Combination of cpg-oligodeoxynucleotides with immunomodulators for cancer immunotherapy

Abstract

The present disclosure provides a combination of CpG-oligodeoxynucleotide and immunomodulator for cancer immunotherapy, which can enhance the efficacy of immune checkpoint blockade against tumors in an immunosuppressive microenvironment. More specifically, the combination therapy involves treating cancer by an immune checkpoint inhibitor and a CpG-oligodeoxynucleotide.

Description

Technical Field

[0001] Generally speaking, this invention relates to the field of cancer treatment, particularly cancer immunotherapy. Background Technology

[0002] PD-1 is one of the most thoroughly studied immune checkpoint regulators, playing a crucial role in maintaining immune system homeostasis and preventing diseases caused by overactivation of the immune response. PD-1 controls the late immune response of peripheral tissue T cells because its ligands are primarily expressed in non-lymphoid tissues. Various PD-1 / PD-L1 monoclonal antibodies that block immune checkpoints to combat tumors have been developed, and currently, six FDA-approved PD-1 or PD-L1 antibodies are used for immunotherapy in various cancers. While cancer treatments using these immune checkpoint inhibitors have demonstrated significant efficacy, the response rate for patients with solid tumors is typically less than 30%.

[0003] CpG-oligodeoxynucleotides (CpG-ODNs) are synthetic activators of toll-like receptor 9 (TLR9) and TLR21 in various species. Mammals express TLR9 but lack TLR21. TLR9 activation in mammals by CpG-ODN induces immune responses, including an innate immune response within hours of CpG-ODN stimulation and a subsequent phase II adaptive immune response several days later.

[0004] Because activated immune responses help eliminate cancer cells in the body, the anti-tumor effects of CpG-ODN have been widely studied and confirmed in various animal models of cancer. Furthermore, CpG-ODN is being investigated in clinical trials as a cancer treatment drug; however, to date, no CpG-ODN has been approved for cancer treatment. Summary of the Invention

[0005] Therefore, one aspect of the present invention relates to a method for treating cancer, comprising administering an immune checkpoint blocker and an adjuvant composition to a patient in need; wherein the adjuvant composition comprises at least one CpG-oligodeoxynucleotide (CpG-ODN).

[0006] In some embodiments, the immune checkpoint blocker is selected from anti-PD1, anti-PD-L1, and anti-CTLA4 antibodies.

[0007] In some preferred embodiments, the CpG-ODN is selected from CpG-1585 (SEQ ID NO:1), CpG-2216 (SEQ ID NO:2), CpG-1826 (SEQ ID NO:3), CpG-2006 (SEQ ID NO:4), CpG-2722 (SEQ ID NO:5) and CpG-M362 (SEQ ID NO:6).

[0008] In some embodiments, the cancer is resistant to immunotherapy.

[0009] In some embodiments, the cancer is selected from melanoma, non-small cell lung cancer, renal cell carcinoma, Hodgkin lymphoma, head and neck cancer, urothelial carcinoma, hepatocellular carcinoma, and small cell lung cancer.

[0010] Another aspect of the present invention relates to a pharmaceutical composition for treating cancer, comprising an immune checkpoint blocker and an adjuvant composition; wherein the adjuvant composition comprises at least one CpG-ODN. Attached Figure Description

[0011] Data are presented as mean ± standard error (SEM), n = 3 independent experiments. Asterisks *, **, and *** indicate statistically significant differences compared to the control group or the indicated individuals, respectively, with p < 0.05, p < 0.01, and p < 0.001.

[0012] Figure 1A and Figure 1B This study investigated the cytokine-inducing effects of CpG-2722 in human cells.

[0013] As shown in the figure, human peripheral blood mononuclear cells (PBMCs) were treated with 0.5 μM CpG-ODN. Figure 1A Four hours later, the cells were lysed, and the relative mRNA expression levels of each cytokine were determined by RT-qPCR, with β-actin expression level serving as a control. Figure 1B 24 hours later, the cytokines secreted into the cell culture medium were measured by ELISA.

[0014] Figure 2A and Figure 2B This study investigated the cytokine-inducing effect of CpG-2722 in mouse cells.

[0015] Will( Figure 2A Mouse bone marrow-derived macrophages (BMDMs) and ( Figure 2B Mouse spleen cells were treated with 0.5 μM of each CpG-ODN for 4 hours. The relative mRNA expression levels of each cytokine were determined by RT-qPCR, and the expression level of β-actin was used as a control.

[0016] Figure 3 This demonstrates the inhibitory effect of CpG-2722 on tumor cell growth.

[0017] C57BL / 6J mice were injected orally with 2×10 6 NHRI-HNC1 cells were used to establish a head and neck squamous cell carcinoma (HNSCC) model. After 21 days, when the tumor reached 250-550 mm... 3 At that time, the control vector, 50 μg, or 100 μg of CpG-2722 were injected intratumorally twice a week, respectively. Tumor size was measured on days 21, 24, and 31 (each group contained 3 mice and 3 tumors).

[0018] Figures 4A-4E CpG-2722 enhances the inhibitory effect of immune checkpoint inhibitors on tumor cell growth.

[0019] ( Figure 4A C57BL / 6J mice were injected orally with 2×10 6 NHRI-HNC1 cells were used to establish an HNSCC model. Nine days later, when the tumor reached 100 mm... 3 Every 3 days, mice were injected intratumorally with either the control vector or 50 μg of CpG-2722, with or without weekly intraperitoneal injections of 10 μg of anti-PD-1 antibody, for a total of 2 weeks, as shown in Figure 4B. Tumor size was measured every 3 days (each group contained 5 mice and 5 tumors). Figure 4C The tumor's growth endpoint is shown in the figure. Figure 4D Tumor samples were stained with H&E (Figure 40× above) and CD8 levels were measured by immunohistochemical staining. + T cell infiltration (middle image 20× and bottom image 40×), scale bar 100μm. Figure 4E CD8 +Cells were quantified using ImageJ software at 20× magnification.

[0020] Figure 5 This study investigated the cytokine-inducing effects of CpG-2722 in tumor cells.

[0021] C57BL / 6J mice were injected orally with 2×10 6 NHRI-HNC1 cells were used to establish an HNSCC model. When the tumor reached 100 mm... 3 At the same time, the tumor was injected intratumorally with either the control vector or 50 μg of CpG-2722, and euthanasia was performed the following day. Whole RNA was isolated from the tumor tissue samples using TRIzol reagent, and the mRNA expression levels of various cytokines were measured by RT-qPCR, with β-actin expression level serving as a control.

[0022] Figure 6 CpG-2722, alone and in combination with immune checkpoint inhibitors, can increase the expression of cytokine genes in tumor cells.

[0023] Mice with tumors were treated with CpG-2722 and anti-PD-1 alone or in combination for 15 days, followed by euthanasia and tumor sample collection. Whole RNA was isolated from the tumor tissue samples using TRIzol reagent, and the mRNA expression levels of cytokines were measured by RT-qPCR. The expression level of β-actin was used as a control.

[0024] Figure 7A and Figure 7B CpG-2722, alone and in combination with immune checkpoint inhibitors, can increase CD8+ in tumor cells. + Accumulation of T cells and M1 macrophages.

[0025] Mice with tumors were treated with CpG-2722 and anti-PD-1 alone or in combination for 15 days, followed by euthanasia to collect tumor samples. Whole RNA was isolated from the tumor tissue samples using TRIzol reagent, and then analyzed by RT-qPCR (Figure 7A) for various T cells and (…). Figure 7B The expression of various macrophage markers was measured, with the expression level of β-actin serving as a control. Detailed Implementation

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, and in the event of any conflict of meanings, this specification shall prevail.

[0027] Reagents, antibodies, and human peripheral blood mononuclear cells

[0028] All CpG-oligodeoxynucleotides (CpG-ODN) were purchased from Integrated DNA Technologies, Inc. CpG-ODN was dissolved in DNase / RNase-free water, aliquoted, and stored at -20°C. The anti-PD-1 antibody for in vivo therapy was purchased from InvivoGen (Cat. No. mpd1-mab15-10). The anti-mouse CD8 antibody for immunohistochemistry was purchased from Invitrogen (Cat. No. 14-0808-82). Trizol reagents and the SuperScript™ IV reagent kit were purchased from Invitrogen. Green PCR reagent kits were purchased from Qiagen. Human peripheral blood mononuclear cells (PBMCs) were purchased from ZenBio, Inc.

[0029] Preparation of mouse spleen cells

[0030] Mouse spleen cells were isolated from 6- to 8-week-old C57BL / 6J mice (Taiwan Experimental Animal Center, China). In short, mouse spleens were collected and fragmented using a syringe plunger. The fragments were introduced into a 40 μm nylon cell filter (BDFalcon™), and single cells were excised from the spleen fragments, passed through the filter, and centrifuged at 1500 rpm for 5 minutes. The cell pellet was resuspended in RBC lysis buffer for 2 minutes, followed by the addition of 30 ml PBS to terminate the lysis reaction. Spleen cells were centrifuged at 1500 rpm for 5 minutes and cultured in RPMI 1640 complete medium at 37°C and 5% CO2.

[0031] Preparation of mouse bone marrow-derived macrophages

[0032] Bone marrow-derived macrophages (BMDMs) were isolated from 6- to 8-week-old C57BL / 6J mice. Bone marrow cells were washed from the tibia and femur using a syringe with a 27G needle, passed through a 40 μm nylon cell filter, and centrifuged at 1500 rpm for 5 minutes. The cell pellet was resuspended in RBC lysis buffer for 2 minutes, and lysis was immediately terminated by adding 30 ml of PBS. The bone marrow cells were centrifuged at 1500 rpm for 5 minutes and cultured in 70% DMEM complete medium containing 10% FBS, L-glutamine, antibiotics, 10 mM HEPES buffer, and 30% L929 conditioned medium at 37°C and 5% CO2 for 7 days. Non-adherent cells were removed, and BMDMs were gently scraped off. 106 After BMDM was inoculated onto a six-well plate and cultured for 3 days, it was stimulated with various CpG-ODNs.

[0033] RNA extraction

[0034] Whole RNA from mouse spleen cells, BMDM, and human PBMCs was isolated using the illustra™ RNAspin Mini Kit (GE Healthcare) according to the manufacturer's protocol. TRIzol reagent was used to isolate RNA samples from NHRI-HN1-derived tumors.

[0035] Reverse transcription-quantitative PCR analysis

[0036] Cells were treated with various CpG-ODNs at 0.5 μM for 4 hours, and RNA samples were isolated and reverse transcribed using the SuperScript™ IV First-Strand Synthesis System (Invitrogen). Gene expression analysis was performed using QuantiNova™ Quantitative PCR was performed using the Green PCR Kit (Qiagen) and the Applied Biosystems ViiATM7 Real-Time PCR System, with the expression level of β-actin serving as a control.

[0037] Determination of cytokine production using enzyme immunosorbent assay

[0038] Human PBMCs were treated with the specified CpG-ODN for 24 hours, and cell culture medium was collected. Cytokine production was measured using ELISA kits (eBioscience, San Diego, CA, USA) according to the manufacturer's protocol.

[0039] Animal model of syngeneic in situ carcinoma

[0040] A specified number of NHRI-HN1 cells and matrix gel (BD Biosciences) were subcutaneously injected into the oral cavity of 4- to 6-week-old C57BL / 6J mice to induce tumor growth. When the tumors reached a specified size, a specified amount of CpG-2722 was injected intratumorally twice weekly, with or without weekly injection of 10 μg of anti-PD-1 antibody. Each group contained 5 mice and 5 tumors. Tumor volume in mice carrying NHRI-HN1-derived tumors is expressed as length × (width). 2 Measured at ×0.5.

[0041] Immunohistochemistry

[0042] NHRI-NH1-derived tumors were sectioned into 5 μm tissue slices, which were then rehydrated in PBS using gradient concentrations of ethanol and subjected to 3% hydrogen peroxide to block endogenous peroxidase activity for 5 minutes. CD8 staining was performed using a 1:50 dilution of a rat monoclonal antibody against mouse CD8, followed by incubation at room temperature for 1 hour. Immunostaining was performed using the UltraView Universal DAB Detection Kit (Ventana Medical System, Inc. Tucson) and the Discovery XT automated IHC / ISH slide staining system (Ventana Medical System, Inc. Tucson) according to the manufacturer's protocol, with hematoxylin counterstaining for observation.

[0043] The species-specific activity of CpG-ODNs is determined by their nucleotide sequence and length. For example, CpG-2006 is more effective than CpG-1826 in activating human cells, while CpG-1826 is more effective in activating mouse cells. As shown in Table 1, CpG-2006 contains 24 nucleotides with three repeating GTCGTT-hexamethylene motifs. CpG-1826 contains 20 nucleotides with two repeating GTCGTT-hexamethylene motifs. CpG-2722 contains 19 nucleotides with two repeating GTCGTT-hexamethylene motifs and four thymidines between these two hexamethylene motifs. This CpG-ODN was previously developed for activating TLR21 in grouper species, but it is also active against mouse TLR9 and human TLR9, making it a universal CpG-ODN suitable for multiple species. To explore its potential use as an immunostimulant in mammals, this invention first compares the cytokine-inducing capabilities of various CpG-ODNs. CpG-2006, similar to CpG-2722, is a type B CpG-ODN containing a phosphorothiolate backbone and three CpG motifs throughout its sequence. CpG-2216 is a type A CpG-ODN with preferred activity against human cells, containing a central phosphodiester palindrome region with a CpG motif and poly-G sequences connecting the phosphorothiolate backbone at both the 5' and 3' ends. CpG-M362 is a type C CpG-ODN active against both human and mouse cells, containing a phosphorothiolate backbone with one or two CpG motifs and a palindrome sequence at the 3' end (Table 1).

[0044] Table 1 Structural features of CpG-ODN used in this invention

[0045]

[0046] Example

[0047] Example 1: CpG-2722 induces cytokine expression in human cells.

[0048] Human PBMCs were stimulated with CpG-ODN, and the expression of various cytokine genes was analyzed by RT-qPCR. The results showed that, similar to CpG-2006 and CpG-M362, CpG-2722 exhibited activity in inducing the expression of inflammatory cytokines, including TNF-α, IL-1β, IL-6, IL-12β, and IFN-γ. However, CpG-2722 also activated the expression of type I IFNs, including IFNα2 and IFN-β, similar to type A CpG-ODN (…). Figure 1A IL-12p70 is a heterodimer of IL-12A and IL-12B. This cytokine, along with IFN-γ, plays a crucial role in promoting T cell proliferation and activating anti-tumor responses. Therefore, the production of these two cytokines in cell culture medium was verified using an ELISA assay. Consistent with its ability to induce cytokine expression, CpG-2722 also showed higher activity in inducing IL-12 and IFN-γ production than other CpG-ODNs. Figure 1B ).

[0049] Example 2: CpG-2722 induces cytokine expression in mouse cells.

[0050] The immunostimulatory activity of CpG-2722 in mouse cells has been further investigated compared to other types of CpG-ODN. In this invention, type A CpG-1585 and type B CpG-1826 (Table 1), with nucleotide sequences designed for activating mouse cells, were used instead of CpG-2216 and CpG-2006 used in human cell studies. The expression levels of various genes induced in CpG-ODN-treated mouse BMDM were analyzed by RT-qPCR. In these cells, CpG-1826 exhibited better activity than other CpG-ODNs in inducing the expression of inflammatory cytokines (including TNF-α, IL-1β, IL-6, and IL-12B). In contrast, CpG-2722 showed comparable activity to CpG-1826 in inducing the expression of IL-12A and IFN-γ. Furthermore, CpG-2722, like type A CpG-ODN, activates the expression of IFN-α2 and IFN-β. Figure 2AFurthermore, the gene expression of various cytokines in mouse spleen cells stimulated by CpG-ODN was analyzed. Generally, these two B-type CpG-2722 and CpG-1826 showed better activity in inducing the expression of these inflammatory cytokines (including IL-12A, IL-12B, and IFN-γ) compared to other tested A and C-type CpG-ODNs. Figure 2B ).

[0051] Example 3: Antitumor activity of CpG-2722

[0052] CpG-2722, as a type B CpG-ODN, can induce the expression of inflammatory cytokines IL-12 and IFN-γ in human and mouse cells, and can also induce the expression of type 1 interferon (IL-12) similar to type A CpG-ODN. Figure 1A Figure 1B and Figure 2A , Figure 2B These cytokines play a crucial role in enhancing the immune response to eradicate cancer cells. The cancer cell line NHRI-NH1, established from oral squamous cell carcinoma cells derived from C56BL / 6J mice, can be used to study cancer immunobiology. These NHRI-NH1 cells (2 × 10⁻⁶ cells) 6 Cells / mouse cells were injected with the same gene into the buccal mucosa of mice to form in situ tumors. After 21 days, when the tumor size reached 250-550 mm... 3 At the same time, 50 μg or 100 μg of CpG-2722 was injected intratumorally every 3 days for a total of 3 times, and tumor growth was monitored. The results showed that the effect of CpG-2722 on inhibiting tumor growth was similar at both doses. Figure 3 Therefore, in the following examples, a dose of 50 μg CpG-2722 per mouse was used.

[0053] Example 4: Synergistic effect of CpG-2722 combined with anti-PD-1 to inhibit tumor growth

[0054] Although nivolumab and pembrolizumab are approved for use in patients with head and neck cancer, less than 20% of patients respond to these immune checkpoint inhibitors. Therefore, different approaches are still needed to improve the efficacy of immune checkpoint blockade therapy. This invention also investigated the effect of the combination of CpG-2722 and anti-PD-1 antibody on inhibiting tumor growth using an NHRI-NH1 isogenetic in situ cancer animal model.

[0055] The animal experiments were divided into two groups. In the first group, the tumor grew to approximately 100 mm in 9 days. 3Subsequently, CpG-2722 was injected intratumorally every 3 days, and anti-PD-1 antibody was injected intraperitoneally on day 0 and day 6 after CpG-2722 injection. Figure 4A Tumor growth monitoring in these mice showed that both CpG-2722 and anti-PD-1 antibody alone could inhibit tumor growth; however, the combination of CpG-2722 and anti-PD-1 antibody showed a more effective inhibition of tumor growth than either drug alone. Figure 4B Mice were euthanized on day 15 after treatment with CpG-2722 and anti-PD-1 antibody, and tumors were harvested for size analysis. Figure 4C Further histochemical analysis of the tumor tissue showed that both CpG-2722 and anti-PD-1 antibody treatment increased CD8+ in the tumor. + T cell infiltration, however, the combination of CpG-2722 and anti-PD-1 antibody further increased CD8. + The degree of T cell infiltration ( Figure 4D ).

[0056] The second group of experiments was similar to the first, except that the CpG-2722 injections were changed from every three days to every four days, and the anti-PD-1 antibody administration route and schedule were changed to intravenous injection on days 8 and 16 after CpG-2722 administration. The results also showed a synergistic effect of CpG-2722 and the anti-PD-1 antibody in inhibiting tumor growth. Similar to the first group of experiments, the degree of CD8 T cell accumulation in the tumor was correlated with the therapeutic effect of CpG-2722 and / or anti-PD-1 treatment in inhibiting tumor growth. Figure 4E ).

[0057] Example 5: Immunological response in the tumor microenvironment activated by CpG-2722

[0058] The mechanism by which the combination of CpG-2722 and anti-PD-1 antibody enhances the anti-tumor effect is further investigated in this invention. Head and neck cancer was established by injecting mice with NHRI-NH1 cells; when the tumor reached approximately 100 mm... 3 At that time, CpG-2722 was injected intratumorally, and the patient was euthanized 24 hours later. The expression levels of various cytokines within the tumor, obtained by RT-qPCR, showed that the expression of TNF-α, IFNα2, and IFN-γ was induced the day after intratumoral injection of CpG-2722, while the induction of other cytokines was not significant. Furthermore, Figures 4A-4EIn the experiment, mice treated with CpG-2722, anti-PD-1 antibody, and CpG-2722 plus anti-PD-1 antibody for 15 consecutive days had their tumor specimens analyzed for cytokine expression profiles by RT-qPCR. In mouse tumors treated with CpG-2722 alone and with CpG-2722 plus anti-PD-1 antibody, the gene expression of TNF-α, IL-12A, IL-12B, IFN-β, and IFN-γ was significantly increased. Figure 6 The ability of CpG-2722 to induce the expression of these cytokines in tumors is consistent with its ability to induce them in immune cells. Figure 1A , Figure 1B and Figure 2A , Figure 2B In addition, the tumor cytokine induction curves on day 2 after CpG-2722 injection and on day 15 after continuous CpG-2722 injection ( Figure 5 and Figure 6 The results showed that multiple injections of CpG-ODN were required to effectively induce cytokine expression in tumors.

[0059] This invention also further investigated the accumulation of immune cells in tumors of mice treated with CpG-2722 and anti-PD-1 antibody through RT-qPCR analysis of different cell markers. The results showed that mice treated with CpG-2722 alone and with CpG-2722 plus anti-PD-1 antibody had increased CD3 and CD8 positive T cells in their tumors. Figure 7A Macrophages are the most abundant white blood cells in the tumor microenvironment. These tumor-associated macrophages can generally be divided into two subsets: inflammatory M1 macrophages and anti-inflammatory M2 macrophages. Analysis of markers such as F4 / 80 for all macrophages, CCR7, iNOS, and CD86 for M1 macrophages, and ARG1 and CD206 for M2 macrophages showed that CpG-2722 alone and CpG-2722 plus anti-PD-1 antibody enhanced macrophage accumulation in the tumor. Furthermore, the accumulation of M1 macrophages trended in the same direction as the increase in total macrophage numbers, while M2 macrophages did not. Figure 7B In summary, these results demonstrate that CpG-2722 can induce key anti-tumor cytokines, including IL-12, IFN-γ, and type 1 IFN, and increase inflammatory M1 macrophages and CD8+. + The accumulation of T cells improves the tumor microenvironment. These immune responses in the tumor microenvironment activate anti-PD-1 effector T cells, thus releasing the brakes on tumor killing.

[0060] Immune checkpoint blockade using anti-PD-1 antibodies has been approved by the US FDA for the treatment of recurrent and metastatic tumors. Nevertheless, most patients do not respond to treatment, highlighting the need for strategies to mitigate the resistance of the suppressive microenvironment to immunotherapy. CpG-ODN monotherapy has generally shown good activity in inducing tumor regression in animal cancer models; however, to date, no CpG-ODN has been approved for cancer treatment, indicating that CpG-ODN alone may not be sufficient to enhance an effective anti-tumor immune response in humans. The synergistic effect of CpG-ODN and immune checkpoint inhibitors in inhibiting tumor growth has been demonstrated in this invention, and this combination therapy has also proven effective in treating tumors with an immunosuppressive microenvironment. sequence list <110> Taiwan National Health Research Institutes <120> Combinations of CpG-oligonucleotides with immunomodulators for cancer immunotherapy <130> GAI21TW6169 <150> US 17 / 467,390 <151> 2021-09-06 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CpG-Oligodeoxynucleotides <400> 1 ggggt caacg ttgag ggggg 20 <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CpG-Oligodeoxynucleotides <400> 2 ggggg acgat cgtcg ggggg 20 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> CpG-Oligodeoxynucleotides <400> 3 tccat gacgt tcctg acgtt 20 <210> 4 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> CpG-Oligodeoxynucleotides <400> 4 tcgtc gtttt gtcgt tttgt cgtt 24 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> CpG-Oligodeoxynucleotides <400> 5 gttgt cgttt tttgt cgtt 19 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <220> <223> CpG-Oligodeoxynucleotides <400> 6 tcgtc gtcgt tcgaa cgacg ttgat 25

Claims

1. Use of an immune checkpoint inhibitor and CpG-oligodeoxynucleotide (CpG-ODN) in the preparation of a medicament for treating cancer, wherein the immune checkpoint inhibitor is an anti-PD1 antibody; wherein the CpG-oligodeoxynucleotide is CpG-2722 as shown in SEQ ID NO:5; and wherein the cancer is head and neck cancer.

2. The use as described in claim 1, wherein the cancer is resistant to immunotherapy.

3. A pharmaceutical composition for treating cancer, comprising an immune checkpoint inhibitor and an adjuvant composition; wherein the immune checkpoint inhibitor is an anti-PD1 antibody; wherein the adjuvant composition comprises at least one CpG-oligonucleotide; wherein the CpG-oligonucleotide is CpG-2722 as shown in SEQ ID NO:5; wherein the cancer is head and neck cancer.

4. The pharmaceutical composition of claim 3, wherein the cancer is resistant to immunotherapy.

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