RU2782528C1 - Adenoviruses and methods for adenovirus application - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology. A group of inventions is described, including a recombinant oncolytic adenovirus (Ad) based on the human Ad6 strain Ad6, a method for treating a mammal with a malignant neoplasm, the use of a recombinant oncolytic adenovirus for the treatment of malignant neoplasms, and the use of a recombinant oncolytic adenovirus as a drug for the treatment of malignant neoplasms. In one embodiment, the recombinant oncolytic adenovirus comprises capsid hexon polypeptides wherein at least two hexon hypervariable region (HVR) polypeptides of a human Ad6 capsid hexon polypeptide are replaced with at least two HVR polypeptides from human Ad57 strain Ad57.

EFFECT: invention expands the arsenal of recombinant oncolytic adenoviruses.

27 cl, 71 dwg, 2 tbl, 12 ex

Description

BACKGROUND OF THE INVENTION

Field of invention

[0001] The present invention relates to methods and materials for nucleic acid delivery, vaccination and/or treatment of malignant neoplasms. For example, the present invention relates to adenoviruses (Ad) and methods of using adenoviruses for the treatment of diseases such as malignant neoplasms. In one aspect of the invention, the adenovirus described herein can be used as an oncolytic agent.

[0002] Despite tremendous efforts, cancer remains a major health problem in the US 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). Conventional therapies such as chemotherapy, radiation therapy and surgery are often unsuccessful, especially for advanced cancers. One reason for this is that malignant cells can eliminate or modify the components targeted by these therapies and effectively avoid destruction.

[0003] Oncolytic virotherapy may represent an alternative approach for the treatment of malignancies using selectively replicating viruses to kill tumors, activate adaptive immune responses, and provide lifelong immunity against tumors (Russell et al., 2017 Molecular Therapy 25:1107-1116).

[0004] The present invention relates to methods and materials for nucleic acid delivery, vaccination, and/or cancer treatment. For example, the present invention relates to methods and materials for treating cancer by administering one or more recombinant Ads (eg, one or more of Ad657 and variants thereof) as an oncolytic agent. In an embodiment, a recombinant Ad may be derived from a first Ad (eg, may include the genome of a first Ad, such as Ad6, also referred to as a recombinant Ad backbone), and may include a hexon HVR from a second Ad, such as Ad57. If the recombinant Ad includes the Ad6 genome and the HVR hexon Ad57, the recombinant Ad may be a chimeric Ad, referred to as Ad657 (see Nguyen, et al. Oncolytic Virotherapy 7:43-51, 2018).

[0005] In one aspect, the present invention provides methods for vaccinating against an infectious disease using one or more recombinant Ads (eg, one or more Ad657 and variants thereof). In one aspect, the present invention provides methods for treating cancer using one or more recombinant Ads (eg, one or more of Ad657 and variants thereof) as an oncolytic agent. In some instances, one or more recombinant Ads (eg, one or more Ad657s) can be used to reduce cancer cells (eg, by infecting and killing cancer cells) in a mammal. In some instances, one or more recombinant Ads (eg, one or more Ad657 and variants thereof) can be used to stimulate antitumor immune responses in a mammal. In some instances, one or more recombinant Ads (eg, one or more Ad657 and variants thereof) can be used to stimulate immune responses against infectious diseases in a mammal.

[0006] As shown herein, when Ad657 is intravenously injected into mice bearing subcutaneous DU145 human prostate cancer tumors, Ad657 first infects the liver and then reaches distant tumors. Both Ad6 and Ad657 mediated a significant delay in tumor growth and prolongation of survival, with Ad6 mediating higher efficacy.

[0007] The present invention relates to methods and materials for nucleic acid delivery, vaccination, and/or cancer treatment. For example, the present invention relates to methods and materials for treating cancer by administering one or more recombinant Ads (eg, one or more Ad657 and variants thereof) as an oncolytic agent. In an embodiment, a recombinant Ad may be derived from a first Ad (eg, it may include the genome of a first Ad, such as Ad6, also referred to as a recombinant Ad backbone) and may include a hexon HVR from a second Ad, such as Ad57. In cases where the recombinant Ad includes the Ad6 genome and the HVR hexon Ad57, the recombinant Ad may be a chimeric Ad, referred to as Ad657. In one aspect, the present invention provides methods for vaccinating against an infectious disease using one or more recombinant Ads (eg, one or more Ad657 and variants thereof). In one aspect, the present invention provides methods for treating cancer using one or more recombinant Ads (eg, one or more Ad657 and variants thereof) as an oncolytic agent. In some instances, one or more recombinant Ads (eg, one or more Ad657s) can be used to reduce cancer cells (eg, by infecting and killing cancer cells) in a mammal. In some instances, one or more recombinant Ads (eg, one or more Ad657 and variants thereof) can be used to stimulate antitumor immune responses in a mammal. In some instances, one or more recombinant Ads (eg, one or more Ad657 and variants thereof) can be used to stimulate immune responses against infectious diseases in a mammal.

[0008] As shown herein, when Ad657 is intravenously injected into mice bearing DU145 subcutaneous human prostate cancer tumors, Ad657 first infects the liver and then reaches distant tumors. Both Ad6 and Ad657 mediated a significant delay in tumor growth and prolongation of survival, with Ad6 mediating higher efficacy.

[0009] In addition, liver sequestration is a significant problem for essentially any oncolytic virus when used as an intravenous systemic therapy. If the virus infects and destroys hepatocytes, it will cause liver damage at low doses and death at higher doses. It is noteworthy that the introduction of Ad according to the invention, i. chimeric vector Ad657 and its variants, mediated an unexpected lower liver damage than in the case of Ad5 or Ad6. Thus, the unique combination of the Ad6 platform with HVR 1-7 from Ad657 mediated changes in biodistribution and therapy not seen with natural viruses.

[00010] In addition, as shown herein, immunization of rhesus monkeys using replicating single-cycle adenovirus (SC-Ad657) vaccines expressing clade B HIV-1 gp160 only, intranasal (IN) and intramuscular (IM) routes was compared to mucosal and systemic routes of vaccination. SC-Ad vaccines alone result in significant titers of circulating anti-Env antibodies after only a single immunization. Animals immunized by IM alone had high levels of peripheral follicular T helper (pTfh) in the blood, but low levels of Tfh in the lymph nodes, and had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity. Animals immunized with IN had high Tfh levels in the lymph nodes but low levels of pTfh in the blood, and had higher ADCC antibodies. If immunized animals were challenged rectally with SHIV _SF162P3 they all became infected, but mucosally primed animals had a significantly lower viral load in their gastrointestinal tract. Similarly, Ad657, carrying genes for hepatitis C antigens, can lead to cytotoxic T-lymphocyte (CTL) responses against hepatitis and cytomegalovirus. Ad657 can deliver and express therapeutic genes, including cytokines like 4-1BBL, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-21. The results presented herein indicate that recombinant Ads can be used as vehicles for local or systemic delivery of nucleic acids, vaccines, and/or oncolytic virotherapy for cancer.

SUMMARY OF THE INVENTION

[00011] In general, one aspect of the present invention provides 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 hypervariable regions (HVR) hexon polypeptide differ from the HVRs encoded by the Ad genome. The first Ad strain may be a first human Ad strain, and the second Ad strain may be a second human Ad strain different from the first human Ad strain. The first Ad strain and the second Ad strain may be serotypically different. The first Ad strain may be a human Ad6 strain and the second Ad strain may be a human Ad57 strain. The recombinant Ad may also include one or more targeting polypeptides, antigenic polypeptides, enzymes, amino acid substitutions, pegylated regions, ligands, labels, and the like.

[00012] Recombinant Ad can be used as a vector for gene vaccination, administration/delivery of a gene therapy agent, or oncolytic virotherapy.

[00013] In a further embodiment, the recombinant Ad comprises (a) the Ad genome from the 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 polypeptide from one or more Ad strains are different from the HVRs encoded by the first Ad genome.

[00014] In a further embodiment, the recombinant Ad may be a replication-competent or conditionally-replicative Ad (eg, CRAd).

[00015] In another aspect, the present invention provides 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 differ from the amino acid sequence of the hypervariable region of the second hexon polypeptide. The nucleic acid may be from the first Ad strain and the second hexon polypeptide may be from the 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 different from the first human Ad strain. The first Ad strain and the second Ad strain may be serotypically different. 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 include a targeting polypeptide. The targeting polypeptide may include the amino acid sequence TARGEHKEEELI (SEQ ID NO: 1).

[00016] In a further 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 one or more more strains of Ad.

[00017] In a further embodiment, the recombinant Ad may be a replication competent Ad or a conditionally replicative Ad (eg, CRAd).

[00018] In another aspect, the invention relates to materials and methods for treating a mammal having cancer. The methods may comprise or consist essentially of administering to a mammal having a 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 a hexon polypeptide are different from HVRs encoded by the Ad genome and/or Ad containing (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 differs from the amino acid sequence second hexon polypeptide. The mammal may be a human. The malignancy may be prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain malignancy, colon cancer, lymphoma, myeloma, lymphocytic leukemia, or myelogenous leukemia. Administration may include systemic or local administration (eg, intravenous, intratumoral, intramuscular, intraorganic, and lymph node administration).

[00019] As described herein, Ad657 and variants thereof can be used to deliver therapeutic genes to cells for the expression of therapeutic polypeptides. Thus, recombinant Ads, including chimeric Ads, can be used as vehicles for local or systemic delivery of nucleic acids, vaccines, and/or oncolytic virotherapy for cancer.

[00020] Details of one or more embodiments of the invention are given in the examples and in the accompanying drawings, as well as in the description below. 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 from one or more other Ad strains, wherein at least one hypervariable region (HVR) of the hexon polypeptide differs from the HVRs encoded by the Ad genome of the first Ad strain.

[00021] A further aspect of the invention relates to such a recombinant Ad, wherein the first Ad strain and one or more other Ad strains are serotypically different.

[00022] A further aspect of the invention relates to such a recombinant Ad, wherein the first Ad strain is a human Ad6 strain and where the second Ad strain is a human Ad57 strain.

[00023] A further aspect of the invention relates to such a recombinant Ad further comprising a nucleic acid encoding a targeting polypeptide, antigen, enzyme, receptor, ligand, or label.

[00024] An additional aspect of the invention relates to such a recombinant Ad, where the targeting polypeptide contains an amino acid sequence selected from SEQ ID NO: 1-41 and SEQ ID NO: 46-47.

[00025] A further aspect of the invention relates to such a recombinant Ad, wherein said recombinant Ad is a replication competent Ad.

[00026] A further aspect of the invention relates to such a recombinant Ad, wherein the replication competent Ad is a single cycle Ad or conditionally replicative Ad (CRAd).

[00027] A further aspect of the invention relates to such a recombinant adenovirus (Ad) comprising (a) Ad capsid polypeptides from a first Ad strain and (b) at least one hexon polypeptide from one or more other Ad strains, wherein the hypervariable regions (HVRs) the hexon polypeptide or capsid polypeptides differ from the HVR or capsid polypeptides of the first Ad strain.

[00028] A further aspect of the invention relates to such 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.

[00029] A further aspect of the invention relates to such a recombinant Ad, wherein the nucleic acid encoding at least one hexon polypeptide differs from the amino acid sequence of the hypervariable region of said second hexon polypeptide.

[00030] A further aspect of the invention relates to such a recombinant Ad, wherein said nucleic acid is derived from a first Ad strain, and wherein said second hexon polypeptide is derived from a second Ad strain.

[00031] A further aspect of the invention relates to such a recombinant Ad, wherein said first Ad strain is a first human Ad strain, and wherein said second Ad strain is a second human Ad strain different from said first human Ad strain.

[00032] A further aspect of the invention relates to such a recombinant Ad, wherein said first Ad strain and said second Ad strain are serotypically different.

[00033] A further aspect of the invention relates to such a recombinant Ad, wherein said first Ad strain is a human Ad6 strain, and wherein said second Ad strain is a human Ad57 strain.

[00034] An additional aspect of the invention relates to such a recombinant Ad, additionally containing a targeting polypeptide.

[00035] An additional aspect of the invention relates to such a recombinant Ad, where the specified targeting polypeptide contains the amino acid sequence TARGEHKEEELI (SEQ ID NO: 1).

[00036] A further aspect of the invention relates to such a recombinant Ad, wherein said recombinant Ad is a replication competent Ad.

[00037] A further aspect of the invention relates to such a recombinant Ad, wherein said replication competent Ad is a single cycle Ad or conditionally replicative Ad (CRAd).

[00038] A further aspect of the invention relates to a method for treating a mammal having cancer, said method comprising administering to said mammal a recombinant adenovirus (Ad) as described herein.

[00039] A further aspect of the invention relates to such a method, wherein said cancer is selected from the group consisting of prostate cancer, ovarian cancer, lung cancer, hepatic cell carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer intestine, lymphoma, myeloma, lymphocytic leukemia and myelogenous leukemia.

[00040] An additional aspect of the invention relates to such a method, where the specified introduction includes systemic administration.

[00041] An additional aspect of the invention relates to such a method, where systemic administration includes intramuscular, intranasal or intravenous administration.

[00042] An additional aspect of the invention relates to such a method, where the specified introduction includes local administration.

[00043] An additional aspect of the invention relates to such a method, where the specified local introduction includes intratumoral injection.

[00044] Unless otherwise defined, all technical and scientific terms used in the present description have the meaning generally understood by experts in the field to which the present invention belongs. Although methods and materials similar or equivalent to those described herein may be used to practice the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in the present description are incorporated herein by reference in their entirety. In the event of a conflict, the present description, including definitions, shall take precedence. In addition, the materials, methods, and examples are illustrative only and are not intended to limit the invention.

[00045] Details of one or more embodiments of the invention are shown in the accompanying drawings and in the description below. Other features, objects and advantages of the invention will be apparent from the description, drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[00046] Figure 1 shows translation of context-specific peptides from phage into adenovirus. A) Diagram of a phage display library containing Ad5 fiber HI β-sheets that structurally impede a library of random 12-mer peptides. Below is an image of a structurally similar region between β7- and β8-sheets in HVR5 of the Ad5 hexon. B) Primary alignments of amino acids 12.51 and 12.52 in the HI library and their localization upon insertion into the HVR5 hexon. C) Presentation of GFP-Luc-expressing Ad5 viruses modified with peptides.

[00047] Figure 2 shows in vitro transduction. A) GFP expression by fluorescence microscopy of C2C12 cells infected with 10 ⁴ vp/cell of the indicated vectors 2 days after infection. C) Luciferase activity in C2C12 cells 2 days after infection with various MOIs of the indicated Ad.

[00048] Figure 3 shows in vivo transduction in mice. A) Luciferase imaging of mice 1 day after intravenous (IV) or intramuscular (IM) injection. Mice injected with IM were injected with 10 ⁹ vp in each quadriceps muscle. IV injected mice were injected with 10 ¹⁰ vp via the tail vein. C) Quantitative analysis of luciferase activity by imaging on the indicated days after injection. *p<0.05 based on one-way ANOVA. ***p<0.001 based on one-way ANOVA.

[00049] Figure 4 shows in vivo transduction in hamsters. Luciferase activity in hamster muscles 1 day after 10 ¹⁰ vp injection by IM route. **p<0.01 using Student's t-test.

[00050] Figure 5 shows immune responses to gene immunization 16 weeks after a single IM immunization. The mice shown in Fig. 3 were bled 16 weeks after IM injection and their sera were analyzed at serial dilutions by ELISA for detection of antibodies against the GFP protein. *p<0.05, **p<0.01, ***p<0.001 based on one-way ANOVA. ****p<0.0001 based on one-way ANOVA. All soaps injected with Ad produced significant anti-GFP antibodies compared to the PBS group at serum dilutions of 1:10,000 to 1:1,000. Comparison of Ad5-GL-HVR-12.51 and 12.52 with Ad5-GL is shown with black asterisks. Comparison of Ad5-GL-HVR-12.51 and Ad5-GL-HVR-12.52 is shown with a gray asterisk. The gray dashed and dotted lines at OD 0.06 show the 95% confidence interval to distinguish between antibodies other than the PBS group.

[00051] Figure 6 shows a phylogenetic tree of genome-wide sequences of human adenovirus serotypes.

[00052] Figure 7 shows the alignment of Ad5, 6, and 57 showing variations in hexon and E3 regions. (A) Pustell DNA alignment of the Ad6 and Ad57 genomes. The boxes indicate the hexon and E3 regions where the variation is highest between the two viruses. (B) Alignment of ClustalW hypervariable region amino acids in hexon proteins from Ad5, Ad6 and Ad57. Alignments were performed using MacVector.

[00053] Figure 8 shows an image of the Ad657 design when replacing HVR Ad6 with HVR Ad57. Abbreviations: HVR, hypervariable regions.

[00054] Figure 9 shows oncolytic activity in vitro . LNCaP and DU145 cells were treated with the indicated viruses at the indicated vp/cell for 5 days. Cells were stained with crystal violet and cell viability was measured by OD595. Cell viability (%) was calculated by dividing the OD of samples by the average OD of untreated control cells in the same 96-well plate and multiplying this number by 100. (A) Destruction of LNCaP cells. (B) Destruction of DU145 cells. Abbreviations: vp, virus particle.

[00055] Figure 10 shows the effects of oncolytic Ad on liver injury. C57BL/6 mice (n=6 per group) were injected with 10 ¹¹ vp of each virus via the tail vein. (A) Survival by Kaplan-Meier analysis. (B) Blood was collected for ALT measurements 3 days after injection (****p<0.001 by ANOVA). Abbreviations: ALT, alanine aminotransferase; vp, virus particle.

[00056] Figure 11 shows the antitumor activity of Ad6 and Ad657 in DU145 tumor xenografts in nude mice after a single iv injection. Nude mice (n=9 per group) bearing mature DU145 tumors were injected iv with a single dose of 3×10 ¹⁰ vp of the indicated viruses or PBS. (A) Effect of a single iv injection on tumor growth. Tumor dimensions were measured with a caliper and the tumor volume was calculated as width ² × length × 1/2. Data are presented as mean±SE. *p<0.05, ****p=0.0001 when analyzed by ANOVA or Student's t-test as described. Black asterisks with a black arrow pointing up show statistical differences between the Ad6 group and the PBS group on the selected day described in the text. Gray asterisks and an upward pointing arrow show the differences between the Ad657 group and the PBS group on the indicated day. Shaded white asterisks with a gray arrow pointing down show statistical differences between the Ad6 and Ad657 groups on the indicated day. (B) Effect of a single iv injection on survival. Animals were sacrificed when tumor volume reached 2000 µl or when other criteria for sacrifice were met (eg, ulceration) and Kaplan-Meier survival curves were constructed (*p<0.05, **p<0.01 using log-rank test ). Abbreviations: iv, intravenous.

[00057] Figure 12 shows luciferase imaging of nude mice. Four days after a single iv injection of 3×10 ¹⁰ vp Ad6 and Ad657-GFP-Luc with deletions of part 12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K ( RIDβ) and 14.7K and partial deletion of E4 34K. Abbreviations: iv, intravenous; vp, virus particle.

[00058] Figure 13 shows luciferase imaging of nude mice. Notes: Fourteen days after a single iv injection of 3×10 ¹⁰ vp Ad657-GFP-Luc. Abbreviations: iv, intravenous; vp, virus particle.

[00059] Figure 14 shows plasma HIV Env binding titers. Immunizations with different SC-Ads and gp140 proteins are indicated by large arrows above the graph. ELISA midpoint gp140 F8 binding titers are shown for each animal before and after each immunization. The dashed dotted line indicates the lower detection limit for antibodies in this assay. The symbols are distributed along the x-axis at each point in time so that individual measurements can be observed. SC-Ad6-Ebov is a negative control Ad vaccine. This group of animals did not receive a booster dose of gp140. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 based on one-way ANOVA compared to the SC-Ad6-Ebov group.

[00060] Figure 15 shows plasma HIV neutralization titers. Neutralization of these viruses was performed using a TZM-bl neutralization assay. All values were calculated in comparison to wells containing only virus. Each point corresponds to the mean value for each animal.

[00061] Figure 16 shows plasma ADCC activity. Plasma samples were tested using CD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc target cells infected with SHIV _SF162P3 . Each point corresponds to the mean value for each animal. *p<0.05, ***p<0.001, ****p<0.0001 based on one-way ANOVA compared to the SC-Ad6-Ebov group.

[00062] Figure 17 shows ADCC activity in the mucosa. Vaginal washes and saliva samples were tested using CD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc target cells infected with SHIV _SF162P3 . Each point corresponds to the mean value for each animal. *p<0.05 by one-way ANOVA compared to the SC-Ad6-Ebov group.

[00063] Figure 18 shows IFNγ-secreting cells from PBMCs and lymph nodes. PBMC cells and lymph nodes were analyzed by ELISPOT using staining for IFNγ. The designation "against Env" refers to cells that have been stimulated with conserved HIV Env peptides and SC-Ad. The total number of spotting cells (SFCs) for each of the stimulated cell types was calculated and corrected for the control medium as background. Each point corresponds to the mean value for each animal. *p<0.05 based on one-way ANOVA.

[00064] The figure 19 shows the transport and activation of T cells in the mucous membranes. T cells were collected from rectal biopsies obtained after the second protein boost and analyzed by flow cytometry for CD4, CD8, α4β7 integrin, CD69 and FoxP3. Each point corresponds to the mean value for each animal.

[00065] Figure 20 shows the response of Tfh cells in the blood and lymph nodes. PBMC and lymph node cells harvested 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 corresponds to the mean value for each animal. *p<0.05 based on one-way ANOVA.

[00066] Figure 21 shows protection against repeated SHIV _SF162P3 rectal challenge. These groups were challenged rectally with 4.3 TCID ₅₀ (on rhesus PBMC) SHIV _SF162P3 on a weekly basis. Plasma samples were analyzed for copies of SHIV viral RNA. Animals with more than 10 RNA copies were considered infected and the number of infections required to infect that animal were used as events for the Kaplan-Meier survival analysis.

[00067] Figure 22 shows infection and viral load with SHIV _SF162P3 . A) Animals shown in Figure 8 were grouped according to their original SC-Ad priming pathway (IM or IN) into groups of 8 and analyzed by the Kaplan-Meier method. B) Plasma levels of SHIV _SF162P3 viral RNA during the challenge study.

[00068] Figure 23 shows SHIV viral load in tissues. RNA from PBMC and cadaveric tissues was collected and qPCR was performed to detect SHIV viral RNA.

[00069] Figure 24 shows the single cycle adenovirus vaccines used in Example 7. A) Depiction of SC-Ad serotypes 6 and 657 carrying the F8 and G4 envelope genes of HIV clade B. B) Alignment of clade C Ad hexons, including Ad6 and 57 hexons exhibited on vaccines.

[00070] Figure 25 shows the titration of HIV env binding in saliva and vaginal washings. OD450 levels by ELISA results are shown for the indicated samples at the indicated dilutions when tested against F8. The low level of antibodies in these mucosal samples prevents the assay from reaching saturation. For this reason, EC ₅₀ values cannot be easily calculated for most animals. Rhesus monkey Rh13-091 in the IN-IM-IM group was the only animal in which EC _{50 could be calculated (EC 50 =} 4580). Similar results were observed in ELISA using gp140 SF162.

[00071] Figure 26 shows Ad657 expressing hepatitis C and cytomegalovirus (CMV) gB antigen genes leading to in vivo cytotoxic T-lymphocyte (CTL) activity. Destruction of HCV peptide-loaded target cells was shown in mice vaccinated with Ad657-HCV rather than gB CMV.

[00072] Figure 27 shows Ad657 human GM-CSF expression inducing proliferation of human GM-CSF-dependent TF-1 erythroblasts.

[00073] Figure 28 is a graph showing a single IV injection of Ad6 versus A549 lung tumors.

[00074] Figure 29 is a graph showing a single IV or IT injection of Ad6 versus Panc1 pancreatic tumors.

[00075] Figure 30 is a graph showing a single IV injection of Ad6 for kidney cancer in immunocompetent hamsters.

[00076] Figure 31 is a graph showing luciferase activity in B16 melanoma cells and A549 lung cancer cells exposed to Ad exhibiting cell-binding peptides 12.51 in the HVR5 hexon.

[00077] Figure 32 is a graph showing luciferase activity in hepatic cell carcinoma and kidney cancer when exposed to Ad exhibiting cell-binding peptides 12.51 in the HVR5 hexon.

[00078] Figure 33 shows an image combining insertion of individual HVRs from different Ad serotypes with insertion of cell-targeting/non-targeting peptides or novel amino acids such as cysteine into a hexon for targeted chemical modification and shielding. Chimeric HVR constructs are shown that combine different HVRs from different Ad serotypes to modulate natural interactions with cells and blood factors that improve pharmacology, when combined with insertion of cell-binding and cell-non-targeting peptides in different HVRs to alter cell entry and cellular avoidance. . If one HVR is replaced out of 100 Ad, then 100 different hexon chimeras are obtained. If each of all 7 HVRs is contributed by a different HVR Ad, this combinatorial library will be equivalent to ^7,100 variants. If 1 peptide is inserted into 7 HVRs, this will be equivalent to 7× ^7,100 variants. If 10 different peptides are inserted into 7 HVRs, this will equal 10 x 7 x 7 ¹⁰⁰ variants, and so on.

[00079] Figure 34 shows plasmid maps for exemplary combinatorial hexons and peptide combinations. Shown are hexons with HVR1 from Ad6 and HVR 2-7 from Ad57, as well as insertions of cell-targeting peptides into individual HVRs.

[00080] Figure 35 shows chimeric HVR constructs that combine different HVRs from different Ad serotypes to modulate natural pharmacology-enhancing interactions with cells and blood factors when combined with insertion of cell-binding and cell-non-targeting peptides in different HVRs to modify cell entry and cellular escape. In this example, the single amino acid cysteine is inserted into HVR1 and HVR5 Ad657 to modulate pharmacology and allow targeted conjugation of polymers like polyethylene glycol or other residues like imaging agents like fluorophores.

[00081] Figure 36 shows the conjugation of polyethylene glycol (PEG) with Ad657-HVR1-C. A) PAAG electrophoresis with SDS of Ad proteins with or without PEGylation. The arrow indicates the increase in size due to the chemical attachment of PEG to the hexon. B) Effects of targeted PEGylation by maleimide-PEG and non-targeting NHS-PEG on virus infection.

[00082] Figure 37 shows the conjugation of polyethylene glycol (PEG) with Ad657-HVR5-C. A) PAAG electrophoresis with SDS of Ad proteins with or without PEGylation. B) Near-IR imaging of PAAG with SDS of Ad proteins with or without PEGylation and with and without IR800 near-IR imaging label. C) In vivo transduction after intraperitoneal injection of maleimide-pegylated Ad657-HVR5-C by luciferase imaging.

[00083] Figure 38 shows luciferase imaging of nude mice. A) 1, 4, 7, 14, 28 and 42 days after a single I.V. Ad6 injections of relatively distant DU145 prostate tumors. B) 3, 7 and 19 days after I.V. injection of replicating Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

[00084] Figure 39 shows a diagram of dl1101+dl1107 tumor-specific conditionally replicating Ad (CRAd) having a modification in the E1A gene.

[00085] Figure 40 is a graph showing that both Ad6 and Ad657 can be used as CRAd for targeted cancer therapy.

[00086] Figure 41 is a diagram showing Ad therapeutic cycles. A) Scheme of switching serotype with Ad. B) Schematic of an example therapeutic cycle where Ad6 and Ad657 can be used for multiple rounds of treatment via serotype switching in combination with polymer covalent conjugation.

[00087] Figure 42 shows serotype switching and targeted luciferase activity in DU145 prostate tumors after a single IV injection of Ad6 and Ad6-F35 with deletions in the E3A genes (12.5K, 6.7K, 19K, 11.6K) but retained E3B genes (10.4K, 14.5K, and 14.7K) and retention of E4 34K. Mice whose tumors were resistant to a previous single IV injection of Ad657 and CRAd657, both with intact E3 genes, were injected with these vectors via a single IV injection.

[00088] Figure 43 is a diagram of a replication competent Ad (RC-Ad) where E1 expression is controlled by a native E1 promoter; variant CRAd-probasin-E1A (Ad-PB), where E1 expression is controlled by a prostate-specific probasin promoter; CRAd-dl1101, where p300 pathway binding, susceptible to the IFN pathway in normal cells, was eliminated; CRAd-dl1107, where elimination of pRB binding allows the virus to kill malignant cells with RB pathway disruptions, but is repressed in RB ⁺ normal cells; CRAd-dl1101/07, where the binding of the p300 pathway susceptible to the IFN pathway is eliminated, the elimination of pRB binding allows the virus to kill malignant cells with RB pathway disturbances, but it is repressed in RB ⁺ normal cells.

[00089] Figure 44 (A and B) shows the effect of infection of replication competent Ad5, Ad6, Ad657 on non-cancerous cells and modification of Ad6 and Ad657 to become conditionally replicative Ad (CRAd).

[00090] Figure 45 shows the destruction of malignant cells by replication competent Ad5, Ad6, Ad657 and the indicated CRAd.

[00091] Figure 46 shows the modification of Ad6 and Ad657 to become conditionally replicated Ad (CRAd).

[00092] Figure 47 shows the modification of Ad6 and Ad657 to become conditionally replicated Ads (CRAds).

[00093] Figure 48 shows the in vivo effects of replication competent Ad6 or indicated CRAds on the growth of DU145 tumors in mice.

[00094] Figure 49 shows the in vivo effects of replication competent Ad657 and conditionally replicating Ad657-dl1101/07 (both with intact E3 regions) in vivo after a single intravenous injection in mice bearing human prostate tumors.

[00095] Figure 50 shows that pegylation results in adenovirus detargeting to the liver in vivo.

[00096] Figure 51 shows shorter fiber modification of Ad657 from chimpanzee AdC68 and addition of the E4 34K gene with a wobble codon alters in vitro potency.

[00097] Figure 52 shows virus 6/56/6 killing of a human prostate cancer cell with and without CRAd modifications.

[00098] Figure 53 shows antibody production in response to human cancer antigen folate receptor alpha after a single intramuscular immunization of BALB/c mice with CRAd657-dl1101/07-FOLR with an intact E3 region.

[00099] Figure 54 shows regions on HVR Ad that can be modified, for example, by pegylation or "bapilation" using biotin acceptor peptides (BAPs).

[000100] Figure 55 is a diagram of Ad variants having mutations in the E1 protein to convert the virus to conditionally replicating Ad (CRAd).

[000101] Figure 56 shows the amino acid sequences of the N-terminus of the wild type E1A polypeptide and the N-terminus of the E1A variants of CRAd, dl1101, dl1107 and dl1101/1107.

[000102] Figure 57 shows a diagram of the various E3 immunosurveillance evasion genes in Ad species of clade C of instance Ad6 and species of Ad clade D of instance Ad26. Both Ads express variants with different sizes and sequences of the 12.5K, 6.7K, 19K, 10.4K (RIDα), 14.5K (RIDβ) and 14.7K E3 genes, as well as depicting the functions of these E3-encoded proteins.

[000103] Figure 58 shows the effects of pegylation and E3 deletion on the oncolytic viral antitumor activity of Ad6-Luc viruses in immunocompetent hamsters. Both Ad6-Luc and Ad6-Luc-20K PEG have all E3 and E4 34K genes intact. Ad6-deltaE3-Luc has a partial deletion of E3 12.5K and E4 34K and a complete deletion of the E3 genes 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7K . In this immunocompetent animal model, oncolytic efficacy is lost if these immunosurveillance evasion genes are absent in the oncolytic adenovirus.

[000104] Figure 59 is a map of plasmid Ad657 with partial deletion of E3 12.5K and E4 34K and complete deletion of E3 genes 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ) and 14.7K.

[000105] Figure 60 shows 657 CRAd constructs with and without dl1101/1107 CRAd modifications and with and without deletions of selected E3 immunosurveillance evasion genes.

[000106] Figure 61 shows CRAd657 with an E3 insertion site. It is CRAd657 with and without the dl1101/1107 CRAd modifications provided herein and with and without E3 immunosurveillance evasion gene modifications.

[000107] Figure 62 shows CRAd657 +/- Ad35 fiber or Chimpanzee C68 fiber +/- K7 peptide. They are CRAd657 with and without the dl1101/1107 CRAd modifications shown in the preceding images and with and without E3 immunosurveillance evasion gene modifications. In some cases, the E4 34K gene with a "wobble" codon is included after the E4 and before the fiber to compensate for the partial deletion of the E4 34K if the E3B genes are deleted.

[000108] Figure 63 shows CRAd657 +/- Ad35 fiber or chimpanzee C68 strand +/- K7 peptide. They are CRAd657 with and without the CRAd dl1101/1107 modifications shown in previous images and with or without immunosurveillance evasion gene modifications E3.

[000109] Figure 64 shows CRAd657 +/- Ad35 fiber or Chimpanzee C68 fiber +/- K7 peptide expressing folic acid receptor alpha.

[000110] Figure 65 shows CRAd657 +/- Ad35 fiber or Chimpanzee C68 fiber +/- K7 peptide expressing granulocyte-macrophage colony-stimulating factor (GM-CSF).

[000111] Figure 66 shows CRAd657 +/- Ad35 fiber or Chimpanzee C68 fiber +/- K7 peptide expressing 4-1BBL or GM-CSF or IL-21 or CD40L and combinations in a single virus.

[000112] Figure 67 shows Ad6/57 with HVR1 Ad6 and HVR 2-7 Ad57 +/- Ad35 fiber or Chimpanzee C68 fiber +/- K7 peptide.

[000113] Figure 68 shows Ad6/57/6 with HVR1Ad6, HVR 2-6 Ad57, HVR7 Ad6 +/- chimpanzee Ad35 fiber or C68 strand +/- K7 peptide.

[000114] Figure 69 shows Ad6/57/6 with HVR1Ad6, HVR 2-6 Ad57, HVR7 Ad6 +/- Ad35 fiber or Chimpanzee C68 fiber +/- K7 peptide expressing GFP-luciferase.

[000115] Figure 70 shows luciferase imaging after serotype switching. Mice bearing 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 of indicated Ad6/57/6 variants expressing GFP-luciferase with and without fiber variants and codon-optimized E4 34K gene. These Ad6/57/6 variants include the Ad6/57/6 virus having various fiber modifications including added 7 lysines per fiber (K7), a chimpanzee C68 fiber grafted onto an Ad6 fiber after its KKTK flexibility domain, and the Ad6/57 virus /6 with Ad35 fiber. After 7 days, mice were visualized for luciferase activity.

[000116] Figure 71 shows A549 human lung cancer cells treated with indicated viral particles (vp) per Ad6/57/6 cell with and without fiber variants and codon-optimized E4 34K gene. After 7 days, the cells were stained with crystal violet and the wells analyzed with a plate reader spectrophotometer. A high OD indicates the presence of viable cells. Low OD indicates death and loss of adherent cells.

DETAILED DESCRIPTION OF THE INVENTION

[000117] The present invention relates to methods and materials for nucleic acid delivery, vaccination, and/or cancer treatment. For example, the present invention provides methods and materials for protein/polypeptide nucleic acid delivery, vaccination, and/or cancer treatment using one or more recombinant Ad (eg, Ad657 and variants thereof) as an oncolytic agent.

[000118] The adenovirus icosahedron consists of up to 720 copies of its hexon protein. The virus does not use this protein to bind receptors, but this nano-lattice of repetitive proteins provides a template for interactions (eg, natural interactions and non-natural interactions) with proteins, cells, and drugs. Antibodies that can neutralize Ad can be targeted to the hypervariable regions (HVRs) of the hexon polypeptide on Ad.

[000119] In some cases, the present invention relates to recombinant Ad having oncolytic antitumor activity. For example, a recombinant Ad may be derived from the first Ad and may include a hexon HVR from one or more different Ads. HVR can be derived from any Ad clade C species, such as Ad1, Ad2, Ad5, Ad6 and Ad57. In an embodiment, the recombinant Ad may be derived from the first Ad and may include one or more hexon HVRs from at least one other Ad, wherein at least one hexon HVR is different from the HVR of the first Ad. The first Ad strain may be a human Ad6 strain and the second Ad strain may be a human Ad57 strain. The hexon shuttle plasmid map (Figure 34) shows the combination of insertion of individual HVRs from different Ad serotypes with the insertion of cell-targeting/non-targeting peptides or novel amino acids such as cysteine into the hexon for targeted chemical modification and shielding. In an embodiment, the recombinant Ads contain amino acid substitutions, such as cysteine substitutions in polypeptides, and modifications such as pegylation and bapilation. This description shows the ability to target polymer modifications and other chemical modifications to cysteines embedded in the Ad657 hexon.

[000120] Demonstrated Ad657 as an oncolytic agent against human prostate cancer. The Ad6 HVRs were replaced with the Ad57 HVRs to generate the chimeric clade C oncolytic virus Ad657. Ad657 and Ad6 were tested as systemic oncolytic therapeutics via a single i.v. injections to "naked" mice bearing malignant human tumors. Ad657 can be used as a local or systemic oncolytic virotherapy for malignancy. These data also suggest unexpected effects of serotype switching with oncolytic Ad clades C.

[000121] In some instances, the present invention relates to methods of using one or more of the recombinant Ads provided herein for the treatment of a mammal having or at risk of developing a cancer, infectious disease, and/or genetic disease. For example, one or more recombinant Ads can be administered to a mammal having or at risk of developing cancer to reduce the number of cancer cells (eg, by infecting and killing cancer cells) in the mammal (eg, human). For example, one or more recombinant Ads can be administered to a mammal having or at risk of developing cancer to stimulate antitumor immune responses in the mammal (eg, human).

[000122] In some instances, the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) are not degraded by the mammalian immune system. For example, recombinant Ad is not degraded by antigen presenting cells (APCs), macrophages and/or other immune cells of a mammal receiving recombinant Ad.

[000123] In some instances, the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) may be administered over multiple (eg, two or more) treatment rounds. For example, the first recombinant Ad provided herein may avoid antibodies that can neutralize the second recombinant Ad provided herein, and vice versa. In cases where a mammal having a cancer is treated with one or more of the recombinant Ads provided herein, the mammal may be subjected to a first round of treatment with the first recombinant Ad and then may be subjected to a second round of treatment with the second recombinant Ad.

[000124] In some instances, the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) may be replication-competent Ads (RC-Ads). For example, an RC-Ad may be an RC-Ad comprising a nucleic acid encoding an E1 polypeptide (eg, E1 ⁺ RC-Ad). For example, an RC-Ad may be a single cycle Ad (SC-Ad) comprising a deletion of one or more nucleic acids encoding one or more polypeptides associated with the production of infectious viral progeny (eg, pIIIa and E3). For example, an RC-Ad may be a Conditionally Replicative Ad (CRAd). Examples of single cycle Ads and how to make and use them are given elsewhere (International Patent Application Publication No. WO2009/111738).

[000125] In some instances, the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) may be replication defective Ads (RD-Ads). For example, an RD-Ad may be an RD-Ad comprising a deletion of the nucleic acid encoding an E1 polypeptide (eg, an E1-deleted RD-Ad).

[000126] The examples herein show that CRAd 657 and its variants are conditionally replicating Ad (CRAd) in malignant cells, and that infection of cells with CRAd 657 and its variants reduces cell viability and tumor volume. Thus, CRAd 657 and its variants can be used as a local or systemic oncolytic virotherapy for individuals with malignant neoplasms.

[000127] In addition, it has been shown that CRAd can be used to express antigens and as a vaccine for vaccination against viruses, for example, human immunodeficiency virus (HIV), human papillomavirus (HPV) and hepatitis C virus (HCV).

[000128] In some cases, the recombinant Ads provided herein (e.g., recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) can bind to a cell surface receptor (e.g., to facilitate entry of the virus into the cell). For example, the recombinant Ad provided herein can bind to coxsackie-adenoviral receptors (CAR) and/or Fc receptors (eg FcμR and FcγR), complement receptors (eg CR3 and/or C2qR).

[000129] In one aspect of the invention, CRAds may comprise nucleic acids encoding polypeptides that are heterologous to Ad, such as antigens, cell surface receptors, cell-targeting polypeptides, and the like. For example, CRAd-657-dl1101/1107-FolR is a recombinant Ad containing intact E3 and expressing the human folic acid receptor alpha. The present description shows that CRAd can be used to generate antibodies against known cancer antigens, for example, folic acid receptor alpha.

[000130] In some instances, the recombinant Ads provided herein (e.g., recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) may avoid (e.g., do not bind) the scavenger receptor (e.g., to facilitate virus entry in a cell). For example, the recombinant Ad provided herein avoids binding to SREC receptors and/or SR-A receptors.

[000131] In some instances, the recombinant Ads described herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) can avoid phagocytosis.

[000132] In some instances, the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) are non-pathogenic (eg, in a mammal being treated as described herein).

[000133] In some instances, the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) can infect dividing cells (eg, can only infect dividing cells).

[000134] The recombinant Ad provided herein may be any suitable recombinant Ad (eg, a recombinant Ad having oncolytic antitumor activity) produced by recombinant DNA technology and methods known to those skilled in the art. The recombinant Ad may be any Ad obtained by recombination of a material (eg, nucleic acid and/or polypeptide) from any organism other than the Ad from which the recombinant Ad is derived. For example, a recombinant Ad may include one or more materials not naturally occurring in that Ad (eg, not naturally occurring in that Ad prior to recombination). In some instances, the recombinant Ad provided herein may be a chimeric Ad (eg, may include viral elements from two or more (eg, two, three, four, five or more) different Ad genomes).

[000135] These embodiments also refer to Ads in which different HVRs from different Ads are combined (i.e., an HVR permutation is performed). For example, HVR1 from Ad6 with HVR 2-7 from Ad57, or HVR1 and 7 from Ad6 with HVR 2-6 from Ad57, or HVR 1 and 7 from Ad6 and HVR 2-6 from Ad657.

[000136] Nucleic acid and/or polypeptides not naturally occurring in Ad may be from any suitable source. In some cases, the nucleic acid and/or polypeptide not naturally occurring in this Ad may be from a non-viral organism. In some instances, the nucleic acid and/or polypeptide not naturally occurring in the Ad may be from a virus other than the Ad. In some cases, the nucleic acid and/or polypeptide not naturally occurring in this Ad may be from an Ad derived from another species. In some instances, the nucleic acid and/or polypeptide not naturally occurring in the Ad may be from a different strain of the Ad (eg, serotypically different strains). In some cases, the nucleic acid and/or polypeptide not naturally occurring in this Ad may be a synthetic nucleic acid and/or a synthetic polypeptide.

[000137] A recombinant Ad as provided herein (eg, a recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657) can be generated (eg, it may include a genomic backbone) from any suitable Ad. In some cases, the recombinant Ad provided herein can be obtained from an Ad having a low seroprevalence. 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 be exposed to Ad, from which the recombinant Ad presented in the present description is obtained. In terms of seroprevalence, clade C Ad6 and Ad657 adenoviruses have a lower prevalence than the Ad5 archetype virus. In some instances, the recombinant Ad provided herein can be obtained from an Ad having reduced or eliminated side effects (eg, phagocytosis and liver damage). Recombinant Ad can be obtained from Ad isolated from any suitable animal species. For example, Ad can be isolated from non-human primates (eg, monkeys such as marmoset species like the rhesus monkey), fish, frogs, and snakes. In some instances, the recombinant Ad provided herein can be derived from a human Ad (HAd or HAdV). Recombinant Ad can be obtained from any kind of Ad (eg, A, B, C, D, E, F or G). In some instances, the recombinant Ad provided herein can be derived from an Ad species of clade C (eg, human Ad of clade C (HAd-C)). Recombinant Ad can be derived from any suitable Ad serotype (eg 2, 5, 6 or 57). In some instances, the recombinant Ad provided herein can be derived from Ad serotype 6 (Ad6; eg, human Ad6).

[000138] In some instances, a recombinant Ad as provided herein (e.g., a recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) may include an Ad genome containing one or more modifications in one or more nucleic acids, encoding a polypeptide (or fragments thereof), and/or one or more viral elements of the Ad genome. One or more modifications may be any suitable modification. In some cases, the modification may be effective in inhibiting the ability of the modified polypeptide to bind to another polypeptide, such as p300 and/or pRB. In some cases, the modification may be effective in neutralizing one or more interferon pathways. Examples of modifications that can be made to a nucleic acid encoding a polypeptide or viral element include, but are not limited to, substitutions, deletions, insertions, and mutations.

[000139] Ad, for example, Ad657 and its variants, can be modified to retain all E1A genes or modified to delete selected regions and functions of the encoded proteins.

[000140] Figure 57 shows a diagram of the various E3 immunosurveillance evasion genes in Ad6 clade C Ad6 species Ad6 and Ad26 D clade D species, and depicts the functions of these E3-encoded proteins. Both Ads express variants of different sizes and sequences of the 12.5K, 6.7K, 19K, 10.4K (RIDα), 14.5K (RIDβ) and 14.7K E3 genes. 19K reduces the exposure of MHC I and MIC proteins on the cell surface to protect infected cells from T cells and NK cells. RID proteins protect infected cells from death-inducing ligands (FAS, TRAIL, TNFR and EGFR). 14.7K inhibits intrinsic activation of apoptosis in infected cells. Clade C Ad species also express 11.6K, known as adenovirus-infected cell death protein (ADP). Overexpression of ADP accelerates cell death, but in general, cell death remains the same. Clade D viruses also express two new variants, named 49K and 31K. The secreted form of 49K binds to CD46 on T cells and NK cells, resulting in downregulation of these cells and less effective killing of MHC class I deficient cells by NK cells. Ad657 plasmids are modified to retain all native E3 immune surveillance evasion genes (12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ) and 14.7K) and E4 34K or for deletion of selected areas. Ad657 and its variants are also modified with the addition of 49K and 31K to provide these clade C viral platforms with these additional features.

[000141] Ad, eg Ad657 and variants thereof, can be modified to retain all E3 immune surveillance evasion genes or to delete selected regions and functions of encoded proteins. Regarding E3 mutations: 19k downregulates MHCI and MIC proteins on infected cells; overexpression of ADP accelerates cell death, but does not increase the number of cells destroyed; 10k and 14k proteins (RIDα and RIDβ) are combined to block cell killing by extrinsic apoptotic proteins like FAS, TRAIL, TNF, TNFR and EGFR; 14.7k protein inhibits intrinsic apoptosis signaling.

[000142] The retention of these E3 proteins may allow the oncolytic agent to persist longer, and their deletion may increase immune system stimulation.

[000143] Oncolytic efficacy testing data suggests that intact E3 mediates better efficacy.

[000144] DE3 constructs have a 12.5k to 14.7k deleted portion, DE3A constructs have a 12.5k to 19k deleted E3 portion, and DE3ADP constructs have a 12.5k deleted E3 portion up to and including ADP.

[000145] Surprisingly, deletion of all E3 genes makes the oncolytic virus less effective in repressing tumor growth.

[000146] In an embodiment, the present invention relates to a single cycle adenovirus, for example, SC-Ad657 and its variants. Recombinant SC-Ad viruses with heterologous nucleic acids encoding polypeptides have been evaluated for use as a vaccine. The SC-Ad657 vaccines themselves resulted in significant titers of circulating anti-HIV envelope protein antibodies after only a single immunization.

[000147] Similarly, Ad657 carrying hepatitis B and C antigen genes can lead to cytotoxic T-lymphocyte (CTL) responses against hepatitis and cytomegalovirus.

[000148] Ad657 was modified by inserting synthetic peptides from human papillomavirus into HVR5. In an embodiment, the amino acid sequence of the Ad657-HVR5-HPV hexon variant is defined in SEQ ID NO: 57. The modification allows this antigen to be exposed for vaccination purposes as well as redirection via binding to proteins interacting with HPV peptides.

[000149] In an additional embodiment, the present description shows the expression of Ad657 human granulocyte-macrophage colony-stimulating factor (GM-CSF).

[000150] Thus, the examples herein demonstrate that recombinant Ads, eg, Ad657 and variants thereof, can be used to express heterologous proteins, eg, polypeptide antigens and cell-targeting polypeptides.

[000151] In some instances, a recombinant Ad as provided herein (eg, a recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657) may include an Ad genome containing one or more substitutions. For example, one or more nucleic acids encoding a polypeptide (or fragments thereof) and/or one or more viral elements encoded by the Ad genome can be substituted. The replacement may be any suitable replacement. In some instances, one or more nucleic acids encoding a capsid polypeptide of the first Ad genome can be replaced with one or more nucleic acids encoding a second Ad capsid polypeptide to produce a chimeric Ad. For example, if the recombinant Ad includes a genome from a first Ad, where a nucleic acid encoding a capsid polypeptide in the genome is replaced with a nucleic acid encoding a capsid polypeptide from a second Ad (e.g., a different Ad than backbone Ad), a nucleic acid encoding a capsid polypeptide from the second The Ad may express one or more capsid polypeptides, and the expressed capsid polypeptides may be inserted into the capsid of the recombinant Ad. Non-limiting examples of capsid polypeptides include hexon polypeptides, fiber polypeptides, pentone base polypeptides, IIIa polypeptides, IX polypeptides, and pVI polypeptides.

[000152] The Ad fiber protein is a complex of three apparently identical subunits that mediate the initial stage of cell attachment. The native Ad6 fiber protein contains the amino acid sequence shown in SEQ ID NO: 60 and binds to CAR.

[000153] In one aspect of the invention, fiber-modified recombinant and chimeric Ads are prepared having fiber proteins that are not native to the parent Ad or Ad backbone.

[000154] Chimeric Ad, an AdF35 fiber chimera, has the amino acid sequence of SEQ ID NO: 61 and is shorter than the Ad5 and Ad6 fiber proteins and redirects the virus to CD46.

[000155] Fiber-modified recombinant Ad containing fiber K7 having the sequence of SEQ ID NO: 62 targets the virus to heparin sulfate proteoglycans and negative charges on cells.

[000156] Recombinant, chimeric Ad, fiber 6/FC68, containing the sequence of SEQ ID NO: 63, is a chimeric Ad having a fiber protein from chimpanzee adenovirus C68. The fiber protein is shorter than the Ad5 or Ad6 fiber proteins and binds to CAR.

[000157] Recombinant, chimeric Ad, fiber 6/FC68-K7, containing the sequence of SEQ ID NO: 64, is a chimeric Ad having a fiber protein from chimpanzee adenovirus C68. The fiber protein is shorter than the Ad5 or Ad6 fiber proteins. Fiber 6/FC68-K7 binds to CAR and redirects to heparin sulfate and negative charges.

[000158] Recombinant, chimeric Ad, fiber 6/FC68-HI-K7, containing the sequence of SEQ ID NO: 65, is a chimeric Ad having a fiber protein from chimpanzee adenovirus C68. The fiber protein is shorter than the Ad5 or Ad6 fiber proteins. Fiber 6/FC68-HI-K7 binds to CAR and redirects to heparin sulfate and negative charges.

[000159] In some cases, the recombinant Ad may include a genome from the first Ad, where a nucleic acid encoding a hexon polypeptide (e.g., an HVR nucleic acid encoding a hexon polypeptide) in the genome is replaced with a nucleic acid encoding a hexon polypeptide (e.g., an HVR nucleic acid, encoding a hexon polypeptide) from the second Ad. In some instances, the recombinant Ad provided herein may include the genome from the first Ad having one or more HVRs replaced by one or more HVRs from the second Ad. For example, the recombinant Ad may be a chimera, in particular Ad657 (eg, may include the Ad6 genome, where the hexon HVR is replaced with the Ad57 hexon HVR). In cases where the recombinant Ad includes the genome from the first Ad, where the nucleic acid encoding the hexon polypeptide in the genome is replaced with the nucleic acid encoding the hexon polypeptide from the second Ad, the recombinant Ad may include from about 1 to about 720 hexon polypeptides from the second Ad. For example, if the recombinant Ad is Ad657, Ad657 may include the Ad6 genome and 720 hexon polypeptides, including the Ad57 hexon HVR.

[000160] In some instances, a recombinant Ad as provided herein (eg, a recombinant Ad having oncolytic antitumor 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. A nucleic acid deletion may be a complete deletion (eg, a deletion of a nucleic acid encoding a polypeptide) or a partial deletion (eg, a deletion of one or more nucleotides in a nucleic acid encoding a polypeptide). Deletion of a nucleic acid can reduce or eliminate transcription and translation of the polypeptide encoded by the deleted nucleic acid. Deletions can be made with any suitable nucleic acid. In some cases, a nucleic acid encoding a polypeptide associated with the production of infectious progeny can be deleted. Examples of nucleic acids that can be deleted and/or modified in the recombinant Ad provided herein can encode for E1 (e.g., E1A and E1B), E2, E3, E4, pIIIA, fiber, E1B, and include viral enhancers and promoters. For example, a recombinant Ad as provided herein (eg, a recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657) may include an Ad genome containing a deletion of one or more nucleotides in a nucleic acid encoding an E1 polypeptide. In some instances, the recombinant Ad provided herein may include one or more substitutions in the nucleic acid encoding the E1 polypeptide.

[000161] In specific embodiments, the recombinant Ad provided herein is modified to contain a probasin promoter comprising, for example, the nucleic acid of SEQ ID NO: 48; the recombinant Ad provided herein is modified to contain a dl1101 deletion in the nucleic acid encoding the E1 polypeptide; the recombinant Ad provided herein is modified to contain a dl1107 deletion in the nucleic acid encoding the E1 polypeptide; the recombinant Ad provided herein is modified to contain a dl1101 deletion and a dl1107 deletion. See the examples herein and Figure 56 for the N-terminal amino acid sequences of the E1A polypeptide, eg wild-type E1A Ad, and the CRAd-657-dl1101, CRAd-657-dl1107 and CRAd-657-dl1101/1107 variants.

[000162] In an embodiment, the CRAd-657-dl1101/1107-FolR variant contains intact E3 and expresses the human folic acid receptor alpha found on malignant cells.

[000163] Basically, Ad can be modified to include modifications of CRAd presented in the present description.

[000164] In some instances, a recombinant Ad as provided herein (eg, a recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657) may include an Ad genome containing one or more nucleic acid insertions. For example, a nucleic acid insertion may include a nucleic acid encoding a polypeptide. The nucleic acid can be inserted at any suitable location in the genome of the recombinant Ad described herein. In some instances, a nucleic acid encoding a polypeptide can be inserted into an HVR (eg, HVR 5 loop) of the recombinant Ad genome provided herein. For example, if a nucleic acid encoding a polypeptide is inserted into the HVR of the genome of the recombinant Ad provided herein, one or more polypeptides can be expressed from the nucleic acid encoding the polypeptide, and the expressed polypeptides can be inserted into the capsid of the recombinant Ad. In cases where a nucleic acid encoding a polypeptide is inserted into the HVR of the genome of a recombinant Ad as described herein, the recombinant Ad can present from about 1 to about 720 polypeptides encoded by the inserted nucleic acid on its surface. The nucleic acid insertion may be a nucleic acid encoding any suitable polypeptide. In some cases, the nucleic acid insertion may encode a polypeptide antigen.

[000165] In some cases, the nucleic acid insertion may encode a targeting polypeptide. Non-limiting examples of targeting polypeptides that can be included in the recombinant Ad provided herein include 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), alpha-4 integrin binding peptide (NMSLDVNRKA; SEQ ID NO: 6), Met 3-4 (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), 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), XS.1 (GPEDTSRAPENQQKTFHRRW; SEQ ID NO: 16), REDVmyc (MGREDVGEQKLISEEDLGGS; SEQ ID NO: 17), RGD-4C (ACDCRGDCFCG; SEQ ID NO: 18), REDV- 4C (ACDCREDVCFCG; SEQ ID NO: 19), SKBR5C1 (GQIPITEPELCCVPWTEAF Y; 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 (CAAARWKKAFIAVSAANRFKKIS; 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 (QSTPPTKHLTIPRHLRNTL; 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 peptides, PD-1, GLA polypeptides (e.g. factor X), antigen genes, fused e proteins, fusogenic glycoproteins, single-chain antibodies and capsid proteins of other viruses. The targeting polypeptide can target any suitable cell type. Non-limiting examples of cell types that can be targeted by the targeting polypeptide included in the recombinant Ad provided herein include muscle cells (e.g., skeletal muscle cells), tumors, malignant cells, kidney cells, liver cells, mucosal cells, carbohydrates. and cell membranes.

[000166] This example demonstrates that peptides selected in a compatible structural context on phage libraries can be translated into the Ad hexon protein. For example, in the case of peptide 12.51, this insertion site increases transduction in muscle while reducing non-specific infection in the liver. Thus, such recombinant muscle-targeted Ad can be used as a vector for genetic muscle vaccination or gene therapy/delivery to muscle.

[000167] In some cases, the insertion of a nucleic acid can lead to non-targeting of the virus (for example, through disruption of cell-protein interactions occurring in said HVR). In some cases, the nucleic acid insertion may encode a detectable label. Non-limiting examples of detectable labels include fluorophores (eg, green fluorescent protein (GFP), mCherry, and mBFP) and enzymes (eg, luciferase, DNases, proteases, transporters, and polymerases).

[000168] The present invention also relates to expression vectors containing recombinant Ad as described herein (eg, recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof). Expression vectors can carry the recombinant Ad provided herein to another cell (eg, a cancer cell) where it can be replicated and/or expressed. An expression vector, also often referred to as an expression construct, is typically a plasmid or vector having an enhancer/promoter region that controls the expression of a particular nucleic acid. When inserted into a cell, the expression vector can use the cellular machinery for protein synthesis to produce the virus in the cell. In some instances, the expression vectors containing the recombinant Ads described herein may be viral vectors. For example, the expression vector containing the recombinant Ad provided herein may be a retroviral vector. In some instances, expression vectors comprising the recombinant Ad provided herein can also be designed to allow insertion of one or more transgenes (eg, in a multiple cloning site). For example, expression vectors comprising the recombinant Ad provided herein may also include a nucleic acid encoding a detectable label. Non-limiting examples of detectable labels include fluorophores (eg, green fluorescent protein (GFP), mCherry, and mBFP), and enzymes (eg, luciferase, recombinases, nucleases, and transcription factors).

[000169] The present invention also relates to methods and materials for using one or more of the recombinant Ads described herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657). In some instances, the recombinant Ad provided herein can be used to treat a mammal that has or is at risk of developing cancer. For example, methods of treating a mammal having or at risk of developing cancer may include administering one or more of the recombinant Ads provided herein to the mammal. In some instances, methods of treating a mammal having or at risk of developing cancer may include administering one or more expression vectors encoding a recombinant Ad as provided herein or a nucleic acid encoding a recombinant Ad as provided herein to the mammal. In some instances, one or more of the recombinant Ads provided herein may be administered to a mammal to reduce the number of malignant cells in the mammal (eg, suppress and/or delay tumor growth) and/or improve the survival of the mammal.

[000170] Cancer targeting by serotype-switching oncolytic adenoviruses has been shown. Mice bearing DU145 or LNCaP prostate tumors in their flanks were treated once with intravenous (IV) injection of Ad657. These mice were injected a second time with alternative variants of the oncolytic viruses Ad6 or Ad6/57/6 having fiber modifications and expressing GFP-luciferase, and the luciferase activity was measured by imaging. Ad6 has an Ad6 hexon and an Ad6 fiber targeted to CAR. Ad6-F35 has an Ad6 hexon and an Ad35 fiber targeted to CD46. Ad6/57/6 has HVR1 and 7 from Ad6 and HVR 2-6 from Ad57. Ad6/57/6 viruses have an Ad6 fiber, an AdC68 fiber, or an Ad35 fiber. These data, presented in Figure 9, demonstrate the unexpected possibility of serotype switching of oncolytic agents using tumor-targeting viruses with lower off-target liver infection.

[000171] In another example of serotype switching, mice bearing LNCaP prostate tumors on their flanks were subjected to a single intravenous (IV) injection of Ad657 or CRAd657. These mice were injected a second time, 5 months later, with an alternative oncolytic virus Ad6/57/6 expressing GFP-luciferase and fiber variants K7 (with the addition of 7 lysines), F35 (with an Ad35 fiber) or KKTK-C68 (chimpanzee C68 fiber, fused after the flexibility domain KKTK Ad6). The KKTK-C68 virus also has an added codon-optimized E4 34K gene to increase viral productivity. Luciferase activity was measured by imaging. All Ad6/57/6 have a hexon with HVR1 and 7 from Ad6 and HVR 2-6 from Ad57. Ad6/57/6 and KKTK-C68 have CAR-targeted fibers. Ad6/57/6-F35 has an Ad35 fiber targeted to CD46. K7 increases binding to negative charges on cells, including binding to heparin sulfate proteoglycans. Figure 70 shows the ability to switch serotype of oncolytic agents using tumor-targeting viruses with lower off-target liver infection.

[000172] Any suitable mammal having or at risk of developing cancer, infectious disease, and/or genetic disease can be treated as described herein. For example, humans, non-human primates (e.g., monkeys), horses, cattle, pigs, dogs, cats, mice, rats, and food animals having cancer, an infectious disease, and/or a genetic disease can be treated for cancer. , as presented in the present description. In some cases, a person who has a malignant neoplasm can be treated. In some instances, the mammal (e.g., human) being treated as described herein is not the natural host of the Ad used to produce the recombinant Ad described herein (e.g., recombinant Ad having oncolytic antitumor activity, such as recombinant Ad657 ). For example, a human being treated with the recombinant Ad657 provided herein may lack any pre-existing adaptive immunity against Ad6.

[000173] A mammal having any type of cancer may be treated as described herein. In some cases, a malignant neoplasm may include one or more solid tumors. In some cases, a malignant neoplasm may be hemoblastosis. Non-limiting examples of cancers that can be treated as described herein include prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, and leukemias. (eg, lymphocytic leukemias and myelogenous leukemias).

[000174] In some instances, the methods provided herein may also include identifying a mammal as having a cancer. Non-limiting examples of methods for identifying a mammal as having a malignancy include physical examination, laboratory tests (eg, blood and/or urine tests), biopsy, imaging techniques (eg, radiography, PET/CT, MRI, and/or ultrasound), scintigraphy (eg, bone scintigraphy). ), endoscopy and/or genetic testing. Once identified as having a cancer, infectious disease, and/or genetic disease, a mammal may be administered or instructed to self-administer one or more of the recombinant Ads provided herein (e.g., recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 ), or a nucleic acid (eg, an expression vector) encoding one or more of the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657).

[000175] One or more of the recombinant Ads provided herein (eg, recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) may be administered by any suitable route. In some cases, the introduction may be a local introduction. In some cases, the administration may be systemic administration. Non-limiting examples of routes of administration include intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, parenteral, intratumoral, rectourethral, subcapsular, vaginal, and rectal administration. In cases where multiple rounds of treatment are given, the first round of treatment may include administering one or more of the recombinant Ads provided herein to a mammal (e.g., human) by first route (e.g., intravenously), and the second round of treatment may include administering one or more recombinant Ads provided herein to a mammal (eg, human) via a second route (eg, intramuscularly).

[000176] As used herein, the term "pharmaceutical composition" refers to the combination of one or more recombinant and/or chimeric Ads of the present invention with a carrier, inert or active, making the composition particularly suitable for therapeutic use. One or more of the recombinant Ads provided herein (e.g., recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) can be formulated into a composition (e.g., pharmaceutical composition) for administration to a mammal (e.g., a mammal having cancer or having the risk of its development). For example, one or more recombinant Ads can be formulated into a pharmaceutically acceptable composition for administration to a mammal having or at risk of developing cancer. In some instances, one or more recombinant Ads may be formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents. The pharmaceutical composition may be formulated for administration in solid or liquid form, including, but not limited to, sterile solutions, suspensions, sustained release formulations, tablets, capsules, pills, powders, cachets, and granules. Pharmaceutically acceptable carriers, excipients, and carriers that can be used in the pharmaceutical composition provided herein include, but are not limited to, saline (e.g., phosphate buffered saline, ion exchange material, alumina, aluminum stearate, lecithin, whey proteins , such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glycerides of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen orthophosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block copolymers, 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 is described in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20 ^th Ed. (Lippincott Williams & Wilkins, 2003).

[000177] A composition (e.g., a pharmaceutical composition) comprising one or more of the recombinant Ads described herein (e.g., recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657) can be administered to a mammal (e.g., a mammal having cancer or at risk of developing it) as a vaccine. The vaccine may be prophylactic or therapeutic.

[000178] In some instances, the methods provided herein may also include administering to a mammal (eg, a mammal having cancer) one or more additional agents used to treat cancer. One or more additional agents used in the treatment of cancer may include any suitable anticancer agent. In some cases, cancer treatment may include surgery. In some cases, cancer treatment may include radiation therapy. In some cases, cancer treatment may include drug therapy such as chemotherapy, hormonal therapy, targeted therapy, and/or cytotoxic therapy. For example, a mammal having cancer may be administered one or more of the recombinant Ads described herein (e.g., recombinant Ads having oncolytic antitumor activity, such as recombinant Ad657 and variants thereof) and one or more additional agents used to treatment of malignant neoplasms. In cases where a mammal having a cancer is treated with one or more of the recombinant Ads provided herein and is treated with one or more additional agents used to treat a cancer, an infectious disease, and/or a genetic disease, the additional agents used for the treatment of malignancy, infectious disease and/or genetic disease, you can enter simultaneously or independently. For example, one or more of the recombinant Ads provided herein and one or more additional agents used to treat cancer, an infectious disease, and/or a genetic disease can be formulated together to form a single composition. In some instances, one or more of the recombinant Ads provided herein may be administered first, and one or more additional agents used to treat cancer, infectious disease, and/or genetic disease may be administered second, or vice versa.

EXAMPLES

Example 1. Adenoviruses.

[000179] For 50 years, only four human Ad species of clade C have been known, including Ad1, Ad2, Ad5 and Ad6 (see, for example, Weaver et al., 2011 Virology . 412:19-27). In 2001, a fifth adenovirus clade C was identified as field isolate strain #16700, and virus neutralization assays using antisera against Ad1, 2, 5, and 6 showed high levels of neutralization (reciprocal titers 500-16000) when each antiserum was used against a cognate virus. (Lukashev et al., 2008 J Gen Virol . 89:380-388). In contrast, antibodies against Ad1, 2 and 5 have weak cross-reactivity against #16700 (reciprocal titers 32-64). Serum against Ad6 showed higher cross-reactivity against #16700, but neutralization required 10 times higher concentrations of sera to neutralize #16700 compared to Ad6 itself. Subsequent sequence comparisons confirmed that #16700 is a new clade C adenovirus species and was renamed Ad57 (see, for example, Walsh et al., 2011 J. Clin Microbiol. 49:3482-3490).

[000180] 15 Ad human was evaluated for oncolytic activity against malignant neoplasms of the breast, ovary, liver, prostate, kidney and B-cell malignancies. In tests against human prostate tumors DU145, clade C Ad6 species was more active after a single intratumoral or intravenous (i.v.) injection than clade C Ad5 species or clade B virus species Ad11 and Ad35. Ad6 was also more potent than Ad5 and Ad11. in immunocompetent Syrian hamsters.

Construction of recombinant adenoviruses.

[000181] Recombinant adenoviruses were constructed using recombinant DNA technology by methods known to those skilled in the art. The recombinant Ad is derived from the first Ad (eg it may include the genome of the first Ad such as Ad6) and it may include the hexon HVR from the second Ad such as Ad57. In cases where the recombinant Ad includes the Ad6 genome and the HVR hexon Ad57, the recombinant Ad may be a chimeric Ad, referred to as Ad657.

[000182] To obtain Ad657, the Ad57 HVR sequence was synthesized and inserted into the Ad6 hexon in a plasmid in the FRT-Zeocin®-FRT cassette between pVI and hexon. They were recombined into various pAd6 plasmids to produce Ad657 and its variants. The amino acid sequence of the Ad657 hexon is shown in SEQ ID NO: 49. See Ad657 variant plasmid maps in Figures 34 and 59-65.

[000183] For the Ad657 variants, the HVR Ad57 sequence was synthesized using HVR1 modified with cysteine, amino acids that confer flexibility, and restriction sites to allow insertion of other peptides. It was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRT cassette between pVI and hexon. They were recombined into various pAd6 plasmids to generate Ad657 cysteine variants in HVR1, the variant designated as Ad657-HVR1-XXA contains a hexon having the amino acid sequence of SEQ ID NO: 50.

[000184] For the Ad657 variants, the HVR Ad57 sequence was synthesized using HVR5 modified with cysteine, amino acids that confer flexibility, and restriction sites to allow insertion of other peptides. It was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRT cassette between pVI and hexon. These were recombined into various pAd6 plasmids to generate cysteine variants of Ad657 in HVR5, the variant referred to as Ad657-HVR5-XXA contains a hexon having the amino acid sequence of SEQ ID NO: 51.

[000185] For the Ad657 variants, the HVR Ad57 sequence was synthesized using HVR1 modified with cysteine, amino acids that confer flexibility, and restriction sites to allow insertion of other peptides. It was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRT cassette between pVI and hexon. These were recombined into various pAd6 plasmids to produce Ad657 variants without cysteine in HVR1 but with restriction sites allowing peptide insertions into HVR1, the variant, designated Ad657-HVR1-XA, contains a hexon having the amino acid sequence of SEQ ID NO: 52.

[000186] For the Ad657 variants, the HVR Ad57 sequence was synthesized using HVR5 modified with cysteine, amino acids that confer flexibility, and restriction sites to allow insertion of other peptides. It was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin®-FRT cassette between pVI and hexon. These were recombined into various pAd6 plasmids to produce Ad657 variants without cysteine in HVR5 but with restriction sites allowing insertion of peptides into HVR5, the variant, designated Ad657-HVR5-XA, contains a hexon having the amino acid sequence of SEQ ID NO: 53.

[000187] For the Ad657 variants, the Ad657 HVR1-XA sequence was modified by inserting a biotin acceptor peptide into HVR1. These were recombined into various pAd6 plasmids to generate Ad657 BAP variants in HVR1, the variant designated Ad657-HVR1-PSTCD containing a hexon having the amino acid sequence of SEQ ID NO: 54.

[000188] Insertion of a biotin acceptor peptide results in non-targeting of virus variants to the liver, allows virus redirection using avidin or streptavidin and biotinylated ligands, and allows virus purification on monomeric avidin or streptavidin columns.

[000189] For the Ad657 variants, the HVR1-XA Ad657 sequence was modified by inserting a biotin acceptor peptide into HVR1. These were recombined into different pAd6 plasmids to generate Ad657 variants with BAP in HVR1, the variant designated as Ad657-HVR5-PSTCD contains a hexon having the amino acid sequence of SEQ ID NO: 55.

[000190] For the Ad657 variants, the HVR5-XA Ad657 sequence was modified by inserting a V1/V2 synthetic loop from the HIV envelope into HVR5, the variant referred to as Ad657-HVR5-V1/V2 contains a hexon having the amino acid sequence of SEQ ID NO: 56 .

[000191] Insertion of the V1/V2 synthetic loop from the HIV envelope allows the antigen to be exposed to serve as a vaccine, as well as redirected by binding to proteins interacting with the HIV envelope.

[000192] With respect to the Ad657 variants, the HVR5-XA Ad657 sequence was modified by inserting synthetic peptides from human papillomavirus into HVR5, the variant referred to as Ad657-HVR5-HPV contains a hexon having the amino acid sequence of SEQ ID NO: 57.

[000193] The insertion of synthetic peptides from human papillomavirus allows the exposure of HPV peptides as antigens for vaccination purposes, as well as redirection through binding to proteins interacting with HPV peptides.

[000194] In another aspect of the invention, chimeric Ads having HVR1 Ad6 and HVR 2-7 Ad57 were prepared, the chimera, referred to as the HVR Ad6/57 chimera, contains a hexon having the amino acid sequence of SEQ ID NO: 58.

[000195] In another aspect of the invention, chimeric Ads having HVR1 and 7 Ad6 and HVR 2-6Ad57 were prepared, the chimera, referred to as the HVR Ad6/57/6 chimera, contains a hexon having the amino acid sequence of SEQ ID NO: 59.

Example 2 Retargeted and untargeted recombinant adenovirus for gene delivery.

[000196] Adenoviruses are reliable vectors for gene delivery and gene immunization. The adenovirus archetype used in most cases is human adenovirus serotype 5 clade C (HAdV-C5 or Ad5). In vitro , Ad5 binds to and enters cells through the combined interactions of its fiber proteins and pentone base with cell surface receptors. The trimeric fiber binds to the coxsackie-adenoviral receptor (CAR), and cells lacking CAR are relatively resistant to infection unless they also express α _v integrins that can bind to the pentone-based RGD motif.

[000197] In vivo , these interactions are also used, but their importance varies depending on the route of injection. When directly injected into a solid tissue or tumor, CAR and integrin interactions dominate. With intravenous (IV) injection, these interactions become secondary due to the effects of Ad5 binding to vitamin K-dependent clotting factors. Coagulation factor X (FX) binds with subnanomolar affinity to Ad5 hexons and thus allows Ad5 to efficiently transduce hepatocytes after IV injection. In the absence of FX, liver transduction is significantly reduced.

[000198] Adenovirus vectors are somewhat unique due to their ability to carry very large cDNA sequences up to 36 kbp compared to other vectors like adeno-associated virus (AAV) vectors with a DNA sequence of only 4.5 kb This loading capacity justifies early studies of Ad vectors for muscle gene therapy to deliver very large transgenes like the 14 kb dystrophin cDNA. IV administration to neonatal mice can mediate muscle gene therapy, but this possibility is lost in the case of adult mice. The decrease in transfection with age is partly a result of the very large size of Ad virions (ie, 100 nm) as well as the loss of the CAR receptor on muscle cells with age. Currently, the intramuscular (IM) route is the most commonly used route for gene vaccines using Ad5 and other serotypes, despite the fact that CAR is absent on skeletal muscle cells.

[000199] Thus, muscle transduction with Ad5 and another serotype may be sufficient for gene therapy or gene vaccination. However, the lack of a primary receptor for the virus in muscle reduces the effectiveness of the virus and requires delivery of more vector to achieve the desired effects.

Construction of peptide-modified hexons in adenovirus.

[000200] Peptides of 12 amino acids (12-mer) on C2C12 mouse muscle cells were selected from a library of random peptides exposed between H and I β-sheets from the Ad5 "ledge" region (Figure 1A). Peptides 12.51 (TARGEHKEEELI; SEQ ID NO: 1) and 12.52 (LRQTGAASAVWG; SEQ ID NO: 2) were selected against myoblasts with prior purification against non-target cells to obtain peptides that would be specific for muscle cell binding. In most cases, small peptides have a relatively low affinity. Thus, it is hypothesized that displaying these muscle-selected peptides on 720 copies of the Ad5 hexon may allow better delivery of genes to muscle. This insertion site may also have the advantage of inactivating FX binding to hexon to "non-target" the vector to the liver if any vector enters the circulation after IM injection.

[000201] Insertion of these muscle-selected peptides between two other β-sheets, which also impedes a hypervariable loop to the virus, was evaluated for the ability of the modified Ad to modulate tropism. Peptides 12.51 and 12.52 were inserted into the hypervariable region (HVR) 5 loop obstructed by β7 and β8 sheets in the Ad5 hexon. The ability of these viruses to transduce tissues in vivo after intravenous and intramuscular injections in mice and hamsters was evaluated.

Adenoviruses

[000202] E1-deleted Ad5-GL (RD-Ad5-GL) expresses the 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). Muscle-selected peptides 12.51 and 12.52 were inserted in place of HVR5 on the Ad5 hexon between β7 and β8 sheets structurally similar to β-sheets H and I from the Ad5 "ledge" region (Figure 1A) by methods known to those skilled in the art. this area. Flexibility leader peptides are substituted for the entire loop of HVR5 in Ad5 (FIG. 1B). These modified hexon sequences were inserted into a replication-deficient Ad5 expressing a green fluorescent protein-luciferase (RD-Ad5-GL) fusion protein via Red-dependent recombination in bacteria (see, e.g., Campos et al., 2004 Hum. Gene Ther . , 15:1125-1130). Peptide-modified Ads were constructed by inserting annealable oligonucleotides encoding peptides 12.51 and 12.52 (Figure 1B) into the pHVR5 display plasmid (Figure 1A bottom) to generate recombinant Ads, Ad5-HVR5-12.51 and Ad5-HVR5-12.52.

[000203] The resulting plasmids were digested and used for Red-dependent recombination into pAd5-GL. These vectors were rescued in 293 cells purified on two successive CsCl gradients, desalted on Econopac 10-DG (Bio-Rad) chromatographic columns in 50 mM Tris, pH 8, with 0.5 M sucrose, and stored at -80°C. C.

In vitro testing of the virus

[000204] C2C12 mouse myoblasts were purchased from American Type Tissue Culture (Manassas, VA). 293 cells were obtained from Microbix, Toronto, Ontario, Canada. Cells were maintained in DMEM with 10% FBS, Invitrogen.

[000205] C2C12 muscle cells were seeded in 6-well plates (Corning) the day before infection. Viruses were used to infect cells in DMEM with 5% FBS. Cells were incubated for 2 days until green fluorescence was observed.

[000206] Animal experiments were performed with the approval of the Mayo Clinic Institutional Animal Care and Use Committee in accordance with the provisions of the Animal Welfare Act, the PHS Animal Welfare Policy, and the principles of the NIH Laboratory Animal Care and Use Guidelines. Female CD-1 mice (Charles River) were anesthetized and injected intramuscularly (IM) or intravenously (IV) with 10 ¹⁰ vp of the indicated viruses at the indicated time points. Animals were anesthetized, injected with 3 mg D-luciferin (Molecular Imaging Products) and imaged using the Xenogen system. Animals were later anesthetized and blood was collected in tubes to separate serum for ELISA.

[000207] ELISA was performed as follows. Immulon 4 HBX plates (Thermo) were incubated with 100 ng per well of GFP protein in 1x phosphate buffered saline (PBS) at 4°C overnight, washed and blocked with 5% milk in 0.1 Tris buffered saline. % Tween 20 (TBST) at room temperature for 2 hours. Prepared dilutions from 1:100,000 to 1:11,000 of each serum sample in blocking buffer. The wells were washed and 100 μl of each sample was added to GFP-coated plates in triplicate and incubated for 3 hours at room temperature. The wells were washed and a 1/10,000 dilution of HRP-conjugated goat anti-mouse secondary antibody (Pierce Chemical) was added to each well. The plates were incubated for 2 hours at room temperature, washed and 50 μl 1 Step Ultra TMB ELISA (Thermo Fisher Scientific Inc.) for HRP detection was added, followed by 50 μl 2 MH ₂ SO ₄ . Optical density at 450 nm was determined using a Beckman Coulter DTX 880.

[000208] Statistical analyzes were performed using Prism (Graphpad). Statistical significance was calculated by one-way ANOVA followed by Tukey's HSD.

In vitro transduction in C2C12 mouse muscle cells

[000209] RD-Ad5-GL, RD-Ad5-GL-HVR5-12.51, and RD-Ad5-HVR5-12.52 were used to infect C2C12 mouse myoblasts at various multiplicity of infection (MOI) in terms of viral particles (vp)/cell. If green fluorescence of the GFP fusion protein was observed by fluorescence microscopy, both of the peptide-modified vectors mediated significantly better transduction than RD-Ad5-GL (Figure 2A). When luciferase activity was measured, significant increases were observed in RD-Ad5-GL-12.51- and 12.52-infected cells (Figure 2B). When one of the peptides, 12.51, was inserted back into the Ad5 'knob' region, the peptide increased C2C12 myoblast transduction in vitro by 14-fold.

In vivo transduction after intramuscular injection in mice

[000210] 10 ⁹ vp Ad5-GL, Ad5-GL-HVR5-12.51 and Ad5-GL-HVR5-12.52 mice were injected with the IM pathway into both quadriceps and luciferase imaging was performed at different time points (Figure 3A, top). Ad5-GL-HVR5-12.51 resulted in 2-3 times higher luciferase activity than Ad5-GL at all time points (p<0.05 on day 1 by one-way ANOVA) (Figure 3B left). Ad5-GL-HVR5-12.52 activity in muscle was lower than Ad5-GL and HVR5-12.51, in contrast to its higher activity in vitro.

In vivo transduction after intravenous injection in mice

[000211] 3x10 ¹⁰ vp Ad5-GL, Ad5-GL-HVR5-12.51 and Ad5-GL-HVR5-12.52 were injected into mice by IV route and luciferase imaging was performed (Figure 3A bottom). In contrast to the results in muscle, only unmodified Ad5-GL mediated strong liver transduction. Liver transduction of Ad5-GL was 60-fold higher than Ad5-GL-HVR5-12.51 and Ad5-GL-HVR5-12.52 (p<0.001 on day 1 by one-way ANOVA) (Figure 3B right), indicating that recombinant Ads target muscle tissue while reducing off-target infection in the liver.

In vivo transduction after intramuscular injection in hamsters

[000212] To test whether the 12.51-modified vector works in species other than mice, 10 ¹⁰ vp Ad5-GL and Ad5-GL-HVR5-12.51 were injected with the IM pathway into both quadriceps muscles of larger Syrian hamsters and luciferase imaging was performed. after 24 hours (figure 4). In this case, Ad5-GL-HVR5-12.51 mediated 7-fold higher luciferase activity than Ad5-GL (p<0.01 on day 1 by one-way ANOVA).

Gene immunization after intramuscular injection in mice

[000213] 16 weeks after IM injection, sera from mice treated as described above and as shown in Figure 3 were collected and analyzed in serial dilutions by ELISA for antibodies against transgene-encoded GFP (Figure 5). All mice injected with Ad generated significant levels of anti-GFP antibodies compared to the PBS group at serum dilutions from 1:10,000 to 1:1,000 (p<0.0001 by one-way ANOVA). However, Ad5-GL-HVR-12.51 resulted in higher antibody levels than either Ad5 or Ad5-HVR5-12.52. At serum dilutions from 1:1000 to 1:10000, Ad5-GL-HVR-12.51 was significantly higher than Ad5-GL (p<0.01-0.0001 by one-way ANOVA). At a serum dilution of 1:1000, 12.51 was significantly higher than 12.52 (p<0.05). At serum dilutions of 1:1000 and 1:10000, Ad5-GL-12.52 was significantly higher than Ad5-GL (p<0.05-0.001).

[000214] This example shows that peptides selected in a compatible structural context on phage libraries can be translated into the Ad hexon protein. For example, in the case of peptide 12.51, this insertion site increases muscle transduction while reducing off-target infection in the liver. Thus, such recombinant muscle-targeted Ad can be used as a vector for muscle gene vaccination or muscle gene therapy/delivery.

[000215] A further aspect of the invention relates to recombinant and/or chimeric Ads containing other cell-targeting peptides inserted into the Ad657 HVR and the Ad6 and C68 HI loops described in Table 1.

Table 1

Other cell-targeting peptides embedded in HVR Ad657 and HI loops Ad6 and C68

Cell-binding peptide VSV GTWLNPGFPPQSCGYATVT (SEQ ID NO: 4) Integrin binding peptide RGD-4C CDCRGDCFC (SEQ ID NO: 5) Phage-selected peptide 12.51 TARGEHKEEELI (SEQ ID NO: 1) Phage-selected peptide 12.52 LRQTGAASAVWG (SEQ ID NO: 2) Phage-selected peptide 12.53 ARRADTQWRGLE (SEQ ID NO: 3) Alpha 4 binding peptide NMSLDVNRKA (SEQ ID NO: 6) Lung cell binding peptide, L10.1 WTMGLDQLRDSSWAHGGFSA (SEQ ID NO: 9) Lung cell binding peptide, L10.2 RSVSGTEWVPMNEQHRGAIW (SEQ ID NO: 10) Lung cell binding peptide, L10.5 TELRTHTSKELTIRTAASSD(SEQ ID NO: 11) Muscle cell binding peptide, S5.1 DRAIGWQDKLYKLPLGSIHN (SEQ ID NO: 12) Prostate cancer cell binding peptide DU9C.1 MGSWEKAALWNRVSASSGGA (SEQ ID NO: 13) Prostate cancer cell binding peptide, DU9C.2 MAMGGKPERPADSDNVQVRG (SEQ ID NO: 14) Prostate cancer cell binding peptide, DU9A.7 MASRGDAGEGSTQSNTNVPS (SEQ ID NO: 15) Dendritic cell binding peptide XS.1 GPEDTSRAPENQQKTFHRRW (SEQ ID NO: 17) REDV endothelial cell binding peptide REVY (SEQ ID NO: 46) Breast cancer cell binding peptide, SKBR5C1 GQIPITEPELCCCVPWTEAFY (SEQ ID NQ: 20) Breast cancer cell binding peptide, 231R10.1 PQPPNSTAHPNPHKAPPNTT (SEQ ID NO: 21) Hepatic cell carcinoma binding peptide, HepaCD8 VRWFPGGEWGVTHPESLPPP (SEQ ID NO: 22) Breast cancer cell binding peptide, HI Met 231 3-4 ISLSSHRATWVV (SEQ ID NO: 47) Selected peptides of B-cell malignancies: 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) 1-6 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) Small biotin acceptor peptide BAP GLNDIFEAQKIEWH (SEQ ID NO: 24) calmodulin-binding peptide CAAARWKKAFIAVSAANRFKKIS (SEQ ID NO: 25)

[000216] DNA encoding these peptides and its complementary DNA flanked with sticky ends for ligation into Ad plasmids, eg hexon XA plasmids or pAd6-NdePfl fiber shuttle plasmids, were synthesized. These annealable oligonucleotides were ligated into the HVR or HI Ad loop. These plasmids were used for recombination into various Ad backbone plasmids.

[000217] Some peptides are designed to target new receptors on cells. Other peptides like the small BAP can be used for avidin targeting and purification if the virus is grown in cells expressing the bacterial BirA biotin ligase. The calmodulin peptide allows the virus to bind to calmodulin or calmodulin fusion proteins to redirect or clear the virus.

[000218] Thus, such recombinant Ads targeting specific tissues/cell receptors can be used as a vector for gene vaccination or gene therapy in target cells and/or tissues.

Example 3 Insertion of individual HVRs from different Ad serotypes with insertion of cell-targeting/non-targeting peptides or novel amino acids.

[000219] The hexon shuttle plasmid map (Figure 34) shows the combination of insertion of individual HVRs from different Ad serotypes with the insertion of cell-targeting/non-targeting peptides or novel amino acids such as cysteine into the hexon for targeted chemical modification and shielding.

[000220] In certain embodiments, cell-binding peptides 12.51, VSV, RGD (see Table 1) are inserted into HVR 1 or HVR 5, where the embodiments serve as examples of insertion of these and other peptides into any of the HVR Ad (Figure 34) . In another example, these HVRs are shown to insert a biotin acceptor peptide (BAP) allowing vector redirection with avidin or streptavidin and biotinylated ligands or with avidin or streptavidin fusion proteins. BAP insertion also allows the purification of viruses on monomeric avidin or streptavidin columns to obtain a vector. Similarly, Ad57-HVR1-XXA and XA are an example of cysteine insertion at this site to allow targeted chemical modification with maleimide or other cysteine-reactive agents (Figure 34).

[000221] These embodiments also refer to Ads in which different HVRs from different Ads are combined (ie, HVR permutation). For example, HVR1 from Ad6 and HVR 2-7 from Ad57 or HVR1 and 7 from Ad6 with HVR 2-6 from Ad57. In a further embodiment, the 6/56/6 virus has HVR 1 and 7 from Ad6 and HVR 2-6 from Ad657.

Example 4 Targeted Chemical Conjugation of Cysteine Modified Hexon Modified Ad657-HVR5C.

[000222] Figure 35 is a depiction of Ad variants showing the combination of insertion of individual HVRs from different Ad serotypes with insertion of new amino acids such as cysteine into a hexon for targeted chemical modification and shielding.

[000223] Comparison of the effects of non-targeted chemical conjugation and directed chemical conjugation on shielding and function of cysteine-modified hexon-modified Ad657-HVR1C (Figure 36). This example demonstrates the possibility of targeting polymer and other chemical modifications to cysteines embedded in the Ad hexon. Non-targeted PEG inactivates the viral infection, while cysteine-targeted PEGylation preserves the functions of the virus.

[000224] In one aspect of the invention, the use of polymers or embedded peptides/proteins is contemplated for detargeting, retargeting, and shielding from antibodies, proteins, cells. Figure 54 shows HVR Ad regions that can be modified, for example, by pegylation or "bapilling".

[000225] In an embodiment, the different serotypes and/or Ad variants include polymer shielding to allow multiple administration of Ad6 and Ad657 variants. An example of a therapeutic cycle is given where Ad6 and Ad657 can be used for multiple rounds of treatment due to serotype switching in combination with covalent polymer conjugation (Figure 41B).

[000226] Ad657-HVR1C expressing GFP-luciferase was obtained from cells and purified using CsCl gradients. The virus was covalently modified with 5 kDa polyethylene glycol (PEG). Virus was treated with NHS-PEG randomly reacting with amines/lysines on viral proteins or maleimide-PEG reacting specifically with cysteine inserted into HVR1 using the shuttle plasmid XXA. These unmodified or modified viruses were then purified by final centrifugation in CsCl followed by desalting. The specified virus was separated using PAAG in SDS, stained with SyproRuby and visualized (figure 36A). It has been shown that NHS-pegylation allows random modification of many viral proteins, as evidenced by increases in the apparent mass of proteins (shown by arrows). In contrast, the targeted reaction of maleimide-PEG with cysteine in HVR1 allows modification of only the hexon and does not damage other viral capsomeric proteins. The effects of pegylation on virus function were evaluated.

[000227] These viruses were incubated with A549 cells and their ability to infect cells was measured by luciferase assay. This indicates that random NHS-pegylation reduces viral activity by more than 90%, while maleimide-PEG does not (Figure 36B).

[000228] Immunocompetent Syrian hamsters were engrafted with subcutaneous HaK kidney cancer tumors. When they reached a volume of 200 μl, the hamsters were injected once intravenously with the indicated Ad6 viruses engineered with and without E3 (DE3) and with or without random NHS-pegylation. Tumor size was measured over time. Evidence suggests that deletion of all E3 genes in the Ad6-deltaE3-Luc oncolytic virus renders the virus less effective in reducing tumor volume than the parental Ad6-Luc oncolytic virus. The data also suggests that Ad6 can be pegylated and retain their efficacy (see Ad6-Luc vs. Ad6-Luc-20 kDa PEG) (Figure 58).

[000229] Targeted chemical conjugation of cysteine-modified hexon-modified Ad, eg Ad657-HVR5C. Ad657-HVR5C expressing GFP-luciferase was obtained from cells and purified using CsCl gradients. The virus was covalently modified with the near-IR fluorophore maleimide-IR800, maleimide-biotin or 5 kDa maleimide-PEG reacting specifically with cysteine inserted into HVR5 using the XXA shuttle plasmid. Said Ad and modified Ad were separated by SDS-PAGE, stained with SyproRuby and visualized (FIG. 37A). SDS-PAGE of viral proteins followed by near-IR imaging indicated that HVR-C could be labeled with an imaging agent (FIG. 37B). The effects of pegylation on Ad virus function in vivo were demonstrated by intraperitoneal injection of pegylated Ad virus. The ability to infect tumor-bearing mouse cells was demonstrated by detectable luciferase activity by imaging. Figure 37C shows the possibility of polymer targeting and other chemical modifications to cysteines embedded in the region of the Ad657 hexon. In addition, pegylation has been shown to cause non-targeting of adenovirus to the liver in vivo (Figure 50).

Example 5 Ad657 expression of human granulocyte-macrophage colony-stimulating factor (GM-CSF).

[000230] Ad657 carrying human GM-CSF cDNA was used to infect A549 cells and various amounts of supernatant were added to GM-CSF dependent TF-1 cells. An increase in the number of cells indicates the expression of a functional human cytokine (figure 27). The data suggest that recombinant Ads can be used to express heterologous proteins.

Example 6 Ad657 oncolytic adenovirus for systemic virotherapy against malignant cells.

[000231] An alignment of the selected full genomes of Ad that generated a phylogenetic tree in which Ad57 clusters with other clade C virus species with the highest homology to Ad6 is shown in Figure 6.

[000232] Ad57 appears almost identical to Ad6 with sequence divergence in hexon hypervariable regions (HVRs) and E3 immunosurveillance evasion genes (Figure 7). Other exposed viral capsid proteins, including fiber, pentone base, IIIa and IX, are essentially identical between Ad6 and Ad57. Neutralization assay data is consistent with most adenovirus neutralizing antibodies targeting HVR Ad. The reason for the low cross-reactivity between Ad6 antiserum and Ad57 is thought to be antibodies that can target the common fiber protein (Lukashev et al., 2008 J Gen Virol . 89:380-388).

[000233] This example demonstrates the usefulness of Ad657 as an oncolytic agent against human prostate cancer. Ad6 HVRs were substituted for HVRs from Ad57 to generate a chimeric clade C oncolytic virus named Ad657. Ad657 and Ad6 were tested as systemic oncolytic therapy via a single i.v. injections into nude mice bearing human prostate cancer tumors. The liver and tumor tropism of this virus was evaluated in mouse models of prostate cancer as follows.

[000234] DU145 human prostate carcinoma cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and tested free of specific pathogens by IMPACT RADIL testing. 293 cells were obtained from Microbix, Toronto, Ontario, Canada. Cells were maintained in DMEM with 10% FBS (Invitrogen, Grand Island, NY, USA).

[000235] The Ad6 genome of strain Tonsil 99 (ATCC VR-1083) was cloned as described elsewhere (see, for example, Weaver et al., 2013 PLoS One . 8:e73313). The cassette corresponding to the Ad57 hexon between the natural regions of ApaI and SacI was synthesized in Genscript. This fragment was cloned into the pUC57-Hexon Ad6-FZF shuttle plasmid containing the pVI and Ad6 hexon genes with the FRT FRT- ^Zeocin® resistance gene cassette between them for homologous recombination in bacteria 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 fragment of ApaI-SacI was replaced with the Ad57 fragment, resulting in plasmid pUC57-Hexon Ad6/57-FZF. It has been recombined into the Ad6 genome by Red-dependent recombination (Campos et al., 2004 Hum Gene Ther . 15:1125-1130). Figure 59 shows a map of plasmid Ad657 with an E3 deletion. Viruses were rescued by transfection in 293 cells and produced in CellStack 10 culture chambers (Corning Life Sciences, Lowell, MA, USA). Viruses expressing the green fluorescent protein-luciferase (GFP-Luc) fusion protein contain the CMV-GFP-Luc expression cassette inserted between the Ad and E4 fiber and an E3 deletion to make room for this insertion. Viruses were purified using two CsCl gradients and the number of viral particles (vp) was calculated from OD260.

[000236] To study oncolytic activity in vitro , cells were treated at the indicated multiplicity of infection (MOI) in terms of vp/cell in DMEM with 5% FBS and an antibiotic-antifungal agent (Invitrogen, Grand Island, NY, USA). After five days, media was removed and 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. Cells were washed twice with PBS and then incubated overnight at 37° C. in 0.1% sodium dodecyl sulfate in PBS to solubilize crystal violet. Crystal violet absorbance was measured at OD595 using a Beckman Coulter DTX 880 plate reader spectrophotometer. Cell viability (%) was calculated by dividing the OD of samples by the mean OD of untreated control cells in the same 96-well plate and multiplying this number by 100.

[000237] Animals were kept in the Mayo Clinic vivarium according to the guidelines of the International Association for the Evaluation and Accreditation of Laboratory Animal Research. The studies were approved by the Mayo Clinic Institutional Animal Care and Use Committee in accordance with the provisions of the Animal Welfare Act, PHS Animal Welfare Policy. Subcutaneous tumor development in 4 week old nude mice (Harlan Sprague Dawley, Indianapolis, IN, USA) was initiated by subcutaneous injection (sc) of 1 x 10 ⁷ DU145 cells in 100 μl DMEM/50% Matrigel (BD Biosciences, San Jose, CA, USA). Tumor volumes were calculated using the equation width ² ×length×1/2. When the tumors reached a volume of ~200 μl, mice were divided into different groups and plunged them with a single iv injection into the tail vein. Animals were sacrificed when the tumor volume reached 2000 μl, or if the animals were in agony, distress, or if the skin had ruptured over the tumor.

[000238] For blood alanine aminotransferase (ALT) measurements, groups of six C57BL/6 mice were injected with iv 10 ¹¹ vp Ad5, Ad6, or Ad657 in the tail vein and blood was collected 3 days later for ALT measurement using an ALT activity assay (Sigma-Aldrich, St. Louis, MO, USA).

[000239] 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 built and compared using a logarithmic rank test.

[000240] The Ad57 capsomeric protein genes are almost identical to Ad6 except for the HVR hexon (Figures 6 and 7). To obtain a chimeric Ad57 and Ad6 virus, the cassette corresponding to the Ad57 hexon HVR was recombined into the wild-type Ad6 genome (Figure 8). This virus was rescued, produced in 293 cells and purified on CsCl gradients. Given that the backbone of the viral genome was Ad6, these hexon-chimeric viruses are referred to as Ad657 (Figure 59).

[000241] In vitro oncolytic activity was assessed by infecting LNCap and DU145 cells using 10, 100 or 1000 vp/cell. To compare liver damage to Ad5, Ad6 and Ad657, a high dose of 10 ¹¹ vp of each virus was injected into the tail vein of immunocompetent C57BL/6 mice. Animals injected with Ad5 experienced agony within 2 days and had to be sacrificed (FIG. 10A). Survival of Ad5 and Ad6 was significantly lower compared to PBS ( p = 0.0001 and 0.0009, respectively, by log-rank analysis). Survival of Ad657 was also reduced compared to PBS ( p =0.0578). Survival after exposure to Ad6 or Ad657 was significantly better than mice treated with Ad5 ( p =0.0001 and 0.0001, respectively). Survival was not significantly different between Ad6 and Ad657 ( p =0.248).

[000242] ALT was measured in blood 3 days after injection in animals surviving Ad6 and Ad657. Animals treated with Ad5 were not tested because the main part of the group had to be killed. This analysis showed that Ad6 provoked relatively low levels of liver damage in terms of the release of the liver ALT enzyme into the bloodstream (Figure 10B). Both Ad6 and Ad657 groups had low, but significant, ALT levels compared to PBS-treated mice ( p <0.001 by Tukey's one-way ANOVA for both viruses). Ad657 resulted in lower ALT levels than Ad6 ( p <0.001 by ANOVA). This corresponded to higher levels of Ad6 infection in the liver than Ad657 after iv injection of luciferase-expressing viruses (Figure 12). One animal in the Ad657 group was lost after blood sampling on day 3. After 6 days, most of the animals in the Ad6 group went into agony (figure 10A). In contrast, 50% of the animals in the Ad657 group survived more than 2 weeks after administration.

[000243] To compare the oncolytic activity of Ad6 and Ad657 against human DU145 prostate tumors, DU145 sc cells were engrafted in nude mice. Animals were grouped according to similar tumor sizes averaging 200 μl, and groups of nine mice were given a single iv dose of 3×10 ¹⁰ vp Ad6 or Ad657 (FIG. 11).

[000244] This single iv injection of Ad6 and Ad657 resulted in a reduction in tumor size compared to control animals injected with PBS. Tumors were significantly smaller in the Ad6 group at 7 days compared to the PBS group ( p < 0.05 by two-way ANOVA using Tukey's multiple comparison test). Tumors in the Ad657 group were significantly different from tumors in the PBS group by day 14 ( p <0.01 by ANOVA). Both Ad6 and Ad657 maintained a significant difference with PBS until day 38 ( p < 0.0001 by two-way ANOVA). This comparison ended on day 38 when the first animal in the PBS group had to be euthanized and the subsequent comparison would be skewed due to changes in the number of animals. Tumor sizes in the Ad6 and Ad657 groups did not differ significantly until day 38, when the Ad657 group had a significantly larger tumor volume ( p < 0.05) based on two-way ANOVA (Figure 11A). This difference between tumor sizes in the Ad6 and Ad657 groups persisted until day 52 (p=0.04, Student's t-test), after which the tumors no longer differed significantly.

[000245] When overall survival was assessed, both Ad6 and Ad657 significantly extended survival compared to PBS-treated animals (Figure 11B, p <0.01 and 0.05, respectively, by log-rank analysis). Overall survival in the Ad6 group was significantly better than in the Ad657 group ( p <0.05). However, this was a survival artifact attributed entirely to the fact that three of the animals in the Ad657 group had to be euthanized according to Institutional Animal Care and Use Committee (IACUC) guidelines due to skin ulceration over the tumor rather than to excess tumor size. In some cases, ulceration is specifically associated with effective tumor control. Like Ad6, Ad657 expressing GFP-luciferase resulted in significant luciferase activity in distant subcutaneous DU145 tumors after a single iv injection (Figure 12 and Figure 13). This suggests that both Ad6 and Ad657 can mediate oncolytic effects in prostate tumors after a single systemic injection.

[000246] This example shows that Ad657 can be used as a local or systemic oncolytic virotherapy for prostate cancer. These data also suggest unexpected effects of serotype switching in the oncolytic Ad clade C.

[000247] The oncolytic activity of Ad was evaluated in tumor cells and/or malignant tumors. A single IV injection of Ad6 was evaluated against A549 lung tumor cells (FIG. 28); single IV or intratumoral (IT) injection of Ad6 was evaluated for Panc1 pancreatic tumors (Figure 29), and single IV injection of Ad6 was evaluated for kidney cancer in immunocompetent hamsters (Figure 30).

[000248] Figure 38 shows luciferase imaging of nude mice. A) 1, 4, 7, 14, 28 and 42 days after a single I.V. Ad6 injections for distant DU145 prostate tumors. B) 3, 7 and 19 days after I.V. injection of replicating Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

[000249] It can be concluded that after IV injection, Ad6 reaches distant target cells.

[000250] In another embodiment, luciferase-expressing Ads with or without peptide library-derived peptides 12.51 and 12.52 inserted into the HVR5 hexon were incubated on the indicated cell lines, B16 melanoma cells and A549 lung carcinoma cells, with the indicated number of viral particles (vp) and measured luciferase activity. Shows improved infection of malignant cells with Ad bearing peptide-modified hexons (figure 31).

Improved infection of malignant Ad cells bearing peptide-modified hexons.

[000251] Luciferase-expressing Ads with peptides 12.51 and 12.52 derived from a peptide library inserted into the HVR5 hexon were incubated on the indicated hepatic cell carcinoma cell lines using 10 ⁴ vp of each virus and luciferase activity was measured. Shows improved infection of malignant cells with Ad bearing peptide-modified hexons (figure 32).

EXAMPLE 7 Divergent HIV-1 driven immune responses achieved by systemic and mucosal immunization with replicating single cycle adenoviruses in rhesus monkeys.

[000252] Most adenovirus gene vaccines are replication defective Ad (RD-Ad) vectors in which the E1 gene has been deleted to prevent replication and initiate Ad infections. In helper-dependent adenoviruses (HD-Ad), all Ad genes are deleted and they are also replication defective. An E1-deleted Ad vaccine can infect a cell, deliver one copy of the antigen gene, and express one copy (eg, "1X") of that antigen. They are safe but do not replicate or express transgenes.

[000253] In contrast, an E1 ⁺ replication-competent Ad (RC-Ad) vaccine can infect the same cell, replicate the antigen gene DNA 10,000 times, produce significantly more antigen, and elicit stronger immune responses than E1-deleted vectors. Although RC-Ad is more potent than RD-Ad, replication-competent Ads may pose a significant risk for severe adenoviral infections in humans.

[000254] To take advantage of transgene DNA replication but avoid the risk of adenoviral infections, single-cycle Ad (SC-Ad) vectors were developed with a gene deletion for a key viral late protein, pIIIa (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-Ads retain their E1 genes, allowing their genome to replicate, but the absence of pIIIa blocks the production of infectious viral progeny. SC-Ads replicate their genomes and transgenes, as well as RC-Ads (up to 10,000 times; Crosby et al., 2014. Virology 462-463:158-165). RC- and SC-Ad produce more transgenic protein than RD-Ad vectors (Crosby et al., 2014. Virology 462-463:158-165). SC-Ads generate stronger and more persistent immune responses than RD-Ads or RC-Ads (Crosby et al., 2015 J Virol 89:669-675). When compared side by side, SC-Ad results in significantly more antibodies and better protection against influenza virus (Crosby et al., 2017 J Virol 91:e00720-16).

[000255] In this study, rhesus monkeys were immunized with SC-Ad expressing clade B envelope sequences obtained from an HIV-1 patient before and after their humoral response underwent an increase in neutralization latitude. Macaques were immunized by a single systemic IM immunization or a single mucosal intranasal (IN) immunization. Animals were then boosted by the same or alternative routes of SC-6 Ad followed by a protein boost. This example describes how these different SC-Ad immunization strategies affected the formation of HIV-binding, antibody-dependent cellular cytotoxicity (ADCC) and neutralizing antibodies, as well as their effects on cellular immune responses, including follicular T helper (pTfh) in blood and lymph nodes.

Single cycle adenovirus expressing the HIV-1 envelope protein gp140.

[000256] Clade B gp160 coat protein sequences occurring prior to (G4) and just prior to the peak of increased antibody neutralization latitude (F8) from HIV patient VC10014 (Malherbe et al., 2014 J Virol 88:12949-12967) were used as immunogens. Motif-optimized gp160 G4 and F8 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; Anguiano-Zarate et al., 2018 J Infectious Dis 218:1883-1889; and Nguyen et al., 2018 Oncolytic Therapy 7:43-51). A control SC-Ad expressing Ebola virus glycoprotein was also used. Viruses were rescued and purified as described elsewhere (see, for example, 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).

[000257] The F8 envelope gene cloned from an HIV-infected clade B individual used in the SC-Ad vector was subjected to motif optimization and modified by site-directed mutagenesis to express uncleaved, trimeric gp140. Details of expression, purification, and antigenic characterization are described elsewhere (see, for example, Malherbe et al., 2014 J Virol 88:12949-12967).

[000258] Adult female rhesus monkeys ( Macaca mulatta ) of Indian origin were kept at the Michael Keeling Center for Comparative Medicine and Research at the University of Texas MD Anderson Cancer Center, Bastrop TX in a specific pathogen-free juvenile colony. All animals were handled 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 Animal Care and Use Manual.

[000259] Macaques were anesthetized with ketamine and immunized by the intranasal (IN) or intramuscular (IM) route using 2×10 ¹⁰ viral particles (vp) of the indicated SC-Ad vaccine. Animals were boosted with 50 μg of purified, trimeric, recombinant gp140 F8 combined with Adjuplex™ adjuvant, IM, as described elsewhere (see, e.g., Malherbe et al., 2014 J Virol 88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078).

[000260] Peripheral venous blood samples were collected in EDTA. Before isolation of peripheral blood mononuclear cells (PBMC), plasma was separated and stored at -80°C. PBMC was obtained from blood in a ficoll-hypac density gradient. Saliva and vaginal swabs collected with Weck-Cel swabs in 1 ml PBS containing protease inhibitors were vortexed and supernatants were collected after centrifugation at 2000 rpm as described elsewhere (see e.g. Kozlowski et al., 1997 Infect Immun 65:1387-1394). Samples were stored frozen at -80°C until further use.

ELISPOT assay for detection of antigen-specific, IFNγ-producing cells

[000261] Freshly isolated PBMCs were stimulated with gp140 F8 protein (1 μg/ml) or thermally inactivated Ad6 (7.0 x 10 ⁸ vp/well) to quantify IFNγ-producing cells by enzyme immunostaining (ELISPOT) method using the methodology described elsewhere. sources (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 of PBMC (10 ⁵ /well) were seeded in duplicate in wells of 96-well plates (polyvinylidene difluoride antihalation plates, MAIP S45, Millipore, Bedford, MA) pre-coated with anti-IFNγ primary antibody, and lymphocytes were stimulated Con A, gp140 F8 protein, or thermally inactivated Ad6. After incubation for 30-36 hours at 37°C, the cells were removed, the wells were thoroughly washed with PBS and developed according to the manufacturer's instructions. The results were expressed as IFNγ spotted cells (SFC) per 10 ⁵ PBMC after subtraction of parallel wells with media only (negative control) and were considered positive at 2-fold background and more than 5 SFC/10 ⁵ PBMC.

ELISA of antibodies

[000262] HIV-1 envelope-binding antibody titers were measured in plasma samples collected at regular intervals against gp140 F8 or gp140 SF162 as described elsewhere (Malherbe et al., 2014 J Virol 88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078).

Neutralization analysis

[000263] Neutralization of HIV was performed using TZM-bl neutralization assays 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 in comparison to virus-only wells.

Antibody dependent cellular cytotoxicity (ADCC)

[000264] CEM.NKR.CCR5.CD4+-Luc target cells were infected with 50 ng of SHIV _SF162P3 and cultured 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 infected targets over 20 minutes at room temperature. CD16-KHYG-1 effector cells were added at a 10:1 effector-target ratio and incubated for an additional 8 hours. Cells were lysed and luciferase activity was measured using a Bio-Tek plate reader spectrophotometer.

flow cytometry

[000265] Cells harvested from rectal and lymph node biopsies were incubated overnight with 0.2 μg gp140 or media alone in the presence of GolgiPlug™ (BD Biosciences, San Jose, CA, USA) for the last 4 hours. After culture, cells were harvested and incubated on ice for 45 minutes with a panel of human antibodies cross-reacting with rhesus monkey samples. The panels included the following fluorochrome-labeled anti-CD8 (Qdot655), anti-α4β7 (PE), and anti-CXCR5 (PE) antibodies, all obtained from the Nonhuman Primate Reagent Resource; anti-CD69 (BV737, clone FN50) and FoxP3 (PECy5, clone: PCH101) derived from eBioscience, anti-IL-21 (BV421, clone: 3A3-N2.1), anti-CD45 (BV786, D058-1283) and anti-CD3 (clone SP34-2 labeled with PE-Cy7), all from BD Bioscience (San Jose, CA); against CD4 (Pacific Blue, clone OKT4) from ThermoFisher Scientific (Waltham, MA). Antibody dilutions were determined according to the manufacturer's instructions. Dead cells were excluded using the LIVE/DEAD® fixed dead cell staining kit obtained from Invitrogen (Carlsbad, CA). The cells were then washed twice with PBS containing 2% FBS and 2 mM EDTA and then fixed and permeabilized with the FoxP3 Fix/Perm kit (ThermoFisher Scientific, Waltham, MA). Intracellular markers FoxP3 and IL-21 were stained in permeabilization buffer. Both compensatory controls (OneComp eBeads, (ThermoFisher Scientific, Waltham, MA) and fluorescence controls with combination of detectable labels without one (FMO) were used. All samples were collected using an LSR Fortessa X-20 analyzer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (FlowJo, LLC, Ashland, Oregon) Approximately 2×10 ⁵ to 1×10 ⁶ events per sample were collected.

Rectal infection with SHIV _SF162P3

[000266] SHIV _SF162P3 virus was obtained from collection R157 3 (3.16.12). This stock had a P27 content of 66 ng/ml, a Log RNA content of ~9.35, a TCID ₅₀ in Indian rhesus PBMC: 1288/ml, and a TCID ₅₀ in TZM-bl cells: 4.1 x 10 ⁴ /ml. A 1 ml dilution of 1:300 stock was used. This is equivalent to 4.3 TCID ₅₀ in rhesus PBMCs and 137 TCID ₅₀ in TZM-bl cells. This dose was used for weekly intrarectal (IR) challenge. Plasma samples were analyzed for SHIV RNA copy number at Leidos Biomedical Research, Inc., Frederick National Laboratory. Animals with an RNA copy number greater than 10 were considered infected and the number of infections required to infect that animal were used as events for the Kaplan-Meier survival analysis. After infection, the animal was no longer infected. Plasma viral load was subjected to periodic monitoring in the same way until the end of the study.

Viral load of SHIV _SF162P3 in tissues

[000267] At the end of the study, PBMC and cadaveric tissues were collected. PBMC and intestinal samples were analyzed for RNA virus SHIV _SF162P3 by qPCR. Graph Pad Prism 7 software was used for all statistical analysis.

SC-Ad expressing HIV-1 gp160

[000268] Clade B (gp160G4) virus envelope protein sequences were identified before and just before the peak of increasing antibody neutralization latitude (F8 gp160) from HIV patient VC10014. These gp160 sequences were inserted into SC-Ad6 and SC-Ad657 under the control of a strong cytomegalovirus promoter (Figure 24A). Ad57 is a human Ad of clade C, almost identical to Ad6, with a change in hexon hypervariable regions (HVRs) and in E3 immunosurveillance evasion genes (Figure 24B). Most Ad-neutralizing antibodies are targeted against the Ad hexon HVR (Pichla-Gollon et al., 2007 J Virol 81:1680-1689; and Sumida et al., 2005 J Immunol 174:7179-7185). With this in mind, Ad6 HVRs were replaced with Ad57 HVRs to generate a chimeric Ad clade C vector, referred to as Ad657. Both SC-Ad6 and SC-Ad657 retain all Ad genes, including E1, and lack functional pIIIA and E3 genes (Figure 24A). Thus, both SC-Ads can replicate their genomes to amplify gp160 expression, but do not result in progeny of Ad viruses. Both viruses were rescued, produced in 293-IIIA cells and purified with CsCl gradients. When used to infect A549 cells, both vectors resulted in gp160 production as determined by Western blotting.

[000269] Various Ad vectors have previously been tested in rhesus monkeys via systemic intramuscular (IM) administration and administration by various mucosal routes, including gavage, enteric-coated capsules, intranasal (IN), and intravaginal (IVAG) routes. Testing of SC-Ad-G4 by the IM, IN and IVAG pathways in small animals showed that IVAG priming resulted in negligible humoral responses. In contrast, immunization with the IN pathway in mice and hamsters resulted in strong humoral responses. Given these data and the potential difficulties in performing IVAG immunization in humans, the IN route was chosen for the mucosal route of immunization in subsequent monkey studies.

Single mucosal and systemic immunization of rhesus monkeys

[000270] Used 2×10 ¹⁰ vp SC-Ad6-G4 Env to vaccinate groups of 8 female rhesus monkeys via a single IM or IN immunization (Figure 14). This dose is relatively low, being approximately 7.5 times lower than when using HIV coated RC-Ad vaccines delivered by mixed IN and IM immunization. The negative control vector group was immunized with IN with SC-Ad6 expressing Ebola virus glycoprotein (gp). Four weeks later, plasma samples were analyzed for Env-binding antibodies against gp140 F8 (figure 14). Significantly higher midpoint binding titers were observed in the IM immunized group after a single immunization (p<0.01 by ANOVA). Titers of SF162-neutralizing antibodies (NAb) also increased at this point in time, but did not reach significance according to the results of ANOVA for groups of individual routes of administration.

Booster IM versus IN with SC-Ad6 at week 4

[000271] It has been shown that neutralizing antibodies against adenovirus produced by a single IM immunization with Ad can be avoided by boosting by another route (Xiang et al., 2003 J Virol 77:10780-10789). To test the concept of this route of administration to allow reuse of the same Ad serotype in macaques, each SC-Ad6-primed group was divided into 2 groups of 4. Each animal was boosted with SC-Ad6 expressing alternative Env F8 at week 4 IM or IN by. Plasma samples collected 3 weeks after the boost showed an increase in midpoint binding titers in animals that were primed and boosted in the IM-IM, IM-IN and IN-IM groups. No detectable antibodies were observed in the IN-IN group (FIG. 14).

SC-Ad657 Booster at Week 13

[000272] Animals were then boosted by serotype switching using SC-Ad657 expressing Env G4 at week 13. The same route was used in the previous boost. Titers at week 15 indicated that IM-primed animals had increased Env binding titers of approximately 350, but these levels did not differ significantly from controls (Figure 14). In contrast, antibodies in the IN-IM-IM group were significantly higher than in the control vector group and the IN-IN-IN group (p<0.01). Again, no anti-Env antibodies were observed in the IN-IN-IN group even after 3 immunizations.

Booster administration of recombinant trimeric Env protein at week 24

[000273] In most HIV vaccine research, Ad immunization is expanded using protein boosters to amplify humoral responses. For example, in a recent study, RD-Ad26 vectors were used twice and boosted three times with adjuvant gp140 protein (Barouch et al., 2015 Science 349(6245):320-4). To determine if this strategy would enhance SC-Ad vaccines, all SC-Ad-Env groups were boosted with 50 μg of recombinant gp140 F8 trimer protein mixed with ADJUPLEX™ adjuvant, IM by. The F8 trimeric protein increased midpoint binding titers by two orders of magnitude in all groups (Figure 14). This protein immunization also increased IN-IN-IN to levels comparable to other groups, even though Env-binding antibodies were not detected after earlier SC-Ad immunizations.

Plasma binding and neutralizing antibodies after a second protein boost.

[000274] Animals were given a second protein boost at week 38. This raised plasma F8-binding antibody titers to about ¹⁰⁵ by week 40, and all groups were significantly different from controls (Figure 14). Titers of neutralizing antibodies (NAb) against the virus SF162 group 1A increased from 100 to 10,000 at week 40 (figure 15). NAb against group 1B SS1196 virus and group 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).

ADCC activity after the second protein boost

[000275] Plasma antibody-dependent cellular cytotoxicity (ADCC) activity at week 40 was tested against infected SHIV _SF162P3 cells. ADCC activity was generally higher in animals subjected to at least one mucosal IN immunization with SC-Ad (FIG. 16). All mucosal immunized animals had a significantly higher maximum % ADCC than control animals treated with SC-Ad-Ebola (p<0.05, 0.0001, 0.0001 for IM-IN-IN, IN-IM -IM and IN-IN-IN, respectively). When compared across 50% ADCC titers, only IN-primed SC-Ad groups had significantly higher ADCC activity than controls (p<0.05 and 0.001 by ANOVA for IN-IM-IM and IN-IN-IN group). ).

Humoral responses in saliva and vaginal washings after a second protein boost

[000276] Plasma systemic humoral responses were monitored using the above data. Samples of saliva and vaginal washings were also collected at week 40 and antibodies were measured in these mucosal sites. When saliva and vaginal washes were analyzed for env F8 and SF162 binding by ELISA, these responses were observed in most groups except for the SC-Ad-Ebola control group (Figure 25).

[000277] Apparently, in this case, there was a regional effect in relation to these antibodies. In animals immunized with SC-Ad, mainly by the IN pathway (IM-IN-IN and IN-IN-IN), binding antibodies were higher in the saliva near this immunization site, but lower in the more distant site in the vagina (figure 25). When ADCC activity was measured in these mucosal samples, these responses varied greatly (Figure 17). Despite this, higher ADCC activity was observed in the IN-IN-IN group compared to control animals (p<0.05 by ANOVA).

Systemic cellular immune responses after a single protein booster

[000278] At week 38, PBMCs were analyzed by ELISPOT for anti-Env and anti-adenoviral T cells on samples collected immediately prior to the second Env F8 protein boost. All Env-immunized animals had Env-specific IFNγ-secreting cells in PBMC (Figure 18A). Env-specific IFNγ-secreting SFCs were generally elevated in animals that received at least one mucosal immunization. However, IFNγ-secreting SFCs were only significantly higher in the SC-Ad IN-IN-IN group when compared to control animals immunized with SC-Ad Ebola (p<0.05 by ANOVA). SFCs against Ad were relatively low in all groups compared to SFCs against Env at this point in time.

Systemic cellular immune responses after the second protein booster

[000279] At week 40, PBMCs and inguinal lymph node cells were analyzed for Env-specific IFNγ-secreting SFCs by ELISPOT (Figure 18B). This protein boost increased Env-specific SFCs in PBMCs and lymph nodes to levels similar to those in all Env-immunized animals.

Cellular transport in mucous membranes

[000280] Flow cytometry of rectal biopsies at week 40 determined similar numbers of α4β7 CD4 ⁺ and CD8 ⁺ cells in rectal areas (Figure 19A). An upward trend was observed in the IN-primed groups, but did not reach significance. The number of activated CD69 ⁺ CD4 ⁺ cells in the tissues of the rectum were similar between groups (figure 19B). Similarly, FoxP3 ⁺ CD4 ⁺ cells in this area of the mucosa did not differ significantly (figure 19B).

Distribution of antigen-specific Tfh cells

[000281] CXCR5 ⁺ IL-21 ⁺ CD4 ⁺ follicular T helpers (Tfh) were measured in PBMC and lymph node samples at week 40 (Figure 20). Animals immunized with Ad and protein only by the IM route had significantly higher levels of peripheral Tfh cells (pTfh) in PBMC than in the other groups (Figure 20). In the lymph nodes, Tfh cells had the lowest level in the control group and in the IM-only group. In contrast, approximately half of the animals subjected to at least one IN mucosal immunization had detectable Tfh cells in their lymph nodes after the last protein boost (Figure 20).

Rectal infection with SHIV _SF162P3

[000282] Immunized monkeys were challenged rectally with HIV isolate SHIV _SF162P3 . Four non-immunized control animals were added to the study and each group was challenged weekly by rectal inoculation with 1 ml dilution of 1:300 stock _{SHIV SF162P3} provided by NIH. This infection is equivalent to 4.3 TCID ₅₀ on PBMC rhesus monkeys and 137 TCID ₅₀ on TZM-bl cells. After the first challenge, 2 animals in the non-immunized control group and 2 animals in the IM-IM-IM group were challenged (FIG. 21). One animal in each of the mixed administration groups (IM-IN-IN and IN-IM-IM) were challenged after the first challenge. None of the animals in the IN-IN-IN group became infected after the first infection. Plasma viral load indicated that all animals, with the exception of the animal in the Ebola group, reached a high viral load after 3 infections (figure 22B). Animals in the IN-IN-IN group had a delay in achieving this high viral load.

[000283] As infections continued, animals in all groups were challenged except for one animal in the Ebola group remaining uninfected after 7 infections. Trim5α and MHC alleles were analyzed retrospectively (Table 2). In this analysis, no apparently protective genes were observed in the resistant animal in the Ebola group. Most animals cannot be classified by alleles that would make them moderately protected, but most groups included at least one animal with a higher chance of being protected by these alleles. It should be noted that 2/4 of the animals in the IM-IM-IM and IN-IN-IN groups had the Trim5a and MHC alleles, which can predict a higher probability of innate protection against SIVsmm and possibly SHIV _SF162P3 (Table 2).

[000284] Table 2. Retrospective screening for protective alleles of the SIV genes.

Vaccination group Animal number MHC typing TRIM5 alpha Degree of virus protection Non-immunized RHJ663 Not carried out Cyp A/TFP High RH3-39 Not carried out Q/TFP Moderate RHJ403 Not carried out Cyp A/Q Moderate RHJ791 Not carried out Q/TFP Moderate SC-Ad-Ebov RH13-005 A11, B01, B17, A08, A11, B01 Q/TFP Moderate RH13-007 B17 Q/TFP Moderate RH13-043 A08, A11, B17 Q/TFP Moderate RH13-135 A08, A11, B17 Q/TFP Moderate IM-IM-IM RH13-027 A11, B01, B17 TFP/TFP High RH13-031 A08, A11, B01 Cyp A/Q Moderate RH13-051 A08, A11, B17 Cyp A/Q Moderate RH13-139 A08, A11, B17 TFP/TFP High IM-IN-IN RH13-039 A11, B01, B17, A08, A11, B01 Cyp A/Q Moderate RH13-045 B17 Q/Q unstable RH13-095 A08, A11, B17 Q/TFP Moderate RH13-159 A08, A11 Cyp A/TFP High IN-IM-IM RH13-013 A11, B17
A08, A11, B01 Cyp A/Q Moderate RH13-067 B17 Q/TFP Moderate RH13-091 A11, B01, B17 TFP/TFP High RH13-121 A08, A11, B17 Q/TFP Moderate IN-IN-IN RH13-025 A11, B17 Cyp A/Q Moderate RH13-033 A08, A11, B17 Cyp A/TFP High RH13-087 B17 TFP/TFP High RH13-125 A08, A11, B17 Q/TFP Moderate

[000285] When animals were classified based on whether they were primed by IM or IN SC-Ad and survival was analyzed by the Kaplan-Meier method, infection of eight IM-primed animals was consistent with that of control animals (Figure 22A). In contrast, delayed infections were observed in eight animals primed with SC-Ad-Env by the mucosal IN pathway.

Viral load in cadaveric tissues

[000286] The challenge study was terminated 9 weeks after the first challenge. PBMC and intestinal tissues were isolated, RNA purified and evaluated for SHIV viral genomes (Figure 22B). Cadaveric PBMCs had varying levels of SHIV viral RNA with somewhat lower levels in the IN-IN-IN group than in the IM-IM-IM group. Mean colonic viral RNA levels were 15 times lower in the IN-IN-IN group than in the IM-IM-IM group (Figure 23). This difference did not reach significance in ANOVA, but a two-tailed Student's t-test yielded a p value of 0.0079.

[000287] This example demonstrates that replicating SC-Ad vectors can be used as a reliable and safe platform for vaccination against HIV-1 and other infectious diseases. SC-Ad can amplify antigen and cytokine genes up to 10,000-fold in infected human cells. The immune response is amplified to levels well above those mediated by the RD-Ad vectors currently being tested as HIV-1 vaccines in humans. HIV vaccines can be converted into vaccine platforms where HIV antigen genes are amplified using SC-Ad vectors, eg recombinant Ad SC-Ad vectors having low seroprevalence.

Example 8 In vivo assay of cytotoxic T lymphocytes (CTL) for immune responses against hepatitis C virus (HCV) antigen.

[000288] Mice were immunized with Ad657 expressing cytomegalovirus (CMV) glycoprotein B (gB) cDNA or HCV antigen 2.4. Syngeneic cells were injected with HCV peptide and labeled with carboxyfluorescein succinimidyl ester (CFSE) prior to injection into immunized mice. Anti-HCV cognate CTL activity was observed by loss of labeled cells in animals immunized with HCV but not CMV (FIG. 26).

Example 9 Conditionally Replicating Ads (CRAds).

[000289] The scheme of mutations in Ad6, Ad657 and its variants, including mutations in the E1 protein to convert the virus to conditionally replicating Ad (CRAd), is shown in Figure 39 and Figure 43. These include dl1101 and/or dl1107, blocking binding to p300 and pRB, respectively.

[000290] Figure 56 shows the N-terminal amino acid sequences of E1A in wild-type Ad, as well as E1A dl1101, E1A dl1107 and E1A dl1101/1107 Ad variants.

[000291] Also shown is the replacement of the E1 Ad promoter with the prostate-specific probazin-E1 promoter DNA sequence of SEQ ID NO: 44 to produce CRAd, Ad-PB (Figure 55). The probasin promoter is androgen dependent, so it will act in androgen sensitive tumors like LNCaP but not in androgen resistant tumors like DU145.

[000292] A549 cells were infected with the indicated Ad6 or Ad657 variants at the indicated virus concentrations (vp/cell) and cell viability was measured by crystal violet staining after 5 days (Figure 40).

[000293] Destruction of non-malignant cells with replication-deficient Ad (RD-Ad), Ad6, CRAd6-dl1101/dl1107 or CRAd6-PB. Modification of Ad6 and Ad657 to conditionally replicative Ad (CRAd) is shown (Figure 44).

[000294] Figure 45 shows the killing of malignant cells by replication competent Ad5, Ad6, Ad657 and the indicated CRAds. The modification of Ad6 and Ad657 to conditionally replicative Ad (CRAd) is shown.

[000295] The results shown in Figure 46 demonstrate the modification of Ad6 and Ad657 to conditionally replicative Ad (CRAd) in breast cancer cells.

[000296] The results shown in Figure 47 demonstrate the modification of Ad6 and Ad657 to conditionally replicative Ad (CRAd) in prostate cancer cells and lung cancer cells.

[000297] In vivo effects of replication competent Ad6 or indicated CRAds on the growth of DU145 tumors in mice. Figure 48 shows the modification of Ad6 and Ad657 to conditionally replicating Ad (CRAd) in vivo after a single intravenous injection in mice bearing human prostate tumors.

[000298] In vivo effects of replication competent Ad6 or indicated CRAds on survival in mice with DU145 tumors. Figure 49 shows the modification of Ad6 and Ad657 to conditionally replicating Ad (CRAd) in vivo after a single intravenous injection in mice bearing human prostate tumors.

[000299] Figure 51 shows that modifying Ad657 with a shorter fiber from chimpanzee AdC68 reduces efficiency.

[000300] In an embodiment, the Ad 6/56/6 virus has HVR 1 and 7 from Ad6 and HVR 2-6 from Ad657. Figure 52 shows human lung cancer cell-killing Ad 6/56/6 virus with and without CRAd modifications.

[000301] Destruction of tumor cells by Ad variants including mutations in the E3 protein. Immunocompetent Syrian hamsters were engrafted with subcutaneous tumors of kidney cancer HaK. When they reached a volume of 200 μl, the hamsters were injected once intravenously with the indicated Ad6 viruses engineered with and without E3 (DE3) and with or without random NHS-pegylation. Tumor sizes were measured over time. The data indicated that deletion of all E3 genes renders the oncolytic virus less effective (Ad6-deltaE3-Luc vs. Ad6-Luc) (Figure 58).

[000302] The Ad fiber protein is a complex of three apparently identical subunits mediating the initial attachment step. The native Ad6 fiber protein contains the amino acid sequence shown in SEQ ID NO: 60 and binds to CAR.

[000303] In a further aspect of the invention, fiber-modified recombinant Ads were made having different fiber proteins not native to the parent Ad. Recombinant Ads were prepared, including CRAds containing capsid proteins from different Ad strains, eg recombinant Ads containing heterologous Ad35 fiber polypeptide or chimpanzee C68 fiber polypeptide +/- K7 peptide (Figures 62-69).

[000304] Chimeric Ad, an AdF35 fiber chimera, has the amino acid sequence of SEQ ID NO: 61 and is shorter than the Ad5 and Ad6 fiber proteins and redirects the virus to CD46.

[000305] Fiber-modified recombinant Ad containing fiber K7 having the sequence of SEQ ID NO: 62 targets the virus to heparin sulfate proteoglycans and negative charges on cells.

[000306] Recombinant, chimeric Ad, fiber 6/FC68, containing the sequence of SEQ ID NO: 63, is a chimeric Ad having a fiber protein from chimpanzee adenovirus C68. The fiber protein is shorter than the Ad5 or Ad6 fiber proteins and binds to CAR.

[000307] Recombinant, chimeric Ad, fiber 6/FC68-K7, containing the sequence of SEQ ID NO: 64, is a chimeric Ad having a fiber protein from chimpanzee adenovirus C68. The fiber protein is shorter than the Ad5 or Ad6 fiber proteins. Fiber 6/FC68-K7 binds to CAR and redirects to heparin sulfate and negative charges.

[000308] Recombinant, chimeric Ad, fiber 6/FC68-HI-K7, containing the sequence of SEQ ID NO: 65, is a chimeric Ad having a fiber protein from chimpanzee adenovirus C68. The fiber protein is shorter than the Ad5 or Ad6 fiber proteins. Fiber 6/FC68-HI-K7 binds to CAR and redirects to heparin sulfate and negative charges.

Example 10 Adenovirus serotype switching.

[000309] Figure 41 is a diagram showing Ad therapeutic cycles. In an embodiment, an example of serotype switching using different Ads over the course of treatment is provided (Figure 41A).

Prostate tumor targeting after serotype switching of oncolytic adenoviruses.

[000310] Mice bearing DU145 prostate tumors on their flanks were subjected to a single intravenous (IV) injection of Ad657 or CRAd657. These mice were injected with an alternative oncolytic virus Ad6 or Ad6-F35 expressing GFP-luciferase for the second time and the luciferase activity was measured by imaging. Ad6 has an Ad6 hexon and an Ad6 fiber targeted to CAR. Ad6-F35 has an Ad6 hexon and an Ad35 fiber targeted to CD46. Figure 42 shows the possibility of serotype switching of oncolytic agents using tumor targeting viruses with lower off-target liver infection.

[000311] In another example of serotype switching, mice carrying LNCaP prostate tumors on their bulls were subjected to a single intravenous (IV) injection using 3e10 viral particles (vp) of Ad657 or CRAd657. These mice were injected a second time 5 months later with 3e10 vp of an alternative oncolytic virus Ad6/57/6 expressing GFP-luciferase and fiber variants K7 (supplemented with 7 lysines), F35 (with Ad35 fiber) or KKTK-C68 (chimpanzee C68 fiber, fused after the flexibility-giving domain KKTK Ad6). The KKTK-C68 virus also contains an added codon-optimized E4 34K gene to increase viral productivity. Luciferase activity was measured after 7 days by imaging. All Ad6/57/6 had a hexon with HVR1 and 7 from Ad6 and HVR 2-6 from Ad57. Ad6/57/6 and KKTK-C68 had CAR-targeted fibers. Ad6/57/6-F35 had an Ad35 fiber targeted to CD46. K7 increases binding to negative charges on cells, including the binding of heparin sulfate proteoglycans. 70 shows the possibility of serotype switching of oncolytic agents using tumor targeting viruses with lower off-target liver infection.

Serotype switching during vaccination of non-human primates.

[000312] Figures 14-25 show that rhesus monkeys were immunized with replicating single cycle Ad6 expressing HIV envelope and then boosted by serotype switching using single cycle Ad657 expressing HIV envelope. After these immunizations, each animal was subjected to a coat protein booster. Each figure shows the formation of adaptive humoral or cellular immune responses and how animals overcame rectal challenge with SHIV _{SF162P3 virus} . Figure 14 shows the significance of serotype switching, where a change to Ad657 results in a significant increase in humoral responses.

Example 11 Oncolytic antitumor vaccines.

[000313] BALB/c mice were immunized intramuscularly with 10 ¹⁰ intact E3 CRAd-657-dl1101/1107-FolR virus particles expressing the human folic acid receptor alpha, or PBS. Sera were collected 2 weeks after a single immunization and analyzed for antibodies against folic acid receptor alpha by ELISA using an anti-IgM antibody for detection (all antibodies are IgM at this type of early post-immunization time point). The data indicate the formation of antibodies against a known cancer antigen, folic acid receptor alpha, using this CRAd. p - 0.07 using Student's t-test (Figure 53).

Example 12 Effects of E3 Surveillance Evasion Genes on Oncolytic Activity.

[000314] Figure 57 shows a diagram of the various E3 immunosurveillance evasion genes in Ad. E3 19K protects infected cells from T cells and NK cells. RID proteins protect infected cells from death-inducing ligands (FAS, TRAIL, TNFR and EGFR). 14.7K inhibits intrinsic activation of apoptosis in infected cells. Ad clade C also express 11.6K, known as adenovirus-infected cell death protein (ADP). Overexpression of ADP accelerates cell death, but overall cell death is the same. 49K species bind to CD46 on T cells and NK cells, resulting in downregulation of these cells and less efficient killing of MHC class I deficient cells by NK cells.

[000315] Figure 58 shows partial deletion of E3 12.5K and complete deletion of E3 genes 6.7K, 19K, 11.6K (ADP), 10.4K (RIDα), 14.5K (RIDβ), and 14.7K reduces oncolytic efficacy in an immunocompetent hamster kidney cancer model when these immunosurveillance evasion genes are absent in oncolytic adenovirus.

Other embodiments

[000316] It should be understood that although the present invention has been described in combination with the detailed description, the foregoing description is for illustrative purposes and not to limit the scope of the invention as defined by the scope of the claims. Other aspects, advantages and modifications are within the scope of the following claims.

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SEQUENCE LIST

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<210> 16

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 16

Gly Pro Glu Asp Thr Ser Arg Ala Pro Glu Asn Gln Gln Lys Thr Phe

1.5 10 15

His Arg Arg Trp

twenty

<210> 17

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 17

Met Gly Arg Glu Asp Val Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp

1.5 10 15

Leu Gly Gly Ser

twenty

<210> 18

<211> 11

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 18

Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly

1.5 10

<210> 19

<211> 12

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 19

Ala Cys Asp Cys Arg Glu Asp Val Cys Phe Cys Gly

1.5 10

<210> 20

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 20

Gly Gln Ile Pro Ile Thr Glu Pro Glu Leu Cys Cys Val Pro Trp Thr

1.5 10 15

Glu Ala Phe Tyr

twenty

<210> 21

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 21

Pro Gln Pro Pro Asn Ser Thr Ala His Pro Asn Pro His Lys Ala Pro

1.5 10 15

Pro Asn Thr Thr

twenty

<210> 22

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 22

Val Arg Trp Phe Pro Gly Gly Glu Trp Gly Val Thr His Pro Glu Ser

1.5 10 15

Leu Pro Pro

twenty

<210> 23

<211> 19

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 23

Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys

1.5 10 15

Lys Lys Lys

<210> 24

<211> 14

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 24

Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His

1.5 10

<210> 25

<211> 23

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 25

Cys Ala Ala Ala Arg Trp Lys Lys Ala Phe Ile Ala Val Ser Ala Ala

1.5 10 15

Asn Arg Phe Lys Lys Ile Ser

twenty

<210> 26

<211> 14

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 26

Glu Asp Pro Gly Phe Phe Asn Val Glu Ile Pro Glu Phe Pro

1.5 10

<210> 27

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 27

Gly Gly His Gly Arg Val Leu Trp Pro Asp Gly Trp Phe Ser Leu Val

1.5 10 15

Gly Ile Ser Pro

twenty

<210> 28

<211> 21

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 29

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 30

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 31

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 32

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 33

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 34

<211> 8

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<400> 34

Phe Ser Leu Val Gly Ile Ser Pro

fifteen

<210> 35

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 36

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 37

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 38

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 39

<211> 19

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 41

<211> 20

<212> protein

<213> Artificial sequence

<220>

<223> Targeting polypeptide

<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

twenty

<210> 42

<211> 132

<212> protein

<213> Artificial sequence

<220>

<223> N-terminal adenovirus E1A 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> protein

<213> Artificial sequence

<220>

<223> N-terminal E1A 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 Ser

100 105 110

<210> 44

<211> 119

<212> protein

<213> Artificial sequence

<220>

<223> N-terminal E1A 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> protein

<213> Artificial sequence

<220>

<223> N-terminal E1A polypeptide

<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> protein

<213> Artificial sequence

<220>

<223> Cell-binding peptide

<400> 46

Arg Glu Asp Val Tyr

fifteen

<210> 47

<211> 12

<212> protein

<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> DNA

<213> Artificial sequence

<220>

<223> Probasin-E1 nucleic acid

<400> 48

tcgagcgacg gtatcgataa gcttggagct tatgatagca tcttgttctt agtctttttc 60

ttaataggga cataaagccc acaaataaaa atatgcctga agaatgggac aggcattggg 120

cattgtccat gcctagtaaa gtactccaag aacctatttg tatactagat gacacaatgt 180

tctagccaag cttggtagtc atcatgttta aacatctacc attccagtta agaaaatatg 240

atagcatctt gttcttagtc tttttcttaa tagggacata aagcccacaa ataaaaatat 300

gcctgaagaa tgggacaggc attgggcatt gtccatgcct agtaaagtac tccaagaacc 360

tatttgtata ctagatgaca caatgtcaat gtctgtgtac aactgccaac tgggatgcaa 420

gacactgccc atgccaatca tcctgaaaag cagctataaa aagcaggaag ctactctgca 480

ccttgtcagt gaggtccaga tacctccctc gagcggccgc gacgcgcagt gtatttatac 540

ccggtgagtt cctcaagagg ccactcttga gtgccagcga gtagagtttt ctcctccgag 600

ccgctccgac accgggactg aaaatgagac atattatctg ccacgggaggt gttattaccg 660

aagaaatggc cgccagtctt ttggaccagc tgatcgaaga ggtactggct gataatcttc 720

cacctcctag ccattttgaa ccacctaccc ttcacgaact gtatgattta gacgtgacgg 780

cccccgaaga tcccaacgag gaggcggttt cgcagatttt tcccgagtct gtaatgttgg 840

cggtgcagga agggattgac ttattcactt ttccgccggc gcccggttct ccggagccgc 900

ctcacctttc ccggcagccc gagcagccgg agcagagagc cttgggtccg gtttctatgc 960

caaaccttgt gccgggaggtg atcgatctta cctgccacga ggctggcttt ccacccagtg 1020

1080

gttgcaggtc ttgtcattat caccggagga atacggggga cccagatatt atgtgttcgc 1140

tttgctatat gaggacctgt ggcatgtttg tctacagtaa gtgaaaatta tgggcagtcg 1200

1260

1320

agccagaacc ggagcctgca agacctaccc ggcgtcctaa attggtgcct gctatcctga 1380

1440

gtccttctaa cacacctcct gagatacacc cggtggtccc gctgtgcccc attaaaccag 1500

ttgccgtgag agttggtggg cgtcgccagg ctgtggaatg tatcgaggac ttgcttaacg 1560

agtctgggca acctttggac ttgagctgta aacgccccag gccataag 1608

<210> 49

<211> 959

<212> protein

<213> Artificial sequence

<220>

<223> Hexon 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 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> protein

<213> Artificial sequence

<220>

<223> Hexon 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 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> protein

<213> Artificial sequence

<220>

<223> Hexon polypeptide

<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 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

Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val

915 920 925

Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu

930 935 940

Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr

945 950 955

<210> 52

<211> 964

<212> protein

<213> Artificial sequence

<220>

<223> Hexon polypeptide

<400> 52

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 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> protein

<213> Artificial sequence

<220>

<223> Hexon 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 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> protein

<213> Artificial sequence

<220>

<223> Hexon 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 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> protein

<213> Artificial sequence

<220>

<223> Hexon 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 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> protein

<213> Artificial sequence

<220>

<223> Hexon polypeptide

<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 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> protein

<213> Artificial sequence

<220>

<223> Hexon polypeptide

<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 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> protein

<213> Artificial sequence

<220>

<223> Hexon polypeptide

<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 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> protein

<213> Artificial sequence

<220>

<223> Hexon 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

Thr Thr

<210> 60

<211> 528

<212> protein

<213> Artificial sequence

<220>

<223> Fiber polypeptide

<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> protein

<213> Artificial sequence

<220>

<223> Fiber 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> protein

<213> Artificial sequence

<220>

<223> Fiber polypeptide

<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> protein

<213> Artificial sequence

<220>

<223> Fiber 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> protein

<213> Artificial sequence

<220>

<223> Fiber 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> protein

<213> Artificial sequence

<220>

<223> Fiber polypeptide

<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 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

<210> 66

<211> 27

<212> protein

<213> Mus musculus

<400> 66

Pro Val Thr Leu Thr Ile Thr Leu Thr Ala Arg Gly Glu His Lys Glu

1.5 10 15

Glu Glu Leu Ile Gly Ala Tyr Tyr Ser Met Ser

20 25

<210> 67

<211> 27

<212> protein

<213> Mus musculus

<400> 67

Pro Val Thr Leu Thr Ile Thr Leu Leu Arg Gln Thr Gly Ala Ala Ser

1.5 10 15

Ala Val Trp Gly Gly Ala Tyr Tyr Ser Met Ser

20 25

<210> 68

<211> 23

<212> protein

<213> Adenovirus type 5

<400> 68

Glu Met Gln Phe Phe Ser Thr Thr Glu Ala Thr Ala Gly Asn Gly Asp

1.5 10 15

Asn Leu Thr Pro Lys Val Val

twenty

<210> 69

<211> 26

<212> protein

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 69

Glu Met Gln Phe Phe Ser Gly Ser Thr Ala Arg Gly Glu His Lys Glu

1.5 10 15

Glu Glu Leu Ile Gly Thr Pro Lys Val Val

20 25

<210> 70

<211> 27

<212> protein

<213> Artificial sequence

<220>

<223> Artificial sequence

<400> 70

Glu Met Gln Phe Phe Ser Gly Ser Met Leu Arg Gln Thr Gly Ala Ala

1.5 10 15

Ser Ala Val Trp Gly Gly Thr Pro Lys Val Val

20 25

<210> 71

<211> 407

<212> protein

<213> Adenovirus type 5

<400> 71

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Pro Cys Glu Trp Asp

1.5 10 15

Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp

20 25 30

Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His Val

35 40 45

Phe Ser Gln Ala Pro Tyr Ser Gly Ile Asn Ile Thr Lys Glu Gly Ile

50 55 60

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

65 70 75 80

Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Tyr Glu Thr Glu Ile

85 90 95

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

100 105 110

Cys Tyr Gly Ser Tyr Ala Lys Pro Thr Asn Glu Asn Gly Gly Gln Gly

115 120 125

Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met

130 135 140

Gln Phe Phe Ser Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu

145 150 155 160

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

165 170 175

Asp Thr His Ile Ser Tyr Met Pro Thr Ile Lys Glu Gly Asn Ser Arg

180 185 190

Glu Leu Met Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala

195 200 205

Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn

210 215 220

Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp

225 230 235 240

Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser

245 250 255

Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp

260 265 270

Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp

275 280 285

Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Val Ile Asn Thr Glu

290 295 300

Thr Leu Thr Lys Val Lys Pro Lys Thr Gly Gln Glu Asn Gly Trp Glu

305 310 315 320

Lys Asp Ala Thr Glu Phe Ser Asp Lys Asn Glu Ile Arg Val Gly Asn

325 330 335

Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe

340 345 350

Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Ser

355 360 365

Pro Ser Asn Val Lys Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met

370 375 380

Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu

385 390 395 400

Gly Ala Arg Trp Ser Leu Asp

405

<210> 72

<211> 418

<212> protein

<213> Adenovirus type 6

<400> 72

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Glu

1.5 10 15

Gln Asn Glu Thr Ala Gln Val Asp Ala Gln Glu Leu Asp Glu Glu Glu

20 25 30

Asn Glu Ala Asn Glu Ala Gln Ala Arg Glu Gln Glu Gln Ala Lys Lys

35 40 45

Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Ile Lys Ile Thr Lys

50 55 60

Glu Gly Leu Gln Ile Gly Thr Ala Asp Ala Thr Val Ala Gly Ala Gly

65 70 75 80

Lys Glu Ile Phe Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Val Gly

85 90 95

Glu Ser Gln Trp Asn Glu Ala Asp Ala Thr Ala Ala Gly Gly Arg Val

100 105 110

Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg

115 120 125

Pro Thr Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly

130 135 140

Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Thr Asn

145 150 155 160

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

165 170 175

Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro

180 185 190

Lys Met Gly Asp Lys Asn Ala Lys Val Met Leu Gly Gln Gln Ala Met

195 200 205

Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu

210 215 220

Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala

225 230 235 240

Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu

245 250 255

Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe

260 265 270

Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile

275 280 285

Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro

290 295 300

Leu Gly Gly Ile Gly Ile Thr Asp Thr Phe Gln Ala Val Lys Thr Thr

305 310 315 320

Ala Ala Asn Gly Asp Gln Gly Asn Thr Thr Trp Gln Lys Asp Ser Thr

325 330 335

Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu

340 345 350

Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile

355 360 365

Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu

370 375 380

Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val

385 390 395 400

Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser

405 410 415

Leu Glu

<210> 73

<211> 414

<212> protein

<213> Adenovirus type 57

<400> 73

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Asp

1.5 10 15

Glu Asp Asp Thr Gln Val Gln Val Ala Ala Glu Asp Asp Gln Asp Asp

20 25 30

Asp Glu Glu Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys Lys Thr

35 40 45

His Val Tyr Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn Lys Asn

50 55 60

Gly Leu Gln Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn Lys Glu

65 70 75 80

Ile Tyr Ala Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser

85 90 95

Gln Trp Asn Glu Ala Glu Ser Val Ala Gly Gly Arg Val Leu Lys

100 105 110

Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr

115 120 125

Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly Lys Leu

130 135 140

Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn Ala Met

145 150 155 160

Asn Glu Ala Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser Glu Asp

165 170 175

Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys

180 185 190

Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn

195 200 205

Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr

210 215 220

Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln

225 230 235 240

Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr

245 250 255

Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met

260 265 270

Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu

275 280 285

Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly

290 295 300

Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly

305 310 315 320

Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg

325 330 335

Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn

340 345 350

Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu

355 360 365

Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn

370 375 380

Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu

385 390 395 400

Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp

405 410

<210> 74

<211> 348

<212> protein

<213> Adenovirus type 1

<400> 74

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Glu

1.5 10 15

Gln Glu Glu Pro Thr Gln Glu Met Ala Glu Glu Leu Glu Asp Glu Glu

20 25 30

Glu Ala Glu Glu Glu Glu Glu Ala Glu Glu Glu Ala Glu Ala Pro Gln Ala

35 40 45

Asp Gln Lys Val Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ala

50 55 60

Gly Glu Lys Ile Thr Ala Asn Gly Leu Gln Ile Val Ser Asp Thr Gln

65 70 75 80

Thr Glu Gly Asn Pro Val Phe Ala Asp Pro Thr Tyr Gln Pro Glu Pro

85 90 95

Gln Val Gly Glu Ser Gln Trp Asn Glu Ala Glu Ala Thr Ala Ser Gly

100 105 110

Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser

115 120 125

Tyr Ala Arg Pro Thr Asn Lys Asn Gly Gly Gln Gly Ile Leu Val Ala

130 135 140

Asn Asn Gln Gly Ala Leu Glu Ser Lys Val Glu Met Gln Phe Phe Ala

145 150 155 160

Pro Ser Gly Thr Ala Met Asn Glu Arg Asn Ala Val Gln Pro Ser Ile

165 170 175

Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Ile

180 185 190

Ser Tyr Lys Pro Ser Lys Thr Asp Glu Asn Ser Lys Ala Met Leu Gly

195 200 205

Gln Gln Ala Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn

210 215 220

Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu

225 230 235 240

Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg

245 250 255

Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg

260 265 270

Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro

275 280 285

Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn

290 295 300

Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Gly

305 310 315 320

Ile Lys Ser Asn Gly Asn Gly Asn Pro Gln Asn Trp Thr Lys Asn Asp

325 330 335

Asp Phe Ala Ala Arg Asn Glu Ile Gly Val Gly Asn

340 345

<210> 75

<211> 352

<212> protein

<213> Adenovirus type 2

<400> 75

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Glu

1.5 10 15

Gln Thr Glu Asp Ser Gly Arg Ala Val Ala Glu Asp Glu Glu Glu Glu

20 25 30

Asp Glu Asp Glu Glu Glu Glu Glu Glu Glu Glu Gln Asn Ala Arg Asp Gln

35 40 45

Ala Thr Lys Lys Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Glu

50 55 60

Thr Ile Thr Lys Ser Gly Leu Gln Ile Gly Ser Asp Asn Ala Glu Thr

65 70 75 80

Gln Ala Lys Pro Val Tyr Ala Asp Pro Ser Tyr Gln Pro Glu Pro Gln

85 90 95

Ile Gly Glu Ser Gln Trp Asn Glu Ala Asp Ala Asn Ala Ala Gly Gly

100 105 110

Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr

115 120 125

Ala Arg Pro Thr Asn Pro Phe Gly Gly Gln Ser Val Leu Val Pro Asp

130 135 140

Glu Lys Gly Val Pro Leu Pro Lys Val Asp Leu Gln Phe Phe Ser Asn

145 150 155 160

Thr Thr Ser Leu Asn Asp Arg Gln Gly Asn Ala Thr Lys Pro Lys Val

165 170 175

Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu

180 185 190

Ser Tyr Lys Pro Gly Lys Gly Asp Glu Asn Ser Lys Ala Met Leu Gly

195 200 205

Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn

210 215 220

Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu

225 230 235 240

Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg

245 250 255

Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg

260 265 270

Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro

275 280 285

Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn

290 295 300

Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala

305 310 315 320

Ile Lys Ala Asn Gly Asn Gly Ser Gly Asp Asn Gly Asp Thr Thr Trp

325 330 335

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

340 345 350

<210> 76

<211> 336

<212> protein

<213> Adenovirus type 5

<400> 76

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Pro Cys Glu Trp Asp

1.5 10 15

Glu Ala Ala Thr Ala Leu Glu Ile Asn Leu Glu Glu Glu Asp Asp Asp

20 25 30

Asn Glu Asp Glu Val Asp Glu Gln Ala Glu Gln Gln Lys Thr His Val

35 40 45

Phe Ser Gln Ala Pro Tyr Ser Gly Ile Asn Ile Thr Lys Glu Gly Ile

50 55 60

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

65 70 75 80

Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Tyr Glu Thr Glu Ile

85 90 95

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

100 105 110

Cys Tyr Gly Ser Tyr Ala Lys Pro Thr Asn Glu Asn Gly Gly Gln Gly

115 120 125

Ile Leu Val Lys Gln Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met

130 135 140

Gln Phe Phe Ser Thr Glu Ala Thr Ala Gly Asn Gly Asp Asn Leu

145 150 155 160

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

165 170 175

Asp Thr His Ile Ser Tyr Met Pro Thr Ile Lys Glu Gly Asn Ser Arg

180 185 190

Glu Leu Met Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala

195 200 205

Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn

210 215 220

Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp

225 230 235 240

Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser

245 250 255

Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp

260 265 270

Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp

275 280 285

Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Val Ile Asn Thr Glu

290 295 300

Thr Leu Thr Lys Val Lys Pro Lys Thr Gly Gln Glu Asn Gly Trp Glu

305 310 315 320

Lys Asp Ala Thr Glu Phe Ser Asp Lys Asn Glu Ile Arg Val Gly Asn

325 330 335

<210> 77

<211> 347

<212> protein

<213> Adenovirus type 6

<400> 77

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Glu

1.5 10 15

Gln Asn Glu Thr Ala Gln Val Asp Ala Gln Glu Leu Asp Glu Glu Glu

20 25 30

Asn Glu Ala Asn Glu Ala Gln Ala Arg Glu Gln Glu Gln Ala Lys Lys

35 40 45

Thr His Val Tyr Ala Gln Ala Pro Leu Ser Gly Ile Lys Ile Thr Lys

50 55 60

Glu Gly Leu Gln Ile Gly Thr Ala Asp Ala Thr Val Ala Gly Ala Gly

65 70 75 80

Lys Glu Ile Phe Ala Asp Lys Thr Phe Gln Pro Glu Pro Gln Val Gly

85 90 95

Glu Ser Gln Trp Asn Glu Ala Asp Ala Thr Ala Ala Gly Gly Arg Val

100 105 110

Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg

115 120 125

Pro Thr Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly

130 135 140

Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Thr Asn

145 150 155 160

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

165 170 175

Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro

180 185 190

Lys Met Gly Asp Lys Asn Ala Lys Val Met Leu Gly Gln Gln Ala Met

195 200 205

Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu

210 215 220

Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala

225 230 235 240

Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu

245 250 255

Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe

260 265 270

Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile

275 280 285

Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro

290 295 300

Leu Gly Gly Ile Gly Ile Thr Asp Thr Phe Gln Ala Val Lys Thr Thr

305 310 315 320

Ala Ala Asn Gly Asp Gln Gly Asn Thr Thr Trp Gln Lys Asp Ser Thr

325 330 335

Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn

340 345

<210> 78

<211> 343

<212> protein

<213> Adenovirus type 57

<400> 78

Tyr Asn Ala Leu Ala Pro Lys Gly Ala Pro Asn Ser Cys Glu Trp Asp

1.5 10 15

Glu Asp Asp Thr Gln Val Gln Val Ala Ala Glu Asp Asp Gln Asp Asp

20 25 30

Asp Glu Glu Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys Lys Thr

35 40 45

His Val Tyr Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn Lys Asn

50 55 60

Gly Leu Gln Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn Lys Glu

65 70 75 80

Ile Tyr Ala Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser

85 90 95

Gln Trp Asn Glu Ala Glu Ser Val Ala Gly Gly Arg Val Leu Lys

100 105 110

Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr

115 120 125

Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly Lys Leu

130 135 140

Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn Ala Met

145 150 155 160

Asn Glu Ala Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser Glu Asp

165 170 175

Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys

180 185 190

Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn

195 200 205

Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr

210 215 220

Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln

225 230 235 240

Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr

245 250 255

Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met

260 265 270

Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu

275 280 285

Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly

290 295 300

Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly

305 310 315 320

Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg

325 330 335

Asn Glu Ile Gly Val Gly Asn

340

<---

Claims (37)

1. Recombinant oncolytic adenovirus (Ad) based on human Ad strain Ad6, which contains capsid hexon polypeptides, wherein at least two hexon hypervariable region (HVR) polypeptides of human Ad6 capsid hexon polypeptide are replaced by at least two HVR polypeptides from human Ad strain Ad57 Ad57 . 2. Recombinant ocolitic Ad according to claim 1, wherein the HVR1 Ad polypeptide of the Ad6 strain, containing amino acids 16-52 of SEQ ID NO:77, is replaced by the HVR1 Ad polypeptide of the Ad57 strain, containing amino acids 16-51 of SEQ ID NO:78, the HVR 2 Ad polypeptide of strain Ad6 containing amino acids 68-83 of SEQ ID NO: 77 is replaced by the HVR 2 Ad polypeptide of strain Ad57 containing amino acids 67-81 of SEQ ID NO: 78, the HVR 3 Ad polypeptide of strain Ad6 containing amino acids 101-107 of SEQ ID NO: 77 is replaced by the HVR 3 Ad polypeptide of strain Ad57 containing amino acids 99-105 of SEQ ID NO: 78, the HVR 4 Ad polypeptide of strain Ad6 containing amino acids 137-149 of SEQ ID NO: 77 is replaced by the HVR 4 Ad polypeptide of strain Ad57 containing amino acids 135-147 of SEQ ID NO: 78, the HVR 5 Ad polypeptide of strain Ad6 containing amino acids 156-171 of SEQ ID NO: 77 is replaced by the HVR 5 Ad polypeptide of strain Ad57 containing amino acids 154-169 of SEQ ID NO: 78, the HVR 6 Ad polypeptide of strain Ad6 containing amino acids 193-202 of SEQ ID NO: 77 is replaced by the HVR 6 Ad polypeptide of strain Ad57 containing amino acids 191-200 of SEQ ID NO: 78, and the HVR 7 Ad polypeptide of strain Ad6 containing amino acids 307-347 of SEQ ID NO: 77 is replaced by the HVR 7 Ad polypeptide of strain Ad57 containing amino acids 305-343 of SEQ ID NO: 78. 3. Recombinant ocolitic Ad according to claim 1, containing capsid hexon polypeptides containing the amino acid sequence shown in SEQ ID NO:49. 4. A recombinant ocolitic Ad according to claim 2 or 3 which is Ad657. 5. Recombinant ocolitic Ad according to claim 1, wherein the HVR 2 Ad polypeptide of the Ad6 strain, containing amino acids 68-83 of SEQ ID NO: 77, is replaced by the HVR 2 Ad polypeptide of the Ad57 strain, containing amino acids 67-81 of SEQ ID NO: 78, the HVR 3 Ad polypeptide of strain Ad6 containing amino acids 101-107 of SEQ ID NO: 77 is replaced by the HVR 3 Ad polypeptide of strain Ad57 containing amino acids 99-105 of SEQ ID NO: 78, the HVR 4 Ad polypeptide of strain Ad6 containing amino acids 137-149 of SEQ ID NO: 77 is replaced by the HVR 4 Ad polypeptide of strain Ad57 containing amino acids 135-147 of SEQ ID NO: 78, the HVR 5 Ad polypeptide of strain Ad6 containing amino acids 156-171 of SEQ ID NO: 77 is replaced by the HVR 5 Ad polypeptide of strain Ad57 containing amino acids 154-169 of SEQ ID NO: 78, and the HVR 6 Ad polypeptide of strain Ad6 containing amino acids 193-202 of SEQ ID NO: 77 is replaced by the HVR 6 Ad polypeptide of strain Ad57 containing amino acids 191-200 of SEQ ID NO: 78. 6. Recombinant ocolitic Ad according to claim 1, containing capsid hexon polypeptides containing the amino acid sequence shown in SEQ ID NO:59. 7. Recombinant ocolitic Ad according to claim 5 or 6, which is Ad6/57/6. 8. Recombinant okolitic Ad according to any one of paragraphs. 1-7 containing one or more targeting polypeptides, antigenic polypeptides, enzymes, receptors or ligands. 9. The recombinant ocolitic Ad of claim 8, wherein the targeting polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-41 and SEQ ID NOs: 46-47. 10. Recombinant okolitic Ad according to any one of paragraphs. 1-9, wherein the amino acid substitution is a cysteine substitution and the amino acid modification is selected from pegylation and bapilation. 11. Recombinant okolitic Ad according to any one of paragraphs. 1-10, where the recombinant Ad is a conditionally replicative Ad (CRAd). 12. Recombinant okolitic Ad according to any one of paragraphs. 1-10 containing nucleic acids encoding the amino acid sequence of SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45. 13. Recombinant okolitic Ad according to any one of paragraphs. 1-11 containing one or more modified fiber polypeptides. 14. The recombinant oncolytic adenovirus (Ad) according to claim 13, wherein the modified fiber polypeptides comprise a heterologous Ad35 fiber polypeptide optionally containing 7 added lysines (K7 peptide), or a chimpanzee C68 fiber polypeptide optionally containing 7 added lysines (K7 peptide), or their combinations. 15. The recombinant oncolytic adenovirus (Ad) according to claim 14, wherein the modified fiber polypeptides comprise the amino acid sequence of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, or their combinations. 16. Recombinant okolitic Ad according to any one of paragraphs. 1-15 containing one or more nucleic acids encoding one or more heterologous proteins. 17. The recombinant ocolitic Ad of claim 16 wherein the heterologous proteins are selected from therapeutic polypeptides, targeting polypeptides, antigenic polypeptides, antibodies, enzymes, receptors and ligands. 18. The recombinant oncolytic adenovirus (Ad) according to claim 17, wherein the antigenic polypeptide is a human folate receptor alpha polypeptide. 19. Recombinant ocolitic Ad according to claim 17, wherein the heterologous proteins are selected from antibodies, 4-1BB ligand (4-1BBL), granulocyte-macrophage colony stimulating factor (hGMCSF), CD40 ligand (CD40L), programmed death protein 1 (PD-1) ), interleukin-21 (IL-21), and chronic lymphocytic leukemia (CLL) peptides. 20. A method of treating a mammal having a malignant neoplasm, wherein said method comprises administering to said mammal a recombinant oncolytic adenovirus (Ad) according to any one of paragraphs. 1-19. 21. The method of claim 20, wherein said 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 myelogenous leukemia. 22. The method according to any one of paragraphs. 20-21, where said administration includes systemic administration. 23. The method of claim 22, wherein systemic administration includes intramuscular, intranasal, or intravenous administration. 24. The method according to any one of paragraphs. 20-21, where said administration includes topical administration. 25. The method of claim 24, wherein said topical administration comprises intratumoral injection. 26. The use of recombinant oncolytic Ad according to any one of paragraphs. 1-19 for the treatment of malignant neoplasms. 27. Use of a recombinant oncolytic adenovirus (Ad) based on the human Ad strain Ad6, which contains capsid hexon polypeptides, wherein at least two hexon hypervariable region (HVR) polypeptides of the human Ad6 capsid hexon polypeptide are replaced by at least two HVR polypeptides from the human Ad strain Ad57, as a drug for the treatment of malignant neoplasms.

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