Patent Grant
12247052
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 26, 2020 is named 51026-027002 Sequence Listing 8.26.20 ST25 and is 4,993 bytes in size.
Human papillomavirus (HPV)-related cancer is one of the fastest growing cancers in the world. Overall, 5% of all cancers world-wide can be attributed to HPV infections. There continues to be a need for further and more effective cancer treatments.
The invention provides compounds that can be used in therapeutic methods.
Accordingly, in the first aspect, the invention features a compound consisting of the nucleotide sequence 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:1), at its 5′ end, bonded or linked by a linker to the following lipid:
or a salt thereof,
where X is O or S.
In one embodiment of the first aspect of the invention, the nucleotide sequence is bonded to the lipid.
In another embodiment of the first aspect of the invention, all internucleoside groups connecting the nucleosides in 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ (SEQ ID NO:1) are phosphorothioates.
In the second aspect, the invention features a method of treating a cancer in a human patient. This method includes administering to the patient the compound of the first aspect of the invention, a protein including the amino acid sequence:
In one embodiment of the second aspect of the invention, the cancer is human papillomavirus (HPV) positive (e.g., HPV type 16 positive).
In another embodiment of the second aspect of the invention, the cancer is a head or neck squamous cell carcinoma.
In an additional embodiment of the second aspect of the invention, the patient is receiving or has received platinum-containing chemotherapy. In a further embodiment, an anti-PD-1 antibody (e.g., pembrolizumab or nivolumab) is administered to the patient.
In another embodiment of the second aspect of the invention, the compound of the first aspect of the invention and the proteins including the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 are administered concurrently.
In a further embodiment of the second aspect of the invention, the compound of the first aspect of the invention and the proteins including the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3 are administered sequentially.
In a further aspect, the invention features another method of treating a cancer in a human patient. This method includes administering to the patient the compound of the first aspect of the invention, a protein including the amino acid sequence:
In the third aspect, the invention features a pharmaceutical composition including a compound of the first aspect of the invention and a pharmaceutically acceptable carrier.
In one embodiment of the third aspect, the pharmaceutical composition further includes a protein including the amino acid sequence: MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIV YRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQ RFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2), and a protein including the amino acid sequence:
In the fourth aspect, the invention features a kit including (i) a compound of the first aspect of the invention or a composition of the second aspect of the invention and (ii) a protein including the amino acid sequence: MHQKRTAMFQ DPQERPRKLP QLCTELQTTI HDIILECVYC KQQLLRREVY DFAFRDLCIV YRDGNPYAVG DKCLKFYSKI SEYRHYCYSL YGTTLEQQYN KPLCDLLIRC INGQKPLCPE EKQRHLDKKQ RFHNGRGRWT GRCMSCCRSS RTRRETQL (SEQ ID NO:2), and a protein including the amino acid sequence: MHGDTPTLHE YMLDLQPETT DLYGYGQLND SSEEEDEIDG PAGQAEPDRA HYNIVTFCCK CDSTLRLCVQ STHVDIRTLE DLLMGTLGIV CPICSQKP (SEQ ID NO:3).
A “linker,” as used herein, refers to a monovalent or divalent group, in which one valency is covalently bonded to one biologically functional group, and the other valency is covalently bonded to another biologically functional group. In one example, a linker connects a nucleotide sequence of, e.g., 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 linkers can optionally include one or more nucleotides, for example, a dinucleotide (e.g., GG).
A “pharmaceutically acceptable carrier,” as used herein, refers to a vehicle capable of suspending or dissolving the active compound, and having the properties of being nontoxic and non-inflammatory in a patient. Moreover, a pharmaceutically acceptable carrier may include a pharmaceutically acceptable additive, such as a preservative, antioxidant, fragrance, emulsifier, dye, or excipient known or used in the field of drug formulation and that does not significantly interfere with the therapeutic effectiveness of the biological activity of the active agent, and that is non-toxic to the patient.
The terms “treat,” “treatment,” and “treating” refer to therapeutic approaches in which the goal is to reverse, alleviate, ameliorate, inhibit, slow down, or stop the progression or severity of a condition associated with a disease or disorder, e.g., cancer. These terms include reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced, or if a desired response (e.g., a specific immune response) is induced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
The invention provides several advantages. For example, in including lipid moieties and, optionally, a linker, certain compounds of the invention bind to endogenous albumin in subjects to whom they are administered, which enhances delivery of the compounds to the lymph nodes of the subjects. This facilitates the induction of a therapeutic immune response against, for example, HPV proteins administered to the subject, leading to effective cancer treatment.
Other features and advantages of the invention will be apparent from the following detailed description, the drawings, and the claims.
The invention provides compounds that can be used 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, at its 5′ end, to a lipid, such as the following:
where X is O or S. Preferably, X is S. The CpG oligonucleotide may be directly bonded to the lipid. Alternatively, the CpG may 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 may be phosphorothioates).
The CpG ODN can function as an adjuvant to elicit an immune response in a subject, such as an immune response against a cancer antigen (e.g., a HPV antigen). As such, the compounds and compositions of the invention can be used in therapeutic methods. In particular, if the CpG ODN containing compound is administered in combination with one or more HPV proteins the compound can induce an immune response to HPV positive cancer cells. Accordingly, the invention provides methods of treating cancer in a subject (e.g., a human patient) by administering one or more compounds or compositions of the invention to the subject. In various examples, the cancer is a HPV positive (e.g., a HPV type 16 positive) cancer.
The HPV positive cancer may be a head or neck squamous cell carcinoma, a cervical cancer, anal cancer, vulvar cancer, head and neck cancer, oropharyngeal cancer, penile cancer, vaginal cancer, virally induced cancer, bladder cancer, pancreatic cancer, lung cancer, liver cancer, ovarian cancer, colon cancer, stomach cancer, neuroblastoma, breast cancer, prostate cancer, renal cancer, leukemia, sarcoma, carcinoma, basal cell carcinoma, non-small cell lung carcinoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), B-cells chronic lymphocytic leukemia (B-CLL), multiple myeloma (MM), erythroleukemia, renal cell carcinoma, sarcoma, melanoma, astrocytoma, oligoastrocytoma, biliary tract cancer, choriocarcinoma, CNS cancer, larynx cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), adenocarcinoma, giant (or oat) cell carcinoma, squamous cell carcinoma, oral cavity cancer, skin cancer, basal cell cancer, squamous cell cancer, testicular cancer, thyroid cancer, uterine cancer, rectal cancer, a cancer of the respiratory system, or a cancer of the urinary system.
Optionally, the methods of the invention can further include administering a compound or composition of the invention in combination with a second (or further) different approach to treatment.
The invention also provides kits that each contain, for example, a first vessel that includes one or more compounds of the invention, optionally together with a second vessel that includes a cancer antigen, such as an HPV protein described herein.
CpG
CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides in particular sequence contexts. CpG ODNs possess a partially or completely phosphorothioated (PS) backbone, as opposed to the natural phosphodiester (PO) backbone in DNA molecules. Three major classes of stimulatory CpG ODNs have been identified based on structural characteristics and activity on human peripheral blood mononuclear cells (PBMCs), in particular B cells and plasmacytoid dendritic cells (pDCs). These three classes are Class A (Type D), Class B (Type K), and Class C.
CpG1826 and CpG7909 both are in CpG class B. Class B CpG ODNs contain a full 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.
Mutated HPV
Point mutations at 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 performance of this component as an antigen is dictated by the sequence of the protein, with the structure of the protein being inconsequential to that intended function.
Point mutations at C24G and E26G (underlined below) can be introduced into the wild-type HPV16 E7 viral protein to prevent stereochemical interaction with human Rb1. Again, the performance of this component as an antigen is dictated by the sequence of the protein, with the structure of the protein being inconsequential to that intended function.
CpG ODNs may be bonded directly or linked by a linker to the lipid. These compounds may be produced using the ordinary phosphoramidite chemistry known in the art. In some examples, the CpG ODN or CpG ODN-GG may be reacted with the following compound:
to produce an intermediate, which upon oxidation with (e.g., phosphite oxidation methods known in the art, e.g., a sulfurizing agent, such as 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione) and hydrolysis of the cyanoethyl group may produce a compound of the invention.
In order that this invention be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
Mice were immunized prophylactically and the immune response generated was recorded via E7-Tetramer stain and IFNγ-intracellular cytokine staining (ICS) upon HPV16 E6 and HPV16 E7 (E6/E7) stimulation.
The experimental design included the following 6 groups of mice (n=10 for each group)
Protein stock solutions were dissolved in 8M urea. Adjuvant stock solutions are dissolved in H2O. Final injections are diluted with 1× phosphate buffered saline (PBS) (CF of urea <1 M).
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′-ggtccatgacgttcctgacgtt-3′; SEQ ID NO:5). A concentration of 5 nmol for each 100 μl injection was used for both soluble CpG1826 and aCpG1826. CpG1826 is an optimal mouse sequence while CpG7909 is optimal for humans and poorly active in mice. CpG1826 and CpG7909 are in the same CpG class (class B) and generally have similar activity profiles in their respective species.
For both aCpG7909 and soluble CpG7909, the sequence used was 5′-tcgtcgttttgtcgttttgtcgtt-3′ (SEQ ID NO:6) at a concentration of 5 nmol for each 100 μl injection.
Mutated HPV16 E6 with point mutations at C70G, C113G, and I135G (underlined below) was used for immunization. The amino acid sequence used is provided below.
Mutated HPV16 E7 with point mutations at C24G and E26G (underlined below) was used for immunization. The amino acid sequence used is provided below.
For E6/E7, 10 μg each of mutated HPV16 E6 and mutated HPV16 E7 was used per 100 μl injection.
Female C57BL/6J mice (B6) were immunized subcutaneously (s.c.) with the primer dose (E6/7+aCpG) and one booster dose after 2 weeks.
Tetramer analysis for H-2Db HPV16 E7 (RAHYNIVTF; SEQ ID NO:7) was performed 7 days after the booster dose (
Intracellular cytokine staining (ICS) for IFNγ was performed on peripheral blood 7 days after the booster dose to analyze immune responses to E6/E7.
The E6 stimulation used the following peptides: (E6-10: EVYDFAFRDL (SEQ ID NO:8); E6 49-57: VYDFAFRDL (SEQ ID 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 the data shown in
Deconvolution of the E6 stimuli revealed that E49-57 was the only peptide that resulted in stimulation.
For E7 stimulation the following peptide was used: RAHYNIVTF (SEQ ID NO:13). This peptide was used to generate the data shown in
As shown in
As also shown in
E6/E7+aCpG decreased tumor growth compared to either aCpG alone or no treatment with a corresponding increase in percent survival (
To determine an optimal dosing schedule for E7+aCpG with respect to anti-tumor efficacy in female C57BL/6U (B6) mice implanted with TC-1 tumors, weekly dosing was compared to dosing every 2 weeks and to baseline (prime only). E7+aCpG was compared to E7+soluble CpG. All vaccines were administered 3 times (prime and 2 boosts).
Female C57BL/6J mice (B6) were inoculated with 50,000 TC-1 cells subcutaneously in the flank on Day 0 and 12 days later; mice were separated into treatment groups and treated as indicated in Table 1.
aProtein stock solutions were dissolved in 8M urea. Adjuvant stock solutions were dissolved in H2O. Final injections were diluted with 1X PBS (CF of urea <1M).
b8 μg equivalent
The HPV-tetramer specific T cell response to the protein/amphiphilic CpG vaccine was superior to that of the protein/soluble CpG vaccine after both a single dose and repeated doses (
To evaluate the antitumor efficacy of E7 protein in combination with either soluble or amphiphilic CpG and with or without the addition of an anti-PD-1 antibody, female C57BL/6J mice (B6) were inoculated in the flank at baseline with 50,000 TC-1 cells. Eleven days post-inoculation the mice were divided into 5 groups as shown in Table 2. The comparison group was untreated.
aProtein stock solutions were dissolved in 8M urea. Adjuvant stock solutions were dissolved in H2O.
Throughout the study tumor sizes were measured every other day up to day 40 post inoculation and animal survival was monitored. Tetramer analysis for H-2Db HPV16 E7 (RAHYNIVTF) was performed 7 days after each vaccine administration.
Administration of E7/Amph-CpG vaccine, without or without anti-PD-1 antibody, caused a robust increase in HPV Tetramer+CD8-cells specific to the HPV16 E7 (RAHYNIVTF; SEQ ID NO:13) peptide (
Corresponding to these strong HPV Tetramer+CD8 responses, tumor growth was halted around Day 24 and reversed after the first dose of E7/Amph-CpG (with or without anti-PD-1) and tumor size remained small and stable out to the end of the study, in contrast to the other groups where growth progressed (
Also, corresponding to the effects on tumor size, treatment with E7/aCpG vaccine (with or without the anti-PD-1 antibody) had a significant effect on survival and resulted in 6/7 (85%) cures for E7/aCpG without antibody and 8/10 (80%) cures for E7/aCpG plus anti-PD-1 antibody (
To determine a dose of aCpG that produces the highest antigen-specific Tetramer+CD8 response over the course of 6 doses, a dose escalation study was conducted using a fixed dose of 10 μg ovalbumin (OVA) as the antigen. Soluble CpG was used as a comparator. Tolerability (based on body weight and general observations) was also assessed. The study design is outlined in Table 3.
Up to 6 doses of vaccine were administered at 2-week intervals for a total study length of 11 weeks.
Peripheral blood samples were collected 7 days after each injection and flow cytometric analyses of tetramer on CD8+ cells were performed using H-2KbOVA (SIINFEKL; SEQ ID NO:14).
Significant increases in tetramer+CD8+cells were observed only in the groups treated with aCpG+OVA, with 6 nmol producing the greatest pharmacological effect (
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.
All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Some embodiments of the invention are within the following numbered paragraphs.
Other embodiments are within the following claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/020398 | 3/1/2019 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2019/169328 | 9/6/2019 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 6168778 | Janjic | Jan 2001 | B1 |
| 10940201 | Kugimiya | Mar 2021 | B2 |
| 20070298449 | Saito | Dec 2007 | A1 |
| 20080299138 | Duffy | Dec 2008 | A1 |
| 20120121606 | Ruben et al. | May 2012 | A1 |
| 20120129199 | Daftarian et al. | May 2012 | A1 |
| 20120264810 | Lin | Oct 2012 | A1 |
| 20120328701 | Edelson | Dec 2012 | A1 |
| 20140162944 | Tiberg et al. | Jun 2014 | A1 |
| 20140294932 | Kim et al. | Oct 2014 | A1 |
| 20150044208 | Castanheira Aires da Silva et al. | Feb 2015 | A1 |
| 20150232514 | Dockal et al. | Aug 2015 | A1 |
| 20150283205 | Phipps et al. | Oct 2015 | A1 |
| 20160000906 | Diamond | Jan 2016 | A1 |
| 20160220669 | Hoves et al. | Aug 2016 | A1 |
| 20160264810 | Okamoto | Sep 2016 | A1 |
| 20170246288 | Li | Aug 2017 | A1 |
| 20200061172 | Kulangara | Feb 2020 | A1 |
| 20210060149 | Demuth | Mar 2021 | A1 |
| Number | Date | Country |
|---|---|---|
| 2477315 | Mar 2013 | RU |
| WO-2006134423 | Dec 2006 | WO |
| WO-2008068638 | Jun 2008 | WO |
| WO-2010008782 | Jan 2010 | WO |
| WO-2010071852 | Jun 2010 | WO |
| WO-2016040887 | Mar 2016 | WO |
| WO-2018102584 | Jun 2018 | WO |
| Entry |
|---|
| Liu et al. Structure-based programming of lymph-node targeting in molecular vaccines. Nature. Mar. 27, 2014, vol. 507, pp. 519-522. (Year: 2014). |
| Appelbe et al., “Radiation-enhanced delivery of systemically administered amphiphilic-CpG oligodeoxynucleotide,” J Control Release. 266:248-255 (2017). |
| Extended European Search Report for European Patent Application No. 19761384.7, dated Nov. 16, 2021 (9 pages). |
| International Preliminary Report on Patentability for International Patent Application No. PCT/US2019/020398, issued Sep. 8, 2020 (6 pages). |
| International Preliminary Report on Patentability for International Patent Application No. PCT/US2019/020404 issued Sep. 8, 2020 (4 pages). |
| International Search Report for International Patent Application No. PCT/US2019/020398, mailed Jun. 13, 2019 (4 pages). |
| International Search Report for International Patent Application No. PCT/US2019/020404, mailed Jun. 24, 2019 (4 pages). |
| Liu et al., “Membrane anchored immunostimulatory oligonucleotides for in vivo cell modification and localized immunotherapy,” Angew Chem Int Ed Engl. 50(31):7052-5 (2011) (12 pages). |
| Written Opinion of the International Searching Authority for International Patent Application No. PCT/US2019/020398, mailed Jun. 13, 2019 (5 pages). |
| Written Opinion of the International Searching Authority for International Patent Application No. PCT/US2019/020404, mailed Jun. 24, 2019 (3 pages). |
| Yu et al., “Immunostimulatory Properties of Lipid Modified CpG Oligonucleotides,” Mol Pharm. 14(8):2815-2823 (2017). |
| Examination Report for Saudi Arabian Patent Application No. 520420065 dated Feb. 27, 2022 (9 pages). |
| Written Opinion for Singaporean Patent Application No. 11202008432X dated Feb. 21, 2022 (5 pages). |
| Examination Report for Saudi Arabian Patent Application No. 520420065 dated Jan. 31, 2023 (6 pages). |
| Notice of Deficiencies for Israeli Patent Application No. 277101, dated Mar. 1, 2023 (3 pages). |
| Notice of Reasons for Refusal for Japanese Patent Application No. 2020-568945 mailed Feb. 14, 2023 (6 pages). |
| Office Action for Chinese Patent Application No. 201980028828.9, issued Apr. 21, 2023 (12 pages). |
| Wang et al., “In vitro and in vivo evaluations of human papillomavirus type 16 (HPV16)-derived peptide-loaded dendritic cells (DCs) with a CpG oligodeoxynucleotide (CpG-ODN) adjuvant as tumor vaccines for immunotherapy of cervical cancer,” Arch Gynecol Obstet. 289(1):155-62 (Epub Aug. 2013). |
| You et al., Chapter 24: Drug chemical structure modification and new drug research and development. Medicinal Chemistry, Second Edition. The Medical Science and Technology Press of China, 570-572 (Feb. 2011) (7 pages). |
| Zhang et al., Chapter 5: Design of Drugs Acting on Nucleic Acids. Modern Drug Design, First Edition. The Medical Science and Technology Press of China, 272 (Dec. 2005) (5 pages). |
| Office Action and Search Report for Russian Patent Application No. 2020132295, issued Sep. 7, 2022 (16 pages). |
| Marciani, D. J., “Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity,” Drug Discov Today. 8(20):934-43. (Oct. 2003). |
| De Vries et al. “Drug delivery systems based on nucleic acid nanostructures,” Journal of Controlled Release. 172: 467-483. Dec. 2013. (19 pages). |
| Number | Date | Country | |
|---|---|---|---|
| 20210040159 A1 | Feb 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62637824 | Mar 2018 | US |
