CPG amphiphilic molecules and uses thereof

Abstract

The present invention provides compounds comprising CpG oligodeoxynucleotide sequences linked to a lipid via a linker, and related compositions and methods. The invention features a compound consisting of the nucleotide sequence of SEQ ID NO: 1, which is bonded or linked to a lipid at its 5'end via a linker. Furthermore, the invention features a method of treating cancer in a human patient comprising administering the compound to the patient. Furthermore, the invention features a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. The invention features a kit comprising (I) a compound or a composition comprising the compound; (ii) a protein comprising SEQ ID NO: 2 or SEQ ID NO: 3.

Description

[0001] Sequence Listing

[0002] This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on ______________, named __________________________, and is _____ bytes in size. Background of the Invention

[0003] Cancers associated with human papillomavirus (HPV) are among the fastest growing cancers in the world. Overall, 5% of all cancers worldwide can be attributed to HPV infection. There remains a need for further and more effective cancer treatments. Summary of the Invention

[0004] The present invention provides compounds useful in therapeutic methods.

[0005] Thus, in a first aspect, the present invention features a compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1) which is bonded or linked at its 5'-end via a linker to the following lipid:

[0006]

[0007] or a salt thereof,

[0008] wherein X is O or S.

[0009] In one embodiment of the first aspect of the present invention, the nucleotide sequence is bonded to the lipid.

[0010] In another embodiment of the first aspect of the present invention, all internucleoside groups linking the nucleosides in 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1) are phosphorothioates.

[0011] In a second aspect, the present invention features a method of treating cancer in a human patient. The method comprises administering to the patient a compound of the first aspect of the present invention, comprising a protein having the following amino acid sequence:

[0012] MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIVYRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQRFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2), and a protein comprising the following amino acid sequence:

[0013] MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3).

[0014] In one embodiment of the second aspect of the present invention, the cancer is human papillomavirus (HPV)-positive (e.g., HPV16-positive).

[0015] In another embodiment of the second aspect of the present invention, the cancer is head or neck squamous cell carcinoma.

[0016] In another embodiment of the second aspect of the present invention, the patient is receiving or has received platinum-containing chemotherapy. In another embodiment, an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) is administered to the patient.

[0017] In another embodiment of the second aspect of the present invention, the compound of the first aspect of the present invention and the protein comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 are administered simultaneously.

[0018] In another embodiment of the second aspect of the present invention, the compound of the first aspect of the present invention and the protein comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 are administered sequentially.

[0019] In another aspect, the present invention features another method for treating cancer in a human patient. The method comprises administering to the patient a compound of the first aspect of the present invention, a protein comprising the following amino acid sequence:

[0020] MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIVYRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQRFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2), including the following amino acid sequence protein:

[0021] MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3), and

[0022] an anti-PD-1 antibody (such as pembrolizumab or nivolumab).

[0023] In a third aspect, the invention features a pharmaceutical composition comprising a compound of the first aspect of the invention and a pharmaceutically acceptable carrier.

[0024] In one embodiment of the third aspect, the pharmaceutical composition further comprises a protein having the following amino acid sequence: MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIVYRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQRFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2), and a protein having the following amino acid sequence: MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3).

[0025] In a fourth aspect, the present invention features a kit that includes (i) a compound of the first aspect of the present invention or a composition of the second aspect of the present invention, and (ii) a protein comprising the following amino acid sequence: MHQKRTAMFQDPQERPRKLPQLCTELQTTIHDIILECVYCKQQLLRREVYDFAFRDLCIVYRDGNPYAVGDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLLIRCINGQKPLCPEEKQRHLDKKQRFHNGRGRWTGRCMSCCRSSRTRRETQL (SEQ ID NO:2),

[0026] and a protein comprising the following amino acid sequence: MHGDTPTLHEYMLDLQPETTDLYGYGQLNDSSEEEDEIDGPAGQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP (SEQ ID NO:3).

[0027] Definitions

[0028] As used herein, "linker" refers to a monovalent or divalent group in which one valence is covalently bonded to one biofunctional group and the other valence is covalently bonded to another biofunctional group. In one example, the linker connects a nucleotide sequence of, for example, a CpG oligonucleotide to a lipid (e.g., -P(X)(OH)-O-CH(CH2NHCO-(CH2) 16 -CH3)2 or a salt thereof, where X is O or S, as described herein). Such a linker can optionally include one or more nucleotides, such as a dinucleotide (e.g., GG).

[0029] As used herein, "pharmaceutically acceptable carrier" refers to a vehicle capable of suspending or dissolving an active compound and having non-toxic and non-inflammatory properties in a patient. In addition, a pharmaceutically acceptable carrier can include pharmaceutically acceptable additives, such as preservatives, antioxidants, flavorings, emulsifiers, dyes, or excipients known or used in the field of pharmaceutical formulations, and does not significantly interfere with the therapeutic effect of the bioactivity of the active agent and is non-toxic to the patient.

[0030] The terms “treat”, “treatment” and “treating” refer to a treatment method that aims to reverse, mitigate, improve, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (such as cancer). These terms include reducing or alleviating at least one adverse effect or symptom of the condition, disease or disorder. A treatment is generally “effective” if one or more symptoms or clinical signs are reduced, or if a desired response (such as a specific immune response) is induced. Alternatively, a treatment is “effective” if the progression of the disease is reduced or stopped.

[0031] The present invention has several advantages. For example, in the case of including a lipid moiety and an optional linker, certain compounds of the present invention bind to endogenous albumin in the subject to which they are administered, which enhances the delivery of the compound to the lymph nodes of the subject. This promotes the induction of a therapeutic immune response against, for example, the administration of an HPV protein to the subject, resulting in effective cancer treatment.

[0032] Other features and advantages of the present invention will become apparent from the following detailed description, drawings and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a graph showing the immune response against HPV16 E6.

[0034] Figure 2 is a graph showing the immune response against HPV16 E7.

[0035] Figure 3 is a graph showing the tetramer staining analysis of HPV16 E7.

[0036] Figure 4 is a series of graphs showing that co - administration of HPV16 E6 and HPV16 E7 with aCpG can reduce tumor size compared to amphiphile - CpG (aCpG) alone or no treatment (no Tx).

[0037] Figure 5 is a graph showing that the aCpG vaccine has a better HPV tetramer response compared to the soluble CpG vaccine at different time points.

[0038] Figure 6 is a graph showing the change over time of the persistent HPV tetramer response with weekly or bi - weekly administration of aCpG.

[0039] Figure 7 is a graph showing the tumor size response to E7 vaccine treatment.

[0040] Figure 8Is a graph showing improved survival in C57BL6 mice implanted with TC-1 tumor cells and treated with the E7 vaccine.

[0041] Figure 9 Is a series of graphs that show changes in serum cytokine levels after administration.

[0042] Figure 10 Is a graph showing the tetramer response of the HPV16 E7 / aCpG vaccine over time.

[0043] Figure 11 Is a graph showing the tumor growth response to vaccination with the HPV16 E7 / aCpG vaccine plus or minus administration of an anti-PD-1 antibody.

[0044] Figure 12 Is a graph showing the effect of vaccination with the HPV16 E7 / aCpG vaccine plus or minus administration of an anti-PD-1 antibody on survival in mice bearing TC-1 tumors.

[0045] Figure 13 Is a graph showing the tetramer assay for increasing doses of aCpG.

[0046] Figure 14 Shows the structure of the amphiphile-CpG-7909; 5'-(diacyl lipid)TCGTCG TTT TGT CGT TTT GTC GTT-3'(SEQ ID NO:1). All bases are DNA. All linkages are phosphoramidites, including the linkage between the diacyl lipid and the oligodeoxynucleotide. DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides compounds useful in therapeutic methods. The compounds include CpG oligodeoxynucleotides (ODNs) (e.g., a CpG ODN having the sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1)). The CpG ODN is linked to a lipid at its 5' end, such as:

[0049]

[0050] or a salt thereof,

[0051] wherein X is O or S. Preferably, X is S. The CpG oligonucleotide can be directly bonded to the lipid. Alternatively, CpG can be linked to the lipid through a linker such as GG. In the CpG oligonucleotide, all internucleoside groups are phosphorothioates (e.g., all internucleoside groups in the compound can be phosphorothioates).

[0052] CpG ODNs can act as adjuvants to elicit an immune response in a subject, such as an immune response against a cancer antigen (e.g., an HPV antigen). Thus, the compounds and compositions of the invention can be used in therapeutic methods. In particular, if a compound containing CpG ODN is administered in combination with one or more HPV proteins, the compound can induce an immune response against HPV-positive cancer cells. Accordingly, the invention provides a method of treating cancer in a subject (e.g., a human patient) by administering to the subject one or more compounds or compositions of the invention. In various examples, the cancer is HPV-positive (e.g., HPV type 16-positive) cancer.

[0053] HPV-positive cancers can be squamous cell carcinoma of the head or neck, cervical cancer, anal cancer, vulvar cancer, head and neck cancer, oropharyngeal cancer, penile cancer, vaginal cancer, virus-induced cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, colon cancer, gastric cancer, neuroblastoma, breast cancer, prostate cancer, kidney cancer, leukemia, sarcoma, carcinoma, basal cell carcinoma, non-small cell lung carcinoma, non-Hodgkin lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), B-cell chronic lymphocytic leukemia (B-CLL), multiple myeloma (MM), erythroleukemia, renal cell carcinoma, melanoma, astrocytoma, oligoastrocytoma, cholangiocarcinoma, choriocarcinoma, CNS cancer, laryngeal cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, giant (or oat) cell carcinoma, squamous cell carcinoma, oral cancer, skin cancer, basal cell cancer, squamous cell cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, respiratory system cancer, or urinary system cancer.

[0054] Optionally, the methods of the invention can further comprise administering a combination of a compound or composition of the invention with a second (or additional) different therapeutic method.

[0055] The invention also provides kits, each kit comprising, for example, a first container that comprises one or more compounds of the invention, optionally together with a second container that comprises a cancer antigen, such as an HPV protein as described herein.

[0056] CpG

[0057] CpG ODNs are short synthetic single-stranded DNA molecules that include unmethylated CpG dinucleotides within a specific sequence range. In contrast to the natural phosphodiester (PO) backbone in DNA molecules, CpG ODNs have a partial or complete phosphorothioate (PS) backbone. Based on the structural features and activities of human peripheral blood mononuclear cells (PBMCs), particularly B cells and plasmacytoid dendritic cells (pDCs), three major classes of stimulatory CpG ODNs have been identified. These three classes are class A (type D), class B (type K), and class C.

[0058] Both CpG1826 and CpG7909 belong to class B CpGs. Class B CpG ODNs include a complete PS backbone with one or more CpG dinucleotides. They strongly activate B cells and TLR9-dependent NF-κB signaling but weakly stimulate IFN-α secretion.

[0059] Mutated HPV

[0060] Point mutations of C70G, C113G, and I135G (underlined below) can be introduced into the wild-type HPV16 E6 viral protein to prevent stereochemical interaction with human p53. The antigenic properties of this component are determined by the sequence of the protein, and the structure of the protein is irrelevant to the expected function.

[0061] mHPV 16E6 (158aa; SEQ ID NO:2)

[0062] MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC

[0063] KQQLLRREVY DFAFRDLCIV YRDGNPYAV G DKCLKFYSKI

[0064] SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC IN G QKPLCPE

[0065] EKQRHLDKKQ RFHN G RGRWT GRCMSCCRSS RTRRETQL

[0066] Point mutations of C24G and E26G (underlined below) can be introduced into the wild-type HPV16 E7 viral protein to prevent stereochemical interaction with human Rb1. Similarly, the antigenic properties of this component are determined by the sequence of the protein, and the structure of the protein is irrelevant to the expected function.

[0067] mHPV 16E7 (98aa; SEQ ID NO:3)

[0068] MHGDTPTLHE YMLDLQPETT DLY G Y G QLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP.

[0069] CpG ODN can be directly bonded or connected to lipids through a linker. These compounds can be prepared using common phosphoramidite chemistry methods known in the art. In some examples, CpG ODN or CpG ODN-GG can react with the following compounds:

[0070]

[0071] An intermediate is generated, which may produce the compounds of the present invention after being oxidized (e.g., by a phosphorous acid oxidation method known in the art) with a sulfurizing agent (e.g., 3-((N,N-dimethylaminomethylene)amino)-3H-1,2,4-dithiazole-5-thione) and hydrolyzed by a cyanoethyl group.

[0072] To more fully understand the present invention, the following examples are set forth. These examples are for illustrative purposes only and should not be construed in any way as limiting the scope of the present invention. Examples

[0073] Example 1: The combination of HPV16 E6 and HPV16 E7 proteins with a CpG amphiphile adjuvant generates an immune response

[0074] Mice were prophylactically immunized, and the generated immune response was recorded by E7-tetramer staining and IFNγ intracellular cytokine staining (ICS) under the stimulation of HPV16 E6 and HPV16 E7 (E6 / E7).

[0075] The experimental design included the following 6 groups of mice (n = 10 per group)

[0076] 1. Non-immunized

[0077] 2. E6 / E7 + soluble CpG1826

[0078] 3. E6 / E7 + amphiphilic CpG1826 (aCpG1826)

[0079] 4. E6 / E7 + soluble CpG7909

[0080] 5. E6 / E7+ amphiphilic molecule CpG7909 (aCpG7909; Figure 14 )

[0081] 6. E6 / E7+ polyIC (pIC)

[0082] pIC was used as a benchmark adjuvant control.

[0083] The protein stock solution was dissolved in 8M urea. The adjuvant stock solution was dissolved in water. The final injection solution was diluted with 1X phosphate-buffered saline (PBS) (urea C F <1M).

[0084] For aCpG1826, the sequence used was the soluble CpG1826 sequence (5’-tccatgacgttcctgacgtt-3’; SEQ ID NO: 4), with two guanines added at the 5’ end (5’-gg tccatgacgttcctgacgtt-3’; SEQ ID NO: 5). The concentrations of soluble CpG1826 and aCpG1826 used were 5 nmol per 100 μl of injection solution. CpG1826 is the optimal mouse sequence, while CpG7909 is optimal for humans and has weaker activity in mice. CpG1826 and CpG7909 belong to the same CpG class (class B) and generally have similar activity profiles in their respective species.

[0085] For aCpG7909 and soluble CpG7909, the sequence used was 5’-tcgtcgttttgtcgttttgtcgtt-3’ (SEQ ID NO: 6), and the concentration was 5 nmol per 100 μl of injection solution.

[0086] Immunization was carried out using mutant HPV16 E6 with point mutations at C70G, C113G, and I135G (underlined below). The amino acid sequences used are provided below. mHPV 16E6 (158aa; SEQ ID NO:2)

[0087] MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIVYRDGNPYAV G DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC IN G QKPLCPE EKQRHLDKKQRFHN G RGRWT GRCMSCCRSS RTRRETQL

[0088] Immunization was performed using mutant HPV16 E7 with point mutations at C24G and E26G (underlined below). The amino acid sequences used are provided below.

[0089] mHPV 16E7 (98aa; SEQ ID NO:3)

[0090] MHGDTPTLHE YMLDLQPETT DLY G Y G QLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP

[0091] For E6 / E7, 10 μg of mutant HPV16 E6 and 10 μg of mutant HPV16 E7 were used per 100 μl of injection solution.

[0092] Female C57BL / 6J mice (B6) were immunized subcutaneously (s.c.) with a primary dose (E6 / 7 + aCpG) and boosted once 2 weeks later.

[0093] Seven days after the boost dose, tetramer analysis was performed on H-2Db HPV16 E7 (RAHYNIVTF; SEQ ID NO:7) Figure 3 )

[0094] Seven days after the boost dose, intracellular cytokine staining (ICS) for IFNγ was performed on peripheral blood to analyze the immune response to E6 / E7.

[0095] The following peptides were used for E6 stimulation: (E6-10: EVYDFAFRDL (SEQ ID NO:8); E649-57: VYDFAFRDL (SEQID NO:9); E6 37-45: CVYCKQQLL; (SEQ ID NO:10); E6 72-80: KCLKFYSKI (SEQ ID NO:11); and E6 100-108: NKPLCDLLI (SEQ ID NO:12) to generate Figure 1 the data shown.

[0096] Deconvolution of E6 stimulation showed that E49-57 was the only peptide responsible for the stimulation.

[0097] For E7 stimulation, the following peptide was used: RAHYNIVTF (SEQ ID NO:13). This peptide was used to generate Figure 2 the data shown.

[0098] As Figure 1 and 2 shown, strong immune responses against mutant HPV16 E6 and mutant HPV16 E7 were generated using aCpG1826.

[0099] Similarly as Figure 1 and 2 shown, the use of aCpG7909, which is optimal for humans and generally performs poorly in mice, surprisingly generated strong immune responses against mutant HPV16 E6 and mutant HPV16 E7.

[0100] Compared to the use of aCpG alone or no treatment, E6 / E7 + aCpG reduced tumor growth and correspondingly increased survival rates ( Figure 4 ).

[0101] Example 2: Determination of the dosing schedule of HPV 16E7 and aCPG

[0102] To determine the optimal dosing regimen for the anti-tumor efficacy of E7 + aCpG in female C57BL / 6U (B6) mice implanted with TC-1 tumors, weekly dosing was compared with bi-weekly dosing and baseline (only primary immunization). E7 + aCpG was compared with E7 + soluble CpG. All vaccines were administered 3 times (primary immunization and two booster immunizations).

[0103] On day 0 and 12 days later, 50,000 TC-1 cells were subcutaneously inoculated on the flanks of female C57BL / 6J mice (B6); the mice were divided into treatment groups and treated as shown in Table 1.

[0104] Table 1

[0105]

[0106]

[0107] a Dissolve the protein stock solution in 8M urea. Dissolve the adjuvant stock solution

[0108] in water. The final injection solution is diluted with 1X PBS (urea C F <1M)

[0109] diluted.

[0110] b Equivalent to 8 μg

[0111] NA = Not applicable; PBS = Phosphate-buffered saline; ROA = Route of administration; SC = Subcutaneous

[0112] · Throughout the study, up to day 40 post-inoculation, measurements were taken every two days

[0113] Tumor size was measured, and the survival of the animals was monitored. Seven days after each vaccination,

[0114] A tetramer assay for H-2Db HPV16 E7 (RAHYNIVTF) was performed.

[0115] · Serum samples of the aCpG group were collected at 1 hour and 4 hours after each vaccination

[0116] and the cytokine expression (IFNγ, TNFα, IL-6, IL-10, IL-12p70,

[0117] MCP-1) was analyzed by flow cytometric bead array.

[0118] · Anti-E7 serum antibody titers were analyzed 14 days after the primary vaccination. The ELISA

[0119] plate was coated with whole protein E7, on which serum antibodies were captured and

[0120] detected with anti-Fc antibody.

[0121] After single and repeated doses, the HPV-tetramer specific T cell responses to the protein / amphiphilic CpG vaccine were superior to those to the protein / soluble CpG vaccine ( Figure 5 ). After administration of the booster vaccination, the HPV-tetramer responses to the protein / aCpG were further enhanced, and in the once-weekly and twice-weekly regimens, this increase continued until day 28 and day 35, respectively ( Figure 6 ). Compared with the animals vaccinated with soluble CpG, the strong HPV-tetramer responses in the aCpG group were associated with a reduction in tumor size ( Figure 7 ) and improved survival ( Figure 8 ). Between the soluble and aCpG groups, the increase in treatment-related systemic cytokines was comparable, except for IL-10 (which was lower for aCpG compared to soluble CpG) and IFNγ (which was higher for aCpG compared to soluble CpG) ( Figure 9 ).

[0122] Example 3: Antitumor efficacy of the combination of E7 protein with soluble or amphiphilic CpG with or without anti-PD-1 antibody

[0123] To evaluate the anti-tumor efficacy of the combination of E7 protein with soluble or amphiphilic CpG with or without anti-PD-1 antibody, 50,000 TC-1 cells were inoculated subcutaneously at the baseline of female C57BL / 6J mice (B6). Eleven days after inoculation, the mice were divided into 5 groups as shown in Table 2. The comparison group was untreated.

[0124] Table 2

[0125]

[0126] a The stock protein solution was dissolved in 8M urea. The stock adjuvant solution was dissolved in water. The final injection was diluted with 1X PBS (urea C F <1M).

[0127] IP = intraperitoneal; NA = not applicable; PBS = phosphate-buffered saline; ROA = route of administration; SC = subcutaneous

[0128] Throughout the study, tumor size was measured every other day until day 40 after inoculation, and animal survival was monitored. Seven days after each vaccination, tetramer analysis of H-2Db HPV16 E7 (RAHYNIVTF) was performed.

[0129] Administration of the E7 / amphiphile-CpG vaccine with or without anti-PD-1 antibody led to a substantial increase in HPV tetramer+ CD8 cells specific for the HPV16 E7 (RAHYNIVTF; SEQ ID NO:13) peptide ( Figure 10 ). These responses were clearly visible as early as after the first dose, peaked at the second dose, and persisted until the third dose (in contrast to the lower responses observed with E7 / CpG, which did not persist despite enhanced responses with co-administered anti-PD-1).

[0130] Corresponding to these strong HPV tetramer+ CD8 responses, tumor growth stopped around day 24 and regressed after the first administration of E7 / amphiphile-CpG (with or without anti-PD-1), and tumor size remained small and stable until the end of the study, in contrast to the other groups, where growth continued ( Figure 11 ).

[0131] Similarly, corresponding to the effect on tumor size, treatment with the E7 / aCpG vaccine (with or without anti-PD-1 antibody) had a significant effect on survival, resulting in 6 / 7 (85%) cures for E7 / aCpG without antibody and 8 / 10 (80%) cures for E7 / aCpG with anti-PD-1 antibody ( Figure 12 ).

[0132] Example 4: aCpG dose escalation study

[0133] To determine the aCpG dose that generates the highest antigen-specific tetramer + CD8 response during a 6-dose regimen, a dose-escalation study was conducted using a fixed dose of 10 μg ovalbumin (OVA) as the antigen. Soluble CpG was used as the comparator. Tolerance (based on body weight and general observations) was also evaluated. Table 3 outlines the study design.

[0134] Table 3

[0135]

[0136] ROA = Route of administration; SC = Subcutaneous

[0137] Up to 6 doses of the vaccine were administered at 2-week intervals, with a total study time of 11 weeks.

[0138] Peripheral blood samples were collected 7 days after each injection, and flow cytometry analysis of tetramers on CD8+ cells was performed using H-2Kb OVA (SIINFEKL; SEQ ID NO: 14).

[0139] A significant increase in tetramer + CD8+ cells was only observed in the group treated with aCpG + OVA, with 6 nmol producing the greatest pharmacological effect ( Figure 13 ). No weight loss, loss of interest / appetite, or wounds / injuries were observed.

[0140] Other embodiments

[0141] Although the present invention has been described in connection with specific embodiments thereof, it is to be understood that the invention is capable of further modifications, and this application is intended to cover any variations, uses, or modifications of the invention that generally follow the principles of the invention. The invention and such departures from the present disclosure fall within the known or customary practice within the field to which the invention pertains and can be applied to the basic features set forth above.

[0142] All publications, patents, and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application were specifically and individually incorporated by reference in its entirety.

[0143] Some embodiments of the invention are in the following numbered paragraphs.

[0144] 1. A compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO: 1), bonded or linked at its 5' end via a linker to the following lipid:

[0145]

[0146] or a salt thereof,

[0147] wherein X is O or S.

[0148] 2. The compound according to paragraph 1, wherein the nucleotide sequence is bonded to a lipid.

[0149] 3. The compound according to paragraph 1 or 2, wherein all internucleoside groups linking the nucleosides in 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1) are phosphorothioates.

[0150] 4. A method of treating cancer in a human patient, comprising administering to the patient a compound according to any one of paragraphs 1 to 3, a protein comprising the following amino acid sequence:

[0151] MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIVYRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQRFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2),

[0152] and a protein comprising the following amino acid sequence:

[0153] MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3).

[0154] 5. The method according to paragraph 4, wherein the cancer is human papillomavirus (HPV)-positive.

[0155] 6. The method according to paragraph 5, wherein the cancer is HPV type 16-positive.

[0156] 7. The method according to any one of paragraphs 4 to 6, wherein the cancer is head or neck squamous cell carcinoma.

[0157] 8. The method according to any one of paragraphs 4 to 7, wherein the patient is receiving or has received platinum-containing chemotherapy.

[0158] 9. The method according to paragraph 4, wherein the compound described in paragraph 1 and the protein comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 are administered simultaneously.

[0159] 10. The method according to paragraph 4, wherein the compound described in paragraph 1 and the protein comprising the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 3 are administered sequentially.

[0160] 11. A pharmaceutical composition comprising the compound according to any one of paragraphs 1 to 3 and a pharmaceutically acceptable carrier.

[0161] 12. The pharmaceutical composition according to paragraph 11, wherein the composition further comprises a protein comprising the following amino acid sequence:

[0162] MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIVYRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQRFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO: 2),

[0163] and a protein comprising the following amino acid sequence:

[0164] MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO: 3).

[0165] 13. A kit comprising (i) the compound according to any one of paragraphs 1 to 3 or the composition according to paragraph 11, and (ii) a protein comprising the following amino acid sequence: MHQKRTAMFQ DPQERPRKLP QLCTELQTTIHDIILECVYC KQQLLRREVY DFAFRDLCIV YRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYNKPLCDLLIRC INGQKPLCPE EKQRHLDKKQ RFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO: 2),

[0166] and a protein comprising the following amino acid sequence:

[0167] MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3).

[0168] Other embodiments are within the following claims.

Claims

1. A compound consisting of the nucleotide sequence 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1), which is bonded or linked at its 5'-end via a linker to the following lipid: or a salt thereof, wherein X is O or S.

2. The compound according to claim 1, wherein the nucleotide sequence is bonded to the lipid.

3. The compound according to claim 1 or 2, wherein all internucleoside groups linking the nucleosides in 5'-TCGTCGTTTTGTCGTTTTGTCGTT-3' (SEQ ID NO:1) are phosphorothioates.

4. A method of treating cancer in a human patient, which comprises administering to the patient the compound of claim 1 or 2, a protein comprising the following amino acid sequence: MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYCKQQLLRREVY DFAFRDLCIVYRDGNPYAVG DKCLKFYSKISEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPEEKQRHLDKKQRFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2), and a protein comprising the following amino acid sequence: MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCKCDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3).

5. The method according to claim 4, wherein the cancer is human papillomavirus HPV positive.

6. The method according to claim 5, wherein the cancer is HPV type 16 positive.

7. The method according to claim 4, wherein the cancer is head or neck squamous cell carcinoma.

8. The method according to claim 4, wherein the patient is receiving or has received platinum-containing chemotherapy.

9. The method according to claim 4, wherein the compound and the proteins comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 are administered simultaneously.

10. The method according to claim 4, wherein the compound and the proteins comprising the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 are administered sequentially.

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