Human papillomavirus vaccine and its use

Abstract

Multiantigenic human papillomavirus (HPV) molecular vaccine constructs for use and treatment of HPV-related disorders and conditions, e.g., HPV molecular vaccines targeting HPV16, HPV18, and HPV-45-related conditions.

Description

[Technical Field]

[0001] Background technology [Background technology]

[0002] Human papillomavirus (HPV) infects millions of individuals and causes more than 600,000 cases of HPV-related malignancies worldwide each year. Int J Cancer. 2017;141(4):664-670. Approximately 47,000 new cases of HPV-related malignancies are diagnosed in the United States each year. While many papillomavirus infections are benign and resolve spontaneously, some persistent infections can develop into epithelial cell dysplasia, leading to cancers of the cervix, vulva, penis, oropharynx, head and neck, and anal cavity. Int J Oncol. 2018;52(3):637-655. Generally, HPV is thought to be responsible for over 90% of anal and cervical cancers, about 70% of vaginal and vulvar cancers, and over 60% of penile cancers. While oropharyngeal cancer has traditionally been attributed to tobacco and alcohol, recent studies suggest that approximately 70% of oropharyngeal cancers may be linked to HPV. https: / / www.cdc.gov / cancer / hpv / statistics / index.htm Over 200 types of HPV are classified into high-risk and low-risk groups according to their carcinogenic potential. Among the high-risk HPV types, HPV types 16 and 18 are the most common and most carcinogenic. J Gynecol Oncol. 2016;27(2):e21-e21;N Engl J Med. 2003;348(6):518-527. Together, HPV 16 and 18 account for approximately 70% of cervical cancer cases. J Biomed Sci. 2016;23(1):75. Early (E) HPV genes (E1-E8) regulate viral expression and replication, while late (L) genes control the viral protein coding. Virology. 1977;76(2):569-580;J Virol. 1977;24(1):108-120;Virology. 1979;96(2):547-552.

[0003] Several prophylactic HPV vaccines, developed to target the major capsid protein L1 of the HPV virus particle, have shown great success in reducing HPV-related malignancies in developed countries. (J Biomed Sci. 2016;23(1):75.) Prophylactic vaccines prevent healthy individuals from acquiring HPV infection and prevent reinfection in previously infected individuals. Unfortunately, prophylactic vaccines are not effective against established HPV infections and the resulting HPV-related malignancies or precancerous lesions. (Discov Med. 2010;10(50):7-17;Expert Opin Emerg Drugs. 2012;17(4):469-492.) Unlike prophylactic HPV vaccines, which are used to generate neutralizing antibodies against the viral capsid protein L1 to prevent infection, therapeutic HPV vaccines are used to stimulate a cell-mediated immune response that specifically targets and kills infected cells. Clin Diagn Lab Immunol. 2001;8(2):209-220.

[0004] The E6 and E7 proteins of HPV16 and 18 are responsible for the maintenance of malignant tumor phenotypes and are constitutively expressed in tumors, thus representing potential targets for therapeutic vaccines. Expert Opin Emerg Drugs. 2012;17(4):469-492. Furthermore, they are not endogenously expressed in any human tissue, resulting in a very low risk of inducing autoimmune events with vaccines targeting these proteins. Several CD8+ T cell epitopes of E6 and E7 that can induce cytotoxic T lymphocyte (CTL) responses have been previously identified, and clinical studies using diverse vaccine platforms have demonstrated varying degrees of efficacy in terms of inducing HPV-specific responses and clinical benefits. These include live vectors, peptide or protein vaccines, cell-based vaccines, and nucleic acid vaccines. J Immunol. 1994;152(8):3904-3912;Virus Res. 1998;54(1):23-29;Clin Cancer Res. 2001;7(3 Suppl):788s-795s;Expert Opin Emerg Drugs. 2012;17(4):469-492;Clin Exp Immunol. 2015;181(1):164-178;Cancers (Basel). 2011;3(3):3461-3495;Int J Cancer. 2000;86(5):725-730;J Formos Med Assoc. 2010;109(1):4-24;Oncologist. 2005;10(7):528-538;BMC Cancer. 2014;14:748;Expert Rev Vaccines. 2007;6(2):227-239;Expert Rev Vaccines. 2016;15(8):989-1007.

[0005] Naturally occurring sequence mutations in numerous HPV strains present a significant hurdle to the development of effective, broad-spectrum HPV vaccines. For example, recurrent / metastatic cervical cancer associated with HPV16 / 18 has a poor prognosis, with approximately 70% response to chemotherapy ± bevacizumab + pembrolizumab and an overall survival of approximately 10.4 months. Keynote-826; N Engl J Med 2021; 385:1856-1867.

[0006] As a solution to this problem, this vaccine design approach utilizes advanced bioinformatics and protein manipulation approaches to select and design HPV16 / 18 antigen sequences with broad T-cell epitope coverage, novel mutations, and enhancer agonist peptides. Based on the expanded coverage of antigen regions with CTL-specific epitopes and the available information from in silico prediction results, the HPV vaccine antigens designed in this invention aim to induce robust HPV-16 and HPV-18 specific responses and provide therapeutic benefits to individuals at risk of HPV-derived cancers. [Overview of the Initiative]

[0007] Provided herein are polynucleotides, which are not naturally occurring, that encode polypeptide constructs containing immune response-inducing human papillomavirus (HPV) peptides.

[0008] In some embodiments, a polynucleotide that does not exist in nature encodes a polypeptide construct comprising two or more HPV peptides. In some embodiments, the two or more HPV peptides comprise one or more HPV-16 immune response-inducing peptide sequences (HPV-16 peptides).

[0009] In some embodiments, the polypeptide construct comprises HPV-16 E5 peptide, HPV-16 E6 peptide, and / or HPV-16 E7 peptide. In some embodiments, the polypeptide construct comprises HPV-16 E5 peptide having the sequence of SEQ ID NO: 47. In some embodiments, the polypeptide construct comprises HPV-16 E6 peptide having the sequence of SEQ ID NO: 45. In some embodiments, the polypeptide construct comprises HPV-16 E7 peptide having the sequence of SEQ ID NO: 46.

[0010] In some embodiments, the polypeptide construct includes the HPV-18 peptide. In some embodiments, the polypeptide construct includes the HPV-18 E5 peptide, the HPV-18 E6 peptide, and / or the HPV-18 E7 peptide. In some embodiments, the polypeptide construct includes the HPV-18 E5 peptide having the sequence of SEQ ID NO: 50. In some embodiments, the polypeptide construct includes the HPV-16 E6 peptide having the sequence of SEQ ID NO: 48. In some embodiments, the polypeptide construct includes the HPV-18 E7 peptide having the sequence of SEQ ID NO: 49.

[0011] In some embodiments, the polypeptide has the sequence of sequence number 51.

[0012] In some embodiments, at least one of one or more HPV peptides is linked to an agonist peptide (also referred to as an enhancer agonist peptide). In some embodiments, the agonist peptide has the sequence shown in Table 5. In some embodiments, the polypeptide has the sequence of SEQ ID NO: 53.

[0013] Vectors comprising any of the polynucleotides provided herein are further provided herein. In some embodiments, the vector is an adenovirus vector. In some embodiments, the adenovirus vector is a gorilla adenovirus vector.

[0014] Provided herein are E6 peptides comprising, compared to wild-type E6 peptide, the E18A amino acid substitution and at least one of the L50G, E148A, T149A, Q150A, or L151A amino acid substitutions. In some embodiments, the E6 peptide comprises the E18A, L50G, E148A, T149A, Q150A, and L151A amino acid substitutions. In some embodiments, the E6 peptide has the sequence of SEQ ID NO: 45. In some embodiments, the E6 peptide is fused to an agonist peptide. In some embodiments, the agonist peptide is fused to at least one of the C-terminus and N-terminus of the E6 peptide. In some embodiments, the wild-type E6 peptide is derived from HPV-16.

[0015] Provided herein are E6 peptides comprising a deletion compared to the wild-type E6 peptide, wherein the deletion comprises the C-terminus of the wild-type E6 peptide. In some embodiments, the deletion is an amino acid deletion from the 121st residue to the C-terminus of the wild-type E6 peptide. In some embodiments, the E6 peptide comprises at least one of the E18A and L50G substitutions compared to the wild-type E6 peptide. In some embodiments, the wild-type E6 peptide is derived from HPV-18. In some embodiments, the E6 peptide has the sequence of SEQ ID NO: 48.

[0016] Provided herein are E7 peptides comprising a deletion at the N-terminus of the wild-type E7 peptide. In some embodiments, the deletion is an amino acid deletion at positions 1-39 of the wild-type E7 peptide. In some embodiments, the E7 peptide comprises at least one of the E55A and L74R substitutions compared to the wild-type E7 peptide. In some embodiments, the wild-type E7 peptide is derived from HPV-18. In some embodiments, the E7 peptide has the sequence of SEQ ID NO: 49.

[0017] Provided herein are E5 peptides comprising the deletion of amino acids at positions 41-57 of the wild-type E5 peptide. In some embodiments, the E5 peptide has the sequence of SEQ ID NO: 47. In some embodiments, the wild-type E5 peptide is derived from HPV-16.

[0018] Provided herein are E5 peptides comprising deletions of amino acids at positions 27–40 and / or 54–57 of the wild-type E5 peptide. In some embodiments, the E5 peptide has the sequence of SEQ ID NO: 50. In some embodiments, the wild-type E5 peptide is derived from HPV-18.

[0019] Polypeptide constructs comprising any one of the E5, E6, and E7 peptides described herein are provided herein.

[0020] Polypeptide constructs comprising the HPV-16 E6 peptide are provided herein, comprising the E18A amino acid substitution and at least one of the L50G, E148A, T149A, Q150A, or L151A amino acid substitutions compared to the wild-type HPV-16 E6 peptide. In some embodiments, the HPV-16 E6 peptide comprises the E18A, L50G, E148A, T149A, Q150A, and L151A amino acid substitutions. In some embodiments, the HPV-16 E6 peptide has the sequence of SEQ ID NO: 45. In some embodiments, the polypeptide construct further comprises the HPV-16 E7 peptide, comprising at least one of the H2P, C24G, E46A, or L67R amino acid substitutions compared to the wild-type HPV-16 E7 peptide. In some embodiments, the HPV-16 E7 peptide comprises the H2P, C24G, E46A, and L67R amino acid substitutions. In some embodiments, the HPV-16 E7 peptide has the sequence of SEQ ID NO: 46. In some embodiments, the polypeptide construct further comprises the HPV-16 E5 peptide. In some embodiments, the HPV-16 E5 peptide contains one or more amino acid deletions compared to the wild-type HPV-16 E5 peptide. In some embodiments, the deletions consist of amino acids at positions 41-57 of the wild-type HPV-16 E5 peptide. In some embodiments, the HPV-16 E5 peptide has the sequence of SEQ ID NO: 47.

[0021] In some embodiments, the polypeptide construct comprises (a) an HPV-16 E6 peptide comprising an E18A amino acid substitution and at least one of the L50G, E148A, T149A, Q150A, or L151A amino acid substitutions compared to the wild-type HPV-16 E6 peptide; and (b) an HPV-18 E6 peptide. In some embodiments, the HPV-18 E6 peptide comprises the E18A and L50G substitutions compared to the wild-type HPV-18 E6 peptide. In some embodiments, the HPV-18 E6 peptide comprises at least one C-terminal amino acid deletion compared to the wild-type HPV-18 E6 peptide. In some embodiments, the deletion comprises amino acids from the C-terminus of the residue at position 121 of the wild-type HPV-18 E6 peptide. In some embodiments, the HPV-18 E6 peptide has the sequence of SEQ ID NO: 48. In some embodiments, the polypeptide construct further comprises an HPV-18 E7 peptide. In some embodiments, the HPV-18 E7 peptide includes E55A and L74R substitutions compared to the wild-type HPV-18 E7 peptide. In some embodiments, the HPV-18 E7 peptide includes a deletion of at least one amino acid from the N-terminus of the HPV-18 E7 peptide. In some embodiments, the deletion includes an amino acid from the residues at positions 1-40 of the wild-type HPV-18 E7 peptide. In some embodiments, the HPV-18 E7 peptide has the sequence of SEQ ID NO: 49. In some embodiments, the polypeptide construct further includes the HPV-18 E5 peptide. In some embodiments, the HPV-18 E5 peptide includes a deletion of at least one amino acid compared to the wild-type HPV-18 E5 peptide. In some embodiments, the deletion includes an amino acid from the residues at positions 27-40 or from positions 54-57 of the wild-type HPV-18 E5 peptide. In some embodiments, the HPV-18 E5 peptide has the sequence of SEQ ID NO: 50. In some embodiments, the polypeptide construct has the sequence of SEQ ID NO: 51. In some embodiments, the polypeptide construct further comprises at least one agonist peptide. In some embodiments, the at least one agonist peptide has the agonist peptide sequence shown in Table 5.In some embodiments, the polypeptide construct has the sequence of SEQ ID NO: 53.

[0022] Polypeptide constructs comprising an ankyrin-like repeat domain and HPV peptides are provided herein. In some embodiments, the ankyrin-like repeat protein is a human ankyrin-like repeat protein. In some embodiments, the HPV peptide is linked to the ankyrin-like repeat protein by a linker. In some embodiments, the polypeptide construct comprises an HPV-16 peptide and / or an HPV-18 peptide. In some embodiments, the polypeptide construct comprises an HPV-16 E5 peptide, an HPV-16 E6 peptide, and / or an HPV-16 E7 peptide. In some embodiments, the polypeptide construct comprises an HPV-18 E6 peptide and / or an HPV-18 E7 peptide. In some embodiments, the polypeptide construct comprises the HPV-16 E5 sequence, an HPV-16 E6 sequence, an HPV-16 E7 sequence, an HPV-18 E6 sequence, and / or an HPV-18 E7 sequence, as shown in Table 5. In some embodiments, the polypeptide construct has the sequence of SEQ ID NO: 52. In some embodiments, the polypeptide construct further comprises at least one agonist peptide. In some embodiments, the polypeptide construct comprises three agonist peptides. In some embodiments, the polypeptide construct has the sequence of SEQ ID NO: 54.

[0023] Polypeptide constructs comprising at least two HPV peptides shown in Table 5, linked by a KK linker, are provided herein. In some embodiments, the polypeptide construct comprises at least one of the HPV-16 peptide or HPV-18 peptide shown in Table 5. In some embodiments, the polypeptide construct comprises the HPV-16 E5 peptide, the HPV-16 E6 peptide, and / or the HPV-16 E7 peptide, as shown in Table 5. In some embodiments, the polypeptide construct comprises the HPV-18 E6 peptide and / or the HPV-18 E7 peptide, as shown in Table 5. In some embodiments, the polypeptide construct comprises each of the peptides shown in Table 5. In some embodiments, each of such peptides is linked to another such peptide by a KK linker. In some embodiments, the polypeptide construct has the sequence of SEQ ID NO: 55.

[0024] Polynucleotides encoding any of the polypeptide constructs described herein are provided herein. Vectors comprising polynucleotides are also provided herein. In some embodiments, the vector is an adenovirus vector. In some embodiments, the adenovirus vector is a gorilla adenovirus vector.

[0025] A vector is provided herein that is an adenovirus vector comprising a polynucleotide encoding at least one HPV peptide.

[0026] A vector is provided herein that is a gorilla adenovirus vector containing a polynucleotide encoding at least one HPV peptide.

[0027] In some embodiments, any of the polynucleotides and polypeptide constructs described herein are for use in vaccines. Vectors containing polynucleotides are also provided herein. In some embodiments, the vector is an adenovirus vector. In some embodiments, the adenovirus vector is a gorilla adenovirus vector.

[0028] In some embodiments, the compositions and methods of the present invention can be combined with at least one additional therapy. In some embodiments, the combination therapy involves administering a composition or vector expressing a novel HPV antigen design disclosed herein before, during, or after the administration of at least one other distinct therapeutic agent to enhance the effectiveness of treating various medical conditions, such as cancer. Such additional therapies include radiotherapy, surgery (e.g., weight loss), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the aforementioned therapies. The additional therapy may take the form of an adjuvant or neoadjuvant therapy.

[0029] In some embodiments, additional therapies include the administration of anti-inflammatory agents, analgesics, bioresponse modifiers, cytokines, interferons, interleukins, CAR-T cells, bifunctional proteins, colony-stimulating factors, tumor necrosis factors, surfactants, small molecule enzyme inhibitors, or anti-metastatic agents.

[0030] In some embodiments, the HPV vaccine antigens provided herein are delivered in combination with at least one interleukin. In one embodiment, the interleukin is IL-12. In some embodiments, IL-12 is delivered intratumorally.

[0031] In some embodiments, the HPV vaccine antigens provided herein are delivered in combination with a bifunctional fusion protein. In one embodiment, the bifunctional fusion protein is vintrafusp alfa (i.e., a bifunctional fusion protein consisting of the extracellular domain of transforming growth factor (TGF)-βRII (TGF-β "trap") fused to a human IgG1 mAb that blocks programmed cell death ligand 1).

[0032] In some embodiments, the HPV vaccine antigens provided herein are delivered in combination with at least one histone deacetylase inhibitor.

[0033] In some embodiments, the HPV vaccine antigens provided herein are delivered in combination with pembrolizumab.

[0034] In some embodiments, the HPV vaccine antigen provided herein is delivered in combination with docetaxel.

[0035] In some embodiments, the HPV vaccine antigens provided herein are delivered in combination with cisplatin.

[0036] In some embodiments, the HPV vaccine antigens provided herein are delivered in combination with docetaxel and cisplatin.

[0037] (a) HPV-16 E5 peptide or HPV-18 E5 peptide; and (b) polynucleotides encoding a fusion protein comprising HPV-16 E6 peptide, HPV-16 E7 peptide, HPV-18 E6 peptide, or HPV-18 E7 peptide are provided herein.

[0038] In some embodiments, any of the fusion proteins described herein comprises an HPV-16 E5 peptide containing the amino acid sequence of SEQ ID NO: 130. In some embodiments, any of the fusion proteins described herein comprises an HPV-16 E6 peptide containing any one of the amino acid sequences from SEQ ID NOs: 113 to 121. In some embodiments, any of the fusion proteins described herein comprises an HPV-18 E6 peptide containing any one of the amino acid sequences from SEQ ID NOs: 131 to 138. In some embodiments, any of the fusion proteins described herein comprises an HPV-16 E7 peptide containing any one of the amino acid sequences from SEQ ID NOs: 122 to 129. In some embodiments, any of the fusion proteins described herein comprises an HPV-18 E7 peptide containing any one of the amino acid sequences from SEQ ID NOs: 139 to 144.

[0039] In some embodiments, any of the fusion proteins described herein further comprises an agonist peptide. In some embodiments, the agonist peptide comprises one of the amino acid sequences of SEQ ID NOs. 145-147.

[0040] In some embodiments, any of the fusion proteins described herein further comprises an ankyrin-like repeat domain. In some embodiments, the ankyrin-like repeat domain is located between two HPV peptides.

[0041] In some embodiments, any of the fusion proteins described herein include three or more HPV peptides and an ankyrin-like repeat domain between each of the HPV peptides. In some embodiments, any of the fusion proteins described herein include an amino acid sequence having at least 90% identity with SEQ ID NO: 243. In some embodiments, any of the fusion proteins described herein include an amino acid sequence having at least 95% identity with SEQ ID NO: 243. In some embodiments, any of the fusion proteins described herein include an amino acid sequence having at least 97% identity with SEQ ID NO: 243. In some embodiments, any of the fusion proteins described herein include an amino acid sequence having at least 98% identity with SEQ ID NO: 243. In some embodiments, any of the fusion proteins described herein include an amino acid sequence having at least 99% identity with SEQ ID NO: 243. In some embodiments, any of the fusion proteins described herein include the amino acid sequence of SEQ ID NO: 243 or a conservatively substituted variant thereof. In some embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 243.

[0042] In some embodiments, any of the fusion proteins described herein includes an amino acid sequence having at least 90% identity with SEQ ID NO: 51. In some embodiments, any of the fusion proteins described herein includes an amino acid sequence having at least 90% identity with SEQ ID NO: 52. In some embodiments, any of the fusion proteins described herein includes an amino acid sequence having at least 90% identity with SEQ ID NO: 53.

[0043] In some embodiments, any of the fusion proteins described herein may be operably ligated to at least one of the following: a promoter; a 5' untranslated region (UTR); a transcription start site (TSS); a 3' UTR; a tetracycline response element; and a Kozak region. In some embodiments, the promoter is operably ligated to a promoter-enhancer region.

[0044] A vector comprising any one of the polynucleotides described herein is provided herein. In some embodiments, the vector is a plasmid, a viral vector, or a non-viral vector. In some embodiments, the viral vector is an adenovirus vector. In some embodiments, the adenovirus vector is deficient in one or more elements selected from the E1-E4 region and the L1-L5 region. In some embodiments, the adenovirus vector comprises one or more elements selected from E2B, L1, L2, L3, E2A, L4, E3, L5, an inverted terminal repeat (ITR), a poly(a) site, and a spacer. In some embodiments, the adenovirus vector is a gorilla adenovirus vector. In some embodiments, the adenovirus vector is a GC46 gorilla adenovirus vector.

[0045] In some embodiments, any of the vectors described herein includes a nucleic acid sequence having at least 90% identity with SEQ ID NO: 244.

[0046] A method for inducing an anti-HPV immune response in a subject requiring such response is provided herein, comprising the step of administering a therapeutically effective amount of any of the polynucleotides described herein to the subject. In some embodiments, the therapeutically effective amount is about 0.1 × 10⁻⁶ 9 ~About 10×10 12 Includes vectors at the particle level.

[0047] A method for treating an HPV-related disease or disorder in a subject requiring such treatment is provided herein, comprising the step of administering to the subject a therapeutically effective amount of one of the polynucleotides described herein. In some embodiments, the HPV-related disease or disorder is an HPV-16-related disease or disorder, an HPV-18-related disease or disorder, or an HPV-45-related disease or disorder. In some embodiments, the HPV-related disease or disorder is an HPV-related cancer. In some embodiments, the HPV-related cancer is a lower genital neoplasm, cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer.

[0048] In any of the methods for treating HPV-related diseases or disorders provided herein, the therapeutically effective dose is approximately 0.1 × 10⁻⁶. 9 ~About 10×10 12 Includes vectors at the particle level.

[0049] In any of the methods for treating HPV-related diseases or disorders provided herein, the method further includes the step of administering an additional therapy. In some embodiments, the additional therapy includes the administration of at least one of the following: anti-inflammatory agents; analgesics; bio-response modifiers; cytokines; interferons; interleukins; CAR-T cells; bifunctional proteins; colony-stimulating factors; tumor necrosis factors; surfactants; small molecule enzyme inhibitors; chemotherapeutic agents; and anti-metastatic agents. In some embodiments, the additional therapy includes the administration of a bio-response modifier. In some embodiments, the bio-response modifier is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the additional therapy includes the administration of a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a histone deacetylase (HDAC) inhibitor.

[0050] A fusion protein encoded by any of the polynucleotides provided herein is provided herein.

[0051] A composition comprising any of the polynucleotides provided herein is provided herein.

[0052] Any of the compositions provided herein, comprising polynucleotides, polypeptide constructs, and vectors, are intended for use in the treatment of diseases or disorders in subjects requiring such treatment.

[0053] Any of the compositions provided herein, comprising a polynucleotide, a polypeptide construct, or a vector, is intended for use in the manufacture of a pharmaceutical product for use in the treatment of a disease or disorder in a subject requiring it.

[0054] A kit comprising any of the polynucleotides, polypeptide constructs, or vectors provided herein is provided herein.

[0055] A vaccine comprising any of the polynucleotides, polypeptide constructs, or vectors provided herein is provided herein.

[0056] In some embodiments, any of the vaccines provided herein are intended for use in the treatment of a disease or disorder in a subject that requires it. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 is a schematic diagram of the HPV genome. The HPV genome consists of seven early genes (El~E7) and two late genes (Ll and L2), each with a specific function. The E5, E6, and E7 genes are associated with cancer development. [Figure 2] Figure 2 shows a schematic overall workflow performed to design an HPV vaccine antigen. [Figure 3]Figure 3 shows schematic diagrams of HPV16 and HPV18 antigenic designs for HPV Design 1 and HPV Design 3. Using consensus sequence information, HPV16 / HPV18 reference sequences were selected for designs containing all major variants. Vaccine compositions containing different E6, E7, and E5 protein components with domain boundaries, as well as mutation information, are shown. These different domains contain the most dominant peptides estimated from Immune Epitope Database (IEDB) predictions for MHC-I binding. HPV Design 3 is similar to HPV Design 1, with the addition of an enhancer agonist peptide. [Figure 4] Figures 4A and 4B illustrate homology models of HPV design 2 and HPV design 4, respectively. The homology models are used to evaluate the overall structural features and compare the HPV designs to the native ankyrin repeat. The HPV designs are shown in the same orientation and maintain the same overall fold, but suggest different structural conformations due to the shuffled peptide. [Figure 5-1] Figure 5A shows HPV design 5 (target) (SEQ ID NO: 322) mapped to HPV design 4 (query) (SEQ ID NO: 321) using protein blast. Strong and weak binding agents were identified using netMHC. Figure 5B shows density plots of HPV designs 4 and 5 extracted based on the mapped locations. Similar patterns were observed in the predicted strongly / weakly binding peptides. The binding affinity predictions in the matched regions of HPV designs 4 and 5 were similar. [Figure 5-2] Same as above. [Figure 6] Figure 6 is a schematic diagram showing the short and long primer and probe sets prepared for the RNA qPCR relative expression assay. Specific primers were designed for each HPV antigen design. [Figure 7-1]Figure 7A shows the NetMHC 4.0 antigenicity prediction. The predicted strongly and weakly binding peptide indices are plotted against the peptide location. Figure 7B shows the NetMHC 4.0 antigenicity prediction density plot. The difference between the first and second order was used to identify the peaks. Figure 7C shows the amino acid sequences aligned to the consensus sequence to determine coverage across HPV subtypes. Figure 7C discloses sequence numbers 288–314, respectively, in order of appearance. [Figure 7-2] Same as above. [Figure 7-3] Same as above. [Figure 8] Figure 8 shows a comparison between HPV design 1 (sequence number 323) and HPV design 3 (sequence number 324). [Figure 9-1] Figure 9A shows the sequence alignment of HPV16 E6 (SEQ ID NO: 45) to wild-type HPV16 E6 from UP P03126 (SEQ ID NO: 315). Figure 9B shows the sequence alignment of HPV16 E7 (SEQ ID NO: 46) to wild-type HPV16 E7 from UP P03129 (SEQ ID NO: 316). Figure 9C shows the sequence alignment of HPV16 E5 (SEQ ID NO: 47) to wild-type HPV16 E5 from UP P06927 (SEQ ID NO: 317). [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 10-1] Figure 10A shows the sequence alignment of HPV18 E6 (SEQ ID NO: 48) to wild-type HPV18E6 from UP P06463 (SEQ ID NO: 318). Figure 10B shows the sequence alignment of HPV18 E7 (SEQ ID NO: 49) to wild-type HPV18E7 from UP P06788 (SEQ ID NO: 319). Figure 10C shows the sequence alignment of HPV18 E5 (SEQ ID NO: 50) to wild-type HPV18E5 from UP P06792 (SEQ ID NO: 320). [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 11]Figures 11A and 11B show the design of the recombinant gorilla adenovirus vaccine AdV-HPV16 / 18 (including the antigen construct of SEQ ID NO: 243), where Figure 11A is a schematic diagram of the gorilla adeno vector GC46 with multiple gene deletions, and Figure 11B is a schematic diagram of the HPV antigen construct structure. [Figure 12] This figure illustrates a GC46 vector modified with deletions in the E1 and E4 regions and a CMV HPV16 / 18 expression cassette (antigen) in the E1 region. [Figure 13-1] Figures 13A and 13B are diagrams of the AdV-HPV16 / 18 antigen, where Figure 13A shows the predicted structure of the AdV-HPV16 / 18 antigen, and Figure 13B shows the AdV-HPV16 / 18 antigen with the noted HPV epitope / peptide and ankyrin-like linker / scaffold. Figure 13B discloses Sequence ID No. 243. [Figure 13-2] Same as above. [Figure 13-3] Same as above. [Figure 14]Figures 14A–G illustrate data collected from antitumor studies of AdV-HPV16 / 18 in a humanized mouse model. In Figures 14A and 14B, HPV-specific IFN-γ production from healthy donor PBMCs was stimulated for two IVS cycles by autologous dendritic cells infected with HPV vaccine constructs numbers 1–5 and restimulated twice with 15-mer peptides of duplicated HPV16 E6 / E7. IFN-γ levels in the supernatant 24 and 48 hours after the second restimulation are shown for HPV16 E6 (Figure 14A) and HPV16 E7 (Figure 14B). In Figures 14C and 14D, NSG-β2m- / - mice carrying HPV+ cervical cancer (SiHa) were reconstituted with human PBMCs (day 7, blue arrow) and treated twice weekly with PBS (100 μL), an empty vector (1 × 10⁹ VP), or HPV vaccine constructs (numbers 1, 3, and 4; 1 × 10⁹ VP) in sc (red arrow). Figure 14C shows the tumor volume, and Figure 14D shows the tumor weight at day 31. In Figures 14E and 14F, humanized NSG-β2m- / - mice carrying SiHa tumors were vaccinated four times with AdV-HPV16 / 18 (construct number 4). Figure 14E shows the tumor volume, and Figure 14F shows the tumor weight at day 43. In Figure 14G, tumors from Figure 14C were dissected and stained using the Perkin Elmer Opal IHC kit. The central tumor section is shown for representative mice from the blank vector and AdV-HPV16 / 18 groups. Red: CD8+ T cells; Green: CD4+ T cells; Blue: DAPI. Median values ​​are shown. Kruskarwallis and repeated measures ANOVA. *P<0.05, **P<0.01, ****P<0.0001. EV, blank vector; IVS, in vitro stimulation; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; VP, viral particles. Scale bar: 100 μm. [Figure 15]In Figures 15A-15E, C57BL / 6 mice carrying scTC-1 HPV16- tumors were treated with three weekly injections of sc PBS control (100 μL) or AdV-HPV16 / 18 (1 × 10⁹ VP) starting on day 4 post-tumor transplantation (red arrow). Figure 15A shows the mean tumor volume of AdV-HPV16 / 18 versus control-treated mice; P < 0.0001. Figure 15B shows the median tumor weight at the end of the study; P < 0.05. Mice number 10 and 13 were defined as the best responders with tumor weights below the group median, and mice number 11 and 14 were defined as non-responders with tumor weights above the group median. Figures 15C and 15D show flow cytometry of single-cell suspensions of tumors. In Figure 15C, CD8+ and CD4+ T-cell tumor infiltration is shown from representative mice from each group. Figure 15D shows the infiltration of multifunctional (IFN-γ+GzmB+)CD8+ T cells from one representative mouse from each group. In Figure 15E, splenocytes from six mice from each group were stimulated with the 15-mer peptide of duplicated HPV16 E6 in the ELIspot assay. Figure 15E shows the number of SFCs per 2.5 × 10⁵ splenocytes in AdV-HPV16 / 18 versus control-treated mice after subtracting negative controls. Mann-Whitney U test and repeated measures ANOVA were used. *P<0.05, **P<0.01, ****P<0.0001. SFC, spot-forming cell; VP, viral particle. [Figure 16]In Figures 16A–F, C57BL / 6 mice (n=7–8 per group) carrying scTC-1 HPV16+ mouse tumors were treated with either a blank vector control (1 × 10⁹ VP, sc), PBS (100 μL, sc), or AdV-HPV16 / 18 (1 × 10⁹ VP, sc) at 7 and 14 days post-tumor transplantation in two separate studies. Figure 16A shows the tumor weight at the end of the study for individual mice in Study 1. Figure 16B shows the tumor weight at the end of the study for individual mice in Study 2. Figure 16C shows a meta-analysis of both studies showing the tumor weight at the end of the study. Figures 16D–18F show flow cytometry of single-cell suspensions of tumor tissue. T cell subsets are shown per 1 mg of tumor. Ki67 staining was performed in Study 1 only. Figures 16A–F show median values. Mann-Whitney U and Krus-Kalwallis tests were used. *P<0.05, **P<0.01, ***P<0.001. VP, virus particle. [Figure 17-1]In Figures 17A-I, C57BL / 6 mice (n=6-7 per group) carrying scTC-1 HPV16 tumors were treated with either an empty vector control (1×10⁹VP, sc) or AdV-HPV16 / 18 (1×10⁹VP, sc) on days 7 and 14 after tumor transplantation. Tumors were harvested at the end of the study (day 23). CD45+ TILs were isolated and stimulated overnight in triple wells with a combination of duplicated HPV16 E6 / E7 15mer peptide or DMSO (negative control), and then evaluated by flow cytometry. Figures 17A–F show the cell count / mg tumor for total CD8 (Figure 17A), IFN-γ-producing CD8 (Figure 17B), IFN-γ and GzmB-producing CD8 (Figure 17C), total CD4 (Figure 17D), IFN-γ-producing CD4 (Figure 17E), and IFN-γ and GzmB-producing CD4 (Figure 17F). In Figure 17G, unstimulated TILs were also evaluated for CD8 and HPV16 E7 tetramers. Figures 17H and 17I show ELIspot assays performed on splenocytes using duplicated 15-mer peptides for HPV16 E6 (Figure 17H) and HPV18 E6 (Figure 17I). Median values ​​are shown. Mann-Whitney U test was used. *P<0.05, **P<0.01. TIL, tumor-infiltrating lymphocytes; TME, tumor microenvironment; VP, viral particles. [Figure 17-2] Same as above. [Figure 18] Figure 18 shows the tumor volume when C57BL / 6 mice carrying TC-1 tumors were treated with AdV-HPV16 / 18 alone or in combination with vintrafusp alfa and anti-PD-L1 / TGF-β trap protein, and primary tumor growth was evaluated. [Figure 19] Figure 19 shows a scheme for the administration of AdV-HPV16 / 18 alone or in combination with vintrafusp alfa, an anti-PD-L1 / TGF-β trap protein, in subjects with HPV-related cancers. [Figure 20]Figure 20 is a bar graph showing the time to treatment response and duration of response after administration of AdV-HPV16 / 18 alone and in combination with vintrafusp alfa in patients with recurrent / metastatic HPV-related cancer. [Figure 21] Figure 21A is a dot plot graph showing the titer of neutralizing anti-GC46 antibodies against the number of AdV-HPV16 / 18 vaccines administered between administrations of AdV-HPV16 / 18 monotherapy. Figure 21B is a dot plot graph showing the titer of neutralizing anti-GC46 antibodies against the number of AdV-HPV16 / 18 vaccines administered after administration of AdV-HPV16 / 18 and vintrafusp alfa. [Figure 22] Figure 22A shows CT scans of the cervix of individual cervical cancer patients who achieved a complete response to combination therapy with AdV-HPV16 / 18 and vintrafusp alfa. Figure 22B shows a bar graph showing the T-cell activity of the same patients as a percentage of the number of AdV-HPV16 / 18 vaccines administered. [Figure 23] Figure 23 shows a scheme for the administration of AdV-HPV16 / 18 alone or in combination with vintrafusp alfa, an anti-PD-L1 / TGF-β trap protein, in subjects with HPV-related cancers. 18 vaccinations were administered after administration of AdV-HPV16 / 18 and vintrafusp alfa. [Figure 24] Figure 24 shows the development of HPV-specific T cells in the HPV-specific T cell response in patients in whom no such pre-existing response was detected for (A) HPV16 and (B) HPV18. The time point (day) at which each patient was evaluated is shown along the x-axis, and the CD4+ or CD8+ phenotype is shown in the legend. [Figure 25]Figure 25 shows the absolute number of T cells producing cytokines and / or CD107a (left y-axis) in response to (A) HPV16 and (B) HPV18 peptides in patients who experienced a complete response to treatment for more than one year after treatment. Vector neutralizing antibody titers at available time points are plotted on the right y-axis (black squares). Production of anti-GC46 vector neutralizing antibody did not negate the development of the HPV-specific T cell response. Time points (days) are shown along the x-axis, and CD4+ or CD8+ phenotype readings are shown in the legend. [Figure 26] Figure 26 shows the increase in both activated CD8+ memory T cells and mature NK cells, observed only in patients who achieved complete response (CR), approximately two weeks after the first dose of the AdV-HPV16 / 18 vaccine. The percentage changes in (A) total CD8+ T cell frequency and (B) total NK cell frequency at D15 were compared to baseline. [Figure 27] Figure 27 shows the levels of (A) IL-8 and (B) TNFα during the first 8-week period in the study. IL-8 and TNFα levels significantly increased 2 weeks after the initial treatment with AdV-HPV16 / 18 and vintrafusp alfa. [Figure 28] Figure 28 shows Kaplan-Meier survival curves, calculated using the Mantel-Cox log-rank test, for (A) progression-free survival (PFS) and (B) overall survival (OS) in patients treated with AdV-HPV16 / 18 in combination with vintrafusp alfa, indicating that lower serum IL-8 concentrations before the start of treatment were associated with longer-term survival. The cutoff was set at 23.0 pg / ml. [Figure 29] Figure 29 shows the changes in circulating HPV tumor DNA (ctDNA) during treatment with AdV-HPV16 / 18. (A) Absolute copies of HPV16 or HPV18 ctDNA per ml of plasma. (B) Percentage change in ctDNA levels during treatment compared to baseline. The best response rate (BOR) and HPV genotype observed are shown in the legend. [Figure 30]Figure 30 shows the sequence alignment of the HPV45 E6 protein (SEQ ID NO: 325) compared to the following HPV16 E6 or HPV18 E6 epitopes: HPV16 E6 peptide 5 (pep5) (SEQ ID NO: 117), HPV16 E6 peptide 7 (SEQ ID NO: 119), HPV16 E6 peptide 8 (SEQ ID NO: 120), HPV18 E6 peptide 1 (SEQ ID NO: 131), HPV18 E6 peptide 2 (SEQ ID NO: 132), HPV18 E6 peptide 4 (SEQ ID NO: 184), HPV18 E6 peptide 5 (SEQ ID NO: 135), and agonist peptide 1 (SEQ ID NO: 176). [Figure 31] Figure 31 shows the sequence alignment of the HPV45 E7 protein (SEQ ID NO: 326) compared to the following HPV18 E7 epitopes: HPV18 E7 peptide 1 (pep1) (SEQ ID NO: 226), HPV18 E7 peptide 2 (SEQ ID NO: 194), HPV18 E7 peptide 3 (SEQ ID NO: 192), HPV18 E7 peptide 4 (SEQ ID NO: 186), HPV18 E7 peptide 5 (SEQ ID NO: 240), and HPV18 E7 peptide 6 (SEQ ID NO: 182). [Figure 32] Figure 32 shows the sequence alignment of the HPV45 E7 protein (SEQ ID NO: 326) compared to the following HPV18 E7 epitopes: HPV18 E7 peptide 1 (SEQ ID NO: 226), HPV18 E7 peptide 4 (SEQ ID NO: 186), and HPV18 E7 peptide 6 (SEQ ID NO: 182). [Modes for carrying out the invention]

[0058] It should be understood that this disclosure is not limited to the specific embodiments described herein and is subject to modification. While various features of this disclosure may be described in the context of a single embodiment, features may also be provided separately or in any preferred combination. Those skilled in the art will recognize that variations and modifications of this disclosure exist that fall within their scope.

[0059] All terms are intended to be understood in the same way as they are understood by those skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as they are generally understood by those skilled in the art in which this disclosure relates.

[0060] Section headings used herein are for organizational purposes only and should not be construed as limitations on the subject matter described. definition

[0061] The following definitions are supplementary to the definitions in the Art and apply to this application; they should not be attributed to any related or unrelated cases, such as any generally owned patent or application. Therefore, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0062] In this application, the use of the singular form includes the plural form unless otherwise specifically indicated. It should be noted that, as used herein, the singular forms "a," "an," and "the" include multiple referents unless the context explicitly indicates otherwise.

[0063] In this application, the use of “or” means “and / or” unless otherwise specified. The terms “and / or” and “any combination thereof,” as well as their grammatical equivalents, can be used interchangeably as used herein. These terms can convey that any combination is specifically intended. For illustrative purposes only, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof” may mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjugate or disjunctively unless the context specifically refers to a disjunctive use.

[0064] The use of the term "including," as well as other forms such as "include," "includes," and "included," is not restrictive; that is, "including" does not mean "limited to."

[0065] References to “some embodiments,” “a certain embodiment,” “one embodiment,” or “other embodiments” in this specification mean that certain features, structures, or characteristics described in conjunction with embodiments are included in at least some embodiments of this disclosure, but not necessarily in all embodiments of this disclosure.

[0066] As used herein and in the claims, the terms “comprising” (and any form of “comprising,” e.g., “comprise” and “comprises”), “having” (and any form of “having,” e.g., “have” and “has”), “including” (and any form of “including,” e.g., “includes” and “include”), or “containing” (and any form of “containing,” e.g., “contains” and “contain”) are inclusive or open-ended and do not exclude additional possible components, elements, or steps of the method. Any embodiment discussed herein is intended to be performed with respect to any method or composition of the Disclosure, and vice versa. Furthermore, compositions of the Disclosure may be used to achieve the methods of the Disclosure.

[0067] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which in part depends on the method of measuring or determining the value, i.e., the limits of the measuring system. For example, “about” may mean within 1 or a standard deviation greater than 1, according to practice in the art. Alternatively, “about” may mean a range of up to 20%, 10%, 5%, or 1% of a given value. In another example, the quantity “about 10” includes 10 and any quantities from 9 to 11. In yet another example, the term “about” in relation to a reference number may also include a range of values ​​of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or plus or minus 1% from that value. Alternatively, particularly in relation to biological systems or processes, the term “about” may mean within one order of magnitude of the value, preferably within five times, more preferably within two times. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” means within an acceptable margin of error for the particular value to be assumed.

[0068] With respect to the numerical range limitations specified herein, each intervening number between them is explicitly intended to be of the same precision. For example, in the range of 6 to 9, the numbers 7 and 8 are intended in addition to 6 and 9, and in the range of 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0069] The term “isolated” and its grammatical equivalent, as used herein, refers to the removal of nucleic acids, proteins, polypeptides, cells, or other materials from their natural environment. The term “purified” and its grammatical equivalent, as used herein, refers to molecules or compositions whose purity has been increased, whether removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA), and / or amplified under laboratory conditions, where “purity” is a relative term, not “absolute purity.” However, it should be understood that nucleic acids and proteins can be formulated with diluents or adjuvants and still isolated for practical purposes. For example, nucleic acids are typically mixed with an acceptable carrier or diluent when used for introduction into cells. The term “substantially purified” and its grammatical equivalent, as used herein, refers to nucleic acid sequences, polypeptides, proteins, or other compounds that essentially do not contain, i.e., no more than about 50%, no more than about 70%, or no more than about 90% of the polynucleotides, proteins, polypeptides, and other molecules to which the nucleic acid, polypeptide, protein, or other compound relates in nature.

[0070] "Nucleic acid," "nucleic acid molecule," "polynucleotide," "polynucleotide construct," "oligonucleotide," and their grammatical equivalents, as used herein, refer to polymeric forms of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of molecules. Therefore, this term includes double-stranded and single-stranded DNA, triple-stranded DNA, and double-stranded and single-stranded RNA. This also includes polynucleotides modified, for example, by methylation and / or capping, as well as polynucleotides in their unmodified forms. This term also means that it includes molecules, including synthetic and semi-synthetic nucleotides and polynucleotides that do not exist in nature, as well as nucleotide analogs. When discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein in accordance with the usual convention of giving the sequence only in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to mRNA). "Recombinant polynucleotide" is a polynucleotide that has undergone molecular biological manipulation. The polynucleotide sequences and vectors disclosed or intended herein can be introduced into cells, for example, by transfection, transformation, or transduction.

[0071] When applied to a polynucleotide or nucleic acid sequence, the term "fragment" refers to a nucleotide sequence that is shorter than a reference nucleic acid and, across its common portion, contains the same nucleotide sequence as the reference nucleic acid. Such a nucleic acid fragment according to the present invention may, where appropriate, be contained within a longer polynucleotide of which it is a component. Such fragments include, or alternatively consist of, oligonucleotides having a length range of at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or longer consecutive nucleotides of nucleic acid according to the present invention.

[0072] As used herein, “isolated polynucleotide” or “isolated nucleic acid fragment” refers to a polymer of RNA or DNA that is single-stranded or double-stranded, optionally containing synthetic, non-natural, or modified nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may constitute one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0073] The term “gene” and its grammatical equivalent refer to polynucleotides containing nucleotides that code for functional molecules, including functional molecules produced solely by transcription (e.g., bioactive RNA species) or functional molecules produced by both transcription and translation (e.g., polypeptides). The term “gene” encompasses cDNA and genomic DNA nucleic acids. “Genes” also refer to nucleic acid fragments that express specific RNA, proteins, or polypeptides, including regulatory sequences preceding the coding sequence (5' non-coding sequence) and subsequent regulatory sequences (3' non-coding sequence). “Native gene” refers to a gene found naturally together with its own regulatory sequences. “Chimera gene” refers to any gene that is not a native gene and contains regulatory sequences and / or coding sequences that are not found together in nature. Thus, a chimeric gene may contain regulatory sequences and coding sequences from different origins, or regulatory sequences from the same origin but arranged in a different manner than those found in nature. A chimeric gene may contain coding sequences and / or regulatory sequences from different origins. “Endogenous gene” refers to a native gene in its natural location within the genome of an organism. An "external" gene or "other" gene refers to a gene that is not normally found in the host organism but has been introduced into the host organism through gene transfer. External genes can include native genes inserted into non-native organisms or chimeric genes. A "transgene" is a gene that has been introduced into the genome through a transformation procedure.

[0074] The term "genome" includes chromosomes, as well as mitochondria, chloroplasts, and viral DNA or RNA. The term "probe" refers to a single-stranded nucleic acid molecule that can base-pair with a complementary single-stranded target nucleic acid to form a double-stranded molecule.

[0075] "Heterogeneous DNA" refers to DNA that is not naturally located in a cell or in a chromosomal region of a cell. Heterogeneous DNA may include exogenous genes. "Exogenous genes" means genes that are foreign to the subject, i.e., genes introduced into the subject by a transformation process, an unmutated version of an endogenous mutated gene, or a mutated version of an endogenous unmutated gene. Exogenous genes may be either native or synthetic genes introduced into the subject in the form of DNA or RNA that can function by a DNA intermediate, for example by reverse transcriptase. Such genes may be introduced into target cells, directly into the subject, or indirectly by the transfer of transformed cells into the subject.

[0076] A "primer" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to create a double-stranded nucleic acid region that can function as an initiation site for DNA synthesis under favorable conditions. Such primers can be used in polymerase chain reactions or for DNA sequencing.

[0077] A DNA "coding sequence" or "coding region" refers to a double-stranded DNA sequence that codes for a polypeptide and, under the control of a suitable regulatory sequence, can be transcribed and translated into a polypeptide in cells, ex vivo, in vitro, or in vivo. A "suitable regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), internally, or downstream (3' non-coding sequence) of the coding sequence that influences transcription, RNA processing, stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translational leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of a coding sequence are determined by a 5' (amino) terminal start codon and a 3' (carboxy) terminal translation termination codon. Codextic sequences may include, but are not limited to, prokaryotic sequences, mRNA-derived cDNA, genomic DNA sequences, and synthetic DNA sequences. When a coding sequence is intended for expression in eukaryotic cells, the polyadenylation signal and transcription termination sequence are typically located at 3' of the coding sequence.

[0078] An "open reading frame," abbreviated as ORF, refers to a nucleic acid sequence of a certain length, whether DNA, cDNA, or RNA, that contains a translation start signal or start codon, e.g., ATG or AUG, and a stop codon, and is potentially translateable into a polypeptide sequence.

[0079] The term "downstream" refers to a nucleotide sequence located 3' relative to a reference nucleotide sequence. In particular, downstream nucleotide sequences generally refer to sequences that follow the transcription start site. For example, the translation start codon of a gene is located downstream of the transcription start site.

[0080] The term "upstream" refers to a nucleotide sequence located 5' relative to a reference nucleotide sequence. In particular, upstream nucleotide sequences generally refer to sequences located 5' to the coding sequence or the transcription start site. For example, most promoters are located upstream of the transcription start site.

[0081] The term "response element" refers to one or more cis-acting DNA elements that confer responsiveness to a promoter mediated by interaction with the DNA-binding domain of a transcription factor. This DNA element may be a palindrom (complete or incomplete) in its sequence, or it may consist of a sequence motif or half-regions separated by a variable number of nucleotides. The half-regions may be similar or identical and may be arranged as serial or reverse repeat sequences, as a single half-region, or as a serial multimer of adjacent half-regions. Depending on the nature of the cell or organism into which the response element is incorporated, the response element may contain minimal promoters isolated from different organisms. The DNA-binding domain of the transcription factor binds to the DNA sequence of the response element, in the presence or absence of a ligand, to initiate or repress the transcription of downstream genes regulated by this response element. Examples of DNA sequences for response elements of natural ecdysone receptors include RRGG / TTCANTGAC / ACYY (SEQ ID NO: 275) (see Cherbas et. al., Genes Dev. 1991); AGGTCAN (n) AGGTCA(where N (n) Examples include (which may be one or more spacer nucleotides) (SEQ ID NO: 276) (see D'Avino et al., Mol. Cell. Endocrinol. 113:1 1995); and GGGTTGAATGAATTT (SEQ ID NO: 277) (see Antoniewski et al., Mol. Cell Biol. 14:4465 1994).

[0082] The term “operably ligated,” as used herein, refers to the physical and / or functional ligation of a DNA segment to another DNA segment in such a manner that the segments function in their intended manner. A DNA sequence encoding a gene product is operably ligated to a regulatory sequence if it is ligated to a regulatory sequence, such as a promoter, enhancer, and / or silencer, in a manner that allows for the modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably ligated to a promoter if it is ligated to a promoter downstream of the transcription start site of the promoter in the correct reading frame relative to the transcription start site, allowing transcription elongation to proceed by the DNA sequence. An enhancer or silencer is operably ligated to a DNA sequence encoding a gene product if it is ligated to the DNA sequence in a manner that increases or decreases the transcription of the DNA sequence, respectively. Enhancers and silencers may be located upstream, downstream, or embedded within the coding region of the DNA sequence. The DNA for the signal sequence is operably ligated to the DNA encoding the polypeptide, if the signal sequence is expressed as a preprotein that participates in polypeptide secretion. Ligation of the DNA sequence to the regulatory sequence is typically achieved by ligation at a suitable restriction site or via an adapter or linker inserted into the sequence using a restriction endonuclease known to those skilled in the art.

[0083] As used herein, the term “codon degenerate variant” refers to a modified nucleic acid sequence that codes for the same amino acid sequence as the original sequence, but with different specific nucleotides containing codons. The genetic code is degenerate, meaning that multiple codons can code for the same amino acid. For example, the amino acid leucine can be coded by six different codons: CTG, CTT, CTC, CTA, TTG, and TTA. A codon degenerate table, also known as a genetic code table or codon table, is a chart that provides information about the relationships between codons (sequences of three nucleotides) and the corresponding amino acids they code for. The table lists 64 possible codons and indicates which amino acid each codon represents. Table 1 is an example of a codon degenerate table.

[0084] [Table 1]

[0085] The following definitions are supplementary to the definitions in the Art and apply to this application; they should not be attributed to any related or unrelated cases, such as any generally owned patent or application. Therefore, the technical terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0086] Furthermore, publicly available software resources are readily available for the “reverse translation” of polypeptide sequences, also known as computer-generated “reverse transcription,” which is the conversion of a polypeptide sequence into the nucleotide sequence that encodes it. See, for example, Madeira, F., et al., Nucleic Acids Res, 47(Wl), W636-W641 (2019); Madeira, F., et al., Curr Protoc in Bioinformatics, 66(1):e74 (2019); Chojnacki, S, et al., Nucleic Acids Res. 2017 Jul 3;45(Wl):W550-W553 (2017); Athey, J., et al., BMC Bioinformatics 18:391 (2017).

[0087] As used herein, codon degenerate variants may be utilized to optimize gene expression or enhance protein production. By modifying codons within a nucleic acid sequence, it is possible to utilize codons that are more frequently used or preferred by the translational mechanisms of the host organism. This may result in increased efficiency of protein expression or improved compatibility with specific host organisms.

[0088] As used herein, the term "expression" refers to the transcription and stable accumulation of sense RNA (mRNA) or antisense RNA derived from nucleic acids or polynucleotides. Expression may also refer to the translation of mRNA into proteins or polypeptides.

[0089] The terms “cassette,” “expression cassette,” and “gene expression cassette” refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide by specific restriction sites or by homologous recombination. The DNA segment contains a polynucleotide encoding the polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in a proper reading frame for transcription and translation. A “transformation cassette” refers to a specific vector containing a polynucleotide encoding the polypeptide of interest, and having elements in addition to the polynucleotide that promotes transformation of a particular host cell. The cassettes, expression cassettes, gene expression cassettes, and transformation cassettes of the present invention may also include elements that enable enhanced expression of the polynucleotide encoding the polypeptide of interest in a host cell. These elements may include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, and polyadenylation sequences. The expression cassettes described herein are approximately 500-10,000 bp, 1,000-5,000 bp, 1,500-4,500 bp, 1,800-4,400 bp, 2,000-4,500 bp, 2,100-4,400 bp, 2,200-4,300 bp, 2,300-4,200 bp, 2,400-4,100 bp, and 2,500-4,000 bp. The expression cassette may contain a total length of 0 bp, approximately 2,600 to 3,900 bp, approximately 2,700 to 3,800 bp, approximately 2,800 to 3,800 bp, approximately 2,900 to 3,700 bp, approximately 3,000 to 3,600 bp, approximately 3,100 to 3,500 bp, approximately 3,150 to 3,450 bp, approximately 3,200 to 3,400 bp, approximately 3,250 to 3,350 bp, or approximately 3,300 bp. Alternatively, the expression cassette may contain any number of base pairs within these ranges.For example, the expression cassettes are approximately 500 bp, 750 bp, 1,000 bp, 1,250 bp, 1,500 bp, 1,750 bp, 2,000 bp, 2,250 bp, 2,500 bp, 2,550 bp, 2,600 bp, 2,650 bp, 2,700 bp, 2,750 bp, 2,800 bp, 2,850 bp, 2,900 bp, 2,950 bp, 3,000 bp, 3,050 bp, 3,100 bp, 3,150 bp, 3,200 bp, 3,250 bp, and 3,300 bp. The expression may contain approximately 3,350 bp, approximately 3,400 bp, approximately 3,450 bp, approximately 3,500 bp, approximately 3,550 bp, approximately 3,600 bp, approximately 3,650 bp, approximately 3,700 bp, approximately 3,750 bp, approximately 3,800 bp, approximately 3,850 bp, approximately 3,900 bp, approximately 3,950 bp, approximately 4,000 bp, approximately 4,050 bp, approximately 4,100 bp, approximately 4,150 bp, approximately 4,200 bp, approximately 4,250 bp, approximately 4,300 bp, approximately 4,350 bp, approximately 4,400 bp, approximately 4,450 bp, or approximately 4,500 bp. In one embodiment, the expression cassette contains approximately 2,800 bp. In another embodiment, the expression contains 2,825 bp.

[0090] As used herein, the term “vector” refers to any vehicle for cloning and / or transferring nucleic acids into host cells. A vector may be a replicon to which another DNA segment can be attached, resulting in the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., is capable of replicating under its own control. The term “vector” includes both viral and nonviral vehicles for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Numerous vectors known in the art can be used to manipulate nucleic acids, to incorporate response elements and promoters into genes, etc. Possible vectors include, for example, bacteriophages, e.g., lambda derivatives, or plasmids, e.g., pBR322 or pUC plasmid derivatives, or Bluescript vectors, e.g., plasmids or modified viruses. Another example of a vector useful in the present invention is the ULTRAVECTOR® Production System (Intrexon Corp., Blacksburg, VA), described in International Publication No. 2007 / 038276. For example, insertion of DNA fragments corresponding to response elements and promoters into a suitable vector can be achieved by ligating the appropriate DNA fragments into a selected vector having complementary adherent ends. Alternatively, the ends of the DNA molecule may be enzymatically modified, or any site may be generated by ligating a nucleotide sequence (linker) to the DNA ends. Such vectors may be engineered to contain a selection marker gene that provides selection of cells into which the marker has been incorporated into the cellular genome. Such markers enable the identification and / or selection of host cells that incorporate and express the protein encoded by the marker.

[0091] As used herein, the term “plasmid” refers to an extrachromosomal element, often containing genes, that is typically in the form of a circular double-stranded DNA molecule, rather than being part of the cell’s central metabolism. Such elements may be single-stranded or double-stranded DNA or RNA, of any origin, autologous replication sequences, genomic integration sequences, phages or nucleotide sequences, linear, circular, or higher-order coils, where some nucleotide sequences are conjugated to or recombined into a unique construct that can introduce into the cell a promoter fragment and DNA sequence for a selected gene product, along with a suitable 3’ untranslated sequence.

[0092] As used herein, the terms “cloning vector” and “replicon” refer to a unit-length nucleic acid, preferably DNA, such as a plasmid, phage, or cosmid, which replicates sequentially and contains an origin of replication, to which another nucleic acid segment may be attached in such a way that it results in replication of the attached segment. A cloning vector may be capable of replicating in one cell type and expressing in another cell type (a “shuttle vector”). A cloning vector may contain one or more sequences that can be used as a vector for insertion of a sequence of interest and / or for selection of cells containing one or more multiple cloning sites.

[0093] As used herein, the term “viral vector” refers to a virus, viral particle, or derivative thereof that can transfer nucleic acids into cells or into the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. Viral vectors, in particular retroviral vectors, are used in a wide variety of gene delivery applications in cells and living animals. Viral vectors that can be used include, but are not limited to, retroviruses, adeno-associated viruses, pox, baculoviruses, vaccinia, herpes simplex, Epstein-Barr, adenoviruses, geminiviruses, and karimovirus vectors. Non-viral vectors include plasmids, liposomes, charged lipids (cytofectins), DNA-protein complexes, and biopolymers. In addition to nucleic acids, vectors may also include one or more regulatory regions, and / or selection markers useful for selection, measurement, and monitoring of nucleic acid transfer outcomes (e.g., which tissues to transfer into, duration of expression).

[0094] As used herein, the terms “adenovirus” and “adenovirus vector” mean, as used herein, an adenovirus that retains the ability to participate in the adenovirus life cycle and / or has been physically inactivated by, for example, destruction (e.g., sonication), denaturation (e.g., using heat or a solvent), or crosslinking (e.g., via formalin crosslinking). The “adenovirus life cycle” includes (1) virus binding and entry into a cell, (2) transcription of the adenovirus genome and translation of adenovirus proteins, (3) replication of the adenovirus genome, and (4) construction of a viral particle (e.g., Fields Virology, 5 thSee, Knipe et al. (eds.), Lippincott Williams & Wilkins, Philadelphia, PA (2006). Adenoviruses may also be deficient in replication by the deletion of one or more portions of the naturally occurring viral genome (i.e., they do not retain the ability to participate in the adenovirus life cycle), as used and described herein. "Adenovirus" and "adenovirus vector" may include adenoviruses whose adenovirus genome has been engineered to accommodate nucleic acid sequences that are non-native with respect to the adenovirus genome. Typically, adenovirus vectors are constructed, for example, by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenovirus genome of an adenovirus to accommodate the insertion of a non-native nucleic acid sequence into the adenovirus for gene transfer.

[0095] As used herein, "AdV-HPV16 / 18" refers to a vector containing the antigen construct of Sequence ID No. 243.

[0096] As used herein, the terms “MOI” or “infection multiplicity” refer to the average number of viral particles (e.g., recombinant virus or control virus) that infect a single cell in a given experiment.

[0097] As used herein, the term “transfection” refers to the uptake of exogenous or heterologous RNA or DNA by a cell. A cell is “transfected” with exogenous or heterologous RNA or DNA if such RNA or DNA is introduced into the cell. A cell is “transformed” with exogenous or heterologous RNA or DNA if the transfected RNA or DNA causes a phenotypic alteration. The RNA or DNA that transforms a cell may be incorporated (covalently linked) into the chromosomal DNA that makes up the cell’s genome.

[0098] As used herein, the term “transformation” refers to the introduction of a nucleic acid fragment into the genome of a host organism that results in genetically stable inheritance. A host organism containing a transformed nucleic acid fragment is referred to as a “transgenic,” “recombinant,” or “transformed” organism.

[0099] As used herein, the term “electroporation” refers to the use of transmembrane electric field pulses that transiently increase the permeability of the cell membrane, enabling the introduction of exogenous biological materials into cells, such as DNA, RNA, peptides, polypeptides, proteins, enzymes, or ribonucleoproteins (RNPs). The electric field pulses create transient pores in the cell membrane, facilitating the uptake of biological materials. Electroporation can be performed using specialized buffers and devices that control pH, conductivity, osmolality, and other parameters to optimize the process and enhance transfection efficiency while minimizing cell damage. Electroporation can be used to introduce exogenous materials (e.g., biomolecules, plasmids, oligonucleotides, expression cassettes, siRNAs, drugs, and ions) into various cell types, including primary human hematopoiesis, immune cells, pluripotent progenitor cells, fibroblasts, and endothelial cells, for applications in gene therapy, cell therapy, and biotechnology research.

[0100] As used herein, the terms “inducing,” “inducing,” and their grammatical equivalents refer to an increase in nucleic acid sequence transcription, promoter activity, and / or expression, brought about by a transcription regulator, compared to some baseline level of transcription.

[0101] As used herein, the terms “promoter” and “promoter sequence” are interchangeable and refer to DNA sequences capable of controlling the expression of coding sequences or functional RNA. Generally, coding sequences are located 3' relative to promoter sequences. Promoters may be entirely derived from native genes, or they may consist of different elements derived from different promoters found in nature, or they may further include synthetic DNA segments. Those skilled in the art will understand that different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that express genes in most cell types are generally referred to as “constitutive promoters.” Promoters that express genes in specific cell types are generally referred to as “cell-specific promoters” or “tissue-specific promoters.” Promoters that express genes at specific stages of development or cell differentiation are generally referred to as “development-specific promoters” or “cell differentiation-specific promoters.” Promoter induces and triggers gene expression after exposure or treatment of cells with virulence factors, biomolecules, chemicals, ligands, light, etc., which induce the promoter; these are generally referred to as "inducible promoters" or "regulatory promoters." It is further recognized that, in most cases, the precise boundaries of regulatory sequences are not fully defined, and DNA fragments of different lengths may have the same promoter activity.

[0102] The promoter sequence typically extends upstream (5' direction) at its 3' end, bordered by the transcription start site, to contain the minimum number of bases or elements necessary to initiate transcription at a level detectable above the background. Within the promoter sequence, the transcription start site (conveniently defined, e.g., by mapping to a nuclease SI) and the protein-binding domain (consensus sequence) that leads to RNA polymerase binding are found.

[0103] The origin of the promoter may be natural or synthetic, and the origin of the promoter should not limit the scope of the invention as described herein. In other words, the promoter may be cloned directly from cells, or it may have been previously cloned from a different origin, or it may be synthetic.

[0104] As used herein, the term “transcriptional regulator” refers to a biological element that acts to prevent or inhibit the transcription of promoter-driven DNA sequences under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein), or to allow or stimulate the transcription of promoter-driven DNA sequences under certain environmental conditions (e.g., an inducer or enhancer).

[0105] As used herein, the term “enhancer” refers to a DNA sequence that increases transcription, for example, of a operably linked nucleic acid sequence. Enhancers can be located several kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, the pattern of DNA methylation, or changes in DNA structure. Numerous enhancers from various different origins are well known in the art and are available as cloned polynucleotides or within them (e.g., from contract laboratories, e.g., ATCC, and other commercial or personal origins). Some polynucleotides containing promoters (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, within, or downstream of the coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region mapped within an immunoglobulin (Ig) gene locus (such enhancers include, for example, heavy chain (mu) 5' enhancers, light chain (kappa) 5' enhancers, kappa and mu intron enhancers, and 3' enhancers (see, in general, Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353–363; and U.S. Patent No. 5,885,827)).

[0106] As used herein, the term “therapeutic switch promoter” (”TSP”) refers to a promoter that controls the expression of gene switch components. Gene switches and their various components are described in detail elsewhere herein. In certain embodiments, the TSP is constitutive, i.e., continuously active. A constitutive TSP may be constitutive-ubiquitous (i.e., generally functioning in any tissue or cell without the need for additional factors or regulators) or constitutive-tissue or cell-specific (i.e., generally functioning in a specific tissue or cell type without the need for additional factors or regulators). In certain embodiments of the present invention, the TSP is activated under conditions associated with a disease, disorder, or condition. In certain embodiments of the present invention involving two or more TSPs, the promoter may be a combination of a constitutive promoter and an activatable promoter. As used herein, “promoters activated under conditions associated with a disease, disorder, or condition” include, but are not limited to, disease-specific promoters, promoters that respond to specific physiological, developmental, differentiation, or pathological conditions, promoters that respond to specific biomolecules, and promoters that are specific to specific tissues or cell types associated with a disease, disorder, or condition, such as tumor tissue or malignant cells. TSPs may include sequences of naturally occurring promoters, modified sequences derived from naturally occurring promoters, or synthetic sequences (e.g., insertion of a response element into a minimal promoter sequence to alter the promoter's responsiveness).

[0107] A therapeutic switch promoter useful in the present invention may include any promoter useful for treating, restoring, or preventing a specific disease, disorder, or condition. Examples include, but are not limited to, promoters of genes that show increased expression only during a specific disease, disorder, or condition, and promoters of genes that show increased expression under specific cellular conditions (e.g., proliferation, apoptosis, pH changes, oxidative states, oxygen levels). In some embodiments in which the gene switch includes two or more transcription factor sequences, the specificity of the therapeutic method can be increased by combining the disease or condition-specific promoter with a tissue or cell-type-specific promoter that restricts the expression of the therapeutic product to the tissue in which it is expressed. Thus, tissue or cell-type-specific promoters are encompassed within the definition of a therapeutic switch promoter.

[0108] The term “ecdysone receptor-based” refers to a gene switch that includes at least a functional portion of a naturally occurring or synthetic ecdysone receptor ligand-binding domain and modulates gene expression in response to a ligand that binds to the ecdysone receptor ligand-binding domain. Examples of ecdysone response systems are described in U.S. Patent Nos. 7,091,038 and 6,258,603. In one embodiment, the system is the RheoSwitch® therapeutic system (RTS), which contains two fusion proteins: the DEF domain of a mutagenic ecdysone receptor (EcR) fused to a Gal4 DNA-binding domain, and the EF domain of a chimeric RXR fused to a VP16 transcriptional activation domain, both expressed under a constitutive promoter.

[0109] When RNA polymerase transcribes the coding sequence into mRNA, it is "under the control" of transcriptional and translational regulatory sequences in the cell, which are then trans-spliced ​​(if the coding sequence contains introns) and translated into the protein encoded by the coding sequence.

[0110] "Transcriptional and translational regulatory sequences" refer to DNA regulatory sequences, such as promoters, enhancers, and terminators, that provide expression of coding sequences in host cells. In eukaryotic cells, polyadenylation signals are regulatory sequences. Enhancers that may be used in embodiments of the present invention include, but are not limited to, SV40 enhancers, cytomegalovirus (CMV) enhancers, elongation factor 1 (EF 1) enhancers, yeast enhancers, and viral gene enhancers.

[0111] The terms “3' non-coding sequence” and “3' untranslated region (UTR)” refer to DNA sequences located downstream (3') of the coding sequence and may include polyadenylation [poly(A)] recognition sequences and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. Polyadenylation signals are typically characterized by influencing the addition of polyadenylate tracts to the 3' end of mRNA precursors.

[0112] As used herein, the term “regulatory region” refers to a nucleic acid sequence that regulates the expression of a second nucleic acid sequence. A regulatory region may include a sequence that is naturally responsible for the expression of a particular nucleic acid (homologous region), or it may include a sequence of a different origin that is responsible for the expression of a different protein or even a synthetic protein (heterologous region). In particular, the sequence may be a sequence of a prokaryotic, eukaryotic, or viral gene, or an induced sequence, that stimulates or represses the transcription of a gene in a specific or nonspecific manner, and in an inducible or non-inducible manner. A regulatory region may include an origin of replication, an RNA splice site, a promoter, an enhancer, a transcription termination sequence, and a signal sequence that directs a polypeptide to the secretory pathway of a target cell.

[0113] As used herein, the term “modulate” means to induce, reduce, or inhibit nucleic acid or gene expression, resulting in the induction, reduction, or inhibition of protein or polypeptide production, respectively.

[0114] As used herein, the term “inducible promoter” refers to a promoter whose activity is induced by the presence or absence of a transcription factor, such as a biological or abiotic factor. Inducible promoters are useful because the expression of a gene operably linked to them can be turned on or off at a particular stage of development of an organism or a particular tissue. Non-limiting examples of inducible promoters include alcohol-regulating promoters, tetracycline-regulating promoters, steroid-regulating promoters, metal-regulating promoters, pathogenesis-regulating promoters, temperature-regulating promoters, and light-regulating promoters. An inducible promoter may be part of a gene switch or a gene switch. An inducible promoter may be a gene switch ligand-inducible promoter. In some cases, an inducible promoter may be an ecdysone receptor-based gene switch of two small molecule ligand-inducible polypeptides.In some cases, gene switches are, but are not limited to, International Publication Nos. 2001 / 070816; International Publication Nos. 2002 / 029075; International Publication Nos. 2002 / 066613; International Publication Nos. 2002 / 066614; International Publication Nos. 2002 / 066612; International Publication Nos. 2002 / 066615; International Publication Nos. 2003 / 027266; ​​International Publication Nos. 2003 / 027289; International Publication Nos. 2005 / 108 Publication No. 617; International Publication No. 2009 / 045370; International Publication No. 2009 / 048560; International Publication No. 2010 / 042189; International Publication No. 2010 / 042189; International Publication No. 2011 / 119773; and International Publication No. 2012 / 122025; and U.S. Publication No. 7,091,038; U.S. Publication No. 7,776,587; U.S. Publication No. 7,807,417; U.S. Publication No. 8,202,718 Specification; Specification No. 8,105,825; Specification No. 8,168,426; Specification No. 7,531,326; Specification No. 8,236,556; Specification No. 8,598,409; Specification No. 8,715 ,959 specification; 7,601,508 specification; 7,829,676 specification; 7,919,269 specification; 8,030,067 specification; 7,563,879 specification; You can choose from ecdysone-based receptor components described in any of the systems described in Specification No. 8,021,878; Specification No. 8,497,093; Specification No. 7,935,510; Specification No. 8,076,454; Specification No. 9,402,919; Specification No. 9,493,540; Specification No. 9,249,207; and Specification No. 9,492,482.

[0115] As used herein, two or more individually operable gene regulatory systems are referred to as “orthogonal” if a) each modulation of a given system by its respective ligand at a selected concentration results in a measurable change in the magnitude of gene expression in that system, and b) regardless of whether the actual modulations are synchronous or sequential, the change is statistically significantly different from the change in expression of all other simultaneously operable systems in the cell, tissue, or organism. Preferably, the modulation of each individual operable gene regulatory system results in a change in gene expression that is at least twice as large, e.g., at least five times, ten times, 100 times, or 500 times larger than all other operable systems in the cell, tissue, or organism. Ideally, each modulation of a given system by its respective ligand at a selected concentration results in a measurable change in the magnitude of gene expression in that system, and no measurable change in expression of all other operable systems in the cell, tissue, or organism. In such cases, multiple inducible gene regulatory systems are referred to as “fully orthogonal.” Useful orthogonal ligand and orthogonal receptor-based gene expression systems are described in U.S. Patent Application Publication No. 2002 / 0110861.

[0116] As used herein, the term “gene switch” refers to a combination of a promoter-related response element and a ligand-dependent transcription factor-based system that modulates the expression of the gene into which the response element and promoter are incorporated in the presence of one or more ligands. The term “polynucleotide encoding a gene switch” refers to a combination of a promoter-related response element and a ligand-dependent transcription factor-based system that modulates the expression of the gene into which the response element and promoter are incorporated in the presence of one or more ligands. Closely regulated inducible gene expression systems or gene switches, such as EcR-based systems, are useful in a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, and functional genomics and regulation of traits in transgenic plants and animals. Such inducible gene expression systems may include ligand-induced heterologous gene expression systems.

[0117] As used herein, the term "CAP" or "cap" refers to a modified nucleotide, generally 7-methylguanosine (7meG-ppp-G) ligated at the 5' end of eukaryotic mRNA, typically from 3' to 5', which plays a necessary element in the normal translation initiation pathway during protein expression from that mRNA.

[0118] As used herein, the term “Sleeping Beauty (SB) transposon system” refers to a synthetic DNA transposon system for introducing DNA sequences into vertebrate chromosomes. Some exemplary embodiments of the system are described, for example, in U.S. Patent Nos. 6,489,458, 8,227,432, 9,228,180 and International Publication No. 2016 / 145146. The Sleeping Beauty transposon system comprises a Sleeping Beauty (SB) transposase and an SB transposon. In embodiments, the Sleeping Beauty transposon system may include an SB11 transposon system, an SB100X transposon system, or an SB110 transposon system.

[0119] As used herein, the terms “transposon” or “transposition element” (TE) refer to vector DNA sequences that can alter their position within the genome, sometimes by producing or reversing mutations, thereby changing the size of the cell’s genome. Transposition often results in duplication of TEs. Class I TEs are copied in two steps: first, they are transcribed from DNA to RNA, and the resulting RNA is then reverse-transcribed back into DNA. This copied DNA is then inserted into the genome at the new location. The reverse transcription step is catalyzed by a reverse transcriptase, which can be encoded by the TE itself. The characteristics of retrotransposons are similar to those of retroviruses, e.g., HIV. The cut-and-paste transposition mechanism of Class II TEs does not involve an RNA intermediate. Transposition is catalyzed by several transposase enzymes. Some transposases bind nonspecifically to any target site in DNA, while others bind to specific DNA sequence targets. Transposases perform alternating cuts at the target site, resulting in a single-stranded 5' or 3' DNA overhang (sticky end). This step excises the DNA transposon, which is then ligated to a new target site. This process involves the activity of DNA polymerase to fill the gap and DNA ligase to close the sugar-phosphate backbone. This results in duplication of the target site. The insertion site of a DNA transposon can be identified by a short series of repeat sequences that can be created by alternating cuts and filling by DNA polymerase in the target DNA, followed by a series of reverse repeat sequences that are crucial for subsequent transposase-mediated TE excision. Cut-and-paste TEs can overlap if their transposition occurs during the S phase of the cell cycle, when the donor site has already replicated but the target site has not yet. Transpositions can be classified as either autonomous or non-autonomous in both class I and class II TEs. Autonomous TEs can move on their own, while non-autonomous TEs require the presence of another TE to move. This is often because non-autonomous TEs lack transposases (for class II) or reverse transcriptases (for class I).

[0120] As used herein, the term “transposase” refers to an enzyme that binds to the end of a transposon and catalyzes the transposon’s movement to another part of the genome via a cut-and-paste mechanism or a replication-transposition mechanism.

[0121] As used herein, the terms “polypeptide,” “peptide,” “polypeptide construct,” and “peptide construct,” as well as their grammatical equivalents, refer to polymer compounds composed of covalently linked amino acid residues. “Mature protein” is a full-length protein, optionally including glycosylation or other modifications typical of a protein in a given cellular environment. Embodiments of the present invention, as disclosed herein, include the HPV antigen / antigenic polypeptides, peptides, and mature proteins described herein, and also include polynucleotides (DNA or RNA) encoding them. Polypeptides and proteins disclosed herein (including their functional fragments and functional variants) may contain synthetic amino acids instead of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, and indoline. Examples include -2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0122] As used herein, the term “polypeptide fragment” means a polypeptide whose amino acid sequence is shorter than that of a reference polypeptide and which contains the same amino acid sequence throughout the entire portion having these reference polypeptides. Such fragments may, where appropriate, be contained within a larger polypeptide of which they are part. Such fragments of polypeptides according to the present invention may have amino acid lengths of at least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300, or longer.

[0123] As used herein, the terms “isolated polypeptide,” “isolated peptide,” or “isolated protein” refer to polypeptides or proteins that are substantially free from compounds normally associated with them in their natural state (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). “Isolated” does not mean the exclusion of artificial or synthetic mixtures with other compounds, or the presence of impurities that do not impede biological activity, for example, resulting from incomplete purification, the addition of stabilizers, or formulation into pharmaceutically acceptable preparations.

[0124] As used herein, the terms “identical” or “sequence identity” in the context of two nucleic acid or amino acid sequences of polypeptides refer to residues in two sequences that are identical when aligned for maximum match across a defined comparison window. “Comparison window,” as used herein, refers to a segment of at least about 20, typically about 50 to about 200, and more commonly about 100 to about 150 consecutive positions over which the sequences can be compared to the same number of consecutive positions of a reference sequence after the two sequences have been optimally aligned. Methods for aligning sequences for comparison are well known in the art.The optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the alignment algorithm of Needleman and Wunsch, J Mal. Biol., 48:443 (1970); by the similarity search method of Pearson and Lipman, Proc. Nat. Acad Sci US.A., 85:2444 (1988); or by computer execution of these algorithms (including, but not limited to, CLUSTAL in the PC / Gene program from Intelligentics, Mountain View Calif, Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA, GAP, BESTFIT, BLAST, FASTA, and TFASTA); the CLUSTAL program is from Higgins and Sharp, Gene, Alignment is also adequately described in 73:237-244 (1988) and Higgins and Sharp, CABIOS, 5:151-153 (1989); Corpet et al., Nucleic Acids Res., 16:10881-10890 (1988); Huang et al., Computer Applications in the Biosciences, 8:155-165 (1992); and Pearson et al., Methods in Molecular Biology, 24:307-331 (1994). Alignment is also often performed by laboratory and manual alignment.

[0125] In one class of embodiments, the polypeptides herein are at least 80%, 85%, 90%, 98%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to a reference polypeptide or fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example. Similarly, nucleic acids can also be described by reference to an initiating nucleic acid, for example, a nucleic acid may be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identical to a reference nucleic acid or fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters, for example. When a molecule is said to have a certain percentage of sequence identity with a larger molecule, this means that, when the two molecules are optimally aligned, the percentage of residues in the smaller molecule will find matching residues in the larger molecule in the order in which the two molecules are optimally aligned.

[0126] As used herein, the term “identity percentage” is the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing their sequences, as is known in the art. In the art, “identity” also means the degree of sequence relevance between polypeptide or polynucleotide sequences, determined by matching strings of such sequences. "Identity" and "similarity" are not limited to those described above, or can be readily calculated by known methods, including, for example, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM, and Griffin, HG, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991). Methods for determining identity and similarity are systematized in publicly available computer programs. Sequence alignment and identity percentage calculations may be performed using sequence analysis software, such as the MegAlign (or more recently, MegAlign Pro) program from the LASERGENE Bioinformatics Computer Suite (DNASTAR Inc., Madison, Wis.).Multiple alignment of sequences may be performed using Clustal's alignment method (Higgins et al., CABIOS. 5:151 1989) with default parameters (gap penalty=10, gap length penalty=10). Default parameters for pairwise alignment using Clustal's method may be selected: KTUPLE 1, gap penalty=3, window=5, and DIAGONALS SAVED=5.

[0127] As used herein, the term “substantially similar” and its grammatical equivalents, applied to nucleic acids or amino acid sequences, mean that the nucleic acid or amino acid sequence has sequence identity of at least 90% or higher relative to a reference sequence, e.g., at least 95%, at least 98%, at least 99%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and at least 99.99%, relative to a reference sequence, using standard parameters and comparison programs described above, e.g., BLAST. The term “substantially identical” and its grammatical equivalent, applied to nucleic acids or amino acid sequences, means that a nucleic acid or amino acid sequence contains a sequence that, using standard parameters and a comparison program described above, e.g., BLAST, has at least 99% sequence identity with respect to a reference sequence, e.g., at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and at least 99.99%. For example, the BLASTN program (for nucleotide sequences) uses, by default, a word length (W) of 11, an expected value (E) of 10, M=5, N=-4, and comparisons of both strands. For amino acid sequences, the BLASTP program uses, by default, a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992)). The percentage of sequence identity is determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (which does not contain additions or deletions) for optimal alignment of the two sequences.The percentage is calculated by determining the number of positions that give the number of matched positions, where the same nucleic acid base or amino acid residue is present in both sequences, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. In embodiments, substantial identity exists over a region of sequence that is at least about 50 residues long, over a region of at least about 100 residues, and in embodiments, the sequences are substantially identical over at least about 150 residues. In embodiments, the sequences are substantially identical over the entire length of the coding region.

[0128] Where used herein, the term “functional fragment” or its grammatical equivalent is used herein to mean a portion, fragment, or segment of a biomolecule that retains essential functional characteristics or activity of the original biomolecule. The term “functional variant” or its grammatical equivalent is used herein to mean a modified form of a biomolecule that retains essential functional characteristics or activity of the original molecule, while exhibiting some degree of variation. This includes biomolecules that have been modified, for example, by genetic engineering or mutagenesis techniques, to introduce specific changes while preserving the overall functionality of the biomolecule. Functional variants may have one or more amino acid substitutions, insertions, or deletions compared to the original molecule, while still maintaining the desired biological activity or function. Techniques for obtaining these variants, including genetic (repression, deletion, mutation, etc.), chemical, and enzymatic techniques, are known to those skilled in the art. In one embodiment, a biological variant comprises at least about 14 monomers (e.g., nucleotides or amino acids).

[0129] As used herein, the term “homology” in all its grammatical and spelling variations refers to the percentage of identity between two polynucleotides or two polypeptide segments. The degree of sequence agreement between one segment and another can be determined by techniques known in the art. For example, homology can be determined by a direct comparison of sequence information between two polypeptide molecules by aligning the sequence information using readily available computer programs. Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form a stable double helix between homologous regions, followed by digestion by a single-strand specific nuclease and sizing of the digested fragments.

[0130] When used in the context of amino acid sequences, the term "substitution" refers to a variation in an amino acid sequence in which one amino acid is replaced by another. The nomenclature used to represent amino acid substitutions follows a standard format. For example, in "L50G," "L" represents the original amino acid leucine (abbreviated as "L"), "50" indicates the position of the amino acid in the amino acid sequence relative to its N-terminus (in this case, the amino acid is the 50th amino acid from the N-terminus of the sequence), and "G" indicates the substituted amino acid, in this example, glycine (abbreviated as "G"). Therefore, "L50G" represents a substitution in which leucine at position 50 (relative to its N-terminus) of the amino acid sequence is replaced by glycine.

[0131] As used herein, the terms “conservative amino acid substitution” or “conservative mutation” refer to the substitution of one amino acid with another amino acid that shares common properties. A functional method for defining the common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (see Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). According to such analysis, a group of amino acids can be defined as those within a group in which the amino acids preferentially exchange with each other, and therefore their effects on the overall protein structure are most similar to each other (Schulz, GE and Schirmer, RH, above). Examples of conservative mutations include amino acid substitutions of amino acids within the above subgroups, e.g., lysine and arginine, and vice versa, in which a positive charge can be maintained; glutamic acid and aspartic acid, and vice versa, in which a negative charge can be maintained; serine and threonine, in which free -OH can be maintained; and glutamine and asparagine, in which free -NH2 can be maintained. Examples of conservative amino acid substitutions are shown in the chart below.

[0132] [Table 2]

[0133] An amino acid sequence that differs from a reference amino acid sequence solely through conservative amino acid substitutions is referred to herein as a “conservatively substituted variant” of the reference sequence. Given the established knowledge and well-known techniques in protein science, determining the functional impact of a “conservatively substituted variant” compared to a reference amino acid sequence is well within the skill of those skilled in the art.

[0134] In some embodiments, the functional variant may be a conservatively substituted variant of the reference sequence. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 100 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 90 or fewer amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 80 or fewer amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 70 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 60 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 50 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference protein by 40 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 30 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 20 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by 10 or fewer conservative amino acid substitutions. In some embodiments, the conservatively substituted variant may differ from the reference sequence by 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 conservative amino acid substitution.In some embodiments, the conservatively substituted variant may differ from the amino acid sequence of the reference sequence by at least 100 and 150 conservative amino acid substitutions.

[0135] An amino acid sequence that differs from a reference amino acid sequence by at least one non-conservative amino acid substitution is referred to herein as a “non-conservatively substituted variant” of the reference sequence. As used herein, the term “non-conservative amino acid substitution” refers to amino acid substitutions between different groups, such as the substitution of tryptophan by lysine or serine by phenylalanine. In this case, it is preferable that the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, resulting in an increased biological activity of the functional variant compared to the homologous parent protein. The substitutability of amino acids is discussed in more detail, for example, LY Yampolsky and A. Stoltzfus, “The Exchangeability of Amino acids in Proteins,” Genetics 2005 Aug.; 170(4):1459-1472. Given the established knowledge and well-known techniques in protein science, determining the functional effect of non-conserved amino acid substitutions in a functional variant compared to a reference amino acid sequence is well within the skill of those skilled in the art.

[0136] In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least one non-conserved amino acid substitution. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 10 to 20 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 21 to 30 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 31 to 40 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 41 to 50 non-conserved amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 51 to 60 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 61 to 70 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 71 to 80 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 81 to 90 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by 91 to 100 non-conservative amino acid substitutions. In some embodiments, the functional variant may differ from the amino acid sequence of the reference sequence by at least 100 non-conservative amino acid substitutions.

[0137] As used herein, the term “antibody” refers to either a monoclonal antibody or a polyclonal antibody. “Monoclonal antibody,” as used herein, refers to an antibody produced by a single clone of a B cell and bound to the same epitope. In contrast, “polyclonal antibody” refers to a group of antibodies produced by different B cells and bound to different epitopes of the same antigen. A total antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, while each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have similar general structures, and each region contains four framework regions whose sequences are relatively conserved. The framework region is connected by three complementarity-determining regions (CDRs). These three CDRs, known as CDR1, CDR2, and CDR3, form the antibody's "hypervariable region," which is responsible for antigen binding.

[0138] As used herein, the terms “functional antibody fragment” and “functional fragment of an antibody” or their grammatical equivalents are used interchangeably to mean a portion, fragment, or segment of an antibody that retains essential functional characteristics or activity of the original antibody. In one embodiment, this activity is the ability to specifically bind to an antigen (see Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005) for general reference). A functional antibody fragment may include, for example, one or more CDRs, variable regions (or portions thereof), constant regions (or portions thereof), or a combination thereof. Non-limiting examples of functional antibody fragments include: (i) an antigen-binding fragment (Fab), which is a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide crosslinking in the stem region; (iii) a variable fragment ("Fv") consisting of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment joined by a synthetic linker that allows the two domains to synthesize a single polypeptide chain (e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778). Examples include dimers of polypeptide chains, (see 1998), and (v) dimers of polypeptide chains, where each polypeptide chain contains a VH connected to the VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimer molecule having two functional antigen-binding sites. Functional antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent No. 8,603,950.

[0139] As used herein, the term “antibody-like molecule” can be, for example, a protein that is a member of the Ig superfamily capable of selectively binding to a partner. MHC molecules and T cell receptors are such molecules. In one embodiment, the antibody-like molecule is a TCR. In one embodiment, the TCR is modified to increase its MHC binding affinity.

[0140] As used herein, the terms “antigen-recognizing moiety” or “antigen-recognizing domain” refer to a molecule or part of a molecule that specifically binds to an antigen. In one embodiment, the antigen-recognizing moiety is an antibody, an antibody-like molecule, or a fragment thereof, and the antigen is a tumor antigen.

[0141] As used herein, the term “immune cells” includes dendritic cells, macrophages, neutrophils, mast cells, eosinophils, basophils, natural killer cells, and lymphocytes (e.g., B and T cells).

[0142] As used herein, the terms “T cell” or “T lymphocyte” refer to a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on their cell surface. The “TCR” is a protein molecule found on the surface of T cells, and it is a type of leukocyte involved in adaptive immune responses. The variable domain of the TCR contains a highly pleomorphic loop called the complementarity-determining region (CDR), which is responsible for binding to peptide-presenting MHC. There are two main forms of TCR: αβ TCR and γδ TCR. Both forms consist of two protein chains, known as alpha (α) and beta (β) chains for αβ TCR and gamma (γ) and delta (δ) chains for γδ TCR. These chains together form a heterodimer structure. The majority of T cells in the human immune system express αβ TCR. The α and β chains of αβ TCRs are encoded by separate gene segments, which undergo recombination during T cell development to produce diverse TCR specificities. The α and β chains contain variable (V), diverse (D), and conjugating (J) gene segments, respectively, similar to the antibody gene rearrangement process. The combination of the V, D, and J gene segments contributes to the unique antigen-binding specificity of αβ TCRs. αβ TCRs recognize antigenic peptides presented in the context of major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells. In contrast to αβ TCRs, γδ TCRs are less dominant in the immune system but still play an important role. The γ and δ chains of γδ TCRs are also encoded by separate gene segments and undergo recombination during T cell development. The γδ TCR gene rearrangement process differs from that of αβ TCRs. γδ T cells often exhibit tissue-specific distributions and are found in epithelial tissues, such as the skin and intestines. γδ TCRs can recognize various antigens, including certain peptides and non-peptide molecules, independently of MHC presentation. Both αβ TCRs and γδ TCRs participate in and respond to immune surveillance, but they have different functions and specificities.While αβ TCRs are primarily involved in the recognition of peptides presented by major histocompatibility complex (MHC) molecules, γδ TCRs may possess a wider range of antigen recognition capabilities.

[0143] TCRs and constructs encoding TCRs that recognize MHC-antigen complexes can be created and introduced into T cells (known as TCR T cells), and the following TCR-peptide-MHC interactions can be utilized to induce an immune response. Greenbaum et al., Cancer Immunol Res 1 November 2021; 9 (11): 1252-1261. There is interest in the use of TCRs with higher affinity than the normal range for peptide-MHC antigens (type I), referred to as high-affinity TCRs, to generate soluble TCRs that can be used directly against target cells, either 1) to drive the activity of CD4 helper T cells (which lack the CD8 coreceptor), or 2) by attaching to "effector" molecules (e.g., antibody Fc region, toxic drug, or antibody scFv for forming bispecific proteins, e.g., anti-CD3 antibody) (Ashfield and Jakobsen, IDrugs, 9, 554-9 (2006); Foote and Eisen Proc Natl Acad Sci USA, 97:10679-81 (2000); Holler et al., Proc Natl Acad Sci USA, 97:5387-92 (2000); Molloy et al., Curr Opin Pharmacol, 5:438-43 (2005); Richman and Kranz, Biomol Eng, 24:361-73 (2007)). This approach may also overcome the problem faced by some cancer patients due to their T cells not expressing TCRs with sufficient specificity and binding affinity to basic tumor antigens. For example, more than 300 MHC-restricted T cells that define tumor antigens have been identified (Cheever et al., Clin Cancer Res. 2009;15(17):5323-5337). These tumor antigens include mutated peptides, differentiation antigens, and overexpressed antigens, all of which function as targets for therapy.Since most cancer antigens described to date are derived from intracellular proteins that can only be targeted on the cell surface in the context of MHC molecules, TCRs are ideal candidates for therapy because they have evolved to recognize this class of antigens. Similarly, TCRs can detect peptides derived from viral proteins that are processed naturally in infected cells and displayed on the cell surface by MHC molecules. However, patients with these diseases may not have optimized TCRs that bind to and destroy infected cells. Finally, in a highly specific manner, TCRs can be used as receptor antagonists for autoimmune targets or as delivery agents to immunosuppress local immune cell responses, thereby avoiding general immunosuppression.

[0144] As used herein, the term “helper T cells” (TH or Th cells) refers to cells that assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ T cells because they express the CD4 glycoprotein on their surface. Helper T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist in the active immune response. These cells can differentiate into one of several subtypes, including THI, TH2, TH3, TH9, TH17, TH22, or TFH (follicular helper T cells), which secrete different cytokines that promote different types of immune responses. Signaling from APCs directs T cells to specific subtypes.

[0145] As used herein, the terms "cytotoxic T cells" (TC cells, or CTLs) or "cytotoxic T lymphocytes" are cells that destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. By IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated into anergic states that prevent autoimmune diseases.

[0146] As used herein, the term "memory T cells" refers to a subset of antigen-specific T cells that persist for long periods after an infection has resolved. Upon re-exposure to their cognate antigen, they rapidly expand into large numbers of effector T cells, thus providing an immune system with memory of past infections. Memory T cells include three subtypes: central memory T cells (TcM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface proteins CD45RO, CD45RA, and / or CCR7.

[0147] As used herein, the term "regulatory T cells" (Treg cells), previously known as suppressor T cells, refers to T cells that play a role in maintaining immune tolerance. Their primary roles are to shut down T cell-mediated immunity in the direction of ending an immune response and to suppress autoreactive T cells that have avoided the process of negative selection in the thymus.

[0148] As used herein, the term "natural killer T cell" (NKT cell, which should not be confused with natural killer cells of the innate immune system) refers to cells that bridge the adaptive immune system to the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CD. Ibid. When activated, these cells can perform functions attributable to both helper T cells (TH) and cytotoxic T cells (TC) (i.e., cytokine production and release of cytolytic / cell-killing molecules). They can also recognize and eliminate some tumor cells and cells infected with herpes viruses.

[0149] As used herein, the term "proliferative disorder" refers to a unified concept that includes cancer and in which excessive cell growth and / or turnover of the intracellular matrix significantly contributes to the pathogenesis of the disorder.

[0150] "Patient" or "subject" as used herein refers to a mammalian subject diagnosed with, having, or suspected of having a disease or disorder such as cancer. In some embodiments, the term "patient" refers to a mammalian subject having a higher likelihood of developing a proliferative disorder such as cancer than the average likelihood. Exemplary patients can be humans, apes, dogs, pigs, cows, cats, horses, goats, sheep, rodents, and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female. "A patient in need thereof" or "a subject in need thereof" is herein referred to as a patient diagnosed with or suspected of having a disease or disorder, for example, but not limited to human papillomavirus (HPV) infection.

[0151] "Administering" is used herein to refer to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administration of a composition, e.g., by injection, may be carried out by intravenous, subcutaneous, intradermal, intraperitoneal, or intramuscular injection. One or more such routes may be used. Parenteral administration may be, for example, by bolus injection or by progressive perfusion over time. Alternatively, or in combination, administration may be by an oral route. In addition, administration may also be by surgical deposition or placement of a medical device. Pharmaceutical compositions may include the compositions of the present invention described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers, such as neutral buffered saline or phosphate-buffered saline; carbohydrates, such as glucose, mannose, sucrose, or dextran, or mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0152] As used herein, the term “therapeutic product” refers to a therapeutic polypeptide or therapeutic polynucleotide that confers a beneficial function to a host cell on which such product is expressed. Therapeutic polypeptides may include, but are not limited to, small peptides of approximately three amino acids in length, single-stranded or multi-stranded proteins, and fusion proteins. Therapeutic polynucleotides may include, but are not limited to, antisense oligonucleotides, small interfering RNAs, ribozymes, and RNA external guide sequences. Therapeutic products may include naturally occurring sequences, synthetic sequences, or combinations of natural and synthetic sequences.

[0153] As used herein, the terms “treatment,” “to treat,” or their grammatical equivalents refer to obtaining a desired pharmacological and / or physiological effect. In embodiments, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms or pathological signs that may contribute to the disease. For this purpose, the method of the present invention comprises the step of administering a therapeutically effective amount of a composition of the present invention expressing the nucleic acid sequence of the present invention, or a vector comprising the nucleic acid sequence of the present invention.

[0154] As used herein, “treatment interval” refers to a treatment cycle, for example, a course of administration of a therapeutic agent that may be repeated on a regular schedule. In some embodiments, the dosage regimen may have one or more periods during the treatment interval in which no therapeutic agent is administered.

[0155] As used herein, “dosage regimen” or “medication regimen” includes a treatment regimen based on a determined set of doses. The terms “dose” and “medicate” as used herein refer to the administration of a substance to achieve a therapeutic objective (e.g., treatment of a tumor).

[0156] The terms “administered in combination,” “simultaneously administered,” “to administer simultaneously,” or “to provide simultaneously,” as used herein, mean that two (or more) different treatments are delivered to a subject during the course of the subject’s suffering due to a disease or disorder, for example, that two or more treatments are delivered after the subject has been diagnosed with a disease or disorder and before the disease or disorder is cured or eliminated, or before the treatments are discontinued for any other reason. In some embodiments, the delivery of one treatment is still taking place when the delivery of a second treatment is initiated, resulting in an overlap in the duration of administration. This is sometimes referred to herein as “simultaneous” or “simultaneous delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment is initiated. In some embodiments of either case, the treatments are more effective due to the combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is observed with less of the second treatment, or the second treatment reduces symptoms to a greater extent than would be observed if the second treatment were administered in the absence of the first treatment, or a similar situation is observed with the first treatment. In some embodiments, the delivery is such that the reduction of symptoms or other parameters relating to the impairment is greater than that observed with a treatment delivered in the absence of the other. The effects of the two treatments may be partially additive, entirely additive, or more than additive. The delivery may be such that the effect of the delivered first treatment is still detectable when the second treatment is delivered.

[0157] In some embodiments of the present invention, the first and second treatments may be administered simultaneously (e.g., at the same time) or sequentially, using the same or separate compositions. Sequential administration refers to the administration of one treatment prior to the administration of an additional (e.g., secondary) treatment (e.g., immediately before; less than 5, 10, 15, 30, 45, or 60 minutes before; 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 48, 72, 96 hours or longer before; 4, 5, 6, 7, 8, 9 days or longer before; or 1, 2, 3, 4, 5, 6, 7, 8 weeks or longer before). The order of administration of the first and secondary treatments may also be reversed.

[0158] The terms “therapeutic effective dose,” “therapeutic dose,” “immunological effective dose,” “antiotumor effective dose,” and “tumor inhibitory effective dose,” or their grammatical equivalents, refer to an effective dose, which is the amount and duration of medication required to achieve the desired therapeutic outcome. The therapeutic effective dose may vary depending on factors such as the disease state, the age, sex, and weight of the individual, and the ability of the compositions described herein to induce a desired response in one or more subjects.

[0159] Alternatively, the pharmacological and / or physiological effects of administering one or more compositions described herein to a patient or subject may be “preventive,” meaning the effect completely or partially prevents the disease or its symptoms. “Preventive effective dose” refers to the effective dose and duration required to achieve the desired preventive outcome (e.g., prevention of disease or prevention of signs of a target condition). I. Vectors

[0160] Gene therapy, which involves introducing a transgene expressing an exogenous protein into a target (e.g., via vaccination), has demonstrated its usefulness in treating diseases and disorders in those who need it. For such introduction, vectors, such as viral vectors, containing the transgene encoding such a protein are typically used.

[0161] In part, this invention relates to vectors containing expression cassettes and expression cassettes containing transgenes that encode HPV antigen design.

[0162] In certain embodiments, the vector is a plasmid.

[0163] Another suitable vector is an integrated expression vector. Such a vector may be randomly integrated into the DNA of a host cell or may contain a recombination site that enables specific recombination between the expression vector and the chromosomes of the host cell. Such an integrated expression vector can utilize endogenous expression regulatory sequences on the chromosomes of the host cell to produce the expression of the desired protein. Examples of vectors that integrate in a site-specific manner include, for example, the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNATM5 / FRT), or components of the cre-lox system, which can be found in, for example, the pExchange-6 Core vector from Stratagene (La Jolla, Calif.). Examples of vectors that integrate randomly into host cell chromosomes include, for example, pcDNA3.1 from Invitrogen (Carlsbad, Calif.) (when introduced in the absence of the T antigen), and pCI or pFN10A(ACT)FLEXITM from Promega (Madison, Wis.). A. Methods for introducing nucleic acids into cells

[0164] Methods for introducing and expressing genes in cells are well known. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means. Biological methods for introducing a desired polynucleotide into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, for example. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362. 1.Physical method

[0165] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle guns, microinjection, and electroporation. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (2001). In embodiments, the method for introducing polynucleotides into host cells is calcium phosphate transfection or polyethyleneimine (PEI) transfection.

[0166] In some embodiments, the method for introducing polynucleotides into host cells is electroporation. Electroporation is a technique that uses electrical pulses to temporarily increase the permeability of the cell membrane, allowing the uptake of nucleic acid molecules into the cell. This process enhances the delivery and expression of biological materials (e.g., peptides or nucleic acids) in the target cells, potentially improving the immune response to HPV. In some embodiments, the biological material is the HPV antigen. In other embodiments, the biological material is the nucleic acid encoding the HPV antigen.

[0167] The electroporation buffer may contain water, sugar, sugar alcohol, chloride salt, and buffering agent. The pH, conductivity, and osmolality of the buffer are carefully controlled. The buffer may be used with UltraPorator® electroporation apparatus and cartridges. UltraPorator® electroporation apparatuses are designed for the rapid production of gene and cell therapies and can be used as a scale-up and commercialization solution for decentralized cell production. See, for example, PCT / US20 / 59984 (filed November 11, 2020) and U.S. Patent Application No. 17 / 095,028 (filed November 11, 2020).

[0168] In some embodiments, a suspension is formed by combining human-derived cells with exogenous biological material in a buffer, and then an electric current is applied to the suspension to facilitate the introduction of the biological material into the cells. The voltage pulse has an electric field strength of 1–10 kV / cm, a duration of 5–250 μs, and at least 2 A / cm 2It may have a current density of. Using the method, biological materials, such as nucleic acids, peptides, polypeptides, proteins, enzymes, or RNPs, can be introduced into primary human blood cells, pluripotent progenitor cells, fibroblasts, and endothelial cells. In some embodiments, the method can be used to introduce bioactive materials into primary human blood cells, pluripotent progenitor cells of human blood, as well as primary human fibroblasts and endothelial cells. In some embodiments, the cells are human blood cells, such as immune cells. In certain embodiments, the immune cells are neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes (B cells and T cells), or some combination thereof. In some embodiments, the lymphocytes are T cells. In certain embodiments, the cells are obtained from a patient.

[0169] In some embodiments, the transfection yield and the recovery yield of transfected cells using the electroporation buffer may be significantly higher than those obtained using the control buffer. In some embodiments, the transfection yield using the buffer of the present invention is at least about 1.1 times the transfection yield by the control buffer, and is, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 2.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0 times higher than the transfection yield of the control buffer.

[0170] In some of the methods described herein, the HPV antigen is administered to a subject. In some embodiments, these methods include the step of introducing the nucleic acid molecule of the present invention to a subject, followed by electroporation. In specific embodiments, the nucleic acid molecule (e.g., a plasmid encoding the antigen of interest or a therapeutic protein) is injected into the target tissue of the subject, e.g., skin or muscle, using a conventional needle or needle-free injection device. Immediately after injection, a handheld electroporation device is applied to the injection site, e.g., by bringing it into contact with the skin or tissue. The device delivers a short, controlled electrical pulse to the tissue, creating transient pores in the cell membrane, making them more permeable to the nucleic acid molecule, which then enters the cell through the pores created by electroporation. Once inside the cell, the nucleic acid molecule is translated into the desired antigen or therapeutic protein. The produced antigen stimulates an immune response, which may provide protection against the targeted pathogen. In the case of therapeutic proteins, they may exert their intended effects within the cell or tissue.

[0171] In some embodiments, the method may include a step of administering a single nucleic acid molecule, e.g., multiple copies of a single plasmid, or two or more different nucleic acid molecules, e.g., multiple copies of two or more different plasmids. The number of different nucleic acid molecules administered may vary depending on the specific application and may include two, three, four, five, six, seven, eight, nine, ten, or more distinct nucleic acid sequences. This approach enhances the immune response by enabling the delivery of multiple HPV antigens or the simultaneous delivery of additional immunostimulators.

[0172] Gene constructs containing nucleic acids encoding the HPV antigen can be administered using a variety of methods, including electroporation devices, conventional syringes, standard needles, side-port needles (as described in U.S. Patent Application Publication No. 2023 / 0017972), needle-free injection devices, or "microprojectile bombardment gene guns." Each of these methods has its advantages and can be selected based on factors such as target tissue, desired level of gene expression, and specific application.

[0173] Several minimally invasive electroporation devices and methods have been described in the literature. These include devices and methods disclosed in U.S. Patent Publication No. 20080234655; U.S. Patent No. 6,520,950; U.S. Patent No. 7,171,264; U.S. Patent No. 6,208,893; U.S. Patent No. 6,009,347; U.S. Patent No. 6,120,493; U.S. Patent No. 7,245,963; U.S. Patent No. 7,328,064; U.S. Patent No. 6,763,264; and U.S. Patent No. 20240123052. These devices and methods are designed to efficiently deliver nucleic acid molecules to cells while minimizing tissue damage and discomfort to the target. By using these minimally invasive electroporation techniques, nucleic acids encoding HPV antigens can be efficiently introduced into target cells, potentially leading to HPV antigen production and stimulation of an immune response. 2.Chemical method

[0174] Chemical methods for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). B. Virus-based delivery systems

[0175] Virus-based delivery systems for delivering nucleic acids, such as viral vectors, are also provided herein. Representative viral vectors include adeno-associated virus vectors, adenovirus vectors, retrovirus vectors, and herpesvirus-based vectors. Viral vectors may be used as delivery vehicles for nucleic acids encoding therapeutic molecules, such as anti-inflammatory agents, while also avoiding immune surveillance by host cells. Retroviruses, adenoviruses, adeno-associated viruses (AAVs), and herpes simplex viruses are all suitable for viral vector applications. Robbins et al., Pharmacology & Therapeutics, 80:35-47 (1998). In particular, recombinant adenovirus vectors provide high levels of transgene expression, where the vector remains as episomal DNA without integration into the host genome. High transduction efficiency and high levels of short-term gene expression make adenovirus vectors ideal for gene therapy and vaccine applications. Furthermore, these viral vectors may also be deficient in replication by deleting essential viral genes and replacing them with expression cassettes containing exogenous therapeutic genes.

[0176] Viral vectors that are not infectious in humans, or have been engineered to remove or inactivate their infectious properties, are desirable for use in gene therapy because they are efficient in delivering transgenes and can deliver high nucleic acid payloads to dendritic cells.

[0177] The effectiveness of treatments using viral vectors, however, is limited by their immunogenicity. For example, human adenovirus vectors are commonly used in gene therapy; however, since the majority of the US population is exposed to the wild-type form of such viruses, many in the population have pre-existing immunity to them. As a result, such vectors and the transgenes they carry are rapidly removed from the bloodstream. Furthermore, the immunogenicity of such vectors limits their effectiveness in the case of repeated administration. 1. Retroviral vectors

[0178] In certain embodiments, the viral vector is a retroviral vector, such as a lentiviral vector. Retroviral vectors are preferred tools for achieving long-term gene transfer because they allow for the long-term stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as mouse leukemia virus, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of lower immunogenicity. 2. Adeno-associated virus vector

[0179] In certain embodiments, the viral vector is an adeno-associated virus vector. Such vectors are derived from adeno-associated viruses. An advantage of using such vectors is that they have low immunogenicity in humans. Another advantage of using such vectors is that they are small and compact, and therefore can be used efficiently when delivering genes to cells. However, their size also limits the capacity of their payload compared to the use of adenovirus vectors. They are also more difficult to produce compared to adenovirus vectors. In addition, they have a narrow tissue tropism. 3. Herpesvirus-based vectors

[0180] In certain embodiments, the viral vector is a herpesvirus-based vector. Such vectors are derived from herpes simplex virus (HSV). Such vectors are known to be able to infect a wide variety of cell types and to have long persistence in the host. However, they are more difficult to manipulate compared to adenovirus vectors. 4. Adenovirus-based vectors

[0181] In certain embodiments, the viral vector is an adenovirus vector. Such vectors are derived from adenoviruses, such as human adenovirus (e.g., human Ad5 adenovirus), triadenovirus, or gorilla adenovirus. Adenoviruses are generally associated with benign conditions in humans, and the genomes of adenoviruses isolated from various species, including humans, have been extensively studied. Adenovirus vectors are advantageous because they can infect a wide variety of cell types. They are also relatively easy to handle and can carry high payloads.

[0182] Adenovirus vectors can be produced with high titers and efficiently transfer DNA into replicating and non-replicating cells. Adenovirus vector genomes can be constructed using any species, strain, subtype, mixture of species, strains, or subtypes, or chimeric adenoviruses as the source of vector DNA. Adenovirus stocks that can be used as the source of adenoviruses can be amplified from adenovirus serotypes 1 to 51, currently available from the United States Cell Culture Lineage Preservation Center (ATCC, Manassas, Va.), or from any other serotype of adenovirus available from any other source. For example, an adenovirus could belong to subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenovirus serotype.

[0183] An adenovirus vector can be any adenovirus vector that can grow in a cell, which lies in some prominent (but not necessarily substantial) part of the adenovirus genome derived from or based on. An adenovirus vector can be based on the genome of any suitable wild-type adenovirus. In certain embodiments, the adenovirus vector is derived from the genome of a wild-type adenovirus of group C, particularly serotype 2 or 5. Adenovirus vectors are well known in the art, for example, in U.S. Patent Nos. 5,559,099, 5,712,136, 5,731,190, 5,837,511, 5,846,782, 5,851,806, 5,962,311, 5,965,541, 5,981,225, 5,994,106, 6,020,191, and 6,113,913, International Publication Nos. 95 / 34671, 97 / 21826, and 00 / 00628, as well as in Thomas Shenk, “Adenoviridae and their Replication,” and MS This is described in Horwitz, “Adenoviruses,” Chapters 67 and 68, Virology, BN Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996).

[0184] Adenovirus is a medium-sized (90 - 100 nm), non-enveloped icosahedral virus that contains approximately 36 kb of double-stranded DNA. The adenovirus capsid mediates important interactions at the initial stage of cell infection by the virus and is required to package the adenovirus genome at the end of the adenovirus life cycle. The capsid contains 252 capsomeres, which include 240 hexons, 12 penton base proteins, and 12 fibers. Ginsberg et al., Virology, 28: 782 - 783 (1966). The hexon contains three identical proteins, i.e., polypeptide II. Roberts et al., Science, 232: 1148 - 1151 (1986). The penton base contains five identical proteins, and the fiber contains three identical proteins. Proteins IIIa, VI, and IX are present in the adenovirus coat and are thought to stabilize the virus capsid. Stewart et al., Cell, 67: 145 - 54 (1991) and Stewart et al., EMBO J., 12(7): 2589 - 99 (1993). Except for pIX, the expression of capsid proteins is dependent on the adenovirus polymerase protein. Thus, the major components of adenovirus particles are expressed from the genome only when the polymerase protein gene is present and expressed.

[0185] Some features of adenoviruses make them ideal as vehicles for transferring genetic material into cells for therapeutic applications. For example, adenoviruses can be produced at high titers (e.g., about 10 13Adenoviruses can be produced in particle units (PUs) and can deliver genetic material to non-replicating and replicating cells. In addition, adenovirus genomes can be engineered to carry large amounts of exogenous DNA (up to approximately 8 kb), and adenovirus capsids can further enhance the delivery of longer sequences. (Curiel et al., Hum. Gene Ther., 3: 147-154 (1992)). Furthermore, adenoviruses generally do not integrate into host cell chromosomes but are instead maintained as linear episomes, thereby minimizing the possibility that recombinant adenoviruses may interfere with normal cellular function.

[0186] Adenoviruses may be modified, for example, by methods known in the art, to be used as adenovirus vectors, for example, gene delivery vehicles. Adenoviruses and adenovirus vectors may be replicable, conditionally replicable, or lacking in replication.

[0187] A replicable adenovirus or adenovirus vector can replicate in a typical host cell, i.e., a cell typically susceptible to adenovirus infection. A replicable adenovirus or adenovirus vector may have one or more mutations (e.g., one or more deletions, insertions, and / or substitutions) in its adenovirus genome compared to wild-type adenovirus, without inhibiting viral replication in the host cell. For example, an adenovirus or adenovirus vector may have a partial or complete deletion of the early adenovirus region, known as the E3 region, which is not essential for the replication of the adenovirus or adenovirus genome.

[0188] A conditionally replicating adenovirus or adenovirus vector is an adenovirus or adenovirus vector that is engineered to replicate under certain conditions. For example, a replication-essential gene function, such as a gene function encoded by an adenovirus early region, can be operably linked to an inducible, repressive, or tissue-specific transcriptional regulatory sequence, such as a promoter. In such embodiments, replication requires the presence or absence of a specific factor that interacts with the transcriptional regulatory sequence. Conditionally replicating adenovirus vectors are further described in U.S. Patent No. 5,998,205.

[0189] A replication-deficient adenovirus or adenovirus vector is an adenovirus or adenovirus vector that requires the completion of one or more gene functions or regions of the adenovirus genome necessary for replication, for example, as a result of a deletion in one or more replication-essential gene functions or regions, so that the adenovirus or adenovirus vector does not replicate in typical host cells, particularly human cells, to which it infects.

[0190] Deletion of gene function or genomic region, as used herein, is defined as a disruption (e.g., deletion) of sufficient genetic material in the adenovirus genome, such as when the nucleic acid sequence is disrupted (e.g., deleted) in whole or in part, eliminating or impairing the function of the gene (e.g., reducing the function of the gene product to about half, one-fifth, one-tenth, one-twentieth, one-thirtieth, or one-fiftieth or less). Deletion of an entire gene region is often not necessary for disrupting the function of replication-essential genes. However, removal of a large portion of one or more gene regions may be desirable for the purpose of providing sufficient space in the adenovirus genome for one or more transgenes. While deletion of gene material is preferred, mutation of gene material by addition or substitution is also appropriate for disrupting gene function. Essential replication gene functions are gene functions required for adenovirus replication (e.g., proliferation) and are encoded by, for example, early adenovirus regions (e.g., E1, E2, and E4 regions), late regions (e.g., L1, L2, L3, L4, and L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA-1 and / or VA-RNA-2).

[0191] Whether an adenovirus or adenovirus vector is replicable or replication-deficient, an adenovirus or adenovirus vector typically holds at least a portion of the adenovirus genome. An adenovirus or adenovirus vector may contain any portion of the adenovirus genome, including protein-coding regions and / or non-protein-coding regions. An adenovirus or adenovirus vector may contain, for example, at least one nucleic acid sequence encoding an adenovirus protein. An adenovirus or adenovirus vector may contain a nucleic acid sequence encoding any suitable adenovirus protein, for example, a protein encoded by any one of the early region genes (i.e., E1A, E1B, E2A, E2B, E3, and / or E4 regions), or a protein encoded by any one of the late region genes encoding viral structural proteins (i.e., L1, L2, L3, L4, and L5 regions).

[0192] It should be recognized that deletions in different regions of adenovirus vectors can alter the mammalian immune response. In particular, deletions in different regions can reduce the inflammatory response induced by adenovirus vectors. Furthermore, the coat protein of adenovirus vectors can be modified to reduce the ability or inability of the adenovirus vector to be recognized by neutralizing antibodies against the wild-type coat protein, as described in International Publication No. 98 / 40509.

[0193] In certain embodiments, the adenovirus or adenovirus vector comprises one or more nucleic acid sequences encoding a pIX protein, a DNA polymerase protein, a penton protein, a hexon protein, and / or a fiber protein. The adenovirus or adenovirus vector may include a full-length nucleic acid sequence encoding the full-length amino acid sequence of an adenovirus protein. Alternatively, the adenovirus or adenovirus vector may include a portion of a full-length nucleic acid sequence encoding a portion of the full-length amino acid sequence of an adenovirus protein. The “portion” of the amino acid sequence comprises at least three amino acids (e.g., about 3 to about 1,200 amino acids). Preferably, the "part" of the amino acid sequence contains 3 or more amino acids (for example, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, or 50 or more), but includes fewer than 1,200 amino acids (for example, 1,000 or less, 800 or less, 700 or less, 600 or less, 500 or less, 400 or less, 300 or less, 200 or less, or 100 or less). Preferably, a portion of the amino acid sequence is defined by approximately 3 to approximately 500 amino acids (e.g., approximately 10, 100, 200, 300, 400, or 500 amino acids), approximately 3 to approximately 300 amino acids (e.g., approximately 20, 50, 75, 95, 150, 175, or 200 amino acids), or approximately 3 to approximately 100 amino acids (e.g., approximately 15, 25, 35, 40, 45, 60, 65, 70, 80, 85, 90, 95, or 99 amino acids), or any two of the aforementioned values. More preferably, a “portion” of the amino acid sequence includes approximately 500 amino acids or less (e.g., approximately 3 to approximately 400 amino acids, approximately 10 to approximately 250 amino acids, or approximately 50 to approximately 100 amino acids, or any two of the aforementioned values).

[0194] The adenovirus pIX protein is present in the adenovirus capsid and has been shown to enhance hexon nocamer interactions, making it essential for full-length genome packaging. For example, Boulanger et al., J Gen. Virol., 44: 783-800 (1979); Horwitz MS, “Adenoviridae and their replication” in Virology, 2 nd See, ed., BN Fields et al. (eds.), Raven Press, Ltd., New York, pp. 1679-1721 (1990), Ghosh-Choudhury et al., EMBO J., 6: 1733-1739 (1987), and van Oostrum et al., J. Virol., 56: 439-448 (1985). In addition to its contribution to the adenovirus structure, pIX has also been shown to exhibit transcriptional properties, such as stimulation of adenovirus major late promoter (MLP) activity. See, for example, Lutz et al., J. Virol., 71(7): 5102-5109 (1997). The nucleic acid sequences encoding all or part of the adenovirus pIX protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470.

[0195] The adenovirus DNA polymerase protein is essential for viral DNA replication both in vitro and in vivo. The polymerase is co-purified in complex with a terminal protein (TP) precursor (pTP), which is covalently attached to the 5' end of adenovirus DNA. (Field et al., J. Biol. Chem., 259: 9487-9495 (1984)). Both adenovirus DNA polymerase and pTP are encoded by the E2 region. The polymerase protein is required for the expression of all structural proteins except pIX. Without the gene sequence for the polymerase protein, the polymerase protein cannot be produced. As a result, the viral genome will not replicate, the major late promoter will not be activated, and the capsid protein will not be expressed. The nucleic acid sequences encoding all or part of the adenovirus DNA polymerase protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470. Nucleic acid sequences encoding all or part of the adenovirus DNA polymerase protein include, for example, SEQ ID NOs: 7 and SEQ ID NOs: 2. Amino acid sequences containing full-length adenovirus DNA polymerase or a portion thereof include, for example, SEQ ID NOs: 17 and SEQ ID NOs: 12.

[0196] The adenovirus hexon protein is the largest and most abundant protein in the adenovirus capsid. The hexon protein is essential for the construction of the viral capsid, the determination of the icosahedral symmetry of the capsid (which then defines limitations on capsid volume and DNA packaging size), and the integrity of the capsid. In addition, the hexon is a primary target for modification to reduce the neutralization of adenovirus vectors. See, for example, Gall et al., J. Virol., 72: 10260-264 (1998) and Rux et al., J. Virol., 77(17): 9553-9566 (2003). While the main structural features of the hexon protein are shared by adenoviruses across serotypes, the hexon protein differs in size and immunological properties between serotypes. Jornvall et al., J. Biol. Chem., 256(12): 6181-6186 (1981). A comparison of 15 adenovirus hexon proteins revealed that the main antigenic and serotype-specific regions of the hexon appear to be located in loops 1 and 2 (i.e., LI or l1 and LII or l2, respectively), where there are seven distinct hypervariable regions (HVR1-HVR7) of varying lengths and sequences among adenovirus serotypes. Crawford-Miksza et al., J. Virol., 70(3): 1836-1844 (1996). Nucleic acid sequences encoding all or part of the adenovirus hexon proteins are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470. Nucleic acid sequences encoding all or part of the adenovirus hexone protein include, for example, SEQ ID NOs: 9 and SEQ ID NOs: 4. Amino acid sequences containing the full-length adenovirus hexone protein or a portion thereof include, for example, SEQ ID NOs: 19 and SEQ ID NOs: 14.

[0197] Adenovirus fiber protein is a homotrimer of adenovirus polypeptide IV, possessing three domains: tail, shaft, and knob. Devaux et al., J. Molec. Biol., 215: 567-88 (1990), Yeh et al., Virus Res., 33: 179-98 (1991). Fiber protein primarily mediates viral binding to receptors on the cell surface via the knob and shaft domains. Henry et al., J. Virol., 68(8): 5239-46 (1994). The amino acid sequence for trimerization appears to be necessary for the amino terminus of the fiber (tail) to be located in the knob and to properly associate with penton bases. Novelli et al., Virology, 185: 365-76 (1991). In addition to cell receptor recognition and penton base binding, fiber contributes to serotype identity. Fiber proteins from different adenovirus serotypes are quite different. See, for example, Green et al., EMBO J., 2: 1357-65 (1983), Chroboczek et al., Virology, 186: 280-85 (1992), and Signas et al., J. Virol., 53: 672-78 (1985). Therefore, fiber proteins have multiple important functions in the adenovirus life cycle. Nucleic acid sequences encoding all or part of adenovirus fiber proteins are described, for example, in International Publication Nos. 2019 / 173465 and International Publication Nos. 2022 / 115470. Examples of nucleic acid sequences encoding all or part of adenovirus fiber proteins include SEQ ID NOs. 10 and SEQ ID NOs. 5. Examples of amino acid sequences containing full-length adenovirus fiber proteins or parts thereof include SEQ ID NOs. 20 and SEQ ID NOs. 15.

[0198] The adenovirus penton base protein is located at the vertices of the icosahedral capsid and contains five identical monomers. The penton base protein provides the structure for crosslinking hexon proteins on multiple faces of the icosahedral capsid and provides an essential interface for the incorporation of fiber proteins into the capsid. Each monomer of the penton base contains an RGD tripeptide motif. Neumann et al., Gene, 69: 153-157 (1988). The RGD tripeptide mediates binding to αv integrin, and adenoviruses with point mutations in the RGD sequence of the penton base limit their ability to infect cells. Bai et al., J. Virol., 67: 5198-5205 (1993). Therefore, the penton base protein is essential for the structure of the capsid and for the maximum efficiency of viral-cellular interaction. Nucleic acid sequences encoding all or part of the adenovirus penton base protein are described, for example, in International Publication No. 2019 / 173465 and International Publication No. 2022 / 115470. Examples of nucleic acid sequences encoding all or part of the adenovirus penton base protein include SEQ ID NOs. 8 and SEQ ID NOs. 3. Examples of amino acid sequences containing the full-length adenovirus penton base protein or a portion thereof include SEQ ID NOs. 18 and SEQ ID NOs. 13.

[0199] An adenovirus or adenovirus vector may contain one, two, three, four, or all five of the aforementioned sequences, either alone or in any combination. In this regard, an adenovirus or adenovirus vector may contain any combination of any two of the aforementioned sequences, any combination of any three of the aforementioned sequences, any combination of any four of the aforementioned sequences, or all five of the aforementioned sequences.

[0200] In certain embodiments, the adenovirus or adenovirus vector is replication-deficient, and consequently, the replication-deficient adenovirus or adenovirus vector requires the complementation of the replication-essential gene function of at least one region of the adenovirus genome for replication (e.g., to form adenovirus vector particles).

[0201] A replication-deficient adenovirus or adenovirus vector can be modified in any preferred manner to induce a deficiency in the function of one or more replication-essential genes in one or more regions of the adenovirus genome for replication. Complementation of the deficiency in the function of one or more replication-essential genes in one or more regions of the adenovirus genome refers to the use of exogenous means to provide the deficiency in the replication-essential gene function. Such compensation can be brought about in any preferred manner, for example, by using complementary cells and / or exogenous DNA (e.g., helper adenovirus) encoding the disrupted replication-essential gene function.

[0202] In some embodiments, the adenovirus or adenovirus vector may lack the function of one or more replication-essential genes in the early region of the adenovirus genome (i.e., E1–E4 region), the late region of the adenovirus genome (i.e., L1–L5 region), both the early and late regions of the adenovirus genome, or all adenovirus genes (i.e., high-volume adenovirus vector (HC-Ad)). See Morsy et al., Proc. Natl. Acad. Sci. USA, 95: 965–976 (1998); Chen et al., Proc. Natl. Acad. Sci. USA, 94: 1645–1650 (1997); and Kochanek et al., Hum. Gene Ther., 10: 2451–2459 (1999). Adenovirus vectors can also essentially have the entire adenovirus genome removed, in which case at least one of the viral inverted terminal repeat sequences (ITRs) and one or more promoters, or the viral ITRs and packaging signals, remain intact (i.e., the adenovirus amplicon). The larger the region of the adenovirus genome removed, the larger the piece of exogenous nucleic acid sequence that can be inserted into the genome. For example, given that the adenovirus genome is 36kb, the capacity of the adenovirus exogenous insert, by leaving the viral ITRs and one or more promoters intact, is approximately 35kb. Alternatively, an augmented-deficient adenovirus vector containing only the ITRs and packaging signals can efficiently allow for the insertion of approximately 37-38kb of exogenous nucleic acid sequences. Naturally, the inclusion of spacer elements in any or all of the deficient adenovirus regions reduces the capacity of the adenovirus vector for larger inserts.

[0203] In certain embodiments, an adenovirus vector is an "enhanced deletion," meaning that the adenovirus vector lacks the function of one or more genes required for viral replication in each of two or more regions of the adenovirus genome. For example, the aforementioned E1-deficient or E1 / E3-deficient adenovirus vector may further lack the function of at least one replication-essential gene in the E4 region (represented as an E1 / E4-deficient adenovirus vector). An adenovirus vector with an entire E4 region deleted may induce a lower host immune response.

[0204] Examples of replication-deficient adenovirus vectors are disclosed in U.S. Patent Nos. 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, as well as in International Publication Nos. 1994 / 028152, 1995 / 002697, 1995 / 016772, 1995 / 034671, 1996 / 022378, 1997 / 012986, 1997 / 021826, and 2003 / 022311.

[0205] The early region of the adenovirus genome includes the E1, E2, E3, and E4 regions. The E1 region includes the E1A and E1B subregions, and one or more deletions of replication-essential gene function in the E1 region may include one or more deletions of replication-essential gene function in either or both of the E1A and E1B subregions, thereby requiring the completion of the E1A and / or E1B subregions of the adenovirus genome for the adenovirus or adenovirus vector to grow (e.g., to form adenovirus vector particles). The E2 region includes the E2A and E2B subregions, and one or more deletions of replication-essential gene function in the E2 region may include one or more deletions of replication-essential gene function in either or both of the E2A and E2B subregions, thereby requiring the completion of the E2A and / or E2B subregions of the adenovirus genome for the adenovirus or adenovirus vector to grow (e.g., to form adenovirus vector particles).

[0206] The E3 region does not contain any replication-essential gene function, and consequently, deletion of the E3 region does not require the replacement of any gene function in the E3 region for the adenovirus or adenovirus vector to replicate (e.g., to form adenovirus vector particles), either partially or entirely. In the context of this disclosure, the E3 region is defined as a region beginning with an open reading frame encoding a protein with high homology to the 12.5K protein derived from the E3 region of human adenovirus 5 (NCBI reference sequence AP_000218) and ending with an open reading frame encoding a protein with high homology to the 14.7K protein derived from the E3 region of human adenovirus 5 (NCBI reference sequence AP_000224.1). The E3 region may be deleted entirely or partially, or retained entirely or partially. The size of the deletion may be adapted so that the genome holds an adenovirus or adenovirus vector that closely matches the optimal genome packaging size. Larger deletions are adapted to the insertion of larger heterologous nucleic acid sequences in adenoviruses or adenovirus genomes. In some embodiments of this disclosure, the L4 polyadenylation signal sequence located in the E3 region is retained.

[0207] The E4 region contains multiple open reading frames (ORFs). An adenovirus or adenovirus vector having deletions of all open reading frames in the E4 region except ORF6, and in some cases ORF3, does not require any gene function in the E4 region to replicate (e.g., to form adenovirus vector particles). Conversely, adenoviruses or adenovirus vectors having disruption or deletion of ORF6 in the E4 region, and in some cases ORF3 (e.g., having a deficiency in replication-essential gene function based on ORF6 and / or ORF3 in the E4 region), require the completion of the E4 region (specifically, ORF6 and / or ORF3 in the E4 region) with or without disruption or deletion of any other open reading frame in the E4 region, or the native E4 promoter, polyadenylation sequence, and / or right-side inverted terminal repeat (ITR), for the adenovirus or adenovirus vector to replicate (e.g., to form adenovirus vector particles).

[0208] The late regions of the adenovirus genome include the L1, L2, L3, L4, and L5 regions. Adenoviruses or adenovirus vectors may also have mutations in the major late promoter (MLP), as discussed in International Publication No. 2000 / 000628, which can optionally result in replication defects in the adenovirus or adenovirus vector.

[0209] The one or more regions of the adenovirus genome containing the deletion of one or more replication-essential gene functions are preferably one or more early regions of the adenovirus genome, namely the E1, E2, and / or E4 regions. Therefore, in certain embodiments, the adenovirus vector lacks all or part of such regions.

[0210] A replication-deficient adenovirus or adenovirus vector may have one or more mutations (e.g., one or more deletions, insertions, and / or substitutions) in the adenovirus genome compared to wild-type adenovirus, even if these mutations do not inhibit viral replication in host cells. Therefore, in addition to the deletion of one or more replication-essential gene functions, an adenovirus or adenovirus vector may also have deletions of other elements that are not essential for replication. For example, an adenovirus or adenovirus vector may have a partial or complete deletion of the early adenovirus region, known as the E3 region, which is not essential for the proliferation of the adenovirus or adenovirus genome.

[0211] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most the E1 or E4 region of the adenovirus genome for replication (e.g., to form adenovirus vector particles). In some such embodiments, the adenovirus vector may lack all or part of the E1 and / or E4 regions. Thus, the replication-deficient adenovirus or adenovirus vector requires the complementation of at least one replication-essential gene function in the E1A subregion and / or E1B region of the adenovirus genome (represented as an E1-deficient adenovirus vector) or the E4 region of the adenovirus genome (represented as an E4-deficient adenovirus vector) for replication (e.g., to form adenovirus vector particles). Adenoviruses or adenovirus vectors can lack the function of at least one replication-essential gene in the E1 region of the adenovirus genome (preferably all replication-essential gene functions) and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E1 / E3-deficient adenovirus vector). Adenoviruses or adenovirus vectors can lack the function of at least one replication-essential gene in the E4 region of the adenovirus genome (preferably all replication-essential gene functions) and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E3 / E4-deficient adenovirus vector).

[0212] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires complementation of at most the E2 region, preferably the E2A subregion, of the adenovirus genome for replication (e.g., to form adenovirus vector particles). Therefore, the replication-deficient adenovirus or adenovirus vector requires complementation of at least one replication-essential gene function in the E2A subregion of the adenovirus genome for replication (e.g., to form adenovirus vector particles) (represented as an E2A-deficient adenovirus vector). The adenovirus or adenovirus vector may be replication-deficient (preferably all replication-essential gene functions) of at least one replication-essential gene function in the E2A region of the adenovirus genome, and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E2A / E3-deficient adenovirus vector).

[0213] In some embodiments, the adenovirus or adenovirus vector is replication-deficient and requires at most the E1 and E4 regions of the adenovirus genome to replicate (e.g., to form adenovirus vector particles). In some such embodiments, the adenovirus vector may lack all or part of the E1 and / or E4 regions. Thus, the replication-deficient adenovirus or adenovirus vector requires the complementation of at least one replication-essential gene function in both the E1 and E4 regions of the adenovirus genome to replicate (e.g., to form adenovirus vector particles) (represented as an E1 / E4-deficient adenovirus vector). The adenovirus or adenovirus vector may lack at least one replication-essential gene function in the E1 region of the adenovirus genome (preferably all replication-essential gene functions), at least one replication-essential gene function in the E4 region of the adenovirus genome, and at least one gene function in the non-essential E3 region of the adenovirus genome (represented as an E1 / E3 / E4-deficient adenovirus vector). The adenovirus or adenovirus vector preferably requires, at most, completion of the E1 region of the adenovirus genome for replication, and does not require completion of any other deletions in the adenovirus genome for replication. More preferably, the adenovirus or adenovirus vector requires, at most, completion of the E1 and E4 regions of the adenovirus genome for replication, and does not require completion of any other deletions in the adenovirus genome for replication.

[0214] Adenoviruses or adenovirus vectors may contain spacer sequences to provide viral growth in complementary cell lines, similar to what is achieved by adenoviruses or adenovirus vectors lacking a single essential replication gene function (e.g., E1-deficient adenovirus vectors), if multiple essential replication gene functions of the adenovirus genome are deficient (e.g., E1 / E4-deficient adenovirus vectors). The spacer sequences may contain any one or more nucleotide sequences of a desired length, for example, at least about 15 base pairs (e.g., about 15 to about 12,000 nucleotides), preferably about 100 to about 10,000 nucleotides, more preferably about 500 to about 8,000 nucleotides, even more preferably about 1,500 to about 6,000 nucleotides, most preferably about 2,000 to about 3,000 nucleotides, or sequences within a range defined by any two of the aforementioned values. Spacer sequences may be coding or noncoding with respect to the adenovirus genome, and may be native or non-native, but the replication-essential function of the deleted region is not restored. Spacers may also contain expression cassettes. More preferably, spacers include polyadenylated sequences and / or genes that are non-native with respect to the adenovirus or adenovirus vector. The use of spacers in adenovirus vectors is further described, for example, in U.S. Patent No. 5,851,806 and International Publication No. 1997 / 021826.

[0215] By removing all or part of the adenovirus genome, for example, the E1, E3, and E4 regions of the adenovirus genome, the resulting adenovirus or adenovirus vector can accept the insertion of an exogenous nucleic acid sequence while retaining the ability to be packaged into an adenovirus capsid. The exogenous nucleic acid sequence can be inserted at any location in the adenovirus genome, insofar as the insertion at that location enables the formation of an adenovirus or adenovirus vector particle. Preferably, the exogenous nucleic acid sequence is located in the E1, E3, or E4 region of the adenovirus genome.

[0216] The replication-deficient adenoviruses or adenovirus vectors of this disclosure can be produced in complementary cell lines that provide gene functions not present in the replication-deficient adenoviruses or adenovirus vectors but required for viral replication, in order to produce high-titer viral vector stocks. Such complementary cell lines are known and include human embryonic kidney (HEK) 293 cells (e.g., described in Graham et al., J. Gen. Virol., 36: 59-72 (1977)), PER.C6 cells (e.g., described in International Publication No. 1997 / 000326, and U.S. Patent Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (e.g., described in International Publication No. 95 / 34671 and Broough et al., J. Virol., 71: 9206-9213 (1997)). Other suitable complementary cell lines for producing replication-deficient adenoviruses or adenovirus vectors of this disclosure include complementary cells prepared to grow adenovirus vectors whose expression encodes a transgene that inhibits viral growth in host cells (see, for example, U.S. Patent Application Publication No. 2008 / 0233650). Additional suitable complementary cells are described, for example, in U.S. Patent Nos. 6,677,156 and 6,682,929, and International Publication No. 2003 / 020879.

[0217] In some cases, the cell genome does not need to contain a nucleic acid sequence, and its gene product compensates for all the deficiencies in the replication-deficient adenovirus vector. One or more replication-essential gene functions that are missing in the replication-deficient adenovirus vector can be supplied by a helper virus, for example, an adenovirus vector that trans-supplies one or more essential gene functions required for replication of the replication-deficient adenovirus or adenovirus vector. Alternatively, the adenovirus or adenovirus vector of the present invention may contain non-native replication-essential genes that compensate for one or more replication-essential gene functions that are missing in the replication-deficient adenovirus or adenovirus vector of the present invention. For example, the E1 / E4-deficient adenovirus vector can be manipulated to contain a nucleic acid sequence encoding E4 ORF 6 obtained from or derived from a different adenovirus (e.g., an adenovirus of a different serotype than the adenovirus or adenovirus vector of the present invention, or an adenovirus of a different species than the adenovirus or adenovirus vector of the present invention). a. Gorilla-based adenovirus vector

[0218] In some embodiments, the adenoviruses described herein are isolated from gorillas. Western gorillas include the subspecies of Western lowland gorillas (Gorilla gorilla gorilla) and Cross River gorillas (Gorilla gorilla diehli), and eastern gorillas include the subspecies of mountain gorillas (Gorilla beringei beringei) and eastern lowland gorillas (Gorilla beringei graueri). For example, Wilson and Reeder, eds., Mammalian Species of the World, 3 rdSee ed., Johns Hopkins University Press, Baltimore, Md. (2005). In some embodiments, the adenoviruses of this disclosure have been isolated from mountain gorillas (Gorilla beringei beringei). Previous studies have characterized numerous gorilla adenoviruses and their genome sequences (see, for example, International Publication Nos. 2013 / 052832, 2013 / 052811, 2013 / 052799, 2019 / 173465, and 2022 / 115470).

[0219] Gorilla adenoviruses share similarities with human adenoviruses in terms of vector design and safety, offering advantages such as efficient transgene delivery and non-replicability through targeted deletions. Importantly, pre-existing human immunity to gorilla adenoviruses is minimal compared to human adenoviruses. This lack of recognition by the human immune system minimizes potential pre-existing immunity hurdles in gene therapy and vaccine application.

[0220] In certain embodiments, the adenovirus vector is derived from gorilla adenovirus type 40 (GAd40), e.g., GC44, GC45, or GC46. In certain embodiments, the adenovirus vector exhibits the functional adaptations described above. These adaptations may encompass their constituent elements, e.g., sequences encoding functional variants of the E2B, E2A, E3, and L1-L5 regions, as well as inverted terminal repeat sequences. Functional adaptations of such vectors featuring codon degenerate variants of the sequences encoding the E2B, E2A, E3, and L1-L5 regions are also conceivable.

[0221] In a particularly preferred embodiment, the adenovirus vector is derived from GC46, a newly isolated, intrinsic gorilla adenovirus strain isolated from fecal samples of healthy African gorillas. This adenovirus is closely related to and phylogenetically clusters with human adenovirus type C based on comparisons of hexon, DNA polymerase, and exon 4 ORF6 protein sequences. Duncan et al., Virology, 444:119-123 (2013). The seroprevalence of gorilla adenovirus type GC46 is less than approximately 6% in the United States. In comparison, the seroprevalence of type Ad5 is approximately 57%, and most seropositive individuals have high titers (IC90 above 200). Johnson et al., Molecular Therapy, 22:196-205 (2014). Therefore, compared to conventional adenovirus therapies based on the Ad5 serotype, existing neutralizing activity against gorilla adenovirus type GC46 is rare and weak in the United States. In addition, comparative studies from human serum samples originating from sub-Saharan Africa confirmed the rare and weak existing neutralizing activity in human populations. These data suggest that the existing neutralizing activity against GC46 does not significantly interfere with the structure of molecular vaccines and therapeutics on this platform, making gorilla adenovirus GC46 sufficiently suitable as a viral vector scaffold.

[0222] In some embodiments, the gorilla adenovirus vaccine may encode approximately 1 to approximately 200 non-HLA-restricted epitopes derived from HPV16 / 18. For example, a gorilla adenovirus vaccine may encode approximately 1, 5, 10, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 non-HLA-restricted epitopes derived from HPV16 / 18.

[0223] In certain embodiments of the present invention, the gorilla adenovirus vaccine encodes approximately 35 non-HLA-restricted epitopes derived from HPV16 / 18.

[0224] In certain embodiments, the adenovirus vector is a gorilla adenovirus vector manipulated to delete part or all of the E1 and / or E4 regions. A deletion in the E1 region, for example, makes the adenovirus vector a replication defect (Figure 11A), and may include bases 459–3411, resulting in the deletion of the E1A and E1B promoters and open reading frames. A deletion in the E4 region, for example, may include bases 34144–36824, removing all E4 open reading frames (ORFs) and thus eliminating essential elements for gorilla adenovirus replication. (The gorilla adenovirus coordinates provided herein are based on a wild-type adenovirus genome of size 37,213 base pairs.)

[0225] Modified gorilla adenovirus vectors having deletions in and / or within the E1 and / or E4 regions may offer one or more advantages over unmodified vector scaffolds. For example, extended deletions in the adenovirus genome may provide enhanced payload capacity for the adenovirus vector. A second potential advantage is a reduced risk of replicative adenovirus (RCA) development during adenovirus vector production. A third advantage is that the exclusion of E1 and E4 expression products may work to further silence other regions of the viral genome.

[0226] Therefore, in one embodiment, the gorilla adenovirus vector described herein has a deletion in or a portion of the E1 region. In another embodiment, the gorilla adenovirus vector described herein has a deletion in or a portion of the E4 region. In yet another embodiment, the gorilla adenovirus vector described herein has a deletion in or a portion of both the E1 and E4 regions. In one embodiment, the deletion in the E1 and / or E4 region comprises about 100 to about 5,000 base pairs (bp) in length compared to the wild type. For example, the deletion in the E1 and / or E4 region may comprise about 100 bp, about 500 bp, about 1,000 bp, about 1,500 bp, about 2,000 bp, about 2,500 bp, about 3,000 bp, about 3,500 bp, about 4,000 bp, about 4,500 bp, or about 5,000 bp in length compared to the wild type. In some embodiments, deletions in the E1 and / or E4 regions include approximately 100 bp to approximately 5,000 bp, or approximately 500 bp to approximately 4,500 bp, approximately 750 bp to approximately 4,000 bp, or approximately 1,000 bp to approximately 3,750 bp, or approximately 1,250 bp to approximately 3,500 bp, or approximately 1,500 bp to approximately 3,500 bp, or approximately 1,750 bp to approximately 3,500 bp, or approximately 2,000 bp to approximately 3,500 bp, or approximately 2,000 bp to approximately 3,000 bp. In specific embodiments, deletions in the E1 and / or E4 regions include a length of approximately 3,000 bp compared to the wild type.

[0227] In certain embodiments, deletion of the E4 region removes all predictive open reading frames (ORFs) within it. To avoid the possibility of low-level production of adenovirus vectors with E4 deletions, a spacer sequence may be inserted into the E4-deleted region, as shown in Figure 1, to halt any potential transcription initiated from the retained E4 promoter. In one embodiment, the gorilla adenovirus vector described herein includes a spacer sequence inserted in place of the deleted portion of the E4 region. In one embodiment, the spacer sequence includes a bovine growth hormone polyadenylation (BGH polyA) signal sequence inserted in place of the deleted E4 ORF, but any suitable spacer sequence may be used. In some embodiments, the spacer sequence is about 10 to about 500 base pairs (bp) in length. For example, the spacer array is approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, The spacer sequence may be approximately 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or approximately 500 bp in length. Alternatively, the spacer sequence may be approximately 50 bp to 100 bp, 100 bp to 150 bp, 150 bp to 200 bp, 200 bp to 250 bp, 250 bp to 300 bp, 300 bp to 350 bp, 350 bp to 400 bp, 400 bp to 450 bp, or approximately 450 bp to 500 bp compared to the wild type. The spacer may also be of any length within these ranges. For example, the spacer may be approximately 250 bp to 350 bp, approximately 260 bp to 340 bp, approximately 270 bp to 330 bp, approximately 280 bp to 320 bp, or approximately 290 bp to 310 bp in length. In one embodiment, the spacer sequence is approximately 300 base pairs long. In yet another embodiment, the spacer sequence is 278 bp.

[0228] In another embodiment, the spacer sequence is located at approximately 34,000 to 34,500 base pairs in the vector genome compared to the wild type. In yet another embodiment, the spacer sequence is located at 34,040 to 34,317 base pairs in the vector genome compared to the wild type. In yet another embodiment, the spacer sequence contains the nucleic acid sequence of SEQ ID NO: 157.

[0229] In certain embodiments, the vector is missing both the E1 and E4 regions. In some such embodiments, the E1 or E4 region is replaced by an expression cassette containing a transgene or spacer. In some such embodiments, the E1 region is replaced by an expression cassette, and the E4 region is replaced by a spacer.

[0230] In one embodiment, the E1 / E4-deficient GC46 adenovirus vector can be produced in any complementary cell line providing E1 and E4 ORF6 functionality, e.g., engineered 293 cells. Such cells may be cultured, for example, in a serum-free suspension in a shaker flask and infected with a master virus bank at an infection multiplicity (MOI) of 100 PU / cell. The culture sample may be subjected to downstream processing and purified using three rounds of cesium chloride density gradient ultracentrifugation to obtain a highly purified material. This material may then be frozen, then thawed, sterile filtered, and packed into vials, which can be stored in a freezer at approximately -60 to approximately -90°C.

[0231] In some embodiments, the vectors of the present invention may be constructed by isolating a GC46 gorilla adenovector from a non-human primate origin, cloning the isolated GC46 genome, deleting the E1 and E4 regions of GC46, and expressing a human papillomavirus (HPV) 16 / 18 antigen design (e.g., any antigen design construct described herein, including, but not limited to, SEQ ID NO: 243) and inserting an expression cassette in the E1 region under the control of a cytomegalovirus (CMV) pre-early promoter. In one embodiment described below, the CMV-HPV 16 / 18 antigen design contains 35 non-HLA-restricted epitopes of HPV 16 and 18. The overall workflow of the HPV vaccine design disclosed herein is shown in Figure 2 and will be further detailed in the examples.

[0232] In some embodiments, the vector includes a nucleic acid sequence encoding a fusion protein containing an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity with SEQ ID NO: 243. In some embodiments, the fusion protein includes the amino acid sequence of SEQ ID NO: 243 or a conserved variant thereof.

[0233] In some embodiments, the vector includes a nucleic acid sequence having at least 80% identity with SEQ ID NO: 244 (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or higher). In some embodiments, the vector includes a nucleic acid sequence having at least 90% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 95% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 96% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 97% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 98% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 99% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 99.5% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having at least 99.9% identity with SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having 1 to about 500 nucleotide substitutions (e.g., 1 to about 450, 1 to about 450, 1 to about 350, 1 to about 300, 1 to about 250, 1 to about 200, 1 to about 150, 1 to about 100, 1 to about 90, 1 to about 80, 1 to about 70, 1 to about 60, 1 to about 50, 1 to about 40, 1 to about 30, 1 to about 25, 1 to about 20, 1 to about 15, or 1 to about 10) compared to SEQ ID NO: 244. In some embodiments, the vector includes a nucleic acid sequence having SEQ ID NO: 244. C. Non-virus-based delivery systems

[0234] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles).

[0235] The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linking molecules that associate with both liposomes and oligonucleotides, captured within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or in "broken-down" structures. They can also simply be dispersed in solution and, if applicable, form aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include a class of compounds containing naturally occurring lipid droplets in the cytoplasm, as well as long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0236] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -200°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol.

[0237] "Liposome" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-rearrangement before the formation of a closed structure, as well as the capture of water and dissolved solutes between the lipid bilayers (Ghosh et al., Glycobiology 5: 505-10 (1991)). However, compositions that have structures different in solution from the usual vesicular structure are also included. For example, lipids can be assumed to have a micelle structure, or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0238] In some examples, polynucleotides encoding polypeptides can also be introduced into cells using non-viral-based delivery systems, such as the Sleeping Beauty (SB) transposon system. In embodiments, the modified effector cells and other genetic elements described herein are delivered to cells using the SB11 transposon system, the SB100X transposon system, the SB110 transposon system, the piggyBac transposon system (see, e.g., Wilson et al., “PiggyBac Transposon-mediated Gene Transfer in Human Cells,” Molecular Therapy 15:139-145 (2007)) and / or the piggyBac transposon system (see, e.g., Mitra et al., “Functional characterization of piggyBac from the bat Myotis lucifugus unveils an active mammalian DNA transposon,” Proc. Natl. Acad. Sci USA 110:234-239 (2013)). Additional transposase and transposon systems are described in U.S. Patent Nos. 6,489,458; 6,613,752; 7,148,203; 7,985,739; 8,227,432; 9,228,180; U.S. Patent Application Publication No. 2011 / 0117072; Mates et al., Nat Genet, 41(6): 753-61 (2009). doi: 10.1038 / ng.343. Epub 2009 May 3, Gene Ther., 18(9):849-56 (2011). doi: 10.1038 / gt.2011.40. Epub 2011 Mar. 31 and Ivies et al., Cell, It is available in 91(4):501-10, (1997).

[0239] Additional suitable nonviral systems may include embedded expression vectors, which may contain recombination sites that can be randomly integrated into the host cell's DNA or that allow for specific recombination between the expression vector and the host cell's chromosomes. Targeted integration of a transgene to a given locus is a desirable goal for many applications. First, a first recombination site for a site-specific recombinase is inserted into a genomic site, either randomly or at a predetermined location. Subsequently, the cell is transfected with a plasmid containing the gene or DNA of interest, a second recombination site, and an origin for the recombinase (expression plasmid, RNA, protein, or viral expression recombinase). Recombination between the first and second recombination sites results in the integration of plasmid DNA. Such embedded expression vectors can utilize endogenous expression regulatory sequences on the host cell's chromosomes to produce the expression of the desired protein.

[0240] In some embodiments, targeted integration is facilitated by the presence of a sequence on a donor polynucleotide homologous to the sequence flanking at the integration site. For example, targeted integration using donor polynucleotides as described herein can be achieved according to conventional transfection techniques, such as those used to produce gene knockout or knock-in by homologous recombination. In other embodiments, targeted integration is facilitated both by the presence of a sequence on a donor polynucleotide homologous to the sequence flanking at the integration site, and by contacting the cell with the donor polynucleotide in the presence of a site-specific recombinase. Site-specific recombinase, or simply recombinase, means a polypeptide that catalyzes conserved site-specific recombination between its compatible recombination sites. As used herein, site-specific recombinase includes not only native polypeptides but also derivatives, variants, and / or fragments that retain activity, as well as native polynucleotides, derivatives, variants, and / or fragments that encode the recombinase that retains activity.

[0241] Also provided herein are systems for incorporating heterologous genes into host cells, wherein the system comprises one or more gene expression cassettes. In some examples, the system comprises a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide construct. In other examples, the system may include a second gene expression cassette comprising a second polynucleotide encoding a second polypeptide construct. In yet another example, the system may include a third gene expression cassette. In one embodiment, one of the gene expression cassettes may include a gene switch polynucleotide encoding one or more of the following: (i) a transactivation domain; (ii) a nuclear receptor ligand binding domain; (iii) a DNA-binding domain; and (iv) an ecdysone receptor binding domain. In another embodiment, the system further comprises a recombination attachment site and a serine recombinase, wherein the heterologous gene is incorporated into the host cell in the presence of the serine recombinase when the host cell comes into contact with at least the first gene expression cassette.

[0242] In some examples, the system further includes a ligand, and as a result, upon contact with the host cell, the heterologous gene is expressed in the host cell in the presence of the ligand. In one example, the system also includes a recombination attachment site. In some examples, one recombination attachment site is a phage genome recombination attachment site (attP) or a bacterial genome recombination attachment site (attB). In one example, the host cell is a eukaryotic cell. In another example, the host cell is a human cell. In yet another example, the host cell is a T cell or an NK cell. II. Expression Cassette

[0243] Any of the expression cassettes described herein, or a polynucleotide comprising any of the expression cassettes described herein, can be used as a component in a vaccine, such as an HPV vaccine. A. Introduced gene

[0244] The vector of the present invention may include an expression cassette for expressing a transgene. The “transgene” comprises a non-native nucleic acid sequence operably linked to a suitable regulatory element (e.g., a promoter), as a result of the expression of the non-native nucleic acid sequence, which may produce a protein (e.g., a peptide or polypeptide). The regulatory element (e.g., a promoter) may be native or non-native to the adenovirus or adenovirus vector.

[0245] A “non-native” nucleic acid sequence is any nucleic acid sequence (e.g., a DNA, RNA, or cDNA sequence) in which there is no naturally occurring nucleic acid sequence in the adenovirus at its naturally occurring location. Therefore, a non-native nucleic acid sequence may be found naturally in the adenovirus but may be located at a non-native position within the adenovirus genome and / or may be operably linked to a non-native promoter. The terms “non-native nucleic acid sequence,” “heterogeneous nucleic acid sequence,” and “exogenous nucleic acid sequence” are synonymous and can be used interchangeably in the context of this disclosure. A non-native nucleic acid sequence is preferably DNA and preferably encodes a protein (i.e., one or more nucleic acid sequences encoding one or more proteins).

[0246] Non-native nucleic acid sequences can encode therapeutic proteins that can be used to treat mammals preventively or therapeutically against disease. Suitable examples of therapeutic proteins include anti-inflammatory agents, such as cytokines, toxins, tumor suppressor proteins, growth factors, hormones, receptors, mitogens, immunoglobulins, neuropeptides, neurotransmitters, and enzymes. Alternatively, non-native nucleic acid sequences can encode antigens of pathogens (e.g., bacteria or viruses), and adenoviruses or adenovirus vectors can be used as vaccines. B. Promoter

[0247] The gene regulatory components of a therapeutic expression cassette are selected to confer high levels of expression of the transgene. Therefore, another aspect of this disclosure is an expression cassette further comprising a promoter. A promoter is a region of polynucleotides that initiates transcription of a coding sequence. Promoters are located on the same strand of DNA and upstream (in the direction of the 5' region of the sense strand), near the transcription start site of the gene. Some promoters are constitutive, so they are active under all circumstances in the cell, while others are regulated to become active in response to specific stimuli, such as inductive promoters. Further promoters include, but are not limited to, tissue-specific promoters or activation promoters, and include T cell-specific promoters.

[0248] The term "promoter activity" and its grammatical equivalent, as used herein, refer to the degree of expression of a nucleotide sequence operably ligated to a promoter whose activity is being measured. Promoter activity can be measured directly, for example, by determining the amount of RNA transcript produced by Northern blot analysis, or indirectly, by determining the amount of product encoded by a ligated nucleic acid sequence, such as a reporter nucleic acid sequence ligated to the promoter. 1. Inducible promoter

[0249] In certain embodiments, the promoter is an inductive promoter. An inductive promoter is a promoter whose activity is induced by the presence or absence of transcription factors, such as biological or abiotic factors. Inductive promoters are useful because the expression of a gene operably linked to an inductive promoter can be turned on or off at a particular stage of development of an organism or a particular tissue. Examples of inductive promoters include alcohol-regulating promoters, tetracycline-regulating promoters, steroid-regulating promoters, metal-regulating promoters, pathogenesis-regulating promoters, temperature-regulating promoters, and light-regulating promoters. In some embodiments, the inductive promoter is part of a gene switch. The inductive promoter may be a gene switch ligand-inductive promoter. In some cases, the inductive promoter may be an ecdysone receptor-based gene switch of two small molecule ligand-inductive polypeptides, such as the RHEOSWITCH® gene switch, for example, the system described in International Publication No. 2018 / 132494. In some cases, gene switches are, but are not limited to, PCT / US2001 / 009050 (International Publication No. 2001 / 070816); U.S. Patent No. 7,091,038; U.S. Patent No. 7,776,587; U.S. Patent No. 7,807,417; U.S. Patent No. 8,202,718; PCT / US2001 / 030608 (International Publication No. 2002 / 029075); U.S. Patent No. 8,105,825; U.S. Patent No. 8,168,426; PCT / US2002 / 005235 (International Publication No. 2002 / 066613); U.S. Patent Application No. 1 0 / 468,200 (US Patent Application Publication No. 20120167239); PCT / US2002 / 005706 (International Publication No. 2002 / 066614); US Patent No. 7,531,326; US Patent No. 8,236,556; US Patent No. 8,598,409; PCT / US2002 / 005090 (International Publication No. 2002 / 066612); US Patent No. 8,715,959 (US Patent Application Publication No. 20060100416); PCT / US2002 / 005234 (International Publication No. 2003 / 027266);U.S. Patent No. 7,601,508; U.S. Patent No. 7,829,676; U.S. Patent No. 7,919,269; U.S. Patent No. 8,030,067; PCT / US2002 / 005708 (International Publication No. 2002 / 066615); U.S. Patent Application No. 10 / 468,192 (U.S. Patent Application Publication No. 20110212528); PCT / US2002 / 005026 (International Publication No. 2003 / 027289); United States Japanese Patent No. 7,563,879; Japanese Patent No. 8,021,878; Japanese Patent No. 8,497,093; PCT / US2005 / 015089 (International Publication No. 2005 / 108617); U.S. Patent No. 7,935,510; U.S. Patent No. 8,076,454; PCT / US2008 / 011270 (International Publication No. 2009 / 045370); U.S. Patent Application No. 12 / 241,018 (U.S. Patent Application Publication No. 200 90136465 specification); PCT / US2008 / 011563 (International Publication 2009 / 048560); US Patent Application No. 12 / 247,738 specification (US Patent Application Publication 20090123441 specification); PCT / US2009 / 005510 (International Publication 2010 / 042189); US Patent Application No. 13 / 123,129 specification (US Patent Application Publication 20110268766 specification); PCT / US2 The system can be selected from any of the ecdysone-based receptor components described in any of the systems described in Patent No. 011 / 029682 (International Publication No. 2011 / 119773); U.S. Patent Application No. 13 / 636,473 (U.S. Patent Application Publication No. 20130195800); PCT / US2012 / 027515 (International Publication No. 2012 / 122025); and U.S. Patent No. 9,402,919.

[0250] Inducible promoters typically utilize ligands for dose-regulatory control of the expression of at least two of the aforementioned genes. In some cases, ligands include ecdysteroids, 9-cis-retinoic acid, synthetic analogs of retinoic acid, N,N'-diacylhydrazine, oxadiazoline, dibenzoylalkylcyanohydrazine, N-alkyl-N,N'-dialoylhydrazine, N-acyl-N-alkylcarbonylhydrazine, N-aloyl-N-alkyl-N'-aloylhydrazine, arnidoketones, 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide, 8-O-acetylhalpadide, oxysterol, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25-epoxycholesterol, T0901317, and 5-alpha-6-alpha-epoxycholesterol-3-sulfate. The following can be selected: ethyl phosphate (ECHS), 7-ketocholesterol-3-sulfate, framesol, bile acid, 1,1-biphosphonate ester, juvenile hormone III, RG-115819 (3,5-dimethylbenzoate N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazide), RG-115932 ((R)-3,5-dimethylbenzoate N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and RG-115830 (3,5-dimethylbenzoate N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide), and any combination thereof. 2. Non-inducible promoters

[0251] In certain embodiments, the promoter is a non-inducible promoter, including, for example, tissue-specific promoters, strongly constitutive promoters, or minimal promoters known in the Art. Suitable non-inducible promoters include, for example, the CMV promoter, the SV40 promoter, the CAG promoter, or others. In certain embodiments, the promoter is the CMV promoter. 3. Tissue-Specific Promoters

[0252] In certain embodiments, the promoter may be a tissue-specific promoter. Herein, “tissue-specific” refers to the regulated expression of a gene in a subset of tissues or cell types. In some cases, a tissue-specific promoter may be spatially regulated so that the promoter drives expression only in a particular tissue or cell type of an organism. In other cases, a tissue-specific promoter may be temporally regulated so that the promoter drives expression in different cell types or tissues over time, including during the development of an organism. In some cases, a tissue-specific promoter is regulated both spatially and temporally. In certain embodiments, a tissue-specific promoter is activated in a particular cell type either constitutively or intermittently at a specific time or stage of the cell type. For example, a tissue-specific promoter may be a promoter that is activated when a specific cell, such as a T cell or NK cell, is activated. T cells may be activated in various ways, for example, when presented with a peptide antigen by an MHC class II molecule. 4. Synthetic promoters and manipulated promoters

[0253] Synthetic promoters for use in expression cassettes described herein are also intended and may be manipulated to improve expression characteristics. Synthetic promoters may include various subcomponents, including, but are not limited to, blocking sequences, enhancers, and various response elements.

[0254] In certain embodiments, the promoter is an engineered promoter or a variant thereof. As described herein, the promoter may incorporate an IL-2-derived minimal promoter sequence and one or more of the following: NFAT (Nuclear Factor-Action-T) response elements of activated T cells; NFIL2D response elements, NF-κB / TCF response elements, NFAT / NFIL2B response elements, or NFIL2A / OCT response elements. The NFAT transcription factor is a key modulator of effector T cell state. NFAT is an early transcriptional checkpoint that progressively drives depletion. After TCR stimulation, NFAT is rapidly activated in T cells and forms a protein complex with AP-1, which is induced by appropriate co-stimulatory signaling, thereby regulating effector gene and T cell function. NFAT response elements can fusion with other minimal promoter sequences (e.g., the IL2 minimal promoter) to drive transgene expression in response to T cell activation. Further examples of response elements are described in Mattila et al., EMBO J., 9(13):4425-33 (1990). 5. Activation-Specific Promoter

[0255] In certain embodiments, the promoters are activation-specific promoters, such as the interleukin-2 (IL2) promoter and the programmed death (PD)-1 (CD279) promoter. Gene switch components may also be conditionally expressed upon immune cell activation by fusing binding sites for other nuclear factors of the pro-inflammatory signaling pathway, such as NF-KB, to a minimal promoter sequence (e.g., IL2).

[0256] In certain embodiments, the promoter includes an IL-2 core promoter (SEQ ID NO: 26). In some embodiments, at least one promoter includes an IL-2 minimal promoter (SEQ ID NO: 27). In other embodiments, at least one promoter includes an IL-2 enhancer and promoter variant (SEQ ID NOs: 26-28). In yet another embodiment, at least one promoter includes an NF-κB binding site (SEQ ID NOs: 30-32). In some embodiments, at least one promoter includes an (NF-κB)1-IL2 promoter variant (SEQ ID NO: 30). In some embodiments, at least one promoter includes an (NF-κB)3-IL2 promoter variant (SEQ ID NO: 31). In some embodiments, at least one promoter includes an (NF-κB)6-IL2 promoter variant (SEQ ID NO: 32). In some embodiments, at least one promoter includes a 1× activated T cell nuclear factor (NFAT) response element-IL2 promoter variant (SEQ ID NO: 33). In another embodiment, at least one promoter includes a 3× NFAT response element (SEQ ID NOs: 34-35). In yet another embodiment, at least one promoter includes a 6×NFAT response element-IL2 promoter variant (SEQ ID NOs. 36-39). In some embodiments, at least one promoter includes a human EF1A1 promoter variant (SEQ ID NOs. 40-41). In some embodiments, at least one promoter includes a human EF1A1 promoter and enhancer (SEQ ID NOs. 42). In some embodiments, at least one promoter includes a human UBC promoter (SEQ ID NOs. 43). In some embodiments, at least one promoter includes a 6-site GAL4 inducible proximal factor-binding element (PFB). In some embodiments, at least one promoter includes a synthetic minimal promoter 1 (inducible promoter) (SEQ ID NOs. 44). Sequences for such promoters are described, for example, in International Publication Nos. 2019 / 173465 and International Publication Nos. 2022 / 115470.

[0257] In certain embodiments, the promoter may be one or more of the following: IL-2 core promoter, IL-2 minimal promoter, IL-2 enhancer and promoter variant, (NF-κB)1-IL2 promoter variant, (NF-κB)3-IL2 promoter variant, (NF-κB)6-IL2 promoter variant, 1×NFAT response element-IL2 promoter variant, 3×NFAT response element-IL2 promoter variant, 6×NFAT response element-IL2 promoter variant, human EEF1A1 promoter variant, human EEF1A1 promoter and enhancer, human UBC promoter, and synthetic minimal promoter 1. In certain embodiments, the promoter nucleotide may include SEQ ID NOs: 26-44. 6. Constitutive promoters

[0258] In certain embodiments, the promoter is a constitutive promoter. Examples of such promoters include the monkey virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) terminal repeat sequence (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters, such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. 7. Virus promoter

[0259] Exemplary promoters used in the vectors described herein include viral promoters operably combined with heterologous nucleic acid sequences encoding cytokines. Exemplary viral promoters may be derived from, but are not limited to, several known viruses, and include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors (AAVs), and alphavirus vectors. Non-exclusive examples of potentially useful viral vectors include human immunodeficiency virus (HIV), respiratory syncytial virus (RSV), cytomegalovirus (CMV), simian virus 40, herpes simplex virus (HSV), adenovirus (AV), adeno-associated virus (AAV), or lentivirus (LV). For example, specific viral promoters intended herein include the cytomegalovirus (CMV) pre-early promoter, the CAG promoter (a combination of the CMV early enhancer element and the chicken beta-actin promoter), the monkey virus 40 (SV40) promoter, the cauliflower mosaic virus (CaMV) 35S RNA and 19S RNA promoters, the coat protein promoter for tobacco mosaic virus (TMV), and any variants thereof. Examples of mammalian promoters include the human elongation factor 1α-subunit (EF1-1α) promoter, the human ubiquitin C (UCB) promoter, the mouse phosphoglycerate kinase-1 (PGK) promoter, and any variants thereof. 8. Other promoter elements

[0260] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, although some promoters have recently been shown to also contain functional elements downstream of the initiation site. Spacing between promoter elements is often flexible, and as a result, promoter function is conserved when elements are inverted or moved relative to each other. In thymidine kinase (TK) promoters, the spacing between promoter elements can increase to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0261] A synthetic promoter useful in the present invention may include an enhancer sequence. In one embodiment, the enhancer may be an mCMV enhancer sequence. In another embodiment, the mCMV enhancer sequence is about 200 to about 1,000 bp in length. In yet another embodiment, the enhancer sequence is about 365 bp in length. In yet another embodiment, the enhancer sequence includes a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the nucleic acid sequence of SEQ ID NO: 148 or a functional variant thereof, for example, SEQ ID NO: 148, or a conservatively substituted variant of SEQ ID NO: 148, or a non-conservatively substituted variant of SEQ ID NO: 148.

[0262] In another embodiment, the mCMV enhancer includes a transcription factor binding site. In yet another embodiment, the transcription factor binding site includes Sp1, Ebox, ETS, TRE, CREB, and GATA binding sites.

[0263] Response elements may also be included in the promoters described herein. Various response elements are known in the art. For example, to reduce the expression of a transgene during adenovirus production, which may negatively affect the overall production titer, a tetracycline response element (TRE, 2X TetO) may be located within the promoter element of a promoter, between the TATA box and the transcription start site. Thus, when a vector is produced in a cell line expressing a tetracycline (Tet) repressor, the expression of a transgene driven by a promoter containing a TRE is reduced. Gall et al., Molecular Biotechnology, 35:263-273 (2007). In the absence of tetracycline, the Tet repressor interacts with the TRE element and blocks the initiation of transcription. Normal expression levels are observed when infecting producing cells that do not express a Tet repressor. Therefore, in one embodiment described herein, the synthetic promoter includes a TRE. The TRE contains a length of about 10 to about 100 bp. In another embodiment, the TRE is in the range of 40 bp to 50 bp in length. In yet another embodiment, the TRE includes a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the nucleic acid sequence of SEQ ID NO: 150 or a conservedly substituted variant of SEQ ID NO: 150, or a non-conservatively substituted variant of SEQ ID NO: 150.

[0264] In certain embodiments, the promoter includes a transcription blocker, an enhancer sequence, and a response element. In some such embodiments, the promoter includes an mCMV enhancer and a TRE. B. Untranslated region

[0265] In some embodiments, the expression cassette may include an untranslated region (UTR) to regulate or enhance transgene expression. In one embodiment, the expression cassette may include an artificial untranslated region. 5'UTRs with splice units have been shown to enhance the expression of the transgene cassette. Therefore, in some embodiments, the cassette includes a 5'UTR with splice units. In a particular embodiment, the 5'UTR is engineered to include a synthetic splice site sequence extending from canine ATP2A2 intron 2 to the subsequent 5'UTR of the bovine CSN2 gene. In another embodiment, the 5'UTR includes a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the nucleic acid sequence of SEQ ID NO: 151 or a conservedly substituted variant of SEQ ID NO: 151 or a non-conservatively substituted variant of SEQ ID NO: 151. C. Terminal Sequence

[0266] In another embodiment, the expression cassette further comprises a stop sequence. The open reading frame of the cytokine transgene may be followed by the stop sequence. Like promoter sequences, the stop sequence may also contain various regulatory elements to ensure proper 3' transcription end processing. In one embodiment, the stop sequence comprises a partial human growth hormone (HGH) 3' untranslated region. In another embodiment, the stop sequence comprises a polyadenylation signal, including, but not limited to, an SV40 polyadenylation signal and / or an LTR polyadenylation signal. In yet another embodiment, the stop sequence comprises a human beta-actin (ACTb) transcription termination signal sequence. In yet another embodiment, the stop sequence comprises an HGH 3' untranslated region, a polyadenylation signal, and a human beta-actin transcription termination sequence. In another embodiment, the stop sequence comprises a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with the nucleic acid sequence of SEQ ID NO: 156 or a conservedly substituted variant of SEQ ID NO: 156 or a non-conservatively substituted variant of SEQ ID NO: 156. D. Polynucleotide linker

[0267] Expression cassettes and constructs containing polynucleotide linkers that promote the expression of polynucleotides and the functionality of polypeptides described herein are also intended herein.

[0268] In some cases, the linker may be a cleavable linker. The polynucleotide linker may be an oligomer. The polynucleotide linker may be a double-stranded, single-stranded, or a combination thereof DNA. In some cases, the linker may be RNA. The polynucleotide linker may be a double-stranded segment of DNA containing a desired restriction site, which can be added to create a terminal structure compatible with the polynucleotide-containing vectors described herein.

[0269] In some cases, polynucleotide linkers may be useful for modifying vectors containing polynucleotides as described herein. For example, vector modification involving a polynucleotide linker may involve alteration of multiple cloning sites or addition of polyhistidine tails. Polynucleotide linkers can also be used to adapt the ends of blunt-ended insert DNA for cloning into vectors cleaved with restriction enzymes that have adherent ends. The use of polynucleotide linkers may be more efficient than blunt-ended ligation to vectors and may provide a method for releasing the insert from the vector in downstream applications. The insert may be a polynucleotide sequence encoding a polypeptide useful for therapeutic applications.

[0270] In some embodiments, the polynucleotide linker may be ligated, in some cases, to a vector containing the polynucleotide described herein by a T4 ligase. To facilitate ligation, an excess of polynucleotide linker may be added to the composition containing the insert and vector. In some cases, the insert and vector are pre-treated before the linker is introduced. For example, pre-treatment with methylase can prevent unwanted cleavage of the insert DNA.

[0271] In some cases, the polynucleotides or genes described herein may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers. 1. IRES array

[0272] In some embodiments, the polynucleotides described herein may be linked by “intrasequence ribosome entry sites” or “IRES” elements. IRESs can enable the simultaneous expression of multiple genes. For example, an IRES sequence can enable the production of multiple proteins from a single mRNA transcript. Ribosomes can bind to IRESs in a 5'-cap independent manner and initiate translation.

[0273] In expression cassettes containing IRES sequences, the first gene can be translated by cap-dependent ribosome scanning, a mechanism mediated by its own 5'-UTR, while the translation of subsequent genes can be achieved in a cap-independent manner by direct recruitment of ribosomes to the IRES. IRES sequences can enable eukaryotic ribosomes to bind and initiate translation without binding to the 5' cap end. IRES sequences can enable the expression of multiple genes from a single transcript (Mountford and Smith, Trends Genet. 11(5):179-84 (1995)).

[0274] In certain cases, the IRES region may be derived from the IRES sequences of viruses, such as picornaviruses, encephalomyocarditis viruses, and hepatitis C viruses. In other cases, the IRES sequence may be derived from encephalomyocarditis viruses. The terms "EMCV" or "encephalomyocarditis virus," as used herein, refer to any member of an isolate or strain of a encephalomyocarditis virus species of the genus Picornaviridae. Examples include the EMCV-R(Rueckert) virus and the Columbian SK virus. In some cases, cellular IRES elements, such as eukaryotic initiation factor 4G, immunoglobulin heavy chain binding protein, c-myc proto-oncogene, vascular endothelial growth factor, fibroblast growth factor-I, or any combination or modification thereof may be used. In some cases, cellular IRESs may have increased gene expression compared to viral IRESs.

[0275] IRES sequences from viruses, cells, or combinations thereof can be used in vectors. IRESs may be derived from encephalomyelitis virus (EMCV) or poliovirus (PV). In some cases, IRES elements may be derived from poliovirus (PV), encephalomyelitis virus (EMCV), foot-and-mouth disease virus (FMDV), porcine leukemia virus-1 (PTV-1), Aichi virus (AiV), Seneca Barley virus (SVV), hepatitis C virus (HCV), classical swine fever virus (CSFV), human immunodeficiency virus-2 (HIV-2), human immunodeficiency virus-1 (HIV-I), Moloney's mouse leukemia virus (MoMLV), feline immunodeficiency virus (FIV), mouse mammary tumor virus (MMTV), latent human cytomegalovirus (pUL138), Epstein-Barr virus (EBNA-1), herpesvirus Marek's disease (MDV RLORF9), SV40 polycistronic 19S (SV40 The virus is selected from the group consisting of 19S), wheat aphid virus (RhPV), cricket paralysis virus (CrPV), Ectropis obliqua picorna-like virus (EoPV), Plautia stali enteric virus (PSIV), Triatoma virus (TrV), honeybee paralysis discistrovirus (IAPV, KBV), blackcurrant reversion virus (BRV), pelargonium variegation virus (PFBV), hibiscus chlorosis virus (HCRSV), rapeseed infective tobamovirus (CrTMV), potato leaf curl borerovirus (PLRV), tobacco etch virus (TEV), giardiavirus (GLV), leishmania RNA virus-I (LRV-1), and combinations or modifications thereof.

[0276] In some cases, IRES is selected from the group consisting of Apaf-1, XIAP, HIAP2 / c-IAP1, DAP5, Bcl-2, c-myc, CAT-I, INR, differentiated LEF-1, PDGF2, HIF-la, VEGF, FGF2, BiP, BAG-I, CIRP, p53, SHMTI, PITSLREp58, CDKI, Rpr, hid, hsp70, grim, skl, Antennapedia, dFoxO, dinR, Adh-Adhr, HSPI0I, ADH, URE-2, GPRI, NCE102, YMR18la, MSNI, BOil, FLO8, GICI, and any combination or modification thereof. When an IRES element is contained between two open reading frames (ORFs), translation initiation can occur via a classical 5'-m7GpppN cap-dependent mechanism in the first ORF and a cap-independent mechanism in the second ORF downstream of the IRES element.

[0277] In some cases, an IRES sequence can be approximately 9 to 1,000 base pairs long. For example, an IRES sequence can be approximately 9 to 150 base pairs, or approximately 150 to 400 base pairs, or approximately 400 to 600 base pairs, or approximately 600 to 1,000 base pairs. In some embodiments, the IRES sequence is approximately 9, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 275, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 750, 800, 850, 900, 950, or 1,000 base pairs.

[0278] In some cases, the expression of downstream genes within a vector containing an IRES sequence may be reduced. For example, the expression of a gene following an IRES sequence may be reduced compared to the gene preceding the IRES sequence. This reduction in expression can range from 1% to 99.9% compared to the preceding gene, including reductions of 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9% compared to the preceding gene. 2. Virus 2A sequence

[0279] In some embodiments, the polynucleotides described herein may be linked by a viral 2A element or sequence. The 2A element may have 5 to 100 base pairs and may be shorter than the IRES. In some cases, the 2A sequence may contain 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 base pairs. The gene linked to 2A may be expressed in a single open reading frame, and "self-cleavage" may occur co-translationally between the last two amino acids of the GP at the C-terminus of the 2A polypeptide, resulting in equal amounts of co-expressed protein.

[0280] The viral 2A sequence can consist of approximately 20 amino acids. In some cases, the viral 2A sequence may contain the consensus motif Asp-Val / Ile-Glu-X-Asn-Pro-Gly-Pro (SEQ ID NO: 278). The consensus motif sequence can act in a co-translational manner. For example, the normal formation of peptide bonds between glycine and proline residues may be prevented, which can lead to ribosome skipping and cleavage of nascent polypeptides. This effect can produce multiple genes at equimolar levels.

[0281] 2A peptides can enable the translation of multiple proteins into polypeptides within a single open reading frame, which can then be cleaved into individual polypeptides via a ribosome skipping mechanism (Funston et al., J Gen. Viral. 89(Pt 2):389-96 (2008)). In some embodiments, 2A sequences may include F / T2A, T2A, p2A, 2A, T2A, E2A, F2A, and BmCPV2A, BmIFV2A, as well as any combination thereof.

[0282] In some cases, a vector may contain an IRES sequence and a 2A linker sequence. In other cases, the expression of multiple genes linked to a 2A peptide may be promoted by a spacer sequence (GSG) preceding the 2A peptide. In some cases, a construct may combine spacers, linkers, adapters, promoters, or combinations thereof. For example, a linker may have a spacer (SGSG (SEQ ID NO: 84) or GSG, or a Whitlow linker) with a different 2A peptide, and a furin linker (RAKR (SEQ ID NO: 86)) cleavage site. The spacer may be I-Ceui. In some cases, the linker may be manipulated. For example, the linker may be designed to include chemical features such as hydrophobicity. In some cases, at least two linker sequences may produce the same protein. In other cases, multiple linkers may be used in a vector. For example, the gene of interest may be separated by at least two linkers. 3. Polynucleotide-encoded polypeptide linkers

[0283] In certain embodiments, the polynucleotides described herein may encode two or more polypeptides. In some of these embodiments, the polynucleotides may be separated by an intervening sequence that encodes an intervening linker polypeptide. As used herein, the term “intervening linker polypeptide” means an amino acid sequence that separates two or more polypeptides encoded by a polynucleotide and can be distinguished from the term “peptide linker,” which refers to an amino acid sequence optionally included in the polypeptide constructs disclosed herein for linking a transmembrane domain to a cell surface polypeptide (e.g., including truncated variants of native polypeptides).

[0284] In certain cases, the interlinker polypeptide is a cleavage-sensitive interlinker polypeptide. In some embodiments, the interlinker polypeptide is a cleavable linker or a ribosome-skipping linker. In some embodiments, the cleavable linker or ribosome-skipping linker sequence is selected from the group consisting of 2A, GSG-2A, GSG linker, SGSG linker (SEQ ID NO: 84), furinlink variants and their derivatives. In some embodiments, the 2A linker is a p2A linker, T2A linker, F2A linker, or E2A linker. In some embodiments, the polypeptide of interest is expressed as a fusion protein linked by a cleavage-sensitive interlinker polypeptide. In certain embodiments, the cleavage-sensitive interlinker polypeptide may be one or more of F / T2A, T2A, p2A, 2A, GSG-p2A, GSG linker, and furinlink variants. Linkers (polynucleotide and polypeptide sequences), such as those disclosed in PCT / US2016 / 061668 (International Publication No. 2017083750), were published on May 18, 2017. In certain cases, the furin-intervened linker polypeptide may be encoded by a polynucleotide sequence containing "CGTGCAAAGCGT (SEQ ID NO: 69)" or "AGAGCTAAGAGG (SEQ ID NO: 112)".

[0285] In some embodiments, the intervening linker polypeptides include the Whitlow linker (SEQ ID NO: 81), the linker of SEQ ID NO: 82, the GSG linker, the SGSG linker (SEQ ID NO: 84), the (G4S)3 linker (SEQ ID NO: 85), the Furin cleavage site / Furlink1 (SEQ ID NO: 86), the Fmdv linker (SEQ ID NO: 87), the Thosea asigna virus 2A region (T2A) (SEQ ID NO: 88), Furin-GSG-T2A (SEQ ID NO: 89), Furin-SGSG-T2A (SEQ ID NO: 90), and the porcine scourge virus-1. It includes an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identity with the amino acid sequence of the 2A region (P2A) (SEQ ID NO: 91), GSG-P2A (SEQ ID NO: 92), equine rhinitis A virus 2A region (E2A) (SEQ ID NO: 93), foot-and-mouth disease virus 2A region (F2A) (SEQ ID NO: 94), FP2A (SEQ ID NO: 95), linker-GSG (SEQ ID NO: 96), or the linker of SEQ ID NO: 97. In some cases, the intervening linker polypeptide contains an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identity with the amino acid sequence of SEQ ID NOs. 82, 96, and / or 97.

[0286] In some embodiments, the intervening linker polypeptides include the Whitlow linker (SEQ ID NO: 81), the linker of SEQ ID NO: 82, the GSG linker, the SGSG linker (SEQ ID NO: 84), the (G4S)3 linker (SEQ ID NO: 85), the Furin cleavage site / Furlink1 (SEQ ID NO: 86), the Fmdv linker (SEQ ID NO: 87), the Thosea asigna virus 2A region (T2A) (SEQ ID NO: 88), Furin-GSG-T2A (SEQ ID NO: 89), Furin-SGSG-T2A (SEQ ID NO: 90), and the porcine scourge virus-1. The intervening linker polypeptide includes the 2A region (P2A) (SEQ ID NO: 91), GSG-P2A (SEQ ID NO: 92), equine rhinitis A virus 2A region (E2A) (SEQ ID NO: 93), foot-and-mouth disease virus 2A region (F2A) (SEQ ID NO: 94), FP2A (SEQ ID NO: 95), linker-GSG (SEQ ID NO: 96), or a conservatively substituted or non-conservatively substituted variant of the linker of SEQ ID NO: 97. In some cases, the intervening linker polypeptide contains an amino acid sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.1%, 99.5%, 99.9%, 99.99%, or 100% identity with the amino acid sequence of SEQ ID NO: 82, 96, and / or 97.

[0287] In some embodiments, the intervening linker polypeptide comprises a furin polypeptide and a 2A polypeptide linked by a polypeptide linker comprising at least three hydrophobic amino acids. In some cases, the at least three hydrophobic amino acids are selected from the list consisting of glycine (Gly) (G), alanine (Ala) (A), valine (Val) (V), leucine (Leu) (L), isoleucine (Ile) (I), proline (Pro) (P), phenylalanine (Phe) (F), methionine (Met) (M), and tryptophan (Trp) (W). In some cases, the polypeptide linker may also comprise one or more GS linker sequences, e.g., (GS)n (SEQ ID NO: 279), (SG)n (SEQ ID NO: 280), (GSG)n (SEQ ID NO: 281), and (SGSG)n (SEQ ID NO: 282), where n can be any number from 0 to 15.

[0288] The linkers described herein can, in certain cases, improve biological activity, increase expression yield, and achieve a desired pharmacokinetic profile. The linkers may also include hydrazones, peptides, disulfides, or thioesters.

[0289] In some cases, the interlinker polypeptides described herein are flexible linkers. Flexible linkers can be applied when the joined domains require a certain degree of movement or interaction. Flexible linkers may consist of small, nonpolar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr). Flexible linkers may have sequences that are predominant from stretches of Gly and Ser residues ("GS" linkers). An example of a flexible linker may have the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 283). By adjusting the copy number "n", the length of this exemplary GS linker can be optimized to achieve proper separation of functional domains or to maintain the required internal domain interactions. In addition to GS linkers, other flexible linkers can be utilized for recombinant fusion proteins. In some cases, flexible linkers may also be rich in small or polar amino acids such as Gly and Ser, but may contain additional amino acids such as Thr and Ala to maintain flexibility. In other cases, polar amino acids such as Lys and Glu can be used to improve solubility.

[0290] Flexible linkers useful in this invention may be rich in small or polar amino acids such as Gly and Ser to provide good flexibility and solubility. Flexible linkers may be a suitable choice when certain movements or interactions are desired for the fusion protein domains. In addition, although flexible linkers cannot have a rigid structure, they can function as passive linkers to maintain distance between functional domains. The length of the flexible linker can be adjusted to enable proper folding of the fusion protein or to achieve its optimal biological activity.

[0291] In some cases, the interlinker polypeptides described herein are rigid linkers. Rigid linkers can be used to maintain fixed distances between polypeptide domains. Examples of rigid linkers include, to name a few, alpha-helix-forming linkers, Pro-rich sequences, (XP)n, X-Pro skeletons, and A(EAAAK)nA (n=2~5) (SEQ ID NO: 284). Rigid linkers can exhibit a relatively rigid structure in some cases by employing an α-helix structure or by containing multiple Pro residues.

[0292] In some embodiments, the intervening linker polypeptide may be non-cleavable. The non-cleavable linker can covalently join its functional domains together and act as a single molecule throughout the entire in vivo or ex vivo process.

[0293] In other embodiments, the intervening linker polypeptide may be cleavable. A cleavable linker may be introduced to release a free functional domain in vivo. A cleavable linker may be cleaved in the presence of a reducing agent, a protease, to name a few examples. For example, a cleavable linker can be produced by utilizing the reduction of a disulfide bond. In the case of a disulfide linker, a cleavage event by disulfide exchange with a thiol, such as glutathione, can result in cleavage. In other cases, in vivo cleavage of a linker in a recombinant fusion protein may also be carried out by a protease that can be expressed in vivo in specific cells or tissues under pathological conditions (e.g., cancer or inflammation), or by a protease that can be confined within a particular cellular compartment. In some cases, a cleavable linker may enable targeted cleavage. For example, the specificity of many proteases can provide slower linker cleavage in a confined compartment. The cleavable linker may also include a hydrazone, a peptide, a disulfide, or a thioester. For example, hydrazones can confer serum stability. In other cases, hydrazones can enable cleavage in the acidic compartment. The acidic compartment can have a pH of up to 7. The linker may also contain a thioether. The thioether may be non-reducing. The thioether can be designed for intracellular proteolysis. 4. Operated linkers and designed linkers

[0294] In some embodiments, the polynucleotide linker may be manipulated or designed. The method for designing the linker may be computer-based. In some cases, the computer-based method may include graphic techniques. The computer-based method can be used to search for suitable peptides from a library of three-dimensional peptide structures derived from a database. For example, the Brookhaven Protein Data Bank (PDB) can be used to measure the spatial distances between selected amino acids in the linker. C. Adenovirus Expression Cassette

[0295] When an adenovirus vector is used, the expression cassette may be located at the E1 region deletion junction or the E4 deletion junction. In certain embodiments, the expression cassette is located at the E1 region deletion junction.

[0296] In a particular embodiment, the expression cassette is cloned from right to left with respect to the adenovirus viral genome.

[0297] In a particular embodiment, an expression cassette cloned from right to left within the viral genome of an adenovirus includes the nucleic acid sequence of SEQ ID NO: 242 or a functional variant thereof (e.g., a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 242, or a codon degenerate variant of SEQ ID NO: 242, or a conservedly substituted variant of SEQ ID NO: 242, or a non-conservatively substituted variant of SEQ ID NO: 242). D. Packaging sequence

[0298] In addition to the expression cassette, the vector may further include a packaging sequence. As used herein, the term “packaging sequence” refers to a sequence located within the gorilla adenovirus genome that is required for the insertion of viral DNA into a viral capsid or particle. See Ostapchuk et al., Curr. Topics in Microbiology and Immunology, 272:165-185 (1995) and Ahi et al., Frontiers in Microbiology, 7:150 (2016). E. HPV initial region protein

[0299] Constitutive or inducible expression of the early (E) region protein of HPV provides a target for an effective HPV vaccine. See Figure 1.

[0300] HPV genes (E1-E8) regulate viral expression and replication, while late (L) genes control the viral protein coding. The functions of HPV early region proteins include: El and E2 have functions in viral replication / transcription (e.g., E2 regulates the expression of E6 and E7; El / E2 interaction is essential for viral replication); E4 and E5 have increased expression during the later stages of the viral replication cycle; and E6 and E7 act concurrently during replication (E6 is required for episomal genome maintenance, and E7 expands the compartment of epithelial cell activity in DNA replication).

[0301] HPV16 and 18 E6 and E7 proteins are responsible for maintaining the malignant tumor phenotype and are constitutively expressed in tumors, thus representing potential targets for therapeutic vaccines. Furthermore, they are not endogenously expressed in any human tissue, resulting in a very low risk of inducing autoimmune events with vaccines targeting these proteins. Several CD8+ T cell epitopes of E6 and E7 that can induce cytotoxic T lymphocyte (CTL) responses have been previously identified, and clinical studies using diverse vaccine platforms have demonstrated varying degrees of efficacy in terms of inducing HPV-specific responses and clinical benefits. These include live vectors, peptide or protein vaccines, cell-based vaccines, and nucleic acid vaccines. The majority of these vaccines target HPV oncoplasmic proteins E6 and E7 by targeting the activation of HPV antigen-specific CD8+ cytotoxic T cells or CD4+ helper T cells. These therapeutic vaccines also differ by their route of administration.

[0302] An exemplary embodiment of the present invention is an HPV16 / 18 vaccine that delivers a multiepitope antigen design containing 35 non-HLA-restricted epitopes of HPV16 and 18, namely 32 key immunogenic (CTL-specific) peptides derived from E6(HPV16 / 18), E7(HPV-16 / 18), and E5(HPV16), as well as three unique agonist peptides.

[0303] III. Antigenic Bioinformatics Workflow for HPV Vaccine Design

[0304] [ka] A. Identification of new HPV antigenic components

[0305] Based on the crucial roles that HPV E2 and E4 gene components play in essential HPV function, the locations of corresponding proteins, and in-silico predictions, E2 and E4-derived antigens were identified for HPV therapeutic vaccines. Non-oncogenic and viral inactivation gene modifications were also applied to eliminate viral and oncogenic biological activity from HPV proteins, for example, in HPV E2 and E6 proteins.

[0306] By applying genetic engineering, we achieved the production of rearranged protein sequences that maintain the immunological characteristics of the peptides while eliminating the oncogenic and viral amplification functions of E7 and E4, respectively.

[0307] Therefore, some of the innovative aspects of the designs illustrated in this specification include: (1) the use of gene constructs encoding fusion proteins containing four or more different HPV proteins; (2) the combination of amino acid point mutation and duplicate polypeptide sequence shuffling techniques to inactivate oncogenic and essential viral functions; (3) the incorporation of HPV proteins containing multiple antigenic components derived from HPV proteins highly expressed in host-infected cells; (4) the design of hybrid antigens known for the first time; (5) the combination of epitopes derived from high-cancer-risk and low-cancer-risk HPV strains; (6) the use of mixed and adjustable repeat linkers; (7) the use of rigid linkers to stabilize polypeptide subunits and prevent undesirable intramolecular interactions; (8) the use of cleavable linkers between epitopes; and / or (9) the dual use of linker sequences that themselves provide protein-protein linker(-) function as well as both antigen and epitope (i.e., antigenicity conferred by the linker sequence).

[0308] Antigenicity is the ability to stimulate antibody production or a cell-mediated immune response. The antigenicity of the final design sequences was predicted by Vaxjen software, an alignment-independent model of antigen recognition based on the main chemical properties of the amino acid sequence. The results show that five antigenic sequences are antigenic. See Table 4 (Virus and Tumor Antigenicity).

[0309] [Table 3]

[0310] Allergens are generally small antigens that elicit an antibody response. Allergenicity was predicted by ALLERTOP, a bioinformatics-based allergen prediction software that uses machine learning methods for classification, regardless of whether the antigen is allergen or non-allergen. This includes logistic regression (LR), decision trees (DT), simple Bayes (NB), random forest (RF), multilayer perceptron (MLP), and k-nearest neighbors (kNN). The results show that five antigen sequences are non-allergenic. See Table 4 (Allergenicity).

[0311] Cross-reactivity or the onset of autoimmune side effects in various tissues poses a significant safety risk in adoptive immunotherapy. Sequence homology analysis was performed using BLAST search, a basic local alignment search tool, to evaluate whether these novel antigens exhibit cross-reactivity with the human proteome. No host cross-reactivity was identified for these five antigen sequences. See Table 4 (Host Cross-Reactivity). B. Software / Tools

[0312] Software tools used in implementing the designs described herein include, but are not limited to, the following: See ALLERTOP (AllerTOP v.2 -- a server for in silico prediction of allergens; J Mol Model. 2014 Jun; 20(6):2278. doi: 10.1007 / s00894-014-2278-5. Epub 2014 May 31). • ANN (See Reliable prediction of T-cell epitopes using neural networks with novel sequence representations; Protein Sci. 2003 May; 12(5):1007-17). • BLAST (a basic local alignment search tool; NCBI, National Center for Biotechnology Information, US National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA). ·CLUSTALW2 (EMBL-EBI, Wellcome Genome Campus, Hinxton, Cambridgeshire, CB10 1SD, UK. +44(0)1223 49 44 44). • GENEIOUS VI 1.1.5 (See geneious.com / biopharma / ) • See the IEDB consensus (A consensus epitope prediction approach identifies the breadth of murine T(CD8+)-cell responses to vaccinia virus; Nat Biotechnol. 2006 Jul; 24(7):817-9. Epub 2006 Jun 11). See NETMHCPAN 4.0 (Gapped sequence alignment using artificial neural networks: application to the MHC class I system; Bioinformatics. 2016 Feb 15;32(4):511-7. doi: 10.1093 / bioinformatics / btv639. Epub 2015 Oct 29). • PHYRE2 (The Phyre2 web portal for protein modeling, prediction and analysis; 07 May 2015; see nature.com / articles / nprot.2015.053; doi.org / 10.1038 / nprot.2015.053). • PYMOL MOLECULAR GRAPHICS system V2.ll (See pymol.org / 2 / ; sourceforge.net / projects / pymol / support) • VAXJEN (VaxiJen: a server for prediction of protective antigens, tumor antigens and subunit vaccines; see BMC Bioinformatics. 2007 Jan 5;8:4) III. Exemplary HPV antigen design variants

[0313] In exemplary embodiments, the polypeptide construct of the present invention has the sequence of SEQ ID NO: 243 or a functional variant thereof (for example, an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 99.99% sequence identity with SEQ ID NO: 243, or a conservatively substituted variant of SEQ ID NO: 243, or a non-conservatively substituted variant of SEQ ID NO: 243).

[0314] In certain embodiments, the polypeptide constructs of the present invention include functional variants of SEQ ID NO: 243 that have a similar or enhanced binding affinity to HLA proteins associated with HPV16 / 18 and / or produce a similar or enhanced immunogenic response compared to SEQ ID NO: 243. Such variants can be readily determined by sequence alignment software such as ClustalW (see also Example 1 below).

[0315] In a particular embodiment, the variant has the same ankyrin scaffold as SEQ ID NO: 243, where the various HPV and agonist peptides of SEQ ID NO: 243 are shuffled in a different order than those of SEQ ID NO: 243. In such an embodiment, the variant has one of the sequences from SEQ ID NOs: 250 to 261.

[0316] In certain embodiments, the variant has the same ankyrin scaffold as SEQ ID NO: 243, as well as fewer HPV and agonist peptides than SEQ ID NO: 243. In some embodiments, the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fewer HPV / agonist peptides compared to SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 262, SEQ ID NO: 265, SEQ ID NO: 266, or SEQ ID NO: 267.

[0317] In certain embodiments, the variant has the same ankyrin scaffold as SEQ ID NO: 243, as well as more HPV and agonist peptides than SEQ ID NO: 243. In some embodiments, the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more HPV / agonist peptides compared to SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 263, SEQ ID NO: 268, SEQ ID NO: 269, or SEQ ID NO: 270.

[0318] In certain embodiments, the variant differs from SEQ ID NO: 243 in that the variant has a conserved amino acid substitution within the ankyrin-like repeat domain of SEQ ID NO: 243. In some embodiments, one or more conserved amino acid substitutions within the ankyrin-like repeat domain of SEQ ID NO: 243 are 1-5, 15-21, 31-33, 43-45, 55-63, 73-79, 100-102, 112-121, 137-141, 151-153, 163-173, 184-187, 197-199, 209-220, 231-233, 249-251, 262-2 This corresponds to a position selected from 64, 275-295, 304-311, 322-324, 335-337, 349-357, 373-379, 389-391, 402-411, 422-426, 437-439, 450-459, 475-478, 488-490, 500-511, 521-523, 534-536, 546-548, 559-561, or 571-572. In one such embodiment, the variant has one of the sequences of sequence numbers 245-249.

[0319] In certain embodiments, the variant differs from SEQ ID NO: 243 in that the variant has a conserved amino acid substitution within the ankyrin-like repeat domain of SEQ ID NO: 243, as well as more HPV and agonist peptides than SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 271.

[0320] In certain embodiments, the variant differs from SEQ ID NO: 243 in that the variant has a conserved amino acid substitution within the ankyrin-like repeat domain of SEQ ID NO: 243, as well as fewer HPV and agonist peptides than SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 272.

[0321] In certain embodiments, the variant differs from SEQ ID NO: 243 in that the variant has a conserved amino acid substitution within the ankyrin-like repeat domain of SEQ ID NO: 243, as well as various HPV and agonist peptides of SEQ ID NO: 243 that are shuffled in a different order than that of SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 264.

[0322] In certain embodiments, the variant is identical to SEQ ID NO: 243, except that the variant has a conserved amino acid substitution within the ankyrin-like repeat domain of SEQ ID NO: 243, various HPVs and agonist peptides of SEQ ID NO: 243 shuffled in a different order than that of SEQ ID NO: 243, and contains more HPVs and agonist peptides than SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 274.

[0323] In certain embodiments, the variant is identical to SEQ ID NO: 243, except that the variant has a conserved amino acid substitution within the ankyrin-like repeat domain of SEQ ID NO: 243, various HPVs and agonist peptides of SEQ ID NO: 243 shuffled in a different order than that of SEQ ID NO: 243, and contains fewer HPVs and agonist peptides than SEQ ID NO: 243. In some embodiments, the variant has the sequence of SEQ ID NO: 273.

[0324] Either a variant HPV antigen design or its polypeptide construct can be used as a component in a vaccine, such as an HPV vaccine. IV. Treatment Methods

[0325] The present invention relates, in part, to a method for treating a disease or disorder in a subject requiring such treatment, comprising the step of administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to the subject. In certain embodiments, the method comprises the step of administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to a subject having anogenital wart, lower genital neoplasms (e.g., intraepithelial neoplasms of the cervix, vagina, and vulva), cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer. In certain embodiments, the method comprises the step of administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the present invention to a subject having a malignant disease caused by HPV 16 / 18.

[0326] The present invention relates, in part, to HPV infection (e.g., HPV 16 / 18) in subjects who require it (e.g., subjects with penile cancer, cervical cancer, anal cancer, or oropharyngeal cancer). + The invention also relates to a method for priming a T cell response to cells, the method comprising the step of administering the vector of the invention to a subject. In certain embodiments, the method comprises administering the polynucleotides, polypeptides, vectors, compositions, vaccines, or cells of the invention to a subject having a malignant disease caused by HPV 16 / 18.

[0327] In certain embodiments, the method of the present invention prevents disease progression with a lower PU dose than prior methods known in the art. For example, in some embodiments, the method is 5e 9 In some embodiments, the method prevents disease protection by a PU dose of a vector, composition, or vaccine. In other embodiments, the method prevents disease protection by a 5e10 PU dose of a vector, composition, or vaccine. In some embodiments, the method of the present invention prevents disease progression by administering a smaller amount of therapeutic composition than previous methods known in the art. For example, in some embodiments, the method prevents disease protection by administering only a single dose of a vector, composition, or vaccine.

[0328] In certain embodiments, the subject to be treated is a mammal, such as a primate. In some embodiments, the subject to be treated is a human.

[0329] The method may involve the administration of polynucleotides, polypeptides, vectors, compositions, vaccines, or cells in therapeutically effective amounts to treat a disease or disorder, or to increase the activity of the T cell response to a specific HPV protein or antigen. The effective amount may vary depending on the subject's condition, age, sex, medical history, and / or weight. The amount may also vary depending on the condition to be treated, the anti-inflammatory agent encoded, the vector used for administration, the type of cells and / or vaccine, and the route of administration.

[0330] In certain embodiments, the vector, composition, or vaccine is administered in multiple doses. The amount of the vector, composition, or vaccine administered in a dose is the therapeutically effective dose of the vector, composition, or vaccine. In certain embodiments, the amount of medication in one dose is approximately 0.1 × 10⁻⁶ 9 ~About 10×10 12 Particle unit, 0.1 × 10⁻⁶ 9 ~Approx. 1.0×10 12 Particle size, approximately 0.1 × 10⁻⁶ 9 ~About 10×10 11 Particle size, approximately 0.1 x 10⁻¹⁶9 ~about 1.0x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 9 ~about 0.5x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 9 ~approximately 0.1 x 10 11 Particle size, approximately 1.0 x 10⁻¹⁶ 10 ~about 10x10 11 Particle size, approximately 1.0 x 10⁻¹⁶ 10 ~approximately 0.1 x 10 11 Particle size, approximately 0.1 x 10⁻¹⁶ 11 ~about 10x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~approximately 9x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~about 8x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~approximately 7x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 11 ~about 6x10 11 Particle size, approximately 0.5 x 10⁻¹⁶ 12 ~about 10x10 12 Particle size, approximately 0.5 x 10⁻¹⁶ 12 ~about 1.0x10 12 Particle size, approximately 1.0 x 10⁻¹⁶ 11 ~approximately 0.1 x 10 12 Particle size, approximately 0.1 x 10⁻¹⁶ 12 ~about 10x10 12 Particle unit, approximately 1 x 10⁻⁶ 10 Particle unit, approximately 5 x 10 10 Particle unit, approximately 5 x 10 11 Particle unit, approximately 6 x 10 11 Particle unit, approximately 7 x 10 11 Particle unit, approximately 8 x 10 11 Particle unit, approximately 9 x 10 11 Particle unit, approximately 10x10 11 Particle unit, approximately 1 x 10⁻⁶ 12 Particle unit, approximately 2 x 10⁻¹⁶ 12 Particle unit, approximately 3 x 10⁻¹⁶ 12 Particle unit, approximately 4 x 10 12 Particle unit, approximately 5 x 10 12 Particle unit, approximately 6 x 10 12 Particle unit, approximately 7 x 10 12 Particle unit, approximately 8 x 10 12 Particle unit, approximately 9 x 1012 Particle units, or about 10×10 12 may contain particle units.

[0331] In certain embodiments, the dosage may be about 1.0×10 5 ~ about 1.0×10 10 plaque-forming units (PFU), for example, about 0.5×10 5 ~ about 0.5×10 10 PFU, about 0.1x10 5 x0.1x10 10 PFU, about 1x10 6 ~ about 1x10 9 PFU, about 0.5x10 6 ~ about 0.5x10 9 PFU, about 0.1x10 6 ~ about 0.1x10 9 PFU, about 1x10 7 ~ about 1x10 8 PFU, about 0.5x10 7 ~ about 0.5x10 8 PFU, about 0.1x10 7 ~ about 0.1x10 8 PFU, about 1.0x10 6 ~ about 1.0x10 9 PFU, about 0.5x10 6 ~ about 0.5x10 9 PFU, about 1.0x10 7 ~ about 1x10 8 PFU, about 1.0x10 6 ~ about 1.0x10 8 PFU, about 0.5×10 6 ~ about 0.5×10 8 PFU, or about 0.1×10 6 ~ about 0.1×10 8 may contain PFU.

[0332] In certain embodiments, the dosage of any of the polynucleotides encoding any of the fusion proteins described herein may contain about 1×10 -5 ~ about 10 micrograms (μg) of polynucleotide. For example, the dosage of any of the polynucleotides encoding any of the fusion proteins described herein may be about 5×10-5 ~ about 5 μg, about 1 × 10 -4 ~ about 1, about 5 × 10 -4 ~ about 5 μg, about 5 × 10 -3 ~ about 5 μg, about 0.05 - about 5 μg, about 0.25 - about 5 μg, about 0.5 - about 5 μg, about 0.75 - about 5 μg, about 5 × 10 -4 ~ about 1 μg, about 5 × 10 -4 ~ about 0.5 μg, about 5 × 10 -4 ~ about 0.05 μg, or about 5 × 10 -4 ~ about 5 × 10 -3 It may contain polynucleotides of μg.

[0333] In some embodiments, the viral vector and / or polynucleotide encoding any of the fusion proteins described herein can be quantified by quantitative PCT analysis (Q-PCR) or analytical HPLC.

[0334] For the treatment of HPV-related conditions, the dosage of the vector is, for example, about 1 × 10 9 ~ about 1 × 10 13 particle units, about 5x10 9 ~ about 5x10 12 particle units, about 1x10 10 ~ about 1x10 12 particle units, about 1x10 11 ~ about 9x10 11 particle units, about 1x10 11 ~ about 9x10 11 particle units, about 1x10 11 ~ about 9x10 11 particle units, about 1x10 10 ~ about 1x10 12 particle units, about 1x10 11 ~ about 9x10 11 particle units, about 2x10 11 ~ about 8x10 11 particle units, about 3x10 11 ~ about 7x10 11 particle units, about 4 × 10 11 ~ about 6 × 10 11 particle units, or about 5 × 10 11 It can be particle units.

[0335] The dosage can be adjusted during the treatment process, for example, after monitoring the expression level of the transgene. If the level is higher or lower than the desired level, the amount or frequency of the dose can be adjusted accordingly.

[0336] The specific initial and ongoing medication regimens for each patient will vary depending on the nature and severity of the condition as determined by the diagnosing physician, the activity of the therapeutic agent, the patient's age, diet, frequency of administration, route of administration, drug excretion rate, and drug combinations.

[0337] The preferred treatment mode, dosage, route of administration, and medication schedule can be determined and / or adjusted according to methodologies known in the art.

[0338] The methods disclosed herein intend to utilize any route of administration known in the art for the delivery of polynucleotides, polypeptides, vectors, compositions, vaccines, or cells. For example, administration may be oral, subcutaneous, intramuscular, intravenous, intracranial, intra-articular, intradermal, or transdermal. In certain embodiments, subcutaneous or intra-articular administration is by syringe. In some such embodiments, the dose of the vector is contained in a composition in the form of an injectable formulation.

[0339] In certain embodiments, the dosage is contained in a composition having a volume of about 0.1 to about 5 ml, about 0.1 to about 4 ml, about 0.1 to about 3 ml, about 0.1 to about 2 ml, about 0.25 to about 1.75 ml, about 0.5 to about 1.5 ml, about 0.75 to about 1.25 ml, or about 1.0 ml. In some embodiments, the dosage is approximately 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1.0 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.1 ml, 2.2 ml, 2.3 ml, 2.4 ml, 2.5 ml, and 2.6 ml. It is contained in compositions having a volume of approximately 1, 2.7 ml, 2.8 ml, 2.9 ml, 3.0 ml, 3.1 ml, 3.2 ml, 3.3 ml, 3.4 ml, 3.5 ml, 3.6 ml, 3.7 ml, 3.8 ml, 3.9 ml, 4.0 ml, 4.1 ml, 4.2 ml, 4.3 ml, 4.4 ml, 4.5 ml, 4.6 ml, 4.7 ml, 4.8 ml, 4.9 ml, or 5.0 ml.

[0340] Administration of polynucleotides, polypeptides, vectors, compositions, or vaccines may be administered to any preferred site in the subject. The choice of administration site will depend on factors such as the volume of the dose to be administered, the age of the subject, the sex of the subject, and the type of active agent to be administered. Subcutaneous administration may be, for example, administered to the limbs, buttocks, or abdomen of the subject. For doses with larger volumes, intramuscular administration is preferred. Such administration may be, for example, administered to the deltoid muscle, vastus lateralis muscle, ventral gluteus, or dorsal gluteus muscle of the subject. Intravenous administration may be administered to, for example, the arm (e.g., the flexor of the arm), the back of the hand, or the top of the foot of the subject. Intra-articular administration may be administered to, for example, the knee, hip, shoulder, or ankle of the subject.

[0341] The medication regimen will vary depending on the patient's age, sex, and the type of active drug to be administered. Dosage may be once every hour, once daily, once weekly, once monthly, or once year.

[0342] In certain embodiments, doses are delivered at intervals of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. In certain embodiments, doses are delivered at intervals of approximately twice a day, approximately once every day, approximately twice a week, approximately once every week, approximately once every two weeks, approximately once every three weeks, approximately once every four weeks, or approximately once every five weeks. In a particular embodiment, the second dose is administered approximately one week, two weeks, three weeks, four weeks, or five weeks after the first dose; the third dose is administered two weeks, three weeks, four weeks, five weeks, or six weeks after the second dose; and the fourth dose is administered approximately three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, or twelve weeks after the third dose. In one embodiment, the second dose is administered approximately two weeks after the first dose, the third dose is administered approximately six weeks after the second dose, and the fourth dose is administered approximately twelve weeks after the third dose.

[0343] In some embodiments, the procedure includes surgical weight loss, typically by debridement, vasolytic laser, cryotherapy, or carbon dioxide laser. In some embodiments, the surgical procedure is performed via microscopic or endoscopic rigid laryngoscopy, using either a laser or a microdebrider to remove the papilloma, for example.

[0344] Prior to and / or following this, the polynucleotides, polypeptides, vectors, compositions, or vaccines of the present invention may be administered (alone or in combination with another therapeutic agent). In some embodiments, treatment of a patient with the polynucleotides, polypeptides, vectors, vaccines, or cells described herein, or pharmaceutical compositions containing them, reduces and / or eliminates the need for repeated surgical weight loss. V. Gene switch system

[0345] The gene switch may be any gene switch that modulates gene expression by adding or removing a specific ligand. In one embodiment, the gene switch is such that the level of gene expression depends on the level of ligand present. Examples of ligand-dependent transcription factor complexes that may be used in the gene switch of the present invention include, but are not limited to, members of the nuclear receptor superfamily activated by their respective ligands (e.g., glucocorticoids, estrogens, progestins, retinoids, ecdysones, and their analogs and mimetics), and rTTA activated by tetracyclines. In one aspect of the present invention, the gene switch is an EcR-based gene switch. Examples of such systems include, but are not limited to, the systems described in U.S. Patent No. 6,258,603, No. 7,045,315, U.S. Patent Application Publication No. 2006 / 0014711, U.S. Patent Application Publication No. 2007 / 0161086, and International Publication No. 01 / 70816. Examples of chimeric ecdysone receptor systems are described in U.S. Patent Publication No. 7,091,038, U.S. Patent Publication Nos. 2002 / 0110861, 2004 / 0033600, 2004 / 0096942, 2005 / 0266457, and 2006 / 0100416, as well as International Publication Nos. 01 / 70816, 02 / 066612, 02 / 066613, 02 / 066614, 02 / 066615, 02 / 29075, and 2005 / 108617. VII. Manufacturing of pharmaceuticals

[0346] The present invention also relates, in part, to the use of polynucleotides, polypeptides, vectors, vaccines, or cells, or compositions comprising them, as described herein, in the manufacture of pharmaceuticals for use in treating diseases or disorders in subjects requiring such treatment. In certain embodiments, the disease or disorder may be a proliferative disorder or disorder, such as cancer (e.g., HPV 16 / 18 malignancy). IX. Combination Therapy

[0347] In certain embodiments, the compositions and methods of the present invention can be combined with at least one additional active agent or therapy. Such additional therapies include radiotherapy, surgery (e.g., weight loss), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the aforementioned therapies. The additional therapy may be in the form of an adjuvant or neoadjuvant therapy.

[0348] In some embodiments, combination therapy includes the administration of polynucleotides, polypeptides, vectors, vaccines or cells, or compositions comprising them, as described herein, and the accompanying administration of one or more additional compounds, molecules, compositions or agents. The present invention also relates, in part, to combination therapy including the administration of polynucleotides, polypeptides, vectors, vaccines or cells, or compositions comprising them, as described herein, and the accompanying use of surgical or non-surgical procedures. A. Administration of combination therapy

[0349] In certain embodiments, the compositions of the present invention may be administered before, during, or after additional therapies, such as immune checkpoint therapy, or in various combinations with additional therapies. Administration may occur at intervals ranging from simultaneous to minutes, days, or weeks. In embodiments where the compositions are provided to the patient separately from additional therapeutic agents, the operator may generally ensure that no significant time elapses between each delivery time so that the two compositions can continue to exert a beneficial combined effect on the patient. Thus, the two therapies may be provided to the patient within approximately 12–24 hours, 48 ​​hours, or 72 hours from each other, and more particularly, within approximately 6–12 hours from each other. In some situations, the treatment period is a significant period between each dose, ranging from days (2, 3, 4, 5, 6, or 7 days) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8 weeks). Extending the period may also be desirable.

[0350] Various combinations can be used. In the following example, the composition of the present invention is "A" and the additional therapy is "B". A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0351] The administration of any compound or therapy to a patient will follow the general protocol for compound administration, taking into account any drug toxicity present. Therefore, some embodiments include a step to monitor toxicity resulting from the combination therapy.

[0352] In certain embodiments, at least one additional therapy includes co-administration of an additional agent. In some embodiments, the additional agent may be contained in the same composition containing polynucleotides, polypeptides, vectors, vaccines, or cells described herein. Such combination therapies may be useful for enhancing the treatment of a disease or disorder (e.g., improving the response of the subject, extending the effect of the treatment) and / or reducing any side effects of treatment with an anti-inflammatory agent. In some embodiments, HPV 16 / 18 malignancies will be treated.

[0353] Any suitable agent that can be combined with or with polynucleotides, polypeptides, vectors, vaccines, or cells described herein may be used. For example, the agent may be a therapeutic agent, such as a chemotherapeutic agent, an anti-inflammatory agent, an analgesic, a bio-response modifier, a vector containing such an agent, or a cell containing a therapeutic agent or nucleic acid encoding it.

[0354] In certain embodiments, the additional agent is administered at or near the same site as the composition containing the vector of the present invention. In certain other embodiments, the additional agent is administered at a different site, for example, on the opposite or opposite limb.

[0355] The administration of an additional agent may occur simultaneously with the administration of the composition containing the vector of the present invention. In certain embodiments, the additional agent may be contained in the same formulation as the formulation containing the vector and administered together with the vector in a single unit dose. In certain other embodiments, the additional agent may not be contained in the same formulation but may be administered simultaneously with the administration of the vector or within a limited time frame (e.g., one day, one hour, or fraction of an hour) after the administration of the vector.

[0356] Alternatively, the administration of additional agents may be sequential in relation to the administration of the composition containing the vector of the present invention. Such administration may be preferred in cases where it is desirable to minimize adverse reactions. In such embodiments, the additional agents may be administered according to a schedule based on the approved dosing regimen for those agents. Alternatively, the agents may be administered according to a schedule that helps to better maximize the therapeutic effect of the combination therapy while minimizing adverse reactions.

[0357] The timing of administration can be aligned with the specific mechanism of action and pharmacokinetics of each therapy, thereby maximizing synergistic effects and minimizing overlapping potential toxicity. Furthermore, treatment regimens can be adapted based on the individual patient's response and disease progression, thus providing flexibility in individualized treatment strategies. For example, if interleukins, such as IL-12, are one type of immunotherapy, in an embodiment, the initial dose may be administered before other agents to prime the immune system for response enhancement, followed by subsequent therapies to amplify the activated immune response and direct it toward the activated immune response. Alternatively, parallel administration of interleukins and immunotherapy may produce a synergistic immediate boost in antitumor activity, while continuous interleukin treatment supports sustained immune engagement. Sequential or parallel administration of interleukins and other immunotherapies presents a dynamic approach to orchestrate a robust and long-lasting antitumor immune response, thereby offering greater therapeutic potential compared to the administration of individual immunotherapies or agents alone.

[0358] In some embodiments, a dose greater than one dose of the first therapy is administered to the subject. In certain embodiments, a dose greater than one dose of the second therapy is administered to the subject. In even further embodiments, a dose greater than one dose of the third therapy is administered to the subject.

[0359] In some embodiments, subsequent doses of the first therapy are administered once every 1, 2, 3, or 4 weeks after the initial dose of the first therapy. In certain embodiments, subsequent doses of the second therapy are administered once every 1, 2, 3, or 4 weeks after the initial dose of the second therapy. In even further embodiments, subsequent doses of the third therapy are administered once every 1, 2, 3, or 4 weeks after the initial dose of the third therapy.

[0360] In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before the administration of the second therapy. In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before the administration of the second therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks before the administration of the second therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, or 6 months before the administration of the second therapy.

[0361] In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the administration of the second therapy. In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the administration of the second therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the administration of the second therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, or 6 months after the administration of the second therapy.

[0362] In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before the administration of the third therapy. In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before the administration of the third therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks before the administration of the third therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, or 6 months before the administration of the third therapy.

[0363] In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the administration of the third therapy. In some embodiments, the initial dose of the first therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the administration of the third therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the administration of the third therapy. In some embodiments, the initial dose of the first therapy is administered approximately 2, 3, 4, 5, or 6 months after the administration of the third therapy.

[0364] In some embodiments, the initial dose of the second therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours before the administration of the third therapy. In some embodiments, the initial dose of the second therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before the administration of the third therapy. In some embodiments, the initial dose of the second therapy is administered approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks before the administration of the third therapy. In some embodiments, the initial dose of the second therapy is administered approximately 2, 3, 4, 5, or 6 months before the administration of the third therapy.

[0365] In some embodiments, the initial dose of the second therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours after the administration of the third therapy. In some embodiments, the initial dose of the second therapy is administered approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the administration of the third therapy. In some embodiments, the initial dose of the second therapy is administered approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks after the administration of the third therapy. In some embodiments, the initial dose of the second therapy is administered approximately 2, 3, 4, 5, or 6 months after the administration of the third therapy. B. Exemplary combination therapies

[0366] Anti-inflammatory agents for use in such combination therapies include steroids and glucocorticoids, including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone; non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide, and mimicophenolates; and sphingosine 1-phosphate receptor modulators, including fingolimod (Gilenya®), ozanimod (Zeposia®), and amicelimod. In some embodiments, the NSAID is selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox inhibitors such as VIOXX® (rofecoxib) and CELEBREX® (celecoxib), and sialates.

[0367] Examples of analgesics for use in combination therapy include acetaminophen, oxycodone, tramadol, or propoxifen hydrochloride.

[0368] The HPV vaccine antigen of the present invention may be administered in combination with a second therapeutic agent, such as a bioresponse modifier. Examples of bioresponse modifiers suitable for use in combination therapy according to the present invention include, for example, molecules with directional properties towards cell surface markers (e.g., CD4, CD5); cytokine inhibitors, such as TNF inhibitors (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)); chemokine inhibitors; and cell signaling inhibitors, such as EGFR inhibitors (e.g., gefitinib (I)). RESSA® and erlotinib (TARCEVA®), nucleotide analogs (e.g., cidofovir), angiogenesis inhibitors (e.g., bevacizumab (AVASTIN®)), nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., COX-2 selective agents (e.g., celecoxib (CELEBREX®)), immune checkpoint inhibitors (e.g., PD-1 inhibitors (e.g., pembrolizumab (KEYTRUDA®), nivolumab (O)) Examples include PDIVO® and semiprimab (LIBTAYO®), PD-L1 inhibitors (e.g., atezolizumab (TECENTRIQ®), avelumab (BAVENCIO® and durvalumab (IMFINZI®)), adhesion molecule inhibitors, and other adjuvant therapies. In some embodiments, the second therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the second therapeutic agent is a PD-1 inhibitor. In some embodiments, the second therapeutic agent is pembrolizumab (KEYTRUDA®). Bio-response modifiers include not only monoclonal antibodies but also recombinant molecules. Exemplary disease-modifying antirheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0369] In some embodiments, the bio-response modifier is administered in doses ranging from about 0.1 mg / kg to about 10 mg / kg. In specific embodiments, the bio-response modifier is administered in doses of about 0.1 mg / kg, about 0.3 mg / kg, about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 10 mg / kg. In specific embodiments, the bio-response modifier is administered in a dose of 2 mg / kg. In other embodiments, the bio-response modifier is administered in a dose of about 10 mg / kg.

[0370] In some embodiments, the bioresponse modifier is administered in doses of 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, or 1,000 mg. In a particular embodiment, the bioresponse modifier is administered in a dose of 200 mg. In another embodiment, the bioresponse modifier is administered in a dose of 400 mg.

[0371] The dosing regimen for bioresponse modifiers can be adjusted based on individual patient factors, such as body weight, renal function, and hepatic function. Those skilled in the art can determine the most appropriate dosing schedule for each patient.

[0372] Bio-response modifiers may be administered before, concurrently with, or after the administration of the HPV vaccine antigen. For example, bio-response modifiers may be administered approximately 1 day, 3 days, 1 week, 2 weeks, or 1 month before or after the administration of the HPV vaccine antigen.

[0373] In some embodiments, the bioresponse modifier may be administered multiple times, including, but not limited to, twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, or once every ten weeks.

[0374] The duration of treatment with bio-modifiers may vary depending on the type of cancer, the response to the treatment, and tolerance. In some cases, treatment may be continued until disease progression or unacceptable toxicity occurs, while in others, a fixed treatment duration (e.g., 1-2 years) may be recommended. In some embodiments, the duration of treatment with bio-modifiers may be up to 12, 18, 24, 30, or 36 months. In certain embodiments, the duration of treatment with bio-modifiers may be up to 24 months.

[0375] The HPV vaccine antigen of the present invention may be administered in combination with a bio-response modifier within the dosage range disclosed herein for the treatment of various cancers. Exceptional cancers that can be treated with a combination of HPV vaccine antigens and bio-response modifiers include, but are not limited to, cervical cancer, vulvar cancer, vaginal cancer, anal cancer, penile cancer, oropharyngeal cancer (throat cancer), recurrent respiratory papillomatosis (RRP), melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck squamous cell carcinoma (HNSCC), classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite instability-high frequency (MSI-H) or mismatch repair deficiency (dMMR) solid tumors, gastric cancer, esophageal cancer, hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), renal cell carcinoma (RCC), endometrial cancer, high tumor mutational load (TMB-H) solid tumors, cutaneous squamous cell carcinoma (cSCC), and triple-negative breast cancer (TNBC).

[0376] In some embodiments of the present invention, the bio-response modifier administered in combination with the HPV vaccine antigen is pembrolizumab. Pembrolizumab is a humanized monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. Pembrolizumab may be administered within the dosing range disclosed herein and may depend on the cancer being treated and the individual characteristics of the patient. In some embodiments, the dosing regimen of pembrolizumab may be a) 200 mg administered as an intravenous infusion over 30 minutes every three weeks (Q3W); b) 400 mg administered as an intravenous infusion over 30 minutes every six weeks (Q6W); c) 2 mg / kg administered as an intravenous infusion over 30 minutes every three weeks (Q3W); or d) 10 mg / kg administered as an intravenous infusion over 30 minutes every two weeks (Q2W) or every three weeks (Q3W).

[0377] In certain embodiments, a combination of an HPV vaccine antigen and a bioresponse modifier, such as pembrolizumab, is administered for the treatment of cervical cancer, HPV-related cancers, HPV-related malignancies, and / or oropharyngeal squamous cell carcinoma. These cancers are known to be associated with HPV infection, and the combination therapies disclosed herein may provide enhanced therapeutic efficacy compared to either the HPV vaccine antigen or the bioresponse modifier alone.

[0378] In certain embodiments, the HPV vaccine antigens provided herein are co-delivered and / or co-expressed together with other cytokines (e.g., as part of the same HPV antigen delivery vector or by a different vector). In certain embodiments, the HPV vaccine antigens provided herein are polynucleotides encoding gene switch polypeptides and cytokines, or variants or derivatives thereof, as well as methods and systems incorporating them. Cytokines are a category of small molecules between approximately 5 and 20 kDa that are involved in cell signaling. In some examples, cytokines include chemokines, interferons, interleukins, colony-stimulating factors, or tumor necrosis factors. In some embodiments, chemokines act as chemotaxis that guide cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. Exemplary chemokines include the CC subfamily: CCLI, CCL2 (MCP-1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCLI0), CCLI 1, CCL12, CCL13, CCL14, CCL15, CCL16, CCLI 7, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; the CXC subfamily: CCXCLI, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CCXCLI0, CXCLII, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CCXCLI 7. Examples include chemokines from the XC subfamily: XCLI and XCL2, and the CX3C subfamily: CX3CL1.

[0379] In certain embodiments, the HPV vaccine antigens provided herein are co-delivered and / or co-expressed together with a cyclin-dependent kinase inhibitor (CKI). In some embodiments, the CKI specifically inhibits CDK4 and CDK6 (e.g., p16INK4a). In some embodiments, the CKI consists of one or more 21Cip1, p27Kip1, or p57Kip2. In some embodiments, the CKI is delivered by administration of palbociclib (Ibrance), ribociclib (Kisqali), or abemaciclib (Verzenio).

[0380] In some embodiments, the dose of CKI administered is approximately 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or 1000 mg.

[0381] In certain embodiments, the HPV vaccine antigens provided herein are co-delivered and / or co-expressed together with interferon (e.g., as part of the same HPV antigen delivery vector or by a different vector). Interferon (IFN) includes interferon type I (e.g., IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω), interferon type II (e.g., IFN-γ), and interferon type 111. In some embodiments, IFN-α is further classified into about 13 subtypes, including IFNAI, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNAI 7, and IFNA21.

[0382] In certain embodiments, the HPV vaccine antigens provided herein are co-delivered and / or co-expressed together with interleukins (for example, as part of the same HPV antigen delivery vector or by a different vector). Interleukins are expressed by leukocytes or white blood cells and promote the development and differentiation of T and B lymphocytes and hematopoietic cells. Examples of interleukins include IL-I, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (CXCL8), IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, and IL-36, as well as their functional fragments or variants. For some applications, the interleukin is IL-2, IL-12, IL-15, IL-21, or their functional fragments or variants. In some embodiments, the interleukin is IL-15 or a functional fragment or variant thereof, and is contained in a fusion protein comprising IL-15 or a functional variant thereof, and IL-15α or a functional fragment or variant thereof.

[0383] In certain embodiments, the interleukin is IL-12, or a functional fragment or variant thereof.

[0384] IL-12 is spontaneously produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen stimulation. IL-12 is composed of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two distinct genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (called p70) and homodimer of p40 are formed after protein synthesis. IL-12 is a major regulator of the immune system. IL-12 induces local and systemic production, initiating a cytokine cascade that results in the downstream production of endogenous interferon-γ (IFN-γ), activating innate (i.e., NK cell) and adaptive (i.e., T lymphocyte) immunity through these signaling pathways. See Figure 11. The adaptive immune system induces T cells to change from a naive phenotype to an effector or memory phenotype. The Th1 / Th2 phenotype indicates the result of naive T cell activation. IL-12 also acts to remodel the tumor microenvironment (TME) and has anti-angiogenic effects. IL-12 binds to the IL-12 receptor (IL-12R), which is a heterodimeric receptor formed by IL-12R-β1 and IL-12R-β2. This receptor complex is primarily expressed by T cells, but is also expressed by other lymphocyte subpopulations that have been found to be responsive to IL-12.

[0385] IL-12 is a candidate for tumor immunotherapy in humans because it plays a bridging role between innate and adaptive immunity. In fact, IL-12 has demonstrated efficacy in animal models of tumor treatment. However, in human therapeutic studies, systemic administration of IL-12 is often associated with clinically serious side effects. Despite these problems, IL-12 remains of great interest for use in human (clinical) oncology because its full therapeutic potential, particularly when used alone or in combination with other tumor-treating compounds and treatments, and especially when administered locally rather than systemically, has not been sufficiently investigated, let alone realized.

[0386] In certain embodiments, IL-12 is single-stranded IL-12 (scIL-12), protease-sensitive IL-12, destabilized IL-12, membrane-bound IL-12, or intercalated IL-12. In some examples, IL-12 variants are as described in International Publication Nos. 2015 / 095249, 2016 / 048903, and 2017 / 062953.

[0387] In certain embodiments, the HPV vaccine antigen provided herein is delivered to and / or expressed in a subject in conjunction with the delivery and / or expression of IL-12 or a functional fragment or variant thereof. In some embodiments, the IL-12 polypeptide, or a functional fragment or variant thereof, is expressed from the same HPV vaccine antigen expression vector. In other embodiments, the IL-12 polypeptide, or a functional fragment or variant thereof, is expressed from a different vector in conjunction with the delivery or expression of the HPV vaccine antigen. In some embodiments, the vector expressing IL-12 or a functional fragment or variant thereof is a replication-deficient adenovirus vector (e.g., a GC46 gorilla adenovector).

[0388] In certain embodiments, in conjunction with the delivery or expression of the present invention (i.e., the novel HPV antigen design disclosed herein), interleukin expression in the subject is controlled by constitutive or inductive regulation of expression. In preferred embodiments, in conjunction with the delivery or expression of the present invention, interleukin expression in the subject is controlled by inductive regulation of expression (also known as inductively regulated expression of interleukin). See Figure 12.

[0389] In certain embodiments, IL-12 is expressed in a gene construct comprising a polynucleotide encoding IL12p40 or a functional fragment or variant, to which IL-12 is linked by an IRES (e.g., EMCV IRES) to a polynucleotide encoding IL12p35 or a functional fragment or variant. In certain other embodiments, IL-12 is expressed as a fusion protein comprising IL12p40 or a functional fragment or variant, and IL12p35 or a functional fragment or variant. In certain such embodiments, IL12p40 or a functional fragment or variant is linked to IL12p35 or a functional fragment or variant by a peptide linker.

[0390] In certain embodiments, upon delivery or expression of the present invention, IL-12 is expressed as single-stranded IL-12p70 embedded in a GC46 gorilla adenovector (either the same adenovector that delivers the HPV vaccine antigen of the present invention, or a different adenovector) capable of producing dose-dependent bioactive IL-12. In further embodiments, there is no pre-existing immunity or the presence of neutralizing antibodies against the GC46 gorilla adenovector(s), which may limit the usefulness of treating patients with the present invention in combination with IL-12. In certain embodiments, the single-stranded IL-12p70 has bioactivity similar to that of the naturally occurring recombinant protein and does not tend to produce regulatory IL-12p40 homodimers.

[0391] In certain embodiments, interleukins are delivered into the tumor in accordance with the present invention. In other embodiments, interleukins are delivered locally to the tumor site or to lymph nodes associated with the tumor.

[0392] In a particular embodiment, the vector expressing interleukin is approximately 1 × 10⁻⁶ 11 , 2×10 11 , 3 x 10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , 9×10 11 , or 1 × 10 12 , or 2 × 10 12 It is administered in unit doses of viral particles (VP). In some embodiments, the vector is approximately 2 × 10⁻¹⁶ 11 It is administered in a dose of vp. In other embodiments, the vector is approximately 5 × 10 11 It is administered at the VP dose.

[0393] The initial dose of a composition or vector expressing a novel HPV antigen design disclosed herein and the initial dose of interleukin are administered in parallel or simultaneously. For example, the initial dose of the composition or vector expressing the HPV antigen may be administered some time after the initial dose of interleukin. Alternatively, the initial dose of the composition or vector expressing the HPV antigen may be administered some time before the initial dose of interleukin. In some embodiments, the initial dose of interleukin is administered about 1, 2, 3, 4, 5, 6, or 7 days or more before the administration of the composition or vector expressing the HPV antigen. In some embodiments, one or more subsequent doses of interleukin are administered after the administration of the initial dose of the composition or vector expressing the HPV antigen. In some embodiments, one or more subsequent doses of interleukin are administered within 7 to 28 days after the administration of the composition or vector expressing the HPV antigen. In some embodiments, one or more subsequent doses of interleukin are administered at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days after administration of the composition or vector expressing the HPV antigen. In some embodiments, one of the subsequent doses of interleukin is administered 15 days after administration of the composition or vector expressing the HPV antigen.

[0394] In other embodiments, subsequent doses of interleukin are administered once every 1, 2, 3, or 4 weeks after the first dose of interleukin. In further such embodiments, subsequent doses of interleukin are administered once every 2 weeks or once every 4 weeks after the first dose of interleukin.

[0395] In some embodiments, the interleukin is membrane-bound IL-15. In certain such embodiments, membrane-bound IL-15 (mbIL-15) comprises full-length IL-15 (e.g., native IL-15 polypeptide) or a functional fragment or variant thereof, fused in frame with full-length IL-15Rα or a functional fragment or variant thereof. In some cases, IL-15 is indirectly linked to IL-15Rα by a linker. In some embodiments, mbIL-15 is as described in Hurton et al., “Tethered IL-15 augments antitumor activity and promotes a stem-cell memory subset in tumor-specific T cells,” PNAS 2016.

[0396] In certain embodiments, the HPV vaccine antigens provided herein are co-delivered and / or co-expressed together with tumor necrosis factor (e.g., as part of the same HPV antigen delivery vector or by a different vector). Tumor necrosis factor (TNF) is a group of cytokines that modulate apoptosis. In some examples, the TNF family includes about 19 members, including TNFα, lymphotoxin-alpha (LT-alpha), lymphotoxin-beta (LT-beta), T cell antigen gp39 (CD40L), CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-associated apoptosis-inducing ligand (TRAIL).

[0397] In certain embodiments, the HPV vaccine antigens provided herein are co-delivered and / or co-expressed together with colony-stimulating factors (e.g., as part of the same HPV antigen delivery vector or by another vector). Colony-stimulating factors (CSFs) are secreted glycoproteins that interact with receptor proteins on the surface of hematopoietic stem cells, thereby modulating cell proliferation and differentiation into specific types of blood cells. In some examples, CSFs include macrophage colony-stimulating factor, granulocyte-macrophage colony-stimulating facto...

Claims

1. A pharmaceutical composition for use in combination with an immune checkpoint inhibitor to treat an HPV-related disease or disorder, comprising a polynucleotide encoding a fusion protein containing two or more HPV peptides selected from HPV-16 E5, HPV-16 E6, HPV-16 E7, HPV-18 E6, and HPV-18 E7 peptides.

2. The fusion protein comprises: (a) HPV-16 E5 peptide containing the amino acid sequence of SEQ ID NO: 130; (b) HPV-16 E6 peptide containing any one of the amino acid sequences from SEQ ID NOs: 113 to 121; (c) HPV-16 E7 peptide containing any one of the amino acid sequences from SEQ ID NOs: 122 to 129; (d) HPV-18 E6 peptide containing any one of the amino acid sequences from SEQ ID NOs: 131 to 138; and (e) HPV-18 E7 peptide containing any one of the amino acid sequences from SEQ ID NOs: 139 to 144. A pharmaceutical composition according to claim 1, comprising:

3. The pharmaceutical composition according to claim 1, wherein the fusion protein further comprises an agonist peptide.

4. The pharmaceutical composition according to claim 3, wherein the agonist peptide comprises any one amino acid sequence from SEQ ID NOs: 145 to 147.

5. The pharmaceutical composition according to claim 1, wherein two or more HPV peptides are grafted onto a human ankyrin-like repeat protein scaffold.

6. The pharmaceutical composition according to claim 1, wherein the fusion protein comprises one of the amino acid sequences of SEQ ID NOs. 52, 243, and 250-261 or a conservatively substituted variant thereof.

7. The pharmaceutical composition according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 243 or a conservatively substituted variant thereof.

8. The pharmaceutical composition according to claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:

243.

9. The pharmaceutical composition according to claim 1, wherein the polynucleotide is contained in the vector.

10. The pharmaceutical composition according to claim 9, wherein the vector is an adenovirus vector.

11. The pharmaceutical composition according to claim 10, wherein the adenovirus vector lacks one or more elements selected from the E1-E4 region and the L1-L5 region.

12. The pharmaceutical composition according to claim 10, wherein the adenovirus vector is a gorilla adenovirus vector.

13. The pharmaceutical composition according to claim 10, wherein the adenovirus vector is a GC46 gorilla adenovirus vector.

14. The pharmaceutical composition according to claim 1, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody.

15. The pharmaceutical composition according to claim 14, wherein the anti-PD-1 antibody is nivolumab or pembrolizumab.

16. The pharmaceutical composition according to claim 15, wherein the anti-PD-1 antibody is pembrolizumab.

17. The pharmaceutical composition according to claim 1, wherein the HPV-related disease or disorder is a lower genital neoplasm, cervical cancer, vulvar cancer, anal cancer, penile cancer, or head and neck cancer.

18. The therapeutically effective dose of the adenovirus vector is approximately 0.1 × 10⁻⁶. ９ ~About 10×10 １２ The pharmaceutical composition according to claim 10, which is in the form of particles.

19. The pharmaceutical composition according to claim 1, wherein the therapeutically effective dose of the immune checkpoint inhibitor is about 10 to about 1000 mg.

20. A kit for use in treating an HPV-related disease or disorder, comprising (a) the pharmaceutical composition according to claim 1 and (b) an immune checkpoint inhibitor.

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