# Patent

## Title
Vectors for expression of prostate-associated antigens

## Abstract

The present disclosure provides (a) vectors comprising a multi-antigen construct encoding two, three, or more immunogenic PAA polypeptides; (b) compositions comprising the vectors, (c) methods relating to uses of the vectors and compositions for eliciting an immune response or for treating prostate cancers.

## Background
REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application No. 61/898,966 filed Nov. 1, 2013, which is incorporated herein by reference in its entirety.


REFERENCE TO SEQUENCE LISTING
This application is being filed along with a sequence listing in electronic format. The sequence listing is provided as a file in .txt format entitled “PC72055A_UPDATED_SEQListing_ST25.txt”, created on Feb. 2, 2016 and having a size of 492 KB. The sequence listing contained in the .txt file is part of the specification and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates generally to immunotherapy and specifically to vaccines and methods for treating or preventing neoplastic disorders.
BACKGROUND OF THE INVENTION
Prostate cancer is the second most commonly diagnosed cancer and the fourth leading cause of cancer-related death in men in the developed countries worldwide. Various prostate-associated antigens (PAA), such as prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), and prostate stem cell antigen (PSCA) have been shown to be overexpressed by prostate cancer cells as compared to normal counterparts. These antigens, therefore, represent possible targets for inducing specific immune responses against cancers expressing the antigens via the use of vaccine-based immunotherapy. (See e.g. Marrari, A., M. Iero, et al. (2007). “Vaccination therapy in prostate cancer.” Cancer Immunol Immunother 56(4): 429-45)
PSCA is a 123-amino acid membrane protein. The native full length human PSCA consists of amino adds 1 and 4-125 of SEQ ED NO:21 (without the alanine and serine residues at the second and third position respectively). PSCA has high tissue specificity and is expressed on more than 85% of prostate cancer specimens, with expression levels increasing with higher Gleason scores and androgen independence. It is expressed in 80-100% of bone metastasis of prostate cancer patients.
PSA is a kallikrein-like serine protease that is produced exclusively by the columnar epithelial cells lining the acini and ducts of the prostate gland. PSA mRNA is translated as an inactive 261-amino acid preproPSA precursor. PreproPSA has 24 additional residues that constitute the pre-region (the signal polypeptide) and the propolypeptide. Release of the propolypeptide results in the 237-amino acid, mature extracellular form, which is enzymatically active. The full length sequence of the native human PSA consists of amino acids 4-263 of SEQ ID NO: 15. PSA is organ-specific and, as a result, it is produced by the epithelial cells of benign prostatic hyperplastic (BPH) tissue, primary prostate cancer tissue, and metastatic prostate cancer tissue.
PSMA, also known as Folate hydrolase 1 (FOLH1), is composed of 750 amino acids. The amino acid sequence of the full length human PSMA is provided in SEQ ID NO:1. PSMA includes a cytoplasmic domain (amino acids 1-19), a transmembrane domain (amino acids 20-43), and an extracellular domain (amino acids 44-750). PSMA was found to be expressed in prostate cancer cells it at 1000-fold higher levels than normal tissues. It is abundantly expressed on neovasculature of a variety of other solid tumors such as colon, breast, liver, bladder, pancreas, lung, renal cancers as well as melanoma and sarcomas. Thus, PSMA is considered a target not only specific for prostate cancer cells but also a pan-carcinoma target for other cancers.
While a large number of tumor-associated antigens have been identified and many of these antigens have been explored as protein-based or DNA-based vaccines for the treatment or prevention of cancers, most clinical trials so far have failed to produce a therapeutic product. One of the challenges in developing cancer vaccines resides in the fact that the cancer antigens are usually self-derived and, therefore, poorly immunogenic because the immune system is self-regulated not to recognize self-proteins. Accordingly, a need exists for a method to enhance the immunogenicity or therapeutic effect of cancer vaccines.
Numerous approaches have been explored for enhancing the immunogenicity or enhancing anti-tumor efficacy of cancer vaccines. One of such approach involves the use of various immune modulators, such as TLR agonists, TNFR agonists, CTLA-4 inhibitors, and protein kinase inhibitors.
Toll-like receptors (TLRs) are type 1 membrane receptors that are expressed on hematopoietic and non-hematopoietic cells. At least 11 members have been identified in the TLR family. These receptors are characterized by their capacity to recognize pathogen-associated molecular patterns (PAMP) expressed by pathogenic organisms. These receptors in the innate immune systems exert control over the polarity of the ensuing acquired immune response. Among the TLRs, TLR9 has been extensively investigated for its functions in immune responses. Stimulation of the TLR9 receptor directs antigen-presenting cells (APCs) towards priming potent, TH1-dominated T-cell responses, by increasing the production of pro-inflammatory cytokines and the presentation of co-stimulatory molecules to T cells. CpG oligonucleotides, ligands for TLR9, were found to be a class of potent immunostimulatory factors. CpG therapy has been tested against a wide variety of tumor models in mice, and has consistently been shown to promote tumor inhibition or regression.
Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) is a member of the immunoglobulin superfamily and is expressed on the surface of Helper T cells. CTLA-4 is a negative regulator of CD28 dependent T cell activation, and acts as an inhibitory checkpoint for the adaptive immune response. Similar to the T-cell costimulatory protein CD28, CTLA-4 binds to CD80 and CD86 on antigen-presenting cells. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. Human antibodies against human CTLA-4 have been described as immunostimulation modulators in a number of disease conditions, such as treating or preventing viral and bacterial infection and for treating cancer (WO 01/14424 and WO 00/37504). Various preclinical studies have shown that CTLA-4 blockade by monoclonal antibodies enhances the host immune response against immunogenic tumors, and can even reject established tumors. Two fully human anti-human CTLA-4 monoclonal antibodies (mAbs), ipilimumab (MDX-010) and Tremelimumab (also known as CP-675206), have been investigated in clinical trials in the treatment of various types of solid tumors.
The tumor necrosis factor (TNF) superfamily is a group of cytokines that engage specific cognate cell surface receptors, the TNF receptor (TNFR) superfamily. Members of the tumor necrosis factor superfamily act through ligand-mediated trimerization, causing recruitment of several intracellular adaptors to activate multiple signal transduction pathways, such as apoptosis, NF-kB pathway, JNK pathway, as well as immune and inflammatory responses. Examples of the TNF superfamily include CD40 ligands, OX40 ligands, 4-1BB ligands, CD27, CD30 ligand (CD153), TNF-alpha, TNF-beta, RANK ligands, LT-alpha, LT-beta, GITR ligands, and LIGHT. The TNFR superfamily includes, for example, CD40, OX40, 4-1BB, CD70 (CD27 ligand), CD30, TNFR2, RANK, LT-beta R, HVEM, GITR, TROY, and RELT. Among the TNF members, CD40 agonists, including various CD40 agonistic antibodies, such as the fully human agonist CD40 monoclonal antibody CP870893, have been extensively explored for usage in therapies.
Protein kinases are a family of enzymes that catalyze the phosphorylation of specific residues in proteins. A number of kinase inhibitors have been investigated in clinical investigation for use in anti-cancer therapies, which includes, for example, MK0457, VX-680, ZD6474, MLN8054, AZD2171, SNS-032, PTK787/ZK222584, Sorafenib (BAY43-9006), SU5416, SU6668 AMG706, Zactima (ZD6474), MP-412, Dasatinib, CEP-701, (Lestaurtinib), XL647, XL999, Tykerb, (Lapatinib), MLN518, (formerly known as CT53518), PKC412, ST1571, AMN107, AEE 788, OSI-930, OSI-817, Sunitinib malate (Sutent; SU11248), Vatalanib (PTK787/ZK 222584), SNS-032, SNS-314 and Axitinib (AG-013736). Gefitinib and Erlotinib are two orally available EGFR-TKIs.
SUMMARY OF THE INVENTION
The present disclosure relates to vectors constructed from chimpanzee adenovirus ChAd68 genomic sequences for expression of two or more immunogenic PAA polypeptides. The vector comprises (1) a C68 DNA sequence, (2) a multi-antigen construct for expression of two or more immunogenic PAA polypeptides, and (3) regulatory sequences that control the transcription and translation of the antigen products (i.e., the immunogenic PAA polypeptides). The C68 DNA sequence included in the vector is derived from C68 genomic sequence by functional deletion of one or more viral genes but is sufficient for production of an infectious viral particle. In a particular embodiment, the C68 DNA sequence used in the vector is the entire C68 genome with only functional deletions in the E1 and E3 regions.
The multi-antigen construct carried by the vector comprises nucleotide sequences encoding two or more immunogenic PAA polypeptides selected from immunogenic PSMA polypeptide, immunogenic PSA polypeptide, and immunogenic PSCA polypeptide. In some embodiments, the multi-antigen construct carried by the vector comprises (1) a nucleotide sequence encoding at least one immunogenic PSMA polypeptide, (2) a nucleotide sequence encoding at least one immunogenic PSA polypeptide, and (3) a nucleotide sequence encoding at least one immunogenic PSCA polypeptide. The multi-antigen constructs may also include separator sequences that enable expression of separate PAA polypeptides encoded by the construct. Examples of separator sequences include 2A peptide sequences and IRESs. In some embodiments, the vector comprises a multi-antigen construct having one of the following structures:
(1) PSA-F2A-PSMA-mIRES-PSCA,
(2) PSA-F2A-PSMA-T2A-PSCA;
(3) PSA-T2A-PSCA-F2A-PSMA; and
(4) PSCA-F2A-PSMA-mIRES-PSA.
In some embodiments, the nucleotide sequence encoding the immunogenic PSA polypeptide comprises nucleotides 1115-1825 of SEQ ID NO:58 or comprises nucleotides 1106-1825 of SEQ ID NO:58, the nucleotide sequence encoding the immunogenic PSCA polypeptide comprises nucleotides 1892-2257 of SEQ ID NO:58 or comprises nucleotides 1886-2257 of SEQ ID NO:58, and the nucleotide sequence encoding the immunogenic PSMA polypeptide comprises nucleotides 2333-4543 of SEQ ID NO:58 or comprises nucleotides 2324-4543 of SEQ ID NO:58. In some specific embodiments, the multi-antigen construct comprises nucleotide sequence selected from the group consisting of SEQ ID NOs:33, 34, 35, and 36. In a particular embodiment, the multi-antigen construct comprises a nucleotide sequence that encodes a polypeptide sequence of SEQ ID NO:60. In another particular embodiment, the multi-antigen construct comprises a nucleotide sequence of SEQ ID NO:61.
The present disclosure also provides compositions comprising the vectors. In some embodiments, the composition is an immunogenic composition useful for eliciting an immune response against a PAA in a mammal, such as a mouse, dog, monkey, or human. In some embodiments, the composition is a vaccine composition useful for immunization of a mammal, such as a human, for inhibiting abnormal cell proliferation, for providing protection against the development of cancer (used as a prophylactic), or for treatment of disorders (used as a therapeutic) associated with PAA over-expression, such as cancer, particularly prostate cancer.
The present disclosure further relates to methods of using the vectors or compositions for eliciting an immune response against a PAA, or for treating cancers, such as prostate cancers, in a mammal, particularly a human. In some embodiments, the vectors or compositions, including vaccine compositions, are administered to the mammal, particularly human, in combination with one or more immune modulators that enhance the immunogenicity or effect of the vectors or compositions. In some particular embodiments, the method involves co-administration of a vaccine provided by the present invention in combination with at least one immune-suppressive-cell inhibitor and at least one immune-effector-cell enhancer.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Schematic illustration of PJV7563 vector.
FIG. 2. Amino acid alignment of five viral 2A cassettes (FMDV 2A, ERAV 2A, PTV 2A, EMCV 2A, and TAV 2A). The skipped glycine-proline bonds are indicated by asterisks. The amino acid sequence of FMDV 2A, ERAV 2A, PTV 2A, EMCV 2A, and TAV 2A is set forth in SEQ ID NOs: 67, 68, 69, 70, and 74, respectively.
FIG. 3. Sequence of the preferred EMCV IRES (SEQ ID NO:290). The translation initiation site is indicated by the asterisk. The minimal IRES element excludes the underlined first 5 codons of the EMCV L protein.
FIG. 4. Graph showing the Kaplan-Meier survival curves of the groups of mice from a representative study evaluating the effect of sunitinib malate (Sutent) and an anti-murine CTLA-4 monoclonal antibody (clone 9D9) on the anti-tumor efficacy of a cancer vaccine (vaccine) in subcutaneous TUBO tumor bearing BALB/neuT mice.
FIG. 5. Graph depicting the IFNγ ELISPOT results from a representative study evaluating the effect of CpG7909 and an anti-CD40 antibody (Bioxcell #BE0016-2) on the antigen specific T cell responses induced by a cancer vaccine (rHER2).
FIG. 6. Graphs depicting results of a representative study that evaluates the immunomodulatory activity of CpG7909 on the quality of the immune responses induced by a cancer vaccine (PMED) using intracellular cytokine staining assay, in which cytokine positive CD8 T cells were measured. (*indicates P<0.05 by Student's T-test).
FIG. 7. Graphs depicting results of a representative study that evaluates the immunomodulatory activity of CpG7909 on the quality of the immune responses induced by a cancer vaccine (PMED) using intracellular cytokine staining assay, in which cytokine positive CD4 T cells (FIG. 7) were measured. (*indicates P<0.05 by Student's T-test).
FIG. 8. Graphs depicting results of a representative study that evaluates the immunomodulatory activity of an agonistic anti-murine CD40 monoclonal antibody on the quality of the immune responses induced by a cancer vaccine (PMED) using intracellular cytokine staining assay, in which cytokine positive CD8 T cells were measured. (*indicates P<0.05 by Student's T-test)
FIG. 9. Graphs depicting results of a representative study that evaluates the immunomodulatory activity of an agonistic anti-murine CD40 monoclonal antibody on the quality of the immune responses induced by a cancer vaccine (PMED) using intracellular cytokine staining assay, in which cytokine positive CD4 T cells were measured. (*indicates P<0.05 by Student's T-test)
FIG. 10. Graph showing the Kaplan-Meier survival curves of the groups of mice from a representative study that evaluates the effect of low dose sunitinib malate (Sutent) on the anti-tumor efficacy of a cancer vaccine in spontaneous mammary tumor bearing BALB/neuT mice.
FIG. 11. Graph showing the genomic organization of the AdC68-734 vector. CMV Enh/pro=human cytomegalovirus immediate early enhancer and promoter; tet op=tetracycline operator; T2A=Thosea asigna virus 2A; F2A=Foot and Mouth Disease Virus 2A; SV40 pA=Simian Virus 40 polyadenylation signal; LITR=left inverted terminal repeat; RITR=right inverted terminal repeat.
FIG. 12. Dot plots showing expression of PSMA and PSCA on the surface of A549 cells transduced with triple antigen expressing AdC68 vectors by flow cytometry.
FIG. 13. Western blot from lysates of A549 infected by AdC68 vectors.



DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
The term “adjuvant” refers to a substance that is capable of enhancing, accelerating, or prolonging an immune response elicited by a vaccine immunogen.
The term “agonist” refers to a substance which promotes (induces, causes, enhances or increases) the activity of another molecule or a receptor. The term agonist encompasses substances which bind receptor (e.g., an antibody, a homolog of a natural ligand from another species) and substances which promote receptor function without binding thereto (e.g., by activating an associated protein).
The term “antagonist” or “inhibitor” refers to a substance that partially or fully blocks, inhibits, or neutralizes a biological activity of another molecule or receptor.
The term “co-administration” refers to administration of two or more agents to the same subject during a treatment period. The two or more agents may be encompassed in a single formulation and thus be administered simultaneously. Alternatively, the two or more agents may be in separate physical formulations and administered separately, either sequentially or simultaneously, to the subject. The term “administered simultaneously” or “simultaneous administration” means that the administration of the first agent and that of a second agent overlap in time with each other, while the term “administered sequentially” or “sequential administration” means that the administration of the first agent and that of a second agent does not overlap in time with each other.
The term “cytosolic” means that, after a nucleotide sequence encoding a particular polypeptide is expressed by a host cell, the expressed polypeptide is retained inside the host cell.
The terms “degenerate variant” refers to a nucleotide sequence that has substitutions of bases as compared to a reference nucleotide sequence but, due to degeneracy of the genetic code, encodes the same amino add sequence as the reference nucleotide sequence.
The term “effective amount” refers to an amount administered to a mammal that is sufficient to cause a desired effect in the mammal.
The term “fragment” of a given polypeptide refers to a polypeptide that is shorter than the given polypeptide and shares 100% identity with the sequence of the given polypeptide.
The term “identical” or percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence.
The term “immune-effector-cell enhancer” or “IEC enhancer” refers to a substance capable of increasing or enhancing the number, quality, or function of one or more types of immune effector cells of a mammal. Examples of immune effector cells include cytolytic CD8 T cells, CD40 T cells, NK cells, and B cells.
The term “immune modulator” refers to a substance capable of altering (e.g., inhibiting, decreasing, increasing, enhancing or stimulating) the working of any component of the innate, humoral or cellular immune system of a mammal. Thus, the term “immune modulator” encompasses the “immune-effector-cell enhancer” as defined herein and the “immune-suppressive-cell inhibitor” as defined herein, as well as substance that affects other components of the immune system of a mammal.
The term “immune response” refers to any detectable response to a particular substance (such as an antigen or immunogen) by the immune system of a host vertebrate animal, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells, such as antigen-specific T cells, and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids). Examples of immune responses include an alteration (e.g., increase) in Toll-like receptor activation, lymphokine (e.g., cytokine (e.g., Th1, Th2 or Th17 type cytokines) or chemokine) expression or secretion, macrophage activation, dendritic cell activation, T cell (e.g., CD4+ or CD8+ T cell) activation, NK cell activation, B cell activation (e.g., antibody generation and/or secretion), binding of an immunogen (e.g., antigen (e.g., immunogenic polypolypeptide)) to an MHC molecule, induction of a cytotoxic T lymphocyte (“CTL”) response, induction of a B cell response (e.g., antibody production), and, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells and B cells), and increased processing and presentation of antigen by antigen presenting cells. The term “immune response” also encompasses any detectable response to a particular substance (such as an antigen or immunogen) by one or more components of the immune system of a vertebrate animal in vitro.
The term “immunogenic” refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response, or to improve, enhance, increase or prolong a pre-existing immune response, against a particular antigen, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.
The term “immunogenic PSA polypeptide” refers to a polypeptide that is immunogenic against human PSA protein or against cells expressing human PSA protein.
The term “immunogenic PSCA polypeptide” refers to a polypeptide that is immunogenic against human PSCA protein or against cells expressing human PSCA protein.
The term “immunogenic PSMA polypeptide” refers to a polypeptide that is immunogenic against human PSMA protein or against cells expressing human PSMA protein.
The term “immunogenic PAA polypeptide” refers to an “immunogenic PSA polypeptide,” an “immunogenic PSCA polypeptide,” or an “immunogenic PSMA polypeptide” as defined herein above.
The term “immune-suppressive-cell inhibitor” or “ISC inhibitor” refers to a substance capable of reducing or suppressing the number or function of immune suppressive cells of a mammal. Examples of immune suppressive cells include regulatory T cells (“T regs”), myeloid-derived suppressor cells, and tumor-associated macrophages.
The term “intradermal administration,” or “administered intradermally,” in the context of administering a substance, such as a therapeutic agent or an immune modulator, to a mammal including a human, refers to the delivery of the substance into the dermis layer of the skin of the mammal. The skin of a mammal is composed of three layers—the epidermis, dermis, and subcutaneous layer. The epidermis is the relatively thin, tough, outer layer of the skin. Most of the cells in the epidermis are keratinocytes. The dermis, the skin's next layer, is a thick layer of fibrous and elastic tissue (made mostly of collagen, elastin, and fibrillin) that gives the skin its flexibility and strength. The dermis contains nerve endings, sweat glands and oil (sebaceous) glands, hair follicles, and blood vessels. The dermis varies in thickness depending on the location of the skin. In humans it is about 0.3 mm on the eyelid and about 3.0 mm on the back. The subcutaneous layer is made up of fat and connective tissue that houses larger blood vessels and nerves. The thickness of this layer varies throughout the body and from person to person. The term “intradermal administration” refers to delivery of a substance to the inside of the dermis layer. In contrast, “subcutaneous administration” refers to the administration of a substance into the subcutaneous layer and “topical administration” refers to the administration of a substance onto the surface of the skin.
The term “local administration” or “administered locally” encompasses “topical administration,” “intradermal administration,” and “subcutaneous administration,” each as defined herein above. This term also encompasses “intratumoral administration,” which refers to administration of a substance to the inside of a tumor. Local administration is intended to allow for high local concentrations around the site of administration for a period of time until systemic biodistribution has been achieved with of the administered substance, while “systemic administration” is intended for the administered substance to be absorbed into the blood and attain systemic exposure rapidly by being distributed through the circulatory system to organs or tissues throughout the body.
The term “mammal” refers to any animal species of the Mammalia class. Examples of mammals include: humans; non-human primates such as monkeys; laboratory animals such as rats, mice, guinea pigs; domestic animals such as cats, dogs, rabbits, cattle, sheep, goats, horses, and pigs; and captive wild animals such as lions, tigers, elephants, and the like.
The term “membrane-bound” means that after a nucleotide sequence encoding a particular polypeptide is expressed by a host cell, the expressed polypeptide is bound to, attached to, or otherwise associated with, the membrane of the cell.
The term “neoplastic disorder” refers to a condition in which cells proliferate at an abnormally high and uncontrolled rate, the rate exceeding and uncoordinated with that of the surrounding normal tissues. It usually results in a solid lesion or lump known as “tumor.” This term encompasses benign and malignant neoplastic disorders. The term “malignant neoplastic disorder”, which is used interchangeably with the term “cancer” in the present disclosure, refers to a neoplastic disorder characterized by the ability of the tumor cells to spread to other locations in the body (known as “metastasis”). The term “benign neoplastic disorder” refers to a neoplastic disorder in which the tumor cells lack the ability to metastasize.
The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence “operably linked” to a transgene is ligated in such a way that expression of the transgene is achieved under conditions compatible with the control sequences.
The term “pharmaceutically acceptable excipient” refers to a substance in an immunogenic or vaccine composition, other than the active ingredients (e.g., the antigen, antigen-coding nucleic acid, immune modulator, or adjuvant) that is compatible with the active ingredients and does not cause significant untoward effect in subjects to whom it is administered.
The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically, or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones.
The term “preventing” or “prevent” refers to (a) keeping a disorder from occurring or (b) delaying the onset of a disorder or onset of symptoms of a disorder.
The term “prostate-associated-antigen” (or PAA) refers to the TAA (as defined herein) that is specifically expressed on prostate tumor cells or expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. Examples of PAA include PSA, PSCA, and PSMA.
The term “secreted” in the context of a polypeptide means that after a nucleotide sequence encoding the polypeptide is expressed by a host cell, the expressed polypeptide is secreted outside of the host cell.
The term “suboptimal dose” when used to describe the amount of an immune modulator, such as a protein kinase inhibitor, refers to a dose of the immune modulator that is below the minimum amount required to produce the desired therapeutic effect for the disease being treated when the immune modulator is administered alone to a patient.
The term “treating,” “treatment,” or “treat” refers to abrogating a disorder, reducing the severity of a disorder, or reducing the severity or occurrence frequency of a symptom of a disorder.
The term “tumor-associated antigen” or “TAA” refers to an antigen which is specifically expressed by tumor cells or expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. Tumor-associated antigens may be antigens not normally expressed by the host; they may be mutated, truncated, misfolded, or otherwise abnormal manifestations of molecules normally expressed by the host; they may be identical to molecules normally expressed but expressed at abnormally high levels; or they may be expressed in a context or milieu that is abnormal. Tumor-associated antigens may be, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or any combination of these or other biological molecules.
The term “vaccine” refers to an immunogenic composition for administration to a mammal for eliciting an immune response against a particular antigen.
The term “vector” refers to a nucleic acid molecule capable of transporting or transferring a foreign nucleic acid molecule. The foreign nucleic acid molecule is referred to as “insert” or “transgene.” A vector generally consists of an insert and a larger sequence that serves as the backbone of the vector. The term “vector” encompasses both expression vectors and transcription vectors. The term “expression vector” refers to a vector capable of expressing the insert in the target cell. It generally contains control sequences, such as enhancer, promoter, and terminator sequences, that drive expression of the insert. The term “transcription vector” refers to a vector capable of being transcribed but not translated. Transcription vectors are used to amplify their insert. Based on the structure or origin of vectors, major types of vectors include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus (Ad) vectors, and artificial chromosomes.
B. Vectors Containing a Multi-Antigen Construct
In one aspect, the present disclosure provides a voral vector constructed from the genome of chimpanzee adenovirus ChAd68 for expression of two or more immunogenic PAA polypeptides. Chimpanzee adenovirus ChAd68 is also referred in the literature as simian adenovirus 25, C68, Chad68, SAdV25, PanAd9, or Pan9. For convenience, the chimpanzee adenovirus ChAd68 may be referred to in this specification as “C68” and the viral vector constructed from the genome of chimpanzee adenovirus ChAd68 is referred to as “C68 vector.” The full length genomic sequence of C68 is available from Genbank (Accession Number AC_000011.1) and is provided in SEQ ID NO:57. In addition, the full length genomic sequence of C68 and location of the adenovirus genes E1a, E1b, E2a, E2b, E3, E4, 11, 12, L3, L4, and L5 are also provided in U.S. Pat. No. 6,083,716.
The C68 vector provided by the present disclosure comprises (1) a C68 DNA sequence, and (2) a multi-antigen construct for expression of two or more immunogenic PAA polypeptides. The vector may also contain non-native regulatory sequences that control the transcription and translation of the antigen products. The non-native regulatory sequences refer to sequences that are not part of the C68 genome. The C68 DNA sequence, multi-antigen construct, and regulatory sequences are operably linked to each other.
The C68 vector can be replication-competent, conditionally replication-competent, or replication-deficient. A replication-competent C68 vector can replicate in typical host cells, i.e., cells typically capable of being infected by an adenovirus. A replication-competent viral vector can have one or more mutations as compared to the 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. A conditionally-replicating C68 vector is viral vector that has been engineered to replicate under pre-determined conditions. For example, replication-essential gene functions, e.g., gene functions encoded by the adenoviral early regions, can be operably linked to an inducible, repressible, or tissue-specific transcription control sequence, e.g., promoter. A replication-deficient C68 vector is a viral vector that requires complementation of one or more gene functions or regions of the viral genome that are required for replication, as a result of, for example, a deficiency in one or more replication-essential gene function or regions, such that the vector does not replicate in typical host cells, especially those in a human to be infected by the vector.
The vectors are useful for cloning or expressing the immunogenic PAA polypeptides, or for delivering the multi-antigen construct in a composition, such as a vaccine, to a host cell or to a host animal, such as a human. In some particular embodiments, the present disclosure provides a vector selected from the group consisting of (i) a vector comprising or consisting of the nucleotide sequence of SEQ ID NO:58; (ii) a vector comprising or consisting of nucleotides 9-34811 of SEQ ID NO:58; and (iii) a vector comprising or consisting of the nucleotide sequence of SEQ ID NO:63.
The C68 vector provided by the present disclosure also encompasses functional variants of the vectors specifically described or exemplified in the present disclosure. A “functional variant” refers a vector that contains mutations (e.g., additions, deletions, or substitutions) relative to the sequence of a vector (“parent vector”) specifically described or exemplified in the present disclosure but retains the function or property of the parent vector. For example, functional variant may comprise codon-optimized sequence corresponding to a parent vector exemplified in the present disclosure.
B1. The C68 DNA Sequence
The term “C68 DNA sequence” refers to a DNA sequence that is part of the C68 genomic sequence. The C68 DNA sequence included in the vector is derived from C68 genomic sequence by functional deletion of one or more viral genes or genomic regions. The term “functional deletion” means that a sufficient amount of the gene region of the virus is removed or otherwise changed, e.g., by mutation or modification, so that the gene region is no longer capable of producing functional products of gene expression or is otherwise performing its normal function. Deletion of an entire gene region often is not required for disruption of a replication-essential gene function. However, for the purpose of providing sufficient space in the C68 genome for one or more transgenes, removal of a majority of one or more gene regions may be desirable. While deletion of genetic material is preferred, mutation of genetic material by addition or substitution also is appropriate for disrupting gene function.
In some embodiments, the C68 DNA sequence of the vector is derived from the C68 genomic sequence by functionally deleting the entire, or a sufficient portion of, the adenoviral immediate early gene E1a and delayed early gene E1b. In other embodiments, in addition to the functional deletion of E1a and E1b, functional deletion may also be made to one or more other genes, such as the delayed early gene E2a, delayed early gene E3, E4, any of the late genes L1 through L5, the intermediate genes IX, and IVa2. Thus, the C68 DNA sequence for use in the construction of the vector of the invention may contain deletions in E1 only. Alternatively, deletions of entire genes or portions thereof effective to destroy their biological activity may be used in any combination. For example, in one exemplary vector, the C68 DNA sequence is derived from the C68 genomic sequence by functional deletions of the E1 genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 and E3 genes, or of E1, E2a and E4 genes, with or without deletion of E3, and so on. In addition, such deletions may be used in combination with other mutations, such as temperature-sensitive mutations, to achieve a desired result. In a particular embodiment, the C68 DNA sequence is the entire C68 genome with only functional deletions in the E1 and E3 regions.
In some particular embodiments, the functional deletion of E1 gene is accomplished by deletion of nucleotides 577-3403 of SEQ ID NO:57 or by deletion of nucleotides 456-3012 of SEQ ID NO:57, and the functional deletion of E3 gene is accomplished by deletion of nucleotides 27125-31831 of SEQ ID NO:57 or by deletion of nucleotides 27812-31330 of SEQ ID NO:57. In other particular embodiments, the C68 DNA sequence included in the vector comprises nucleotides 3013-27811 of SEQ ID NO:57. In still other particular embodiments, the C68 DNA sequence included in the vector comprises nucleotides 3013-27811 and 31331-36519 of SEQ ID NO:57.
The multi-antigen construct may be inserted into any deleted region of the adenovirus genome. The multi-antigen construct may also be inserted into an existing gene region to disrupt the function of that region. In some embodiments, the multi-antigen construct is inserted in the place of the deleted E1 gene.
B2. The Multi-Antigen Constructs
The term “multi-antigen construct” refers to a nucleic acid molecule or sequence that encodes two or more PAA polypeptides. Such molecules or sequences may also be referred to as “multi-antigen vaccine” or “multi-antigen plasmid” in the present disclosure. A multi-antigen construct can carry two coding nucleotide sequences wherein each of the coding nucleotide sequences expresses an individual immunogenic PAA polypeptide. Such a construct is also referred to as “dual antigen construct,” “dual antigen vaccine,” or “dual antigen plasmid” in this disclosure. A multi-antigen construct can also carry three coding nucleotide sequences wherein each of the coding nucleotide sequences expresses an individual immunogenic PAA polypeptide. Such a construct is also referred to as “triple antigen construct,” “triple antigen vaccine,” or “triple antigen plasmid” in this disclosure. The individual PAA polypeptides encoded by a multi-antigen construct may be immunogenic against the same antigen, such as PSMA, PSA, or PSCA. For example, a dual antigen construct may express two different PAA antigens that are both immunogenic against PSMA. The individual PAA polypeptides encoded by a multi-antigen construct may be immunogenic against different antigens, for example, a dual antigen construct may express two PAA polypeptides that are immunogenic against PSMA and PSA, respectively. It is preferred that a multi-antigen construct encodes two or more individual PAA polypeptides that are immunogenic against different antigens.
In some embodiments, the multi-antigen construct encodes at least two immunogenic PAA polypeptides in any one of the following combinations:
1) an immunogenic PSMA polypeptide and an immunogenic PSA polypeptide;
2) an immunogenic PSMA polypeptide and an immunogenic PSCA polypeptide; and
3) an immunogenic PSA polypeptide and an immunogenic PSCA polypeptide.
In some particular embodiments, the multi-antigen construct encodes at least one immunogenic PSMA polypeptide, at least one immunogenic PSA polypeptide, and at least one immunogenic PSCA polypeptide.
The immunogenic PSMA polypeptide expressed by a multi-antigen construct may be cytosolic, secreted, or membrane-bound, but preferably membrane-bound. In some embodiments, the immunogenic PSMA polypeptide comprises an amino acid sequence selected from the group consisting of:
1) the amino acid sequence of SEQ ID NO:1,
2) amino acids 15-750 of SEQ ID NO:1;
3) the amino acid sequence of SEQ ID NO:3;
4) the amino acid sequence of SEQ ID NO:5;
5) the amino acid sequence of SEQ ID NO:7;
6) amino acids 4-739 of SEQ ID NO:3;
7) amino acids 4-739 of SEQ ID NO:5;
8) amino acids 4-739 of SEQ ID NO:7;
9) the amino acid sequence of SEQ ID NO:9; and
10) amino acids 4-739 of SEQ ID NO:9.
The immunogenic PSA polypeptide expressed by a multi-antigen construct may be cytosolic, secreted, or membrane-bound, but preferably cytosolic. In some embodiments, the immunogenic PSA polypeptide comprises an amino acid sequence selected from the group consisting of:
1) amino acids 27-263 of SEQ ID NO: 15;
2) the amino acid sequence of SEQ ID NO:17; and
3) amino acids 4-240 of SEQ ID NO:17.
The immunogenic PSCA polypeptide expressed by a multi-antigen construct may be the full length human PSCA protein. In some embodiments, the immunogenic PSCA polypeptide comprises an amino acid sequence selected from the group consisting of:
1) the amino acid sequence of SEQ ID NO:21;
2) amino acids 2-125 of SEQ ID NO; 21, and

    
    
        3) amino acids 4-125 of SEQ ID NO:21.
    
    


In some other embodiments, the multi-antigen construct encodes at least one immunogenic PSA polypeptide, at least one immunogenic PSCA polypeptide, and at least one immunogenic PSMA polypeptide, wherein the immunogenic PSA polypeptide comprises the amino acid sequence of SEQ ID NO:17 or amino acids 4-240 of SEQ ID NO:17, wherein the immunogenic PSCA polypeptide comprises the amino acid sequence of SEQ ID NO:21 or amino acids 2-125 of SEQ ID NO:21, and wherein the immunogenic PSMA polypeptide comprises an amino acid sequence selected from the group consisting of:
1) amino acids 15-750 of SEQ ID NO: 1;
2) amino acids 4-739 of SEQ ID NO:9; and
3) the amino acid sequence of SEQ ID NO: 9.
In some particular embodiments, the multi-antigen construct comprises a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:60 or 64.
In some particular embodiments, the multi-antigen construct comprises: (i) a nucleotide sequence encoding an immunogenic PSA polypeptide, (ii) a nucleotide sequence encoding an immunogenic PSCA polypeptide, and (iii) a nucleotide sequence encoding an immunogenic PSMA polypeptide, wherein:
(1) the nucleotide sequence encoding the immunogenic PSA polypeptide is selected from the group consisting of: (i) nucleotide sequence of SEQ ID NO:18; (ii) nucleotide sequence of SEQ ID NO:20; (iii) nucleotide sequence comprising nucleotides 10-720 of SEQ ID NO:18; (iv) nucleotide sequence comprising nucleotides 1115-1825 of SEQ ID NO:58 or SEQ ID NO:63; (v) nucleotide sequence comprising nucleotides 1106-1825 of SEQ ID NO:58 or SEQ ID NO:63; and (vi) a degerate variant of any of the nucleotide sequences provided in (i)-(v) above.
(2) the nucleotide sequence encoding the immunogenic PSCA polypeptide is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO:22; (ii) a nucleotide sequence comprising nucleotides 10-375 of SEQ ID NO:22; (iii) a nucleotide sequence comprising nucleotides 1892-2257 of SEQ ID NO:58 or SEQ ID NO:63; (iv) a nucleotide sequence comprising nucleotides 1886-2257 of SEQ ID NO:58 or SEQ ID NO:63; and (v) a degerate variant of any of the nucleotide sequences provided in (i)-(iv) above; and
(3) the nucleotide sequence encoding the immunogenic PSMA polypeptide is selected from the group consisting of: (i) the nucleotide sequence comprising nucleotides 43-2250 of SEQ ID NO:2; (ii) the nucleotide sequence of SEQ ID NO:4; (iii) the nucleotide sequence of SEQ ID NO:6; (iv) the nucleotide sequence of SEQ ID NO:8; (v) the nucleotide sequence of SEQ ID NO:10; (vi) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:4; (vii) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:6; (viii) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:8; (ix) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:10; (x) the nucleotide sequence comprising nucleotides 2333-4543 of SEQ ID NO:58 or SEQ ID NO:63; (xi) the nucleotide sequence comprising nucleotides 2324-4543 of SEQ ID NO:58 or SEQ ID NO:63; and (xii) a degerate variant of any of the nucleotide sequences provided in (i)-(xi) above.
In another specific embodiment, the multi-antigen construct comprises a nucleotide sequence encoding an immunogenic PSA polypeptide, a nucleotide sequence encoding an immunogenic PSCA polypeptide, and a nucleotide sequence encoding an immunogenic PSMA polypeptide, wherein: the nucleotide sequence encoding the immunogenic PSA polypeptide comprises nucleotides 1106-1825 of SEQ ID NO:58 or SEQ ID NO:63; the nucleotide sequence encoding the immunogenic PSCA polypeptide comprises nucleotides 1886-2257 of SEQ ID NO:58 or SEQ ID NO:62; and the nucleotide sequence encoding the immunogenic PSMA polypeptide comprises nucleotides 2324-4543 of SEQ ID NO:58 or SEQ ID NO:63.
In order to enable expression of separate immunogenic PAA polypeptides from a single multi-antigen construct carried by the vector, intervening sequences are included between the sequences that encode the individual immunogenic PAA polypeptides (i.e., PSA, PSCA, and PSMA polypeptides). These intervening sequences enable the separate translation of the downstream immunogenic PAA polypeptide. Such an intervening sequence is referred to as “separator sequence” in the specification. Any sequences that can be used for the co-expression of multiple polypeptides from a single vector may be used as separator sequences in the vector provided by the present disclosure. Examples of useful separator sequences includes internal ribosomal entry sites (IRESs) and 2A peptide sequences.
2A peptide and 2A peptide-like sequences, also referred to as cleavage cassettes or CHYSELs (cis-acting hydrolase elements), are approximately 20 amino acids long with a highly conserved carboxy terminal D-V/I-EXNPGP motif (FIG. 2). The sequences are rare in nature, most commonly found in viruses such as Foot-and-mouth disease virus (FMDV), Equine rhinitis A virus (ERAV), Encephalomyocarditis virus (EMCV), Porcine teschovirus (PTV), and Thosea asigna virus (TAV) (Luke, G. A., P. de Felipe, et al. (2008). “Occurrence, function and evolutionary origins of ‘2A-like’ sequences in virus genomes.” J Gen Virol 89(Pt 4): 1036-1042). With a 2A-based multi-antigen expression strategy, genes encoding multiple target antigens are linked together in a single open reading frame, separated by 2A sequences. The entire open reading frame is cloned into a vector with a single promoter and terminator. Upon delivery of the constructs to a host cell, mRNA encoding the multiple antigens is transcribed and translated as a single polyprotein. During translation of the 2A sequences, ribosomes skip the bond between the C-terminal glycine and proline. The ribosomal skipping acts like a cotranslational autocatalytic “cleavage” that releases upstream from downstream proteins. General information regarding use of various 2A peptide sequences in vectors co-expressing multiple polypeptides may be found in Andrea L. Szymczak & Darrio AA Vignali: Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opinion Biol. Ther. (2005)5(5) 627-638, the disclosure of which is incorporated herein by reference. The incorporation of a 2A sequence between two protein antigens results in the addition of  ̃20 amino acids onto the C-terminus of the upstream polypeptide and 1 amino acid (proline) to the N-terminus of downstream protein. In an adaptation of this methodology, protease cleavage sites can be incorporated at the N terminus of the 2A cassette such that ubiquitous proteases will cleave the cassette from the upstream protein (Fang, J., S. Yi, et al. (2007). “An antibody delivery system for regulated expression of therapeutic levels of monoclonal antibodies in vivo.” Mol Ther 15(6): 1153-1159).
Examples of specific 2A-peptide sequences that may be used in the present invention are disclosed in Andrea L. Szymczak & Darrio AA Vignali: Development of 2A peptide-based strategies in the design of multicistronic vectors. Expert Opinion Biol. Ther. (2005)5(5) 627-638, and are provided in Table 1.







TABLE 1







2A-peptide Sequences









Virus
2A-peptide Sequence
SEQ ID NO












Foot and mouse disease virus (FMDV)
VKQTLNFDLLKLAGDVESNPG
67


 


Equine rhinitis A virus (ERAV)
QCTNYALLKLAGDVESNPG
68


 


Porcine teschovirus-1 (PTV1)
ATNF-SLLKQAGDVEENPG
69


 


Encephalomyocarditis virus (EMCV)
HYAGYFADLLIHDIETNPG
70


 


Encephalomyocarditis B variant (EMC-B)
GIFN-AHYAGYFADLLIHDIETNPG
71


 


Theiler murine encephalomyelitis GD7
KAVRGYHADYYKQRLIHDVEMNPG
72








(TME-GD7)



 









Equine rhinitis B virus (ERBV)
GATNF-SLLKLAGDVELNPG
73


 


Thosea asigna virus (TAV)
EGRGSLLTCGDVEENPG
74


 


Drosophilia C (DrosC)
AARQMLLLLSGDVETNPG
75


 


Cricket paralysis virus (CrPV)
FLRKRTQLLMSGDVESNPG
76


 


Acute bee paralysis virus (ABPV)
GSWTDILLLLSGDVETNPG
77


 


Infectious flacherie virus (IFV)
TRAEUEDELIRAGIESNPG
78


 


Porcine rotavirus
AKFQIDKILISGDVELNPG
79


 


Human rotavirus
SKFQIDKILISGDIELNPG
80


 


T. brucei TSR1
SSIIRTKMLVSGDVEENPG
81


 


T. cruzi AP endonuclease
CDAQRQKLLLSGDIEQNPG
82









Internal ribosomal entry sites (IRESs) are RNA elements (FIG. 3) found in the 5′ untranslated regions of certain RNA molecules (Bonnal, S., C. Boutonnet, et al. (2003). “IRESdb: the Internal Ribosome Entry Site database.” Nucleic Acids Res 31(1): 427-428). They attract eukaryotic ribosomes to the RNA to facilitate translation of downstream open reading frames. Unlike normal cellular 7-methylguanosine cap-dependent translation, IRES-mediated translation can initiate at AUG codons far within an RNA molecule. The highly efficient process can be exploited for use in multi-cistronic expression vectors (Bochkov, Y. A. and A. C. Palmenberg (2006). “Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location.” Biotechniques 41(3): 283-284, 286, 288). The RNA sequence of a preferred EMCV IRES (pIRES) is provided in FIG. 3 and SEQ ID NO:290, which has the corresponding DNA sequence of SEQ ID NO:59. The minimal EMCV IRES (mIRES) excludes the underlined first five codons of the EMCV L protein as shown in FIG. 3. Typically, two transgenes are inserted into a vector between a promoter and transcription terminator as two separate open reading frames separated by an IRES. Upon delivery of the constructs to a host cell, a single long transcript encoding both transgenes will be transcribed. The first ORF will be translated in the traditional cap-dependent manner, terminating at a stop codon upstream of the IRES. The second ORF will be translated in a cap-independent manner using the IRES. In this way, two independent proteins can be produced from a single mRNA transcribed from a vector with a single expression cassette. In some embodiments, the multi-antigen construct comprises a EMCV IRES comprising nucleotides 1-553 of SEQ ID NO:59.
Typically, only one separator sequence is needed between two immunogenic PAA polypeptide-coding sequences on a multi-antigen construct. The order of the separator sequences and the nucleotide sequences encoding the PAA polypeptides on a multi-antigen construct is shown in formula (I):

PAA1-SS1-PAA2-SS2-PAA3  (I)

Wherein: (i) PAA1, PAA2, and PAA3 each is a nucleotide sequence encoding an immunogenic PSA polypeptide, a nucleotide sequence encoding immunogenic PSCA polypeptide, or a nucleotide sequence encoding immunogenic PSMA polypeptide, provided that PAA1, PAA2, and PAA3 encode different PAA polypeptides, and (ii) SS1 and SS2 are separator sequences and can be same or different.

In some embodiments, the vector comprises a multi-antigen construct of formula (I) wherein:
(i) PAA1 is a nucleotide sequence encoding an immunogenic PSA polypeptide;
(ii) PAA2 is a nucleotide sequence encoding an immunogenic PSCA or PSMA polypeptide. (where PAA2 is nucleotide sequence encoding an immunogenic PSCA, then PAA3 is a nucleotide sequence encoding an immunogenic PSMA, or Vice Versa);
(iii) SS1 is a 2A-peptide sequence; and
(iv) SS2 is a 2A-peptide sequence or an IRES.
In some particular embodiments, the multi-antigen construct has a structure selected from the group consisting of:
(1) PSA-F2A-PSMA-mIRES-PSCA,
(2) PSA-F2A-PSMA-T2A-PSCA;
(3) PSA-T2A-PSCA-F2A-PSMA; and
(4) PSCA-F2A-PSMA-mIRES-PSA
In a specific embodiment, the vector comprises a multi-antigen construct having a structure of formula (I):

PAA1-SS1-PAA2-SS2-PAA3  (I)

wherein:

(i) PAA1 is a nucleotide sequence encoding an immunogenic PSA polypeptide and comprises nucleotides 1115-1825 SEQ ID NO: 58 or comprises 1106-1114 of SEQ ID NO: 58 or 63;
(ii) PAA2 is a nucleotide sequence encoding an immunogenic PSCA polypeptide and comprises nucleotides 1892-2257 of SEQ ID NO: 58 or comprises 1886-2257 of SEQ ID NO: 58 or 63;
(iii) PAA3 is a nucleotide sequence encoding an immunogenic PSMA polypeptide and comprises nucleotides 2333-4543 SEQ ID NO: 58 or comprises 2324-4543 of SEQ ID NO: 58 or 63;
(iv) SS1 is a nucleotide sequence encoding T2A; and
(v) SS2 is a nucleotide sequence encoding F2A.
The multi-antigen construct may also include a linker sequence positioned between a nucleotide sequence encoding an immunogenic PAA polypeptide (i.e, an immunogenic PSA, PSCA, or PSMA polypeptide) and a down-stream separator sequence. One example of such a linker sequence is a nucleotide sequence encoding glycine-serine.
In some particular embodiments, the multi-antigen construct comprises a nucleotide sequence that encodes an amino acid sequence of SEQ ID NO:60 or encodes an amino acid sequence of SEQ ID NO:61. In a particular embodiment, the multi-antigen construct comprises a nucleotide sequence selected from the groups consisting of nucleotide sequence of SEQ ID NO:61, nucleotide sequence of SEQ ID NO:65, nucleotide sequence of SEQ ID NO:66, and degenerate variant of any of the nucleotide sequences.
B3. Regulatory Sequences
In addition to the separator sequences and linker sequences described herein above, the vector may comprise other non-native regulatory sequences to drive the efficient expression of the encoded PAA polypeptides. Examples of the regulatory sequences includes (1) transcription initiation, termination, promoter, and enhancer sequences; (2) efficient RNA processing signals such as splicing and polyadenylation signals; (3) sequences that stabilize cytoplasmic mRNA; (4) sequences that enhance translation efficiency (i.e., Kozak consensus sequence); (5) sequences that enhance protein stability; and (6) sequences that enhance protein secretion. Examples of promoter systems that can be used in the vectors provided by the present disclosure to drive efficient expression in mammalian cells include SV40 promoter, chicken B actin promoter, human elongation factor promoter, human cytomegalovirus (CMV) promoter, simian CMV promoter, murine CMV promoter, psudorabies promoter, Rous Sarcoma Virus promoter, phosphoglycerate kinase promoter, murine leukemia virus LTR promoter, avian leukosis virus LTR promoter, mouse mammary tumor virus LTR promoter, moloney murine leukemia virus LTR promoter, plasminogen activator inhibitor promoter, CYR61, adenovirus major late promoter, mouse metallothionein promoter, mouse phosphoenol-pyruvate carboxykinase promoter, bovine B-lactoglobulin promoter, bovine prolactin promoter, ubiquitin C promoter, and herpes simplex virus thymidine kinase promoter. Examples of transcription termination signals include SV40 polyadenylation (polyA); bovine growth hormone polyA; rabbit B globin polyA; HSV thymidine kinase, glycoprotein B, and glycoprotein HPV E and L, and synthetic terminators.
In some embodiments, the C68 vectors comprise a human cytomegalovirus (CMV) promoter, optionally with the CMV enhancer, and a SV40 polyA.
C. Compositions Comprising a Vector Carrying a Multi-Antigen Construct (Vector Compositions)
The present disclosure also provides a composition comprising a vector provided by the present disclosure (herein “vector composition’). The vector compositions are useful for eliciting an immune response against a PAA protein in vitro or in vivo in a mammal, including a human. The vector composition may comprise the vectors alone, or may further comprise an excipient.
In some embodiments, the vector composition is a pharmaceutical composition, which comprises a vector provided by the present disclosure and a pharmaceutically acceptable excipient. Suitable excipients for pharmaceutical compositions are known in the arts. The excipients may include aqueous solutions, non aqueous solutions, suspensions, and emulsions. Examples of non-aqueous excipients include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous excipient include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Suitable excipients also include agents that assist in cellular uptake of the vector.
In some embodiments, the pharmaceutical composition is a vaccine composition for administration to humans for inhibiting abnormal cell proliferation, providing protection against the development of cancer (used as a prophylactic), or for treatment of cancer (used as a therapeutic) associated with a PAA over-expression, or for eliciting an immune response to a particular human PAA, such as PSMA, PSA, and PSCA. The vaccine composition may further comprise one or more adjuvants. Examples of adjuvants that may be included in the vaccine compositions are provided herein below.
D. Uses of the Vectors and Vector Compositions
In other aspects, the present disclosure provides methods of using the vector or composition comprising the vectors described herein above.
In one aspect, the present disclosure provides a method of eliciting an immune response against a PAA in a mammal, particularly a human, comprising administering to the mammal an effective amount of (1) a vector containing a multi-antigen construct, or (2) a composition comprising such vectors.
In another aspect, the present disclosure provides a method of inhibiting abnormal cell proliferation in a human, wherein the abnormal cell proliferation is associated with over-expression of a PAA. The method comprises administering to the mammal an effective amount of (1) a vector containing a multi-antigen construct encoding two or more immunogenic PAA polypeptides, or (2) a composition comprising such vectors. In some embodiments, the method is for inhibiting abnormal cell proliferation in prostate in a human. In a particular embodiment, the present disclosure provides a method of inhibiting abnormal cell proliferation in prostate over-expressing PSMA. In some embodiments, the disclosure provides a method of treating prostate cancer in a human, comprising administering to the human an effective amount of a (1) a vector containing a multi-antigen construct or (2) a composition comprising such vectors. In a preferred embodiment, the multi-antigen construct is a triple antigen construct that encodes an immunogenic PSMA polypeptide, an immunogenic PSA polypeptide, and an immunogenic PSCA polypeptide.
The vectors or vector compositions can be administered to an animal, including human, by a number of methods known in the art. Examples of suitable methods include: (1) intramuscular, intradermal, intraepidermal, intravenous, intraarterial, subcutaneous, or intraperitoneal administration, (2) oral administration, and (3) topical application (such as ocular, intranasal, and intravaginal application). One particular method of intradermal or intraepidermal administration of a nucleic acid vaccine composition involves the use of gene gun delivery technology, such the Particle Mediated Epidermal Delivery (PMEDTM) vaccine delivery device marketed by PowderMed. Another particular method for intramuscular administration of a nucleic acid vaccine is injection followed by electroporation.
The effective amount of the vector or vector composition to be administered in a given method can be readily determined by a person skilled in the art and will depend on a number of factors. In a method of treating cancer, such as prostate cancer, factors that may be considered in determining the effective amount include, but not limited: (1) the subject to be treated, including the subject's immune status and health, (2) the severity or stage of the cancer to be treated, (3) the specific immunogenic PAA polypeptides expressed, (4) the degree of protection or treatment desired, (5) the administration method and schedule, (6) formulations used, and (7) co-administration of other therapeutic agents (such as adjuvants or immune modulators). For example, the effective amounts of the vector may be in the range of 2 μg/dose-10 mg/dose when the nucleic acid vaccine composition is formulated as an aqueous solution and administered by hypodermic needle injection or pneumatic injection, whereas only 16 ng/dose-16 μg/dose may be required when the nucleic acid is prepared as coated gold beads and delivered using a gene gun technology.
The vectors or vector compositions, including vaccine compositions, provided by the present disclosure may be used together with one or more adjuvants. Examples of suitable adjuvants include: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl polypeptides or bacterial cell wall components), such as MF59TM (containing 5% Squalene, 0.5% Tween 80, and 0.5% sorbitan trioleate) and SAF (containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP); (2) saponin adjuvants, such as QS21, STIMULONTM (Cambridge Bioscience, Worcester, Mass.), Abisco® (Isconova, Sweden), or Iscomatrix® (Commonwealth Serum Laboratories, Australia); (3) complete Freund's Adjuvant (CFA) and incomplete Freund's Adjuvant (IFA); (4) oligonucleotides comprising CpG motifs, i.e. containing at least one CG dinucleotide, where the cytosine is unmethylated (e.g., Krieg, Vaccine (2000) 19:618-622; Krieg, Curr Opin Mol Ther (2001) 3:15-24; WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581); and (5) metal salt including aluminum salts (such as alum, aluminum phosphate, aluminum hydroxide); (12) a saponin and an oil-in-water emulsion (e.g. WO 99/11241).
The vectors or vector compositions provided by the present disclosure may be used together with one or more immune modulators. In a further aspect, the present disclosure provides a method of treating prostate cancer in a mammal, particularly a human, the method comprising administering to the mammal: (1) an effective amount of a vector, vector composition, or vaccine provided by the present invention; (2) an effective amount of at least one immune-suppressive-cell inhibitor (ISC inhibitor); and (3) an effective amount of at least one immune-effector-cell enhancer (IEC enhancer). This method is also referred to as “vaccine-based immunotherapy regimen” (or “VBIR”) in the present disclosure.
The IEC enhancers and ISC inhibitors may be administered by any suitable methods and routes, including (1) systemic administration such as intravenous, intramuscular, or oral administration, and (2) local administration such intradermal and subcutaneous administration. Where appropriate or suitable, local administration is generally preferred over systemic administration. Local administration of any IEC enhancer and ISC inhibitor can be carried out at any location of the body of the mammal that is suitable for local administration of pharmaceuticals; however, it is more preferable that these immune modulators are administered locally at close proximity to the vaccine draining lymph node.
Two or more specific IEC enhancers from a single class of IEC enhancers (for examples, two CTLA-antagonists) may be administered in combination with the ISC inhibitors. In addition, two or more specific IEC enhancers from two or more different classes of IEC enhancers (for example, one CTLA-4 antagonist and one TLR agonist, or one CTLA-4 antagonist and one PD-1 antagonist) may be administered together. Similarly, two or more specific ISC inhibitors from a single class of ISC inhibitors (for examples, two or more protein kinase inhibitors) may be administered in combination with the IEC enhancers. In addition, two or more specific ISC inhibitors from two or more different classes of ISC inhibitors (for example, one protein kinase inhibitor and one COX-2 inhibitor) may be administered together.
The vectors or vector compositions may be administered simultaneously or sequentially with any or all of the immune modulators (i.e., ISC inhibitors and IEC enhancers) used. Similarly, when two or more immune modulators are used, they may be administered simultaneously or sequentially with respect to each other. In some embodiments, a vector or vector composition is administered simultaneously (e.g., in a mixture) with respect to one immune modulator, but sequentially with respect to one or more additional immune modulators. Co-administration of the vector or vector composition and the immune modulators can include cases in which the vaccine and at least one immune modulator are administered so that each is present at the administration site, such as vaccine draining lymph node, at the same time, even though the antigen and the immune modulators are not administered simultaneously. Co-administration of the vaccine and the immune modulators also can include cases in which the vaccine or the immune modulator is cleared from the administration site, but at least one cellular effect of the cleared vaccine or immune modulator persists at the administration site, such as vaccine draining lymph node, at least until one or more additional immune modulators are administered to the administration site. In cases where a nucleic acid vaccine is administered in combination with a CpG, the vaccine and CpG may be contained in a single formulation and administered together by any suitable method. In some embodiments, the nucleic acid vaccine and CpG in a co-formulation (mixture) is administered by intramuscular injection in combination with electroporation.
Any ISC inhibitors may be used in combination with the vectors or vector compositions provided by the present invention. Examples of classes of ISC inhibitors include PD-1/PD-L1 antagonists, protein kinase inhibitors, cyclooxygenase-2 (COX-2) inhibitors, phosphodiesterase type 5 (PDE5) inhibitors, and DNA crosslinkers. Examples PD-1/PD-L1 antagonists include anti-PD-1 and PD-L1 monoclonal antibodies Examples of COX-2 inhibitors include celecoxib and rofecoxib. Examples of PDE5 inhibitors include avanafil, lodenafil, mirodenafil, sildenafil, tadalafil, vardenafil, udenafil, and zaprinast. An example of DNA crosslinkers is cyclophosphamide. Examples of specific protein kinase inhibitors are described in details below.
The term “protein kinase inhibitor” refers to any substance that acts as a selective or non-selective inhibitor of a protein kinase. The term “protein kinases” refers to the enzymes that catalyze the transfer of the terminal phosphate of adenosine triphosphate to tyrosine, serine or threonine residues in protein substrates. Protein kinases include receptor tyrosine kinases and non-receptor tyrosine kinases. Examples of receptor tyrosine kinases include EGFR (e.g., EGFR/HER1/ErbB1, HER2/Neu/ErbB2, HER3/ErbB3, HER4/ErbB4), INSR (insulin receptor), IGF-IR, IGF-II1R, IRR (insulin receptor-related receptor), PDGFR (e.g., PDGFRA, PDGFRB), c-KIT/SCFR, VEGFR-1/FLT-1, VEGFR-2/FLK-1/KDR, VEGFR-3/FLT-4, FLT-3/FLK-2, CSF-1R, FGFR 1-4, CCK4, TRK A-C, MET, RON, EPHA 1-8, EPHB 1-6, AXL, MER, TYRO3, TIE, TEK, RYK, DDR 1-2, RET, c-ROS, LTK (leukocyte tyrosine kinase), ALK (anaplastic lymphoma kinase), ROR 1-2, MUSK, AATYK 1-3, and RTK 106. Examples of non-receptor tyrosine kinases include BCR-ABL, Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack, and LIMK. In the vaccine-based immunotherapy regimen provided by the present disclosure, the protein kinase inhibitors are administered to the mammal at a suboptimal dose. The term “suboptimal dose” refers to the dose amount that is below the minimum effective dose when the tyrosine kinase inhibitor is administered in a monotherapy (i.e., where the protein kinase inhibitor is administered alone without any other therapeutic agents) for the target neoplastic disorder.
Examples of specific protein kinase inhibitors suitable for use in the vaccine-based immunotherapy regimen include lapatinib, AZD 2171, ET180CH 3, indirubin-3′-oxime, NSC-154020, PD 169316, quercetin, roscovitine, triciribine, ZD1839, 5-lodotubercidin, adaphostin, aloisine, alsterpaullone, aminogenistein, API-2, apigenin, arctigenin, ARRY-334543, axitinib, AY-22989, AZD 2171, Bisindolylmaleimide IX, CCI-779, chelerythrine, DMPQ, DRB, edelfosine, ENMD-981693, erbstatin analog, erlotinib, fasudil, gefitinib (ZD1839), H-7, H-8, H-89, HA-100, HA-1004, HA-1077, HA-1100, hydroxyfasudil, kenpaullone, KN-62, KY12420, LFM-A13, luteolin, LY294002, LY-294002, mallotoxin, ML-9, MLN608, NSC-226080, NSC-231634, NSC-664704, NSC-680410, NU6102, olomoucine, oxindole I, PD 153035, PD 98059, phloridzin, piceatannol, picropodophyllin, PKI, PP1, PP2, PTK787/ZK222584, PTK787/ZK-222584, purvalanol A, rapamune, rapamycin, Ro 31-8220, rottlerin, SB202190, SB203580, sirolimus, SL327, SP600125, staurosporine, STI-571, SU1498, SU4312, SU5416, semaxanib, SU6656, SU6668, syk inhibitor, TBB, TCN, tyrphostin AG 1024, tyrphostin AG 490, tyrphostin AG 825, tyrphostin AG 957, U0126, W-7, wortmannin, Y-27632, zactima, ZM 252868, gefitinib, sunitinib malate, erlotinib, lapatinib, canertinib, semaxinib, vatalanib, sorafenib, imatinib, dasatinib, leflunomide, vandetanib, and nilotinib.
In some embodiments, the protein kinase inhibitor is a multi-kinase inhibitor, which is an inhibitor that acts on more than one specific kinase. Examples of multi-kinase inhibitors include imatinib, sorafenib, lapatinib, BIRB-796, and AZD-1152, AMG706, zactima, MP-412, sorafenib, dasatinib, lestaurtinib, XL647, XL999, lapatinib, MLN518, (also known as CT53518), PKC412, ST1571, AEE 788, OSI-930, OSI-817, sunitinib malate, erlotinib, gefitinib, axitinib, bosutinib, temsirolismus and nilotinib. In some particular embodiments, the tyrosine kinase inhibitor is sunitinib, sorafenib, or a pharmaceutically acceptable salt or derivative (such as a malate or a tosylate) of sunitinib or sorafenib.
Sunitinib malate, which is marketed by Pfizer Inc. under the trade name SUTENT, is described chemically as butanedioic acid, hydroxy-, (2S)-, compound with N-[2-(diethylamino)ethyl]-5-[(Z)-(5-fluoro-1,2-dihydro-2-oxo-3H-indol-3-ylidine)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxamide (1:1). The compound, its synthesis, and particular polymorphs are described in U.S. Pat. No. 6,573,293, U.S. Patent Publication Nos. 2003-0229229, 2003-0069298 and 2005-0059824, and in J. M. Manley, M. J. Kalman, B. G. Conway, C. C. Ball, J. L Havens and R. Vaidyanathan, “Early Amidation Approach to 3-[(4-amido)pyrrol-2-yl]-2-indolinones,” J. Org. Chew. 68, 6447-6450 (2003). Formulations of sunitinib and its L-malate salt are described in PCT Publication No. WO 2004/024127. Sunitinib malate has been approved in the U.S. for the treatment of gastrointestinal stromal tumor, advanced renal cell carcinoma, and progressive, well-differentiated pancreatic neuroendocrine tumors in patients with unresectable locally advanced or metastatic disease. The recommended dose of sunitinib malate for gastrointestinal stromal tumor (GIST) and advanced renal cell carcinoma (RCC) for humans is 50 mg taken orally once daily, on a schedule of 4 weeks on treatment followed by 2 weeks off (Schedule 4/2). The recommended dose of sunitinib malate for pancreatic neuroendocrine tumors (pNET) is 37.5 mg taken orally once daily.
In the vaccine-based immunotherapy regimen, sunitinib malate may be administered orally in a single dose or multiple doses. Typically, sunitinib malate is delivered for two, three, four or more consecutive weekly doses followed by a “off” period of about 1 or 2 weeks, or more where no sunitinib malate is delivered. In one embodiment, the doses are delivered for about 4 weeks, with 2 weeks off. In another embodiment, the sunitinib malate is delivered for two weeks, with 1 week off. However, it may also be delivered without a “off’ period for the entire treatment period. The effective amount of sunitinib malate administered orally to a human in the vaccine-based immunotherapy regimen is typically below 40 mg per person per dose. For example, it may be administered orally at 37.5, 31.25, 25, 18.75, 12.5, 6.25 mg per person per day. In some embodiments, sunitinib malate is administered orally in the range of 1-25 mg per person per dose. In some other embodiments, sunitinib malate is administered orally in the range of 6.25, 12.5, or 18.75 mg per person per dose. Other dosage regimens and variations are foreseeable, and will be determined through physician guidance.
Sorafenib tosylate, which is marketed under the trade name NEXAVAR, is also a multi-kinase inhibitor. Its chemical name is 4-(4-{3-[4-Chloro-3-(trifluoromethyl) phenyl]ureido}phenoxy)-N-methylpyrid-ine-2-carboxamide. It is approved in the U.S. for the treatment of primary kidney cancer (advanced renal cell carcinoma) and advanced primary liver cancer (hepatocellular carcinoma). The recommended daily dose is 400 mg taken orally twice daily. In the vaccine-based immunotherapy regimen provided by the present disclosure, the effective amount of sorafenib tosylate administered orally is typically below 400 mg per person per day. In some embodiments, the effective amount of sorafenib tosylate administered orally is in the range of 10-300 mg per person per day. In some other embodiments, the effective amount of sorafenib tosylate administered orally is between 10-200 mg per person per day, such as 10, 20, 60, 80, 100, 120, 140, 160, 180, or 200 mg per person per day.
Axitinib, which is marketed under the trade name INLYTA, is a selective inhibitor of VEGF receptors 1, 2, and 3. Its chemical name is (N-Methyl-2-[3-((E)-2-pyridin-2-yl-vinyl)-1H-indazol-6-ylsulfanyl]-benzamide. It is approved for the treatment of advanced renal cell carcinoma after failure of one prior systemic therapy. The starting dose is 5 mg orally twice daily. Dose adjustments can be made based on individual safety and tolerability. In the vaccine-based immunotherapy regimen provided by the present disclosure, the effective amount of axitinib administered orally is typically below 5 mg twice daily. In some other embodiments, the effective amount of axitinib administered orally is between 1-5 mg twice daily. In some other embodiments, the effective amount of axitinib administered orally is between 1, 2, 3, 4, or 5 mg twice daily.
In the vaccine-based immunotherapy regimens any IEC enhancers may be used. They may be small molecules or large molecules (such as protein, polypeptide, DNA, RNA, and antibody). Examples of IEC enhancers that may be used include TNFR agonists, CTLA-4 antagonists, TLR agonists, programmed cell death protein 1 (PD-1) antagonists (such as anti-PD-1 antibody CT-011), and programmed cell death protein 1 ligand 1 (PD-L1) antagonists (such as BMS-936559), lymphocyte-activation gene 3 (LAG3) antagonists, and T cell Immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) antagonists. Examples of specific TNFR agonists, CTLA-4 antagonists, and TLR agonists are provided in details herein below.
TNFR Agonists.
Examples of TNFR agonists include agonists of OX40, 4-1BB (such as BMS-663513), GITR (such as TRX518), and CD40. Examples of specific CD40 agonists are described in details herein below.
CD40 agonists are substances that bind to a CD40 receptor on a cell and are capable of increasing one or more CD40 or CD40L associated activities. Thus, CD40 “agonists” encompass CD40 “ligands”.
Examples of CD40 agonists include CD40 agonistic antibodies, fragments CD40 agonistic antibodies, CD40 ligands (CD40L), and fragments and derivatives of CD40L such as oligomeric (e.g., bivalent, trimeric CD40L), fusion proteins containing and variants thereof.
CD40 ligands for use in the present invention include any peptide, polypeptide or protein, or a nucleic acid encoding a peptide, polypeptide or protein that can bind to and activate one or more CD40 receptors on a cell. Suitable CD40 ligands are described, for example, in U.S. Pat. No. 6,482,411; U.S. Pat. No. 6,410,711; U.S. Pat. No. 6,391,637; and U.S. Pat. No. 5,981,724, all of which patents and application and the CD40L sequences disclosed therein are incorporated by reference in their entirety herein. Although human CD40 ligands will be preferred for use in human therapy, CD40 ligands from any species may be used in the invention. For use in other animal species, such as in veterinary embodiments, a species of CD40 ligand matched to the animal being treated will be preferred. In certain embodiments, the CD40 ligand is a gp39 peptide or protein oligomer, including naturally forming gp39 peptide, polypeptide or protein oligomers, as well as gp39 peptides, polypeptides, proteins (and encoding nucleic acids) that comprise an oligomerization sequence. While oligomers such as dimers, trimers and tetramers are preferred in certain aspects of the invention, in other aspects of the invention larger oligomeric structures are contemplated for use, so long as the oligomeric structure retains the ability to bind to and activate one or more CD40 receptor(s).
In certain other embodiments, the CD40 agonist is an anti-CD40 antibody, or antigen-binding fragment thereof. The antibody can be a human, humanized or part-human chimeric anti-CD40 antibody. Examples of specific anti-CD40 monoclonal antibodies include the G28-5, mAb89, EA-5 or S2C6 monoclonal antibody, and CP870893. In a particular embodiment, the anti-CD40 agonist antibody is CP870893 or dacetuzumab (SGN-40).
CP-870,893 is a fully human agonistic CD40 monoclonal antibody (mAb) that has been investigated clinically as an anti-tumor therapy. The structure and preparation of CP870,893 is disclosed in WO2003041070 (where the antibody is identified by the internal identified “21.4.1”). The amino acid sequences of the heavy chain and light chain of CP-870,893 are set forth in SEQ ID NO: 40 and SEQ ID NO: 41, respectively. In clinical trials, CP870,893 was administered by intravenous infusion at doses generally in the ranges of 0.05-0.25 mg/kg per infusion. In a phase I clinical study, the maximum tolerated dose (MTD) of CP-870893 was estimated to be 0.2 mg/kg and the dose-limiting toxicities included grade 3 CRS and grade 3 urticaria. [Jens Ruter et al.: Immune modulation with weekly dosing of an agonist CD40 antibody in a phase I study of patients with advanced solid tumors. [Cancer Biology & Therapy 10:10, 983-993; Nov. 15, 2010.]. In the vaccine-based immunotherapy regimen provided by the present disclosure, CP-870,893 can be administered intradermally, subcutaneously, or topically. It is preferred that it is administered intradermally. The effective amount of CP870893 to be administered in the regimen is generally below 0.2 mg/kg, typically in the range of 0.01 mg-0.15 mg/kg, or 0.05-0.1 mg/kg.
Dacetuzumab (also known as SGN-40 or huS2C6; CAS number 88-486-59-9) is another anti-CD40 agonist antibody that has been investigated in clinical trials for indolent lymphomas, diffuse large B cell lymphomas and Multiple Myeloma. In the clinical trials, dacetuzumab was administered intravenously at weekly doses ranging from 2 mg/kg to 16 mg/kg. In the vaccine-based immunotherapy regimen provided by the present disclosure, dacetuzumab can be administered intradermally, subcutaneously, or topically. It is preferred that it is administered intradermally. The effective amount of dacetuzumab to be administered in the vaccine-based immunotherapy regimen is generally below 16 mg/kg, typically in the range of 0.2 mg-14 mg/kg, or 0.5-8 mg/kg, or 1-5 mg/kg.
CTLA-4 Inhibitors.
Suitable anti-CTLA-4 antagonist agents for use in the vaccine-based immunotherapy regimen provided by the disclosure include, without limitation, anti-CTLA-4 antibodies (such as human anti-CTLA-4 antibodies, mouse anti-CTLA-4 antibodies, mammalian anti-CTLA-4 antibodies, humanized anti-CTLA-4 antibodies, monoclonal anti-CTLA-4 antibodies, polyclonal anti-CTLA-4 antibodies, chimeric anti-CTLA-4 antibodies, anti-CTLA-4 domain antibodies), fragments of anti-CTLA-4 antibodies (such as (single chain anti-CTLA-4 fragments, heavy chain anti-CTLA-4 fragments, and light chain anti-CTLA-4 fragments), and inhibitors of CTLA-4 that agonize the co-stimulatory pathway. In some embodiments, the CTLA-4 inhibitor is Ipilimumab or Tremelimumab.
Ipilimumab (also known as MEX-010 or MDX-101), marketed as YERVOY, is a human anti-human CTLA-4 antibody. Ipilimumab can also be referred to by its CAS Registry No. 477202-00-9, and is disclosed as antibody 10DI in PCT Publication No. WO01/14424, which is incorporated herein by reference in its entirety. Examples of pharmaceutical composition comprising Ipilimumab are provided in PCT Publication No. WO2007/67959. Ipilimumab is approved in the U.S. for the treatment of unresectable or metastatic melanoma. The recommended dose of Ipilimumab as monotherapy is 3 mg/kg by intravenous administration every 3 weeks for a total of 4 doses. In the methods provided by the present invention, Ipilimumab is administered locally, particularly intradermally or subcutaneously. The effective amount of Ipilimumab administered locally is typically in the range of 5-200 mg/dose per person. In some embodiments, the effective amount of Ipilimumab is in the range of 10-150 mg/dose per person per dose. In some particular embodiments, the effective amount of Ipilimumab is about 10, 25, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.
Tremelimumab (also known as CP-675,206) is a fully human IgG2 monoclonal antibody and has the CAS number 745013-59-6. Tremelimumab is disclosed as antibody 11.2.1 in U.S. Pat. No. 6,682,736, incorporated herein by reference in its entirety and for all purposes. The amino acid sequences of the heavy chain and light chain of Tremelimumab are set forth in SEQ IND NOs:42 and 43, respectively. Tremelimumab has been investigated in clinical trials for the treatment of various tumors, including melanoma and breast cancer; in which Tremelimumab was administered intravenously either as single dose or multiple doses every 4 or 12 weeks at the dose range of 0.01 and 15 mg/kg. In the regimens provided by the present invention, Tremelimumab is administered locally, particularly intradermally or subcutaneously. The effective amount of Tremelimumab administered intradermally or subcutaneously is typically in the range of 5-200 mg/dose per person. In some embodiments, the effective amount of Tremelimumab is in the range of 10-150 mg/dose per person per dose. In some particular embodiments, the effective amount of Tremelimumab is about 10, 25, 37.5, 40, 50, 75, 100, 125, 150, 175, or 200 mg/dose per person.
Toll-Like Receptor (TLR) Agonists.
The term “toll-like receptor agonist” or “TLR agonist” refers to a compound that acts as an agonist of a toll-like receptor (TLR). This includes agonists of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and TLR11 or a combination thereof. Unless otherwise indicated, reference to a TLR agonist compound can include the compound in any pharmaceutically acceptable form, including any isomer (e.g., diastereomer or enantiomer), salt, solvate, polymorph, and the like. In particular, if a compound is optically active, reference to the compound can include each of the compound's enantiomers as well as racemic mixtures of the enantiomers. Also, a compound may be identified as an agonist of one or more particular TLRs (e.g., a TLR7 agonist, a TLR8 agonist, or a TLR7/8 agonist).
In some embodiments, the TLR agonists are TLR9 agonists, particularly CpG oligonucleotides (or CpG.ODN). A CpG oligonucleotide is a short nucleic acid molecule containing a cytosine followed by a guanine linked by a phosphate bond in which the pyrimidine ring of the cytosine is unmethylated. A CpG motif is a pattern of bases that include an unmethylated central CpG surrounded by at least one base flanking (on the 3′ and the 5′ side of) the central CpG. CpG oligonucleotides include both D and K oligonucleotides. The entire CpG oligonucleotide can be unmethylated or portions may be unmethylated. Examples of CpG oligonucleotides useful in the methods provided by the present disclosure include those disclosed in U.S. Pat. Nos. 6,194,388, 6,207,646, 6,214,806, 6,283,71, 6,239,116, and 6,339,068.
Examples of particular CpG oligonucleotides useful in the methods provided by the present disclosure include:









5′ TCGTCGTTTTGTCGTTTTGTCGTT3′ (CpG 7909);



 



5′ TCGTCGTTTTTCGGTGCTTTT3′ (CpG 24555);



and



 



5′ TCGTCGTTTTTCGGTCGTTTT3′ (CpG 10103).






CpG7909, a synthetic 24mer single stranded oligonucleotide, has been extensively investigated for the treatment of cancer as a monotherapy and in combination with chemotherapeutic agents, as well as an adjuvant for vaccines against cancer and infectious diseases. It was reported that a single intravenous dose of CpG 7909 was well tolerated with no clinical effects and no significant toxicity up to 1.05 mg/kg, while a single dose subcutaneous CpG 7909 had a maximum tolerated dose (MTD) of 0.45 mg/kg with dose limiting toxicity of myalgia and constitutional effects. [See Zent, Clive S, et al: Phase I clinical trial of CpG 7909 (PF-03512676) in patients with previously treated chronic lymphocytic leukemia. Leukemia and Lymphoma, 53(2):211-217(7)(2012)]. In the regimens provided by the present disclosure, CpG7909 may be administered by injection into the muscle or by any other suitable methods. It is preferred that it is administered locally in proximity to the vaccine draining lymph node, particularly by intradermal or subcutaneous administration. For use with a nucleic acid vaccine, such as a DNA vaccine, a CpG may be preferably co-formulated with the vaccine in a single formulation and administered by intramuscular injection coupled with electroporation. The effective amount of CpG7909 by intramuscular, intradermal, or subcutaneous administration is typically in the range of 10 μg/dose-10 mg/dose. In some embodiments, the effective amount of CpG7909 is in the range of 0.05 mg-14 mg/dose. In some particular embodiments, the effective amount of CpG7909 is about 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 05 1 mg/dose. Other CpG oligonucleotides, including CpG 24555 and CpG 10103, may be administered in similar manner and dose levels.
In some particular embodiments, the present disclosure provides a method of enhancing the immunogenicity or therapeutic effect of a vaccine for the treatment of a neoplastic disorder in a human, comprising administering the human (1) an effective amount of at least one ISC inhibitor and (2) an effective amount of at least one IEC enhancer, wherein the at least one ISC inhibitor is protein kinase inhibitor selected from sorafenib tosylate, sunitinib malate, axitinib, erlotinib, gefitinib, axitinib, bosutinib, temsirolismus, or nilotinib and wherein the at least one IEC enhancer is selected from a CTLA-4 inhibitor, a TLR agonist, or a CD40 agonist. In some preferred embodiments, regimen comprises administering to the human (1) an effective amount of at least one ISC inhibitor and (2) effective amount of at least one IEC enhancer, wherein the at least one ISC inhibitor is a protein kinase inhibitor selected from axitinib, sorafenib tosylate, or sunitinib malate and wherein the at least one IEC enhancer is a CTLA-4 inhibitor selected from Ipilimumab or Tremelimumab. In some further preferred embodiments, the regimen comprises administering to the human (1) an effective amount of at least one ISC inhibitor and (2) an effective amount of at least two IEC enhancers, wherein the at least one ISC inhibitor is a protein kinase inhibitor selected from sunitinib or axitinib and wherein the at least two IEC enhancers are Tremelimumab and a TLR agonist selected from CpG7909, CpG2455, or CpG10103.
In some other embodiments, the present disclosure provides a method of treating prostate cancer in a human, comprising administering to the human (1) an effective amount of a vaccine capable of eliciting an immune response against a human PAA, (2) an effective amount of at least one ISC inhibitor, and (3) an effective amount of at least one IEC enhancer, wherein the at least one ISC inhibitor is a protein kinase inhibitor selected from sorafenib tosylate, sunitinib malate, axitinib, erlotinib, gefitinib, axitinib, bosutinib, temsirolismus, or nilotinib, and wherein the at least one IEC enhancer is selected from a CTLA-4 inhibitor, a TLR agonist, or a CD40 agonist. In some preferred embodiments, the method comprises administering to the human (1) an effective amount of a vaccine capable of eliciting an immune response against a human PAA, (2) an effective amount of at least one ISC inhibitor, and (3) an effective amount of at least one IEC enhancer, wherein the at least one ISC inhibitor is a protein kinase inhibitor selected from sorafenib tosylate, sunitinib malate, or axitinib and wherein the at least one IEC enhancer is a CTLA-4 inhibitor selected from Ipilimumab or Tremelimumab.
In some further specific embodiments, the method comprises administering to the human (1) an effective amount of at least one ISC inhibitor and (2) an effective amount of at least two IEC enhancers, wherein the at least one ISC inhibitor is a protein kinase inhibitor selected from sunitinib or axitinib and wherein the at least two IEC enhancers are Tremelimumab and a TLR agonist selected from CpG7909, CpG2455, or CpG10103.
Additional Therapeutic Agents.
The vaccine-based immunotherapy regimen provided by the present disclosure may further comprise an additional therapeutic agent. A wide variety of cancer therapeutic agents may be used, including chemotherapeutic agents and hormone therapeutic agents. The term “chemotherapeutic agent” refers to a chemical or biological substance that can cause death of cancer cells, or interfere with growth, division, repair, and/or function of cancer cells. Examples of particular chemotherapeutic agents include: abiraterone acetate, cabazitaxel, degarelix, denosumab, docetaxel, enzalutamide, leuprolide acetate, prednisone, sipuleucel-T, and radium 223 dichloride. The term “hormone therapeutic agent” refers to a chemical or biological substance that inhibits or eliminates the production of a hormone, or inhibits or counteracts the effect of a hormone on the growth and/or survival of cancer cells. Examples of particular hormone therapeutic agents include leuprolide, goserelin, triptorelin, histrelin, bicalutamide, flutamide, and nilutamide. The VBIR provided by this disclosure may also be used in combination with other therapies, including (1) surgical methods that remove all or part of the organs or glands which participate in the production of the hormone, such as the ovaries, the testicles, the adrenal gland, and the pituitary gland, and (2) radiation treatment, in which the organs or glands of the patient are subjected to radiation in an amount sufficient to inhibit or eliminate the production of the targeted hormone.
E. Examples
The following examples are provided to illustrate certain embodiments of the invention. They should not be construed to limit the scope of the invention in any way. From the above discussion and these examples, one skilled in the art can ascertain the essential characteristics of the invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usage and conditions.
Example 1
Antigens in Cytosolic, Secreted, and Membrane-Bound Formats Derived from the Human PSMA Protein
1A. Design of Immunogenic PSMA Polypeptides
DNA constructs encoding immunogenic PSMA polypeptides in cytosolic, secreted, and modified formats were constructed based on the native human PSMA protein sequence and tested for their ability to induce anti-tumor effector immune responses. The structure and preparation of each of the human PSMA antigen formats are provided as follows.
1A1. Human PSMA Cytosolic Antigen.
An immunogenic PSMA polypeptide in cytosolic form was designed to retain the immunogenic polypeptide inside the cell once it is expressed. The cytoplasmic domain (amino acids 1-19) and the transmembrane domain (amino acids 20-43) of the human PSMA were removed, resulting in a cytosolic PSMA polypeptide that consists of amino acids 44-750 (extracellular domain or ECD) of the human PSMA of SEQ ID NO: 1. The optimal Kozak sequence “MAS” may be added to the N-terminus of the polypeptide for enhancing the expression or to facilitate cloning.
1A2. Human PSMA Secreted Antigen.
An immunogenic PSMA polypeptide in secreted form was designed to secret the polypeptide outside of the cell once it is expressed. The secreted polypeptide is made with amino acids 44-750 (ECD) of the human PSMA of SEQ ID NO:1 and the Ig Kappa secretory element that has the amino acid sequence ETDTLLLWVLLLWVPGSTGD and a two-amino acid linker (AA) in the N-terminal in order to maximize the secretion of the PSMA antigen once it is expressed.
1A3. Human PSMA Membrane-Bound Antigen.
An immunogenic PSMA membrane-bound polypeptide was designed to stabilize the polypeptide on the cell surface. The first 14 amino adds of the human PSMA protein were removed and the resultant immunogenic polypeptide consists of amino adds 15-750 of the human PSMA protein of SEQ ID NO:1. The immunogenic polypeptide that consists of amino adds 15-750 of the native human PSMA protein of SES ID NO: 1 and share 100% sequence identity with the native human PSMA protein is also referred to as “human PSMA modified,” “hPSMA modified,” or “hPSMAmod” antigen in the present disclosure. The following three immunogenic PSMA polypeptides (referred to as “shuffled PSMA modified antigens”) that are variants of the human PSMA modified antigen (SEQ ID NO:9) were also generated:
(1) shuffled PSMA modified antigen 1 having the amino acid sequence of SEQ ID NO:3;
(2) shuffled PSMA modified antigen 2 having the amino acid sequence of SEQ ID NO:5; and
(3) shuffled PSMA modified antigen 3 having the amino acid sequence of SEQ ID NO:7.
The nucleodie sequences encoding the shuffled PSMA modified antigens 1, 2, and 3 are set forth in SEQ ID NOs: 4, 6, and 8, respectively.
1B. Preparation of DNA Plasmids for Expressing the PSMA Antigens
DNA constructs encoding the PSMA cytosolic, PSMA secreted, and PSMA modified antigens were cloned individually into PJV7563 vector that was suitable for in vivo testing in animals (FIG. 1). Both strands of the DNA in the PJV7563 vectors were sequenced to confirm the design integrity.
A large scale plasmid DNA preparation (Qiagen/CsCl) was produced from a sequence confirmed clone. The quality of the plasmid DNA was confirmed by high 260/280 ratio, high super coiled/nicked DNA ratio, low endotoxin levels (<10 U/mg DNA) and negative bio burden.
1C. Expression of PSMA Constructs in Mammalian Cells
The expression of the PSMA cytosolic, secreted, and modified antigens was determined by FACS. Mammalian 293 cells were transfected with the PJV7563 PMED vectors encoding the various immunogenic PSMA polypeptides. Three days later, the 293 cells were stained with mouse anti-PSMA antibody, followed with a fluorescent conjugated (FITC) rat anti-mouse secondary antibody. The results are presented tin Table 2. The data were reported as mean fluorescent intensity (MFI) over negative controls, confirmed that human PSMA modified antigen is expressed on the cell surface.







TABLE 2







Expression of Human PSMA Modified antigen on Cell Surface









Average mean fluorescent


Samples
intensity











Untransfected 293 cells
231


293 cells transfected with full length
6425


human PSMA (SEQ ID NO: 1)


293 cells transfected with human PSMA
12270


modified antigen (SEQ ID NO: 9)









Example 2
Design of Various Immunogenic PSA Polypeptides
3A. Construction of Immunogenic PSA Polypeptides
Similar to what was described in Example 1 for the three different immunogenic PSMA polypeptide forms (e.g., the cytosolic, membrane-bound, and secreted forms), immunogenic PSA polypeptides in the three forms were also designed based on the human PSA sequence. An immunogenic PSA polypeptide in cytosolic form, which consists of amino acids 25-261 of the native human PSA, is constructed by deleting the secretory signal and the pro domain (amino acids 1-24). The amino acid sequence of this cytosolic immunogenic PSA polypeptide is provided in SEQ ID NO: 17. The secreted form of the PSA polypeptide is the native full length human PSA (amino acids 1-261). An immunogenic PSA polypeptide in membrane-bound form is constructed by linking the immunogenic PSA polypeptide cytosolic form (amino acids 25-261 of the native human PSA) to the human PSMA transmembrane domain (amino acids 15-54 of the human PSMA).
38. Immune Responses in Pasteur and HLA A24 Mice
Study Design.
Eight to 10 week old HLA A2 Pasteur mice or HLA A24 mice were immunized with DNA expressing the various PSA antigens using PMED provided in Example 3A in a prime/boost/boost regimen with two week intervals between each vaccination as described in Example 1. The antigen specific T and B cell responses were measured 7 days after the last immunization in an interferon-gamma (IFNγ) ELISPOT assay and sandwich ELISA.
ELISpot data shown in Table 3 indicates that immunogenic PSA polypeptides in both cytosolic and membrane-bound forms are capable of inducing T cells that recognize human tumor cells transduced with adenovirus to express the cytosolic PSA antigen (SKmel5-Ad-PSA) but not cells transduced with adenovirus to express eGFP (SKmel5-Ad-eGFP). These two antigens also elicited response to PSA protein. The PSA secreted antigen failed to induce T cells to both SKmel5-Ad-PSA or PSA protein. SFC>50 is considered positive.







TABLE 3







The induction of T cell responses by PSA antigens in Pasteur mice


to PSA + HLA A2.1 + SKmel5 human cancer cells








HLA A2.1 + human
IFN-γ SFC/1 × 106 splenocytes (SD)










cancer cells or

PSA membrane-



protein
PSA cytosolic
bound
PSA secreted





SKmel5-Ad-eGFP
7.7 (9.6)
1.2 (1.4)
2.9 (2.7)


SKmel5-Ad-PSA
112.0 (169.3)
546.1 (379.6)
18.7 (18.5)


PSA protein
108.8 (161.0)
536.9 (380.9)
20.6 (21) 
















TABLE 4







The induction of anti-PSA antibody response


as measured by a sandwich ELISA assay












ELISA (OD = 1.0)




Antigen Forms
Average (SD)
# of positive







PSA cytosolic
99 (0)
0/6



PSA membrane-bound
4838 (835)
6/6



PSA secreted
 1151 2410)
2/6










Data in Table 4 demonstrates that immunogenic PSA polypeptides in both secreted and membrane-bound forms are capable of inducing anti-PSA antibody responses.
Example 3
Construction of Multi-Antigen Vaccine Constructs
In this Example, constructions of plasmids comprising a multi-antigen construct using different strategies are described. These plasmids share the same general plasmid backbone as pPJV7563. Unless otherwise specified, the genes included in the multi-antigen constructs encode (1) an immunogenic PSMA polypeptide of SEQ ID NO:9, (2) an immunogenic PSCA polypeptide comprising amino acids 2-125 of SEQ ID NO:21, and (3) an immunogenic PSA polypeptide of SEQ ID NO:17.
Example 3A
Plasmids Comprising a Dual Antigen Construct
3a 1. Construction of Plasmid Utilizing Multiple Promoters
Plasmid 460 (PSMA/PSCA Dual Promoter).
Plasmid 460 was constructed using the techniques of site-directed mutagenesis, PCR, and restriction fragment insertion. First, a Kpn I restriction site was introduced upstream of the CMV promoter in plasmid 5259 using site-directed mutagenesis with MD5 and MD6 primers according to manufacturer's protocol (Quickchange kit, Agilent Technologies, Santa Clara, Calif.). Second, an expression cassette consisting of a minimal CMV promoter, human PSMA, and rabbit B globulin transcription terminator was amplified by PCR from plasmid 5166 using primers that carried Kpn I restriction sites (MD7 and MD8). The PCR amplicon was digested with Kpn I and inserted into the newly introduced Kpn I site of calf intestinal alkaline phosphatase (CIP)-treated plasmid 5259.
3A2. Construction of Dual Antigen Constructs Utilizing 2A Peptides
Plasmid 451 (PSMA-T2A-PSCA).
Plasmid 451 was constructed using the techniques of overlapping PCR and restriction fragment exchange. First, the gene encoding human PSMA amino acids 15-750 was amplified by PCR using plasmid 5166 as a template with primers 119 and 117. The gene encoding full-length human PSCA was amplified by PCR using plasmid 5259 as a template with primers 118 and 120. PCR resulted in the addition of overlapping TAV 2A (T2A) sequences at the 3′ end of PSMA and 5′ end of PSCA. The amplicons were mixed together and amplified by PCR with primers 119 and 120. The PSMA-T2A-PSCA amplicon was digested with Nhe I and Bgl II and inserted into similarly digested plasmid 5166. A glycine-serine linker was included between PSMA and the T2A cassette to promote high cleavage efficiency.
Plasmid 454 (PSCA-F2A-PSMA).
Plasmid 454 was created using the techniques of PCR and restriction fragment exchange. First, the gene encoding full-length human PSCA was amplified by PCR using plasmid 5259 as a template with primers 42 and 132. The amplicon was digested with BamH I and inserted into similarly digested, CIP-treated plasmid 5300. A glycine-serine linker was included between PSCA and the FMDV 2A (F2A) cassette to promote high cleavage efficiency.
Plasmid 5300 (PSA-F2A-PSMA)
Plasmid 5300 was constructed using the techniques of overlapping PCR and restriction fragment exchange. First, the gene encoding PSA amino acids 25-261 was amplified by PCR from plasmid 5297 with primers MD1 and MD2. The gene encoding human PSMA amino acids 15-750 was amplified by PCR from plasmid 5166 with primers MD3 and MD4. PCR resulted in the addition of overlapping F2A sequences at the 3′ end of PSA and 5′ end of PSMA. The amplicons were mixed together and extended by PCR. The PSA-F2A-PSMA amplicon was digested with Nhe I and Bgl II and inserted into similarly digested plasmid pPJV7563.
3A3. Dual Antigen Constructs Utilizing Internal Ribosomal Entry Sites
Plasmid 449 (PSMA-mIRES-PSCA).
Plasmid 449 was constructed using the techniques of overlapping PCR and restriction fragment exchange. First, the gene encoding full length human PSCA was amplified by PCR from plasmid 5259 with primers 124 and 123. The minimal EMCV IRES was amplified by PCR from pShuttle-IRES with primers 101 and 125. The overlapping amplicons were mixed together and amplified by PCR with primers 101 and 123. The IRES-PSCA amplicon was digested with Bgl II and BamH I and inserted into Bgl II-digested, CIP-treated plasmid 5166. In order to fix a spontaneous mutation within the IRES, the IRES containing Avr II to Kpn I sequence was replaced with an equivalent fragment from pShuttle-IRES.
Plasmid 603 (PSCA-pIRES-PSMA).
Plasmid 603 was constructed using the techniques of PCR and seamless cloning. The gene encoding full length human PSCA attached at its 3′end to a preferred EMCV IRES was amplified from plasmid 455 by PCR with primers SD546 and SD547. The gene encoding human PSMA amino acids 15-750 was amplified by PCR from plasmid 5166 using primers SD548 and SD550. The two overlapping PCR amplicons were inserted into Nhe I and Bgl II-digested pPJV7563 by seamless cloning according to manufacturer's instructions (Invitrogen, Carlsbad, Calif.).
Plasmid 455 (PSCA-mIRES-PSA).
Plasmid 455 was constructed using the techniques of overlapping PCR and restriction fragment exchange. First, the gene encoding human PSA amino acids 25-261 was amplified by PCR from plasmid 5297 with primers 115 and 114. The minimal EMCV IRES was amplified by PCR from pShuttle-IRES with primers 101 and 116. The overlapping amplicons were mixed together and amplified by PCR with primers 101 and 114. The IRES-PSA amplicon was digested with Bgl II and BamH I and inserted into Bgl II-digested, CIP-treated plasmid 5259. In order to fix a spontaneous mutation within this clone, the Bgl II to BstE II sequence was replaced with an equivalent fragment from a fresh overlapping PCR reaction.
Example 3B
Plasmids Comprising a Triple Antigen Construct
General Strategy.
A number of dual antigen plasmids, including PSA-F2A-PSMA, PSMA-mIRES-PSCA, PSMA-T2A-PSCA, PSA-T2A-PSCA, PSCA-F2A-PSMA, PSCA-pIRES-PSMA, and PSMA-mIRES-PSA, were selected for incorporation in various combinations into triple antigen plasmid vectors. In all cases, the plasmid vectors were based on the parental pPJV7563 plasmid backbone. Four plasmid vectors (plasmids 456, 457, 458, and 459) utilized a single full CMV promoter with a rabbit B globulin transcription terminator to drive expression of all three antigens. Two other plasmid vectors (plasmids 846 and 850) incorporated a dual promoter strategy in combination with either an IRES or 2A to drive expression of the three antigens. Plasmids with multiple 2A cassettes were engineered to carry different cassettes to minimize the likelihood of recombination between the first and second cassette during plasmid/vector amplification. Antigen expression was demonstrated by flow cytometry (FIGS. 7A and 7B) and western blotting (FIGS. 8A and 8B).
Plasmid 456 (PSA-F2A-PSMA-mIRES-PSCA).
Plasmid 456 was constructed by restriction fragment exchange. Plasmid 5300 was digested with Nhe I and Hpa I and the  ̃1.8 kb insert was ligated into similarly digested plasmid 449.
Plasmid 457 (PSA-F2A-PSMA-T2A-PSCA).
Plasmid 457 was constructed by restriction fragment exchange. Plasmid 5300 was digested with Nhe I and Hpa I and the  ̃1.8 kb insert was ligated into similarly digested plasmid 451.
Plasmid 458 (PSA-T2A-PSCA-F2A-PSMA).
Plasmid 458 was constructed using the techniques of PCR and restriction fragment exchange. The gene encoding human PSA amino acids 25-261 was amplified by PCR from plasmid 5297 with primers 119 and 139, resulting in the addition of a T2A sequence and Nhe I restriction site at the 3′ end. The amplicon was digested with Nhe I and inserted into similarly digested plasmid 454.
Plasmid 459 (PSCA-F2A-PSMA-mIRES-PSA).
Plasmid 459 was constructed by restriction fragment exchange. Plasmid 454 was digested with Nhe I and Bgl II and the PSCA-F2A-PSMA containing insert was ligated into similarly digested plasmid 455.
Plasmid 846 (CBA-PSA, CMV-PSCA-pIRES-PSMA).
Plasmid 846 was constructed using the techniques of PCR and seamless cloning. First, an expression cassette was synthesized that consisted of 1) the promoter and 5′ untranslated region from the chicken beta actin (CBA) gene, 2) a hybrid chicken beta actin/rabbit beta globin intron, 3) the gene encoding human PSA amino acids 25-261, and 4) the bovine growth hormone terminator. This PSA expression cassette was amplified by PCR from plasmid 796 with primers 3SaIICBA and 5SaIIBGH. The amplicon was cloned into the SaII site of plasmid 603 using a GeneArt Seamless Cloning and Assembly Kit (Invitrogen, Carlsbad, Calif.). Upon delivery of this plasmid into a cell, PSA expression will be driven off the CBA promoter while PSCA and PSMA expression will be driven off the CMV promoter.
Plasmid 850 (CBA-PSA, CMV-PSCA-F2A-PSMA).
Plasmid 850 was constructed using the techniques of PCR and seamless cloning. First, the CBA promoter-driven PSA expression cassette was amplified by PCR from plasmid 796 with primers 3SaIICBA and 5SaIIBGH. The amplicon was cloned into the SaII site of plasmid 454 using GeneArt Seamless Cloning. Upon delivery of this plasmid into a cell, PSA expression will be driven off the CBA promoter while PSCA and PSMA expression will be driven off the CMV promoter.
Plasmid 916 ((PSA-T2A-PSCA-F2A-PSMA).
Plasmid 916 was constructed using the techniques of PCR and Gibson cloning. The genes encoding the three PAA polypeptides were amplified by PCR and ligated into the Nhe I/Bgl II sites of pPJV7563 by Gibson cloning techniques. The complete nucleotide sequence of Plasmid 916 is set forth in SEQ ID NO:62. Plasmid 458 and Plasmid 916 encode the same amino acid sequence that comprises the three immunogenic PAA polypeptides, which amino acid sequence is set forth in SEQ ID NO:60. The nucleotide sequence in Plasmid 916 that encodes the amino acid sequence comprising the three PAA polypeptides is codon-optimized and is also set forth in SEQ ID NO:61.







TABLE 21







List of Primers Used in the Construction of the Multi-antigen Plasmids










Primer
Sequence (5′ to 3′)
Strand
SEQ ID NO





 42
CGTTGACGCAAATGGGCGGTAGG
Sense
 83


 


101
TCAGAGATCTGACCCCCTAACGTTACTGGC
Sense
 84


 


114
TATAGGATCCTCAGGGGTTGGCCACGATG
Antisense
 85


 


115
GAAAAACACGATGATAATATGGCCAGCATTGTGGGAG
Sense
 86










GCTGGGAGTG



 










116
CCACAATGCTGGCCATATTATCATCGTGTTTTTCAAAG
Antisense
 87



GAAAACCACGTCC




 


117
CATCTCCACAGGTCAATAATGAACCCCTACCTTCGGAT
Antisense
 88










CCGGCTACTTCACTCAAAGTC



 










118
GTTCATTATTGACCTGTGGAGATGTCGAAGAAAACCCA
Sense
 89










GGACCCGCAAGCAAGGCTGTGCTGCTTGCCCTG



 










119
TTGCCTCTCACATCTCGTCAATCTCCGCGAGGAC
Sense
 90


 


120
GATCTTTTGTACAATATGATCTTGTGGCAATGTCCC
Antisense
 91


 


123
TATAGGATCCCTATAGCTGGCCGGGTCC
Antisense
 92


 


124
CACGATGATAATATGGCCAGCAAGGCTGTGCTGCTTG
Sense
 93










CC



 










125
CACAGCCTTGCTGGCCATATTATCATCGTGTTTTTCAAA
Antisense
 94










GGAAAACCACGTCC



 










132
TATAGGATCCTAGCTGGCCGGGTCCCCAGAG
Antisense
 95


 


139
ATATGCTAGCGGGTCCTGGGTTTTCTTCGACATCTCCA
Antisense
 96










CAGGTCAATAATGAACCCCTACCTTCGGATCCGGGG




TTGGCCACGATGGTGTCC



 










SD546
CTGTGACGAACATGGCTAGCAAGG
Sense
 97


 


SD547
ATTATCATCGTGTTTTTCAAAGGAAAACC
Antisense
 98


 


SD548
AAACACGATGATAATATGGCCACAACCATGGCGCGCC
Sense
 99










GCCCGC



 










SD550
TTTTGTTAGGGCCCAGATCTTTAGGC
Antisense
100


 


MD1
GACGAACATGGCTAGCATTGTGGGAGGCTG
Sense
101


 


MD2
CCACATCGCCTGCCAGTTTCAGCAGATCAAAGTTCAGG
Antisense
102










GTCTGGGATCCGGGGTTGGCCACGATGGTGTC



 










MD3
GATCTGCTGAAACTGGCAGGCGATGTGGAAAGCAACC
Sense
103










CAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTG



 










MD4
GTTAGGGCCCAGATCTTTAGGCTACTTCACTCAAAGTC
Antisense
104


 


MD5
CTTGTATTACTGTTTATGTAAGCAGACAGGGTACCAAT
Sense
105










ATTGGCTATTGGCCATTGCATAC



 










MD6
GTATGCAATGGCCAATAGCCAATATTGGTACCCTGTCT
Antisense
106










GCTTACATAAACAGTAATACAAG



 










MD7
CATGCATGGGTACCAATCTTCCGAGTGAGAGACACAAA
Sense
107










AAATTCC



 










MD8
GATCGATCGGTACCCTGCAGGTCGAGCACCAAAATCA
Antisense
108










ACGGG



 










5SalIB
GTTTATGTAAGCAGACAGGTCGACCCATAGAGCCCAC
Antisense
109









GH
CGCATCCCCAGC



 










3SalIC
TGGCCAATAGCCAATATTGTCGACTGGGTCGAGGTGA
Sense
110









BA
GCCCCACGTTCTG










Example 3C
Triple Antigen Adenovirus Vectors
General Strategy.
As with DNA plasmids, viral vectors can be engineered to deliver multiple prostate cancer antigens. The three multi-antigen expression strategies described above for multi-antigen constructs—dual promoters, 2A peptides, and internal ribosome entry sites—were incorporated in various combinations to create triple antigen adenovirus vectors. Briefly, the multi-antigen expression cassettes were cloned into a pShuttle-CMV plasmid modified to carry two copies of the tetracycline operator sequence (TetO2). Recombinant adenovirus serotype 5 vectors were created using the AdEasy Vector System according to manufacturer's protocols (Agilent Technologies, Inc., Santa Clara, Calif.). Viruses were amplified in HEK293 cells and purified by double cesium chloride banding according to standard protocols. Prior to in vivo studies, viral stocks were thoroughly characterized for viral particle concentration, infectivity titer, sterility, endotoxin, genomic and transgene integrity, transgene identity and expression.
Adenovirus-733 (PSA-F2A-PSMA-T2A-PSCA).
Ad-733 is the viral equivalent of plasmid 457. Expression of the three antigens is driven off a single CMV promoter with a tetracycline operator for repressing transgene expression during large scale production in Tet repressor expressing HEK293 lines. Multi-antigen expression strategies include two different 2A sequences.
Adenovirus-734 (PSA-T2A-PSCA-F2A-PSMA).
Ad-734 is the viral equivalent of plasmid 458. Expression of the three antigens is driven off a single CMV promoter with a tetracycline operator for repressing transgene expression during large scale production in Tet repressor expressing HEK293 lines. Multi-antigen expression strategies include two different 2A sequences.
Adenovirus-735 (PSCA-F2A-PSMA-mIRES-PSA).
Ad-735 is the viral equivalent of plasmid 459. Expression of the three antigens is driven off a single CMV promoter with a tetracycline operator for repressing transgene expression during large scale production in Tet repressor expressing HEK293 lines. Multi-antigen expression strategies include a 2A sequence and an IRES.
Adenovirus-796 (CBA-PSA, CMV-PSCA-pIRES-PSMA).
Ad-796 is the viral equivalent of plasmid 846. Expression of PSA is driven off the chicken beta actin promoter while PSCA and PSMA expression is driven off the CMV-TetO2 promoter. Multi-antigen expression strategies include two promoters and an IRES.
Adenovirus-809 (CBA-PSA, CMV-PSCA-F2A-PSMA).
Ad-809 is the viral equivalent of plasmid 850. Expression of PSA is driven off the chicken beta actin promoter while PSCA and PSMA expression is driven off the CMV-TetO2 promoter. Multi-antigen expression strategies include two promoters and a 2A sequence.
Example 4
Anti-Cancer Efficacy of Vaccine in Combination with Sunitinib and Anti-CTLA-4 Antibody
The anti-tumor efficacy of a cancer vaccine in combination with sunitinib and anti-CTLA-4 monoclonal antibody (clone 9D9) was investigated in subcutaneous TUBO tumor bearing BALB/neuT mice.
Study Procedure.
Briefly, ten mice per each group were daily orally dosed with either vehicle or sunitinib malate at 20 mg/kg starting at day 10 post tumor implant until day 64. Vaccination with DNA constructs that either encode no antigen (control vaccine) or a rat Her-2 antigen of SEQ Id NO: 54 (cancer vaccine) as adenovirus vectors initiated on day 13 subsequently followed by two weekly immunizations, two biweekly immunizations, and seven weekly immunizations of respective antigens (HBV antigens or rHer-2) by DNA. The groups of mice (closed circle and open triangle) that were treated with anti-murine CTLA-4 monoclonal antibody were intraperitoneally injected with 250 μg of the antibody on day 20, 27, 41, 55, 62, 69, 76, 83, 90, and 97 right after the PMED injections.
Results.
FIG. 4 shows the Kaplan-Meier survival curve of the groups of mice from a representative study evaluating the anti-tumor efficacy of sunitinib and anti-murine CTLA-4 monoclonal antibody (clone 9D9) in combination with a cancer vaccine. Increased survival time was observed in mice treated with Sutent with control vaccine (open circle), anti-murine CTLA-4 monoclonal antibody (open triangle) or cancer vaccine (closed triangle). A further increase of survival was observed in mice treated with Sutent and cancer vaccine in combination with anti-murine CTLA-4 (closed circle). P values were calculated by log-rank test.
Example 5
Effect of CPG or CD40 Agonist on the Immune Responses Induced by Cancer Vaccine
Immunogenicity Studies in BALB/c Mice
The effect of local administration of immune modulators on the magnitude and quality of antigen specific immune responses induced by a cancer was investigated in BALB/c mice, in which the immune response was assessed by measuring rHER2 specific T cell responses using the IFNγ ELISPOT assay or intracellular cytokine staining assay. Briefly, 4 to 6 female BALB/c mice per group as indicated were immunized with DNA plasmid expression constructs encoding rHER2 antigen sequences (SEQ ID NO:54) by PMED delivery system. The immune modulators, CpG7909 (PF-03512676) and anti-CD40 monoclonal agonistic antibody, were administered locally by intradermal injections in proximity to the vaccine draining inguinal lymph node subsequently after the PMED actuations. Antigen specific T cell responses were measured by IFNγ ELISPOT or intracellular cytokine staining assay according to the procedure described below.
Intracellular Cytokine Staining (ICS) Assay
The rHer-2 specific polyfunctional (multi-cytokine positive) T cell immune responses were measured from splenocytes or PBMCs isolated from individual animals by ICS assay. Typically 1e6 splenocytes were incubated with Brefeldin A at 1 μg/ml and peptide stimulant (rHer-2 specific CD8 p66, rHer-2 specific CD4 p169 or irrelevant HBV p87) at 10 μg/ml for 5 hr at 37° C. in a 5% CO2 incubator. After the stimulation, the splenocytes were washed and blocked with Fc□ block (anti-mouse CD16/CD32) for 10 min. at 4° C. followed by a 20 min staining with Live/dead aqua stain, anti-mouse CD3ePE-Cy7, anti-mouse CD8a Pacific blue, and anti-mouse CD45R/B220 PerCP-Cy5.5. The cells were washed, fixed with 4% paraformaldehyde overnight at 4° C., permeabilized with BD fix/perm solution for 30 min at RT and incubated with anti-mouse IFNγ APC, anti-mouse TNF□ Alexa488 and anti-mouse IL-2 PE for 30 min at RT. The cells were washed and 20,000 CD4 or CD8 T cells were acquired for analysis by flow cytometry. The total number of antigen specific single, double or triple cytokine positive T cells per total spleen of each animal is calculated by subtracting the rHer-2 specific responses to the irrelevant peptide HBV from the vaccine specific responses and normalized to the total number of splenocytes isolated from the spleen.
IFNγ ELISPOT Assay Results
FIG. 5 shows the IFNy ELISPOT results from groups of mice from a representative study evaluating the magnitude of antigen specific T cell responses induced by the rHER2 vaccine when given with the immune modulators as indicated. Briefly, each mouse per treatment group (n=4) was immunized with DNA plasmid expression constructs encoding rHER2 antigen sequences (SEQ ID NO:54) by PMED immediately followed by either 100 ug of control rat IgG monoclonal antibody (Bioxcell #BE0089: control mAb) or 50 □g CpG7909 or 100 ug of anti-CD40 monoclonal antibody (Bioxcell #BE0016-2: a-CD40 mAb) as indicated. The antigen specific immune responses were measured by IFNy ELISPOT assay from 5e5 splenocytes mixed with control or rHer-2 specific p66 peptides at 10 μg/ml concentration, 7 days after the PMED actuation. The number of total IFNy secreting cells from splenocytes containing 1e5 CD8 T cells was calculated from the ELISPOT results from individual animals and the % of CD8 T cells in splenocytes and mean and standard error of mean of each group are plotted. As shown, both CpG7909 and the anti-CD40 monoclonal antibody significantly enhanced the magnitude of antigen specific immune responses induced by rHer-2 DNA compared to mice that received control antibodies.
Intracellular Cytokine Staining (ICS) Assay Results.
FIGS. 6 and 7 show the results of a representative study that evaluates the immunomodulatory activity of CpG 7909 on the quality of the vaccine induced immune responses by intracellular cytokine staining assay. Briefly, each animal was immunized twice with the DNA plasmid expression constructs encoding rHER2 antigen sequences (SEQ ID NO:54) delivered by PMED with a 4-week interval. The mice in each group (n=5) were given intradermal injections of either PBS (PMED group) or 50 □g of CpG 7909 (PMED+CpG group) in proximity to the right side vaccine draining inguinal node immediately following both DNA immunizations by PMED. Seven days after the last immunization by PMED, an ICS assay was performed on the splenocytes isolated from each individual mice to detect antigen specific polyfunctional CD8 or CD4 T cells that secrete IFNy, TNF□ and/or IL-2. A significant increase in rHer-2 specific multi-cytokine positive CD8 and CD4 T cell responses were detected from mice treated with the local delivery of CpG 7909 compared to PBS. An increase in the single cytokine positive CD8 population was observed in the animals that received local delivery of CpG7909 administration compared to PBS.
FIGS. 8 and 9 show the results of a representative study that evaluates the immunomodulatory activity of an agonistic anti-CD40 monoclonal antibody on the quality of the vaccine induced immune responses by intracellular cytokine staining assay. Briefly, each animal was immunized twice by DNA plasmid expression constructs encoding rHER2 antigen sequences (SEQ ID NO:54) delivered by PMED with a 4 week interval. The mice in each group (n=6) were given 100 □g of intradermal injections of either isotype IgG control (PMED with IgG) or anti-CD40 monoclonal antibody (PMED with aCD40) in proximity to the right side vaccine draining inguinal node, one day after the first immunization was administered by PMED. Seven days after the last PMED, an ICS assay was performed on the splenocytes isolated from each individual mice to detect rHer-2 specific polyfunctional CD8 or CD4 T cells that secrete IFN□, TNF□ and/or IL-2. A significant increase in the rHer-2 specific triple-cytokine positive CD8 and CD4 T cell responses were detected from mice treated with the local delivery of anti-CD40 monoclonal antibody compared to isotype IgG control. There were also significant increases in rHer-2 specific single and double cytokine positive CD4 T cells by anti-CD40 monoclonal antibody given locally.
Example 6
Anti-Cancer Efficacy of Cancer Vaccine in Combination with Low Dose Sunitinib
Anti-tumor efficacy of anti-cancer vaccine in combination with low dose sunitinib was investigated in BALB/neuT mice with spontaneous mammary pad tumors.
Animal Treatment.
Briefly, 13-14 weeks old female mice were orally given sunitinib malate (Sutent) at 5 mg/kg for 112 days twice a day. The control vaccine, which delivers no antigen, and cancer vaccine which delivers a rat Her-2 antigen of SEQ ID NO: 54 (rHer-2), were given by adenovirus injections on day 3 as a prime followed by 7 biweekly administrations by PMED of DNA delivering HBV antigens (control vaccine) or rHer-2 (cancer vaccine) respectively. The survival end point was determined when all ten mammary pads became tumor positive or when the volume of any of the mammary tumors reached 2000 mm3.
Results.
The results are presented in FIG. 10. Compared to previously published pharmacokinetic profile of Sutent (Mendel, D., Laird, D., et al.: “In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: determination of a pharmacokinetic/pharmacodynamic relationship”. Clinical Cancer Research, 203, 9:327-337), the CMax of Sutent in mice dosed twice a day at 5 mg/kg is expected to be significantly lower than the minimum blood levels necessary to achieve efficient anti-tumor efficacy in mice and man. The data shows a quick and temporary improvement in the survival of the mice treated with low dose Sutent monotherapy. However when given with the cancer vaccine, a more persistent and significant improvement of survival was observed (P<0.0001 by Log rank test).
Example 7
Enhancement of Vaccine-Induced Immune Responses by Local Administration of CPG
The immune enhancement of local administration of CpG (PF-03512676) on the immune responses induced by a human PSMA nucleic acid provided by the invention was investigated in a monkey study, in which the immune response was assessed by measuring PSMA specific T cell responses using an IFNγ ELISPOT assay.
Animal Treatment and Sample Collection.
Six groups of Chinese cynomolgus macaques, six (#1 to 6) per each test group, were immunized with a plasmid DNA encoding the human PSMA modified antigen (the polypeptide of SEQ ID NO:9) delivered by electroporation. Briefly, all animals received bilateral intramuscular injections of 5 mg of plasmid DNA followed by electroporation (DNA EP) on day 0. Subsequently right after the electroporation, group 2 received bilateral intramuscular injections of 2 mg of CpG mixed with 1 mg Alum in proximity to the DNA injection sites. Groups 3 and 4 received bilateral intramuscular injections of 2 mg of CpG delivered without alum in proximity to the DNA injection sites either on day 0 or day 3, respectively. Group 5 received 2 mg of bilateral intradermal injections of CpG delivered in proximity to the vaccine draining inguinal nodes on day 3. Group 6 received bilateral injections of 200 □g of CpG mixed with the DNA solution which was co-electroporated into the muscle on day 0.
IFNγ ELISPOT Assay Procedure.
Peripheral blood samples were collected from each animal fifteen days after the DNA immunization. Peripheral blood mononuclear cells (PBMCs) were isolated from the blood samples and were subjected to an IFNγ ELISPOT assay to measure the PSMA specific T cell responses. Briefly, 4e5 PBMCs from individual animals were plated per well with pools of PSMA specific peptides or nonspecific control peptides (human HER2 peptide pool) each at 2 ug/ml in IFNγ ELISPOT plates. The composition of each of the PSMA specific peptide pool is provided in Table 24A. The plates were incubated for 16 hrs at 37° C. and 5% CO2 and washed and developed after incubation as per manufacturer's instruction. The number of IFNγ spot forming cells (SFC) was counted by CTL reader. Each condition was performed in duplicates.
Results.
Table 6 shows the result of a representative IFNγ ELISPOT assay that evaluates and compares the IFNγ T cell responses induced by the vaccine without (group 1) or with CpG (PF-03512676) given locally by intramuscular (groups 2, 3, 4, and 5) or intradermal injections (group 6). The reported PSMA specific response was calculated by subtracting the average number of the SFC to the nonspecific control peptides (human HER2 peptide pool) from the average number of SFC to the PSMA peptide pools and normalized to the SFC observed with 1e6 PBMCs. A indicates that the count is not accurate because the numbers of spots were too numerous to count. ND indicates not determined.
The PSMA specific IFNγ T cell responses were detected to multiple PSMA specific peptide pools in the absence of CpG (PF-03512676) in all six animals (group 1). The total responses to the PSMA peptides measured were modestly higher in a few animals that additionally received CpG (PF-03512676) either by intramuscular (group 4: 3/6) or intradermal (group 5: 2/6) injections 3 days after DNA electroporation. However, when CpG was delivered subsequently right after electroporation on the same day (groups 2 and 3), there were several animals that failed to produce high responses (group 2: 4/6 and group 3: 3/6) whether mixed or not mixed with Alum. However, higher net responses were detected in 4/6 animals when a ten-fold lower dose of CpG was co-electroporated with the DNA solution into the muscle (group 6) with a statistically higher response (P<0.05) to peptide pools H1 and R1 compared to animals that did not receive CpG (group 1). The data shows that low dose of CpG can effectively enhance IFNγ T cell responses induced by a DNA vaccine when co-electroporated into the muscle.







TABLE 6







PSMA specific IFNγ T cell responses induced by


the DNA vaccine without (Group 1) or with CpG (Groups


2, 3, 4, 5 and 6) is measured by IFNγ ELISPOT


assay from PBMCs, 15 days after DNA electroporation









Recall Antigen















Group
Animal ID
P1
P2
P3
H1
H2
R1
R2


















1
#1
36
31
1
126
183
5
14



#2
6
3
13
61
524
6
141



#3
11
4
8
108
1049
3
56



#4
10
0
13
20
151
13
10



#5
8
6
11
39
469
14
18



#6
26
5
0
145
356
8
30


2
#1
3
10
0
15
35
0
0



#2
0
0
8
4
6
13
0



#3
3
0
0
0
10
11
0



#4
6
209
4
111
414
23
9



#5
15
5
30
171
104
68
6



#6
0
0
0
9
9
6
8


3
#1
14
19
8
123
1066
10
60



#2
14
16
20
384
393
104
8



#3
0
0
15
0
6
0
0



#4
0
0
0
33
21
0
4



#5
4
91
1
875
{circumflex over ( )}1235
233
109



#6
0
0
0
0
3
0
0


4
#1
0
33
15
1025
{circumflex over ( )}1209
280
90



#2
0
313
3
23
656
6
31



#3
61
120
61
428
1190
143
53



#4
0
0
8
599
870
34
111



#5
0
1
8
19
226
10
36



#6
111
55
39
231
613
121
99


5
#1
21
9
0
355
1131
73
5



#2
0
0
0
118
233
0
0



#3
0
0
0
18
129
0
0



#4
0
28
78
68
294
58
8



#5
25
0
28
329
1125
134
5



#6
0
0
0
23
39
4
0


6
#1
0
0
13
650
1096
270
5



#2
34
1
74
124
474
29
15



#3
0
3
14
684
1074
126
64



#4
8
9
0
136
321
49
1



#5
13
23
35
ND
{circumflex over ( )}1235
333
195



#6
0
0
0
421
{circumflex over ( )}1201
138
29









Example 8
Enhancement of Vaccine-Induced Immune Responses by Local Administration of Anti-CTLA-4 Antibody
The effect of low dose subcutaneous administration of anti-CTLA-4 monoclonal antibody (CP-675, 206) on the immune responses induced by a rhesus PSMA nucleic acid was investigated in a monkey study, in which the immune response was assessed by measuring PSMA specific T cell responses using an IFNγ ELISPOT assay. The rhesus PSMA nucleic acid used in the study has the sequence as set forth in SEQ ID NO: 56) and encodes an immunogenic PSMA polypeptide of SEQ ID NO: 55.
Animal Treatment and Sample Collection.
Five groups of male Indian rhesus macaques, seven (#1 to 7) per each test group, were immunized with an adenovirus encoding a rhesus PSMA modified polypeptide delivered by bilateral intramuscular injections (2×5e10 V.P.). Immediately following the adenovirus injections, group 1 received vehicle, and groups 2 to 4 received bilateral subcutaneous injections of anti-CTLA-4 antibody (CP-675, 206) at doses 2×25 mg, 2×16.7 mg and 2×8.4 mg respectively in proximity to the vaccine draining lymph node.
Nine days after the immunization, peripheral blood mononuclear cells (PBMCs) were isolated from each animal and were subjected to an IFNγ ELISPOT assay to measure the rhesus PSMA specific T cell responses. Briefly, 4e5 PBMCs from individual animals were plated per well with pools of rhesus PSMA specific peptides (P1, P2, P3 or R1+R2 defined in Table 24A) or nonspecific control peptides (human HER2 peptide pool) each at 2 ug/ml in IFN□ ELISPOT plates. The plates were incubated for 16 hrs at 37° C. and 5% CO2 and washed and developed after incubation as per manufacturer's instruction. The number of IFNγ spot forming cells (SFC) was counted by CTL reader. Each condition was performed in duplicates. The average of the duplicates from the background adjusted SFC of the rhesus PSMA specific peptide pools was normalized to the response in 1e6 PBMCs. The individual and sum responses to the peptide pools from each individual animal are presented in Table 29.
IFNγ ELISPOT Assay Procedure.
A capture antibody specific to IFNγ (□BD Bioscience, #51-2525kc) is coated onto a polyvinylidene fluoride (PVDF) membrane in a microplate overnight at 4° C. The plate is blocked with serum/protein to prevent nonspecific binding to the antibody. After blocking, effector cells (such as splenocytes isolated from immunized mice or PBMCs isolated from rhesus macaques) and targets (such as PSMA peptides from peptide library, target cells pulsed with antigen specific peptides or tumor cells expressing the relevant antigens) are added to the wells and incubated overnight at 37° C. in a 5% CO2 incubator. Cytokine secreted by effector cells are captured by the coating antibody on the surface of the PVDF membrane. After removing the cells and culture media, 100 μl of a biotinylated polyclonal anti-humanlFNγ antibody was added to each of the wells for detection. The spots are visualized by adding streptavidin-horseradish peroxidase and the precipitate substrate, 3-amino-9-ethylcarbazole (AEC), to yield a red color spot as per manufacturer's (Mabtech) protocol. Each spot represents a single cytokine producing T cell.
Results.
Table 7 shows the results of a representative IFNγ ELISPOT assay that compares the T cell responses induced by the vaccine without (group 1) or with (groups 2-4) anti-CTLA-4 monoclonal antibody (CP-675,206) given locally by subcutaneous injections in proximity to the vaccine draining lymph node. The vaccine generated an immune response (group 1) that was significantly enhanced by the local administration of the anti-CTLA-4 antibody (CP-675, 206) at a dose of 50 mg (group 2, P=0.001 by Student's T-test using underestimated values). The response was also significantly enhanced by low doses of anti-CTLA-4 antibody at 33.4 mg (group 3: P=0.004 by Student T-test using underestimated values) and 16.7 mg (group 4: P=0.05 by Student T-test) respectively. The data suggests that low doses of anti-CTLA-4 delivered by subcutaneous injection can significantly enhance the vaccine induced immune responses.







TABLE 7







IFNγ T cell responses induced by the vaccine without (Group 1)


or with subcutaneous injections of anti-CTLA-4 antibody (CP-675, 206).












aCTLA4

peptide pool















Group
dose, mg
animal ID
P1
P2
P3
R1 + R2
Sum

















1
NA
1
21
0
0
108
129




2
59
480
28
353
920




3
133
29
359
305
826




4
0
28
1
35
64




5
41
6
30
99
176




6
1
0
849
169
1019




7
0
0
0
23
23


2
50.0
1
{circumflex over ( )}1105
704
{circumflex over ( )}1116
{circumflex over ( )}1116
{circumflex over ( )}4041




2
371
26
661
779
1837




3
393
559
216
198
1366




4
{circumflex over ( )}1100
{circumflex over ( )}1100
406
1078
{circumflex over ( )}3684




5
778
325
554
419
2076




6
{circumflex over ( )}1079
{circumflex over ( )}1079
844
{circumflex over ( )}1079
{circumflex over ( )}4081




7
423
103
535
398
1459


3
33.4
1
{circumflex over ( )}425
{circumflex over ( )}425
{circumflex over ( )}425
{circumflex over ( )}425
{circumflex over ( )}1700




2
{circumflex over ( )}580
{circumflex over ( )}580
{circumflex over ( )}580
{circumflex over ( )}580
{circumflex over ( )}2320




3
TNTC
TNTC
TNTC
TNTC
TNTC




4
321
778
370
409
1878




5
331
466
311
446
1554




6
545
121
{circumflex over ( )}631
{circumflex over ( )}1194
{circumflex over ( )}2491




7
446
299
{circumflex over ( )}1078
{circumflex over ( )}1060
{circumflex over ( )}2883


4
16.7
1
{circumflex over ( )}964
296
{circumflex over ( )}964
{circumflex over ( )}964
{circumflex over ( )}3188




2
76
76
76
76
304




3
{circumflex over ( )}984
{circumflex over ( )}984
{circumflex over ( )}984
{circumflex over ( )}984
{circumflex over ( )}3936




4
260
489
648
{circumflex over ( )}1109
{circumflex over ( )}2506




5
119
45
28
140
332




6
55
76
43
198
372




7
146
726
141
400
1413





{circumflex over ( )}indicates that the count is underestimated due to the high spot numbers.


TNTC means too numerous to count.






Example 9
Immunomodulation of Myeloid Derived Suppressor Cells by Low Dose Sunitinib
The following example is provided to illustrate the immunomodulatory effects of low dose sunitinib on Myeloid Derived Suppressor Cells (MDSC) in vivo, in a non-tumor mouse model.
Study Procedures.
To generate MDSC enriched splenocytes, TUBO cells (1×106) were implanted into the flanks of 5 BALB/neuT mice, and left for approx. 20-30 days until tumor volume reached between 1000-1500 mm3. Mice were then sacrificed, spleens removed and the MDSC enriched splenocytes recovered. Splenocytes were labeled for 10 minutes with 5 μM CFSE, washed with PBS and counted. Labeled cells were subsequently resuspended at 5×107 splenocytes/ml in PBS solution and adoptively transferred via an i.v. tail vein injection into naïve BALB/c recipient mice. Three days prior to adoptive transfer, the recipient mice began bi-daily dosing with vehicle or sunitinib malate (Sutent) at 5 mg/kg, 10 mg/kg and 20 mg/kg. Following adoptive transfer, recipient mice continued to receive bi-daily dosing of Vehicle or sunitinib for two further days, after which point the mice were sacrificed, spleens removed, splenocytes recovered and processed for phenotypic analysis.
Splenocytes were counted and resuspended at 5×106 cells/ml in FACS staining buffer (PBS, 0.2% (w/v) bovine serum albumin, and 0.02% (w/v) Sodium Azide). For flow cytometry staining of splenocytes, 2.5×106 cells were first incubated with anti-bodies to CD16/CD32, 10 minutes at 4° C., to block Fc receptors and minimize non-specific binding. Splenocytes were then stained for 20 minutes at 4° C. with appropriate fluorophore conjugated antibodies (Biolegend) to murine cell surface markers. For T cells (anti-CD3 (Pacific Blue), clone 17A2) and for MDSC (anti-GR-1 (APC), clone RB6-8C5 and anti-CD11 b (PerCp Cy5.5), clone M1/70). A live/dead stain was also included. Following antibody incubation, stained splenocytes were washed with 2 mls of FACS buffer, pelleted by centrifugation and resuspended in 0.2 ml of FACS buffer prior to data acquisition on a BD CANTO 11 flow cytometer. To monitor the effect of Sunitinib or Vehicle on the adoptively transferred MDSC survival, we calculated the percentage of CFSE+, CD3−, GR1+, CD11 b+ in the live, singlet gate. We then determined the number of adoptively transferred MDSC per spleen by calculating what actual cell number the percentage represented of total splenocytes count. Data was analyzed by FloJo and Graph pad software.
Results.
The data presented in Table 27 represents the mean number of adoptively transferred CSFE+, CD3−, GR1+, CD11b+ cells recovered per spleen (n=7/group), 2 days post adoptive transfer, from mice bi-daily dosed with either Vehicle or 5 mg/kg, 10 mg/kg and 20 mg/kg Sunitinib. Statistical significance was determined by one-way ANOVA using the Dunnett's multiple comparison test, comparing the Sunitinib dosed groups against the 0 mg/kg (vehicle) group. The data demonstrates that Sunitinib, dosed bi-daily, in vivo, has an immunomodulatory effect on MDSCs, even when dosed as low as 5 mg/kg, resulting in a statistically significant reduction in the numbers recovered when compared to the vehicle treated control group.







TABLE 8







Mean number of CFSE+, CD3−, GR1+, CD11b+


MDSCs recovered from spleen









Sunitinib Dose (mg/kg)












0 (Vehicle)
5
10
20















MDSC #/spleen
17470 +/−
10980 +/−
4207 +/−
4440 +/−


Mean +/− SEM
2017
1082
338
440


Statistical
NA
Yes
Yes
Yes


significance,


p < 0.05









Example 10
Immunogenicity of Triple Antigen Adenovirus and DNA Constructs
The following example is provided to illustrate the capability of triple antigen vaccine constructs (either in the form of adenovirus vector or DNA plasmid) expressing three antigens PSMA, PSCA and PSA provided by the invention to elicit specific T cell responses to all three encoded antigens in nonhuman primates.
In Vivo Study Procedures.
The T cell immunogenicity of five adenovirus vectors each expressing three antigens (PSMA, PSCA and PSA; Ad-733, Ad-734, Ad-735, Ad-796 and Ad-809) provided by the invention were compared to the mix of three adenovirus vectors each only expressing a single antigen (PSMA, PSA or PSCA), 9 days post prime. The response to single adenovirus expressing a single antigen (groups 1-3) was evaluated to demonstrate the specificity. Briefly, Indian rhesus macaques (n=6 for groups 1 and 3, n=7 for group 2 and n=8 for groups 4-9) were intramuscularly injected with a total of 1e11 V.P. followed by intradermal injections of anti-CTLA-4 at 10 mg/kg on the same day. Nine days after the injections, peripheral blood mononuclear cells (PBMCs) were isolated from each animal and were subjected to an IFN□ ELISPOT assay to measure the PSMA, PSA and PSCA specific T cell responses.
Thirteen weeks after the adenovirus and anti-CTLA-4 injections when the T cell responses have contracted, the monkeys received DNA (Group 1: PSMA, plasmid 5166; Group 2: PSA, plasmid 5297; Group 3: PSCA, plasmid 5259; Group 4: mix of PSMA, PSA and PSCA, plasmids 5166, 5259 and 5297; Group 4: plasmid 457; Group 6: plasmid 458; Group 7: plasmid 459; Group 8: plasmid 796 and Group 9: plasmid 809) boost vaccinations delivered by electroporation. In summary, each animal received a total 5 mg of plasmid DNA provided by the invention which delivers the same expression cassette encoded in the adenovirus used in the prime. Nine days after the boost vaccination, peripheral blood mononuclear cells (PBMCs) were isolated from each animal and were subjected to an IFNγ ELISPOT assay.
IFNγ ELISPOT Assay.
Briefly, 4e5 PBMCs from individual animals were plated per well with PSMA specific peptide pools P1, P2, P3 or H1 and H2 (Table 9A), PSA specific pool 1 or 2 (Table 9B), PSCA specific pool (Table 10) or nonspecific control peptides (human HER2 peptide pool) each at 2 ug/ml in IFNγ ELISPOT plates. The plates were incubated for 16 hrs at 37° C. and 5% CO2 and washed and developed after incubation as per manufacturer's instruction. The number of IFNγ spot forming cells (SFC) was counted by CTL reader. Each condition was performed in duplicates. The average of the duplicates from the background adjusted SFC of the antigen specific peptide pools was normalized to the response in 1e6 PBMCs. The antigen specific responses in the tables present the sum of the responses to the corresponding antigen specific peptides or peptide pools.
Results:
Table 11 represents a study that evaluates the T cell immunogenicity of five different adenoviruses each expressing all three antigens in comparison to the mixture of three adenoviruses each expressing a single antigen in Indian rhesus macaques by IFNγ ELISPOT. The majority of animals that only received Ad-PSMA (group 1) injections induced specific responses to PSMA but not to PSA or PSCA (Student's T-test, P<0.03. One animal (#4) that induced responses to PSCA preferentially was removed from the statistical analysis). The animals that only received injections of Ad-PSA (group 2) induced specific responses to PSA but not to PSMA or PSCA (Student's T-test, P<0.02). The animals that only received injections of Ad-PSCA (group 3) induced specific responses to PSCA but not to PSMA or PSA (Student's T-test, P<0.03). All five triple-antigen expressing adenovirus vectors (groups 5-9) induced IFN□ T cell responses to all three antigens which the magnitude varied by animal. The magnitude of the responses to PSCA induced by the triple antigen expressing adenoviruses was similar to the mix of individual vectors (group 4). However the magnitude of responses to PSMA induced by Ad-809 (group 9) and responses to PSA induced by Ad-796 (group 8) were each significantly superior to the mix (Student's T-test, P=0.04 and P=0.02) respectively. These results indicate that vaccinating with an adenovirus expressing triple antigens can elicit equivalent or superior T cell immune responses to vaccinating with the mix of individual adenoviruses in nonhuman primates.
Table 12 shows the IFNγ ELISPOT results represents a study that evaluates the immunogenicity of the five different triple antigen expression cassettes provided in the invention delivered by an adenovirus prime in combination with anti-CTLA-4 followed by an electroporation boost of the corresponding plasmid DNA. The immune responses are compared to the mix of three constructs expressing a single antigen delivered similarly by an adenovirus prime with anti-CTLA-4 and DNA electroporation boost immunizations.
All of the animals that only received Ad-PSMA with anti-CTLA-4 followed by plasmid-PSMA (group 1) immunizations induced specific responses to PSMA but not to PSA or PSCA. Similarly all of the animals that only received Ad-PSA with anti-CTLA-4 followed by plasmid-PSA immunizations (group 2) induced specific responses to PSA but not to PSMA or PSCA and finally all of the animals that only received Ad-PSCA with anti-CTLA-4 followed by plasmid-PSCA (group 3) immunizations induced specific responses to PSCA but not to PSMA or PSA (Student's T-test, P<0.01).
All animals that have been immunized with either the triple-antigen expressing vectors (groups 5-9) or the mix (group 4) induced IFNγ T cell responses to all three antigens. The frequency of PSCA or PSA specific IFγ T cells detected were similar in all of these groups (groups 4-9) respectively. However construct groups 7 and 9 that received triple antigen expression vector vaccinations produced significantly higher frequency of responses to PSMA than the mix of three single antigen expressing constructs (group 4). These results indicate that adenovirus and DNA vaccines expressing triple antigens in one cassette can elicit equivalent or superior IFNγ T cell responses to the mix of adenoviruses and DNAs expressing the single antigens in nonhuman primates.







TABLE 9A







PSMA peptide pools*













P1
P2
P3
H1
H2
R1
R2





h 1-15
h 249-263
h 449-463
h 33-47
h 465-479
r 33-47
r 465-479


h 5-19
h 253-267
h 453-467
h 37-51
h 469-483
r 37-51
r 469-483


h 9-23
h 257-271
h 457-471
h 41-55
h 473-487
r 41-55
r 473-487


h 13-27
h 261-275
h 485-499
h 45-59
h 477-491
r 45-59
r 477-491


h 17-31
h 265-279
h 489-503
h 61-75
h 481-495
r 61-75
r 481-495


h 21-35
h 269-283
h 493-507
h 65-79
h 537-551
r 65-79
r 537-551


h 25-39
h 273-287
h 497-511
h 69-83
h 541-555
r 69-83
r 541-555


h 29-43
h 277-291
h 501-515
h 73-87
h 545-559
r 73-87
r 545-559


h 49-63
h 281-295
h 505-519
h 97-111
h 577-591
r 97-111
r 577-591


h 53-67
h 285-299
h 509-523
h 101-115
h 581-595
r 101-115
r 581-595


h 57-71
h 289-303
h 513-527
h 105-119
h 585-599
r 105-119
r 585-599


h 77-91
h 293-307
h 517-531
h 109-123
h 589-603
r 109-123
r 589-603


h 81-95
h 297-311
h 521-535
h 137-151
h 601-615
r 137-151
r 601-615


h 85-99
h 317-331
h 525-539
h 141-155
h 605-619
r 141-155
r 605-619


h 89-103
h 321-335
h 529-543
h 145-159
h 609-623
r 145-159
r 609-623


h 93-107
h 325-339
h 533-547
h 149-163
h 613-627
r 149-163
r 613-627


h 113-127
h 329-343
h 549-563
h 209-223
h 637-651
r 209-223
r 637-651


h 117-131
h 333-347
h 553-567
h 213-227
h 641-655
r 213-227
r 641-655


h 121-135
h 353-367
h 557-571
h 217-231
h 645-659
r 217-231
r 645-659


h 125-139
h 357-371
h 561-575
h 221-235
h 649-663
r 221-235
r 649-663


h 129-143
h 361-375
h 565-579
h 301-315
h 653-667
r 301-315
r 653-667


h 133-147
h 365-379
h 569-583
h 305-319
h 657-671
r 305-319
r 657-671


h 153-167
h 369-383
h 573-587
h 309-323
h 709-723
r 309-323
r 709-723


h 157-171
h 373-387
h 593-607
h 313-327
h 713-727
r 313-327
r 713-727


h 161-175
h 377-391
h 597-611
h 337-351
h 717-731
r 337-351
r 717-731


h 165-179
h 381-395
h 617-631
h 341-355
h 721-735
r 341-355
r 721-735


h 169-183
h 385-399
h 621-635
h 345-359
h 725-739
r 345-359
r 725-739


h 173-187
h 389-403
h 625-639
h 349-363
h 729-743
r 349-363
r 729-743


h 177-191
h 393-407
h 629-643
h 461-475
h 733-747
r 461-475
r 733-747


h 181-195
h 397-411
h 633-647


h 185-199
h 401-415
h 661-675


h 189-203
h 405-419
h 665-679


h 193-207
h 409-423
h 669-683


h 197-211
h 413-427
h 673-687


h 201-215
h 417-431
h 677-691


h 205-219
h 421-435
h 681-695


h 225-239
h 425-439
h 685-699


h 229-243
h 429-443
h 689-703


h 233-247
h 433-447
h 693-707


h 237-251
h 437-451
h 697-711


h 241-255
h 441-455
h 701-715


h 245-259
h 445-459
h 705-719




h 737-750
















TABLE 9B







PSA peptide pools: the amino acid position and


sequence of fifteen amino acid peptides over-


lapping by thirteen amino acids from PSA peptide


library is shown.








PSA peptide pool 1
PSA peptide pool 2












amino
PSA peptide

amino
PSA peptide



acid no.
sequence
SEQ ID NO
acid no.
sequence
SEQ ID NO





  5-19
VVFLTLSVTWIGAAP
111
129-143
PAELTDAVKVMDLPT
172


 


  9-23
TLSVTWIGAAPLILS
112
131-145
ELTDAVKVMDLPTQE
173


 


 11-25
SVTWIGAAPLILSRI
113
133-147
TDAVKVMDLPTQEPA
174


 


 13-27
TWIGAAPLILSRIVG
114
135-149
AVKVMDLPTQEPALG
175


 


 15-29
IGAAPLILSRIVGGW
115
137-151
KVMDLPTQEPALGTT
176


 


 17-31
AAPLILSRIVGGWEC
116
139-153
MDLPTQEPALGTTCY
177


 


 19-33
PLILSRIVGGWECEK
117
141-155
LPTQEPALGTTCYAS
178


 


 21-35
ILSRIVGGWECEKHS
118
143-157
TQEPALGTTCYASGW
179


 


 23-37
SRIVGGWECEKHSQP
119
145-159
EPALGTTCYASGWGS
180


 


 25-39
IVGGWECEKHSQPWQ
120
147-161
ALGTTCYASGWGSIE
181


 


 27-41
GGWECEKHSQPWQVL
121
149-163
GTTCYASGWGSIEPE
182


 


 29-43
WECEKHSQPWQVLVA
122
151-165
TCYASGWGSIEPEEF
183


 


 31-45
CEKHSQPWQVLVASR
123
153-167
YASGWGSIEPEEFLT
184


 


 33-47
KHSQPWQVLVASRGR
124
155-169
SGWGSIEPEEFLTPK
185


 


 35-49
SQPWQVLVASRGRAV
125
157-171
WGSIEPEEFLTPKKL
186


 


 37-51
PWQVLVASRGRAVCG
126
159-173
SIEPEEFLTPKKLQC
187


 


 39-53
QVLVASRGRAVCGGV
127
161-175
EPEEFLTPKKLQCVD
188


 


 41-55
LVASRGRAVCGGVLV
128
163-177
EEFLTPKKLQCVDLH
189


 


 43-57
ASRGRAVCGGVLVHP
129
165-179
FLTPKKLQCVDLHVI
190


 


 45-59
RGRAVCGGVLVHPQW
130
167-181
TPKKLQCVDLHVISN
191


 


 47-61
RAVCGGVLVHPQWVL
131
169-183
KKLQCVDLHVISNDV
192


 


 49-63
VCGGVLVHPQWVLTA
132
171-185
LQCVDLHVISNDVCA
193


 


 51-65
GGVLVHPQWVLTAAH
133
173-187
CVDLHVISNDVCAQV
194


 


 53-67
VLVHPQWVLTAAHCI
134
175-189
DLHVISNDVCAQVHP
195


 


 55-69
VHPQWVLTAAHCIRN
135
177-191
HVISNDVCAQVHPQK
196


 


 57-71
PQWVLTAAHCIRNKS
136
179-193
ISNDVCAQVHPQKVT
197


 


 59-73
WVLTAAHCIRNKSVI
137
181-195
NDVCAQVHPQKVTKF
198


 


 61-75
LTAAHCIRNKSVILL
138
183-197
VCAQVHPQKVTKFML
199


 


 63-77
AAHCIRNKSVILLGR
139
185-199
AQVHPQKVTKFMLCA
200


 


 65-79
HCIRNKSVILLGRHS
140
187-201
VHPQKVTKFMLCAGR
201


 


 67-81
IRNKSVILLGRHSLF
141
189-203
PQKVTKFMLCAGRWT
202


 


 69-83
NKSVILLGRHSLFHP
142
191-205
KVTKFMLCAGRWTGG
203


 


 71-85
SVILLGRHSLFHPED
143
193-207
TKFMLCAGRWTGGKS
204


 


 73-87
ILLGRHSLFHPEDTG
144
195-209
FMLCAGRWTGGKSTC
205


 


 75-89
LGRHSLFHPEDTGQV
145
197-211
LCAGRWTGGKSTCSG
206


 


 77-91
RHSLFHPEDTGQVFQ
146
199-213
AGRWTGGKSTCSGDS
207


 


 79-93
SLFHPEDTGQVFQVS
147
201-215
RWTGGKSTCSGDSGG
208


 


 81-95
FHPEDTGQVFQVSHS
148
203-217
TGGKSTCSGDSGGPL
209


 


 83-97
PEDTGQVFQVSHSFP
149
205-219
GKSTCSGDSGGPLVC
210


 


 85-99
DTGQVFQVSHSFPHP
150
207-221
STCSGDSGGPLVCNG
211


 


 87-101
GQVFQVSHSFPHPLY
151
209-223
CSGDSGGPLVCNGVL
212


 


 89-103
VFQVSHSFPHPLYDM
152
211-225
GDSGGPLVCNGVLQG
213


 


 91-105
QVSHSFPHPLYDMSL
153
213-227
SGGPLVCNGVLQGIT
214


 


 93-107
SHSFPHPLYDMSLLK
154
215-229
GPLVCNGVLQGITSW
215


 


 95-109
SFPHPLYDMSLLKNR
155
217-231
LVCNGVLQGITSWGS
216


 


 97-111
PHPLYDMSLLKNRFL
156
219-233
CNGVLQGITSWGSEP
217


 


 99-113
PLYDMSLLKNRFLRP
157
221-235
GVLQGITSWGSEPCA
218


 


101-115
YDMSLLKNRFLRPGD
158
223-237
LQGITSWGSEPCALP
219


 


103-117
MSLLKNRFLRPGDDS
159
225-239
GITSWGSEPCALPER
220


 


105-119
LLKNRFLRPGDDSSH
160
227-241
TSWGSEPCALPERPS
221


 


107-121
KNRFLRPGDDSSHDL
161
229-243
WGSEPCALPERPSLY
222


 


109-123
RFLRPGDDSSHDLML
162
231-245
SEPCALPERPSLYTK
223


 


111-125
LRPGDDSSHDLMLLR
163
233-247
PCALPERPSLYTKVV
224


 


113-127
PGDDSSHDLMLLRLS
164
235-249
ALPERPSLYTKVVHY
225


 


115-129
DDSSHDLMLLRLSEP
165
237-251
PERPSLYTKVVHYRK
226


 


117-131
SSHDLMLLRLSEPAE
166
239-253
RPSLYTKVVHYRKWI
227


 


119-133
HDLMLLRLSEPAELT
167
241-255
SLYTKVVHYRKWIKD
228


 


121-135
LMLLRLSEPAELTDA
168
243-257
YTKVVHYRKWIKDTI
229


 


123-137
LLRLSEPAELTDAVK
169
245-259
KVVHYRKWIKDTIVA
230


 


125-139
RLSEPAELTDAVKVM
170
247-261
VHYRKWIKDTIVANP
231


 


127-141
SEPAELTDAVKVMDL
171
249-261
YRKWIKDTIVANP
232


 





251-261
KWIKDTIVANP
233
















TABLE 10







PSCA peptide pool: The amino acid position and


sequence of fifteen amino acid peptides over-


lapping by thirteen amino acids from PSCA peptide


library is shown.









amino acid no.
PSCA peptide sequence
SEQ ID NO





  1-15
MKAVLLALLMAGLAL
234


 


  3-17
AVLLALLMAGLALQP
235


 


  5-19
LLALLMAGLALQPGT
236


 


  7-21
ALLMAGLALQPGTAL
237


 


  9-23
LMAGLALQPGTALLC
238


 


 11-25
AGLALQPGTALLCYS
239


 


 13-27
LALQPGTALLCYSCK
240


 


 15-29
LQPGTALLCYSCKAQ
241


 


 17-31
PGTALLCYSCKAQVS
242


 


 19-33
TALLCYSCKAQVSNE
243


 


 21-35
LLCYSCKAQVSNEDC
244


 


 23-37
CYSCKAQVSNEDCLQ
245


 


 25-39
SCKAQVSNEDCLQVE
246


 


 27-41
KAQVSNEDCLQVENC
247


 


 29-43
QVSNEDCLQVENCTQ
248


 


 31-45
SNEDCLQVENCTQLG
249


 


 33-47
EDCLQVENCTQLGEQ
250


 


 35-49
CLQVENCTQLGEQCW
251


 


 37-51
QVENCTQLGEQCWTA
252


 


 39-53
ENCTQLGEQCWTARI
253


 


 41-55
CTQLGEQCWTARIRA
254


 


 43-57
QLGEQCWTARIRAVG
255


 


 45-59
GEQCWTARIRAVGLL
256


 


 47-61
QCWTARIRAVGLLTV
257


 


 49-63
WTARIRAVGLLTVIS
258


 


 51-65
ARIRAVGLLTVISKG
259


 


 53-67
IRAVGLLTVISKGCS
260


 


 55-69
AVGLLTVISKGCSLN
261


 


 57-71
GLLTVISKGCSLNCV
262


 


 59-73
LTVISKGCSLNCVDD
263


 


 61-75
VISKGCSLNCVDDSQ
264


 


 63-77
SKGCSLNCVDDSQDY
265


 


 65-79
GCSLNCVDDSQDYYV
266


 


 67-81
SLNCVDDSQDYYVGK
267


 


 69-83
NCVDDSQDYYVGKKN
268


 


 71-85
VDDSQDYYVGKKNIT
269


 


 73-87
DSQDYYVGKKNITCC
270


 


 75-89
QDYYVGKKNITCCDT
271


 


 77-91
YYVGKKNITCCDTDL
272


 


 79-93
VGKKNITCCDTDLCN
273


 


 81-95
KKNITCCDTDLCNAS
274


 


 83-97
NITCCDTDLCNASGA
275


 


 85-99
TCCDTDLCNASGAHA
276


 


 87-101
CDTDLCNASGAHALQ
277


 


 89-103
TDLCNASGAHALQPA
278


 


 91-105
LCNASGAHALQPAAA
279


 


 93-107
NASGAHALQPAAAIL
280


 


 95-109
SGAHALQPAAAILAL
281


 


 97-111
AHALQPAAAILALLP
282


 


 99-113
ALQPAAAILALLPAL
283


 


101-115
QPAAAILALLPALGL
284


 


103-117
AAAILALLPALGLLL
285


 


105-119
AILALLPALGLLLWG
286


 


107-121
LALLPALGLLLWGPG
287


 


109-123
LLPALGLLLWGPGQL
288


 


111-125
PALGLLLWGPGQL
289
















TABLE 11







IFNγ T cell responses induced by the single antigen (Group


1: Ad-PSMA; Group 2: Ad-PSA; Group 3: Ad-PSCA; Group 4: mix


of Ad-PSMA, Ad-PSA and Ad-PSCA) or triple antigen expressing


adenovirus vectors (Group 4: Ad-733; Group 6: Ad-734; Group


7: Ad-735; Group 8: Ad-796 and Group 9: Ad-809) after adenovirus


prime with anti-CTLA-4 analyzed by ELISPOT assay.









animal ID
















1
2
3
4
5
6
7
8













Response to




PSMA peptides
















Group
1
2356
988
1505
335
501
2145
NA
NA


No.
2
342
1776
154
329
158
438
321
NA



3
0
1276
40
126
20
0
NA
NA



4
304
1198
774
2007
1277
1310
1159
2774



5
943
2670
2757
780
1082
2251
1566
544



6
472
2092
4248
1369
1760
2964
1447
263



7
2161
2202
939
869
3513
1654
3424
900



8
1166
799
2566
663
1043
497
1334
560



9
1621
3247
2031
980
2942
1882
1918
3805















Response to










PSA peptides
















Group
1
0
0
0
48
0
42
NA
NA


No.
2
1419
1426
298
1223
1346
1120
1694
NA



3
6
462
91
0
77
0
NA
NA



4
790
1093
1611
790
186
783
2016
1964



5
101
510
955
665
336
1512
1052
119



6
236
673
2155
724
504
1600
930
83



7
0
1086
494
663
2265
117
1712
84



8
1893
2060
1490
1759
2352
1700
2232
1326



9
1193
1432
207
1738
1886
949
492
1940















Response to










PSCA peptides
















Group
1
795
425
874
1069
219
203
NA
NA


No.
2
669
713
391
199
164
560
461
NA



3
510
1234
1099
1115
1194
339
NA
NA



4
778
528
680
1101
165
531
1175
1009



5
378
1061
1161
143
71
756
766
204



6
118
380
1190
403
829
1225
148
261



7
615
1141
794
564
1175
490
856
204



8
968
1136
745
290
550
976
955
841



9
929
434
1150
745
1120
246
1195
970
















TABLE 12







IFNγ T cell responses induced by the single antigen (Group


1: PSMA; Group 2: PSA; Group 3: PSCA; Group 4: mix of PSMA,


PSA and PSCA) or triple antigen expressing vectors (Groups


5-9) after adenovirus prime with anti-CTLA-4 and DNA electroporation


boost immunizations analyzed by ELISPOT assay.









animal ID
















1
2
3
4
5
6
7
8













Response to




PSMA peptides
















Group
1
1327
1535
1643
535
1506
1267
NA
NA


No.
2
15
266
26
191
10
46
1305
NA



3
0
445
5
75
4
6
NA
NA



4
365
675
731
1134
244
714
999
1683



5
270
1623
2254
626
860
2245
1453
1046



6
541
1151
2923
1094
1061
1746
691
489



7
1183
1183
1453
1649
2844
1470
2321
991



8
486
69
399
216
351
758
416
1389



9
1430
2631
2015
475
1368
1826
1851
3141








Response to



PSA peptides
















Group
1
0
0
0
1
0
26
NA
NA


No.
2
1883
1236
1574
393
461
941
1565
NA



3
33
30
9
13
8
11
NA
NA



4
571
1129
1180
210
88
274
924
360



5
50
1255
1344
628
210
638
948
1161



6
88
228
1390
489
1006
908
683
51



7
0
211
321
156
1509
56
199
85



8
414
611
85
105
544
1080
331
1883



9
434
821
556
343
1160
510
144
1115















Response to










PSCA peptides
















Group
1
615
799
533
74
258
61
NA
NA


No.
2
194
170
133
133
8
66
405
NA



3
819
1071
873
839
1045
724
NA
NA



4
543
506
664
470
70
673
761
1235



5
154
455
1218
109
218
1094
285
569



6
56
293
603
506
745
911
63
165



7
429
298
939
589
1226
263
803
451



8
279
214
871
61
144
511
193
963



9
379
191
1196
73
699
198
616
836









Example 11
Construction of C68 Vectors
11A. Vector AdC68-734 Construction
AdC68-734 is a replication incompetent adenovirus vector based upon the chimpanzee adenovirus C68 that encodes three immunogenic PAA polypeptides—an immunogenic PSA polypeptide, immunogenic PSCA polypeptide, and immunogenic PSMA polypeptide. The vector sequence was designed in silico. First, the baseline full length C68 sequence was obtained from Genbank (Definition: Simian adenovirus 25, complete genome; accession number AC_000011.1). Five point mutations described in the literature were introduced into the sequence. (Roshorm, Y., M. G. Cottingham, et al. (2012). “T cells induced by recombinant chimpanzee adenovirus alone and in prime-boost regimens decrease chimeric EcoHIV/NDK challenge virus load.” Eur J Immunol 42(12): 3243-3255) Next, 2.6 kilobases of the viral early transcription region 1 (E1) were deleted to render the vector replication incompetent, and 3.5 kilobases of the early transcription region 3 (E3) were removed to create space in the vector for the transgene expression cassette. (Tatsis, N., L. Tesema, et al. (2006). Chimpanzee-origin adenovirus vectors as vaccine carriers. Gene Ther. 13: 421-429) A highly efficient eukaryotic expression cassette was then introduced into the E1 region. The expression cassette included the following components: (A) Cytomegalovirus (CMV) immediate early enhancer/promoter, (B) Tet operator (binding site for the tetracycline repressor), (C) the multi-antigen construct comprising (1) nucleotide sequence encoding amino acids 25 through 261 of the human PSA, (2) Cis acting hydrolase element encoding a glycine-serine linker and Thosea asigna virus 2A peptide (T2A), (3) nucleotide sequence encoding amino acids 2 through 123 of the human PSCA, (4) Cis acting hydrolase element encoding a glycine-serine linker and Foot and Mouth Disease Virus 2A peptide (F2A), and (5) nucleotide sequence encoding amino acids 15 through 750 the human PSMA, and (D) SV40 polyA transcription termination signal. Finally, Pacl restriction sites were inserted at each end of the viral genome to facilitate the release of the genome from the parent Bacmid. Nucleotides from the Pacl restriction sites are removed during viral propagation and, therefore, are not incorporated into the genome of the vector product itself. A nucleotid sequence of the entire vector AdC68-734, including the Pacl restriction sites, is set forth in SEQ ID NO:58. The multi-antigen construct (PSA-T2A-PSCA-F2A-PSMA) incorporated in vector AdC68-734 (as well as in Plasmid 458) is also set forth in SEQ ID NO:61. The amino acid sequence encoded by the multi-antigen construct of SEQ ID NO:61 is set forth in SEq ID NO:60. The components of vector AdC68-734 are provided in Table 13.







TABLE 13







Components of Vector AdC68-734








Base Numbers
Feature





1-8
PacI restriction site


 9-463
Bases 1-455 of AC000011.1 (SEQ ID NO: 57)


 464-1096
CMV enhancer/promoter


1031-1070
Tetracycline operator/represser binding site


1106-1825
Sequence encoding amino acids 25 through 261 of the human



PSA and the preceding methionine-alanine-serine linker


1826-1831
Linker encoding glycine - serine


1832-1885
Cis acting hydrolase element encoding a Thosea asigna virus



2A peptide


1886-2257
Sequence encoding amino acids 2 through 123 of the human



PSCA and the preceding alanine-serine linker


2258-2263
Linker encoding glycine - serine


2264-2323
Cis acting hydrolase element encoding a Foot and Mouth



Disease Virus 2A peptide


2324-4543
Sequence encoding amino acids 15 through 750 of the human



PSMA and the preceding methionine-alanine-serine linker


4541-4543
Stop codon


4596-4823
SV40 polyA transcription termination signal


 4824-29622
Bases 3013-27811 of AC000011.1 (SEQ ID NO: 57)


29623-34811
Bases 31331-36519 of AC000011.1 (SEQ ID NO: 57)


10730
C to G substitution at base 89 19 of AC000011.1 (SEQ ID



NO: 57)


17569
G to C substitution at base 15758 of AC000011.1 (SEQ ID



NO: 57)


18967
A to T substitution at base 17156 of AC000011.1 (SEQ ID



NO: 57)


19245
C to A substitution at base 17434 of AC000011.1 (SEQ ID



NO: 57)


33520
G to C substitution at base 35228 of AC000011.1 (SEQ ID



NO: 57)


34812-34819
PacI restriction site









Following in silico design, the 34,819 base-pair sequence was biochemically synthesized in a multi-stage process utilizing in vitro oligo synthesis and subsequent recombination-mediated intermediate assembly in E. coli and yeast. The viral genome was ultimately inserted into a bacterial artificial chromosome (pCC1BAC-LCyeast-TRP Trunc) for propagation. Next generation sequencing (MiSeq technology) was performed at multiple steps in the production process, including the final Bacmid 17.3.3.22 lot that was used to create the viral seed stock. Viral seed stock was generated by digesting Bacmid 17.3.3.22 with Pacl to release the AdC68-734 genome from the BAC backbone. The linearized nucleic acid was transfected into an E1 complimenting adherent HEK293 cell line and upon visible cytopathic effects and adenovirus foci formation, cultures were harvested by multiple rounds of freezing/thawing to release virus from the cells. Viruses were amplified and purified by standard techniques. The genetic organization of Bacmid 17.3.3.22 is provided in FIG. 11.
11B. Constructions of Additional C68 Vectors
Additional triple antigen C68 vectors were constructed in a similar fashion to AdC68-734. Some of the additional vectors involve functional deletions in the C68 genome that are slightly different from those in Vector AdC68-734, while others incorporate different multi-antigen constructs. Based on these examples and other description of the present disclosure, a person skilled in the art would be able construct additional vectors from C68 for expressing various multi-antigen constructs, all of which are within the scope of the present invention.
(1) AdC68X-734 and AdC68W-734
Vector AdC68X-734 was constructed from C68 by functional deletion of the E1 and E3 regions of the C68 genome through deletions of nucleotides 577-3403 (E1 region) and 27125-31831 (E2 region) of the C68 genome of SEQ ID NO:57 and by insertion of the triple antigen construct (PSA-T2A-PSCA-F2A-PSMA) of SEQ ID NO:61 in the deleted E1 region. Vector AdC68W-734 is identical to Vector vector AdC68-734 except that AdC68W-734 contains one or more mutations in the C68 NDA sequence.
(2) AdC68X-733 and AdC68X-735
Vectors AdC68X-733 and AdC68X-735 were created by replacing the triple antigen-construct incorporated in the AdC68X-734 vector with the triple antigen construct of SEQ ID NOs:65 and 66, respectively. The multi-antigen construct incorporated in vector AdC68X-733 (i.e, PSA-F2A-PSMA-T2A-PSCA) is the same as that incorporated in Plasmid 457 and the multi-antigen construct incorporated in vector AdC68X-735 (i.e., PSCA-F2A-PSMA-mIRES-PSA) is the same as that in Plasmid 459.
11C. Research Productivity Characterization
Various research grade lots of AdC68-734 were produced and tested for productivity. Bacmid was digested with Pacl to release the vector genome from the BAC backbone and the linearized nucleic acid was transfected into E1 complimenting adherent HEK293 cell lines. When extensive cytopathic effects and adenovirus foci were visible, cultures were harvested by multiple rounds of freezing/thawing to release virus from the cells. Viruses from these Passage 0 (P0) cultures were amplified at least one additional passage in tissue culture flasks and then used as seed stocks for research scale production runs ( ̃0.5 to 3e13 total viral particles per lot). In total, 11 production runs were executed (five in HEK293 suspension cells and six in HEK293 adherent cells). The average specific productivity was 15,000+/−6,000 viral particles purified per initial infected cell, with a viral particle:infectious unit ratio of 55. Research scale productivities are summarized in Table 14.







TABLE 14







Specific productivities and infectivities


of research scale production lots










Specific productivity (purified
Viral particle:infectious


Lot
viral particles/cell)
unit ratio












 20039
17000
33


 20424
19000
49


 20542
12000
76


 20609
25000
54


 20626
16000
58


 20671
19000
ND


130502
17000
51


 130718*
3500
52


130820
7400
55


130821
9300
70


130822
19000
54





*Late passage HEK293 suspension cells used in production






11 D. Antigen Expression
The surface expression of PSMA and PSCA was measured by flow cytometry (FIG. 12) and total cellular expression of PSMA, PSCA and PSA was measured by western blot analysis (FIG. 13) from AdC68-vector infected A549 cells at an M01=10,000. Mock and AdC68 infected cells were stained with anti-PSCA (fluorescein isothiocyanate-conjugated monoclonal antibody 1G8 [1:200]) and PSMA antibodies (allophycocyanin-conjugated monoclonal antibody J591 [1:200]) for flow cytometric analysis, 2 days post infection. Surface expression of PSCA and PSMA were detected from majority of the cells infected with the different triple antigen-expressing AdC68 vectors with varying levels. Relatively higher levels of expression of PSCA and PSMA were detected from AdC68X-809 infected cells and lower levels were detected from AdC68X-733 infected cell. Two days after infection, total cellular lysates from approximately 1×105 infected cells were loaded onto each lane of a sodium dodecyl sulfate polyacrylamide gel. The gel was subsequently transferred to a membrane for the detection of PSA, PSMA, and PSCA proteins using primary antibodies specific to PSA, PSMA, and PSCA by western blot analysis. The expressions of all three antigens were detected in the infected cells to varying degrees. While relatively similar levels of PSMA and PSCA were detected from AdC68-734 and AdC68X-735 infected lysates, higher levels of PSA were detected from AdC68-734 lysates compared to those from AdC68X-735
11E. Immunogenicity
A head-to head comparison of the CD8 IFNγ responses induced by various triple antigen AdC68 vectors was performed. Each group of mice (n=5 per group) was immunized with AdC68-734, AdC68X-735, AdC68X-809, or Ad5-734 at 1e9 or 1e10 VP in the quadriceps. IFNγ CD8+ T cell responses in the mice were measured by collecting the spleens from each animal on day 13 post immunization. Splenocytes were isolated and subjected to an IFNγ ELISPOT assay to measure the PSMA, PSCA, and PSA-specific T cell responses. Briefly, 2.5 to 5×105 splenocytes from immunized animals were cultured in the presence of individual human PSMA, PSCA, or PSA-specific peptides at 10 μg/ml. The 15-mer peptides were previously defined to contain CD8+ T cell epitopes to each prostate antigen. Splenocytes cultured with medium alone served as a control. Each condition was performed in triplicate. The plates were incubated for 20 h at 37° C. and 5% CO2, washed, and developed after incubation as per the manufacturer's instructions. The number of IFNγ SFC was counted by a CTL reader. The results show the average number of PSMA, PSCA, and PSA-specific SFCs with the medium alone background values subtracted, and normalized to 1×106 splenocytes.
In summary, all triple antigen expressing AdC68 vectors induced immune responses to all three antigens but to different magnitude. At 1e9 VP, the response to PSMA by the AdC68 vectors was similar to Ad5. The response to PSCA by the three AdC68 vectors was similar or lower than the response induced by Ad5 while the response to PSA was lower with Ad68-735 compared to all of the vectors tested. However at 1e10VP, AdC68-809 induced similar or better responses to all three antigens compared to AdC68-734, AdC68-735 or Ad5. Results are presented in Table 15.







TABLE 15







IFNγ T cellular Immunogenicity by AdC68 vectors co-expressing


PSMA, PSA and PSCA in C57BL6 mice by IFNγ ELISPOT assay









Construct












Ad5-734
AdC68-734
AdC68-809
AdC68-735















Titer, vp
1e9
1e10
1e9
1e10
1e9
1e10
1e9
1e10


















PSMA
473
1221
699
296
489
684
288
503



491
831
143
513
221
687
203
261



435
740
149
607
315
809
256
745



248
596
224
116
347
317
317
1197



709
711
269
681
296
536
320
368


PSA
1299
1472
1180
1741
1973
1979
533
695



939
1025
1327
1985
841
1532
313
1615



1096
797
672
780
1869
1979
277
1420



989
933
904
635
1009
1669
535
616



1971
1047
1309
1901
907
1920
824
403


PSCA
104
64
228
61
115
197
148
92



160
80
11
41
59
92
80
897



163
52
15
116
25
235
47
39



119
223
32
57
24
96
107
33



207
100
8
53
17
35
32
16









Select Raw Sequences








SEQ ID NO: 1. AMINO ACID SEQUENCE OF THE FULL LENGTH HUMAN



PSMA


MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNM


 


KAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLS


 


YPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVN


 


YARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAP


 


GVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPV


 


HPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNE


 


VTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAWHEIVRSFGTLKKEGW


 


RPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPL


 


MYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQR


 


LGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVF


 


ELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASK


 


FSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGES


 


FPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA


 


SEQ ID NO: 2. NUCLEOTIDE SEQUENCE ENCODING THE FULL LENGTH


HUMAN PSMA OF SEQ ID NO: 1


atgtggaatctccttcacgaaaccgactcggctgtggccaccgcgcgccgcccgcgctggctgtgcgctggggcgctggt


 


gctggcgggtggcttctttctcctcggcttcctcttcgggtggtttataaaatcctccaatgaagctactaacattactccaaagc


 


ataatatgaaagcatttttggatgaattgaaagctgagaacatcaagaagttcttatataattttacacagataccacatttag


 


caggaacagaacaaaactttcagcttgcaaagcaaattcaatcccagtggaaagaatttggcctggattctgttgagctag


 


cacattatgatgtcctgttgtcctacccaaataagactcatcccaactacatctcaataattaatgaagatggaaatgagatttt


 


caacacatcattatttgaaccacctcctccaggatatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaag


 


gaatgccagagggcgatctagtgtatgttaactatgcacgaactgaagacttctttaaattggaacgggacatgaaaatca


 


attgctctgggaaaattgtaattgccagatatgggaaagttttcagaggaaataaggttaaaaatgcccagctggcagggg


 


ccaaaggagtcattctctactccgaccctgctgactactttgctcctggggtgaagtcctatccagatggttggaatcttcctgg


 


aggtggtgtccagcgtggaaatatcctaaatctgaatggtgcaggagaccctctcacaccaggttacccagcaaatgaat


 


atgcttataggcgtggaattgcagaggctgttggtcttccaagtattcctgttcatccaattggatactatgatgcacagaagct


 


cctagaaaaaatgggtggctcagcaccaccagatagcagctggagaggaagtctcaaagtgccctacaatgttggacct


 


ggctttactggaaacttttctacacaaaaagtcaagatgcacatccactctaccaatgaagtgacaagaatttacaatgtgat


 


aggtactctcagaggagcagtggaaccagacagatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtatt


 


gaccctcagagtggagcagctgttgttcatgaaattgtgaggagctttggaacactgaaaaaggaagggtggagacctag


 


aagaacaattttgtttgcaagctgggatgcagaagaatttggtcttcttggttctactgagtgggcagaggagaattcaagac


 


tccttcaagagcgtggcgtggcttatattaatgctgactcatctatagaaggaaactacactctgagagttgattgtacaccgc


 


tgatgtacagcttggtacacaacctaacaaaagagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaa


 


gttggactaaaaaaagtccttccccagagttcagtggcatgcccaggataagcaaattgggatctggaaatgattttgaggt


 


gttcttccaacgacttggaattgcttcaggcagagcacggtatactaaaaattgggaaacaaacaaattcagcggctatcc


 


actgtatcacagtgtctatgaaacatatgagttggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccag


 


gttcgaggagggatggtgtttgagctagccaattccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtat


 


gctgacaaaatctacagtatttctatgaaacatccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcag


 


taaagaattttacagaaattgcttccaagttcagtgagagactccaggactttgacaaaagcaacccaatagtattaagaat


 


gatgaatgatcaactcatgtttctggaaagagcatttattgatccattagggttaccagacaggcctttttataggcatgtcatct


 


atgctccaagcagccacaacaagtatgcaggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagt


 


ggacccttccaaggcctggggagaagtgaagagacagatttatgttgcagccttcacagtgcaggcagctgcagagactt


 


tgagtgaagtagcc


 


SEQ ID NO: 3. AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 1


 


SEQ ID NO: 4. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID


 


SEQUENCE OF PSMA SHUFFLED ANTIGEN 1 OF SEQ ID NO: 3


 


SEQ ID NO: 5. AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 2


 


SEQ ID NO: 6. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID


 


SEQUENCE OF PSMA SHUFFLED ANTIGEN 2 OF SEQ ID NO:5


 


SEQ ID NO: 7. AMINO ACID SEQUENCE OF PSMA SHUFFLED ANTIGEN 3


 


SEQ ID NO: 8. NUCLEOTIDE SEQEUNCE ENCODING AMINO ACID


 


SEQUENCE OF PSMA SHUFFLED ANTIGEN 3 OF SEQ ID NO:7


 


SEQ ID NO: 9. AMINO ACID SEQUENCE OF A MEMBRANE-BOUND PSMA


ANTIGEN


MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENI


 


KKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISI


 


INEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLE


 


RDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWN


 


LPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLL


 


EKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRG


 


AVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWD


 


AEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKEL


 


KSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTK


 


NWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDC


 


RDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKS


 


NPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIE


 


SKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA


 


SEQ ID NO: 10. NUCLEOTIDE SEQEUNCE ENCODING AMINO ACID


SEQUENCE OF THE MEMBRANE-BOUND PSMA ANTIGEN OF SEQ ID NO: 9


atggctagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttc


 


gggtggtttataaaatcctccaatgaagctactaacattactccaaagcataatatgaaagcatttttggatgaattgaaagct


 


gagaacatcaagaagttcttatataattttacacagataccacatttagcaggaacagaacaaaactttcagcttgcaaag


 


caaattcaatcccagtggaaagaatttggcctggattctgttgagctggcacattatgatgtcctgttgtcctacccaaataag


 


actcatcccaactacatctcaataattaatgaagatggaaatgagattttcaacacatcattatttgaaccacctcctccagg


 


atatgaaaatgtttcggatattgtaccacctttcagtgctttctctcctcaaggaatgccagagggcgatctagtgtatgttaact


 


atgcacgaactgaagacttctttaaattggaacgggacatgaaaatcaattgctctgggaaaattgtaattgccagatatgg


 


gaaagttttcagaggaaataaggttaaaaatgcccagctggcaggggccaaaggagtcattctctactccgaccctgctg


 


actactttgctcctggggtgaagtcctatccagatggttggaatcttcctggaggtggtgtccagcgtggaaatatcctaaatct


 


gaatggtgcaggagaccctctcacaccaggttacccagcaaatgaatatgcttataggcgtggaattgcagaggctgttgg


 


tcttccaagtattcctgttcatccaattggatactatgatgcacagaagctcctagaaaaaatgggtggctcagcaccacca


 


gatagcagctggagaggaagtctcaaagtgccctacaatgttggacctggctttactggaaacttttctacacaaaaagtca


 


agatgcacatccactctaccaatgaagtgacaagaatttacaatgtgataggtactctcagaggagcagtggaaccagac


 


agatatgtcattctgggaggtcaccgggactcatgggtgtttggtggtattgaccctcagagtggagcagctgttgttcatgaa


 


attgtgaggagctttggaacactgaaaaaggaagggtggagacctagaagaacaattttgtttgcaagctgggatgcaga


 


agaatttggtcttcttggttctactgagtgggcagaggagaattcaagactccttcaagagcgtggcgtggcttatattaatgct


 


gactcatctatagaaggaaactacactctgagagttgattgtacaccgctgatgtacagcttggtacacaacctaacaaaa


 


gagctgaaaagccctgatgaaggctttgaaggcaaatctctttatgaaagttggactaaaaaaagtccttccccagagttc


 


agtggcatgcccaggataagcaaattgggatctggaaatgattttgaggtgttcttccaacgacttggaattgcttcaggcag


 


agcacggtatactaaaaattgggaaacaaacaaattcagcggctatccactgtatcacagtgtctatgaaacatatgagtt


 


ggtggaaaagttttatgatccaatgtttaaatatcacctcactgtggcccaggttcgaggagggatggtgtttgagctggcca


 


attccatagtgctcccttttgattgtcgagattatgctgtagttttaagaaagtatgctgacaaaatctacagtatttctatgaaac


 


atccacaggaaatgaagacatacagtgtatcatttgattcacttttttctgcagtaaagaattttacagaaattgcttccaagttc


 


agtgagagactccaggactttgacaaaagcaacccaatagtattaagaatgatgaatgatcaactcatgtttctggaaaga


 


gcatttattgatccattagggttaccagacaggcctttttataggcatgtcatctatgctccaagcagccacaacaagtatgca


 


ggggagtcattcccaggaatttatgatgctctgtttgatattgaaagcaaagtggacccttccaaggcctggggagaagtga


 


agagacagatttatgttgcagccttcacagtgcaggcagctgcagagactttgagtgaagtagcc


 


SEQ ID NO: 11. AMINO ACID SEQUENCE OF A CYTOSOLIC PSMA


 


ANTIGEN


 


SEQ ID NO: 12. NUCLEOTIDE SEQEUNCE ENCODING AMINO ACID


 


SEQUENCE OF THE CYTOSOLIC PSMA ANTIGEN OF SEQ ID NO: 11


 


SEQ ID NO: 13. AMINO ACID SEQUENCE OF A SECRETED PSMA ANTIGEN


 


SEQ ID NO: 14. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID


 


SEQUENCE OF THE SECRETED PSMA ANTIGEN OF SEQ ID NO:13


 


SEQ ID NO: 15. AMINO ACID SEQUENCE OF THE FULL LENGTH HUMAN


PSA


MASWVPVVFLTLSVTWIGAAPLILSRIVGGWECEKHSQPWQVLVASRGRAVCGGVLV


 


HPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRP


 


GDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKL


 


QCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTCSGDSGGPLVCNGVLQG ITS


 


WGSEPCALPERPSLYTKVVHYRKWIKDTIVANP


 


SEQ ID NO: 16. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID


SEQUENCE OF THE FULL LENGTH HUMAN PSA OF SEQ ID NO: 15


atggctagctgggtcccggttgtcttcctcaccctgtccgtgacgtggattggcgctgcgcccctcatcctgtctcggattgtgg


 


gaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgtt


 


ctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagctt


 


gtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaag


 


aatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggat


 


gctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctcaggctggggcagcatt


 


gaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttca


 


ccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggg


 


gcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctg


 


tacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccc


 


SEQ ID NO: 17. AMINO ACID SEQUENCE OF A CYTOSOLIC PSA ANTIGEN


MASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRH


 


SLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAV


 


KVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTK


 


FMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR


 


KWIKDTIVANP


 


SEQ ID NO: 18. NUCLEOTIDE SEQEUNCE ENCODING AMINO ACID


SEQUENCE OF THE CYTOSOLIC PSA ANTIGEN OF SEQ ID NO: 17


atggctagcattgtgggaggctgggagtgcgagaagcattcccaaccctggcaggtgcttgtggcctctcgtggcagggc


 


agtctgcggcggtgttctggtgcacccccagtgggtcctcacagctgcccactgcatcaggaacaaaagcgtgatcttgct


 


gggtcggcacagcttgtttcatcctgaagacacaggccaggtatttcaggtcagccacagcttcccacacccgctctacgat


 


atgagcctcctgaagaatcgattcctcaggccaggtgatgactccagccacgacctcatgctgctccgcctgtcagagcct


 


gccgagctcacggatgctgtgaaggtcatggacctgcccacccaggagccagcactggggaccacctgctacgcctca


 


ggctggggcagcattgaaccagaggagttcttgaccccaaagaaacttcagtgtgtggacctccatgttatttccaatgacg


 


tgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctgtgtgctggacgctggacagggggcaaaagcacctgc


 


tcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaaggtatcacgtcatggggcagtgaaccatgtgccctgcc


 


cgaaaggccttccctgtacaccaaggtggtgcattaccggaagtggatcaaggacaccatcgtggccaacccc


 


SEQ ID NO: 19. AMINO ACID SEQUENCE OF A MEMBRANE-BOUND PSA


ANTIGEN


MASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPGIVGGWECEKHSQP


 


WQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRHSLFHPEDTGQVFQVSH


 


SFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAVKVMDLPTQEPALGTTC


 


YASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTKFMLCAGRWTGGKSTC


 


SGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYRKWIKDTIVANP


 


SEQ ID NO: 20. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID


SEQUENCE OF THE MEMBRANE-BOUND PSA ANTIGEN OF SEQ ID NO:19


atggctagcgcgcgccgcccgcgctggctgtgcgctggggcgctggtgctggcgggtggcttctttctcctcggcttcctcttc


 


gggtggtttataaaatcctccaatgaagctactaacattactccaggaattgtgggaggctgggagtgcgagaagcattcc


 


caaccctggcaggtgcttgtggcctctcgtggcagggcagtctgcggcggtgttctggtgcacccccagtgggtcctcaca


 


gctgcccactgcatcaggaacaaaagcgtgatcttgctgggtcggcacagcttgtttcatcctgaagacacaggccaggta


 


tttcaggtcagccacagcttcccacacccgctctacgatatgagcctcctgaagaatcgattcctcaggccaggtgatgact


 


ccagccacgacctcatgctgctccgcctgtcagagcctgccgagctcacggatgctgtgaaggtcatggacctgcccacc


 


caggagccagcactggggaccacctgctacgcctcaggctggggcagcattgaaccagaggagttcttgaccccaaag


 


aaacttcagtgtgtggacctccatgttatttccaatgacgtgtgtgcgcaagttcaccctcagaaggtgaccaagttcatgctg


 


tgtgctggacgctggacagggggcaaaagcacctgctcgggtgattctgggggcccacttgtctgtaatggtgtgcttcaag


 


gtatcacgtcatggggcagtgaaccatgtgccctgcccgaaaggccttccctgtacaccaaggtggtgcattaccggaagt


 


ggatcaaggacaccatcgtggccaacccctga


 


SEQ ID NO: 21. AMINO ACID SEQUENCE OF THE FULL LENGTH HUMAN


PSCA


MASKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDCLQVENCTQLGEQCWTARIRAV


 


GLLTVISKGCSLNCVDDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAILALLPALGLL


 


LWGPGQL


 


SEQ ID NO: 22. NUCLEOTIDE SEQUENCE ENCODING AMINO ACID


SEQUENCE OF THE FULL LENGTH HUMAN PSCA OF SEQ ID NO: 21


atggctagcaaggctgtgctgcttgccctgttgatggcaggcttggccctgcagccaggcactgccctgctgtgctactcctg


 


caaagcccaggtgagcaacgaggactgcctgcaggtggagaactgcacccagctgggggagcagtgctggaccgcg


 


cgcatccgcgcagttggcctcctgaccgtcatcagcaaaggctgcagcttgaactgcgtggatgactcacaggactacta


 


cgtgggcaagaagaacatcacgtgctgtgacaccgacttgtgcaacgccagcggggcccatgccctgcagccggctgc


 


cgccatccttgcgctgctccctgcactcggcctgctgctctggggacccggccagcta


 


SEQ ID NO: 23. NUCLEOTIDE SEQUENCE OF PLASMID 5166


 


SEQ ID NO: 24. NUCLEOTIDE SEQUENCE OF PLASMID 5259


 


SEQ ID NO: 25. NUCLEOTIDE SEQUENCE OF PLASMID 5297


 


SEQ ID NO: 26. NUCLEOTIDE SEQUENCE OF PLASMID 460


 


SEQ ID NO: 27. NUCLEOTIDE SEQUENCE OF PLASMID 451


 


SEQ ID NO: 28. NUCLEOTIDE SEQUENCE OF PLASMID 454


 


SEQ ID NO: 29. NUCLEOTIDE SEQUENCE OF PLASMID 5300


 


SEQ ID NO: 30. NUCLEOTIDE SEQUENCE OF PLASMID 449


 


SEQ ID NO: 31. NUCLEOTIDE SEQUENCE OF PLASMID 603


 


SEQ ID NO: 32. NUCLEOTIDE SEQUENCE OF PLASMID 455


 


SEQ ID NO: 33. NUCLEOTIDE SEQUENCE OF PLASMID 456


 


SEQ ID NO: 34. NUCLEOTIDE SEQUENCE OF PLASMID 457


 


SEQ ID NO: 35. NUCLEOTIDE SEQUENCE OF PLASMID 458


GGCGTAATGCTCTGCCAGTGTTACAACCAATTAACCAATTCTGATTAGAAAAACTCA


 


TCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTT


 


GAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATG


 


GCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTAT


 


TAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGAC


 


TGAATCCGGTGAGAATGGCAAAAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGG


 


CCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCG


 


TGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAA


 


CAGGAATCAAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCA


 


CCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGT


 


GGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAG


 


GCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAA


 


CGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAT


 


CGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATA


 


TAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGAGCAAGACGTTTCCCGTT


 


GAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGGTCGACAA


 


TATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATT


 


GGCTCATGTCCAATATGACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAG


 


TAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA


 


CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT


 


CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT


 


GGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG


 


CCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG


 


CCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCA


 


TCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCG


 


GTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGT


 


TTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTG


 


ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTT


 


TAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGA


 


AGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGG


 


ATTCCCCGTGCCAAGAGTGACTCACCGTCCGGATCTCAGCAAGCAGGTATGTACTC


 


TCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAGGGATTTGAGGGACGCTGT


 


GGGCTCTTCTCTTACATGTACCTTTTGCTTGCCTCAACCCTGACTATCTTCCAGGTC


 


AGGATCCCAGAGTCAGGGGTCTGTATTTTCCTGCTGGTGGCTCCAGTTCAGGAACA


 


GTAAACCCTGCTCCGAATATTGCCTCTCACATCTCGTCAATCTCCGCGAGGACTGG


 


GGACCCTGTGACGAACATGGCTAGCATTGTGGGAGGCTGGGAGTGCGAGAAGCAT


 


TCCCAACCCTGGCAGGTGCTTGTGGCCTCTCGTGGCAGGGCAGTCTGCGGCGGT


 


GTTCTGGTGCACCCCCAGTGGGTCCTCACAGCTGCCCACTGCATCAGGAACAAAA


 


GCGTGATCTTGCTGGGTCGGCACAGCTTGTTTCATCCTGAAGACACAGGCCAGGT


 


ATTTCAGGTCAGCCACAGCTTCCCACACCCGCTCTACGATATGAGCCTCCTGAAGA


 


ATCGATTCCTCAGGCCAGGTGATGACTCCAGCCACGACCTCATGCTGCTCCGCCT


 


GTCAGAGCCTGCCGAGCTCACGGATGCTGTGAAGGTCATGGACCTGCCCACCCAG


 


GAGCCAGCACTGGGGACCACCTGCTACGCCTCAGGCTGGGGCAGCATTGAACCA


 


GAGGAGTTCTTGACCCCAAAGAAACTTCAGTGTGTGGACCTCCATGTTATTTCCAAT


 


GACGTGTGTGCGCAAGTTCACCCTCAGAAGGTGACCAAGTTCATGCTGTGTGCTG


 


GACGCTGGACAGGGGGCAAAAGCACCTGCTCGGGTGATTCTGGGGGCCCACTTG


 


TCTGTAATGGTGTGCTTCAAGGTATCACGTCATGGGGCAGTGAACCATGTGCCCTG


 


CCCGAAAGGCCTTCCCTGTACACCAAGGTGGTGCATTACCGGAAGTGGATCAAGG


 


ACACCATCGTGGCCAACCCCGGATCCGAAGGTAGGGGTTCATTATTGACCTGTGG


 


AGATGTCGAAGAAAACCCAGGACCCGCTAGCAAGGCTGTGCTGCTTGCCCTGTTG


 


ATGGCAGGCTTGGCCCTGCAGCCAGGCACTGCCCTGCTGTGCTACTCCTGCAAAG


 


CCCAGGTGAGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGGGG


 


AGCAGTGCTGGACCGCGCGCATCCGCGCAGTTGGCCTCCTGACCGTCATCAGCAA


 


AGGCTGCAGCTTGAACTGCGTGGATGACTCACAGGACTACTACGTGGGCAAGAAG


 


AACATCACGTGCTGTGACACCGACTTGTGCAACGCCAGCGGGGCCCATGCCCTGC


 


AGCCGGCTGCCGCCATCCTTGCGCTGCTCCCTGCACTCGGCCTGCTGCTCTGGG


 


GACCCGGCCAGCTAGGATCCCAGACCCTGAACTTTGATCTGCTGAAACTGGCAGG


 


CGATGTGGAAAGCAACCCAGGCCCAATGGCAAGCGCGCGCCGCCCGCGCTGGCT


 


GTGCGCTGGGGCGCTGGTGCTGGCGGGTGGCTTCTTTCTCCTCGGCTTCCTCTTC


 


GGGTGGTTTATAAAATCCTCCAATGAAGCTACTAACATTACTCCAAAGCATAATATG


 


AAAGCATTTTTGGATGAATTGAAAGCTGAGAACATCAAGAAGTTCTTATATAATTTTA


 


CACAGATACCACATTTAGCAGGAACAGAACAAAACTTTCAGCTTGCAAAGCAAATTC


 


AATCCCAGTGGAAAGAATTTGGCCTGGATTCTGTTGAGCTGGCACATTATGATGTC


 


CTGTTGTCCTACCCAAATAAGACTCATCCCAACTACATCTCAATAATTAATGAAGAT


 


GGAAATGAGATTTTCAACACATCATTATTTGAACCACCTCCTCCAGGATATGAAAAT


 


GTTTCGGATATTGTACCACCTTTCAGTGCTTTCTCTCCTCAAGGAATGCCAGAGGG


 


CGATCTAGTGTATGTTAACTATGCACGAACTGAAGACTTCTTTAAATTGGAACGGGA


 


CATGAAAATCAATTGCTCTGGGAAAATTGTAATTGCCAGATATGGGAAAGTTTTCAG


 


AGGAAATAAGGTTAAAAATGCCCAGCTGGCAGGGGCCAAAGGAGTCATTCTCTACT


 


CCGACCCTGCTGACTACTTTGCTCCTGGGGTGAAGTCCTATCCAGATGGTTGGAAT


 


CTTCCTGGAGGTGGTGTCCAGCGTGGAAATATCCTAAATCTGAATGGTGCAGGAGA


 


CCCTCTCACACCAGGTTACCCAGCAAATGAATATGCTTATAGGCGTGGAATTGCAG


 


AGGCTGTTGGTCTTCCAAGTATTCCTGTTCATCCAATTGGATACTATGATGCACAGA


 


AGCTCCTAGAAAAAATGGGTGGCTCAGCACCACCAGATAGCAGCTGGAGAGGAAG


 


TCTCAAAGTGCCCTACAATGTTGGACCTGGCTTTACTGGAAACTTTTCTACACAAAA


 


AGTCAAGATGCACATCCACTCTACCAATGAAGTGACAAGAATTTACAATGTGATAGG


 


TACTCTCAGAGGAGCAGTGGAACCAGACAGATATGTCATTCTGGGAGGTCACCGG


 


GACTCATGGGTGTTTGGTGGTATTGACCCTCAGAGTGGAGCAGCTGTTGTTCATGA


 


AATTGTGAGGAGCTTTGGAACACTGAAAAAGGAAGGGTGGAGACCTAGAAGAACA


 


ATTTTGTTTGCAAGCTGGGATGCAGAAGAATTTGGTCTTCTTGGTTCTACTGAGTGG


 


GCAGAGGAGAATTCAAGACTCCTTCAAGAGCGTGGCGTGGCTTATATTAATGCTGA


 


CTCATCTATAGAAGGAAACTACACTCTGAGAGTTGATTGTACACCGCTGATGTACA


 


GCTTGGTACACAACCTAACAAAAGAGCTGAAAAGCCCTGATGAAGGCTTTGAAGGC


 


AAATCTCTTTATGAAAGTTGGACTAAAAAAAGTCCTTCCCCAGAGTTCAGTGGCATG


 


CCCAGGATAAGCAAATTGGGATCTGGAAATGATTTTGAGGTGTTCTTCCAACGACT


 


TGGAATTGCTTCAGGCAGAGCACGGTATACTAAAAATTGGGAAACAAACAAATTCA


 


GCGGCTATCCACTGTATCACAGTGTCTATGAAACATATGAGTTGGTGGAAAAGTTTT


 


ATGATCCAATGTTTAAATATCACCTCACTGTGGCCCAGGTTCGAGGAGGGATGGTG


 


TTTGAGCTGGCCAATTCCATAGTGCTCCCTTTTGATTGTCGAGATTATGCTGTAGTT


 


TTAAGAAAGTATGCTGACAAAATCTACAGTATTTCTATGAAACATCCACAGGAAATG


 


AAGACATACAGTGTATCATTTGATTCACTTTTTTCTGCAGTAAAGAATTTTACAGAAA


 


TTGCTTCCAAGTTCAGTGAGAGACTCCAGGACTTTGACAAAAGCAACCCAATAGTA


 


TTAAGAATGATGAATGATCAACTCATGTTTCTGGAAAGAGCATTTATTGATCCATTA


 


GGGTTACCAGACAGGCCTTTTTATAGGCATGTCATCTATGCTCCAAGCAGCCACAA


 


CAAGTATGCAGGGGAGTCATTCCCAGGAATTTATGATGCTCTGTTTGATATTGAAAG


 


CAAAGTGGACCCTTCCAAGGCCTGGGGAGAAGTGAAGAGACAGATTTATGTTGCA


 


GCCTTCACAGTGCAGGCAGCTGCAGAGACTTTGAGTGAAGTAGCCTAAAGATCTG


 


GGCCCTAACAAAACAAAAAGATGGGGTTATTCCCTAAACTTCATGGGTTACGTAATT


 


GGAAGTTGGGGGACATTGCCACAAGATCATATTGTACAAAAGATCAAACACTGTTTT


 


AGAAAACTTCCTGTAAACAGGCCTATTGATTGGAAAGTATGTCAAAGGATTGTGGG


 


TCTTTTGGGCTTTGCTGCTCCATTTACACAATGTGGATATCCTGCCTTAATGCCTTT


 


GTATGCATGTATACAAGCTAAACAGGCTTTCACTTTCTCGCCAACTTACAAGGCCTT


 


TCTAAGTAAACAGTACATGAACCTTTACCCCGTTGCTCGGCAACGGCCTGGTCTGT


 


GCCAAGTGTTTGCTGACGCAACCCCCACTGGCTGGGGCTTGGCCATAGGCCATCA


 


GCGCATGCGTGGAACCTTTGTGGCTCCTCTGCCGATCCATACTGCGGAACTCCTA


 


GCCGCTTGTTTTGCTCGCAGCCGGTCTGGAGCAAAGCTCATAGGAACTGACAATTC


 


TGTCGTCCTCTCGCGGAAATATACATCGTTTCGATCTACGTATGATCTTTTTCCCTC


 


TGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAAT


 


AAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGG


 


AAGGAATTCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT


 


TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG


 


CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAG


 


GGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCG


 


TAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT


 


CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA


 


CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG


 


CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAG


 


CTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT


 


GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC


 


TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAA


 


CAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGG


 


CCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCC


 


AGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG


 


GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCT


 


CAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTC


 


ACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTT


 


AAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC


 


AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCA


 


TCCATAGTTGCCTGACTC


 


SEQ ID NO: 36. NUCLEOTIDE SEQUENCE OF PLASMID 459


 


SEQ ID NO: 37. NUCLEOTIDE SEQUENCE OF PSHUTTLE IRES


 


SEQ ID NO: 38. Amino acid sequence of Her-2 antigen:


 


SEQ ID NO: 39. Nucleic acid sequence encoding the Her-2 antigen amino


 


acid sequence of SEQ ID NO: 38


 


SEQ ID NO: 40. Amino acid sequence of heavy chain of the anti-CD40


antibody CP870,893:


MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV


 


RQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAV


 


YYCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAA


 


LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYT


 


CNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTC


 


VVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK


 


EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD


 


IAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH


 


NHYTQKSLSLSPGK.


 


SEQ ID NO: 41. Acid sequence of the light chain of the anti-CD40 antibody


CP870,893:


MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQ


 


KPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTF


 


GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS


 


GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.


 


SEQ ID NO: 42. Acid sequence of the heavy chain of the anti-CTLA-4


antibody Tremelimumab


QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGS


 


NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDPRGATLYYYYYGMD


 


VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA


 


LTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCC


 


VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGV


 


EVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTK


 


GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPM


 


LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK


 


SEQ ID NO: 43. Acid sequence of the light chain of the anti-CTLA-4


antibody Tremelimumab


DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVP


 


SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFP


 


PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS


 


STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC


 


SEQ ID NO: 44. Nucleotide sequence of CpG 7909


5′ TCGTCGTTTTGTCGTTTTGTCGTT3′


 


SEQ ID NO: 45. Nucleotide sequence of CpG 24555


5′ TCGTCGTTTTTCGGTGCTTTT3′


 


SEQ ID NO: 46. Nucleotide sequence of CpG 10103


5′ TCGTCGTTTTTCGGTCGTTTT3′


 


SEQ ID NO: 47. Amino acid sequence of eGFP


 


SEQ ID NO: 48. Amino acid sequence of HBV core antigen


 


SEQ ID NO: 49. Amino acid sequence of HBV surface antigen


 


SEQ ID NO: 50. Amino acid sequence of Rhesus PSMA ECD protein:


 


SEQ ID NO: 51. Amino acid sequence of rat Her-2 p66 peptide (H-2d T cell


 


epitope)


 


SEQ ID NO: 52. Amino acid sequence of rat Her-2 p169 peptide (H-2d T cell


 


epitope)


 


SEQ ID NO: 53. Amino acid sequence of HBV core antigen p87 peptide


 


SEQ ID NO: 54. Amino acid sequence of a Rat Her-2 Antigen (rHer-2):


 


SEQ ID NO: 55. Amino Acid Sequence of Rhesus PSMA antigen:


 


SEQ ID NO: 56. Nucleotide sequence encoding the rhesus PSMA antigen of


 


SEQ ID NO: 55′′


 


SEQ ID NO: 57. Complete Genome of Simian Adenovirus 25 (C68)


ccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaagggcggtgatt


 


ggtcgagggatgagcgaccgttaggggcggggcgagtgacgttttgatgacgtggttgcgaggaggagccagtttgcaa


 


gttctcgtgggaaaagtgacgtcaaacgaggtgtggtttgaacacggaaatactcaattttcccgcgctctctgacaggaaa


 


tgaggtgtttctgggcggatgcaagtgaaaacgggccattttcgcgcgaaaactgaatgaggaagtgaaaatctgagtaa


 


tttcgcgtttatggcagggaggagtatttgccgagggccgagtagactttgaccgattacgtgggggtttcgattaccgtgttttt


 


cacctaaatttccgcgtacggtgtcaaagtccggtgtttttacgtaggtgtcagctgatcgccagggtatttaaacctgcgctct


 


ccagtcaagaggccactcttgagtgccagcgagaagagttttctcctccgcgccgcgagtcagatctacactttgaaagat


 


gaggcacctgagagacctgcccgatgagaaaatcatcatcgcttccgggaacgagattctggaactggtggtaaatgcc


 


atgatgggcgacgaccctccggagccccccaccccatttgagacaccttcgctgcacgatttgtatgatctggaggtggat


 


gtgcccgaggacgatcccaatgaggaggcggtaaatgatttttttagcgatgccgcgctgctagctgccgaggaggcttcg


 


agctctagctcagacagcgactcttcactgcatacccctagacccggcagaggtgagaaaaagatccccgagcttaaag


 


gggaagagatggacttgcgctgctatgaggaatgcttgcccccgagcgatgatgaggacgagcaggcgatccagaacg


 


cagcgagccagggagtgcaagccgccagcgagagctttgcgctggactgcccgcctctgcccggacacggctgtaagt


 


cttgtgaatttcatcgcatgaatactggagataaagctgtgttgtgtgcactttgctatatgagagcttacaaccattgtgtttaca


 


gtaagtgtgattaagttgaactttagagggaggcagagagcagggtgactgggcgatgactggtttatttatgtatatatgttct


 


ttatataggtcccgtctctgacgcagatgatgagacccccactacaaagtccacttcgtcacccccagaaattggcacatct


 


ccacctgagaatattgttagaccagttcctgttagagccactgggaggagagcagctgtggaatgtttggatgacttgctac


 


agggtggggttgaacctttggacttgtgtacccggaaacgccccaggcactaagtgccacacatgtgtgtttacttgaggtg


 


atgtcagtatttatagggtgtggagtgcaataaaaaatgtgttgactttaagtgcgtggtttatgactcaggggtggggactgtg


 


agtatataagcaggtgcagacctgtgtggttagctcagagcggcatggagatttggacggtcttggaagactttcacaaga


 


ctagacagctgctagagaacgcctcgaacggagtctcttacctgtggagattctgcttcggtggcgacctagctaggctagt


 


ctacagggccaaacaggattatagtgaacaatttgaggttattttgagagagtgttctggtctttttgacgctcttaacttgggcc


 


atcagtctcactttaaccagaggatttcgagagcccttgattttactactcctggcagaaccactgcagcagtagccttttttgct


 


tttattcttgacaaatggagtcaagaaacccatttcagcagggattaccagctggatttcttagcagtagctttgtggagaaca


 


tggaagtgccagcgcctgaatgcaatctccggctacttgccggtacagccgctagacactctgaggatcctgaatctccag


 


gagagtcccagggcacgccaacgtcgccagcagcagcagcaggaggaggatcaagaagagaacccgagagccg


 


gcctggaccctccggcggaggaggaggagtagctgacctgtttcctgaactgcgccgggtgctgactaggtcttcgagtg


 


gtcgggagagggggattaagcgggagaggcatgatgagactaatcacagaactgaactgactgtgggtctgatgagtc


 


gcaagcgcccagaaacagtgtggtggcatgaggtgcagtcgactggcacagatgaggtgtcggtgatgcatgagaggtt


 


ttctctagaacaagtcaagacttgttggttagagcctgaggatgattgggaggtagccatcaggaattatgccaagctggct


 


ctgaggccagacaagaagtacaagattactaagctgataaatatcagaaatgcctgctacatctcagggaatggggctg


 


aagtggagatctgtctccaggaaagggtggctttcagatgctgcatgatgaatatgtacccgggagtggtgggcatggatg


 


gggttacctttatgaacatgaggttcaggggagatgggtataatggcacggtctttatggccaataccaagctgacagtcca


 


tggctgctccttctttgggtttaataacacctgcatcgaggcctggggtcaggtcggtgtgaggggctgcagtttttcagccaa


 


ctggatgggggtcgtgggcaggaccaagagtatgctgtccgtgaagaaatgcttgtttgagaggtgccacctgggggtgat


 


gagcgagggcgaagccagaatccgccactgcgcctctaccgagacgggctgctttgtgctgtgcaagggcaatgctaag


 


atcaagcataatatgatctgtggagcctcggacgagcgcggctaccagatgctgacctgcgccggcgggaacagccata


 


tgctggccaccgtacatgtggcttcccatgctcgcaagccctggcccgagttcgagcacaatgtcatgaccaggtgcaata


 


tgcatctggggtcccgccgaggcatgttcatgccctaccagtgcaacctgaattatgtgaaggtgctgctggagcccgatgc


 


catgtccagagtgagcctgacgggggtgtttgacatgaatgtggaggtgtggaagattctgagatatgatgaatccaagac


 


caggtgccgagcctgcgagtgcggagggaagcatgccaggttccagcccgtgtgtgtggatgtgacggaggacctgcg


 


acccgatcatttggtgttgccctgcaccgggacggagttcggttccagcggggaagaatctgactagagtgagtagtgttct


 


ggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgcagcagcatgagcggaag


 


cggctcctttgagggaggggtattcagcccttatctgacggggcgtctcccctcctgggcgggagtgcgtcagaatgtgatg


 


ggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaaccctgagctcttcgtcgttg


 


gacgcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatgggcgccggctactacggc


 


actctggtggccaactcgagttccaccaataatcccgccagcctgaacgaggagaagctgttgctgctgatggcccagct


 


cgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagcagacgcgggccgcggttg


 


ccacggtgaaatccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaacacagagtctgaatctttatt


 


tgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtggatcttttccaggacccggtagag


 


gtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattgcagggcctcgtgctcggg


 


ggtggtgttgtaaatcacccagtcatagcaggggcgcagggcatggtgttgcacaatatctttgaggaggagactgatggc


 


cacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcggggggagatgaggtgcatcttg


 


gcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcaggaccaccagcacggtgtatc


 


cggtgcacttggggaatttatcatgcaacttggaagggaaggcgtgaaagaatttggcgacgcctttgtgcccgcccaggtt


 


ttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacgtttcgggggtcggacacat


 


catagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgccggactgggggacaaaggt


 


accctcgatcccgggggcgtagttcccctcacagatctgcatctcccaggctttgagctcggagggggggatcatgtccac


 


ctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaagcaagttccggagcagctgg


 


gacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttgagggagagacagctgccg


 


tcctcccggaggaggggggccacctcgttcatcatctcgcgcacgtgcatgttctcgcgcaccagttccgccaggaggcg


 


ctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggcttgagtccgtcggccatgggcattttggag


 


agggtttgttgcaagagttccaggcggtcccagagctcggtgatgtgctctacggcatctcgatccagcagacctcctcgttt


 


cgcgggttgggacggctgcgggagtagggcaccagacgatgggcgtccagcgcagccagggtccggtccttccagggt


 


cgcagcgtccgcgtcagggtggtctccgtcacggtgaaggggtgcgcgccgggctgggcgcttgcgagggtgcgcttca


 


ggctcatccggctggtcgaaaaccgctcccgatcggcgccctgcgcgtcggccaggtagcaattgaccatgagttcgtag


 


ttgagcgcctcggccgcgtggcctttggcgcggagcttacctttggaagtctgcccgcaggcgggacagaggagggactt


 


gagggcgtagagcttgggggcgaggaagacggactcgggggcgtaggcgtccgcgccgcagtgggcgcagacggtc


 


tcgcactccacgagccaggtgaggtcgggctggtcggggtcaaaaaccagtttcccgccgttctttttgatgcgtttcttacctt


 


tggtctccatgagctcgtgtccccgctgggtgacaaagaggctgtccgtgtccccgtagaccgactttatgggccggtcctc


 


gagcggtgtgccgcggtcctcctcgtagaggaaccccgcccactccgagacgaaagcccgggtccaggccagcacga


 


aggaggccacgtgggacgggtagcggtcgttgtccaccagcgggtccaccttttccagggtatgcaaacacatgtccccc


 


tcgtccacatccaggaaggtgattggcttgtaagtgtaggccacgtgaccgggggtcccggccgggggggtataaaagg


 


gtgcgggtccctgctcgtcctcactgtcttccggatcgctgtccaggagcgccagctgttggggtaggtattccctctcgaag


 


gcgggcatgacctcggcactcaggttgtcagtttctagaaacgaggaggatttgatattgacggtgccggcggagatgcctt


 


tcaagagcccctcgtccatctggtcagaaaagacgatctttttgttgtcgagcttggtggcgaaggagccgtagagggcgtt


 


ggagaggagcttggcgatggagcgcatggtctggtttttttccttgtcggcgcgctccttggcggcgatgttgagctgcacgta


 


ctcgcgcgccacgcacttccattcggggaagacggtggtcagctcgtcgggcacgattctgacctgccagccccgattatg


 


cagggtgatgaggtccacactggtggccacctcgccgcgcaggggctcattagtccagcagaggcgtccgcccttgcgc


 


gagcagaaggggggcagggggtccagcatgacctcgtcgggggggtcggcatcgatggtgaagatgccgggcagga


 


ggtcggggtcaaagtagctgatggaagtggccagatcgtccagggcagcttgccattcgcgcacggccagcgcgcgctc


 


gtagggactgaggggcgtgccccagggcatgggatgggtaagcgcggaggcgtacatgccgcagatgtcgtagacgt


 


agaggggctcctcgaggatgccgatgtaggtggggtagcagcgccccccgcggatgctggcgcgcacgtagtcataca


 


gctcgtgcgagggggcgaggagccccgggcccaggttggtgcgactgggcttttcggcgcggtagacgatctggcgga


 


aaatggcatgcgagttggaggagatggtgggcctttggaagatgttgaagtgggcgtggggcagtccgaccgagtcgcg


 


gatgaagtgggcgtaggagtcttgcagcttggcgacgagctcggcggtgactaggacgtccagagcgcagtagtcgag


 


ggtctcctggatgatgtcatacttgagctgtcccttttgtttccacagctcgcggttgagaaggaactcttcgcggtccttccagt


 


actcttcgagggggaacccgtcctgatctgcacggtaagagcctagcatgtagaactggttgacggccttgtaggcgcag


 


cagcccttctccacggggagggcgtaggcctgggcggccttgcgcagggaggtgtgcgtgagggcgaaagtgtccctg


 


accatgaccttgaggaactggtgcttgaagtcgatatcgtcgcagcccccctgctcccagagctggaagtccgtgcgcttct


 


tgtaggcggggttgggcaaagcgaaagtaacatcgttgaagaggatcttgcccgcgcggggcataaagttgcgagtgat


 


gcggaaaggttggggcacctcggcccggttgttgatgacctgggcggcgagcacgatctcgtcgaagccgttgatgttgtg


 


gcccacgatgtagagttccacgaatcgcggacggcccttgacgtggggcagtttcttgagctcctcgtaggtgagctcgtcg


 


gggtcgctgagcccgtgctgctcgagcgcccagtcggcgagatgggggttggcgcggaggaaggaagtccagagatc


 


cacggccagggcggtttgcagacggtcccggtactgacggaactgctgcccgacggccattttttcgggggtgacgcagt


 


agaaggtgcgggggtccccgtgccagcgatcccatttgagctggagggcgagatcgagggcgagctcgacgagccgg


 


tcgtccccggagagtttcatgaccagcatgaaggggacgagctgcttgccgaaggaccccatccaggtgtaggtttccac


 


atcgtaggtgaggaagagcctttcggtgcgaggatgcgagccgatggggaagaactggatctcctgccaccaattggag


 


gaatggctgttgatgtgatggaagtagaaatgccgacggcgcgccgaacactcgtgcttgtgtttatacaagcggccacag


 


tgctcgcaacgctgcacgggatgcacgtgctgcacgagctgtacctgagttcctttgacgaggaatttcagtgggaagtgg


 


agtcgtggcgcctgcatctcgtgctgtactacgtcgtggtggtcggcctggccctcttctgcctcgatggtggtcatgctgacg


 


agcccgcgcgggaggcaggtccagacctcggcgcgagcgggtcggagagcgaggacgagggcgcgcaggccgga


 


gctgtccagggtcctgagacgctgcggagtcaggtcagtgggcagcggcggcgcgcggttgacttgcaggagtttttcca


 


gggcgcgcgggaggtccagatggtacttgatctccaccgcgccattggtggcgacgtcgatggcttgcagggtcccgtgc


 


ccctggggtgtgaccaccgtcccccgtttcttcttgggcggctggggcgacgggggcggtgcctcttccatggttagaagcg


 


gcggcgaggacgcgcgccgggcggcaggggcggctcggggcccggaggcaggggcggcaggggcacgtcggcg


 


ccgcgcgcgggtaggttctggtactgcgcccggagaagactggcgtgagcgacgacgcgacggttgacgtcctggatct


 


gacgcctctgggtgaaggccacgggacccgtgagtttgaacctgaaagagagttcgacagaatcaatctcggtatcgttg


 


acggcggcctgccgcaggatctcttgcacgtcgcccgagttgtcctggtaggcgatctcggtcatgaactgctcgatctcctc


 


ctcttgaaggtctccgcggccggcgcgctccacggtggccgcgaggtcgttggagatgcggcccatgagctgcgagaag


 


gcgttcatgcccgcctcgttccagacgcggctgtagaccacgacgccctcgggatcgccggcgcgcatgaccacctggg


 


cgaggttgagctccacgtggcgcgtgaagaccgcgtagttgcagaggcgctggtagaggtagttgagcgtggtggcgat


 


gtgctcggtgacgaagaaatacatgatccagcggcggagcggcatctcgctgacgtcgcccagcgcctccaaacgttcc


 


atggcctcgtaaaagtccacggcgaagttgaaaaactgggagttgcgcgccgagacggtcaactcctcctccagaagac


 


ggatgagctcggcgatggtggcgcgcacctcgcgctcgaaggcccccgggagttcctccacttcctcttcttcctcctccact


 


aacatctcttctacttcctcctcaggcggcagtggtggcgggggagggggcctgcgtcgccggcggcgcacgggcagac


 


ggtcgatgaagcgctcgatggtctcgccgcgccggcgtcgcatggtctcggtgacggcgcgcccgtcctcgcggggccg


 


cagcgtgaagacgccgccgcgcatctccaggtggccgggggggtccccgttgggcagggagagggcgctgacgatgc


 


atcttatcaattgccccgtagggactccgcgcaaggacctgagcgtctcgagatccacgggatctgaaaaccgctgaacg


 


aaggcttcgagccagtcgcagtcgcaaggtaggctgagcacggtttcttctggcgggtcatgttggttgggagcggggcgg


 


gcgatgctgctggtgatgaagttgaaataggcggttctgagacggcggatggtggcgaggagcaccaggtctttgggccc


 


ggcttgctggatgcgcagacggtcggccatgccccaggcgtggtcctgacacctggccaggtccttgtagtagtcctgcat


 


gagccgctccacgggcacctcctcctcgcccgcgcggccgtgcatgcgcgtgagcccgaagccgcgctggggctggac


 


gagcgccaggtcggcgacgacgcgctcggcgaggatggcttgctggatctgggtgagggtggtctggaagtcatcaaag


 


tcgacgaagcggtggtaggctccggtgttgatggtgtaggagcagttggccatgacggaccagttgacggtctggtggccc


 


ggacgcacgagctcgtggtacttgaggcgcgagtaggcgcgcgtgtcgaagatgtagtcgttgcaggtgcgcaccaggt


 


actggtagccgatgaggaagtgcggcggcggctggcggtagagcggccatcgctcggtggcgggggcgccgggcgc


 


gaggtcctcgagcatggtgcggtggtagccgtagatgtacctggacatccaggtgatgccggcggcggtggtggaggcg


 


cgcgggaactcgcggacgcggttccagatgttgcgcagcggcaggaagtagttcatggtgggcacggtctggcccgtga


 


ggcgcgcgcagtcgtggatgctctatacgggcaaaaacgaaagcggtcagcggctcgactccgtggcctggaggctaa


 


gcgaacgggttgggctgcgcgtgtaccccggttcgaatctcgaatcaggctggagccgcagctaacgtggtattggcactc


 


ccgtctcgacccaagcctgcaccaaccctccaggatacggaggcgggtcgttttgcaacttttttttggaggccggatgaga


 


ctagtaagcgcggaaagcggccgaccgcgatggctcgctgccgtagtctggagaagaatcgccagggttgcgttgcggt


 


gtgccccggttcgaggccggccggattccgcggctaacgagggcgtggctgccccgtcgtttccaagaccccatagcca


 


gccgacttctccagttacggagcgagcccctcttttgttttgtttgtttttgccagatgcatcccgtactgcggcagatgcgcccc


 


caccaccctccaccgcaacaacagccccctccacagccggcgcttctgcccccgccccagcagcaacttccagccacg


 


accgccgcggccgccgtgagcggggctggacagagttatgatcaccagctggccttggaagagggcgaggggctggc


 


gcgcctgggggcgtcgtcgccggagcggcacccgcgcgtgcagatgaaaagggacgctcgcgaggcctacgtgccc


 


aagcagaacctgttcagagacaggagcggcgaggagcccgaggagatgcgcgcggcccggttccacgcggggcgg


 


gagctgcggcgcggcctggaccgaaagagggtgctgagggacgaggatttcgaggcggacgagctgacggggatca


 


gccccgcgcgcgcgcacgtggccgcggccaacctggtcacggcgtacgagcagaccgtgaaggaggagagcaactt


 


ccaaaaatccttcaacaaccacgtgcgcaccctgatcgcgcgcgaggaggtgaccctgggcctgatgcacctgtgggac


 


ctgctggaggccatcgtgcagaaccccaccagcaagccgctgacggcgcagctgttcctggtggtgcagcatagtcggg


 


acaacgaagcgttcagggaggcgctgctgaatatcaccgagcccgagggccgctggctcctggacctggtgaacattct


 


gcagagcatcgtggtgcaggagcgcgggctgccgctgtccgagaagctggcggccatcaacttctcggtgctgagtttgg


 


gcaagtactacgctaggaagatctacaagaccccgtacgtgcccatagacaaggaggtgaagatcgacgggttttacat


 


gcgcatgaccctgaaagtgctgaccctgagcgacgatctgggggtgtaccgcaacgacaggatgcaccgtgcggtgag


 


cgccagcaggcggcgcgagctgagcgaccaggagctgatgcatagtctgcagcgggccctgaccggggccgggacc


 


gagggggagagctactttgacatgggcgcggacctgcactggcagcccagccgccgggccttggaggcggcggcagg


 


accctacgtagaagaggtggacgatgaggtggacgaggagggcgagtacctggaagactgatggcgcgaccgtattttt


 


gctagatgcaacaacaacagccacctcctgatcccgcgatgcgggcggcgctgcagagccagccgtccggcattaact


 


cctcggacgattggacccaggccatgcaacgcatcatggcgctgacgacccgcaaccccgaagcctttagacagcagc


 


cccaggccaaccggctctcggccatcctggaggccgtggtgccctcgcgctccaaccccacgcacgagaaggtcctgg


 


ccatcgtgaacgcgctggtggagaacaaggccatccgcggcgacgaggccggcctggtgtacaacgcgctgctggag


 


cgcgtggcccgctacaacagcaccaacgtgcagaccaacctggaccgcatggtgaccgacgtgcgcgaggccgtggc


 


ccagcgcgagcggttccaccgcgagtccaacctgggatccatggtggcgctgaacgccttcctcagcacccagcccgcc


 


aacgtgccccggggccaggaggactacaccaacttcatcagcgccctgcgcctgatggtgaccgaggtgccccagagc


 


gaggtgtaccagtccgggccggactacttcttccagaccagtcgccagggcttgcagaccgtgaacctgagccaggcttt


 


caagaacttgcagggcctgtggggcgtgcaggccccggtcggggaccgcgcgacggtgtcgagcctgctgacgccga


 


actcgcgcctgctgctgctgctggtggcccccttcacggacagcggcagcatcaaccgcaactcgtacctgggctacctg


 


attaacctgtaccgcgaggccatcggccaggcgcacgtggacgagcagacctaccaggagatcacccacgtgagccg


 


cgccctgggccaggacgacccgggcaacctggaagccaccctgaactttttgctgaccaaccggtcgcagaagatccc


 


gccccagtacgcgctcagcaccgaggaggagcgcatcctgcgttacgtgcagcagagcgtgggcctgttcctgatgcag


 


gagggggccacccccagcgccgcgctcgacatgaccgcgcgcaacatggagcccagcatgtacgccagcaaccgcc


 


cgttcatcaataaactgatggactacttgcatcgggcggccgccatgaactctgactatttcaccaacgccatcctgaatccc


 


cactggctcccgccgccggggttctacacgggcgagtacgacatgcccgaccccaatgacgggttcctgtgggacgatgt


 


ggacagcagcgtgttctccccccgaccgggtgctaacgagcgccccttgtggaagaaggaaggcagcgaccgacgcc


 


cgtcctcggcgctgtccggccgcgagggtgctgccgcggcggtgcccgaggccgccagtcctttcccgagcttgcccttct


 


cgctgaacagtatccgcagcagcgagctgggcaggatcacgcgcccgcgcttgctgggcgaagaggagtacttgaatg


 


actcgctgttgagacccgagcgggagaagaacttccccaataacgggatagaaagcctggtggacaagatgagccgct


 


ggaagacgtatgcgcaggagcacagggacgatccccgggcgtcgcagggggccacgagccggggcagcgccgcc


 


cgtaaacgccggtggcacgacaggcagcggggacagatgtgggacgatgaggactccgccgacgacagcagcgtgt


 


tggacttgggtgggagtggtaacccgttcgctcacctgcgcccccgtatcgggcgcatgatgtaagagaaaccgaaaata


 


aatgatactcaccaaggccatggcgaccagcgtgcgttcgtttcttctctgttgttgttgtatctagtatgatgaggcgtgcgtac


 


ccggagggtcctcctccctcgtacgagagcgtgatgcagcaggcgatggcggcggcggcgatgcagcccccgctgga


 


ggctccttacgtgcccccgcggtacctggcgcctacggaggggcggaacagcattcgttactcggagctggcacccttgta


 


cgataccacccggttgtacctggtggacaacaagtcggcggacatcgcctcgctgaactaccagaacgaccacagcaa


 


cttcctgaccaccgtggtgcagaacaatgacttcacccccacggaggccagcacccagaccatcaactttgacgagcgc


 


tcgcggtggggcggccagctgaaaaccatcatgcacaccaacatgcccaacgtgaacgagttcatgtacagcaacaag


 


ttcaaggcgcgggtgatggtctcccgcaagacccccaatggggtgacagtgacagaggattatgatggtagtcaggatg


 


agctgaagtatgaatgggtggaatttgagctgcccgaaggcaacttctcggtgaccatgaccatcgacctgatgaacaac


 


gccatcatcgacaattacttggcggtggggcggcagaacggggtgctggagagcgacatcggcgtgaagttcgacacta


 


ggaacttcaggctgggctgggaccccgtgaccgagctggtcatgcccggggtgtacaccaacgaggctttccatcccgat


 


attgtcttgctgcccggctgcggggtggacttcaccgagagccgcctcagcaacctgctgggcattcgcaagaggcagcc


 


cttccaggaaggcttccagatcatgtacgaggatctggaggggggcaacatccccgcgctcctggatgtcgacgcctatg


 


agaaaagcaaggaggatgcagcagctgaagcaactgcagccgtagctaccgcctctaccgaggtcaggggcgataat


 


tttgcaagcgccgcagcagtggcagcggccgaggcggctgaaaccgaaagtaagatagtcattcagccggtggagaa


 


ggatagcaagaacaggagctacaacgtactaccggacaagataaacaccgcctaccgcagctggtacctagcctaca


 


actatggcgaccccgagaagggcgtgcgctcctggacgctgctcaccacctcggacgtcacctgcggcgtggagcaagt


 


ctactggtcgctgcccgacatgatgcaagacccggtcaccttccgctccacgcgtcaagttagcaactacccggtggtggg


 


cgccgagctcctgcccgtctactccaagagcttcttcaacgagcaggccgtctactcgcagcagctgcgcgccttcacctc


 


gcttacgcacgtcttcaaccgcttccccgagaaccagatcctcgtccgcccgcccgcgcccaccattaccaccgtcagtga


 


aaacgttcctgctctcacagatcacgggaccctgccgctgcgcagcagtatccggggagtccagcgcgtgaccgttactg


 


acgccagacgccgcacctgcccctacgtctacaaggccctgggcatagtcgcgccgcgcgtcctctcgagccgcacctt


 


ctaaatgtccattctcatctcgcccagtaataacaccggttggggcctgcgcgcgcccagcaagatgtacggaggcgctc


 


gccaacgctccacgcaacaccccgtgcgcgtgcgcgggcacttccgcgctccctggggcgccctcaagggccgcgtgc


 


ggtcgcgcaccaccgtcgacgacgtgatcgaccaggtggtggccgacgcgcgcaactacacccccgccgccgcgccc


 


gtctccaccgtggacgccgtcatcgacagcgtggtggcggacgcgcgccggtacgcccgcgccaagagccggcggcg


 


gcgcatcgcccggcggcaccggagcacccccgccatgcgcgcggcgcgagccttgctgcgcagggccaggcgcacg


 


ggacgcagggccatgctcagggcggccagacgcgcggcttcaggcgccagcgccggcaggacccggagacgcgcg


 


gccacggcggcggcagcggccatcgccagcatgtcccgcccgcggcgagggaacgtgtactgggtgcgcgacgccg


 


ccaccggtgtgcgcgtgcccgtgcgcacccgcccccctcgcacttgaagatgttcacttcgcgatgttgatgtgtcccagcg


 


gcgaggaggatgtccaagcgcaaattcaaggaagagatgctccaggtcatcgcgcctgagatctacggccctgcggtg


 


gtgaaggaggaaagaaagccccgcaaaatcaagcgggtcaaaaaggacaaaaaggaagaagaaagtgatgtgga


 


cggattggtggagtttgtgcgcgagttcgccccccggcggcgcgtgcagtggcgcgggcggaaggtgcaaccggtgctg


 


agacccggcaccaccgtggtcttcacgcccggcgagcgctccggcaccgcttccaagcgctcctacgacgaggtgtacg


 


gggatgatgatattctggagcaggcggccgagcgcctgggcgagtttgcttacggcaagcgcagccgttccgcaccgaa


 


ggaagaggcggtgtccatcccgctggaccacggcaaccccacgccgagcctcaagcccgtgaccttgcagcaggtgct


 


gccgaccgcggcgccgcgccgggggttcaagcgcgagggcgaggatctgtaccccaccatgcagctgatggtgccca


 


agcgccagaagctggaagacgtgctggagaccatgaaggtggacccggacgtgcagcccgaggtcaaggtgcggcc


 


catcaagcaggtggccccgggcctgggcgtgcagaccgtggacatcaagattcccacggagcccatggaaacgcaga


 


ccgagcccatgatcaagcccagcaccagcaccatggaggtgcagacggatccctggatgccatcggctcctagtcgaa


 


gaccccggcgcaagtacggcgcggccagcctgctgatgcccaactacgcgctgcatccttccatcatccccacgccggg


 


ctaccgcggcacgcgcttctaccgcggtcataccagcagccgccgccgcaagaccaccactcgccgccgccgtcgccg


 


caccgccgctgcaaccacccctgccgccctggtgcggagagtgtaccgccgcggccgcgcacctctgaccctgccgcg


 


cgcgcgctaccacccgagcatcgccatttaaactttcgccagctttgcagatcaatggccctcacatgccgccttcgcgttcc


 


cattacgggctaccgaggaagaaaaccgcgccgtagaaggctggcggggaacgggatgcgtcgccaccaccaccgg


 


cggcggcgcgccatcagcaagcggttggggggaggcttcctgcccgcgctgatccccatcatcgccgcggcgatcggg


 


gcgatccccggcattgcttccgtggcggtgcaggcctctcagcgccactgagacacacttggaaacatcttgtaataaacc


 


catggactctgacgctcctggtcctgtgatgtgttttcgtagacagatggaagacatcaatttttcgtccctggctccgcgacac


 


ggcacgcggccgttcatgggcacctggagcgacatcggcaccagccaactgaacgggggcgccttcaattggagcagt


 


ctctggagcgggcttaagaatttcgggtccacgcttaaaacctatggcagcaaggcgtggaacagcaccacagggcag


 


gcgctgagggataagctgaaagagcagaacttccagcagaaggtggtcgatgggctcgcctcgggcatcaacggggtg


 


gtggacctggccaaccaggccgtgcagcggcagatcaacagccgcctggacccggtgccgcccgccggctccgtgga


 


gatgccgcaggtggaggaggagctgcctcccctggacaagcggggcgagaagcgaccccgccccgatgcggagga


 


gacgctgctgacgcacacggacgagccgcccccgtacgaggaggcggtgaaactgggtctgcccaccacgcggccc


 


atcgcgcccctggccaccggggtgctgaaacccgaaaagcccgcgaccctggacttgcctcctccccagccttcccgcc


 


cctctacagtggctaagcccctgccgccggtggccgtggcccgcgcgcgacccgggggcaccgcccgccctcatgcga


 


actggcagagcactctgaacagcatcgtgggtctgggagtgcagagtgtgaagcgccgccgctgctattaaacctaccgt


 


agcgcttaacttgcttgtctgtgtgtgtatgtattatgtcgccgccgccgctgtccaccagaaggaggagtgaagaggcgcgt


 


cgccgagttgcaagatggccaccccatcgatgctgccccagtgggcgtacatgcacatcgccggacaggacgcttcgga


 


gtacctgagtccgggtctggtgcagtttgcccgcgccacagacacctacttcagtctggggaacaagtttaggaaccccac


 


ggtggcgcccacgcacgatgtgaccaccgaccgcagccagcggctgacgctgcgcttcgtgcccgtggaccgcgagg


 


acaacacctactcgtacaaagtgcgctacacgctggccgtgggcgacaaccgcgtgctggacatggccagcacctacttt


 


gacatccgcggcgtgctggatcggggccctagcttcaaaccctactccggcaccgcctacaacagtctggcccccaagg


 


gagcacccaacacttgtcagtggacatataaagccgatggtgaaactgccacagaaaaaacctatacatatggaaatgc


 


acccgtgcagggcattaacatcacaaaagatggtattcaacttggaactgacaccgatgatcagccaatctacgcagata


 


aaacctatcagcctgaacctcaagtgggtgatgctgaatggcatgacatcactggtactgatgaaaagtatggaggcaga


 


gctcttaagcctgataccaaaatgaagccttgttatggttcttttgccaagcctactaataaagaaggaggtcaggcaaatgt


 


gaaaacaggaacaggcactactaaagaatatgacatagacatggctttctttgacaacagaagtgcggctgctgctggcc


 


tagctccagaaattgttttgtatactgaaaatgtggatttggaaactccagatacccatattgtatacaaagcaggcacagat


 


gacagcagctcttctattaatttgggtcagcaagccatgcccaacagacctaactacattggtttcagagacaactttatcgg


 


gctcatgtactacaacagcactggcaatatgggggtgctggccggtcaggcttctcagctgaatgctgtggttgacttgcaa


 


gacagaaacaccgagctgtcctaccagctcttgcttgactctctgggtgacagaacccggtatttcagtatgtggaatcagg


 


cggtggacagctatgatcctgatgtgcgcattattgaaaatcatggtgtggaggatgaacttcccaactattgtttccctctgga


 


tgctgttggcagaacagatacttatcagggaattaaggctaatggaactgatcaaaccacatggaccaaagatgacagtg


 


tcaatgatgctaatgagataggcaagggtaatccattcgccatggaaatcaacatccaagccaacctgtggaggaacttc


 


ctctacgccaacgtggccctgtacctgcccgactcttacaagtacacgccggccaatgttaccctgcccaccaacaccaa


 


cacctacgattacatgaacggccgggtggtggcgccctcgctggtggactcctacatcaacatcggggcgcgctggtcgc


 


tggatcccatggacaacgtgaaccccttcaaccaccaccgcaatgcggggctgcgctaccgctccatgctcctgggcaa


 


cgggcgctacgtgcccttccacatccaggtgccccagaaatttttcgccatcaagagcctcctgctcctgcccgggtcctac


 


acctacgagtggaacttccgcaaggacgtcaacatgatcctgcagagctccctcggcaacgacctgcgcacggacggg


 


gcctccatctccttcaccagcatcaacctctacgccaccttcttccccatggcgcacaacacggcctccacgctcgaggcc


 


atgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcggccaacatgctctaccccatcccggcca


 


acgccaccaacgtgcccatctccatcccctcgcgcaactgggccgccttccgcggctggtccttcacgcgtctcaagacca


 


aggagacgccctcgctgggctccgggttcgacccctacttcgtctactcgggctccatcccctacctcgacggcaccttcta


 


cctcaaccacaccttcaagaaggtctccatcaccttcgactcctccgtcagctggcccggcaacgaccggctcctgacgc


 


ccaacgagttcgaaatcaagcgcaccgtcgacggcgagggctacaacgtggcccagtgcaacatgaccaaggactgg


 


ttcctggtccagatgctggcccactacaacatcggctaccagggcttctacgtgcccgagggctacaaggaccgcatgtac


 


tccttcttccgcaacttccagcccatgagccgccaggtggtggacgaggtcaactacaaggactaccaggccgtcaccct


 


ggcctaccagcacaacaactcgggcttcgtcggctacctcgcgcccaccatgcgccagggccagccctaccccgccaa


 


ctacccctacccgctcatcggcaagagcgccgtcaccagcgtcacccagaaaaagttcctctgcgacagggtcatgtgg


 


cgcatccccttctccagcaacttcatgtccatgggcgcgctcaccgacctcggccagaacatgctctatgccaactccgcc


 


cacgcgctagacatgaatttcgaagtcgaccccatggatgagtccacccttctctatgttgtcttcgaagtcttcgacgtcgtc


 


cgagtgcaccagccccaccgcggcgtcatcgaggccgtctacctgcgcacccccttctcggccggtaacgccaccacct


 


aagctcttgcttcttgcaagccatggccgcgggctccggcgagcaggagctcagggccatcatccgcgacctgggctgcg


 


ggccctacttcctgggcaccttcgataagcgcttcccgggattcatggccccgcacaagctggcctgcgccatcgtcaaca


 


cggccggccgcgagaccgggggcgagcactggctggccttcgcctggaacccgcgctcgaacacctgctacctcttcg


 


accccttcgggttctcggacgagcgcctcaagcagatctaccagttcgagtacgagggcctgctgcgccgcagcgccctg


 


gccaccgaggaccgctgcgtcaccctggaaaagtccacccagaccgtgcagggtccgcgctcggccgcctgcgggctc


 


ttctgctgcatgttcctgcacgccttcgtgcactggcccgaccgccccatggacaagaaccccaccatgaacttgctgacg


 


ggggtgcccaacggcatgctccagtcgccccaggtggaacccaccctgcgccgcaaccaggaggcgctctaccgcttc


 


ctcaactcccactccgcctactttcgctcccaccgcgcgcgcatcgagaaggccaccgccttcgaccgcatgaatcaaga


 


catgtaaaccgtgtgtgtatgttaaatgtctttaataaacagcactttcatgttacacatgcatctgagatgatttatttagaaatc


 


gaaagggttctgccgggtctcggcatggcccgcgggcagggacacgttgcggaactggtacttggccagccacttgaact


 


cggggatcagcagtttgggcagcggggtgtcggggaaggagtcggtccacagcttccgcgtcagttgcagggcgccca


 


gcaggtcgggcgcggagatcttgaaatcgcagttgggacccgcgttctgcgcgcgggagttgcggtacacggggttgca


 


gcactggaacaccatcagggccgggtgcttcacgctcgccagcaccgtcgcgtcggtgatgctctccacgtcgaggtcct


 


cggcgttggccatcccgaagggggtcatcttgcaggtctgccttcccatggtgggcacgcacccgggcttgtggttgcaatc


 


gcagtgcagggggatcagcatcatctgggcctggtcggcgttcatccccgggtacatggccttcatgaaagcctccaattg


 


cctgaacgcctgctgggccttggctccctcggtgaagaagaccccgcaggacttgctagagaactggttggtggcgcacc


 


cggcgtcgtgcacgcagcagcgcgcgtcgttgttggccagctgcaccacgctgcgcccccagcggttctgggtgatcttgg


 


cccggtcggggttctccttcagcgcgcgctgcccgttctcgctcgccacatccatctcgatcatgtgctccttctggatcatggt


 


ggtcccgtgcaggcaccgcagcttgccctcggcctcggtgcacccgtgcagccacagcgcgcacccggtgcactccca


 


gttcttgtgggcgatctgggaatgcgcgtgcacgaagccctgcaggaagcggcccatcatggtggtcagggtcttgttgcta


 


gtgaaggtcagcggaatgccgcggtgctcctcgttgatgtacaggtggcagatgcggcggtacacctcgccctgctcggg


 


catcagctggaagttggctttcaggtcggtctccacgcggtagcggtccatcagcatagtcatgatttccatacccttctccca


 


ggccgagacgatgggcaggctcatagggttcttcaccatcatcttagcgctagcagccgcggccagggggtcgctctcgt


 


ccagggtctcaaagctccgcttgccgtccttctcggtgatccgcaccggggggtagctgaagcccacggccgccagctcc


 


tcctcggcctgtctttcgtcctcgctgtcctggctgacgtcctgcaggaccacatgcttggtcttgcggggtttcttcttggcggc


 


agcggcggcggagatgttggagatggcgagggggagcgcgagttctcgctcaccactactatctcttcctcttcttggtccg


 


aggccacgcggcggtaggtatgtctcttcgggggcagaggcggaggcgacgggctctcgccgccgcgacttggcggat


 


ggctggcagagccccttccgcgttcgggggtgcgctcccggcggcgctctgactgacttcctccgcggccggccattgtgtt


 


ctcctagggaggaacaacaagcatggagactcagccatcgccaacctcgccatctgcccccaccgccgacgagaagc


 


agcagcagcagaatgaaagcttaaccgccccgccgcccagccccgccacctccgacgcggccgtcccagacatgca


 


agagatggaggaatccatcgagattgacctgggctatgtgacgcccgcggagcacgaggaggagctggcagtgcgcttt


 


tcacaagaagagatacaccaagaacagccagagcaggaagcagagaatgagcagagtcaggctgggctcgagcat


 


gacggcgactacctccacctgagcgggggggaggacgcgctcatcaagcatctggcccggcaggccaccatcgtcaa


 


ggatgcgctgctcgaccgcaccgaggtgcccctcagcgtggaggagctcagccgcgcctacgagttgaacctcttctcgc


 


cgcgcgtgccccccaagcgccagcccaatggcacctgcgagcccaacccgcgcctcaacttctacccggtcttcgcggt


 


gcccgaggccctggccacctaccacatctttttcaagaaccaaaagatccccgtctcctgccgcgccaaccgcacccgc


 


gccgacgcccttttcaacctgggtcccggcgcccgcctacctgatatcgcctccttggaagaggttcccaagatcttcgagg


 


gtctgggcagcgacgagactcgggccgcgaacgctctgcaaggagaaggaggagagcatgagcaccacagcgccct


 


ggtcgagttggaaggcgacaacgcgcggctggcggtgctcaaacgcacggtcgagctgacccatttcgcctacccggct


 


ctgaacctgccccccaaagtcatgagcgcggtcatggaccaggtgctcatcaagcgcgcgtcgcccatctccgaggacg


 


agggcatgcaagactccgaggagggcaagcccgtggtcagcgacgagcagctggcccggtggctgggtcctaatgct


 


agtccccagagtttggaagagcggcgcaaactcatgatggccgtggtcctggtgaccgtggagctggagtgcctgcgcc


 


gcttcttcgccgacgcggagaccctgcgcaaggtcgaggagaacctgcactacctcttcaggcacgggttcgtgcgccag


 


gcctgcaagatctccaacgtggagctgaccaacctggtctcctacatgggcatcttgcacgagaaccgcctggggcaga


 


acgtgctgcacaccaccctgcgcggggaggcccggcgcgactacatccgcgactgcgtctacctctacctctgccacac


 


ctggcagacgggcatgggcgtgtggcagcagtgtctggaggagcagaacctgaaagagctctgcaagctcctgcagaa


 


gaacctcaagggtctgtggaccgggttcgacgagcgcaccaccgcctcggacctggccgacctcattttccccgagcgc


 


ctcaggctgacgctgcgcaacggcctgcccgactttatgagccaaagcatgttgcaaaactttcgctctttcatcctcgaacg


 


ctccggaatcctgcccgccacctgctccgcgctgccctcggacttcgtgccgctgaccttccgcgagtgccccccgccgct


 


gtggagccactgctacctgctgcgcctggccaactacctggcctaccactcggacgtgatcgaggacgtcagcggcgag


 


ggcctgctcgagtgccactgccgctgcaacctctgcacgccgcaccgctccctggcctgcaacccccagctgctgagcg


 


agacccagatcatcggcaccttcgagttgcaagggcccagcgaaggcgagggttcagccgccaaggggggtctgaaa


 


ctcaccccggggctgtggacctcggcctacttgcgcaagttcgtgcccgaggactaccatcccttcgagatcaggttctacg


 


aggaccaatcccatccgcccaaggccgagctgtcggcctgcgtcatcacccagggggcgatcctggcccaattgcaag


 


ccatccagaaatcccgccaagaattcttgctgaaaaagggccgcggggtctacctcgacccccagaccggtgaggagc


 


tcaaccccggcttcccccaggatgccccgaggaaacaagaagctgaaagtggagctgccgcccgtggaggatttggag


 


gaagactgggagaacagcagtcaggcagaggaggaggagatggaggaagactgggacagcactcaggcagagg


 


aggacagcctgcaagacagtctggaggaagacgaggaggaggcagaggaggaggtggaagaagcagccgccgc


 


cagaccgtcgtcctcggcgggggagaaagcaagcagcacggataccatctccgctccgggtcggggtcccgctcgacc


 


acacagtagatgggacgagaccggacgattcccgaaccccaccacccagaccggtaagaaggagcggcagggata


 


caagtcctggcgggggcacaaaaacgccatcgtctcctgcttgcaggcctgcgggggcaacatctccttcacccggcgct


 


acctgctcttccaccgcggggtgaactttccccgcaacatcttgcattactaccgtcacctccacagcccctactacttccaa


 


gaagaggcagcagcagcagaaaaagaccagcagaaaaccagcagctagaaaatccacagcggcggcagcaggt


 


ggactgaggatcgcggcgaacgagccggcgcaaacccgggagctgaggaaccggatctttcccaccctctatgccatc


 


ttccagcagagtcgggggcaggagcaggaactgaaagtcaagaaccgttctctgcgctcgctcacccgcagttgtctgtat


 


cacaagagcgaagaccaacttcagcgcactctcgaggacgccgaggctctcttcaacaagtactgcgcgctcactcttaa


 


agagtagcccgcgcccgcccagtcgcagaaaaaggcgggaattacgtcacctgtgcccttcgccctagccgcctccacc


 


catcatcatgagcaaagagattcccacgccttacatgtggagctaccagccccagatgggcctggccgccggtgccgcc


 


caggactactccacccgcatgaattggctcagcgccgggcccgcgatgatctcacgggtgaatgacatccgcgcccacc


 


gaaaccagatactcctagaacagtcagcgctcaccgccacgccccgcaatcacctcaatccgcgtaattggcccgccgc


 


cctggtgtaccaggaaattccccagcccacgaccgtactacttccgcgagacgcccaggccgaagtccagctgactaac


 


tcaggtgtccagctggcgggcggcgccaccctgtgtcgtcaccgccccgctcagggtataaagcggctggtgatccggg


 


gcagaggcacacagctcaacgacgaggtggtgagctcttcgctgggtctgcgacctgacggagtcttccaactcgccgg


 


atcggggagatcttccttcacgcctcgtcaggccgtcctgactttggagagttcgtcctcgcagccccgctcgggtggcatcg


 


gcactctccagttcgtggaggagttcactccctcggtctacttcaaccccttctccggctcccccggccactacccggacga


 


gttcatcccgaacttcgacgccatcagcgagtcggtggacggctacgattgaatgtcccatggtggcgcagctgacctagc


 


tcggcttcgacacctggaccactgccgccgcttccgctgcttcgctcgggatctcgccgagtttgcctactttgagctgcccga


 


ggagcaccctcagggcccggcccacggagtgcggatcgtcgtcgaagggggcctcgactcccacctgcttcggatcttc


 


agccagcgtccgatcctggtcgagcgcgagcaaggacagacccttctgactctgtactgcatctgcaaccaccccggcct


 


gcatgaaagtctttgttgtctgctgtgtactgagtataataaaagctgagatcagcgactactccggacttccgtgtgttcctga


 


atccatcaaccagtctttgttcttcaccgggaacgagaccgagctccagctccagtgtaagccccacaagaagtacctcac


 


ctggctgttccagggctccccgatcgccgttgtcaaccactgcgacaacgacggagtcctgctgagcggccctgccaacc


 


ttactttttccacccgcagaagcaagctccagctcttccaacccttcctccccgggacctatcagtgcgtctcgggaccctgc


 


catcacaccttccacctgatcccgaataccacagcgtcgctccccgctactaacaaccaaactaacctccaccaacgcca


 


ccgtcgcgacctttctgaatctaatactaccacccacaccggaggtgagctccgaggtcaaccaacctctgggatttactac


 


ggcccctgggaggtggttgggttaatagcgctaggcctagttgcgggtgggcttttggttctctgctacctatacctcccttgct


 


gttcgtacttagtggtgctgtgttgctggtttaagaaatggggaagatcaccctagtgagctgcggtgcgctggtggcggtgtt


 


gctttcgattgtgggactgggcggtgcggctgtagtgaaggagaaggccgatccctgcttgcatttcaatcccaacaaatgc


 


cagctgagttttcagcccgatggcaatcggtgcgcggtactgatcaagtgcggatgggaatgcgagaacgtgagaatcg


 


agtacaataacaagactcggaacaatactctcgcgtccgtgtggcagcccggggaccccgagtggtacaccgtctctgtc


 


cccggtgctgacggctccccgcgcaccgtgaataatactttcatttttgcgcacatgtgcgacacggtcatgtggatgagca


 


agcagtacgatatgtggccccccacgaaggagaacatcgtggtcttctccatcgcttacagcctgtgcacggcgctaatca


 


ccgctatcgtgtgcctgagcattcacatgctcatcgctattcgccccagaaataatgccgaaaaagaaaaacagccataa


 


cgttttttttcacacctttttcagaccatggcctctgttaaatttttgcttttatttgccagtctcattgccgtcattcatggaatgagtaa


 


tgagaaaattactatttacactggcactaatcacacattgaaaggtccagaaaaagccacagaagtttcatggtattgttattt


 


taatgaatcagatgtatctactgaactctgtggaaacaataacaaaaaaaatgagagcattactctcatcaagtttcaatgtg


 


gatctgacttaaccctaattaacatcactagagactatgtaggtatgtattatggaactacagcaggcatttcggacatggaa


 


ttttatcaagtttctgtgtctgaacccaccacgcctagaatgaccacaaccacaaaaactacacctgttaccactatgcagct


 


cactaccaataacatttttgccatgcgtcaaatggtcaacaatagcactcaacccaccccacccagtgaggaaattcccaa


 


atccatgattggcattattgttgctgtagtggtgtgcatgttgatcatcgccttgtgcatggtgtactatgccttctgctacagaaa


 


gcacagactgaacgacaagctggaacacttactaagtgttgaattttaattttttagaaccatgaagatcctaggccttttaatt


 


ttttctatcattacctctgctctatgcaattctgacaatgaggacgttactgtcgttgtcggatcaaattatacactgaaaggtcca


 


gcgaagggtatgctttcgtggtattgctattttggatctgacactacagaaactgaattatgcaatcttaagaatggcaaaattc


 


aaaattctaaaattaacaattatatatgcaatggtactgatctgatactcctcaatatcacgaaatcatatgctggcagttaca


 


cctgccctggagatgatgctgacagtatgattttttacaaagtaactgttgttgatcccactactccacctccacccaccacaa


 


ctactcacaccacacacacagatcaaaccgcagcagaggaggcagcaaagttagccttgcaggtccaagacagttcat


 


ttgttggcattacccctacacctgatcagcggtgtccggggctgctagtcagcggcattgtcggtgtgctttcgggattagcag


 


tcataatcatctgcatgttcatttttgcttgctgctatagaaggctttaccgacaaaaatcagacccactgctgaacctctatgttt


 


aattttttccagagtcatgaaggcagttagcgctctagttttttgttctttgattggcattgttttttgcaatcctattcctaaagttagct


 


ttattaaagatgtgaatgttactgaggggggcaatgtgacactggtaggtgtagagggtgctgaaaacaccacctggaca


 


aaataccacctcaatgggtggaaagatatttgcaattggagtgtattagtttatacatgtgagggagttaatcttaccattgtca


 


atgccacctcagctcaaaatggtagaattcaaggacaaagtgtcagtgtatctaatgggtattttacccaacatacttttatcta


 


tgacgttaaagtcataccactgcctacgcctagcccacctagcactaccacacagacaacccacactacacagacaacc


 


acatacagtacattaaatcagcctaccaccactacagcagcagaggttgccagctcgtctggggtccgagtggcatttttga


 


tgtgggccccatctagcagtcccactgctagtaccaatgagcagactactgaatttttgtccactgtcgagagccacaccac


 


agctacctccagtgccttctctagcaccgccaatctctcctcgctttcctctacaccaatcagtcccgctactactcctagcccc


 


gctcctcttcccactcccctgaagcaaacagacggcggcatgcaatggcagatcaccctgctcattgtgatcgggttggtca


 


tcctggccgtgttgctctactacatcttctgccgccgcattcccaacgcgcaccgcaagccggtctacaagcccatcattgtc


 


gggcagccggagccgcttcaggtggaagggggtctaaggaatcttctcttctcttttacagtatggtgattgaactatgattcct


 


agacaattcttgatcactattcttatctgcctcctccaagtctgtgccaccctcgctctggtggccaacgccagtccagactgta


 


ttgggcccttcgcctcctacgtgctctttgccttcaccacctgcatctgctgctgtagcatagtctgcctgcttatcaccttcttcca


 


gttcattgactggatctttgtgcgcatcgcctacctgcgccaccacccccagtaccgcgaccagcgagtggcgcggctgct


 


caggctcctctgataagcatgcgggctctgctacttctcgcgcttctgctgttagtgctcccccgtcccgtcgacccccggtcc


 


cccacccagtcccccgaggaggtccgcaaatgcaaattccaagaaccctggaaattcctcaaatgctaccgccaaaaat


 


cagacatgcatcccagctggatcatgatcattgggatcgtgaacattctggcctgcaccctcatctcctttgtgatttacccctg


 


ctttgactttggttggaactcgccagaggcgctctatctcccgcctgaacctgacacaccaccacagcaacctcaggcaca


 


cgcactaccaccactacagcctaggccacaatacatgcccatattagactatgaggccgagccacagcgacccatgctc


 


cccgctattagttacttcaatctaaccggcggagatgactgacccactggccaacaacaacgtcaacgaccttctcctgga


 


catggacggccgcgcctcggagcagcgactcgcccaacttcgcattcgccagcagcaggagagagccgtcaaggagc


 


tgcaggatgcggtggccatccaccagtgcaagagaggcatcttctgcctggtgaaacaggccaagatctcctacgaggtc


 


actccaaacgaccatcgcctctcctacgagctcctgcagcagcgccagaagttcacctgcctggtcggagtcaaccccat


 


cgtcatcacccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtccacactctgat


 


caagaccctctgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagatcatattgatga


 


tgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgatgatttgagtttaacaaaaaaataa


 


agaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttcccagctctggta


 


ctgcaggccccggcgggctgcaaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaatcttcattttatc


 


ttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgcagacaacgcacc


 


gaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccctgcgactgg


 


ccgaccccgtcaccaccaagaacggggaaatcaccctcaagctgggagagggggtggacctcgattcctcgggaaaa


 


ctcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatggatcacccctt


 


ttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaacacactagcttt


 


aggttttggatcaggtttaggactccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatggaaaca


 


taaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggctaaaggtttaa


 


aatttgaagatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaacaggtgttgat


 


gatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaacaaagaag


 


acgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgcaaaactaaca


 


ctttgcttgactaaatgtggtagtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaaaccccattactg


 


gcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattctacactaaaaaaatactg


 


ggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaatttaaaagcttatcc


 


aaagtcacaaagttctactactaaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatgcttctca


 


ctataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaatggaagctatgttg


 


gagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccctgcatgccaaccct


 


tcccaccccactctgtggaacaaactctgaaacacaaaataaaataaagttcaagtgttttattgattcaacagttttacagg


 


attcgagcagttatttttcctccaccctcccaggacatggaatacaccaccctctccccccgcacagccttgaacatctgaat


 


gccattggtgatggacatgcttttggtctccacgttccacacagtttcagagcgagccagtctcgggtcggtcagggagatg


 


aaaccctccgggcactcccgcatctgcacctcacagctcaacagctgaggattgtcctcggtggtcgggatcacggttatct


 


ggaagaagcagaagagcggcggtgggaatcatagtccgcgaacgggatcggccggtggtgtcgcatcaggccccgc


 


agcagtcgctgccgccgccgctccgtcaagctgctgctcagggggtccgggtccagggactccctcagcatgatgccca


 


cggccctcagcatcagtcgtctggtgcggcgggcgcagcagcgcatgcggatctcgctcaggtcgctgcagtacgtgca


 


acacagaaccaccaggttgttcaacagtccatagttcaacacgctccagccgaaactcatcgcgggaaggatgctaccc


 


acgtggccgtcgtaccagatcctcaggtaaatcaagtggtgccccctccagaacacgctgcccacgtacatgatctccttg


 


ggcatgtggcggttcaccacctcccggtaccacatcaccctctggttgaacatgcagccccggatgatcctgcggaacca


 


cagggccagcaccgccccgcccgccatgcagcgaagagaccccgggtcccggcaatggcaatggaggacccaccg


 


ctcgtacccgtggatcatctgggagctgaacaagtctatgttggcacagcacaggcatatgctcatgcatctcttcagcactc


 


tcaactcctcgggggtcaaaaccatatcccagggcacggggaactcttgcaggacagcgaaccccgcagaacagggc


 


aatcctcgcacagaacttacattgtgcatggacagggtatcgcaatcaggcagcaccgggtgatcctccaccagagaag


 


cgcgggtctcggtctcctcacagcgtggtaagggggccggccgatacgggtgatggcgggacgcggctgatcgtgttcgc


 


gaccgtgtcatgatgcagttgctttcggacattttcgtacttgctgtagcagaacctggtccgggcgctgcacaccgatcgcc


 


ggcggcggtctcggcgcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagcagatctagg


 


gcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtggaatgggccagacccagcc


 


agatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaacttttaatccaaac


 


ggtctcggagtacttcaaaatgaagatcgcggagatggcacctctcgcccccgctgtgttggtggaaaataacagccagg


 


tcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaacaagacaat


 


agcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatttttccagcctt


 


gaatgattcgaactagttcgtgaggtaaatccaagccagccatgataaagagctcgcgcagagcgccctccaccggcatt


 


cttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatcaaaatctctg


 


ccgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccataggaccacca


 


ggaataagattagggcaagccacagtacagataaaccgaagtcctccccagtgagcattgccaaatgcaagactgctat


 


aagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaagaaaatcaaca


 


aaagaaaaatcctccaggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgttccagcatg


 


gttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaacaggtgggtaaatcgttctct


 


ccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccatcacagaga


 


gacgttcccggtggccggcgtgaatgattcgacaagatgaatacacccccggaacattggcgtccgcgagtgaaaaaa


 


agcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatgaagcaca


 


aaattctcaggtgcgtacaaaatgtaattactcccctcctgcacaggcagcaaagcccccgatccctccaggtacacatac


 


aaagcctcagcgtccatagcttaccgagcagcagcacacaacaggcgcaagagtcagagaaaggctgagctctaacc


 


tgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccgccaaataa


 


tcacacacgcccagcacacgcccagaaaccggtgacacactcaaaaaaatacgcgcacttcctcaaacgcccaaaa


 


ctgccgtcatttccgggttcccacgctacgtcatcaaaacacgactttcaaattccgtcgaccgttaaaaacgtcacccgcc


 


ccgcccctaacggtcgcccgtctctcagccaatcagcgccccgcatccccaaattcaaacacctcatttgcatattaacgc


 


gcacaaaaagtttgaggtatattattgatgatgg


 


SEQ ID NO: 58. Complete Sequence of the AdC68-734 Vector


TTAATTAAccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaa


 


gggcggtgattggtcgagggatgagcgaccgttaggggcggggcgagtgacgttttgatgacgtggttgcgaggaggag


 


ccagtttgcaagttctcgtgggaaaagtgacgtcaaacgaggtgtggtttgaacacggaaatactcaattttcccgcgctctc


 


tgacaggaaatgaggtgtttctgggcggatgcaagtgaaaacgggccattttcgcgcgaaaactgaatgaggaagtgaa


 


aatctgagtaatttcgcgtttatggcagggaggagtatttgccgagggccgagtagactttgaccgattacgtgggggtttcg


 


attaccgtgtttttcacctaaatttccgcgtacggtgtcaaagtccggtgtttttactactgtaatagtaatcaattacggggtcatt


 


agttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccg


 


cccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacg


 


gtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggccc


 


gcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggt


 


gatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtca


 


atgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcg


 


gtaggcgtgtacggtgggaggtctatataagcagagctgtccctatcagtgatagagatctccctatcagtgatagagagttt


 


agtgaaccgtcagatccgctagggtaccaacATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAA


 


AAGCACTCACAGCCATGGCAGGTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGT


 


GGAGGTGTGCTGGTCCACCCGCAGTGGGTGTTGACTGCGGCCCATTGCATCAGAA


 


ATAAGTCCGTGATCCTCTTGGGGAGACATTCCCTGTTTCACCCCGAAGATACTGGA


 


CAGGTGTTCCAAGTGAGCCACTCCTTCCCGCATCCACTGTACGACATGAGCCTGCT


 


GAAGAACCGCTTTCTGCGGCCAGGGGACGACTCATCACACGATTTGATGCTGCTT


 


CGGCTCTCGGAACCGGCCGAGCTCACCGACGCAGTGAAGGTCATGGACCTCCCTA


 


CGCAAGAGCCTGCTCTCGGTACCACTTGTTACGCATCGGGATGGGGCTCCATCGA


 


GCCGGAAGAATTCCTGACCCCGAAAAAGCTGCAGTGCGTGGATCTGCACGTGATT


 


TCGAATGACGTGTGCGCGCAAGTGCATCCACAAAAGGTCACTAAGTTCATGCTGTG


 


CGCCGGAAGGTGGACCGGCGGAAAATCGACCTGTTCCGGCGACAGCGGAGGCCC


 


ACTCGTGTGCAACGGTGTGCTGCAGGGCATCACTAGCTGGGGATCAGAACCGTGC


 


GCGCTTCCGGAGCGGCCCTCGCTCTACACGAAGGTGGTGCACTACCGCAAATGGA


 


TTAAAGATACCATCGTCGCAAACCCTggatccgaaggtaggggttcattattgacctgtggagatgtcga


 


agaaaacccaggacccGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCG


 


CGCTGCAGCCTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAA


 


TGAGGACTGTTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACT


 


GCACGGATCCGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGA


 


ACTGCGTGGACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGC


 


GACACGGATCTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCC


 


ATTCTGGCCCTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTCg


 


gatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGC


 


GCTCGCAGACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTC


 


TTTTTGCTCGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAAT


 


ATCACCCCGAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACAT


 


TAAGAAGTTCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGA


 


ACTTTCAGTTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTC


 


CGTCGAGCTGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGA


 


ACTATATCTCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCG


 


AGCCGCCACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGC


 


CTTCTCGCCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGG


 


ACCGAGGACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGAT


 


CGTCATCGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAG


 


TTGGCAGGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTC


 


CTGGCGTGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAG


 


GGGAAATATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCG


 


GCCAACGAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCC


 


ATCCCGGTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGG


 


GAGGCAGCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACA


 


ACGTGGGACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATT


 


CACTCCACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAG


 


CGGTGGAACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGT


 


TCGGAGGAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTC


 


CTTTGGTACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCT


 


CGTGGGATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAA


 


CTCCCGCCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATC


 


GAAGGAAACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGC


 


ACAACCTGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTG


 


TACGAGTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGG


 


ATCTCAAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAAT


 


TGCGTCGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGA


 


TACCCGCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGA


 


TCCTATGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCG


 


AGTTGGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTG


 


AGAAAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAA


 


AACCTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGA


 


TCGCGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTC


 


CTCCGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACT


 


GGGACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCAT


 


AACAAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGA


 


GTCCAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTG


 


GCGGCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTTGActcga


 


gcctaagcttctagataagatatccgatccaccggatctagataactgatcataatcagccataccacatttgtagaggtttta


 


cttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagc


 


ttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa


 


actcatcaatgtatcttatatgctggccaccgtacatgtggcttcccatgctcgcaagccctggcccgagttcgagcacaatg


 


tcatgaccaggtgcaatatgcatctggggtcccgccgaggcatgttcatgccctaccagtgcaacctgaattatgtgaaggt


 


gctgctggagcccgatgccatgtccagagtgagcctgacgggggtgtttgacatgaatgtggaggtgtggaagattctgag


 


atatgatgaatccaagaccaggtgccgagcctgcgagtgcggagggaagcatgccaggttccagcccgtgtgtgtggat


 


gtgacggaggacctgcgacccgatcatttggtgttgccctgcaccgggacggagttcggttccagcggggaagaatctga


 


ctagagtgagtagtgttctggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgca


 


gcagcatgagcggaagcggctcctttgagggaggggtattcagcccttatctgacggggcgtctcccctcctgggcggga


 


gtgcgtcagaatgtgatgggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaa


 


ccctgagctcttcgtcgttggacgcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatg


 


ggcgccggctactacggcactctggtggccaactcgagttccaccaataatcccgccagcctgaacgaggagaagctgt


 


tgctgctgatggcccagctcgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagc


 


agacgcgggccgcggttgccacggtgaaatccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaa


 


cacagagtctgaatctttatttgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtggatctt


 


ttccaggacccggtagaggtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattg


 


cagggcctcgtgctcgggggtggtgttgtaaatcacccagtcatagcaggggcgcagggcatggtgttgcacaatatctttg


 


aggaggagactgatggccacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcgggg


 


ggagatgaggtgcatcttggcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcagga


 


ccaccagcacggtgtatccggtgcacttggggaatttatcatgcaacttggaagggaaggcgtgaaagaatttggcgacg


 


cctttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacg


 


tttcgggggtcggacacatcatagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgcc


 


ggactgggggacaaaggtaccctcgatcccgggggcgtagttcccctcacagatctgcatctcccaggctttgagctcgg


 


agggggggatcatgtccacctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaag


 


caagttccggagcagctgggacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttg


 


agggagagacagctgccgtcctcccggaggaggggggccacctcgttcatcatctcgcgcacgtgcatgttctcgcgcac


 


cagttccgccaggaggcgctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggcttgagtccgt


 


cggccatgggcattttggagagggtttgttgcaagagttccaggcggtcccagagctcggtgatgtgctctacggcatctcg


 


atccagcagacctcctcgtttcgcgggttgggacggctgcgggagtagggcaccagacgatgggcgtccagcgcagcc


 


agggtccggtccttccagggtcgcagcgtccgcgtcagggtggtctccgtcacggtgaaggggtgcgcgccgggctggg


 


cgcttgcgagggtgcgcttcaggctcatccggctggtcgaaaaccgctcccgatcggcgccctgcgcgtcggccaggtag


 


caattgaccatgagttcgtagttgagcgcctcggccgcgtggcctttggcgcggagcttacctttggaagtctgcccgcagg


 


cgggacagaggagggacttgagggcgtagagcttgggggcgaggaagacggactcgggggcgtaggcgtccgcgc


 


cgcagtgggcgcagacggtctcgcactccacgagccaggtgaggtcgggctggtcggggtcaaaaaccagtttcccgc


 


cgttctttttgatgcgtttcttacctttggtctccatgagctcgtgtccccgctgggtgacaaagaggctgtccgtgtccccgtaga


 


ccgactttatgggccggtcctcgagcggtgtgccgcggtcctcctcgtagaggaaccccgcccactccgagacgaaagc


 


ccgggtccaggccagcacgaaggaggccacgtgggacgggtagcggtcgttgtccaccagcgggtccaccttttccag


 


ggtatgcaaacacatgtccccctcgtccacatccaggaaggtgattggcttgtaagtgtaggccacgtgaccgggggtccc


 


ggccgggggggtataaaagggtgcgggtccctgctcgtcdcactgtcttccggatcgctgtccaggagcgccagctgttg


 


gggtaggtattccctctcgaaggcgggcatgacctcggcactcaggttgtcagtttctagaaacgaggaggatttgatattg


 


acggtgccggcggagatgcctttcaagagcccctcgtccatctggtcagaaaagacgatctttttgttgtcgagcttggtggc


 


gaaggagccgtagagggcgttggagaggagcttggcgatggagcgcatggtctggtttttttccttgtcggcgcgctccttg


 


gcggcgatgttgagctgcacgtactcgcgcgccacgcacttccattcggggaagacggtggtcagctcgtcgggcacgat


 


tctgacctgccagccccgattatgcagggtgatgaggtccacactggtggccacctcgccgcgcaggggctcattagtcca


 


gcagaggcgtccgcccttgcgcgagcagaaggggggcagggggtccagcatgacctcgtcgggggggtcggcatcg


 


atggtgaagatgccgggcaggaggtcggggtcaaagtagctgatggaagtggccagatcgtccagggcagcttgccatt


 


cgcgcacggccagcgcgcgctcgtagggactgaggggcgtgccccagggcatgggatgggtaagcgcggaggcgta


 


catgccgcagatgtcgtagacgtagaggggctcctcgaggatgccgatgtaggtggggtagcagcgccccccgcggat


 


gctggcgcgcacgtagtcatacagctcgtgcgagggggcgaggagccccgggcccaggttggtgcgactgggcttttcg


 


gcgcggtagacgatctggcggaaaatggcatgcgagttggaggagatggtgggcctttggaagatgttgaagtgggcgt


 


ggggcagtccgaccgagtcgcggatgaagtgggcgtaggagtcttgcagcttggcgacgagctcggcggtgactagga


 


cgtccagagcgcagtagtcgagggtctcctggatgatgtcatacttgagctgtcccttttgtttccacagctcgcggttgagaa


 


ggaactcttcgcggtccttccagtactcttcgagggggaacccgtcctgatctgcacggtaagagcctagcatgtagaactg


 


gttgacggccttgtaggcgcagcagcccttctccacggggagggcgtaggcctgggcggccttgcgcagggaggtgtgc


 


gtgagggcgaaagtgtccctgaccatgaccttgaggaactggtgcttgaagtcgatatcgtcgcagcccccctgctcccag


 


agctggaagtccgtgcgcttcttgtaggcggggttgggcaaagcgaaagtaacatcgttgaagaggatcttgcccgcgcg


 


gggcataaagttgcgagtgatgcggaaaggttggggcacctcggcccggttgttgatgacctgggcggcgagcacgatct


 


cgtcgaagccgttgatgttgtggcccacgatgtagagttccacgaatcgcggacggcccttgacgtggggcagtttcttgag


 


ctcctcgtaggtgagctcgtcggggtcgctgagcccgtgctgctcgagcgcccagtcggcgagatgggggttggcgcgga


 


ggaaggaagtccagagatccacggccagggcggtttgcagacggtcccggtactgacggaactgctgcccgacggcc


 


attttttcgggggtgacgcagtagaaggtgcgggggtccccgtgccagcgatcccatttgagctggagggcgagatcgag


 


ggcgagctcgacgagccggtcgtccccggagagtttcatgaccagcatgaaggggacgagctgcttgccgaaggaccc


 


catccaggtgtaggtttccacatcgtaggtgaggaagagcctttcggtgcgaggatgcgagccgatggggaagaactgg


 


atctcctgccaccaattggaggaatggctgttgatgtgatggaagtagaaatgccgacggcgcgccgaacactcgtgcttg


 


tgtttatacaagcggccacagtgctcgcaacgctgcacgggatgcacgtgctgcacgagctgtacctgagttcctttgacga


 


ggaatttcagtgggaagtggagtcgtggcgcctgcatctcgtgctgtactacgtcgtggtggtcggcctggccctcttctgcct


 


cgatggtggtcatgctgacgagcccgcgcgggaggcaggtccagacctcggcgcgagcgggtcggagagcgaggac


 


gagggcgcgcaggccggagctgtccagggtcctgagacgctgcggagtcaggtcagtgggcagcggcggcgcgcgg


 


ttgacttgcaggagtttttccagggcgcgcgggaggtccagatggtacttgatctccaccgcgccattggtggcgacgtcga


 


tggcttgcagggtcccgtgcccctggggtgtgaccaccgtcccccgtttcttcttgggcggctggggcgacgggggcggtg


 


cctcttccatggttagaagcggcggcgaggacgcgcgccgggcggcaggggcggctcggggcccggaggcaggggc


 


ggcaggggcacgtcggcgccgcgcgcgggtaggttctggtactgcgcccggagaagactggcgtgagcgacgacgcg


 


acggttgacgtcctggatctgacgcctctgggtgaaggccacgggacccgtgagtttgaacctgaaagagagttcgacag


 


aatcaatctcggtatcgttgacggcggcctgccgcaggatctcttgcacgtcgcccgagttgtcctggtaggcgatctcggtc


 


atgaactgctcgatctcctcctcttgaaggtctccgcggccggcgcgctccacggtggccgcgaggtcgttggagatgcgg


 


cccatgagctgcgagaaggcgttcatgcccgcctcgttccagacgcggctgtagaccacgacgccctcgggatcgcGg


 


gcgcgcatgaccacctgggcgaggttgagctccacgtggcgcgtgaagaccgcgtagttgcagaggcgctggtagagg


 


tagttgagcgtggtggcgatgtgctcggtgacgaagaaatacatgatccagcggcggagcggcatctcgctgacgtcgcc


 


cagcgcctccaaacgttccatggcctcgtaaaagtccacggcgaagttgaaaaactgggagttgcgcgccgagacggtc


 


aactcctcctccagaagacggatgagctcggcgatggtggcgcgcacctcgcgctcgaaggcccccgggagttcctcca


 


cttcctcttcttcctcctccactaacatctcttctacttcctcctcaggcggcagtggtggcgggggagggggcctgcgtcgcc


 


ggcggcgcacgggcagacggtcgatgaagcgctcgatggtctcgccgcgccggcgtcgcatggtctcggtgacggcgc


 


gcccgtcctcgcggggccgcagcgtgaagacgccgccgcgcatctccaggtggccgggggggtccccgttgggcagg


 


gagagggcgctgacgatgcatcttatcaattgccccgtagggactccgcgcaaggacctgagcgtctcgagatccacgg


 


gatctgaaaaccgctgaacgaaggcttcgagccagtcgcagtcgcaaggtaggctgagcacggtttcttctggcgggtca


 


tgttggttgggagcggggcgggcgatgctgctggtgatgaagttgaaataggcggttctgagacggcggatggtggcgag


 


gagcaccaggtctttgggcccggcttgctggatgcgcagacggtcggccatgccccaggcgtggtcctgacacctggcca


 


ggtccttgtagtagtcctgcatgagccgctccacgggcacctcctcctcgcccgcgcggccgtgcatgcgcgtgagcccga


 


agccgcgctggggctggacgagcgccaggtcggcgacgacgcgctcggcgaggatggcttgctggatctgggtgagg


 


gtggtctggaagtcatcaaagtcgacgaagcggtggtaggctccggtgttgatggtgtaggagcagttggccatgacgga


 


ccagttgacggtctggtggcccggacgcacgagctcgtggtacttgaggcgcgagtaggcgcgcgtgtcgaagatgtagt


 


cgttgcaggtgcgcaccaggtactggtagccgatgaggaagtgcggcggcggctggcggtagagcggccatcgctcgg


 


tggcgggggcgccgggcgcgaggtcctcgagcatggtgcggtggtagccgtagatgtacctggacatccaggtgatgcc


 


ggcggcggtggtggaggcgcgcgggaactcgcggacgcggttccagatgttgcgcagcggcaggaagtagttcatggt


 


gggcacggtctggcccgtgaggcgcgcgcagtcgtggatgctctatacgggcaaaaacgaaagcggtcagcggctcg


 


actccgtggcctggaggctaagcgaacgggttgggctgcgcgtgtaccccggttcgaatctcgaatcaggctggagccgc


 


agctaacgtggtattggcactcccgtctcgacccaagcctgcaccaaccctccaggatacggaggcgggtcgttttgcaac


 


ttttttttggaggccggatgagactagtaagcgcggaaagcggccgaccgcgatggctcgctgccgtagtctggagaaga


 


atcgccagggttgcgttgcggtgtgccccggttcgaggccggccggattccgcggctaacgagggcgtggctgccccgtc


 


gtttccaagaccccatagccagccgacttctccagttacggagcgagcccctcttttgttttgtttgtttttgccagatgcatcccg


 


tactgcggcagatgcgcccccaccaccctccaccgcaacaacagccccctccacagccggcgcttctgcccccgcccc


 


agcagcaacttccagccacgaccgccgcggccgccgtgagcggggctggacagagttatgatcaccagctggccttgg


 


aagagggcgaggggctggcgcgcctgggggcgtcgtcgccggagcggcacccgcgcgtgcagatgaaaagggacg


 


ctcgcgaggcctacgtgcccaagcagaacctgttcagagacaggagcggcgaggagcccgaggagatgcgcgcggc


 


ccggttccacgcggggcgggagctgcggcgcggcctggaccgaaagagggtgctgagggacgaggatttcgaggcg


 


gacgagctgacggggatcagccccgcgcgcgcgcacgtggccgcggccaacctggtcacggcgtacgagcagaccg


 


tgaaggaggagagcaacttccaaaaatccttcaacaaccacgtgcgcaccctgatcgcgcgcgaggaggtgaccctgg


 


gcctgatgcacctgtgggacctgctggaggccatcgtgcagaaccccaccagcaagccgctgacggcgcagctgttcct


 


ggtggtgcagcatagtcgggacaacgaagcgttcagggaggcgctgctgaatatcaccgagcccgagggccgctggct


 


cctggacctggtgaacattctgcagagcatcgtggtgcaggagcgcgggctgccgctgtccgagaagctggcggccatc


 


aacttctcggtgctgagtttgggcaagtactacgctaggaagatctacaagaccccgtacgtgcccatagacaaggaggt


 


gaagatcgacgggttttacatgcgcatgaccctgaaagtgctgaccctgagcgacgatctgggggtgtaccgcaacgac


 


aggatgcaccgtgcggtgagcgccagcaggcggcgcgagctgagcgaccaggagctgatgcatagtctgcagcggg


 


ccctgaccggggccgggaccgagggggagagctactttgacatgggcgcggacctgcactggcagcccagccgccg


 


ggccttggaggcggcggcaggaccctacgtagaagaggtggacgatgaggtggacgaggagggcgagtacctggaa


 


gactgatggcgcgaccgtatttttgctagatgcaacaacaacagccacctcctgatcccgcgatgcgggcggcgctgcag


 


agccagccgtccggcattaactcctcggacgattggacccaggccatgcaacgcatcatggcgctgacgacccgcaac


 


cccgaagcctttagacagcagccccaggccaaccggctctcggccatcctggaggccgtggtgccctcgcgctccaacc


 


ccacgcacgagaaggtcctggccatcgtgaacgcgctggtggagaacaaggccatccgcggcgacgaggccggcct


 


ggtgtacaacgcgctgctggagcgcgtggcccgctacaacagcaccaacgtgcagaccaacctggaccgcatggtga


 


ccgacgtgcgcgaggccgtggcccagcgcgagcggttccaccgcgagtccaacctgggatccatggtggcgctgaacg


 


ccttcctcagcacccagcccgccaacgtgccccggggccaggaggactacaccaacttcatcagcgccctgcgcctgat


 


ggtgaccgaggtgccccagagcgaggtgtaccagtccgggccggactacttcttccagaccagtcgccagggcttgcag


 


accgtgaacctgagccaggctttcaagaacttgcagggcctgtggggcgtgcaggccccggtcggggaccgcgcgacg


 


gtgtcgagcctgctgacgccgaactcgcgcctgctgctgctgctggtggcccccttcacggacagcggcagcatcaaccg


 


caactcgtacctgggctacctgattaacctgtaccgcgaggccatcggccaggcgcacgtggacgagcagacctaccag


 


gagatcacccacgtgagccgcgccctgggccaggacgacccgggcaacctggaagccaccctgaactttttgctgacc


 


aaccggtcgcagaagatcccgccccagtacgcgctcagcaccgaggaggagcgcatcctgcgttacgtgcagcagag


 


cgtgggcctgttcctgatgcaggagggggccacccccagcgccgcgctcgacatgaccgcgcgcaacatggagccca


 


gcatgtacgccagcaaccgcccgttcatcaataaactgatggactacttgcatcgggcggccgccatgaactctgactattt


 


caccacgccatcctgaatccccactggctcccgccgccggggttctacacgggcgagtacgacatgcccgaccccaat


 


gacgggttcctgtgggacgatgtggacagcagcgtgttctccccccgaccgggtgctaacgagcgccccttgtggaagaa


 


ggaaggcagcgaccgacgcccgtcctcggcgctgtccggccgcgagggtgctgccgcggcggtgcccgaggccgcc


 


agtcctttcccgagcttgcccttctcgctgaacagtatccgcagcagcgagctgggcaggatcacgcgcccgcgcttgctg


 


ggcgaagaggagtacttgaatgactcgctgttgagacccgagcgggagaagaacttccccaataacgggatagaaag


 


cctggtggacaagatgagccgctggaagacgtatgcgcaggagcacagggacgatccccgggcgtcgcagggggcc


 


acgagccggggcagcgccgcccgtaaacgccggtggcacgacaggcagcggggacagatgtgggacgatgagga


 


ctccgccgacgacagcagcgtgttggacttgggtgggagtggtaacccgttcgctcacctgcgcccccgtatcgggcgcat


 


gatgtaagagaaaccgaaaataaatgatactcaccaaggccatggcgaccagcgtgcgttcgtttcttctctgttgttgttgta


 


tctagtatgatgaggcgtgcgtacccggagggtcctcctccctcgtacgagagcgtgatgcagcaggcgatggcggcggc


 


ggcgatgcagcccccgctggaggctccttacgtgcccccgcggtacctggcgcctacggaggggcggaacagcattcgt


 


tactcggagctggcacccttgtacgataccacccggttgtacctggtggacaacaagtcggcggacatcgcctcgctgaa


 


ctaccagaacgaccacagcaacttcctgaccaccgtggtgcagaacaatgacttcacccccacggaggccagcaccc


 


agaccatcaactttgacgagcgctcgcggtggggcggccagctgaaaaccatcatgcacaccaacatgcccaacgtga


 


acgagttcatgtacagcaacaagttcaaggcgcgggtgatggtctcccgcaagacccccaatggggtgacagtgacag


 


aggattatgatggtagtcaggatgagctgaagtatgaatgggtggaatttgagctgcccgaaggcaacttctcggtgaccat


 


gaccatcgacctgatgaacaacgccatcatcgacaattacttggcggtggggcggcagaacggggtgctggagagcga


 


catcggcgtgaagttcgacactaggaacttcaggctgggctgggaccccgtgaccgagctggtcatgcccggggtgtaca


 


ccaacgaggctttccatcccgatattgtcttgctgcccggctgcggggtggacttcaccgagagccgcctcagcaacctgct


 


gggcattcgcaagaggcagcccttccaggaaggcttccagatcatgtacgaggatctggaggggggcaacatccccgc


 


gctcctggatgtcgacgcctatgagaaaagcaaggaggatgcagcagctgaagcaactgcagccgtagctaccgcctct


 


accgaggtcaggggcgataattttgcaagcgccgcagcagtggcagcggccgaggcggctgaaaccgaaagtaagat


 


agtcattcagccggtggagaaggatagcaagaacaggagctacaacgtactaccggacaagataaacaccgcctacc


 


gcagctggtacctagcctacaactatggcgaccccgagaagggcgtgcgctcctggacgctgctcaccacctcggacgt


 


cacctgcggcgtggagcaagtctactggtcgctgcccgacatgatgcaagacccggtcaccttccgctccacgcgtcaag


 


ttagcaactacccggtggtgggcgccgagctcctgcccgtctactccaagagcttcttcaacgagcaggccgtctactcgc


 


agcagctgcgcgccttcacctcgcttacgcacgtcttcaaccgcttccccgagaaccagatcctcgtccgcccgcccgcgc


 


ccaccattaccaccgtcagtgaaaacgttcctgctctcacagatcacgggaccctgccgctgcgcagcagtatccgggga


 


gtccagcgcgtgaccgttactgacgccagacgccgcacctgcccctacgtctacaaggccctgggcatagtcgcgccgc


 


gcgtcctctcgagccgcaccttctaaatgtccattctcatctcgcccagtaataacaccggttggggcctgcgcgcgcccag


 


caagatgtacggaggcgctcgccaacgctccacgcaacaccccgtgcgcgtgcgcgggcacttccgcgctccctgggg


 


cgccctcaagggccgcgtgcggtcgcgcaccaccgtcgacgacgtgatcgaccaggtggtggccgacgcgcgcaact


 


acacccccgccgccgcgcccgtctccaccgtggacgccgtcatcgacagcgtggtggcCgacgcgcgccggtacgcc


 


cgcgccaagagccggcggcggcgcatcgcccggcggcaccggagcacccccgccatgcgcgcggcgcgagccttg


 


ctgcgcagggccaggcgcacgggacgcagggccatgctcagggcggccagacgcgcggcttcaggcgccagcgcc


 


ggcaggacccggagacgcgcggccacggcggcggcagcggccatcgccagcatgtcccgcccgcggcgagggaa


 


cgtgtactgggtgcgcgacgccgccaccggtgtgcgcgtgcccgtgcgcacccgcccccctcgcacttgaagatgttcact


 


tcgcgatgttgatgtgtcccagcggcgaggaggatgtccaagcgcaaattcaaggaagagatgctccaggtcatcgcgc


 


ctgagatctacggccctgcggtggtgaaggaggaaagaaagccccgcaaaatcaagcgggtcaaaaaggacaaaa


 


aggaagaagaaagtgatgtggacggattggtggagtttgtgcgcgagttcgccccccggcggcgcgtgcagtggcgcg


 


ggcggaaggtgcaaccggtgctgagacccggcaccaccgtggtcttcacgcccggcgagcgctccggcaccgcttcca


 


agcgctcctacgacgaggtgtacggggatgatgatattctggagcaggcggccgagcgcctgggcgagtttgcttacggc


 


aagcgcagccgttccgcaccgaaggaagaggcggtgtccatcccgctggaccacggcaaccccacgccgagcctca


 


agcccgtgaccttgcagcaggtgctgccgaccgcggcgccgcgccgggggttcaagcgcgagggcgaggatctgtac


 


cccaccatgcagctgatggtgcccaagcgccagaagctggaagacgtgctggagaccatgaaggtggacccggacgt


 


gcagcccgaggtcaaggtgcggcccatcaagcaggtggccccgggcctgggcgtgcagaccgtggacatcaagattc


 


ccacggagcccatggaaacgcagaccgagcccatgatcaagcccagcaccagcaccatggaggtgcagacggatcc


 


ctggatgccatcggctcctagtcgaagaccccggcgcaagtacggcgcggccagcctgctgatgcccaactacgcgctg


 


catccttccatcatccccacgccgggctaccgcggcacgcgcttctaccgcggtcataccagcagccgccgccgcaaga


 


ccaccactcgccgccgccgtcgccgcaccgccgctgcaaccacccctgccgccctggtgcggagagtgtaccgccgcg


 


gccgcgcacctctgaccctgccgcgcgcgcgctaccacccgagcatcgccatttaaactttcgccTgctttgcagatcaat


 


ggccctcacatgccgccttcgcgttcccattacgggctaccgaggaagaaaaccgcgccgtagaaggctggcggggaa


 


cgggatgcgtcgccaccaccaccggcggcggcgcgccatcagcaagcggttggggggaggcttcctgcccgcgctgat


 


ccccatcatcgccgcggcgatcggggcgatccccggcattgcttccgtggcggtgcaggcctctcagcgccactgagac


 


acacttggaaacatcttgtaataaaccAatggactctgacgctcctggtcctgtgatgttttcgtagacagatggaagaca


 


tcaatttttcgtccctggctccgcgacacggcacgcggccgttcatgggcacctggagcgacatcggcaccagccaactg


 


aacgggggcgccttcaattggagcagtctctggagcgggcttaagaatttcgggtccacgcttaaaacctatggcagcaa


 


ggcgtggaacagcaccacagggcaggcgctgagggataagctgaaagagcagaacttccagcagaaggtggtcgat


 


gggctcgcctcgggcatcaacggggtggtggacctggccaaccaggccgtgcagcggcagatcaacagccgcctgga


 


cccggtgccgcccgccggctccgtggagatgccgcaggtggaggaggagctgcctcccctggacaagcggggcgag


 


aagcgaccccgccccgatgcggaggagacgctgctgacgcacacggacgagccgcccccgtacgaggaggcggtg


 


aaactgggtctgcccaccacgcggcccatcgcgcccctggccaccggggtgctgaaacccgaaaagcccgcgaccct


 


ggacttgcctcctccccagccttcccgcccctctacagtggctaagcccctgccgccggtggccgtggcccgcgcgcgac


 


ccgggggcaccgcccgccctcatgcgaactggcagagcactctgaacagcatcgtgggtctgggagtgcagagtgtga


 


agcgccgccgctgctattaaacctaccgtagcgcttaacttgcttgtctgtgtgtgtatgtattatgtcgccgccgccgctgtcc


 


accagaaggaggagtgaagaggcgcgtcgccgagttgcaagatggccaccccatcgatgctgccccagtgggcgtac


 


atgcacatcgccggacaggacgcttcggagtacctgagtccgggtctggtgcagtttgcccgcgccacagacacctacttc


 


agtctggggaacaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgcagccagcggctgac


 


gctgcgcttcgtgcccgtggaccgcgaggacaacacctactcgtacaaagtgcgctacacgctggccgtgggcgacaac


 


cgcgtgctggacatggccagcacctactttgacatccgcggcgtgctggatcggggccctagcttcaaaccctactccggc


 


accgcctacaacagtctggcccccaagggagcacccaacacttgtcagtggacatataaagccgatggtgaaactgcc


 


acagaaaaaacctatacatatggaaatgcacccgtgcagggcattaacatcacaaaagatggtattcaacttggaactg


 


acaccgatgatcagccaatctacgcagataaaacctatcagcctgaacctcaagtgggtgatgctgaatggcatgacatc


 


actggtactgatgaaaagtatggaggcagagctcttaagcctgataccaaaatgaagccttgttatggttcttttgccaagcc


 


tactaataaagaaggaggtcaggcaaatgtgaaaacaggaacaggcactactaaagaatatgacatagacatggctttc


 


tttgacaacagaagtgcggctgctgctggcctagctccagaaattgttttgtatactgaaaatgtggatttggaaactccagat


 


acccatattgtatacaaagcaggcacagatgacagcagctcttctattaatttgggtcagcaagccatgcccaacagacct


 


aactacattggtttcagagacaactttatcgggctcatgtactacaacagcactggcaatatgggggtgctggccggtcagg


 


cttctcagctgaatgctgtggttgacttgcaagacagaaacaccgagctgtcctaccagctcttgcttgactctctgggtgaca


 


gaacccggtatttcagtatgtggaatcaggcggtggacagctatgatcctgatgtgcgcattattgaaaatcatggtgtggag


 


gatgaacttcccaactattgtttccctctggatgctgttggcagaacagatacttatcagggaattaaggctaatggaactgat


 


caaaccacatggaccaaagatgacagtgtcaatgatgctaatgagataggcaagggtaatccattcgccatggaaatca


 


acatccaagccaacctgtggaggaacttcctctacgccaacgtggccctgtacctgcccgactcttacaagtacacgccg


 


gccaatgttaccctgcccaccaacaccaacacctacgattacatgaacggccgggtggtggcgccctcgctggtggactc


 


ctacatcaacatcggggcgcgctggtcgctggatcccatggacaacgtgaaccccttcaaccaccaccgcaatgcgggg


 


ctgcgctaccgctccatgctcctgggcaacgggcgctacgtgcccttccacatccaggtgccccagaaatttttcgccatca


 


agagcctcctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggacgtcaacatgatcctgcagagctcc


 


ctcggcaacgacctgcgcacggacggggcctccatctccttcaccagcatcaacctctacgccaccttcttccccatggcg


 


cacaacacggcctccacgctcgaggccatgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcg


 


gccaacatgctctaccccatcccggccaacgccaccaacgtgcccatctccatcccctcgcgcaactgggccgccttccg


 


cggctggtccttcacgcgtctcaagaccaaggagacgccctcgctgggctccgggttcgacccctacttcgtctactcggg


 


ctccatcccctacctcgacggcaccttctacctcaaccacaccttcaagaaggtctccatcaccttcgactcctccgtcagct


 


ggcccggcaacgaccggctcctgacgcccaacgagttcgaaatcaagcgcaccgtcgacggcgagggctacaacgtg


 


gcccagtgcaacatgaccaaggactggttcctggtccagatgctggcccactacaacatcggctaccagggcttctacgt


 


gcccgagggctacaaggaccgcatgtactccttcttccgcaacttccagcccatgagccgccaggtggtggacgaggtca


 


actacaaggactaccaggccgtcaccctggcctaccagcacaacaactcgggcttcgtcggctacctcgcgcccaccat


 


gcgccagggccagccctaccccgccaactacccctacccgctcatcggcaagagcgccgtcaccagcgtcacccaga


 


aaaagttcctctgcgacagggtcatgtggcgcatccccttctccagcaacttcatgtccatgggcgcgctcaccgacctcgg


 


ccagaacatgctctatgccaactccgcccacgcgctagacatgaatttcgaagtcgaccccatggatgagtccacccttct


 


ctatgttgtcttcgaagtcttcgacgtcgtccgagtgcaccagccccaccgcggcgtcatcgaggccgtctacctgcgcacc


 


cccttctcggccggtaacgccaccacctaagctcttgcttcttgcaagccatggccgcgggctccggcgagcaggagctc


 


agggccatcatccgcgacctgggctgcgggccctacttcctgggcaccttcgataagcgcttcccgggattcatggccccg


 


cacaagctggcctgcgccatcgtcaacacggccggccgcgagaccgggggcgagcactggctggccttcgcctggaa


 


cccgcgctcgaacacctgctacctcttcgaccccttcgggttctcggacgagcgcctcaagcagatctaccagttcgagtac


 


gagggcctgctgcgccgcagcgccctggccaccgaggaccgctgcgtcaccctggaaaagtccacccagaccgtgca


 


gggtccgcgctcggccgcctgcgggctcttctgctgcatgttcctgcacgccttcgtgcactggcccgaccgccccatggac


 


aagaaccccaccatgaacttgctgacgggggtgcccaacggcatgctccagtcgccccaggtggaacccaccctgcgc


 


cgcaaccaggaggcgctctaccgcttcctcaactcccactccgcctactttcgctcccaccgcgcgcgcatcgagaaggc


 


caccgccttcgaccgcatgaatcaagacatgtaaaccgtgtgtgtatgttaaatgtctttaataaacagcactttcatgttaca


 


catgcatctgagatgatttatttagaaatcgaaagggttctgccgggtctcggcatggcccgcgggcagggacacgttgcg


 


gaactggtacttggccagccacttgaactcggggatcagcagtttgggcagcggggtgtcggggaaggagtcggtccac


 


agcttccgcgtcagttgcagggcgcccagcaggtcgggcgcggagatcttgaaatcgcagttgggacccgcgttctgcgc


 


gcgggagttgcggtacacggggttgcagcactggaacaccatcagggccgggtgcttcacgctcgccagcaccgtcgc


 


gtcggtgatgctctccacgtcgaggtcctcggcgttggccatcccgaagggggtcatcttgcaggtctgccttcccatggtgg


 


gcacgcacccgggcttgtggttgcaatcgcagtgcagggggatcagcatcatctgggcctggtcggcgttcatccccgggt


 


acatggccttcatgaaagcctccaattgcctgaacgcctgctgggccttggctccctcggtgaagaagaccccgcaggact


 


tgctagagaactggttggtggcgcacccggcgtcgtgcacgcagcagcgcgcgtcgttgttggccagctgcaccacgctg


 


cgcccccagcggttctgggtgatcttggcccggtcggggttctccttcagcgcgcgctgcccgttctcgctcgccacatccat


 


ctcgatcatgtgctccttctggatcatggtggtcccgtgcaggcaccgcagcttgccctcggcctcggtgcacccgtgcagc


 


cacagcgcgcacccggtgcactcccagttcttgtgggcgatctgggaatgcgcgtgcacgaagccctgcaggaagcgg


 


cccatcatggtggtcagggtcttgttgctagtgaaggtcagcggaatgccgcggtgctcctcgttgatgtacaggtggcagat


 


gcggcggtacacctcgccctgctcgggcatcagctggaagttggctttcaggtcggtctccacgcggtagcggtccatcag


 


catagtcatgatttccatacccttctcccaggccgagacgatgggcaggctcatagggttcttcaccatcatcttagcgctag


 


cagccgcggccagggggtcgctctcgtccagggtctcaaagctccgcttgccgtccttctcggtgatccgcaccggggggt


 


agctgaagcccacggccgccagctcctcctcggcctgtctttcgtcctcgctgtcctggctgacgtcctgcaggaccacatg


 


cttggtcttgcggggtttcttcttgggcggcagcggcggcggagatgttggagatggcgagggggagcgcgagttctcgctc


 


accactactatctcttcctcttcttggtccgaggccacgcggcggtaggtatgtctcttcgggggcagaggcggaggcgacg


 


ggctctcgccgccgcgacttggcggatggctggcagagccccttccgcgttcgggggtgcgctcccggcggcgctctgac


 


tgacttcctccgcggccggccattgtgttctcctagggaggaacaacaagcatggagactcagccatcgccaacctcgcc


 


atctgcccccaccgccgacgagaagcagcagcagcagaatgaaagcttaaccgccccgccgcccagccccgccacc


 


tccgacgcggccgtcccagacatgcaagagatggaggaatccatcgagattgacctgggctatgtgacgcccgcggag


 


cacgaggaggagctggcagtgcgcttttcacaagaagagatacaccaagaacagccagagcaggaagcagagaat


 


gagcagagtcaggctgggctcgagcatgacggcgactacctccacctgagcgggggggaggacgcgctcatcaagca


 


tctggcccggcaggccaccatcgtcaaggatgcgctgctcgaccgcaccgaggtgcccctcagcgtggaggagctcag


 


ccgcgcctacgagttgaacctcttctcgccgcgcgtgccccccaagcgccagcccaatggcacctgcgagcccaacccg


 


cgcctcaacttctacccggtcttcgcggtgcccgaggccctggccacctaccacatctttttcaagaaccaaaagatccccg


 


tctcctgccgcgccaaccgcacccgcgccgacgcccttttcaacctgggtcccggcgcccgcctacctgatatcgcctcctt


 


ggaagaggttcccaagatcttcgagggtctgggcagcgacgagactcgggccgcgaacgctctgcaaggagaaggag


 


gagagcatgagcaccacagcgccctggtcgagttggaaggcgacaacgcgcggctggcggtgctcaaacgcacggtc


 


gagctgacccatttcgcctacccggctctgaacctgccccccaaagtcatgagcgcggtcatggaccaggtgctcatcaa


 


gcgcgcgtcgcccatctccgaggacgagggcatgcaagactccgaggagggcaagcccgtggtcagcgacgagcag


 


ctggcccggtggctgggtcctaatgctagtccccagagtttggaagagcggcgcaaactcatgatggccgtggtcctggtg


 


accgtggagctggagtgcctgcgccgcttcttcgccgacgcggagaccctgcgcaaggtcgaggagaacctgcactacc


 


tcttcaggcacgggttcgtgcgccaggcctgcaagatctccaacgtggagctgaccaacctggtctcctacatgggcatctt


 


gcacgagaaccgcctggggcagaacgtgctgcacaccaccctgcgcggggaggcccggcgcgactacatccgcgac


 


tgcgtctacctctacctctgccacacctggcagacgggcatgggcgtgtggcagcagtgtctggaggagcagaacctgaa


 


agagctctgcaagctcctgcagaagaacctcaagggtctgtggaccgggttcgacgagcgcaccaccgcctcggacct


 


ggccgacctcattttccccgagcgcctcaggctgacgctgcgcaacggcctgcccgactttatgagccaaagcatgttgca


 


aaactttcgctctttcatcctcgaacgctccggaatcctgcccgccacctgctccgcgctgccctcggacttcgtgccgctga


 


ccttccgcgagtgccccccgccgctgtggagccactgctacctgctgcgcctggccaactacctggcctaccactcggac


 


gtgatcgaggacgtcagcggcgagggcctgctcgagtgccactgccgctgcaacctctgcacgccgcaccgctccctgg


 


cctgcaacccccagctgctgagcgagacccagatcatcggcaccttcgagttgcaagggcccagcgaaggcgagggtt


 


cagccgccaaggggggtctgaaactcaccccggggctgtggacctcggcctacttgcgcaagttcgtgcccgaggacta


 


ccatcccttcgagatcaggttctacgaggaccaatcccatccgcccaaggccgagctgtcggcctgcgtcatcacccagg


 


gggcgatcctggcccaattgcaagccatccagaaatcccgccaagaattcttgctgaaaaagggccgcggggtctacct


 


cgacccccagaccggtgaggagctcaaccccggcttcccccaggatgccccgaggaaacaagaagctgaaagtgga


 


gctgccgcccgtggaggatttggaggaagactgggagaacagcagtcaggcagaggaggaggagatggaggaaga


 


ctgggacagcactcaggcagaggaggacagcctgcaagacagtctggaggaagacgaggaggaggcagaggagg


 


aggtggaagaagcagccgccgccagaccgtcgtcctcggcgggggagaaagcaagcagcacggataccatctccgc


 


tccgggtcggggtcccgctcgaccacacagtagatgggacgagaccggacgattcccgaaccccaccacccagaccg


 


gtaagaaggagcggcagggatacaagtcctggcgggggcacaaaaacgccatcgtctcctgcttgcaggcctgcggg


 


ggcaacatctccttcacccggcgctacctgctcttccaccgcggggtgaactttccccgcaacatcttgcattactaccgtca


 


cctccacagcccctactacttccaagaagaggcagcagcagcagaaaaagaccagcagaaaaccagcagctagaa


 


aatccacagcggcggcagcaggtggactgaggatcgcggcgaacgagccggcgcaaacccgggagctgaggaacc


 


ggatctttcccaccctctatgccatcttccagcagagtcgggggcaggagcaggaactgaaagtcaagaaccgttctctgc


 


gctcgctcacccgcagttgtctgtatcacaagagcgaagaccaacttcagcgcactctcgaggacgccgaggctctcttca


 


acaagtactgcgcgctcactcttaaagagtagcccgcgcccgcccagtcgcagaaaaaggcgggaattacgtcacctgt


 


gcccttcgccctagccgcctccacccatcatcatgagcaaagagattcccacgccttacatgtggagctaccagccccag


 


atgggcctggccgccggtgccgcccaggactactccacccgcatgaattggctcagcgccgggcccgcgatgatctcac


 


gggtgaatgacatccgcgcccaccgaaaccagatactcctagaacagtcagcgctcaccgccacgccccgcaatcacc


 


tcaatccgcgtaattggcccgccgccctggtgtaccaggaaattccccagcccacgaccgtactacttccgcgagacgcc


 


caggccgaagtccagctgactaactcaggtgtccagctggcgggcggcgccaccctgtgtcgtcaccgccccgctcagg


 


gtataaagcggctggtgatccggggcagaggcacacagctcaacgacgaggtggtgagctcttcgctgggtctgcgacc


 


tgacggagtcttccaactcgccggatcggggagatcttccttcacgcctcgtcaggccgtcctgactttggagagttcgtcctc


 


gcagccccgctcgggtggcatcggcactctccagttcgtggaggagttcactccctcggtctacttcaaccccttctccggct


 


cccccggccactacccggacgagttcatcccgaacttcgacgccatcagcgagtcggtggacggctacgattgaatgtcc


 


catggtggcgcagctgacctagctcggcttcgacacctggaccactgccgccgcttccgctgcttcgctcgggatctcgccg


 


agtttgcctactttgagctgcccgaggagcaccctcagggcccggcccacggagtgcggatcgtcgtcgaagggggcct


 


cgactcccacctgcttcggatcttcagccagcgtccgatcctggtcgagcgcgagcaaggacagacccttctgactctgta


 


ctgcatctgcaaccaccccggcctgcatgaaagtctttgttgtctgctgtgtactgagtataataaaagctgagatcagcgac


 


tactccggacttccgtgtgttcctgaatccatcaaccagtctttgttcttcaccgggaacgagaccgagctccagctccagtgt


 


aagccccacaagaagtacctcacctggctgttccagggctccccgatcgccgttgtcaaccactgcgacaacgacggag


 


tcctgctgagcggccctgccaaccttactttttccacccgcagaagcaagctccagctcttccaacccttcctccccgggacc


 


tatcagtgcgtctcgggaccctgccatcacaccttccacctgatcccgaataccacagcgtcgctccccgctactaacaac


 


caaactaacctccaccaacgccaccgtcgctaggccacaatacatgcccatattagactatgaggccgagccacagcg


 


acccatgctccccgctattagttacttcaatctaaccggcggagatgactgacccactggccaacaacaacgtcaacgac


 


cttctcctggacatggacggccgcgcctcggagcagcgactcgcccaacttcgcattcgccagcagcaggagagagcc


 


gtcaaggagctgcaggatgcggtggccatccaccagtgcaagagaggcatcttctgcctggtgaaacaggccaagatct


 


cctacgaggtcactccaaacgaccatcgcctctcctacgagctcctgcagcagcgccagaagttcacctgcctggtcgga


 


gtcaaccccatcgtcatcacccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtc


 


cacactctgatcaagaccctctgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagat


 


catattgatgatgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgatgatttgagtttaaca


 


aaaaaataaagaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttccc


 


agctctggtactgcaggccccggcgggctgcaaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaat


 


cttcattttatcttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgcagac


 


aacgcaccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccc


 


tgcgactggccgaccccgtcaccaccaagaacggggaaatcaccctcaagctgggagagggggtggacctcgattcct


 


cgggaaaactcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatgg


 


atcaccccttttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaaca


 


cactagctttaggttttggatcaggtttaggactccgtggctctgccttggcagtacagttagtctctccacttacatttgatactg


 


atggaaacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggct


 


aaaggtttaaaatttgaagatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaac


 


aggtgttgatgatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacaggagccataatggctggta


 


acaaagaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgc


 


aaaactaacactttgcttgactaaatgtggtagtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaa


 


accccattactggcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattctacact


 


aaaaaaatactgggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaattt


 


aaaagcttatccaaagtcacaaagttctactactaaaaataatatagtagggcaagtatacatgaatggagatgtttcaaa


 


acctatgcttctcactataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaat


 


ggaagctatgttggagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccct


 


gcatgccaacccttcccaccccactctgtggaacaaactctgaaacacaaaataaaataaagttcaagtgttttattgattca


 


acagttttacaggattcgagcagttatttttcctccaccctcccaggacatggaatacaccaccctctccccccgcacagcctt


 


gaacatctgaatgccattggtgatggacatgcttttggtctccacgttccacacagtttcagagcgagccagtctcgggtcgg


 


tcagggagatgaaaccctccgggcactcccgcatctgcacctcacagctcaacagctgaggattgtcctcggtggtcggg


 


atcacggttatctggaagaagcagaagagcggcggtgggaatcatagtccgcgaacgggatcggccggtggtgtcgca


 


tcaggccccgcagcagtcgctgccgccgccgctccgtcaagctgctgctcagggggtccgggtccagggactccctcag


 


catgatgcccacggccctcagcatcagtcgtctggtgcggcgggcgcagcagcgcatgcggatctcgctcaggtcgctgc


 


agtacgtgcaacacagaaccaccaggttgttcaacagtccatagttcaacacgctccagccgaaactcatcgcgggaag


 


gatgctacccacgtggccgtcgtaccagatcctcaggtaaatcaagtggtgccccctccagaacacgctgcccacgtaca


 


tgatctccttgggcatgtggcggttcaccacctcccggtaccacatcaccctctggttgaacatgcagccccggatgatcctg


 


cggaaccacagggccagcaccgccccgcccgccatgcagcgaagagaccccgggtcccggcaatggcaatggagg


 


acccaccgctcgtacccgtggatcatctgggagctgaacaagtctatgttggcacagcacaggcatatgctcatgcatctct


 


tcagcactctcaactcctcgggggtcaaaaccatatcccagggcacggggaactcttgcaggacagcgaaccccgcag


 


aacagggcaatcctcgcacagaacttacattgtgcatggacagggtatcgcaatcaggcagcaccgggtgatcctccac


 


cagagaagcgcgggtctcggtctcctcacagcgtggtaagggggccggccgatacgggtgatggcgggacgcggctg


 


atcgtgttcgcgaccgtgtcatgatgcagttgctttcggacattttcgtacttgctgtagcagaacctggtccgggcgctgcaca


 


ccgatcgccggcggcggtctcggcgcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagc


 


agatctagggcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtggaatgggccag


 


acccagccagatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaactttta


 


atccaaacggtctcggagtacttcaaaatgaagatcgcggagatggcacctctcgcccccgctgtgttggtggaaaataa


 


cagccaggtcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaa


 


caagacaatagcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatt


 


tttccagccttgaatgattcgaactagttcCtgaggtaaatccaagccagccatgataaagagctcgcgcagagcgccctc


 


caccggcattcttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatc


 


aaaatctctgccgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccatag


 


gaccaccaggaataagattagggcaagccacagtacagataaaccgaagtcctccccagtgagcattgccaaatgca


 


agactgctataagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaaga


 


aaatcaacaaaagaaaaatcctccaggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgt


 


tccagcatggttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaacaggtgggta


 


aatcgttctctccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccat


 


cacagagagacgttcccggtggccggcgtgaatgattcgacaagatgaatacacccccggaacattggcgtccgcgagt


 


gaaaaaaagcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatg


 


aagcacaaaattctcaggtgcgtacaaaatgtaattactcccctcctgcacaggcagcaaagcccccgatccctccaggt


 


acacatacaaagcctcagcgtccatagcttaccgagcagcagcacacaacaggcgcaagagtcagagaaaggctga


 


gctctaacctgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccg


 


ccaaataatcacacacgcccagcacacgcccagaaaccggtgacacactcaaaaaaatacgcgcacttcctcaaacg


 


cccaaaactgccgtcatttccgggttcccacgctacgtcatcaaaacacgactttcaaattccgtcgaccgttaaaaacgtc


 


acccgccccgcccctaacggtcgcccgtctctcagccaatcagcgccccgcatccccaaattcaaacacctcatttgcata


 


ttaacgcgcacaaaaagtttgaggtatattattgatgatggTTAATTAA


 


SEQ ID NO: 59: Nucleotide Seqeunce of Preferred EMCV IRES (pIRES)


TAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGT


 


TATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCT


 


GTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGG


 


TCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAA


 


CGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCT


 


CTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAG


 


TGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGT


 


ATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATC


 


TGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAG


 


GCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGC


 


CACAACCATG


 


(The minimal EMCV IRES (mIRES) lacks the underlined 15 nucleotides)


 


SEQ ID NO: 60. Amino Acid Sequence Comprising an Immunogenic PSA,


PSCA, and PSMA Polypeptide (Encoded by by Plasmid 916 and Vectors AdC68-


734 and AdC68W-734)


MASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRH


 


SLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAV


 


KVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTK


 


FMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR


 


KWIKDTIVANPGSEGRGSLLTCGDVEENPGPASKAVLLALLMAGLALQPGTALLCYSCK


 


AQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNIT


 


CCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQLGSQTLNFDLLKLAGDVESNP


 


GPMASARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKA


 


ENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPN


 


YISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFF


 


KLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDG


 


WNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQ


 


KLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGT


 


LRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFA


 


SWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNL


 


TKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRA


 


RYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVL


 


PFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQD


 


FDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDA


 


LFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA


 


SEQ ID NO: 61. Nucleotide Sequence Encoding the Amino Acid Sequence


of SEQ ID NO: 60.


atggctagcatcgtcggagggtgggagtgcgaaaagcactcacagccatggcaggtcctggtcgcctcgcgcggacgc


 


gccgtgtgtggaggtgtgctggtccacccgcagtgggtgttgactgcggcccattgcatcagaaataagtccgtgatcctctt


 


ggggagacattccctgtttcaccccgaagatactggacaggtgttccaagtgagccactccttcccgcatccactgtacgac


 


atgagcctgctgaagaaccgctttctgcggccaggggacgactcatcacacgatttgatgctgcttcggctctcggaaccg


 


gccgagctcaccgacgcagtgaaggtcatggacctccctacgcaagagcctgctctcggtaccacttgttacgcatcggg


 


atggggctccatcgagccggaagaattcctgaccccgaaaaagctgcagtgcgtggatctgcacgtgatttcgaatgacg


 


tgtgcgcgcaagtgcatccacaaaaggtcactaagttcatgctgtgcgccggaaggtggaccggcggaaaatcgacctg


 


ttccggcgacagcggaggcccactcgtgtgcaacggtgtgctgcagggcatcactagctggggatcagaaccgtgcgcg


 


cttccggagcggccctcgctctacacgaaggtggtgcactaccgcaaatggattaaagataccatcgtcgcaaaccctgg


 


atccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccgctagcaaagcagtgctgctgg


 


cgctcctgatggctggactcgcgctgcagcctggaaccgccctgctctgttactcgtgcaaggcccaagtctcgaatgagg


 


actgtttgcaagtggaaaactgcacccagctcggagaacaatgctggactgcacggatccgcgctgtcggcctgctgacc


 


gtgatctccaaagggtgctcattgaactgcgtggacgatagccaggactactacgtgggaaagaagaatatcacttgttgc


 


gacacggatctttgcaacgcgtccggagcgcacgccctgcagccagcagccgccattctggccctgcttccggccctggg


 


gttgctgctctggggtccgggccagctcggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaag


 


caacccaggcccaatggctagcgctcgcagaccgcggtggctgtgtgcaggggcgctcgtcctggcgggtggcttcttttt


 


gctcggctttcttttcggatggttcatcaaatcgtcaaacgaagctaccaatatcaccccgaagcacaacatgaaggcctttc


 


tggatgagctgaaggctgagaacattaagaagttcctctacaacttcacccagatcccacatttggcgggcactgagcag


 


aactttcagttggctaagcagatccagagccagtggaaggaattcggcctggactccgtcgagctggcgcattacgatgtg


 


ctgctgagctaccctaataagactcatccgaactatatctcgattatcaatgaggacggaaacgaaatctttaacacgtccct


 


cttcgagccgccaccgcctggatacgagaacgtgtcagatatcgtgcctccgttctcggccttctcgccccagggaatgcc


 


cgaaggggacctggtgtacgtgaactacgcaaggaccgaggacttcttcaagttggagcgggatatgaagatcaattgc


 


agcggaaagatcgtcatcgcccgctacggcaaagtgttccgcggcaacaaggtgaagaatgcacagttggcaggcgc


 


caagggcgtcatcctctactcggatcctgccgactacttcgctcctggcgtgaaatcctaccctgatggttggaatctgccag


 


gaggaggggtgcagaggggaaatatcctgaacctgaacggtgccggtgacccacttactccgggttacccggccaacg


 


aatacgcgtacaggcggggtatcgcggaagccgtcggactgccgtccatcccggtccatccgattggttactacgacgcc


 


cagaagctcctcgaaaagatgggaggcagcgcccctccggactcgtcatggagaggctcgctgaaggtgccatacaac


 


gtgggacccggattcactggaaatttcagcactcaaaaagtgaagatgcacattcactccactaacgaagtcaccaggat


 


ctacaacgtcatcggaaccctccggggagcggtggaaccggaccgctacgtgatcctcggtggacaccgggatagctg


 


ggtgttcggaggaatcgatcctcaatcgggcgcagccgtcgtccatgaaatcgtcaggtcctttggtactcttaagaaggag


 


ggctggcgccctagacgcactattctgttcgcctcgtgggatgccgaagaatttggtctgctcggcagcaccgaatgggctg


 


aggaaaactcccgcctgctccaagaacgcggagtggcgtacatcaatgccgactcatccatcgaaggaaactacacgc


 


tgcgggtggactgcactccactgatgtactcgctcgtgcacaacctgaccaaagaactcaaatccccagacgaaggattc


 


gagggaaaatcgctgtacgagtcgtggaccaagaagagcccatccccggagttcagcgggatgccgcggatctcaaa


 


gctcggatcaggaaatgatttcgaagtgttctttcagaggctgggaattgcgtcgggaagggctcggtacacgaaaaactg


 


ggaaactaacaagttctcgggatacccgctgtaccactcggtgtatgaaacttacgaactggtggagaaattctacgatcct


 


atgtttaagtaccacctgactgtggcccaagtgagaggcggaatggtgttcgagttggccaattcaattgtgctgccattcgat


 


tgccgcgactacgccgtggtgctgagaaagtacgcagacaaaatctactcaatcagcatgaagcacccacaagagatg


 


aaaacctactcagtctccttcgactccctcttctccgcggtgaagaacttcaccgagatcgcgagcaaattctcggagcgcc


 


ttcaagattttgacaaatccaatccgatcgtcctccgcatgatgaatgaccagctcatgtttctcgaacgggccttcatcgatc


 


cactgggacttccggaccggccgttttaccgccacgtgatctacgcgccctcgtcgcataacaagtatgctggagagagct


 


tcccgggtatctacgacgcattgttcgacattgagtccaaggtggatccgtccaaagcctggggtgaagtgaagcgccaa


 


atctacgtggcggcctttaccgtccaggcggcagcagaaaccttgagcgaggtggct


 


SEQ ID NO: 62. Nucleotide Sequence of Plasmid 916


ggcgtaatgctctgccagtgttacaaccaattaaccaattctgattagaaaaactcatcgagcatcaaatgaaactgcaattt


 


attcatatcaggattatcaataccatatttttgaaaaagccgtttctgtaatgaaggagaaaactcaccgaggcagttccata


 


ggatggcaagatcctggtatcggtctgcgattccgactcgtccaacatcaatacaacctattaatttcccctcgtcaaaaata


 


aggttatcaagtgagaaatcaccatgagtgacgactgaatccggtgagaatggcaaaagcttatgcatttctttccagactt


 


gttcaacaggccagccattacgctcgtcatcaaaatcactcgcatcaaccaaaccgttattcattcgtgattgcgcctgagc


 


gagacgaaatacgcgatcgctgttaaaaggacaattacaaacaggaatcaaatgcaaccggcgcaggaacactgcca


 


gcgcatcaacaatattttcacctgaatcaggatattcttctaatacctggaatgctgttttcccggggatcgcagtggtgagtaa


 


ccatgcatcatcaggagtacggataaaatgcttgatggtcggaagaggcataaattccgtcagccagtttagtctgaccatc


 


tcatctgtaacatcattggcaacgctacctttgccatgtttcagaaacaactctggcgcatcgggcttcccatacaatcgatag


 


attgtcgcacctgattgcccgacattatcgcgagcccatttatacccatataaatcagcatccatgttggaatttaatcgcggc


 


ctcgagcaagacgtttcccgttgaatatggctcataacaccccttgtattactgtttatgtaagcagacaggtcgacaatattg


 


gctattggccattgcatacgttgtatctatatcataatatgtacatttatattggctcatgtccaatatgaccgccatgttgacattg


 


attattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggt


 


aaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaa


 


tagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaa


 


gtccgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttacgggactttcctactt


 


ggcagtacatctacgtattagtcatcgctattaccatggtgatgcgOttggcagtacaccaatgggcgtggatagcggtttg


 


actcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaa


 


tgtcgtaataaccccgccccgttgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctcgttta


 


gtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccgatccagcctccg


 


cggccgggaacggtgcattggaacgcggattccccgtgccaagagtgactcaccgtccggatctcagcaagcaggtatg


 


tactctccagggtgggcctggcttccccagtcaagactccagggatttgagggacgctgtgggctcttctcttacatgtaccttt


 


tgcttgcctcaaccctgactatcttccaggtcaggatcccagagtcaggggtctgtattttcctgctggtggctccagttcagga


 


acagtaaaccctgctccgaatattgcctctcacatctcgtcaatctccgcgaggactggggaccctgtgacgaacatggct


 


agcatcgtcggagggtgggagtgcgaaaagcactcacagccatggcaggtcctggtcgcctcgcgcggacgcgccgtg


 


tgtggaggtgtgctggtccacccgcagtgggtgttgactgcggcccattgcatcagaaataagtccgtgatcctcttgggga


 


gacattccctgtttcaccccgaagatactggacaggtgttccaagtgagccactccttcccgcatccactgtacgacatgag


 


cctgctgaagaaccgctttctgcggccaggggacgactcatcacacgatttgatgctgcttcggctctcggaaccggccga


 


gctcaccgacgcagtgaaggtcatggacctccctacgcaagagcctgctctcggtaccacttgttacgcatcgggatggg


 


gctccatcgagccggaagaattcctgaccccgaaaaagctgcagtgcgtggatctgcacgtgatttcgaatgacgtgtgc


 


gcgcaagtgcatccacaaaaggtcactaagttcatgctgtgcgccggaaggtggaccggcggaaaatcgacctgttccg


 


gcgacagcggaggcccactcgtgtgcaacggtgtgctgcagggcatcactagctggggatcagaaccgtgcgcgcttcc


 


ggagcggccctcgctctacacgaaggtggtgcactaccgcaaatggattaaagataccatcgtcgcaaaccctggatcc


 


gaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccgctagcaaagcagtgctgctggcgct


 


cctgatggctggactcgcgctgcagcctggaaccgccctgctctgttactcgtgcaaggcccaagtctcgaatgaggactg


 


tttgcaagtggaaaactgcacccagctcggagaacaatgctggactgcacggatccgcgctgtcggcctgctgaccgtga


 


tctccaaagggtgctcattgaactgcgtggacgatagccaggactactacgtgggaaagaagaatatcacttgttgcgaca


 


cggatctttgcaacgcgtccggagcgcacgccctgcagccagcagccgccattctggccctgcttccggccctggggttgc


 


tgctctggggtccgggccagctcggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaac


 


ccaggcccaatggctagcgctcgcagaccgcggtggctgtgtgcaggggcgctcgtcctggcgggtggcttctttttgctcg


 


gctttcttttcggatggttcatcaaatcgtcaaacgaagctaccaatatcaccccgaagcacaacatgaaggcctttctggat


 


gagctgaaggctgagaacattaagaagttcctctacaacttcacccagatcccacatttggcgggcactgagcagaacttt


 


cagttggctaagcagatccagagccagtggaaggaattcggcctggactccgtcgagctggcgcattacgatgtgctgct


 


gagctaccctaataagactcatccgaactatatctcgattatcaatgaggacggaaacgaaatctttaacacgtccctcttcg


 


agccgccaccgcctggatacgagaacgtgtcagatatcgtgcctccgttctcggccttctcgccccagggaatgcccgaa


 


ggggacctggtgtacgtgaactacgcaaggaccgaggacttcttcaagttggagcgggatatgaagatcaattgcagcg


 


gaaagatcgtcatcgcccgctacggcaaagtgttccgcggcaacaaggtgaagaatgcacagttggcaggcgccaag


 


ggcgtcatcctctactcggatcctgccgactacttcgctcctggcgtgaaatcctaccctgatggttggaatctgccaggagg


 


aggggtgcagaggggaaatatcctgaacctgaacggtgccggtgacccacttactccgggttacccggccaacgaatac


 


gcgtacaggcggggtatcgcggaagccgtcggactgccgtccatcccggtccatccgattggttactacgacgcccaga


 


agctcctcgaaaagatgggaggcagcgcccctccggactcgtcatggagaggctcgctgaaggtgccatacaacgtgg


 


gacccggattcactggaaatttcagcactcaaaaagtgaagatgcacattcactccactaacgaagtcaccaggatctac


 


aacgtcatcggaaccctccggggagcggtggaaccggaccgctacgtgatcctcggtggacaccgggatagctgggtgt


 


tcggaggaatcgatcctcaatcgggcgcagccgtcgtccatgaaatcgtcaggtcctttggtactcttaagaaggagggct


 


ggcgccctagacgcactattctgttcgcctcgtgggatgccgaagaatttggtctgctcggcagcaccgaatgggctgagg


 


aaaactcccgcctgctccaagaacgcggagtggcgtacatcaatgccgactcatccatcgaaggaaactacacgctgc


 


gggtggactgcactccactgatgtactcgctcgtgcacaacctgaccaaagaactcaaatccccagacgaaggattcga


 


gggaaaatcgctgtacgagtcgtggaccaagaagagcccatccccggagttcagcgggatgccgcggatctcaaagct


 


cggatcaggaaatgatttcgaagtgttctttcagaggctgggaattgcgtcgggaagggctcggtacacgaaaaactggg


 


aaactaacaagttctcgggatacccgctgtaccactcggtgtatgaaacttacgaactggtggagaaattctacgatcctat


 


gtttaagtaccacctgactgtggcccaagtgagaggcggaatggtgttcgagttggccaattcaattgtgctgccattcgattg


 


ccgcgactacgccgtggtgctgagaaagtacgcagacaaaatctactcaatcagcatgaagcacccacaagagatga


 


aaacctactcagtctccttcgactccctcttctccgcggtgaagaacttcaccgagatcgcgagcaaattctcggagcgcctt


 


caagattttgacaaatccaatccgatcgtcctccgcatgatgaatgaccagctcatgtttctcgaacgggccttcatcgatcc


 


actgggacttccggaccggccgttttaccgccacgtgatctacgcgccctcgtcgcataacaagtatgctggagagagctt


 


cccgggtatctacgacgcattgttcgacattgagtccaaggtggatccgtccaaagcctggggtgaagtgaagcgccaaa


 


tctacgtggcggcctttaccgtccaggcggcagcagaaaccttgagcgaggtggcttaaagatctgggccctaacaaaac


 


aaaaagatggggttattccctaaacttcatgggttacgtaattggaagttgggggacattgccacaagatcatattgtacaaa


 


agatcaaacactgttttagaaaacttcctgtaaacaggcctattgattggaaagtatgtcaaaggattgtgggtcttttgggcttt


 


gctgctccatttacacaatgtggatatcctgccttaatgcctttgtatgcatgtatacaagctaaacaggctttcactttctcgcca


 


acttacaaggcctttctaagtaaacagtacatgaacctttaccccgttgctcggcaacggcctggtctgtgccaagtgtttgct


 


gacgcaacccccactggctggggcttggccataggccatcagcgcatgcgtggaacctttgtggctcctctgccgatccat


 


actgcggaactcctagccgcttgttttgctcgcagccggtctggagcaaagctcataggaactgacaattctgtcgtcctctc


 


gcggaaatatacatcgtttcgatctacgtatgatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcat


 


ctgacttctggctaataaaggaaatttattttcattgcaatagtgtgttggaattttttgtgtctctcactcggaaggaattctgcatt


 


aatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctc


 


ggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgca


 


ggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccatag


 


gctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagat


 


accaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctccc


 


ttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtg


 


cacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgactta


 


tcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtg


 


gcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggt


 


agctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaa


 


ggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatg


 


agattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaactt


 


ggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactc


 


SEQ ID NO: 63. Complete Sequence of the AdC68W-734 Vector


ccatcttcaataatatacctcaaactttttgtgcgcgttaatatgcaaatgaggcgtttgaatttggggaggaagggcggtgatt


 


ggtcgagggatgagcgaccgttaggggcggggcgagtgacgttttgatgacgtggttgcgaggaggagccagtttgcaa


 


gttctcgtgggaaaagtgacgtcaaacgaggtgtggtttgaacacggaaatactcaattttcccgcgctctctgacaggaaa


 


tgaggtgtttctgggcggatgcaagtgaaaacgggccattttcgcgcgaaaactgaatgaggaagtgaaaatctgagtaa


 


tttcgcgtttatggcagggaggagtatttgccgagggccgagtagactttgaccgattacgtgggggtttcgattaccgtgttttt


 


cacctaaatttccgcgtacggtgtcaaagtccggtgtttttactactgtaatagtaatcaattacggggtcattagttcatagccc


 


atatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtc


 


aataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgccc


 


acttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattat


 


gcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttg


 


gcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgt


 


tttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtac


 


ggtgggaggtctatataagcagagctgtccctatcagtgatagagatctccctatcagtgatagagagtttagtgaaccgtc


 


agatccgctagggtaccaacATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAAAAGCACTC


 


ACAGCCATGGCAGGTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGTGGAGGTGT


 


GCTGGTCCACCCGCAGTGGGTGTTGACTGCGGCCCATTGCATCAGAAATAAGTCC


 


GTGATCCTCTTGGGGAGACATTCCCTGTTTCACCCCGAAGATACTGGACAGGTGTT


 


CCAAGTGAGCCACTCCTTCCCGCATCCACTGTACGACATGAGCCTGCTGAAGAAC


 


CGCTTTCTGCGGCCAGGGGACGACTCATCACACGATTTGATGCTGCTTCGGCTCT


 


CGGAACCGGCCGAGCTCACCGACGCAGTGAAGGTCATGGACCTCCCTACGCAAG


 


AGCCTGCTCTCGGTACCACTTGTTACGCATCGGGATGGGGCTCCATCGAGCCGGA


 


AGAATTCCTGACCCCGAAAAAGCTGCAGTGCGTGGATCTGCACGTGATTTCGAATG


 


ACGTGTGCGCGCAAGTGCATCCACAAAAGGTCACTAAGTTCATGCTGTGCGCCGG


 


AAGGTGGACCGGCGGAAAATCGACCTGTTCCGGCGACAGCGGAGGCCCACTCGT


 


GTGCAACGGTGTGCTGCAGGGCATCACTAGCTGGGGATCAGAACCGTGCGCGCTT


 


CCGGAGCGGCCCTCGCTCTACACGAAGGTGGTGCACTACCGCAAATGGATTAAAG


 


ATACCATCGTCGCAAACCCTggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacc


 


caggacccGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGC


 


AGCCTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGA


 


CTGTTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACTGCACGG


 


ATCCGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGAACTGCG


 


TGGACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGCGACACG


 


GATCTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGG


 


CCCTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTCggatcccaga


 


ccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGCGCTCGCA


 


GACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTCTTTTTGCT


 


CGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAATATCACCCC


 


GAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACATTAAGAAGT


 


TCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGAACTTTCAG


 


TTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTCCGTCGAGC


 


TGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGAACTATATC


 


TCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCGAGCCGCC


 


ACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGCCTTCTCG


 


CCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGGACCGAG


 


GACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGATCGTCAT


 


CGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAGTTGGCA


 


GGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTCCTGGCG


 


TGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAGGGGAAA


 


TATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCGGCCAAC


 


GAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCCATCCCG


 


GTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGGGAGGCA


 


GCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACAACGTGG


 


GACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATTCACTCC


 


ACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAGCGGTGG


 


AACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGTTCGGAG


 


GAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTCCTTTGGT


 


ACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCTCGTGGG


 


ATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAACTCCCG


 


CCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATCGAAGGA


 


AACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGCACAACC


 


TGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTGTACGA


 


GTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGGATCTC


 


AAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAATTGCGT


 


CGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGATACCC


 


GCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGATCCTA


 


TGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCGAGTT


 


GGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTGAGA


 


AAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAAAAC


 


CTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGATCG


 


CGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTCCTC


 


CGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACTGGG


 


ACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCATAAC


 


AAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGAGTC


 


CAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTGGCG


 


GCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTTGActcgagccta


 


agcttctagataagatatccgatccaccggatctagataactgatcataatcagccataccacatttgtagaggttttacttgct


 


ttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttata


 


atggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactc


 


atcaatgtatcttatatgctggccaccgtacatgtggcttcccatgctcgcaagccctggcccgagttcgagcacaatgtcat


 


gaccaggtgcaatatgcatctggggtcccgccgaggcatgttcatgccctaccagtgcaacctgaattatgtgaaggtgct


 


gctggagcccgatgccatgtccagagtgagcctgacgggggtgtttgacatgaatgtggaggtgtggaagattctgagata


 


tgatgaatccaagaccaggtgccgagcctgcgagtgcggagggaagcatgccaggttccagcccgtgtgtgtggatgtg


 


acggaggacctgcgacccgatcatttggtgttgccctgcaccgggacggagttcggttccagcggggaagaatctgacta


 


gagtgagtagtgttctggggcgggggaggacctgcatgagggccagaataactgaaatctgtgcttttctgtgtgttgcagc


 


agcatgagcggaagcggctcctttgagggaggggtattcagcccttatctgacggggcgtctcccctcctgggcgggagt


 


gcgtcagaatgtgatgggatccacggtggacggccggcccgtgcagcccgcgaactcttcaaccctgacctatgcaacc


 


ctgagctcttcgtcgttggacgcagctgccgccgcagctgctgcatctgccgccagcgccgtgcgcggaatggccatggg


 


cgccggctactacggcactctggtggccaactcgagttccaccaataatcccgccagcctgaacgaggagaagctgttgc


 


tgctgatggcccagctcgaggccttgacccagcgcctgggcgagctgacccagcaggtggctcagctgcaggagcaga


 


cgcgggccgcggttgccacggtgaaatccaaataaaaaatgaatcaataaataaacggagacggttgttgattttaacac


 


agagtctgaatctttatttgatttttcgcgcgcggtaggccctggaccaccggtctcgatcattgagcacccggtggatcttttcc


 


aggacccggtagaggtgggcttggatgttgaggtacatgggcatgagcccgtcccgggggtggaggtagctccattgca


 


gggcctcgtgctcgggggtggtgttgtaaatcacccagtcatagcaggggcgcagggcatggtgttgcacaatatctttgag


 


gaggagactgatggccacgggcagccctttggtgtaggtgtttacaaatctgttgagctgggagggatgcatgcgggggg


 


agatgaggtgcatcttggcctggatcttgagattggcgatgttaccgcccagatcccgcctggggttcatgttgtgcaggacc


 


accagcacggtgtatccggtgcacttggggaatttatcatgcaacttggaagggaaggcgtgaaagaatttggcgacgcc


 


tttgtgcccgcccaggttttccatgcactcatccatgatgatggcgatgggcccgtgggcggcggcctgggcaaagacgttt


 


cgggggtcggacacatcatagttgtggtcctgggtgaggtcatcataggccattttaatgaatttggggcggagggtgccgg


 


actgggggacaaaggtaccctcgatcccgggggcgtagttcccctcacagatctgcatctcccaggctttgagctcggag


 


ggggggatcatgtccacctgcggggcgataaagaacacggtttccggggcgggggagatgagctgggccgaaagca


 


agttccggagcagctgggacttgccgcagccggtggggccgtagatgaccccgatgaccggctgcaggtggtagttgag


 


ggagagacagctgccgtcctcccggaggaggggggccacctcgttcatcatctcgcgcacgtgcatgttctcgcgcacca


 


gttccgccaggaggcgctctccccccagggataggagctcctggagcgaggcgaagtttttcagcggcttgagtccgtcg


 


gccatgggcattttggagagggtttgrtgcaagagttccaggcggtcccagagctcggtgatgtgctctacggcatctcgatc


 


cagcagacctcctcgtttcgcgggttgggacggctgcgggagtagggcaccagacgatgggcgtccagcgcagccagg


 


gtccggtccttccagggtcgcagcgtccgcgtcagggtggtctccgtcacggtgaaggggtgcgcgccgggctgggcgct


 


tgcgagggtgcgcttcaggctcatccggctggtcgaaaaccgctcccgatcggcgccctgcgcgtcggccaggtagcaat


 


tgaccatgagttcgtagttgagcgcctcggccgcgtggcctttggcgcggagcttacctttggaagtctgcccgcaggcggg


 


acagaggagggacttgagggcgtagagcttgggggcgaggaagacggactcgggggcgtaggcgtccgcgccgca


 


gtgggcgcagacggtctcgcactccacgagccaggtgaggtcgggctggtcggggtcaaaaaccagtttcccgccgttct


 


ttttgatgcgtttcttacctttggtctccatgagctcgtgtccccgctgggtgacaaagaggctgtccgtgtccccgtagaccga


 


ctttatgggccggtcctcgagcggtgtgccgcggtcctcctcgtagaggaaccccgcccactccgagacgaaagcccgg


 


gtccaggccagcacgaaggaggccacgtgggacgggtagcggtcgttgtccaccagcgggtccaccttttccagggtat


 


gcaaacacatgtccccctcgtccacatccaggaaggtgattggcttgtaagtgtaggccacgtgaccgggggtcccggcc


 


gggggggtataaaagggtgcgggtccctgctcgtcctcactgtcttccggatcgctgtccaggagcgccagctgttggggt


 


aggtattccctctcgaaggcgggcatgacctcggcactcaggttgtcagtttctagaaacgaggaggatttgatattgacggt


 


gccggcggagatgcctttcaagagcccctcgtccatctggtcagaaaagacgatctttttgttgtcgagcttggtggcgaag


 


gagccgtagagggcgttggagaggagcttggcgatggagcgcatggtctggtttttttccttgtcggcgcgctccttggcggc


 


gatgttgagctgcacgtactcgcgcgccacgcacttccattcggggaagacggtggtcagctcgtcgggcacgattctgac


 


ctgccagccccgattatgcagggtgatgaggtccacactggtggccacctcgccgcgcaggggctcattagtccagcag


 


aggcgtccgcccttgcgcgagcagaaggggggcagggggtccagcatgacctcgtcgggggggtcggcatcgatggt


 


gaagatgccgggcaggaggtcggggtcaaagtagctgatggaagtggccagatcgtccagggcagcttgccattcgcg


 


cacggccagcgcgcgctcgtagggactgaggggcgtgccccagggcatgggatgggtaagcgcggaggcgtacatg


 


ccgcagatgtcgtagacgtagaggggctcctcgaggatgccgatgtaggtggggtagcagcgccccccgcggatgctg


 


gcgcgcacgtagtcatacagctcgtgcgagggggcgaggagccccgggcccaggttggtgcgactgggcttttcggcgc


 


ggtagacgatctggcggaaaatggcatgcgagttggaggagatggtgggcctttggaagatgttgaagtgggcgtgggg


 


cagtccgaccgagtcgcggatgaagtgggcgtaggagtcttgcagcttggcgacgagctcggcggtgactaggacgtcc


 


agagcgcagtagtcgagggtctcctggatgatgtcatacttgagctgtcccttttgtttccacagctcgcggttgagaaggaa


 


ctcttcgcggtccttccagtactcttcgagggggaacccgtcctgatctgcacggtaagagcctagcatgtagaactggttga


 


cggccttgtaggcgcagcagcccttctccacggggagggcgtaggcctgggcggccttgcgcagggaggtgtgcgtgag


 


ggcgaaagtgtccctgaccatgaccttgaggaactggtgcttgaagtcgatatcgtcgcagcccccctgctcccagagctg


 


gaagtccgtgcgcttcttgtaggcggggttgggcaaagcgaaagtaacatcgttgaagaggatcttgcccgcgcggggca


 


taaagttgcgagtgatgcggaaaggttggggcacctcggcccggttgttgatgacctgggcggcgagcacgatctcgtcg


 


aagccgttgatgttgtggcccacgatgtagagttccacgaatcgcggacggcccttgacgtggggcagtttcttgagctcctc


 


gtaggtgagctcgtcggggtcgctgagcccgtgctgctcgagcgcccagtcggcgagatgggggttggcgcggaggaa


 


ggaagtccagagatccacggccagggcggtttgcagacggtcccggtactgacggaactgctgcccgacggccatttttt


 


cgggggtgacgcagtagaaggtgcgggggtccccgtgccagcgatcccatttgagctggagggcgagatcgagggcg


 


agctcgacgagccggtcgtccccggagagtttcatgaccagcatgaaggggacgagctgcttgccgaaggaccccatc


 


caggtgtaggtttccacatcgtaggtgaggaagagcctttcggtgcgaggatgcgagccgatggggaagaactggatctc


 


ctgccaccaattggaggaatggctgttgatgtgatggaagtagaaatgccgacggcgcgccgaacactcgtgcttgtgttta


 


tacaagcggccacagtgctcgcaacgctgcacgggatgcacgtgctgcacgagctgtacctgagttcctttgacgaggaa


 


tttcagtgggaagtggagtcgtggcgcctgcatctcgtgctgtactacgtcgtggtggtcggcctggccctcttctgcctcgatg


 


gtggtcatgctgacgagcccgcgcgggaggcaggtccagacctcggcgcgagcgggtcggagagcgaggacgagg


 


gcgcgcaggccggagctgtccagggtcctgagacgctgcggagtcaggtcagtgggcagcggcggcgcgcggttgact


 


tgcaggagtttttccagggcgcgcgggaggtccagatggtacttgatctccaccgcgccattggtggcgacgtcgatggctt


 


gcagggtcccgtgcccctggggtgtgaccaccgtcccccgtttcttcttgggcggctggggcgacgggggcggtgcctcttc


 


catggttagaagcggcggcgaggacgcgcgccgggcggcaggggcggctcggggcccggaggcaggggcggcag


 


gggcacgtcggcgccgcgcgcgggtaggttctggtactgcgcccggagaagactggcgtgagcgacgacgcgacggtt


 


gacgtcctggatctgacgcctctgggtgaaggccacgggacccgtgagtttgaacctgaaagagagttcgacagaatca


 


atctcggtatcgttgacggcggcctgccgcaggatctcttgcacgtcgcccgagttgtcctggtaggcgatctcggtcatgaa


 


ctgctcgatctcctcctcttgaaggtctccgcggccggcgcgctccacggtggccgcgaggtcgttggagatgcggcccat


 


gagctgcgagaaggcgttcatgcccgcctcgttccagacgcggctgtagaccacgacgccctcgggatcgcGggcgcg


 


catgaccacctgggcgaggttgagctccacgtggcgcgtgaagaccgcgtagttgcagaggcgctggtagaggtagttg


 


agcgtggtggcgatgtgctcggtgacgaagaaatacatgatccagcggcggagcggcatctcgctgacgtcgcccagc


 


gcctccaaacgttccatggcctcgtaaaagtccacggcgaagttgaaaaactgggagttgcgcgccgagacggtcaact


 


cctcctccagaagacggatgagctcggcgatggtggcgcgcacctcgcgctcgaaggcccccgggagttcctccacttc


 


ctcttcttcctcctccactaacatctcttctacttcctcctcaggcggcagtggtggcgggggagggggcctgcgtcgccggc


 


ggcgcacgggcagacggtcgatgaagcgctcgatggtctcgccgcgccggcgtcgcatggtctcggtgacggcgcgcc


 


cgtcctcgcggggccgcagcgtgaagacgccgccgcgcatctccaggtggccgggggggtccccgttgggcagggag


 


agggcgctgacgatgcatcttatcaattgccccgtagggactccgcgcaaggacctgagcgtctcgagatccacgggatc


 


tgaaaaccgctgaacgaaggcttcgagccagtcgcagtcgcaaggtaggctgagcacggtttcttctggcgggtcatgttg


 


gttgggagcggggcgggcgatgctgctggtgatgaagttgaaataggcggttctgagacggcggatggtggcgaggag


 


caccaggtctttgggcccggcttgctggatgcgcagacggtcggccatgccccaggcgtggtcctgacacctggccaggt


 


ccttgtagtagtcctgcatgagccgctccacgggcacctcctcctcgcccgcgcggccgtgcatgcgcgtgagcccgaag


 


ccgcgctggggctggacgagcgccaggtcggcgacgacgcgctcggcgaggatggcttgctggatctgggtgagggtg


 


gtctggaagtcatcaaagtcgacgaagcggtggtaggctccggtgttgatggtgtaggagcagttggccatgacggacca


 


gttgacggtctggtggcccggacgcacgagctcgtggtacttgaggcgcgagtaggcgcgcgtgtcgaagatgtagtcgtt


 


gcaggtgcgcaccaggtactggtagccgatgaggaagtgcggcggcggctggcggtagagcggccatcgctcggtgg


 


cgggggcgccgggcgcgaggtcctcgagcatggtgcggtggtagccgtagatgtacctggacatccaggtgatgccgg


 


cggcggtggtggaggcgcgcgggaactcgcggacgcggttccagatgttgcgcagcggcaggaagtagttcatggtgg


 


gcacggtctggcccgtgaggcgcgcgcagtcgtggatgctctatacgggcaaaaacgaaagcggtcagcggctcgact


 


ccgtggcctggaggctaagcgaacgggttgggctgcgcgtgtaccccggttcgaatctcgaatcaggctggagccgcag


 


ctaacgtggtattggcactcccgtctcgacccaagcctgcaccaaccctccaggatacggaggcgggtcgttttgcaactttt


 


ttttggaggccggatgagactagtaagcgcggaaagcggccgaccgcgatggctcgctgccgtagtctggagaagaatc


 


gccagggttgcgttgcggtgtgccccggttcgaggccggccggattccgcggctaacgagggcgtggctgccccgtcgttt


 


ccaagaccccatagccagccgacttctccagttacggagcgagcccctcttttgttttgtttgtttttgccagatgcatcccgtac


 


tgcggcagatgcgcccccaccaccctccaccgcaacaacagccccctccacagccggcgcttctgcccccgccccagc


 


agcaacttccagccacgaccgccgcggccgccgtgagcggggctggacagagttatgatcaccagctggccttggaag


 


agggcgaggggctggcgcgcctgggggcgtcgtcgccggagcggcacccgcgcgtgcagatgaaaagggacgctcg


 


cgaggcctacgtgcccaagcagaacctgttcagagacaggagcggcgaggagcccgaggagatgcgcgcggcccg


 


gttccacgcggggcgggagctgcggcgcggcctggaccgaaagagggtgctgagggacgaggatttcgaggcggac


 


gagctgacggggatcagccccgcgcgcgcgcacgtggccgcggccaacctggtcacggcgtacgagcagaccgtga


 


aggaggagagcaacttccaaaaatccttcaacaaccacgtgcgcaccctgatcgcgcgcgaggaggtgaccctgggc


 


ctgatgcacctgtgggacctgctggaggccatcgtgcagaaccccaccagcaagccgctgacggcgcagctgttcctggt


 


ggtgcagcatagtcgggacaacgaagcgttcagggaggcgctgctgaatatcaccgagcccgagggccgctggctcct


 


ggacctggtgaacattctgcagagcatcgtggtgcaggagcgcgggctgccgctgtccgagaagctggcggccatcaac


 


ttctcggtgctgagtttgggcaagtactacgctaggaagatctacaagaccccgtacgtgcccatagacaaggaggtgaa


 


gatcgacgggttttacatgcgcatgaccctgaaagtgctgaccctgagcgacgatctgggggtgtaccgcaacgacagg


 


atgcaccgtgcggtgagcgccagcaggcggcgcgagctgagcgaccaggagctgatgcatagtctgcagcgggccct


 


gaccggggccgggaccgagggggagagctactttgacatgggcgcggacctgcactggcagcccagccgccgggcc


 


ttggaggcggcggcaggaccctacgtagaagaggtggacgatgaggtggacgaggagggcgagtacctggaagact


 


gatggcgcgaccgtatttttgctagatgcaacaacaacagccacctcctgatcccgcgatgcgggcggcgctgcagagc


 


cagccgtccggcattaactcctcggacgattggacccaggccatgcaacgcatcatggcgctgacgacccgcaacccc


 


gaagcctttagacagcagccccaggccaaccggctctcggccatcctggaggccgtggtgccctcgcgctccaacccca


 


cgcacgagaaggtcctggccatcgtgaacgcgctggtggagaacaaggccatccgcggcgacgaggccggcctggtg


 


tacaacgcgctgctggagcgcgtggcccgctacaacagcaccaacgtgcagaccaacctggaccgcatggtgaccga


 


cgtgcgcgaggccgtggcccagcgcgagcggttccaccgcgagtccaacctgggatccatggtggcgctgaacgccttc


 


ctcagcacccagcccgccaacgtgccccggggccaggaggactacaccaacttcatcagcgccctgcgcctgatggtg


 


accgaggtgccccagagcgaggtgtaccagtccgggccggactacttcttccagaccagtcgccagggcttgcagaccg


 


tgaacctgagccaggctttcaagaacttgcagggcctgtggggcgtgcaggccccggtcggggaccgcgcgacggtgtc


 


gagcctgctgacgccgaactcgcgcctgctgctgctgctggtggcccccttcacggacagcggcagcatcaaccgcaac


 


tcgtacctgggctacctgattaacctgtaccgcgaggccatcggccaggcgcacgtggacgagcagacctaccaggag


 


atcacccacgtgagccgcgccctgggccaggacgacccgggcaacctggaagccaccctgaactttttgctgaccaac


 


cggtcgcagaagatcccgccccagtacgcgctcagcaccgaggaggagcgcatcctgcgttacgtgcagcagagcgt


 


gggcctgttcctgatgcaggagggggccacccccagcgccgcgctcgacatgaccgcgcgcaacatggagcccagca


 


tgtacgccagcaaccgcccgttcatcaataaactgatggactacttgcatcgggcggccgccatgaactctgactatttcac


 


caacgccatcctgaatccccactggctcccgccgccggggttctacacgggcgagtacgacatgcccgaccccaatgac


 


gggttcctgtgggacgatgtggacagcagcgtgttctccccccgaccgggtgctaacgagcgccccttgtggaagaagga


 


aggcagcgaccgacgcccgtcctcggcgctgtccggccgcgagggtgctgccgcggcggtgcccgaggccgccagtc


 


ctttcccgagcttgcccttctcgctgaacagtatccgcagcagcgagctgggcaggatcacgcgcccgcgcttgctgggcg


 


aagaggagtacttgaatgactcgctgttgagacccgagcgggagaagaacttccccaataacgggatagaaagcctgg


 


tggacaagatgagccgctggaagacgtatgcgcaggagcacagggacgatccccgggcgtcgcagggggccacga


 


gccggggcagcgccgcccgtaaacgccggtggcacgacaggcagcggggacagatgtgggacgatgaggactccg


 


ccgacgacagcagcgtgttggacttgggtgggagtggtaacccgttcgctcacctgcgcccccgtatcgggcgcatgatgt


 


aagagaaaccgaaaataaatgatactcaccaaggccatggcgaccagcgtgcgttcgtttcttctctgttgttgttgtatctag


 


tatgatgaggcgtgcgtacccggagggtcctcctccctcgtacgagagcgtgatgcagcaggcgatggcggcggcggcg


 


atgcagcccccgctggaggctccttacgtgcccccgcggtacctggcgcctacggaggggcggaacagcattcgttactc


 


ggagctggcacccttgtacgataccacccggttgtacctggtggacaacaagtcggcggacatcgcctcgctgaactacc


 


agaacgaccacagcaacttcctgaccaccgtggtgcagaacaatgacttcacccccacggaggccagcacccagacc


 


atcaactttgacgagcgctcgcggtggggcggccagctgaaaaccatcatgcacaccaacatgcccaacgtgaacgag


 


ttcatgtacagcaacaagttcaaggcgcgggtgatggtctcccgcaagacccccaatggggtgacagtgacagaggatt


 


atgatggtagtcaggatgagctgaagtatgaatgggtggaatttgagctgcccgaaggcaacttctcggtgaccatgacca


 


tcgacctgatgaacaacgccatcatcgacaattacttggcggtggggcggcagaacggggtgctggagagcgacatcg


 


gcgtgaagttcgacactaggaacttcaggctgggctgggaccccgtgaccgagctggtcatgcccggggtgtacaccaa


 


cgaggctttccatcccgatattgtcttgctgcccggctgcggggtggacttcaccgagagccgcctcagcaacctgctgggc


 


attcgcaagaggcagcccttccaggaaggcttccagatcatgtacgaggatctggaggggggcaacatccccgcgctcc


 


tggatgtcgacgcctatgagaaaagcaaggaggatgcagcagctgaagcaactgcagccgtagctaccgcctctaccg


 


aggtcaggggcgataattttgcaagcgccgcagcagtggcagcggccgaggcggctgaaaccgaaagtaagatagtc


 


attcagccggtggagaaggatagcaagaacaggagctacaacgtactaccggacaagataaacaccgcctaccgca


 


gctggtacctagcctacaactatggcgaccccgagaagggcgtgcgctcctggacgctgctcaccacctcggacgtcac


 


ctgcggcgtggagcaagtctactggtcgctgcccgacatgatgcaagacccggtcaccttccgctccacgcgtcaagttag


 


caactacccggtggtgggcgccgagctcctgcccgtctactccaagagcttcttcaacgagcaggccgtctactcgcagc


 


agctgcgcgccttcacctcgcttacgcacgtcttcaaccgcttccccgagaaccagatcctcgtccgcccgcccgcgccca


 


ccattaccaccgtcagtgaaaacgttcctgctctcacagatcacgggaccctgccgctgcgcagcagtatccggggagtc


 


cagcgcgtgaccgttactgacgccagacgccgcacctgcccctacgtctacaaggccctgggcatagtcgcgccgcgcg


 


tcctctcgagccgcaccttctaaatgtccattctcatctcgcccagtaataacaccggttggggcctgcgcgcgcccagcaa


 


gatgtacggaggcgctcgccaacgctccacgcaacaccccgtgcgcgtgcgcgggcacttccgcgctccctggggcgc


 


cctcaagggccgcgtgcggtcgcgcaccaccgtcgacgacgtgatcgaccaggtggtggccgacgcgcgcaactaca


 


cccccgccgccgcgcccgtctccaccgtggacgccgtcatcgacagcgtggtggcCgacgcgcgccggtacgcccgc


 


gccaagagccggcggcggcgcatcgcccggcggcaccggagcacccccgccatgcgcgcggcgcgagccttgctgc


 


gcagggccaggcgcacgggacgcagggccatgctcagggcggccagacgcgcggcttcaggcgccagcgccggca


 


ggacccggagacgcgcggccacggcggcggcagcggccatcgccagcatgtcccgcccgcggcgagggaacgtgt


 


actgggtgcgcgacgccgccaccggtgtgcgcgtgcccgtgcgcacccgcccccctcgcacttgaagatgttcacttcgc


 


gatgttgatgtgtcccagcggcgaggaggatgtccaagcgcaaattcaaggaagagatgctccaggtcatcgcgcctga


 


gatctacggccctgcggtggtgaaggaggaaagaaagccccgcaaaatcaagcgggtcaaaaaggacaaaaagga


 


agaagaaagtgatgtggacggattggtggagtttgtgcgcgagttcgccccccggcggcgcgtgcagtggcgcgggcgg


 


aaggtgcaaccggtgctgagacccggcaccaccgtggtcttcacgcccggcgagcgctccggcaccgcttccaagcgc


 


tcctacgacgaggtgtacggggatgatgatattctggagcaggcggccgagcgcctgggcgagtttgcttacggcaagcg


 


cagccgttccgcaccgaaggaagaggcggtgtccatcccgctggaccacggcaaccccacgccgagcctcaagcccg


 


tgaccttgcagcaggtgctgccgaccgcggcgccgcgccgggggttcaagcgcgagggcgaggatctgtaccccacca


 


tgcagctgatggtgcccaagcgccagaagctggaagacgtgctggagaccatgaaggtggacccggacgtgcagccc


 


gaggtcaaggtgcggcccatcaagcaggtggccccgggcctgggcgtgcagaccgtggacatcaagattcccacgga


 


gcccatggaaacgcagaccgagcccatgatcaagcccagcaccagcaccatggaggtgcagacggatccctggatg


 


ccatcggctcctagtcgaagaccccggcgcaagtacggcgcggccagcctgctgatgcccaactacgcgctgcatccttc


 


catcatccccacgccgggctaccgcggcacgcgcttctaccgcggtcataccagcagccgccgccgcaagaccaccac


 


tcgccgccgccgtcgccgcaccgccgctgcaaccacccctgccgccctggtgcggagagtgtaccgccgcggccgcgc


 


acctctgaccctgccgcgcgcgcgctaccacccgagcatcgccatttaaactttcgccTgctttgcagatcaatggccctca


 


catgccgccttcgcgttcccattacgggctaccgaggaagaaaaccgcgccgtagaaggctggcggggaacgggatgc


 


gtcgccaccaccaccggcggcggcgcgccatcagcaagcggttggggggaggcttcctgcccgcgctgatccccatca


 


tcgccgcggcgatcggggcgatccccggcattgcttccgtggcggtgcaggcctctcagcgccactgagacacacttgga


 


aacatcttgtaataaaccAatggactctgacgctcctggtcctgtgatgtgttttcgtagacagatggaagacatcaatttttcg


 


tccctggctccgcgacacggcacgcggccgttcatgggcacctggagcgacatcggcaccagccaactgaacggggg


 


cgccttcaattggagcagtctctggagcgggcttaagaatttcgggtccacgcttaaaacctatggcagcaaggcgtggaa


 


cagcaccacagggcaggcgctgagggataagctgaaagagcagaacttccagcagaaggtggtcgatgggctcgcct


 


cgggcatcaacggggtggtggacctggccaaccaggccgtgcagcggcagatcaacagccgcctggacccggtgcc


 


gcccgccggctccgtggagatgccgcaggtggaggaggagctgcctcccctggacaagcggggcgagaagcgaccc


 


cgccccgatgcggaggagacgctgctgacgcacacggacgagccgcccccgtacgaggaggcggtgaaactgggtc


 


tgcccaccacgcggcccatcgcgcccctggccaccggggtgctgaaacccgaaaagcccgcgaccctggacttgcctc


 


ctccccagccttcccgcccctctacagtggctaagcccctgccgccggtggccgtggcccgcgcgcgacccgggggcac


 


cgcccgccctcatgcgaactggcagagcactctgaacagcatcgtgggtctgggagtgcagagtgtgaagcgccgccg


 


ctgctattaaacctaccgtagcgcttaacttgcttgtctgtgtgtgtatgtattatgtcgccgccgccgctgtccaccagaagga


 


ggagtgaagaggcgcgtcgccgagttgcaagatggccaccccatcgatgctgccccagtgggcgtacatgcacatcgc


 


cggacaggacgcttcggagtacctgagtccgggtctggtgcagtttgcccgcgccacagacacctacttcagtctgggga


 


acaagtttaggaaccccacggtggcgcccacgcacgatgtgaccaccgaccgcagccagcggctgacgctgcgcttcg


 


tgcccgtggaccgcgaggacaacacctactcgtacaaagtgcgctacacgctggccgtgggcgacaaccgcgtgctgg


 


acatggccagcacctactttgacatccgcggcgtgctggatcggggccctagcttcaaaccctactccggcaccgcctac


 


aacagtctggcccccaagggagcacccaacacttgtcagtggacatataaagccgatggtgaaactgccacagaaaa


 


aacctatacatatggaaatgcacccgtgcagggcattaacatcacaaaagatggtattcaacttggaactgacaccgatg


 


atcagccaatctacgcagataaaacctatcagcctgaacctcaagtgggtgatgctgaatggcatgacatcactggtactg


 


atgaaaagtatggaggcagagctcttaagcctgataccaaaatgaagccttgttatggttcttttgccaagcctactaataaa


 


gaaggaggtcaggcaaatgtgaaaacaggaacaggcactactaaagaatatgacatagacatggctttctttgacaaca


 


gaagtgcggctgctgctggcctagctccagaaattgttttgtatactgaaaatgtggatttggaaactccagatacccatattg


 


tatacaaagcaggcacagatgacagcagctcttctattaatttgggtcagcaagccatgcccaacagacctaactacattg


 


gtttcagagacaactttatcgggctcatgtactacaacagcactggcaatatgggggtgctggccggtcaggcttctcagct


 


gaatgctgtggttgacttgcaagacagaaacaccgagctgtcctaccagctcttgcttgactctctgggtgacagaacccgg


 


tatttcagtatgtggaatcaggcggtggacagctatgatcctgatgtgcgcattattgaaaatcatggtgtggaggatgaactt


 


cccaactattgtttccctctggatgctgttggcagaacagatacttatcagggaattaaggctaatggaactgatcaaaccac


 


atggaccaaagatgacagtgtcaatgatgctaatgagataggcaagggtaatccattcgccatggaaatcaacatccaa


 


gccaacctgtggaggaacttcctctacgccaacgtggccctgtacctgcccgactcttacaagtacacgccggccaatgtt


 


accctgcccaccaacaccaacacctacgattacatgaacggccgggtggtggcgccctcgctggtggactcctacatca


 


acatcggggcgcgctggtcgctggatcccatggacaacgtgaaccccttcaaccaccaccgcaatgcggggctgcgcta


 


ccgctccatgctcctgggcaacgggcgctacgtgcccttccacatccaggtgccccagaaatttttcgccatcaagagcctc


 


ctgctcctgcccgggtcctacacctacgagtggaacttccgcaaggacgtcaacatgatcctgcagagctccctcggcaa


 


cgacctgcgcacggacggggcctccatctccttcaccagcatcaacctctacgccaccttcttccccatggcgcacaacac


 


ggcctccacgctcgaggccatgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggcggccaacatg


 


ctctaccccatcccggccaacgccaccaacgtgcccatctccatcccctcgcgcaactgggccgccttccgcggctggtc


 


cttcacgcgtctcaagaccaaggagacgccctcgctgggctccgggttcgacccctacttcgtctactcgggctccatcccc


 


tacctcgacggcaccttctacctcaaccacaccttcaagaaggtctccatcaccttcgactcctccgtcagctggcccggca


 


acgaccggctcctgacgcccaacgagttcgaaatcaagcgcaccgtcgacggcgagggctacaacgtggcccagtgc


 


aacatgaccaaggactggttcctggtccagatgctggcccactacaacatcggctaccagggcttctacgtgcccgaggg


 


ctacaaggaccgcatgtactccttcttccgcaacttccagcccatgagccgccaggtggtggacgaggtcaactacaagg


 


actaccaggccgtcaccctggcctaccagcacaacaactcgggcttcgtcggctacctcgcgcccaccatgcgccaggg


 


ccagccctaccccgccaactacccctacccgctcatcggcaagagcgccgtcaccagcgtcacccagaaaaagttcctc


 


tgcgacagggtcatgtggcgcatccccttctccagcaacttcatgtccatgggcgcgctcaccgacctcggccagaacatg


 


ctctatgccaactccgcccacgcgctagacatgaatttcgaagtcgaccccatggatgagtccacccttctctatgttgtcttc


 


gaagtcttcgacgtcgtccgagtgcaccagccccaccgcggcgtcatcgaggccgtctacctgcgcacccccttctcggc


 


cggtaacgccaccacctaagctcttgcttcttgcaagccatggccgcgggctccggcgagcaggagctcagggccatcat


 


ccgcgacctgggctgcgggccctacttcctgggcaccttcgataagcgcttcccgggattcatggccccgcacaagctgg


 


cctgcgccatcgtcaacacggccggccgcgagaccgggggcgagcactggctggccttcgcctggaacccgcgctcg


 


aacacctgctacctcttcgaccccttcgggttctcggacgagcgcctcaagcagatctaccagttcgagtacgagggcctg


 


ctgcgccgcagcgccctggccaccgaggaccgctgcgtcaccctggaaaagtccacccagaccgtgcagggtccgcg


 


ctcggccgcctgcgggctcttctgctgcatgttcctgcacgccttcgtgcactggcccgaccgccccatggacaagaaccc


 


caccatgaacttgctgacgggggtgcccaacggcatgctccagtcgccccaggtggaacccaccctgcgccgcaacca


 


ggaggcgctctaccgcttcctcaactcccactccgcctactttcgctcccaccgcgcgcgcatcgagaaggccaccgcctt


 


cgaccgcatgaatcaagacatgtaaaccgtgtgtgtatgttaaatgtctttaataaacagcactttcatgttacacatgcatctg


 


agatgatttatttagaaatcgaaagggttctgccgggtctcggcatggcccgcgggcagggacacgttgcggaactggtac


 


ttggccagccacttgaactcggggatcagcagtttgggcagcggggtgtcggggaaggagtcggtccacagcttccgcgt


 


cagttgcagggcgcccagcaggtcgggcgcggagatcttgaaatcgcagttgggacccgcgttctgcgcgcgggagttg


 


cggtacacggggttgcagcactggaacaccatcagggccgggtgcttcacgctcgccagcaccgtcgcgtcggtgatgc


 


tctccacgtcgaggtcctcggcgttggccatcccgaagggggtcatcttgcaggtctgccttcccatggtgggcacgcaccc


 


gggcttgtggttgcaatcgcagtgcagggggatcagcatcatctgggcctggtcggcgttcatccccgggtacatggccttc


 


atgaaagcctccaattgcctgaacgcctgctgggccttggctccctcggtgaagaagaccccgcaggacttgctagagaa


 


ctggttggtggcgcacccggcgtcgtgcacgcagcagcgcgcgtcgttgttggccagctgcaccacgctgcgcccccag


 


cggttctgggtgatcttggcccggtcggggttctccttcagcgcgcgctgcccgttctcgctcgccacatccatctcgatcatgt


 


gctccttctggatcatggtggtcccgtgcaggcaccgcagcttgccctcggcctcggtgcacccgtgcagccacagcgcgc


 


acccggtgcactcccagttcttgtgggcgatctgggaatgcgcgtgcacgaagccctgcaggaagcggcccatcatggtg


 


gtcagggtcttgttgctagtgaaggtcagcggaatgccgcggtgctcctcgttgatgtacaggtggcagatgcggcggtaca


 


cctcgccctgctcgggcatcagctggaagttggctttcaggtcggtctccacgcggtagcggtccatcagcatagtcatgatt


 


tccatacccttctcccaggccgagacgatgggcaggctcatagggttcttcaccatcatcttagcgctagcagccgcggcc


 


agggggtcgctctcgtccagggtctcaaagctccgcttgccgtccttctcggtgatccgcaccggggggtagctgaagccc


 


acggccgccagctcctcctcggcctgtctttcgtcctcgctgtcctggctgacgtcctgcaggaccacatgcttggtcttgcgg


 


ggtttcttcttgggcggcagcggcggcggagatgttggagatggcgagggggagcgcgagttctcgctcaccactactatc


 


tcttcctcttcttggtccgaggccacgcggcggtaggtatgtctcttcgggggcagaggcggaggcgacgggctctcgccg


 


ccgcgacttggcggatggctggcagagccccttccgcgttcgggggtgcgctcccggcggcgctctgactgacttcctccg


 


cggccggccattgtgttctcctagggaggaacaacaagcatggagactcagccatcgccaacctcgccatctgccccca


 


ccgccgacgagaagcagcagcagcagaatgaaagcttaaccgccccgccgcccagccccgccacctccgacgcgg


 


ccgtcccagacatgcaagagatggaggaatccatcgagattgacctgggctatgtgacgcccgcggagcacgaggag


 


gagctggcagtgcgcttttcacaagaagagatacaccaagaacagccagagcaggaagcagagaatgagcagagtc


 


aggctgggctcgagcatgacggcgactacctccacctgagcgggggggaggacgcgctcatcaagcatctggcccgg


 


caggccaccatcgtcaaggatgcgctgctcgaccgcaccgaggtgcccctcagcgtggaggagctcagccgcgcctac


 


gagttgaacctcttctcgccgcgcgtgccccccaagcgccagcccaatggcacctgcgagcccaacccgcgcctcaact


 


tctacccggtcttcgcggtgcccgaggccctggccacctaccacatctttttcaagaaccaaaagatccccgtctcctgccg


 


cgccaaccgcacccgcgccgacgcccttttcaacctgggtcccggcgcccgcctacctgatatcgcctccttggaagagg


 


ttcccaagatcttcgagggtctgggcagcgacgagactcgggccgcgaacgctctgcaaggagaaggaggagagcat


 


gagcaccacagcgccctggtcgagttggaaggcgacaacgcgcggctggcggtgctcaaacgcacggtcgagctgac


 


ccatttcgcctacccggctctgaacctgccccccaaagtcatgagcgcggtcatggaccaggtgctcatcaagcgcgcgt


 


cgcccatctccgaggacgagggcatgcaagactccgaggagggcaagcccgtggtcagcgacgagcagctggcccg


 


gtggctgggtcctaatgctagtccccagagtttggaagagcggcgcaaactcatgatggccgtggtcctggtgaccgtgga


 


gctggagtgcctgcgccgcttcttcgccgacgcggagaccctgcgcaaggtcgaggagaacctgcactacctcttcaggc


 


acgggttcgtgcgccaggcctgcaagatctccaacgtggagctgaccaacctggtctcctacatgggcatcttgcacgag


 


aaccgcctggggcagaacgtgctgcacaccaccctgcgcggggaggcccggcgcgactacatccgcgactgcgtcta


 


cctctacctctgccacacctggcagacgggcatgggcgtgtggcagcagtgtctggaggagcagaacctgaaagagctc


 


tgcaagctcctgcagaagaacctcaagggtctgtggaccgggttcgacgagcgcaccaccgcctcggacctggccgac


 


ctcattttccccgagcgcctcaggctgacgctgcgcaacggcctgcccgactttatgagccaaagcatgttgcaaaactttc


 


gctctttcatcctcgaacgctccggaatcctgcccgccacctgctccgcgctgccctcggacttcgtgccgctgaccttccgc


 


gagtgccccccgccgctgtggagccactgctacctgctgcgcctggccaactacctggcctaccactcggacgtgatcga


 


ggacgtcagcggcgagggcctgctcgagtgccactgccgctgcaacctctgcacgccgcaccgctccctggcctgcaac


 


ccccagctgctgagcgagacccagatcatcggcaccttcgagttgcaagggcccagcgaaggcgagggttcagccgcc


 


aaggggggtctgaaactcaccccggggctgtggacctcggcctacttgcgcaagttcgtgcccgaggactaccatcccttc


 


gagatcaggttctacgaggaccaatcccatccgcccaaggccgagctgtcggcctgcgtcatcacccagggggcgatcc


 


tggcccaattgcaagccatccagaaatcccgccaagaattcttgctgaaaaagggccgcggggtctacctcgaccccca


 


gaccggtgaggagctcaaccccggcttcccccaggatgccccgaggaaacaagaagctgaaagtggagctgccgcc


 


cgtggaggatttggaggaagactgggagaacagcagtcaggcagaggaggaggagatggaggaagactgggacag


 


cactcaggcagaggaggacagcctgcaagacagtctggaggaagacgaggaggaggcagaggaggaggtggaag


 


aagcagccgccgccagaccgtcgtcctcggcgggggagaaagcaagcagcacggataccatctccgctccgggtcgg


 


ggtcccgctcgaccacacagtagatgggacgagaccggacgattcccgaaccccaccacccagaccggtaagaagg


 


agcggcagggatacaagtcctggcgggggcacaaaaacgccatcgtctcctgcttgcaggcctgcgggggcaacatct


 


ccttcacccggcgctacctgctcttccaccgcggggtgaactttccccgcaacatcttgcattactaccgtcacctccacagc


 


ccctactacttccaagaagaggcagcagcagcagaaaaagaccagcagaaaaccagcagctagaaaatccacagc


 


ggcggcagcaggtggactgaggatcgcggcgaacgagccggcgcaaacccgggagctgaggaaccggatctttccc


 


accctctatgccatcttccagcagagtcgggggcaggagcaggaactgaaagtcaagaaccgttctctgcgctcgctcac


 


ccgcagttgtctgtatcacaagagcgaagaccaacttcagcgcactctcgaggacgccgaggctctcttcaacaagtact


 


gcgcgctcactcttaaagagtagcccgcgcccgcccagtcgcagaaaaaggcgggaattacgtcacctgtgcccttcgc


 


cctagccgcctccacccatcatcatgagcaaagagattcccacgccttacatgtggagctaccagccccagatgggcctg


 


gccgccggtgccgcccaggactactccacccgcatgaattggctcagcgccgggcccgcgatgatctcacgggtgaatg


 


acatccgcgcccaccgaaaccagatactcctagaacagtcagcgctcaccgccacgccccgcaatcacctcaatccgc


 


gtaattggcccgccgccctggtgtaccaggaaattccccagcccacgaccgtactacttccgcgagacgcccaggccga


 


agtccagctgactaactcaggtgtccagctggcgggcggcgccaccctgtgtcgtcaccgccccgctcagggtataaagc


 


ggctggtgatccggggcagaggcacacagctcaacgacgaggtggtgagctcttcgctgggtctgcgacctgacggagt


 


cttccaactcgccggatcggggagatcttccttcacgcctcgtcaggccgtcctgactttggagagttcgtcctcgcagcccc


 


gctcgggtggcatcggcactctccagttcgtggaggagttcactccctcggtctacttcaaccccttctccggctcccccggc


 


cactacccggacgagttcatcccgaacttcgacgccatcagcgagtcggtggacggctacgattgaatgtcccatggtgg


 


cgcagctgacctagctcggcttcgacacctggaccactgccgccgcttccgctgcttcgctcgggatctcgccgagtttgcct


 


actttgagctgcccgaggagcaccctcagggcccggcccacggagtgcggatcgtcgtcgaagggggcctcgactccc


 


acctgcttcggatcttcagccagcgtccgatcctggtcgagcgcgagcaaggacagacccttctgactctgtactgcatctg


 


caaccaccccggcctgcatgaaagtctttgttgtctgctgtgtactgagtataataaaagctgagatcagcgactactccgg


 


acttccgtgtgttcctgaatccatcaaccagtctttgttcttcaccgggaacgagaccgagctccagctccagtgtaagcccc


 


acaagaagtacctcacctggctgttccagggctccccgatcgccgttgtcaaccactgcgacaacgacggagtcctgctg


 


agcggccctgccaaccttactttttccacccgcagaagcaagctccagctcttccaacccttcctccccgggacctatcagt


 


gcgtctcgggaccctgccatcacaccttccacctgatcccgaataccacagcgtcgctccccgctactaacaaccaaact


 


aacctccaccaacgccaccgtcgctaggccacaatacatgcccatattagactatgaggccgagccacagcgacccat


 


gctccccgctattagttacttcaatctaaccggcggagatgactgacccactggccaacaacaacgtcaacgaccttctcct


 


ggacatggacggccgcgcctcggagcagcgactcgcccaacttcgcattcgccagcagcaggagagagccgtcaag


 


gagctgcaggatgcggtggccatccaccagtgcaagagaggcatcttctgcctggtgaaacaggccaagatctcctacg


 


aggtcactccaaacgaccatcgcctctcctacgagctcctgcagcagcgccagaagttcacctgcctggtcggagtcaac


 


cccatcgtcatcacccagcagtctggcgataccaaggggtgcatccactgctcctgcgactcccccgactgcgtccacact


 


ctgatcaagaccctctgcggcctccgcgacctcctccccatgaactaatcacccccttatccagtgaaataaagatcatatt


 


gatgatgattttacagaaataaaaaataatcatttgatttgaaataaagatacaatcatattgatgatttgagtttaacaaaaa


 


aataaagaatcacttacttgaaatctgataccaggtctctgtccatgttttctgccaacaccacttcactcccctcttcccagctc


 


tggtactgcaggccccggcgggctgcaaacttcctccacacgctgaaggggatgtcaaattcctcctgtccctcaatcttcat


 


tttatcttctatcagatgtccaaaaagcgcgtccgggtggatgatgacttcgaccccgtctacccctacgatgcagacaacg


 


caccgaccgtgcccttcatcaacccccccttcgtctcttcagatggattccaagagaagcccctgggggtgttgtccctgcg


 


actggccgaccccgtcaccaccaagaacggggaaatcaccctcaagctgggagagggggtggacctcgattcctcgg


 


gaaaactcatctccaacacggccaccaaggccgccgcccctctcagtttttccaacaacaccatttcccttaacatggatca


 


ccccttttacactaaagatggaaaattatccttacaagtttctccaccattaaatatactgagaacaagcattctaaacacact


 


agctttaggttttggatcaggtttaggactccgtggctctgccttggcagtacagttagtctctccacttacatttgatactgatgg


 


aaacataaagcttaccttagacagaggtttgcatgttacaacaggagatgcaattgaaagcaacataagctgggctaaag


 


gtttaaaatttgaagatggagccatagcaaccaacattggaaatgggttagagtttggaagcagtagtacagaaacaggt


 


gttgatgatgcttacccaatccaagttaaacttggatctggccttagctttgacagtacaggagccataatggctggtaacaa


 


agaagacgataaactcactttgtggacaacacctgatccatcaccaaactgtcaaatactcgcagaaaatgatgcaaaa


 


ctaacactttgcttgactaaatgtggtagtcaaatactggccactgtgtcagtcttagttgtaggaagtggaaacctaaacccc


 


attactggcaccgtaagcagtgctcaggtgtttctacgttttgatgcaaacggtgttcttttaacagaacattctacactaaaaa


 


aatactgggggtataggcagggagatagcatagatggcactccatataccaatgctgtaggattcatgcccaatttaaaag


 


cttatccaaagtcacaaagttctactactaaaaataatatagtagggcaagtatacatgaatggagatgtttcaaaacctatg


 


cttctcactataaccctcaatggtactgatgacagcaacagtacatattcaatgtcattttcatacacctggactaatggaagc


 


tatgttggagcaacatttggggctaactcttataccttctcatacatcgcccaagaatgaacactgtatcccaccctgcatgcc


 


aacccttcccaccccactctgtggaacaaactctgaaacacaaaataaaataaagttcaagtgttttattgattcaacagtttt


 


acaggattcgagcagttatttttcctccaccctcccaggacatggaatacaccaccctctccccccgcacagccttgaacat


 


ctgaatgccattggtgatggacatgcttttggtctccacgttccacacagtttcagagcgagccagtctcgggtcggtcaggg


 


agatgaaaccctccgggcactcccgcatctgcacctcacagctcaacagctgaggattgtcctcggtggtcgggatcacg


 


gttatctggaagaagcagaagagcggcggtgggaatcatagtccgcgaacgggatcggccggtggtgtcgcatcaggc


 


cccgcagcagtcgctgccgccgccgctccgtcaagctgctgctcagggggtccgggtccagggactccctcagcatgat


 


gcccacggccctcagcatcagtcgtctggtgcggcgggcgcagcagcgcatgcggatctcgctcaggtcgctgcagtac


 


gtgcaacacagaaccaccaggttgttcaacagtccatagttcaacacgctccagccgaaactcatcgcgggaaggatgc


 


tacccacgtggccgtcgtaccagatcctcaggtaaatcaagtggtgccccctccagaacacgctgcccacgtacatgatct


 


ccttgggcatgtggcggttcaccacctcccggtaccacatcaccctctggttgaacatgcagccccggatgatcctgcgga


 


accacagggccagcaccgccccgcccgccatgcagcgaagagaccccgggtcccggcaatggcaatggaggaccc


 


accgctcgtacccgtggatcatctgggagctgaacaagtctatgttggcacagcacaggcatatgctcatgcatctcttcag


 


cactctcaactcctcgggggtcaaaaccatatcccagggcacggggaactcttgcaggacagcgaaccccgcagaaca


 


gggcaatcctcgcacagaacttacattgtgcatggacagggtatcgcaatcaggcagcaccgggtgatcctccaccaga


 


gaagcgcgggtctcggtctcctcacagcgtggtaagggggccggccgatacgggtgatggcgggacgcggctgatcgt


 


gttcgcgaccgtgtcatgatgcagttgctttcggacattttcgtacttgctgtagcagaacctggtccgggcgctgcacaccga


 


tcgccggcggcggtctcggcgcttggaacgctcggtgttgaaattgtaaaacagccactctctcagaccgtgcagcagatc


 


tagggcctcaggagtgatgaagatcccatcatgcctgatggctctgatcacatcgaccaccgtggaatgggccagaccca


 


gccagatgatgcaattttgttgggtttcggtgacggcgggggagggaagaacaggaagaaccatgattaacttttaatcca


 


aacggtctcggagtacttcaaaatgaagatcgcggagatggcacctctcgcccccgctgtgttggtggaaaataacagcc


 


aggtcaaaggtgatacggttctcgagatgttccacggtggcttccagcaaagcctccacgcgcacatccagaaacaaga


 


caatagcgaaagcgggagggttctctaattcctcaatcatcatgttacactcctgcaccatccccagataattttcatttttcca


 


gccttgaatgattcgaactagttcCtgaggtaaatccaagccagccatgataaagagctcgcgcagagcgccctccacc


 


ggcattcttaagcacaccctcataattccaagatattctgctcctggttcacctgcagcagattgacaagcggaatatcaaaa


 


tctctgccgcgatccctgagctcctccctcagcaataactgtaagtactctttcatatcctctccgaaatttttagccataggacc


 


accaggaataagattagggcaagccacagtacagataaaccgaagtcctccccagtgagcattgccaaatgcaagact


 


gctataagcatgctggctagacccggtgatatcttccagataactggacagaaaatcgcccaggcaatttttaagaaaatc


 


aacaaaagaaaaatcctccaggtggacgtttagagcctcgggaacaacgatgaagtaaatgcaagcggtgcgttccag


 


catggttagttagctgatctgtagaaaaaacaaaaatgaacattaaaccatgctagcctggcgaacaggtgggtaaatcgt


 


tctctccagcaccaggcaggccacggggtctccggcgcgaccctcgtaaaaattgtcgctatgattgaaaaccatcacag


 


agagacgttcccggtggccggcgtgaatgattcgacaagatgaatacacccccggaacattggcgtccgcgagtgaaa


 


aaaagcgcccgaggaagcaataaggcactacaatgctcagtctcaagtccagcaaagcgatgccatgcggatgaagc


 


acaaaattctcaggtgcgtacaaaatgtaattactcccctcctgcacaggcagcaaagcccccgatccctccaggtacac


 


atacaaagcctcagcgtccatagcttaccgagcagcagcacacaacaggcgcaagagtcagagaaaggctgagctct


 


aacctgtccacccgctctctgctcaatatatagcccagatctacactgacgtaaaggccaaagtctaaaaatacccgccaa


 


ataatcacacacgcccagcacacgcccagaaaccggtgacacactcaaaaaaatacgcgcacttcctcaaacgccca


 


aaactgccgtcatttccgggttcccacgctacgtcatcaaaacacgactttcaaattccgtcgaccgttaaaaacgtcaccc


 


gccccgcccctaacggtcgcccgtctctcagccaatcagcgccccgcatccccaaattcaaacGcctcatttgcatattaa


 


cgcgcacaaaaagtttgaggtatattattgatgatgg


 


SEQ ID NO: 64. Amino Acid Sequence Comprising an Immunogenic PSA,


PSMA, and PSCA Polypeptide (Encoded by Plasmid 457 and Vector AdC68X-733)


MASIVGGWECEKHSQPWQVLVASRGRAVCGGVLVHPQWVLTAAHCIRNKSVILLGRH


 


SLFHPEDTGQVFQVSHSFPHPLYDMSLLKNRFLRPGDDSSHDLMLLRLSEPAELTDAV


 


KVMDLPTQEPALGTTCYASGWGSIEPEEFLTPKKLQCVDLHVISNDVCAQVHPQKVTK


 


FMLCAGRWTGGKSTCSGDSGGPLVCNGVLQGITSWGSEPCALPERPSLYTKVVHYR


 


KWIKDTIVANPGSQTLNFDLLKLAGDVESNPGPMASARRPRWLCAGALVLAGGFFLLG


 


FLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQI


 


QSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVS


 


DIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKV


 


KNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPG


 


YPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNV


 


GPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDP


 


QSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERG


 


VAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPE


 


FSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVE


 


KFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQE


 


MKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLG


 


LPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTV


 


QAAAETLSEVAGSEGRGSLLTCGDVEENPGPASKAVLLALLMAGLALQPGTALLCYSC


 


KAQVSNEDCLQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCVDDSQDYYVGKKNI


 


TCCDTDLCNASGAHALQPAAAILALLPALGLLLWGPGQL


 


SEQ ID NO: 65. Nucleotide Sequence Encoding the Amino Acid Sequence


of SEQ ID NO: 64


ATGGCTAGCATCGTCGGAGGGTGGGAGTGCGAAAAGCACTCACAGCCATGGCAG


 


GTCCTGGTCGCCTCGCGCGGACGCGCCGTGTGTGGAGGTGTGCTGGTCCACCCG


 


CAGTGGGTGTTGACTGCGGCCCATTGCATCAGAAATAAGTCCGTGATCCTCTTGGG


 


GAGACATTCCCTGTTTCACCCCGAAGATACTGGACAGGTGTTCCAAGTGAGCCACT


 


CCTTCCCGCATCCACTGTACGACATGAGCCTGCTGAAGAACCGCTTTCTGCGGCC


 


AGGGGACGACTCATCACACGATTTGATGCTGCTTCGGCTCTCGGAACCGGCCGAG


 


CTCACCGACGCAGTGAAGGTCATGGACCTCCCTACGCAAGAGCCTGCTCTCGGTA


 


CCACTTGTTACGCATCGGGATGGGGCTCCATCGAGCCGGAAGAATTCCTGACCCC


 


GAAAAAGCTGCAGTGCGTGGATCTGCACGTGATTTCGAATGACGTGTGCGCGCAA


 


GTGCATCCACAAAAGGTCACTAAGTTCATGCTGTGCGCCGGAAGGTGGACCGGCG


 


GAAAATCGACCTGTTCCGGCGACAGCGGAGGCCCACTCGTGTGCAACGGTGTGCT


 


GCAGGGCATCACTAGCTGGGGATCAGAACCGTGCGCGCTTCCGGAGCGGCCCTC


 


GCTCTACACGAAGGTGGTGCACTACCGCAAATGGATTAAAGATACCATCGTCGCAA


 


ACCCTggatcccagaccctgaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGG


 


CTAGCGCTCGCAGACCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTG


 


GCTTCTTTTTGCTCGGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTA


 


CCAATATCACCCCGAAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGA


 


GAACATTAAGAAGTTCCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTG


 


AGCAGAACTTTCAGTTGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCT


 


GGACTCCGTCGAGCTGGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACT


 


CATCCGAACTATATCTCGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCC


 


CTCTTCGAGCCGCCACCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGT


 


TCTCGGCCTTCTCGCCCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTA


 


CGCAAGGACCGAGGACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGC


 


GGAAAGATCGTCATCGCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGA


 


ATGCACAGTTGGCAGGCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTA


 


CTTCGCTCCTGGCGTGAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGG


 


GTGCAGAGGGGAAATATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGG


 


GTTACCCGGCCAACGAATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGAC


 


TGCCGTCCATCCCGGTCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGA


 


AAAGATGGGAGGCAGCGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGT


 


GCCATACAACGTGGGACCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGA


 


TGCACATTCACTCCACTAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTC


 


CGGGGAGCGGTGGAACCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGC


 


TGGGTGTTCGGAGGAATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCG


 


TCAGGTCCTTTGGTACTCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTG


 


TTCGCCTCGTGGGATGCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTG


 


AGGAAAACTCCCGCCTGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTC


 


ATCCATCGAAGGAAACTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCG


 


CTCGTGCACAACCTGACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAA


 


AATCGCTGTACGAGTCGTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGAT


 


GCCGCGGATCTCAAAGCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGC


 


TGGGAATTGCGTCGGGAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTT


 


CTCGGGATACCCGCTGTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAAT


 


TCTACGATCCTATGTTTAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATG


 


GTGTTCGAGTTGGCCAATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGT


 


GGTGCTGAGAAAGTACGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAG


 


AGATGAAAACCTACTCAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTC


 


ACCGAGATCGCGAGCAAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCC


 


GATCGTCCTCCGCATGATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCG


 


ATCCACTGGGACTTCCGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTC


 


GTCGCATAACAAGTATGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCG


 


ACATTGAGTCCAAGGTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAAT


 


CTACGTGGCGGCCTTTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCT


 


ggatccgaaggtaggggttcattattgacctgtggagatgtcgaagaaaacccaggacccGCTAGCAAAGCAG


 


TGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGCAGCCTGGAACCGCCCTGCT


 


CTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGACTGTTTGCAAGTGGAAAACT


 


GCACCCAGCTCGGAGAACAATGCTGGACTGCACGGATCCGCGCTGTCGGCCTGCT


 


GACCGTGATCTCCAAAGGGTGCTCATTGAACTGCGTGGACGATAGCCAGGACTAC


 


TACGTGGGAAAGAAGAATATCACTTGTTGCGACACGGATCTTTGCAACGCGTCCGG


 


AGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGGCCCTGCTTCCGGCCCTGGG


 


GTTGCTGCTCTGGGGTCCGGGCCAGCTC


 


SEQ ID NO: 66. Nucleotide Sequence of the Multi-antigen Construct (PSCA-


F2A-PSMA-mIRES-PSA) Incorporated in Plasmid 459 and Vector AdC68X-735


ATGGCTAGCAAAGCAGTGCTGCTGGCGCTCCTGATGGCTGGACTCGCGCTGCAGC


 


CTGGAACCGCCCTGCTCTGTTACTCGTGCAAGGCCCAAGTCTCGAATGAGGACTG


 


TTTGCAAGTGGAAAACTGCACCCAGCTCGGAGAACAATGCTGGACTGCACGGATC


 


CGCGCTGTCGGCCTGCTGACCGTGATCTCCAAAGGGTGCTCATTGAACTGCGTGG


 


ACGATAGCCAGGACTACTACGTGGGAAAGAAGAATATCACTTGTTGCGACACGGAT


 


CTTTGCAACGCGTCCGGAGCGCACGCCCTGCAGCCAGCAGCCGCCATTCTGGCC


 


CTGCTTCCGGCCCTGGGGTTGCTGCTCTGGGGTCCGGGCCAGCTCggatcccagaccct


 


gaactttgatctgctgaaactggcaggcgatgtggaaagcaacccaggcccaATGGCTAGCGCTCGCAGA


 


CCGCGGTGGCTGTGTGCAGGGGCGCTCGTCCTGGCGGGTGGCTTCTTTTTGCTC


 


GGCTTTCTTTTCGGATGGTTCATCAAATCGTCAAACGAAGCTACCAATATCACCCCG


 


AAGCACAACATGAAGGCCTTTCTGGATGAGCTGAAGGCTGAGAACATTAAGAAGTT


 


CCTCTACAACTTCACCCAGATCCCACATTTGGCGGGCACTGAGCAGAACTTTCAGT


 


TGGCTAAGCAGATCCAGAGCCAGTGGAAGGAATTCGGCCTGGACTCCGTCGAGCT


 


GGCGCATTACGATGTGCTGCTGAGCTACCCTAATAAGACTCATCCGAACTATATCT


 


CGATTATCAATGAGGACGGAAACGAAATCTTTAACACGTCCCTCTTCGAGCCGCCA


 


CCGCCTGGATACGAGAACGTGTCAGATATCGTGCCTCCGTTCTCGGCCTTCTCGC


 


CCCAGGGAATGCCCGAAGGGGACCTGGTGTACGTGAACTACGCAAGGACCGAGG


 


ACTTCTTCAAGTTGGAGCGGGATATGAAGATCAATTGCAGCGGAAAGATCGTCATC


 


GCCCGCTACGGCAAAGTGTTCCGCGGCAACAAGGTGAAGAATGCACAGTTGGCAG


 


GCGCCAAGGGCGTCATCCTCTACTCGGATCCTGCCGACTACTTCGCTCCTGGCGT


 


GAAATCCTACCCTGATGGTTGGAATCTGCCAGGAGGAGGGGTGCAGAGGGGAAAT


 


ATCCTGAACCTGAACGGTGCCGGTGACCCACTTACTCCGGGTTACCCGGCCAACG


 


AATACGCGTACAGGCGGGGTATCGCGGAAGCCGTCGGACTGCCGTCCATCCCGG


 


TCCATCCGATTGGTTACTACGACGCCCAGAAGCTCCTCGAAAAGATGGGAGGCAG


 


CGCCCCTCCGGACTCGTCATGGAGAGGCTCGCTGAAGGTGCCATACAACGTGGGA


 


CCCGGATTCACTGGAAATTTCAGCACTCAAAAAGTGAAGATGCACATTCACTCCAC


 


TAACGAAGTCACCAGGATCTACAACGTCATCGGAACCCTCCGGGGAGCGGTGGAA


 


CCGGACCGCTACGTGATCCTCGGTGGACACCGGGATAGCTGGGTGTTCGGAGGA


 


ATCGATCCTCAATCGGGCGCAGCCGTCGTCCATGAAATCGTCAGGTCCTTTGGTAC


 


TCTTAAGAAGGAGGGCTGGCGCCCTAGACGCACTATTCTGTTCGCCTCGTGGGAT


 


GCCGAAGAATTTGGTCTGCTCGGCAGCACCGAATGGGCTGAGGAAAACTCCCGCC


 


TGCTCCAAGAACGCGGAGTGGCGTACATCAATGCCGACTCATCCATCGAAGGAAA


 


CTACACGCTGCGGGTGGACTGCACTCCACTGATGTACTCGCTCGTGCACAACCTG


 


ACCAAAGAACTCAAATCCCCAGACGAAGGATTCGAGGGAAAATCGCTGTACGAGTC


 


GTGGACCAAGAAGAGCCCATCCCCGGAGTTCAGCGGGATGCCGCGGATCTCAAA


 


GCTCGGATCAGGAAATGATTTCGAAGTGTTCTTTCAGAGGCTGGGAATTGCGTCGG


 


GAAGGGCTCGGTACACGAAAAACTGGGAAACTAACAAGTTCTCGGGATACCCGCT


 


GTACCACTCGGTGTATGAAACTTACGAACTGGTGGAGAAATTCTACGATCCTATGTT


 


TAAGTACCACCTGACTGTGGCCCAAGTGAGAGGCGGAATGGTGTTCGAGTTGGCC


 


AATTCAATTGTGCTGCCATTCGATTGCCGCGACTACGCCGTGGTGCTGAGAAAGTA


 


CGCAGACAAAATCTACTCAATCAGCATGAAGCACCCACAAGAGATGAAAACCTACT


 


CAGTCTCCTTCGACTCCCTCTTCTCCGCGGTGAAGAACTTCACCGAGATCGCGAGC


 


AAATTCTCGGAGCGCCTTCAAGATTTTGACAAATCCAATCCGATCGTCCTCCGCAT


 


GATGAATGACCAGCTCATGTTTCTCGAACGGGCCTTCATCGATCCACTGGGACTTC


 


CGGACCGGCCGTTTTACCGCCACGTGATCTACGCGCCCTCGTCGCATAACAAGTA


 


TGCTGGAGAGAGCTTCCCGGGTATCTACGACGCATTGTTCGACATTGAGTCCAAG


 


GTGGATCCGTCCAAAGCCTGGGGTGAAGTGAAGCGCCAAATCTACGTGGCGGCCT


 


TTACCGTCCAGGCGGCAGCAGAAACCTTGAGCGAGGTGGCTTGAagatctgaccccctaa


 


cgttactggccgaagccgcttggaataaggccggtgtgcgtttgtctatatgttattttccaccatattgccgtcttttggcaatgt


 


gagggcccggaaacctggccctgtcttcttgacgagcattcctaggggtctttcccctctcgccaaaggaatgcaaggtctg


 


ttgaatgtcgtgaaggaagcagttcctctggaagcttcttgaagacaaacaacgtctgtagcgaccctttgcaggcagcgg


 


aaccccccacctggcgacaggtgcctctgcggccaaaagccacgtgtataagatacacctgcaaaggcggcacaacc


 


ccagtgccacgttgtgagttggatagttgtggaaagagtcaaatggctctcctcaagcgtattcaacaaggggctgaagga


 


tgcccagaaggtaccccattgtatgggatctgatctggggcctcggtgcacatgctttacatgtgtttagtcgaggttaaaaaa


 


cgtctaggccccccgaaccacggggacgtggttttcctttgaaaaacacgatgataatATGGCTAGCATCGTCG


 


GAGGGTGGGAGTGCGAAAAGCACTCACAGCCATGGCAGGTCCTGGTCGCCTCGC


 


GCGGACGCGCCGTGTGTGGAGGTGTGCTGGTCCACCCGCAGTGGGTGTTGACTG


 


CGGCCCATTGCATCAGAAATAAGTCCGTGATCCTCTTGGGGAGACATTCCCTGTTT


 


CACCCCGAAGATACTGGACAGGTGTTCCAAGTGAGCCACTCCTTCCCGCATCCACT


 


GTACGACATGAGCCTGCTGAAGAACCGCTTTCTGCGGCCAGGGGACGACTCATCA


 


CACGATTTGATGCTGCTTCGGCTCTCGGAACCGGCCGAGCTCACCGACGCAGTGA


 


AGGTCATGGACCTCCCTACGCAAGAGCCTGCTCTCGGTACCACTTGTTACGCATCG


 


GGATGGGGCTCCATCGAGCCGGAAGAATTCCTGACCCCGAAAAAGCTGCAGTGCG


 


TGGATCTGCACGTGATTTCGAATGACGTGTGCGCGCAAGTGCATCCACAAAAGGTC


 


ACTAAGTTCATGCTGTGCGCCGGAAGGTGGACCGGCGGAAAATCGACCTGTTCCG


 


GCGACAGCGGAGGCCCACTCGTGTGCAACGGTGTGCTGCAGGGCATCACTAGCT


 


GGGGATCAGAACCGTGCGCGCTTCCGGAGCGGCCCTCGCTCTACACGAAGGTGG


 


TGCACTACCGCAAATGGATTAAAGATACCATCGTCGCAAACCCT

## Claims

1. A C68 vector comprising:
(a) a C68 nucleotide sequence; and
(b) a multi-antigen construct that comprises at least one nucleotide sequence encoding an immunogenic PSA polypeptide, at least one nucleotide sequence encoding an immunogenic PSCA polypeptide, and at least one nucleotide sequence encoding an immunogenic PSMA polypeptide.

2. The C68 vector according to claim 1, wherein the C68 nucleotide sequence is the sequence of SEQ ID NO: 57 lacking at least one gene selected from the group consisting of E1A, E1B, E2A, E2B, E3, E4, L1, L2, L3, L4 and L5 genes, wherein the immunogenic PSA polypeptide comprises amino acids 27-263 of SEQ ID NO:15 or amino acids 4-240 of SEQ ID NO:17, wherein the immunogenic PSCA polypeptide comprises an amino acid sequence selected from the group consisting of:
(1) the amino acid sequence of SEQ ID NO:21,
(2) amino acids 2-125 of SEQ ID NO:21, and
(3) amino acids 4-125 Of SEQ ID NO:21,

and wherein the immunogenic PSMA polypeptide is selected from the group consisting of:
(1) a polypeptide comprising amino acids 15-750 of SEQ ID NO: 1;
(2) a polypeptide comprising the amino acid sequence of SEQ ID NO:3;
(3) a polypeptide comprising the amino acid sequence of SEQ ID NO:5;
(4) a polypeptide comprising the amino acid sequence of SEQ ID NO:7;
(5) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:9;
(6) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:3;
(7) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:5;
(8) a polypeptide comprising the amino acids 4-739 of SEQ ID NO:7; and
(9) a polypeptide comprising the amino acid sequence of SEQ ID NO: 9.

3. The C68 vector according to claim 2, wherein the C68 nucleotide sequence is the sequence of SEQ ID NO: 57 lacking the genes of E1A, E1B, and E3, and wherein the multi-antigen construct further comprises a separator sequence between two nucleotide sequences encoding two different immunogenic polypeptides and has the structure of formula (I):


PAA1-SS1-PAA2-SS2-PAA3  (I)

wherein in formula (I):
(i) PAA1, PAA2, and PAA3 each is a nucleotide sequence encoding an immunogenic PSA polypeptide, a nucleotide sequence encoding immunogenic PSCA polypeptide, or a nucleotide sequence encoding immunogenic PSMA polypeptide, provided the PAA1, PAA2, and PAA3 encode different PAA polypeptides, and
(ii) SS1 and SS2 are separator sequences and can be the same or different.

4. The C68 vector according to claim 3, wherein the separator sequences are selected from 2A peptide sequences and IRESs.

5. The C68 vector according to claim 4, wherein the 2A peptide sequence is selected from the group consisting of the 2A-peptide sequence of FMDV, ERAV, PTV1, EMC-B, EMCV, TME-GD7, ERBV, TaV, DrosC, CrPV, ABPV, IFV, Porcine rotavirus, human rotavirus, T brucei TSR1, and T cruzi AP endonuclease; and wherein the IRES is a EMCV IRES.

6. The vector according to claim 5, wherein PAA1 in formula (I) is a nucleotide sequence encoding the immunogenic PSA polypeptide or a nucleotide sequence encoding the immunogenic PSCA polypeptide.

7. The vector according to claim 6, wherein:
(i) PAA1 is a nucleotide sequence encoding the immunogenic PSA polypeptide;
(ii) PAA2 is a nucleotide sequence encoding an immunogenic PSCA or PSMA polypeptide;
(iii) SS1 is a 2A-peptide sequence; and
(iv) SS2 is a 2A-peptide sequence or an EMCV IRES.

8. The C68 vector according to claim 7, wherein the 2A-peptide sequence is the FMDV 2A-peptide sequence or the TAV 2A-peptide sequence.

9. The C68 vector according to claim 8, wherein:
(1) the nucleotide sequence encoding the immunogenic PSA polypeptide is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO: 18; (ii) the nucleotide sequence of SEQ ID NO: 20; (iii) the nucleotide sequence comprising nucleotides 10-720 of SEQ ID NO:18; (iv) the nucleotide sequence comprising nucleotides 1115-1825 of SEQ ID NO:58; and (v) the nucleotide sequence comprising nucleotides 1106-1825 of SEQ ID NO:58;
(2) the nucleotide sequence encoding the immunogenic PSCA polypeptide is selected from the group consisting of: (i) the nucleotide sequence of SEQ ID NO:22; (ii) a nucleotide sequence comprising nucleotides 10-372 of SEQ ID NO:22; (iii) a nucleotide sequence comprising nucleotides 1892-2257 of SEQ ID NO:58; and (iv) a nucleotide sequence comprising nucleotides 1886-2257 of SEQ ID NO:58; and
(3) the nucleotide sequence encoding the immunogenic PSMA polypeptide is selected from the group consisting of: (i) the nucleotide sequence comprising nucleotides 43-2250 of SEQ ID NO:2; (ii) the nucleotide sequence of SEQ ID NO:4; (iii) the nucleotide sequence of SEQ ID NO:6; (iv) the nucleotide sequence of SEQ ID NO:8; (v) the nucleotide sequence of SEQ ID NO:10; (vi) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:4; (vii) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:6; (viii) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:8; (ix) a nucleotide sequence comprising nucleotides 10-2217 of SEQ ID NO:10; (x) a nucleotide sequence comprising nucleotides 2333-4543 of SEQ ID NO:58; and (xi) a nucleotide sequence comprising nucleotides 2324-4543 of SEQ ID NO:58.

10. The C68 vector according to claim 3, wherein in formula (I):
(1) PAA1 is a nucleotide sequence encoding an immunogenic PSA polypeptide and comprises nucleotides 1115-1825 of SEQ ID NO: 58 or comprises 1106-1114 of SEQ ID NO: 58;
(2) PAA2 is a nucleotide sequence encoding an immunogenic PSCA polypeptide and comprises nucleotides 1892-2257 of SEQ ID NO: 58 or comprises 1886-2257 of SEQ ID NO: 58;
(3) PAA3 is a nucleotide sequence encoding an immunogenic PSMA polypeptide and comprises nucleotides 2333-4543 of SEQ ID NO: 58 or comprises 2324-4543 of SEQ ID NO: 58;
(4) SS1 is a nucleotide sequence encoding T2A; and
(5) SS2 is a nucleotide sequence encoding F2A.

11. The C68 vector according to claim 1, wherein the multi-antigen construct comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO:60 or SEQ ID NO:64.

12. The C68 vector according to claim 1, wherein the multi-antigen construct comprises a nucleotide sequence of SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66, or a degenerate variant of the nucleotide sequence of SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:66.

13. The C68 vector according to claim 11 or claim 12, further comprising a CMV promoter.

14. The C68 vector according to claim 1, which comprises the nucleotide sequence of SEQ ID NO:58, nucleotides 9-34811 of SEQ ID NO:58, or the nucleotide sequence of SEQ ID NO:63.

15. A composition comprising a C68 vector according to claim 1.

16. A cell comprising a C68 vector according to claim 1.

17. A pharmaceutical composition comprising a C68 vector according to claim 1 and a pharmaceutically acceptable excipient.

18. A method of treating prostate cancer in a human, comprising administering to the human an effective amount of the pharmaceutical composition according to claim 17.

19. The method according to claim 18, further comprising administering to the human an effective amount of an immune modulator.

20. The method according to claim 18, further comprising administering to the human (a) an effective amount of at least one immune-suppressive-cell inhibitor and (b) an effective amount of at least one immune-effector-cell enhancer.

21. The method according to claim 20, wherein the immune-suppressive-cell inhibitor is selected from the group consisting of a protein kinase inhibitor, a COX-2 inhibitor, and a PDE5 inhibitor, and wherein the immune-effector-cell enhancer is selected from the group consisting of a CTLA-4 inhibitor, a CD40 agonist, a TLR agonist, a 4-1 BB agonist, a OX40 agonist, a GITR agonist, a PD-1 antagonist, and a PD-L1 antagonist.

22. The method according to claim 21, wherein:
(1) the protein kinase inhibitor is selected from the group consisting of imatinib, sorafenib, lapatinib, zactima MP-412, dasatinib, lestaurtinib, sunitinib malate, axitinib, erlotinib, gefitinib, bosutinib, temsirolismus, and nilotinib;
(2) the CTLA-4 inhibitor is selected from the group consisting of ipilimumab and tremelimumab;
(3) the CD40 agonist is an anti-CD40 antibody selected from the group consisting of G28-5, mAb89, EA-5, S2C6, CP870893, and dacetuzumab; and
(4) the TLR agonist is a CpG oligonucleotide selected from the group consisting of CpG 24555, CpG 10103, CpG7909, and CpG1018.

23. The method according to claim 22, wherein the immune-suppressive-cell inhibitor is a protein kinase inhibitor selected from the group consisting of sorafenib, dasatinib, imatinib, axitinib, and sunitinib malate, and wherein the immune-effector-cell enhancer is tremelimumab.

24. The method according to claim 22, wherein the immune-suppressive-cell inhibitor is a protein kinase inhibitor selected from the group consisting of sorafenib, dasatinib, imatinib, axitinib, and sunitinib malate, and wherein the immune-effector-cell enhancer is a CpG oligonucleotide selected from the group consisting of CpG24555, CpG10103, CpG7909, and CpG1018.