Adenoviruses and methods of using adenoviruses

Abstract

The present invention relates to methods and materials for nucleic acid delivery, vaccination and / or treatment of cancer. More specifically, methods and materials for nucleic acid delivery, vaccination and / or treatment of cancer using one or more recombinant adenoviruses (Ad) as oncolytic agents are provided.

Description

Background of the Invention

[0002] Technical field of the invention

[0003] The present invention relates to methods and materials for nucleic acid delivery, vaccination, and / or treatment of cancer. For example, the present invention includes adenoviruses (Ad) and methods of using adenoviruses to treat medical conditions such as cancer. In one aspect of the invention, the adenoviruses provided herein can be used as oncolytic agents.

[0004] Despite significant efforts, cancer remains a major public health problem in the United States, with over 1.6 million new cases in 2017 alone (National Cancer Institute, “Cancer Stat Facts: Cancer of Any Site,” seer.cancer.gov / statfacts / html / all.html). Traditional treatments, such as chemotherapy, radiation therapy, and surgery, often fail, especially in the late stages of cancer. One reason is that cancer cells can eliminate or modify the components targeted by these therapies and effectively avoid being killed.

[0005] Oncolytic virotherapy may offer an alternative approach to cancer treatment by utilizing selectively replicating viruses to destroy tumors, activate adaptive immune responses, and ensure lifelong immunity against tumors (Russell et al., 2017 Molecular Therapy 25:1107-1116).

[0006] The present invention provides methods and materials for nucleic acid delivery, vaccination and / or treatment of cancer. For example, the present invention provides methods and materials for treating cancer by administering one or more recombinant Ad (e.g., one or more of Ad657 and its variants) as oncolytic agents. In one embodiment, the recombinant Ad may be derived from a first Ad (e.g., may include a genome of a first Ad such as Ad6, also known as a recombinant Ad backbone), and may include hexon HVRs from a second Ad such as Ad57. In the case where the recombinant Ad includes the Ad6 genome and the Ad57 hexon HVRs, the recombinant Ad may be a chimeric Ad referred to as Ad657 (see Nguyen, et al. Oncolytic Virotherapy 7:43-51, 2018).

[0007] In one aspect, the present invention provides methods for vaccinating against infectious diseases using one or more recombinant Ad (e.g., one or more of Ad657 and its variants). In one aspect, the present invention provides methods for treating cancer using one or more recombinant Ad (e.g., one or more of Ad657 and its variants) as oncolytic agents. In some cases, one or more recombinant Ad (e.g., one or more Ad657) can be used to reduce the number of cancer cells in a mammal (e.g., by infecting and killing cancer cells). In some cases, one or more recombinant Ad (e.g., one or more of Ad657 and its variants) can be used to stimulate an anti-cancer immune response in a mammal. In some cases, one or more recombinant Ad (e.g., one or more of Ad657 and its variants) can be used to stimulate an immune response against an infectious disease in a mammal.

[0008] As demonstrated herein, when Ad657 was delivered intravenously to mice bearing subcutaneous human DU145 prostate cancer tumors, it first infected the liver and then reached distant tumors. Both Ad6 and Ad657 mediated a significant delay in tumor growth and prolonged survival, with Ad6-mediated greater efficacy.

[0009] The present invention provides methods and materials for nucleic acid delivery, vaccination and / or treatment of cancer. For example, the present invention provides methods and materials for treating cancer by administering one or more recombinant Ad (e.g., one or more of Ad657 and its variants) as oncolytic agents. In one embodiment, the recombinant Ad may be derived from a first Ad (e.g., may include the genome of a first Ad such as Ad6, also referred to as a recombinant Ad backbone) and may include hexon HVRs from a second Ad such as Ad57. In the case where the recombinant Ad includes the Ad6 genome and the Ad57 hexon HVRs, the recombinant Ad may be a chimeric Ad referred to as Ad657. In one aspect, the present invention provides methods for vaccinating against infectious diseases using one or more recombinant Ad (e.g., one or more of Ad657 and its variants). In one aspect, the present invention provides methods for treating cancer using one or more recombinant Ad (e.g., one or more of Ad657 and its variants) as oncolytic agents. In some cases, one or more recombinant Ad (e.g., one or more Ad657) may be used to reduce the number of cancer cells in a mammal (e.g., by infecting and killing cancer cells). In some cases, one or more recombinant Ad (e.g., one or more of Ad657 and its variants) can be used to stimulate an anti-cancer immune response in a mammal. In some cases, one or more recombinant Ad (e.g., one or more of Ad657 and its variants) can be used to stimulate an immune response against an infectious disease in a mammal.

[0010] As demonstrated herein, when Ad657 was delivered intravenously to mice bearing subcutaneous human DU145 prostate cancer tumors, it first infected the liver and then reached distant tumors. Both Ad6 and Ad657 mediated a significant delay in tumor growth and prolonged survival, with Ad6-mediated greater efficacy.

[0011] In addition, for almost any oncolytic virus, liver retention is a considerable problem if it is used as an intravenous systemic therapy. If the virus infects and kills hepatocytes, this can cause liver damage at low doses and death at high doses. It is worth noting that compared to Ad5 or Ad6, the administration of the Ad of the present invention (ie, Ad657 chimeric vectors and variants thereof) mediated unexpectedly lower liver damage. Therefore, the unique combination of HVR 1-7 of the Ad6 platform and Ad657 mediates changes in biodistribution and therapy that are not observed in natural viruses.

[0012] Similarly, as demonstrated herein, rhesus macaques were immunized with a replicative, single-cycle adenovirus (SC-Ad657) vaccine expressing only clade B HIV-1 gp160 via the intranasal (IN) and intramuscular (IM) routes and compared to mucosal and systemic routes of vaccination. The SC-Ad vaccine alone produced significant circulating antibody titers against Env after only one immunization. Animals immunized only by the IM route had high peripheral T follicular helper (pTfh) cells in the blood, low Tfh in the lymph nodes, and lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity. Animals immunized by the IN route had high Tfh in the lymph nodes, low pTfh in the blood, and higher ADCC antibodies. When immunized with SHIV SF162P3 After rectal attack immune animals, they are all infected, but the gastrointestinal viral load of mucosal primary immunity (prime) animals is significantly lower. Similarly, Ad657 carrying hepatitis C antigen genes can produce cytotoxic T lymphocytes (CTL) responses for hepatitis and cytomegalovirus. Ad657 can deliver and express therapeutic genes, including cytokines such as 4-1BBL, granulocyte macrophage stimulating factor (GMCSF) and IL-21. The results provided herein demonstrate that recombinant Ad can be used as a local or systemic delivery vehicle for nucleic acid, vaccine and / or oncolytic virus therapy for cancer. Summary of the Invention

[0013] In general, one aspect of the present invention features a recombinant Ad comprising (a) an Ad genome from a first Ad strain, and (b) a nucleic acid encoding a hexon polypeptide from a second Ad strain, wherein one or more of the hypervariable regions (HVRs) of the hexon polypeptide are different from the HVRs encoded by the Ad genome. The first Ad strain can be a first human Ad strain, and the second Ad strain can be a second human Ad strain that is different from the first human Ad strain. The first Ad strain and the second Ad strain can be different in serotype. The first Ad strain can be a human Ad6 strain, and the second Ad strain can be a human Ad57 strain. The recombinant Ad can also include one or more targeting polypeptides, antigenic polypeptides, enzymes, amino acid substitutions, PEGylation, ligands, tags, and the like.

[0014] Recombinant Ad can be used as vectors for gene-based vaccination, for gene therapy applications / delivery, or for oncolytic virotherapy.

[0015] In another embodiment, the recombinant Ad comprises (a) an Ad genome from a first Ad strain, and (b) a nucleic acid encoding at least one hexon polypeptide from one or more Ad strains, wherein the hypervariable regions (HVRs) of the hexon polypeptides from the one or more Ad strains are different from the HVRs encoded by the first Ad genome.

[0016] In another embodiment, the recombinant Ad can be a replication-competent Ad or a conditionally replicating Ad (eg, CRAd).

[0017] In another aspect, the present invention features a recombinant and / or chimeric Ad comprising (a) a nucleic acid encoding a first hexon polypeptide, and (b) a second hexon polypeptide, wherein the amino acid sequence of the first hexon polypeptide is different from the amino acid sequence of the second hexon polypeptide. The amino acid sequence of the hypervariable region (HVR) of the first hexon polypeptide may be different from the amino acid sequence of the hypervariable region of the second hexon polypeptide. The nucleic acid may be from a first Ad strain, and the second hexon polypeptide may be from a second Ad strain. The first Ad strain may be a first human Ad strain, and the second Ad strain may be a second human Ad strain that is different from the first human Ad strain. The first Ad strain and the second Ad strain may be different in serotype. The Ad strain may be a human Ad6 strain, and the second Ad strain may be a human Ad57 strain. The recombinant Ad may also comprise a targeting polypeptide. The targeting polypeptide may comprise the amino acid sequence TARGEHKEEELI (SEQ ID NO: 1).

[0018] In another embodiment, the recombinant Ad comprises (a) a nucleic acid encoding a first hexon polypeptide, and (b) a second hexon polypeptide from one or more Ad strains, wherein the amino acid sequence of the first hexon polypeptide is different from the amino acid sequence of the second hexon polypeptide from the one or more Ad strains.

[0019] In another embodiment, the recombinant Ad can be a replication-competent Ad or a conditionally replicating Ad (eg, CRAd).

[0020] In another aspect, the present invention provides materials and methods for treating a mammal with cancer. The method may comprise or consist essentially of administering to a mammal with cancer a recombinant Ad comprising (a) an Ad genome from a first Ad strain and (b) at least one hexon polypeptide from one or more Ad strains, wherein one or more hypervariable regions (HVRs) of the hexon polypeptide are different from the HVRs encoded by the Ad genome, and / or administering to a mammal with cancer an Ad comprising (a) a nucleic acid encoding a first hexon polypeptide and (b) a second hexon polypeptide, wherein the amino acid sequence of the first hexon polypeptide is different from the amino acid sequence of the second hexon polypeptide. The mammal may be human. The cancer may be prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, lymphocytic leukemia, or myeloid leukemia. Administration may comprise systemic or local administration (e.g., intravenous, intratumoral, intramuscular, intra-organ, intra-lymph node administration).

[0021] This paper demonstrates that Ad657 and its variants can deliver therapeutic genes to cells to express therapeutic polypeptides. Therefore, recombinant Ad, including chimeric Ad, can be used as a local or systemic delivery vehicle for nucleic acid, vaccine and / or oncolytic virus therapy for cancer.

[0022] The details of one or more embodiments of the invention are set forth in the examples and drawings and in the description that follows. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. A recombinant adenovirus (Ad) comprising (a) an Ad genome encoding a hexon polypeptide from a first Ad strain, and (b) a nucleic acid encoding at least one hexon polypeptide nucleic acid from one or more different Ad strains, wherein at least one hypervariable region (HVR) of the hexon polypeptide is different from an HVR encoded by the Ad genome of the first Ad strain.

[0023] Another aspect of the invention relates to recombinant Ad wherein the first Ad strain and the one or more different Ad strains differ in serotype.

[0024] Another aspect of the present invention relates to recombinant Ad, wherein the first Ad strain is a human Ad6 strain, and wherein the second Ad strain is a human Ad57 strain.

[0025] Another aspect of the present invention relates to such a recombinant Ad, which further comprises a nucleic acid encoding a targeting polypeptide, antigen, enzyme, receptor, ligand or tag.

[0026] Another aspect of the present invention relates to a recombinant Ad, wherein the targeting polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-41 and SEQ ID NOs: 46-47.

[0027] Another aspect of the present invention relates to such a recombinant Ad, wherein the recombinant Ad is a replication-competent Ad.

[0028] Another aspect of the present invention relates to such recombinant Ad, wherein the replication-competent Ad is a single-cycle Ad or a conditionally replicating Ad (CRAd).

[0029] Another aspect of the present invention relates to a recombinant adenovirus (Ad) comprising (a) an Ad capsid polypeptide from a first Ad strain and (b) at least one hexon polypeptide from one or more different Ad strains, wherein the hypervariable region (HVR) or capsid polypeptide of the hexon polypeptide is different from the HVR or capsid polypeptide of the first Ad strain.

[0030] Another aspect of the present invention relates to a recombinant adenovirus (Ad) comprising (a) a nucleic acid encoding a first hexon polypeptide and (b) a nucleic acid encoding a second hexon polypeptide, wherein the amino acid sequence of the first hexon polypeptide is different from the amino acid sequence of the second hexon polypeptide.

[0031] Another aspect of the present invention relates to recombinant Ad wherein the nucleic acid encodes at least one hexon polypeptide that differs from the amino acid sequence of the hypervariable region of the second hexon polypeptide.

[0032] Another aspect of the invention relates to recombinant Ad, wherein the nucleic acid is from a first Ad strain, and wherein the second hexon polypeptide is from a second Ad strain.

[0033] Another aspect of the present invention relates to recombinant Ad, wherein the first Ad strain is a first human Ad strain, and wherein the second Ad strain is a second human Ad strain different from the first human Ad strain.

[0034] Another aspect of the present invention relates to recombinant Ad, wherein the first Ad strain and the second Ad strain are different in serotype.

[0035] Another aspect of the present invention relates to recombinant Ad, wherein the first Ad strain is a human Ad6 strain, and wherein the second Ad strain is a human Ad57 strain.

[0036] Another aspect of the present invention relates to such a recombinant Ad, which further comprises a targeting polypeptide.

[0037] Another aspect of the present invention relates to a recombinant Ad, wherein the targeting polypeptide comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 1).

[0038] Another aspect of the present invention relates to such a recombinant Ad, wherein the recombinant Ad is a replication-competent Ad.

[0039] Another aspect of the present invention relates to such recombinant Ad, wherein the replication-competent Ad is a single-cycle Ad or a conditionally replicating Ad (CRAd).

[0040] Another aspect of the present invention relates to a method of treating a mammal having cancer, wherein the method comprises administering to the mammal a recombinant adenovirus (Ad) described herein.

[0041] Another aspect of the invention relates to such a method, wherein the cancer is selected from the group consisting of prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, lymphocytic leukemia and myeloid leukemia.

[0042] Another aspect of the invention relates to such a method, wherein said administering comprises systemic administration.

[0043] Another aspect of the invention relates to such methods, wherein systemic administration comprises intramuscular, intranasal or intravenous administration.

[0044] Another aspect of the present invention relates to such a method, wherein said administering comprises topical administration.

[0045] Another aspect of the invention relates to such a method, wherein said local administration comprises intratumoral injection.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to those described herein can be used to implement the methods of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, the present specification, including definitions, will prevail. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

[0047] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1Illustration of translation of context-specific peptides from phage to adenovirus. A) Schematic diagram of a phage display library containing the Ad5 fimbriae HI β sheet, which structurally constrains a random 12-mer peptide library. Below is a diagram of the structurally similar sites between the β7 and β8 sheets in the Ad5 HVR5 hexon. B) Primary amino acid alignment of 12.51 and 12.52 in the HI library and their positions upon insertion into HVR5 of the hexon. C) Representative Ad5 GFP-Luc expressing virus modified with the peptide.

[0050] Figure 2 In vitro transduction is shown. A) 10 4 C2C12 cells were infected with the indicated vectors at 100 vp / cell and GFP expression was examined by fluorescence microscopy 2 days after infection. B) Luciferase activity from C2C12 cells 2 days after infection with different MOIs of the indicated Ad.

[0051] Figure 3 In vivo transduction in mice is shown. A) Luciferase imaging of mice 1 day after injection by intravenous (IV) or intramuscular (IM) routes. IM injected mice received 10 9 vp. IV-injected mice received 10 10 vp. B) Luciferase activity was quantified by imaging at the indicated days after injection. *p<0.05 by one-way ANOVA. ***p<0.001 by one-way ANOVA.

[0052] Figure 4 In vivo transduction in hamsters is shown. A) Injection of 10 10 Luciferase activity in hamster muscle 1 day after VP. **p<0.01 by T-test.

[0053] Figure 5 Figure 16 shows the gene-based immune response 16 weeks after a single IM immunization. Figure 3 Mice were bled and their sera were analyzed by ELISA in serial dilutions to detect antibodies against the GFP protein. By one-way ANOVA, *p<0.05, **p<0.01, ***p<0.001. By one-way ANOVA, ****p<0.0001. All Ad-injected mice produced significant anti-GFP antibodies compared to the PBS group at serum dilutions of 1:10,000 to 1:1000. The comparison of Ad5-GL-HVR-12.51 and 12.52 with Ad5-GL is shown with a black asterisk. The comparison between Ad5-GL-HVR-12.51 and Ad5-GL-HVR-12.52 is shown with a gray asterisk. The gray dotted line at OD0.06 represents the 95% confidence interval for distinguishing antibodies from the PBS group.

[0054] Figure 6 Phylogenetic tree showing the complete genome sequences of human adenovirus serotypes.

[0055] Figure 7 An alignment of Ad5, Ad6, and Ad57 showing variation in the hexon and E3 regions is shown. (A) Pustell DNA alignment of the Ad6 and Ad57 genomes. Boxes indicate the hexon and E3 regions with the highest variation between the two viruses. (B) ClustalW amino acid alignment of the hypervariable regions in the hexon proteins from Ad5, Ad6, and Ad57. Alignment was performed using MacVector.

[0056] Figure 8 Cartoon showing the construction of Ad657 using Ad57 HVRs in place of Ad6 HVRs. Abbreviation: HVR, hypervariable region.

[0057] Figure 9 In vitro oncolytic activity is shown. LNCaP and DU145 cells were treated with the indicated viruses at the indicated volumes per cell for 5 days. Cells were stained with crystal violet, and cell viability was measured by OD595. Cell viability (%) was calculated by dividing the sample OD value by the mean OD value of untreated control cells grown on the same 96-well plate and multiplying this value by 100. (A) LNCaP cell killing. (B) DU145 cell killing. Abbreviation: vp, viral particles.

[0058] Figure 10 The effect of oncolytic Ad on liver damage is shown. C57BL / 6 mice (n=6 per group) were injected with 10 μg of each virus via the tail vein. 11 vp. (A) Kaplan-Meier survival analysis. (B) Blood was drawn 3 days after injection for ALT measurement (by ANOVA, ****p < 0.001). Abbreviations: ALT, alanine aminotransferase; vp, viral particles.

[0059] Figure 11 The anticancer activity of Ad6 and Ad657 in DU145 tumor xenografts in nude mice after a single iv administration is shown. 10 Nude mice with established DU145 tumors were injected with a single dose of either iv or PBS (n=9 per group). (A) Effect of a single iv injection on tumor growth. Tumor dimensions were measured with a caliper and tumor volume was calculated as the width 2× length × 1 / 2. Data are shown as mean ± SE. *p<0.05, ****p=0.0001 by ANOVA as described herein or by T test. Black asterisks and upward black arrows indicate statistical differences between the Ad6 group and the PBS group on the selected day described herein. Gray asterisks and upward arrows indicate differences between the Ad657 group and the PBS group on the indicated day. Shaded white asterisks and downward gray arrows indicate statistical differences between the Ad6 and Ad657 groups on the indicated day. (B) Effect of a single iv injection on survival. When the tumor volume reached 2000 μL or met other sacrifice criteria (e.g., ulcer), the animals were euthanized and Kaplan-Meier survival curves were plotted (by log-rank analysis, *p<0.05, **p<0.01). Abbreviation: iv, intravenous.

[0060] Figure 12 Nude mice were imaged with luciferase. A single iv injection of 3 × 10 10 4 days after vp of Ad6 and Ad657-GFP-Luc (deletions of 12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7K, and a partial deletion of E4 34K). Abbreviations: iv, intravenous; vp, viral particle.

[0061] Figure 13 Nude mice were imaged for luciferase. Note: A single iv injection of 3 × 10 10 Abbreviations: iv, intravenous; vp, viral particles.

[0062] Figure 14 Plasma HIV Env binding titers are shown. Immunizations with different SC-Ad and gp140 proteins are indicated above the graph by large arrows. Midpoint F8 gp140 binding titers for each animal before and after each immunization are shown by ELISA. The dashed line represents the minimum detection limit of the antibody in this assay. Symbols are distributed along the x-direction at each time point to allow observation of individual measurements. SC-Ad6-Ebov is a negative control Ad vaccine. This group of animals was not boosted with gp140. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to the SC-Ad6-Ebov group by one-way ANOVA.

[0063] Figure 15 Plasma HIV neutralization titers are shown. Neutralization of the indicated viruses was performed using the TZM-b1 neutralization assay. All values ​​are calculated relative to virus-only wells. Each point represents the mean for each animal.

[0064] Figure 16 Plasma ADCC activity is shown. Plasma samples were tested with CD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc, which were induced with SHIV SF162P3 Infected target cells. Each point represents the mean of each animal. *p<0.05, ***p<0.001, ****p<0.0001 compared with the SC-Ad6-Ebov group by one-way ANOVA.

[0065] Figure 17 Mucosal ADCC activity is shown. Vaginal wash and saliva samples were tested with CD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc target cells infected with SHIVSF162P3. Each point represents the mean for each animal. *p<0.05 compared to the SC-Ad6-Ebov group by one-way ANOVA.

[0066] Figure 18 IFN-γ secreting cells from PBMC and lymph nodes are shown. PBMC and lymph node cells were analyzed by ELISPOT staining for IFN-γ. Anti-Env represents cells stimulated with conserved HIV Env peptides and SC-Ad. The total number of spot-forming cells (SFC) in each stimulation well was counted and adjusted for control medium as a background. Each point represents the mean of each animal. *p<0.05 by one-way ANOVA.

[0067] Figure 19 Mucosal T cell trafficking and activation are shown. T cells were harvested from rectal biopsies collected after the second protein boost and analyzed by flow cytometry for CD4, CD8, α4β7 integrin, CD69, and FoxP3. Each point represents the mean for each animal.

[0068] Figure 20 Figure 2. Tfh cell responses in blood and lymph nodes. PBMCs and lymph node cells collected at week 40 were stimulated with HIV-1 Env protein and then examined for co-expression of CD3+, CD4+, CXCR5+, and IL-21. Each point represents the mean for each animal. *p<0.05 by one-way ANOVA.

[0069] Figure 21 Shown for repeated rectal SHIV SF162P3 Protection from challenge. Weekly administration of 4.3 TCID50 (for rhesus monkey PBMC) of SHIV SF162P3The indicated groups were challenged rectally. Plasma samples were analyzed for SHIV viral RNA copies. Animals with RNA copies greater than 10 were considered infected, and the number of challenges required to infect the animal was used as the event for Kaplan-Meier survival analysis.

[0070] Figure 22 Shown SHIV SF162P3 A) will Figure 8 Animals in the challenge study were divided into 8 groups according to their initial SC-Ad priming route (IM or IN) and subjected to Kaplan-Meier analysis. B) Plasma SHIVSF162P3 viral RNA levels during the challenge study.

[0071] Figure 23 SHIV viral load in tissues is shown. RNA from PBMCs and postmortem tissues was collected and analyzed by qPCR to detect SHIV viral RNA.

[0072] Figure 24 Shown are the single-cycle adenovirus vaccines used in Example 7. A) Diagram of SC-Ad serotypes 6 and 657 carrying F8 and G4 clade B HIV envelope genes. B) Alignment of clade C Ad hexons, including Ad6 and 57 hexons displayed on the vaccine.

[0073] Figure 25 Saliva and vaginal HIVenv binding titrations are shown. When tested against F8, the ELISA OD450 levels of the samples shown are shown at the dilutions shown. Antibody levels in these mucosal samples were low and did not reach saturation in the assay. For this reason, EC50 values ​​could not be reliably calculated for most animals. The rhesus monkey Rh13-091 in the IN-IM-IM group was the only animal for which an EC50 could be calculated (EC50=4580). Similar results were observed in the ELISA using SF162gp140.

[0074] Figure 26 Ad657 expressing antigen genes from hepatitis C and cytomegalovirus (CMV) gB generates in vivo cytotoxic T lymphocyte (CTL) activity. Shown is the killing of hepatitis C peptide-loaded target cells in mice vaccinated with Ad657-HCV but not CMV gB.

[0075] Figure 27 It was shown that expression of human GMCSF from Ad657 induced the proliferation of GMCSF-dependent TF-1 human erythroblasts.

[0076] Figure 28 is a graph showing a single IV injection of Ad6 versus A549 lung tumors.

[0077] Figure 29 is a graph showing the effect of a single IV or IT injection of Ad6 on Pancl pancreatic tumors.

[0078] Figure 30 is a graph showing a single IV injection of Ad6 versus renal carcinoma in immunocompetent hamsters.

[0079] Figure 31 is a graph showing luciferase activity in B16 melanoma and A549 lung tumor / cancer cells by Ad displaying the 12.51 cell-binding peptide in HVR5 of hexon.

[0080] Figure 32 Graph showing luciferase activity in hepatocellular carcinoma and renal carcinoma by Ad displaying the 12.51 cell-binding peptide in HVR5 of hexon.

[0081] Figure 33 Figure 2 shows the combination of inserting individual HVRs from different Ad serotypes with the insertion of cell targeting / detargeting peptides or novel amino acids such as cysteine ​​into hexon for targeted chemical modification and shielding. Depicted are chimeric HVR constructs that combine different HVRs from different Ad serotypes to modulate natural interactions with cells and blood factors to improve pharmacology, and combined with the insertion of cell binding and cell detargeting peptides in different HVRs to alter cell entry and cell avoidance. If one HVR is replaced from 100 Ads, this would result in 100 different hexon chimeras. If all 7 HVRs each received a different Ad HVR, this combinatorial library would be equivalent to 7 100 If a peptide is introduced into 7 HVRs, this would be equal to 7X 7 100 If 10 different peptides were introduced into 7 HVRs, this would be equivalent to 10X 7X7 100 variants, and so on.

[0082] Figure 34 Plasmid maps of representative combined hexon and peptide combinations are shown. Shown is a hexon with HVR1 from Ad6 and HVRs 2-7 from Ad57 with cell targeting peptides inserted into the individual HVRs.

[0083] Figure 35 Chimeric HVR constructs are shown that combine different HVRs from different Ad serotypes to modulate natural interactions with cells and blood factors to improve pharmacology, and combine the insertion of cell binding and cell detargeting peptides in different HVRs to alter cell entry and cell avoidance. In this example, a single cysteine ​​amino acid is inserted into HVR1 and HVR5 of Ad657 to modulate pharmacology and allow targeted conjugation of polymers (e.g., polyethylene glycol) or other moieties (e.g., imaging agents such as fluorophores).

[0084] Figure 36 Conjugation of polyethylene glycol (PEG) to Ad657-HVR1-C is shown. A) SDS-PAGE of Ad proteins with and without PEGylation. Arrows indicate the size increase due to chemical attachment of PEG to the hexon. B) Effects of targeted PEGylation via maleimide-PEG and non-targeted NHS-PEG on viral infection.

[0085] Figure 37 Conjugation of polyethylene glycol (PEG) to Ad657-HVR5-C is shown. A) SDS-PAGE of Ad proteins with and without PEGylation. B) Near-infrared imaging of SDS-PAGE of Ad proteins with and without PEGylation and with and without the near-infrared fluorescent imaging tag IR800. C) In vivo transduction following intraperitoneal injection of maleimide-PEGylated Ad657-HVR5-C as shown by luciferase imaging.

[0086] Figure 38 Luciferase imaging of nude mice is shown. A) 1, 4, 7, 14, 28, and 42 days after a single IV injection of Ad6 treatment relative to a distant DU145 prostate tumor. B) 3, 7, and 19 days after IV injection of replicative Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

[0087] Figure 39 Schematic diagram showing cancer-specific conditionally replicative Ad (CRAd) dl1101+dl1107 with modifications in the E1A gene.

[0088] Figure 40 is a diagram showing that both Ad6 and Ad657 can be used as CRAds for targeted cancer therapy.

[0089] Figure 41 Schematic diagrams illustrating Ad treatment cycles. A) Schematic diagram of serotype switching using Ad. B) Schematic diagram of an exemplary treatment cycle in which Ad6 and Ad657 can be used for multiple rounds of treatment through serotype switching combined with covalent polymer conjugation.

[0090] Figure 42 Shown are serotype switching and on-target luciferase activity in DU145 prostate tumors following a single IV injection of Ad6 and Ad6-F35 with deletions in the E3A gene (12.5K, 6.7K, 19K, 11.6K) but retaining the E3B gene (10.4K, 14.5K, and 14.7K) and retaining E4 34K. Mice whose tumors were resistant to a previous single IV injection of Ad657 and CRAd657 with an intact E3 gene were injected with the indicated vectors by a single IV injection.

[0091] Figure 43 Schematic diagram of replication-competent Ad (RC-Ad), in which E1 expression is controlled by the native E1 promoter; the variant CRAd-Probasin-E1A (Ad-PB), in which E1 expression is controlled by the prostate-specific probasin promoter; CRAd-dl1101, in which p300 pathway binding is abolished, susceptible to the IFN pathway in normal cells; CRAd-dl1107, in which abolition of pRB binding allows the virus to kill cancer cells with RB pathway disruption but is suppressed in RB+ normal cells; CRAd-dl1101 / 07, in which abolition of p300 pathway binding is susceptible to the IFN pathway, and abolition of pRB binding allows the virus to kill cancer cells with RB pathway disruption but is suppressed in RB+ normal cells.

[0092] Figure 44 (A and B) show the effects of infection of non-cancer cells with replication-competent Ad5, Ad6, and Ad657, as well as the effects of modification of Ad6 and Ad657 to conditionally replicating Ad (CRAd).

[0093] Figure 45 Killing of cancer cells by replication-competent Ad5, Ad6, Ad657, and the indicated CRAds is shown.

[0094] Figure 46 Modification of Ad6 and Ad657 into conditionally replicating Ad (CRAd) is shown.

[0095] Figure 47 Modification of Ad6 and Ad657 into conditionally replicating Ad (CRAd) is shown.

[0096] Figure 48 Shown are the in vivo effects of replication-competent Ad6 or the indicated CRAds on DU145 tumor growth in mice.

[0097] Figure 49 Shown are the in vivo effects of replication-competent Ad657 with an intact E3 region and conditionally replicating Ad657-dl1101 / 07 following a single intravenous injection in mice bearing human prostate tumors.

[0098] Figure 50 PEGylation was shown to detarget adenovirus to the liver in vivo.

[0099] Figure 51 Modification of Ad657 with shorter cilia from chimpanzee AdC68 and addition of the codon wobble E4 34.K gene was shown to alter in vitro efficacy.

[0100] Figure 52 Killing of human prostate cancer cells by 6 / 56 / 6 virus with and without CRad modification is shown.

[0101] Figure 53 We show that a single intramuscular immunization of BALB / c mice with CRAd657-dl1101 / 07-FOLR harboring an intact E3 region generates an antibody response against the human cancer antigen folate receptor α.

[0102] Figure 54 Depicted are sites on the AdHVR that can be modified, for example, by PEGylation or "BAPylation" with a biotin acceptor peptide (BAP).

[0103] Figure 55 is a schematic diagram of variants of Ad with mutations in the E1 protein to convert the virus into a conditionally replicating Ad (CRAd).

[0104] Figure 56 The amino acid sequences of the N-terminal portion of the wild-type E1A polypeptide and the E1AN termini of the CRAd variants dl1101, dl1107, and dl1101 / 1107 are shown.

[0105] Figure 57 Schematic diagram showing different E3 immune evasion genes in Ad6, an example of Ad class C, and Ad26, an example of Ad class D. Both Ads express size and sequence variants of the E3 12.5K, 6.7K, 19K, 10.4K (RIDα), 14.5K (RIDβ), and 14.7K genes, and the functions of these E3-encoded proteins are described.

[0106] Figure 58 The effects of PEGylation and E3 deletion of Ad6-Luc virus on the anti-tumor activity of oncolytic viruses in immunocompetent hamsters are shown. Both Ad6-Luc and Ad6-Luc-20K PEG have all E3 genes and E434K intact. Ad6-deltaE3-Luc has partial deletions of E3 12.5K and E4 34K, as well as complete deletions of E3 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ) and 14.7K genes. When these immune escape genes are not present in the oncolytic adenovirus, oncolytic efficacy is lost in this immunocompetent animal model.

[0107] Figure 59 This is the plasmid map of Ad657, in which E3 12.5K and E4 34K are partially deleted, and E3 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ) and 14.7K genes are completely deleted.

[0108] Figure 60Depicted are CRAd 657 constructs with and without dl1101 / 1107 CRAd modifications and with and without deletions of selected E3 immune evasion genes.

[0109] Figure 61 Depicted are CRAd657 with E3 insertion sites. These have and do not have the dl1101 / 1107 CRAd modifications described herein, and have and do not have E3 immune evasion modifications.

[0110] Figure 62 Depicted are CRAd657 + / - Ad35 pili or chimpanzee C68 pili + / - K7 peptides. These have and do not have the dl1101 / 1107 CRAd modifications described in the previous figures, and have and do not have the E3 immune evasion modification. In some cases, the codon wobble E4 34K gene is included after E4 and before the pili to compensate for the partial loss of E4 34K when the E3B gene is deleted.

[0111] Figure 63 Depicted are CRAd657 + / - Ad35 pili or chimpanzee C68 pili + / - K7 peptide. These have and do not have the dl1101 / 1107 CRAd modifications described in the previous figures, and have and do not have the E3 immune evasion modification.

[0112] Figure 64 Folate receptor α-expressing CRAd657 + / - Ad35 fibrils or chimpanzee C68 fibrils + / - K7 peptide are depicted.

[0113] Figure 65 Depicted are CRAd657 + / - Ad35 fibrils or chimpanzee C68 fibrils + / - K7 peptide expressing granulocyte macrophage colony-stimulating factor (GMCSF).

[0114] Figure 66 Depicted are the expression of 4-1BBL or GMCSF or IL21 or CD40L in combination with CRAd657 + / - Ad35 fibrils or chimpanzee C68 fibrils + / - K7 peptide in one virus.

[0115] Figure 67 Depicted are Ad6 / 57 + / - Ad35 fimbriae or chimpanzee C68 fimbriae + / - K7 peptides with Ad6 HVR1 and Ad57 HVR2-7.

[0116] Figure 68 Depicted are Ad6 / 57 / 6 + / - Ad35 fimbriae or chimpanzee C68 fimbriae + / - K7 peptides with Ad6 HVR1, Ad57 HVR2-6, Ad6 HVR7.

[0117] Figure 69Depicted are Ad6 / 57 / 6 + / - Ad35 fimbriae with Ad6 HVR1, Ad57 HVR2-6, Ad6 HVR7 or chimpanzee C68 fimbriae + / - K7 peptide expressing GFP luciferase.

[0118] Figure 70 Luciferase imaging after serotype switching is shown. Mice with LNCaP prostate tumors in their flanks were treated with a single IV injection of Ad657 or CRAd657. Mice with residual tumors 5 months after a single IV injection were treated by serotype switching to the indicated Ad6 / 57 / 6 variants (with and without variant pili and codon-optimized E4 34K genes) expressing GFP luciferase. The indicated Ad6 / 57 / 6 variants include Ad6 / 57 / 6 viruses with different pili protein modifications, including the addition of 7 lysines (K7) to the pili protein, grafting of chimpanzee C68 pili protein onto the Ad6 pili protein after its KKTK flexible domain, and with Ad35 pili protein. Mice were imaged for luciferase activity 7 days later.

[0119] Figure 71 A549 human lung cancer cells treated with Ad549 / 57 / 6 with and without variant pilin and codon-optimized E4 34K genes at the indicated viral particles (vp) / cell are shown. After 7 days, cells were stained with crystal violet and wells were analyzed in a plate reader. High OD values ​​indicate the presence of viable cells. Low OD values ​​indicate adherent cell death and loss. Detailed Description of the Invention

[0121] The present invention provides methods and materials for nucleic acid delivery, vaccination, and / or treatment of cancer. For example, the present invention provides methods and materials for nucleic acid delivery, vaccination, and / or treatment of cancer using one or more recombinant Ad (e.g., Ad657 and its variants) as oncolytic agents.

[0122] The adenovirus icosahedron is composed of 720 copies of its hexon protein. The virus does not use this protein to bind to receptors, but this nanolattice of repeating proteins provides a matrix for interactions (e.g., both natural and non-natural) with proteins, cells, and drugs. Antibodies capable of neutralizing Ad can target the hypervariable regions (HVRs) of the hexon polypeptide on Ad.

[0123] In some cases, the present invention provides recombinant Ad with oncolytic anticancer activity. For example, the recombinant Ad can be derived from a first Ad and can include hexon HVRs from one or more different Ads. HVRs can be derived from any type of C Ad, such as Ad1, Ad2, Ad5, Ad6, and Ad57. In an embodiment, the recombinant Ad can be derived from a first Ad and can include one or more hexon HVRs from at least one other Ad, wherein at least one hexon HVR is different from the HVR(s) of the first Ad. The first Ad strain can be a human Ad6 strain, and the second Ad strain can be a human Ad57 strain. Hexon shuttle plasmid map ( Figure 34 ) demonstrates the insertion of individual HVRs from different Ad serotypes and the combination of cell targeting / detargeting peptides or novel amino acids (e.g., cysteine) into hexon for targeted chemical modification and shielding. In one embodiment, the recombinant Ad comprises amino acid substitutions, such as cysteine ​​substitutions in the polypeptide, as well as modifications such as PEGylation and BAPylation. The ability to target polymers and other chemical modifications to the inserted cysteine ​​in the Ad657 hexon is demonstrated herein.

[0124] Ad657 was demonstrated as an oncolytic agent against human prostate cancer. The Ad6 HVRs were replaced with HVRs from Ad57 to generate a chimeric species C oncolytic virus called Ad657. Ad657 and Ad6 were tested as systemic oncolytic therapies by a single iv injection of nude mice bearing human carcinomas. Ad657 can be used as a local or systemic oncolytic virus therapy for cancer. These data also demonstrate the surprising effect of serotype switching with oncolytic species C Ad.

[0125] In some cases, the present invention provides methods for treating mammals having cancer, infectious diseases and / or genetic diseases or at risk of having these diseases using one or more recombinant Ads provided herein. For example, one or more recombinant Ads can be administered to a mammal having cancer or at risk of cancer to reduce the number of cancer cells in the mammal (e.g., human) (e.g., by infecting and killing cancer cells). For example, one or more recombinant Ads can be administered to a mammal having cancer or at risk of cancer to stimulate an anti-cancer immune response in a mammal (e.g., human).

[0126] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anticancer activity, such as recombinant Ad657 and its variants) is not destroyed by the immune system of a mammal. For example, the recombinant Ad is not destroyed by antigen presenting cells (APCs), macrophages and / or other immune cells in a mammal to which the recombinant Ad is administered.

[0127] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity, such as recombinant Ad657 and its variants) can be administered for multiple rounds (e.g., two or more rounds) of treatment. For example, the first recombinant Ad described herein can avoid antibodies that can neutralize the second recombinant Ad described herein, and vice versa. In the case of treating a mammal with cancer with one or more recombinant Ads described herein, the first recombinant Ad can be administered to the mammal for a first round of treatment, and then the second recombinant Ad can be administered for a second round of treatment.

[0128] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anticancer activity, such as recombinant Ad657 and its variants) can be a replicable Ad (RC-Ad). For example, RC-Ad can be an RC-Ad comprising a nucleic acid encoding an E1 polypeptide (e.g., E1+RC-Ad). For example, RC-Ad can be a single-cycle Ad (SC-Ad), which includes a deletion of one or more nucleic acids encoding one or more polypeptides associated with infectious viral progeny (e.g., pIIIa and E3). For example, RC-Ad can be a conditionally replicable Ad (CRAd). Examples of single-cycle Ads and examples of how to prepare and use them are provided elsewhere (International Patent Application Publication No. WO2009 / 111738).

[0129] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anticancer activity, such as recombinant Ad657 and its variants) can be a replication-deficient Ad (RD-Ad). For example, the RD-Ad can be an RD-Ad that lacks the nucleic acid encoding the E1 polypeptide (e.g., an E1-deficient RD-Ad).

[0130] The examples herein demonstrate that CRAd 657 and its variants are conditionally replicating Ads (CRAds) in cancer cells, and that infection of cells with CRAd 657 and its variants reduces cell viability and tumor volume. Therefore, CRAd 657 and its variants can be used as local or systemic oncolytic virotherapy in subjects with cancer.

[0131] Furthermore, it has been demonstrated that CRAd can be used to express antigens and used as vaccines for vaccination against viruses such as human immunodeficiency virus (HIV), human papillomavirus (HPV), and hepatitis C virus (HCV).

[0132] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anticancer activity, such as recombinant Ad657 and its variants) can bind to cell surface receptors (e.g., to promote viral entry into cells). For example, the recombinant Ad described herein can bind to Coxsackie-adenovirus receptors (CAR) and / or Fc receptors (e.g., FcμR and FcγR), complement receptors (e.g., CR3 and / or C2qR).

[0133] In one aspect of the present invention, CRAd may comprise a nucleic acid encoding a polypeptide heterologous to Ad, such as an antigen, a cell surface receptor, a cell targeting polypeptide, etc. For example, CRAd-657-dl1101 / 1107-FolR is a recombinant Ad containing an intact E3 and expressing human folate receptor α. It is demonstrated herein that CRAd can be used to generate antibodies against known cancer antigens, such as folate receptor α.

[0134] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad with oncolytic anticancer activity, such as recombinant Ad657) can avoid binding (e.g., not binding) to a scavenger receptor (e.g., to facilitate viral entry into cells). For example, the recombinant Ad described herein avoids binding to a SREC receptor and / or a SR-A receptor.

[0135] In some cases, the recombinant Ad described herein (eg, recombinant Ad with oncolytic anticancer activity, such as recombinant Ad657) can avoid phagocytosis.

[0136] In some cases, a recombinant Ad described herein (eg, a recombinant Ad having oncolytic anti-cancer activity, such as recombinant Ad657) is non-pathogenic (eg, to a mammal being treated as described herein).

[0137] In some cases, the recombinant Ad described herein (eg, a recombinant Ad having oncolytic anti-cancer activity, such as recombinant Ad657) can infect dividing cells (eg, can only infect dividing cells).

[0138] The recombinant Ad described herein can be any suitable recombinant Ad (e.g., recombinant Ad with oncolytic anti-cancer activity) produced by recombinant DNA technology and methods known to those skilled in the art. Recombinant Ad can be any Ad produced by recombining substances (e.g., nucleic acids and / or polypeptides) from any organism other than the Ad from which the recombinant Ad is derived. For example, the recombinant Ad may include one or more substances that are not naturally present in the Ad (e.g., not naturally present in the Ad before recombination). In some cases, the recombinant Ad provided herein may be a chimeric Ad (e.g., may include viral elements from two or more (e.g., two, three, four, five or more) different Ad genomes).

[0139] These embodiments have been applied in the context of combining Ads with different HVRs from different Ads (i.e., shuffling HVRs), for example, HVR1 of Ad6 with HVR2-7 of Ad57, or HVR1 and 7 of Ad6 with HVR2-6 of Ad57, or HVR1 and 7 from Ad6 with HVR2-6 from Ad657.

[0140] The non-naturally occurring nucleic acids and / or polypeptides in Ad may be from any suitable source. In some cases, the non-naturally occurring nucleic acids and / or polypeptides in Ad may be from non-viral organisms. In some cases, the non-naturally occurring nucleic acids and / or polypeptides in Ad may be from viruses other than Ad. In some cases, the non-naturally occurring nucleic acids and / or polypeptides in Ad may be from Ads obtained from different species. In some cases, the non-naturally occurring nucleic acids and / or polypeptides in Ad may be from different Ad strains (e.g., strains with different serotypes). In some cases, the non-naturally occurring nucleic acids and / or polypeptides in Ad may be synthetic nucleic acids and / or synthetic polypeptides.

[0141] The recombinant Ad described herein (e.g., recombinant Ad with oncolytic anticancer activity, such as recombinant Ad657) can be derived from any suitable Ad (e.g., can include a genomic backbone from any suitable Ad). In some cases, the recombinant Ad described herein can be derived from an Ad with a low seropositivity rate. For example, 50% or less (e.g., 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less) of mammals (e.g., humans) may have been exposed to the Ad from which the recombinant Ad described herein is derived. Regarding seropositivity, the seropositivity rates of class C adenoviruses Ad6 and Ad657 are lower than those of the prototype Ad5 virus. In some cases, the recombinant Ad described herein can be derived from an Ad with reduced or eliminated side effects (e.g., phagocytosis and liver damage). The recombinant Ad can be derived from an Ad isolated from any suitable animal species. For example, Ad can be isolated from humans, non-human primates (e.g., monkeys such as Old World monkey species, e.g., rhesus macaques), fish, frogs, and snakes. In some cases, the recombinant Ad described herein can be derived from human Ad (HAd or HAdV). Recombinant Ad can be derived from any class of Ad (e.g., A, B, C, D, E, F, or G). In some cases, the recombinant Ad described herein can be derived from class C Ad (e.g., human class C Ad (HAd-C)). Recombinant Ad can be derived from any suitable Ad serotype (e.g., 2, 5, 6, or 57). In some cases, the recombinant Ad described herein can be derived from Ad serotype 6 (Ad6; e.g., human Ad6).

[0142] In some cases, the recombinant Ad described herein (e.g., recombinant Ad having oncolytic anti-cancer activity, such as recombinant Ad657 and variants thereof) may comprise an Ad genome comprising one or more modifications to one or more nucleic acids encoding a polypeptide (or fragment thereof) and / or one or more viral elements of the Ad genome. The one or more modifications may be any appropriate modifications. In some cases, the modification may effectively inhibit the ability of the modified polypeptide to bind to another polypeptide such as p300 and / or pRB. In some cases, the modification may effectively neutralize one or more interferon pathways. Examples of modifications that may be made to nucleic acids encoding polypeptides or viral elements include, but are not limited to, substitutions, deletions, insertions, and mutations.

[0143] Ad, such as Ad657 and its variants, can be modified to retain the entire E1A gene, or modified to delete selected regions and functions of its encoded protein.

[0144] Figure 57 Schematic diagram showing the different E3 immune evasion genes in Ad species C example Ad6 and Ad species D example Ad26, as well as describing the functions of these E3 encoded proteins. Both Ads express size and sequence variants of E3 12.5K, 6.7K, 19K, 10.4K (RIDα), 14.5K (RIDβ) and 14.7K genes. 19K reduces the display of MHC I and MIC proteins on the cell surface, thereby protecting infected cells from damage by T cells and NK cells. RID proteins protect infected cells from the effects of death-inducing ligands (FAS, TRAIL, TNFR and EGFR). 14.7K inhibits the intrinsic activation of apoptosis in infected cells. Species C Ad also expresses 11.6K, known as adenovirus death protein (ADP). Overexpression of ADP accelerates cell death, but overall cell death is equal. Species D viruses also express two new variants, known as 49K and 31K. The secreted form of 49K binds to CD46 on T cells and NK cells, resulting in downregulation of these cells and lower efficiency of NK cell killing of class I MHC-deficient cells. The Ad657 plasmid has been modified to retain all natural E3 immune evasion genes (12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ) and 14.7K) and E4 34K or to delete selected regions. Ad657 and its variants have also been modified by adding 49K and 31K to provide these additional functions to these C virus platforms.

[0145] Ad, such as Ad657 and its variants, can be modified to retain all E3 immune evasion genes or to delete selected regions and the function of their encoded proteins. Regarding E3 mutations: 19k downregulates MHCI and MIC proteins on infected cells; overexpression of ADP accelerates cell death without increasing the number of cells killed; the 10k and 14k proteins (RIDα and RIDβ) combine to block cell killing by exogenous apoptotic proteins (e.g., FAS, TRAIL, TNF, TNFR, and EGFR); and the 14.7k protein inhibits intrinsic apoptotic signaling.

[0146] Retaining these E3 proteins allows for longer-lasting oncolysis, whereas their deletion increases immune stimulation.

[0147] Data testing oncolytic efficacy suggest that intact E3 mediates better efficacy.

[0148] The DE3 construct has a portion of 12.5k deleted and includes 14.7k, the DE3A construct has a portion of E3 12.5k deleted and includes 19k, and the DE3ADP construct has a portion of E3 12.5k deleted and includes ADP.

[0149] Surprisingly, deletion of all E3 genes made the oncolytic virus less effective in suppressing tumor growth.

[0150] In embodiments, the present invention encompasses single-cycle adenoviruses, such as SC-Ad657 and variants thereof. Recombinant SC-Ad viruses harboring heterologous nucleic acid encoding polypeptides are evaluated for use as vaccines. The SC-Ad657 vaccine itself produces significant circulating antibody titers against HIV envelope proteins after only a single immunization.

[0151] Similarly, Ad657 carrying genes for hepatitis B and C antigens can generate cytotoxic T lymphocyte (CTL) responses against hepatitis and cytomegalovirus.

[0152] Ad657 is modified by inserting a synthetic peptide from human papillomavirus into HVR 5. In one embodiment, the amino acid sequence of the variant Ad657-HVR5-HPV hexon is defined by SEQ ID NO: 57. The modification allows for display of the antigen for vaccine purposes and retargeting by binding to proteins that interact with the HPV peptide.

[0153] In another embodiment, Ad657 is demonstrated herein to express human granulocyte-macrophage colony stimulating factor (GMCSF).

[0154] Thus, the examples herein demonstrate that recombinant Ad such as Ad657 and its variants can be used to express heterologous proteins such as polypeptide antigens and cell-targeting polypeptides.

[0155] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anticancer activity, such as recombinant Ad657) may comprise an Ad genome containing one or more substitutions. For example, one or more nucleic acids encoding a polypeptide (or fragment thereof) and / or one or more viral elements encoded by the Ad genome may be substituted. The substitution may be any appropriate substitution. In some cases, one or more nucleic acids encoding a capsid polypeptide of the genome of a first Ad may be substituted with one or more nucleic acids encoding a capsid polypeptide of a second Ad to produce a chimeric Ad. For example, when a recombinant Ad comprises a genome from a first Ad, wherein the nucleic acid encoding a capsid polypeptide in the genome is substituted with a nucleic acid encoding a capsid polypeptide from a second Ad (e.g., an Ad with a different Ad backbone), the nucleic acid encoding the capsid polypeptide forming the second Ad may express one or more capsid polypeptides, and the expressed capsid polypeptide(s) may be incorporated into the capsid of the recombinant Ad. Examples of capsid polypeptides include, but are not limited to, hexon polypeptides, pilin polypeptides, penton matrix polypeptides, IIIa polypeptides, IX polypeptides, and pVI polypeptides.

[0156] Ad fimbriae is a complex of three distinct subunits that mediates the initial cell attachment step. The native Ad6 fimbriae comprises the amino acid sequence shown in SEQ ID NO:60 and binds to CAR.

[0157] In one aspect of the invention, pili-modified recombinant and chimeric Ads are generated that possess pili proteins that are not native to the parent or "backbone" Ad.

[0158] One chimeric Ad, the AdF35 fimbriae chimera, has the amino acid sequence of SEQ ID NO: 61 and is shorter than the Ad5 and Ad6 fimbriae proteins and retargets the virus to CD46.

[0159] A fiber-modified recombinant Ad comprising a K7 pili having the sequence of SEQ ID NO: 62 targets the virus to heparan sulfate proteoglycans and negative charges on cells.

[0160] A recombinant chimeric Ad, 6 / FC68, comprising the sequence of SEQ ID NO: 63, is a chimeric Ad with a pilin from chimpanzee adenovirus C68. This pilin is shorter than Ad5 or Ad6 pilins and binds to CAR.

[0161] A recombinant chimeric Ad, 6 / FC68-K7, comprising the sequence of SEQ ID NO:64, is a chimeric Ad with a pilin protein from chimpanzee adenovirus C68. This pilin protein is shorter than that of Ad5 or Ad6. The 6 / FC68-K7 pilin binds CAR and is retargeted to heparin sulfate and negative charge.

[0162] A recombinant chimeric Ad, 6 / FC68-HI-K7 pili, comprising the sequence of SEQ ID NO:65, is a chimeric Ad with a pili protein from chimpanzee adenovirus C68. This pili protein is shorter than the pili proteins of Ad5 or Ad6. The 6 / FC68-HI-K7 pili bind to CAR and are retargeted to heparin sulfate and negative charges.

[0163] In some cases, the recombinant Ad may comprise a genome from a first Ad in which a nucleic acid encoding a hexon polypeptide (e.g., an HVR of a nucleic acid encoding a hexon polypeptide) is replaced by a nucleic acid encoding a hexon polypeptide from a second Ad (e.g., an HVR of a nucleic acid encoding a hexon polypeptide). In some cases, the recombinant Ad described herein may comprise a genome from a first Ad having one or more HVRs replaced by one or more HVRs from a second Ad. For example, the recombinant Ad may be a chimera, particularly Ad657 (e.g., may comprise an Ad6 genome in which the hexon HVRs are replaced by Ad57 hexon HVRs). In cases where the recombinant Ad comprises a genome from a first Ad in which a nucleic acid encoding a hexon polypeptide is replaced by a nucleic acid encoding a hexon polypeptide from a second Ad, the recombinant Ad may comprise from about 1 to about 720 hexon polypeptides from the second Ad. For example, when the recombinant Ad is Ad657, Ad657 may comprise an Ad6 genome and 720 hexon polypeptides comprising the Ad57 hexon HVRs.

[0164] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad with oncolytic anti-cancer activity, such as recombinant Ad657) may include an Ad genome containing one or more nucleic acid deletions. The nucleic acid deletion may be any suitable nucleic acid deletion. The nucleic acid deletion may be a complete deletion (e.g., a deletion of a nucleic acid encoding a polypeptide) or a partial deletion (e.g., a deletion of one or more nucleotides in a nucleic acid encoding a polypeptide). The nucleic acid deletion may reduce or eliminate the transcription and translation of the polypeptide encoded by the deleted nucleic acid. Any suitable nucleic acid may be deleted. In some cases, nucleic acids encoding polypeptides associated with the production of infectious offspring may be deleted. Examples of nucleic acids that may be deleted and / or modified in the recombinant Ad described herein may encode E1 (e.g., E1A and E1B), E2, E3, E4, pIIIA, cilia, E1B, and include viral enhancers and promoters. For example, the recombinant Ad described herein (e.g., a recombinant Ad with oncolytic anti-cancer activity, such as recombinant Ad657) may include an Ad genome comprising a deletion of one or more nucleotides in a nucleic acid encoding an E1 polypeptide. In some cases, the recombinant Ad described herein may comprise one or more substitutions in a nucleic acid encoding an E1 polypeptide.

[0165] In certain embodiments, the recombinant Ad described herein is modified to include a probasin promoter comprising, for example, a nucleic acid of SEQ ID NO: 48; the recombinant Ad described herein is modified to include a dl1101 deletion in the nucleic acid encoding the E1 polypeptide; the recombinant Ad described herein is modified to include a dl1107 deletion in the nucleic acid encoding the E1 polypeptide; the recombinant Ad described herein is modified to include a dl1101 deletion and a dl1107 deletion. For N-terminal amino acid sequences of E1A polypeptides (e.g., wild-type Ad E1A and CRAd-657-dl1101, CRAd-657-dl1107, and CRAd-657-dl1101 / 1107 variants), see Examples and Figure 56 .

[0166] In one embodiment, the variant CRAd-657-dl1101 / 1107-FolR comprises an intact E3 and expresses the human folate receptor alpha found on cancer cells.

[0167] Typically, Ad can be modified to include the CRAd modifications described herein.

[0168] In some cases, the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anticancer activity, such as recombinant Ad657) may include an Ad genome containing one or more nucleic acid insertions. For example, a nucleic acid insert may include a nucleic acid encoding a polypeptide. The nucleic acid may be inserted into any suitable position of the genome of the recombinant Ad described herein. In some cases, the nucleic acid encoding the polypeptide may be inserted into the HVR (e.g., HVR5 loop) of the genome of the recombinant Ad described herein. For example, when a nucleic acid encoding a polypeptide is inserted into the HVR of the genome of the recombinant Ad described herein, the nucleic acid encoding the polypeptide may express one or more polypeptides, and the expressed polypeptide (one or more) may be incorporated into the capsid of the recombinant Ad. In the case of inserting a nucleic acid encoding a polypeptide into the HVR of the genome of the recombinant Ad described herein, the recombinant Ad may present on its surface about 1 to about 720 polypeptides encoded by the inserted nucleic acid. The nucleic acid insert may be a nucleic acid encoding any suitable polypeptide. In some cases, the nucleic acid insert may encode a polypeptide antigen.

[0169] In some cases, the nucleic acid insert may encode a targeting polypeptide. Examples of targeting polypeptides that can be included in the recombinant Ad described herein include, but are not limited to, peptide 12.51 (TARGEHKEEELI; SEQ ID NO: 1), peptide 12.52 (LRQTGAASAVWG; SEQ ID NO: 2), 12.53 (ARRADTQWRGLE; SEQ ID NO: 3), VSV (GTWLNPGFPPQSCGYATVT; SEQ ID NO: 4), RGD (CDCRGDCFC; SEQ ID NO: 5), α4 integrin binding peptide (NMSLDVNRKA; SEQ ID NO: 6), Met3-4 (ISLSSHRATWVV; SEQ ID NO: 7), L10.1F (WTMGLDQLRDSSWAHGGFSA; SEQ ID NO: 8), L10.1RGDF (WTMGLDQLRGDSSWAHGGFS; SEQ ID NO: 9), L10.2F (RSVSGTEWVPMNEQHRGAIW; SEQ ID NO: 10). NO:10), L10.5F (TELRTHTSKELTIRTAASSD; SEQ ID NO:11), S5.1 (DRAIGWQDKLYKLPLGSIHN; SEQ ID NO:12), DU9C.1 (MGSWEKAALWNRVSASSGGA; SEQ ID NO:13), DU9C.2 (MAMGGKPERPADSDNVQVRG; SEQ ID NO: 14), DU9A.7 (MASRGDAGEGSTQSNTNVPS; SEQ ID NO: 15), NO:18), REDV-4C (ACDCREDVCFCG; SEQ ID NO:19), SKBR5C1 (GQIPITEPELCCPWTEAFY; SEQ ID NO:20), 231R10.1 (PQPPNSTAHPNPHKAPPNTT; SEQ ID NO:21), HepaCD8 (VRWFPGGEWGVTHPESLPPP; SEQ ID NO:22), K20 (KKKKKKKKKKKKKKKKKK; SEQ ID NO:23), BAP (GLNDIFEAQKIEWH; SEQ ID NO:24), CALM BP (CAAARWKKAFIAAVSAANRFKKIS; SEQ ID NO:25), EBV (EDPGFFNVEIPEFP; SEQ ID NO:26), #1-5 (GGHGRVLWPDGWFSLVGISP; SEQ ID NO:27), ##4*-5 (MARTVTANVPGMGEGMVVVPC; SEQ ID NO:28), 1-1 (GVSKRGLQCHDFISCSGVPW; SEQ ID NO: 29), 1-2 (NQSIPKVAGDSKVFCWWCAL; SEQ ID NO: 30), 1-3 (QSTPPTKHLTIPRHLRNTLI; SEQ ID NO: 31), 1-4 (DMSFQLVTPFLKALPTGWRG; SEQ ID NO: 32), 1-5 (GGHGRVLWPDGWFSLVGISP; SEQ ID NO: 33), 1-5con (FSLVGISP; SEQ ID NO: 34), 1-6 (QIMMGPSLGYYMPSESIFAY; SEQ ID NO: 35), 2-11 (ISWDIWRWWYTSEDRDAGSA; SEQ ID NO: 36), 2-14 (VWGMTTSDHQRKTERLDSPE; SEQ ID NO: 37), 2-20 (MTSAQTSEKLKAETDRHTAE; SEQ ID NO: 38), 2-9 (MGSRSAVGDFESAEGSRRP; SEQ ID NO:39), 3b-6 (MGRTVQSGDGTPAQTQPSVN; SEQ ID NO:40), 4*-5 (MARTVTANVPGMGEGMVVVP; SEQ ID NO:41), CLL peptide, PD-1, GLA polypeptide (e.g., Factor X), antigen genes, fusion proteins, fusion-producing glycoproteins, single-chain antibodies, and capsid proteins from other viruses. Targeting polypeptides can target any suitable cell type. Examples of cell types that can be targeted by targeting polypeptides included in the recombinant Ad described herein include, but are not limited to, muscle cells (e.g., skeletal muscle cells), tumors, cancer cells, kidney cells, hepatocytes, mucosal cells, carbohydrates, and cell membranes.

[0170] Examples demonstrate that peptides selected from a phage library within a compatible structural context can be translated into the Ad hexon protein. For example, for the 12.51 peptide, this insertion site increased muscle transduction while reducing off-target infection in the liver. Therefore, this muscle-targeted recombinant Ad can be used as a vector for gene-based intramuscular vaccination or for gene therapy application / delivery to muscle.

[0171] In some cases, the nucleic acid insert can detarget the virus (e.g., by disrupting cell and protein interactions occurring at a given HVR). In some cases, the nucleic acid insert can encode a detectable label. Examples of detectable labels include, but are not limited to, fluorophores (e.g., green fluorescent protein (GFP), mCherry, and mBFP) and enzymes (e.g., luciferase, DNA enzyme, protease, transporter, and polymerase).

[0172] Also provided herein are expression vectors containing recombinant Ad described herein (e.g., recombinant Ad with oncolytic anticancer activity, such as recombinant Ad657 and its variants). The expression vector can carry the recombinant Ad described herein to another cell (e.g., cancer cell), where it can be replicated and / or expressed. An expression vector, also commonly referred to as an expression construct, is typically a plasmid or vector with an enhancer / promoter region that controls the expression of a specific nucleic acid. After the expression vector is introduced into a cell, it can utilize the cell protein synthesis mechanism to produce a virus in the cell. In some cases, the expression vector containing the recombinant Ad described herein can be a viral vector. For example, the expression vector containing the recombinant Ad described herein can be a retroviral vector. In some cases, the expression vector containing the recombinant Ad described herein can also be designed to allow insertion of one or more transgenes (e.g., at a multiple cloning site). For example, the expression vector containing the recombinant Ad described herein can also include a nucleic acid encoding a detectable marker. Examples of detectable markers include, but are not limited to, fluorophores (e.g., green fluorescent protein (GFP), mCherry, and mBFP) and enzymes (e.g., luciferase, recombinase, nuclease, and transcription factor).

[0173] The present invention also provides methods and materials utilizing one or more recombinant Ad described herein (e.g., recombinant Ad with oncolytic anti-cancer activity, such as recombinant Ad657). In some cases, the recombinant Ad provided herein may be used to treat a mammal having cancer or at risk of cancer. For example, a method for treating a mammal having cancer or at risk of cancer may include administering one or more recombinant Ad described herein to the mammal. In some cases, a method for treating a mammal having cancer or at risk of cancer may include administering to the mammal one or more expression vectors encoding the recombinant Ad described herein or nucleic acids encoding the recombinant Ad described herein. In some cases, one or more recombinant Ad described herein may be administered to a mammal to reduce the number of cancer cells in the mammal (e.g., to suppress and / or delay tumor growth) and / or to increase the survival of the mammal.

[0174] Targeting cancerous tumors by serotype conversion of oncolytic adenoviruses was demonstrated. Mice with DU145 or LNCaP prostate tumors on their flanks were treated once with an intravenous (IV) injection of Ad657. These mice were treated a second time with alternative Ad6 or Ad6 / 57 / 6 oncolytic virus variants that had cilia modifications and expressed GFP luciferase, and luciferase activity was measured by imaging. Ad6 has an Ad6 hexon and cilia targeting CAR. Ad6-F35 has an Ad6 hexon and Ad35 cilia targeting CD46. Ad6 / 57 / 6 has HVR1 and 7 from Ad6, and HVR2-6 from Ad57. The Ad6 / 57 / 6 virus has Ad6 cilia, AdC68 cilia, or Ad35 cilia. Figure 9 These data in demonstrate the surprising ability to serotype-switch oncolysis with a virus that targets tumors with reduced off-target infection of the liver.

[0175] In another example of serotype conversion, mice with LNCaP prostate tumors on their flanks were treated with a single intravenous (IV) injection of Ad657 or CRAd657. Five months later, these mice were treated again with alternative Ad6 / 57 / 6 oncolytic viruses expressing GFP luciferase and cilia variants K7 (7 lysines added), F35 (with Ad35 cilia), or KKTK-C68 (Ad6 KKTK flexible domains fused to chimpanzee C68 cilia). The KKTK-C68 virus also adds a codon-optimized E434K gene to increase viral productivity. Luciferase activity was measured by imaging. All Ad6 / 57 / 6 have hexons, which have HVR1 and 7 from Ad6 and HVR 2-6 from Ad57. Ad6 / 57 / 6 and KKTK-C68 have cilia targeting CAR. Ad6 / 57 / 6-F35 has Ad35 cilia targeting CD46. K7 increases binding to negatively charged cellular structures, including heparan sulfate proteoglycans. Figure 70 The ability to serotype-switch oncolysis with a virus that targets tumors with reduced off-target infection of the liver was demonstrated.

[0176] Any suitable mammal having or at risk of having cancer, infectious diseases, and / or genetic diseases can be treated as described herein. For example, as described herein, humans, non-human primates (e.g., monkeys), horses, cattle, pigs, dogs, cats, mice, rats, and farmed animals having cancer, infectious diseases, and / or genetic diseases can be treated for cancer. In some cases, humans having cancer can be treated. In some cases, the mammal (e.g., human) treated as described herein is not the natural host of the Ad used to produce the recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity, such as recombinant Ad657). For example, a human treated with the recombinant Ad657 described herein may lack any pre-existing acquired immunity to Ad6.

[0177] Mammals with any type of cancer can be treated as described herein. In some cases, the cancer can include one or more solid tumors. In some cases, the cancer can be a blood cancer. Examples of cancers that can be treated as described herein include, but are not limited to, prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, and leukemia (e.g., lymphocytic leukemia and myeloid leukemia).

[0178] In some cases, the methods described herein may further include identifying that a mammal has cancer. Examples of methods for identifying that a mammal has cancer include, but are not limited to, physical examinations, laboratory tests (e.g., blood and / or urine), biopsies, imaging studies (e.g., X-rays, PET / CT, MRI and / or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy and / or genetic testing. Once identified as having cancer, infectious diseases and / or genetic diseases, the mammal may be administered or instructed to self-administer one or more recombinant Ad described herein (e.g., recombinant Ad with oncolytic anticancer activity, e.g., recombinant Ad657) or nucleic acids (e.g., expression vectors) encoding one or more recombinant Ad described herein (e.g., recombinant Ad with oncolytic anticancer activity, e.g., recombinant Ad657).

[0179] One or more recombinant Ad described herein (e.g., recombinant Ad with oncolytic anticancer activity, such as recombinant Ad657) can be administered by any suitable route. In some cases, administration can be topical administration. In some cases, administration can be systemic administration. Examples of routes of administration include, but are not limited to, intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, parenteral, intratumoral, retro-ureteral, subcapsular, vaginal, and rectal administration. In the case of administering multiple rounds of treatment, a first round of treatment may comprise administering one or more recombinant Ad described herein to a mammal (e.g., a human) by a first route (e.g., intravenous), and a second round of treatment may comprise administering one or more recombinant Ad described herein to a mammal by a second route (e.g., intramuscular).

[0180] As used herein, the term "pharmaceutical composition" refers to a combination of one or more recombinant and / or chimeric Ads of the present invention and an inert or active carrier, such that the composition is particularly suitable for therapeutic use. One or more recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity, such as recombinant Ad657) can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a mammal suffering from or at risk of suffering from cancer). For example, one or more recombinant Ads can be formulated into a pharmaceutically acceptable composition for administration to a mammal suffering from cancer or at risk of suffering from cancer. In some cases, one or more recombinant Ads can be formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. Pharmaceutical compositions for administration in solid or liquid form can be formulated, including but not limited to sterile solutions, suspensions, sustained-release preparations, tablets, capsules, pills, powders, wafers, and granules. Pharmaceutically acceptable carriers, fillers, and vehicles that can be used in the pharmaceutical compositions described herein include, but are not limited to, saline (e.g., phosphate-buffered saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based materials, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol, and lanolin. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" (EW Martin, 18th edition). The selection and use of suitable excipients are taught in Remington: The Science and Practice of Pharmacy (Gennaro ed., 20th edition, Lippincott Williams & Wilkins 2003).

[0181] A composition (e.g., a pharmaceutical composition) comprising one or more recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity, such as recombinant Ad657) can be administered as a vaccine to a mammal (e.g., a mammal having cancer or at risk of developing cancer). The vaccine can be prophylactic or therapeutic.

[0182] In some cases, the methods described herein may further include administering one or more other agents / means for treating cancer to a mammal (e.g., a mammal with cancer). The one or more other agents / means for treating cancer may include any suitable cancer treatment. In some cases, cancer treatment may include surgery. In some cases, cancer treatment may include radiotherapy. In some cases, cancer treatment may include the administration of drug therapy, such as chemotherapy, hormone therapy, targeted therapy and / or cytotoxic therapy. For example, one or more recombinant Ad described herein (e.g., recombinant Ad with oncolytic anticancer activity, such as recombinant Ad657 and its variants) may be administered to a mammal with cancer, and one or more other agents / means for treating cancer may be administered. In the case of treating a mammal with cancer with one or more recombinant Ad described herein and with one or more other agents / means for treating cancer, infectious diseases and / or genetic diseases, the other agents / means for treating cancer, infectious diseases and / or genetic diseases may be administered simultaneously or independently. For example, one or more recombinant Ad described herein and one or more other agents for treating cancer, infectious diseases and / or genetic diseases may be formulated together to form a single composition. In some cases, one or more recombinant Ads described herein may be administered first, followed by one or more other agents for treating cancer, infectious diseases, and / or genetic diseases, or vice versa. Example

[0183] Example 1. Adenovirus

[0184] For 50 years, there have been only four known species C human Ads, including Ad1, Ad2, Ad5, and Ad6 (see, e.g., Weaver et al., 2011 Virology. 412: 19–27). In 2001, field isolate #16700 was identified as the fifth species C adenovirus, and virus neutralization tests using antisera against Ad1, 2, 5, and 6 showed high levels of neutralization (reciprocal titers of 500–16,000) when each antiserum was used against its homologous virus (Lukashev et al., 2008 J Gen Virol. 89: 380–388). In contrast, anti-Ad1, 2, and 5 antibodies had weak cross-reactivity against #16700 (reciprocal titers of 32–64). Anti-Ad6 serum showed higher cross-reactivity against #16700, but compared to Ad6 itself, a 10-fold higher serum concentration was required to neutralize #16700. Subsequent sequence comparison confirmed #16700 to be a new species C adenovirus and renamed it Ad57 (see, eg, Walsh et al., 2011 J. Clin Microbiol. 49:3482-3490).

[0185] Fifteen human Ad strains were evaluated for oncolytic activity against breast, ovarian, liver, prostate, kidney, and B-cell malignancies. In tests against DU145 human prostate tumors, species C Ad6 was more effective than species C Ad5 or species B viruses Ad11 and Ad35 after a single intratumoral or intravenous (iv) injection. In immunocompetent Syrian hamsters, Ad6 was also more effective than Ad5 and Ad11.

[0186] Construction of recombinant adenovirus

[0187] Recombinant adenoviruses are constructed by recombinant DNA technology using methods known to those skilled in the art. The recombinant Ad is derived from a first Ad (e.g., can include the genome of a first Ad, such as Ad6), and can include hexon HVRs from a second Ad (e.g., Ad57). In the case where the recombinant Ad includes the Ad6 genome and the Ad57 hexon HVRs, the recombinant Ad can be a chimeric Ad referred to as Ad657.

[0188] To obtain Ad657, the Ad57 HVR sequence was synthesized and inserted into the Ad6 hexon in a plasmid with a 5′-HVR located between pVI and the hexon. It was recombined into various pAd6 plasmids to produce Ad657 and its variants. The amino acid sequence of Ad657 hexon is shown in SEQ ID NO: 49. The plasmid maps of Ad657 variants are shown in Figure 34 and Figures 59 to 65 .

[0189] Regarding the Ad657 variant, the Ad57 HVR sequence was synthesized with HVR1 modified with cysteine, flexible amino acids, and restriction sites to allow insertion of other peptides. This was inserted into the Ad6 hexon of a plasmid with a 1:1 region between pVI and the hexon. This was recombined into various pAd6 plasmids to generate an Ad657 variant with a cysteine ​​in HVR1, designated Ad657-HVR1-XXA, comprising a hexon having the amino acid sequence of SEQ ID NO:50.

[0190] Regarding the Ad657 variant, the Ad57 HVR sequence was synthesized with HVR5 modified with cysteine, flexible amino acids, and restriction sites to allow insertion of other peptides. This was inserted into the Ad6 hexon of a plasmid with a pVI located between the pVI and hexon. It was recombined into various pAd6 plasmids to generate an Ad657 variant with a cysteine ​​in HVR5, designated Ad657-HVR5-XXA, comprising a hexon having the amino acid sequence of SEQ ID NO:51.

[0191] Regarding the Ad657 variant, the Ad57 HVR sequence was synthesized with HVR1 modified with cysteine, flexible amino acids, and restriction sites to allow insertion of other peptides. It was inserted into the Ad6 hexon of a plasmid with a 5′-pVI located between the pVI and hexon. It was recombined into various pAd6 plasmids to generate an Ad657 variant lacking cysteine ​​in HVR1 but having a restriction site allowing peptide insertion into HVR1, termed Ad657-HVR1-XA, comprising a hexon having the amino acid sequence of SEQ ID NO:52.

[0192] Regarding the Ad657 variant, the Ad57 HVR sequence was synthesized with HVR5 modified with cysteine, flexible amino acids, and restriction sites to allow insertion of other peptides. This was inserted into the Ad6 hexon of a plasmid with a pVI located between the pVI and hexon. It was recombined into various pAd6 plasmids to generate an Ad657 variant lacking cysteine ​​in HVR5 but having a restriction site allowing peptide insertion into HVR5, which was designated Ad657-HVR5-XA and contains a hexon having the amino acid sequence of SEQ ID NO:53.

[0193] Regarding variants of Ad657, the Ad657 HVR1-XA sequence was modified by inserting a biotin acceptor peptide into HVR1. This was recombined into various pAd6 plasmids to generate an Ad657 variant with a BAP in HVR1, designated Ad657-HVR1-PSTCD, containing a hexon having the amino acid sequence of SEQ ID NO: 54.

[0194] Insertion of the biotin acceptor peptide detargets the viral variant from the liver, allows retargeting of the virus with avidin or streptavidin and a biotinylated ligand, and permits purification of the virus on a monomeric avidin or streptavidin column.

[0195] Regarding variants of Ad657, the Ad657 HVR1-XA sequence was modified by inserting a biotin acceptor peptide into HVR1. This was recombined into various pAd6 plasmids to generate an Ad657 variant with a BAP in HVR1, designated Ad657-HVR5-PSTCD, containing a hexon having the amino acid sequence of SEQ ID NO: 55.

[0196] Regarding a variant of Ad657, the Ad657 HVR5-XA sequence was modified by inserting a synthetic V1 / V2 loop from the HIV envelope into HVR5. This variant, designated Ad657-HVR5-V1 / V2, comprises a hexon having the amino acid sequence of SEQ ID NO:56.

[0197] Insertion of a synthetic V1 / V2 loop from the HIV envelope allows display of this antigen for use as a vaccine as well as retargeting by binding to proteins that interact with the HIV envelope.

[0198] Regarding a variant of Ad657, the Ad657 HVR5-XA sequence was modified by inserting a synthetic peptide from human papillomavirus into HVR5. This variant, designated Ad657-HVR5-HPV, comprises a hexon having the amino acid sequence of SEQ ID NO:57.

[0199] Insertion of synthetic peptides from human papillomavirus allows for display of HPV peptides as antigens for vaccine purposes and for retargeting by binding to proteins that interact with the HPV peptides.

[0200] In another aspect of the present invention, a chimeric Ad having Ad6 HVR1 and Ad57 HVRs 2-7 was generated. This chimera is referred to as the Ad6 / 57 HVR chimera and comprises a hexon having the amino acid sequence of SEQ ID NO:58.

[0201] In yet another aspect of the present invention, a chimeric Ad having Ad6 HVR1 and 7 and Ad57 HVR2-6 was generated, which chimera is referred to as the Ad6 / 57 / 6 HVR chimera, comprising a hexon having the amino acid sequence of SEQ ID NO:59.

[0202] Example 2. Retargeted and Detargeted Recombinant Adenoviruses for Gene Delivery

[0203] Adenovirus is a robust vector for gene delivery and gene-based immunity. The prototype adenovirus for the vast majority of these applications is human adenovirus serotype C serotype 5 (HAdV-C5 or Ad5). In vitro, Ad5 binds and enters cells through a combination of its pili and penton matrix proteins that interact with cell surface receptors. This trimeric pili binds to the coxsackie-adenovirus receptor (CAR), and cells lacking CAR are relatively resistant to infection unless they also express an α-receptor that can be bound by the RGD motif on the penton matrix. v Integrin.

[0204] In vivo, these interactions are still exploited, but their importance varies depending on the route of injection. If injected directly into solid tissue or tumors, the CAR and integrin interactions predominate. If injected intravenously (IV), these interactions become secondary due to the influence of Ad5 binding to vitamin K-dependent coagulation factors. Blood factor X (FX) binds to the hexon of Ad5 with subnanomolar affinity, thus enabling Ad5 to efficiently transduce liver hepatocytes after IV injection. In the absence of FX, liver transduction is dramatically reduced.

[0205] Compared to other vectors such as adeno-associated virus (AAV) vectors, which have only 4.5 kb of DNA sequence, adenoviral vectors are somewhat unique in their ability to carry very large cDNA sequences of up to 36 kilobase pairs (kbp). This payload capacity was justified for early exploration of Ad vectors for muscle gene therapy when delivering very large transgenes such as the 14 kbp dystrophin cDNA. IV administration can mediate muscle gene delivery in newborn mice, but this ability is lost in adult mice. Part of the reason for the decrease in transfection with age is the very large size of the Ad virus particles (i.e., 100 nm) and the loss of CAR receptors on muscle cells with age. Despite the fact that CAR is not present on skeletal muscle cells, the intramuscular (IM) route is by far the most popular route for gene-based vaccines using Ad5 and other serotypes.

[0206] Therefore, Ad5 and other Ad serotype transduction of muscle may be sufficient for gene therapy or gene-based vaccination. However, the lack of the primary viral receptor in muscle reduces viral efficacy and requires the delivery of larger amounts of vector to achieve the desired effect.

[0207] Construction of peptide-modified hexons in adenovirus

[0208] The 12-amino acid (12-mer) peptide on C2C12 mouse myocytes was selected from a random peptide library displayed between the knot region H and the Iβ sheet of Ad5 ( Figure 1 A). Peptides 12.51 (TARGEHKEEELI; SEQ ID NO: 1) and 12.52 (LRQTGAASAVWG; SEQ ID NO: 2) were selected for myoblasts and pre-cleared against non-target cells to obtain peptides that specifically bind to myocytes. In most cases, small peptides have relatively low affinity. Therefore, it was hypothesized that displaying these muscle-selected peptides on 720 copies of Ad5 hexon might better achieve muscle gene delivery. This insertion site may also have the benefit of inactivating hexon-bound FX, thereby "de-targeting" the vector from the liver, if any vector leaks into the blood after IM injection.

[0209] The ability of the modified Ad to modulate tropism was assessed by inserting these muscle-selective peptides between two other β sheets that also constrain the hypervariable loops on the virus. Peptides 12.51 and 12.52 were introduced into the hypervariable region (HVR) 5 loop, which is constrained by the β7 and β8 sheets of Ad5 hexon. The ability of these viruses to transduce tissues in vivo following intravenous and intramuscular injection in mice and hamsters was assessed.

[0210] Adenovirus

[0211] Ad5-GL lacking E1 (RD-Ad5-GL) expresses a green fluorescent protein-luciferase (GFP-luciferase, GL) fusion protein as described elsewhere (see, e.g., Crosby et al., 2002 J. Virol., 76:6893-6899; Khare et al., 2011 Mol. Ther., 19:1254-1262; and Khare et al., 2012 J. Virol., 86:2293-2301). According to methods known to those skilled in the art, muscle-selective peptides 12.51 and 12.52 were inserted between the β7 and β8 sheets of the Ad5 hexon at the HVR5 position, which is similar in structure to the H and I β sheets from the knob region of Ad5 ( Figure 1 A). Peptide with flexible leader sequence replaces the entire HVR5 loop in Ad5 ( Figure 1 B). These modified hexon sequences were introduced into replication-defective Ad5 expressing green fluorescent protein-luciferase fusion protein (RD-Ad5-GL) by red recombination in bacteria (see, e.g., Campos et al., 2004 Hum. Gene Ther., 15: 1125-1130). Figure 1 B) Annealed oligonucleotides inserted into plasmid pHVR5 display ( Figure 1 A below) to construct peptide-modified Ad to generate recombinant Ad, Ad5-HVR5-12.51 and Ad5-HVR5-12.52.

[0212] The resulting plasmids were digested and used for red recombination into pAd5-GL. These vectors were rescued in 293 cells, purified on two consecutive CsCl gradients, and desalted on an Econopac 10-DG column (Bio-Rad) into 50 mM Tris pH 8 containing 0.5 M sucrose and stored at -80°C.

[0213] In vitro virus testing

[0214] C2C12 mouse myoblasts were purchased from the American Type Culture Collection (Manassas, VA). 293 cells were obtained from Microbix (Toronto, Ontario, Canada). Cells were maintained in DMEM containing 10% FBS (Invitrogen).

[0215] C2C12 myocytes were plated in 6-well plates (Corning) one day before injection. Viruses were used to infect cells in DMEM with 5% FBS. Cells were incubated for 2 days before observation under green fluorescence.

[0216] Animal experiments were performed in accordance with the Animal Protection Act, the PHS Animal Care Policy, and the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Mayo Clinic Institutional Animal Care and Use Committee. Female CD-1 mice (Charles River) were anesthetized and injected intramuscularly (IM) or intravenously (IV) for 10 10 vp of the indicated viruses. The animals were anesthetized and injected with 3 mg of d-luciferin (Molecular Imaging Products) and imaged on a Xenogen imaging system. Afterwards, the animals were anesthetized and blood was collected in a serum separator for ELISA.

[0217] The ELISA was performed as follows. Immulon 4HBX plates (Thermo) were incubated with 100 ng / well GFP protein in 1X phosphate-buffered saline (PBS) at 4°C overnight, washed, and blocked with 5% milk in TRIS-buffered saline containing 0.1% Tween 20 (TBST) at room temperature for 2 hours. A 1:100,000 to 1:1,1000 dilution of each serum sample was prepared in blocking buffer. The wells were washed, and 100 μL of each was added to the GFP-coated plate in triplicate and incubated at room temperature for 3 hours. The wells were washed and a 1 / 10,000 dilution of goat anti-mouse-HRP secondary antibody (Pierce Chemical) was added to each well. The plate was incubated at room temperature for 2 hours, washed, and then 50 μL of 1Step Ultra TMB ELISA (Thermo Fisher Scientific Inc.) was added for HRP detection, followed by 50 μL of 2M H2SO4. The absorbance at 450 nm was measured using Beckman Coulter DTX880.

[0218] Statistical analysis was performed using Prism (Graphpad). Statistical significance was calculated by one-way ANOVA followed by Tukey's HSD.

[0219] In vitro transduction in mouse C2C12 myocytes

[0220] RD-Ad5-GL, RD-Ad5-GL-HVR5-12.51 and RD-Ad5-HVR5-12.52 were used to infect mouse C2C12 myoblasts at different multiplicities of infection (MOI) expressed as viral particles (vp) / cell. When the green fluorescence from the GFP fusion protein was observed by fluorescence microscopy, the transduction mediated by the two peptide-modified vectors was significantly better than that of RD-Ad5-GL ( Figure 2 A). When luciferase activity was measured, a significant increase was observed in cells infected with RD-Ad5-GL-12.51 and 12.52 ( Figure 2 B) When one of the peptides, 12.51, was inserted back into the knob region of Ad5, the peptide increased transduction of C2C12 myoblasts in vitro by 14-fold.

[0221] In vivo transduction of mice following intramuscular injection

[0222] Put 10 9 vp of Ad5-GL, Ad5-GL-HVR5-12.51, and Ad5-GL-HVR5-12.52 were injected into both quadriceps muscles of mice via the IM route, and luciferase imaging was performed at different times ( Figure 3 A, upper panel). At all time points, Ad5-GL-HVR5-12.51 produced 2- to 3-fold higher luciferase activity than Ad5-GL (one-way ANOVA, p<0.05 on day 1) ( Figure 3 B (left panel). Ad5-GL-HVR5-12.52 was less active in muscle than Ad5-GL and HVR5-12.51, but more active in vitro.

[0223] In vivo transduction of mice following intravenous injection

[0224] 3×10 10 vp of Ad5-GL, Ad5-GL-HVR5-12.51, and Ad5-GL-HVR5-12.52 were injected into mice via IV route, and luciferase imaging was performed ( Figure 3 A below). In contrast to the results in muscle, only unmodified Ad5-GL mediated strong liver transduction. Liver transduction by Ad5-GL was 60-fold higher than that by Ad5-GL-HVR5-12.51 and Ad5-GL-HVR5-12.52 (one-way ANOVA, p<0.001 on day 1) ( Figure 3 B (right panel) shows that the recombinant Ad targets muscle tissue while reducing off-target infection in the liver.

[0225] In vivo transduction after intramuscular injection in hamsters

[0226] To test whether the 12.51-modified vector works in species other than mice, 10 10 Ad5-GL and Ad5-GL-HVR5-12.51 were injected IM into both quadriceps muscles of older Syrian hamsters and luciferase imaging was performed 24 hours later ( Figure 4 In this case, Ad5-GL-HVR5-12.51 mediated 7-fold higher luciferase activity than Ad5-GL (one-way ANOVA, p < 0.01 on day 1).

[0227] Gene-based immunization after intramuscular injection in mice

[0228] Sixteen weeks after IM injection, the samples were collected as described above and as Figure 3 Sera from mice treated as indicated were collected and serial dilutions were analyzed by ELISA for antibodies against transgene-encoded GFP ( Figure 5 ). Compared with the PBS group, all Ad-injected mice produced significant anti-GFP antibodies at serum dilutions of 1:10,000 to 1:1000 (p<0.0001 by one-way ANOVA). However, Ad5-GL-HVR-12.51 produced higher antibodies than Ad5 or Ad5-HVR5-12.52. In serum dilutions of 1:1000 to 1:10,000, Ad5-GL-HVR-12.51 was significantly higher than Ad5-GL (p<0.01 to 0.0001 by one-way ANOVA). In serum dilutions of 1:1000, 12.51 was significantly higher than 12.52 (p<0.05). In serum dilutions of 1:1000 and 1:10,000, Ad5-GL-12.52 was significantly higher than Ad5-GL (p<0.05 to 0.001).

[0229] This example demonstrates that peptides selected from a phage library can be translated into the Ad hexon protein within a compatible structural context. For example, for the 12.51 peptide, this insertion site increased muscle transduction while reducing off-target infection in the liver. Therefore, this muscle-targeted recombinant Ad can be used as a vector for gene-based intramuscular vaccination or for gene therapy application / delivery to muscle.

[0230] Another aspect of the present invention relates to recombinant and / or chimeric Ad comprising additional cell targeting peptides inserted into the Ad657 HVR and the Ad6 and C68 HI loops, as described in Table 1.

[0231] Table 1.

[0232] Other cell-targeting peptides inserted into the Ad657 HVR and the Ad6 and C68 HI loops

[0233]

[0234] Selected peptides for B-cell cancers:

[0235]

[0236] DNA encoding the indicated peptides and their complementary DNA were synthesized, flanked by cohesive ends for ligation into Ad plasmids (e.g., XA hexon plasmid or pAd6-NdePfl pilinary shuttle plasmid). These annealed oligonucleotides were ligated into the HVR or HI loop of Ad. These plasmids were used for recombination into various Ad backbone plasmids.

[0237] Some peptides are used to target new receptors on cells. Others, such as the small BAP, can be used for avidin targeting and purification if the virus is grown in cells expressing the bacterial biotin ligase BirA. Calmodulin peptides allow the virus to bind to calmodulin or calmodulin fusion proteins for retargeting or virus purification.

[0238] Therefore, such recombinant Ads targeted to specific tissue / cell receptors can be used as vectors for gene-based vaccination in targeted cells and / or tissues or for gene therapy applications.

[0239] Example 3. Insertion of individual HVRs from different Ad serotypes and insertion of cell targeting / detargeting peptides or novel amino acids

[0240] Hexon shuttle plasmid map ( Figure 34 ) shows the insertion of individual HVRs from different Ad serotypes into hexon and a combination of insertion of cell targeting / detargeting peptides or novel amino acids such as cysteine ​​for targeted chemical modification and shielding.

[0241] In certain embodiments, the cell binding peptides 12.51, VSV, RGD (see Table 1) are inserted into HVR1 or HVR5, and these embodiments serve as examples for inserting these and other peptides into any HVR of Ad ( Figure 34 ). Another example shows the insertion of a biotin acceptor peptide (BAP) into these HVRs, allowing retargeting of the vector using avidin or streptavidin and a biotinylated ligand, or using an avidin fusion protein or a streptavidin fusion protein. The BAP insertion also allows purification of the virus on a monomeric avidin or streptavidin column for vector production. Similarly, Ad57-HVR1-XXA and XA show an example of the insertion of a cysteine ​​into this site to allow for targeted chemical modification using maleimide or other cysteine-reactive agents ( Figure 34 ).

[0242] These embodiments also apply to cases where Ads are combined with different HVRs from different Ads (i.e., HVR shuffling). For example, HVR1 of Ad6 with HVR2-7 of Ad57 or HVR1 and 7 of Ad6 with HVR2-6 of Ad57. In another embodiment, a 6 / 56 / 6 virus has HVR1 and 7 from Ad6 with HVR2-6 from Ad657.

[0243] Example 4. Targeted chemical conjugation of cysteine-modified hexon-modified Ad657-HVR5C

[0244] Figure 35 Ad variants are depicted showing the combined insertion of individual HVRs from different Ad serotypes and novel amino acids such as cysteine ​​into the hexon for targeted chemical modification and shielding.

[0245] Comparison of the effects of non-targeted and targeted chemical conjugation on the shielding and function of cysteine-modified hexon-modified Ad657-HVR1C ( Figure 36 This example demonstrates the ability to target polymers and other chemical modifications to the cysteine ​​residues of the Ad hexon. Non-targeted PEGylation inactivates viral infection, while PEGylation targeted to the cysteine ​​residues preserves viral function.

[0246] In one aspect, the present invention contemplates the use of polymers or intercalated peptides / proteins for detargeting, retargeting, and shielding of antibodies, proteins, and cells. Figure 54 Sites of the Ad HVR that can be modified, for example, by PEGylation or "BAPylation" are described.

[0247] In embodiments, different Ad serotypes and / or variants include polymer shielding to allow multiple administrations of Ad6 and Ad657 variants. An exemplary treatment cycle is shown where Ad6 and Ad657 can be used for multiple rounds of treatment by combining serotype switching with covalent polymer conjugation ( Figure 41 B).

[0248] Ad657-HVR1C expressing GFP luciferase was produced by cells and purified on a CsCl gradient. The virus was covalently modified with 5 kDa polyethylene glycol (PEG). The virus was treated with NHS-PEG that reacts randomly with amines / lysines on viral proteins or with maleimide PEG that reacts specifically with cysteines inserted into HVR1 using the XXA shuttle plasmid. These unmodified or modified viruses were then purified by a final CsCl spin followed by desalting. The viruses shown were separated on SDS-PAGE gels, stained with SyproRuby, and visualized by imaging ( Figure 36 A). This indicates that NHS-PEGylation randomly modifies many viral proteins, as evidenced by an increase in protein apparent mass (indicated by arrows). In contrast, targeted maleimide-PEG reaction with cysteine ​​in HVR1 modifies only the hexon, leaving other capsomere proteins unaffected. The effect of PEGylation on viral function was assessed.

[0249] The indicated viruses were incubated with A549 cells and their ability to infect cells was measured by luciferase assay. This showed that random NHS-PEGylation reduced viral activity by more than 90%, while maleimide-PEG did not ( Figure 36 B).

[0250] Subcutaneous HaK renal carcinoma tumors were implanted in immunocompetent Syrian hamsters. When these reached a volume of 200 μl, they were injected once with the indicated Ad6 virus by the intravenous route, wherein the indicated Ad6 virus was constructed with and without E3 (DE3) and with or without random NHS-PEGylation. Tumor size was measured over time. The data showed that the deletion of all E3 genes in the oncolytic virus Ad6-deltaE3-Luc made the virus less effective in reducing tumor volume than the oncolytic parent virus Ad6-Luc. The data also showed that Ad6 can be PEGylated and maintain efficacy (see Ad6-Luc compared to Ad6-Luc-20kDa PEG) ( Figure 58 ).

[0251] Targeted chemical conjugation of cysteine-modified hexon-modified Ad (e.g., Ad657-HVR5C). Ad657-HVR5C expressing GFP luciferase was produced by cells and purified on a CsCl gradient. The virus was covalently modified with a maleimide-IR800 near-infrared fluorophore specifically reactive with cysteine ​​(inserted into HVR5 using its XXA shuttle plasmid), maleimide-biotin, or 5 kDa maleimide-PEG. The Ad and modified Ad shown were separated on SDS-PAGE gels, stained with SyproRuby, and visualized by imaging ( Figure 37 A). SDS-PAGE of viral proteins followed by near-infrared imaging showed that HVR-C can be labeled with an imaging agent ( Figure 37 B) The effect of PEGylation on Ad virus function in vivo was demonstrated by intraperitoneal injection of PEGylated Ad virus. The ability to infect cells in tumor-bearing mice was demonstrated by imaging of detectable luciferase activity. Figure 37 C demonstrates the ability to target polymers and other chemical modifications to the cysteine ​​inserted into the hexon region of Ad657. Furthermore, PEGylation was demonstrated to detarget adenovirus to the liver in vivo ( Figure 50 ).

[0252] Example 5. Ad657 expresses human granulocyte-macrophage colony-stimulating factor (GMCSF)

[0253] Ad657 carrying the cDNA of human GMCSF was used to infect A549 cells, and different amounts of supernatant were added to GMCSF growth-dependent TF-1 cells. Increased cell numbers indicate the expression of functional human cytokine ( Figure 27). The data show that recombinant Ad can be used to express heterologous proteins.

[0254] Example 6. Oncolytic adenovirus Ad657 for systemic viral therapy against cancer cells

[0255] Figure 6 Shown is a phylogenetic tree generated by aligning selected complete Ad genomes, which clusters Ad57 with other species C viruses that are most homologous to Ad6.

[0256] Ad57 appears to be nearly identical to Ad6, but with sequence differences in the hexon hypervariable region (HVR) and the E3 immune evasion gene ( Figure 7 Other exposed viral capsid proteins, including the pili, penton matrix, IIIa, and IX, are nearly identical between Ad6 and Ad57. Neutralization data are consistent with the fact that most adenovirus neutralizing antibodies target the HVRs on Ad. The low cross-reactivity between Ad6 antiserum and Ad57 is thought to be due to the antibodies potentially targeting their common pili proteins (Lukashev et al., 2008 J Gen Virol. 89:380-388).

[0257] In this example, the utility of Ad657 as an oncolytic agent against human prostate cancer is demonstrated. The Ad6 HVR was replaced with the HVR from Ad57 to generate a chimeric class C oncolytic virus called Ad657. Ad657 and Ad6 were tested as systemic oncolytic therapies in nude mice bearing human prostate cancer tumors by a single IV injection. The liver and tumor tropism of the virus were evaluated in a mouse model of prostate cancer as follows.

[0258] DU145 human prostate cancer cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and confirmed to be specific pathogen-free by RADIL's IMPACT assay. 293 cells were obtained from Microbix (Toronto, Ontario, Canada). Cells were maintained in DMEM (Invitrogen, Grand Island, NY, USA) supplemented with 10% FBS.

[0259] The genome of Ad6, Tonsil 99 strain (ATCC VR-1083), was cloned as described elsewhere (see, e.g., Weaver et al., 2013 PLoS One. 8:e73313). A cassette corresponding to the Ad57 hexon between the native ApaI and Sad sites was synthesized by Genscript. This fragment was cloned into a vector containing the Ad6 pVI and hexon genes with a Homologous recombination in bacteria was performed in the shuttle plasmid pUC57-Ad6 Hexon-FZF (containing a 57-resistant gene-FRT cassette) as described elsewhere (see, e.g., Campos et al., 2004 Hum Gene Ther. 15:1125–1130; and Khare et al., 2012 J Virol. 86:2293–2301). The Ad6 ApaI-SacI fragment was replaced with the Ad57 fragment, generating the plasmid pUC57-Ad6 / 57Hexon-FZF. This was recombined into the Ad6 genome by red recombination (Campos et al., 2004 Hum Gene Ther. 15:1125–1130). Figure 59 The plasmid map of Ad657 with E3 deletion is shown. Virus was rescued by transfection into 293 cells and produced from a 10-plate CellStack (Corning Life Sciences, Lowell, MA, USA). Viruses expressing green fluorescent protein-luciferase (GFP-Luc) fusion protein have a CMV-GFP-Luc expression cassette inserted between the Ad pili and E4, and lack E3 to make room for the insert. Viruses were purified on two CsCl gradients, and the number of viral particles (vp) was calculated by OD260.

[0260] To examine in vitro oncolytic activity, cells were treated with the indicated multiplicity of infection (MOI) expressed in vp / cell in DMEM (Invitrogen, Grand Island, NY, USA) with 5% FBS and antibiotic-antimycotic agents. After 5 days, the culture medium was removed and the cells were treated with crystal violet (0.05% crystal violet, 3.7% formaldehyde in phosphate-buffered saline; Invitrogen, Grand Island, NY, USA) for 10 minutes. The cells were washed twice with PBS and then incubated overnight at 37°C in 0.1% sodium dodecyl sulfate in PBS to dissolve the crystal violet. The crystal violet absorbance was measured at OD595 on a Beckman Coulter DTX 880 plate reader. Cell viability (%) was calculated by dividing the OD of the sample by the mean OD of the untreated control cells on the same 96-well plate and multiplying the number by 100.

[0261] Animals were housed in the Mayo Clinic Research Animal Facility in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. This study was approved by the Mayo Clinic Research Animal Use and Care Committee in accordance with the Animal Protection Act and the PHS Animal Care Policy. Four-week-old nude mice (Harlan Sprague Dawley, Indianapolis, IN, USA) were injected subcutaneously with 1 × 10 dapoxetine (dapoxetine) in 100 μL DMEM / 50% Matrigel (BD Biosciences, San Jose, CA, USA). 7 DU145 cells initiated subcutaneous tumors. Tumor volume was calculated using the equation 2 =Calculated as x length x 1 / 2. When tumor volume reached approximately 200 μL, mice were divided into different groups and treated by a single iv injection into the tail vein. Animals were euthanized when tumor volume reached approximately 2000 μL or when the animals were moribund, in distress, or when the skin over the tumor was broken.

[0262] For blood alanine aminotransferase (ALT) measurement, 6 C57BL / 6 mice in each group were injected iv via the tail vein for 10 11 vp of Ad5, Ad6, or Ad657, and blood was collected 3 days later for ALT measurement using an ALT activity assay (Sigma-Aldrich, St. Louis, MO, USA).

[0263] Statistical analysis was performed using Prism (Graphpad) by repeated measures ANOVA or one-way ANOVA followed by Tukey's HSD test. Kaplan-Meier survival curves were plotted and compared by log-rank test.

[0264] Except for their hexon HVRs, the viral capsid genes of Ad57 are almost identical to those of Ad6 ( Figure 6 and Figure 7 To generate chimeric viruses of Ad57 and Ad6, a cassette corresponding to the Ad57 hexon HVR was recombined into the wild-type Ad6 genome ( Figure 8 The virus was rescued and produced in 293 cells and purified on a CsCl gradient. Considering that the base viral genome is Ad6, these hexon chimeric viruses are called Ad657 ( Figure 59 ).

[0265] In vitro oncolytic activity was assessed by infecting LNCap and DU145 cells with 10, 100, or 1000 vp / cell. To compare the liver damage caused by Ad5, Ad6, and Ad657, each virus was inoculated at 10 11High doses of vp were injected intravenously into immunocompetent C57BL / 6 mice. Animals injected with Ad5 became moribund within 2 days and had to be euthanized ( Figure 10 A). Survival was significantly reduced for Ad5 and Ad6 compared to PBS (p = 0.0001 and 0.0009, respectively, by log-rank analysis). Survival was also reduced for Ad657 compared to PBS (p = 0.0578). Survival after exposure to Ad6 or Ad657 was significantly better than that of mice treated with Ad5 (p = 0.0001 and 0.0001, respectively). There was no statistical difference in survival between Ad6 and Ad657 (p = 0.248).

[0266] ALT was measured in the blood of surviving Ad6 and Ad657 animals 3 days after injection. Ad5-treated animals were not tested because most of this group needed to be sacrificed. This assay showed that the level of liver damage caused by Ad6 was relatively low in terms of the release of hepatic ALT enzyme in the blood ( Figure 10 B). Both Ad6 and Ad657 groups had low but significant ALT levels compared to PBS-treated mice (p < 0.001 for both viruses by one-way ANOVA with Tukey's multiple comparison test). ALT levels were lower for Ad657 than for Ad6 (ANOVA, p < 0.001). This is consistent with the higher levels of Ad6 infection in the liver after iv injection of luciferase-expressing virus than for Ad657 ( Figure 12 One Ad657 animal was lost after bleeding on day 3. By day 6, most Ad6 animals had become moribund ( Figure 10 A). In contrast, 50% of Ad657 animals survived 2 weeks after treatment.

[0267] To compare the oncolytic activity of Ad6 and Ad657 against human DU145 prostate tumors, nude mice were transplanted sc with DU145 cells. The animals were divided into groups with similar tumor sizes averaging 200 μL, and each group of 9 mice was treated with 3 × 10 10 vp dose of Ad6 or Ad657 single treatment ( Figure 11 ).

[0268] This single iv injection of Ad6 and Ad657 reduced tumor size compared to control animals injected with PBS. Tumors in the Ad6 group were significantly smaller over 7 days compared to the PBS group (p<0.05 by two-way ANOVA and Tukey's multiple comparison test). By day 14, tumors in the Ad657 group were significantly different from those in the PBS group (ANOVA, p<0.01). By day 38, both Ad6 and Ad657 remained significantly different from PBS (p<0.0001 by two-way ANOVA). The comparison ended at day 38, when the first animal in the PBS group had to be sacrificed because subsequent comparisons would be biased by changes in animal numbers. Tumor sizes in the Ad6 and Ad657 groups did not differ significantly until day 38, when tumor volumes in the Ad657 group were significantly larger (p<0.05 by two-way ANOVA) ( Figure 11 A) This difference in tumor size between Ad6 and Ad657 persisted until day 52 (p=0.04 by T-test), after which the tumors were not significantly different.

[0269] When survival due to all causes was assessed, both Ad6 and Ad657 significantly prolonged survival compared to animals treated with PBS ( Figure 11 B, p < 0.01 and 0.05, respectively, by log-rank analysis). Ad6 survival due to all causes was significantly better than Ad657 (p < 0.05). However, this is an artifact of survival due to all causes, as three Ad657 animals had to be sacrificed due to ulceration of the skin overlying the tumors rather than due to excessive tumor size, in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. In some cases, ulceration was actually associated with effective tumor control. Like Ad6, Ad657 expressing GFP luciferase produced significant luciferase activity in distant DU145 subcutaneous tumors after a single iv injection ( Figure 12 and Figure 13 This suggests that both Ad6 and Ad657 can mediate oncolysis in prostate tumors after a single systemic treatment.

[0270] This example demonstrates that Ad657 can be used as a local or systemic oncolytic virus therapy for prostate cancer. These data also demonstrate the surprising effect of serotype switching using the oncolytic species C Ad.

[0271] The oncolytic activity of Ad was evaluated in tumor cells and / or cancerous tumors. In immunocompetent hamsters, a single IV injection of Ad6 was evaluated for the oncolytic activity of A549 lung tumor cells ( Figure 28 ); evaluated the efficacy of a single IV or intratumoral (IT) injection of Ad6 for the treatment of Panc1 pancreatic tumors ( Figure 29 ), and evaluated a single IV injection of Ad6 for renal cancer ( Figure 30 ).

[0272] Figure 38 Luciferase imaging of nude mice is shown. A) 1, 4, 7, 14, 28, and 42 days after a single IV injection of Ad6 into a distant DU145 prostate tumor. B) 3, 7, and 19 days after IV injection of replicative Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

[0273] It can be concluded that Ad6 reaches distant target cells after IV injection.

[0274] In another embodiment, luciferase-expressing Ads with and without peptide library-generated peptides 12.51 and 12.52 inserted into HVR5 of hexon were incubated with the indicated number of viral particles (vp) on the indicated cell lines B16 melanoma and A549 lung cancer cells, and luciferase activity was measured. Improved infection of cancer cells by Ads with peptide-modified hexons was demonstrated ( Figure 31 ).

[0275] Ad with peptide-modified hexon improves infection of cancer cells

[0276] Luciferase-expressing Ad was cloned with peptides 12.51 and 12.52 generated from the peptide library with and without insertion into HVR5 of the hexon at a rate of 10 4 vp were incubated on the indicated hepatocellular carcinoma cell lines and luciferase activity was measured. Figure 32 ).

[0277] Example 7. Different HIV-1-directed immune responses in rhesus monkeys generated by systemic and mucosal immunization with replicating single-cycle adenovirus

[0278] Most gene-based adenovirus vaccines are replication-defective Ad (RD-Ad) vectors, which have a deletion of the E1 gene to prevent them from replicating and causing Ad infection. Helper-dependent adenovirus (HD-Ad) has all Ad genes deleted and is also replication-defective. E1-deleted Ad vaccines can infect cells, deliver one copy of their antigen gene, and express a single copy of that antigen (e.g., "1X"). They are safe but do not replicate the transgene or its expression.

[0279] In contrast, E1+ replication-competent Ad (RC-Ad) vaccines can infect the same cells, replicate antigen gene DNA 10,000-fold more than E1-deficient vectors, produce substantially more antigen, and stimulate a stronger immune response. Although RC-Ad is more potent than RD-Ad, replication-competent Ad may pose a real risk of causing significant adenoviral infection in humans.

[0280] In order to utilize transgenic DNA replication but avoid the risk of adenovirus infection, a single-cycle Ad (SC-Ad) vector with a deletion of the key viral late protein pIIIa gene was developed (Crosby et al., 2014. Virology 462-463:158-165; Crosby et al., 2015 J Virol 89:669-675; Anguiano-Zarate et al., 2018 J Infectious Dis 218:1883-1889; and Crosby et al., 2017 Genes (Basel) 8:E79). SC-Ad retains its E1 gene to allow it to replicate its genome, but the absence of pIIIa blocks the production of infectious progeny viruses. SC-Ad replicates its genome and transgene as well as RC-Ad (up to 10,000 times; Crosby et al., 2014. Virology 462-463:158-165). RC-Ad and SC-Ad produce more transgenic protein than RD-Ad vectors (Crosby et al., 2014. Virology 462-463:158-165). SC-Ad produces a more robust and longer-lasting immune response than RD-Ad or RC-Ad (Crosby et al., 2015 J Virol 89:669-675). In a head-to-head comparison, SC-Ad produces significantly higher antibodies and better protection against influenza virus (Crosby et al., 2017 J Virol 91:e00720-16).

[0281] In this study, rhesus macaques were immunized with SC-Ad expressing clade B envelope sequences obtained from HIV-1 patients (before and after their antibody responses underwent expansion neutralization breadth). Rhesus macaques were immunized by a single systemic IM immunization or by a single mucosal intranasal (IN) immunization. The animals were then boosted with SC-6Ad by the same or alternative routes, followed by a protein boost. This example describes how these different SC-Ad immunization strategies affect HIV-bound antibody-dependent cellular cytotoxicity (ADCC) and the production of neutralizing antibodies, as well as their effects on cellular immune responses, including T follicular helper (pTfh) cells in the blood and lymph nodes.

[0282] Single-cycle adenovirus expressing HIV-1 envelope protein gp140

[0283] The evolutionary branch B gp160 envelope sequence that appeared before (G4) and immediately before (F8) the peak of antibody neutralization from HIV patient VC10014 was used as an immunogen (Malherbe et al., 2014 J Virol 88: 12949-12967). The motif-optimized G4 and F8 gp160 sequences were recombined into SC-Ad based on human Ad serotypes 6 and 57 (see, e.g., Crosby et al., 2014. Virology 462-463: 158-165; Crosby et al., 2015 J Virol 89: 669-675; Anguiiano-Zarate et al., 2018 J Infectious Dis 218: 1883-1889; and Nguyen et al., 2018 Oncolytic Virotherapy 7: 43–51). A control SC-Ad expressing Ebola virus glycoprotein was also used. Viruses were rescued and purified as described elsewhere (see, e.g., Crosby et al., 2014. Virology 462-463: 158-165; Crosby et al., 2015 J Virol 89: 669-675; and Anguiano-Zarate et al., 2018 J Infectious Dis 218: 1883-1889).

[0284] The envelope gene F8 used in the SC-Ad vector, cloned from a subject infected with HIV clade B, was sequence-optimized and modified by site-directed mutagenesis to express uncleaved trimeric gp140. Details of expression, purification, and antigen characterization have been described elsewhere (see, e.g., Malherbe et al., 2014 J Virol 88:12949-12967).

[0285] Adult female rhesus macaques (Macaca mulatta) of Indian origin were housed in a specific pathogen-free breeding colony at the Michael Keeling Center for Comparative Medicine Research at the University of Texas MD Anderson Cancer Center (Bastrop, TX). All animal handling was performed in accordance with the policies and procedures of the Mayo Clinic and the University of Texas MD Anderson Cancer Center, the provisions of the Animal Welfare Act, the PHS Animal Welfare Policy, and the principles of the NIH Guide for the Care and Use of Laboratory Animals.

[0286] Rhesus monkeys were anesthetized with ketamine and treated with 2x10 10 The SC-Ad vaccine containing viral particles (vp) was administered intranasally (IN) or intramuscularly (IM). TMAdjuvanted with 50 μg of purified trimeric recombinant F8 gpl40 was boosted by the IM route as described elsewhere (see, eg, Malherbe et al., 2014 J Virol 88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078).

[0287] Peripheral venous blood samples were collected in EDTA. Before isolating peripheral blood mononuclear cells (PBMC), plasma was separated and immediately stored at -80 ° C. PBMC was prepared from blood on a Ficoll-Hypaque density gradient. Saliva and vaginal swabs were collected in 1 ml of PBS containing protease inhibitors with Wek-Cel Spears, vortexed, and collected after centrifugation at 2,000 rpm as described elsewhere (see, e.g., Kozlowski et al., 1997 Infect Immun 65: 1387-1394). Before further use, samples were stored frozen at -80 ° C.

[0288] ELISPOT assay for detecting antigen-specific IFN-γ-producing cells

[0289] Freshly isolated PBMCs were treated with F8 gp140 protein (1 μg / mL) or heat-inactivated Ad6 (7.0×10 8 vp / well) to determine the number of IFN-γ producing cells by enzyme-linked immunospot (ELISPOT) assay using methods described elsewhere (see, e.g., Nehete et al., 2017 Comp Med 67:67-78; Nehete et al., 2013 PLoS One 8:e79836; and Nehete et al., 2017 J Am Assoc Lab Anim Sci 56:509-519). Briefly, aliquots (10 5 Lymphocytes were plated in duplicate in wells of 96-well plates (polyvinylidene difluoride-backed plates, MAIP S 45, Millipore, Bedford, MA) pre-coated with an anti-IFN-γ antibody. The cells were then stimulated with Con A, F8 gp140 protein, or heat-inactivated Ad6. After incubation at 37°C for 30-36 hours, the cells were removed and the wells were thoroughly washed with PBS and developed according to the manufacturer's protocol. Results are expressed as IFN-γ spot-forming cells (SFC) / 10, minus the number of replicate wells treated with culture medium alone (negative control). 5 PBMCs, if greater than twice the background and greater than 5 SFC / 10 5 PBMCs were considered positive.

[0290] Antibody ELISA

[0291] HIV-1 envelope binding antibody titers were measured against F8 gpl40 or SF162 gpl40 in regularly collected plasma samples as described elsewhere (Malherbe et al., 2014 J Virol 88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078).

[0292] Neutralization test

[0293] HIV neutralization was performed using the TZM-bl neutralization assay as described elsewhere (Malherbe et al., 2014 J Virol 88: 12949-12967; and Hessell et al., 2016 J Immunol 196: 3064-3078). All values ​​were calculated compared to virus-only wells.

[0294] Antibody-dependent cellular cytotoxicity (ADCC)

[0295] CEM.NKR.CCR5.CD4+-Luc target cells were treated with 50ng SHIV SF162P3 Infection and culture for 4 days as described elsewhere (see, e.g., Alpert et al., 2012 PLoS Pathog 8:e1002890). Two-fold serial dilutions of each sample were added to the infected target at room temperature for 20 minutes. CD16-KHYG-1 effector cells were added at a ratio of 10:1 effector cells to targets and incubated for another 8 hours. The cells were lysed and luciferase activity was measured using a Bio-Tek plate reader.

[0296] Flow cytometry

[0297] Cells collected from rectal and lymph node biopsies were incubated with 0.2 μg gp140 or culture medium alone in a GolgiPlug TMAfter incubation, cells were harvested and incubated on ice for 45 minutes with a panel of human antibodies that cross-reacted with rhesus monkey samples. The panel included the following fluorescent dye-labeled antibodies: CD8 (Qdot655), α4β7 (PE), and CXCR5 (PE), all obtained from the Nonhuman Primate Reagent Resource; CD69 (BV737, clone FN50) and FoxP3 (PECy5, clone: ​​PCH101), obtained from eBioscience; IL-21 (BV421, clone: ​​3A3-N2.1), CD45 (BV786, D058-1283), and CD3 (clone SP34-2, PE-Cy7 labeled), all obtained from BD Bioscience (San Jose, CA); and CD4 (Pacific Blue, clone OKT4), from ThermoFisher Scientific (Waltham, MA). Antibody dilutions were determined according to the manufacturer's recommendations. Dead cells were excluded using the live-dead fixable dead cell staining kit from Invitrogen (Carlsbad, CA). Subsequently, the cells were washed twice with PBS containing 2% FBS and 2mM EDTA, and then fixed and permeabilized using the FoxP3 Fix / Perm kit (ThermoFisher Scientific, Waltham, MA). Intracellular markers FoxP3 and IL-21 were stained in permeabilization buffer. Compensation controls (OneComp eBeAds, (ThermoFisher Scientific, Waltham, MA) and fluorescence minus one (FMO) controls were used. All samples were collected on an LSRFortessa X-20 analyzer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo, LLC, Ashland, Oregon). Approximately 2x10 5 Up to 1x10 6 an event.

[0298] SHIV SF162P3 Rectal attack

[0299] SHIV SF162P3The virus was derived from R157 harvest 3 (3.16.12). The stock solution had a P27 content of 66 ng / ml, an RNA content of Log~9.35, a TCID50 in Indian rhesus monkey PBMC: 1288 / ml, and a TCID50 in TZM-bl cells: 4.1x10 4 / ml. Use 1 ml of a 1:300 diluted stock solution. This is equivalent to 4.3 TCID50 on rhesus monkey PBMC and 137 TCID50 on TZM-bl cells. This dose is used for weekly intrarectal (IR) challenges. Plasma samples are analyzed for SHIV viral RNA copy number (by Leidos Biomedical Research, Inc., Frederick National Laboratory). Animals with RNA copy numbers higher than 10 are considered infected, and the number of attacks required to infect the animal is used as an event for Kaplan-Meier survival analysis. Once infected, animals are no longer challenged. Before the end of the study, plasma viral load is regularly monitored by the same method.

[0300] SHIV in tissues SF162P3 viral load

[0301] PBMC and postmortem tissues were collected at the end of the study. PBMC and digestive tract samples were analyzed for SHIV by qPCR. SF162P3 Prism 7 graphics software was used for all statistical analyses.

[0302] SC-Ad expressing HIV-1 gp160

[0303] Clade B envelope protein sequences were identified before (G4 gp160) and immediately before (F8 gp160) the expansion of neutralization breadth in antibodies from HIV patient VC10014. These gp160 sequences were inserted into SC-Ad6 and SC-Ad657 under the control of a strong cytomegalovirus promoter ( Figure 24 A). Ad57 is a class C human Ad that is almost identical to Ad6, but with variations in its hexon hypervariable region (HVR) and E3 immune evasion gene ( Figure 24B). Most Ad neutralizing antibodies target the hexon HVR of Ad (Pichla-Gollon et al., 2007 J Virol 81:1680-1689; and Sumida et al., 2005 J Immunol 174:7179-7185). In view of this, the HVR of Ad6 was replaced with the HVR from Ad57 to generate a chimeric species C Ad vector called Ad657. Both SC-Ad6 and SC-Ad657 retain all Ad genes, including E1, and lack functional pIIIA and E3 genes ( Figure 24 A). Thus, both SC-Ad vectors replicated their genomes to amplify gp160 expression but did not produce progeny Ad viruses. Both viruses were rescued and produced in 293-IIIA cells and purified on a CsCl gradient. When used to infect A549 cells, both vectors produced gp160 as determined by Western blotting.

[0304] Different Ad vectors have been previously tested in rhesus monkeys by the systemic intramuscular (IM) route and multiple mucosal routes, including oral gavage, oral enteric-coated capsules, intranasal (IN), and intravaginal (IVAG). Testing of SC-Ad-G4 by the IM, IN, and IVAG routes in small animals showed that the antibody response elicited by the IVAG route was negligible. In contrast, IN immunization in mice and hamsters both produced strong antibody responses. Given these data and the potential difficulties of IVAG immunization in humans, the IN route was chosen as the mucosal immunization route in subsequent rhesus monkey studies.

[0305] Single mucosal and systemic immunization in rhesus monkeys

[0306] 2x10 10 vp of SC-Ad6-G4 Env was used to vaccinate groups of 8 female rhesus macaques by a single IM or IN immunization ( Figure 14 This dose is relatively low, approximately 7.5-fold lower than the recently used RC-Ad HIV envelope vaccine delivered by mixed IN and IM immunization. The negative control vector group was immunized IN with SC-Ad6 expressing Ebola virus glycoprotein (gp). After 4 weeks, Env binding antibodies against F8 gp140 were measured in plasma samples ( Figure 14 This showed that the midpoint binding titer was significantly higher in the IM immunization group after a single immunization (p < 0.01 by ANOVA). SF162 neutralizing antibody (NAb) titers also increased at this time point, but did not reach significance by ANOVA across the various routes.

[0307] IM vs IN boost with SC-Ad6 at week 4

[0308] It has been reported that the generation of anti-adenovirus neutralizing antibodies by a single Ad IM immunization can be avoided by boosting with a different route (Xiang et al., 2003 J Virol 77:10780-10789). To test this route concept to enable repeated use of the same Ad serotype in rhesus macaques, each SC-Ad6 primed group was divided into two groups of four. Each of these was boosted at week 4 with SC-Ad6 expressing an alternative F8 Env by either the IM or IN route. Plasma samples collected 3 weeks after this boost showed that the prime-boost midpoint binding titers were elevated in animals in the IM-IM, IM-IN, and IN-IM groups. No detectable antibodies were observed in the IN-IN group ( Figure 14 ).

[0309] SC-Ad657 boost at week 13

[0310] Animals were then boosted at week 13 by serotype switching with SC-Ad657 expressing G4 Env. The same route was used as in the previous boost. Titers at week 15 showed that Env binding titers in IM primed animals increased to nearly 350, but these levels were not significantly different from controls ( Figure 14 In contrast, antibodies in the IN-IM-IM group were significantly higher than those in the vehicle control and IN-IN-IN groups (p < 0.01). Even after three immunizations, the IN-IN-IN group still showed no Env antibodies.

[0311] Recombinant trimeric Env protein boost at week 24

[0312] Most HIV vaccine studies use protein boosting to enhance Ad immunity to enhance antibody responses. For example, in a recent study, RD-Ad26 vectors were used twice and boosted three times with adjuvanted gp140 protein (Barouch et al., 2015 Science 349(6245):320-4). To determine whether this strategy would enhance SC-Ad vaccines, all SC-Ad-Env groups were administered with ADJUPLEX® via the IM route. TM The adjuvant was supplemented with 50 μg of recombinant F8 trimeric gp140 protein. The F8 trimeric protein increased the midpoint binding titer by two orders of magnitude in all groups ( Figure 14 This protein immunization also increased IN-IN-IN to levels comparable to those of the other groups, even though no Env-binding antibodies were detected after the earlier SC-Ad immunization.

[0313] Binding and neutralizing antibodies in plasma after the second protein boost

[0314] The animals were boosted with protein a second time at week 38. This increased F8-binding plasma antibody titers to nearly 10 by week 40. 5, and all groups became significantly different from the control ( Figure 14 At week 40, neutralizing antibody (NAb) titers against the grade 1A SF162 virus increased to 100 to 10,000 ( Figure 15 NAbs against the class 1B virus SS1196 and the class 2 JRCSF virus increased to 100 in most animals, except for two animals in the IN-IN-IN group whose titers were at background levels ( Figure 15 ).

[0315] ADCC activity after the second protein boost

[0316] SHIV SF162P3 Antibody-dependent cellular cytotoxicity (ADCC) activity in plasma was tested for infected cells at week 40. ADCC activity was generally higher in animals that had received at least one IN mucosal SC-Ad immunization ( Figure 16 ). The maximum %ADCC of all animals receiving mucosal immunization was significantly higher than that of SC-Ad-Ebola virus control animals (p < 0.05, 0.0001, and 0.0001 for IM-IN-IN, IN-IM-IM, and IN-IN-IN, respectively). When compared by 50% ADCC titer, only the IN SC-Ad prime group had significantly higher ADCC activity than the control (p < 0.05 and 0.001 for (IN-IM-IM and IN-IN-IN groups) by ANOVA).

[0317] Antibody responses in saliva and vaginal washes after the second protein boost

[0318] The above data monitor systemic antibody responses in plasma. Saliva and vaginal wash samples were also collected at week 40 and antibodies were measured in these mucosal sites. When saliva and vaginal washes were measured for F8 and SF162 env binding by ELISA, these responses were observed in most groups except the SC-Ad-Ebola control group ( Figure 25 ).

[0319] There appears to be a local effect on these mucosal antibodies. In animals immunized with SC-Ad primarily by the IN route (IM-IN-IN and IN-IN-IN), binding antibodies were higher in saliva close to the site of immunization but lower in more distal vaginal sites ( Figure 25 When ADCC activity was measured in these mucosal samples, the responses were highly variable ( Figure 17 ). Nevertheless, higher ADCC activity was observed in the IN-IN-IN group compared to control animals (ANOVA, p<0.05).

[0320] Systemic cellular immune response after a single protein boost

[0321] Env- and adenovirus-specific T cells were measured by ELISPOT in PBMC at week 38 for samples collected immediately before the second F8 Env protein boost. All Env-immunized animals had Env-specific IFN-γ secreting cells in their PBMC ( Figure 18 A). In animals that received at least one mucosal immunization, Env-specific IFN-γ SFC levels generally increased. However, when compared to control animals immunized with SC-Ad Ebola virus, IFN-γ SFC was significantly higher only in the IN-IN-IN SC-Ad group (ANOVA, p < 0.05). At this time point, anti-Ad SFC was relatively low compared to anti-Env SFC in all groups.

[0322] Systemic cellular immune response after the second protein boost

[0323] At week 40, Env-specific IFN-γ SFC ( Figure 18 B) This protein boost increased Env-specific SFC in PBMC and lymph nodes to similar levels in all Env-immunized animals.

[0324] Mucosal cell trafficking

[0325] Flow cytometry of rectal biopsy samples at week 40 showed similar numbers of α4β7CD4 and CD8 cells in the rectum ( Figure 19 A). There was a trend toward increased numbers in the IN prime group, but these did not reach significance. The number of activated CD69+CD4+ cells in rectal tissue was similar between the groups ( Figure 19 B). Similarly, there was no significant difference in FoxP3+CD4+ cells in this mucosal site ( Figure 19 B).

[0326] Distribution of antigen-specific Tfh cells

[0327] CXCR5+IL-21+CD4+ follicular helper T (Tfh) cells were measured in PBMC and lymph node samples at week 40 ( Figure 20 Animals immunized with Ad and protein only by IM route had significantly higher peripheral Tfh (pTfh) cells in PBMC compared with other groups ( Figure 20 In the lymph nodes, Tfh cells were lowest in the control and IM-only groups. In contrast, approximately half of the animals that received at least one IN mucosal immunization had detectable Tfh in their lymph nodes after the last protein boost ( Figure 20 ).

[0328] Using SHIV SF162P3 Perform a rectal attack

[0329] HIV isolate SHIV SF162P3 Immunized rhesus monkeys were challenged rectal. Four unimmunized control animals were enrolled in the study and each group was vaccinated weekly with SHIV provided by NIH via rectal administration. SF162P3 The challenge was performed with 1 ml of a 1:300 dilution of the challenge stock solution. This challenge is equivalent to 4.3 TCID on rhesus monkey PBMCs. 50 and 137TCID on TZM-bl cells 50 After the first challenge, 2 animals in the non-immunized control group and 2 animals in the IM-IM-IM group became infected ( Figure 21 One animal in each mixed-route group (IM-IN-IN and IN-IM-IM) became infected after one challenge. No animal in the IN-IN-IN group became infected after the first challenge. Plasma viral loads showed that all animals except the Ebola virus group reached high viral loads after 3 challenges ( Figure 22 B) Animals in the IN-IN-IN group had a delay in reaching these high viral loads.

[0330] As the challenge continued, animals in all groups became infected, with the exception of one animal in the Ebola group which remained uninfected after seven challenges. Trim5α and MHC alleles were retrospectively examined (Table 2). This analysis did not reveal clear protective genes in the animals in the resistant Ebola groups. Most animals could not be classified with alleles that would have made them moderately protected, but most groups had at least one animal with a higher likelihood of protection by virtue of these alleles. It should be noted that 2 / 4 of the animals in the IM-IM-IM and IN-IN-IN groups had Trim5a and MHC alleles that may indicate protection against SIVsmm and possibly SHIV SF162P3 have a higher likelihood of innate protection (Table 2).

[0331] Table 2 Retrospective screening of SIV protective gene alleles

[0332]

[0333] When animals were grouped according to whether they were primed with SC-Ad by the IM or IN route and Kaplan-Meier survival analysis was performed, infection of the eight IM-primed animals was paralleled with infection of the control animals ( Figure 22 A) In contrast, infection was slightly delayed in 8 animals primed with SC-Ad-Env via the mucosal IN route.

[0334] Postmortem viral load in tissues

[0335] The challenge study was terminated 9 weeks after the first challenge. PBMC and digestive tract tissues were isolated, RNA was purified, and the SHIV viral genome was evaluated ( Figure 22 B). Postmortem PBMCs had different levels of SHIV viral RNA, with the level in the IN-IN-IN group slightly lower than that in the IM-IM-IM group. The average viral RNA in the colon was 15-fold lower in the IN-IN-IN group than in the IM-IM-IM group ( Figure 23 The difference did not reach significance by ANOVA, but the p value obtained by two-tailed T test was 0.0079.

[0336] This example demonstrates that replicating SC-Ad vectors can be used as a robust and safe platform for vaccination against HIV-1 and other infectious diseases. SC-Ad is able to amplify antigen and cytokine genes up to 10,000-fold in infected human cells. The degree of enhancement of the immune response is much higher than those mediated by RD-Ad vectors, which are currently being tested in humans as HIV-1 vaccines. HIV vaccines can be transferred to vaccine platforms that utilize SC-Ad vectors (e.g., SC-Ad vectors based on recombinant Ad with low seropositivity) to expand HIV antigen genes.

[0337] Example 8. In vivo cytotoxic T lymphocyte (CTL) assay of immune responses against hepatitis C virus (HCV) antigens

[0338] Mice were immunized with Ad657 expressing CMV cytomegalovirus (CMV) glycoprotein B (gB) cDNA or HCV antigen 2.4. Syngeneic cells were pulsed with HCV peptides and labeled with carboxyfluorescein succinimidyl ester (CFSE) before injection into immunized mice. Cognate CTL activity against HCV was observed by a decrease in labeled cells in animals immunized with HCV but not CMV. Figure 26 ).

[0339] Example 9. Conditionally replicating Ad (CRAd)

[0340] Figure 39 and Figure 43 Schematic diagram showing mutations in Ad6, Ad657, and their variants involving E1 protein mutations to convert the viruses into conditionally replicating Ad (CRAd). These include dl1101 and / or dl1107, which block binding to p300 and pRB, respectively.

[0341] Figure 56Shown are the N-terminal amino acid sequences of E1A in wild-type Ad and the Ad variants E1Ad1101, E1Ad1107, and E1A d1101 / 1107.

[0342] Also shown is the replacement of the AdE1 promoter with the probasin-E1 DNA sequence of SEQ ID NO: 44, a prostate-specific promoter, to generate CRAd, Ad-PB ( Figure 55 The probasin promoter is androgen-dependent and thus will function in androgen-sensitive tumors such as LNCaP, but not in androgen-resistant tumors such as DU145.

[0343] A549 cells were infected with the indicated Ad6 or Ad657 variants at the indicated virus concentrations (vp / cell), and cell viability was measured 5 days later by crystal violet staining ( Figure 40 ).

[0344] Killing of non-cancer cells by replication-defective Ad (RD-Ad), Ad6, CRAd6-d1101 / d1107 or CRAd6-PB. Modification of Ad6 and Ad657 into conditionally replicating Ad (CRAd) ( Figure 44 ).

[0345] Figure 45 Killing of cancer cells by replication-competent Ad5, Ad6, Ad657, and the indicated CRAds is shown. Modification of Ad6 and Ad657 into conditionally replicating Ads (CRAds) is shown.

[0346] Figure 46 Results presented show modification of Ad6 and Ad657 into conditionally replicating Ad (CRAd) in breast cancer cells.

[0347] Figure 47 Results presented show modification of Ad6 and Ad657 into conditionally replicating Ad (CRAd) in prostate and lung cancer cells.

[0348] In vivo effects of replication-competent Ad6 or the indicated CRAds on DU145 tumor growth in mice. Figure 48 Modifications of Ad6 and Ad657 were shown to be conditionally replicating Ad (CRAd) in vivo following a single intravenous injection of mice bearing human prostate tumors.

[0349] In vivo effects of replication-competent Ad6 or the indicated CRAds on the survival of mice bearing DU145 tumors. Figure 49 Modifications of Ad6 and Ad657 were shown to be conditionally replicating Ad (CRAd) in vivo following a single intravenous injection of mice bearing human prostate tumors.

[0350] Figure 51 Modification of Ad657 with shorter cilia from chimpanzee AdC68 was shown to reduce efficacy.

[0351] In one embodiment, the Ad 6 / 56 / 6 virus has HVRs 1 and 7 from Ad6 and HVRs 2-6 from Ad657. Figure 52 Ad 6 / 56 / 6 virus was shown to kill human lung cancer cells with and without CRAd modification.

[0352] Killing of tumor cells by Ad variants involving mutations in the E3 protein. Immunocompetent Syrian hamsters were subcutaneously implanted with HaK renal carcinoma tumors. When it reached a volume of 200 μl, a single injection of the indicated Ad6 virus, which was constructed with and without E3 (DE3) and with or without random NHS-PEGylation, was given by intravenous route. Tumor size was measured over time. The data showed that the deletion of all E3 genes reduced the effectiveness of the oncolytic virus (Ad6-deltaE3-Luc compared to Ad6-Luc) ( Figure 58 ).

[0353] Ad fimbriae is a complex of three distinct subunits that mediate the initial attachment step. The native Ad6 fimbriae comprises the amino acid sequence shown in SEQ ID NO:60 and binds to CAR.

[0354] In another aspect of the present invention, recombinant Ads with pili modifications that have different pili proteins that are not native to the parent Ad are generated. Recombinant Ads containing capsid proteins from different Ad strains are generated, including CRAds, for example, recombinant Ads containing heterologous Ad35 pili polypeptides or chimpanzee C68 pili polypeptides, + / - K7 peptides ( Figures 62 to 69 ).

[0355] The chimeric Ad, AdF35 fimbriae chimera, has the amino acid sequence of SEQ ID NO:61, is shorter than the Ad5 and Ad6 fimbriae proteins, and retargets the virus to CD46.

[0356] The pili-modified recombinant Ad comprises a K7 pili having the sequence of SEQ ID NO: 62, which targets the virus to heparan sulfate proteoglycans and negative charges on cells.

[0357] The recombinant chimeric Ad, 6 / FC68 pili comprises the sequence of SEQ ID NO: 63 and is a chimeric Ad with a pili protein from chimpanzee adenovirus C68. This pili protein is shorter than the Ad5 or Ad6 pili proteins and binds to CAR.

[0358] The recombinant chimeric Ad, 6 / FC68-K7, contains the sequence of SEQ ID NO:64 and is a chimeric Ad with a pilin protein from chimpanzee adenovirus C68. This pilin protein is shorter than the pilin proteins of Ad5 or Ad6. The 6 / FC68-K7 pilin binds CAR and retargets it to heparin sulfate and negative charges.

[0359] The recombinant chimeric Ad, 6 / FC68-HI-K7, contains the sequence of SEQ ID NO: 65 and is a chimeric Ad with a pilin protein from chimpanzee adenovirus C68. This pilin protein is shorter than the Ad5 or Ad6 pilins. The 6 / FC68-HI-K7 pilin binds CAR and retargets it to heparin sulfate and negative charges.

[0360] Example 10. Adenovirus serotype switching

[0361] Figure 41 Schematic diagram showing the Ad treatment cycle. In the embodiment, serotype conversion with different Ad during treatment is exemplified ( Figure 41 A).

[0362] Prostate tumor targeting after oncolytic adenovirus serotype switching

[0363] Mice with DU145 prostate tumors on their flanks were treated with a single intravenous (IV) injection of Ad657 or CRAd657. These mice were treated a second time with an alternative Ad6 oncolytic virus or Ad6-F35 expressing GFP luciferase, and luciferase activity was measured by imaging. Ad6 has an Ad6 hexon and a CAR-targeting cilium. Ad6-F35 has an Ad6 hexon and an Ad35 cilium targeting CD46. Figure 42 The ability to serotype switch oncolysis was demonstrated using a virus that targeted the tumor with reduced off-target infection of the liver.

[0364] In another example of serotype conversion, mice with LNCaP prostate tumors on their flanks were treated with a single intravenous (IV) injection of 3e10 viral particles (vp) of Ad657 or CRAd657. Five months later, these mice were treated for a second time with 3e10 vp of alternative Ad6 / 57 / 6 oncolytic viruses expressing GFP luciferase and cilia variants K7 (adding 7 lysines), F35 (with Ad35 cilia), or KKTK-C68 (Ad6KKTK flexible domain followed by fusion of chimpanzee C68 cilia). The KKTK-C68 virus also added a codon-optimized E4 34K gene to increase viral productivity. Luciferase activity was measured by imaging after 7 days. All Ad6 / 57 / 6 had hexons with HVR1 and 7 from Ad6 and HVR2-6 from Ad57. Ad6 / 57 / 6 and KKTK-C68 had cilia targeting CAR. Ad6 / 57 / 6-F35 has Ad35 fibrils that target CD46. K7 increases binding to negatively charged molecules on cells, including heparan sulfate proteoglycans. Figure 70 The ability to serotype switch oncolysis was demonstrated using a virus that targeted the tumor with reduced off-target infection of the liver.

[0365] Serotype switching during vaccination of nonhuman primates

[0366] exist Figures 14 to 25 In this study, rhesus macaques were immunized with a replicating single-cycle Ad6 expressing the HIV envelope and then boosted by serotype conversion with a single-cycle Ad657 expressing the HIV envelope. Following these immunizations, each animal was boosted with envelope protein. Each figure shows the generation of acquired antibody or cellular immune responses and how the animals resisted SHIV. SF162P3 Rectal attack of the virus. Figure 14 A significant amount of serotype switching was documented, with the change to Ad657 generating a significant increase in the antibody response.

[0367] Example 11. Oncolytic Cancer Vaccine

[0368] BALB / c mice were treated with 10 10 Immunization was performed with CRAd-657-dl1101 / 1107-FolR containing intact E3 and expressing human folate receptor α or PBS. Two weeks after the first immunization, serum was collected and analyzed for anti-folate receptor α antibodies by ELISA using anti-IgM antibodies (all antibodies were IgM at this early time point after immunization). The data showed that this CRAd produced antibodies against the known cancer antigen folate receptor α. By T-test, p-0.07 ( Figure 53 ).

[0369] Example 12. Effect of E3 immune evasion gene on oncolytic activity

[0370] Figure 57 Schematic diagram of different E3 immune evasion genes in Ad. E3 19K protects infected cells from damage by T cells and NK cells. RID protein protects infected cells from the effects of death-inducing ligands (FAS, TRAIL, TNFR and EGFR). 14.7K inhibits intrinsic activation of apoptosis in infected cells. Class C Ad also expresses 11.6K, known as adenovirus death protein (ADP). Overexpression of ADP accelerates cell death, but overall cell death is equal. Class 49K binds to CD46 on T cells and NK cells, resulting in downregulation of these cells and lower cell killing efficiency of NK cells against class I MHC-deficient cells.

[0371] Figure 58 showed that partial deletion of E3 12.5K and complete deletion of E3 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7K genes reduced oncolytic efficacy in an immunocompetent hamster model of renal cancer when these immune evasion genes were not present in the oncolytic adenovirus.

[0372] Other implementation plans

[0373] It should be understood that while the invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims. Sequence Listing <110> MAYO FOUNDATION FOR MEDICAL EDUCATION AND RESEARCH <120> Adenoviruses and methods of using adenoviruses <130> ADZE 1 US SEQ <150> 62 / 770,631 <151> 2018-11-21 <160> 65 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 1 Thr Ala Arg Gly Glu His Lys Glu Glu Glu Leu Ile 1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 2 Leu Arg Gln Thr Gly Ala Ala Ser Ala Val Trp Gly 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 3 Ala Arg Arg Ala Asp Thr Gln Trp Arg Gly Leu Glu 1 5 10 <210> 4 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 4 Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln Ser Cys Gly Tyr Ala 1 5 10 15 Thr Val Thr <210> 5 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 5 Cys Asp Cys Arg Gly Asp Cys Phe Cys 1 5 <210> 6 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 6 Asn Met Ser Leu Asp Val Asn Arg Lys Ala 1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 7 Ile Ser Leu Ser Ser His Arg Ala Thr Trp Val Val 1 5 10 <210> 8 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 8 Trp Thr Met Gly Leu Asp Gln Leu Arg Asp Ser Ser Trp Ala His Gly 1 5 10 15 Gly Phe Ser Ala 20 <210> 9 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 9 Trp Thr Met Gly Leu Asp Gln Leu Arg Gly Asp Ser Ser Trp Ala His 1 5 10 15 Gly Gly Phe Ser 20 <210> 10 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 10 Arg Ser Val Ser Gly Thr Glu Trp Val Pro Met Asn Glu Gln His Arg 1 5 10 15 Gly Ala Ile Trp 20 <210> 11 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 11 Thr Glu Leu Arg Thr His Thr Ser Lys Glu Leu Thr Ile Arg Thr Ala 1 5 10 15 Ala Ser Ser Asp 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sequence <220> <223> Targeting peptides <400> 28 Met Ala Arg Thr Val Thr Ala Asn Val Pro Gly Met Gly Glu Gly Met 1 5 10 15 Val Val Val Pro Cys 20 <210> 29 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 29 Gly Val Ser Lys Arg Gly Leu Gln Cys His Asp Phe Ile Ser Cys Ser 1 5 10 15 Gly Val Pro Trp 20 <210> 30 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 30 Asn Gln Ser Ile Pro Lys Val Ala Gly Asp Ser Lys Val Phe Cys Trp 1 5 10 15 Trp Cys Ala Leu 20 <210> 31 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 31 Gln Ser Thr Pro Pro Thr Lys His Leu Thr Ile Pro Arg His Leu Arg 1 5 10 15 Asn Thr Leu Ile 20 <210> 32 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 32 Asp Met Ser Phe Gln Leu Val Thr Pro Phe Leu Lys Ala Leu Pro Thr 1 5 10 15 Gly Trp Arg Gly 20 <210> 33 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 33 Gly Gly His Gly Arg Val Leu Trp Pro Asp Gly Trp Phe Ser Leu Val 1 5 10 15 Gly Ile Ser Pro 20 <210> 34 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 34 Phe Ser Leu Val Gly Ile Ser Pro 1 5 <210> 35 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 35 Gln Ile Met Met Gly Pro Ser Leu Gly Tyr Tyr Met Pro Ser Glu Ser 1 5 10 15 Ile Phe Ala Tyr 20 <210> 36 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 36 Ile Ser Trp Asp Ile Trp Arg Trp Trp Tyr Thr Ser Glu Asp Arg Asp 1 5 10 15 Ala Gly Ser Ala 20 <210> 37 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 37 Val Trp Gly Met Thr Thr Ser Asp His Gln Arg Lys Thr Glu Arg Leu 1 5 10 15 Asp Ser Pro Glu 20 <210> 38 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 38 Met Thr Ser Ala Gln Thr Ser Glu Lys Leu Lys Ala Glu Thr Asp Arg 1 5 10 15 His Thr Ala Glu 20 <210> 39 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 39 Met Gly Ser Arg Ser Ala Val Gly Asp Phe Glu Ser Ala Glu Gly Ser 1 5 10 15 Arg Arg Pro <210> 40 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 40 Met Gly Arg Thr Val Gln Ser Gly Asp Gly Thr Pro Ala Gln Thr Gln 1 5 10 15 Pro Ser Val Asn 20 <210> 41 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Targeting peptides <400> 41 Met Ala Arg Thr Val Thr Ala Asn Val Pro Gly Met Gly Glu Gly Met 1 5 10 15 Val Val Val Pro 20 <210> 42 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Adenovirus E1A N-terminal polypeptide <400> 42 Met Arg His Ile Ile Cys His Gly Gly Val Ile Thr Glu Glu Met Ala 1 5 10 15 Ala Ser Leu Leu Asp Gln Leu Ile Glu Glu Val Leu Ala Asp Asn Leu 20 25 30 Pro Pro Pro Ser His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp 35 40 45 Leu Asp Val Thr Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln 50 55 60 Ile Phe Pro Glu Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu 65 70 75 80 Phe Thr Phe Pro Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser 85 90 95 Arg Gln Pro Glu Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Ser Met 100 105 110 Pro Asn Leu Val Pro Glu Val Ile Asp Leu Thr Cys His Glu Ala Gly 115 120 125 Phe Pro Pro Ser 130 <210> 43 <211> 112 <212> PRT <213> Artificial sequence <220> <223> E1A N-terminal polypeptide <400> 43 Met Arg His Ile Glu Glu Val Leu Ala Asp Asn Leu Pro Pro Pro Ser 1 5 10 15 His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp Leu Asp Val Thr 20 25 30 Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln Ile Phe Pro Glu 35 40 45 Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu Phe Thr Phe Pro 50 55 60 Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser Arg Gln Pro Glu 65 70 75 80 Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Ser Met Pro Asn Leu Val 85 90 95 Pro Glu Val Ile Asp Leu Thr Cys His Glu Ala Gly Phe Pro Pro Ser 100 105 110 <210> 44 <211> 119 <212> PRT <213> Artificial sequence <220> <223> E1A N-terminal polypeptide <400> 44 Met Arg His Ile Ile Cys His Gly Gly Val Ile Thr Glu Glu Met Ala 1 5 10 15 Ala Ser Leu Leu Asp Gln Leu Ile Glu Glu Val Leu Ala Asp Asn Leu 20 25 30 Pro Pro Pro Ser His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp 35 40 45 Leu Asp Val Thr Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln 50 55 60 Ile Phe Pro Glu Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu 65 70 75 80 Phe Thr Phe Pro Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser 85 90 95 Arg Gln Pro Glu Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Cys His 100 105 110 Glu Ala Gly Phe Pro Pro Ser 115 <210> 45 <211> 99 <212> PRT <213> artificial sequence <220> <223> E1A N terminal multi-peptide <400> 45 Met Arg His Ile Glu Glu Val Leu Ala Asp Asn Leu Pro Pro Pro Ser 1 5 10 15 His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp Leu Asp Val Thr 20 25 30 Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln Ile Phe Pro Glu 35 40 45 Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu Phe Thr Phe Pro 50 55 60 Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser Arg Gln Pro Glu 65 70 75 80 Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Cys His Glu Ala Gly Phe 85 90 95 Pro Pro Ser <210> 46 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Cell-binding peptides <400> 46 Arg Glu Asp Val Tyr 1 5 <210> 47 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Breast cancer binding peptide <400> 47 Ile Ser Leu Ser Ser His Arg Ala Thr Trp Val Val 1 5 10 <210> 48 <211> 1608 <212> PRT <213> Artificial sequence <220> <223> Probasin-E1 nucleic acid <400> 48 Thr Cys Gly Ala Gly Cys Gly Ala Cys Gly Gly Thr Ala Thr Cys Gly 1 5 10 15 Ala Thr Ala Ala Gly Cys Thr Thr Gly Gly Ala Gly Cys Thr Thr Ala 20 25 30 Thr Gly Ala Thr Ala Gly Cys Ala Thr Cys Thr Thr Gly Thr Thr Cys 35 40 45 Thr Thr Ala Gly Thr Cys Thr Thr Thr Thr Thr Cys Thr Thr Ala Ala 50 55 60 Thr Ala Gly Gly Gly Ala Cys Ala Thr Ala Ala Ala Gly Cys Cys Cys 65 70 75 80 Ala Cys Ala Ala Ala Thr Ala Ala Ala Ala Ala Thr Ala Thr Gly Cys 85 90 95 Cys Thr Gly Ala Ala Gly Ala Ala Thr Gly Gly Gly Ala Cys Ala Gly 100 105 110 Gly Cys Ala Thr Thr Gly Gly Gly Cys Ala Thr Thr Gly Thr Cys Cys 115 120 125 Ala Thr Gly Cys Cys Thr Ala Gly Thr Ala Ala Ala Gly Thr Ala Cys 130 135 140 Thr Cys Cys Ala Ala Gly Ala Ala Cys Cys Thr Ala Thr Thr Thr Gly 145 150 155 160 Thr Ala Thr Ala Cys Thr Ala Gly Ala Thr Gly Ala Cys Ala Cys Ala 165 170 175 Ala Thr Gly Thr Thr Cys Thr Ala Gly Cys Cys Ala Ala Gly Cys Thr 180 185 190 Thr Gly Gly Thr Ala Gly Thr Cys Ala Thr Cys Ala Thr Gly Thr Thr 195 200 205 Thr Ala Ala Ala Cys Ala Thr Cys Thr Ala Cys Cys Ala Thr Thr Cys 210 215 220 Cys Ala Gly Thr Thr Ala Ala Gly Ala Ala Ala Ala Thr Ala Thr Gly 225 230 235 240 Ala Thr Ala Gly Cys Ala Thr Cys Thr Thr Gly Thr Thr Cys Thr Thr 245 250 255 Ala Gly Thr Cys Thr Thr Thr Thr Thr Cys Thr Thr Ala Ala Thr Ala 260 265 270 Gly Gly Gly Ala Cys Ala Thr Ala Ala Ala Gly Cys Cys Cys Ala Cys 275 280 285 Ala Ala Ala Thr Ala Ala Ala Ala Ala Thr Ala Thr Gly Cys Cys Thr 290 295 300 Gly Ala Ala Gly Ala Ala Thr Gly Gly Gly Ala Cys Ala Gly Gly Cys 305 310 315 320 Ala Thr Thr Gly Gly Gly Cys Ala Thr Thr Gly Thr Cys Cys Ala Thr 325 330 335 Gly Cys Cys Thr Ala Gly Thr Ala Ala Ala Gly Thr Ala Cys Thr Cys 340 345 350 Cys Ala Ala Gly Ala Ala Cys Cys Thr Ala Thr Thr Thr Gly Thr Ala 355 360 365 Thr Ala Cys Thr Ala Gly Ala Thr Gly Ala Cys Ala Cys Ala Ala Thr 370 375 380 Gly Thr Cys Ala Ala Thr Gly Thr Cys Thr Gly Thr Gly Thr Ala Cys 385 390 395 400 Ala Ala Cys Thr Gly Cys Cys Ala Ala Cys Thr Gly Gly Gly Ala Thr 405 410 415 Gly Cys Ala Ala Gly Ala Cys Ala Cys Thr Gly Cys Cys Cys Ala Thr 420 425 430 Gly Cys Cys Ala Ala Thr Cys Ala Thr Cys Cys Thr Gly Ala Ala Ala 435 440 445 Ala Gly Cys Ala Gly Cys Thr Ala Thr Ala Ala Ala Ala Ala Gly Cys 450 455 460 Ala Gly Gly Ala Ala Gly Cys Thr Ala Cys Thr Cys Thr Gly Cys Ala 465 470 475 480 Cys Cys Thr Thr Gly Thr Cys Ala Gly Thr Gly Ala Gly Gly Thr Cys 485 490 495 Cys Ala Gly Ala Thr Ala Cys Cys Thr Cys Cys Cys Thr Cys Gly Ala 500 505 510 Gly Cys Gly Gly Cys Cys Gly Cys Gly Ala Cys Gly Cys Gly Cys Ala 515 520 525 Gly Thr Gly Thr Ala Thr Thr Thr Ala Thr Ala Cys Cys Cys Gly Gly 530 535 540 Thr Gly Ala Gly Thr Thr Cys Cys Thr Cys Ala Ala Gly Ala Gly Gly 545 550 555 560 Cys Cys Ala Cys Thr Cys Thr Thr Gly Ala Gly Thr Gly Cys Cys Ala 565 570 575 Gly Cys Gly Ala Gly Thr Ala Gly Ala Gly Thr Thr Thr Thr Cys Thr 580 585 590 Cys Cys Thr Cys Cys Gly Ala Gly Cys Cys Gly Cys Thr Cys Cys Gly 595 600 605 Ala Cys Ala Cys Cys Gly Gly Gly Ala Cys Thr Gly Ala Ala Ala Ala 610 615 620 Thr Gly Ala Gly Ala Cys Ala Thr Ala Thr Thr Ala Thr Cys Thr Gly 625 630 635 640 Cys Cys Ala Cys Gly Gly Ala Gly Gly Thr Gly Thr Thr Ala Thr Thr 645 650 655 Ala Cys Cys Gly Ala Ala Gly Ala Ala Ala Thr Gly Gly Cys Cys Gly 660 665 670 Cys Cys Ala Gly Thr Cys Thr Thr Thr Thr Gly Gly Ala Cys Cys Ala 675 680 685 Gly Cys Thr Gly Ala Thr Cys Gly Ala Ala Gly Ala Gly Gly Thr Ala 690 695 700 Cys Thr Gly Gly Cys Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys 705 710 715 720 Cys Ala Cys Cys Thr Cys Cys Thr Ala Gly Cys Cys Ala Thr Thr Thr 725 730 735 Thr Gly Ala Ala Cys Cys Ala Cys Cys Thr Ala Cys Cys Cys Thr Thr 740 745 750 Cys Ala Cys Gly Ala Ala Cys Thr Gly Thr Ala Thr Gly Ala Thr Thr 755 760 765 Thr Ala Gly Ala Cys Gly Thr Gly Ala Cys Gly Gly Cys Cys Cys Cys 770 775 780 Cys Gly Ala Ala Gly Ala Thr Cys Cys Cys Ala Ala Cys Gly Ala Gly 785 790 795 800 Gly Ala Gly Gly Cys Gly Gly Thr Thr Thr Cys Gly Cys Ala Gly Ala 805 810 815 Thr Thr Thr Thr Thr Cys Cys Cys Gly Ala Gly Thr Cys Thr Gly Thr 820 825 830 Ala Ala Thr Gly Thr Thr Gly Gly Cys Gly Gly Thr Gly Cys Ala Gly 835 840 845 Gly Ala Ala Gly Gly Gly Ala Thr Thr Gly Ala Cys Thr Thr Ala Thr 850 855 860 Thr Cys Ala Cys Thr Thr Thr Thr Cys Cys Gly Cys Cys Gly Gly Cys 865 870 875 880 Gly Cys Cys Cys Gly Gly Thr Thr Cys Thr Cys Cys Gly Gly Ala Gly 885 890 895 Cys Cys Gly Cys Cys Thr Cys Ala Cys Cys Thr Thr Thr Cys Cys Cys 900 905 910 Gly Gly Cys Ala Gly Cys Cys Cys Gly Ala Gly Cys Ala Gly Cys Cys 915 920 925 Gly Gly Ala Gly Cys Ala Gly Ala Gly Ala Gly Cys Cys Thr Thr Gly 930 935 940 Gly Gly Thr Cys Cys Gly Gly Thr Thr Thr Cys Thr Ala Thr Gly Cys 945 950 955 960 Cys Ala Ala Ala Cys Cys Thr Thr Gly Thr Gly Cys Cys Gly Gly Ala 965 970 975 Gly Gly Thr Gly Ala Thr Cys Gly Ala Thr Cys Thr Thr Ala Cys Cys 980 985 990 Thr Gly Cys Cys Ala Cys Gly Ala Gly Gly Cys Thr Gly Gly Cys Thr 995 1000 1005 Thr Thr Cys Cys Ala Cys Cys Cys Ala Gly Thr Gly Ala Cys Gly 1010 1015 1020 Ala Cys Gly Ala Gly Gly Ala Thr Gly Ala Ala Gly Ala Gly Gly 1025 1030 1035 Gly Thr Gly Ala Gly Gly Ala Gly Thr Thr Thr Gly Thr Gly Thr 1040 1045 1050 Thr Ala Gly Ala Thr Thr Ala Thr Gly Thr Gly Gly Ala Gly Cys 1055 1060 1065 Ala Cys Cys Cys Cys Gly Gly Gly Cys Ala Cys Gly Gly Thr Thr 1070 1075 1080 Gly Cys Ala Gly Gly Thr Cys Thr Thr Gly Thr Cys Ala Thr Thr 1085 1090 1095 Ala Thr Cys Ala Cys Cys Gly Gly Ala Gly Gly Ala Ala Thr Ala 1100 1105 1110 Cys Gly Gly Gly Gly Gly Ala Cys Cys Cys Ala Gly Ala Thr Ala 1115 1120 1125 Thr Thr Ala Thr Gly Thr Gly Thr Thr Cys Gly Cys Thr Thr Thr 1130 1135 1140 Gly Cys Thr Ala Thr Ala Thr Gly Ala Gly Gly Ala Cys Cys Thr 1145 1150 1155 Gly Thr Gly Gly Cys Ala Thr Gly Thr Thr Thr Gly Thr Cys Thr 1160 1165 1170 Ala Cys Ala Gly Thr Ala Ala Gly Thr Gly Ala Ala Ala Ala Thr 1175 1180 1185 Thr Ala Thr Gly Gly Gly Cys Ala Gly Thr Cys Gly Gly Thr Gly 1190 1195 1200 Ala Thr Ala Gly Ala Gly Thr Gly Gly Thr Gly Gly Gly Thr Thr 1205 1210 1215 Thr Gly Gly Thr Gly Thr Gly Gly Thr Ala Ala Thr Thr Thr Thr 1220 1225 1230 Thr Thr Thr Thr Thr Ala Ala Thr Thr Thr Thr Thr Ala Cys Ala 1235 1240 1245 Gly Thr Thr Thr Thr Gly Thr Gly Gly Thr Thr Thr Ala Ala Ala 1250 1255 1260 Gly Ala Ala Thr Thr Thr Thr Gly Thr Ala Thr Thr Gly Thr Gly 1265 1270 1275 Ala Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Gly Gly Thr Cys 1280 1285 1290 Cys Thr Gly Thr Gly Thr Cys Thr Gly Ala Ala Cys Cys Thr Gly 1295 1300 1305 Ala Gly Cys Cys Thr Gly Ala Gly Cys Cys Cys Gly Ala Gly Cys 1310 1315 1320 Cys Ala Gly Ala Ala Cys Cys Gly Gly Ala Gly Cys Cys Thr Gly 1325 1330 1335 Cys Ala Ala Gly Ala Cys Cys Thr Ala Cys Cys Cys Gly Gly Cys 1340 1345 1350 Gly Thr Cys Cys Thr Ala Ala Ala Thr Thr Gly Gly Thr Gly Cys 1355 1360 1365 Cys Thr Gly Cys Thr Ala Thr Cys Cys Thr Gly Ala Gly Ala Cys 1370 1375 1380 Gly Cys Cys Cys Gly Ala Cys Ala Thr Cys Ala Cys Cys Thr Gly 1385 1390 1395 Thr Gly Thr Cys Thr Ala Gly Ala Gly Ala Ala Thr Gly Cys Ala 1400 1405 1410 Ala Thr Ala Gly Thr Ala Gly Thr Ala Cys Gly Gly Ala Thr Ala 1415 1420 1425 Gly Cys Thr Gly Thr Gly Ala Cys Thr Cys Cys Gly Gly Thr Cys 1430 1435 1440 Cys Thr Thr Cys Thr Ala Ala Cys Ala Cys Ala Cys Cys Thr Cys 1445 1450 1455 Cys Thr Gly Ala Gly Ala Thr Ala Cys Ala Cys Cys Cys Gly Gly 1460 1465 1470 Thr Gly Gly Thr Cys Cys Cys Gly Cys Thr Gly Thr Gly Cys Cys 1475 1480 1485 Cys Cys Ala Thr Thr Ala Ala Ala Cys Cys Ala Gly Thr Thr Gly 1490 1495 1500 Cys Cys Gly Thr Gly Ala Gly Ala Gly Thr Thr Gly Gly Thr Gly 1505 1510 1515 Gly Gly Cys Gly Thr Cys Gly Cys Cys Ala Gly Gly Cys Thr Gly 1520 1525 1530 Thr Gly Gly Ala Ala Thr Gly Thr Ala Thr Cys Gly Ala Gly Gly 1535 1540 1545 Ala Cys Thr Thr Gly Cys Thr Thr Ala Ala Cys Gly Ala Gly Thr 1550 1555 1560 Cys Thr Gly Gly Gly Cys Ala Ala Cys Cys Thr Thr Thr Gly Gly 1565 1570 1575 Ala Cys Thr Thr Gly Ala Gly Cys Thr Gly Thr Ala Ala Ala Cys 1580 1585 1590 Gly Cys Cys Cys Cys Ala Gly Gly Cys Cys Ala Thr Ala Ala Gly 1595 1600 1605 <210> 49 <211> 959 <212> PRT <213> Artificial Sequence <220> <223> Hexamer Polypeptide <400> 49 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala Asn Ala Ile Gln Pro 275 280 285 Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr 290 295 300 His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met 305 310 315 320 Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg 325 330 335 Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly 340 345 350 Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln 355 360 365 Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly 370 375 380 Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr 385 390 395 400 Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu 405 410 415 Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr 420 425 430 Gln Ala Ile Lys Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala 435 440 445 Gln Asp Asn Thr Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn 450 455 460 Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu 465 470 475 480 Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro 485 490 495 Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn 500 505 510 Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly 515 520 525 Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His 530 535 540 His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly 545 550 555 560 Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile 565 570 575 Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe 580 585 590 Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu 595 600 605 Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala 610 615 620 Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met 625 630 635 640 Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala 645 650 655 Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile 660 665 670 Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr 675 680 685 Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro 690 695 700 Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr 705 710 715 720 Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val 725 730 735 Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile 740 745 750 Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met 755 760 765 Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly 770 775 780 Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser 785 790 795 800 Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr 805 810 815 Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn 820 825 830 Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala 835 840 845 Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp 850 855 860 Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile 865 870 875 880 Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly 885 890 895 Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe 900 905 910 Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu 915 920 925 Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu 930 935 940 Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 945 950 955 <210> 50 <211> 967 <212> PRT <213> Artificial Sequence <220> <223> Hexamer Polypeptide <400> 50 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Ser Ser Cys Ser Ser Gly Gly 145 150 155 160 Thr Glu Glu Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys Lys Thr 165 170 175 His Val Tyr Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn Lys Asn 180 185 190 Gly Leu Gln Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn Lys Glu 195 200 205 Ile Tyr Ala Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser 210 215 220 Gln Trp Asn Glu Ala Glu Ser Ser Val Ala Gly Gly Arg Val Leu Lys 225 230 235 240 Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr 245 250 255 Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly Lys Leu 260 265 270 Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn Ala Met 275 280 285 Asn Glu Ala Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser Glu Asp 290 295 300 Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys 305 310 315 320 Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn 325 330 335 Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr 340 345 350 Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln 355 360 365 Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr 370 375 380 Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met 385 390 395 400 Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu 405 410 415 Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly 420 425 430 Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly 435 440 445 Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg 450 455 460 Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn 465 470 475 480 Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu 485 490 495 Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn 500 505 510 Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu 515 520 525 Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met 530 535 540 Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr 545 550 555 560 Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln 565 570 575 Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly 580 585 590 Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu 595 600 605 Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys 610 615 620 Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn 625 630 635 640 Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln 645 650 655 Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro 660 665 670 Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala 675 680 685 Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro 690 695 700 Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile 705 710 715 720 Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val 725 730 735 Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu 740 745 750 Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly 755 760 765 Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln 770 775 780 Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu 785 790 795 800 Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met 805 810 815 Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val 820 825 830 Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala 835 840 845 Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro 850 855 860 Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu 865 870 875 880 Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser 885 890 895 Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser 900 905 910 Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro 915 920 925 Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His 930 935 940 Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe 945 950 955 960 Ser Ala Gly Asn Ala Thr Thr 965 <210> 51 <211> 955 <212> PRT <213> Artificial sequence <220> <223> Hexapeptide <400> 51 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Ser Cys Ser Ser Gly Gly Thr Pro Lys Leu Val Leu 275 280 285 Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr 290 295 300 Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln 305 310 315 320 Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile 325 330 335 Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly 340 345 350 Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr 355 360 365 Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg 370 375 380 Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val 385 390 395 400 Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys 405 410 415 Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys 420 425 430 Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr 435 440 445 Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu 450 455 460 Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile 465 470 475 480 Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu 485 490 495 Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val 500 505 510 Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser 515 520 525 Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala 530 535 540 Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro 545 550 555 560 Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu 565 570 575 Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val 580 585 590 Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly 595 600 605 Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro 610 615 620 Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp 625 630 635 640 Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu 645 650 655 Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser 660 665 670 Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr 675 680 685 Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr 690 695 700 Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr 705 710 715 720 Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly 725 730 735 Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val 740 745 750 Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp 755 760 765 Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe 770 775 780 Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn 785 790 795 800 Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp 805 810 815 Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val 820 825 830 Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn 835 840 845 Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln 850 855 860 Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser 865 870 875 880 Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu 885 890 895 Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro 900 905 910 Methionine, Aspartic acid, Glutamic acid, Proline, Threonine, Leucine, Leucine, Tyrosine, Valine, Leucine, Phenylalanine, Glutamic acid, Valine, Phenylalanine, Aspartic acid, Valine 915 920 925 Valine, Arginine, Valine, Histidine, Glutamine, Proline, Histidine, Arginine, Glycine, Valine, Isoleucine, Glutamic acid, Threonine, Valine, Tyrosine, Leucine 930 935 940 Arginine, Threonine, Proline, Phenylalanine, Serine, Alanine, Glycine, Asparagine, Alanine, Threonine, Threonine 945 950 955 <210> 52 <211> 964 <212> PRT <213> Artificial Sequence <220> <223> Hexon Polypeptide <400> 52 Methionine, Alanine, Threonine, Proline, Serine, Methionine, Methionine, Proline, Glutamine, Tryptophan, Serine, Tyrosine, Methionine, Histidine, Isoleucine, Serine 1 5 10 15 Glycine, Glutamine, Aspartic acid, Alanine, Serine, Glutamic acid, Tyrosine, Leucine, Serine, Proline, Glycine, Leucine, Valine, Glutamine, Phenylalanine, Alanine 20 25 30 Arginine, Alanine, Threonine, Glutamic acid, Threonine, Tyrosine, Phenylalanine, Serine, Leucine, Asparagine, Asparagine, Lysine, Phenylalanine, Arginine, Asparagine, Proline 35 40 45 Threonine, Valine, Alanine, Proline, Threonine, Histidine, Aspartic acid, Valine, Threonine, Threonine, Aspartic acid, Arginine, Serine, Glutamine, Arginine, Leucine 50 55 60 Threonine, Leucine, Arginine, Phenylalanine, Isoleucine, Proline, Valine, Aspartic acid, Arginine, Glutamic acid, Aspartic acid, Threonine, Alanine, Tyrosine, Serine, Tyrosine 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Ser Ser Gly Gly Thr Glu Glu 145 150 155 160 Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr 165 170 175 Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln 180 185 190 Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala 195 200 205 Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn 210 215 220 Glu Ala Glu Ser Ser Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr 225 230 235 240 Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn 245 250 255 Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln 260 265 270 Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala 275 280 285 Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met 290 295 300 Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp 305 310 315 320 Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn 325 330 335 Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser 340 345 350 Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala 355 360 365 Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu 370 375 380 Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln 385 390 395 400 Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly 405 410 415 Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly 420 425 430 Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly Asn Gly Gly 435 440 445 Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg Asn Glu Ile 450 455 460 Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu 465 470 475 480 Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys 485 490 495 Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr 500 505 510 Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys 515 520 525 Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val 530 535 540 Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met 545 550 555 560 Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln 565 570 575 Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr 580 585 590 Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser 595 600 605 Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser 610 615 620 Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser 625 630 635 640 Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn 645 650 655 Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala 660 665 670 Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg 675 680 685 Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly 690 695 700 Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu 705 710 715 720 Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr 725 730 735 Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro 740 745 750 Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val 755 760 765 Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala 770 775 780 Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys 785 790 795 800 Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln 805 810 815 Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile 820 825 830 His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met 835 840 845 Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly 850 855 860 Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg 865 870 875 880 Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala 885 890 895 Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala 900 905 910 Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu 915 920 925 Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Gln Pro His 930 935 940 Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly 945 950 955 960 Asn Ala Thr Thr <210> 53 <211> 952 <212> PRT <213> Artificial Sequence <220> <223> Hexamer Polypeptide <400> 53 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Ser Gly Gly Thr Pro Lys Leu Val Leu Tyr Ser Glu 275 280 285 Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly 290 295 300 Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro 305 310 315 320 Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met 325 330 335 Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser 340 345 350 Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser 355 360 365 Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser 370 375 380 Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile 385 390 395 400 Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu 405 410 415 Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn 420 425 430 Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu 435 440 445 Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu 450 455 460 Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr 465 470 475 480 Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp 485 490 495 Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly 500 505 510 Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr 515 520 525 Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg 530 535 540 Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile 545 550 555 560 Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro 565 570 575 Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val 580 585 590 Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile 595 600 605 Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His 610 615 620 Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp 625 630 635 640 Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile 645 650 655 Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp 660 665 670 Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr 675 680 685 Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser 690 695 700 Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys 705 710 715 720 Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg 725 730 735 Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu 740 745 750 Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val 755 760 765 Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro 770 775 780 Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro 785 790 795 800 Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln 805 810 815 Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu 820 825 830 Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr 835 840 845 Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe 850 855 860 Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met 865 870 875 880 Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn 885 890 895 Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu 900 905 910 Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val 915 920 925 His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro 930 935 940 Phe Ser Ala Gly Asn Ala Thr Thr 945 950 <210> 54 <211> 1033 <212> PRT <213> Artificial Sequence <220> <223> Hexamer Polypeptide <400> 54 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Ser Thr Gly Glu Ile Pro Ala 145 150 155 160 Pro Leu Ala Gly Thr Val Ser Lys Ile Leu Val Lys Glu Gly Asp Thr 165 170 175 Val Lys Ala Gly Gln Thr Val Leu Val Leu Glu Ala Met Lys Met Glu 180 185 190 Thr Glu Ile Asn Ala Pro Thr Asp Gly Lys Val Glu Lys Val Leu Val 195 200 205 Lys Glu Arg Asp Ala Val Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly 210 215 220 Gly Gly Thr Glu Glu Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys 225 230 235 240 Lys Thr His Val Tyr Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn 245 250 255 Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn 260 265 270 Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly 275 280 285 Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser Val Ala Gly Gly Arg Val 290 295 300 Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg 305 310 315 320 Pro Thr Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly 325 330 335 Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn 340 345 350 Ala Met Asn Glu Ala Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser 355 360 365 Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro 370 375 380 Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met 385 390 395 400 Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu 405 410 415 Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala 420 425 430 Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu 435 440 445 Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe 450 455 460 Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile 465 470 475 480 Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro 485 490 495 Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr 500 505 510 Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala 515 520 525 Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn 530 535 540 Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu 545 550 555 560 Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser 565 570 575 Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro 580 585 590 Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp 595 600 605 Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu 610 615 620 Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His 625 630 635 640 Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu 645 650 655 Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met 660 665 670 Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser 675 680 685 Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala 690 695 700 His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn 705 710 715 720 Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro 725 730 735 Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn 740 745 750 Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu 755 760 765 Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly 770 775 780 Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys 785 790 795 800 Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp 805 810 815 Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly 820 825 830 Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu 835 840 845 Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile 850 855 860 Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln 865 870 875 880 Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln 885 890 895 Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr 900 905 910 Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro 915 920 925 Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys 930 935 940 Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe 945 950 955 960 Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala 965 970 975 Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp 980 985 990 Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg 995 1000 1005 Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg 1010 1015 1020 Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 1025 1030 <210> 55 <211> 1021 <212> PRT <213> Artificial Sequence <220> <223> Hexameric Polypeptide <400> 55 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Thr Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val 275 280 285 Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr 290 295 300 Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro 305 310 315 320 Thr Asp Gly Lys Val Glu Lys Val Leu Val Lys Glu Arg Asp Ala Val 325 330 335 Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly Gly Gly Thr Pro Lys Leu 340 345 350 Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu 355 360 365 Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly 370 375 380 Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn 385 390 395 400 Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu 405 410 415 Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg 420 425 430 Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg 435 440 445 Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro 450 455 460 Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn 465 470 475 480 Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala 485 490 495 Ile Lys Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp 500 505 510 Asn Thr Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala 515 520 525 Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser 530 535 540 Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn 545 550 555 560 Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg 565 570 575 Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg 580 585 590 Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg 595 600 605 Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr 610 615 620 Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn 625 630 635 640 Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys 645 650 655 Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val 660 665 670 Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe 675 680 685 Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg 690 695 700 Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn 705 710 715 720 Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile 725 730 735 Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu 740 745 750 Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr 755 760 765 Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn 770 775 780 His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp 785 790 795 800 Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg 805 810 815 Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys 820 825 830 Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln 835 840 845 Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe 850 855 860 Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr 865 870 875 880 Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly 885 890 895 Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro 900 905 910 Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile 915 920 925 Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe 930 935 940 Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn 945 950 955 960 Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val 965 970 975 Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe 980 985 990 Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val 995 1000 1005 Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 1010 1015 1020 <210> 56 <211> 1015 <212> PRT <213> Artificial Sequence <220> <223> Hexapeptide <400> 56 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Ser Ser Asn Phe Thr Arg Glu Gly Asn Val Thr Tyr 275 280 285 Lys Glu Glu Met Asp Lys Val Lys Asn Cys Ser Phe Asn Val Thr Thr 290 295 300 Gly Ile Arg Asp Lys Lys Gln Lys Val Asn Ala Leu Phe Tyr Arg Leu 305 310 315 320 Asp Ile Thr Pro Leu Asp Glu Asn Asn Asn Asn Ser Ser Glu Tyr Arg 325 330 335 Leu Ile Asn Ser Gly Gly Thr Pro Lys Leu Val Leu Tyr Ser Glu Asp 340 345 350 Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys 355 360 365 Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn 370 375 380 Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr 385 390 395 400 Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln 405 410 415 Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr 420 425 430 Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met 435 440 445 Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu 450 455 460 Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly 465 470 475 480 Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly 485 490 495 Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg 500 505 510 Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn 515 520 525 Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu 530 535 540 Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn 545 550 555 560 Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu 565 570 575 Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met 580 585 590 Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr 595 600 605 Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln 610 615 620 Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly 625 630 635 640 Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu 645 650 655 Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys 660 665 670 Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn 675 680 685 Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln 690 695 700 Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro 705 710 715 720 Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala 725 730 735 Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro 740 745 750 Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile 755 760 765 Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val 770 775 780 Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu 785 790 795 800 Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly 805 810 815 Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln 820 825 830 Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu 835 840 845 Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met 850 855 860 Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val 865 870 875 880 Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala 885 890 895 Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro 900 905 910 Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu 915 920 925 Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser 930 935 940 Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser 945 950 955 960 Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro 965 970 975 Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His 980 985 990 Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe 995 1000 1005 Ser Ala Gly Asn Ala Thr Thr 1010 1015 <210> 57 <211> 987 <212> PRT <213> Artificial sequence <220> <223> Hexapeptide <400> 57 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Ser Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile 275 280 285 Val Thr Phe Cys Cys Lys Cys Asp Gln Leu Leu Arg Arg Glu Val Tyr 290 295 300 Asp Phe Ala Phe Arg Asp Leu Ser Gly Gly Thr Pro Lys Leu Val Leu 305 310 315 320 Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr 325 330 335 Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln 340 345 350 Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile 355 360 365 Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly 370 375 380 Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr 385 390 395 400 Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg 405 410 415 Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val 420 425 430 Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys 435 440 445 Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys 450 455 460 Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr 465 470 475 480 Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu 485 490 495 Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile 500 505 510 Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu 515 520 525 Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val 530 535 540 Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser 545 550 555 560 Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala 565 570 575 Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro 580 585 590 Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu 595 600 605 Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val 610 615 620 Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly 625 630 635 640 Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro 645 650 655 Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp 660 665 670 Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu 675 680 685 Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser 690 695 700 Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr 705 710 715 720 Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr 725 730 735 Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr 740 745 750 Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly 755 760 765 Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val 770 775 780 Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp 785 790 795 800 Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe 805 810 815 Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn 820 825 830 Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp 835 840 845 Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val 850 855 860 Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn 865 870 875 880 Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln 885 890 895 Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser 900 905 910 Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu 915 920 925 Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro 930 935 940 Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val 945 950 955 960 Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu 965 970 975 Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 980 985 <210> 58 <211> 959 <212> PRT <213> Artificial Sequence <220> <223> Hexapeptide <400> 58 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140 Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro 145 150 155 160 Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175 Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190 Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205 Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220 Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys 225 230 235 240 Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255 Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270 Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala Asn Ala Ile Gln Pro 275 280 285 Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr 290 295 300 His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met 305 310 315 320 Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg 325 330 335 Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly 340 345 350 Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln 355 360 365 Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly 370 375 380 Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr 385 390 395 400 Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu 405 410 415 Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr 420 425 430 Gln Ala Ile Lys Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala 435 440 445 Gln Asp Asn Thr Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn 450 455 460 Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu 465 470 475 480 Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro 485 490 495 Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn 500 505 510 Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly 515 520 525 Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His 530 535 540 His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly 545 550 555 560 Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile 565 570 575 Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe 580 585 590 Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu 595 600 605 Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala 610 615 620 Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met 625 630 635 640 Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala 645 650 655 Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile 660 665 670 Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr 675 680 685 Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro 690 695 700 Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr 705 710 715 720 Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val 725 730 735 Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile 740 745 750 Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met 755 760 765 Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly 770 775 780 Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser 785 790 795 800 Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr 805 810 815 Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn 820 825 830 Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala 835 840 845 Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp 850 855 860 Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile 865 870 875 880 Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly 885 890 895 Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe 900 905 910 Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu 915 920 925 Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu 930 935 940 Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 945 950 955 <210> 59 <211> 962 <212> PRT <213> Artificial Sequence <220> <223> Hexamer Polypeptide <400> 59 Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser 1 5 10 15 Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30 Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45 Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60 Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr 65 70 75 80 Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95 Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110 Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125 Ala Pro Asn Ser Cys Glu Trp Glu Gln Asn Glu Thr Ala Gln Val Asp 130 135 140 Ala Gln Glu Leu Asp Glu Glu Glu Asn Glu Ala Asn Glu Ala Gln Ala 145 150 155 160 Arg Glu Gln Glu Gln Ala Lys Lys Thr His Val Tyr Ala Gln Ala Pro 165 170 175 Leu Ser Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn 180 185 190 Gly Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr 195 200 205 Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser 210 215 220 Ser Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro 225 230 235 240 Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly 245 250 255 Val Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln 260 265 270 Phe Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala Asn Ala Ile Gln 275 280 285 Pro Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp 290 295 300 Thr His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala 305 310 315 320 Met Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe 325 330 335 Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met 340 345 350 Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu 355 360 365 Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile 370 375 380 Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser 385 390 395 400 Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu 405 410 415 Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Ile Thr Asp Thr 420 425 430 Phe Gln Ala Val Lys Thr Thr Ala Ala Asn Gly Asp Gln Gly Asn Thr 435 440 445 Thr Trp Gln Lys Asp Ser Thr Phe Ala Glu Arg Asn Glu Ile Gly Val 450 455 460 Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg 465 470 475 480 Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys 485 490 495 Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp 500 505 510 Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile 515 520 525 Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro 530 535 540 Phe Asn His Pro Arg His Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu 545 550 555 560 Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe 565 570 575 Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu 580 585 590 Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly 595 600 605 Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys 610 615 620 Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu 625 630 635 640 Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr 645 650 655 Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn 660 665 670 Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp 675 680 685 Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly 690 695 700 Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly 705 710 715 720 Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp 725 730 735 Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu 740 745 750 Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln 755 760 765 Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr 770 775 780 Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg 785 790 795 800 Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val 805 810 815 Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln 820 825 830 His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu 835 840 845 Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr 850 855 860 Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu 865 870 875 880 Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr 885 890 895 Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp 900 905 910 Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val 915 920 925 Leu Phe Glu Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly 930 935 940 Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala 945 950 955 960 Thursday Thursday <210> 60 <211> 528 <212> PRT <213> artificial sequence <220> <223> fibrillar peptide <400> 60 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro Leu Val Thr Ser His Gly Met Leu Ala Leu 50 55 60 Lys Met Gly Ser Gly Leu Ser Leu Asp Gln Ala Gly Asn Leu Thr Ser 65 70 75 80 Asn Thr Ile Thr Val Ser Gln Pro Leu Lys Lys Thr Lys Ser Asn Ile 85 90 95 Thr Leu Glu Thr Ser Ala Pro Leu Thr Val Ser Ser Gly Ala Leu Thr 100 105 110 Met Ala Thr Thr Ser Pro Leu Val Val Ser Asp Asn Thr Leu Thr Met 115 120 125 Gln Ser Gln Ala Pro Leu Thr Val Gln Asp Ser Lys Leu Ser Ile Ala 130 135 140 Thr Lys Glu Pro Leu Thr Val Leu Asp Gly Lys Leu Ala Leu Gln Thr 145 150 155 160 Ser Ala Pro Leu Ser Ala Thr Asp Asn Asn Ala Leu Thr Ile Thr Ala 165 170 175 Ser Pro Pro Leu Thr Thr Ala Asn Gly Ser Leu Ala Val Thr Met Glu 180 185 190 Asn Pro Leu Tyr Asn Asn Asn Gly Lys Leu Gly Leu Lys Ile Gly Gly 195 200 205 Pro Leu Gln Val Ala Thr Asp Ser His Ala Leu Thr Leu Gly Thr Gly 210 215 220 Gln Gly Val Ala Val His Asn Asn Leu Leu His Thr Lys Val Thr Gly 225 230 235 240 Ala Ile Gly Phe Asp Thr Ser Gly Asn Met Glu Leu Lys Thr Gly Asp 245 250 255 Gly Leu Tyr Val Asp Ser Ala Gly Pro Asn Gln Lys Leu His Ile Asn 260 265 270 Leu Asn Thr Thr Lys Gly Leu Ala Phe Asp Asn Thr Ala Ile Thr Ile 275 280 285 Asn Ala Gly Lys Gly Leu Glu Phe Glu Thr Asp Ser Ser Asn Gly Asn 290 295 300 Pro Ile Lys Thr Lys Ile Gly Ser Gly Ile Gln Tyr Asn Thr Asn Gly 305 310 315 320 Ala Met Val Ala Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Ser Gly 325 330 335 Ala Ile Thr Met Gly Ser Ile Asn Asn Asp Arg Leu Thr Leu Trp Thr 340 345 350 Thr Pro Asp Pro Ser Pro Asn Cys Arg Ile Ala Ser Asp Lys Asp Cys 355 360 365 Lys Leu Thr Leu Ala Leu Thr Lys Cys Gly Ser Gln Ile Leu Gly Thr 370 375 380 Val Ser Ala Leu Ala Val Ser Gly Asn Met Ala Ser Ile Asn Gly Thr 385 390 395 400 Leu Ser Ser Val Asn Leu Val Leu Arg Phe Asp Asp Asn Gly Val Leu 405 410 415 Met Ser Asn Ser Ser Leu Asp Lys Gln Tyr Trp Asn Phe Arg Asn Gly 420 425 430 Asp Ser Thr Asn Gly Gln Pro Tyr Thr Tyr Ala Val Gly Phe Met Pro 435 440 445 Asn Leu Lys Ala Tyr Pro Lys Thr Gln Ser Lys Thr Ala Lys Ser Asn 450 455 460 Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Ser Lys Pro Leu His 465 470 475 480 Phe Thr Ile Thr Leu Asn Gly Thr Asp Glu Thr Asn Gln Val Ser Lys 485 490 495 Tyr Ser Ile Ser Phe Ser Trp Ser Trp Asn Ser Gly Gln Tyr Thr Asn 500 505 510 Asp Lys Phe Ala Thr Asn Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 515 520 525 <210> 61 <211> 324 <212> PRT <213> Artificial Sequence <220> <223> Ciliary Polypeptide <400> 61 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Thr 35 40 45 Leu Lys Cys Leu Thr Pro Leu Thr Thr Thr Gly Gly Ser Leu Gln Leu 50 55 60 Lys Val Gly Gly Gly Leu Thr Val Asp Asp Thr Asp Gly Thr Leu Gln 65 70 75 80 Glu Asn Ile Arg Ala Thr Ala Pro Ile Thr Lys Asn Asn His Ser Val 85 90 95 Glu Leu Ser Ile Gly Asn Gly Leu Glu Thr Gln Asn Asn Lys Leu Cys 100 105 110 Ala Lys Leu Gly Asn Gly Leu Lys Phe Asn Asn Gly Asp Ile Cys Ile 115 120 125 Lys Asp Ser Ile Asn Thr Leu Trp Thr Gly Ile Asn Pro Pro Pro Asn 130 135 140 Cys Gln Ile Val Glu Asn Thr Asn Thr Asn Asp Gly Lys Leu Thr Leu 145 150 155 160 Val Leu Val Lys Asn Gly Gly Leu Val Asn Gly Tyr Val Ser Leu Val 165 170 175 Gly Val Ser Asp Thr Val Asn Gln Met Phe Thr Gln Lys Thr Ala Asn 180 185 190 Ile Gln Leu Arg Leu Tyr Phe Asp Ser Ser Gly Asn Leu Leu Thr Asp 195 200 205 Glu Ser Asp Leu Lys Ile Pro Leu Lys Asn Lys Ser Ser Thr Ala Thr 210 215 220 Ser Glu Thr Val Ala Ser Ser Lys Ala Phe Met Pro Ser Thr Thr Ala 225 230 235 240 Tyr Pro Phe Asn Thr Thr Thr Arg Asp Ser Glu Asn Tyr Ile His Gly 245 250 255 Ile Cys Tyr Tyr Met Thr Ser Tyr Asp Arg Ser Leu Phe Pro Leu Asn 260 265 270 Ile Ser Ile Met Leu Asn Ser Arg Met Ile Ser Ser Asn Val Ala Tyr 275 280 285 Ala Ile Gln Phe Glu Trp Asn Leu Asn Ala Ser Glu Ser Pro Glu Ser 290 295 300 Asn Ile Ala Thr Leu Thr Thr Ser Pro Phe Phe Phe Ser Tyr Ile Thr 305 310 315 320 Glu Asp Asp Asn <210> 62 <211> 534 <212> PRT <213> artificial sequence <220> <223> Cellulite <400> 62 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro Leu Val Thr Ser His Gly Met Leu Ala Leu 50 55 60 Lys Met Gly Ser Gly Leu Ser Leu Asp Gln Ala Gly Asn Leu Thr Ser 65 70 75 80 Asn Thr Ile Thr Val Ser Gln Pro Leu Lys Lys Thr Lys Ser Asn Ile 85 90 95 Thr Leu Glu Thr Ser Ala Pro Leu Thr Val Ser Ser Gly Ala Leu Thr 100 105 110 Met Ala Thr Thr Ser Pro Leu Val Val Ser Asp Asn Thr Leu Thr Met 115 120 125 Gln Ser Gln Ala Pro Leu Thr Val Gln Asp Ser Lys Leu Ser Ile Ala 130 135 140 Thr Lys Glu Pro Leu Thr Val Leu Asp Gly Lys Leu Ala Leu Gln Thr 145 150 155 160 Ser Ala Pro Leu Ser Ala Thr Asp Asn Asn Ala Leu Thr Ile Thr Ala 165 170 175 Ser Pro Pro Leu Thr Thr Ala Asn Gly Ser Leu Ala Val Thr Met Glu 180 185 190 Asn Pro Leu Tyr Asn Asn Asn Gly Lys Leu Gly Leu Lys Ile Gly Gly 195 200 205 Pro Leu Gln Val Ala Thr Asp Ser His Ala Leu Thr Leu Gly Thr Gly 210 215 220 Gln Gly Val Ala Val His Asn Asn Leu Leu His Thr Lys Val Thr Gly 225 230 235 240 Ala Ile Gly Phe Asp Thr Ser Gly Asn Met Glu Leu Lys Thr Gly Asp 245 250 255 Gly Leu Tyr Val Asp Ser Ala Gly Pro Asn Gln Lys Leu His Ile Asn 260 265 270 Leu Asn Thr Thr Lys Gly Leu Ala Phe Asp Asn Thr Ala Ile Thr Ile 275 280 285 Asn Ala Gly Lys Gly Leu Glu Phe Glu Thr Asp Ser Ser Asn Gly Asn 290 295 300 Pro Ile Lys Thr Lys Ile Gly Ser Gly Ile Gln Tyr Asn Thr Asn Gly 305 310 315 320 Ala Met Val Ala Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Ser Gly 325 330 335 Ala Ile Thr Met Gly Ser Ile Asn Asn Asp Arg Leu Thr Leu Trp Thr 340 345 350 Thr Pro Asp Pro Ser Pro Asn Cys Arg Ile Ala Ser Asp Lys Asp Cys 355 360 365 Lys Leu Thr Leu Ala Leu Thr Lys Cys Gly Ser Gln Ile Leu Gly Thr 370 375 380 Val Ser Ala Leu Ala Val Ser Gly Asn Met Ala Ser Ile Asn Gly Thr 385 390 395 400 Leu Ser Ser Val Asn Leu Val Leu Arg Phe Asp Asp Asn Gly Val Leu 405 410 415 Met Ser Asn Ser Ser Leu Asp Lys Gln Tyr Trp Asn Phe Arg Asn Gly 420 425 430 Asp Ser Thr Asn Gly Gln Pro Tyr Thr Tyr Ala Val Gly Phe Met Pro 435 440 445 Asn Leu Lys Ala Tyr Pro Lys Thr Gln Ser Lys Thr Ala Lys Ser Asn 450 455 460 Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Ser Lys Pro Leu His 465 470 475 480 Phe Thr Ile Thr Leu Asn Gly Thr Asp Glu Thr Asn Gln Val Ser Lys 485 490 495 Tyr Ser Ile Ser Phe Ser Trp Ser Trp Asn Ser Gly Gln Tyr Thr Asn 500 505 510 Asp Lys Phe Ala Thr Asn Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 515 520 525 Lys Lys Lys Lys Lys Lys 530 <210> 63 <211> 425 <212> PRT <213> Artificial sequence <220> <223> Ciliary polypeptide <400> 63 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro Leu Val Thr Lys Asn Gly Glu Ile Thr Leu 50 55 60 Lys Leu Gly Glu Gly Val Asp Leu Asp Ser Ser Gly Lys Leu Ile Ser 65 70 75 80 Asn Thr Ala Thr Lys Ala Ala Ala Pro Leu Ser Phe Ser Asn Asn Thr 85 90 95 Ile Ser Leu Asn Met Asp His Pro Phe Tyr Thr Lys Asp Gly Lys Leu 100 105 110 Ser Leu Gln Val Ser Pro Pro Leu Asn Ile Leu Arg Thr Ser Ile Leu 115 120 125 Asn Thr Leu Ala Leu Gly Phe Gly Ser Gly Leu Gly Leu Arg Gly Ser 130 135 140 Ala Leu Ala Val Gln Leu Val Ser Pro Leu Thr Phe Asp Thr Asp Gly 145 150 155 160 Asn Ile Lys Leu Thr Leu Asp Arg Gly Leu His Val Thr Thr Gly Asp 165 170 175 Ala Ile Glu Ser Asn Ile Ser Trp Ala Lys Gly Leu Lys Phe Glu Asp 180 185 190 Gly Ala Ile Ala Thr Asn Ile Gly Asn Gly Leu Glu Phe Gly Ser Ser 195 200 205 Ser Thr Glu Thr Gly Val Asp Asp Ala Tyr Pro Ile Gln Val Lys Leu 210 215 220 Gly Ser Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Met Ala Gly Asn 225 230 235 240 Lys Glu Asp Asp Lys Leu Thr Leu Trp Thr Thr Pro Asp Pro Ser Pro 245 250 255 Asn Cys Gln Ile Leu Ala Glu Asn Asp Ala Lys Leu Thr Leu Cys Leu 260 265 270 Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Val Val 275 280 285 Gly Ser Gly Asn Leu Asn Pro Ile Thr Gly Thr Val Ser Ser Ala Gln 290 295 300 Val Phe Leu Arg Phe Asp Ala Asn Gly Val Leu Leu Thr Glu His Ser 305 310 315 320 Thr Leu Lys Lys Tyr Trp Gly Tyr Arg Gln Gly Asp Ser Ile Asp Gly 325 330 335 Thr Pro Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Lys Ala Tyr 340 345 350 Pro Lys Ser Gln Ser Ser Thr Thr Lys Asn Asn Ile Val Gly Gln Val 355 360 365 Tyr Met Asn Gly Asp Val Ser Lys Pro Met Leu Leu Thr Ile Thr Leu 370 375 380 Asn Gly Thr Asp Asp Ser Asn Ser Thr Tyr Ser Met Ser Phe Ser Tyr 385 390 395 400 Thr Trp Thr Asn Gly Ser Tyr Val Gly Ala Thr Phe Gly Ala Asn Ser 405 410 415 Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 420 425 <210> 64 <211> 432 <212> PRT <213> Artificial sequence <220> <223> Ciliary polypeptide <400> 64 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro Leu Val Thr Lys Asn Gly Glu Ile Thr Leu 50 55 60 Lys Leu Gly Glu Gly Val Asp Leu Asp Ser Ser Gly Lys Leu Ile Ser 65 70 75 80 Asn Thr Ala Thr Lys Ala Ala Ala Pro Leu Ser Phe Ser Asn Asn Thr 85 90 95 Ile Ser Leu Asn Met Asp His Pro Phe Tyr Thr Lys Asp Gly Lys Leu 100 105 110 Ser Leu Gln Val Ser Pro Pro Leu Asn Ile Leu Arg Thr Ser Ile Leu 115 120 125 Asn Thr Leu Ala Leu Gly Phe Gly Ser Gly Leu Gly Leu Arg Gly Ser 130 135 140 Ala Leu Ala Val Gln Leu Val Ser Pro Leu Thr Phe Asp Thr Asp Gly 145 150 155 160 Asn Ile Lys Leu Thr Leu Asp Arg Gly Leu His Val Thr Thr Gly Asp 165 170 175 Ala Ile Glu Ser Asn Ile Ser Trp Ala Lys Gly Leu Lys Phe Glu Asp 180 185 190 Gly Ala Ile Ala Thr Asn Ile Gly Asn Gly Leu Glu Phe Gly Ser Ser 195 200 205 Ser Thr Glu Thr Gly Val Asp Asp Ala Tyr Pro Ile Gln Val Lys Leu 210 215 220 Gly Ser Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Met Ala Gly Asn 225 230 235 240 Lys Glu Asp Asp Lys Leu Thr Leu Trp Thr Thr Pro Asp Pro Ser Pro 245 250 255 Asn Cys Gln Ile Leu Ala Glu Asn Asp Ala Lys Leu Thr Leu Cys Leu 260 265 270 Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Val Val 275 280 285 Gly Ser Gly Asn Leu Asn Pro Ile Thr Gly Thr Val Ser Ser Ala Gln 290 295 300 Val Phe Leu Arg Phe Asp Ala Asn Gly Val Leu Leu Thr Glu His Ser 305 310 315 320 Thr Leu Lys Lys Tyr Trp Gly Tyr Arg Gln Gly Asp Ser Ile Asp Gly 325 330 335 Thr Pro Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Lys Ala Tyr 340 345 350 Pro Lys Ser Gln Ser Ser Thr Thr Lys Asn Asn Ile Val Gly Gln Val 355 360 365 Tyr Met Asn Gly Asp Val Ser Lys Pro Met Leu Leu Thr Ile Thr Leu 370 375 380 Asn Gly Thr Asp Asp Ser Asn Ser Thr Tyr Ser Met Ser Phe Ser Tyr 385 390 395 400 Thr Trp Thr Asn Gly Ser Tyr Val Gly Ala Thr Phe Gly Ala Asn Ser 405 410 415 Tyr Thr Phe Ser Tyr Ile Ala Gln Glu Lys Lys Lys Lys Lys Lys Lys 420 425 430 <210> 65 <211> 441 <212> PRT <213> artificial sequence <220> <223> Cellulite <400> 65 Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 15 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30 Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45 Leu Arg Leu Ser Glu Pro Leu Val Thr Lys Asn Gly Glu Ile Thr Leu 50 55 60 Lys Leu Gly Glu Gly Val Asp Leu Asp Ser Ser Gly Lys Leu Ile Ser 65 70 75 80 Asn Thr Ala Thr Lys Ala Ala Ala Pro Leu Ser Phe Ser Asn Asn Thr 85 90 95 Ile Ser Leu Asn Met Asp His Pro Phe Tyr Thr Lys Asp Gly Lys Leu 100 105 110 Ser Leu Gln Val Ser Pro Pro Leu Asn Ile Leu Arg Thr Ser Ile Leu 115 120 125 Asn Thr Leu Ala Leu Gly Phe Gly Ser Gly Leu Gly Leu Arg Gly Ser 130 135 140 Ala Leu Ala Val Gln Leu Val Ser Pro Leu Thr Phe Asp Thr Asp Gly 145 150 155 160 Asn Ile Lys Leu Thr Leu Asp Arg Gly Leu His Val Thr Thr Gly Asp 165 170 175 Ala Ile Glu Ser Asn Ile Ser Trp Ala Lys Gly Leu Lys Phe Glu Asp 180 185 190 Gly Ala Ile Ala Thr Asn Ile Gly Asn Gly Leu Glu Phe Gly Ser Ser 195 200 205 Ser Thr Glu Thr Gly Val Asp Asp Ala Tyr Pro Ile Gln Val Lys Leu 210 215 220 Gly Ser Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Met Ala Gly Asn 225 230 235 240 Lys Glu Asp Asp Lys Leu Thr Leu Trp Thr Thr Pro Asp Pro Ser Pro 245 250 255 Asn Cys Gln Ile Leu Ala Glu Asn Asp Ala Lys Leu Thr Leu Cys Leu 260 265 270 Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Val Val 275 280 285 Gly Ser Gly Asn Leu Asn Pro Ile Thr Gly Thr Val Ser Ser Ala Gln 290 295 300 Val Phe Leu Arg Phe Asp Ala Asn Gly Val Leu Leu Thr Glu His Ser 305 310 315 320 Thr Leu Lys Lys Tyr Trp Gly Tyr Arg Gln Gly Asp Ser Ile Asp Gly 325 330 335 Thr Pro Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Lys Ala Tyr 340 345 350 Pro Lys Ser Gln Ser Ser Thr Thr Lys Asn Asn Ile Val Gly Gln Val 355 360 365 Tyr Met Asn Gly Asp Val Ser Lys Pro Met Leu Leu Thr Ile Thr Leu 370 375 380 Asn Gly Thr Asp Asp Ser Gly Gly Ser Ser Gly Lys Lys Lys Lys Lys 385 390 395 400 Lys Lys Ala Ser Gly Gly Ser Ser Thr Tyr Ser Met Ser Phe Ser Tyr 405 410 415 Thr Trp Thr Asn Gly Ser Tyr Val Gly Ala Thr Phe Gly Ala Asn Ser 420 425 430 Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 435 440

Claims

1. A recombinant adenovirus (Ad) comprising (a) an Ad capsid polypeptide from a human Ad6 strain, and (b) at least two hexon hypervariable region (HVR) polypeptides from a human Ad57 strain, wherein the recombinant Ad further comprises a nucleic acid encoding 4-1BBL, CD40L, or a combination of 4-1BBL and CD40L.

2. A recombinant adenovirus (Ad) comprising (a) an Ad capsid polypeptide from a human Ad6 strain, and (b) at least two hexon hypervariable region (HVR) polypeptides from a human Ad57 strain, wherein the recombinant Ad is a conditionally replicating Ad (CRAd), and the CRAd further comprises a nucleic acid encoding 4-1BBL, CD40L, or a combination of 4-1BBL and CD40L.

3. The recombinant Ad of claim 1 or 2, wherein the capsid hexon polypeptide of the human Ad6 strain comprises capsid hexon hypervariable region (HVR) polypeptides 1-7 from the human Ad57 strain.

4. The recombinant Ad of claim 1 or 2, wherein the capsid hexon polypeptide of the human Ad6 strain comprises capsid hexon hypervariable region (HVR) polypeptides 2-6 from the human Ad57 strain.

5. The recombinant Ad of claim 1 or 2, further comprising a capsid polypeptide from one or more different Ad strains, wherein the capsid polypeptide is a pilus polypeptide different from the pilus polypeptides of the human Ad6 strain and the human Ad57 strain.

6. The recombinant Ad of claim 1 or 2, further comprising an amino acid substitution and / or an amino acid modification, wherein the amino acid substitution is a substitution to cysteine ​​in the HVR polypeptide, and the amino acid modification is a modification of cysteine ​​in the HVR polypeptide selected from PEGylation and BAPylation.

7. The recombinant Ad of claim 2, comprising the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO:

45.

8. A recombinant adenovirus (Ad) comprising (a) an Ad genome encoding a hexon polypeptide from a human Ad6 strain, and (b) a nucleic acid encoding at least two hexon hypervariable region (HVR) polypeptides from a human Ad57 strain, wherein the recombinant Ad further comprises a nucleic acid encoding 4-1BBL, CD40L, or a combination of 4-1BBL and CD40L.

9. A recombinant adenovirus (Ad), comprising (a) an Ad genome encoding a hexon polypeptide from a human Ad6 strain, and (b) a nucleic acid encoding at least two hexon hypervariable region (HVR) polypeptides from a human Ad57 strain, wherein the recombinant Ad is a conditionally replicating Ad (CRAd), and the CRAd further comprises a nucleic acid encoding 4-1BBL, CD40L, or a combination of 4-1BBL and CD40L.

10. The recombinant Ad of claim 8 or 9, wherein the capsid hexon polypeptide of the human Ad6 strain comprises capsid hexon hypervariable region (HVR) polypeptides 1-7 from the human Ad57 strain.

11. The recombinant Ad of claim 8 or 9, wherein the capsid hexon polypeptide of the human Ad6 strain comprises capsid hexon hypervariable region (HVR) polypeptides 2-6 from the human Ad57 strain.

12. The recombinant Ad of claim 8 or 9, further comprising a nucleic acid encoding a capsid polypeptide from one or more different Ad strains, wherein the capsid polypeptide is a pilus polypeptide different from the pilus polypeptides of the human Ad6 strain and the human Ad57 strain.

13. The recombinant Ad of claim 9, which is modified to include a dl1101 deletion in the nucleic acid encoding the E1 polypeptide, modified to include a dl1107 deletion in the nucleic acid encoding the E1 polypeptide, or modified to include a dl1101 deletion and a dl1107 deletion in the nucleic acid encoding the E1 polypeptide.

14. The recombinant Ad of claim 13, comprising a nucleic acid encoding the amino acid sequence of SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO:

45.

15. Use of the recombinant adenovirus (Ad) of any one of claims 1 to 14 in the preparation of a medicament for use in a method for treating a mammal suffering from cancer, wherein the method comprises administering the recombinant adenovirus (Ad) to the mammal.

16. The use of claim 15, wherein the cancer is selected from the group consisting of prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, lymphocytic leukemia, and myeloid leukemia.

17. The use of any one of claims 15 to 16, wherein the administration comprises systemic administration.

18. The use of claim 17, wherein systemic administration comprises intramuscular, intranasal or intravenous administration.

19. The use of any one of claims 15 to 16, wherein the administration comprises topical administration.

20. The use of claim 19, wherein the local administration comprises intratumoral injection.

21. Use of the recombinant Ad according to any one of claims 1 to 14 in the preparation of a medicament for treating cancer.

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