lymphocytes, T cells, B cells, dendritic cells, mast cells, and macrophages.

[0028] In certain embodiments, the cell is a macrophage. In certain embodiments, the macrophage is an M2 macrophage. In certain embodiments, the M2 macrophage expresses one or more cell surface markers, including, but not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, CD206, and / or other M2 macrophage markers generally known in the art.

[0029] In other embodiments, the cells are monocytes. In certain embodiments, the monocytes are M2 monocytes. In certain embodiments, the M2 monocytes express one or more cell surface markers, including, but not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, CD206, and / or other M2 monocyte markers generally known in the art.

[0030] In certain embodiments, the nucleic acid downregulates IGF-1R expression in any cell. In other embodiments, the nucleic acid downregulates IGF-1R expression in M2 cells. In certain embodiments, IGF-1R expression in M2 cells is downregulated by at least about 1%, at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to M2 cells not treated with the nucleic acid. IGF-1R expression in M2 cells may be measured by quantitative RT-PCR.

[0031] In certain embodiments, IGF-1R expression in M2 cells is downregulated within approximately 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours after administration of nucleic acid to the target.

[0032] In some embodiments, IGF-1R expression in M2 cells is maintained downregulated in subjects for at least approximately 1 day, at least approximately 2 days, at least approximately 3 days, at least approximately 4 days, at least approximately 5 days, at least approximately 6 days, at least approximately 7 days, at least approximately 8 days, at least approximately 9 days, at least approximately 10 days, at least approximately 11 days, at least approximately 12 days, at least approximately 13 days, at least approximately 14 days, at least approximately 3 weeks, at least approximately 4 weeks, at least approximately 5 weeks, or at least approximately 6 weeks after administration of one dose of nucleic acid.

[0033] In some embodiments, downregulation of IGF-1R expression in M2 cells leads to a selective reduction of M2 cells compared to M1 cells in the target. In specific embodiments, targeting IGF-1R expression in M2 cells leads to a selective reduction of M2 cells compared to M1 cells in the target.

[0034] In certain embodiments, the M2 cell population in a subject is reduced by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% compared to a subject requiring treatment with nucleic acids that target IGF-1R expression in M2 cells, for example, a subject before treatment. In some embodiments, the M2 cell population in a subject is reduced by at least about 40%. In other embodiments, the M2 cell population is eliminated. For example, after administration of the pharmaceutical composition of the present invention, the M2 cell population may be about 1%, about 2%, about 5%, or about 10% of the population before administration of the pharmaceutical composition. The M2 cell population in a subject may be measured using FACS. In certain embodiments, after treatment, the M2 cells are eliminated, i.e., undetectable by FACS.

[0035] In certain embodiments, a reduction in M2 cells is observed within approximately 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, or 72 hours after administration of nucleic acid to the patient.

[0036] In some embodiments, the reduction of M2 cells in the subject lasts for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after administration of one dose of nucleic acid.

[0037] In some embodiments, targeting IGF-1R expression prevents undifferentiated monocytes from being polarized into M2 cells. In other embodiments, targeting IGF-1R expression within M2 cells causes M2 cells to lose their M2 phenotype and functional characteristics or to undergo cell death. In certain embodiments, the cell death is necrosis. In other embodiments, the cell death is apoptosis. For the purposes of this invention, apoptosis is defined as programmed cell death and includes, but is not limited to, the regression of primary and metastatic tumors. Apoptosis is programmed cell death, a broad phenomenon that plays a vital role in countless physiological and pathological processes. Necrosis, on the other hand, is accidental cell death, a cellular response to various harmful conditions and toxic substances. In yet another embodiment, targeting IGF-1R expression within M2 cells causes M2 cells to undergo cell cycle arrest.

[0038] In certain embodiments, the nucleic acid of the present invention is an antisense deoxynucleotide (IGF-1R AS ODN) specific to IGF-1R. The full-length coding sequence of IGF-1R is shown in SEQ ID NO: 19. In certain embodiments, the nucleic acid may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or 100% identity with respect to IGF-1R AS ODN. Percentage identity can be calculated using the alignment program ClustalW2, available at www.ebi.ac.uk / Tools / msa / clustalw2 / , with default parameters.

[0039] In certain embodiments, the nucleic acid comprises a nucleotide sequence complementary to the IGF-1R signal sequence, which includes either RNA or DNA. The IGF-1R signal sequence is a 30-amino acid sequence. In other embodiments, the nucleic acid comprises a nucleotide sequence complementary to a portion of the IGF-1R signal sequence, which includes either RNA or DNA. In some embodiments, the nucleic acid comprises a nucleotide sequence complementary to codons 1-309 of IGF-1R, which includes either RNA or DNA. In other embodiments, the nucleic acid comprises a nucleotide sequence complementary to a portion of codons 1-309 of IGF-1R, which includes either RNA or DNA.

[0040] In certain embodiments, the nucleic acid is at least about 5 nucleotides long, at least about 10 nucleotides long, at least about 15 nucleotides long, at least about 20 nucleotides long, at least about 25 nucleotides long, at least about 30 nucleotides long, at least about 35 nucleotides long, at least about 40 nucleotides long, at least about 45 nucleotides long, or at least about 50 nucleotides long. In some embodiments, the nucleic acid is about 15 to about 22 nucleotides long. In certain embodiments, the nucleic acid is about 18 nucleotides long.

[0041] In certain embodiments, nucleic acids form a secondary structure at 18°C ​​but not at approximately 37°C. In other embodiments, nucleic acids do not form a secondary structure at approximately 18°C ​​or approximately 37°C. In yet another embodiment, nucleic acids do not form a secondary structure at any temperature. In yet another embodiment, nucleic acids do not form a secondary structure at 37°C. In certain embodiments, the secondary structure is a hairpin loop structure.

[0042] In some embodiments, the nucleic acid comprises any of SEQ ID NOs: 1 to 14 or a fragment thereof. In certain embodiments, the nucleic acid may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or 100% identity with any of SEQ ID NOs: 1 to 14 or a fragment thereof.

[0043] In some embodiments, the nucleic acid consists of one of sequence numbers 1 to 14. In a specific embodiment, the nucleic acid is sequence number 1. Sequence number 1 is called NOBEL. NOBEL is an 18-mer oligodeoxynucleotide with a phosphorothioate backbone and is a sequence complementary to codons 2 to 7 in the IGF-1R gene. Therefore, NOBEL is an antisense oligonucleotide specific to IGF-1R (IGF-1R AS ODN). The NOBEL sequence induced as the complementary sequence of the IGF-1R gene at the 5' end is: 5'-TCCTCCGGAGCCAGACTT-3' That is the case.

[0044] NOBEL has a stable shelf life and, due to its phosphorothioate skeleton, is resistant to nuclease degradation. Administration of NOBEL can be provided in any of the standard methods related to the introduction of oligodeoxynucleotides known to those skilled in the art. Advantageously, the AS ODNs disclosed herein, including NOBEL, can be administered with little to no toxicity. No toxic issues were observed even at (adjusted) levels of approximately 2 g / kg based on mouse studies (40 μg in the tail vein). NOBEL can be prepared according to common procedures known to those skilled in the art.

[0045] The pharmaceutical compositions disclosed herein contain nucleic acids in addition to a pharmaceutically acceptable carrier or diluent, and for example, the composition may contain physiological saline (0.9% sodium chloride). The dose of nucleic acid in human subjects may be approximately 0.025 g / kg, approximately 0.05 g / kg, approximately 0.1 g / kg, approximately 0.15 g / kg, or approximately 0.2 g / kg. In certain embodiments, the nucleic acid is supplied as a lyophilized powder and resuspended before administration. When resuspended, the concentration of nucleic acid may be approximately 50 mg / ml, approximately 100 mg / ml, approximately 200 mg / ml, approximately 500 mg / ml, approximately 1000 mg / ml, or in the range between these amounts.

[0046] In certain embodiments, the subject is an animal. In other embodiments, the subject is a human. In some embodiments, the subject suffers from a disease. In certain embodiments, the disease is cancer. In certain embodiments, cancer includes, but is not limited to, breast cancer, astrocytoma, head and neck squamous cell carcinoma, papillary renal cell carcinoma type II, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, glioma, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer. In certain embodiments, cancer is a glioma. In certain embodiments, the patient has a malignant glioma. In certain embodiments, the malignant glioma is a recurrent malignant glioma.

[0047] In certain embodiments, a subject who is a candidate for nucleic acid-based therapy is identified by measuring the level of circulating monocytes in their blood. In some embodiments, the candidate has an increased number of circulating monocytes compared to a healthy subject. As used herein, the term “healthy subject” refers to a subject who does not have cancer or any other disease and does not require nucleic acid-based therapy according to the present invention. In some embodiments, circulating monocytes include, but are not limited to, CD11b+, CD14+, CD15+, CD23+, CD64+, CD68+, CD163+, CD204+, or CD206+ monocytes. In certain embodiments, the level of one or more circulating monocytes is increased by at least approximately 1.3 times, at least approximately 1.5 times, at least approximately 1.8 times, at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, at least approximately 10 times, at least approximately 20 times, at least approximately 30 times, at least approximately 40 times, at least approximately 50 times, at least approximately 60 times, at least approximately 70 times, at least approximately 80 times, at least approximately 90 times, or at least approximately 100 times compared to healthy subjects. In certain embodiments, the level of one or more circulating monocytes is increased by about 2 times compared to healthy subjects. The level of circulating monocytes in subjects may be measured using a fluorescent cell sorter (FACS).

[0048] In certain embodiments, subjects have an increased number of circulating CD14+ monocytes compared to healthy subjects. In certain embodiments, the level of circulating CD14+ monocytes is increased by at least about 1.3 times, at least about 1.5 times, at least about 1.8 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times compared to healthy subjects. In certain embodiments, the level of circulating CD14+ monocytes is increased by about 2 times compared to healthy subjects.

[0049] In certain embodiments, circulating CD14+ monocytes have elevated levels of CD163 compared to healthy subjects. In some embodiments, the level of CD163 in circulating CD14+ monocytes is at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times higher than in healthy subjects. In certain embodiments, the level of CD163 in circulating CD14+ monocytes is about 2 times higher than in healthy subjects.

[0050] In other embodiments, the subject, a candidate for nucleic acid-based therapy, has serum that polarizes undifferentiated monocytes into M2 cells. In certain embodiments, incubation of the subject serum with undifferentiated monocytes induces the expression of one or more cell surface markers on the monocytes, including, but not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and / or CD206. In other embodiments, incubation of the subject serum with undifferentiated monocytes increases the expression of one or more cell surface markers on the monocytes compared to monocytes not incubated with the subject serum. In certain embodiments, the cell surface markers include, but not limited to, CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and / or CD206. In some embodiments, the level of one or more surface markers is increased by at least about 1.3 times, at least about 1.5 times, at least about 1.8 times, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, or at least about 100 times compared to undifferentiated monocytes not incubated with the serum of the subject. Monocytes polarized by the serum of the subject may be measured using FACS.

[0051] In further embodiments, a candidate subject for nucleic acid-based therapy is identified by performing a tumor biopsy on the subject. In some embodiments, tumors derived from the subject are assayed for the presence of monocytes. In certain embodiments, monocytes include, but are not limited to, CD11b+, CD14+, CD15+, CD23+, CD64+, CD68+, CD163+, CD204+, or CD206+ monocytes. The presence of monocytes in the tumor may be assayed using immunohistochemistry.

[0052] In certain embodiments, the subject, which is a candidate for nucleic acid-based therapy, represents approximately 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the subject's total peripheral blood mononuclear cells (PBMCs) as CD163+M2 cells. In certain embodiments, the subject represents approximately 20% or more of the subject's total PBMCs as CD163+M2 cells.

[0053] In certain embodiments, the subject who is a candidate for nucleic acid-based therapy suffers from a WHO Grade II, WHO Grade III, or WHO Grade IV tumor. In certain embodiments, the subject suffers from a WHO Grade II tumor. In some embodiments, the tumor is an astrocytoma. In certain embodiments, the tumor is selected from Grade II astrocytoma, AIII (IDH1 R132H mutant Grade III astrocytoma), AIII-G (IDH1 wild-type Grade III with features of glioblastoma / astrocytoma), or Grade IV astrocytoma. In some embodiments, Grade IV astrocytoma is glioblastoma.

[0054] In some embodiments, subjects who are candidates for nucleic acid-based therapy are identified by measuring levels of a specific cytokine set. In some embodiments, subjects have elevated levels of these cytokines compared to healthy subjects. In other embodiments, subjects are identified by detecting specific microRNAs (miRNAs) present within tumors. In certain embodiments, subjects have elevated levels of these miRNAs compared to healthy subjects.

[0055] In some embodiments, nucleic acids induce cancer regression in patients. In other embodiments, nucleic acids induce tumor reduction in patients. In yet another embodiment, nucleic acids induce cancer elimination in patients.

[0056] In some embodiments, nucleic acids induce regression of glioma tumors in patients. In other embodiments, nucleic acids induce reduction of glioma tumors in patients. In yet another embodiment, nucleic acids induce removal of glioma tumors in patients.

[0057] In certain embodiments, nucleic acids are formulated into a pharmaceutical composition. In some embodiments, the pharmaceutical composition is formulated in liquid form. In other embodiments, the pharmaceutical composition is formulated in solid form. In certain embodiments, the pharmaceutical composition is formulated for oral administration. In certain embodiments, the pharmaceutical composition is in the form of capsules. In other embodiments, the pharmaceutical composition is in the form of tablets. In some embodiments, the tablets are rapid-release tablets. In other embodiments, the tablets are sustained-release tablets. In other embodiments, the pharmaceutical composition is formulated for intraperitoneal administration. In yet another embodiment, the pharmaceutical composition is formulated for intravenous administration. In yet another embodiment, the pharmaceutical composition is formulated for intramuscular administration.

[0058] In certain embodiments, the pharmaceutical composition is introduced into a diffusion chamber, which is surgically implanted into the rectus abdominis sheath of the target for a therapeutically effective period (see, for example, U.S. Patent No. 6,541,036, which is incorporated herein by reference in its entirety).

[0059] As discussed, this disclosure shows that M2 cells (not M1 cells) greedily take up IGF-1R-specific antisense nucleic acid (IGF-1R AS ODN), and that IGF-1R AS ODN induces dose-dependent selective reduction in cancer patient-derived M2 cells compared to M1 cells. More importantly, this disclosure shows that selective reduction of M2 cells leads to cancer regression in these patients.

[0060] Therefore, in certain embodiments, a method is provided for the selective removal of M2 cells in a patient suffering from cancer, comprising the step of administering an effective amount of a pharmaceutical composition to the patient.

[0061] In another embodiment, a method is provided for treating cancer by targeting the expression of IGF-1R in M2 cells, the method comprising the step of administering an effective amount of a pharmaceutical composition to a patient with cancer.

[0062] In certain embodiments, the method of treating cancer further includes combination therapy. In some embodiments, the combination therapy includes radiotherapy. In other embodiments, the combination therapy includes chemotherapy. In certain embodiments, radiotherapy or chemotherapy is administered to the patient after administration of the pharmaceutical composition. In certain embodiments, radiotherapy or chemotherapy is administered to the patient at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after administration of the pharmaceutical composition.

[0063] In certain embodiments, the pharmaceutical composition is administered to the patient after the administration of radiotherapy or chemotherapy. In certain embodiments, the pharmaceutical composition is administered to the patient at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after the administration of radiotherapy or chemotherapy.

[0064] In certain embodiments, radiotherapy includes, but is not limited to, internal source radiotherapy, external beam radiotherapy, and whole-body isotope radiotherapy. In certain embodiments, radiotherapy is external beam radiotherapy. In some embodiments, external beam radiotherapy includes, but is not limited to, gamma ray therapy, X-ray therapy, intensity-modulated radiation therapy (IMRT), and image-guided radiation therapy (IGRT). In certain embodiments, external beam radiotherapy is gamma ray therapy.

[0065] In certain embodiments, AS ODN may be administered preoperatively, for example, before surgery to reduce tumor volume. For example, AS ODN may be administered within 24 hours, 36 hours, 48 ​​hours, or 72 hours prior to surgery. In certain embodiments, the pharmaceutical composition may be administered approximately 48 to 72 hours prior to surgery. Typically, under such circumstances, administration is via an intravenous bolus.

[0066] As discussed, and without limitation, in patients with certain cancers, including glioma, astrocytoma, breast cancer, head and neck squamous cell carcinoma, papillary renal cell carcinoma type II, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer, M2 cells make the tumor environment immunomodulatory against type 2 immunity and suppress type 1 immunity. This reduces the induction of therapeutic anti-tumor immunity by M2 cells. For example, Table 1 below summarizes possible glioma-induced immunomodulations that may be caused at least in part by M2 cells within gliomas.

[0067] [Table 1] In the treatment of such cancer patients using the nucleic acids of the present invention, M2 cells are removed or modified, resulting in a direct inhibitory effect on tumor growth and a promotion of the immune response. Furthermore, in certain embodiments, it is a priority to treat such patients in combination with nucleic acids and vaccination to promote immunity. Therefore, in certain embodiments, it is advantageous to provide a therapeutic method in which nucleic acids are provided alone or in combination with further agents to selectively target M2 cells. Through the removal of these cells, tumor development and promotion are mitigated and reduced, and immunomodulatory factors are further modified.

[0068] Accordingly, in certain embodiments, a method is provided for enhancing the immune response by targeting the expression of IGF-1R in M2 cells in a patient with cancer, the method comprising the step of administering an effective amount of a pharmaceutical composition to a patient with cancer.

[0069] Combination therapy Reduction in M2 cells may be achieved by stimulating an antitumor immune response, which is collectively referred to herein as vaccination therapy. In certain embodiments, vaccination therapy includes the steps of placing tumor cells cultured in vitro or in vitro in a culture medium supplemented with a pro-apoptotic agent for a period of time, e.g., 3 to 48 hours; washing the tumor cells with a buffer to remove any traces of the pro-apoptotic agent; and subsequently transferring the cells to a diffusion chamber (then transplanted into a subject) (see, for example, U.S. Patent No. 6,541,036, which is incorporated herein by reference in its entirety). In certain embodiments, the diffusion chamber contains tumor cells derived from the same type of tumor in which regression is induced by the pharmaceutical composition of the present invention. In other embodiments, the tumor cells placed in the diffusion chamber are of a different type from the tumor in which regression is induced.

[0070] In certain embodiments, the diffusion chamber is implanted in a subject before systemic administration of the pharmaceutical composition of the present invention to the subject. In other embodiments, the diffusion chamber is implanted in a subject after systemic administration of the pharmaceutical composition. In yet another embodiment, the diffusion chamber implanted in the subject also contains the pharmaceutical composition. In some embodiments, a diffusion chamber containing the pharmaceutical composition and autologous tumor cells (processed in vitro or in vitro as described above) is implanted in a subject. In other embodiments, a diffusion chamber containing the pharmaceutical composition and tumor cells derived from the subject (processed in vitro or in vitro as described above) is implanted in a subject. In certain embodiments, implanting the diffusion chamber in a subject further reduces the number of M2 cells in the subject compared to a subject to which the pharmaceutical composition is administered alone.

[0071] In some embodiments, the chamber is implanted in the abdomen of the subject. In certain embodiments, the diffusion chamber is surgically implanted in the rectus abdominis sheath of the subject for a therapeutically effective period. In some embodiments, the pharmaceutical composition is administered to the subject after the administration of the vaccination therapy. In certain embodiments, the pharmaceutical composition is administered to the subject at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after the administration of the pharmaceutical composition. In certain embodiments, the vaccination therapy is administered to the subject at least about 48 hours after the administration of the vaccination therapy.

[0072] In certain embodiments, vaccination therapy is administered to the subject after administration of the pharmaceutical composition. In certain embodiments, vaccination therapy is administered to the subject at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after administration of the pharmaceutical composition. In certain embodiments, vaccination therapy is administered to the subject at least about 48 hours after administration of the pharmaceutical composition.

[0073] In some embodiments, a method for enhancing an immune response includes the step of administering a second pharmaceutical composition following a vaccination therapy. In certain embodiments, the second pharmaceutical composition is administered to the subject at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after the administration of the vaccination therapy.

[0074] In other embodiments, vaccination therapy is administered to the subject after administration of the second pharmaceutical composition. In specific embodiments, vaccination therapy is administered to the subject at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, or at least about 6 weeks after administration of the second pharmaceutical composition.

[0075] In certain embodiments, the pharmaceutical composition and the second pharmaceutical composition are the same. In other embodiments, the pharmaceutical composition and the second pharmaceutical composition are different. Typically, tumor cells are irradiated before transplantation; for example, cells may be treated with gamma radiation at doses of approximately 1 Gy, 2 Gy, 4 Gy, 5 Gy, 6 Gy, 10 Gy, or up to 15 Gy. In certain embodiments, cells may be irradiated at least once, at least twice, at least three times, at least four times, or at least five times.

[0076] In certain embodiments, a method is provided for preventing or delaying cancer in a subject by targeting the expression of IGF-1R in M2 cells, the method comprising the step of administering an effective amount of a pharmaceutical composition to the subject. In some embodiments, the subject is an animal. In other embodiments, the subject is a human. In certain embodiments, a human is susceptible to cancer because of continuous exposure to one or more carcinogens that increase the risk of cancer. In certain embodiments, these carcinogens include, but are not limited to, tobacco smoke, tobacco, and asbestos. In other embodiments, a human is genetically susceptible to cancer.

[0077] In certain embodiments, a method is provided for treating, preventing, or delaying diseases in a subject, including, but not limited to, Alzheimer's disease, inflammatory bowel disease, insulin resistance in type 2 diabetes, and psoriasis, by targeting IGF-1R expression in M2 cells, the method comprising the step of administering an effective amount of a pharmaceutical composition to the subject.

[0078] (Examples) Example 1: Immunohistochemistry of IGF-1R in glioblastoma polymorphoni specimens The original magnifications were 200× (Panels A, B, C, D) and 400× (Panel E). To evaluate the association with IGF-1R expression in glioblastoma multiforme, 18 ongoing glioblastoma cases were stained with anti-IGF-1Rα (Santa Cruz Biotechnology, Santa Cruz, California). Immunohistochemical analysis for IGF-1Rα was performed on standard formalin-fixed paraffin-embedded sections (Figure 5). Following the retrieval of epitopes induced by vapor heating of sections enhanced with Target Retrieval solution (#1699, Dako Corporation, Carpinteria, California), automated immunostaining (Dako autostainer, Model #LV-1, Dako Corporation) or IGF-1Rα (Santa Cruz Biotechnology, Santa Cruz, California) was performed at a dilution of 1:500. Detection was performed using the rabbit secondary antibody MACH3 Rabbit HRP kit, Biocare Medical, and AB solution (3,3'-diaminobenzidine in dye stock, Dako Corporation). All tumors showed immunoreactivity to IGF-1R.

[0079] Example 2: NOBEL's active pharmaceutical ingredient, formulation, and stability As described above, NOBEL is an 18-mer oligodeoxynucleotide with a phosphorothioate backbone, beginning with the initial methionine codon and consisting of six nucleotides downstream, and is an antisense molecule specific to the insulin-like growth factor 1 receptor (IGF-1R). This corresponds to AS ODN). The molecular weight of the free acid is 5708.71 daltons. The molecular weight of the sodium salt is 6082.40 daltons. The NOBEL sequence derived as the complementary sequence of the IGF-1R gene at the 5' end is 5'-TCCTCCGGAGCCAGACTT-3'.

[0080] NOBEL is prepared by solid-phase organic synthesis using well-established methodologies in a synthesizer equipped with a closed chemical column reactor using flow-through technology. Each synthesis cycle sequence on a solid support consists of multiple steps, which are carried out sequentially until a full-length oligonucleotide is established. The active pharmaceutical ingredient, which is a lyophilized powder, is packaged in an HDPE container with a screw cap and then vacuum-heat-sealed inside a 5 mL Mylar pouch for storage at -80°C.

[0081] The formulation consists of a fresh active pharmaceutical ingredient dissolved in physiological saline until a 100 mg / mL solution is obtained. The resulting solution is sterile filtered through a 0.22 μm membrane filter. Before storage at -80°C, a 1 mL aliquot is filled into a USP Type 1 glass vial and sealed with an appropriate rubber stopper and aluminum cap. This formulation has been shown to be stable for 9 years (to the last test date) in two independent formulations when reconstituted in sterile, standard physiological saline (Figure 6).

[0082] Example 3: Preclinical evaluation of toxicity in mice The purpose of this study was to evaluate the toxicity of the test substance Nobel when administered to mice as a single dose via intravenous injection. Animals were observed for approximately 48 hours (day 3, intermediate sacrifice) or approximately 14 days (day 15, final sacrifice) after administration to assess reversibility, persistence, or delay in the onset of effect. This study was conducted in accordance with best practices and standards established by the Food and Drug Administration. Male and female Crl:CD1 (ICR) mice were assigned to several groups and administered various doses, as shown in the table below. Animals received a single intravenous injection into the tail vein of either a solvent / diluent [0.9% sodium chloride for injection, USP (sterile saline)] or Nobel (provided as 100 mg / mL in sterile saline) in a volume of 100 μL (see Table 2).

[0083] [Table 2] Toxicity was assessed based on mortality, clinical observations, body weight, food intake, and clinical and anatomical pathology. All animals survived until their scheduled autopsy on day 3 or day 15 of the administration period. No clinical observations or changes in food intake related to the test substance occurred. No statistically significant effects on body weight were observed. However, in males administered 0.04 mg / mouse, a slight weight loss (93.8% of controls) occurred by day 15 of the administration period. This trend was observed as early as day 8 in these males, but since no clinical changes in physical condition or hydration status occurred, it was considered not harmful in relation to the test substance. In males or females administered 0.01 mg / mouse, no effect on body weight related to the test substance was observed.

[0084] Nobel administration had no effect on clinicopathological test results. Among intermediate sacrifice animals, lung weight was higher in females given 0.01 mg / mouse, testicular weight was higher in males given 0.01 or 0.04 mg / mouse, and thymus weight was higher in males given 0.04 mg / mouse. Among final sacrifice animals, brain weight was higher in males given 0.01 or 0.04 mg / mouse, and lung weight was higher in females given 0.04 mg / mouse. No microscopic findings correlated with these organ weight changes. The dependence on the test substance was unknown. In any case, no weight differences were considered harmful. No macroscopic or microscopic findings were considered to be related to the test substance.

[0085] In conclusion, NOBEL administered as a single intravenous bolus injection to male and female mice at dose levels of 0, 0.01, or 0.04 mg / mouse was well-tolerated and not associated with any clinical observations, changes in food intake, clinicopathological changes, or macroscopic or microscopic changes. Small changes in body weight related to the test substance, as well as increases in brain and lung weight whose relationship to the test substance was unclear, were not considered harmful. Therefore, the efficacy level at which no harmful effects were observed is considered to be 0.04 mg / mouse.

[0086] Example 4: Selective knockdown of M2 (CD163+) macrophages NOBEL selectively knocks down CD163+ cells in both humans and mice. Serum from patients with malignant gliomas, as well as various other cancers including, but not limited to, astrocytoma, breast cancer, head and neck squamous cell carcinoma, papillary renal cell carcinoma type II, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer, differentiates monocytes into a CD163+ phenotype that absorbs NOBEL at low micromolar concentrations, resulting in the knockdown of this phenotype.

[0087] In the C57B / 6 mouse model, intraperitoneal antisense administration 20 days after tumor inoculation into the flank resulted in tumor-inducible CD163 cell knockdown for at least 14 days (see Figure 7). GL261 cells were transplanted into the flanks of C57BL / 6 and Tbet- / - mice, and then approximately 20 days later, the animals received systemic intraperitoneal injections of either PBS or 4 mg of Nobel alone (Figure 8). To distinguish the effect in the balance of type 1 and type 2 immunity from M2 cell knockdown, Tbet- / - mice that could not initiate antitumor type 1 immunity and reject the GL261 tumor were included. In all cases, a single dose of Nobel alone administered immediately before detection of a palpable tumor delayed tumor formation for a considerable period. Long-term survival was also promoted in 80% of C57 mice that did not develop tumors and 50% of Tbet knockout mice (Figure 8). Mixing NOBEL with GL261 cells had little effect on tumor growth in the absence of an intact immune system (Morin-Brureau et al., Cancer Immunol.Immunother., Vol. 64: pp. 447-457 (2015)), and mixing NOBEL with GL261 cells upon transplantation into Tbet mice also did not interfere with tumor growth. An important conclusion here is that the effect of IGF-R1 AS ODN on GL261 during the induction of tumor immunity is distinct from its effect on tumor growth some time later. While co-administration of the tumor as an antigen and NOBEL as an immunostimulant may be necessary in some cases, systemic effects may occur independently of the response to the antigen. It is clear that knockdown of M2 cells, and possibly other cells involved in promoting tumor growth, is sufficient to inhibit tumor growth even when recipient animals are unable to initiate a therapeutic type 1 response. The data suggest that a reduction in M2 cells from the tumor microenvironment prevents tumor cells from thriving.

[0088] Example 5: Administration of IGR-1R AS ODN to patients with glioma Based on current studies, the inventors have determined suitable doses for administration in a recurrent glioma cohort for the following administration schedules: 0.025 g / kg, 0.05 g / kg, 0.1 g / kg, 0.15 g / kg, and 0.2 g / kg.

[0089] Patients receiving one of the above doses are those with recurrent gliomas who receive a preoperative intravenous bolus infusion of IGF-1R, increasing 48–72 hours prior to surgery. A second bolus infusion may be administered depending on the quantification of the M2 cell population. This dosing schedule is not limited to, but is also effective for other cancers, including astrocytoma, breast cancer, head and neck squamous cell carcinoma, papillary renal cell carcinoma type II, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer.

[0090] Example 6: Different IGF-1R AS ODN sequences Different IGF-1R antisense sequences exhibit bioactivity in some or all of the polymorphic effects of the NOBEL sequence (5'-TCCTCCGGAGCCAGACTT-3' (SEQ ID NO. 1)). The 18-mer NOBEL sequence possesses both IGF-1R receptor downregulatory activity and TLR agonist activity, and further experiments in mice suggest that both activities are necessary for in vivo antitumor immunoactivity. While the AS ODN molecule possesses antitumor activity, the complementary sense sequence, despite also possessing a CpG motif, does not. The entire open reading frame (4104 base pairs) of the IGF-1R exon was investigated, and 10 additional CpG motifs, including IDT1220, were identified (SEQ ID NOs. 2-11) (Table 3). Furthermore, several additional IGF-1R antisense sequences (SEQ ID NOs. 12-14) that do not contain a CpG motif were also identified (Table 3).

[0091] [Table 3] Example 7: Activation of Toll-like receptor 9 by Nobel Frontline defense in the immunological response to invading pathogens involves interactions between pathogen structures and a set of receptors, including Toll-like receptors (TLRs), which activate the innate immune system. This arm of the immune system recognizes a common class of molecules produced by various pathogens, including bacteria, viruses, fungi, and parasites (all of which are essential for the survival of invading pathogens). These pathogen-associated molecular patterns, or PAMPs, are recognized by pathogen-related receptors, or PRRs, expressed in immune effector cells, particularly dendritic cells. These PRRs are called Toll-like receptors based on their homology to the Toll protein characterized in Drosophila, first characterized in 1991 by Nusslein-Volhard (Stein et al., Cell, Vol. 65: pp. 725-735 (1991)). The activating effect of TLRs is important in directing the type of subsequent adaptive immune response. Currently, there are 10 known human TLRs, with TLR9 being of particular interest. TLR9 binds to DNA fragments containing unmethylated cytosine-guanosine sequences specific to bacteria and viruses. Human CpG dinucleotides are always methylated, making autologous human DNA immunotolerogenic when exposed to the immune system. Unmethylated CpG motifs induce a potent innate immune response, especially when nested within a particularly favorable adjacent nucleotide hexamer sequence (Figure 9). The response is observed at drug concentrations 1000 times lower than those that halt antisense gene translation.

[0092] The NOBEL sequence was found to activate both plasmacytoid dendritic cells (DCs) and B cells. In vitro AS ODN uptake experiments were performed using PBMCs to assay the activation of immune cell subsets. The highest uptake of AS ODN occurred in endocytosis antigen-presenting cells: monocytes, dendritic cells (DCs), and B cells, while uptake was negligible in T cells or NK cells. Plasmacytoid dendritic cells (pDCs) and B cells treated with AS ODN increased the expression of co-stimulatory molecules (CD80, 83, and 86) that are important for T cell activation. Despite the highest levels of AS ODN uptake, the expression levels of CD80, 83, and 86 remained unchanged in monocytes and bone marrow dendritic cells (Figure 10).

[0093] Example 8: Activation and maturation of dendritic cells after NOBEL treatment Treatment of monocyte-derived dendritic cells with NOBEL exhibits a marked dose-dependent maturation response (Figure 11). Immature DCs were obtained by culturing CD14+ PBMCs in rGM-CSF and rIL-4 for 4 days. Immature DCs were treated with NOBEL for 36 hours. PolyI:C was used as a positive control for DC maturation. Treated DCs were collected, incubated with fluorescent proteins, and analyzed using a flow cytometer. High endocytotic capacity is characteristic of immature DCs, which rapidly and dramatically decreases upon maturation signaling. NOBEL treatment reduced endocytotic capacity in DCs in a dose-dependent manner.

[0094] Example 9: Optimal AS ODN Sequence As a guideline for further screening of IGF-1R AS ODN sequences, the 5' end sequence of the targeted mRNA transcript has the best probability of binding to the mRNA sequence according to the Watson-Crick base pairing rule. This biological activity may be due to the preferred sequence that generates linear molecules with a higher probability at body temperature, unlike DWA sequences which are more likely to form a stable 5' hairpin loop at body temperature (Figure 13). As mentioned above, the biological activity may be due to the reachability to the targeted mRNA sequence and unmethylated CpG dinucleotide, which should be reachable at body temperature from the 5' end of the molecule, ideally from the 5' end of the linear molecule. Despite these guidelines, DWA sequences that do not have the ability to downregulate IGF-1R appeared to be superior in DC maturation (Figure 12). The biological activities of different IGF-1R AS ODNs are summarized in Table 4 below.

[0095] [Table 4] Example 10: Downregulation of IGF-1R: Summary of Nobel's biological specificity and bioactivity Two assays were designed to evaluate the biological specificity of AS ODN sequences.

[0096] a. Quantitative RT-PCR to evaluate downregulation of IGF-1R mRNA - This assay was designed to confirm Watson-Crick base pairing between intracellular IGF-1R transcription mRNA and AS ODN. GL261 mouse cells were present in RPMI supplemented with 10% FBS, 4 MmL-glutamine (Fisher), 50 μg / mL-gentamicin (GIBCO), and 0.05 MmL-ME (Sigma), and were obtained from NCI-Frederick DTP, DCTD Tumor Bank Repository (Frederick, Maryland). As shown in Figure 14, IGF-1R expression was significantly reduced in GL261 cells treated with 1 mg of Nobel AS-ODN / well (P<0.001) and in cells treated with 0.1 mg of Nobel AS ODN / well (P<0.05).

[0097] b. Quantitative RT-PCR-IGF-1 induces hexokinase RNA expression in cancer cells to evaluate the downstream downregulation of hexokinase isotype 2 mRNA. It was predicted that IGF-1-reduced activation of IGF-1R, resulting from AS-ODN treatment-induced downregulation of IGF-1R, should lead to a similar decrease in hexokinase expression. As shown in Figure 15, this is the case when the decrease in IGF-1R mRNA is highly correlated with the downregulation of hexokinase II. A 90% decrease in IGF-1R copy number corresponds to a 90% decrease in hexokinase II copy number. Downregulation of housekeeping genes was also hypothesized, given that IGF-1R inhibition slows growth rate and metabolism in vitro, when observed as a 75% decrease at L13.

[0098] Example 11: Bioactivity of IGF-1R AS ODN: A mouse ventral model to evaluate the vaccine activity of the NOBEL sequence against tumor burden. C57 / BL6 mice were obtained from Jackson Laboratory (Bar Harbor, Maine) and Taconic Farms, Inc., and were 8-10 weeks old. The mice were anesthetized in a chamber containing isoflurane and 100 μL of PBS. 6 Individual GL261 cells were injected into the flank using a 1 mL BD Falcon syringe and a 21 G BD (Fisher) needle. AS ODN GL261 cell preparation was injected into the left flank, while wild-type GL261 cells were injected into the right flank two weeks later. Mice were examined for tumor production at least twice a week. It is noteworthy that in these studies, it is well known that when GL261 cells are placed in the flank of genetically similar mice, they are immunostimulant to the extent that approximately 50% of such animals are expected to develop antitumor cell immunity in the absence of intervention. As shown in Figure 16, pretreatment with GL261 cells treated with AS-ODN reduced WT GL261 tumor growth from 53% to 13% in control mice, while GL261 AS ODN mixture (4 mg NOBEL / 10 6 Pretreatment with individual GL261 cells reduced WT GL261 tumors to 0%. Interestingly, when GL261 and NOBEL were injected into the opposite flank of mice, vaccine efficacy was lost (Figure 17). These data suggest that GL261 treated with AS ODN and antisense molecules contribute to the antitumor response, but the most effective vaccine involves co-injection of autologous tumor cells with IGF-1R AS ODN. Even more interestingly, the NOBEL sense sequence, a palindromic element surrounding the CpG motif, was not effective upon stimulation of antitumor immunity, suggesting that the biological efficacy of the CpG motif is related to the biological activity of IGF-1R AS ODN, which surpasses that of the CpG motif alone.

[0099] Example 12: Nobel has radiation-sensitizing properties. U118 cells were treated with Nobel (4mg / 107 Cells were incubated for 24 hours in or out of the presence of individual cells. Cells were collected, irradiated, and returned to culture (with or without fresh NOBEL) in the presence of Click-iTedu reagent (final concentration of 10 μM). After 72 hours of incubation, cultures were developed according to the manufacturer's protocol. As shown in Figure 18, NOBEL AS ODN induced radiosensitivity of U118 human glioblastoma cells across the entire radiation dose range of 1–15 Gy, with an iso-effect plateau above 5 Gy.

[0100] Example 13: Treatment of astrocytoma WHO grade IV astrocytoma (glioblastoma) is a uniformly fatal primary intracranial malignancy with a median survival of 18 months. Twelve patients diagnosed with recurrent glioblastoma who were considered good candidates for surgery were enrolled for treatment. Standard treatment, including surgery, temozolomide chemotherapy, and conformal radiotherapy, had not been successful in any of these patients. A summary of the enrolled patients and their disease course is included in Table 5. All patients were treated with subcutaneous enoxaparin at 40 mg / day for 3 months.

[0101] [Table 5] The mixture of autologous tumor cells was removed during surgery, then treated overnight with IGF-1R AS ODN, and subsequently added to a semipermeable chamber for irradiation. The vaccine product contained 18-mer IGF-1R AS with sequence 5'-TCCTCCGGAGCCAGACTT-3'. Using ODNs, a single frameshift was introduced downstream from the preceding sequence, and 2 μg of exogenous antisense was added to each chamber (Cv) based on its immunostimulatory properties. The protocol was also revised to include chambers (Cp) containing PBS. Up to 10 chambers were transplanted into the rectus abdominis sheath. The autologous tumor cell supernatant obtained during the plating stage of the vaccine formulation and the contents of the explanted chambers were rapidly frozen for exploratory research purposes.

[0102] The objective of the study is to assess safety and radiographic response, such as tumor-relative cerebral blood volume (rCBV), apparent diffusion coefficient (ADC), and at 90 and 240 minutes. 18 The study included evaluation of PET / CT with fluorodeoxyglucose dual-time imaging. The exploratory objective was to evaluate the time-course effects of peripheral blood mononuclear cells (PBMCs) and chemokines / cytokines in serum, chamber fluid, and cell cultures using multiplex analysis (Luminex).

[0103] Immunological evaluation To assess immune parameters at baseline, plasma leukocyte ferresis was performed one week prior to surgery. Blood was also examined as previously mentioned. 1 The samples were obtained postoperatively. Serum and cell fractions were separated by centrifugation, cells were treated with erythrocyte lysis buffer, and leukocytes were quantified by flow cytometry or stored in DMSO at -80°C, similar to the serum samples. Flow cytometry was performed using EasyCyte 8HT as described above. The analysis was performed using 8HT (Millipore) and fluorescently conjugated mAbs specific to human CD4, CD8, CD11b, CD14, CD16, CD20, CD45, CD56, CD80, CD83, and CD86 (all from BD Biosciences), and CD163 (R&D Systems). Post-collection analysis was performed using FlowJo software (Tree Star Inc., Ashland, Oregon). Soluble cytokine factors were quantified using Luminex bead arrays (Millipore human cytokine / chemokine panels I, II, and III). To evaluate T-cell pluripotency before treatment and after surgery, patient- and normal control ptomega-costomy cells (PBMCs) were stimulated in vitro with phorbol 13-12-myristate acetate (PMA) and ionomycin (Sigma Aldrich), and cytokines and chemokines released into the culture medium were quantified using Luminex. Tumor tissue sections were evaluated by immunohistochemistry for IGF-1R, CD163, CD14, CD206, CD204, CD3, CD4, and CD8. Where possible, Aperio quantification of immunopositive cells was used. Otherwise, immunohistochemistry was qualitatively evaluated by a qualified neuropathologist (LEK) using an ordinal scale from 0 (no staining) to 6 (strong diffusion staining).

[0104] The levels of cytokines / chemokines in serum preoperatively and for two days postoperatively, the contents of the explanted chamber, and the supernatant derived from overnight tumor cell cultures (SNs) were all quantified using Luminex. Membranes obtained from vaccine and control chamber pairs were embedded in paraffin for immunopathological examination.

[0105] Safety evaluation and clinical course Of the 54 severe adverse events recorded, only one SAE was associated with the protocol, which included thrombosis from the femoral port used for plasma leukophagesis. The incidence of DVT in the trial was 8.3%. Nine patients died due to tumor growth, while three died from other causes, including intracranial hemorrhage and sepsis (Candida glabrata, Klebsiella pneumonia). Five autopsies were performed. All patients followed clinical instructions and underwent steroid discontinuation or maintenance of daily doses during the postoperative period. The median overall survival was 91.4 weeks, which correlated highly with the interval between initial surgery and surgery for recurrence. After recurrent tumor surgery and autologous cell vaccination, two significantly different protocol survival cohorts were identified as the longer-term and shorter-term survival cohorts, at 48.2 weeks and 10 weeks, respectively (Figure 19a-c). After removing one outlier (TJ03), we recorded a significant correlation between protocol survival at registration and the degree of lymphopenia (Figure 19d). Comparison of values ​​at initial diagnosis and protocol registration showed a significant decrease in mean lymphocyte count after standard treatment (65%) (8 available paired samples, p=.012, paired t-test). There was no significant difference between lymphocyte count at registration and final available lymphocyte count after vaccination (data not shown).

[0106] X-ray response Anatomical tumor responses were scored. Examples are shown in Figures 20a and 20b. Anatomical improvements on standard MRI did not correlate with survival, but additional imaging criteria did. Three of the four longer-term survival cohorts (TJ03, TJ06, and TJ09) had a paradoxical increase in rCBV that correlated highly with an increasing apparent diffusion coefficient (ADC, see Figure 20c). This was considered paradoxical because these patients had ADC values ​​that reflected intratumoral cell loss, despite perfusion data suggesting disease progression. In the TJ06 case, a significant and persistent decrease in the CD163+ macrophage population was observed at the time of the second vaccination performed prior to autopsy (see Figure 20d and below). In two of these cases, PET / CT criteria consistent with inflammation were observed, supporting these findings (Figure 24). Summary cytokine plots supported a pro-inflammatory process in these three patients (Figure 20e).

[0107] Examination of explanted chambers and pathological specimens The explanted chambers were structurally intact and contained no viable cells by trypan blue exclusion. Histological analysis of membranes obtained from the chambers showed that CD15+ neutrophils and CD163+ macrophages coated the outer surface of membranes obtained from both Cp and Cv chambers, but were dramatically increased on Cv (Figure 3a). Chamber contents reflected the products of factors diffusing inward from encapsulated cells and the surrounding environment, with the control Cp chamber regulating the latter. Chemokines elevated in Cv compared to Cp included CCL21, CCL20, and CCL19, all of which were significantly elevated above serum levels. CXCL12 was elevated in both Cv and serum compared to Cp (Figure 25 and Table 6). Furthermore, significant increases in HSP-70 and granzyme B were observed in Cv compared to serum levels (3826 pg / ml vs. 327 pg / ml, p=.0015, and 37 pg / ml vs. 12 pg / ml, p=.01, respectively). These results indicate that the method disclosed herein induces a pro-inflammatory immune response that enhances the anti-cancer effect.

[0108] [Table 6] Paraffin sections derived from surgical interventions via autopsy were available for immunohistochemical analysis. In all evaluable cases, the number of tumor-infiltrating CD163+ macrophages, which was significantly increased at vaccination compared to the initial diagnosis, was significantly decreased at autopsy (Figures 21b and 26).

[0109] Levels of IGF-1R-expressing cells were high throughout the tumor tissue from initial diagnosis to surgery for recurrence, but significantly decreased at autopsy. Qualitative measures were used because staining was too diffuse for quantification of immunopositive cells using Aperio. A significant decrease in IGF-1R-positive cells was observed when comparing pre- and post-vaccination levels (Figures 21c and 26).

[0110] Comparing the survival cohorts, we observed significantly lower levels of CD163+TAM in long-term survival compared to the short-term survival cohort, both at initial diagnosis (3.7% vs. 51.5%, p.0075) and at vaccination (26% vs. 53.9%, p=.0402) (Figure 27). TAM levels were highly correlated with circulating M2 cells in the short-term survival cohort (Figure S4b). Several CD3, CD4, or CD8 cells were observed throughout all consecutive control samples (data not shown).

[0111] Chemokine / cytokine content in collected samples It was hypothesized that any significant increase in cytokines / chemokines in Cv could reflect an increased presence of them in either the tumor microenvironment (TME) or serum. To gain further insight into this issue, we explored whether tumor reduction surgery reduced serum levels of these cytokines. Excluding two abnormal values ​​from all serum cytokines / chemokines examined, serum CCL21 was significantly decreased on postoperative day 2 (Table 6), which supported CCL21 production from the TME. Interestingly, post-vaccination levels of T cells tended to closely resemble levels of both CCL21 and CXCL12 in longer-surviving subjects (Figure 4). In contrast, we found no associated patterns between T cells, monocytes, or cytokines in the short-survival cohort (Figure 28).

[0112] High levels of CCL2 in both SN and Cv also significantly increased in serum after vaccination, suggesting a source of this chemokine other than TME. Mean postoperative serum levels of CCL2 were also significantly higher in the short term compared to the long-term survival cohort (3812 pg / ml vs. 1978 pg / ml, p<.0078).

[0113] Following initial and revaccination, longer-survivable subjects showed coordinated changes in circulating levels of T cells, monocytes, and pro-inflammatory chemokines / cytokines. Inverse correlations were observed between T cells and macrophages, and between the CD163+ subset of circulating CD14+CD16-macrophages and the chemokine CCL2. See Table 7 for specific cytokines. In 3 out of 4 subjects, circulating levels of CD163+ and CCL2+ cells also directly correlated (R2=.68, p=.043). Significantly higher CD4 / CD8 ratios were evident in the longer-survivable cohort during the post-vaccination period.

[0114] [Table 7] In vitro T cell activation PBMC samples obtained on days 7 and 14 were nonspecifically stimulated with PMA / ionomycin, and chemokine / cytokine levels were evaluated in the supernatant. After excluding one abnormal value (TJ03) with significant lymphopenia, significant differences were observed in the two survival cohorts on day 14 for six putative cytokines associated with classical Th-1 and Th-2 responses (Figure 5 and Table 8).

[0115] [Table 8] Example 14: Monocytes polarized to overexpression of IGF-1R in M2 cells Immature, undifferentiated human monocytes induced to M2 polarization by IL-4 and IL-13-mediated M2 differentiation overexpress IGF-1R compared to macrophages induced to M1 polarization. Furthermore, treatment with IGF-1R AS ODN selectively blocks the emergence of polarized M2 cells and the survival of existing M2 cells (Figure 29). These observations represent new information about the immune system and support therapeutic interventions, including targeted removal of M2 cells associated with poor prognosis in patients with various cancers. Figure 29a shows that the majority of IGF-1R AS ODN uptake occurs in monocytes and neutrophils. Similar uptake of IGF-1R AS ODN occurs in macrophages differentiated into M1 and M2, but increased IGF-1R AS ODN concentration targets the selective removal of M2 CD163+ cells, with only IGF-1R being upregulated (Figure 29b). The rate of apoptotic cell death in CD163+ cells is directly related to the concentration of IGF-1R AS ODN (Figure 29c).

[0116] Example 15: Polarization of monocytes to M2 by incubation of normal monocytes in the serum of cancer patients. We conducted analyses to observe whether the serum of patients with different types of cancer had the ability to differentiate into CD163+ cells. As shown in Figure 30, CD163+ macrophage differentiation was observed from undifferentiated monocytes co-incubated with serum from head and neck squamous cell carcinoma (N=2), non-small cell lung cancer (N=2), and prostate cancer (N=5). In all cases, this cell population was knocked down by treatment with IGF-1R AS ODN. This confirms that factors present in the serum of patients with various cancers induce the differentiation of monocytes into M2 monocytes that differentially express CD163 and / or various other phenotypic markers, such as CD204 and CD206.

[0117] Example 16: Monocytes polarized to the M2 CD163+ phenotype by treatment with serum from patients with different cancers show upregulation of both CD163 and PDL-1. Figure 31 shows that monocytes polarized to the M2 CD163+ phenotype by treatment with serum from patients with different cancers exhibit upregulation of both CD163 and PDL-1, and in all cases, treatment with AS ODN knocks down both CD163 and PDL-1 by selectively targeting this cell population. Figure 31A shows a comparison of the means in IGF-1R AS ODN (NOBEL, 250 μg) treatment of CD163+ macrophages expressing PDL-1 compared to PBS controls. Figure 31B shows that matched-pair analysis reveals a highly significant reduction in this cell population, reflected as a significant decrease in PDL-1. Removal of the PDL-1 overexpressing cell population releases cytotoxic T cells from the source of inhibition, thereby restoring type 1 immunity in these cancer patients.

[0118] Example 17: Differences in circulating CD163+ monocytes between a normal individual and an astrocytoma patient. We examined the differences in circulating CD163+ monocytes between normal individuals and astrocytoma patients. Normal individuals showed intermediate levels of CD163 along with approximately 6% CD14+ monocytes in their circulation (Figure 33A). In cancer patients, two changes were observed: an increase in monocyte number and higher levels of CD163 in the monocytes (Figure 33A). Other cells (red boxes) lacked CD163 entirely. Normal individuals may have a wide range of monocytes due to infection, etc. (Figure 33B, CD11b+CD14-positive cells), but these are elevated in patients with malignant astrocytoma. The histogram in Figure 33C shows that patient monocytes invariably have higher levels of CD163 on their CD14 monocytes than control cells (red histogram).

[0119] Example 18: Tumor-infiltrating M2 monocytes and wild-type isocitrate dehydrogenase (IDH1) status are associated with MRI-mediated gadolinium enhancement and poor prognosis in patients with undifferentiated astrocytoma.

[0120] Gadolinium enhancement on MRI in patients with tumor-infiltrating M2 monocytes, wild-type IDH1 status, and undifferentiated astrocytoma distinguishes more invasive tumors associated with poor prognosis. Formalin-fixed paraffin-embedded tissue was stained for IDHR1 mutations R132H (A) and CD163 (B). Representative images in FLAIR (C and D, left panel) and gadolinium-enhanced T1-weighted axial MRI (C and D, right panel) show AIII (IDH1 R132H mutant grade III) (C) tumors in the unenhanced state and AIII-G (IDH1 wild-type grade III with glioblastoma-like features) (D) tumors in the enhanced state. Patients were divided into several groups based on these three parameters (A-D), specifically AIII and AIII-G (E, F, and G) which are more similar to the more invasive GBM. The presence of IDH1 mutations (R132H) in 38 randomly selected AA patients using enhanced and unenhanced MRI. + ) or absent (R132H -The results for are shown in Panel E (where n.d. indicates no detection). CD163 + cell contents in the resected tumor specimens were counted using an automated cell counting system and presented for AA specimens sorted by enrichment in Panel F. Box whisker plots show the 75, 50, and 25 percentiles, while the maximum and minimum data values are represented by the upper and lower whiskers. The statistical significance of differences between groups was evaluated by the Mann–Whitney test ( *** , p < 0.001). Kaplan–Meier survival curves for patients stratified based on the invasiveness of their tumors are presented in Panel G. Statistically significant survival differences between groups ( ** ) were determined by the log-rank (p = 0.0019) and Wilcoxon tests (p = 0.0088). According to the results, enhancement of IDH R132H mutant grade III astrocytomas with gadolinium is rare, and as expected, accumulation of CD163 + M2 cells within the tumor tissue is shown to be associated with loss of vascular integrity.

[0121] Example 19: The number of circulating monocytes is elevated in AIII and AIII-G patients and expresses the M2 marker CD163 at elevated levels. PBMC obtained from 18 randomly selected WHO grade III astrocytoma patients and 24 normal donors were stained with antibodies specific for CD11b, CD14, and CD163 and evaluated by flow cytometry. Forward scatter (FSC) and side scatter (SSC) characteristics were used to establish a live gate, and monocytes were defined as live cells expressing CD11b and CD14 (Figure 35A). Representative contour plots in the live gate and analysis of CD11b and CD14 positivity in PBMC obtained from normal and AA donors are shown in Figure 35A, where the axes are shown on a log scale and the numbers indicate the frequency of gated cells. Figure 35B shows CD11b + CD14 +This is a summary chart showing the frequency of monocytes. The statistical significance of the difference in cell percentage between normal individuals and the AA patient subset was evaluated by Student's t-test. ** (p<0.01). The median fluorescence intensity (MFI) of CD163-stained gated monocytes with CD11b+CD14+ is overlaid in Figure 35C from representative histogram plots of AIII, AIII-G, and normal blood samples. The axis is shown as a logarithmic scale. The MFI of CD163-stained gated monocyte subsets in PBMC samples obtained from different donor groups is shown in Figure 35D. Statistical significance was assessed by analysis of variance followed by the Tukey post-test. ** (p<0.05). CD11b + CD14 + While monocytes were present at similarly elevated levels in the circulation of all study patients, cells obtained from the circulation of patients with Grade III tumors that closely resembled GBM (Glioblastoma multiforme; AIII-G) progressively expressed higher levels of CD163 than those from patients with less aggressive Grade AIII tumors and normal controls (Figure 35, C and D). As expected, the increase in circulating monocytes led to the increased CD3 in the blood of Grade III astrocytoma patients. + and CD20 + The frequency of lymphocytes decreases compared to normal individuals.

[0122] Example 20: Antibodies present in the serum of AIII and AIII-G patients that bind to a common antigen on astrocytoma exosomes have different isotype characteristics. Exosomes isolated from primary tumor cell lines of three astrocytoma patients were coated onto 96-well plates and incubated with patient serum (13AIII, 8AIII-G) and normal control serum (4) collected before initial surgery. Binding antibodies were detected with fluorescently conjugated whole IgG (Figure 36A) or IgG isotype-specific secondary antibodies (Figure 36B), and the degree of antibody binding was measured as MFI. Data are shown as values ​​from individual subjects in box plots as described in Example 18. In Figure 36A, asterisks and bars indicate values ​​significantly different from normal control values, determined by ANOVA followed by Dunnett's test (p<0.05). In Figure 36B, groups of values ​​obtained from AIII-G patients that were statistically significantly different from normal control values ​​and AIII patient values, as determined by ANOVA and Tukey's post-test, are shown. ** This was observed (p=0.004). As shown in Figure 36A, IgG antibodies that react with these exosomes are also present in the serum of the majority of grade III astrocytoma patients, regardless of their prognostic category. However, when isotype-specific antibodies were used for detection, we found that exosome-binding antibodies for the Th2-associated IgG2 isotype were significantly elevated in AIII-G patients compared to longer-lived AIII patients (Figure 36B). While IgG1 levels tended to be slightly elevated in the latter patients, and IgG4 levels were slightly elevated in AIII-G patients, neither of these differences was significant.

[0123] Example 21: Soluble factors generally associated with Th1 and Th2 immunity are elevated in the serum of patients with AIII and AIII-G, respectively. Serum from AA patients, separated into AIII (n=17) and AIII-G (n=13) subsets based on gadolinium-enhanced MRI, was evaluated for soluble factor levels using Luminex. Concentrations in individual samples are shown in box plots as described in Example 18. Statistical significance of the difference between the two groups was evaluated by Student's t-test. **** p≦0.001; ** p≦0.01; *(p≦0.05). Serum concentrations of Th2 cytokines IL-10 and CCL4 were significantly elevated in patients with AIII tumors exhibiting GBM characteristics, while IL-9 and Th1-related IL-12 P40, CXCL10, and FLT3L were significantly elevated in the remaining AIII patients (Figure 37).

[0124] Example 22: The expression levels of genes encoding leukocyte phenotypic markers, cytokines, and chemokine receptors, as well as their ligands, in PBMCs differ between AIII and AIII-G patients.

[0125] Copy numbers of genes for monocyte phenotypic markers (Figure 38A), interleukins (Figure 38B), interleukin receptors (Figure 38C), CC chemokines (Figure 38D) and their receptors (Figure 38E), and CXC chemokines (Figure 38F) and their receptors (Figure 38G) in PBMCs obtained from 17 randomly selected AA patients were evaluated by high-throughput quantitative RT-PCR and normalized to the copy number of the housekeeping gene L13a present in each sample. LDA performed on the normalized copy numbers is shown in the left panel (each dot represents data from an individual patient). Dots representing the results of analysis for individual AIII and AIII-G patients are colored blue and red, respectively. The multivariate mean for each group is shown as a + at the center of a similarly colored circle / ellipse, representing the 95% confidence interval of the mean. The mean copy numbers for each gene tested in the two patient cohorts are shown in the accompanying right-hand panel as red and green heatmaps corresponding to high and low expression levels, respectively, and the range of detected gene copy numbers is shown in the corresponding scale bar. Gray boxes represent reactions that did not produce detectable products.

[0126] Linear discriminant analysis (LDA) was used to determine whether the expression of each marker class adequately separated and characterized the two patient cohorts. Figure 38A shows the LDA results for the monocyte phenotypic markers CD11b, CD14, CD15, CD68, CD163, CD204, and CD206, along with a heatmap of the corresponding gene expression levels. Moderate to high elevations (8-fold, 3-fold, and 5-fold, respectively) were observed in PBMC samples obtained from patients with AIII-G in the expression levels of mRNA specific to CD15, CD163, and CD206 compared to conventional AIII tumors, although the elevations observed in CD11b and CD204 transcript levels in the latter were only slight (1.9-fold). Based on the LDA analysis of monocyte phenotypic gene expression levels, 14 out of 17 cases tested were accurately separated into one of the two patient cohorts. Consistent with flow cytometry data, CD163 was identified as the most characteristic phenotypic mRNA marker. Similar LDAs performed on 28 interleukin genes showed that only type 1-related cytokines IL-15 and IL-32 differed significantly between AIII and AIII-G samples (p=0.0111 and p=0.0152, respectively), and despite both being lower in the latter, 100% of patients were accurately classified into their appropriate cohort (Figure 38B). Other genes were either not expressed or expressed at insignificantly different levels. Analysis of mRNA traces of 22 interleukin receptor genes in PBMCs also clearly distinguished AIII and AIII-G patients (Figure 38C). Most transcripts of these genes, when differentially expressed, were lower in AIII-G patients than in AIII PBMCs. In particular, IL-23R and IL31RA were expressed at significantly higher levels in the AIII sample (p=0.0055 and p=0.0360, respectively). For this analysis, the two patient cohorts were also distinguished by mRNA level LDA of 15 of the 21 CCL genes that were sufficiently expressed in PBMCs (Figure 38D).Trends in increased expression were detected in the CCL3, CCL8, CCL13, CCL21, CCL23, and CCL28 genes in AIII samples, and in the CCL2, CCL11, and CCL14 genes in AIII-G samples. However, statistical significance was only obtained for CCL3 mRNA in the available samples, which was present at higher levels in AIII compared to PBMCs in AIII-G. Patients were also clearly distinguished in LDA of CCR gene expression data (Figure 38E). Most CCR mRNAs showed a tendency towards higher expression in conventional AIII samples, with the exception of CCR4, which was slightly elevated in AIII-G samples, but only two genes, CCR1 and CCR5, were significantly overexpressed (p=0.0206 and p=0.0003, respectively). LDA of CXCL gene expression data accurately characterized 15 out of 17 cases tested as belonging to different patient cohorts, despite no statistically significant differences at mRNA levels (Figure 38F). Only CXCL7 (upregulated in PBMCs from AIII-G patients) showed a nearly significant difference (p=0.0673). CXCL2, CXCL10, and CXCL16 transcripts were detected at higher levels in conventional AIII PBMCs, but the differences from AIII-G samples were not significant. Similar results were obtained for LDA based on CXC chemokine receptor expression, where 14 out of 17 AA cases were accurately subgrouped (Figure 38G). CXCR1, CXCR3, CXCR4, CXCR6, and CX3CR1 transcripts were found to be overexpressed in AIII-G, but only CXCR3 and CXCR6 showed significant levels (p=0.0111 and p=0.0206, respectively). In PBMCs derived from patients with classic AIII tumors, only CXCR7 mRNA levels were elevated; however, the difference did not reach statistical significance given the number of samples analyzed.

[0127] Example 23: Patient subsets AIII and AIII-G can be precisely distinguished by the expression of selected immunologically related genes in PBMCs. First, discriminant analysis was used to identify the gene expression data obtained as described in Example 22, which best differentiated PBMCs from AIII and AIII-G patients (Figure 39A). Next, principal component analysis was used to determine which of these genes—CCL3, CCR4, CCR5, CCR7, CXCL7, IL-15, IL-32, IL-15R, IL-21R, IL-23R, IL-31RA, and CD163—was most effective in distinguishing the two patient cohorts (Figure 39B). Both the discriminant and principal component plots were created using gene-specific expression characteristics for each individual's AIII and AIII-G PBMC samples (represented by blue and red dots, respectively). The green dashed line in (A) and the green vector in (B) represent the direction of gene transcripts in the reference and component spaces, respectively. To identify the genes that most reliably describe AIII in AIII-G patients, LDA was performed on mRNA levels of all 93 genes using detectable signals from PBMCs (Figure 39A). Twelve genes were selected for PCA analysis, as detailed in Table 9 (Figure 39B). The total variance expressed by the first principal component was 37%, while the second principal component expressed approximately 20% of the total variance. Based on PCA, evaluation of the expression levels of the M2 marker CD163, the pro-inflammatory cytokine IL-32, and the type 1 cytokine receptors IL-21R and IL-23R in PBMCs is sufficient to obtain clear differentiation between patients with different classes of AIII tumors.

[0128] [Table 9] Reference All patents and publications referenced herein are invoked by reference in their entirety herein.

[0129] The publications discussed herein are provided solely for the purpose of disclosing them prior to the filing date of this application. This disclosure should never be construed as an acknowledgment that no prior rights are granted to such publications on the grounds of prior disclosure. (Note) The technical concepts that can be understood from the above embodiments and modified examples are described below. [Item 1] A pharmaceutical composition comprising an effective amount of insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN), A pharmaceutical composition wherein, when administered to a subject having circulating M2 cells, M2 cells in the tumor microenvironment, or serum that polarizes undifferentiated monocytes into M2 cells, the number of M2 cells in the subject is reduced. [Item 2] The pharmaceutical composition according to item 1, wherein the number of M2 cells in the subject decreases by at least about 40% within about 24 hours after administration of the pharmaceutical composition to the subject, according to measurements by FACS. [Item 3] The pharmaceutical composition according to any one of items 1 to 2, wherein the administration of the pharmaceutical composition does not affect the number of M1 cells in the subject. [Item 4] The pharmaceutical composition according to any one of items 1 to 3, wherein the IGF-1R AS ODN comprises one or more phosphorothioate bonds. [Item 5] The pharmaceutical composition according to any one of items 1 to 4, wherein the IGF-1R AS ODN has at least about 75%, at least about 80%, at least about 90%, at least about 95%, or at least about 98% identity with any of sequence numbers 1 to 14. [Item 6] The pharmaceutical composition according to any one of items 1 to 4, wherein the AS ODN consists of any of sequence numbers 1 to 14. [Item 7] A pharmaceutical composition according to any one of items 1 to 4, wherein the aforementioned AS ODN is Sequence ID No. 1. [Item 8] The pharmaceutical composition according to item 6, wherein one of sequence numbers 1 to 14 is administered to the subject in a dose ranging from approximately 0.025 g / kg to approximately 0.2 g / kg. [Item 9] The pharmaceutical composition described in item 7, wherein Sequence ID No. 1 is administered to the subject in a dose ranging from approximately 0.025 g / kg to approximately 0.2 g / kg. [Item 10] The pharmaceutical composition according to any one of items 1 to 9, wherein the pharmaceutical composition restores type 1 immunity in the subject. [Item 11] A pharmaceutical composition according to any one of items 1 to 10, wherein the IGF-1R AS ODN does not form a hairpin loop structure at approximately 37°C. [Item 12] The pharmaceutical composition according to any one of items 1 to 11, wherein the M2 cells are M2 macrophages expressing one or more cell surface markers selected from the group consisting of CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and CD206. [Item 13] The pharmaceutical composition according to any one of items 1 to 11, wherein the M2 cells are M2 monocytes expressing one or more cell surface markers selected from the group consisting of CD11b, CD14, CD15, CD23, CD64, CD68, CD163, CD204, and CD206. [Item 14] The pharmaceutical composition according to item 12 or 13, wherein the cell surface marker is CD163. [Item 15] The pharmaceutical composition according to item 1, wherein the subject has circulating M2 cells. [Item 16] The pharmaceutical composition according to item 1, wherein the subject includes M2 cells in the tumor microenvironment. [Item 17] A pharmaceutical composition according to any one of items 1 to 16, wherein the subject has cancer. [Item 18] The pharmaceutical composition according to item 17, wherein the cancer is selected from the group consisting of glioma, astrocytoma, breast cancer, squamous cell carcinoma of the head and neck, papillary renal cell carcinoma type 2, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer. [Item 19] The pharmaceutical composition according to item 18, wherein the cancer is a glioma. [Item 20] The pharmaceutical composition according to item 19, which induces regression of glioma tumors in the subject. [Item 21] The pharmaceutical composition described in item 20, which causes a decrease in glioma tumor production in the subject. [Item 22] The pharmaceutical composition according to item 21, which causes the removal of glioma tumor production in the subject. [Item 23] The pharmaceutical composition according to item 18, wherein the cancer is an astrocytoma. [Item 24] The pharmaceutical composition according to item 23, wherein the astrocytoma is selected from the group consisting of grade II astrocytoma, grade III astrocytoma, and grade IV astrocytoma. [Item 25] The pharmaceutical composition according to item 24, wherein the grade III astrocytoma is AIII or AIII-G. [Item 26] The pharmaceutical composition according to item 24, wherein the grade IV astrocytoma is glioblastoma multiforme. [Item 27] A pharmaceutical composition according to any one of items 1 to 26, formulated for oral, intraperitoneal, or intravenous administration. [Item 28] A method for selective removal of M2 cells in a subject, comprising the step of systemically administering to the subject an effective amount of a pharmaceutical composition described in any one of items 1 to 27. [Item 29] A method for treating cancer by reducing the number of M2 cells, comprising the step of systemically administering an effective amount of a pharmaceutical composition described in any one of items 1 to 27 to a subject suffering from the cancer. [Item 30] The method according to item 29, further comprising radiotherapy administered to the subject after administration of the pharmaceutical composition. [Item 31] The radiotherapy described above is selected from the group consisting of internal source radiotherapy, external beam radiotherapy, and whole-body radioisotope radiotherapy, according to the method of item 30. [Item 32] The method according to item 31, wherein the radiotherapy is external beam radiotherapy. [Item 33] The method according to item 32, wherein the external beam radiotherapy is selected from the group consisting of gamma ray therapy, X-ray therapy, intensity-modulated radiotherapy (IMRT), and image-guided radiotherapy (IGRT). [Item 34] The method according to item 33, wherein the external beam radiotherapy is gamma ray therapy. [Item 35] A method for enhancing the immune response in a subject, comprising the step of systemically administering to the subject an effective amount of a pharmaceutical composition described in any one of items 1 to 27. [Item 36] The method according to item 35, further comprising a vaccination therapy administered to the subject at least about 48 hours after administration of the pharmaceutical composition. [Item 37] The method according to any one of items 28 to 36, wherein the pharmaceutical composition is administered to the subject by peritoneal injection. [Item 38] The method according to any one of items 28 to 36, wherein the pharmaceutical composition is administered to the subject by intravenous injection. [Item 39] The method according to any one of items 28 to 36, wherein the pharmaceutical composition is administered to the subject in an oral dosage form.

Claims

1. An immunogenic diffusion chamber comprising tumor cells and irradiated insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN), wherein the IGF-1R AS ODN has the sequence of Sequence ID No.

1.

2. An immunogenic diffusion chamber according to claim 1, comprising 2 μg of the IGF-1R AS ODN.

3. The immunogenic diffusion chamber according to claim 1, further comprising a second amount of irradiated IGF-1R AS ODN.

4. The tumor cells have been irradiated, The immunogenic diffusion chamber according to claim 3, wherein the tumor cells and the second amount of IGF-1R AS ODN are irradiated with gamma rays.

5. The immunogenic diffusion chamber according to claim 4, wherein the tumor cells and the second amount of IGF-1R AS ODN are irradiated with gamma rays at a maximum of approximately 15 Gy.

6. The immunogenic diffusion chamber according to claim 5, wherein the tumor cells and the second amount of IGF-1R AS ODN are irradiated with gamma rays at a rate of about 1 Gy, about 2 Gy, about 4 Gy, about 5 Gy, about 6 Gy, or about 10 Gy.

7. The immunogenic diffusion chamber according to claim 6, wherein the tumor cells are irradiated with gamma rays at approximately 5 Gy.

8. The immunogenic diffusion chamber according to claim 4, wherein the tumor cells are irradiated at least two, at least three, at least four, or at least five times.

9. The immunogenic diffusion chamber according to claim 1, wherein the tumor cells are obtained from a subject and treated in vitro for 3 to 48 hours.

10. An immunogenic diffusion chamber for use in a method for inducing an antitumor immune response in a target that requires it, The method includes implanting the immunogenic diffusion chamber into the subject for a period of therapeutically effective duration. The immunogenic diffusion chamber comprises an insulin-like growth factor 1 receptor antisense oligodeoxynucleotide (IGF-1R AS ODN) having the sequence of SEQ ID NO: 1, tumor cells, and a second amount of the IGF-1R AS ODN.

11. The immunogenic diffusion chamber according to claim 10, wherein the tumor cells and the second amount of IGF-1R AS ODN are irradiated with gamma rays.

12. The immunogenic diffusion chamber according to claim 11, wherein the tumor cells and the second amount of IGF-1R AS ODN are irradiated with gamma rays at a maximum of approximately 15 Gy.

13. The immunogenic diffusion chamber according to claim 12, wherein the tumor cells are irradiated with gamma rays at a dose of approximately 1 Gy, approximately 2 Gy, approximately 4 Gy, approximately 5 Gy, approximately 6 Gy, or approximately 10 Gy.

14. The immunogenic diffusion chamber according to claim 11, wherein the tumor cells are irradiated at least two, at least three, at least four, or at least five times.

15. The immunogenic diffusion chamber according to claim 11, wherein the tumor cells are obtained from the subject and treated in vitro for 3 to 48 hours.

16. The immunogenic diffusion chamber according to claim 10, wherein the subject is suffering from at least one selected from the group consisting of glioma, astrocytoma, breast cancer, squamous cell carcinoma of the head and neck, papillary renal cell carcinoma type 2, lung cancer, pancreatic cancer, gallbladder cancer, rectal cancer, classical Hodgkin lymphoma, ovarian cancer, and colorectal cancer.

17. The immunogenic diffusion chamber according to claim 16, wherein the subject is suffering from a glioma.

18. An immunogenic diffusion chamber according to claim 10, which is implanted in the abdomen of the subject.

19. The immunogenic diffusion chamber according to claim 18, which is implanted into the rectus abdominis muscle sheath of the subject.

20. The immunogenic diffusion chamber according to claim 10, further comprising administering a systemic amount of the pharmaceutical composition containing IGF-1R AS ODN to the subject.

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