RU2850195C1 - Vaccines against human papillomavirus and their use in the treatment of hpv-related diseases - Google Patents

Abstract

FIELD: biotechnology, virology; medicine.

SUBSTANCE: polynucleotide encoding a polypeptide not found in nature, a polypeptide for use in treating a disease or disorder caused by human papillomavirus type 6 and/or human papillomavirus type 11, a vector, a composition containing a pharmaceutically acceptable carrier and a polynucleotide for use in treating a disease or disorder caused by human papillomavirus type 6 and/or human papillomavirus type 11, and the use of the composition in the manufacture of a medicament for treating a disease or disorder caused by human papillomavirus type 6 and/or human papillomavirus type 11.

EFFECT: obtaining a vaccine for the treatment of diseases and pathologies associated with HPV6 and HPV11.

14 cl, 23 dwg, 5 tbl

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to U.S. Provisional Patent Application No. 63/118,222, filed May 25, 2020, which is incorporated herein by reference in its entirety.

LINK TO SEQUENCE LISTING

[2] The contents of the electronically filed sequence listing (name: INX00511US - V1_SEQ - LIST_ST25; size: 71267 bytes; creation date: November 24, 2020), which was filed with this application, are incorporated herein by reference in their entirety.

FIELD OF INVENTION

[3] This invention relates to innovative multi-antigen molecular vaccines against HPV. More specifically, this invention relates to molecular vaccines against HPV targeting pathologies associated with HPV6 and HPV11.

LEVEL OF TECHNOLOGY

[4] Currently, tens of millions of people worldwide are infected with the human papillomavirus (HPV), and millions of people become newly infected each year. Currently, vaccinations can protect against HPV-related diseases when administered to recommended age groups; however, there is a significant need for HPV vaccines with expanded coverage (e.g., against multiple and/or targeted subsets of HPV strains) and functionality against HPV-associated pathologies.

[5] Recurrent respiratory papillomatosis (RRP) is a rare disease characterized by the recurrent growth of wart-like benign papillomas in the respiratory tract. RRP occurs in both children and adults. Juvenile onset is almost always diagnosed by age ten and usually before age five. RRP is caused by the human papillomavirus (HPV), specifically HPV6 and HPV11.

[6] Genital warts are extremely common and may also be known by other names such as anogenital warts or genital condylomata acuminata, with between 500,000 and one million new cases diagnosed annually in the United States alone. HPV6 and HPV11 cause approximately 90% of genital warts. Current treatment options primarily focus on removing the warts rather than eliminating the underlying viral infection. A wide range of treatments are currently used, which vary widely and can have significant differences in effectiveness. However, no treatment has been 100% effective in eliminating warts and preventing their recurrence in all patients.

[7] Currently, the standard treatment for RRP is surgical excision with adjuvant therapy as needed. However, a distinctive aspect of RRP is its tendency to recur, requiring repeat surgeries. Aggressiveness varies among patients: some may require surgery every few weeks, while others may only require surgery twice a year or only a few times during their lifetime.

[8] Unfortunately, previously developed treatments have failed to prevent multiple recurrences of RRP. More effective treatments for patients with RRP are needed. This paper describes an innovative immunotherapy approach, a novel therapeutic HPV vaccine aimed at inducing immunity against HPV6/11 for the treatment and modification of RRP and other HPV6/11-associated diseases such as genital warts.

[9] Vaccines have traditionally been used as preventative measures against infectious diseases. Several HPV vaccines (GARDASIL and CERVARIX) have been developed and approved by the Food and Drug Administration (FDA) to protect women from cervical cancer, which is also associated with infection by HPV of the same subtypes that cause RRP.

[10] GARDASIL is a vaccine against HPV types 6, 11, 16, and 18 (RRP is caused by types 6 and 11), and CERVARIX is a vaccine only against HPV types 16 and 18. Gardasil 9 is designed to protect against high-risk strains 31, 33, 45, 52, 58.

[11] The prophylactic vaccines described above have been successful in preventing HPV infection in healthy individuals and reinfection in previously infected patients; however, they cannot treat or cure patients who are already infected with HPV.

[12] A number of therapeutic HPV vaccines have entered clinical trials, some of which show promising results. MVA E2, a recombinant modified vaccinia virus Ankara, was reported to demonstrate 90 % lesion clearance in female patients and 100 % lesion clearance in male patients. IN0-3106 , a DNA vaccine consisting of plasmids encoding E6 and E7, showed clinical efficacy in a study of two patients with recurrent respiratory papillomatosis. However, despite this apparent positive progress, there is a shortage of approved therapeutic HPV vaccines; indeed, as of the filing date, the patent applicants are not currently aware of any FDA-approved therapeutic HPV vaccine.

INCORPORATION BY LINK

[13] All publications, patents, and patent applications mentioned in this document are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

ESSENCE OF THE INVENTION

[14] Provided herein are non-naturally occurring polynucleotides (and polypeptides expressed therefrom) encoding non-naturally occurring polypeptides comprising human papillomavirus (HPV) immune response-inducing polypeptides. Also provided herein are non-naturally occurring, variably arranged configurations of HPV immune response-inducing polypeptides joined by different polypeptide linker sequences; thus, including fusion proteins useful as vaccine antigens.

[15] The present invention includes, but is not limited to, compositions, methods for making the compositions, and uses of unnaturally occurring polynucleotides encoding unnaturally occurring polypeptides comprising antigens for vaccines directed against human papillomavirus (HPV), particularly HPV6 and HPV11; and therapeutic approaches to treating pathological conditions caused by these particular HPV agents. In particular, the present invention includes polynucleotides and the polypeptides encoded therefrom encoding multiantigen polypeptides derived from HPV6, HPV11, and HPV16, and other polypeptide sequences for use as vaccine components and for treating disorders associated with HPV infection. For example, an "indication" of the present invention is the use of the compositions described herein as therapeutic vaccines against diseases induced by or associated with HPV6 and/or HPV11 (HPV6/11); such as, but not limited to, the treatment of recurrent respiratory papillomatosis (RRP), anogenital warts and other HPV6/11-associated diseases.

[16] The present invention includes unique and innovative configurations of approaches to developing an HPV polypeptide vaccine for HPV6/11-induced diseases. The present invention includes a combination of design strategies; for example, the use of complete protein sequences, the use of peptide "fragments," hybrid polypeptide constructs, the introduction of amino acid substitutions, insertions, deletions, and rearrangements of HPV polypeptide and peptide gene products (proteins). The five exemplary design concepts (Designs Nos. 1-5 ) as described herein can be further modified and/or optimized for various variants as needed.

[17] As noted above, the unique modifications of the antigens described herein were accomplished by introducing point mutations, substitution mutations, and/or reordering of viral polypeptide sequences, in particular to prevent oncogenic gene expression and/or disable essential viral functions (e.g., viral replication).

[18] In some embodiments, early HPV E2 and E4 proteins have been identified as novel antigens for HPV6/11 and are included in the designs described herein. In some embodiments, the present invention includes a novel incorporation of four antigenic components from HPV into a single HPV vaccine design. In some embodiments, the present invention includes a novel integration and combination of epitopes from both high- and low-cancer-risk HPV genotypes in a single HPV antigen design. The antigen designs described herein were carefully designed and selected for potential human antigenicity in vivo without cross-reactivity with endogenous human proteins, as determined by in silico analysis .

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[19] Fig. 1 provides a schematic representation of the HPV6/11 antigen design, including reference to the tools and methods used to develop and evaluate the designs described herein.

[20] Figures 2-6 show schematics, sequences, and structural representations of the polypeptides and modules used therein for HPV6/11 antigen designs #1 through #5.

DETAILED DESCRIPTION OF THE ESSENCE OF THE INVENTION

[21] It should be understood that the present invention is not limited to the specific embodiments described herein and is therefore subject to variation. Those skilled in the art will appreciate that there are variations and modifications of the present invention that are within its scope.

[22] The rigid linker, cleavable linkers, HPV epitope linkers, and HPV agonist enhancer linkers used to connect the antigenic components as described herein and as shown in the figures and sequence listing may be further configured in a variety of other permutations.

[23] Embodiments of the present invention include the use of a rigid linker peptide (EAAAK)n; i.e., (SEQ ID NO:76)n; wherein "n" can range from 1 to 10 (e.g., EAAAK is n=1; EAAAKEAAAK is n=2, EAAAKEAAAKEAAAK is n=3, etc.) and can be encoded by different nucleotide sequences (e.g., but not limited to, SEQ ID Nos: 77-81 ). It is also contemplated that the number of "E" (also known as Glu; glutamic acid), "A" (also known as Ala; alanine), and "K" (also known as Lys; lysine) residues can vary in number and pattern of repetition when creating rigid linker peptide sequences of similar composition.

[24] Similarly, the antigenic fragments as described herein and as shown in the figures can be intermixed in various permutations both within and between each construct.

[25] The vaccine design as described herein and as shown in the figures may be used for the treatment of recurrent respiratory papillomavirus pathologies and disorders, as well as for the treatment of genital, anogenital and other warts, and other diseases, disorders and hyperproliferative pathologies caused by HPV.

[26] Constitutive or induced expression of HPV early (E) region proteins provides targets for an effective HPV6/11 vaccine.

[27] HPV early region protein functions include the following: E1, E2 function in viral replication/transcription (e.g., E2 regulates the expression of E6 and E7, and the E1/E2 interaction is required for viral replication); E4, E5 are overexpressed late in the viral replication cycle (although HPV11 E4 is not essential for viral genome amplification); and E6, E7 act together during replication (E6 is required for episomal genome maintenance, E7 expands the epithelial cell compartment active in DNA replication).

[28] E2 is an excellent target, is expressed in infected cells, elicits a strong immune response, and there is preclinical and clinical data for the use of E2 as a single target in a vaccine (plasmid DNA and MVA E2).

[29] E4 is a unique target, expressed in infected cells, late overexpression, although not essential for HPV11 replication, may play a critical role in establishing an episomal state; no vaccine data available.

[30] E6 and E7 are both expressed in infected cells but are absent from healthy cells, making them ideal antigen targets, and there is clinical data for the use of E6 and E7 as targets in vaccines.

[31] All terms shall be understood as understood by one of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[32] The section headings used in this document are for organizational purposes only and are not intended to limit the scope of the invention described.

[33] Although various features of the present invention may be described in the context of a single embodiment, the features may also be provided individually or in any suitable combination. The following definitions supplement those used in the art and are specific to the current application and are not intended to be interpreted as referring to any related or unrelated instance, such as any patent or application under common ownership. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Definitions

[34] In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used in the present description and the appended claims, the singular forms include references to the identified objects in the plural unless the context clearly dictates otherwise.

[35] In this application, the use of "or" means "and/or" unless otherwise specified. The terms "and/or" and "any combination thereof" and their grammatical equivalents as used herein may be used interchangeably. These terms may mean that any combination is specifically contemplated. 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" may be used in conjunction or singly unless the context specifically indicates a disjunctive use.

[36] Furthermore, the use of the term "including", as well as other forms such as "include", "includes" and "included", is not limiting; i.e., "including" does not mean "limited".

[37] Reference in the description to “some embodiments,” “an embodiment,” “one embodiment,” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the invention.

[38] As used in this specification and claim(s), the words "comprising" (and any form of inclusion, such as "comprise" and "contains"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of inclusion, such as "includes" and "include"), or "containing" (and any form of inclusion, such as "comprises" and "comprise") are inclusive or non-limiting, and do not exclude additional possible components, elements, or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, the compositions of the invention can be used to implement the methods of the invention.

[39] The term "about" in relation to a reference numerical value and its grammatical equivalents, in the context of this document, may include the numerical value itself and a range of values plus or minus 10% of that numerical value.

[40] The term "about" or "approximately" means within an acceptable range of errors for a particular value as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation in accordance with practice in the art. Alternatively, "about" may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. In another example, the quantity "about 10" includes 10 and any quantity from 9 to 11. In yet another example, the term "about" with respect to a reference numerical value may also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that value. Alternatively, particularly with respect to biological systems or processes, the term "about" may mean a value within an order of magnitude, preferably within 5 times , and more preferably within 2 times, the value. If the application and claims describe specific values, unless otherwise stated, the term "about" should be understood to mean within the permissible range of error for the specific value.

[41] The term "isolated" and its grammatical equivalents, as used herein, refer to the removal of a nucleic acid from its natural environment. The term "purified," as used herein, and its grammatical equivalents, refer to a molecule or composition removed from nature (including genomic DNA and mRNA) or synthesized (including cDNA) and/or amplified in a laboratory setting, the purity of which has been increased, where "purity" is a relative term and not "absolute purity." However, it should be understood that nucleic acids and proteins may be prepared with diluents or adjuvants and still be 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 equivalents as used herein refer to a nucleic acid sequence, polypeptide, protein or other compound that is substantially free of, i.e., more than 50% free of, more than 70% free of, or more than 90% free of, polynucleotides, proteins, polypeptides and other molecules with which the nucleic acid, polypeptide, protein or other compound is naturally associated.

[42] "Polynucleotide," "oligonucleotide," "polynucleotide construct," "gene," "gene construct," "heterologous gene," and their grammatical equivalents, as used herein, refer to a polymeric form of nucleotides or nucleic acids of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded and single-stranded DNA, triplex DNA, and double-stranded and single-stranded RNA. It also includes modified, for example, by methylation and/or capping, and unmodified forms of a polynucleotide. The term also includes molecules that include non-naturally occurring or synthetic nucleotides, as well as nucleotide analogs. The nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into a cell, for example, by transfection, transformation, or transduction.

[43] "Polynucleotides encoding polypeptides" of the present invention, and similar or similar terms and phrases used herein, include any polynucleotides that can be used to express (i.e., encode) the polypeptides of the present invention. Indeed, it is well known and understood by those skilled in the art how to construct any number of different polynucleotide sequences that can encode the same polypeptide (i.e., a polypeptide with an identical amino acid sequence, albeit encoded by different codon sequences) (nucleotide triplets)). For example, different codons in the standard genetic code can be used to create a plurality of polynucleotide sequences encoding a particular polypeptide. Furthermore, it is also possible to create a plurality of polynucleotide sequences encoding a particular polypeptide by using codon selection in accordance with guidelines and selected using the "codon usage frequency" observed in a given organism; for example, but not limited to, the Homo sapiens codon usage frequency table as shown below.

[44] Accordingly, it is also well known and understood by those skilled in the art that amino acids are encoded by codons (nucleotide triplets) such as in the table below:

Table 1: Standard genetic code The position of a nucleotide in a codon is encoded by an amino acid. The first nucleotide Second
nucleotide Third nucleotide U C A G U UUU - Phe
UUC - Phe
UUA - Leu
UUG - Leu UCU - Ser
UCC - Ser
UCA - Ser
UCG - Ser UAU - Tyr
UAC - Tyr
UAA - *
UAG - * UGU - Cys
UGC - Cys
UGA - *
UGG - Trp U
C
A
G C CUU - Leu
CUC - Leu
CUA - Leu
CUG - Leu CCU - Pro
CCC - Pro
CCA - Pro
CCG - Pro CAU - His
CAC - His
CAA - Gln
CAG - Gln CGU - Arg
CGC - Arg
CGA - Arg
CGG - Arg U
C
A
G A AUU - Ile
AUC - Ile
AUA - Ile
AUG - Met ACU - Thr
ACC - Thr
ACA - Thr
ACG - Thr AAU - Asn
AAC - Asn
AAA - Lys
AAG - Lys AGU - Ser
AGC - Ser
AGA - Arg
AGG - Arg U
C
A
G G GUU - Val
GUC - Val
GUA - Val
GUG - Val GCU - Ala
GCC - Ala
GCA - Ala
GCG - Ala GAU - Asp
GAC - Asp
GAA - Glu
GAG - Glu GGU - Gly
GGC - Gly
GGA - Gly
GGG - Gly U
C
A
G

(U stands for "uracil," included in mRNA in place of T (thymine) found in DNA; both form base pairs complementary to "A"(adenine);"*" indicates stop codons)

Table 2: Homo sapiens, codon frequency table [frequency per thousand nucleotide triplets in H. sapiens ] UUU [17.6] UCU [15.2] UAU [12.2] UGU [10.6] UUC [20.3] UCC [17.7] UAC [15.3] UGC [12.6] UUA [7,7] UCA [12.2] UAA [1.0] UGA [1.6] UUG [12.9] UCG [4,4] UAG [0.8] UGG [13.2] CUU [13.2] CCU [17.5] CAU [10.9] CGU [4.5] CUC [19.6] CCC [19.8] CAC [15.1] CGC [10.4] CUA [7.2] CCA [16.9] CAA [12.3] CGA [6.2] CUG [39.6] CCG [6.9] CAG [34.2] CGG [11.4] AUU [16.0] ACU [13.1] AAU [17.0] AGU [12.1] AUC [20.8] ACC [18.9] AAC [19.1] AGC [19.5] AUA [7.5] ACA [15.1] AAA [24.4] AGA [12.2] AUG [22.0] ACG [6.1] AAG [31.9] AGG [12.0] GUU [11.0] GCU [18.4] GAU [21.8] GGU [10.8] GUC [14.5] GCC [27.7] GAC [25.1] GGC [22.2] GUA [7.1] GCA [15.8] GAA [29.0] GGA [16.5] GUG [28.1] GCG [7.4] GAG [39.6] GGG [16.5]

[45] Furthermore, publicly available software resources are readily available for computer "reverse translation" (also known as "back translation") of polypeptide sequences (i.e., converting polypeptide sequences into nucleotide sequences encoding them). See, for example:

EMBL-EBI Sequence Search and Analysis Tools API in 2019

Madeira, F., et al., Nucleic Acids Res , 47(W1), W636-W641 (2019)

DOI: 10.1093/nar/gkz268; EuropePMC: 30976793.

Using EMBL-EBI services via a web interface and programmatically via web services

Madeira, F., et al., Curr Protoc in Bioinformatics , 66(1):e74 (2019)

DOI: 10.1002/cpbi.74; EuropePMC: 31039604.

Programmatic access to bioinformatics tools from the EMBL-EBI update: 2017.

Chojnacki, S, et al. , Nucleic Acids Res . 2017 Jul 3;45(W1):W550 - W553 (2017).

DOI: 10.1093/nar/gkx273. PMID: 28431173; PMCID: PMC5570243.

New and updated resource for codon usage tables.

Athey, J., et al., BMC Bioinformatics 18:391 (2017)

DOI 10.1186/s12859 - 017 - 1793 - 7.

MPI Bioinformatics Toolkit for Protein Sequence Analysis

Biegert, A., et al., Nucleic Acids Research , Vol. 34, Iss. Suppl_2, pp. W335-W339 (2006)

An advanced tool for molecular biology

Rawitch, A. B., Science ; Washington Vol. 288, Iss. 5465, pp. 457–458 (2000). Embossed: European Open Software Package for Molecular Biology

Rice, P., et al., Trends Genet. 16(6)276 - 277 (2000)

DOI: 10.1016/S0168 - 9525(00)02024 - 2; PubMed: 10827456.

Microcomputer programs for reverse translation of protein into DNA sequences and analysis of ambiguous DNA sequences

Mount, D. W. and Conrad, B. Nucleic Acids Res. , Vol. 12, Iss.1, Part 2,

pp. 819–823 (1984).

Computer programs for working with nucleic acid sequences

Keller, C. et al ., Nucleic Acids Res., Vol. 12, Iss. 1, Part 1, pp. 379-386 (1984).

[46] "Polypeptide,""peptide,""polypeptideconstruct," and "peptide construct," and their grammatical equivalents, as used herein, refer to a polymer of amino acid residues. A "mature protein" is a full-length protein that optionally includes glycosylation or other modifications typical of a protein in a given cellular environment. As disclosed herein, embodiments of the invention include the HPV antigens/antigen polypeptides, peptides, and mature proteins described herein, as well as the polynucleotides (DNA or RNA) that encode them. The polypeptides and proteins described herein (including functional portions and functional variants thereof) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known to those skilled in the art and include, 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, indoline - 2 - carboxylic acid, 1,2,3,4 - tetrahydroisoquinoline - 3 - carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N' - benzyl - N'- methyllysine, 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.

[47] Nucleic acids and/or nucleic acid sequences are "homologous" if they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Proteins and/or protein sequences are "homologous" when the DNA encoding them is derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules may be referred to as homologues. For example, any naturally occurring proteins, as described herein, can be modified by any available mutagenesis method. When expressed, the mutagenized nucleic acid encodes a polypeptide homologous to the protein encoded by the original nucleic acid. Homology is typically inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The exact percentage of sequence identity useful for establishing homology varies depending on the nucleic acid and protein in question, but as little as 25% sequence identity is typically used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining percentage sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are publicly available.

[48] The term "identical" and its grammatical equivalents as used herein, or "sequence identity" in the context of two nucleic acid sequences or amino acid sequences of polypeptides, refers to residues in the two sequences that are the same when aligned for maximum matching over a given comparison window. A "comparison window," as used herein, refers to a segment of at least about 20 contiguous positions, typically from about 50 to about 200, more typically from about 100 to about 150, in which a sequence can be compared to a reference sequence with the same number of contiguous positions after optimal alignment of the two sequences. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed using the Smith local homology search algorithm-Waterman-Smith and WatermanAdv. Appl. Math., 2:482 (1981); using the Needleman and Wunsch alignment algorithm,J. Mol. Biol., 48:443 (1970); using the Pearson and Lipman similarity search method,Proc. Nat. Acad. Sci USA., 85:2444 (1988); using computerized implementations of these algorithms (including, but not limited to, CLUSTAL in the PC/Gene program from Intelligentics, Mountain View Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI, USA); the CLUSTAL program is well described by Higgins and Sharp, Gene, 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 accomplished by inspection and manual alignment. In one class of embodiments, the polypeptides provided herein are at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or fragment thereof, for example, as determined by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. In one class of embodiments, the polypeptides provided herein are at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or fragment thereof, for example, as determined by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. When one 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 specified percentage of residues in the smaller molecule find a corresponding residue in the larger molecule according to the order in which the two molecules are optimally aligned.

[49] The term "substantially identical" and its grammatical equivalents, when applied to nucleic acid or amino acid sequences, means that the nucleic acid or amino acid sequence comprises a sequence that has at least 90% sequence identity or more, at least 95%, at least 98%, and at least 99%, compared to a reference sequence using the programs described above, such as BLAST, using standard parameters. For example, the BLASTN program (for nucleotide sequences) uses by default a word length (W) of 11, an expectation (E) of 10, M=5, N= - 4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses by default a wordlength (W) of 3, an expectation (E) of 10, and the 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 in a comparison window, where a portion of the polynucleotide sequence in 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 at which an identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, by dividing the number of matching 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 sequences of at least about 50 residues, 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 regions.

[50] An "expression vector" or "vector" is any genetic element, such as a plasmid, chromosome, virus, or transposon, that behaves either as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replication under its own control) or is capable of replication by insertion into a host cell chromosome by attaching another polynucleotide segment to cause replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, transposons, mammalian viruses, bacteriophages, and cosmids. Vectors may contain polynucleotide sequences necessary to effect ligation or insertion of the vector into the desired host cell and to effect expression of the attached segment. Such sequences vary depending on the host organism; they include promoter sequences to affect transcription, enhancer sequences to increase transcription, ribosome binding site sequences, and transcription and translation termination sequences. Alternatively, expression vectors may be capable of directly expressing the nucleic acid sequence products encoded therein without ligation or integration of the vector into host cell DNA sequences. In some embodiments, the vector is an "episomal expression vector" or "episome" that is capable of replicating in the host cell and is maintained as an extrachromosomal DNA segment in the host cell in the presence of appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11 : 1735-1742 (2004)).

[51] The terms "adenovirus" and "adenoviral vector," as used herein, refer to an adenovirus that retains the ability to participate in the adenoviral life cycle and/or that has been physically inactivated, such as by disruption (e.g., sonication), denaturation (e.g., using heat or solvents), or cross-linking (e.g., cross-linking with formalin). The "adenoviral life cycle" includes (1) viral binding and entry into cells, (2) transcription of the adenoviral genome and translation of adenoviral proteins, (3) replication of the adenoviral genome, and (4) assembly of viral particles (see, e.g., Fields Virology, ^5th ed., Knipe et al. (eds.), Lippincott Williams & Wilkins, Philadelphia, PA (2006)). The adenoviruses used and described herein may also be replication-abolished (i.e., lack the ability to participate in the adenovirus life cycle) by deleting one or more portions of the naturally occurring viral genome. "Adenoviruses" and "adenoviral vector" as used and described herein may include an adenovirus in which the adenoviral genome has been altered to accommodate a nucleic acid sequence that is not native to the adenoviral genome. Typically, an adenoviral vector is created by introducing one or more mutations (e.g., deletions, insertions, or substitutions) into the adenoviral genome of an adenovirus to allow for the insertion of a non-native nucleic acid sequence, for example, to transfer a gene into an adenovirus.

[52] The term "selectable marker gene," as used herein, refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected for or against in the presence of an appropriate selective agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publications WO 1992/08796 and WO 1994/28143; Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78 : 1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78 : 2072 (1981); Colberre - Garapin et al., J. Mol. Biol., 150 :1 (1981); Santerre et al., Gene, 30 : 147 (1984); Kent et al., Science, 237 : 901 - 903 (1987); Wigler et al., Cell, 11 : 223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48 : 2026 (1962); Lowy et al., Cell, 22 : 817 (1980); and US Patent Nos. 5122464 and 5770359.

[53] The term "coding sequence" as used herein refers to the segment of a polynucleotide that encodes a protein. The region or sequence is bounded near the 5 ' end by a start codon and near the 3' end by a stop codon. Coding sequences may also be referred to as open reading frames.

[54] The term "operably linked," as used herein, refers to the physical and/or functional association of a DNA segment with another DNA segment such that the segments can function in their intended manner. A DNA sequence encoding a gene product is operably linked to a regulatory sequence when it is linked to a regulatory sequence, such as, for example, promoters, enhancers, and/or silencers, in a manner that allows for the modulation of transcription of the DNA sequence, directly or indirectly. For example, a DNA sequence is operably linked to a promoter when it is ligated to the promoter downstream of the transcription initiation site of the promoter, in the correct reading frame with respect to the transcription initiation site, and allows transcription elongation to occur through the DNA sequence. An enhancer or silencer is operably linked to a DNA sequence encoding a gene product when they are ligated to the DNA sequence in a manner that increases or decreases, respectively, transcription of the DNA sequence. Enhancers and silencers can be located upstream, downstream, or integrated into the coding regions of a DNA sequence. DNA for a signal sequence is operably linked to DNA encoding a polypeptide if the signal sequence is expressed as a preprotein involved in polypeptide secretion. Linking DNA sequences to regulatory sequences is typically accomplished by ligation into appropriate restriction sites or by using adapters or linkers inserted into the sequence using restriction endonucleases known to those skilled in the art.

[55] The terms "induce," "induction," and their grammatical equivalents, as used herein, refer to an increase in transcription of a nucleic acid sequence, promoter activity, and/or expression caused by a transcriptional regulator, compared to some basal level of transcription.

[56] The term "transcriptional regulator" refers to a biochemical element that prevents or inhibits transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., a repressor or nuclear inhibitory protein) or permits or stimulates transcription of a promoter-driven DNA sequence under certain environmental conditions (e.g., an inducer or enhancer).

[57] The term "enhancer," as used herein, refers to a DNA sequence that increases transcription of, for example, a nucleic acid sequence to which it is operably linked. Enhancers may be located many thousands of base pairs from the coding region of a nucleic acid sequence and may mediate the binding of regulatory factors, DNA methylation patterns, or changes in DNA structure. A large number of enhancers are well known in the art from a variety of sources, available as or within cloned polynucleotides (e.g., from depositories such as the ATCC, as well as from other commercial or individual sources). A number of polynucleotides containing promoters (such as the commonly used CMV promoter) also contain enhancer sequences. Enhancers may be located upstream, within, or downstream of coding sequences. The term "Ig enhancers" refers to enhancer elements derived from enhancer regions mapped to the immunoglobulin (Ig) locus (such enhancers include, for example, 5 ' heavy chain (mu) enhancers, 5 ' light chain (kappa) enhancers, kappa and mu intronic enhancers, and 3' enhancers (see generally Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363 ; and U.S. Patent No. 5,885,827).

[58] The term "promoter" refers to the region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand and upstream of DNA (towards the 5 ' region of the sense strand). Some promoters are constitutive, being active under all circumstances in a cell, while others are regulated, becoming active in response to certain stimuli, such as an inducible promoter. The term "promoter activity" and its grammatical equivalents, as used herein, refer to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is being measured. Promoter activity can be measured directly by determining the amount of RNA transcript produced, such as by Northern blotting , or indirectly by determining the amount of product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to a promoter.

[59] An "inducible promoter," as used herein, refers to a promoter whose activity is induced by the presence or absence of transcriptional regulators, such as biotic or abiotic factors. Inducible promoters are useful because the expression of genes functionally linked to them can be turned on or off at specific stages of an organism's development or in a specific tissue. Non-limiting examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, and light-regulated promoters. An inducible promoter may be part of a gene switch or a gene switch. An inducible promoter may be a promoter inducible by a gene switch ligand. In some cases, the inducible promoter may be a small molecule ligand-inducible two-polypeptide ecdysone receptor-based gene switch. In some cases, the gene switch may be selected from components of an ecdysone-based receptor as described in any of the systems described in: International Patent Applications WO 2001/070816; WO 2002/029075; WO 2002/066613; WO 2002/066614; WO 2002/066612; WO 2002/066615; WO 2003/027266; WO 2003/027289; WO 2005/108617; WO 2009/045370; WO 2009/048560; WO 2010/042189; WO 2010/042189; WO 2011/119773; and WO 2012/122025; and US patents No. 7091038; 7776587; 7807417; 8202718; 8105825; 8168426; 7531326; 8236556; 8598409; 8715959; 7601508; 7829676; 7919269; 8030067; 7563879; 8021878; 8497093; 7935510; 8076454; 9402919; 9493540; 9249207; and 9492482, each of which is incorporated by reference in its entirety).

[60] The term "gene switch" or "gene switch" refers to the combination of a response element linked to a promoter and, for example, an EcR-based system that, in the presence of one or more ligands, modulates the expression of a gene in which the response element and promoter are incorporated. Tightly regulated inducible gene expression systems or gene switches are useful for a variety of applications, such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, functional genomics, and trait regulation in transgenic plants and animals. Such inducible gene expression systems may include ligand-inducible heterologous gene expression systems.

[61] The 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 WO/2016/145146. The Sleeping Beauty transposon system is comprised of 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.

[62] A "transposon" or "transposable element" (TE) is a vector DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and changing the size of the cell's genome. Transposition often results in duplication of the TE. Class I TEs are copied in two steps: first, they are transcribed from DNA to RNA, and then the resulting RNA is reverse transcribed into DNA. This copied DNA is then inserted at the new position in the genome. The reverse transcription step is catalyzed by a reverse transcriptase, which may be encoded by the TE itself. The characteristics of retrotransposons are similar to retroviruses such as HIV. The "cut and paste" transposition mechanism of Class II TEs does not involve an RNA intermediate. Transpositions are catalyzed by several transposase enzymes. Some transposases bind non-specifically to any target site in DNA, while others bind to specific target DNA sequences. Transposase makes staggered cuts at the target site, resulting in the formation of single-stranded 5 ' or 3' DNA overhangs (sticky ends). This step excises the transposon DNA, which is then ligated into the new target site ; this process involves the activity of DNA polymerase, which fills in the gaps, and DNA ligase, which closes the sugar - phosphate backbone. This results in duplication of the target site. Insertion sites of DNA transposons can be identified by short direct repeats, which can be created by staggered cutting of the target DNA and filling by DNA polymerase, followed by a series of inverted repeats essential for excision of the TE by transposase. Cut-and-inserted TEs can be duplicated if their transposition occurs during the S phase of the cell cycle, when the donor site has already been replicated and the target site has not yet been replicated. Transposition can be classified as 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 often occurs because non-autonomous TEs lack transposase (for class II) or reverse transcriptase (for class I).

[63] "Tranposase" refers to an enzyme that binds to the end of a transposon and catalyzes the movement of the transposon to another part of the genome via a cut-and-paste mechanism, or replicative transposition mechanism.

[64] A " T cell" or " T lymphocyte," as used herein, is 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 the cell surface.

[65] T helper cells (T _H cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells , and 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 they are presented with peptide antigens 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, which regulate or assist the active immune response. These cells can differentiate into one of several subtypes, including T _H 1, T _H 2, T _H 3, T _H 9, T _H 17, T _H 22, or T _FH ( T follicular helper cells), which secrete different cytokines to facilitate different types of immune responses. Signaling from APCs directs T cells to specific subtypes.

[66] Cytotoxic T cells ( TC cells or CTLs) or cytotoxic T lymphocytes 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 antigen-associated MHC class I molecules, which are present on the surface of all nucleated cells. Through IL - 10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an anergic state, preventing autoimmune diseases.

[67] Memory T cells are a subset of antigen - specific T cells that persist long after infection has resolved. They rapidly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thereby providing the immune system with memory against past infections. Memory T cells include three subtypes: central memory T cells (T _CM cells) and two types of effector memory T cells (T _EM cells and T _EMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface proteins CD45RO, CD45RA, and/or CCR7.

[68] Regulatory T cells ( Treg cells), previously known as suppressor T cells, play a role in maintaining immune tolerance. Their primary role is to shut down T cell- mediated immunity toward the end of the immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[69] Natural killer T cells ( NKT cells - not to be confused with natural killer cells of the innate immune system) link the adaptive immune system with the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize a glycolipid antigen presented by a molecule called CD1d. Once activated, these cells can perform functions attributed to both T helper ( _TH ) and cytotoxic T (TC) cells (i.e., producing cytokines and releasing cytolytic/killer cell molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses.

[70] Adoptive T cell transfer refers to the ex vivo isolation and expansion of tumor-specific T cells to achieve a larger number of T cells than could be achieved through vaccination alone or the patient's natural tumor response. The tumor - specific T cells are then administered to cancer patients to enable their immune system to suppress the remaining tumor with T cells that can attack and kill the cancer. There are many forms of adoptive T cell therapy used to treat cancer; culturing tumor-infiltrating lymphocytes or TILs, isolating and expanding a single specific T cell or clone, and even using T cells that have been engineered to effectively recognize and attack tumors.

[71] The term "antibody," as used herein, refers to monoclonal or polyclonal antibodies. The term "monoclonal antibodies," as used herein, refers to antibodies that are produced by a single clone of B cells and bind to the same epitope. In contrast, "polyclonal antibodies" refers to a population of antibodies that are produced by different B cells and bind to different epitopes of the same antigen. A complete 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 regions (CH1, CH2, and CH3), and each light chain contains one N - terminal variable (VL) region and one C - terminal constant region (CL). The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. The VH and VL regions have a similar overall structure, with each region comprising four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity-determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the antibody's "hypervariable region," responsible for antigen binding.

[72] "Antibody-like molecules" may be, for example, proteins that are members of the Ig superfamily that are 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 has been modified to increase its MHC binding affinity.

[73] The terms "antibody fragment,""antibodyfragment,""functional antibody fragment,""antigen-bindingportion," or grammatical equivalents thereof, are used interchangeably herein to refer to one or more fragments, or portions, of an antibody that retain the ability to specifically bind to an antigen (see, generally, Holliger et al., Nat. Biotech., 23(9) : 1126-1129 (2005)). Desirably, an antibody fragment comprises, for example, one or more CDRs, a variable region (or portions thereof), a constant region (or portions thereof), or combinations thereof. Non-limiting examples of antibody fragments include (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a divalent fragment comprising two Fab fragments linked by a disulfide bridge at the stem region; (iii) an Fv fragment consisting of the VL and VH domains of one arm of an antibody; (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains of an Fv fragment (i.e., VL and VH) joined by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, 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 (1998)) and (v) a diabody, which is a dimer of polypeptide chains, each polypeptide chain comprising a VH joined to a VL by a peptide linker that is too short to allow pairing between VH and VL on the same the same polypeptide chain, thereby leading to pairing between complementary domains on different polypeptide chains VH - VL to form a dimeric molecule with two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent 8,603,950.

[74] An "antigen recognition moiety" or "antigen recognition domain" refers to a molecule or portion of a molecule that specifically binds to an antigen. In one embodiment, the antigen recognition moiety is an antibody, antibody-like molecule, or fragment thereof, and the antigen is a tumor antigen.

[75] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid by another amino acid with a common property. A functional way to determine common properties between individual amino acids is to analyze the normalized frequencies of amino acid substitutions between corresponding proteins of homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer - Verlag, New York (1979)). According to such analyses, groups of amino acids can be identified in which the amino acids within the group preferentially exchange with each other and are therefore most similar to each other in their effect on the overall protein structure (Schulz, GE and Schirmer, RH, see above). Examples of conservative mutations include amino acid substitutions of amino acids in the subgroups mentioned above, e.g., lysine for arginine and vice versa, so that a positive charge can be maintained; glutamic acid for aspartic acid and vice versa, so that a negative charge can be maintained; serine instead of threonine so that free -OH can be maintained; and glutamine for asparagine so that free -NH ₂ can be maintained. Alternatively or additionally, functional variants may comprise the amino acid sequence of the reference protein with at least one non-conservative amino acid substitution.

[76] The term "non-conservative mutations" includes amino acid substitutions between different groups, such as lysine to tryptophan or phenylalanine to serine, etc. 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. A non-conservative amino acid substitution may enhance the biological activity of the functional variant, so that the biological activity of the functional variant is increased compared to the homologous original protein.

[77] The term “proliferative disease” as used in this document refers to the unifying concept that excessive cell proliferation and/or cellular matrix turnover contribute significantly to the pathogenesis of disease, including cancer.

[78] "Patient" or "subject" as used herein refers to a mammalian subject who is diagnosed with or suspected of having or developing a proliferative disorder, such as cancer. In some embodiments, the term "patient" refers to a mammalian subject who is more likely than average to develop a proliferative disorder, such as cancer. Representative patients may include humans, great apes, dogs, pigs, cattle, cats, horses, goats, sheep, rodents, and other mammals that may benefit from the treatment methods described herein. Representative human patients may be male and/or female. A "patient in need thereof" or "subject in need thereof" is referred to herein as a patient diagnosed with or suspected of having a disease or disorder, such as, but not limited to, human papillomavirus (HPV) infection.

[79] "Administration" as used herein refers to the administration of one or more compositions described herein to a patient or subject. By way of example, but not limitation, administration of a composition, such as an injection, can be by intravenous (IV) injection, subcutaneous (SC) injection, intradermal (ID) injection, intraperitoneal (IP) injection, or intramuscular (IM) injection. One or more of these administrations can be used. Parenteral administration can be accomplished, for example, by bolus injection or gradual perfusion over time. Alternatively, or concurrently, administration can be accomplished orally. Additionally, administration can also be accomplished by surgery or positioning of a medical device. A pharmaceutical composition can comprise a composition of the invention as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. aluminum hydroxide); and preservatives.

[80] The term "treatment," "treating," or grammatical equivalents thereof, as used herein, refers to producing 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 an adverse symptom or pathological manifestation associated with the disease. To this end, the method of the invention comprises administering a therapeutically effective amount of a composition of the invention expressing a nucleic acid sequence of the invention or a vector comprising nucleic acid sequences of the invention.

[81] The term "therapeutically effective amount," "therapeutic amount," "immunologically effective amount," "effective antitumor amount," "effective tumor-inhibitory amount," or grammatical equivalents thereof, refers to an amount effective, at the required dosages and at the required intervals of administration, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the composition described herein to elicit a desired response in one or more subjects.

[82] Alternatively, the pharmacological and/or physiological effect of administering one or more compositions described herein to a patient or subject may be "prophylactic," i.e., the effect completely or partially prevents a disease or symptom thereof. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., preventing a disease or preventing the manifestation of a target pathology).

[83] The term "in sequential order" or grammatical equivalents with respect to polypeptide and amino acid sequences refers to the order of the sequences from the N - terminus to the C - terminus. The term "in sequential order" or grammatical equivalents with respect to polynucleotide and nucleic acid sequences refers to the order of the sequences from 5'("fiveprime") to 3'("threeprime").

Molecular vaccine against HPV

[84] Human papillomavirus (HPV) is a group of more than 200 related viruses. Each HPV virus in this large group is assigned a number, called an HPV type (or serotype). HPV is a small, non-enveloped deoxyribonucleic acid (DNA) virus that infects cells of the skin or mucous membranes. The circular, double-stranded viral genome is approximately 8 kbp long. The genome encodes seven early proteins (E1 through E7), responsible for viral replication, and two late proteins (L1 and L2), which are viral structural proteins. Each gene has specific functions. At least 13 of the more than 200 known HPV types can cause cervical cancer and are associated with other anogenital cancers and head and neck cancer. The two most common "high-risk" serotypes (HPV - 16 and HPV - 18) cause approximately 70% of all cervical cancer cases. In HPV16 and HPV - 18, two primary oncoproteins, E6 and E7, are constitutively expressed in HPV-associated tumors and are critical for the induction and maintenance of cellular transformation in HPV-infected cells. Recent evidence also suggests that the E5 protein also influences viral transformation. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82 are considered carcinogenic. Two "low-risk" HPV types, HPV 6 and 11, are known to cause genital warts, a common benign condition of the external genitalia that causes significant morbidity. HPV is easily transmitted, with peak incidence shortly after the onset of sexual activity, and most people become infected at some point in their lives.

[85] Genital warts are extremely common and may also be known by other names such as anogenital warts or genital condylomata acuminata, with between 500,000 and one million new cases diagnosed annually in the United States alone. HPV6 and HPV11 cause approximately 90% of genital warts. Current treatment options primarily focus on removing the warts rather than eliminating the underlying viral infection. A wide range of treatments are currently used, which vary widely and can have significant differences in effectiveness. However, no treatment has been 100% effective in eliminating warts and preventing their recurrence in all patients.

[86] Currently, the standard of treatment for RRP is surgical excision with adjuvant therapy as needed. However, a distinctive aspect of RRP is its tendency to recur, requiring repeat surgeries. Aggressiveness varies among patients: some people may require surgery every few weeks, while others may require surgery only twice a year or only a few times during their lifetime.

[87] Unfortunately, previously developed treatments have failed to prevent multiple recurrences of RRP. More effective treatments for patients with RRP are needed. This paper describes an innovative immunotherapy approach, a novel therapeutic HPV vaccine aimed at inducing immunity against HPV6/11 for the treatment and modification of RRP and other HPV6/11-associated diseases such as genital warts.

[88] Prophylactic vaccines consisting of virus-like particles induce immunity only to the capsid structure and not to the nonstructural proteins responsible for cell transformation. Initial studies of HPV in animal models showed that inoculation with species-specific papillomaviruses induces an immune response that provides protection against challenge with the homologous virus. However, native papillomaviruses are not a good substrate for vaccine development because they are not easily grown in tissue culture. Subsequent studies were initiated to produce viral particles by expressing structural proteins in heterologous expression systems such as yeast or baculovirus vectors. The results showed that expression of L1 alone results in the production of virus-like particles (VLPs) that morphologically resemble true HPV virions but do not contain viral DNA. These VLPs are produced by self-assembly of the L1 protein when expressed in a heterologous cellular substrate. Animal studies have shown that VLPs protect against experimental infection with high doses of a homologous virus. HPV VLPs are highly immunogenic in mice or rabbits, and the resulting antibodies have been shown to be neutralizing and of limited type when tested in a pseudovirion neutralization assay. Immunization with denatured particles does not result in the production of neutralizing antibodies and does not protect against experimental infection with the virus, indicating that neutralizing epitopes are conformation-dependent.

[89] The present document provides compositions, kits, and systems comprising methods for producing recombinant HPV vaccines. The recombinant HPV vaccines (e.g., HPV constructs 1-5 ) in the present invention are constructed using protein engineering.

Delivery system

Shuttle - gorilla adenovirus vector

[90] Certain aspects of the present invention relate to a vector comprising a polynucleotide encoding a polypeptide comprising one or more HPV immune response-inducing polypeptides described herein. In some embodiments, the vector is a viral vector. In particular embodiments, the vector is an adenovirus vector. Adenoviruses are generally associated with benign pathologies in humans, and the genomes of adenoviruses isolated from various species, including humans, have been extensively studied. Adenovirus is a medium-sized (90-100 nm) icosahedral non-enveloped virus containing approximately 36 kb of double-stranded DNA. The adenovirus capsid mediates key interactions early in viral cell infection and is required for packaging of adenovirus genomes late in the adenovirus life cycle. The capsid consists of 252 capsomeres, including 240 hexons, 12 penton base proteins, and 12 fibers (Ginsberg et al., Virology , 28 : 782–83 (1966)). The hexon is composed of three identical proteins, namely polypeptide II (Roberts et al., Science , 232 : 1148–51 (1986)). The penton base is composed of five identical proteins, and the fiber is composed of three identical proteins. Proteins IIIa, VI, and IX are present in the adenovirus envelope and are thought to stabilize the viral capsid (Stewart et al., Cell , 67 : 145–54 (1991), and Stewart et al., EMBO J ., 12 (7): 2589–99 (1993)). Expression of capsid proteins, with the exception of pIX, depends on the adenovirus polymerase protein. Therefore, the key components of the adenovirus particle are expressed from the genome only when the polymerase protein gene is present and expressed.

[91] Several features of adenoviruses make them ideal vehicles for transferring genetic material into cells for therapeutic purposes (e.g., "gene therapy") or for use as antigen delivery systems for vaccine applications. For example, adenoviruses can be produced in high titers (e.g., about 10 ¹³ particle units (p.u.)) and can transfer genetic material into non-replicating and replicating cells. The adenoviral genome can be manipulated to carry large amounts of exogenous DNA (up to approximately 8 kb), and the adenoviral capsid can potentiate the transfer of even longer sequences (Curiel et al., Hum. Gene Ther ., 3 : 147–154 (1992)). Furthermore, adenoviruses do not typically integrate into the host cell chromosome, but rather are maintained as a linear episome, minimizing the likelihood that recombinant adenovirus will interfere with normal cell function.

[92] In some embodiments, the adenovirus described herein is isolated from a gorilla. There are four widely recognized subspecies of gorilla within the two species of Eastern Gorilla ( Gorilla beringei ) and Western Gorilla ( Gorilla gorilla ). The western gorilla species include the subspecies of western lowland gorilla (Gorilla gorilla gorilla) and Cross River gorilla (Gorilla gorilla diehli). The eastern gorilla species include the subspecies of mountain gorilla ( Gorilla beringei beringei ) and eastern lowland gorilla ( Gorilla beringei graueri ) (see, e.g., Wilson and Reeder, eds., Mammalian Species of the World , 3rd ed., Johns Hopkins University Press, Baltimore, MD (2005)). In some embodiments, the adenovirus of the present invention is isolated from a mountain gorilla ( Gorilla beringei beringei ).

[93] Various gorilla adenoviruses or adenoviral vectors are described in International Patent Application Publications WO 2013/052832; WO 2013/052811; and WO 2013 052799, each of which is incorporated herein by reference in its entirety.

[94] Adenovirus can be modified in the same way as previously known adenoviruses for use as an adenoviral vector, such as a gene delivery vehicle. Adenovirus and adenoviral vector can be replication-competent, conditionally replication-competent, or replication-deficient.

[95] A replication-competent adenovirus or adenoviral vector can replicate in typical host cells , i.e., cells normally capable of being infected by adenovirus. A replication-competent adenovirus or adenoviral vector may have one or more mutations compared to wild-type adenovirus (e.g., one or more deletions, insertions, and/or substitutions) in the adenoviral genome that do not inhibit viral replication in host cells . For example, an adenovirus or adenoviral vector may have a partial or complete deletion of an early region of adenovirus known as the E3 region, which is not essential for replication of the adenovirus or adenoviral genome.

[96] A conditionally replicating adenovirus or adenoviral vector is an adenovirus or adenoviral vector that has been engineered to replicate under predetermined conditions. For example, replication-critical gene functions, such as those encoded by the early regions of the adenovirus, may be operably linked to an inducible, repressible, or tissue-specific transcriptional control sequence, such as a promoter. In such an embodiment, replication requires the presence or absence of specific factors that interact with the transcriptional control sequence. Conditionally replicating adenoviral vectors are further described in U.S. Patent 5,998,205.

[97] A replication-deficient adenovirus or adenoviral vector is an adenovirus or adenoviral vector that requires complementation of one or more gene functions or regions of the adenoviral genome necessary for replication, resulting, for example, from a deficiency in one or more replication-critical gene functions or regions, such that the adenovirus or adenoviral vector does not replicate in typical host cells , particularly in cells from a human infected with the adenovirus or adenoviral vector.

[98] A deficiency in gene function or a genomic region, as used herein, is defined as a disruption (e.g., deletion) of the genetic material of the adenoviral genome sufficient to eliminate or disrupt the function of the gene (e.g., such that the function of the gene product is reduced by at least about 2, 5, 10, 20, 30, or 50 times), the nucleic acid sequence of which has been disrupted (e.g., deleted), in whole or in part. Deletion of an entire region of a gene is often not required to disrupt replication-essential gene function. However, in order to provide sufficient space in the adenoviral genome for one or more transgenes, it may be desirable to delete a majority of regions of one or more genes. Although deletion of genetic material is preferred, mutation of genetic material by addition or substitution is also suitable for disrupting gene function. Replication-requiring gene functions are those functions of genes that are required for adenovirus replication (e.g., replication) and are encoded by, for example, adenovirus early regions (e.g., E1, E2, and E4 regions), late regions (e.g., L1, L2, L3, L4, and L5 regions), genes involved in virus packaging (e.g., IVa2 gene), and virus - associated RNAs (e.g., VA - RNA - 1 and/or VA - RNA - 2).

[99] Regardless of whether the adenovirus or adenoviral vector is replication-competent or replication-deficient, the adenovirus or adenoviral vector retains at least a portion of the adenoviral genome. The adenovirus or adenoviral vector may contain any portion of the adenoviral genome, including protein-coding regions and non-protein-coding regions. Desirably, the adenovirus or adenoviral vector contains at least one nucleic acid sequence that encodes an adenoviral protein. The adenovirus or adenoviral vector may contain a nucleic acid sequence that encodes any suitable adenoviral protein, such as, for example, a protein encoded by any of the early region genes (i.e., E1A, E1B, E2A, E2B, E3 and/or E4 regions), or a protein encoded by any of the late region genes that encode the structural proteins of the virus (i.e., L1, L2, L3, L4 and L5 regions).

[100] It is desirable that the adenovirus or adenoviral vector contain one or more nucleic acid sequences that encode a pIX protein, a DNA polymerase protein, a penton protein, a hexon protein, and/or a fiber protein. The adenovirus or adenoviral vector may contain a full-length nucleic acid sequence that encodes a full-length amino acid sequence of an adenoviral protein. Alternatively, the adenovirus or adenoviral vector may contain a portion of a full-length nucleic acid sequence that encodes a portion of a full-length amino acid sequence of an adenoviral protein.

[101] A "region" of a nucleic acid sequence comprises at least ten nucleotides (e.g., from about 10 to about 5000 nucleotides). Preferably, the "region" of a nucleic acid sequence comprises 10 or more (e.g., 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, or 100 or more) nucleotides, but less than 5000 (e.g., 4900 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 800 or less, 500 or less, 300 or less, or 100 or less) nucleotides. Preferably, the portion of the nucleic acid sequence is from about 10 to about 3500 nucleotides (e.g., about 10, 20, 30, 50, 100, 300, 500, 700, 1000, 1500, 2000, 2500, or 3000 nucleotides), from about 10 to about 1000 nucleotides (e.g., about 25, 55, 125, 325, 525, 725, or 925 nucleotides), or from about 10 to about 500 nucleotides (e.g., about 15, 30, 40, 50, 60, 70, 80, 90, 150, 175, 250, 275, 350, 375, 450, 475, 480, 490, 495, or 499 nucleotides), or a range defined by any two of the preceding values. More preferably, the "region" of the nucleic acid sequence comprises no more than about 3200 nucleotides (e.g., from about 10 to about 3200 nucleotides, from about 10 to about 3000 nucleotides, or from about 30 to about 500 nucleotides, or a range defined by any two of the preceding values).

[102] A "region" of an amino acid sequence comprises at least three amino acids (e.g., from about 3 to about 1200 amino acids). Preferably, the "region" of an amino acid sequence comprises 3 or more (e.g., 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) amino acids, but less than 1200 (e.g., 1000 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) amino acids. Preferably, the portion of the amino acid sequence is from about 3 to about 500 amino acids (e.g., about 10, 100, 200, 300, 400, or 500 amino acids), from about 3 to about 300 amino acids (e.g., about 20, 50, 75, 95, 150, 175, or 200 amino acids), or from about 3 to about 100 amino acids (e.g., about 15, 25, 35, 40, 45, 60, 65, 70, 80, 85, 90, 95, or 99 amino acids), or a range defined by any two of the preceding values. More preferably, the "region" of the amino acid sequence comprises no more than about 500 amino acids (e.g., from about 3 to about 400 amino acids, from about 10 to about 250 amino acids, or from about 50 to about 100 amino acids, or a range defined by any two of the foregoing).

[103] The adenovirus pIX protein is present in the adenovirus capsid and has been shown to enhance hexon-nonamer interactions and to be required for packaging of full-length genomes (see, e.g., Boulanger et al., J. Gen. Virol. , 44 : 783–800 (1979); Horwitz MS, “Adenoviridae and their replication” in Virology , ^2nd 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 adenovirus structure, pIX has also been shown to exhibit transcriptional properties, such as stimulation of the adenovirus major late promoter (MLP) activity (see, e.g., Lutz et al., J. Virol ., 71 (7) : 5102–5109 (1997)).

[104] The adenovirus DNA polymerase protein is required for viral DNA replication both in vitro and in vivo . The polymerase copurifies, in a complex with the precursor (pTP), the terminal protein (TP), which is covalently attached to the 5 ' ends of adenovirus DNA (Field et al., J. Biol. Chem ., 259 : 9487–9495 (1984)). Both adenoviral 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 polymerase protein gene sequence, the polymerase protein is not formed. As a result, the viral genome is not replicated, the major late promoter is not activated, and capsid proteins are not expressed.

[105] The adenovirus hexon protein is the largest and most abundant protein in the adenovirus capsid. The hexon protein is required for the assembly of the viral capsid, the determination of capsid icosahedral symmetry (which in turn determines the limits of capsid volume and DNA packaging size), and capsid integrity. Furthermore, hexon is a major target for modification to reduce neutralization of adenoviral vectors (see, e.g., Gall et al., J. Virol ., 72: 10260–264 (1998), and Rux et al., J. Virol ., 77(17): 9553–9566 (2003)). The basic structural features of the hexon protein are common to adenoviruses of all serotypes, but the hexon protein varies in size and immunological properties among serotypes (Jornvall et al., J. Biol. Chem. , 256 (12): 6181–6186 (1981)). Comparison of 15 adenoviral hexon proteins showed that the predominant antigenic and serotype - specific regions of the hexon appear to be in loops 1 and 2 ( i.e. , L1 or l1 , and LII or l2 , respectively), within which are seven distinct hypervariable regions (HVR1 – HVR7) that differ in length and sequence between adenoviral serotypes (Crawford - Miksza et al., J. Virol. , 70 (3): 1836–1844 (1996)).

[106] The adenovirus fiber protein is a homotrimer of adenoviral polypeptide IV that has three domains: a tail, a stem, and a knob (Devaux et al., J. Molec. Biol. , 215 : 567–88 (1990), Yeh et al., Virus Res. , 33 : 179–98 (1991)). The fiber protein mediates primary binding of the virus to cell surface receptors through the knob and stem domains (Henry et al., J. Virol. , 68 (8): 5239–46 (1994)). The amino acid sequences for trimerization are located in the protrusion, which is necessary for the correct association of the amino terminus of the fiber (tail) with the penton base (Novelli et al., Virology , 185 : 365–76 (1991)). In addition to recognizing cellular receptors and binding the penton base, the fiber contributes to serotype identity. The fiber proteins of different adenovirus serotypes differ considerably (see, e.g., 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)). Thus, the fiber protein has several key functions in the adenovirus life cycle.

[107] The adenovirus penton protein is located at the vertices of the icosahedral capsid and consists of five identical monomers. The penton protein provides a structure for the binding of hexon proteins on multiple faces of the icosahedral capsid and provides the necessary interface for the incorporation of the fiber protein into the capsid. Each monomer of the penton protein contains a tripeptide RGD motif (Neumann et al., Gene , 69 : 153–157 (1988)). The RGD tripeptide mediates binding to αv integrins, and adenoviruses that have point mutations in the RGD sequence of the penton protein have a limited ability to infect cells (Bai et al., J. Virol. , 67 : 5198–5205 (1993)). Thus, the penton protein is essential for capsid architecture and for maximal efficiency of virus-cell interaction.

[108] The "identity" of a nucleic acid or amino acid sequence, as described herein, can be determined by comparing the nucleic acid or amino acid sequence of interest to a reference nucleic acid or amino acid sequence. The number of nucleotides or amino acid residues that have been altered and/or modified (e.g., by point mutations, insertions, or deletions) in the reference sequence to produce the sequence of interest is counted. The total number of such changes is subtracted from the total length of the sequence of interest, and the difference is divided by the length of the sequence of interest and expressed as a percentage. A number of mathematical algorithms for obtaining optimal alignment and calculating identity between two or more sequences are known and are included in a number of available programs. Examples of such programs include CLUSTAL - W, T - Coffee, and ALIGN (for aligning nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions), and FASTA programs (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment and sequence similarity searching). Sequence alignment algorithms are also disclosed, for example, in Altschul et al., J. Molecular Biol. , 215 (3): 403–410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA , 106 (10): 3770 - 3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probabilistic Models of Proteins and Nucleic Acids , Cambridge University Press, Cambridge, UK (2009), Soding, Bioinformatics , 21 (7): 951 - 960 (2005), Altschul et al., Nucleic Acids Res. , 25 (17): 3389 - 3402 (1997) and Gusfield, Algorithms on Strings, Trees and Sequences , Cambridge University Press, Cambridge UK (1997).

[109] An adenovirus or adenoviral vector may comprise one, two, three, four, or all five of the above-mentioned sequences individually or in any combination. In this regard, an adenovirus or adenoviral vector may comprise any combination of any two of the above-mentioned sequences, any combination of any three of the above-mentioned sequences, any combination of any four of the above-mentioned sequences, or all five of the above-mentioned sequences.

[110] As discussed herein, the adenovirus or adenoviral vector may be replication - competent, conditionally replicating, or replication - deficient. Preferably, the adenovirus or adenoviral vector is replication-defective such that propagation of the replication-defective adenovirus or adenoviral vector requires complementation of at least one replication-essential gene function of one or more regions of the adenoviral genome for propagation (e.g., for formation of adenoviral vector particles).

[111] A replication-defective adenovirus or adenoviral vector may be modified by any suitable method to cause a deficiency in one or more replication-essential gene functions in one or more regions of the adenoviral genome for propagation. Complementation of the deficiency in one or more replication-essential gene functions in one or more regions of the adenoviral genome refers to the use of exogenous means to provide the deficient replication-essential gene functions. Such complementation may be accomplished by any suitable method, such as using complementing cells and/or exogenous DNA (e.g., helper adenovirus) encoding the defective functions of genes essential for replication.

[112] An adenovirus or adenoviral vector may be defective in one or more replication-essential gene functions of only the early regions (i.e., E1 - E4 regions) of the adenoviral genome, only the late regions (i.e., L1 - L5 regions) of the genome, both the early and late regions of the adenoviral genome, or all adenoviral genes (i.e., a high-throughput adenovector (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). Examples of replication - deficient adenoviral vectors are disclosed in U.S. Patents 5,837,511; 5,851,806; 5,994,106; 6,127,175; 6,482,616; and 7,195,896, as well as international patent application publications WO 1994/028152, WO 1995/002697, WO 1995/016772, WO 1995/034671, WO 1996/022378, WO 1997/012986, WO 1997/021826, and WO 2003/022311.

[113] The early regions of the adenoviral genome include the E1, E2, E3, and E4 regions. The E1 region includes the E1A and E1B subregions, and one or more defects in replication-critical gene functions in the E1 region may include one or more deficiencies in replication-critical gene functions in one or both of the E1A and E1B subregions that require complementation of the E1A subregion and/or the E1B subregion of the adenoviral genome for propagation of the adenovirus or adenoviral vector (e.g., for formation of adenoviral vector particles). The E2 region includes the E2A and E2B subregions, and one or more defects in replication-critical gene functions in the E2 region may include one or more deficiencies in replication-critical gene functions in one or both of the E2A and E2B subregions that require complementation of the E2A subregion and/or the E2B subregion of the adenoviral genome for propagation of the adenovirus or adenoviral vector (e.g., for formation of adenoviral vector particles).

[114] The E3 region does not include any replication - essential gene functions, so deletion of the E3 region, in whole or in part, does not require complementation of any gene functions in the E3 region for propagation of the adenovirus or adenoviral vector (e.g., for formation of adenoviral vector particles). As used herein, the E3 region is defined as a region that begins with an open reading frame encoding a protein highly homologous to the 12.5K protein from the E3 region of human adenovirus 5 (NCBI reference sequence AP_000218) and ends with an open reading frame encoding a protein highly homologous to the 14.7K protein from the E3 region of human adenovirus 5 (NCBI reference sequence AP_000224.1). The E3 region may be deleted in whole or in part, or retained in whole or in part. The deletion size can be adjusted to retain an adenovirus or adenoviral vector whose genome closely matches the optimal genome packaging size. A larger deletion will facilitate the insertion of larger heterologous nucleic acid sequences into the adenovirus or adenoviral genome. In one embodiment of the present invention, the L4 polyadenylation signal sequences, which are located in the E3 region, are retained.

[115] The E4 region contains several open reading frames (ORFs). An adenovirus or adenoviral vector with a deletion of all E4 region open reading frames except ORF6 and, in some cases, ORF3, does not require complementation of any gene functions in the E4 region for adenovirus or adenoviral vector propagation (e.g., for the formation of adenoviral vector particles). Conversely, an adenovirus or adenoviral vector with a disruption or deletion of ORF6 and, in some cases, ORF3 of the E4 region (e.g., a deficiency in a replication-essential gene function based on ORF6 and/or ORF3 of the E4 region), with or without a disruption or deletion of any of the other open reading frames of the E4 region or the native E4 promoter, the polyadenylation sequence, and/or the right-hand inverted terminal repeat (ITR), requires complementation of the E4 region (particularly ORF6 and/or ORF3 of the E4 region) for propagation of the adenovirus or adenoviral vector (e.g., for the formation of adenoviral vector particles). The late regions of the adenoviral genome include the L1, L2, L3, L4, and L5 regions. An adenovirus or adenoviral vector may also have a mutation in the major late promoter (MLP), as discussed in International Patent Application Publication WO 2000/000628, which may, if desired, render the adenovirus or adenoviral vector replication deficient.

[116] The one or more regions of the adenoviral genome that contain one or more defects in replication-essential gene functions are desirably one or more early regions of the adenoviral genome, i.e., the E1, E2 and/or E4 regions, optionally with a deletion of part or all of the E3 region.

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

[118] In one embodiment, the adenovirus or adenoviral vector is replication deficient and requires, at most, complementation of the E1 region or the E4 region of the adenoviral genome for propagation (e.g., to form adenoviral vector particles). Thus, the replication - deficient adenovirus or adenoviral vector requires complementation of at least one replication-essential gene function of the E1A subregion and/or the E1B region of the adenoviral genome (referred to as an E1 - deficient adenoviral vector) or the E4 region of the adenoviral genome (referred to as an E4 - deficient adenoviral vector) for propagation (e.g., to form adenoviral vector particles). An adenovirus or adenoviral vector may be defective in at least one replication-essential gene function (preferably in all replication-essential gene functions) of the E1 region of the adenoviral genome and at least one gene function in the nonessential E3 region of the adenoviral genome (referred to as an E1/E3 - deficient adenoviral vector). An adenovirus or adenoviral vector may be defective in at least one replication-essential gene function (preferably in all replication-essential gene functions) of the E4 region of the adenoviral genome and at least one gene function in the nonessential E3 region of the adenoviral genome (referred to as an E3/E4 - deficient adenoviral vector).

[119] In one embodiment, the adenovirus or adenoviral vector is replication deficient and requires, at most, complementation of the E2 region, preferably the E2A subregion, of the adenoviral genome for propagation (e.g., to form adenoviral vector particles). Thus, the replication - deficient adenovirus or adenoviral vector requires complementation of at least one replication-essential gene function of the E2A subregion of the adenoviral genome (referred to as an E2A - deficient adenoviral vector) for propagation (e.g., to form adenoviral vector particles). The adenovirus or adenoviral vector may be defective in at least one replication-essential gene function (preferably all replication-essential gene functions) of the E2A region of the adenoviral genome and at least one gene function of the nonessential E3 region of the adenoviral genome (referred to as an E2A/E3 - deficient adenoviral vector).

[120] In one embodiment, the adenovirus or adenoviral vector is replication deficient and requires, at most, complementation of the E1 and E4 regions of the adenoviral genome to propagate (e.g., to form adenoviral vector particles). Thus, the replication - deficient adenovirus or adenoviral vector requires complementation of at least one replication-essential gene function in both the E1 and E4 regions of the adenoviral genome (referred to as an E1/E4 - deficient adenoviral vector) to propagate (e.g., to form adenoviral vector particles). An adenovirus or adenoviral vector may be defective in at least one replication-essential gene function (preferably in all replication-essential gene functions) of the E1 region of the adenoviral genome, at least one replication-essential gene function of the E4 region of the adenoviral genome, and at least one function of the nonessential gene function of the E3 region of the adenoviral genome (referred to as an E1/E3/E4 - deficient adenoviral vector). The adenovirus or adenoviral vector preferably requires, at most, complementation of the E1 region of the adenoviral genome for propagation and does not require complementation of any other deficiency of the adenoviral genome for propagation. More preferably, the adenovirus or adenoviral vector requires, at most, complementation of the E1 and E4 regions of the adenoviral genome for propagation and does not require complementation of any other deficiency of the adenoviral genome for propagation.

[121] An adenovirus or adenoviral vector, when deficient in multiple replication-essential gene functions of the adenoviral genome (e.g., an E1/E4 - deficient adenoviral vector), may include a spacer sequence to allow growth of the virus in a complementing cell line similar to that achieved by adenoviruses or adenoviral vectors lacking one replication-essential gene function (e.g., an E1 - deficient adenoviral vector). The spacer sequence may comprise any nucleotide sequence or sequences of the desired length, such as sequences of at least about 15 base pairs in length (e.g., from about 15 nucleotides to about 12,000 nucleotides), preferably from about 100 nucleotides to about 10,000 nucleotides, more preferably from about 500 nucleotides to about 8,000 nucleotides, even more preferably from about 1,500 nucleotides to about 6,000 nucleotides, and most preferably from about 2,000 to about 3,000 nucleotides in length, or a range defined by any two of the preceding values. The spacer sequence may be coding or non-coding, native or non-native with respect to the adenovirus genome, but it does not restore the replication-critical function of the defective region. The spacer may also comprise an expression cassette. More preferably, the spacer comprises a polyadenylation sequence and/or a gene that is not native to the adenovirus or adenoviral vector. The use of a spacer in an adenoviral vector is further described, for example, in U.S. Patent 5,851,806 and International Patent Application Publication WO 1997/021826.

[122] By removing all or part of the adenoviral genome, such as the E1, E3, and E4 regions of the adenoviral genome, the resulting adenovirus or adenoviral vector is capable of accepting inserts of exogenous nucleic acid sequences while retaining the ability to be packaged into adenoviral capsids. The exogenous nucleic acid sequence may be inserted into any position of the adenoviral genome, provided that the insertion into the position allows the formation of an adenovirus or adenoviral vector particle. The exogenous nucleic acid sequence is preferably located in the E1 region, the E3 region, or the E4 region of the adenoviral genome.

[123] The replication - deficient adenovirus or adenoviral vector of the present invention can be produced in complementary cell lines that provide gene functions that are absent in the replication - deficient adenovirus or adenoviral vector, but are necessary for viral propagation, at appropriate levels to obtain high titers of the original viral vector. Such complementing cell lines are known and include, but are not limited to, 293 cells (described, for example, in Graham et al., J. Gen. Virol. , 36 : 59-72 (1977)), PER.C6 cells (described, for example, in International Patent Application Publication No. WO 1997/000326 and U.S. Patents 5,994,128 and 6,033,908), and 293 - ORF6 cells (described, for example, in International Patent Application Publication No. WO 95/34671 and Brough et al., J. Virol ., 71 : 9206-9213 (1997)). Other suitable complementing cell lines for producing the replication-deficient adenovirus or adenoviral vector of the present invention include complementing cells that have been engineered to propagate adenoviral vectors encoding transgenes whose expression inhibits viral growth in host cells (see, e.g., U.S. Patent Application Publication No. 2008/0233650). Additional suitable complementing cells are described, e.g., in U.S. Patents 6,677,156 and 6,682,929 and International Patent Application Publication No. WO 2003/020879. In some cases, the cellular genome need not contain nucleic acid sequences whose gene products compensate for all of the deficiencies of the replication - deficient adenoviral vector. One or more replication-essential gene functions lacking in a replication - deficient adenoviral vector can be provided by a helper virus , such as an adenoviral vector, that delivers in trans one or more essential gene functions required for replication of the replication-deficient adenovirus or adenoviral vector. Alternatively, the adenovirus or adenoviral vector of the present invention may contain a non-native replication-essential gene that complements one or more functions of a replication-essential gene lacking in the replication-defective adenovirus or adenoviral vector of the present invention. For example, an E1/E4-deficient adenovirus vector can be engineered to contain a nucleic acid sequence encoding E4 ORF 6 that is derived or produced from a different adenovirus (e.g., an adenovirus of a different serotype than the adenovirus or adenovirus vector of the invention, or an adenovirus of a different species than the adenovirus or adenovirus vector of the invention).

[124] An adenovirus or adenoviral vector may further comprise a transgene. The term "transgene" is defined herein as a non-native nucleic acid sequence that is operably linked to appropriate regulatory elements (e.g., a promoter) such that the non-native nucleic acid sequence can be expressed to produce a protein (e.g., a peptide or polypeptide). The regulatory elements (e.g., a promoter) may be native or non-native to the adenovirus or adenoviral vector.

[125] A "non-native" nucleic acid sequence is any nucleic acid sequence (e.g., a DNA, RNA, or cDNA sequence) that is not a native nucleic acid sequence of an adenovirus in a natural position. Thus, a non-native nucleic acid sequence may be naturally found in an adenovirus, but located in a non-native position within the adenoviral genome and/or operably linked to a non-native promoter. The terms "non-native nucleic acid sequence," "heterologous nucleic acid sequence," and "exogenous nucleic acid sequence" are synonymous and may be used interchangeably in the context of the present invention. 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).

[126] A non-native nucleic acid sequence may encode a therapeutic protein that can be used to prophylactically or therapeutically treat a mammal for a disease. Examples of suitable therapeutic proteins include cytokines, toxins, tumor suppressor proteins , growth factors, hormones, receptors, mitogens, immunoglobulins, neuropeptides, neurotransmitters, and enzymes. Alternatively, a non-native nucleic acid sequence may encode an antigen of a pathogen (e.g., a bacterium or virus), and an adenovirus or adenoviral vector can be used as a vaccine.

Viral delivery system

[127] The present invention also relates to delivery systems, such as virus-based systems, into which the nucleic acid described herein is incorporated. Representative viral expression vectors include, but are not limited to, adeno-associated viral vectors, adenovirus-based vectors, lentivirus-based vectors, retroviral vectors, and herpesvirus-based vectors. In one embodiment, the viral vector is a lentiviral vector. Vectors derived from retroviruses, such as lentivirus, are suitable tools for achieving long-term gene transfer because they provide long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have an additional advantage over vectors derived from oncoretroviruses, such as murine leukemia viruses, in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of low immunogenicity. In a further embodiment, the viral vector is an adeno-associated viral vector. In another embodiment, the viral vector is a retroviral vector. In general, and in some embodiments, a suitable vector comprises an origin of replication functional in at least one organism, a promoter sequence, suitable restriction endonuclease sites, and one or more selectable markers.

[128] Additional suitable vectors include integrating expression vectors, which can integrate randomly into the host cell DNA or can include a recombination site to ensure specific recombination between the expression vector and the host cell chromosome. Such integrating expression vectors can utilize endogenous expression control sequences of the host cell chromosomes to drive expression of the desired protein. Examples of vectors that integrate site - specifically include, for example, components of the flp - in system from Invitrogen (Carlsbad, CA) (e.g., pcDNATM5/FRT) or the cre - lox system, such as can be found in the pExchange - 6 core vectors from Stratagene (La Jolla , CA). Examples of vectors that randomly integrate into host cell chromosomes include pcDNA3.1 (when administered in the absence of T antigen) from Invitrogen (Carlsbad, CA) and pCI or pFN10A (ACT) FLEXITM from Promega (Madison, WI). Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. They are typically located in the region 30 to 110 bp upstream of the start site, although a number of promoters have recently been shown to also contain functional elements downstream of the start site. The distance between promoter elements is often flexible, so that promoter function is maintained when the elements are inverted or moved relative to each other. In the thymidine kinase (tk) promoter, the distance between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, it appears that individual elements can function together or independently to activate transcription.

[129] One example of a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it.

[130] However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, the murine mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the Epstein - Barr virus early promoter, the Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence to which it is operably linked when such expression is desired or turning it off when expression is undesired. Examples of inducible promoters include, but are not limited to, the metallothionein gene promoter, the glucocorticoid receptor gene promoter, the progesterone receptor gene promoter, and the tetracycline resistance gene promoter.

[131] Methods for introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, such as a mammalian cell, a bacterial cell, a yeast cell, or an insect cell, using any method in the art. For example, the expression vector can be transferred into the host cell by physical, chemical, or biological means.

[132] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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 of introducing a polynucleotide into a host cell is calcium phosphate transfection or polyethyleneimine (PEI) transfection.

[133] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for transferring genes into mammalian cells, such as human cells. Other viral vectors can be obtained from lentivirus, poxvirus, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patents 5,350,674 and 5,585,362.

Non-viral delivery system

[134] Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems including oil - in - water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system for use as a delivery vehicle in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle).

[135] The use of lipid formulations for introducing nucleic acids into a host cell ( in vitro, ex vivo or in vivo ) is provided. In another aspect of the invention, the nucleic acid may be linked to a lipid. The lipid-linked nucleic acid may be encapsulated in the aqueous interior of a liposome, dispersed throughout the lipid bilayer of a liposome, attached to a liposome via a linker molecule that is linked to both the liposome and the oligonucleotide, contained within a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise linked to a lipid. Lipid-, lipid/DNA-, or lipid/expression vector-linked compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, such as micelles, or have a "broken" structure. They may also be simply dispersed in solution, presumably forming aggregates that are not uniform in size or shape. Lipids are fats, which can be natural or synthetic lipids. For example, lipids include fatty droplets, which are naturally found in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[136] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("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; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about - 20 °C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a general term encompassing a variety of unilamellar and multilamellar lipid carriers formed by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an internal aqueous environment. Multilamellar liposomes have multiple lipid layers separated by an aqueous environment. They form spontaneously upon suspension of phospholipids in excess aqueous solution. The lipid components undergo self-rearrangement before forming closed structures and entrapping water and solutes between the lipid bilayers (Ghosh et al., Glycobiology 5 : 505–10 (1991)). However, compositions whose structure in solution differs from the normal vesicular structure are also encompassed. For example, lipids may have a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine – nucleic acid complexes are also considered.

[137] In some cases, polynucleotides encoding polypeptides may also be introduced into cells using non-viral delivery systems, such as the Sleeping Beauty (SB) transposon system, which 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 and 8,227,432. The Sleeping Beauty transposon system consists of 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.

[138] DNA transposons move from one DNA site to another by a simple "cut and paste" process. Transposition is a precise process in which a specific segment of DNA is excised from one DNA molecule and moved to another site on the same or a different DNA molecule or genome. Like other Tc1/Mariner-type transposases, SB transposase inserts the transposon into a pair of TA dinucleotides in the recipient DNA sequence. The insertion site may be elsewhere on the same DNA molecule or on a different DNA molecule (or chromosome). There are approximately 200 million TA sites in the genomes of mammals, including humans. The TA insertion site is duplicated during transposon integration. This duplication of the TA sequence is a hallmark of transposition and is used to elucidate the mechanism in some experiments. A transposase can be encoded either within a transposon or supplied from another source, such as DNA or mRNA, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons are most useful as genetic tools because, once inserted, they cannot independently continue excision and reinsertion. SB transposons are proposed for use as non-viral vectors for introducing genes into vertebrate genomes and for gene therapy.

[139] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose the cell to an inhibitor of the invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, molecular assays well known to those skilled in the art, such as Southern and Northern blotting, RT - PCR and PCR; "biochemical" assays, such as detecting the presence or absence of a particular peptide, for example, by immunological means (ELISA and Western blotting ) or the assays described herein to identify agents within the scope of the invention.

[140] In embodiments, the modified effector cell described herein and other genetic elements are delivered to the cell 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), incorporated herein by reference in its entirety) 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 transposases and transposon systems are disclosed in U.S. patents Nos. 6,489,458; 6,613,752, 7,148,203; 7,985,739; 8,227,432; 9,228,180; U.S. Patent 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 in Ivics et al . , Cell, 91(4) : 501–10 (1997), each of which is incorporated herein in its entirety by link.

[141] Additional suitable non-viral systems may include integrating expression vectors that can integrate randomly into host cell DNA or may include a recombination site to ensure specific recombination between the expression vector and the host cell chromosome. Targeted integration of transgenes into predetermined genetic loci is a desirable goal for many applications. First , a first recombination site for a site - specific recombinase is inserted into a genomic site at either a random or a predetermined location. Cells are then transfected with a plasmid carrying the gene or DNA of interest, a second recombination site, and a recombinase source (expression plasmid, RNA, protein, or virus-expressing recombinase). Recombination between the first and second recombination sites results in integration of the plasmid DNA.

[142] Such integrating expression vectors may utilize endogenous expression control sequences of the host cell chromosomes to achieve expression of the desired protein. In some embodiments, targeted integration is facilitated by the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site. For example, targeted integration using the donor polynucleotides described herein can be achieved using conventional transfection methods, such as methods used to create gene knockouts or knockouts by homologous recombination. In other embodiments, targeted integration is facilitated by both the presence of sequences on the donor polynucleotide that are homologous to sequences flanking the integration site and by contacting the cells with the donor polynucleotide in the presence of a site - specific recombinase. By site - specific recombinase or simply recombinase is meant a polypeptide that catalyzes conserved site - specific recombination between its compatible recombination sites. As used herein, the term "site - specific recombinase" includes native polypeptides, as well as derivatives, variants and/or fragments that retain activity, and native polynucleotides, derivatives, variants and/or fragments that encode a recombinase that retains activity.

[143] Also provided herein is a system for integrating heterologous genes into a host cell, the system comprising one or more gene expression cassettes. In some cases, the system comprises a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide construct. In other cases, the system may comprise a second gene expression cassette comprising a second polynucleotide encoding a second polypeptide construct. In yet other cases, the system may comprise a third gene expression cassette. In one embodiment, one of the gene expression cassettes may comprise a gene switch polynucleotide encoding one or more of: (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 recombinant binding sites; and a serine recombinase; in such a way that upon contact of said host cell with at least said first gene expression cassette in the presence of said serine recombinase, said heterologous genes are integrated into said host cell .

[144] In some cases, the system further comprises a ligand; such that upon contact with said host cell in the presence of said ligand, said heterologous gene is expressed in said host cell. In one case, the system also includes recombinant attachment sites. In some cases, one recombination attachment site is a phage genomic recombination attachment site (attP) or a bacterial genomic recombination attachment site (attB). In one case, the host cell is a eukaryotic cell. In another case, the host cell is a human cell. In other cases, the host cell is a T cell or an NK cell.

Promoters

[145] The term "promoter" refers to the region of a polynucleotide that initiates transcription of a coding sequence. Promoters are located near the transcription start sites of genes, on the same strand, and upstream of DNA (toward the 5' region of the sense strand). Some promoters are constitutive, being active under all circumstances in the cell, while others are regulated, becoming active in response to specific stimuli, such as an inducible promoter. Still other promoters are tissue-specific or activated promoters, including, but not limited to, T - cell-specific promoters.

[146] The term "promoter activity" and its grammatical equivalents as used herein refer to the degree of expression of a nucleotide sequence operably linked to a promoter whose activity is being measured. Promoter activity may be measured directly by determining the amount of RNA transcript produced, such as by Northern blotting , or indirectly by determining the amount of product encoded by a linked nucleic acid sequence, such as a reporter nucleic acid sequence linked to the promoter.

[147] The term "inducible promoter" as used herein refers to a promoter whose activity is induced by the presence or absence of transcriptional regulators, such as biotic or abiotic factors. Inducible promoters are useful because the expression of genes functionally linked to them can be turned on or off at specific stages of an organism's development or in a specific tissue. Examples of inducible promoters include alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenesis-regulated promoters, temperature-regulated promoters, and light-regulated promoters. In one embodiment, the inducible promoter is part of a genetic switch. The inducible promoter can be a promoter inducible by a gene switch ligand. In some cases, the inducible promoter may be a small molecule ligand-inducible two-polypeptide ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch. In some cases, the gene switch may be selected from components of an ecdysone-based receptor as described in any of the systems described in: PCT/US2001/009050 (WO 2001/070816); U.S. Patent Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT/US2001/030608 (WO 2002/029075); U.S. Patent Nos. 8,105,825; 8,168,426; PCT/1J52002/005235 (WO 2002/066613); U.S. Patent Application Ser. No. 10/468,200 (U.S. Patent Publication No. 20120167239); PCT/US2002/005706 (WO 2002/066614); U.S. Patent Nos. 7531326; 8236556; 8598409; PCT/U52002/005090 (WO 2002/066612); U.S. Patent No. 8715959 (U.S. Patent Publication No. 20060100416); PCT/US2002/005234 (WO 2003/027266); U.S. Patent Nos. 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT/U52002/005708 (WO 2002/066615); U.S. Patent Application No. 10/468,192 (U.S. Patent Publication No. 20110212528); PCT/US2002/005026 (WO 2003/027289); U.S. Patent Nos. 7,563,879; 8,021,878; 8,497,093; PCT/US2005/015089 (WO 2005/108617); U.S. Patent Nos. 7,935,510; 8,076,454; PCT/U52008/011270 (WO 2009/045370); U.S. Patent Application Ser. No. 12/241,018 (U.S. Patent Publication No. 20090136465); PCT/US2008/011563 (WO 2009/048560); U.S. Patent Application Ser. No. 12/247,738 (U.S. Patent Publication No. 20090123441); PCT/US2009/005510 (WO 2010/042189); U.S. Patent Application Ser. No. 13/123,129 (U.S. Patent Publication No. 20110268766); PCT/US2011/029682 (WO 2011/119773); U.S. Patent Application No. 13/636473 (U.S. Patent Publication No. 20130195800); PCT/US2012/027515 (WO 2012/122025); and U.S. Patent No. 9,402,919, each of which is incorporated by reference in its entirety).

[148] Provided herein are methods comprising administering to a subject at least one non-viral vector comprising a polynucleotide encoding a polypeptide sequence described herein comprising at least two functional proteins or portions thereof; at least one promoter; and at least one engineered recombination site; wherein said at least one promoter directs the expression of said at least two functional proteins. In some cases, at least one promoter may be constitutive. In some cases, at least one promoter may be tissue-specific. In some cases, at least one promoter may be inducible. In some cases, the inducible promoter is a small molecule ligand-inducible two-polypeptide gene switch based on the ecdysone receptor. In other cases, a combination of promoters may be used, wherein at least one promoter may be inducible and at least one promoter may be activation-specific.

[149] An inducible promoter uses a ligand to dose-regulate the expression of at least two specified genes. In some cases, the ligand may be selected from the group consisting of ecdysteroid, 9 - cis - retinoic acid, synthetic analogs of retinoic acid, N,N' - diacylhydrazines, oxadiazolines, dibenzoylalkyl cyanohydrazines, N - alkyl - N,N' - diaroylhydrazines, N - acyl - N - alkylcarbonylhydrazines, N - aroyl - N - alkyl - N“ -aroylhydrazines , arnido ketones, 3,5 - di - tert - butyl - 4 - hydroxy - N - isobutylbenzamide, 8 - O - acetylharpagide, oxysterols, 22(R)hydroxycholesterol, 24(S)hydroxycholesterol, 25 - epoxycholesterol, T0901317, 5 - alpha - 6 - alpha- epoxycholesterol - 3 - sulfate (ECHS), 7 - ketocholesterol - 3 - sulfate, frameol, bile acids, 1,1 - bisphosphonate esters, juvenile hormone III, RG - 115819 (3,5 - dimethyl - benzoic acid N - (1 - ethyl - 2,2 - dimethyl - propyl) - N' - (2 - methyl - 3 - methoxybenzoyl) - hydrazide - ), RG - 115932 ((R) - 3,5 - dimethylbenzoic acid N - (1 - tert - butylbutyl) - N' - (2 - ethyl - 3 - methoxybenzoyl) - hydrazide), and RG - 115830 (3,5 - dimethyl - benzoic acid, N - (1 - tert - butyl - butyl) - N' - (2 - ethyl - 3 - methoxy - benzoyl) - hydrazide), and any combinations thereof.

[150] In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a non-inducible promoter. In some cases, the promoter may be a tissue-specific promoter. As used herein, the term "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 such that the promoter drives expression only in certain tissues or cell types of the organism. In other cases, a tissue-specific promoter may be temporally regulated such that the promoter differentially drives expression in a cell type or tissue over time, including during development of the organism. In some cases, a tissue-specific promoter is regulated both spatially and temporally. In some embodiments, a tissue-specific promoter is activated in certain cell types continuously or periodically at certain times or stages of the cell type. For example, a tissue-specific promoter can be one that is activated when a specific cell, such as a T cell or NK cell, is activated. T cells can be activated in a variety of ways, such as by the presentation of peptide antigens by MHC class II molecules.

[151] In one case, at least one promoter is an engineered promoter or variants thereof. As described herein, the promoter may include minimal promoter sequences from IL - 2 and one or more of the following elements: nuclear factor of activated T cells (NFAT) response element(s); an NFIL2D response element, an NFkB/TCF response element, an NF_AT/NFIL2B response element, or an NFIL2A/OCT response element. Examples of response elements are described in Mattila et al., EMBO J. 9(13) : 4425-33 (1990); incorporated herein in its entirety.

[152] The use of a gene switch for ligand-induced control of IL - 12 expression described herein may improve the safety profile of IL - 12, for example, by providing regulated expression and improving the therapeutic index. However, a prerequisite for ligand dose-dependent expression of IL - 12 using the gene switch(es) is the presence or absence of an activator ligand (e.g. , veledimex). In some embodiments, an additional conditional control of the induction of IL - 12 expression is provided. Gene switch components are provided under the control of specific T - cell-activated promoters. This results in the conditional expression (e.g., T - cell activation) of the gene switch components required for veledimex-controlled expression of the transgene(s) under the control of the gene switch. In some embodiments, this results in preferential expression of cytokines, such as IL - 12 or IL - 15, by tumor-specific T cells when veledimex is present and the T cells are activated. This can lead to increased localized levels of transgene expression controlled by gene switching.

[153] For example, T cell activation-specific expression of gene switch components can be controlled by a promoter containing nuclear factor of activated T cells (NFAT) response element(s). NFAT transcription factors are key modulators of effector T cell state. NFATs represent an early transcriptional checkpoint that gradually leads to exhaustion. NFATs are rapidly activated in T cells upon TCR stimulation and form a protein complex with AP - 1, induced by appropriate costimulatory signaling, and regulate effector genes and T cell functions. NFAT response element(s) can be fused to other minimal promoter sequences (e.g. , the IL2 minimal promoter) to drive transgene expression in response to T cell activation.

[154] Other examples of activation-specific promoters include, but are not limited to, the interleukin - 2 (IL2) promoter and the programmed death (PD) -1 promoter (CD279). Gene switch components can also be conditionally expressed upon immune cell activation by fusing binding sites for other nuclear factors, such as the NF - κB proinflammatory signaling pathway, to a minimal promoter sequence (e.g., IL2).

[155] In some embodiments, the promoter may be any one or more of: the IL - 2 core promoter, the IL - 2 minimal promoter, the IL - 2 enhancer and variant promoter, the (NF - kB) ₁ - IL2 variant promoter, the (NF - kB) ₃ - IL2 variant promoter, the (NF - kB) ₆ - IL2 variant promoter, the 1X NFAT response elements - IL2 variant promoter, the 3X NFAT response elements - IL2 variant promoter, the 6X NFAT response elements - IL2 variant promoter, the human EEF1A1 promoter variant, the human EEF1A1 promoter and enhancer, the human UBC promoter, and the synthetic minimal promoter 1.

Gene switch

[156] Provided herein are gene switch polypeptides, polynucleotides encoding ligand-inducible gene switch polypeptides, and methods and systems comprising these polypeptides and/or polynucleotides. In some aspects, the present invention relates to a polynucleotide comprising one or more polynucleotides encoding a gene switch system for inducibly controlling the expression of a heterologous gene, wherein the expression of the heterologous gene is regulated by said gene switch system; and wherein said heterologous gene comprises a polynucleotide encoding a polypeptide comprising one or more human papillomavirus (HPV) immune response-inducing polypeptides disclosed herein.

[157] The term "gene switch" refers to the combination of a response element linked to a promoter and, for example, an EcR-based system that, in the presence of one or more ligands, modulates the expression of a gene in which the response element and promoter are incorporated. Tightly regulated inducible gene expression systems or gene switches are useful for a variety of applications such as gene therapy, large-scale protein production in cells, cell-based high-throughput screening assays, functional genomics, and trait regulation in transgenic plants and animals. Such inducible gene expression systems may include ligand-inducible heterologous gene expression systems.

[158] An early version of the EcR-based gene switch used the Drosophila melanogaster EcR (DmEcR) and Mus musculus RXR (MmRXR) polypeptides and showed that these receptors, in the presence of the steroid ponasterone A, transactivate reporter genes in mammalian cell lines and transgenic mice (Christopherson et al., Proc. Natl. Acad. Sci. USA 89(14):6314–18 (1992); No et al., Proc. Natl. Acad . Sci. USA 93(8): 3346–51 (1996)). Later, Suhr et al. ( Proc. Natl. Acad. Sci. USA 95(14):7999 - 8004 (1998)) showed that the nonsteroidal ecdysone agonist, tebufenozide, induces high levels of transactivation of reporter genes in mammalian cells via Bombyx mori EcR (BmEcR) in the absence of an exogenous heterodimeric partner.

[159] International Patent Application Nos. PCT/US97/05330 (WO 97/38117) and PCT/US99/08381 (WO99/58155) disclose methods for modulating the expression of an exogenous gene, wherein a DNA construct containing the exogenous gene and an ecdysone response element is activated by a second DNA construct containing an ecdysone receptor, which, in the presence of its ligand and, optionally, in the presence of a receptor capable of acting as a silencing partner, binds to the ecdysone response element to induce gene expression. In this example, the ecdysone receptor was isolated from Drosophila melanogaster . Typically, such systems require the presence of a silencing partner, preferably the retinoid X receptor (RXR), to ensure optimal activation. In mammalian cells, the insect ecdysone receptor (EcR) is capable of heterodimerization with the mammalian retinoid X receptor (RXR) and can thus be used to regulate the expression of target genes or heterologous genes in a ligand - dependent manner. International Patent Application No. PCT/US98/14215 (WO 99/02683) discloses that the ecdysone receptor, isolated from the silkworm Bombyx mori , functions in mammalian systems without the need for an exogenous dimer partner.

[160] U.S. Patent No. 6,265,173 discloses that various members of the steroid/thyroid receptor superfamily can be combined with the Drosophila melanogaster ultracoiled receptor (USP) or fragments thereof containing at least the USP dimerization domain for use in a gene expression system. U.S. Patent No. 5,880,333 describes a heterodimeric EcR and Drosophila melanogaster ultracoiled protein (USP) system for use in plants, in which the transactivation domain and DNA binding domain are located on two different fusion proteins. In each of these cases, the transactivation domain and the DNA binding domain (either as native EcR, as in International Patent Application No. PCT/US98/14215, or as modified EcR, as in International Patent Application No. PCT/US97/05330) were incorporated into a single molecule, and the other heterodimer partners, either USP or RXR, were used in their native state.

[161] International Patent Application No. PCT/US01/0905 describes an ecdysone receptor-based inducible gene expression system in which the transactivation and DNA - binding domains are separated by placing them on two different proteins, resulting in a significant reduction in background activity in the absence of ligand and a significantly increased activity above background in the presence of ligand. This two-hybrid system represents a significantly improved system for modulating inducible gene expression compared to the two systems disclosed in applications PCT/US97/05330 and PCT/US98/14215. The two-hybrid system is thought to exploit the ability of a pair of interacting proteins to position the transcriptional activation domain favorably relative to the DNA-binding domain, so that when the DNA-binding domain binds to a DNA-binding site on a gene, the transactivation domain more efficiently activates the promoter (see, e.g., U.S. Patent No. 5,283,173). The two-hybrid gene expression system comprises two gene expression cassettes; the first encodes a DNA - binding domain fused to a nuclear receptor polypeptide, and the second encodes a transactivation domain fused to a different nuclear receptor polypeptide. The presence of ligand is thought to induce a conformational change that favors interaction of the first polypeptide with the second polypeptide, resulting in dimerization of the DNA - binding domain and the transactivation domain. Because the DNA-binding and transactivation domains are on two different molecules, background activity is significantly reduced in the absence of ligand.

[162] In some cases, the inducible promoter may be a small molecule ligand-inducible, two-polypeptide ecdysone receptor-based gene switch, such as the RHEOSWITCH® gene switch from Intrexon Corporation. In some cases, the gene switch may be selected from components of an ecdysone-based receptor as described in any of the systems described in: PCT/US2001/009050 (WO 2001/070816); U.S. Patent Nos. 7,091,038; 7,776,587; 7,807,417; 8,202,718; PCT/US2001/030608 (WO 2002/029075); U.S. Patent Nos. 8,105,825; 8,168,426; PCT/1J52002/005235 (WO 2002/066613); U.S. Patent Application Ser. No. 10/468,200 (U.S. Patent Publication No. 20120167239); PCT/US2002/005706 (WO 2002/066614); U.S. Patent Nos. 7531326; 8236556; 8598409; PCT/U52002/005090 (WO 2002/066612); U.S. Patent No. 8715959 (U.S. Patent Publication No. 20060100416); PCT/US2002/005234 (WO 2003/027266); U.S. Patent Nos. 7,601,508; 7,829,676; 7,919,269; 8,030,067; PCT/U52002/005708 (WO 2002/066615); U.S. Patent Application No. 10/468,192 (U.S. Patent Publication No. 20110212528); PCT/US2002/005026 (WO 2003/027289); U.S. Patent Nos. 7,563,879; 8,021,878; 8,497,093; PCT/US2005/015089 (WO 2005/108617); U.S. Patent Nos. 7,935,510; 8,076,454; PCT/U52008/011270 (WO 2009/045370); U.S. Patent Application Ser. No. 12/241,018 (U.S. Patent Publication No. 20090136465); PCT/US2008/011563 (WO 2009/048560); U.S. Patent Application Ser. No. 12/247,738 (U.S. Patent Publication No. 20090123441); PCT/US2009/005510 (WO 2010/042189); U.S. Patent Application Ser. No. 13/123,129 (U.S. Patent Publication No. 20110268766); PCT/US2011/029682 (WO 2011/119773); U.S. Patent Application No. 13/636473 (U.S. Patent Publication No. 20130195800); PCT/US2012/027515 (WO 2012/122025); and U.S. Patent No. 9,402,919, each of which is incorporated by reference in its entirety.

[163] Systems for modulating the expression of a heterologous gene and interleukin in a host cell are proposed, including polynucleotides expressing the gene switching polypeptides described in this document.

[164] In some embodiments, systems are provided for modulating the expression of a heterologous gene and a cytokine in a host cell, comprising a first gene expression cassette comprising a first polynucleotide encoding a first polypeptide; a second gene expression cassette comprising a second polynucleotide encoding a second polypeptide; and a ligand; wherein said first and second polypeptides comprise one or more of: (i) a transactivation domain; (ii) a DNA binding domain; and (iii) a ligand binding domain; (iv) said heterologous gene; and (vi) said cytokine such that upon contacting said host cell with said first gene expression cassette and said second gene expression cassette in the presence of said ligand, said heterologous gene and said cytokine are expressed in said host cell. In some cases, the heterologous gene comprises an antigen-binding polypeptide as described herein. In some cases, the cytokine comprises at least one chemokine, interferon, interleukin, lymphokine, tumor necrosis factor, or variant thereof, or a combination thereof. In some cases, the cytokine is an interleukin. In some cases, the interleukin is at least one of IL12, IL2, IL15, IL21, and functional variants and fragments thereof. In some embodiments, the cytokines may be membrane-associated or secreted. In other embodiments, the cytokines may be intracellular. The interleukin may comprise membrane-associated IL - 15 (mbIL - 15) or a fusion of IL - 15 and IL - 15Rα. In some embodiments, mbIL - 15 is a membrane-associated chimeric IL - 15 that may be co-expressed with the modified effector cell described herein. In some embodiments, mbIL - 15 comprises full-length IL - 15 ( e.g. , a native IL - 15 polypeptide) or a fragment or variant thereof fused in frame to full-length IL - 15Rα, a functional fragment, or variant thereof. In some cases, IL - 15 is indirectly linked to IL - 15Rα via a linker. In some cases, mbIL - 15 is described in Hurton et al., “Tethered IL - 15 enhances antitumor activity and promotes a stem - cell memory subset in tumor - specific T cells,” Proc. Natl. Acad. Sci. USA 113(48) :E7788 – E7797 (2016). In another aspect, the interleukin may comprise IL - 12. In some embodiments, the IL - 12 is single-chain IL - 12 (scIL - 12), protease-sensitive IL - 12, destabilized IL - 12, membrane-associated IL - 12, intercalated IL - 12. In some cases, IL - 12 variants are described in WO 2015/095249, WO 2016/048903, WO 2017/062953, all of which are incorporated by reference in their entirety.

[165] Provided herein are polynucleotides encoding gene switch polypeptides, wherein said gene switch polypeptides comprise: a) a first gene switch polypeptide comprising a DNA binding domain fused to a ligand binding domain of a nuclear receptor, and b) a second gene switch polypeptide comprising a transactivation domain fused to a ligand binding domain of a nuclear receptor, wherein the first gene switch polypeptide and the second gene switch polypeptide are connected by a linker. In some cases, the linker may be a linker as described herein, such as a GSG linker, a furinlink, a 2A linker such as F/T2A, T2A, p2A, GSG - p2A, variants and derivatives thereof. In other cases, the linker may be an IRES.

[166] In some cases, the DNA binding domain (DBD) comprises a DBD described herein, such as at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid/thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD. The transactivation domain may comprise a transactivation domain described herein, such as one of the VP16 transactivation domain, the p53 transactivation domain, and the acid activator B42 transactivation domain. The ligand binding domain of the nuclear receptor may comprise at least one of ecdysone receptor (EcR), ubiquitous receptor, orphan receptor 1, NER - 1, steroid hormone nuclear receptor 1, retinoid X receptor - interacting protein 15, liver X receptor β , steroid hormone receptor-like protein, liver X receptor α, farnesoid X receptor, receptor-interacting protein 14, and famisol receptor.

[167] In some cases, gene switch polypeptides connected by a polypeptide linker or ribosome skipping sequence exhibit improved dose-dependent ligand-inducible control of gene expression compared to a ligand-inducible gene switch where the gene switch polypeptides are connected by non-coding sequences, such as an IRES. In some cases, gene switch polypeptides connected by a 2A linker may exhibit improved dose-dependent ligand-inducible control of heterologous gene expression compared to a gene switch where the gene switch polypeptides are separated by an IRES.

[168] In some embodiments, the gene switch comprises a VP16 transactivation domain. In one embodiment, the gene switch comprises at least one of ecdysone receptor (EcR), ubiquitous receptor, orphan receptor 1, NER - 1, steroid hormone nuclear receptor 1, retinoid X receptor interacting protein 15 , liver X receptor β, steroid hormone receptor - like protein, liver X receptor α, farnesoid X receptor, receptor interacting protein 14, and famisol receptor. In another embodiment, the DNA binding domain (DBD) of the gene switch comprises at least one of GAL4 (GAL4 DBD), LexA DBD, transcription factor DBD, steroid/thyroid hormone nuclear receptor superfamily member DBD, bacterial LacZ DBD, and yeast DBD. In yet another case, the gene switch further comprises at least one of ultraspiracular protein (USP), retinoid X receptor (RXR), functional fragments and variants thereof, wherein said functional fragments and variants are capable of binding to EcR.

[169] The polypeptides and polynucleotides as described herein can be expressed in an engineered cell. As used herein, an engineered cell is a cell that has been modified compared to its natural or endogenous state. An example of an engineered cell is a cell described herein that has been modified ( e.g. , by transfecting a polynucleotide into the cell) to encode, for example, gene switch polypeptides, a gene of interest (GOI), cellular labels, heterologous genes, and any other polypeptides and polynucleotides described herein.

Ligands

[170] In some embodiments, the ligand used to regulate the inducible gene switch may be selected from any of the following, but is not limited to: N - [(1R) -1- ( 1,1 - dimethylethyl)butyl] -N ′ - (2 - ethyl - 3 - methoxybenzoyl) -3,5 - dimethylbenzohydrazide (also called Weledimex), (2S,3R, 5R,9R,10R,13R,14S,17R) -17 - [(2S,3R) -3,6 - dihydroxy - 6 - methylheptan - 2 - yl] -2,3,14 - trihydroxy - 10,13 - dimethyl - 2,3,4,5,9,11,12,15,16,17 - decahydro - 1H - cyclopenta[a] phenanthrene- 6 - she; N′ - (3,5 - dimethylbenzoyl) - N′ - [(3R) - 2,2 - dimethyl - 3 - hexanyl] - 2 - ethyl - 3 - methoxybenzohydrazide; 5 - methyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid N′ - (3,5 - dimethylbenzoyl) - N′ - (1 - ethyl - 2,2 - dimethylpropyl) - hydrazide; 5 - methyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid N′ - (3,5 - dimethoxy - 4 - methylbenzoyl) - N′ - (1 - ethyl - 2,2 - dimethylpropyl) - hydrazide; N′ - (1 - tert - butylbutyl) - N′ - (3,5 - dimethylbenzoyl)hydrazide 5 - methyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid; 5 - methyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid N′ - (1 - tert - butylbutyl) - N′ - (3,5 - dimethoxy - 4 - methylbenzoyl) - hydrazide; 5 - ethyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid N′ - (3,5 - dimethylbenzoyl) - N′ - (1 - ethyl - 2,2 - dimethylpropyl) - hydrazide; 5 - ethyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid N′ - (3,5 - dimethoxy - 4 - methylbenzoyl) - N′ - (1 - ethyl - 2,2 - dimethylpropyl) - hydrazide; N′ - (1 - tert - butylbutyl) - N′ - (3,5 - dimethylbenzoyl)hydrazide 5 - ethyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid; 5 - ethyl - 2,3 - dihydrobenzo[1,4]dioxin - 6 - carboxylic acid N′ - (1 - tert - butylbutyl) - N′ - (3,5 - dimethoxy - 4 - methylbenzoyl) - hydrazide; N - (1 - ethyl - 2,2 - dimethylpropyl) - N' - (3 - methoxy - 2 - methylbenzoyl) hydrazide of 3,5 - dimethylbenzoic acid; N - (1 - ethyl - 2,2 - dimethylpropyl) - N' - (3 - methoxy - 2 - methylbenzoyl) hydrazide of 3,5 - dimethoxy - 4 - methylbenzoic acid; N - (1 - tert - butylbutyl) - N' - (3 - methoxy - 2 - methylbenzoyl) hydrazide of 3,5 - dimethylbenzoic acid; N - (1 - tert - butylbutyl) - N' - (3 - methoxy - 2 - methylbenzoyl) hydrazide of 3,5 - dimethoxy - 4 - methylbenzoic acid; N - (1 - ethyl - 2,2 - dimethylpropyl) - N′ - (2 - ethyl - 3 - methoxybenzoyl) 3,5 - dimethylbenzoic acid hydrazide; N - (1 - ethyl - 2,2 - dimethylpropyl) - N′ - (2 - ethyl - 3 - methoxybenzoyl) 3,5 - dimethoxy - 4 - methylbenzoic acid hydrazide; N - (1 - tert - butylbutyl) - N′ - (2 - ethyl - 3 - methoxybenzoyl) 3,5 - dimethylbenzoic acid hydrazide; N - (1 - tert - butylbutyl) - N′ - (2 - ethyl - 3 - methoxybenzoyl) 3,5 - dimethoxy - 4 - methylbenzoic acid hydrazide; N- (1 - tert - butylpentyl) -N ′ - (4 - ethylbenzoyl)hydrazide of 2 - methoxynicotinic acid; N- (2,2 - dimethyl - 1 - phenylpropyl) -N “ - (4 - ethylbenzoyl)hydrazide of 3,5 - dimethylbenzoic acid; N- (1 - tert - butylpentyl) -N ′ - (3 - methoxy - 2 - methylbenzoyl)hydrazide of 3,5 - dimethylbenzoic acid; and N- (1 - tert - butylpentyl) -N ′ - (3 - methoxy - 2 - methylbenzoyl)hydrazide of 3,5 - dimethoxy - 4 - methylbenzoic acid.

[171] In some cases, the ligand used for dose-controlled control of ecdysone receptor-based inducible gene switching may be selected from any, but not limited to, an ecdysteroid such as ecdysone, 20 - hydroxyecdysone, ponasterone A, muristerone A, etc., 9 - cis - retinoic acid, synthetic retinoic acid analogs, N,N' - diacylhydrazines such as those disclosed in U.S. Patent Nos. 6,013,836; 5,117,057; 5,530,028; and 5,378,726 and U.S. Patent Application Publications Nos. 2005/0209283 and 2006/0020146; oxadiazolines such as those described in U.S. Published Patent Application No. 2004/0171651; dibenzoylalkyl cyanohydrazines such as those described in European Patent Application No. 461809; N - alkyl - N,N'' - diaroylhydrazines such as those disclosed in U.S. Patent No. 5,225,443; N - acyl - N - alkylcarbonylhydrazines such as those described in European Patent Application No. 234994; N - aroyl - N - alkyl - N' - aroylhydrazines such as those described in U.S. Patent No. 4,985,461; arnido ketones such as those described in U.S. Published Patent Application No. 2004/0049037; each of which is incorporated herein by reference, and other similar materials, including 3,5 - di - tert - butyl - 4 - hydroxy - N - isobutylbenzamide, 8 - O - acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S) hydroxycholesterol, 25 - epoxycholesterol, T0901317, 5 - alpha - 6 - alpha - epoxycholesterol - 3 - sulfate (ECHS), 7 - ketocholesterol - 3 - sulfate, frameol, bile acids, 1,1 - bisphosphonate esters, juvenile hormone III, and the like. Examples of diacylhydrazine ligands useful in the present invention include RG - 115819 (3,5 - dimethylbenzoic acid, N- (1 - ethyl - 2,2 - dimethylpropyl) -N ' - (2 - methyl - 3 - methoxy - benzoyl) -hydrazide- ), RG - 115932 ( (R) -3,5 - dimethyl - benzoic acid N- (1 - tert - butyl - butyl - 1) -N ' - (2 - ethyl - 3 - methoxy - benzoyl ) -hydrazide ) and RG - 115830 ( N- (1 - tert - butylbutyl) -N ' - (2 - ethyl - 3 - methoxybenzoyl) -3,5 - dimethylbenzoic acid hydrazide). See, for example , U.S. Patent Application No. 12/155,111 and PCT Application No. PCT/US2008/006757, both of which are incorporated herein by reference in their entireties.

Cytokines

[172] In some embodiments, the HPV vaccine antigens provided herein are delivered and/or co-expressed ( e.g. , as part of the same HPV antigen delivery vector or via a separate vector) with other cytokines. In some embodiments, the HPV vaccine antigens provided herein are polynucleotides encoding gene switch polypeptides and a cytokine, or a variant or derivative thereof, and methods and systems comprising them. A cytokine is a category of small proteins with a molecular weight of about 5-20 kDa that are involved in cell signaling. In some cases, cytokines include chemokines, interferons, interleukins, colony-stimulating factors, or tumor necrosis factors. In some embodiments, chemokines act as chemoattractants that direct cell migration and are classified into four subfamilies: CXC, CC, CX3C, and XC. Examples of chemokines include those of the CC subfamily: CCL1, CCL2 (MCP - 1), CCL3, CCL4, CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (or CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, and CCL28; CXC subfamily: CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, and CXCL17; XC subfamily: XCL1 and XCL2; and CX3C subfamily CX3CL1.

[173] In some embodiments, the HPV vaccine antigens provided herein are delivered and/or co-expressed (e.g., as part of the same HPV antigen delivery vector or via a separate vector) with interferons. Interferons (IFNs) include type I interferon (e.g., IFN - α, IFN - β, IFN - ε, IFN - κ, and IFN - ω), type II interferon (e.g., IFN - γ), and type III interferon. In some embodiments, IFN - α is further subdivided into about 13 subtypes, including IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21.

[174] In some embodiments, the HPV vaccine antigens provided herein are delivered and/or co-expressed (e.g., as part of the same HPV antigen delivery vector or via a separate vector) along with other interleukins. Interleukins are expressed by white blood cells or leukocytes and promote the development and differentiation of T- and B - lymphocytes and hematopoietic cells. Examples of interleukins include IL - 1, 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. In some embodiments, the interleukins are IL - 2, IL - 12, IL - 15, IL - 21, or a fusion of IL - 15 and IL - 15α.

[175] In some aspects, the interleukin may comprise IL - 12. In some embodiments, the IL - 12 is single-chain IL - 12 (scIL - 12), protease-sensitive IL - 12, destabilized IL - 12, membrane-associated IL - 12, intercalated IL - 12. In some cases, IL - 12 variants are described in WO 2015/095249, WO 2016/048903, WO 2017/062953, all of which are incorporated by reference in their entireties.

[176] In some embodiments, the interleukin comprises mbIL - 15. In some embodiments, mbIL - 15 is a membrane-bound chimeric IL - 15 that can be co-expressed with a modified effector cell described herein. In some embodiments, mbIL - 15 comprises full-length IL - 15 ( e.g. , a native IL - 15 polypeptide) or a fragment or variant thereof fused in frame to full-length IL - 15Rα, a functional fragment or variant thereof. In some cases, IL - 15 is indirectly linked to IL - 15Rα via a linker. In some cases, mbIL - 15 is 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.

[177] In some embodiments, the HPV vaccine antigens provided herein are delivered and/or co-expressed (e.g., as part of the same HPV antigen delivery vector or via a separate vector) with tumor necrosis factors. Tumor necrosis factors (TNF) are a group of cytokines that modulate apoptosis. In some cases, the TNF family includes approximately 19 members, including, but not limited to, 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).

[178] In some embodiments, the HPV vaccine antigens provided herein are delivered and/or co-expressed (e.g., as part of the same HPV antigen delivery vector or via a separate vector) with colony stimulating factors. Colony stimulating factors (CSFs) are secreted glycoproteins that interact with receptor proteins on the surface of hematopoietic stem cells, which subsequently modulates cellular proliferation and differentiation into certain blood cell types. In some cases, the CSF includes macrophage colony stimulating factor, granulocyte - macrophage colony stimulating factor (GM - CSF), granulocyte colony stimulating factor (G - CSF), or promegapoietin.

[179] In some embodiments, the cytokine is a membrane-bound cytokine that is co-expressed with a chimeric antigen receptor described herein. In some embodiments, one or more methods described herein further comprise administering a cytokine. In some cases, the cytokine comprises a chemokine, an interferon, an interleukin, a colony-stimulating factor, or a tumor necrosis factor. In some cases, one or more methods described herein further comprise administering a cytokine selected from a chemokine, an interferon, an interleukin, a colony-stimulating factor, or a tumor necrosis factor. In some cases, one or more methods described herein further comprise administering a cytokine selected from IL2, IL7, IL12, IL15, a fusion of IL - 15 and IL - 15Rα, IL21, IFNγ, or TNF - α.

Interleukin - 12

[180] In particular embodiments, the HPV vaccine antigens provided herein are delivered to and/or expressed in a subject in combination with the delivery and/or expression of the cytokine interleukin - 12 ( e.g. , IL - 12 polypeptides are expressed from the same HPV vaccine antigen expression vector or expressed from a separate vector in combination with the delivery or expression of the HPV vaccine antigen). In some embodiments, in combination with the delivery or expression of the HPV vaccine antigen, the expression of IL - 12 in the subject is controlled by constitutive or inducible regulation of expression. In a preferred embodiment, in combination with the delivery or expression of the HPV vaccine antigen, the expression of IL - 12 in the subject is controlled by inducible regulation of expression (also referred to as inducible regulated expression of IL - 12). Interleukin 12 (IL - 12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC - 37) in response to antigen stimulation. IL - 12 consists of a bundle of four alpha helices. It is a heterodimeric cytokine encoded by two separate genes, IL - 12A (p35) and IL - 12B (p40). The active heterodimer (designated p70) and the p40 homodimer are formed after protein synthesis. IL - 12 is a master regulator of the immune system. IL - 12 promotes the immune response by activating NK cells and T cells ( Fig. 11 ).

[181] This document provides compositions, kits, and systems including methods for producing recombinant HPV vaccines. This document also provides polynucleotides encoding gene switch polypeptides and IL - 12 or a variant or derivative thereof, as well as methods and systems including them.

Linkers

[182] Also disclosed are constructs containing a linker to facilitate the expression and functionality of the polynucleotides and polypeptides described herein. In some embodiments, a polynucleotide linker may be used in a polynucleotide described herein. The polynucleotide linker may be a double-stranded DNA segment containing desired restriction sites that can be added to create end structures compatible with a vector containing the polynucleotide described herein. In some cases, the polynucleotide linker may be useful for modifying vectors containing the polynucleotides described herein. For example, a modification of a vector containing a polynucleotide linker may be a change in a multiple cloning site or the addition of a polyhistidine tail. Polynucleotide linkers may also be used to adapt blunt-ended DNA ends for cloning into a restriction enzyme-digested vector with sticky ends. The use of polynucleotide linkers can be more efficient than blunt ligation into a vector and can provide a means of releasing the insert from the vector for subsequent applications. In some cases, the insert may be a polynucleotide sequence encoding polypeptides suitable for therapeutic use. In some cases, the linker may be a cleavable linker.

[183] The polynucleotide linker may be an oligomer. The polynucleotide linker may be double-stranded DNA, single-stranded DNA, or a combination of both. In some cases, the linker may be RNA. In some cases, the polynucleotide linker may be ligated into a vector containing a polynucleotide described herein using T4 ligase. To facilitate ligation, excess polynucleotide linkers may be added to the composition containing the insert and the vector. In some cases, the insert and vector are pretreated before the linker is introduced. For example, pretreatment with methylase may prevent unwanted cleavage of the insert DNA .

[184] In some embodiments, two or more polypeptides encoded by a polynucleotide described herein may be separated by an intervening sequence encoding an intervening linker polypeptide. As used herein, the term "intervening linker polypeptide," which refers to an amino acid sequence separating two or more polypeptides encoded by a polynucleotide, is distinguished from the term "peptide linker," which refers to an amino acid sequence that is optionally included in a polypeptide construct described herein for attaching a transmembrane domain to a cell surface polypeptide (e.g., comprising a truncated version of a naturally occurring polypeptide). In some cases, the intervening linker is a cleavage-sensitive intervening linker polypeptide. In some embodiments, the linker is a cleavable linker or a ribosome-permeable linker. In some embodiments, the sequence of the cleavable linker or ribosome-permeable linker is selected from the group consisting of 2A, GSG - 2A, GSG linker, SGSG linker, furinlink variants, and derivatives thereof. In some embodiments, the 2A linker is a p2A linker, a T2A linker, an F2A linker, or an E2A linker. In some embodiments, the polypeptides of interest are expressed as fusion proteins linked by a cleavage-sensitive intermediate linker polypeptide. In some embodiments, the cleavage-sensitive intermediate linker polypeptide(s) may be any one or more of: F/T2A variants, T2A, p2A, 2A, GSG - p2A, GSG - linker, and furinlink. The linkers (polynucleotide and polypeptide sequences) disclosed in document PCT/US2016/061668 (WO2017083750), published May 18, 2017, are incorporated herein by reference.

[185] In some embodiments, the linker polypeptide comprises the following:

Table 3: Amino acid sequences of linkers SEQ ID NO: Linker type Amino acid sequence 34, 76 HARD LINKER EAAAK _(n)
(where n = a sequence that repeats any number of times, such as 1, 2, 3, 4, 5 times, etc.) 40 HPV6 E6 epitope/linker TAEIYSYAYKQLKVL 42 HPV11 E6 epitope/linker TAEIYAYAYKNLKVV 45 HPV11 E7 epitope/linker HCYEQLEDSSEDEVD 52 HPV16 E7 agonist RTLEDLLMGV 48 HPV16 E6 agonist 1 KLPQLCTEV 50 HPV16 E6 agonist 2 QLYNKPLCDV 57 Flexible linker1 VSQTSKLTR

[186] In some cases, the viral 2A sequence may be used. 2A elements may be shorter than IRESs and range from 5 to 100 base pairs. In some cases, the 2A sequence may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 nucleotides long. Linked 2A genes may be expressed in a single open reading frame, and "self-cleavage" may occur cotranslationally between the last two amino acids, GP, at the C - terminus of the 2A polypeptide, resulting in equal amounts of co - expressed proteins.

[187] The 2A virus sequence may consist of approximately 20 amino acids. In some cases, the 2A virus sequence may contain a consensus motif Asp - Val/Ile - Glu - X - Asn - Pro - Gly - Pro. The consensus motif sequence can act cotranslationally. For example, it can prevent the formation of a normal peptide bond between glycine and proline residues, which can lead to ribosome skipping and cleavage of the nascent polypeptide. This effect can produce multiple genes at equimolar levels.

[188] Peptide 2A can provide for the translation of multiple proteins in a single open reading frame into a polypeptide, which can subsequently be cleaved into a single polypeptide via the ribosome skipping mechanism (Funston et al., J. Gen. Virol. 89(Pt 2) : 389-96 (2008)). In some embodiments, the 2A sequence may include: F/T2A, T2A, p2A, 2A, T2A, E2A, F2A and BmCPV2A, BmIFV2A and any combination thereof.

[189] In some cases, the vector may contain an IRES sequence and a 2A linker sequence. In other cases, expression of multiple genes associated with 2A peptides may be promoted by a spacer sequence (GSG) upstream of the 2A peptides. In some cases, constructs may combine spacers, linkers, adapters, promoters, or combinations thereof. For example, a linker may have a spacer cleavage site (SGSG or GSG or Whitlow linker) and a furin linker (R - A - K - R) with different 2A peptides. The spacer may be I - CeuI. In some cases, the linker may be designed. For example, the linker may be designed to have chemical characteristics such as hydrophobicity. In some cases, at least two linker sequences can produce the same protein. In other cases, multiple linkers may be used in a vector. For example, genes of interest may be separated by at least two linkers.

[190] In some embodiments, two or more polypeptides encoded by a polynucleotide described herein may be separated by an intervening sequence encoding a linker polypeptide. In some cases, the linker is a cleavage-sensitive linker. In some embodiments, the polypeptides of interest are expressed as fusion proteins linked by a cleavage-sensitive linker polypeptide. In some embodiments, the cleavage-sensitive linker polypeptide(s) may be any one or two of Furinlink, fmdv, p2a, GSG - p2a, and/or fp2a, described below. In some cases, the linker is APVKQGSG.

[191] In some cases, the linker polypeptide may comprise the amino acid sequence "RAKR". In some cases, the furin linker polypeptide may be encoded by a polynucleotide sequence comprising "CGTGCAAAGCGT" or "AGAGCTAAGAGG".

[192] In some embodiments, a linker may be used in a polynucleotide described herein. The linker may be a flexible linker, a rigid linker, an in vivo cleavable linker, or any combination thereof. In some cases, the linker may link functional domains together (as in flexible and rigid linkers) or release a free functional domain in vivo , as in in vivo cleavable linkers.

[193] Linkers can improve biological activity, increase expression yield, and achieve desired pharmacokinetic profiles. A linker can also contain a hydrazone, peptide, disulfide, or thioester.

[194] In some cases, the linker sequence described herein may include a flexible linker. Flexible linkers may be used when the domain being attached requires a certain degree of movement or interaction. Flexible linkers may be composed of small non-polar ( e.g. , Gly) or polar ( e.g. , Ser or Thr) amino acids. A flexible linker may have sequences consisting primarily of stretches of Gly and Ser residues (a GS linker). An example of a flexible linker may have the sequence (Gly - Gly - Gly - Gly - Ser)n. By adjusting the copy number of "n", the length of this exemplary GS linker can be optimized to achieve proper separation of functional domains or to maintain desired interdomain interactions. In addition to GS linkers, other flexible linkers can be used 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.

[195] Flexible linkers included in the linker sequences described herein can be enriched in small or polar amino acids, such as Gly and Ser, to ensure good flexibility and solubility. Flexible linkers may be a suitable choice when specific movements or interactions are desired for the domains of the fusion proteins. Furthermore, although flexible linkers cannot have rigid structures, they can serve as passive linkers to maintain the distance between functional domains. The length of flexible linkers can be adjusted to ensure proper folding or to achieve optimal biological activity of the fusion proteins.

[196] The linker described herein may, in some cases, further comprise a rigid linker. A rigid linker may be used to maintain a fixed distance between domains of a polypeptide. Examples of rigid linkers include: Linkers forming an alpha helix, a Pro-rich sequence, (XP)n, an X - Pro backbone, A(EAAAK)nA (n = 2-5 ), to name a few. Rigid linkers may have relatively rigid structures, adopting α - helical structures or, in some cases, containing multiple Pro residues.

[197] The linker described herein may be cleavable in some cases. In other cases, the linker is not cleavable. Linkers that are not cleavable may covalently join functional domains together to act as a single molecule during in vivo or ex vivo processes. The linker may also be cleavable in vivo . A cleavable linker may be introduced to release free functional domains in vivo . A cleavable linker may be cleaved in the presence of reducing reagents, such as proteases. For example, disulfide bond reduction can be used to generate a cleavable linker. In the case of a disulfide linker, a cleavage event via disulfide exchange with a thiol such as glutathione may result in cleavage. In other cases, linker cleavage in a recombinant fusion protein in vivo can also be accomplished by proteases that may be expressed in vivo under pathological conditions (e.g. , cancer or inflammation), in specific cells or tissues, or restricted to specific cellular compartments. In some cases, a cleavable linker can provide targeted cleavage. For example, the specificity of many proteases can ensure slower cleavage of the linker in restricted compartments. A cleavable linker may also contain a hydrazone, peptides, disulfide, or thioester. For example, a hydrazone may provide serum stability. In other cases, a hydrazone may permit cleavage in an acidic environment. The acidic compartment may have a pH of up to 7. The linker may also include a thioether. The thioether may be non-reducible. The thioether may be targeted for intracellular proteolytic degradation.

[198] In some cases, genes may be separated by 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 linkers.

[199] The linker may be a designed linker. Linker design methods may be computational. In some cases, computational methods may include graphical methods. Computational methods can be used to search for suitable peptides in libraries of three-dimensional peptide structures obtained from databases. For example, the Brookhaven Protein Data Bank (PDB) can be used to cover the spatial distance between selected linker amino acids.

[200] In some embodiments, the polynucleotides are polynucleotides encoding a polypeptide construct comprising a furin polypeptide and a 2A polypeptide, wherein the furin polypeptide and the 2A polypeptide are connected 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), tryptophan (Trp)(W). In some cases, the polypeptide linker may also include one or more GS linker sequences, such as (GS)n, (SG)n, (GSG)n, and (SGSG)n, where n may be any number from zero to fifteen.

[201] Methods are provided for obtaining improved expression of a polypeptide construct, comprising: providing a polynucleotide encoding said polypeptide construct, comprising a first functional polypeptide and a second functional polypeptide, wherein said first functional polypeptide and second functional polypeptide are connected by a linker polypeptide comprising a sequence with at least 60% sequence identity to APVKQ; and expressing said polynucleotide in a host cell, wherein said expression results in improved expression of the polypeptide construct compared to a corresponding polypeptide construct that does not have a linker polypeptide comprising a sequence that is at least 60% identical to the sequence of APVKQ.

IRES elements

[202] Also disclosed herein are constructs containing an IRES element to facilitate the expression and functionality of the polynucleotides and polypeptides described herein. The term "internal ribosome entry site (IRES)" as used herein may refer to an internal ribosome entry site. In a vector containing an IRES sequence, the first gene can be translated by a cap - dependent ribosome scanning mechanism with its own 5 ' UTR, while translation of the subsequent gene can be accomplished by direct recruitment of the ribosome to the IRES in a cap - independent manner. The IRES sequence can allow eukaryotic ribosomes to bind and begin translation without binding to the 5 ' capped end. The IRES sequence can provide for the expression of multiple genes from a single transcript (Mountford and Smith, Trends Genet. 11(5) : 179-84 (1995)).

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

[204] In some cases, the IRES region may be derived from the IRES sequence of a virus such as picornavirus, encephalomyocarditis virus, hepatitis C virus. In other cases, the IRES sequence may be derived from encephalomyocarditis virus. The term "EMCV" or "encephalomyocarditis virus" as used herein refers to any member of an isolate or strain of the encephalomyocarditis virus species of the genus Picornaviridae family. Examples: EMCV strain virus - R (Rueckert), Columbia virus - SK. In some cases, a cellular IRES element may be used, such as eukaryotic initiation factor 4G, immunoglobulin heavy chain binding protein, proto-oncogene c - myc, vascular endothelial growth factor, fibroblast growth factor - 1 IRES, or any combination or modification thereof. In some cases, a cellular IRES may have increased gene expression compared to a viral IRES.

[205] The IRES sequence of a virus, a cell, or a combination of both can be used in a vector. The IRES can be from encephalomyocarditis virus (EMCV) or poliovirus (PV). In some cases, the IRES element is selected from the group consisting of elements from poliovirus (PV), encephalomyelitis virus (EMCV), foot-and-mouth disease virus (FMDV), porcine teschovirus - 1 (PTV - 1), aichivirus (AiV), Seneca Valley virus (SVV), hepatitis C virus (HCV), classical swine fever virus (CSFV), human immunodeficiency virus - 2 (HIV - 2), human immunodeficiency virus - 1 (HIV - 1), Moloney murine leukemia virus (MoMLV), feline immunodeficiency virus (FIV), mouse mammary tumor virus (MMTV), human latent cytomegalovirus (pUL138), Epstein - Barr virus (EBNA - 1), herpes virus, Marek's disease (MDV RLORF9), SV40 polycistronic 19S (SV40 19S), Rhopalosiphum padi (RhPV), cricket paralysis virus (CrPV), Ectropis obliqua picorna - like virus (EoPV), Plautia stali intestinal virus (PSIV), triatoma virus (TrV), bee paralysis dicistrovirus (IAPV, KBV), blackcurrant reversion virus (BRV), pelargonium flower burst virus (PFBV), hibiscus chlorotic ringspot virus (HCRSV), cruciferous tobamovirus (CrTMV), potato leafroll polerovirus (PLRV), tobacco etch virus (TEV), Giardiavirus (GLV), leishmania RNA virus - 1 (LRV - 1) and combinations or modifications thereof. In some cases, the IRES is selected from the group consisting of Apaf - 1, XIAP, HIAP2/c - IAP1, DAP5, Bcl - 2, c - myc, CAT - 1, INR, LEF differentiation - 1, PDGF2, HIF - 1a, VEGF, FGF2, BiP, BAG - 1, CIRP, p53, SHMT1, PITSLREp58, CDK1, Rpr, hid, hsp70, grim, skl, Antennapedia, dFoxO, dInR, Adh - Adhr, HSP101, ADH, URE - 2, GPR1, NCE102, YMR181a, MSN1, BOI1, FLO8, GIC1 and any combination or modification thereof. When an IRES element is included between two open reading frames (ORFs), translation initiation can occur via the canonical 5' - m7GpppN - dependent cap - dependent mechanism in the first ORF and a cap - independent mechanism in the second ORF downstream of the IRES element.

[206] In some cases, genes may be linked by an internal ribosome entry site (IRES). An IRES may allow for the simultaneous expression of multiple genes. For example, an IRES sequence may mediate the production of multiple proteins from a single mRNA transcript. The ribosome can bind to the IRES in a 5' cap - independent manner and initiate translation.

[207] In some cases, the IRES sequence may be approximately 500 base pairs long. An IRES sequence may contain between 300 base pairs and 1000 base pairs. For example, an IRES may be 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 base pairs long.

[208] In some cases, the expression of a downstream gene in a vector containing an IRES sequence may be reduced. For example, a gene following an IRES sequence may have reduced expression compared to a gene preceding the IRES sequence. The reduction in expression can range from 1% to 99% of the reduction compared to the preceding gene.

Methods of expression regulation

[209] In one embodiment, a method for regulating the expression of a heterologous gene in an engineered cell is provided. Polynucleotides encoding gene switch polypeptides for ligand-inducible control of the expression of a heterologous gene, an antigen-binding polypeptide, and a heterologous gene are provided. In some cases, the polynucleotides are in one or more gene expression cassettes as shown in any of Figs. 1 to 16. In another case, the polynucleotides are introduced into the engineered cell using viral or non-viral vectors. Viral vectors may include lentiviral vectors, retroviral vectors, or adenoviral vectors. Non-viral vectors may include Sleeping Beauty transposons. In other cases, the polynucleotides are introduced into the engineered cell using recombinases or gene editing methods. Examples of recombinases include serine recombinases, as described herein. Examples of gene editing methods may include CRISPR or Argonaute systems. As used herein, "CRISPR gene editing system" or "CRISPR system" refers to any Cas protein-mediated, RNA-guided process for targeting DNA sequence changes to a specific region of the genome. As used herein, "Argonaute gene editing system" refers to any Argonaute endonuclease-mediated, single-stranded DNA - guided process for targeting DNA sequence changes to a specific region of the genome.

Pharmaceutical compositions and dosage

[210] This disclosure provides a composition comprising an adenovirus or adenoviral vector described herein and a carrier ( e.g. , a pharmaceutically acceptable carrier) thereof. The composition is desirably a physiologically acceptable (e.g., pharmaceutically acceptable) composition that comprises a carrier, preferably a physiologically ( e.g. , pharmaceutically) acceptable carrier, and an adenovirus or adenoviral vector. Any suitable carrier can be used in the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined in part by the specific use of the composition (e.g., administration to an animal) and the specific method used to administer the composition. Ideally, in the context of replication - deficient adenoviral vectors, the pharmaceutical composition preferably does not contain replication - competent adenovirus. The pharmaceutical composition may optionally be sterile.

[211] Suitable compositions include aqueous and non-aqueous isotonic sterile solutions which may contain antioxidants, buffers and bacteriostats, and aqueous and non-aqueous sterile suspensions which may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. The composition may be presented in single-dose or multi-dose sealed containers such as ampoules and vials and may be stored in a lyophilized (lyophilized) state requiring only the addition of a sterile liquid carrier, such as water, immediately before use. Solutions and suspensions for extemporaneous use can be prepared from sterile powders, granules and tablets. Preferably, the carrier is a buffered saline solution. More preferably, the adenovirus or adenoviral vector is part of a composition formulated to protect the adenovirus or adenoviral vector from damage prior to administration. For example, the composition can be formulated to reduce the loss of adenovirus or adenoviral vector on devices used to produce, store, or administer the adenovirus or adenoviral vector, such as glassware, syringes, or needles. The composition can be formulated to reduce the photosensitivity and/or temperature sensitivity of the adenovirus or adenoviral vector. For this purpose, the composition preferably comprises a pharmaceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of polysorbate 80, L - arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such a composition will extend the shelf life of the adenovirus or adenoviral vector and facilitate its administration. Formulations of compositions containing an adenovirus or an adenovirus vector are further described, for example, in U.S. Patent 6,225,289, U.S. Patent 6,514,943, and International Patent Application Publication WO 2000/034444.

[212] The composition may also be formulated to enhance transduction efficiency. Furthermore, one skilled in the art will appreciate that the adenovirus or adenoviral vector may be present in a composition with other therapeutic or biologically active agents. For example, inflammation-controlling factors such as ibuprofen or steroids may be part of the composition to reduce swelling and inflammation associated with in vivo administration of the adenovirus or adenoviral vector. If the adenovirus or adenoviral vector is used to deliver a nucleic acid sequence encoding an antigen to the host, immune system stimulants or adjuvants, such as interleukins, lipopolysaccharide, or double-stranded RNA, may be administered to enhance or modify any immune response to the antigen. Antibiotics, i.e., microbicides and fungicides, may be present to treat an existing infection and/or reduce the risk of a future infection, such as an infection associated with gene transfer procedures.

[213] In some embodiments, compositions comprising a polynucleotide or polypeptide disclosed herein are disclosed herein for administration to a subject. In some cases, these are modified effector cell compositions encoding a polynucleotide or polypeptide disclosed herein and optionally comprising a cytokine and/or an additional therapeutic agent. In some cases, vectors encoding gene switch polypeptides for regulating the expression of a chimeric antigen receptor for modifying an effector cell are also included.

[214] In some cases, pharmaceutical compositions of the modified effector cell or vector encoding gene switch polypeptides and a chimeric antigen receptor are prepared in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries that facilitate processing of the active compounds into preparations suitable for pharmaceutical purposes. The proper formulation depends on the chosen route of administration. Summaries of the pharmaceutical compositions described herein can be found in, for example, Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[215] The pharmaceutical compositions are optionally prepared in a conventional manner such as, by way of example only, by conventional mixing, dissolving, granulating, drageeing, shaking, emulsifying, encapsulating, entrapping or pressing techniques.

[216] In some embodiments, the compositions may also include one or more pH adjusting or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric, and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris - hydroxymethylaminomethane; and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers are included in an amount necessary to maintain the pH of the composition within an acceptable range.

[217] In other embodiments, the compositions may also include one or more salts in an amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include salts containing sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[218] The pharmaceutical compositions described herein are administered by any suitable route, including, but not limited to, oral, parenteral ( e.g. , intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial), intranasal, buccal, sublingual, or rectal administration. In some cases, the pharmaceutical composition is prepared for parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, or intracranial) administration.

[219] The pharmaceutical compositions described herein are prepared in any suitable dosage form, including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like dosage forms, aerosols, controlled release formulations, fast melting formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations. In some embodiments, the pharmaceutical compositions are prepared as capsules. In some embodiments, the pharmaceutical compositions are prepared as solutions (e.g., for intravenous administration). In some cases, the pharmaceutical composition is prepared as an infusion. In some cases, the pharmaceutical composition is prepared as an injection.

[220] The pharmaceutical solid dosage forms described herein optionally include a compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filler, suspending agent, flavoring agent, sweetener, disintegrating agent, dispersing agent, surfactant , lubricant, colorant, diluent, solubilizer, wetting agent, plasticizer, stabilizer, penetration enhancer, wetting agent, antifoaming agent, antioxidant, preservative, or one or more combinations thereof.

[221] In other aspects, using standard coating procedures, such as those described in Remington's Pharmaceutical Sciences, 20th Edition (2000), the compositions are film-coated. In some embodiments, the compositions are formulated as particles (e.g., for administration as capsules) and some or all of the particles are coated. In some embodiments, the compositions are formulated as particles (e.g., for administration as capsules) and some or all of the particles are microencapsulated. In some embodiments, the compositions are formulated as particles (e.g., for administration as capsules) and some or all of the particles are not microencapsulated or coated.

[222] In some embodiments, the compositions provided herein may also include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merphen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[223] "Defoamers" reduce foaming during processing, which can lead to coagulation of aqueous dispersions, formation of bubbles in the finished film, or generally poor processing. Examples of defoamers include silicone emulsions or sorbitan sescoleate.

[224] "Antioxidants" include, for example, butylated hydroxytoluene (BHT), sodium ascorbate, ascorbic acid, sodium metabisulfite, and tocopherol. In some embodiments, antioxidants enhance chemical stability where needed.

[225] The compositions described herein can be enhanced with antioxidants, metal chelating agents, thiol-containing compounds, and other general-purpose stabilizers. Examples of such stabilizers include, but are not limited to: (a) from about 0.5% to about 2% w/v glycerol, (b) from about 0.1% to about 1% w/v methionine, (c) from about 0.1% to about 2% w/v monothioglycerol, (d) from about 1 mM to about 10 mM EDTA, (e) from about 0.01% to about 2% w/v ascorbic acid, (f) from about 0.003% to about 0.02% w/v polysorbate 80, (g) about 0.001% to about 0.05% w/v polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[226] "Binding" agents impart cohesive qualities and include, for example, alginic acid and its salts; cellulose derivatives such as carboxymethylcellulose, methylcellulose (e.g., Methocel®), hydroxypropylmethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose (e.g., Klucel®), ethylcellulose (e.g., Ethocel®), and microcrystalline cellulose (e.g., Avicel®); microcrystalline dextrose; amylose; magnesium aluminum silicate; acid polysaccharides; bentonites; gelatin; polyvinylpyrrolidone/vinyl acetate copolymer; crospovidone; povidone; starch; pregelatinized starch; Tragacanth, dextrin, sugar such as sucrose (e.g. Dipac®), glucose, dextrose, molasses, mannitol, sorbitol, xylitol (e.g. Xylitab®) and lactose; natural or synthetic gum such as acacia, tragacanth, gum ghatti, isapol husk mucilage, polyvinylpyrrolidone (e.g. Polyvidone® CL, Kollidon® CL, Polyplasdone® XL - 10), larch arabogalactan, Veegum®, polyethyleneglycol, waxes, sodium alginate and the like.

[227] "Carrier" or " carrier materials" include any excipients commonly used in pharmaceuticals and should be selected based on compatibility with the compounds disclosed herein, such as ibrutinib and anticancer agent compounds, and the release profile properties of the desired dosage form. Examples of carrier materials include, for example, binders, suspending agents, disintegrants, fillers, surfactants , solubilizers, stabilizers, lubricants, wetting agents, diluents, and the like. "Pharmaceutically compatible carrier materials" may include, but are not limited to, gum arabic, gelatin, colloidal silicon dioxide, calcium glycerophosphate, calcium lactate, maltodextrin, glycerin, magnesium silicate, polyvinylpyrrolidone (PVP), cholesterol, cholesterol esters, sodium caseinate, soy lecithin, taurocholic acid, phosphatidylcholine, sodium chloride, tricalcium phosphate, dipotassium phosphate, cellulose and cellulose conjugates, sugars, sodium stearoyl lactylate, carrageenan, monoglyceride, diglyceride, pregelatinized starch, and the like. See, e.g., Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins1999).

[228] "Dispersing agents" and/or "viscosity modulating agents" include materials that control the diffusion and homogeneity of the drug through liquid media or a granulation process or a mixing process. In some embodiments, these agents also enhance the effectiveness of the coating or disruptive matrix. Examples of diffusion facilitating/dispersing agents include, for example, hydrophilic polymers, electrolytes, Tween® 60 or 80, PEG, polyvinylpyrrolidone (PVP; commercially known as Plasdone®), and carbohydrate-based dispersing agents such as, for example, hydroxypropyl celluloses (e.g., HPC, HPC - SL, and HPC - L), hydroxypropyl methylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M), sodium carboxymethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate stearate (HPMCAS), non-crystalline cellulose, magnesium aluminum silicate, triethanolamine, polyvinyl alcohol (PVA), copolymer vinylpyrrolidone/vinyl acetate (S630), polymer of 4- (1,1,3,3 - tetramethylbutyl)phenol with ethylene oxide and formaldehyde (also known as tyloxapol), poloxamers (e.g. Pluronics F68®, F88® and F108®, which are block copolymers of ethylene oxide and propylene oxide); and poloxamines (e.g., Tetronic 908®, also known as Poloxamine 908®, which is a tetrafunctional block copolymer prepared by the sequential addition of propylene oxide and ethylene oxide to ethylenediamine (BASF Corporation, Parsippany, NJ )), polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer (S - 630), polyethylene glycol, e.g., polyethylene glycol may have a molecular weight of from about 300 to about 6,000, or from about 3,350 to about 4,000, or from about 7,000 to about 5,400, sodium carboxymethylcellulose, methylcellulose, polysorbate 80, sodium alginate, gums such such as gum tragacanth, acacia, guar gum, xanthans including xanthan gum, sugars, celluloses such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, polysorbate 80 , sodium alginate, polyethoxylated sorbitan monolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates, chitosans, and combinations thereof. Plasticizers such as cellulose or triethylcellulose may also be used as dispersing agents. Dispersing agents particularly useful in liposomal dispersions and self-emulsifying dispersions include dimyristoylphosphatidylcholine, natural phosphatidylcholine from eggs, natural phosphatidylglycerol from eggs, cholesterol, and isopropyl myristate.

[229] Combinations of one or more erosion aids with one or more diffusion aids may also be used in the compositions of the present invention.

[230] The term "diluent" refers to chemical compounds that are used to dilute the compound of interest prior to delivery. Diluents can also be used to stabilize compounds because they can provide a more stable environment. Salts dissolved in buffered solutions (which can also provide pH control or maintenance) are used as diluents in the art, including, but not limited to, phosphate buffered saline. In some embodiments, diluents increase the volume of the composition to facilitate compression or to create sufficient volume for a homogeneous mixture for filling into capsules. Such compounds include, for example, lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose such as Avicel®; dibasic calcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate, calcium phosphate; anhydrous lactose, spray-dried lactose; pregelatinized starch, compressible sugar such as Di - Pac® (Amstar); Mannitol, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose acetate stearate, sucrose-based diluents, confectioners'sugar; calcium sulfate monobasic monohydrate, calcium sulfate dihydrate; calcium lactate trihydrate, dextrates; hydrolyzed cereal solids, amylose; cellulose powder, calcium carbonate; glycine, kaolin; mannitol, sodium chloride; inositol, bentonite, etc.

[231] "Fillers" include compounds such as lactose, calcium carbonate, calcium phosphate, dibasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrose, dextrates, dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride, polyethylene glycol, etc.

[232] "Lubricants" and "glidants" are compounds that prevent, reduce, or inhibit the sticking or friction of materials. Examples of lubricants include, for example, stearic acid, calcium hydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineral oil or hydrogenated vegetable oil such as hydrogenated soybean oil (Sterotex®), higher fatty acids and their alkali metal and alkaline earth metal salts such as aluminum, calcium, magnesium, zinc, stearic acid, sodium stearates, glycerol, talc, waxes, Stearowet®, boric acid, sodium benzoate, sodium acetate, sodium chloride, leucine, polyethylene glycol (e.g., PEG - 4000) or methoxypolyethylene glycol such as Carbowax™, sodium oleate, sodium benzoate, glycerol behenate, polyethylene glycol, magnesium or sodium lauryl sulfate, colloidal silicon dioxide such as Syloid™, Cab - O - Sil®, starch such as corn starch, silicone oil, surfactant , etc.

[233] "Plasticizers" are compounds used to soften the material of microcapsules or film coatings to make them less brittle. Suitable plasticizers include, for example, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG 1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid, triethylcellulose, and triacetin. In some embodiments, plasticizers can also act as dispersing agents or wetting agents.

[234] “Solubilizers” include compounds such as triacetin, triethyl citrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate, sodium docusate, vitamin E TPGS, dimethylacetamide, N - methylpyrrolidone, N - hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, n - butanol, isopropyl alcohol, cholesterol, bile salts, polyethylene glycol 200-600 , glycofurol, transcutol, propylene glycol and dimethyl isosorbide, etc.

[235] "Stabilizers" include compounds such as any antioxidants, buffers, acids, preservatives, etc.

[236] “Suspending agents” include compounds such as polyvinylpyrrolidone, such as polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, or polyvinylpyrrolidone K30, vinylpyrrolidone/vinyl acetate copolymer (S630), polyethylene glycol, such as polyethylene glycol may have a molecular weight of about 300 to 6,000, or about 3,350 to 4,000, or about 7,000 to 5,400, sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, hydroxymethylcellulose acetate stearate, polysorbate 80 , hydroxyethylcellulose, sodium alginate, gums such as, for example, gum tragacanth and gum acacia, guar gum, xanthans including xanthan gum, sugars, celluloses such as sodium carboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxyethylcellulose, polysorbate 80 , sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitan monolaurate, povidone and the like.

[237] " Surfactants " include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, ethylene oxide-propylene oxide copolymers such as Pluronic® (BASF), and the like. Some other surfactants include polyoxyethylene fatty acid glycerides and vegetable oils such as polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkyl ethers and alkyl phenyl ethers such as octoxynol 10, octoxynol 40. In some embodiments , surfactants may be included to enhance physical stability or for other purposes.

[238] “Viscosity enhancers” include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetate stearate, hydroxypropylmethylcellulose phthalate, carbomer, polyvinyl alcohol, alginates, gum arabic, chitosans, and combinations thereof.

[239] "Wetting agents" include compounds such as oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate, sodium lauryl sulfate, sodium docusate, triacetin, Tween 80, vitamin E TPGS, ammonium salts, and the like.

Kits/final products

[240] In some embodiments, kits and articles for use with one or more of the methods described herein are disclosed herein. Such kits include a carrier, package, or container divided into compartments to accommodate one or more containers, such as vials, test tubes, and the like, wherein each of the containers contains one of the individual elements used in the method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are made of various materials, such as glass or plastic.

[241] The articles provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, vials, tubes, bags, containers, bottles, and any packaging material suitable for the selected formulation and the intended route of administration and treatment.

[242] The kit typically includes labels listing the contents and/or instructions for use, as well as package inserts containing instructions for use. An instruction set will usually also be included.

[243] In some embodiments, the label is on or associated with the container. In one embodiment, the label is on the container when the letters, numbers, or other symbols that form the label are affixed, molded, or engraved on the container itself; the label is associated with the container when it is present in a vessel or carrier that also contains the container, such as in the form of a package insert. In one embodiment, the label is used to indicate that the contents are to be used for a specific therapeutic application. The label also provides directions for using the contents, such as in the methods described herein.

Bioinformatic antigenicity workflow for HPV vaccine development

Identification of new antigenic components of HPV

[244] Based on the important roles that the HPV E2 and E4 genetic components play in essential HPV functions, the arrangement of the corresponding proteins, and in silico prediction, E2- and E4-derived antigens were identified for a therapeutic HPV vaccine. See Fig. 1. Non-oncogenic and viral inactivation genetic modifications have also been used to eliminate the viral and oncogenic biological activities of HPV proteins, such as the HPV E2 and E6 proteins. Genetic manipulation has also been used to produce protein sequences rearranged in a way that retains the immunogenic properties of the peptides but eliminates the oncogenic and viral amplification functions of E7 and E4, respectively.

[245] Thus, the innovative aspects of the five constructs presented in this specification include: (1) the use of gene constructs encoding fusion proteins comprising four or more different HPV proteins; (2) a combination of amino acid point mutations and polypeptide sequence shuffling techniques to inactivate oncogenic and essential viral functions; (3) the incorporation of HPV proteins containing multiple antigenic components from HPV proteins that are highly expressed in infected host cells; (4) the first known hybrid antigen constructs; (5) the fusion of epitopes from high- and low-risk HPV strains; (6) the use of mixed and regularly repeated linkers; (7) the use of rigid linkers to stabilize polypeptide subunits and prevent unwanted intramolecular interactions; (8) the use of cleavable linkers between epitopes; (9) dual use of the linker sequence to provide the function of both a protein - protein linker ( - ) and antigens and epitopes as such (i.e., antigenicity conferred by linker sequences).

Table 4: Assessment in - silico Design No. Antigenicity Allergenicity Cross-reactivity with the host cell Virus Tumor 1 0.5288 0.6134 Non-allergenic No hits 2 0.5252 0.5814 Non-allergenic No hits 3 0.5587 0.6322 Non-allergenic No hits 4 0.5451 0.5809 Non-allergenic No hits 5 0.4964 0.5997 Non-allergenic No hits

[246] Antigenicity is the ability to stimulate the production of antibodies or cell - mediated immune responses. The antigenicity of the final design sequences was predicted using Vaxjen software, which is an alignment-independent antigen recognition model based on the basic chemical properties of amino acid sequences. The results indicate that five antigenic sequences are antigenic. See Table 4 (antigenicity/virus and tumor).

[247] Allergens are small antigens that typically elicit an antibody response. Allergenicity, whether an antigen is allergenic or non-allergenic, was predicted using ALLERTOP, a bioinformatics-based allergen prediction software with machine learning methods for classification. It includes logistic regression (LR), decision tree (DT), naive Bayes (NB), random forest (RF), multilayer perceptron (MLP), and k-nearest neighbors (kNN). The results indicate that five antigen sequences are non-allergenic. See Table 4 (Allergenicity).

[248] Cross-reactivity or the occurrence of autoimmune side effects in different tissues has important safety implications for adoptive immunotherapy. Sequence homology analyses were performed to assess whether these new antigens were cross-reactive with the human proteome using blast search, a basic local alignment search tool. No cross-reactivity in host cells was detected in these five antigen sequences. See Table 4 (cross-reactivity in host cells).

Software/Tools:

[249] The software tools used to carry out the projects described in this document include:

ALLERTOP (see 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 (Basic Local Alignment Search Tool; NCBI, National Center for Biotechnology Information, U.S. 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 V11.1.5 ( see genious.com/biopharma/)

IEDB CONSENSUS ( see 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.)

NETMHCPAN 4.0 ( see 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 ( see The Phyre2 web portal for protein modeling, prediction and analysis; 07 May 2015; nature.com/articles/nprot.2015.053; doi.org/10.1038/nprot.2015.053.)

PYMOL V2.1.1 MOLECULAR GRAPHICS SYSTEM ( see pymol.org/2/; sourceforge.net/projects/pymol/support)

VAXJEN ( see VaxiJen: a server for prediction of protective antigens, tumor antigens and subunit vaccines ; BMC Bioinformatics. 2007 Jan 5;8:4.)

Table 5: Nomenclature of antigen designs and components Symbol of the designated component SEQ ID NO
and Sequence Type Descriptive title Sequence type A 1
AA HPV6 E2* Amino acid 2
DNA HPV6 E2* Nucleic acid B 3
AA HPV6 E4 1-56 Amino acid 4
DNA HPV6 E4 1-56 Nucleic acid C 5
AA HPV6 E7 70-98 Amino acid 6
DNA HPV6 E7 70-98 Nucleic acid D 7
AA HPV6 E4 36-90 Amino acid 8
DNA HPV6 E4 36-90 Nucleic acid E 9
AA HPV6 E7 1-58 Amino acid 10
DNA HPV6 E7 1-58 Nucleic acid F 11
AA HPV6 E6* Amino acid 12
DNA HPV6 E6* Nucleic acid G 13
AA HPV6 E2* 1-368 Del_206-306 Amino acid 14
DNA HPV6 E2* 1-368 Del_206-306 Nucleic acid H 15
AA HPV6 E4 1-70 Del_21-50 Amino acid 16
DNA HPV6 E4 1-70 Del_21-50 Nucleic acid I 17
AA HPV6 E4 51-70 Amino acid 18
DNA HPV6 E4 51-70 Nucleic acid 19
DNA HPV6 E4 51-70 Nucleic acid J 20
AA HPV6 E6* 1-120 Del C-end Amino acid 21
DNA HPV6 E6* 1-120 Del C-end Nucleic acid K 22
AA HPV6 E7 1-98 Del_40-75 Amino acid 23
DNA HPV6 E7 1-98 Del_40-75 Nucleic acid L 24
AA HPV6 E2* 1-205 Amino acid 25
DNA HPV6 E2* 1-205 Nucleic acid M 26
AA HPV6 E4 1-20 Amino acid 27
DNA HPV6 E4 1-20 Nucleic acid N 28
AA HPV6 E2* 307-368 Amino acid 29
DNA HPV6 E2* 307-368 Nucleic acid O 30
AA HPV6 E4 Epitope No. 1 Amino acid 31
DNA HPV6 E4 Epitope No. 1 Nucleic acid P 32
AA HPV6 E4 Epitope No. 2 Amino acid 33
DNA HPV6 E4 Epitope No. 2 Nucleic acid L1 34
AA Hard linker Amino acid 35
DNA Hard linker Nucleic acid 36
DNA Hard linker Nucleic acid 37
DNA Hard linker Nucleic acid 38
DNA Hard linker Nucleic acid 39
DNA Hard linker Nucleic acid L2 40
AA HPV6 E6 epitope linker Amino acid 41
DNA HPV6 E6 epitope linker Nucleic acid L3 42
AA HPV11 E6 epitope linker Amino acid 43
DNA HPV11 E6 epitope linker Nucleic acid 44
DNA HPV11 E6 epitope linker Nucleic acid L4 45
AA HPV11 E7 epitope linker Amino acid 46
DNA HPV11 E7 epitope linker Nucleic acid 47
DNA HPV11 E7 epitope linker Nucleic acid L5 48
AA HPV16 E6 Agonist Booster No. 1 Amino acid 49
DNA HPV16 E6 Agonist Booster No. 1 Nucleic acid L6 50
AA HPV16 E6 Agonist Booster No. 2 Amino acid 51
DNA HPV16 E6 Agonist Booster No. 2 Nucleic acid L7 52
AA HPV16 E7 agonist booster Amino acid 53
DNA HPV16 E7 agonist booster Nucleic acid 54
DNA HPV16 E7 agonist booster Nucleic acid 55
DNA HPV16 E7 agonist booster Nucleic acid 56
DNA HPV16 E7 agonist booster Nucleic acid L8 57
AA Flexible linker Amino acid 58
DNA Flexible linker Nucleic acid 59
DNA Flexible linker Nucleic acid 60
DNA Flexible linker Nucleic acid 61
DNA Flexible linker Nucleic acid 62
DNA Flexible linker Nucleic acid 63
DNA Flexible linker Nucleic acid 64
DNA Flexible linker Nucleic acid 65
DNA Flexible linker Nucleic acid D1 66
AA Design No. 1 Amino acid 67
DNA Design No. 1 Nucleic acid D2 68
AA Design No. 2 Amino acid 69
DNA Design No. 2 Nucleic acid D3 70
AA Design No. 3 Amino acid 71
DNA Design No. 3 Nucleic acid D4 72
AA Design No. 4 Amino acid 73
DNA Design No. 4 Nucleic acid D5 74
AA Design No. 5 Amino acid 75
DNA Design No. 5 Nucleic acid L9 76
AA Rigid linker polypeptide Amino acid 77
DNA Rigid linker DNA Nucleic acid 78
DNA Rigid linker DNA Nucleic acid 79
DNA Rigid linker DNA Nucleic acid 80
DNA Rigid linker DNA Nucleic acid 81
DNA Rigid linker DNA Nucleic acid

Additional embodiments of the invention

[250] Embodiments (“ E ”) of the present invention include, but are not limited to:

E1 . A polynucleotide encoding a non-naturally occurring polypeptide comprising at least one epitope derived from a low-risk human papillomavirus (HPV) protein and at least one epitope derived from a high-risk HPV protein.

E2 . A polynucleotide encoding a non-naturally occurring polypeptide comprising at least one epitope derived from human papillomavirus 6 (HPV6) and at least one epitope derived from human papillomavirus 11 (HPV11).

E3 . An E2 polynucleotide in which the non-naturally occurring polypeptide further comprises at least one epitope derived from human papillomavirus 16 (HPV16).

E4 . A polynucleotide encoding a non-naturally occurring polypeptide containing HPV epitopes derived from any four of:

HPV6 E2 protein;

HPV6 E4 protein;

HPV6 E6 protein;

HPV6 E7 protein;

HPV11 E6 protein; and/or

HPV11 E7 protein;

wherein the polypeptide comprises at least one HPV6 epitope and at least one HPV11 epitope.

E5 . An E4 polynucleotide in which the non-naturally occurring polypeptide further comprises at least one epitope derived from HPV16 proteins.

E6 . A polynucleotide encoding a non-naturally occurring polypeptide containing HPV epitopes derived from any three of:

HPV6 E2 protein;

HPV6 E4 protein;

HPV6 E6 protein; or

HPV6 E7 protein;

Moreover, HPV epitopes are linked by amino acid sequences that are not naturally encoded by the HPV genome.

E7 . A polynucleotide encoding a non-naturally occurring polypeptide containing HPV epitopes derived from each of:

HPV6 E2 protein;

HPV6 E4 protein;

HPV6 E6 protein; and

HPV6 E7 protein,

Moreover, HPV epitopes are linked by amino acid sequences that are not naturally encoded by the HPV genome.

E8 . An E6 or E7 polynucleotide in which the amino acid sequence binding at least two HPV epitopes comprises the sequence of SEQ ID NO: 34.

E9 . A polynucleotide encoding a non-naturally occurring polypeptide, wherein the non-naturally occurring polypeptide contains HPV epitopes derived from:

a) HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV6 E7 protein, HPV11 E6 protein and HPV11 E7 protein; or,

b) HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV6 E7 protein, HPV11 E6 protein, HPV11 E7 protein; HPV16 E6 protein and HPV16 E7 protein.

E10 . The polynucleotide of E9 , further comprising one or more polynucleotide sequences encoding one or more polypeptide linkers, wherein the one or more linkers comprise the amino acid sequence:

a) SEQ ID NO: 45;

b) SEQ ID NO: 42;

c) SEQ ID NO: 40;

d) SEQ ID NO: 34;

e) SEQ ID NO: 52;

f) SEQ ID NO: 48;

g) SEQ ID NO: 50; and/or,

h) SEQ ID NO: 57.

E11 . A polynucleotide encoding a non-naturally occurring polypeptide, wherein the non-naturally occurring polypeptide contains HPV epitopes derived from:

a) HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV6 E7 protein, HPV11 E6 protein and HPV11 E7 protein, wherein the polypeptide further comprises at least one linker sequence of SEQ ID NO: 45, SEQ ID NO: 42, or SEQ ID NO: 40;

b) HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV6 E7 protein, HPV11 E6 protein and HPV11 E7 protein, wherein the polypeptide further comprises at least one linker sequence of SEQ ID NO: 34;

c) HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV11 E6 protein, HPV11 E7 protein, HPV16 E6 protein; HPV16 E7 protein, wherein the polypeptide further comprises at least one linker sequence of SEQ ID NO: 52, SEQ ID NO: 48, or SEQ ID NO: 50; or,

d) HPV6 E2 protein, HPV6 E4 protein, HPV6 E6 protein, HPV11 E6 protein, HPV11 E7 protein, HPV16 E6 protein; and HPV16 E7 protein, wherein the polypeptide further comprises at least one linker sequence of SEQ ID NO: 57.

E12. A polynucleotide comprising a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical:

a) SEQ ID NO: 67;

b) SEQ ID NO: 69;

c) SEQ ID NO: 71;

d) SEQ ID NO: 73; or,

e) SEQ ID NO: 75.

E13 . A polynucleotide containing a sequence encoding a non-naturally occurring polypeptide containing an amino acid sequence at least 80% identical to:

a) SEQ ID NO: 66;

b) SEQ ID NO: 68;

c) SEQ ID NO: 70;

d) SEQ ID NO: 72; or,

e) SEQ ID NO: 74.

E14 . Polynucleotide E13 , in which the amino acid sequence is at least 85% identical, at least 90% identical, at least 95% identical, at least 98% identical, or 100% identical to any of a), b), c), d) or e).

E15 . A polynucleotide according to any one of items E1 to E14 , wherein one or more HPV epitopes comprise amino acid substitutions, amino acid insertions, amino acid sequence deletions, amino acid sequence separations, or amino acid sequence rearrangements compared to the corresponding wild-type HPV sequences, wherein said amino acid sequence substitutions, insertions, deletions, separations, or rearrangements abolish the natural oncogenic biological activity of HPV or essential viral replication functions.

E16 . The polynucleotide of any one of paragraphs E1-E15 , wherein said polynucleotide is useful for generating an immune response in vivo to HPV6 or HPV11 antigens in a mammal, or wherein said polynucleotide is useful for generating an immune response in vivo to HPV6 and HPV11 antigens in a mammal.

E17 . A polypeptide encoded by a polynucleotide according to any one of paragraphs E1-E16 .

E18 . A vector encoding a polynucleotide according to any one of paragraphs E1-E16.

E19 . The vector according to E18 , wherein said vector is a plasmid, a viral vector or a non-viral vector.

E20 . The vector according to E19 , wherein said viral vector is an adenoviral vector.

E21 . The vector according to E20 , wherein said adenoviral vector is a viral vector of a non-human primate.

E22 . A composition or vaccine comprising a pharmaceutically acceptable carrier and a polynucleotide according to any one of paragraphs E1-E16 , a polypeptide according to paragraph E17 or a vector according to paragraphs E18-E21 .

E23 . A method for creating a composition, vaccine, or HPV6 and HPV11 polynucleotide encoding a non-naturally occurring polypeptide for use in the composition or vaccine, comprising engineering HPV polynucleotide sequences to encode derivatives of naturally occurring HPV polypeptides, wherein at least one of the viral polypeptides has oncogenic biological activity or an essential viral replication function, wherein a combination of one or more amino acid sequence substitutions, deletions, insertions, divisions, or rearrangements is expressed through the composition, vaccine, or encoded by the polynucleotide to form the derivative viral polypeptides, wherein the oncogenic biological activity of HPV or the essential HPV replication function is eliminated from the derivative viral polypeptides, but wherein the host immunoreactivity and/or immunogenic activity towards the derivative viral polypeptides is maintained or enhanced compared to the corresponding naturally occurring viral polypeptides.

E24 . The method of claim E23 , wherein the polynucleotide or viral polypeptide derivative is constructed using steps comprising identifying viral antigens in combination with any two or more of: in silico prediction of in vivo antigen epitope recognition, in silico prediction of in vivo allergen recognition, sequence homology analysis of human proteome cross-reactivity, analysis of physicochemical properties of amino acid sequence, analysis of three-dimensional (3D) structure, identification of oncogenic and/or viral inactivating mutations, deletions, substitutions and/or rearrangements.

E25 . The method according to paragraph E24 , further comprising assessing in vitro or in vivo the immunogenicity, immunoprotective effects, oncogenicity or viral function of the derivatives of viral polypeptides.

E26 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more components from Table 5.

E27 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises sequences of any two or more of components A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, or P of Table 5.

E28 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any one or more of components L1, L2, L3, L4, L5, L6, L7, L8, or L9 from Table 5.

E29 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more components A, B, C, D, E, or F from Table 5.

E30 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more components A, B, C, D, E, F, L1, L2, L3 or L4 from Table 5.

E31 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more components A, B, C, D, E, F or L1 from Table 5.

E32 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more of components A, B, C, D, E, F, L2, L3, or L4 from Table 5.

E33 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components A, B, C, D, E, F, and L1 from Table 5.

E34 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components A, B, C, D, E, F, L2, L3, and L4 from Table 5.

E35 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more of the G, H, I, J, or K components from Table 5.

E36 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more of the components G, H, I, J, K, L1, L2, or L4 from Table 5.

E37 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises components of the G, H, I, J, and K sequences from Table 5.

E38 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components G, H, I, J, K, L1, L2, and L4 from Table 5.

E39 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more components I, L, M, or N from Table 5.

E40 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more of components I, L, M, N, L2, L4, L5, L6, or L7 from Table 5.

E41 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components I, L, M, and N from Table 5.

E42 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components I, L, M, N, L2, L4, L5, L6, and L7 from Table 5.

E43 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises sequences of any two or more N, O, or P components from Table 5.

E44 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of any two or more of the components N, O, P, L2, L3, L4, L5, L6, L7, or L8 from Table 5.

E45 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the N, O, and P component sequences from Table 5.

E46 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components N, O, P, L2, L3, L4, L5, L6, L7, and L8 from Table 5.

E47 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components A, B, C, D, E, F, and L1 from Table 5 in any order.

E48 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises, in sequential order, the sequence components A, L1, B, L1, C, L1, D, L1, E, L1, and F of Table 5.

E49 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components A, B, C, D, E, F, L2, L3, and L4 from Table 5 in any order.

E50 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises, in sequential order, the sequence components A, L4, B, L3, C, L2, D, L3, E, L4, and F of Table 5.

E51 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components G, H, I, J, K, L1, L2, and L4 from Table 5 in any order.

E52 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises, in sequential order, the sequence components G, L1, L2, L1, H, L1, L4, L1, I, L1, J, L1, and K from Table 5.

E53 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components I, L, M, N, L2, L4, L5, L6, and L7 from Table 5 in any order.

E54 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises, in sequential order, the sequence components L, L7, L2, L7, M, L5, L4, L6, I, L7, L2, L7, and N of Table 5.

E55 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises the sequences of components N, O, P, L2, L3, L4, L5, L6, L7, and L8 from Table 5 in any order.

E56 . A non-naturally occurring polypeptide or polynucleotide, wherein the polypeptide or polynucleotide comprises, in sequential order, the sequence components N, L8, L2, L8, O, L8, P, L8, L4, L8, L3, L8, L5, L8, L6, L8, and L7 of Table 5.

E57 . A polynucleotide or polypeptide according to any one of paragraphs E26-E56 , wherein any one or more components of the L1 sequence are replaced by components of the L9 sequence from Table 5.

E58. A polypeptide comprising any two or more sequences: Subset AA #1, Subset AA #2, Subset AA #3, Subset AA #4, or Subset AA #5, with the sequences being in any order or in sequential order as shown below.

E59 . A polypeptide comprising the sequences: AA Subset #1, AA Subset #2, AA Subset #3, AA Subset #4, or AA Subset #5, wherein the sequences are in any order or in sequential order as shown below.

E60 . A polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising any two or more of the following sequences: AA Subset 1, AA Subset 2, AA Subset 3, AA Subset 4, or AA Subset 5, wherein the sequences are arranged in any order or in a sequential order as shown below.

E61 . A polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising the sequences: AA Subset 1, AA Subset 2, AA Subset 3, AA Subset 4, or AA Subset 5, wherein the sequences are arranged in any order or in a sequential order as shown below.

AA Subset No. 1:

SEQ ID NO: 1 (HPV6 E2*);

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 3 (HPV6 E4 1 - 56);

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 5 (HPV6 E7 70 - 98);

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 7 (HPV6 E4 36 - 90);

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 9 (HPV6 E7 1 - 58);

SEQ ID NO: 34 (rigid linker); and/or,

SEQ ID NO: 11 (HPV6 E6*).

Subset AA No. 2:

SEQ ID NO: 1 (HPV6 E2*);

SEQ ID NO: 45 (HPV11 E7 epitope linker);

SEQ ID NO: 3 (HPV6 E4);

SEQ ID NO: 42 (HPV11 E6 linker);

SEQ ID NO: 5 (HPV6 E7);

SEQ ID NO: 40 (HPV6 E6 linker);

SEQ ID NO: 7 (HPV6 E4);

SEQ ID NO: 42 (HPV11 E6 linker);

SEQ ID NO: 9 (HPV6 E7);

SEQ ID NO: 45 (HPV11 E7 epitope linker); and/or,

SEQ ID NO: 11 (HPV6 E6*).

AA Subset No. 3:

SEQ ID NO: 13 (HPV6 E2* 1 - 368 (Del 206 - 306));

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 42 (HPV11 E6 epitope, "linker");

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 15 (HPV6 E4 1 - 70 (Del 21 - 50));

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 45 (HPV11 E7 epitope, "linker");

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 17 (HPV6 E4 51 - 70);

SEQ ID NO: 34 (rigid linker);

SEQ ID NO: 20 (HPV6 E6* 1 - 120 (Del C - end));

SEQ ID NO: 34 (rigid linker); and/or,

SEQ ID NO: 22 (HPV6 E7 1 - 98 (Del 40 - 75)).

AA Subset No. 4:

SEQ ID NO: 24 (HPV6 E2* 1 - 205);

SEQ ID NO: 52 (HPV16 E7 agonist enhancer linker);

SEQ ID NO: 42 (HPV11 E6 epitope linker);

SEQ ID NO: 52 (HPV16 E7 agonist enhancer linker);

SEQ ID NO: 26 (HPV6 E4 1 - 20);

SEQ ID NO: 48 (linker #1, HPV16 E6 agonist enhancer);

SEQ ID NO: 45 (HPV11 E7 epitope linker);

SEQ ID NO: 50 (linker #2, HPV16 E6 agonist enhancer);

SEQ ID NO: 17 (HPV6 E4 51 - 70);

SEQ ID NO: 52 (HPV16 E7 agonist enhancer linker);

SEQ ID NO: 40 (HPV6 E6 epitope linker);

SEQ ID NO: 52 (HPV16 E7 agonist enhancer linker); and/or,

SEQ ID NO: 28 (HPV6 E2* 307 - 368).

AA Subset No. 5:

SEQ ID NO: 28 (HPV6 E2*);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 40 (HPV6 E6 epitope linker);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 30 (HPV6 E4 epitope 1);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 32 (HPV6 E4 epitope #2);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 45 (HPV11 E7 epitope linker);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 42 (HPV11 E6 epitope linker);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 48 (HPV16 E6 agonist enhancer #1);

SEQ ID NO: 57 (flexible linker);

SEQ ID NO: 50 (HPV16 E6 agonist enhancer #2);

SEQ ID NO: 57 (flexible linker); and/or,

SEQ ID NO: 52 (HPV16 E7 agonist enhancer).

E62 . A polynucleotide comprising any two or more of the nucleic acid sequences DNA Subset 1, DNA Subset 2, DNA Subset 3, DNA Subset 4, or DNA Subset 5, wherein the sequences are arranged in any order or in a sequential order as shown below.

E63 . A polynucleotide comprising the nucleic acid sequences of DNA Subset 1, DNA Subset 2, DNA Subset 3, DNA Subset 4, or DNA Subset 5, wherein the sequences are arranged in any order or in a sequential order as shown below.

DNA subset #1:

SEQ ID NO: 2 (HPV6 E2*);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 4 (HPV6 E4 1 - 56);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 6 (HPV6 E7 70 - 98);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 8 (HPV6 E4 36 - 90);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 10 (HPV6 E7 1 - 58);

SEQ ID NO: 35, 36, 37, 38, or 39 (rigid linker); and/or,

SEQ ID NO: 12 (HPV6 E6*).

DNA Subset #2:

SEQ ID NO: 2 (HPV6 E2*);

SEQ ID NO: 46 or 47 (HPV11 E7 epitope linker);

SEQ ID NO: 4 (HPV6 E4);

SEQ ID NO: 43 or 44 (HPV11 E6 linker);

SEQ ID NO: 6 (HPV6 E7);

SEQ ID NO: 41 (HPV6 E6 linker);

SEQ ID NO: 8 (HPV6 E4);

SEQ ID NO: 43 or 44 (HPV11 E6 linker);

SEQ ID NO: 10 (HPV6 E7 1 - 58);

SEQ ID NO: 46 or 47 (HPV11 E7 epitope linker); and/or,

SEQ ID NO: 12 (HPV6 E6*).

DNA Subset #3:

SEQ ID NO: 14 (HPV6 E2* 1 - 368 (Del 206 - 306));

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 43 or 44 (HPV11 E6 epitope linker);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 16 (HPV6 E4 1 - 70 (Del 21 - 50));

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 46 or 47 (HPV11 E7 epitope linker);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 18 or 19 (HPV6 E4 51 - 70);

SEQ ID NO: 35, 36, 37, 38 or 39 (rigid linker);

SEQ ID NO: 21 (HPV6 E6* 1 - 120 (Del C - end));

SEQ ID NO: 35, 36, 37, 38, or 39 (rigid linker); and/or,

SEQ ID NO: 23 (HPV6 E7 1 - 98 (Del 40 - 75)).

DNA Subset #4:

SEQ ID NO: 25 (HPV6 E2* 1 - 205);

SEQ ID NO: 53, 54, 55 or 56 (HPV16 E7 agonist enhancer linker);

SEQ ID NO: 43 or 44 (HPV11 E6 epitope linker);

SEQ ID NO: 53, 54, 55 or 56 (HPV16 E7 agonist enhancer linker);

SEQ ID NO: 27 (HPV6 E4 1 - 20);

SEQ ID NO: 49 (linker #1, HPV16 E6 agonist enhancer);

SEQ ID NO: 46 or 47 (HPV11 E7 epitope linker);

SEQ ID NO: 51 (linker #2, HPV16 E6 agonist enhancer);

SEQ ID NO: 18 or 19 (HPV6 E4 51 - 70);

SEQ ID NO: 53, 54, 55 or 56 (HPV16 E7 agonist enhancer linker);

SEQ ID NO: 41 (HPV6 E6 epitope linker);

SEQ ID NO: 53, 54, 55, or 56 (HPV16 E7 agonist enhancer linker); and/or,

SEQ ID NO: 29 (HPV6 E2* 307 - 368).

DNA Subset #5:

SEQ ID NO: 29 (HPV6 E2*);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 41 (HPV6 E6 epitope linker);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 31 (HPV6 E4 epitope 1);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 33 (HPV6 E4 epitope #2);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 46 or 47 (HPV11 E7 epitope linker);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 43 or 44 (HPV11 E6 epitope linker);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 49 (HPV16 E6 agonist enhancer #1);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64 or 65 (flexible linker);

SEQ ID NO: 51 (HPV16 E6 agonist enhancer #2);

SEQ ID NO: 58, 59, 60, 61, 62, 63, 64, or 65 (flexible linker); and/or

SEQ ID NO: 53, 54, 55 or 56 (HPV16 E7 agonist enhancer).

E64 . A vector encoding a polynucleotide according to any one of paragraphs E1-E16 and E26-E63.

E65 . The vector according to E64 , wherein the vector is a plasmid, a viral vector or a non-viral vector.

E66 . The vector according to E65 , wherein the viral vector is an adenovirus vector.

E67 . The vector according to E66 , wherein the adenoviral vector is a viral vector of a non-human primate.

E68 . A composition or vaccine comprising a pharmaceutically acceptable carrier and a polynucleotide according to any one of paragraphs E1-E16 and E26-E63 , a polypeptide according to any one of paragraphs E17 and E26-E59 or a vector according to any one of paragraphs E18-E21 and E64-E67 .

E69 . A polynucleotide, polypeptide, vector, composition or vaccine according to any one of paragraphs E1-E22 and E26-E68 , wherein the polynucleotide, polypeptide, vector, composition or vaccine is capable of inducing an immune response against HPV.

E70 . Use of a polynucleotide, polypeptide, vector, composition or vaccine according to E69 for the treatment of a disease or disorder associated with HPV.

E72 . Use of a polynucleotide, polypeptide, vector, composition or vaccine according to E70 , wherein the disease or disorder is a disease or disorder associated with HPV6, HPV11 or HPV16.

E72 . Use of a polynucleotide, polypeptide, vector, composition or vaccine according to E70 , wherein the disease or disorder is recurrent respiratory papillomatosis (RRP).

Bibliography

[251] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. To the extent that any disclosure made in references incorporated by reference conflicts with the disclosure in this application, the disclosure in this application shall be deemed to control and supersede such references.

Abbate, E. A., Berger, J. M., & Botchan, M. R. (2004). The X - ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2. Genes & Development , 18(16), 1981-1996. doi:10.1101/gad.1220104.

Ahn, J., Peng, S., Hung, C. F., Roden, R., Wu, T. C., & Best, S. R. (2017). Immunologic responses to a novel DNA vaccine targeting human papillomavirus - 11 E6E7. The Laryngoscope , 127(12), 2713-2720. doi:10.1002/lary.26737.

Anderson, Karen S., et al . (2015). Biological predictors of serologic responses to HPV in oropharyngeal cancer: The HOTSPOT study. Oral Oncology , 51(8): 751-758. doi: 10.1016/j.oraloncology.2015.05.007

Brokaw, J. L., Blanco, M., & McBride, A. A. (1996). Amino acids critical for the functions of the bovine papillomavirus type 1 E2 transactivator. Journal of Virology, 70(1), 23-29.

Graham, S. (2017). The human papillomavirus replication cycle, and its links to cancer progression: a comprehensive review. Clinical Science , 131(17), 2201 - 2221. doi: 10.1042/CS20160786.

Holm, A., Nagaeva, O., Nagaev, I., Loizou, C., Laurell, G., Mincheva - Nilsson, L., Olofsson, K. (2017). Lymphocyte profile and cytokine mRNA expression in peripheral blood mononuclear cells of patients with recurrent respiratory papillomatosis suggest dysregulated cytokine mRNA response and impaired cytotoxic capacity. Immunity, Inflammation and Disease , 5(4), 541-550. doi:10.1002/iid3.188.

Oh, ST, Longworth, MS, & Laimins, LA. (2004) Roles of the E6 and E7 proteins in the life cycle of low - risk human papillomavirus type 11. Journal of Virology , 78(5), 2620-2626. doi:10.1128/jvi.78.5.2620 - 2626.2004.

Seedat, R., Combrinck, C., Bester, P., Lee, J., & Burt, F. (2016). Determination of the complete genome and functional analysis of HPV6 isolate VBD19/10 from a patient with aggressive recurrent respiratory papillomatosis. Epidemiology and Infection , 144(10), 2128 - 2135. doi:10.1017/S0950268816000388.

Shin, T. H., Pankhong, P., Yan, J., Khan, A. S., Sardesai, N. Y., & Weiner, D. B. (2012). Induction of robust cellular immunity against HPV6 and HPV11 in mice by DNA vaccine encoding for E6/E7 antigen. Human Vaccines & Immunotherapeutics , 8(4), 470-478. doi:10.4161/hv.19180.

Trimble, C. L., Morrow, M. P., Kraynyak, K. A., Shen, X., Dallas, M., Yan, J., Bagarazzi, M. L. (2015). Safety, efficacy, and immunogenicity of VGX - 3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2/3: a randomised, double - blind, placebo - controlled phase 2b trial. Lancet (London, England), 386(10008), 2078-2088. doi:10.1016/S0140 - 6736(15)00239 - 1.

Wang, Y., Coulombe, R., Cameron, D.R., Thauvette, L., Massariol, M., Amon, L.M., Fink, D., Titolo, S., Welchner, E., Yoakim, C., Arachambault, J. & White, P.W. (2004). Crystal structure of the E2 domain of human papillomavirus type 11 bound to a protein interaction inhibitor. Journal of Biological Chemistry . 279(8), 6976 - 6985. doi:10.1074/jbc.M311376200.

Yiu, Y., Fayson, S., Smith, H., & Matrka, L. (2019). Implementation of Routine HPV Vaccination in the Management of Recurrent Respiratory Papillomatosis. Annals of Otology, Rhinology & Laryngology , 128(4), 309-315.

--->

SEQUENCE LIST

<110> Precigen Inc.

<120> HUMAN PAPILLOMA VIRUS VACCINES AND USES OF THE SAME FOR HPV

Associated Diseases

<130> 2584.166PC01

<150> US 63/118,222

<151> 2020-11-25

<160> 81

<170> PatentIn version 3.5

<210> 1

<211> 368

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 1

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile

195 200 205

Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser

210 215 220

Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala

225 230 235 240

Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile

245 250 255

Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp

260 265 270

Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val

275 280 285

Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu

290 295 300

Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His

305 310 315 320

Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr

325 330 335

Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile

340 345 350

Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu

355 360 365

<210> 2

<211> 1104

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 2

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagaggtcag cattcccgaa agcacaacct atacccctgc ccagacctcc 660

accctcgtgt ccagctccac caaagaggat gccgtccaga caccccctag aaaaagagct 720

agaggagtgc aacagtcccc ctgtaacgct ctgtgtgtgg ctcacattgg ccctgtggat 780

agcggaaacc ataacctcat cacaaacaat cacgatcagc atcagaggag aaataactcc 840

aactccagcg ctacccctat cgtccagttt cagggagagt ccaactgtct gaaatgcttt 900

agatatagac tcaacgatag acatagacat ctgtttgacc tcatctccag cacatggcat 960

tgggccagct ccaaggctcc ccataagcac gccattgtga cagtgacata cgatagcgaa 1020

gagcaaagac aacagtttct ggatgtggtc aagattcccc ctaccattag ccataagctc 1080

ggctttatgt ccctgcatct gctc 1104

<210> 3

<211> 56

<212> PRT

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 3

Met Ala Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu

1 5 10 15

Leu His Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro

20 25 30

Arg Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser

35 40 45

Asn Ser Pro Leu Ala Thr Pro Cys

50 55

<210> 4

<211> 168

<212> DNA

<213> Artificial Sequence

<220>

<223> Synthetic

<400> 4

atggcagacg attctgcact gcataaaaag tacccattcc tgaacctgct gcatacccca 60

ccgcatcgcc caccgccact gtgtccgcaa gctccacgca agacccaatg caagcgccgt 120

ctgggtaacg agcacgagga atccaactcc ccgctggcta ctccgtgt 168

<210> 5

<211> 29

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 5

Gln Cys Thr Glu Thr Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly

1 5 10 15

Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr

20 25

<210> 6

<211> 87

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 6

caatgcacag agacagacat tagagaagtg caacagctcc tgctcggcac actgaatatc 60

gtctgcccta tctgtgcccc taagaca 87

<210> 7

<211> 55

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 7

Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro

1 5 10 15

Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val Glu

20 25 30

Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp Gly Thr

35 40 45

Thr Val Thr Val Gln Leu Arg

50 55

<210> 8

<211> 165

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 8

caatgcaagc gccgtctggg taacgagcac gaggaatcca actccccgct ggctactccg 60

tgtgtttggc cgactctgga cccgtggacc gtggaaacta ccacttcttc cctgactatc 120

actacctcca ccaaggacgg caccactgtt actgttcaac tgcgt 165

<210> 9

<211> 58

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 9

Met His Gly Arg His Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln

1 5 10 15

Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser

20 25 30

Ser Glu Asp Glu Val Asp Glu Val Asp Gly Gln Asp Ser Gln Pro Leu

35 40 45

Lys Gln His Phe Gln Ile Val Thr Cys Cys

50 55

<210> 10

<211> 174

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 10

atgcacggca ggcacgtcac cctcaaggat atcgtcctgg atctgcaacc ccctgaccct 60

gtgggactgc attgctatga gcaactggtg gattccagcg aagacgaagt ggatgaggtg 120

gacggacagg atagccaacc cctcaagcaa cactttcaga ttgtgacatg ctgt 174

<210> 11

<211> 150

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 11

Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu

1 5 10 15

Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val

20 25 30

Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr

35 40 45

Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys

50 55 60

Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Ala His Phe

65 70 75 80

Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp

85 90 95

Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Gln Cys

100 105 110

Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile Lys

115 120 125

Leu Asn Cys Thr Arg Lys Gly Arg Cys Leu His Cys Trp Thr Thr Cys

130 135 140

Met Glu Asp Met Leu Pro

145 150

<210> 12

<211> 450

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 12

atggaaagcg ctaacgccag cacaagcgct accacaatcg accagctctg caaaaccttt 60

aacctctcca tgcacacact gcaaatcaac tgcgtcttct gtaagaatgc cctcaccaca 120

gccgaaatct atagctatgc ctataagcat ctgaaagtgc tcttcagggg cggataccct 180

tacgctgcct gtgcctgttg cctggagttt cacggaaaga ttaaccaata cgctcacttt 240

gactatgccg gatacgctac cacagtggaa gaggaaacca aacaggatat cctcgacgtg 300

ctgattagat gttacctctg ccataagcct cagtgtgagg tcgagaaagt gaaacacatt 360

ctgacaaagg ctagatttat caaactgaat tgcacaagaa aaggcaggtg cctccactgt 420

tggacaacct gtatggaaga catgctgcct 450

<210> 13

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 13

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Arg His Arg

195 200 205

His Leu Phe Asp Leu Ile Ser Ser Thr Trp His Trp Ala Ser Ser Lys

210 215 220

Ala Pro His Lys His Ala Ile Val Thr Val Thr Tyr Asp Ser Glu Glu

225 230 235 240

Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile Pro Pro Thr Ile Ser

245 250 255

His Lys Leu Gly Phe Met Ser Leu His Leu Leu

260 265

<210> 14

<211> 1104

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 14

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagaggtcag cattcccgaa agcacaacct atacccctgc ccagacctcc 660

accctcgtgt ccagctccac caaagaggat gccgtccaga caccccctag aaaaagagct 720

agaggagtgc aacagtcccc ctgtaacgct ctgtgtgtgg ctcacattgg ccctgtggat 780

agcggaaacc ataacctcat cacaaacaat cacgatcagc atcagaggag aaataactcc 840

aactccagcg ctacccctat cgtccagttt cagggagagt ccaactgtct gaaatgcttt 900

agatatagac tcaacgatag acatagacat ctgtttgacc tcatctccag cacatggcat 960

tgggccagct ccaaggctcc ccataagcac gccattgtga cagtgacata cgatagcgaa 1020

gagcaaagac aacagtttct ggatgtggtc aagattcccc ctaccattag ccataagctc 1080

ggctttatgt ccctgcatct gctc 1104

<210> 15

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 15

Met Ala Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu

1 5 10 15

Leu His Thr Pro Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp

20 25 30

Pro Trp Thr Val Glu Thr Thr Thr

35 40

<210> 16

<211> 120

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 16

atggcagacg attctgcact gcataaaaag tacccattcc tgaacctgct gcatacccca 60

ccgctggcta ctccgtgtgt ttggccgact ctggacccgt ggaccgtgga aactaccact 120

<210> 17

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 17

Met Ala Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu

1 5 10 15

Leu His Thr Pro Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp

20 25 30

Pro Trp Thr Val Glu Thr Thr Thr

35 40

<210> 18

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 18

ccgctcgcaa caccatgcgt ttggcccaca ctcgatccct ggactgtcga gacaaccaca 60

<210> 19

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 19

ccgctggcta ctccgtgtgt ttggccgact ctggacccgt ggaccgtgga aactaccact 60

<210> 20

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 20

Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln Leu

1 5 10 15

Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys Val

20 25 30

Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr

35 40 45

Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala Cys

50 55 60

Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Ala His Phe

65 70 75 80

Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln Asp

85 90 95

Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Gln Cys

100 105 110

Glu Val Glu Lys Val Lys His Ile

115 120

<210> 21

<211> 360

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 21

atggaaagcg ctaacgccag cacaagcgct accacaatcg accagctctg caaaaccttt 60

aacctctcca tgcacacact gcaaatcaac tgcgtcttct gtaagaatgc cctcaccaca 120

gccgaaatct atagctatgc ctataagcat ctgaaagtgc tcttcagggg cggataccct 180

tacgctgcct gtgcctgttg cctggagttt cacggaaaga ttaaccaata cgctcacttt 240

gactatgccg gatacgctac cacagtggaa gaggaaacca aacaggatat cctcgacgtg 300

ctgattagat gttacctctg ccataagcct cagtgtgagg tcgagaaagt gaaacacatt 360

<210> 22

<211> 62

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 22

Met His Gly Arg His Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln

1 5 10 15

Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Val Asp Ser

20 25 30

Ser Glu Asp Glu Val Asp Glu Ile Arg Glu Val Gln Gln Leu Leu Leu

35 40 45

Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro Lys Thr

50 55 60

<210> 23

<211> 186

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 23

atgcacggca ggcacgtcac cctcaaggat atcgtcctgg atctgcaacc ccctgaccct 60

gtgggactgc attgctatga gcaactggtg gattccagcg aagacgaagt ggatgagatt 120

agagaagtgc aacagctcct gctcggcaca ctgaatatcg tctgccctat ctgtgcccct 180

aagaca 186

<210> 24

<211> 205

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 24

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu

195 200 205

<210> 25

<211> 615

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 25

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagag 615

<210> 26

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 26

Met Ala Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu

1 5 10 15

Leu His Thr Pro

20

<210> 27

<211> 60

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 27

atggcagacg attctgcact gcataaaaag tacccattcc tgaacctgct gcatacccca 60

<210> 28

<211> 62

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 28

Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His Trp Ala

1 5 10 15

Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr Tyr Asp

20 25 30

Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile Pro Pro

35 40 45

Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu

50 55 60

<210> 29

<211> 186

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 29

agacatagac atctgtttga cctcatctcc agcacatggc attgggccag ctccaaggct 60

ccccataagc acgccattgt gacagtgaca tacgatagcg aagagcaaag acaacagttt 120

ctggatgtgg tcaagattcc ccctaccatt agccataagc tcggctttat gtccctgcat 180

ctgctc 186

<210> 30

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 30

Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr Pro Pro

1 5 10 15

His Arg Pro Pro Pro Leu

20

<210> 31

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 31

gcactgcata aaaagtaccc attcctgaac ctgctgcata ccccaccgca tcgcccaccg 60

ccactg 66

<210> 32

<211> 23

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 32

Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val

1 5 10 15

Glu Thr Thr Thr Ser Ser Leu

20

<210> 33

<211> 69

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 33

ccgctggcta ctccgtgtgt ttggccgact ctggacccgt ggaccgtgga aactaccact 60

tcttccctg 69

<210> 34

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 34

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10

<210> 35

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 35

gaggctgccg ctaaggaagc tgccgccaaa 30

<210> 36

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 36

gaggctgccg ccaaggaggc cgccgctaag 30

<210> 37

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 37

gaggccgcgg ctaaagaggc ggccgcgaaa 30

<210> 38

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 38

gaggcagccg caaaggaggc tgccgcaaaa 30

<210> 39

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 39

gaggcagccg caaaggaggc cgcagctaag 30

<210> 40

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 40

Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys Gln Leu Lys Val Leu

1 5 10 15

<210> 41

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 41

acagccgaaa tctatagcta tgcctataag caactgaaag tgctc 45

<210> 42

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 42

Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr Lys Asn Leu Lys Val Val

1 5 10 15

<210> 43

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 43

acagccgaaa tctatgccta tgcctataag aatctgaaag tggtc 45

<210> 44

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 44

accgctgaga tttacgctta cgcttacaaa aacctcaagg tcgtg 45

<210> 45

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 45

His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val Asp

1 5 10 15

<210> 46

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 46

cattgctatg agcaactgga agactccagc gaagacgaag tggat 45

<210> 47

<211> 45

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 47

cactgttacg aacagctgga ggatagctcc gaggatgagg tggac 45

<210> 48

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 48

Lys Leu Pro Gln Leu Cys Thr Glu Val

1 5

<210> 49

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 49

aagctccccc aactgtgtac cgaagtg 27

<210> 50

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 50

Gln Leu Tyr Asn Lys Pro Leu Cys Asp Val

1 5 10

<210> 51

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 51

cagctctaca ataagcctct gtgtgacgtg 30

<210> 52

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 52

Arg Thr Leu Glu Asp Leu Leu Met Gly Val

1 5 10

<210> 53

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 53

agaacactgg aagacctcct gatgggcgtc 30

<210> 54

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 54

agaaccctgg aggatcttct catggggtg 30

<210> 55

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 55

agaacgctag aagatcttct catgggtgtg 30

<210> 56

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 56

cgaacactgg aagacttgct gatgggagtc 30

<210> 57

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 57

Val Ser Gln Thr Ser Lys Leu Thr Arg

1 5

<210> 58

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 58

gtgtcccaga caagcaaact gacaaga 27

<210> 59

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 59

gtaagccaga caagcaagct taccaga 27

<210> 60

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 60

gttagccaaa ccagtaagtt gaccagg 27

<210> 61

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 61

gtatcccaga ctagcaagct caccagg 27

<210> 62

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 62

gttagtcaga ccagtaagtt gacaagg 27

<210> 63

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 63

gtgtcccaga catcaaagct gactaga 27

<210> 64

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 64

gtctctcaga cctctaaact cacccga 27

<210> 65

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 65

gtgtcccaga caagcaaact gacaaga 27

<210> 66

<211> 766

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 66

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile

195 200 205

Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser

210 215 220

Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala

225 230 235 240

Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile

245 250 255

Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp

260 265 270

Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val

275 280 285

Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu

290 295 300

Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His

305 310 315 320

Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr

325 330 335

Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile

340 345 350

Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu

355 360 365

Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Met Ala Asp Asp Ser Ala

370 375 380

Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr Pro Pro His

385 390 395 400

Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg Lys Thr Gln Cys Lys

405 410 415

Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn Ser Pro Leu Ala Thr

420 425 430

Pro Cys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Gln Cys Thr Glu

435 440 445

Thr Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile

450 455 460

Val Cys Pro Ile Cys Ala Pro Lys Thr Glu Ala Ala Ala Lys Glu Ala

465 470 475 480

Ala Ala Lys Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser

485 490 495

Asn Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp

500 505 510

Thr Val Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys

515 520 525

Asp Gly Thr Thr Val Thr Val Gln Leu Arg Glu Ala Ala Ala Lys Glu

530 535 540

Ala Ala Ala Lys Met His Gly Arg His Val Thr Leu Lys Asp Ile Val

545 550 555 560

Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys Tyr Glu Gln

565 570 575

Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp Gly Gln Asp

580 585 590

Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr Cys Cys Glu Ala

595 600 605

Ala Ala Lys Glu Ala Ala Ala Lys Met Glu Ser Ala Asn Ala Ser Thr

610 615 620

Ser Ala Thr Thr Ile Asp Gln Leu Cys Lys Thr Phe Asn Leu Ser Met

625 630 635 640

His Thr Leu Gln Ile Asn Cys Val Phe Cys Lys Asn Ala Leu Thr Thr

645 650 655

Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu Phe Arg

660 665 670

Gly Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly

675 680 685

Lys Ile Asn Gln Tyr Ala His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr

690 695 700

Val Glu Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys

705 710 715 720

Tyr Leu Cys His Lys Pro Gln Cys Glu Val Glu Lys Val Lys His Ile

725 730 735

Leu Thr Lys Ala Arg Phe Ile Lys Leu Asn Cys Thr Arg Lys Gly Arg

740 745 750

Cys Leu His Cys Trp Thr Thr Cys Met Glu Asp Met Leu Pro

755 760 765

<210> 67

<211> 2298

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 67

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagaggtcag cattcccgaa agcacaacct atacccctgc ccagacctcc 660

accctcgtgt ccagctccac caaagaggat gccgtccaga caccccctag aaaaagagct 720

agaggagtgc aacagtcccc ctgtaacgct ctgtgtgtgg ctcacattgg ccctgtggat 780

agcggaaacc ataacctcat cacaaacaat cacgatcagc atcagaggag aaataactcc 840

aactccagcg ctacccctat cgtccagttt cagggagagt ccaactgtct gaaatgcttt 900

agatatagac tcaacgatag acatagacat ctgtttgacc tcatctccag cacatggcat 960

tgggccagct ccaaggctcc ccataagcac gccattgtga cagtgacata cgatagcgaa 1020

gagcaaagac aacagtttct ggatgtggtc aagattcccc ctaccattag ccataagctc 1080

ggctttatgt ccctgcatct gctcgaggct gccgctaagg aagctgccgc caaaatggca 1140

gacgattctg cactgcataa aaagtaccca ttcctgaacc tgctgcatac cccaccgcat 1200

cgcccaccgc cactgtgtcc gcaagctcca cgcaagaccc aatgcaagcg ccgtctgggt 1260

aacgagcacg aggaatccaa ctccccgctg gctactccgt gtgaggctgc cgccaaggag 1320

gccgccgcta agcaatgcac agagacagac attagagaag tgcaacagct cctgctcggc 1380

acactgaata tcgtctgccc tatctgtgcc cctaagacag aggccgcggc taaagaggcg 1440

gccgcgaaac aatgcaagcg ccgtctgggt aacgagcacg aggaatccaa ctccccgctg 1500

gctactccgt gtgtttggcc gactctggac ccgtggaccg tggaaactac cacttcttcc 1560

ctgactatca ctacctccac caaggacggc accactgtta ctgttcaact gcgtgaggca 1620

gccgcaaagg aggctgccgc aaaaatgcac ggcaggcacg tcaccctcaa ggatatcgtc 1680

ctggatctgc aaccccctga ccctgtggga ctgcattgct atgagcaact ggtggattcc 1740

agcgaagacg aagtggatga ggtggacgga caggatagcc aacccctcaa gcaacacttt 1800

cagattgtga catgctgtga ggcagccgca aaggaggccg cagctaagat ggaaagcgct 1860

aacgccagca caagcgctac cacaatcgac cagctctgca aaacctttaa cctctccatg 1920

cacacactgc aaatcaactg cgtcttctgt aagaatgccc tcaccacagc cgaaatctat 1980

agctatgcct ataagcatct gaaagtgctc ttcaggggcg gataccctta cgctgcctgt 2040

gcctgttgcc tggagtttca cggaaagatt aaccaatacg ctcactttga ctatgccgga 2100

tacgctacca cagtggaaga ggaaaccaaa caggatatcc tcgacgtgct gattagatgt 2160

tacctctgcc ataagcctca gtgtgaggtc gagaaagtga aacacattct gacaaaggct 2220

agatttatca aactgaattg cacaagaaaa ggcaggtgcc tccactgttg gacaacctgt 2280

atggaagaca tgctgcct 2298

<210> 68

<211> 791

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 68

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile

195 200 205

Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser

210 215 220

Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala

225 230 235 240

Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile

245 250 255

Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp

260 265 270

Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val

275 280 285

Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu

290 295 300

Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His

305 310 315 320

Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr

325 330 335

Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile

340 345 350

Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu

355 360 365

His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val Asp Met

370 375 380

Ala Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu

385 390 395 400

His Thr Pro Pro His Arg Pro Pro Pro Leu Cys Pro Gln Ala Pro Arg

405 410 415

Lys Thr Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn

420 425 430

Ser Pro Leu Ala Thr Pro Cys Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr

435 440 445

Lys Asn Leu Lys Val Val Gln Cys Thr Glu Thr Asp Ile Arg Glu Val

450 455 460

Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala

465 470 475 480

Pro Lys Thr Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys Gln Leu Lys

485 490 495

Val Leu Gln Cys Lys Arg Arg Leu Gly Asn Glu His Glu Glu Ser Asn

500 505 510

Ser Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr

515 520 525

Val Glu Thr Thr Thr Ser Ser Leu Thr Ile Thr Thr Ser Thr Lys Asp

530 535 540

Gly Thr Thr Val Thr Val Gln Leu Arg Thr Ala Glu Ile Tyr Ala Tyr

545 550 555 560

Ala Tyr Lys Asn Leu Lys Val Val Met His Gly Arg His Val Thr Leu

565 570 575

Lys Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His

580 585 590

Cys Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val

595 600 605

Asp Gly Gln Asp Ser Gln Pro Leu Lys Gln His Phe Gln Ile Val Thr

610 615 620

Cys Cys His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val

625 630 635 640

Asp Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp Gln

645 650 655

Leu Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile Asn Cys

660 665 670

Val Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr Ser Tyr Ala

675 680 685

Tyr Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr Pro Tyr Ala Ala

690 695 700

Cys Ala Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln Tyr Ala His

705 710 715 720

Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu Glu Thr Lys Gln

725 730 735

Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Gln

740 745 750

Cys Glu Val Glu Lys Val Lys His Ile Leu Thr Lys Ala Arg Phe Ile

755 760 765

Lys Leu Asn Cys Thr Arg Lys Gly Arg Cys Leu His Cys Trp Thr Thr

770 775 780

Cys Met Glu Asp Met Leu Pro

785 790

<210> 69

<211> 2373

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 69

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagaggtcag cattcccgaa agcacaacct atacccctgc ccagacctcc 660

accctcgtgt ccagctccac caaagaggat gccgtccaga caccccctag aaaaagagct 720

agaggagtgc aacagtcccc ctgtaacgct ctgtgtgtgg ctcacattgg ccctgtggat 780

agcggaaacc ataacctcat cacaaacaat cacgatcagc atcagaggag aaataactcc 840

aactccagcg ctacccctat cgtccagttt cagggagagt ccaactgtct gaaatgcttt 900

agatatagac tcaacgatag acatagacat ctgtttgacc tcatctccag cacatggcat 960

tgggccagct ccaaggctcc ccataagcac gccattgtga cagtgacata cgatagcgaa 1020

gagcaaagac aacagtttct ggatgtggtc aagattcccc ctaccattag ccataagctc 1080

ggctttatgt ccctgcatct gctccattgc tatgagcaac tggaagactc cagcgaagac 1140

gaagtggata tggcagacga ttctgcactg cataaaaagt acccattcct gaacctgctg 1200

cataccccac cgcatcgccc accgccactg tgtccgcaag ctccacgcaa gacccaatgc 1260

aagcgccgtc tgggtaacga gcacgaggaa tccaactccc cgctggctac tccgtgtaca 1320

gccgaaatct atgcctatgc ctataagaat ctgaaagtgg tccaatgcac agagacagac 1380

attagagaag tgcaacagct cctgctcggc acactgaata tcgtctgccc tatctgtgcc 1440

cctaagacaa cagccgaaat ctatagctat gcctataagc aactgaaagt gctccaatgc 1500

aagcgccgtc tgggtaacga gcacgaggaa tccaactccc cgctggctac tccgtgtgtt 1560

tggccgactc tggacccgtg gaccgtggaa actaccactt cttccctgac tatcactacc 1620

tccaccaagg acggcaccac tgttactgtt caactgcgta ccgctgagat ttacgcttac 1680

gcttacaaaa acctcaaggt cgtgatgcac ggcaggcacg tcaccctcaa ggatatcgtc 1740

ctggatctgc aaccccctga ccctgtggga ctgcattgct atgagcaact ggtggattcc 1800

agcgaagacg aagtggatga ggtggacgga caggatagcc aacccctcaa gcaacacttt 1860

cagattgtga catgctgtca ctgttacgaa cagctggagg atagctccga ggatgaggtg 1920

gacatggaaa gcgctaacgc cagcacaagc gctaccacaa tcgaccagct ctgcaaaacc 1980

tttaacctct ccatgcacac actgcaaatc aactgcgtct tctgtaagaa tgccctcacc 2040

acagccgaaa tctatagcta tgcctataag catctgaaag tgctcttcag gggcggatac 2100

ccttacgctg cctgtgcctg ttgcctggag tttcacggaa agattaacca atacgctcac 2160

tttgactatg ccggatacgc taccacagtg gaagaggaaa ccaaacagga tatcctcgac 2220

gtgctgatta gatgttacct ctgccataag cctcagtgtg aggtcgagaa agtgaaacac 2280

attctgacaa aggctagatt tatcaaactg aattgcacaa gaaaaggcag gtgcctccac 2340

tgttggacaa cctgtatgga agacatgctg cct 2373

<210> 70

<211> 599

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 70

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Arg His Arg

195 200 205

His Leu Phe Asp Leu Ile Ser Ser Thr Trp His Trp Ala Ser Ser Lys

210 215 220

Ala Pro His Lys His Ala Ile Val Thr Val Thr Tyr Asp Ser Glu Glu

225 230 235 240

Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile Pro Pro Thr Ile Ser

245 250 255

His Lys Leu Gly Phe Met Ser Leu His Leu Leu Glu Ala Ala Ala Lys

260 265 270

Glu Ala Ala Ala Lys Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr Lys Asn

275 280 285

Leu Lys Val Val Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Met Ala

290 295 300

Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His

305 310 315 320

Thr Pro Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp

325 330 335

Thr Val Glu Thr Thr Thr Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

340 345 350

His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val Asp Glu

355 360 365

Ala Ala Ala Lys Glu Ala Ala Ala Lys Pro Leu Ala Thr Pro Cys Val

370 375 380

Trp Pro Thr Leu Asp Pro Trp Thr Val Glu Thr Thr Thr Glu Ala Ala

385 390 395 400

Ala Lys Glu Ala Ala Ala Lys Met Glu Ser Ala Asn Ala Ser Thr Ser

405 410 415

Ala Thr Thr Ile Asp Gln Leu Cys Lys Thr Phe Asn Leu Ser Met His

420 425 430

Thr Leu Gln Ile Asn Cys Val Phe Cys Lys Asn Ala Leu Thr Thr Ala

435 440 445

Glu Ile Tyr Ser Tyr Ala Tyr Lys His Leu Lys Val Leu Phe Arg Gly

450 455 460

Gly Tyr Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly Lys

465 470 475 480

Ile Asn Gln Tyr Ala His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val

485 490 495

Glu Glu Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr

500 505 510

Leu Cys His Lys Pro Gln Cys Glu Val Glu Lys Val Lys His Ile Glu

515 520 525

Ala Ala Ala Lys Glu Ala Ala Ala Lys Met His Gly Arg His Val Thr

530 535 540

Leu Lys Asp Ile Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu

545 550 555 560

His Cys Tyr Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu

565 570 575

Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile Val Cys

580 585 590

Pro Ile Cys Ala Pro Lys Thr

595

<210> 71

<211> 1797

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 71

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagagagaca tagacatctg tttgacctca tctccagcac atggcattgg 660

gccagctcca aggctcccca taagcacgcc attgtgacag tgacatacga tagcgaagag 720

caaagacaac agtttctgga tgtggtcaag attcccccta ccattagcca taagctcggc 780

tttatgtccc tgcatctgct cgaggctgcc gctaaggaag ctgccgccaa aacagccgaa 840

atctatgcct atgcctataa gaatctgaaa gtggtcgagg ctgccgccaa ggaggccgcc 900

gctaagatgg cagacgattc tgcactgcat aaaaagtacc cattcctgaa cctgctgcat 960

accccaccgc tggctactcc gtgtgtttgg ccgactctgg acccgtggac cgtggaaact 1020

accactgagg ccgcggctaa agaggcggcc gcgaaacatt gctatgagca actggaagac 1080

tccagcgaag acgaagtgga tgaggcagcc gcaaaggagg ctgccgcaaa accgctcgca 1140

acaccatgcg tttggcccac actcgatccc tggactgtcg agacaaccac agaggcagcc 1200

gcaaaggagg ccgcagctaa gatggaaagc gctaacgcca gcacaagcgc taccacaatc 1260

gaccagctct gcaaaacctt taacctctcc atgcacacac tgcaaatcaa ctgcgtcttc 1320

tgtaagaatg ccctcaccac agccgaaatc tatagctatg cctataagca tctgaaagtg 1380

ctcttcaggg gcggataccc ttacgctgcc tgtgcctgtt gcctggagtt tcacggaaag 1440

attaaccaat acgctcactt tgactatgcc ggatacgcta cccacagtgga agaggaaacc 1500

aaacaggata tcctcgacgt gctgattaga tgttacctct gccataagcc tcagtgtgag 1560

gtcgagaaag tgaaacacat tgaggctgcc gctaaggaag ctgccgccaa aatgcacggc 1620

aggcacgtca ccctcaagga tatcgtcctg gatctgcaac cccctgaccc tgtgggactg 1680

cattgctatg agcaactggt ggattccagc gaagacgaag tggatgagat tagagaagtg 1740

caacagctcc tgctcggcac actgaatatc gtctgcccta tctgtgcccc taagaca 1797

<210> 72

<211> 411

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 72

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Arg Thr Leu

195 200 205

Glu Asp Leu Leu Met Gly Val Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr

210 215 220

Lys Asn Leu Lys Val Val Arg Thr Leu Glu Asp Leu Leu Met Gly Val

225 230 235 240

Met Ala Asp Asp Ser Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu

245 250 255

Leu His Thr Pro Lys Leu Pro Gln Leu Cys Thr Glu Val His Cys Tyr

260 265 270

Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val Asp Gln Leu Tyr Asn

275 280 285

Lys Pro Leu Cys Asp Val Pro Leu Ala Thr Pro Cys Val Trp Pro Thr

290 295 300

Leu Asp Pro Trp Thr Val Glu Thr Thr Thr Arg Thr Leu Glu Asp Leu

305 310 315 320

Leu Met Gly Val Thr Ala Glu Ile Tyr Ser Tyr Ala Tyr Lys Gln Leu

325 330 335

Lys Val Leu Arg Thr Leu Glu Asp Leu Leu Met Gly Val Arg His Arg

340 345 350

His Leu Phe Asp Leu Ile Ser Ser Thr Trp His Trp Ala Ser Ser Lys

355 360 365

Ala Pro His Lys His Ala Ile Val Thr Val Thr Tyr Asp Ser Glu Glu

370 375 380

Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile Pro Pro Thr Ile Ser

385 390 395 400

His Lys Leu Gly Phe Met Ser Leu His Leu Leu

405 410

<210> 73

<211> 1233

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 73

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagagagaac actggaagac ctcctgatgg gcgtcacagc cgaaatctat 660

gcctatgcct ataagaatct gaaagtggtc agaaccctgg aggatcttct catgggggtg 720

atggcagacg attctgcact gcataaaaag tacccattcc tgaacctgct gcatacccca 780

aagctccccc aactgtgtac cgaagtgcat tgctatgagc aactggaaga ctccagcgaa 840

gacgaagtgg atcagctcta caataagcct ctgtgtgacg tgccgctggc tactccgtgt 900

gtttggccga ctctggaccc gtggaccgtg gaaactacca ctcgaacact ggaagacttg 960

ctgatgggag tcacagccga aatctatagc tatgcctata agcaactgaa agtgctcaga 1020

acgctagaag atcttctcat gggtgtgaga catagacatc tgtttgacct catctccagc 1080

acatggcatt gggccagctc caaggctccc cataagcacg ccattgtgac agtgacatac 1140

gatagcgaag agcaaagaca acagtttctg gatgtggtca agattccccc taccattagc 1200

cataagctcg gctttatgtc cctgcatctg ctc 1233

<210> 74

<211> 559

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 74

Met Glu Ala Ile Ala Lys Arg Leu Asp Ala Cys Ala Glu Gln Leu Leu

1 5 10 15

Glu Leu Ala Glu Glu Asn Ser Thr Asp Leu His Lys His Val Leu His

20 25 30

Trp Lys Cys Met Arg His Ala Ser Val Leu Leu Tyr Lys Ala Lys Gln

35 40 45

Met Gly Leu Ser His Ile Gly Met Gln Val Val Pro Pro Leu Lys Val

50 55 60

Ser Glu Ala Lys Gly His Asn Ala Ile Glu Met Gln Met His Leu Glu

65 70 75 80

Ser Leu Leu Arg Thr Glu Tyr Ser Met Glu Pro Trp Thr Leu Gln Glu

85 90 95

Thr Ser Tyr Ala Met Trp Gln Thr Pro Pro Lys Arg Cys Phe Lys Lys

100 105 110

Arg Gly Lys Thr Val Glu Val Lys Phe Asp Gly Cys Ala Asn Asn Thr

115 120 125

Met Asp Tyr Val Val Trp Thr Asp Val Tyr Val Gln Asp Asn Asp Thr

130 135 140

Trp Val Lys Val His Ser Met Val Asp Ala Lys Gly Ile Tyr Tyr Thr

145 150 155 160

Cys Gly Gln Phe Lys Thr Tyr Tyr Val Asn Phe Val Lys Glu Ala Glu

165 170 175

Lys Tyr Gly Ser Thr Lys His Trp Glu Val Cys Tyr Gly Ser Thr Val

180 185 190

Ile Cys Ser Pro Ala Ser Val Ser Ser Thr Thr Gln Glu Val Ser Ile

195 200 205

Pro Glu Ser Thr Thr Tyr Thr Pro Ala Gln Thr Ser Thr Leu Val Ser

210 215 220

Ser Ser Thr Lys Glu Asp Ala Val Gln Thr Pro Pro Arg Lys Arg Ala

225 230 235 240

Arg Gly Val Gln Gln Ser Pro Cys Asn Ala Leu Cys Val Ala His Ile

245 250 255

Gly Pro Val Asp Ser Gly Asn His Asn Leu Ile Thr Asn Asn His Asp

260 265 270

Gln His Gln Arg Arg Asn Asn Ser Asn Ser Ser Ala Thr Pro Ile Val

275 280 285

Gln Phe Gln Gly Glu Ser Asn Cys Leu Lys Cys Phe Arg Tyr Arg Leu

290 295 300

Asn Asp Arg His Arg His Leu Phe Asp Leu Ile Ser Ser Thr Trp His

305 310 315 320

Trp Ala Ser Ser Lys Ala Pro His Lys His Ala Ile Val Thr Val Thr

325 330 335

Tyr Asp Ser Glu Glu Gln Arg Gln Gln Phe Leu Asp Val Val Lys Ile

340 345 350

Pro Pro Thr Ile Ser His Lys Leu Gly Phe Met Ser Leu His Leu Leu

355 360 365

Val Ser Gln Thr Ser Lys Leu Thr Arg Thr Ala Glu Ile Tyr Ser Tyr

370 375 380

Ala Tyr Lys Gln Leu Lys Val Leu Val Ser Gln Thr Ser Lys Leu Thr

385 390 395 400

Arg Ala Leu His Lys Lys Tyr Pro Phe Leu Asn Leu Leu His Thr Pro

405 410 415

Pro His Arg Pro Pro Pro Leu Val Ser Gln Thr Ser Lys Leu Thr Arg

420 425 430

Pro Leu Ala Thr Pro Cys Val Trp Pro Thr Leu Asp Pro Trp Thr Val

435 440 445

Glu Thr Thr Thr Ser Ser Leu Val Ser Gln Thr Ser Lys Leu Thr Arg

450 455 460

His Cys Tyr Glu Gln Leu Glu Asp Ser Ser Glu Asp Glu Val Asp Val

465 470 475 480

Ser Gln Thr Ser Lys Leu Thr Arg Thr Ala Glu Ile Tyr Ala Tyr Ala

485 490 495

Tyr Lys Asn Leu Lys Val Val Val Ser Gln Thr Ser Lys Leu Thr Arg

500 505 510

Lys Leu Pro Gln Leu Cys Thr Glu Val Val Ser Gln Thr Ser Lys Leu

515 520 525

Thr Arg Gln Leu Tyr Asn Lys Pro Leu Cys Asp Val Val Ser Gln Thr

530 535 540

Ser Lys Leu Thr Arg Arg Thr Leu Glu Asp Leu Leu Met Gly Val

545 550 555

<210> 75

<211> 1677

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 75

atggaagcca ttgccaaaag actcgacgct tgcgccgagc agctgctgga gctggccgaa 60

gagaatagca cagacctcca caaacacgtc ctgcattgga agtgcatgag acatgcctcc 120

gtgctcctgt ataaggccaa gcagatggga ctgtcccaca ttggaatgca ggtcgtgcct 180

cccctcaagg tcagcgaagc caaaggccat aacgctatcg aaatgcaaat gcacctggaa 240

agcctcctga ggaccgaata ctccatggaa ccctggaccc tccaggaaac ctcctacgct 300

atgtggcaga caccccctaa gaggtgcttt aagaaaagag gaaagacagt ggaagtgaaa 360

ttcgatggct gtgccaataa cacaatggat tacgtcgtgt ggaccgatgt gtatgtgcaa 420

gacaatgaca catgggtcaa ggtccactcc atggtggacg ctaagggaat ctattacaca 480

tgcggacagt ttaagacata ctatgtgaat ttcgtcaagg aagccgaaaa gtatggctcc 540

accaaacact gggaggtctg ctatggctcc accgtcatct gtagccctgc ctccgtgtcc 600

agcacaaccc aagaggtcag cattcccgaa agcacaacct atacccctgc ccagacctcc 660

accctcgtgt ccagctccac caaagaggat gccgtccaga caccccctag aaaaagagct 720

agaggagtgc aacagtcccc ctgtaacgct ctgtgtgtgg ctcacattgg ccctgtggat 780

agcggaaacc ataacctcat cacaaacaat cacgatcagc atcagaggag aaataactcc 840

aactccagcg ctacccctat cgtccagttt cagggagagt ccaactgtct gaaatgcttt 900

agatatagac tcaacgatag acatagacat ctgtttgacc tcatctccag cacatggcat 960

tgggccagct ccaaggctcc ccataagcac gccattgtga cagtgacata cgatagcgaa 1020

gagcaaagac aacagtttct ggatgtggtc aagattcccc ctaccattag ccataagctc 1080

ggctttatgt ccctgcatct gctcgtgtcc cagacaagca aactgacaag aacagccgaa 1140

atctatagct atgcctataa gcaactgaaa gtgctcgtaa gccagacaag caagcttacc 1200

agagcactgc ataaaaagta cccattcctg aacctgctgc ataccccacc gcatcgccca 1260

ccgccactgg ttagccaaac cagtaagttg accaggccgc tggctactcc gtgtgtttgg 1320

ccgactctgg acccgtggac cgtggaaact accacttctt ccctggtatc ccagactagc 1380

aagctcacca ggcattgcta tgagcaactg gaagactcca gcgaagacga agtggatgtt 1440

agtcagacca gtaagttgac aaggacagcc gaaatctatg cctatgccta taagaatctg 1500

aaagtggtcg tgtcccagac atcaaagctg actagaaagc tcccccaact gtgtaccgaa 1560

gtggtctctc agacctctaa actcacccga cagctctaca ataagcctct gtgtgacgtg 1620

gtgtcccaga caagcaaact gacaagaaga acactggaag acctcctgat gggcgtc 1677

<210> 76

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> synthetic

<400> 76

Glu Ala Ala Ala Lys

1 5

<210> 77

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 77

gaggctgccg ctaag 15

<210> 78

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 78

gaggctgccg ccaag 15

<210> 79

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 79

gaggccgcgg ctaaa 15

<210> 80

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 80

gaggcagccg caaag 15

<210> 81

<211> 15

<212> DNA

<213> Artificial Sequence

<220>

<223> synthetic

<400> 81

gaggcagccg caaag 15

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Claims (17)

1. A polynucleotide encoding a non-naturally occurring polypeptide containing (a) (i) an epitope consisting of SEQ ID NO: 1; (ii) an epitope consisting of SEQ ID NO: 3; (iii) an epitope consisting of SEQ ID NO: 5; (iv) an epitope consisting of SEQ ID NO: 7; (v) an epitope consisting of SEQ ID NO: 9; and (vi) an epitope consisting of SEQ ID NO: 11; (b) (i) an epitope consisting of SEQ ID NO: 13; (ii) an epitope consisting of SEQ ID NO: 42; (iii) an epitope consisting of SEQ ID NO: 15; (iv) an epitope consisting of SEQ ID NO: 45; (v) an epitope consisting of SEQ ID NO: 17; (vi) an epitope consisting of SEQ ID NO: 20; and (vii) an epitope consisting of SEQ ID NO: 22; or (c) (i) an epitope consisting of SEQ ID NO: 24; (ii) a first epitope consisting of SEQ ID NO: 52; (iii) an epitope consisting of SEQ ID NO: 42; (iv) a second epitope consisting of SEQ ID NO: 52; (v) an epitope consisting of SEQ ID NO: 26; (vi) an epitope consisting of SEQ ID NO: 48; (vii) an epitope consisting of SEQ ID NO: 45; (viii) an epitope consisting of SEQ ID NO: 50; (ix) an epitope consisting of SEQ ID NO: 17; (x) a third epitope consisting of SEQ ID NO: 52; (xi) an epitope consisting of SEQ ID NO: 40; (xii) a fourth epitope consisting of SEQ ID NO: 52; and (xiii) an epitope consisting of SEQ ID NO: 28. 2. The polynucleotide of claim 1, wherein the non-naturally occurring polypeptide comprises the epitopes specified in (a) and further comprises: (vi) an ethitope consisting of SEQ ID NO: 45; (vii) a first ethitope consisting of SEQ ID NO: 42; (viii) an ethitope consisting of SEQ ID NO: 40; (ix) a second ethitope consisting of SEQ ID NO: 42; and (x) a second ethitope consisting of SEQ ID NO: 45. 3. The polynucleotide according to claim 1, characterized in that the polynucleotide contains SEQ ID NO: 69. 4. The polynucleotide of claim 1, wherein the non-naturally occurring polypeptide comprises the amino acid sequence SEQ ID NO: 68. 5. A polypeptide encoded by a polynucleotide according to any one of claims 1 to 4, for use in the treatment of a disease or disorder caused by human papillomavirus type 6 (HPV6) and/or human papillomavirus type 11 (HPV11). 6. A vector comprising a polynucleotide according to any one of claims 1 to 4, for use in the treatment of a disease or disorder caused by human papillomavirus type 6 (HPV6) and/or human papillomavirus type 11 (HPV11). 7. The vector according to paragraph 6, characterized in that it is a viral vector. 8. The vector according to claim 7, characterized in that the viral vector is an adenoviral vector. 9. The vector according to claim 8, characterized in that the adenoviral vector is a viral vector of a primate other than a human. 10. The vector according to claim 8, characterized in that the adenoviral vector is a gorilla adenoviral vector. 11. A composition comprising a pharmaceutically acceptable carrier and a polynucleotide according to any one of claims 1-4, for use in the treatment of a disease or disorder caused by human papillomavirus type 6 (HPV6) and/or human papillomavirus type 11 (HPV11). 12. Use of the composition according to claim 11 in the preparation of a medicinal product for the treatment of a disease or disorder caused by human papillomavirus type 6 (HPV6) and/or human papillomavirus type 11 (HPV11). 13. The use according to claim 12, wherein the disease or disorder is a disease or disorder associated with HPV6 or HPV11. 14. The use according to claim 12, wherein the disease or disorder is recurrent respiratory papillomatosis (RRP).