ADENOVIRUSES AND METHODS FOR USING ADENOVIRUSES

Abstract

This invention relates to methods and materials for nucleic acid delivery, vaccination, and/or treating cancer. More specifically, methods and materials for nucleic acid delivery, vaccination, and/or treating cancer using one or more recombinant adenoviruses (Ads) as an oncolytic agent are provided.

Description

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0001] Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 147,456 Bytes ASCII (Text) file named "SEQUENCE_LISTING.TXT," created on 9 Jun. 2021.

STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTOR OR A JOINT INVENTOR QUALIFYING FOR THE GRACE PERIOD PROVIDED UNDER 35 USC .sctn. 102(b)(1):

[0002] The applicant submits that the following disclosures qualify under the grace period provided as noted above: Nguyen, et al., Oncolytic Virotherapy 8:43-51, May 3, 2018; Nguyen, et al., Virology 514:118-123, 15 Jan. 2018; Matchett, et al., J Virol., 93:1-18, May 1, 2019.

BACKGROUND OF THE INVENTION

Technical Field of the Invention

[0003] This invention relates to methods and materials for nucleic acid delivery, vaccination, and/or treating cancer. For example, the invention encompasses adenoviruses (Ads) and methods for using adenoviruses to treat medical conditions such as cancer. In an aspect of the invention, an adenovirus provided herein can be used as an oncolytic agent.

[0004] Despite vast efforts, cancer remains a major public health issue in the United States with over 1.6 million new cases in 2017 alone (National Cancer Institute, "Cancer Stat Facts: Cancer of Any Site," seer.cancer.gov/statfacts/html/all.html). Traditional therapies, such as chemotherapeutics, radiation therapy and surgery, often fail, especially when cancer is advanced. One of the reasons is for this that cancer cells can eliminate or modify the components that are targeted by these therapies and effectively avoid being killed.

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

[0006] This invention provides methods and materials for nucleic acid delivery, vaccination, and/or treating cancer. For example, this invention provides methods and materials for treating cancer by administering one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) as an oncolytic agent. In an embodiment, a recombinant Ad can be derived from a first Ad (e.g., can include a genome of a first Ad, such as Ad6, also referred to as the recombinant Ad backbone) and can include hexon HVRs from a second Ad such as Ad57. In cases where a recombinant Ad includes an Ad6 genome and Ad57 hexon HVRs, the recombinant Ad can be a chimeric Ad referred to as Ad657. (See Nguyen, et al. Oncolytic Virotherapy 7:43-51, 2018, the disclosure of which is incorporated by reference).

[0007] In an aspect, this invention provides methods for vaccinating against infectious disease using one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof). In an aspect, this invention provides methods for treating cancer using one or more recombinant Ads (ex., one or more of Ad657 and variants thereof) as an oncolytic agent. In some cases, one or more recombinant Ads (e.g., one or more Ad657s) can be used to reduce the number of cancer cells (e.g., by infecting and killing cancer cells) in a mammal. In some cases, one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) can be used to stimulate anti-cancer immune responses in a mammal. In some cases, one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) can be used to stimulate immune responses against infectious diseases in a mammal.

[0008] As demonstrated herein, when Ad657 is delivered by intravenous injection to mice having subcutaneous human DU145 prostate cancer tumors, Ad657 first infects the liver and then reaches distant tumors. Both Ad6 and Ad657 mediated significant delays in tumor growth and extension of survival with Ad6 mediating higher efficacy.

[0009] This invention provides methods and materials for nucleic acid delivery, vaccination, and/or treating cancer. For example, this invention provides methods and materials for treating cancer by administering one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) as an oncolytic agent. In an embodiment, a recombinant Ad can be derived from a first Ad (e.g., can include a genome of a first Ad, such as Ad6, also referred to as the recombinant Ad backbone) and can include hexon HVRs from a second Ad such as Ad57. In cases where a recombinant Ad includes an Ad6 genome and Ad57 hexon HVRs, the recombinant Ad can be a chimeric Ad referred to as Ad657. In an aspect, this invention provides methods for vaccinating against infectious disease using one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof). In an aspect, this invention provides methods for treating cancer using one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) as an oncolytic agent. In some cases, one or more recombinant Ads (e.g., one or more Ad657s) can be used to reduce the number of cancer cells (e.g., by infecting and killing cancer cells) in a mammal. In some cases, one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) can be used to stimulate anti-cancer immune responses in a mammal. In some cases, one or more recombinant Ads (e.g., one or more of Ad657 and variants thereof) can be used to stimulate immune responses against infectious diseases in a mammal.

[0010] As demonstrated herein, when Ad657 is delivered by intravenous injection to mice having subcutaneous human DU145 prostate cancer tumors, Ad657 first infects the liver and then reaches distant tumors. Both Ad6 and Ad657 mediated significant delays in tumor growth and extension of survival with Ad6 mediating higher efficacy.

[0011] Moreover, liver sequestration is a considerable problem for virtually any oncolytic virus if it is used as an intravenous systemic therapy. If the virus infects hepatocytes and kills them, this will result in liver damage at low doses and death at higher doses. Notably, administration of the Ads of the invention, i.e., Ad657 chimeric vector and variants thereof, mediated unexpected lower liver damage than either Ad5 or Ad6. Thus the unique combination of Ad6 platform with the HVRs 1-7 of Ad657 mediated changes in biodistribution and therapy not observed in natural viruses.

[0012] Also, as demonstrated herein, immunization of rhesus macaques with replicating single-cycle adenovirus (SC-Ad657) vaccines expressing only clade B HIV-1 gp160 by intranasal (IN) and intramuscular (IM) routes was compared to mucosal and systemic routes of vaccination. SC-Ad vaccines by themselves generated significant circulating antibody titers against Env after only a single immunization. Animals immunized only by the IM route had high peripheral T follicular helper (pTfh) cells in blood, but low Tfh in lymph nodes, and had lower antibody-dependent cellular cytotoxicity (ADCC) antibody activity. Animals immunized by the IN route had high Tfh in lymph nodes, but low pTfh in the blood, and had higher ADCC antibodies. When immunized animals were challenged rectally with SHIV.sub.SF162P3, they all became infected, but mucosally-primed animals had markedly lower viral loads their gastrointestinal tracts. Similarly, Ad657 carrying genes for hepatitis C antigens is able to generate cytotoxic T lymphocyte (CTL) responses against hepatitis and cytomegalovirus. Ad657 is able to delivery and express therapeutic genes including cytokines like 4-1BBL, granulocyte macrophage stimulating factor (GMCSF), and IL-21. The results provided herein demonstrate that recombinant Ads can be used as a local or systemic delivery vehicle for nucleic acid, vaccines, and/or oncolytic virotherapy for cancers.

BRIEF SUMMARY OF THE INVENTION

[0013] In general, one aspect of this invention features 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, where one or more of the hypervariable regions the hypervariable regions (HVRs) of the hexon polypeptide are different from the HVRs encoded by the Ad genome. The first Ad strain can be a first human Ad strain, and the second Ad strain can be a second human Ad strain which is different from the first human Ad strain. The first Ad strain and the second Ad strain can be serotypically distinct. The first Ad strain can be a human Ad6 strain, and the second Ad strain can be a human Ad57 strain. The recombinant Ad also can include one or more targeting polypeptides, antigenic polypeptides, enzymes, amino acid substitutions, PEGylation, ligands, tags and the like.

[0014] A recombinant Ad may be used as a vector for gene-based vaccination, for gene therapy application/delivery, or for oncolytic virotherapy.

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

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

[0017] In another aspect, this invention features a recombinant and/or chimeric Ad comprising (a) nucleic acid encoding a first hexon polypeptide and (b) a second hexon polypeptide, where 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 a hypervariable region (HVR) of the first hexon polypeptide can be different from the amino acid sequence of a hypervariable region of the second hexon polypeptide. The nucleic acid can be from a first Ad strain, and the second hexon polypeptide can be from a second Ad strain. The first Ad strain can be a first human Ad strain, and the second Ad strain can be a second human Ad strain different from the first human Ad strain. The first Ad strain and the second Ad strain can be serotypically distinct. The Ad strain can be a human Ad6 strain, and the second Ad strain can be a human Ad57 strain. The recombinant Ad also can include a targeting polypeptide. The targeting polypeptide can include the amino acid sequence TARGEHKEEELI (SEQ ID NO:1).

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

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

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

[0021] It is demonstrated herein that Ad657 and variants thereof are able to deliver therapeutic genes to cells for expression of therapeutic polypeptides. Thus, recombinant Ads, including chimeric Ads, can be used as a local or systemic delivery vehicle for nucleic acid, vaccines, and/or oncolytic virotherapy for cancers.

[0022] An aspect of the invention relates to recombinant adenovirus (Ad) comprising (a) an Ad genome encoding hexon polypeptides from a first Ad strain and (b) a nucleic acid encoding at least one hexon polypeptide from one or more different Ad strains, wherein at least one hypervariable region (HVR) of the hexon polypeptide is different from the HVRs encoded by the Ad genome of the first Ad strain.

[0023] A further aspect of the invention relates to such a recombinant Ad, wherein the first Ad strain and the one or more different Ad strains are serotypically distinct.

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

[0025] 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 tag.

[0026] A further aspect of the invention relates to such a recombinant Ad, wherein the targeting polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1-41 and SEQ ID NO:46-47.

[0027] A further aspect of the invention relates to such a recombinant Ad, wherein said recombinant Ad is a replication competent Ad.

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

[0029] 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 different Ad strains, wherein hypervariable regions (HVRs) of the hexon polypeptide or capsid polypeptides are different from the HVRs or capsid polypeptides of the first Ad strain.

[0030] 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.

[0031] A further aspect of the invention relates to such a recombinant Ad, wherein the nucleic acid encoding at least one hexon polypeptide is different from the amino acid sequence of a hypervariable region of said second hexon polypeptide.

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

[0033] 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.

[0034] A further aspect of the invention relates to such a recombinant Ad, wherein said first Ad strain and said second Ad strain are serotypically distinct.

[0035] 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.

[0036] A further aspect of the invention relates to such a recombinant Ad, further comprising a targeting polypeptide.

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

[0038] A further aspect of the invention relates to such a recombinant Ad, wherein said recombinant Ad is a replication competent Ad.

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

[0040] A further aspect of the invention relates to a method for treating a mammal having cancer, wherein said method comprises administering, to said mammal, a recombinant adenovirus (Ad) as described herein.

[0041] 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, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, lymphocytic leukemia, and myelogenous leukemia.

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

[0043] A further aspect of the invention relates to such a method, wherein in systemic administration comprises intramuscular, intranasal, or intravenous administration.

[0044] A further aspect of the invention relates to such a method, wherein said administering comprises local administration.

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

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

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

BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIG. 1 shows a translation of context-specific peptides from phage to adenovirus. A) Diagram of a phage display library containing the Ad5 fiber HI .beta. sheets that structurally constrain a random 12-mer peptide library. Shown below is a depiction of the structurally similar site between the .beta.7 and .beta.8 sheets in the Ad5 HVR5 hexon. B) Primary amino acid alignments of 12.51 and 12.52 in the HI library and their location when inserted into HVR5 of hexon. C) Representation of Ad5 GFP-Luc expressing viruses modified with the peptides.

[0049] FIG. 2 shows in vitro transduction A) GFP expression by fluorescent microscopy of C2C12 cells infected with 10.sup.4 vp/cell of the indicated vectors 2 days after infection. B) Luciferase activity from C2C12 cells 2 days after infection with varied MOIs of the indicated Ads.

[0050] FIG. 3 shows in vivo transduction in mice. A) Luciferase imaging of mice 1 day after injection by the intravenous (IV) or intramuscular (IM) routes. IM injected mice received 10.sup.9 vp into each quadricep. IV injected mice received 10.sup.10 vp by tail vein. B) Quantitation of luciferase activity by imaging on the indicated days after injection. *p<0.05 by one way ANOVA. ***p<0.001 by one way ANOVA.

[0051] FIG. 4 shows in vim transduction in hamsters. A) Luciferase activity in the muscles of hamsters 1 day after injection with 10.sup.10 vp by the IM route. **p<0.01 by T test.

[0052] FIG. 5 shows gene-based immune responses 16 weeks after single IM immunization. Mice from FIG. 3 were bled 16 weeks after IM injection and their sera were analyzed in serial dilutions by ELISA to detect antibodies against GFP protein. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA. ****p<0.0001 by one-way ANOVA. All Ad-injected mice generated significant anti-GFP antibodies when compared to the PBS group at sera dilutions of 1:10,000 to 1:1000. Comparisons of Ad5-GL-HVR-12.51 and 12.52 to Ad5-GL are shown with black asterisks. Comparison between Ad5-GL-HVR-12.51 and Ad5-GL-HVR-12.52 are shown with a gray asterisk. A gray dashed and dotted line at OD 0.06 shows the 95% confidence interval discriminating antibodies that are different from the PBS group.

[0053] FIG. 6 shows a phylogenetic tree of whole genome sequences of human adenovirus serotypes.

[0054] FIG. 7 shows an alignment of Ad5, 6, and 57 showing variation in hexon and E3 regions. (A) A Pustell DNA alignment of the genomes of Ad6 and Ad57. Boxes indicate hexon and E3 regions where variation is highest between the two viruses. (B) ClustalW amino acid alignment of the hypervariable region in hexon proteins from Ad5, Ad6, and Ad57. Alignments were performed on MacVector.

[0055] FIG. 8 shows a cartoon of the construction of Ad657 by replacement of the Ad6 HVRs with Ad57 HVRs. Abbreviation: HVRs, hypervariable regions.

[0056] FIG. 9 shows in vitro oncolytic activity. LNCaP and DU145 cells were treated with the indicated viruses with the indicated vp/cell for 5 days. The cells were stained with crystal violet and cell viability was measured by OD595. Cell viability (%) was calculated by dividing the OD of the samples by the mean OD of untreated control cells on the same 96-well plate and multiplying this number by 100. (A) LNCaP cell killing. (B) DU145 killing. Abbreviation: vp, viral particle.

[0057] FIG. 10 shows effects of oncolytic Ads on liver damage. C57BL/6 mice (n=6 per group) were injected with 1011 vp of each virus by tail vein. (A) Kaplan-Meier survival. (B) Blood was drawn for ALT measurements 3 days after injection (****p<0.001 by ANOVA). Abbreviations: ALT, alanine aminotransferase: vp, viral particle.

[0058] FIG. 11 shows anticancer activity of Ad6 and Ad657 in DU145 tumor xenografts in nude mice after single i.v. administration. Nude mice (n=9 per group) bearing established DU145 tumors were injected i.v. with a single dose of 3.times.10.sup.10 vp of the indicated viruses or with PBS. (A) Effect of a single i.v. injection on tumor growth. Tumor dimensions were measured with calipers and tumor volume was calculated as width.sup.2.times.length.times.1/2. The data are shown as mean.+-.SE. *p<0.05, ****p=0.0001 by ANOVA or by T-test as described in the text. Black asterisks with a black arrow pointing up indicate the statistical difference between the Ad6 group and the PBS group on a selected day described in the text. Gray asterisks and an arrow pointing up indicate differences between the Ad657 group and the PBS group on the indicated day. The shadowed white asterisks with a gray arrow pointing down indicates the statistical difference between the Ad6 and Ad657 groups on the indicated day. (B) Effect of a single i.v. injection on survival. Animals were euthanized when the tumor volume reached 2000 .mu.L or when other sacrifice criteria were met (e.g., ulceration) and Kaplan-Meier survival curves were plotted (*p<0.05, **p<0.01 by log-rank analysis). Abbreviation: i.v., intravenous.

[0059] FIG. 12 shows luciferase imaging nude mice. Four days after single i.v. injection of 3.times.10.sup.10 vp of Ad6 and Ad657-GFP-Luc with deletions of part of 12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RID.alpha.), 14.5K (RID.beta.), and 14.7K and a partial deletion of E4 34K, Abbreviations: i.v., intravenous; vp, viral particle.

[0060] FIG. 13 shows luciferase imaging nude mice. Fourteen days after single i.v. injection of 3.times.10.sup.10 vp of Ad657-GFP-Luc. Abbreviations: i.v., intravenous; vp, viral particle.

[0061] FIG. 14 shows plasma HIV Env binding titers. Immunizations with different SC-Ads and gp140 proteins are shown above the graph with large arrows. Midpoint F8 gp140 binding titers by ELISA are shown for each animal before and after each immunization. The dashed line indicates the minimal detection limit for antibodies in this assay. Symbols are scattered in the x direction at each time point to allow individual measurements to be observed. SC-Ad6-Ebov is a negative control Ad vaccine. This group of animals was not boosted with gp140. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by one way ANOVA in comparison to the SC-Ad6-Ebov group.

[0062] FIG. 15 shows plasma HIV neutralization titers. Neutralization of the indicated viruses was performed using the TZM-bl neutralization assay. All values were calculated as compared to virus-only wells. Each dot represents the mean value for each animal.

[0063] FIG. 16 shows plasma ADCC activity. Plasma samples were tested with CD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc, target cells infected with SHIVSF162P3. Each dot represents the mean value for each animal. *p<0.05, ***p<0.001, ****p<0.0001 by one-way ANOVA vs. the SC-Ad6-Ebov group.

[0064] FIG. 17 shows mucosal ADCC activity. Vaginal wash and saliva samples were tested with CD16-KHYG-1 effector cells to kill CEM.NKR.CCR5.CD4+-Luc, target cells infected with SHIVSF162P3. Each dot represents the mean value for each animal. *p<0.05 by one-way ANOVA vs. the SC-Ad6-Ebov group.

[0065] FIG. 18 shows IFN-.gamma. Secreting Cells from PBMCs and Lymph Nodes. PBMCs and lymph node cells were analyzed by ELISPOT by staining for IFN-.gamma.. Anti-Env indicates cells that were stimulated with conserved HIV Env peptides, and SC-Ads. The total number of spot forming cells (SFCs) in each of the stimulated wells were counted and adjusted to control medium as background. Each dot represents the mean value for each animal, *p<0.05 by one-way ANOVA.

[0066] FIG. 19 shows mucosal T cell trafficking and activation. T cells were harvested from rectal biopsies collected after the second protein boost and analyzed by flow cytometry for CD4, CD8, a4.beta.7 integrin, CD69, and FoxP3. Each dot represents the mean value for each animal.

[0067] FIG. 20 shows Tfh cell response in the blood and in lymph nodes. PBMCs and lymph node cells collected at week 40 were stimulated with HIV-1 Env protein and then examined for co-expression of CD3+, CD4+, CXCR5+, and IL-21. Each dot represents the mean value for each animal. *p<0.05 by one-way ANOVA.

[0068] FIG. 21 shows protection against repeated rectal SHIV.sub.SF162P3 challenge. The indicated groups were challenged rectally with 4.3 TCID50 (on rhesus PBMCs) of SHIVSF162P3 on a weekly basis. Plasma samples were analyzed for SHIV viral RNA copies. Animals with RNA copies above 10 were considered infected and the number of challenges required to infect that animal were used as events for Kaplan-Meier survival analysis.

[0069] FIG. 22 shows SHIV.sub.SF162P3 acquisition and viral loads. A) Animals from FIG. 8 were grouped by their initial SC-Ad priming route (IM or IN) yielding groups of 8 and Kaplan-Meier analysis was performed B) Plasma SHIVSF162P3 viral RNA levels over the course of the challenge study.

[0070] FIG. 23 shows SHIV viral load in tissues. RNA from PBMCs and post-mortem tissues were collected and qPCR was performed to detect analyzed for SHIV viral RNA.

[0071] FIG. 24 shows single-cycle adenovirus vaccines used in Example 7. A) Cartoon of SC-Ad serotypes 6 and 657 carrying F8 and G4 clade B HIV envelope genes. B) Alignment of clade C Ad hexons including the Ad6 and 57 hexons displayed on vaccines.

[0072] FIG. 25 shows saliva and vaginal HIV env binding titrations. ELISA OD450 levels are shown for the indicated samples at the indicated dilutions when tested against F8. The low level of antibodies in these mucosal samples prevent reaching saturation of the assay. For this reason, EC50 values cannot be reliably calculated for most animals, Rhesus macaque Rh13-091 in the IN-IM-IM group was the only animal in which an EC50 could be calculated (EC50=4580). Similar results were observed in ELISAs using SF162 gp140.

[0073] FIG. 26 shows Ad657 expressing antigen genes from hepatitis C and cytomegalovirus (CMV) gB generating in vivo cytotoxic T lymphocyte (CTL) activity. Shown is killing of hepatitis C peptide-loaded target cells in mice vaccinated with Ad657-HCV rather than CMV gB.

[0074] FIG. 27 shows expression of human GMCSF from Ad657 inducing proliferation of GMCSF-dependent TF-1 human erythroblasts.

[0075] FIG. 28 is a graph showing Ad6 single IV injection vs. A549 lung tumors.

[0076] FIG. 29 is a graph showing Ad6 single IV or IT injection vs. Panc1 pancreatic tumors.

[0077] FIG. 30 is a graph showing Ad6 single IV injection vs. kidney cancer in immune competent hamsters.

[0078] FIG. 31 is a graph showing luciferase activity in B16 melanoma and A549 lung tumor/cancer cells by Ads displaying 12.51 cell binding peptides in HVR5 of the hexon.

[0079] FIG. 32 is a graph showing luciferase activity in hepatocellular carcinoma and kidney cancer by Ads displaying 12.51 cell binding peptides in HVR5 of the hexon.

[0080] FIG. 33 shows a cartoon combining the insertion of individual HVRs from different Ad serotypes with the insertion of cell targeting/detargeting peptides or novel amino acids such as cysteine into the hexon for targeted chemical modification and shielding. Depicted are chimeric HVR constructs that combine different HVRs from different Ad serotypes to modulate natural interactions with cells and blood factors improve pharmacology combined with insertion of cell binding and cell detargeting peptides in different HVRs to change cell entry and cell avoidance. If one HVR is substituted from 100 Ads, this would create 100 different hexon chimeras. If all 7 HVRs each receive a different Ad HVR, this combinatorial library would equal 7.sup.100 variants. If one 1 peptide were introduced into 7 HVRs this would equal 7.times.7.sup.100 variants. If 10 different peptides were introduced into 7 HVRs, this would equal 10.times.7.times.7.sup.100 variants. etc.

[0081] FIG. 34 shows plasmid maps for representative combinatorial hexons and peptide combinations. Shown are hexons with HVR1 from Ad6 and HVRs 2-7 from Ad57 as well as insertions of cell targeting peptides into individual HVRs.

[0082] FIG. 35 shows chimeric HVR constructs that combine different HVRs from different Ad serotypes to modulate natural interactions with cells and blood factors improve pharmacology combined with insertion of cell binding and cell detargeting peptides in different HVRs to change cell entry and cell avoidance. In this example, a single cysteine amino acid is inserted into the HVR1 and HVR5 of Ad657 to modulate pharmacology and allow targeted conjugation of polymers like polyethylene glycol or other moieties like imaging agents like fluorophores.

[0083] FIG. 36 shows conjugation of polyethylene glycol (PEG) to Ad657-HVR1-C. A) SDS-PAGE of Ad proteins with and without PEGylation. Arrows show size increases due to chemical attachment of PEG to hexon. B) Effects of targeted PEGylation by maleimide-PEG and non-targeting NHS-PEG on virus infection.

[0084] FIG. 37 shows conjugation of polyethylene glycol (PEG) to Ad657-HVR5-C, A) SDS-PAGE of Ad proteins with and without PEGylation. B) Near infrared imaging of SDS-PAGE of Ad proteins with and without PEGylation and with and without the near infrared fluorescence imaging tag IR800. C) In vivo transduction after intraperitoneal injection of maleimide-PEGylated Ad657-HVR5-C by luciferase imaging.

[0085] FIG. 38 shows luciferase imaging of nude mice. A) 1, 4, 7, 14, 28, and 42 days after single I.V. injection of Ad6 treatment vs. distant DU145 prostate tumors. B) 3, 7, and 19 days after I.V. injection of replicating Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

[0086] FIG. 39 shows a schematic of cancer-specific conditionally-replicating Ads (CRAds) dl1101+dl1107 having a modification in the E1A gene.

[0087] FIG. 40 is a graph showing that Ad6 and Ad657 can both be used as CRAds for targeted cancer therapy.

[0088] FIG. 41 is a schematic showing Ad therapeutic cycles. A) A schematic of serotype-switching with Ads. B) A schematic of an exemplary therapeutic cycle where Ad6 and Ad657 can be used for multiple rounds of treatment by serotype-switching in combination with covalent polymer conjugation.

[0089] FIG. 42 demonstrates serotype-switching and on-target luciferase activity in the DU145 prostate tumors after a single IV injection of Ad6 and Ad6-F35 with deletions in E3A genes (12.5K, 6.7K, 19K, 11.6K), but retention of E3B genes (10.4K, 14.5K, and 14.7K) and retention of E4 34K. Mice whose tumors resisted prior single IV injection with Ad657 and CRAd657 both with intact E3 genes were injected with the indicated vectors by single IV injection.

[0090] FIG. 43 is a schematic of a replication competent Ad (RC-Ad), wherein E1 expression is controlled by the native E1 promoter; a variant CRAd-Probasin-E1A (Ad-PB), wherein E1 expression is controlled by prostate-specific probasin promoter; CRAd-dl1101, wherein p300 pathway binding ablated, susceptible to IFN pathway in normal cells; CRAd-dl11107, wherein pRB binding ablated allows virus to kill cancer cells with RB pathway disruptions, but is repressed in RB+ normal cells; CRAd-dl1101/07, wherein p300 pathway binding ablated, susceptible to IFN pathway pRB binding ablated allows virus to kill cancer cells with RB pathway disruptions, but is repressed in RB+ normal cells.

[0091] FIG. 44 (A and B) shows the effect of infection with replication-competent Ad5, Ad6, Ad657 on non-cancerous cells and modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds).

[0092] FIG. 45 demonstrates killing of cancerous cells by replication-competent Ad5, Ad6, Ad657, and the indicated CRAds.

[0093] FIG. 46 demonstrates modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds).

[0094] FIG. 47 demonstrates modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds).

[0095] FIG. 48 demonstrates in vivo effects of replication-competent Ad6 or the indicated CRAds on growth of DU145 tumors in mice.

[0096] FIG. 49 demonstrates 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.

[0097] FIG. 50 demonstrates that PEGylation de-targets adenovirus to liver in vivo.

[0098] FIG. 51 demonstrates modification of Ad657 with the shorter fiber from chimpanzee AdC68 and the addition of a codon-wobbled E4 34.K gene changes in vitro efficacy.

[0099] FIG. 52 demonstrates 6/56/6 virus killing human prostate cancer cells with and without CRAd modifications.

[0100] FIG. 53 demonstrates the production of antibody responses against the human cancer antigen folate receptor alpha after a single intramuscular immunization of BALM mice by CRAd657-dl1101/07-FOLR with an intact E3 region.

[0101] FIG. 54 depicts sites on Ad HVRs which may be modified, for example, by PEGylation or "BAPylation" with biotin acceptor peptides (BAPs).

[0102] FIG. 55 is a schematic of variants of Ads having mutations in the E1 protein to convert the virus to a conditionally-replicating Ad (CRAd).

[0103] FIG. 56 shows amino acid sequences of the N-terminal portion of the wild-type E1A polypeptide and the E1A N-terminus of the CRAd variants, dl1101, dl1107 and dl1101/1107.

[0104] FIG. 57 shows as schematic of different E3 immune evasion genes in Ad species C exemplar Ad6 and Ad species D exemplar Ad26. Both Ads express size and sequence variants of E3 12.5K, 6.7K, 19K, 10.4K (RID.alpha.), 14.5K (RID.beta.), and 14.7K genes, as well as a depiction of the functions of these E3 encoded proteins.

[0105] FIG. 58 demonstrates the effects of PEGylation and E3 deletion on oncolytic viral anti-tumor activity by Ad6-Luc viruses in immunocompetent hamsters. Ad6-Luc and Ad6-Luc-20K PEG both have all E3 genes and E4 34K intact. Ad6-deltaE3-Luc has partial deletion of E3 12.5K and E4 34K and full deletion of E3 6.7K, 19K, 11.6K (ADP), 10.4K (RID.alpha.), 14.5K (RID.alpha.), and 14.7K genes. Oncolytic efficacy is lost in this immunocompetent animal model when these immune evasion genes are not present in oncolytic adenovirus.

[0106] FIG. 59 is a plasmid map of Ad657 with partial deletion of E3 12.5K and E4 34K and full deletion of E3 6.7K, 19K, 11.6K (ADP), 10.4K (RID.alpha.), 14.5K (RID.beta.), and 14.7K genes.

[0107] FIG. 60 depicts CRAd 657 constructs with and without dl1101/1107 CRAd modifications and with and without deletions of selected E3 immune evasion genes.

[0108] FIG. 61 depicts CRAd657 with E3 insertion site. These are with and without dl1101/1107 CRAd modifications described herein and with and without E3 immune evasion modifications.

[0109] FIG. 62 depicts CRAd657+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide. These are with and without dl1101/1107 CRAd modifications described in previous slides and with and without E3 immune evasion modifications. In some cases, a codon-wobbled E4 34K gene is included after E4 and before fiber to compensate for E4 34K partial deletion when deleted E3B genes.

[0110] FIG. 63 depicts CRAd657+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide. These are with and without dl1101/1107 CRAd modifications described in previous slides and with and without E3 immune evasion modifications.

[0111] FIG. 64 depicts CRAd657+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide Expressing Folate Receptor alpha.

[0112] FIG. 65 depicts CRAd657+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide Expressing Granulocyte Macrophage Colony Stimulating Factor (GMCSF).

[0113] FIG. 66 depicts CRAd657+/-Ad35 Fiber or Chimpanzee C68 Fiber +/-K7 peptide Expressing 4-1BBL or GMCSF or IL21 or CD40L and combinations in one virus.

[0114] FIG. 67 depicts Ad6/57 with Ad6 HVR1 and Ad57 HVRs2-7+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide.

[0115] FIG. 68 depicts Ad6/57/6 with Ad6 HVR1, Ad57 HVRs2-6, Ad6 HVR7+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide.

[0116] FIG. 69 depicts Ad6/57/6 with Ad6 HVR1, Ad57 HVRs2-6, Ad6 HVR7+/-Ad35 Fiber or Chimpanzee C68 Fiber+/-K7 peptide expressing GFPLuciferase.

[0117] FIG. 70 shows luciferase imaging after serotype switching. Mice bearing LNCaP prostate tumors on their flanks were treated by a single IV injection of Ad657 or CRAd657. Mice with residual tumors 5 months after single IV injection were treated by serotype-switching of the indicated Ad6/57/6 variants expressing GFPLuciferase with and without variant fibers and a codon-optimized E4 34K gene. The indicated Ad6/57/6 variants include Ad6/57/6 virus having different fiber modifications including an added 7 lysine on fiber (K7), chimpanzee C68 fiber grafted onto Ad6 fiber after its KKTK flexibility domain and with an Ad35 fiber. Mice were imaged for luciferase activity 7 days later.

[0118] FIG. 71 shows A549 human lung cancer cells which were treated with the indicated viral particles (vp) per cell of Ad6/57/6 with and without variant fibers and a codon-optimized E4 34K gene. 7 days later, the cells were stained with crystal violet and the wells were analyzed in a plate reader. High OD indicates the presence of viable cells. Low OD indicates death and loss of adherent cells.

DETAILED DESCRIPTION OF THE INVENTION

[0119] This invention provides methods and materials for nucleic acid delivery, vaccination, and/or treating cancer. For example, this invention provides methods and materials for nucleic acid delivery of proteins/polypeptides, vaccination, and/or treating cancer using one or more recombinant Ads (e.g., Ad657 and variants thereof) as an oncolytic agent.

[0120] An adenovirus icosahedron is made up of 720 copies of its hexon protein. The virus does not use this protein to bind receptors, but this nano-lattice of repeating proteins provides a matrix for interactions (e.g., natural interactions and unnatural interactions) with proteins, cells, and drugs, Antibodies that can neutralize Ads can target hypervariable regions (HVRs) of the hexon polypeptide on an Ads.

[0121] In some cases, this invention provides recombinant Ads having oncolytic anti-cancer activity. For example, a recombinant Ad can be derived from a first Ad and can include hexon HVRs from one or more different Ads. The HVRs may be derived from any species C Ads, for example Ad1, Ad2, Ad5, Ad6 and Ad57. In an embodiment, a recombinant Ad can be derived from a first Ad and can include one or more hexon HVRs from at least one other Ad, wherein at least one hexon HVR is different from the HVR(s) of the first Ad. The first Ad strain can be a human Ad6 strain, and the second Ad strain can be a human Ad57 strain. Hexon shuttle plasmid maps (FIG. 34) show the combination of the insertion of individual HVRs from different Ad serotypes with the insertion of cell targeting/detargeting peptides or novel amino acids such as cysteine into the hexon for targeted chemical modification and shielding. In an embodiment, the recombinant Ads comprise amino acid substitutions, for example, substitution of cysteines into polypeptides, and modifications such as PEGylation and BAPylation. The ability to target polymer and other chemical modifications to cysteines inserted in Ad657 hexon is demonstrated herein.

[0122] Ad657 as an oncolytic against human prostate cancer is demonstrated. The Ad6 HVRs were replaced with those from Ad57 to generate a chimeric species C oncolytic virus called Ad657. Ad657 and Ad6 were tested as systemic oncolytic therapies by single i.v. injection in nude mice bearing human cancerous tumors. Ad657 may be used as a local or systemic oncolytic virotherapy for cancers. These data also demonstrate surprising effects of serotype-switching with oncolytic species C Ads.

[0123] In some cases, this invention provides methods for using one or more recombinant Ads provided herein to treat a mammal having, or at risk of having, cancer, an infectious disease, and/or a genetic disease. For example, one or more recombinant Ads can be administered to a mammal having, or at risk of having, cancer to reduce the number of cancer cells (e.g., by infecting and killing cancer cells) in the mammal (e.g., a human). For example, one or more recombinant Ads can be administered to a mammal having, or at risk of having, cancer to stimulate anti-cancer immune responses in the mammal (e.g., a human).

[0124] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657 and variants thereof) are not destroyed by a mammal's immune system. For example, a recombinant Ad is not destroyed by antigen presenting cells (APCs), macrophages, and/or other immune cells in a mammal that the recombinant Ad is administered to.

[0125] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657 and variants thereof) can be administered for multiple (e.g., two or more) rounds of treatment. For example, a first recombinant Ad described herein can avoid antibodies that can neutralize a second recombinant Ad described herein, and vice versa. In cases where a mammal having cancer is treated with one or more recombinant Ads described herein, the mammal can be administered a first round of treatment with a first recombinant Ad and can subsequently be administered a second round of treatment with a second recombinant Ad.

[0126] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657 and variants thereof) can be replication competent Ads (RC-Ads). For example, a RC-Ad can be a RC-Ad that includes a nucleic acid encoding an E1 polypeptide (e.g., an E1+RC-Ad). For example, a RC-Ad can be a single-cycle Ad (SC-Ad) that includes a deletion of one or more nucleic acids encoding one or more polypeptides associated with the production of infectious viral progeny (e.g., pIIIa and E3). For example, a RC-Ad can be a conditionally-replicating Ad (CRAd). Examples of single-cycle Ads and how to make and use them are provided elsewhere (International Patent Application Publication No. WO2009/111738).

[0127] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657 and variants thereof) can be replication defective Ads (RD-Ads). For example, a RD-Ad can be a RD-Ad that includes a deletion of a nucleic acid encoding an E1 polypeptide (e.g., an E1-deleted RD-Ad).

[0128] It is demonstrated in the examples herein that CRAd 657 and variants thereof are conditionally-replicating Ads (CRAds) in cancerous cells and that infection of cells with CRAd 657 and variants thereof reduces cell viability and tumor volume. Thus, CRAd 657 and variants thereof may be used as a local or systemic oncolytic virotherapy in subjects with cancer.

[0129] What is more, it is demonstrated that CRAds can be used for expression of antigens and used as a vaccine for vaccinating against viruses, for example, against Human Immunodeficiency Virus (HIV), Human Papilloma Virus (HPV) and Hepatitis C Virus (HCV).

[0130] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657 and variants thereof) can bind to a cell surface receptor (e.g., to facilitate viral entry to a cell). For example, a recombinant Ad described herein can bind to coxsackie-adenovirus receptors (CARS) and/or Fc receptors (e.g., Fc.mu.R and Fe.gamma.R), complement receptors (e.g., CR3 and/or C2qR).

[0131] In an aspect of the invention, CRAds may comprise nucleic acids encoding polypeptides heterologous to the Ad, for example, antigens, cell surface receptors, cell targeting polypeptides and the like. For example, CRAd-657-dl1101/1107-FolR is a recombinant Ad comprising intact E3 and expressing the human folate receptor alpha. It is demonstrated herein that CRAds may be used to generate antibodies against known cancer antigens, for example, folate receptor alpha.

[0132] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) can avoid binding (e.g., do not bind) to a scavenger receptor (e.g., to facilitate viral entry to a cell). For example, a recombinant Ad described herein avoid binding to a SREC receptors and/or SR-A receptors.

[0133] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) can avoid phagocytosis.

[0134] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) are non-pathogenic (e.g., to a mammal being treated as described herein).

[0135] In some cases, recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) can infect dividing cells (e.g., can infect only dividing cells).

[0136] A recombinant Ad described herein can be any appropriate recombinant Ad (e.g., a recombinant Ad having oncolytic anti-cancer activity) generated by recombinant DNA technology and methods known to those skilled in the art. A recombinant Ad can be any Ad generated by recombining material (e.g., nucleic acid and/or polypeptide) from any organism other than the Ad from which the recombinant Ad is derived. For example, a recombinant Ad can include one or more materials that do not naturally occur in that Ad (e.g., do no naturally occur in that Ad prior to recombination). In some cases, a recombinant Ad provided herein can be a chimeric Ad (e.g., can include viral elements from two or more (e.g., two, three, four, five, or more) different Ad genomes).

[0137] These embodiments have been applied also in the context of Ads which combine different HVRs from different Ads (i.e., shuffling HVRs). For example, HVR1 of Ad6 with HVRs 2-7 of Ad57 or HVR1 and 7 of Ad6 with HVRs 2-6 of Ad57, or HVRs 1 and 7 from Ad6 and HVRs 2-6 from Ad657.

[0138] Nucleic acid and/or polypeptides that do not naturally occur in the Ad can be from any appropriate source. In some cases, a nucleic acid and/or a polypeptide that does not naturally occur in that Ad can be from a non-viral organism. In some cases, a nucleic acid and/or a polypeptide that does not naturally occur in that Ad can be from a virus other than an Ad. In some cases, a nucleic acid and/or a polypeptide that does not naturally occur in that Ad can be from an Ad obtained from a different species. In some cases, a nucleic acid and/or a polypeptide that does not naturally occur in that Ad can be from a different strain of Ad (e.g., serotypically distinct strains). In some cases, a nucleic acid and/or a polypeptide that does not naturally occur in that Ad can be a synthetic nucleic acid and/or a synthetic polypeptide.

[0139] A recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657) can be derived from (e.g., can include a genomic backbone from) any appropriate Ad. In some cases, a recombinant Ad described herein can be derived from an Ad having low seroprevalence. For example, 50% or fewer (e.g., 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or fewer) of mammals (e.g., human) can have been exposed to an Ad from which a recombinant Ad described herein is derived. With regard to seroprevalence, species C adenoviruses, Ad6 and Ad657 have lower prevalence than archetype Ad5 virus. In some cases, a recombinant Ad described herein can be derived from an Ad having reduced or eliminated side effects (e.g., phagocytosis and liver damage). A recombinant Ad can be derived from an Ad isolated from any appropriate species of animal. For example, Ads can be isolated from humans, non-human primates (e.g., monkeys such as Old World monkey species like rhesus macaques), fish, frogs, and snakes. In some cases, a recombinant Ad described herein can be derived from a human Ad (HAd or HAdV). A recombinant Ad can be derived from any species of Ad (e.g., A, B, C, D, E, F, or G). In some cases, a recombinant Ad described herein can be derived from an Ad C species (e.g., a human Ad C species (HAd-C)). A recombinant Ad can be derived from any appropriate Ad serotype (e.g., 2, 5, 6, or 57), In some cases, a recombinant Ad described herein can be derived from an Ad serotype 6 (Ad6; e.g., a human Ad6).

[0140] In some cases, a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657 and variants thereof) can include an Ad genome containing one or more modifications to one or more nucleic acids encoding a polypeptide (or fragments thereof) and/or one or more viral elements of the Ad genome. The one or more modifications can be any appropriate modification. In some cases, a modification can be effective to inhibit the ability of the modified polypeptide to bind another polypeptide such as p300 and/or pRB. In some cases, a modification can be effective to neutralize one or more interferon pathways. Examples of modifications that can be made to a nucleic acid encoding a polypeptide or to a viral element include, without limitation, substitutions, deletions, insertions, and mutations.

[0141] Ads, for example Ad657 and variants thereof, may be modified and retain all E1A genes, or modified to delete selected regions and functions of their encoded proteins.

[0142] FIG. 57 shows as schematic of different E3 immune evasion genes in Ad species C exemplar Ad6 and Ad species D exemplar Ad26, as well as a depiction of the functions of these E3 encoded proteins. Both Ads express size and sequence variants of E3 12.5K, 6.7K, 19K, 10.4K (RID.alpha.), 14.5K (RID.beta.), and 14.7K genes. 19K reduces display 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. Species C Ads also express the 11.6K known as the adenovirus death protein (ADP). Over-expression of ADP accelerates cell death, but overall cell death is equal. Species D viruses also express two novel variants called 49K and 31K. The secreted form of 49K binds to CD46 on T cells and NK cells leading to down-regulation of these cells and less-efficient cell killing of cells deficient in class I MHC by NK cells. Ad657 plasmids have been modified to retain all native E3 immune evasion genes (12.5K, 6.7K, 19K, 11.6K (ADP), 10.4K (RID.alpha.), 14.5K (RID.beta.), and 14.7K) and E4 34K or to delete selected regions. Ad657 and its variants are also modified with the addition of 49K and 31K to provide these extra functions to these species C viral platforms.

[0143] Ads, for example Ad657 and variants thereof, may be modified to retain all E3 immune evasion genes, or to delete selected regions and functions of their encoded proteins. With respect to E3 mutations: 19k downregulates MHCI and MIC proteins on infected cells; ADP over expression accelerates cell death, but does not increase the number of cells that are killed; 10k and 14k proteins (RID.alpha. and RID.beta.) combine to block cell killing by extrinsic apoptosis proteins like FAS, TRAIL, TNF, TNFR, and EGFR; 14.7k protein inhibits intrinsic apoptosis signaling.

[0144] Retaining these E3 proteins may allow oncolytic to persist longer, and deleting them may increase immune stimulation.

[0145] Data testing oncolytic efficacy suggests intact E3 mediates better efficacy.

[0146] DE3 constructs have deleted part of 12.5k through and including 14.7k, DE3A constructs have deleted part of E3 12.5k through and including 19k, and DE3ADP constructs have deleted part of E3 12.5k through and including ADP.

[0147] Surprisingly, deleting all E3 genes makes the oncolytic virus less effective in repressing tumor growth.

[0148] In an embodiment, the invention encompasses single-cycle adenovirus, for example SC-Ad657 and variants thereof. Recombinant SC-Ad viruses with heterologous nucleic acids encoding polypeptides were evaluated for use as a vaccine. SC-Ad657 vaccines by themselves generated significant circulating antibody titers against an HIV envelope protein after only a single immunization.

[0149] Similarly, Ad657 carrying genes for hepatitis B and C antigens is able to generate cytotoxic T lymphocyte (CTL) responses against hepatitis and cytomegalovirus.

[0150] Ad657 was modified by insertion of synthetic peptides from human papilloma virus into HVR5. In an embodiment, the amino acid sequence of the variant Ad657-HVR5-HPV hexon is defined in SEQ ID NO:57. The modification allows display of this antigen for vaccine purposes as well as retargeting by binding to proteins that interact with HPV peptides.

[0151] In a further embodiment, expression of Human Granulocyte-Macrophage Colony Stimulating Factor (GMCSF) by Ad657 is demonstrated herein.

[0152] Thus, from the examples herein, it is demonstrated that recombinant Ads, for example Ad657 and variants thereof, may be utilized for expression of heterologous proteins, for example, polypeptide antigens and cell targeting polypeptides.

[0153] In some cases, a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657) can 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. A substitution can be any appropriate substitution. In some cases, one or more nucleic acids encoding a capsid polypeptide of a genome of a first Ad can be substituted with one or more nucleic acids encoding a capsid polypeptide of a second Ad to generate a chimeric Ad. For example, when a recombinant Ad includes a genome from a first Ad where a nucleic acid encoding a capsid polypeptide in the genome is substituted for a nucleic acid encoding a capsid polypeptide from a second Ad (e.g., an Ad different from the Ad backbone), the nucleic acid encoding a capsid polypeptide form the second Ad can express one or more capsid polypeptides, and the expressed capsid polypeptide(s) can be incorporated into the capsid of the recombinant Ad. Examples of capsid polypeptides include, without limitation, hexon polypeptides, fiber polypeptides, penton base polypeptides, IIIa polypeptides, IX polypeptides, and pVI polypeptides.

[0154] The Ad fiber protein is a complex of three apparently identical subunits which mediates the initial cell attachment step. The native Ad6 fiber protein comprises the amino acid sequence set forth in SEQ ID NO:60 and binds CAR.

[0155] In an aspect of the invention, fiber-modified recombinant and chimeric Ads having fiber proteins which are not native to the parental or "backbone" Ad were generated.

[0156] A chimeric Ad, AdF35 fiber chimera, has the amino acid sequence of SEQ ID NO:61 and is shorter than Ad5 and Ad6 fiber proteins and retargets virus to CD46.

[0157] A fiber-modified recombinant Ad, comprising K7 Fiber having the sequence of SEQ ID NO:62, targets virus to heparin sulfate proteoglycans and negative charges on cells.

[0158] A recombinant, chimeric Ad, 6/FC68 Fiber comprising 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 Ad5 or Ad6 fiber proteins and binds CAR.

[0159] A recombinant, chimeric Ad, 6/FC68-K7 Fiber comprising 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 Ad5 or Ad6 fiber proteins. The 6/FC68-K7 Fiber binds CAR and is retargeted to heparin sulfate and negative charges.

[0160] A recombinant, chimeric Ad, 6/FC68-HI-K7 Fiber comprising 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 Ad5 or Ad6 fiber proteins. The 6/FC684-HI-K7 Fiber binds CAR and is retargeted to heparin sulfate and negative charges.

[0161] In some cases, a recombinant Ad can include a genome from a first Ad where a nucleic acid encoding a hexon polypeptide (e.g., HVRs of a nucleic acid encoding a hexon polypeptide) in the genome is substituted for a nucleic acid encoding a hexon polypeptide (e.g., HVRs of a nucleic acid encoding a hexon polypeptide) from a second Ad. In some cases, a recombinant Ad described herein can include a genome from a first Ad that has one or more HVRs substituted for one or more HVRs from a second Ad. For example, a recombinant Ad can be a chimera, in particular Ad657 (e.g., can include an Ad6 genome where the hexon HVRs are substituted for Ad57 hexon HVRs). In cases where a recombinant Ad includes a genome from a first Ad where a nucleic acid encoding a hexon polypeptide in the genome is substituted for a nucleic acid encoding a hexon polypeptide from a second Ad, the recombinant Ad can include from about 1 to about 720 hexon polypeptides from the second Ad. For example, when a recombinant Ad is an Ad657, the Ad657 can include an Ad6 genome and 720 hexon polypeptides including Ad57 hexon HVRs.

[0162] In some cases, a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657) can include an Ad genome containing one or more nucleic acid deletions. A nucleic acid deletion can be any appropriate nucleic acid deletion. A nucleic acid deletion can be a full deletion (e.g., deletion of a nucleic acid encoding a polypeptide) or a partial deletion (e.g., deletion of one or more nucleotides within a nucleic acid encoding a polypeptide). A nucleic acid deletion can reduce or eliminate transcription and translation of a polypeptide encoded by the deleted nucleic acid. Any appropriate nucleic acid can be deleted. In some cases, a nucleic acid encoding a polypeptide associated with production of infectious progeny can be deleted. Examples of nucleic acids that can be deleted and/or modified in a recombinant Ad described herein may encode E1 (e.g., E1A and E1B), E2, E3, E4, pIIIA, fiber, E1B, and include viral enhancers and promoters. For example, a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657) can include an Ad genome containing a deletion of one or more nucleotides within a nucleic acid encoding an E1 polypeptide. In some cases, a recombinant Ad described herein can include one or more substitutions in a nucleic acid encoding an E1 polypeptide.

[0163] In particular embodiments, a recombinant Ad described herein is modified to comprise a probasin promoter comprising, for example, a nucleic acid of SEQ ID NO:48; a recombinant Ad described herein is modified to comprise a dl1101 deletion in a nucleic acid encoding an E1 polypeptide; a recombinant Ad described herein is modified to comprise a dl1107 deletion in a nucleic acid encoding an E1 polypeptide; a recombinant Ad described herein is modified to comprise a dl1101 deletion and a dl1107 deletion. See the examples herein and FIG. 56 for N-terminal amino acid sequences of the E1A polypeptide, for example, wild-type Ad E1A, and CRAd-657-dl1101, CRAd-657-dl1107 and CRAd-657-dl1101/1107 variants.

[0164] In an embodiment, a variant CRAd-657-dl1101/1107-FolR comprises intact E3 and expresses the human folate receptor alpha found on cancer cells.

[0165] In general, Ads may be modified to include CRAd modifications described herein.

[0166] In some cases, a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657) can include an Ad genome containing one or more nucleic acid insertions. For example, a nucleic acid insertion can include a nucleic acid encoding a polypeptide. A nucleic acid can be inserted into any appropriate location within a genome of a recombinant Ad described herein. In some cases, a nucleic acid encoding a polypeptide can be inserted into a HVR (e.g., HVR 5 loop) of a genome of a recombinant Ad described herein. For example, when a nucleic acid encoding a polypeptide is inserted into a HVR of a genome of a recombinant Ad described herein, the nucleic acid encoding a polypeptide can express one or more polypeptides, and the expressed polypeptide(s) can be incorporated into the capsid of the recombinant Ad. In cases where a nucleic acid encoding a polypeptide is inserted into a HVR of a genome of a recombinant Ad described herein, the recombinant Ad can present from about 1 to about 720 polypeptides encoded by the inserted nucleic acid on its surface. A nucleic acid insertion can be nucleic acid encoding any appropriate polypeptide. In some cases, a nucleic acid insertion can encode a polypeptide antigen.

[0167] In some cases, a nucleic acid insertion can encode a targeting polypeptide. Examples of targeting polypeptides that can be included in a recombinant Ad described herein include, without limitation 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), alpha4 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 (GQIPITEPELCCVPWTEAFY; SEQ ID NO:20), 231R10.1 (PQPPNSTAHPNPHKAPPNTT; SEQ ID NO:21), HepaCD8 (VRWFPGGEWGVTHPESLPPP; SEQ ID NO:22), K20 (KKKKKKKKKKKKKKKKKKK; 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 (QSTPPTKHLTIPRHLRNTLI; SEQ ID NO:31), 1-4 (DMSFQLVTPFLKALPTGWRG; SEQ ID NO:32), 1-5 (GGHGRVLWPDGWFSLVGISP; SEQ ID NO:33), 1-5con (FSLVGISP; SEQ ID NO:34), 1-6 (QIMMGPSLGYYMPSESIFAY: SEQ ID NO:35), 2-11 (ISWDIWRWWYTSEDRDAGSA; SEQ ID NO:36), 2-14 (VWGMTTSDHQRKTERLDSPE; SEQ ID NO:37), 2-20 (MTSAQTSEKLKAETDRHTAE; SEQ ID NO:38), 2-9 (MGSRSAVGDFESAEGSRRP; SEQ ID NO:39), 3b-6 (MGRTVQSGDGTPAQTQPSVN; SEQ ID NO:40), 4*-5 (MARTVTANVPGMGEGMVVVP; SEQ ID NO:41), CLL peptides, PD-I, GLA polypeptides (e.g., Factor X), antigen genes, fusion proteins, fusogenic glycoproteins, single-chain antibodies, and capsid proteins from other viruses. A targeting polypeptide can target any appropriate type of cell. Examples of types of cells that can be targeted by a targeting polypeptide included in a recombinant Ad described herein include, without limitation, muscle cells (e.g., skeletal muscle cells), tumors, cancer cells, kidney cells, liver cells, mucosal cells, carbohydrates, and cell membranes.

[0168] This example demonstrates that peptides selected in a compatible structural context on phage libraries can be translated into the Ad hexon protein. For example, for the 12.51 peptide, this insertion site increases muscle transduction while decreasing off target infection in the liver. Thus, such a recombinant Ad which targets muscle tissue may be used as a vector for gene-based muscle vaccination or for gene therapy application/delivery to the muscle.

[0169] In some cases, a nucleic acid insertion can detarget the virus (e.g., by disrupting cell and protein interactions that occur on a given HVR). In some cases, a nucleic acid insertion can encode a detectable label. Examples of detectable labels include, without limitation, fluorophores (e.g., green fluorescent protein (GFP), mCherry, and mBFP), and enzymes (e.g., luciferase, DNAses, proteases, transporters, and polymerases).

[0170] Also provided herein are expression vectors containing a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657 and variants thereof). Expression vectors can carry a recombinant Ad described herein into another cell (e.g., a cancer cell), where it can be replicated and/or expressed. An expression vector, also commonly referred to as an expression construct, is typically a plasmid or vector having an enhancer/promoter region controlling expression of a specific nucleic acid. When introduced into a cell, the expression vector can use cellular protein synthesis machinery to produce the virus in the cell. In some cases, expression vectors containing recombinant Ads described herein can be viral vectors. For example, an expression vector containing a recombinant Ad described herein can be a retroviral vector. In some cases, expression vectors including a recombinant Ad described herein also can be designed to allow insertion of one or more transgenes (e.g., at a multi-cloning site). For example, expression vectors including a recombinant Ad described herein also can include a nucleic acid encoding a detectable label. Examples of detectable labels include, without limitation, fluorophores (e.g., green fluorescent protein (GFP), mCherry, and mBFP), and enzymes (e.g., luciferase, recombinases, nucleases, and transcription factors).

[0171] This invention also provides methods and materials for using one or more recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s). In some cases, a recombinant Ad provided herein can used for treating a mammal having, or at risk of having, cancer. For example, methods for treating a mammal having, or at risk of having, cancer can include administering one or more recombinant Ads described herein to the mammal. In some cases, methods for treating a mammal having, or at risk of having, cancer can include administering one or more expression vectors that encode a recombinant Ad described herein or nucleic acid encoding a recombinant Ad described herein to the mammal. In some cases, one or more recombinant Ads described herein can be administered to a mammal to reduce the number of cancer cells in the mammal (e.g., suppress and/or delay tumor growth) and/or to increase survival of the mammal.

[0172] Targeting a cancerous tumor by serotype-switching oncolytic adenoviruses is demonstrated. Mice bearing DU145 or LNCaP prostate tumors on their flanks were treated one by intravenous (IV) injection with Ad657. These mice were treated a second time with alternate Ad6 or Ad6/57/6 oncolytic virus variants with fiber modifications and expressing GFPLuciferase and luciferase activity was measured by imaging. Ad6 has Ad6 hexon and fiber that targets CAR. Ad6-F35 has Ad6 hexon and the Ad35 fiber that targets CD46. Ad6/57/6 has HVR1 and 7 from Ad6 and HVRs 2-6 from Ad57. Ad6/57/6 viruses have Ad6 fiber, AdC68 fiber, or Ad35 fiber. These data in FIG. 9 show the surprising ability to serotype-switch oncolytics with viruses targeting the tumor with lower off-target infection of the liver.

[0173] In another example of serotype-switching, mice bearing LNCaP prostate tumors on their flanks were treated by a single intravenous (IV) injection with Ad657 or CRAd657. These mice were treated a second time 5 months later with alternate Ad6/57/6 oncolytic virus expressing GFPLuciferase and fiber variants K7 (with 7 lysines added), F35 (with the Ad35 fiber), or KKTK-C68 (chimpanzee C68 fiber fused after the Ad6 KKTK flexibility domain. KKTK-C68 virus also has an added codon-optimized E4 34K gene to enhance viral productivity. Luciferase activity was measured by imaging. All Ad6/57/6's have a hexon with HVR1 and 7 from Ad6 and HVRs 2-6 from Ad57. Ad6/57/6 and KKTK-C68 have fibers that targets CAR. Ad6/57/6-F35 has the Ad35 fiber that targets CD46. K7 increases binding to negative charges on cells including binding heparin sulfate proteoglycans. FIG. 70 demonstrates the capability to serotype-switch oncolytics with viruses targeting a tumor with lower off-target infection of the liver.

[0174] Any appropriate mammal having, or at risk of having, cancer, an infectious disease, and/or a genetic disease can be treated as described herein. For example, humans, non-human primates (e.g., monkeys), horses, bovine species, porcine species, dogs, cats, mice, rats, and feed animals having cancer, an infectious disease, and/or a genetic disease can be treated for cancer as described herein. In some cases, a human having cancer can be treated. In some cases, a mammal (e.g., a human) treated as described herein is not a natural host of an Ad used to generate a recombinant Ad described herein (e.g., a recombinant Ad having oncolytic anti-cancer activity such as a recombinant Ad657). For example, a human being treated with a recombinant Ad657 described herein can lack any pre-existing adaptive immunity to Ad6.

[0175] A mammal having any type of cancer can be treated as described herein. In some cases, a cancer can include one or more solid tumors. In some cases, a cancer can be a blood cancer. Examples of cancers that can be treated as described herein include, without limitation, prostate cancer, ovarian cancer, lung cancer, hepatocellular carcinoma, pancreatic cancer, kidney cancer, melanoma, brain cancer, colon cancer, lymphoma, myeloma, and leukemias (e.g., lymphocytic leukemias and myelogenous leukemias).

[0176] In some cases, methods described herein also can include identifying a mammal as having cancer. Examples of methods for identifying a mammal as having cancer include, without limitation, physical examination, laboratory tests (e.g., blood and/or urine), biopsy, imaging tests (e.g., X-ray, PET/CT, MRI, and/or ultrasound), nuclear medicine scans (e.g., bone scans), endoscopy, and/or genetic tests. Once identified as having cancer, an infectious disease, and/or a genetic disease, a mammal can be administered or instructed to self-administer one or more a recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) or a nucleic acid (e.g., an expression vector) encoding one or more a recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s).

[0177] One or more recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) can be administered by any appropriate route. In some cases, administration can be local administration. In some cases, administration can be systemic administration. Examples of routes of administration include, without limitation, intravenous, intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, parenteral, intratumoral, retro-ureter, sub-capsular, vaginal, and rectal administration. In cases where multiple rounds of treatment are administered, a first round of treatment can include administering one or more recombinant Ads described herein to a mammal (e.g., a human) by a first route (e.g., intravenously), and a second round of treatment can include administering one or more recombinant Ads described herein to a mammal (e.g., a human) by a second route (e.g., intramuscularly).

[0178] 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 especially suitable for therapeutic use. One or more recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) can be formulated into a composition (e.g., a pharmaceutical composition) for administration to a mammal (e.g., a mammal having, or at risk of having, cancer). 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 having, cancer. In some cases, one or more recombinant Ads can be formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents. A pharmaceutical composition can be formulated for administration in solid or liquid form including, without limitation, sterile solutions, suspensions, sustained-release formulations, tablets, capsules, pills, powders, wafers, and granules. Pharmaceutically acceptable carriers, fillers, and vehicles that may be used in a pharmaceutical composition described herein include, without limitation, saline (e.g., phosphate-buffered saline, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

[0179] A composition (e.g., a pharmaceutical composition) including one or more recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657s) can be administered to a mammal (e.g., a mammal having, or at risk of having, cancer) as a vaccine. A vaccine can be prophylactic or therapeutic.

[0180] In some cases, methods described herein also can include administering to a mammal (e.g., a mammal having cancer) one or more additional agents used to treat a cancer. The one or more additional agents used to treat a cancer can include any appropriate cancer treatment. In some cases, a cancer treatment can include surgery. In some cases, a cancer treatment can include radiation therapy. In some cases, a cancer treatment can include administration of a pharmacotherapy such as a chemotherapy, hormone therapy, targeted therapy, and/or cytotoxic therapy. For example, a mammal having cancer can be administered one or more a recombinant Ads described herein (e.g., recombinant Ads having oncolytic anti-cancer activity such as recombinant Ad657 and variants thereof) and administered one or more additional agents used to treat a cancer. In cases where a mammal having cancer is treated with one or more a recombinant Ads described 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 to treat a cancer, an infectious disease, and/or a genetic disease can be administered at the same time or independently. For example, one or more a recombinant Ads described herein and one or more additional agents used to treat a cancer, an infectious disease, and/or a genetic disease can be formulated together to form a single composition. In some cases, one or more a recombinant Ads described herein can be administered first, and the one or more additional agents used to treat a cancer, an infectious disease, and/or a genetic disease administered second, or vice versa.

EXAMPLES

Example 1. Adenoviruses

[0181] For 50 years, there were only four known species C human species Ads including Ad1, Ad2, Ad5, and Ad6 (see, e.g., Weaver et al., 2011 Virology. 412:19-27). In 2001, a fifth species C adenovirus was identified as field isolate strain #16700, and virus neutralization testing with antisera against Ad1, 2, 5, and 6 demonstrated high levels of neutralization (reciprocal titers of 500-16,000) when each antisera was used against its cognate virus (Lukashev et al., 2008 J Gen Virol. 89:380-388). In contrast, anti-Ad1, 2, and 5 antibodies have weak cross-reactivity against #16700 (reciprocal titers of 32-64). Anti-Ad6 sera demonstrated higher cross-reactivity against #16700, but neutralization required 10-fold higher concentrations of sera to neutralize #16700 when compared with Ad6 itself. Subsequent sequence comparisons confirmed #16700 as a novel species C adenovirus and renamed it as Ad57 (see, e.g., Walsh et al., 2011 J. Clin Microbiol. 49:3482-3490).

[0182] 15 human Ads were evaluated for oncolytic activity against breast, ovarian, liver, prostate, kidney, and B cell malignancies. In tests against DU145 human prostate tumors, species C Ad6 was more potent after single intratumoral or intravenous (i.v.) injection than species C Ad5 or species B viruses Ad11 and Ad35. Ad6 was also more effective than Ad5 and Ad11 in immunocompetent Syrian hamsters.

Construction of Recombinant Adenoviruses

[0183] Recombinant adenoviruses were constructed by recombinant DNA technology utilizing methods known to those skilled in the art. A recombinant Ad is derived from a first Ad (e.g., can include a genome of a first Ad, such as Ad6) and may include hexon HVRs from a second Ad such as Ad57. In cases where a recombinant Ad includes an Ad6 genome and Ad57 hexon HVRs, the recombinant Ad can be a chimeric Ad referred to as Ad657.

[0184] To obtain Ad657, an Ad57 HVR sequence was synthesized and inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin.RTM.-FRT cassette between pVI and hexon. This was recombined into various pAd6 plasmids to generate Ad657 and variants thereof. The amino acid sequence of the Ad657 hexon is set forth in SEQ ID NO:49. See FIGS. 34 and 59-65 for plasmid maps of Ad657 variants.

[0185] With respect to variants of Ad657, the Ad57 HVR sequence was synthesized with HVR1 modified with a cysteine, flexibility amino acids, and restriction sites to allow insertions of other peptides. This was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin.RTM.-FRT cassette between pVI and hexon. This was recombined into various pAd6 plasmids to generate Ad657 variants with cysteine in HVR1, the variant referred to as Ad657-HVR1-XXA comprises the hexon having the amino acid sequence of SEQ ID NO:50.

[0186] With respect to variants of Ad657, the Ad57 HVR sequence was synthesized with HVR5 modified with a cysteine, flexibility amino acids, and restriction sites to allow insertions of other peptides. This was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin.RTM.-FRT cassette between pVI and hexon. This was recombined into various pAd6 plasmids to generate Ad657 variants with cysteine in HVR5, the variant referred to as Ad657-HVR5-XXA comprises the hexon having the amino acid sequence of SEQ ID NO:51.

[0187] With respect to variants of Ad657, the Ad57 HVR sequence was synthesized with HVR1 modified with a cysteine, flexibility amino acids, and restriction sites to allow insertions of other peptides. This was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin.RTM.-FRT cassette between pVI and hexon. This was recombined into various pAd6 plasmids to generate Ad657 variants without cysteine in HVR1, but with restriction sites allowing peptide insertions into HVR1, the variant referred to as Ad657-HVR1-XA comprises the hexon having the amino acid sequence of SEQ ID NO:52.

[0188] With respect to variants of Ad657, the Ad57 HVR sequence was synthesized with HVR5 modified with a cysteine, flexibility amino acids, and restriction sites to allow insertions of other peptides. This was inserted into the Ad6 hexon in a plasmid with an FRT-Zeocin.RTM.-FRT cassette between pVI and hexon. This was recombined into various pAd6 plasmids to generate Ad657 variants without cysteine in HVR5, but with restriction sites allowing peptide insertions into HVR5, the variant referred to as Ad657-HVR5-XA comprises the hexon having the amino acid sequence of SEQ ID NO:53.

[0189] With respect to variants of Ad657, the Ad657 HVR1-XA sequence was modified by insertion of a biotin acceptor peptide into HVR1. This was recombined into various pAd6 plasmids to generate Ad657 variants a BAP in HVR1, the variant referred to as Ad657-HVR1-PSTCD comprises the hexon having the amino acid sequence of SEQ ID NO:54.

[0190] The insertion of a biotin acceptor peptide detargets the virus variants from the liver, allows the virus to be retargeted with avidin or streptavidin and biotinylated ligands, and allows the virus to be purified on monomeric avidin or streptavidin columns.

[0191] With respect to variants of Ad657, the Ad657 HVR1-XA sequence was modified by insertion of a biotin acceptor peptide into HVR1. This was recombined into various pAd6 plasmids to generate Ad657 variants a BAP in HVR1, the variant referred to as Ad657-HVR5-PSTCD comprises the hexon having the amino acid sequence of SEQ ID NO:55.

[0192] With respect to variants of Ad657, the Ad657 HVR5-XA sequence was modified by insertion of a synthetic V1/V2 loop from HIV envelope into HVR5, the variant referred to as Ad657-HVR5-V1/V2 comprises the hexon having the amino acid sequence of SEQ ID NO:56.

[0193] The insertion of a synthetic V1/V2 loop from HIV envelope allows display of this antigen to serve as a vaccine as well as retargeting by binding to proteins that interact with HIV envelope.

[0194] With respect to variants of Ad657, the Ad657 HVR5-XA sequence was modified by insertion of synthetic peptides from human papilloma virus into HVR5, the variant referred to as Ad657-HVR5-HPV comprises the hexon having the amino acid sequence of SEQ ID NO:57.

[0195] The insertion of synthetic peptides from human papilloma virus allows display of HPV peptides as antigens for vaccine purposes as well as for retargeting by binding to proteins that interact with HPV peptides.

[0196] In another aspect of the invention, chimeric Ads were generated which have an Ad6 HVR1 and Ad57 HVRs 2-7, the chimera, referred to as Ad6/57 HVR chimera, comprises the hexon having the amino acid sequence of SEQ ID NO:58.

[0197] In yet another aspect of the invention, chimeric Ads were generated which have Ad6 HVR1 and 7 and Ad57 HVRs 2-6, the chimera, referred to as Ad6/57/6 HVR chimera, comprises the hexon having the amino acid sequence of SEQ ID NO:59.

Example 2. Retargeted and Detargeted Recombinant Adenovirus for Gene Delivery

[0198] Adenoviruses are robust vectors for gene delivery and gene-based immunization. The archetype adenovirus used for the vast majority of these application has been human species C adenovirus serotype 5 (HAdV-C5 or Ad5). In vitro, Ad5 binds and enters cells through the combined interactions of its fiber and penton base proteins with cell surface receptors. The trimeric fiber binds the coxsackie-adenovirus receptor (CAR), and cells that lack CAR are relatively resistant to infection unless they also express .alpha..sub.v integrins that can be bound by an RGD motif on the penton base.

[0199] In vivo, these interactions are still utilized, but their importance varies by injection route. If injected directly into a solid tissue or tumor, CAR and integrin interactions dominate. If injected intravenously (IV), these interactions become secondary due to the effects of Ad5 binding to vitamin-K-dependent blood clotting factors. Blood factor X (FX) binds with subnanomolar affinity to the hexons of Ad5 and, consequently, enables Ad5 to efficiently transduce liver hepatocytes after IV injection. In the absence of FX, liver transduction is drastically reduced.

[0200] Adenoviral vectors are somewhat unique in their ability to carry very large cDNA sequences of up to 36 kilobase pairs (kbp) when compared to other vectors like adeno-associated virus (AAV) vectors with only 4.5 kb of DNA sequence. This payload capacity justified early exploration of Ad vectors for muscle gene therapy when delivering very large transgenes like the 14 kbp dystrophin cDNA. IV administration in newborn mice can mediate muscle gene delivery, but this ability is lost in adult mice. The decreased transfection with age is due in part to the very large size of Ad virions (i.e. 100 nm) as well as the loss of CAR receptor on muscle cells with aging. The intramuscular (IM) route is by far the most popular route for gene-based vaccines when using Ad5 and other serotypes despite the fact that CAR is absent on skeletal muscle cells.

[0201] Therefore, Ad5 and other Ad serotype transduction of muscle can be adequate for gene therapy or gene-based vaccination. However, the absence of the virus' primary receptor in the muscle reduces the efficacy of the virus and requires more vector to be delivered to achieve desired effects.

Construction of Peptide-Modified Hexons in Adenovirus

[0202] 12 amino acid (12-mer) peptides on C2C12 mouse muscle cells were selected from a random peptide library displayed between the H and .beta. sheets from the knob region of Ad5 (FIG. 1A). Peptides 12.51 (TARGEHKEEELI; SEQ ID NO:1) and 12.52 (LRQTGAASAVWG; SEQ ID NO:2) were selected against myoblasts with pre-clearing against non-target cells to obtain peptides which would be specific for binding muscle cells. In most cases, small peptides have relatively low affinity. It was therefore reasoned that displaying these muscle-selected peptides on the 720 copies of the Ad5 hexon might enable better muscle gene delivery. This insertion site might also have the benefit of inactivating FX binding to the hexon to "detarget" the vector from the liver if any vector leaked into the blood after IM injection.

[0203] Inserting these muscle-selected peptides between two other .beta. sheets which also constrains a hypervariable loop on the virus, the ability of the modified Ads to modulate tropism was evaluated. Peptides 12.51 and 12.52 were introduced into the hypervariable region (HVR) 5 loop constrained by the .beta.7 and .beta.8 sheets in Ad5 hexon. The in vivo ability of these viruses to transduce tissues after intravenous and intramuscular injections in mice and in hamsters was evaluated.

Adenoviruses

[0204] E1 -deleted Ad5-GL (RD-Ad5-GL) expresses a green fluorescent protein-luciferase (GFP-Luciferase, GL) fusion protein as described elsewhere (see., e.g., Crosby et al., 2002 J. Virol., 76:6893-6899; Khare et al, 2011 Mol. Ther., 19:1254-1262; and Khare et al., 2012 J. Virol., 86:2293-2301). Muscle selected peptides 12.51 and 12.52 were inserted in place of HVR5 on Ad5 hexon between its .beta.7 and .beta.8 sheets which structurally similar to the H and 1.beta. sheets from the knob region of Ad5 (FIG. 1A) according to methods known to those skilled in the art. The peptides with a flexibility leader replaced the entire HVR5 loop in Ad5 (FIG. 1B). These modified hexon sequences were introduced into replication-defective Ad5 expressing a green fluorescent protein-luciferase (RD-Ad5-GL) fusion protein by red recombination in bacteria (see, e.g., Campos et al., 2004 Hum. Gene Ther., 15:1125-1130). Peptide modified Ads were constructed by insertion of annealed oligonucleotides encoding the peptides 12.51 and 12.52 (FIG. 1B) into the plasmid pHVR5 display (FIG. 1A bottom) to yield recombinant Ads, Ad5-HVR5-12.51 and Ad5-HVR5-12.52.

[0205] The resulting plasmids were digested and used for red recombination into pAd5-GL. These vectors were rescued in 293 cells, purified on two consecutive CsCl gradients and were desalted on Econopac 10-DG chromatography columns (Bio-Rad) into 50 mM Iris pH 8 with 0.5 M sucrose and stored at -80.degree. C.

In Vitro Virus Testing

[0206] 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.

[0207] C2C12 muscle cells were plated in 6 well plates (Corning) the day before infection. Viruses were used to infect cells in DMEM with 5% FBS. The cells were incubated for 2 days prior to observation under green fluorescence.

[0208] Animal experiments were performed with approval by the Mayo Clinic Institutional Animal Care and Use Committee under the provisions of the Animal Welfare Act, PHS Animal Welfare Policy and principles of the NIH Guide for the Care and Use of Laboratory Animals. Female CD-1 mice (Charles River) were anesthetized and injected intramuscularly (IM) or intravenously (IV) with 10.sup.10 vp of the indicated viruses at indicated times. The animals were anesthetized and injected with 3 mg of d-luciferin (Molecular Imaging Products) and were imaged on a Xenogen imaging system. At later times, the animals were anesthetized and blood was collected in serum separators for ELISA.

[0209] ELISA was performed as follows. Immulon 4 HBX plates (Thermo) were incubated with 100 ng per well of GFP protein in 1.times. phosphate-buffered saline (PBS) at 4.degree. C. overnight, washed, and blocked with 5% milk in TRIS-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 2 hours. 1:100,000 to 1:1,1000 dilutions of each serum sample were prepared in blocking buffer. Wells were washed and 100 .mu.L of each were added to GFP-coated plates in triplicate and incubated for 3 hours at room temperature. Wells were washed and a 1/10,000 dilution of goat anti-mouse-HRP secondary antibody (Pierce Chemical) was added to each well. Plates were incubated for 2 hours at room temperature, washed, and 50 .mu.L of 1 Step Ultra TMB ELISA (Thermo Fisher Scientific Inc.) was added for HRP detection followed by 50 .mu.L of 2 M H.sub.2SO.sub.4. Absorbance at 450 nm was determined with a Beckman Coulter DTX 880.

[0210] Statistical analyses were performed with Prism (Graphpad). Statistical significance was calculated by one-way ANOVA followed by Tukey's HSD

In Vitro Transduction in Mouse C2C12 Muscle Cells

[0211] RD-Ad5-GL, RD-Ad5-GL-HVR5-12.51, and RD-Ad5-HVR5-12.52 were used to infect mouse C2C12 myoblast cells at varied multiplicities of infection (MOI) in terms of virus particles (vp)/cell. When green fluorescence from the GFP fusion protein was observed by fluorescence microscopy, both of the peptide-modified vectors mediated significantly better transduction than RD-Ad5-GL (FIG. 2A). When luciferase activity was measured, significant increases were observed in RD-Ad5-GL-12.51 and 12.52 infected cells (FIG. 2B). When one of the peptides, 12.51, was inserted back into the knob region of Ad5, the peptide increased in vitro transduction 14-fold on C2C12 myoblasts.

In Vivo Transduction after Intramuscular Injection in Mice

[0212] 10.sup.9 vp of Ad5-GL, Ad5-GL-HVR5-12.51, and Ad5-GL-HVR5-12.52 were injected by the IM route into both quadriceps muscles in mice and luciferase imaging was performed at varied times (FIG. 3A top). Ad5-GL-HVR5-12.51 produced 2 to 3-fold higher luciferase activity than Ad5-GL at all the time points (p<0.05 at day 1 by one-way ANOVA) (FIG. 3B left). Ad5-GL-HVR5-12.52 activity in the muscle was lower than both Ad5-GL and HVR5-12.51 in contrast to its stronger activity in vitro.

In Vivo Transduction after Intravenous Injection in Mice

[0213] 3.times.10.sup.10 vp of Ad5-GL, Ad5-GL-HVR5-12.51, and Ad5-GL-HVR5-12.52 were injected by the IV route in mice and luciferase imaging was performed (FIG. 3A bottom). In contrast to the results in the muscle, only unmodified Ad5-GL mediated strong liver transduction. Liver transduction by Ad5-GL was 60-fold higher than both Ad5-GL-HVR5-12.51 and Ad5-GL-HVR5-12.52 (p<0.001 at day 1 by one-way ANOVA) (FIG. 3B right) demonstrating that the recombinant Ads target muscle tissue while decreasing off target infection in the liver.

In Vivo Transduction after Intramuscular Injection in Hamsters

[0214] To test if the 12.51 modified vector works in other species than mice, 10.sup.10 vp of Ad5-GL and Ad5-GL-HVR5-12.51 were injected IM into both quadriceps of larger Syrian hamsters and luciferase imaging was performed 24 hours later (FIG. 4). In this case, Ad5-GL-HVR5-12.51 mediated 7-fold higher luciferase activity than Ad5-GL (p<0.01 at day 1 by one-way ANOVA).

Gene-Based Immunization after Intramuscular Injection in Mice

[0215] 16 weeks after IM injection, sera were collected the mice treated as described above and as shown in FIG. 3 and analyzed in serial dilutions by ELISA for antibodies against transgene-encoded GFP (FIG. 5). All Ad-injected mice generated significant anti-GFP antibodies when compared to the PBS group at sera dilutions of 1:10,000 to 1:1000 (p<0.0001 by one-way ANOVA). However, Ad5-GL-HVR-12.5 produced higher antibodies than either Ad5 or Ad5-HVR5-12.52. At 1:1000 to 1:10,000 dilutions of sera, Ad5-GL-HVR-12.51 was significantly higher than Ad5-GL (p<0.01 to 0.0001 by one-way ANOVA). At a 1:1000 dilution of sera, 12.51 was significantly higher than 12.52 (p<0,05). Ad5-GL-12.52 was significantly higher than Ad5-GL at 1:1000 and 1:10,000 dilutions of sera (p<0.05 to 0.001).

[0216] This example demonstrates that peptides selected in a compatible structural context on phage libraries can be translated into the Ad hexon protein. For example, for the 12.51 peptide, this insertion site increases muscle transduction while decreasing off target infection in the liver. Thus, such a recombinant Ad which targets muscle tissue may be used as a vector for gene-based muscle vaccination or for gene therapy application/delivery to the muscle.

[0217] A further aspect of the invention relates to recombinant and/or chimeric Ads which comprise other cell targeting peptides inserted into Ad657 HVRs and into Ad6 and C68 HI loops are described in Table 1.

TABLE-US-00001 TABLE 1 Other Cell Targeting Peptides Inserted into Ad657 HVRs and into Ad6 and C68 HI Loops VSV cell binding peptide GTWLNPGFPPQSCGYATVT (SEQ ID NO: 4) RGD-4C integrin binding peptide CDCRGDCFC (SEQ ID NO: 5) 12.51 phage-selected peptide TARGEHKEEELI (SEQ ID NO: 1) 12.52 phage-selected peptide LRQTGAASAVWG (SEQ ID NO: 2) 12.53 phage-selected peptide ARRADTQWRGLE (SEQ ID NO: 3) alpha4 binding peptide NMSLDVNRKA (SEQ ID NO: 6) L10.1 lung binding peptide WTMGLDQLRDSSWAHGGFSA (SEQ ID NO: 9) L10.2 lung binding peptide RSVSGTEWVPMNEQHRGAIW (SEQ ID NO: 10) L10.5 lung binding peptide TELRTHTSKELTIRTAASSD (SEQ ID NO: 11) S5.1 muscle binding peptide DRAIGWQDKLYKLPLGSIHN (SEQ ID NO: 12) DU9C.1 prostate cancer binding peptide MGSWEKAALWNRVSASSGGA (SEQ ID NO: 13) DU9C.2 prostate cancer binding peptide MAMGGKPERPADSDNVQVRG (SEQ ID NO: 14) DU9A.7 prostate cancer binding peptide MASRGDAGEGSTQSNTNVPS (SEQ ID NO: 15) XS.1 dendritic cell binding peptide GPEDTSRAPENQQKTFHRRW (SEQ ID NO: 17) REDV endothelial cell binding peptide REDVY (SEQ ID NO: 46) SKBR5C1 breast cancer cell binding peptide GQIPITEPELCCVPWTEAFY (SEQ ID NO: 20) 231R10.1 breast cancer cell binding peptide PQPPNSTAHPNPHKAPPNTT (SEQ ID NO: 21) HepaCD8 hepatocellular cancer binding peptide VRWFPGGEWGVTHPESLPPP (SEQ ID NO: 22) HI Met 231 3-4 breast cancer binding peptide ISLSSHRATWVV (SEQ ID NO: 47) B Cell Cancer Selected Peptides: 1-1 GVSKRGLQCHDFISCSGVPW (SEQ ID NO: 29) 1-2 NQSIPKVAGDSKWCWWCAL (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 MGRTVQ.SGDGTPAQTQPSVN (SEQ ID NO : 40) 4*-5 MARTVTANVPGMGEGMVVVP (SEQ ID NO: 41) Small BAP biotin acceptor peptide GLNDIFEAQKIEWH (SEQ ID NO: 24) calmodulin binding peptide CAAARWKKAFIAVSAANRFKKIS (SEQ ID NO: 25)

[0218] DNA encoding the indicated peptides and its complementary DNA was synthesized flanked by cohesive ends for ligation into Ad plasmids, for example, XA hexon plasmids or pAd6-NdePfl fiber shuttle plasmids. These annealed oligonucleotides were ligated into HVRs or the HI loop of Ads. These plasmids were used to recombine into various Ad backbone plasmids.

[0219] Some peptides serve to target novel receptors on cells. Others like the small BAP can be used for avidin targeting and purification if the virus is grown in cells expressing bacterial biotin ligase BirA. The calmodulin peptide allows the virus to bind to calmodulin or calmodulin-fusion proteins for retargeting or for virus purification.

[0220] Thus, such a recombinant Ads which targets specific tissues/cell receptors may be used as a vector for gene-based vaccination or for gene therapy application in the targeted cells and/or tissues.

Example 3. Insertion of Individual HVRs from Different Ad Serotypes with the Insertion of Cell Targeting/Detargeting Peptides or Novel Amino Acids

[0221] Hexon shuttle plasmid maps (FIG. 34) show the combination of the insertion of individual HVRs from different Ad serotypes with the insertion of cell targeting/detargeting peptides or novel amino acids such as cysteine into the hexon for targeted chemical modification and shielding.

[0222] In certain embodiments, cell binding peptides 12.51, VSV, RGD (see Table 1) are inserted into HVR 1 or HVR 5, which embodiments serve as examples of inserting these and other peptides in any of the HVRs of an Ad (FIG. 34). Another example shows insertion of a biotin acceptor peptide (BAP) is inserted into these HVRs allowing for vector retargeting with avidin or streptavidin and biotinylated ligands or with avidin- or streptavidin fusion proteins. BAP insertion also allows the viruses to be purified on monomeric avidin or streptavidin columns for vector production. Likewise, Ad57-HVR1-XXA and XA shows the example of inserting a cysteine into this site to allow targeted chemical modification with maleimide or other cysteine-reactive agents (FIG. 34).

[0223] These embodiments have been applied also in the context of Ads which combine different HVRs from different Ads (i.e., shuffling HVRs). For example, HVR1 of Ad6 with HVRs 2-7 of Ad57 or HVR1 and 7 of Ad6 with HVRs 2-6 of Ad57. In a further embodiment, a 6/56/6 virus has HVRs 1 and 7 from Ad6 and HVRs 2-6 from Ad657.

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

[0224] FIG. 35 is a depiction of Ad variants showing the combination of insertion of individual HVRs from different Ad serotypes with the insertion of novel amino acids such as cysteine into the hexon for targeted chemical modification and shielding.

[0225] Comparison of the effects of non-targeted chemical conjugation to targeted chemical conjugation on shielding and function of cysteine-modified hexon-modified Ad657-HVR1C (FIG. 36). This example demonstrates the ability to target polymer and other chemical modifications to cysteines inserted into an Ad hexon. Untargeted PEG inactivates virus infection whereas cysteine-targeting PEGylation retains virus functions.

[0226] In an aspect of the invention, the use of polymers or inserted peptides/proteins to detarget, retarget, and shield from antibodies, proteins, cells is contemplated. FIG. 54 depicts sites of Ad HVRs which may be modified, for example, by PEGylation or "BAPylation".

[0227] In an embodiment, the different Ad serotypes and/or variants comprise polymer shielding to allow multi dosing of Ad6 and Ad657 variants. An exemplary therapeutic cycle where Ad6 and Ad657 can be used for multiple rounds of treatment by serotype-switching in combination with covalent polymer conjugation is shown (FIG. 41B).

[0228] Ad657-HVR1C expressing GFPLuciferase was produced from a cells and purified on CsCl gradients. The virus was covalently modified with 5 kDa polyethylene glycol (PEG). The virus was treated with either NHS-PEG that reacts randomly with aminesflysines on viral proteins or with maleimide PEG that reacts specifically with cysteine that was inserted into HVR1 using the XXA shuttle plasmid. These unmodified or modified viruses were then purified by a final CsCl spin followed by desalting. The indicated virus were separated on SDS-PAGE gels, stained with SyproRuby, and visualized by imaging (FIG. 36A). This shows that NHS-PEGylation randomly modifies many viral proteins as demonstrated by increases in the apparent mass of the proteins (indicated by arrows). In contrast, targeted maleimide PEG reaction with the cysteine in HVR1 modifies only hexon and does not damage other viral capsomer proteins. The effects of PEGylation on virus function was evaluated.

[0229] The indicated viruses were incubated with A549 cells and their ability to infect the cells was measured by luciferase assay. This shows that random NHS-PEGylation reduces virus activity more than 90% whereas maleimide-PEG does not (FIG. 36B).

[0230] Immune competent Syrian hamsters were engrafted with subcutaneous HaK kidney cancer tumors. When these reached 200 .mu.l volume, they were injected a single time by the intravenous route with the indicated Ad6 viruses constructed with and without E3 (DE3) and with or without random NHS-PEGylation. Tumor sizes were measured over time. The data shows that deleting all E3 genes in the oncolytic virus Ad6-deltaE3-Luc makes the virus less effective at reducing tumor volume than the oncolytic parent virus, Ad6-Luc. The data also shows that Ad6 can be PEGylated and retain efficacy (see Ad6-Luc vs. Ad6-Luc-20 kDa PEG) (FIG. 58).

[0231] Targeted chemical conjugation of cysteine-modified hexon-modified Ads, for example Ad657-HVR5C. Ad657-HVR5C expressing GFPLuciferase was produced from cells and purified on CsCl gradients. The virus was covalently modified with maleimide-IR800 near-infrared fluorophore, maleimide-biotin, or 5 kDa maleimide-PEG that reacts specifically with cysteine that was inserted into HVR5 using its XXA shuttle plasmid. The indicated Ads and modified Ads were separated on SDS-PAGE gels, stained with SyproRuby, and visualized by imaging (FIG. 37A). SDS-PAGE of viral proteins followed by near infrared imaging demonstrates that the HVR-C can be tagged with an imaging agent (FIG. 37B). The effects of PEGylation on in vivo Ad virus function was demonstrated by injecting PEGylated Ad virus intraperitoneally. The ability to infect cells in tumor bearing mice is demonstrated by detectable luciferase activity by imaging. FIG. 37C demonstrates the ability to target polymer and other chemical modifications to cysteines inserted into the Ad657 hexon region. What is more, it is demonstrated that PEGylation de-targets adenovirus to liver in vivo (FIG. 50).

Example 5. Expression of Human Granulocyte-Macrophage Colony Stimulating Factor (GMCSF) by Ad657

[0232] Ad657 carrying the cDNA for human GMCSF was used to infect A549 cells and varied amounts of the supernatant were added to GMCSF growth-dependent TF-1 cells. Increased cell number indicates expression of the functional human cytokine (FIG. 27). The data demonstrates that recombinant Ads may be utilized for expression of heterologous proteins.

Example 6. Oncolytic Adenovirus Ad657 for Systemic Virotherapy Against Cancer Cells

[0233] An alignment of selected full Ad genomes produces a phylogenetic tree that clusters Ad57 with other species C viruses with most homology with Ad6 is shown in FIG. 6.

[0234] Ad57 appears nearly identical to Ad6 with sequence divergence in hexon hypervariable regions (HVRs) and in E3 immune evasion genes (FIG. 7). Other exposed viral capsid proteins including fiber, penton base, IIIa, and IX are virtually identical between Ad6 and Ad57. The neutralization data are consistent with the fact that most adenovirus-neutralizing antibodies target the HVRs on Ads. The low cross-reactivity between Ad6 antisera and Ad57 is thought to be due to antibodies that may target their common fiber protein (Lukashev et al., 2008 J Gen Virol. 89:380-388).

[0235] In this example, the utility of Ad657 as an oncolytic against human prostate cancer is demonstrated. The Ad6 HVRs were replaced with those from Ad57 to generate a chimeric species C oncolytic virus called Ad657. Ad657 and Ad6 were tested as systemic oncolytic therapies by single i.v. injection in nude mice bearing human prostate cancer tumors. The liver and tumor tropism of this virus were evaluated in mouse models of prostate cancer as follows.

[0236] DU145 human prostate carcinoma cells were purchased from American Type Culture Collection (ATCC; Manassas, Va., USA) and verified to be specific pathogen free by IMPACT testing by RADIL. 293 cells were obtained from Microbix, Toronto, Ontario, Canada. Cells were maintained in DMEM with 10% FBS (Invitrogen, Grand Island, N.Y., USA).

[0237] The genome of Ad6, Tonsil 99 strain (ATCC VR-1083), was cloned as described elsewhere (see, e.g., Weaver et al., 2013 PLoS One. 8:e73313). A cassette corresponding to the Ad57 hexon between a natural ApaI and SacI sites was synthesized by Genscript. This fragment was cloned into the shuttle plasmid pUC57-Ad6 Hexon-FZF containing the Ad6 pVI and hexon genes with a FRT-Zeocin.RTM. resistance gene-FRT 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 ApaI-SacI fragment was replaced with the Ad57 fragment generating the plasmid pUC57-Ad6/57 Hexon-FZF. This was recombined into the Ad6 genome by red recombination (Campos et al., 2004 Hum Gene Ther. 15:1125-1130). FIG. 59 shows a plasmid map of Ad657 with E3 deletion. Viruses were rescued by transfection into 293 cells and produced from a 10 plate CellStack (Corning Life Sciences, Lowell, Mass., USA). Viruses expressing a green fluorescent protein-luciferase (GFP-Luc) fusion protein have a CMV-GFP-Luc expression cassette inserted between the Ad fiber and E4 and an E3 deletion to make space for this insertion. Viruses were purified on two CsCl gradients, and viral particle (vp) numbers were calculated by OD260.

[0238] To examine in vitro oncolytic activity, cells were treated at the indicated multiplicities of infection (MOI) in terms of vp/cell in DMEM with 5% FBS and antibiotic-antimycotic (Invitrogen, Grand Island, N.Y., USA). Five days later, media was removed and the cells were treated with crystal violet (0.05% crystal violet, 3.7% formaldehyde, in phosphate-buffered saline; Invitrogen, Grand Island, N.Y., USA) for 10 minutes. The cells were washed twice with PBS and then incubated overnight at 37.degree. C. in 0.1% sodium dodecyl sulfate in PBS to solubilize the crystal violet. Crystal violet absorbance was measured at OD595 on a Beckman Coulter DTX 880 plate reader. Cell viability (%) was calculated by dividing the OD of the samples by the mean OD of untreated control cells on the same 96-well plate and multiplying this number by 100.

[0239] Animals were housed in the Mayo Clinic Animal Facility under Association for Assessment and Accreditation of Laboratory Animal Care guidelines. The studies were approved by the Mayo Clinic Animal Use and Care Committee under the provisions of the Animal Welfare Act, PHS Animal Welfare Policy. Subcutaneous tumors were initiated in 4-week-old nude mice (Harlan Sprague Dawley, Indianapolis, Ind., USA) by injecting subcutaneously (s.c.) with 1.times.10.sup.7 DU145 cells in 100 .mu.L of DMEM/50% Matrigel (BD Biosciences, San Jose, Calif., USA). Tumor volumes were calculated using the equation width.sup.2.times.length.times.1/2. When tumors reached .about.200 .mu.L in volume, mice were distributed into different groups and were treated by a single i.v. injection tail vein. Animals were euthanized when the tumor volume reached 2000 .mu.L or if animals were moribund, in distress, or if the skin ruptured over the tumor.

[0240] For blood alanine aminotransferase (ALT) measurements, groups of six C57BL/6 mice were injected i.v. with 10.sup.11 vp of Ad5, Ad6, or Ad657 by tail vein and blood was collected 3 days later for ALT measurement using ALT Activity Assay (Sigma-Aldrich, St. Louis, Mo., USA).

[0241] Statistical analysis was performed with Prism (Graphpad) by repeated measures ANOVA or one-way ANOVA followed by Tukey's HSD test. Kaplan-Meier survival curves were plotted and compared by log rank test.

[0242] The capsomer genes of Ad57 are nearly identical to Ad6 with the exception of their hexon HVRs (FIGS. 6 and 7). To generate a chimeric virus of Ad57 and Ad6, a cassette corresponding to the Ad57 hexon HVRs was recombined into the wild-type Ad6 genome (FIG. 8). This virus was rescued and produced in 293 cells and purified on CsCl wadients. Given that the base viral genome is Ad6, these hexon chimeric viruses are referred to as Ad657 (FIG. 59).

[0243] In vitro oncolytic activity was evaluated by infecting LNCap and DU145 cells with 10, 100 or 1000 vp/cell. To compare liver damage by Ad5, Ad6, and Ad657, a high dose of 10.sup.11 vp of each virus were injected by tail vein into immunocompetent C57BL/6 mice. Ad5-injected animals became moribund within 2 days and had to be euthanized (FIG. 10A). Survival for Ad5 and Ad6 was significantly lower when compared with PBS (p=0.0001 and 0.0009, respectively, by log-rank analysis). Survival for Ad657 was also reduced when compared with PBS (p=0.0578). Survival after exposure to Ad6 or Ad657 was significantly better than in Ad5-treated mice (p=0.0001 and 0.0001, respectively). Ad6 and Ad657 survival were not statistically different (p=0.248).

[0244] ALT was measured in the blood 3 days after injection in surviving Ad6 and Ad657 animals. Ad5-treated animals were not tested, since most of the group needed to be sacrificed. This assay showed that Ad6 provoked relatively low levels of liver damage in terms of liver ALT enzyme release in the blood (FIG. 10B). Both Ad6 and Ad657 groups had low, but significant, ALT levels when compared with PBS-treated mice (p<0.001 by one-way ANOVA with Tukey's multiple comparison test for both viruses). Ad657 had lower ALT levels than Ad6 (p<0.001 by ANOVA). This is consistent with higher levels of Ad6 infection in the liver than Ad657 after i.v. injection of luciferase expressing viruses (FIG. 12). One Ad657 animal was lost following bleeding on day 3. By 6 days, most of the Ad6 animals became moribund (FIG. 10A). In contrast, 50% of Ad657 animals survived beyond 2 weeks of the treatment.

[0245] To compare the oncolytic activity of Ad6 and Ad657 against human DU145 prostate tumors, nude mice were engrafted s.c. with DU145 cells. Animals were distributed into groups with similar tumor sizes averaging 200 .mu.L and groups of nine mice were treated a single time by the i.v. route with a dose of 3.times.10.sup.10 vp of Ad6 or Ad657 (FIG. 11).

[0246] This single i.v. injection of Ad6 and Ad657 reduced tumor sizes when compared with PBS-injected control animals. Tumors were significantly smaller in the Ad6 group within 7 days when compared with the PBS group (p<0.05 by two-way ANOVA with Tukey's multiple comparison test). Tumors in the Ad657 group were significantly different from those in the PBS group by day 14 (p<0.01 by ANOVA). Both Ad6 and Ad657 maintained significant differences with PBS through 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 sacrificed since later comparison would be skewed due to the change in animal numbers. Tumor sizes in the Ad6 and Ad657 groups were not significantly different until day 38, when Ad657 had a significantly higher tumor volume (p<0.05) by two-way ANOVA (FIG. 11A). This difference between Ad6 and Ad657 tumor sizes persisted until day 52 (p=0.04 by T-test), and then the tumors were not significantly different after this time.

[0247] When survival due to all causes was assessed, both Ad6 and Ad657 significantly extended survival when compared with PBS-treated animals (FIG. 11B, p<0.01 and 0.05, respectively, by log-rank analysis). Ad6 survival due to all causes was significantly better than Ad657 (p<0.05). However, this was an artifact of survival attributed to all because three of the Ad657 animals had to be sacrificed per Institutional Animal Care and Use Committee (IACUC) guidelines due to the formation of ulcers on the skin over the tumor rather than due to excess tumor size. In some cases, ulceration is actually associated with effective tumor control. Like Ad6, Ad657 expressing GFP-luciferase produced significant luciferase activity in distant DU145 subcutaneous tumors after a single i.v. injection (FIG. 12 and FIG. 13). This suggests that both Ad6 and Ad657 can mediate oncolytic effects in prostate tumors after a single systemic treatment.

[0248] This example demonstrates that Ad657 may be used as a local or systemic oncolytic virotherapy for prostate cancers. These data also demonstrate surprising effects of serotype-switching with oncolytic species C Ads.

[0249] The oncolytic activity of Ads was evaluated in tumor cells and/or cancerous tumors. Ad6 single IV injection vs. A549 lung tumor cells was evaluated (FIG. 28); Ad6 single IV or intratumoral (IT) injection vs. Panc1 pancreatic tumors was evaluated (FIG. 29), and Ad6 single IV injection vs. kidney cancer in immune competent hamsters was evaluated (FIG. 30).

[0250] FIG. 38 shows luciferase imaging of nude mice. A) 1, 4, 7, 14, 28, and 42 days after single I.V. injection of Ad6 treatment vs. distant DU145 prostate tumors. B) 3, 7, and 19 days after I.V. injection of replicating Ad5-GFPLUC into mice bearing LNCaP prostate tumors.

[0251] It may be concluded that Ad6 gets to distant target cells after IV injection.

[0252] In another embodiment, Ads expressing luciferase with and without peptide library generated peptides 12.51 and 12.52 inserted in HVR5 of hexon were incubated on indicated cell lines, B16 melanoma and A549 lung carcinoma cells, with the indicated numbers of virus particles (vp) and luciferase activity was measured. Improved infection of cancer cells by Ads bearing peptide-modified hexons is demonstrated (FIG. 31).

Improved Infection of Cancer Cells by Ads Bearing Peptide-Modified Hexons

[0253] Ads expressing luciferase with and without peptide library generated peptides 12.51 and 12.52 inserted in HVR5 of hexon were incubated on indicated hepatocellular carcinoma cell lines with 10.sup.4 vp of each virus and luciferase activity was measured. Improved infection of cancer cells by Ads bearing peptide-modified hexons is demonstrated (FIG. 32).

Example 7. Divergent HIV-1 Directed Immune Responses Generated by Systemic and Mucosal Immunization with Replicating Single-Cycle Adenoviruses in Rhesus Macaques

[0254] Most gene-based adenovirus vaccines are replication-defective Ad (RD-Ad) vectors that have their E1 gene deleted to prevent them from replicating and causing Ad infections. Helper-dependent adenoviruses (HD-Ads) have all Ad genes deleted and are also replication-defective. An E1-deleted Ad vaccine can infect a cell, deliver its one copy of an antigen gene, and express a single copy (e.g., "1.times.") of this antigen. They are safe, but do not replicate transgenes or their expression.

[0255] In contrast, an E1+ replication-competent Ad (RC-Ad) vaccine can infect the same cell, replicate the antigen gene DNA 10,000-fold, produce substantially more antigen, and provoke stronger immune responses than E1-deleted vectors. While RC-Ad is more potent than RD-Ad, replication-competent Ads can run the real risk of causing frank adenovirus infections in humans.

[0256] To take advantage of transgene DNA replication, but avoid the risk of adenovirus infections, single-cycle Ad (SC-Ad) vectors with a deletion of a gene for a key viral late protein, pIIIa, were developed (Crosby et al., 2014. Virology 462-463:158-165; Crosby et al., 2015 J Virol 89:669-675; Anguiano-Zarate et al., 2018 J Infectious Dis 218:1883-1889; and Crosby et al., 2017 Genes (Basel) 8:E79). SC-Ads retain their E1 genes to allow it to replicate its genome, but the absence of pIIIa blocks the production of infectious progeny viruses. SC-Ads replicate their genomes and transgenes as well as RC-Ad (up to 10,000-fold; Crosby et al., 2014. Virology 462-463:158-165). RC- and SC-Ad produce more transgene protein than RD-Ad vectors (Crosby et al., 2014. Virology 462-463:158-165). SC-Ads generate more robust and more persistent immune responses than either RD-Ad or RC-Ads (Crosby et al., 2015 J Virol 89:669-675). In head-to-head comparisons, SC-Ad produces significantly higher antibodies and better protection against influenza virus (Crosby et al., 2017 J Virol 91:e00720-16).

[0257] In this study, rhesus macaques were immunized with SC-Ads expressing clade B envelope sequences that were obtained from an HIV-1 patient before and after their antibody response underwent an expansion neutralization breadth. Macaques were immunized by a single systemic IM immunization or by single mucosal intranasal (IN) immunization. The animals were then boosted by the same or alternative routes with SC-6 Ad followed by protein boost. This example describes how these various SC-Ad immunization strategies affected the generation of HIV binding, antibody-dependent cellular cytotoxicity (ADCC) and neutralizing antibodies as well as their effects on cellular immune responses including T follicular helper (pTfh) cells in blood and lymph nodes.

Single-Cycle Adenovirus Expressing HIV-1 Envelope Protein Gp140

[0258] Clade B gp160 envelope sequences that arose before (G4) and immediately preceding a peak in the expansion of antibody neutralization breadth (F8) from HIV patient VC10014 (Malherbe et al., 2014 J Virol 88:12949-12967) were used as immunogens. Motif-optimized G4 and F8 gp160 sequences were recombined into SC-Ads 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 Virotherapy 7:43-51). A control SC-Ad expressing Ebola glycoprotein was also used. Viruses were rescued and purified as described elsewhere (see, e.g., Crosby et al., 2014. Virology 462-463:158-165; Crosby et al., 2015 J Virol 89:669-675; and Anguiano-Zarate et al., 2018 J Infectious Dis 218:1883-1889).

[0259] The envelope gene F8 cloned from an HIV Clade B-infected subject used in the SC-Ad vector was motif-optimized and modified by site-directed mutagenesis to express uncleaved, trimeric gp140. Details of expression, purification, and antigenic characterization have been described elsewhere (see, e.g., Malherbe et al., 2014 J Virol 88:12949-12967).

[0260] Female adult rhesus macaques (Macaca mulatta) of Indian origin were maintained at the Michael Keeling Center for Comparative Medicine and Research at the University of Texas M D Anderson Cancer Center, Bastrop Tex. in the specific pathogen-free breeding colony. All animal handling was carried out in accordance with the policies and procedures of the Mayo Clinic and the University of Texas M D Anderson Cancer Center, the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals.

[0261] Macaques were anesthetized with ketamine and immunized by the intranasal (IN) or intramuscular (IM) route with 2.times.10.sup.10 virus particles (vp) of the indicated SC-Ad vaccine. Animals were boosted with 50 .mu.g of purified, trimeric recombinant F8 gp140 combined with Adjuplex.TM. adjuvant by the IM route 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).

[0262] Peripheral venous blood samples were collected in EDTA. Before isolation of peripheral blood mononuclear cells (PBMC), plasma was separated and stored immediately at -80.degree. C. PBMCs were prepared from the blood on Ficoll-Hypaque density-gradients. Saliva and vaginal swabs collected with Wek-Cel Spears in 1 ml of PBS containing protease inhibitors, vortexed, and supernatants were collected after a 2,000 r.p.m. centrifugation as described elsewhere (see, e.g., Kozlowski et al., 1997 Infect Immun 65:1387-1394). Samples were kept frozen at -80 C until further use.

ELISPOT Assay for Detecting Antigen-Specific IFN-.gamma. Producing Cells

[0263] Freshly-isolated PBMCs were stimulated with either F8 gp140 protein (1 .mu.g/mL) or heat inactivated Ado (7.0.times.10.sup.8 vp/well) to determine the numbers of IFN-.gamma.-producing cells by the Enzyme Linked Immuno Spot (ELISPOT) assay using the methodology described elsewhere (see, e.g., Nehete et al., 2017 Comp Med 67:67-78: Nehete et al., 2013 PLoS One 8:e79836; and Nehete et al., 2017 J Am Assoc Lab Anim Sci 56:509-519). Briefly, aliquots of PBMCs (10.sup.5/well) were seeded in duplicate wells of 96-well plates (polyvinylidene difluoride backed plates, MAIP S 45, Millipore, Bedford, Mass.) pre-coated with the primary IFN-.gamma. antibody and the lymphocytes were stimulated with either Con A, F8 gp140 protein, or heat inactivated Ad6. After incubation for 30-36 hours at 37.degree. C., the cells were removed and the wells were thoroughly washed with PBS and developed as per protocol provided by the manufacturer. Results are expressed as IFN-.gamma. spot-forming cells (SFCs) per 10.sup.5 PBMCs after subtraction of the duplicate wells with medium only (negative control) and are considered positive if greater than twice the background and greater than 5 SFCs/10.sup.5 PBMCs.

Antibody ELISAs

[0264] HIV-1 envelope binding antibody titers were measured in plasma samples collected at regular intervals against F8 gp140 or SF162 gp140 as described elsewhere (Malherbe et al., 2014 J Virol 88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078).

Neutralization Assay

[0265] HIV neutralization was performed using the TZM-bl neutralization assay as described elsewhere (Malherbe et al., 2014 J Virol 88:12949-12967; and Hessell et al., 2016 J Immunol 196:3064-3078). All values were calculated as compared to virus-only wells.

Antibody Dependent Cellular Cytotoxicity (ADCC)

[0266] CEM.NKR.CCRS.CD4+-Luc, target cells were infected with 50 ng SHIV.sub.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 the infected targets for 20 minutes at room temperature. CD16-KHYG-1 effector cells were added at a 10:1 effector to target ratio and these were incubated for additional 8 hours. The cells were lysed and luciferase activity was measured on the Bio-Tek plate reader.

Flow Cytometry

[0267] Cells collected from rectal and lymph node biopsies were incubated overnight with 0.2 .mu.g gp140 or media alone in the presence of GolgiPlug.TM. (BD Biosciences, San Jose, Calif., USA) for the last 4 hours. After culture, cells were harvested and incubated on ice for 45 minutes with a panel of human antibodies that cross-react with rhesus macaque samples. The panels included the following fluorochrome labeled antibodies: CD8 (Qdot655), a4.beta.7 (PE) and CXCR5 (PE), all obtained front the Nonhuman Primate Reagent Resource; CD69 (BV737, clone FN50) and FoxP3 (PECy5, clone: PCH101) obtained from eBioscience, IL-21 (BV421, clone: 3A3-N2.1), CD45 (BV786, D058-1283) and CD3 (clone SP34-2, PE-Cy7-labeled) all from BD Bioscience (San Jose, Calif.); CD4 (Pacific Blue, clone OKT4) from ThermoFisher Scientific (Waltham, Mass.). Dilutions for antibodies were determined by following manufacturer's recommendations. Dead cells were excluded by using live-dead fixable dead cells stain kit obtained from Invitrogen (Carlsbad, Calif.). Subsequently, the cells were washed twice with PBS containing 2% FBS and 2 mM EDTA and then fixed and permeabilized with FoxP3 Fix/Perm Kit (ThermoFisher Scientific, Waltham, Mass.). The intracellular markers FoxP3 and IL-21 were stained in permeabilization buffer. Both compensation controls (OneComp eBeads, (ThermoFisher Scientific, Waltham, Mass.) and fluorescence minus one (FMO) controls were utilized. All the samples were collected on an LSR Fortessa X-20 analyzer (BD Biosciences, San Jose, Calif.) and were analyzed using FlowJo software (FlowJo, LLC, Ashland, Oreg.). Approximately 2.times.10.sup.5 to 1.times.10.sup.6 events were collected per sample.

SHIV.sub.SF162P3 Rectal Challenge

[0268] SHIV.sub.SF162P3 virus was derived from R157 harvest 3 (3.16.12). This stock had a P27 content of 66 ng/ml, RNA content Log 18 9.35, TCID50 in Indian origin rhesus PBMC: 1288/ml, and TCID50 in TZM-bl cells: 4.1.times.10.sup.4/ml. 1 ml of a 1:300 dilution of the stock was used. This equaled 4.3 TCID50 on rhesus PBMCs and 137 TCID50 on TZM-bl cells. This dose was used for weekly intrarectal (IR) challenge. Plasma samples were analyzed for SHIV viral RNA copy numbers by Leidos Biomedical Research, Inc., Frederick National Laboratory. Animals with RNA copies above 10 were considered to be infected and the number of challenges required to infect that animal were used as events for Kaplan-Meier survival analysis. Once infected, the animal was no longer challenged. Plasma viral loads were monitored periodically by the same method until the end of the study.

SHIV.sub.SF162P3 Viral Load in Tissues

[0269] At the end of study PBMCs and post-mortem tissues were collected. PBMC and gut samples were analyzed for SHIV.sub.SF162P3 viral RNA by qPCR. Prism 7 Graphical software was used for all statistical analyses.

SC-Ad Expressing HIV-1 Gp160

[0270] Clade B envelope protein sequences (G4 gp160) were identified before and immediately preceding a peak in the expansion of antibody neutralization breadth (F8 gp160) from HIV patient VC10014. These gp160 sequences were inserted into SC-Ad6 and SC-Ad657 under the control of the strong cytomegalovirus promoter (FIG. 24A). Ad57 is a species C human Ad that is nearly identical to Ad6 with variation in its hexon hypervariable regions (HVRs) and in its E3 immunevasion genes (FIG. 24B). Most Ad neutralizing antibodies target Ad's hexon HVRs (Pichla-Gollon et al., 2007 J Virol 81:1680-1689; and Sumida et al., 2005 J Immunol 174:7179-7185). Given this, Ad6's HVRs were replaced with those from Ad57 to generate a chimeric species C Ad vector termed Ad657. Both SC-Ad6 and SC-Ad657 retain all Ad genes including E1 and lack functional pIIIA and E3 genes (FIG. 24A). Both SC-Ads can therefore replicate their genomes to amplify gp160 expression, but do not generate progeny Ad viruses. Both viruses were rescued and produced in 293-IIIA cells and purified on CsCl gradients. When used to infect A549 cells, both vectors produced gp160 as determined by Western blotting.

[0271] Different Ad vectors were previously tested in rhesus macaques by the systemic intramuscular (IM) route and by a variety of mucosal routes including oral gavage, oral enteric coated capsules, intranasal (IN), and intravaginal (IVAG). Testing of SC-Ad-G4 by IM, IN, and IVAG routes in small animals revealed that priming by IVAG route generated negligible antibody responses. In contrast, IN immunization in both mice and hamsters generated strong antibody responses. Given these data and the potential difficulty in performing IVAG immunizations in humans, the IN route was selected for the mucosal immunization route in the subsequent macaque studies.

Single Mucosal and Systemic Immunization in Rhesus Macaques

[0272] 2.times.10.sup.10 vp of SC-Ad6-G4 Env was used to vaccinate groups of 8 female rhesus macaques by single IM or IN immunization (FIG. 14). This dose is relatively low, being approximately 7.5-fold lower than recent use of RC-Ad HIV envelope vaccines delivered by mixed IN and IM immunization. A negative control vector group was immunized IN with SC-Ad6 expressing Ebola glycoprotein (gp). Four weeks later, plasma samples were assayed for Env binding antibodies against F8 gp140 (FIG. 14). This showed significantly higher midpoint binding titers in the IM immunized route group after single immunization (p<0.01 by ANOVA). SF162 neutralizing antibody (NAb) titers were also elevated at this time point, but did not reach significance by ANOVA for the individual route groups.

IM vs. IN Boost with SC-Ad6 at Week 4

[0273] It was been reported that anti-adenovirus neutralizing antibodies that are produced by one Ad IM immunization can be avoided by boosting by a different route (Xiang et al., 2003 J Virol 77:10780-10789). To test this route concept to enable the re-use of the same Ad serotype in macaques, each SC-Ad6-primed group was divided into 2 groups of 4. These were each boosted with SC-Ad6 expressing the alternate F8 Env at week 4 by either the IM or the IN route. Plasma samples collected 3 weeks after this boost showed elevations in midpoint binding titers in the animals that were prime-boosted by the IM-IM, IM-IN, and IN-IM groups. No detectable antibodies were observed in the IN-IN group (FIG. 14).

SC-Ad657 Boost at Week 13

[0274] The animals were then boosted by serotype-switching with SC-Ad657 expressing G4 Env at week 13. The same route was used as in the previous boost. Week 15 titers showed that IM primed animals had elevated Env binding titers near 350, but these levels were not significantly different than controls (FIG. 14). In contrast, antibodies in the IN-IM-IM group were significantly higher than both the vector control and the IN-IN-IN group (p<0.01). The IN-IN-IN group again showed no Env antibodies even after 3 immunizations.

Recombinant Trimeric Env Protein Boost at Week 24

[0275] Most HIV vaccine studies augment Ad immunizations with protein boosts to amplify antibody 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). In an effort to determine whether this strategy would enhance the SC-Ad vaccines, all of the SC-Ad-Env groups were boosted with 50 .mu.g of recombinant F8 trimeric gp140 protein mixed with ADJUPLEX.TM. adjuvant by the IM route. The F8 trimeric protein boosted midpoint binding titers by two orders of magnitude in all of the groups (FIG. 14). This protein immunization also boosted the IN-IN-IN to levels comparable to the other groups even though Env binding antibodies were not detected after the earlier SC-Ad immunizations.

Binding and Neutralizing Antibodies in Plasma after a Second Protein Boost

[0276] The animals were boosted with protein a second time at week 38. This increased F8 binding plasma antibody titers to nearly 10.sup.5 by week 40 and all groups became significantly different than controls (FIG. 14). Neutralizing antibody (NAb) titers against Tier 1 A SF162 virus were increased to 100 to 10,000 at week 40 (FIG. 15). NAbs against Tier 1B virus SS1196 and Tier 2 JRCSF virus increased to 100 in most animals with the exception of two animals in the IN-IN-IN group whose titers were at background levels (FIG. 15).

ADCC Activity after the Second Protein Boost

[0277] Antibody-dependent cellular cytotoxicity (ADCC) activity in week 40 plasma was tested against SHIV.sub.SF162P3 infected cells. ADCC activity was generally higher in animals that had at least one IN mucosal SC-Ad immunization (FIG. 16). All animals that received a mucosal immunization had significantly higher maximum % ADCC than SC-Ad-Ebola control animals (p<0.05, 0.0001, 0.0001 for IM-IN-IN, IN-IM-IM, and IN-IN-IN, respectively). When compared by 50% ADCC titers, only the IN SC-Ad primed groups had significantly higher ADCC activity than controls (p<0.05 and 0.001 by ANOVA for (IN-IM-IM and IN-IN-IN groups).

Antibody Responses in Saliva and Vaginal Washes after a Second Protein Boost

[0278] The data above monitored systemic antibody responses in plasma. Saliva and vaginal wash samples were also collected at week 40 and measured for antibodies in these mucosal sites. When saliva and vaginal washes were assayed for F8 and SF162 env binding by ELISA, these responses were observed in most groups with the exception of the SC-Ad-Ebola control group (FIG. 25).

[0279] There appeared to be a regional effect on these mucosal antibodies. In animals that were immunized with SC-Ad mostly by the IN route (IM-IN-IN and IN-IN-IN), binding antibodies were higher in the saliva near this site of immunization, but lower in the more distant vaginal site (FIG. 25). When ADCC activity was measured in these mucosal samples, these responses were highly variable (FIG. 17). Despite this, higher ADCC activity was observed in the IN-IN-IN group when compared to control animals (p<0.05 by ANOVA).

Systemic Cellular Immune Responses after One Protein Boost

[0280] Week 38 PBMCs were assayed for T cells against Env and against adenovirus by ELISPOT on samples collected just prior to a second F8 Env protein boost. All Env-immunized animals had Env-specific IFN-.gamma. secreting cells in their PBMCs (FIG. 18A). The level of Env-specific IFN-.gamma. SFCs were generally increased in animals that received at least one mucosal immunization. However, IFN-.gamma. SFCs were only significantly higher only in the IN-IN-IN SC-Ad group when they were compared to SC-Ad Ebola immunized control animals (p<0.05 by ANOVA). Anti-Ad SFCs were relatively low in all groups when compared to anti-Env SFCs at this time point.

Systemic Cellular Immune Responses after a Second Protein Boost

[0281] At week 40, PBMCs and inguinal lymph node cells were assayed for Env-specific IFN-.gamma. SFCs by ELISPOT (FIG. 18B). This protein boost increased Env-specific SFCs in PBMCs and in lymph nodes to similar levels in all of the Env-immunized animals.

Mucosal Cellular Trafficking

[0282] Flow cytometry on rectal biopsy samples at week 40 showed similar numbers of a4.beta.7 CD4 and CD8 cells in rectal sites (FIG. 19A). There was a trend towards increasing numbers in the IN primed groups, but these did not reach significance. The numbers of activated CD69+ CD4+ cells in rectal tissues were similar between the groups (FIG. 19B). Similarly, FoxP3+ CD4+ cells in this mucosal site were not appreciably different (FIG. 19B).

Antigen-Specific Tfh Cell Distributions

[0283] CXCR5+ IL-21+ CD4+ T follicular helper (Tfh) cells were measured in PBMCs and lymph node samples at week 40 (FIG. 20). The animals that were immunized by Ad and protein by only the IM route had significantly higher peripheral Tfh (pTfh) cells in PBMCs than other groups (FIG. 20). In lymph nodes, Tfh cells were lowest in the control and IM only group. In contrast, approximately one half of the animals that received at least one IN mucosal immunization have detectable Tfh in their lymph nodes after the last protein boost (FIG. 20).

Rectal Challenge with SHIV.sub.SF162P3

[0284] The immunized macaques were challenged rectally with HIV isolate SHIV.sub.SF162P3. Four unimmunized control animals were added to the study and each group was challenged weekly by rectal inoculation with 1 ml a 1:300 dilution of SHIV.sub.SF162P3 challenge stock provided by NIH. This challenge equaled 4.3 TCID.sub.50 on rhesus PBMCs and 137 TCID.sub.50 on TZM-bl cells. After the first challenge, 2 animals in an un-immunized control group and 2 animals in the IM-IM-IM group became infected (FIG. 21). One animal in each of the mixed route groups (IM-IN-IN and IN-IM-IM) became infected after one challenge. None of the animals in the IN-IN-IN group were infected after the first challenge. Viral loads in plasma indicated that all animals except the Ebola group animal reached high viral loads after 3 challenges (FIG. 22B). Animals in the IN-IN-IN group had a delay in reaching these high viral loads.

[0285] As challenges continued, animals in all groups became infected with the exception of one animal in the Ebola group that remained uninfected after 7 challenges. Trim5.alpha. and MHC alleles were examined retrospectively (Table 2). This analysis did not reveal overtly protective genes in the resistant Ebola group animal. Most animals could not be classified with alleles that might keep them moderately protected, but most groups had at least one animal with a higher likelihood of protection by virtue of these alleles. It should be noted that 2/4 animals in the IM-IM-IM and IN-IN-IN groups had Trim5a and MHC alleles that might predict a higher likelihood of innate protection against SIVsmm and perhaps SHIV.sub.SF162P3 (Table 2).

TABLE-US-00002 TABLE 2 Retrospective Screening for SIV Protective Gene Alleles. Degree of Vaccine Animal viral Group Number MHC typing TRIM5alpha protection Unimmunized RHJ663 Not done Cyp A/ TFP High RH3-39 Not done Q/TFP Moderate RHJ403 Not done CypA/Q Moderate RHJ791 Not done Q/TFP Moderate SC-Ad-Ebov RH13-005 A11, B01, B17 Q/TFP Moderate A08, A11, B01, RH13-007 B17 Q/TFP Moderate RH13-043 A08, A11, B17 Q/TFP Moderate RH13-135 A08, A11, B17 Q/TFP Mmerate 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 Cyp A/Q Moderate A08, A11, B01, RH13-045 B17 Q/Q Susceptible RH13-095 A08, A11, B17 Q/TFP Moderate RH13-159 A08, A11 Cyp A/TFP High IN-IM-IM RH13-013 A11, B17 Cyp A/Q Moderate A08, A11, B01, 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 A08, A11, B01 RH13-087 B17 TFP/TFP High RH13-125 A08, A11, B17 Q/TFP Moderate

[0286] When the animals were grouped based on whether they were primed by the IM or IN route with SC-Ad and Kaplan-Meier survival was analyzed, infection of the eight IM primed animals paralleled that of control animals (FIG. 22A). In contrast, infection was somewhat delayed in the eight animals that were primed with SC-Ad-Env by the mucosal IN route

Post-Mortem Viral Loads in Tissues

[0287] The challenge study was terminated 9 weeks after first challenge. PBMCs and gut tissues were isolated, RNA was purified, and evaluated for SHIV viral genomes (FIG. 22B). Post-mortem PBMCs had varied levels of SHIV viral RNA with somewhat lower levels in the IN-IN-IN group than in the IM-IM-IM group. Mean viral RNA in the colon was 15-fold lower in the IN-IN-IN group than the IM-IM-IM group (FIG. 23). This difference did not reach significance by ANOVA, but two-tailed T test gave a p value of 0.0079.

[0288] This example demonstrates that replicating SC-Ad vectors can be used as a robust and safe platform for vaccination against HIV-1 and other infectious diseases. SC-Ad is able to amplify antigen and cytokine genes up to 10,000-fold in infected human cells. The immune response is amplified well-above those mediated by RD-Ad vectors that are currently being tested as HIV-1 vaccines in humans. HIV vaccines can be transitioned to vaccine platforms that amplify HIV antigen genes by utilizing SC-Ad vectors, for example, SC-Ad vectors based on recombinant Ads having low seroprevelance.

Example 8. In Vivo Cytotoxic T lymphocyte (CTL) Assay for Immune Responses against Hepatitis C Virus (HCV) Antigen

[0289] Mice were immunized with Ad657 expressing the CMV cytomegalovirus (CMV) glycoprotein B (gB) cDNA or HCV antigen 2.4. Syngeneic cells were pulsed with HCV peptide and labeled with carboxyfluorescein succinimidyl ester (CFSE) prior to injection into the immunized mice. Cognate CTL activity is observed against HCV by loss of labeled cells in the HCV, but not CMV immunized animals (FIG. 26).

Example 9. Conditionally Replicating Ads (CRAds)

[0290] Schematic of mutations in Ad6, Ad657 and variants thereof involving mutations in the E1 protein to convert the virus to a conditionally-replicating Ad (CRAd) is shown in FIG. 39 and FIG. 43. These include dl1101 and/or the dl1107 that block binding to p300 and pRB, respectively.

[0291] FIG. 56 shows the N-terminal amino acid sequences of E1A in a wild-type Ad, as well as Ad variants E1A dl1101, E1A dl1107 and E1A dl1101/1107.

[0292] Also shown is the replacement of the Ad E1 promoter with the prostate-specific promoter probasin-E1 DNA sequence of SEQ ID NO:44 to generate the CRAd, Ad-PB (FIG. 55). The probasin promoter is androgen dependent, so will work in androgen-sensitive tumors like LNCaP, but not in androgen-resistant tumors like DU145.

[0293] A549 cells were infected with the indicated Ad6 or Ad657 variants at the indicated concentrations of virus (vp/cell) and cell viability was measured by crystal violet staining after 5 days (FIG. 40).

[0294] Killing of non-cancerous cells by replication-defective Ad (RD-Ad), Ad6, CRAd6-dl1101/dl1107 or CRAd6-PB. Modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds) is demonstrated (FIG. 44).

[0295] Killing of cancerous cells by replication-competent Ad5, Ad6, Ad657, and the indicated CRAds is shown in FIG. 45. The modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds) is demonstrated.

[0296] The results shown in FIG. 46 demonstrate modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds) in breast cancer cells.

[0297] The results shown in FIG. 47 demonstrate modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds) in prostate cancer cells and lung cancer cells.

[0298] In vivo effects of replication-competent Ad6 or the indicated CRAds on growth of DU145 tumors in mice. FIG. 48 demonstrates modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds) in vivo after a single intravenous injection in mice bearing human prostate tumors.

[0299] In vivo effects of replication-competent Ad6 or the indicated CRAds on survival of mice with DU145 tumors. FIG. 49 demonstrates modification of Ad6 and Ad657 to be conditionally-replicating Ads (CRAds) in vivo after a single intravenous injection in mice bearing human prostate tumors.

[0300] FIG. 51 demonstrates that modification of Ad657 with the shorter fiber from chimpanzee AdC68 reduces efficacy.

[0301] In an embodiment, an Ad 6/56/6 virus has HVRs 1 and 7 from Ad6 and HVRs 2-6 from Ad657. FIG. 52 demonstrates Ad 6/56/6 virus killing human lung cancer cells with and without CRAd modifications.

[0302] Tumor cell killing by Ad variants involving mutations in the E3 protein. Immune competent Syrian hamsters were engrafted with subcutaneous HaK kidney cancer tumors. When these reached 200 .mu.I volume, they were injected a single time by the intravenous route with the indicated Ad6 viruses constructed with and without E3 (DE3) and with or without random NHS-PEGylation. Tumor sizes were measured over time. The data shows that deleting all E3 genes makes the oncolytic virus less effective (Ad6-deltaE3-Luc vs Ad6-Luc) (FIG. 58).

[0303] The Ad fiber protein is a complex of three apparently identical subunits which mediates the initial attachment step. The native Ad6 fiber protein comprises the amino acid sequence set forth in SEQ ID NO:60 and binds CAR.

[0304] In a further aspect of the invention, fiber-modified recombinant Ads having different fiber proteins which are not native to the parental Ad were generated. Recombinant Ads, including CRAds, comprising capsid proteins from different Ad strains were generated, for example, recombinant Ads comprising a heterologous Ad35 fiber polypeptide or Chimpanzee C68 fiber polypeptide, +/-a K7 peptide (FIGS. 62-69).

[0305] A chimeric Ad, AdF35 fiber chimera, has the amino acid sequence of SEQ ID NO:61 and is shorter than Ad5 and Ad6 fiber proteins and retargets virus to CD46.

[0306] A fiber-modified recombinant Ad, comprising K7 Fiber having the sequence of SEQ ID NO:62, targets virus to heparin sulfate proteoglycans and negative charges on cells.

[0307] A recombinant, chimeric Ad, 6/FC68 Fiber comprising 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 Ad5 or Ad6 fiber proteins and binds CAR.

[0308] A recombinant, chimeric Ad, 6/FC68-K7 Fiber comprising 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 Ad5 or Ad6 fiber proteins. The 6/FC68-K7 Fiber binds CAR and is retargeted to heparin sulfate and negative charges.

[0309] A recombinant, chimeric Ad, 6/FC68-HI-K7 Fiber comprising 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 Ad5 or Ad6 fiber proteins. The 6/FC68-HI-K7 Fiber binds CAR and is retargeted to heparin sulfate and negative charges.

Example 10. Serotype-Switching of Adenoviruses

[0310] FIG. 41 is a schematic showing Ad therapeutic cycles. In an embodiment serotype-switching with different Ads over the course of a treatment is exemplified (FIG. 41A).

Prostate Tumor Targeting after Serotype-Switching of Oncolytic Adenoviruses

[0311] Mice bearing DU145 prostate tumors on their flanks were treated by a single intravenous (IV) injection with Ad657 or CRAd657. These mice were treated a second time with alternate Ad6 oncolytic virus or Ad6-F35 expressing GFPLuciferase and luciferase activity was measured by imaging. Ad6 has Ad6 hexon and fiber that targets CAR. Ad6-F35 has Ad6 hexon and the Ad35 fiber that targets CD46. FIG. 42 demonstrates the capability to serotype-switch oncolytics with viruses targeting a tumor with lower off-target infection of the liver.

[0312] In another example of serotype-switching, mice bearing LNCaP prostate tumors on their flanks were treated by a single intravenous (IV) injection with 3e10 viral particles (vp) of Ad657 or CRAd657. These mice were treated a second time 5 months later with 3e10 vp alternate Ad6/57/6 oncolytic virus expressing GFPLuciferase and fiber variants K7 (with 7 lysines added), F35 (with the Ad35 fiber), or KKTK-C68 (chimpanzee C68 fiber fused after the Ad6 KKTK flexibility domain. KKTK-C68 virus also has an added codon-optimized E4 34K gene to enhance viral productivity. Luciferase activity was measured by imaging 7 days later. All Ad6/57/6's have a hexon with HVR1 and 7 from Ad6 and HVRs 2-6 from Ad57. Ad6/57/6 and KKTK-C68 have fibers that targets CAR. Ad6/57/6-F35 has the Ad35 fiber that targets CD46. K7 increases binding to negative charges on cells including binding heparin sulfate proteoglycans. FIG. 70 demonstrates the capability to serotype-switch oncolytics with viruses targeting a tumor with lower off-target infection of the liver.

Serotype-Switching During Vaccination of Non-Human Primates

[0313] In FIGS. 14 through 25, rhesus macaques were immunized with replicating single-cycle Ad6 expressing HIV envelope and then boosted by serotype-switching with single-cycle Ad657 expressing HIV envelope. Following these immunizations, each animal was boosted with envelope protein. Each figure shows the generation of adaptive antibody or cellular immune responses and how the animals repelled rectal challenge with SHIV.sub.SF162P3 virus. FIG. 14 documents the value of the serotype-switch where changing to Ad657 generated marked increases in antibody responses.

Example 11. Oncolytic Cancer Vaccines

[0314] BALB/c mice were immunized with 10.sup.10 virus particles of CRAd-657-dl1101/1107-FolR with intact E3 and expressing the human folate receptor alpha or with PBS by the intramuscular route. Sera was collected 2 weeks after one immunization and analyzed for anti-Folate Receptor alpha antibodies by ELISA using anti-IgM antibody for detection (all antibodies are IgM at this type of early time point after immunization). Data shows the generation of antibodies against the known cancer antigen folate receptor alpha by this CRAd. p-0.07 by T test (FIG. 53).

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

[0315] FIG. 57 shows as schematic of different E3 immune evasion genes in Ads. 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. Species C Ads also express the 11.6K known as the adenovirus death protein (ADP). Over-expression of ADP accelerates cell death, but overall cell death is equal. Species 49K binds to CD46 on T cells and NK cells leading to down-regulation of these cells and less-efficient cell killing of cells deficient in class I MHC by NK cells.

[0316] FIG. 58 demonstrates that partial deletion of E3 12.5K and full deletion of E3 6.7K, 19K, 11.6K (ADP), 10.4K (RID.alpha.), 14.5K (RID.beta.), and 14.7K genes reduces oncolytic efficacy in an immunocompetent hamster model of kidney cancer when these immune evasion genes are not present in oncolytic adenovirus.

Other Embodiments

[0317] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1

1

65112PRTArtificial Sequencetargeting polypeptide 1Thr Ala Arg Gly Glu His Lys Glu Glu Glu Leu Ile1 5 10212PRTArtificial Sequencetargeting polypeptide 2Leu Arg Gln Thr Gly Ala Ala Ser Ala Val Trp Gly1 5 10312PRTArtificial Sequencetargeting polypeptide 3Ala Arg Arg Ala Asp Thr Gln Trp Arg Gly Leu Glu1 5 10419PRTArtificial Sequencetargeting polypeptide 4Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln Ser Cys Gly Tyr Ala1 5 10 15Thr Val Thr59PRTArtificial Sequencetargeting polypeptide 5Cys Asp Cys Arg Gly Asp Cys Phe Cys1 5610PRTArtificial Sequencetargeting polypeptide 6Asn Met Ser Leu Asp Val Asn Arg Lys Ala1 5 10712PRTArtificial Sequencetargeting polypeptide 7Ile Ser Leu Ser Ser His Arg Ala Thr Trp Val Val1 5 10820PRTArtificial Sequencetargeting polypeptide 8Trp Thr Met Gly Leu Asp Gln Leu Arg Asp Ser Ser Trp Ala His Gly1 5 10 15Gly Phe Ser Ala 20920PRTArtificial Sequencetargeting polypeptide 9Trp Thr Met Gly Leu Asp Gln Leu Arg Gly Asp Ser Ser Trp Ala His1 5 10 15Gly Gly Phe Ser 201020PRTArtificial Sequencetargeting polypeptide 10Arg Ser Val Ser Gly Thr Glu Trp Val Pro Met Asn Glu Gln His Arg1 5 10 15Gly Ala Ile Trp 201120PRTArtificial Sequencetargeting polypeptide 11Thr Glu Leu Arg Thr His Thr Ser Lys Glu Leu Thr Ile Arg Thr Ala1 5 10 15Ala Ser Ser Asp 201220PRTArtificial Sequencetargeting polypeptide 12Asp Arg Ala Ile Gly Trp Gln Asp Lys Leu Tyr Lys Leu Pro Leu Gly1 5 10 15Ser Ile His Asn 201320PRTArtificial Sequencetargeting polypeptide 13Met Gly Ser Trp Glu Lys Ala Ala Leu Trp Asn Arg Val Ser Ala Ser1 5 10 15Ser Gly Gly Ala 201420PRTArtificial Sequencetargeting polypeptide 14Met Ala Met Gly Gly Lys Pro Glu Arg Pro Ala Asp Ser Asp Asn Val1 5 10 15Gln Val Arg Gly 201520PRTArtificial Sequencetargeting polypeptide 15Met Ala Ser Arg Gly Asp Ala Gly Glu Gly Ser Thr Gln Ser Asn Thr1 5 10 15Asn Val Pro Ser 201620PRTArtificial Sequencetargeting polypeptide 16Gly Pro Glu Asp Thr Ser Arg Ala Pro Glu Asn Gln Gln Lys Thr Phe1 5 10 15His Arg Arg Trp 201720PRTArtificial Sequencetargeting polypeptide 17Met Gly Arg Glu Asp Val Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp1 5 10 15Leu Gly Gly Ser 201811PRTArtificial Sequencetargeting polypeptide 18Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys Gly1 5 101912PRTArtificial Sequencetargeting polypeptide 19Ala Cys Asp Cys Arg Glu Asp Val Cys Phe Cys Gly1 5 102020PRTArtificial Sequencetargeting polypeptide 20Gly Gln Ile Pro Ile Thr Glu Pro Glu Leu Cys Cys Val Pro Trp Thr1 5 10 15Glu Ala Phe Tyr 202120PRTArtificial Sequencetargeting polypeptide 21Pro Gln Pro Pro Asn Ser Thr Ala His Pro Asn Pro His Lys Ala Pro1 5 10 15Pro Asn Thr Thr 202220PRTArtificial Sequencetargeting polypeptide 22Val Arg Trp Phe Pro Gly Gly Glu Trp Gly Val Thr His Pro Glu Ser1 5 10 15Leu Pro Pro Pro 202319PRTArtificial Sequencetargeting polypeptide 23Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10 15Lys Lys Lys2414PRTArtificial Sequencetargeting polypeptide 24Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His1 5 102523PRTArtificial Sequencetargeting polypeptide 25Cys Ala Ala Ala Arg Trp Lys Lys Ala Phe Ile Ala Val Ser Ala Ala1 5 10 15Asn Arg Phe Lys Lys Ile Ser 202614PRTArtificial Sequencetargeting polypeptide 26Glu Asp Pro Gly Phe Phe Asn Val Glu Ile Pro Glu Phe Pro1 5 102720PRTArtificial Sequencetargeting polypeptide 27Gly Gly His Gly Arg Val Leu Trp Pro Asp Gly Trp Phe Ser Leu Val1 5 10 15Gly Ile Ser Pro 202821PRTArtificial Sequencetargeting polypeptide 28Met Ala Arg Thr Val Thr Ala Asn Val Pro Gly Met Gly Glu Gly Met1 5 10 15Val Val Val Pro Cys 202920PRTArtificial Sequencetargeting polypeptide 29Gly Val Ser Lys Arg Gly Leu Gln Cys His Asp Phe Ile Ser Cys Ser1 5 10 15Gly Val Pro Trp 203020PRTArtificial Sequencetargeting polypeptide 30Asn Gln Ser Ile Pro Lys Val Ala Gly Asp Ser Lys Val Phe Cys Trp1 5 10 15Trp Cys Ala Leu 203120PRTArtificial Sequencetargeting polypeptide 31Gln Ser Thr Pro Pro Thr Lys His Leu Thr Ile Pro Arg His Leu Arg1 5 10 15Asn Thr Leu Ile 203220PRTArtificial Sequencetargeting polypeptide 32Asp Met Ser Phe Gln Leu Val Thr Pro Phe Leu Lys Ala Leu Pro Thr1 5 10 15Gly Trp Arg Gly 203320PRTArtificial Sequencetargeting polypeptide 33Gly Gly His Gly Arg Val Leu Trp Pro Asp Gly Trp Phe Ser Leu Val1 5 10 15Gly Ile Ser Pro 20348PRTArtificial Sequencetargeting polypeptide 34Phe Ser Leu Val Gly Ile Ser Pro1 53520PRTArtificial Sequencetargeting polypeptide 35Gln Ile Met Met Gly Pro Ser Leu Gly Tyr Tyr Met Pro Ser Glu Ser1 5 10 15Ile Phe Ala Tyr 203620PRTArtificial Sequencetargeting polypeptide 36Ile Ser Trp Asp Ile Trp Arg Trp Trp Tyr Thr Ser Glu Asp Arg Asp1 5 10 15Ala Gly Ser Ala 203720PRTArtificial Sequencetargeting polypeptide 37Val Trp Gly Met Thr Thr Ser Asp His Gln Arg Lys Thr Glu Arg Leu1 5 10 15Asp Ser Pro Glu 203820PRTArtificial Sequencetargeting polypeptide 38Met Thr Ser Ala Gln Thr Ser Glu Lys Leu Lys Ala Glu Thr Asp Arg1 5 10 15His Thr Ala Glu 203919PRTArtificial Sequencetargeting polypeptide 39Met Gly Ser Arg Ser Ala Val Gly Asp Phe Glu Ser Ala Glu Gly Ser1 5 10 15Arg Arg Pro4020PRTArtificial Sequencetargeting polypeptide 40Met Gly Arg Thr Val Gln Ser Gly Asp Gly Thr Pro Ala Gln Thr Gln1 5 10 15Pro Ser Val Asn 204120PRTArtificial Sequencetargeting polypeptide 41Met Ala Arg Thr Val Thr Ala Asn Val Pro Gly Met Gly Glu Gly Met1 5 10 15Val Val Val Pro 2042132PRTArtificial SequenceAdenovirus E1A N-terminus polypeptide 42Met Arg His Ile Ile Cys His Gly Gly Val Ile Thr Glu Glu Met Ala1 5 10 15Ala Ser Leu Leu Asp Gln Leu Ile Glu Glu Val Leu Ala Asp Asn Leu 20 25 30Pro Pro Pro Ser His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp 35 40 45Leu Asp Val Thr Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln 50 55 60Ile Phe Pro Glu Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu65 70 75 80Phe Thr Phe Pro Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser 85 90 95Arg Gln Pro Glu Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Ser Met 100 105 110Pro Asn Leu Val Pro Glu Val Ile Asp Leu Thr Cys His Glu Ala Gly 115 120 125Phe Pro Pro Ser 13043112PRTArtificial SequenceE1A N-terminus polypeptide 43Met Arg His Ile Glu Glu Val Leu Ala Asp Asn Leu Pro Pro Pro Ser1 5 10 15His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp Leu Asp Val Thr 20 25 30Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln Ile Phe Pro Glu 35 40 45Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu Phe Thr Phe Pro 50 55 60Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser Arg Gln Pro Glu65 70 75 80Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Ser Met Pro Asn Leu Val 85 90 95Pro Glu Val Ile Asp Leu Thr Cys His Glu Ala Gly Phe Pro Pro Ser 100 105 11044119PRTArtificial SequenceE1A N-terminus polypeptide 44Met Arg His Ile Ile Cys His Gly Gly Val Ile Thr Glu Glu Met Ala1 5 10 15Ala Ser Leu Leu Asp Gln Leu Ile Glu Glu Val Leu Ala Asp Asn Leu 20 25 30Pro Pro Pro Ser His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp 35 40 45Leu Asp Val Thr Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln 50 55 60Ile Phe Pro Glu Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu65 70 75 80Phe Thr Phe Pro Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser 85 90 95Arg Gln Pro Glu Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Cys His 100 105 110Glu Ala Gly Phe Pro Pro Ser 1154599PRTArtificial SequenceE1A N-terminus polypeptide 45Met Arg His Ile Glu Glu Val Leu Ala Asp Asn Leu Pro Pro Pro Ser1 5 10 15His Phe Glu Pro Pro Thr Leu His Glu Leu Tyr Asp Leu Asp Val Thr 20 25 30Ala Pro Glu Asp Pro Asn Glu Glu Ala Val Ser Gln Ile Phe Pro Glu 35 40 45Ser Val Met Leu Ala Val Gln Glu Gly Ile Asp Leu Phe Thr Phe Pro 50 55 60Pro Ala Pro Gly Ser Pro Glu Pro Pro His Leu Ser Arg Gln Pro Glu65 70 75 80Gln Pro Glu Gln Arg Ala Leu Gly Pro Val Cys His Glu Ala Gly Phe 85 90 95Pro Pro Ser465PRTArtificial SequenceCell Binding Peptide 46Arg Glu Asp Val Tyr1 54712PRTArtificial SequenceBreast Cancer Binding Peptide 47Ile Ser Leu Ser Ser His Arg Ala Thr Trp Val Val1 5 10481608PRTArtificial SequenceProbasin-E1 nucleic acid 48Thr Cys Gly Ala Gly Cys Gly Ala Cys Gly Gly Thr Ala Thr Cys Gly1 5 10 15Ala Thr Ala Ala Gly Cys Thr Thr Gly Gly Ala Gly Cys Thr Thr Ala 20 25 30Thr Gly Ala Thr Ala Gly Cys Ala Thr Cys Thr Thr Gly Thr Thr Cys 35 40 45Thr Thr Ala Gly Thr Cys Thr Thr Thr Thr Thr Cys Thr Thr Ala Ala 50 55 60Thr Ala Gly Gly Gly Ala Cys Ala Thr Ala Ala Ala Gly Cys Cys Cys65 70 75 80Ala Cys Ala Ala Ala Thr Ala Ala Ala Ala Ala Thr Ala Thr Gly Cys 85 90 95Cys Thr Gly Ala Ala Gly Ala Ala Thr Gly Gly Gly Ala Cys Ala Gly 100 105 110Gly Cys Ala Thr Thr Gly Gly Gly Cys Ala Thr Thr Gly Thr Cys Cys 115 120 125Ala Thr Gly Cys Cys Thr Ala Gly Thr Ala Ala Ala Gly Thr Ala Cys 130 135 140Thr Cys Cys Ala Ala Gly Ala Ala Cys Cys Thr Ala Thr Thr Thr Gly145 150 155 160Thr Ala Thr Ala Cys Thr Ala Gly Ala Thr Gly Ala Cys Ala Cys Ala 165 170 175Ala Thr Gly Thr Thr Cys Thr Ala Gly Cys Cys Ala Ala Gly Cys Thr 180 185 190Thr Gly Gly Thr Ala Gly Thr Cys Ala Thr Cys Ala Thr Gly Thr Thr 195 200 205Thr Ala Ala Ala Cys Ala Thr Cys Thr Ala Cys Cys Ala Thr Thr Cys 210 215 220Cys Ala Gly Thr Thr Ala Ala Gly Ala Ala Ala Ala Thr Ala Thr Gly225 230 235 240Ala Thr Ala Gly Cys Ala Thr Cys Thr Thr Gly Thr Thr Cys Thr Thr 245 250 255Ala Gly Thr Cys Thr Thr Thr Thr Thr Cys Thr Thr Ala Ala Thr Ala 260 265 270Gly Gly Gly Ala Cys Ala Thr Ala Ala Ala Gly Cys Cys Cys Ala Cys 275 280 285Ala Ala Ala Thr Ala Ala Ala Ala Ala Thr Ala Thr Gly Cys Cys Thr 290 295 300Gly Ala Ala Gly Ala Ala Thr Gly Gly Gly Ala Cys Ala Gly Gly Cys305 310 315 320Ala Thr Thr Gly Gly Gly Cys Ala Thr Thr Gly Thr Cys Cys Ala Thr 325 330 335Gly Cys Cys Thr Ala Gly Thr Ala Ala Ala Gly Thr Ala Cys Thr Cys 340 345 350Cys Ala Ala Gly Ala Ala Cys Cys Thr Ala Thr Thr Thr Gly Thr Ala 355 360 365Thr Ala Cys Thr Ala Gly Ala Thr Gly Ala Cys Ala Cys Ala Ala Thr 370 375 380Gly Thr Cys Ala Ala Thr Gly Thr Cys Thr Gly Thr Gly Thr Ala Cys385 390 395 400Ala Ala Cys Thr Gly Cys Cys Ala Ala Cys Thr Gly Gly Gly Ala Thr 405 410 415Gly Cys Ala Ala Gly Ala Cys Ala Cys Thr Gly Cys Cys Cys Ala Thr 420 425 430Gly Cys Cys Ala Ala Thr Cys Ala Thr Cys Cys Thr Gly Ala Ala Ala 435 440 445Ala Gly Cys Ala Gly Cys Thr Ala Thr Ala Ala Ala Ala Ala Gly Cys 450 455 460Ala Gly Gly Ala Ala Gly Cys Thr Ala Cys Thr Cys Thr Gly Cys Ala465 470 475 480Cys Cys Thr Thr Gly Thr Cys Ala Gly Thr Gly Ala Gly Gly Thr Cys 485 490 495Cys Ala Gly Ala Thr Ala Cys Cys Thr Cys Cys Cys Thr Cys Gly Ala 500 505 510Gly Cys Gly Gly Cys Cys Gly Cys Gly Ala Cys Gly Cys Gly Cys Ala 515 520 525Gly Thr Gly Thr Ala Thr Thr Thr Ala Thr Ala Cys Cys Cys Gly Gly 530 535 540Thr Gly Ala Gly Thr Thr Cys Cys Thr Cys Ala Ala Gly Ala Gly Gly545 550 555 560Cys Cys Ala Cys Thr Cys Thr Thr Gly Ala Gly Thr Gly Cys Cys Ala 565 570 575Gly Cys Gly Ala Gly Thr Ala Gly Ala Gly Thr Thr Thr Thr Cys Thr 580 585 590Cys Cys Thr Cys Cys Gly Ala Gly Cys Cys Gly Cys Thr Cys Cys Gly 595 600 605Ala Cys Ala Cys Cys Gly Gly Gly Ala Cys Thr Gly Ala Ala Ala Ala 610 615 620Thr Gly Ala Gly Ala Cys Ala Thr Ala Thr Thr Ala Thr Cys Thr Gly625 630 635 640Cys Cys Ala Cys Gly Gly Ala Gly Gly Thr Gly Thr Thr Ala Thr Thr 645 650 655Ala Cys Cys Gly Ala Ala Gly Ala Ala Ala Thr Gly Gly Cys Cys Gly 660 665 670Cys Cys Ala Gly Thr Cys Thr Thr Thr Thr Gly Gly Ala Cys Cys Ala 675 680 685Gly Cys Thr Gly Ala Thr Cys Gly Ala Ala Gly Ala Gly Gly Thr Ala 690 695 700Cys Thr Gly Gly Cys Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys705 710 715 720Cys Ala Cys Cys Thr Cys Cys Thr Ala Gly Cys Cys Ala Thr Thr Thr 725 730 735Thr Gly Ala Ala Cys Cys Ala Cys Cys Thr Ala Cys Cys Cys Thr Thr 740 745 750Cys Ala Cys Gly Ala Ala Cys Thr Gly Thr Ala Thr Gly Ala Thr Thr 755 760 765Thr Ala Gly Ala Cys Gly Thr Gly Ala Cys Gly Gly Cys Cys Cys Cys 770 775 780Cys Gly Ala Ala Gly Ala Thr Cys Cys Cys Ala Ala Cys Gly Ala Gly785 790 795 800Gly Ala Gly Gly Cys Gly Gly Thr Thr Thr Cys Gly Cys Ala Gly Ala 805 810 815Thr Thr Thr Thr Thr Cys Cys Cys Gly Ala Gly Thr Cys Thr Gly Thr 820 825 830Ala Ala Thr Gly Thr Thr Gly Gly Cys Gly Gly Thr Gly Cys Ala Gly 835 840 845Gly Ala Ala Gly Gly Gly Ala Thr Thr Gly Ala Cys Thr Thr Ala Thr 850 855 860Thr Cys Ala Cys Thr Thr Thr Thr Cys Cys Gly Cys Cys Gly Gly Cys865 870 875 880Gly Cys Cys Cys Gly Gly Thr Thr Cys Thr Cys Cys Gly Gly Ala Gly 885 890 895Cys Cys Gly Cys Cys Thr Cys Ala Cys Cys Thr Thr Thr Cys Cys Cys 900 905 910Gly Gly Cys Ala Gly Cys Cys Cys Gly Ala Gly Cys Ala Gly Cys Cys 915 920 925Gly Gly Ala Gly Cys Ala Gly Ala Gly Ala Gly Cys Cys Thr Thr Gly 930

935 940Gly Gly Thr Cys Cys Gly Gly Thr Thr Thr Cys Thr Ala Thr Gly Cys945 950 955 960Cys Ala Ala Ala Cys Cys Thr Thr Gly Thr Gly Cys Cys Gly Gly Ala 965 970 975Gly Gly Thr Gly Ala Thr Cys Gly Ala Thr Cys Thr Thr Ala Cys Cys 980 985 990Thr Gly Cys Cys Ala Cys Gly Ala Gly Gly Cys Thr Gly Gly Cys Thr 995 1000 1005Thr Thr Cys Cys Ala Cys Cys Cys Ala Gly Thr Gly Ala Cys Gly 1010 1015 1020Ala Cys Gly Ala Gly Gly Ala Thr Gly Ala Ala Gly Ala Gly Gly 1025 1030 1035Gly Thr Gly Ala Gly Gly Ala Gly Thr Thr Thr Gly Thr Gly Thr 1040 1045 1050Thr Ala Gly Ala Thr Thr Ala Thr Gly Thr Gly Gly Ala Gly Cys 1055 1060 1065Ala Cys Cys Cys Cys Gly Gly Gly Cys Ala Cys Gly Gly Thr Thr 1070 1075 1080Gly Cys Ala Gly Gly Thr Cys Thr Thr Gly Thr Cys Ala Thr Thr 1085 1090 1095Ala Thr Cys Ala Cys Cys Gly Gly Ala Gly Gly Ala Ala Thr Ala 1100 1105 1110Cys Gly Gly Gly Gly Gly Ala Cys Cys Cys Ala Gly Ala Thr Ala 1115 1120 1125Thr Thr Ala Thr Gly Thr Gly Thr Thr Cys Gly Cys Thr Thr Thr 1130 1135 1140Gly Cys Thr Ala Thr Ala Thr Gly Ala Gly Gly Ala Cys Cys Thr 1145 1150 1155Gly Thr Gly Gly Cys Ala Thr Gly Thr Thr Thr Gly Thr Cys Thr 1160 1165 1170Ala Cys Ala Gly Thr Ala Ala Gly Thr Gly Ala Ala Ala Ala Thr 1175 1180 1185Thr Ala Thr Gly Gly Gly Cys Ala Gly Thr Cys Gly Gly Thr Gly 1190 1195 1200Ala Thr Ala Gly Ala Gly Thr Gly Gly Thr Gly Gly Gly Thr Thr 1205 1210 1215Thr Gly Gly Thr Gly Thr Gly Gly Thr Ala Ala Thr Thr Thr Thr 1220 1225 1230Thr Thr Thr Thr Thr Ala Ala Thr Thr Thr Thr Thr Ala Cys Ala 1235 1240 1245Gly Thr Thr Thr Thr Gly Thr Gly Gly Thr Thr Thr Ala Ala Ala 1250 1255 1260Gly Ala Ala Thr Thr Thr Thr Gly Thr Ala Thr Thr Gly Thr Gly 1265 1270 1275Ala Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Gly Gly Thr Cys 1280 1285 1290Cys Thr Gly Thr Gly Thr Cys Thr Gly Ala Ala Cys Cys Thr Gly 1295 1300 1305Ala Gly Cys Cys Thr Gly Ala Gly Cys Cys Cys Gly Ala Gly Cys 1310 1315 1320Cys Ala Gly Ala Ala Cys Cys Gly Gly Ala Gly Cys Cys Thr Gly 1325 1330 1335Cys Ala Ala Gly Ala Cys Cys Thr Ala Cys Cys Cys Gly Gly Cys 1340 1345 1350Gly Thr Cys Cys Thr Ala Ala Ala Thr Thr Gly Gly Thr Gly Cys 1355 1360 1365Cys Thr Gly Cys Thr Ala Thr Cys Cys Thr Gly Ala Gly Ala Cys 1370 1375 1380Gly Cys Cys Cys Gly Ala Cys Ala Thr Cys Ala Cys Cys Thr Gly 1385 1390 1395Thr Gly Thr Cys Thr Ala Gly Ala Gly Ala Ala Thr Gly Cys Ala 1400 1405 1410Ala Thr Ala Gly Thr Ala Gly Thr Ala Cys Gly Gly Ala Thr Ala 1415 1420 1425Gly Cys Thr Gly Thr Gly Ala Cys Thr Cys Cys Gly Gly Thr Cys 1430 1435 1440Cys Thr Thr Cys Thr Ala Ala Cys Ala Cys Ala Cys Cys Thr Cys 1445 1450 1455Cys Thr Gly Ala Gly Ala Thr Ala Cys Ala Cys Cys Cys Gly Gly 1460 1465 1470Thr Gly Gly Thr Cys Cys Cys Gly Cys Thr Gly Thr Gly Cys Cys 1475 1480 1485Cys Cys Ala Thr Thr Ala Ala Ala Cys Cys Ala Gly Thr Thr Gly 1490 1495 1500Cys Cys Gly Thr Gly Ala Gly Ala Gly Thr Thr Gly Gly Thr Gly 1505 1510 1515Gly Gly Cys Gly Thr Cys Gly Cys Cys Ala Gly Gly Cys Thr Gly 1520 1525 1530Thr Gly Gly Ala Ala Thr Gly Thr Ala Thr Cys Gly Ala Gly Gly 1535 1540 1545Ala Cys Thr Thr Gly Cys Thr Thr Ala Ala Cys Gly Ala Gly Thr 1550 1555 1560Cys Thr Gly Gly Gly Cys Ala Ala Cys Cys Thr Thr Thr Gly Gly 1565 1570 1575Ala Cys Thr Thr Gly Ala Gly Cys Thr Gly Thr Ala Ala Ala Cys 1580 1585 1590Gly Cys Cys Cys Cys Ala Gly Gly Cys Cys Ala Thr Ala Ala Gly 1595 1600 160549959PRTArtificial SequenceHexon Polypeptide 49Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala Asn Ala Ile Gln Pro 275 280 285Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr 290 295 300His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met305 310 315 320Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg 325 330 335Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly 340 345 350Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln 355 360 365Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly 370 375 380Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr385 390 395 400Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu 405 410 415Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr 420 425 430Gln Ala Ile Lys Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala 435 440 445Gln Asp Asn Thr Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn 450 455 460Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu465 470 475 480Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro 485 490 495Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn 500 505 510Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly 515 520 525Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His 530 535 540His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly545 550 555 560Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile 565 570 575Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe 580 585 590Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu 595 600 605Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala 610 615 620Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met625 630 635 640Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala 645 650 655Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile 660 665 670Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr 675 680 685Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro 690 695 700Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr705 710 715 720Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val 725 730 735Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile 740 745 750Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met 755 760 765Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly 770 775 780Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser785 790 795 800Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr 805 810 815Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn 820 825 830Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala 835 840 845Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp 850 855 860Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile865 870 875 880Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly 885 890 895Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe 900 905 910Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu 915 920 925Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu 930 935 940Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr945 950 95550967PRTArtificial SequenceHexon Polypeptide 50Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Ser Ser Cys Ser Ser Gly Gly145 150 155 160Thr Glu Glu Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys Lys Thr 165 170 175His Val Tyr Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn Lys Asn 180 185 190Gly Leu Gln Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn Lys Glu 195 200 205Ile Tyr Ala Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser 210 215 220Gln Trp Asn Glu Ala Glu Ser Ser Val Ala Gly Gly Arg Val Leu Lys225 230 235 240Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr 245 250 255Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly Lys Leu 260 265 270Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn Ala Met 275 280 285Asn Glu Ala Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser Glu Asp 290 295 300Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys305 310 315 320Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn 325 330 335Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr 340 345 350Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln 355 360 365Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr 370 375 380Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met385 390 395 400Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu 405 410 415Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly 420 425 430Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly 435 440 445Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg 450 455 460Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn465 470 475 480Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu 485 490 495Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn 500 505 510Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu 515 520 525Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met 530 535 540Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr545 550 555 560Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln 565 570 575Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly 580 585 590Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu 595 600 605Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys 610 615 620Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn625 630 635 640Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln 645 650 655Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro 660 665 670Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala 675 680 685Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro 690 695 700Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile705 710 715 720Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val 725 730 735Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu 740 745 750Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly 755 760 765Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln 770 775 780Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu785 790 795 800Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met 805 810 815Ser Arg Gln Val Val Asp Asp

Thr Lys Tyr Lys Asp Tyr Gln Gln Val 820 825 830Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala 835 840 845Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro 850 855 860Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu865 870 875 880Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser 885 890 895Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser 900 905 910Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro 915 920 925Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His 930 935 940Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe945 950 955 960Ser Ala Gly Asn Ala Thr Thr 96551955PRTArtificial SequenceHexon Polypeptide 51Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Ser Cys Ser Ser Gly Gly Thr Pro Lys Leu Val Leu 275 280 285Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr 290 295 300Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln305 310 315 320Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile 325 330 335Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly 340 345 350Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr 355 360 365Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg 370 375 380Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val385 390 395 400Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys 405 410 415Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys 420 425 430Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr 435 440 445Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu 450 455 460Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile465 470 475 480Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu 485 490 495Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val 500 505 510Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser 515 520 525Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala 530 535 540Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro545 550 555 560Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu 565 570 575Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val 580 585 590Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly 595 600 605Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro 610 615 620Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp625 630 635 640Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu 645 650 655Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser 660 665 670Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr 675 680 685Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr 690 695 700Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr705 710 715 720Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly 725 730 735Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val 740 745 750Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp 755 760 765Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe 770 775 780Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn785 790 795 800Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp 805 810 815Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val 820 825 830Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn 835 840 845Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln 850 855 860Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser865 870 875 880Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu 885 890 895Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro 900 905 910Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val 915 920 925Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu 930 935 940Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr945 950 95552964PRTArtificial SequenceHexon Polypeptide 52Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Ser Ser Gly Gly Thr Glu Glu145 150 155 160Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr 165 170 175Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln 180 185 190Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala 195 200 205Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn 210 215 220Glu Ala Glu Ser Ser Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr225 230 235 240Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn 245 250 255Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln 260 265 270Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala 275 280 285Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met 290 295 300Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp305 310 315 320Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn 325 330 335Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser 340 345 350Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala 355 360 365Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu 370 375 380Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln385 390 395 400Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly 405 410 415Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly 420 425 430Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly Asn Gly Gly 435 440 445Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg Asn Glu Ile 450 455 460Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu465 470 475 480Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys 485 490 495Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr 500 505 510Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys 515 520 525Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val 530 535 540Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met545 550 555 560Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln 565 570 575Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr 580 585 590Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser 595 600 605Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser 610 615 620Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser625 630 635 640Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn 645 650 655Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala 660 665 670Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg 675 680 685Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly 690 695 700Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu705 710 715 720Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr 725 730 735Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro 740 745 750Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val 755 760 765Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala 770 775 780Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys785 790 795 800Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln 805 810 815Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile 820 825 830His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met 835 840 845Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly 850 855 860Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg865 870 875 880Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala 885 890 895Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala 900 905 910Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu 915 920 925Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His Gln Pro His 930 935 940Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly945 950 955 960Asn Ala Thr Thr53952PRTArtificial SequenceHexon Polypeptide 53Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Ser Gly Gly Thr Pro Lys Leu Val Leu Tyr Ser Glu 275 280 285Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly 290 295 300Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro305 310 315 320Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met 325 330 335Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser 340 345 350Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser 355 360 365Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser 370 375 380Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile385 390

395 400Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu 405 410 415Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn 420 425 430Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu 435 440 445Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu 450 455 460Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr465 470 475 480Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp 485 490 495Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly 500 505 510Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr 515 520 525Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg 530 535 540Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile545 550 555 560Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro 565 570 575Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val 580 585 590Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile 595 600 605Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His 610 615 620Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp625 630 635 640Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile 645 650 655Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp 660 665 670Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr 675 680 685Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser 690 695 700Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys705 710 715 720Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg 725 730 735Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu 740 745 750Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val 755 760 765Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro 770 775 780Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro785 790 795 800Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln 805 810 815Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu 820 825 830Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr 835 840 845Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe 850 855 860Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met865 870 875 880Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn 885 890 895Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu 900 905 910Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val 915 920 925His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro 930 935 940Phe Ser Ala Gly Asn Ala Thr Thr945 950541033PRTArtificial SequenceHexon Polypeptide 54Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Ser Thr Gly Glu Ile Pro Ala145 150 155 160Pro Leu Ala Gly Thr Val Ser Lys Ile Leu Val Lys Glu Gly Asp Thr 165 170 175Val Lys Ala Gly Gln Thr Val Leu Val Leu Glu Ala Met Lys Met Glu 180 185 190Thr Glu Ile Asn Ala Pro Thr Asp Gly Lys Val Glu Lys Val Leu Val 195 200 205Lys Glu Arg Asp Ala Val Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly 210 215 220Gly Gly Thr Glu Glu Glu Glu Gln Leu Pro Gln Gln Arg Asn Gly Lys225 230 235 240Lys Thr His Val Tyr Ala Gln Ala Pro Phe Ala Gly Glu Ala Ile Asn 245 250 255Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly Ala Ala Thr Glu Gly Asn 260 265 270Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln Pro Glu Pro Gln Ile Gly 275 280 285Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser Val Ala Gly Gly Arg Val 290 295 300Leu Lys Lys Thr Thr Pro Met Lys Pro Cys Tyr Gly Ser Tyr Ala Arg305 310 315 320Pro Thr Asn Ser Asn Gly Gly Gln Gly Val Met Val Glu Gln Asn Gly 325 330 335Lys Leu Glu Ser Gln Val Glu Met Gln Phe Phe Ser Thr Ser Val Asn 340 345 350Ala Met Asn Glu Ala Asn Ala Ile Gln Pro Lys Leu Val Leu Tyr Ser 355 360 365Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro 370 375 380Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met385 390 395 400Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu 405 410 415Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala 420 425 430Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu 435 440 445Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe 450 455 460Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile465 470 475 480Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro 485 490 495Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr 500 505 510Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala 515 520 525Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn 530 535 540Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu545 550 555 560Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser 565 570 575Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro 580 585 590Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp 595 600 605Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu 610 615 620Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His625 630 635 640Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu 645 650 655Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met 660 665 670Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser 675 680 685Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala 690 695 700His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn705 710 715 720Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro 725 730 735Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn 740 745 750Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu 755 760 765Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly 770 775 780Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys785 790 795 800Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp 805 810 815Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly 820 825 830Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu 835 840 845Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile 850 855 860Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln865 870 875 880Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln 885 890 895Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr 900 905 910Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro 915 920 925Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys 930 935 940Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe945 950 955 960Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala 965 970 975Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp 980 985 990Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg 995 1000 1005Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg 1010 1015 1020Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr1025 1030551021PRTArtificial SequenceHexon Polypeptide 55Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Thr Gly Glu Ile Pro Ala Pro Leu Ala Gly Thr Val 275 280 285Ser Lys Ile Leu Val Lys Glu Gly Asp Thr Val Lys Ala Gly Gln Thr 290 295 300Val Leu Val Leu Glu Ala Met Lys Met Glu Thr Glu Ile Asn Ala Pro305 310 315 320Thr Asp Gly Lys Val Glu Lys Val Leu Val Lys Glu Arg Asp Ala Val 325 330 335Gln Gly Gly Gln Gly Leu Ile Lys Ile Gly Gly Gly Thr Pro Lys Leu 340 345 350Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu 355 360 365Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly 370 375 380Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn385 390 395 400Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu 405 410 415Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg 420 425 430Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg 435 440 445Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro 450 455 460Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn465 470 475 480Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala 485 490 495Ile Lys Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp 500 505 510Asn Thr Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala 515 520 525Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser 530 535 540Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn545 550 555 560Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg 565 570 575Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg 580 585 590Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg 595 600 605Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr 610 615 620Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn625 630 635 640Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys 645 650 655Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val 660 665 670Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe 675 680 685Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg 690 695 700Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn705 710 715 720Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile 725 730 735Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu 740 745 750Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr 755 760 765Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn 770 775 780His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp785 790 795 800Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg 805 810 815Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys 820 825 830Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln 835 840 845Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe 850 855 860Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr865

870 875 880Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly 885 890 895Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro 900 905 910Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile 915 920 925Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe 930 935 940Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn945 950 955 960Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val 965 970 975Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe 980 985 990Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val 995 1000 1005Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 1010 1015 1020561015PRTArtificial SequenceHexon Polypeptide 56Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Ser Ser Asn Phe Thr Arg Glu Gly Asn Val Thr Tyr 275 280 285Lys Glu Glu Met Asp Lys Val Lys Asn Cys Ser Phe Asn Val Thr Thr 290 295 300Gly Ile Arg Asp Lys Lys Gln Lys Val Asn Ala Leu Phe Tyr Arg Leu305 310 315 320Asp Ile Thr Pro Leu Asp Glu Asn Asn Asn Asn Ser Ser Glu Tyr Arg 325 330 335Leu Ile Asn Ser Gly Gly Thr Pro Lys Leu Val Leu Tyr Ser Glu Asp 340 345 350Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr Lys Pro Gly Lys 355 360 365Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln Ser Met Pro Asn 370 375 380Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile Gly Leu Met Tyr385 390 395 400Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly Gln Ala Ser Gln 405 410 415Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr Glu Leu Ser Tyr 420 425 430Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg Tyr Phe Ser Met 435 440 445Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val Arg Ile Ile Glu 450 455 460Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys Phe Pro Leu Gly465 470 475 480Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys Ala Thr Asn Gly 485 490 495Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr Phe Ala Glu Arg 500 505 510Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn 515 520 525Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu 530 535 540Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn545 550 555 560Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu 565 570 575Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met 580 585 590Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala Gly Leu Arg Tyr 595 600 605Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln 610 615 620Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly625 630 635 640Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu 645 650 655Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys 660 665 670Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn 675 680 685Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln 690 695 700Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro705 710 715 720Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala 725 730 735Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro 740 745 750Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile 755 760 765Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val 770 775 780Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu785 790 795 800Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly 805 810 815Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln 820 825 830Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu 835 840 845Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met 850 855 860Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val865 870 875 880Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala 885 890 895Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro 900 905 910Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu 915 920 925Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser 930 935 940Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser945 950 955 960Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro 965 970 975Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val Val Arg Val His 980 985 990Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe 995 1000 1005Ser Ala Gly Asn Ala Thr Thr 1010 101557987PRTArtificial SequenceHexon Polypeptide 57Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Ser Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile 275 280 285Val Thr Phe Cys Cys Lys Cys Asp Gln Leu Leu Arg Arg Glu Val Tyr 290 295 300Asp Phe Ala Phe Arg Asp Leu Ser Gly Gly Thr Pro Lys Leu Val Leu305 310 315 320Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr His Leu Ser Tyr 325 330 335Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met Leu Gly Gln Gln 340 345 350Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg Asp Asn Phe Ile 355 360 365Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly Val Leu Ala Gly 370 375 380Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln Asp Arg Asn Thr385 390 395 400Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly Asp Arg Thr Arg 405 410 415Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr Asp Pro Asp Val 420 425 430Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu Pro Asn Tyr Cys 435 440 445Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr Gln Ala Ile Lys 450 455 460Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala Gln Asp Asn Thr465 470 475 480Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn Phe Ala Met Glu 485 490 495Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu Tyr Ser Asn Ile 500 505 510Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro Thr Asn Val Glu 515 520 525Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn Lys Arg Val Val 530 535 540Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly Ala Arg Trp Ser545 550 555 560Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Ala 565 570 575Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly Arg Tyr Val Pro 580 585 590Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu 595 600 605Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe Arg Lys Asp Val 610 615 620Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu Arg Val Asp Gly625 630 635 640Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala Thr Phe Phe Pro 645 650 655Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met Leu Arg Asn Asp 660 665 670Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala Ala Asn Met Leu 675 680 685Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile Ser Ile Pro Ser 690 695 700Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr Arg Leu Lys Thr705 710 715 720Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro Tyr Tyr Thr Tyr 725 730 735Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Asn His Thr 740 745 750Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val Ser Trp Pro Gly 755 760 765Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile Lys Arg Ser Val 770 775 780Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met Thr Lys Asp Trp785 790 795 800Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly Tyr Gln Gly Phe 805 810 815Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser Phe Phe Arg Asn 820 825 830Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr Lys Tyr Lys Asp 835 840 845Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn Ser Gly Phe Val 850 855 860Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala Tyr Pro Ala Asn865 870 875 880Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp Ser Ile Thr Gln 885 890 895Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile Pro Phe Ser Ser 900 905 910Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly Gln Asn Leu Leu 915 920 925Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe Glu Val Asp Pro 930 935 940Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu Val Phe Asp Val945 950 955 960Val Arg Val His Gln Pro His Arg Gly Val Ile Glu Thr Val Tyr Leu 965 970 975Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr 980 98558959PRTArtificial SequenceHexon Polypeptide 58Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Asp Glu Asp Asp Thr Gln Val Gln Val 130 135 140Ala Ala Glu Asp Asp Gln Asp Asp Asp Glu Glu Glu Glu Gln Leu Pro145 150 155 160Gln Gln Arg Asn Gly Lys Lys Thr His Val Tyr Ala Gln Ala Pro Phe 165 170 175Ala Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn Gly 180 185 190Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr Gln 195 200 205Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser Ser 210 215 220Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro Cys225 230 235 240Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly Val 245 250 255Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln Phe 260 265 270Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala Asn Ala Ile Gln Pro 275 280 285Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp Thr 290

295 300His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala Met305 310 315 320Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe Arg 325 330 335Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met Gly 340 345 350Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu Gln 355 360 365Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile Gly 370 375 380Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser Tyr385 390 395 400Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu Leu 405 410 415Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Val Thr Asp Thr Tyr 420 425 430Gln Ala Ile Lys Ala Thr Asn Gly Asn Gly Gly Ala Thr Thr Trp Ala 435 440 445Gln Asp Asn Thr Phe Ala Glu Arg Asn Glu Ile Gly Val Gly Asn Asn 450 455 460Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg Asn Phe Leu465 470 475 480Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys Tyr Asn Pro 485 490 495Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp Tyr Met Asn 500 505 510Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile Asn Leu Gly 515 520 525Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro Phe Asn His 530 535 540His Arg Asn Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu Gly Asn Gly545 550 555 560Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe Phe Ala Ile 565 570 575Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu Trp Asn Phe 580 585 590Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly Asn Asp Leu 595 600 605Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys Leu Tyr Ala 610 615 620Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu Glu Ala Met625 630 635 640Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr Leu Ser Ala 645 650 655Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn Val Pro Ile 660 665 670Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp Ala Phe Thr 675 680 685Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly Tyr Asp Pro 690 695 700Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly Thr Phe Tyr705 710 715 720Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp Ser Ser Val 725 730 735Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu Phe Glu Ile 740 745 750Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln Cys Asn Met 755 760 765Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr Asn Ile Gly 770 775 780Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg Met Tyr Ser785 790 795 800Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val Asp Asp Thr 805 810 815Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln His Asn Asn 820 825 830Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu Gly Gln Ala 835 840 845Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr Ala Val Asp 850 855 860Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu Trp Arg Ile865 870 875 880Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr Asp Leu Gly 885 890 895Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp Met Thr Phe 900 905 910Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val Leu Phe Glu 915 920 925Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly Val Ile Glu 930 935 940Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala Thr Thr945 950 95559962PRTArtificial SequenceHexon Polypeptide 59Met Ala Thr Pro Ser Met Met Pro Gln Trp Ser Tyr Met His Ile Ser1 5 10 15Gly Gln Asp Ala Ser Glu Tyr Leu Ser Pro Gly Leu Val Gln Phe Ala 20 25 30Arg Ala Thr Glu Thr Tyr Phe Ser Leu Asn Asn Lys Phe Arg Asn Pro 35 40 45Thr Val Ala Pro Thr His Asp Val Thr Thr Asp Arg Ser Gln Arg Leu 50 55 60Thr Leu Arg Phe Ile Pro Val Asp Arg Glu Asp Thr Ala Tyr Ser Tyr65 70 75 80Lys Ala Arg Phe Thr Leu Ala Val Gly Asp Asn Arg Val Leu Asp Met 85 90 95Ala Ser Thr Tyr Phe Asp Ile Arg Gly Val Leu Asp Arg Gly Pro Thr 100 105 110Phe Lys Pro Tyr Ser Gly Thr Ala Tyr Asn Ala Leu Ala Pro Lys Gly 115 120 125Ala Pro Asn Ser Cys Glu Trp Glu Gln Asn Glu Thr Ala Gln Val Asp 130 135 140Ala Gln Glu Leu Asp Glu Glu Glu Asn Glu Ala Asn Glu Ala Gln Ala145 150 155 160Arg Glu Gln Glu Gln Ala Lys Lys Thr His Val Tyr Ala Gln Ala Pro 165 170 175Leu Ser Gly Glu Ala Ile Asn Lys Asn Gly Leu Gln Ile Gly Thr Asn 180 185 190Gly Ala Ala Thr Glu Gly Asn Lys Glu Ile Tyr Ala Asp Lys Thr Tyr 195 200 205Gln Pro Glu Pro Gln Ile Gly Glu Ser Gln Trp Asn Glu Ala Glu Ser 210 215 220Ser Val Ala Gly Gly Arg Val Leu Lys Lys Thr Thr Pro Met Lys Pro225 230 235 240Cys Tyr Gly Ser Tyr Ala Arg Pro Thr Asn Ser Asn Gly Gly Gln Gly 245 250 255Val Met Val Glu Gln Asn Gly Lys Leu Glu Ser Gln Val Glu Met Gln 260 265 270Phe Phe Ser Thr Ser Val Asn Ala Met Asn Glu Ala Asn Ala Ile Gln 275 280 285Pro Lys Leu Val Leu Tyr Ser Glu Asp Val Asn Met Glu Thr Pro Asp 290 295 300Thr His Leu Ser Tyr Lys Pro Gly Lys Ser Asp Asp Asn Ser Lys Ala305 310 315 320Met Leu Gly Gln Gln Ser Met Pro Asn Arg Pro Asn Tyr Ile Ala Phe 325 330 335Arg Asp Asn Phe Ile Gly Leu Met Tyr Tyr Asn Ser Thr Gly Asn Met 340 345 350Gly Val Leu Ala Gly Gln Ala Ser Gln Leu Asn Ala Val Val Asp Leu 355 360 365Gln Asp Arg Asn Thr Glu Leu Ser Tyr Gln Leu Leu Leu Asp Ser Ile 370 375 380Gly Asp Arg Thr Arg Tyr Phe Ser Met Trp Asn Gln Ala Val Asp Ser385 390 395 400Tyr Asp Pro Asp Val Arg Ile Ile Glu Asn His Gly Thr Glu Asp Glu 405 410 415Leu Pro Asn Tyr Cys Phe Pro Leu Gly Gly Ile Gly Ile Thr Asp Thr 420 425 430Phe Gln Ala Val Lys Thr Thr Ala Ala Asn Gly Asp Gln Gly Asn Thr 435 440 445Thr Trp Gln Lys Asp Ser Thr Phe Ala Glu Arg Asn Glu Ile Gly Val 450 455 460Gly Asn Asn Phe Ala Met Glu Ile Asn Leu Asn Ala Asn Leu Trp Arg465 470 475 480Asn Phe Leu Tyr Ser Asn Ile Ala Leu Tyr Leu Pro Asp Lys Leu Lys 485 490 495Tyr Asn Pro Thr Asn Val Glu Ile Ser Asp Asn Pro Asn Thr Tyr Asp 500 505 510Tyr Met Asn Lys Arg Val Val Ala Pro Gly Leu Val Asp Cys Tyr Ile 515 520 525Asn Leu Gly Ala Arg Trp Ser Leu Asp Tyr Met Asp Asn Val Asn Pro 530 535 540Phe Asn His Pro Arg His Ala Gly Leu Arg Tyr Arg Ser Met Leu Leu545 550 555 560Gly Asn Gly Arg Tyr Val Pro Phe His Ile Gln Val Pro Gln Lys Phe 565 570 575Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro Gly Ser Tyr Thr Tyr Glu 580 585 590Trp Asn Phe Arg Lys Asp Val Asn Met Val Leu Gln Ser Ser Leu Gly 595 600 605Asn Asp Leu Arg Val Asp Gly Ala Ser Ile Lys Phe Asp Ser Ile Cys 610 615 620Leu Tyr Ala Thr Phe Phe Pro Met Ala His Asn Thr Ala Ser Thr Leu625 630 635 640Glu Ala Met Leu Arg Asn Asp Thr Asn Asp Gln Ser Phe Asn Asp Tyr 645 650 655Leu Ser Ala Ala Asn Met Leu Tyr Pro Ile Pro Ala Asn Ala Thr Asn 660 665 670Val Pro Ile Ser Ile Pro Ser Arg Asn Trp Ala Ala Phe Arg Gly Trp 675 680 685Ala Phe Thr Arg Leu Lys Thr Lys Glu Thr Pro Ser Leu Gly Ser Gly 690 695 700Tyr Asp Pro Tyr Tyr Thr Tyr Ser Gly Ser Ile Pro Tyr Leu Asp Gly705 710 715 720Thr Phe Tyr Leu Asn His Thr Phe Lys Lys Val Ala Ile Thr Phe Asp 725 730 735Ser Ser Val Ser Trp Pro Gly Asn Asp Arg Leu Leu Thr Pro Asn Glu 740 745 750Phe Glu Ile Lys Arg Ser Val Asp Gly Glu Gly Tyr Asn Val Ala Gln 755 760 765Cys Asn Met Thr Lys Asp Trp Phe Leu Val Gln Met Leu Ala Asn Tyr 770 775 780Asn Ile Gly Tyr Gln Gly Phe Tyr Ile Pro Glu Ser Tyr Lys Asp Arg785 790 795 800Met Tyr Ser Phe Phe Arg Asn Phe Gln Pro Met Ser Arg Gln Val Val 805 810 815Asp Asp Thr Lys Tyr Lys Asp Tyr Gln Gln Val Gly Ile Ile His Gln 820 825 830His Asn Asn Ser Gly Phe Val Gly Tyr Leu Ala Pro Thr Met Arg Glu 835 840 845Gly Gln Ala Tyr Pro Ala Asn Val Pro Tyr Pro Leu Ile Gly Lys Thr 850 855 860Ala Val Asp Ser Ile Thr Gln Lys Lys Phe Leu Cys Asp Arg Thr Leu865 870 875 880Trp Arg Ile Pro Phe Ser Ser Asn Phe Met Ser Met Gly Ala Leu Thr 885 890 895Asp Leu Gly Gln Asn Leu Leu Tyr Ala Asn Ser Ala His Ala Leu Asp 900 905 910Met Thr Phe Glu Val Asp Pro Met Asp Glu Pro Thr Leu Leu Tyr Val 915 920 925Leu Phe Glu Val Phe Asp Val Val Arg Val His Gln Pro His Arg Gly 930 935 940Val Ile Glu Thr Val Tyr Leu Arg Thr Pro Phe Ser Ala Gly Asn Ala945 950 955 960Thr Thr60528PRTArtificial SequenceFiber Polypeptide 60Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro1 5 10 15Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45Leu Arg Leu Ser Glu Pro Leu Val Thr Ser His Gly Met Leu Ala Leu 50 55 60Lys Met Gly Ser Gly Leu Ser Leu Asp Gln Ala Gly Asn Leu Thr Ser65 70 75 80Asn Thr Ile Thr Val Ser Gln Pro Leu Lys Lys Thr Lys Ser Asn Ile 85 90 95Thr Leu Glu Thr Ser Ala Pro Leu Thr Val Ser Ser Gly Ala Leu Thr 100 105 110Met Ala Thr Thr Ser Pro Leu Val Val Ser Asp Asn Thr Leu Thr Met 115 120 125Gln Ser Gln Ala Pro Leu Thr Val Gln Asp Ser Lys Leu Ser Ile Ala 130 135 140Thr Lys Glu Pro Leu Thr Val Leu Asp Gly Lys Leu Ala Leu Gln Thr145 150 155 160Ser Ala Pro Leu Ser Ala Thr Asp Asn Asn Ala Leu Thr Ile Thr Ala 165 170 175Ser Pro Pro Leu Thr Thr Ala Asn Gly Ser Leu Ala Val Thr Met Glu 180 185 190Asn Pro Leu Tyr Asn Asn Asn Gly Lys Leu Gly Leu Lys Ile Gly Gly 195 200 205Pro Leu Gln Val Ala Thr Asp Ser His Ala Leu Thr Leu Gly Thr Gly 210 215 220Gln Gly Val Ala Val His Asn Asn Leu Leu His Thr Lys Val Thr Gly225 230 235 240Ala Ile Gly Phe Asp Thr Ser Gly Asn Met Glu Leu Lys Thr Gly Asp 245 250 255Gly Leu Tyr Val Asp Ser Ala Gly Pro Asn Gln Lys Leu His Ile Asn 260 265 270Leu Asn Thr Thr Lys Gly Leu Ala Phe Asp Asn Thr Ala Ile Thr Ile 275 280 285Asn Ala Gly Lys Gly Leu Glu Phe Glu Thr Asp Ser Ser Asn Gly Asn 290 295 300Pro Ile Lys Thr Lys Ile Gly Ser Gly Ile Gln Tyr Asn Thr Asn Gly305 310 315 320Ala Met Val Ala Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Ser Gly 325 330 335Ala Ile Thr Met Gly Ser Ile Asn Asn Asp Arg Leu Thr Leu Trp Thr 340 345 350Thr Pro Asp Pro Ser Pro Asn Cys Arg Ile Ala Ser Asp Lys Asp Cys 355 360 365Lys Leu Thr Leu Ala Leu Thr Lys Cys Gly Ser Gln Ile Leu Gly Thr 370 375 380Val Ser Ala Leu Ala Val Ser Gly Asn Met Ala Ser Ile Asn Gly Thr385 390 395 400Leu Ser Ser Val Asn Leu Val Leu Arg Phe Asp Asp Asn Gly Val Leu 405 410 415Met Ser Asn Ser Ser Leu Asp Lys Gln Tyr Trp Asn Phe Arg Asn Gly 420 425 430Asp Ser Thr Asn Gly Gln Pro Tyr Thr Tyr Ala Val Gly Phe Met Pro 435 440 445Asn Leu Lys Ala Tyr Pro Lys Thr Gln Ser Lys Thr Ala Lys Ser Asn 450 455 460Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Ser Lys Pro Leu His465 470 475 480Phe Thr Ile Thr Leu Asn Gly Thr Asp Glu Thr Asn Gln Val Ser Lys 485 490 495Tyr Ser Ile Ser Phe Ser Trp Ser Trp Asn Ser Gly Gln Tyr Thr Asn 500 505 510Asp Lys Phe Ala Thr Asn Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 515 520 52561324PRTArtificial SequenceFiber Polypeptide 61Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro1 5 10 15Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Thr 35 40 45Leu Lys Cys Leu Thr Pro Leu Thr Thr Thr Gly Gly Ser Leu Gln Leu 50 55 60Lys Val Gly Gly Gly Leu Thr Val Asp Asp Thr Asp Gly Thr Leu Gln65 70 75 80Glu Asn Ile Arg Ala Thr Ala Pro Ile Thr Lys Asn Asn His Ser Val 85 90 95Glu Leu Ser Ile Gly Asn Gly Leu Glu Thr Gln Asn Asn Lys Leu Cys 100 105 110Ala Lys Leu Gly Asn Gly Leu Lys Phe Asn Asn Gly Asp Ile Cys Ile 115 120 125Lys Asp Ser Ile Asn Thr Leu Trp Thr Gly Ile Asn Pro Pro Pro Asn 130 135 140Cys Gln Ile Val Glu Asn Thr Asn Thr Asn Asp Gly Lys Leu Thr Leu145 150 155 160Val Leu Val Lys Asn Gly Gly Leu Val Asn Gly Tyr Val Ser Leu Val 165 170 175Gly Val Ser Asp Thr Val Asn Gln Met Phe Thr Gln Lys Thr Ala Asn 180 185 190Ile Gln Leu Arg Leu Tyr Phe Asp Ser Ser Gly Asn Leu Leu Thr Asp 195 200 205Glu Ser Asp Leu Lys Ile Pro Leu Lys Asn Lys Ser Ser Thr Ala Thr 210 215 220Ser Glu Thr Val Ala Ser Ser Lys Ala Phe Met Pro Ser Thr Thr Ala225 230 235 240Tyr Pro Phe Asn Thr Thr Thr Arg Asp Ser Glu Asn Tyr Ile His Gly 245 250 255Ile Cys Tyr Tyr Met Thr Ser Tyr Asp Arg Ser Leu Phe Pro Leu Asn 260 265 270Ile Ser Ile Met Leu Asn Ser Arg Met Ile Ser Ser Asn Val Ala Tyr 275 280 285Ala Ile Gln Phe Glu Trp Asn Leu Asn Ala Ser Glu Ser Pro Glu Ser 290 295 300Asn Ile Ala Thr Leu Thr Thr Ser Pro Phe

Phe Phe Ser Tyr Ile Thr305 310 315 320Glu Asp Asp Asn62534PRTArtificial SequenceFiber Polypeptide 62Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro1 5 10 15Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45Leu Arg Leu Ser Glu Pro Leu Val Thr Ser His Gly Met Leu Ala Leu 50 55 60Lys Met Gly Ser Gly Leu Ser Leu Asp Gln Ala Gly Asn Leu Thr Ser65 70 75 80Asn Thr Ile Thr Val Ser Gln Pro Leu Lys Lys Thr Lys Ser Asn Ile 85 90 95Thr Leu Glu Thr Ser Ala Pro Leu Thr Val Ser Ser Gly Ala Leu Thr 100 105 110Met Ala Thr Thr Ser Pro Leu Val Val Ser Asp Asn Thr Leu Thr Met 115 120 125Gln Ser Gln Ala Pro Leu Thr Val Gln Asp Ser Lys Leu Ser Ile Ala 130 135 140Thr Lys Glu Pro Leu Thr Val Leu Asp Gly Lys Leu Ala Leu Gln Thr145 150 155 160Ser Ala Pro Leu Ser Ala Thr Asp Asn Asn Ala Leu Thr Ile Thr Ala 165 170 175Ser Pro Pro Leu Thr Thr Ala Asn Gly Ser Leu Ala Val Thr Met Glu 180 185 190Asn Pro Leu Tyr Asn Asn Asn Gly Lys Leu Gly Leu Lys Ile Gly Gly 195 200 205Pro Leu Gln Val Ala Thr Asp Ser His Ala Leu Thr Leu Gly Thr Gly 210 215 220Gln Gly Val Ala Val His Asn Asn Leu Leu His Thr Lys Val Thr Gly225 230 235 240Ala Ile Gly Phe Asp Thr Ser Gly Asn Met Glu Leu Lys Thr Gly Asp 245 250 255Gly Leu Tyr Val Asp Ser Ala Gly Pro Asn Gln Lys Leu His Ile Asn 260 265 270Leu Asn Thr Thr Lys Gly Leu Ala Phe Asp Asn Thr Ala Ile Thr Ile 275 280 285Asn Ala Gly Lys Gly Leu Glu Phe Glu Thr Asp Ser Ser Asn Gly Asn 290 295 300Pro Ile Lys Thr Lys Ile Gly Ser Gly Ile Gln Tyr Asn Thr Asn Gly305 310 315 320Ala Met Val Ala Lys Leu Gly Thr Gly Leu Ser Phe Asp Ser Ser Gly 325 330 335Ala Ile Thr Met Gly Ser Ile Asn Asn Asp Arg Leu Thr Leu Trp Thr 340 345 350Thr Pro Asp Pro Ser Pro Asn Cys Arg Ile Ala Ser Asp Lys Asp Cys 355 360 365Lys Leu Thr Leu Ala Leu Thr Lys Cys Gly Ser Gln Ile Leu Gly Thr 370 375 380Val Ser Ala Leu Ala Val Ser Gly Asn Met Ala Ser Ile Asn Gly Thr385 390 395 400Leu Ser Ser Val Asn Leu Val Leu Arg Phe Asp Asp Asn Gly Val Leu 405 410 415Met Ser Asn Ser Ser Leu Asp Lys Gln Tyr Trp Asn Phe Arg Asn Gly 420 425 430Asp Ser Thr Asn Gly Gln Pro Tyr Thr Tyr Ala Val Gly Phe Met Pro 435 440 445Asn Leu Lys Ala Tyr Pro Lys Thr Gln Ser Lys Thr Ala Lys Ser Asn 450 455 460Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Ser Lys Pro Leu His465 470 475 480Phe Thr Ile Thr Leu Asn Gly Thr Asp Glu Thr Asn Gln Val Ser Lys 485 490 495Tyr Ser Ile Ser Phe Ser Trp Ser Trp Asn Ser Gly Gln Tyr Thr Asn 500 505 510Asp Lys Phe Ala Thr Asn Ser Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 515 520 525Lys Lys Lys Lys Lys Lys 53063425PRTArtificial SequenceFiber Polypeptide 63Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro1 5 10 15Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45Leu Arg Leu Ser Glu Pro Leu Val Thr Lys Asn Gly Glu Ile Thr Leu 50 55 60Lys Leu Gly Glu Gly Val Asp Leu Asp Ser Ser Gly Lys Leu Ile Ser65 70 75 80Asn Thr Ala Thr Lys Ala Ala Ala Pro Leu Ser Phe Ser Asn Asn Thr 85 90 95Ile Ser Leu Asn Met Asp His Pro Phe Tyr Thr Lys Asp Gly Lys Leu 100 105 110Ser Leu Gln Val Ser Pro Pro Leu Asn Ile Leu Arg Thr Ser Ile Leu 115 120 125Asn Thr Leu Ala Leu Gly Phe Gly Ser Gly Leu Gly Leu Arg Gly Ser 130 135 140Ala Leu Ala Val Gln Leu Val Ser Pro Leu Thr Phe Asp Thr Asp Gly145 150 155 160Asn Ile Lys Leu Thr Leu Asp Arg Gly Leu His Val Thr Thr Gly Asp 165 170 175Ala Ile Glu Ser Asn Ile Ser Trp Ala Lys Gly Leu Lys Phe Glu Asp 180 185 190Gly Ala Ile Ala Thr Asn Ile Gly Asn Gly Leu Glu Phe Gly Ser Ser 195 200 205Ser Thr Glu Thr Gly Val Asp Asp Ala Tyr Pro Ile Gln Val Lys Leu 210 215 220Gly Ser Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Met Ala Gly Asn225 230 235 240Lys Glu Asp Asp Lys Leu Thr Leu Trp Thr Thr Pro Asp Pro Ser Pro 245 250 255Asn Cys Gln Ile Leu Ala Glu Asn Asp Ala Lys Leu Thr Leu Cys Leu 260 265 270Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Val Val 275 280 285Gly Ser Gly Asn Leu Asn Pro Ile Thr Gly Thr Val Ser Ser Ala Gln 290 295 300Val Phe Leu Arg Phe Asp Ala Asn Gly Val Leu Leu Thr Glu His Ser305 310 315 320Thr Leu Lys Lys Tyr Trp Gly Tyr Arg Gln Gly Asp Ser Ile Asp Gly 325 330 335Thr Pro Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Lys Ala Tyr 340 345 350Pro Lys Ser Gln Ser Ser Thr Thr Lys Asn Asn Ile Val Gly Gln Val 355 360 365Tyr Met Asn Gly Asp Val Ser Lys Pro Met Leu Leu Thr Ile Thr Leu 370 375 380Asn Gly Thr Asp Asp Ser Asn Ser Thr Tyr Ser Met Ser Phe Ser Tyr385 390 395 400Thr Trp Thr Asn Gly Ser Tyr Val Gly Ala Thr Phe Gly Ala Asn Ser 405 410 415Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 420 42564432PRTArtificial SequenceFiber Polypeptide 64Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro1 5 10 15Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45Leu Arg Leu Ser Glu Pro Leu Val Thr Lys Asn Gly Glu Ile Thr Leu 50 55 60Lys Leu Gly Glu Gly Val Asp Leu Asp Ser Ser Gly Lys Leu Ile Ser65 70 75 80Asn Thr Ala Thr Lys Ala Ala Ala Pro Leu Ser Phe Ser Asn Asn Thr 85 90 95Ile Ser Leu Asn Met Asp His Pro Phe Tyr Thr Lys Asp Gly Lys Leu 100 105 110Ser Leu Gln Val Ser Pro Pro Leu Asn Ile Leu Arg Thr Ser Ile Leu 115 120 125Asn Thr Leu Ala Leu Gly Phe Gly Ser Gly Leu Gly Leu Arg Gly Ser 130 135 140Ala Leu Ala Val Gln Leu Val Ser Pro Leu Thr Phe Asp Thr Asp Gly145 150 155 160Asn Ile Lys Leu Thr Leu Asp Arg Gly Leu His Val Thr Thr Gly Asp 165 170 175Ala Ile Glu Ser Asn Ile Ser Trp Ala Lys Gly Leu Lys Phe Glu Asp 180 185 190Gly Ala Ile Ala Thr Asn Ile Gly Asn Gly Leu Glu Phe Gly Ser Ser 195 200 205Ser Thr Glu Thr Gly Val Asp Asp Ala Tyr Pro Ile Gln Val Lys Leu 210 215 220Gly Ser Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Met Ala Gly Asn225 230 235 240Lys Glu Asp Asp Lys Leu Thr Leu Trp Thr Thr Pro Asp Pro Ser Pro 245 250 255Asn Cys Gln Ile Leu Ala Glu Asn Asp Ala Lys Leu Thr Leu Cys Leu 260 265 270Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Val Val 275 280 285Gly Ser Gly Asn Leu Asn Pro Ile Thr Gly Thr Val Ser Ser Ala Gln 290 295 300Val Phe Leu Arg Phe Asp Ala Asn Gly Val Leu Leu Thr Glu His Ser305 310 315 320Thr Leu Lys Lys Tyr Trp Gly Tyr Arg Gln Gly Asp Ser Ile Asp Gly 325 330 335Thr Pro Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Lys Ala Tyr 340 345 350Pro Lys Ser Gln Ser Ser Thr Thr Lys Asn Asn Ile Val Gly Gln Val 355 360 365Tyr Met Asn Gly Asp Val Ser Lys Pro Met Leu Leu Thr Ile Thr Leu 370 375 380Asn Gly Thr Asp Asp Ser Asn Ser Thr Tyr Ser Met Ser Phe Ser Tyr385 390 395 400Thr Trp Thr Asn Gly Ser Tyr Val Gly Ala Thr Phe Gly Ala Asn Ser 405 410 415Tyr Thr Phe Ser Tyr Ile Ala Gln Glu Lys Lys Lys Lys Lys Lys Lys 420 425 43065441PRTArtificial SequenceFiber Polypeptide 65Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro1 5 10 15Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro 20 25 30Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser 35 40 45Leu Arg Leu Ser Glu Pro Leu Val Thr Lys Asn Gly Glu Ile Thr Leu 50 55 60Lys Leu Gly Glu Gly Val Asp Leu Asp Ser Ser Gly Lys Leu Ile Ser65 70 75 80Asn Thr Ala Thr Lys Ala Ala Ala Pro Leu Ser Phe Ser Asn Asn Thr 85 90 95Ile Ser Leu Asn Met Asp His Pro Phe Tyr Thr Lys Asp Gly Lys Leu 100 105 110Ser Leu Gln Val Ser Pro Pro Leu Asn Ile Leu Arg Thr Ser Ile Leu 115 120 125Asn Thr Leu Ala Leu Gly Phe Gly Ser Gly Leu Gly Leu Arg Gly Ser 130 135 140Ala Leu Ala Val Gln Leu Val Ser Pro Leu Thr Phe Asp Thr Asp Gly145 150 155 160Asn Ile Lys Leu Thr Leu Asp Arg Gly Leu His Val Thr Thr Gly Asp 165 170 175Ala Ile Glu Ser Asn Ile Ser Trp Ala Lys Gly Leu Lys Phe Glu Asp 180 185 190Gly Ala Ile Ala Thr Asn Ile Gly Asn Gly Leu Glu Phe Gly Ser Ser 195 200 205Ser Thr Glu Thr Gly Val Asp Asp Ala Tyr Pro Ile Gln Val Lys Leu 210 215 220Gly Ser Gly Leu Ser Phe Asp Ser Thr Gly Ala Ile Met Ala Gly Asn225 230 235 240Lys Glu Asp Asp Lys Leu Thr Leu Trp Thr Thr Pro Asp Pro Ser Pro 245 250 255Asn Cys Gln Ile Leu Ala Glu Asn Asp Ala Lys Leu Thr Leu Cys Leu 260 265 270Thr Lys Cys Gly Ser Gln Ile Leu Ala Thr Val Ser Val Leu Val Val 275 280 285Gly Ser Gly Asn Leu Asn Pro Ile Thr Gly Thr Val Ser Ser Ala Gln 290 295 300Val Phe Leu Arg Phe Asp Ala Asn Gly Val Leu Leu Thr Glu His Ser305 310 315 320Thr Leu Lys Lys Tyr Trp Gly Tyr Arg Gln Gly Asp Ser Ile Asp Gly 325 330 335Thr Pro Tyr Thr Asn Ala Val Gly Phe Met Pro Asn Leu Lys Ala Tyr 340 345 350Pro Lys Ser Gln Ser Ser Thr Thr Lys Asn Asn Ile Val Gly Gln Val 355 360 365Tyr Met Asn Gly Asp Val Ser Lys Pro Met Leu Leu Thr Ile Thr Leu 370 375 380Asn Gly Thr Asp Asp Ser Gly Gly Ser Ser Gly Lys Lys Lys Lys Lys385 390 395 400Lys Lys Ala Ser Gly Gly Ser Ser Thr Tyr Ser Met Ser Phe Ser Tyr 405 410 415Thr Trp Thr Asn Gly Ser Tyr Val Gly Ala Thr Phe Gly Ala Asn Ser 420 425 430Tyr Thr Phe Ser Tyr Ile Ala Gln Glu 435 440

Claims

1. A recombinant adenovirus (Ad) strain 6, comprising one or more modified fiber proteins.

2. The recombinant adenovirus (Ad) of claim 1, wherein the modified fiber protein comprises an Ad strain 35 fiber protein.

3. The recombinant adenovirus (Ad) of claim 2, comprising the amino acid sequence of SEQ ID NO:61.

4. The recombinant adenovirus (Ad) of claim 1, wherein the modified fiber protein comprises an extension of seven lysines (K7) on the Ad strain 6 fiber protein.

5. The recombinant adenovirus (Ad) of claim 4, comprising the amino acid sequence of SEQ ID NO:62.

6. The recombinant adenovirus (Ad) of claim 1, wherein the modified fiber protein comprises a chimpanzee adenovirus C68 fiber protein.

7. The recombinant adenovirus (Ad) of claim 6, comprising the amino acid sequence of SEQ ID NO:63.

8. The recombinant adenovirus (Ad) of claim 6, further comprising an extension of 7 lysines (K7) on the fiber protein, wherein the fiber protein comprises the amino acid sequence of SEQ ID NO:64 or SEQ ID NO:65.

9. The recombinant Ad of claim 1, further comprising a modification in the E3 region of the Ad strain 6.

10. The recombinant Ad of claim 9, wherein the modification in the E3 region of the Ad strain 6 comprises a deletion in part of E3 12.5K and full deletion of E3 6.7K, 19K and 11.6K (ADP).

11. The recombinant adenovirus (Ad) of claim 1, comprising nucleic acids which encode a heterologous antigen.

12. The recombinant adenovirus (Ad) of claim 11, wherein the heterologous antigen is selected from a human folate receptor alpha polypeptide, an HIV envelope polypeptide, a hepatitis C virus (HCV) antigen or hepatitis B virus (HBV) antigen, and a Human Papilloma Virus (HPV) antigen.

13. The recombinant adenovirus (Ad) of claim 1, comprising nucleic acids which encode one or more proteins selected from 4-1BBL, human granulocyte macrophage stimulating factor (hGMCSF), CD40L and IL-21.

14. The recombinant adenovirus (Ad) of claim 1, further comprising cysteine amino acid substitutions, and wherein N-Hydroxysuccinimide (NHS) or maleimide polyethylene glycol (PEGylation) or biotin acceptor peptide (BAPylation) modification bind to cysteine substituted Ad capsid hexon hypervariable region (HVR) polypeptides.

15. A method of targeting a cancerous tumor with an Adenovirus (Ad) and lowering infection of the liver by the Ad comprising, administering an Ad strain to a subject in need thereof, and subsequently administering to the subject in one or more rounds of treatment, an alternate Ad strain having a modified fiber protein.

16. The method of claim 15, wherein the Ad comprises a covalent polymer conjugation.

17. The method of claim 15, wherein the alternate Ad strain is the Ad of claim 1.

18. A conditionally-replicating Adenovirus (CRAd) comprising, a recombinant Adenovirus (Ad) strain 6 which has been modified in an E1A gene encoding an E1A polypeptide, wherein the CRAd exhibits amino acid substitutions in the E1A polypeptide relative to wild-type E1A polypeptide of an Ad strain.

19. The CRAd of claim 11, wherein the N-terminal portion of the E1A polypeptide comprises an amino acid sequence set forth in SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45.