WO2006119449A2 - Modified adenovirus containing a stabilized antibody - Google Patents

Abstract

The present invention relates generally to the fields of vector biology and gene therapy. The invention is based, in part, on Applicants' discovery that stabilization of the targeting molecule results in successful incorporation of antibody-related molecules into the adenovirus capsid. Accordingly, the present invention relates to the production of recombinant adenoviral vectors containing stabilized antibodies for cell-specific targeting.

Description

TITLE OF THE INVENTION

MODIFIED ADENOVIRUS CONTAINING A STABILIZED ANTIBODY INCORPORATION BY REFERENCE

This application claims priority to U.S. provisional application Serial No. 60/677,683 filed May 4, 2005.

Reference is made to U.S. application Serial No. 10/944,496 filed September 17, 2004, which is a continuation-in-part of U.S. application Serial No. 09/612,852 filed My 10, 2000, now U.S. Patent No. 6,815,200 issued November 9, 2004, which is a continuation-in- part application of U.S. application Serial No. 09/250,580 filed February 16, 1999, now U.S. Patent No. 6,210,946 issued April 3, 2001, which claims benefit of U.S. provisional application Serial No. 60/074,844 filed February 17, 1998.

The foregoing applications, and all documents cited therein or during their prosecution ("appln cited documents") and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein ("herein cited documents"), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. FEDERAL FUNDING LEGEND This invention was supported in part using federal funds from the National Institutes of Health. Accordingly, the Federal Government has certain rights in this invention. FIELD OF THE INVENTION

The present invention relates generally to the fields of vector biology and gene therapy. More specifically, the present invention relates to the production of recombinant adenoviral vectors containing stabilized antibodies, which encompasses stabilized antibody- related framents/molecules, for cell-specific targeting. BACKGROUND OF THE INVENTION

Adenoviral (Ad) vectors are of utility for a wide range of gene therapy applications. To improve their utility profile strategies have been developed to alter adenoviral tropism to achieve a cell-specific gene delivery capacity. In this regard, modifications of the major viral capsid proteins have been employed as a means to achieve this desired goal of altered tropism. Such strategies have sought to incorporate into the adenoviral capsid targeting motifs that recognize target cell surface markers, hi this regard, targeting motifs derived from antibody molecules represent highly useful agents to achieve this desired goal, as they embody unparalleled affinity and specificity for recognition and binding to target cell surface markers. Thus, it is widely recognized that genetically incorporating antibody-derived targeting motifs into the adenoviral capsid represents the optimal means to achieve the field- wide goal of rendering Ad target cell specific. A number of antibody-related targeting motifs exist for utility in the aforementioned strategy. Many of these species are derived from parental antibody molecules by genetic engineering methods. Thus species such as single chain antibodies (scFv), antibody heavy chain variable domains (dAabs), and others, such as antibody mimics, embody a useful antigen binding profile but also are based upon genetic engineering elements which distinguish them from primary antibodies. In the aggregate, these antibody-related species embrace a wide spectrum of targeting possibilities. Further, their target antigen recognition profile renders them of potential utility for strategies designed to retargeting adenovirus for gene therapy purposes.

Underlying this general concept is the requirement to incorporate the antibody-related species into the adenoviral capsid via genetic capsid modifications. This technical goal requires the identification of capsid locales compatible with incorporation of heterologous proteins of the size and complexity represented by antibody-related species. Whereas small peptides have been incorporable at the major capsid proteins fiber, penton and hexon, identified locales within these capsid proteins have not been shown to be suitable for incorporation of molecules of the complexity as represented by antibody-related molecules. On the other hand, several sites, essentially end-terminal regions of certain capsid proteins, have been identified which are permissive of incorporation of antibody-related molecules such as scFv. Thus, the technical means presently exists to achieve genetic incorporation of antibody-related molecules into the Ad capsid at selected locales to accomplish the goal of adenovirus vector targeting.

Attempts to directly incorporate antibody-related molecules, such as scFv, into the Ad capsid have been problematic to this point (see, e.g., Magnusson et al., 2002, J Gene Med 4, 356-370). Specifically, reports to this point have confirmed incorporation of scFv into the Ad capsid, however, antigen recognition/binding of the scFv is not retained. Consideration of the biology embodied within this strategy has led to an understanding of the basis of this phenomenon. In this regard, antibody-related molecules are generally synthesized via normal antibody synthetic pathways - assembly and folding in the RER followed by secretion. On the other hand, adenoviral capsid proteins are synthesized via a distinct pathway - synthesis in the host cell cytosol followed by cytosol-to-nuclear transport and full virion assembly in the nucleus. Of note, the cytosol environment is potentially deleterious to scFv based upon its redox state, and other possible factors. On this basis, it is clear that the non-native routing imposed on scFv by adenovirus capsid incorporation methods can potentially confound proper scFv folding thus perturbing its assumption of the proper conformation required for antigen recognition. This issue would confound any efforts to exploit antibody-related molecules for Ad targeting purposes by capsid incorporation methods.

Another limitation is the necessity to identify (or modify) capsid proteins that are compatible with the incorporation of heterologeous ligands of comparable complexity to scFv (see, e.g., Nicklin & Baker, 2002, Curr Gene Ther 2, 273-293). A means to circumvent these restrictions would be the utilization of genetically engineered scFv that are resistant to any cytosol-induced alterations, hence "stabilized", in combination for example, with radical reconstructions of Ad fiber allowing restrictions on the size and complexity of incorporable targeting ligands to be reduced. Such antibody-related species would thus embody a "stabilization" specifically relevant to allowing it to accomplish cytosol-to-nuclear transport and nuclear residence as an Ad capsid component, while retaining its key conformational aspects dictating antigen recognition/binding. In this regard, a variety of genetic engineering methods are consistent with this concept of stabilization.

On this basis, it is clear that it is the stabilization of the targeting molecule, per se, that allows it to retain antigen recognition in the adenovirus capsid-incorporated context. Further, it is the recognition of this requirement of stabilization at the antibody-related molecule portion that represents the basis of this invention. Without knowledge or practice of this maneuver, that is the use of antibody-related molecules which are stabilized, the employment of such targeting approaches could not be successful.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. SUMMARY OF THE INVENTION

The invention is based, in part, on Applicants' discovery that stabilization of the targeting molecule results in successful incorporation of functional antibody-related molecules into the adenovirus capsid. The genetic incorporation of a stabilized antibody into an adenoviral capsid protein with the retention of scFv functionality and affinity for a target ligand, therefore enabling cell-specific adenovirus targeting, removes the limitations for global therapeutic gene delivery utilizing adenovirus vectors.

The invention encompasses a modified adenovirus (Ad) which may comprise a stabilized antibody. The modified adenovirus may contain a modified fiber protein in addition to the stabilized antibody. For example, the fiber protein may comprise a fiber shaft and fibritin. Li one embodiment, the modified adenovirus (Ad) may comprise a composite fiber comprising an Ad5 fiber shaft connected to twelvth coiled-coil segment of fibritin and a stabilized antibody. In another embodiment, the modified adenovirus may contain a stabilized antibody inserted into a capsid protein. Advantageously, the stabilized antibodies may be inserted into the"minor" capsid proteins, pllla and pIX of adenovirus, pllla and pIX are present on the adenoviral capsid as monomers and trimers, respectively, and the proteins have an extended amino-terminus and carboxy-terminus parts, respectively. Thus, both locale and structural considerations indicate that pllla and pIX are the ideal capsid proteins for incorporating single chain antibodies and other targeting peptides and achieving genetic modification and retargeting of the adenovirus. The minor capsid protein pllla gene may be modified by inserting a DNA sequence encoding a stabilized antibody into the 5' end of the pllla gene, resulting in a stabilized antibody inserted at the N terminus of the pill protein. Similarly, the minor capsid protein pIX gene may be modified by inserting a DNA sequence encoding a single chain antibody into the 3' end of the pIX gene, resulting in a stabilized antibody inserted at the C terminus of the pIX protein.

The stabilized antibody of the present invention encompasses all stabilized antibody- related molecules/fragments known or developed by one of skill in the art. In a preferred embodiment, the stabilized antibody may be a single chain antibody (scFv). In other preferred embodiments, the stabilized antibody may be a mini antibody or a heavy chain variable domain (dAb).

The invention relates to the above-described adenovirus which may comprise a transgene, which may be inserted anywhere within the adenovirus. The invention also encompasses viral vectors, preferably an adenoviral vector comprising the adenovirus of described herein. The invention also provides for transformed host cells comprising such vectors. In one embodiment, the vector may be introduced into the cell by transfection, electroporation or transformation.

The invention also provides for a method for preparing a transformed cell expressing the adenovirus of the present invention which may comprise transfecting, electroporating or transforming a cell with the adenovirus to produce a transformed host cell and maintaining the transformed host cell under biological conditions sufficient for expression of the adenovirus in the host cell. In another embodiment, the invention encompasses a method for inhibiting tumor cell growth in a subject in need thereof which may comprise administering to the subject in need thereof a therapeutically effective amount of the adenovirus described herein wherein the stabilized antibody targets the tumor cell such that the adenovirus infects the tumor cells and thereby inhibits tumor cell growth in the subject. In one embodiment, the adenovirus may further comprise a transgene.

In another embodiment, the invention encompasses methods of increasing the ability of an adenovirus to transduce a specific cell type relative to an unmodified adenovirus. The modification may comprise modifying a composite fiber comprising an Ad5 fiber shaft connected to twelvth coiled-coil segment of fibritin by introducing a DNA sequence encoding a stabilized antibody into the 3' end of the composite fiber gene, wherein said modification increases the ability of said adenovirus to transduce a specific cell type relative to an unmodified adenovirus. In another embodiment, the modification may comprise modifying a gene encoding an adenoviral capsid protein by introducing a DNA sequence encoding a stabilized antibody into the 3 ' end of the minor capsid protein pIX gene, wherein said modification increases the ability of said adenovirus to transduce a specific cell type relative to an unmodified adenovirus.

In either method of increasing the ability of an adenovirus to transduce a specific cell type relative to an unmodified adenovirus, the stabilized antibody may be directed towards a protein, wherein said protein is specific to a cell type. Advantageously, the cell type is is a dendritic cell, fibroblast, immune cell, keratinocyte or tumor cell. In another embodiment, the protein is a cell-surface protein. In a preferred embodiment, the stabilized antibody is a single chain antibody (scFv).

Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention given for the purpose of disclosure.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of and "consists essentially of have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages and objects- of the invention, as well as others which will become clear, are attained and can be understood in detail, more particular descriptions of the invention briefly summarized above may be had by reference to certain embodiments thereof which are illustrated in the appended drawings. These drawings form a part of the specification. It is to be noted, however, that the appended drawings illustrate preferred embodiments of the invention and therefore are not to be considered limiting in their scope.

FIG. IA shows pKanl 1F-Bael, which is the starting plasmid to insert the cDNA of a stabilized antibody next to the 1 IF fiber.

FIG. IB shows the sequence of pKanl 1F-Bael (SEQ ID NO: 1).

FIG. 1C shows pKanl 1-F-a-ALK, where a-ALK located from base pairs 2108-2890, with the VL domain from base pairs 2108 - 2452 and the V_H domain from base pairs 2513 - 2890.

FIG. ID shows the sequence of pKanl lF-a-ALK (SEQ ID NO: 2)

FIG. IE shows pKanl 1F-FW4.4, where FW4.4 located from base pairs 2108-2890, with the VL domain from base pairs 2108 - 2452 and the V_H domain from base pairs 2513 — 289.

FIG. IF shows the sequence of pKanllF-FW4.4 (SEQ ID NO: 3)

FIG. 2A shows pSILucIXNhe, which is the starting plasmid to insert the cDNA of a stabilized antibody, at the C terminus of pIX.

FIG. 2B shows the sequence of pSILucIXNhe (SEQ ID NO: 4). FIG. 2C shows pSILucIX-a-ALK, where a-ALK located from base pairs 3570 - 4355, with the VL domain from base pairs 3570 - 3914 and the VH domain from base pairs 3975 - 4355.

FIG. 2D shows the sequence of pSILucIX-a-ALK (SEQ ID NO: 5).

FIG. 2E shows pSILucIX-FW4.4, where FW4.4 located from base pairs 3570 - 4355, with the V_L domain from base pairs 3570 - 3914 and the VH domain from base pairs 3975 - 4355.

FIG. 2F shows the sequence of pSILucIX-FW4.4 (SEQ ID NO: 6).

FIG 3 A shows the binding of CsCl purified virions to ALK recombinant antigen. CsCl purified virions from 28 cells (mosaic), 293 cells (non-mosaic) or 293-TAYT (TAYT- mosaic), as indicated in the figure, were tested for ability to bind to GST-ALK recombinant antigen. Ad.1 lF-a-ALK (diamond) and Ad.11F-FW4.4 (square) were compared with control virus Ad5.1ucl (triangle) at the virus particles/well as indicated on the graphs. Absorbance was measured at 450nm. FIG. 3B shows the binding of CsCl purified pIX-a-ALK virions to ALK recombinant antigen. CsCl purified virions from 293 cells were tested for ability to bind to GST-ALK recombinant antigen. Ad.pIX-a-ALK (diamond) and Ad.pIX-FW4.4 (triangle) are shown. Virions were titered from an initial concentration of l.lxlO¹⁰ vp/well in the 1/3 group. Data shown with non-specific background signal subtracted. Absorbance was measured at 490 run.

FIG. 4 shows targeted gene transfer as a function of luciferase activity. Parental or clonal (A 16) cells known to express an artificial receptor were transduced with either lOOOvp/cell of either Ad5.1ucl, Ad.luc.l 1F-FW4.4 or Ad.luc.l lF-a-ALK (for 1 hour on ice). Prior to virus transduction cells virus was coated with GST-ALK (2ug per 1 x 10⁹ vp, white bars, unblocked virus in hatch bars) (for 15 minutes at RT). Cells were then cultured for a further 24 hours before luciferase activity was assessed by standard methods. DETAILED DESCRIPTION

The invention encompasses a modified adenovirus (Ad), which may comprise a stabilized antibody as a targeting ligand. The modified adenovirus may contain a modified fiber protein in addition to the stabilized antibody. For example, the fiber protein may comprise a fiber shaft and fibritin. Accordingly, the present invention encompasses chimeric fiber-fibritin proteins. For example, the modified adenovirus may comprise a composite fiber with a fiber shaft connected to one of the coil segments of fibritin. In one embodiment, the modified adenovirus (Ad) may comprise a composite fiber comprising an Ad5 fiber shaft connected to twelvth coiled-coil segment of fibritin and a stabilized antibody. A cDNA encoding the stabilized antibody may be inserted 3' (downstream) or 5' (upstream) of the cDNA encoding the composite fiber. Advantageously, the cDNA encoding the stabilized antibody is adjacent to the cDNA encoding the composite fiber. In a preferred embodiment, the stabilized antibody may be downstream of the composite fiber.

In another embodiment, the modified adenovirus may contain a stabilized antibody inserted into a capsid protein. Advantageously, the stabilized antibodies may be inserted into the"minor" capsid proteins, pllla and plX of adenovirus, pllla and pFX are present on the adenoviral capsid as monomers and trimers, respectively, and the proteins have an extended amino-terminus and carboxy-termimαs, respectively. Thus, both locale and structural considerations indicate that pllla and pIX are the ideal capsid proteins for incorporating single chain antibodies and other targeting peptides and achieving genetic modification and retargeting of the adenovirus. The minor capsid protein pllla gene may be modified by inserting a DNA sequence encoding a stabilized antibody into the 5' end of the pllla gene, resulting in a stabilized antibody inserted at the N terminus of the pill protein. Similarly, the minor capsid protein pIX gene may be modified by inserting a DNA sequence encoding a single chain antibody into the 3' end of the pIX gene, resulting in a stabilized antibody inserted at the C terminus of the pIX protein. The stabilized antibody of the present invention encompasses all stabilized antibodies known or developed by one of skill in the art. In a preferred embodiment, the stabilized antibody may be a single chain antibody (scFv), such as a humanized scFv (see, e.g., Graff et al. in Protein Eng Des SeI. 2004 Apr;17(4):293-304). The stabilized antibodies of the present invention also encompass disulfide stabilized antibodies, wherein the heavy and light chains of the antibody are associated by disulfide bonds rather than a peptide linker (see, e.g., U.S. Patent Nos. 6,639,057 and 6,538,111). In other preferred embodiments, the stabilized antibody may be a mini antibody or a heavy chain variable domain (dAb) (see, e.g., Jespers et al. in Nat Biotechnol. 2004 Sep;22(9):l 161-5). In yet another embodiment, the stabilized antibody may be a polymer conjugates which exhibits stabilized antibody binding capacity (see, e.g., U.S. Patent Nos. 6,538,104 and 6,491,923). The invention also encompasses stabilized antibodies produced by the method of U.S. Patent No. 6,262,238 wherein stabilized antibodies free of disulfide bridges are obtained by substituting the cysteines which form disulfide bridges by other amino acids and replacing at least one, and preferably two or more amino acids by stability-mediating amino acids. The invention also encompasses the stabilized, divalent antigen-binding antibody fragments of U.S. Patent No. 5,506,342. The only requirement for the stabilized antibodies of the present invention is the ability of the stabilized antibody to accomplish cytosol-to-nuclear transport and nuclear residence as an Ad capsid component, while retaining its key conformational aspects dictating antigen recognition and binding. In another embodiment of the invention, the stabilized scFv ligand comprises mutations in the scFv CDR regions. Any mutations, which preserve an ability of scFv in the context of Ad capsid to bind an antigen are suitable for methods of the invention. Examples of scFv stabilizing mutations include, but are not limited to, those mutations described in Arndt et al., J MoI Biol 2001 Sep 7;312(l):221-8; Bestagno et al., Biochemistry 2001 Sep 4;40(35):10686-92 and Rajpal et al., Proteins 2000 JuI l;40(l):49-57, the disclosures of which are incorporated by reference. A stabilized scFv "framework" is developed via directed mutations in the scFv CDR regions. These stabilized CDRs' framework can then serve as a scaffold onto which scFv variable domains, which embody antigen recognition, can then be grafted by molecular engineering methods. The chimeric scFv thus manifests the desired antigen recognition profile while also embodying the stability of the scaffold CDR domain.

In a preferred embodiment, the stabilized antibody is targeted to a cell surface marker of a tumor cell. Cell surface markers that can be targeted according to the methods of the present invention include, but are not limited to, CD40, DC-SIGN, DEC-205, CEA and PSMA. hi a preferred embodiment, the stabilized scFv ligand is an anti-CD40 scFv.

In one embodiment, the adenovirus carries in its genome a transgene, which can be therapeutic gene. A representative example of a therapeutic gene is a herpes simplex virus thymidine kinase gene. Other target transgenes include, but are not limited to, cytosine deaminase (CD) and a fusion of cytosine deaminase and uracilphosphoribosyltransferase (CD/UPRT). hi another embodiment, the invention encompasses a method for inhibiting tumor cell growth in a subject in need thereof comprising administering to the subject in need thereof a therapeutically effective amount of the adenovirus described herein wherein the scFv ligand targets the tumor cell such that the adenovirus infects the tumor cells and thereby inhibits tumor cell growth in the subject, hi one embodiment, the adenovirus further comprises a transgene. hi an embodiment wherein the transgene is herpes simplex virus thymidine kinase the method for inhibiting tumor cell growth can optionally comprise administering ganciclovir. Another agent that can be co administered in combination with a transgene is 5- fluorocytosine (5FC).

In another embodiment, the invention encompasses methods of increasing the ability of an adenovirus to transduce a specific cell type relative to an unmodified adenovirus. Reference is made to U.S. Patent No. 6,955,808. The modification may comprise modifying a composite fiber comprising an Ad5 fiber shaft connected to twelvth coiled-coil segment of fibritin by introducing a DNA sequence encoding a stabilized antibody into the 3 ' end of the composite fiber gene, wherein said modification increases the ability of said adenovirus to transduce a specific cell type relative to an unmodified adenovirus. In another embodiment, the modification may comprise modifying a gene encoding an adenoviral capsid protein by introducing a DNA sequence encoding a stabilized antibody into the 3' end of the minor capsid protein pIX gene, wherein said modification increases the ability of said adenovirus to transduce a specific cell type relative to an unmodified adenovirus.

In either method of increasing the ability of an adenovirus to transduce a specific cell type relative to an unmodified adenovirus, the stabilized antibody may be directed towards a protein, wherein said protein is specific to a cell type. Advantageously, the cell type is is a dendritic cell, fibroblast, immune cell, keratinocyte or tumor cell. In another embodiment, the protein is a cell-surface protein. In a preferred embodiment, the stabilized antibody is a single chain antibody (scFv).

In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis, Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed. 1985); "Oligonucleotide Synthesis" (MJ. Gait ed. 1984); "Nucleic Acid Hybridization" [B.D. Hames & SJ. Higgins eds. (1985)]; "Transcription and Translation" [B.D. Hames & SJ. Higgins eds. (1984)]; "Animal Cell

Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984). Therefore, .if appearing herein, the following terms shall have the terminology set out below.

A "DNA molecule" refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).

A "vector" is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A "replicon" is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An "origin of replication" refers to those DNA sequences that participate in DNA synthesis. An "expression control sequence" is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is "operably linked" and "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.

In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.

A DNA "coding sequence" is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence. A "cDNA" is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript.

Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A "cis-element" is a nucleotide sequence, also termed a "consensus sequence" or "motif, that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A "signal sequence" can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.

A "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.

The term "oligonucleotide" is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term "primer" as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use for the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.

The primers herein are selected to be "substantially" complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence to hybridize therewith and thereby form the template for the synthesis of the extension product.

As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to enzymes which cut double-stranded DNA at or near a specific nucleotide sequence.

"Recombinant DNA technology" refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include "gene splicing", "molecular cloning" and "genetic engineering". The product of these manipulations results in a "recombinant" or "recombinant molecule". A cell has been "transformed" or "transfected" with exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A "clone" is a population of cells derived from a single cell or ancestor by mitosis. A "cell line" is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed "transgenic". As used herein, the term "host" is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells and plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.

Two DNA sequences are "substantially homologous" when at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, preferably at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at and most preferably at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning, VoIs. I & II, supra; Nucleic Acid Hybridization, supra.

A "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, the coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. For example, a polynucleotide, may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter. In addition, the invention may include portions or fragments of the fiber or fibritin genes. As used herein, "fragment" or "portion" as applied to a gene or a polypeptide, will ordinarily be at least about 10 residues, at least about 11 residues, at least about 12 residues, at least about 13 residues, at least about 14 residues, at least about 15 residues, at least about 16 residues, at least about 17 residues, at least about 18 residues, at least about 19 residues, more typically at least about 20 residues, residues, at least about 21 residues, at least about 22 residues, at least about 23 residues, at least about 24 residues, at least about 25 residues, at least about 26 residues, at least about 27 residues, at least about 28 residues, at least about 29 residues and preferably at least about 30 residues in length, at least 31 residues, at least 32 residues, at least 33 residues, at least 34 residues, at least 35 residues, at least 36 residues, at least 37 residues, at least 38 residues, at least 39 residues, at least 40 residues, at least 41 residues, at least 42 residues, at least 43 residues, at least 44 residues, at least 45 residues, at least 46 residues, at least 47 residues, at least 48 residues, at least 49 residues or at least 50 residues but less than the entire, intact sequence. Fragments of these genes can be generated by methods known to those skilled in the art, e.g., by restriction digestion of naturally occurring or recombinant fiber or fibritin genes, by recombinant DNA techniques using a vector that encodes a defined fragment of the fiber or fibritin gene, or by chemical synthesis.

As used herein, "chimera" or "chimeric" refers to a single transcription unit possessing multiple components, often but not necessarily from different organisms. As used herein, "chimeric" is used to refer to tandemly arranged coding sequence (in this case, that which usually codes for the adenovirus fiber gene) that have been genetically engineered to result in a protein possessing region corresponding to the functions or activities of the individual coding sequences.

The "native biosynthesis profile" of the chimeric fiber protein as used herein is defined as exhibiting correct trimerization, proper association with the adenovirus capsid, ability of the ligand to bind its target, etc. The ability of a candidate chimeric fiber-fibritin- ligand protein fragment to exhibit the "native biosynthesis profile" can be assessed by methods described herein..

A standard northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue in accordance with conventional northern hybridization techniques known to those persons of ordinary skill in the art. Alternatively, a standard Southern blot assay may be used to confirm the presence and the copy number of the gene of interest in accordance with conventional Southern hybridization techniques known to those of ordinary skill in the art. Both the northern blot and Southern blot use a hybridization probe, e.g. radiolabeled cDNA or oligonucleotide of at least about 20, at least about 21 , at least about 22, at least about , at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, preferably at least 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, more preferably at least 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 71, at least about 72, at least about 73, at least about 74, at least about 75, at least about 76, at least about 77, at least about 78, at least about 79, at least about 80, at least about 81, at least about 82, at least about 83, at least about 84, at least about 85, at least about 86, at least about 87, at least about 88, at least about 89, at least about 90, at least about 91, at least about 92, at least about 93, at least about 94, at least about 95, at least about 96, at least about 97, at least about 98, at least about 99, at least about and most preferably at least 100 consecutive nucleotides in length. The DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art.

Hybridization reactions can be performed under conditions of different "stringency." Conditions that increase stringency of a hybridization reaction are well known. See for examples, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al. 1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25⁰C, 37⁰C, 5O⁰C, and 68⁰C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2 or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6 x SSC, 1 x SSC, 0.1 x SSC, or deionized water.

The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope maybe selected from ³H, ¹⁴C, ³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵1, ¹³¹I, and ¹⁸⁶Re.

Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fiuorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β -glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.

As used herein, the terms "fiber gene" and "fiber" refer to the gene encoding the adenovirus fiber protein. As used herein, "chimeric fiber protein" refers to a modified fiber gene as described above. As used herein the term "physiologic ligand" refers to a ligand for a cell surface receptor.

The present invention is directed to a vector system that provides both a highly efficient and specific targeting of adenovirus vector for the purpose of in vivo gene delivery to predefined cell types after administration. In the recombinant adenoviral vector of the present invention, a fiber replacement protein comprising a fiber-fibritin-ligand is employed to target adenoviral vector to a specific cell for gene therapy. This is accomplished by the construction of adenoviral vectors which contain fiber-fibritin-ligand chimeras. These adenoviral vectors are capable of delivering gene products with high efficiency and specificity to cells expressing receptors which recognize the ligand component of the fiber- fibritin-ligand chimera. A person having ordinary skill in this art would recognize that one may exploit a wide variety of genes encoding e.g. receptor ligands or antibody fragments which specifically recognize cell surface proteins unique to a particular cell type to be targeted. The invention also encompasses viral vectors, preferably an adenoviral vector comprising the adenovirus of described herein. In one embodiment, adenovirus is operatively linked to a non- viral promoter.

Methods for making and/or administering a vector or recombinants or plasmid for expression of gene products of genes of the invention either in vivo or in vitro can be any desired method, e.g., a method which is by or analogous to the methods disclosed in, or disclosed in documents cited in: U.S. Patent Nos. 4,603,112; 4,769,330; 4,394,448;

4,722,848; 4,745,051; 4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744,141;

5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941; 5,338,683; 5,494,807;

5,591,639; 5,589,466; 5,677,178; 5,591,439; 5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473; 6,368,603;

6,348,196; 6,306,400; 6,228,846; 6,221,362; 6,217,883; 6,207,166; 6,207,165; 6,159,477;

6,153,199; 6,090,393; 6,074,649; 6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882;

6,312,682; 6,348,450 and 6; 312,683; U.S. patent application Serial No. 920,197, filed

October 16,1986; WO 90/01543; W091/11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785; EP 0 370 573; Andreansky et al., Proc. Natl. Acad. Sci. USA

1996;93:11313-11318; Ballay et al., EMBO J. 1993;4:3861-65; Feigner et al., J. Biol. Chem.

1994;269:2550-2561; Frolov et al., Proc. Natl. Acad. Sci. USA 1996;93:11371-11377;

Graham, Tibtech 1990;8:85-87; Grunhaus et al., Sem. Virol. 1992;3:237-52; Ju et al.,

Diabetologia 1998;41 -.736-739; Kitson et al., J. Virol. 1991;65:3068-3075; McClements et al., Proc. Natl. Acad. Sci. USA 1996;93:11414-11420; Moss, Proc. Natl. Acad. Sci. USA

1996;93:11341-11348; Paoletti, Proc. Natl. Acad. Sci. USA 1996;93:11349-11353; Pennock et al., MoI. Cell. Biol. 1984;4:399-406; Richardson (Ed), Methods in Molecular Biology

1995;39, "Baculovirus Expression Protocols," Humana Press Inc.; Smith et al. (1983) MoI.

Cell. Biol. 1983;3:2156-2165; Robertson et al., Proc. Natl. Acad. Sci. USA 1996;93:11334- 11340; Robinson et al., Sem. Immunol. 1997;9:271; and Roizman, Proc. Natl. Acad. Sci.

USA 1996;93:11307-11312.

According to one embodiment of the invention, the expression vector is a viral vector, in particular an in vivo expression vector. In an advantageous embodiment, the expression vector is an adenovirus vector, such as a human adenovirus (HAV) or a canine adenovirus (CAV). Advantageously, the adenovirus is a human Ad5 vector, an El -deleted adenovirus or an E3 -deleted adenovirus.

The adenovirus vector not need to be limited to the El and E3 region, but can be deleted in any region as long as it is grown in a comlementary cell line to the deletion (eg 293-ORF6 cell line which complements the E4 region; see my patents from GenVec)

The adenovirus can be a CRAd that is able to grow without complementation of the El region which can be grown in other cells (eg A549 or HeLa).

The adenovirus can be a "gutted" or helper dependent (HD) vector that is complemented by an other helper vector and not by the cells. In one embodiment the viral vector is a human adenovirus, in particular a serotype 5 adenovirus, rendered deficient for replication by a deletion in the El region of the viral genome. The deleted adenovirus is propagated in El -expressing cell line. Exemples of this type of cells include, but are not limited to, HEK 293 cells or PER.C6 (F. Falloux et al Human Gene Therapy 1998, 9, 1909-1917). The human adenovirus can be deleted in the E3 region eventually in combination with a deletion in the El region {see, e.g. J. Shriver et al. Nature, 2002, 415, 331-335, F. Graham et al Methods in Molecular Biology VoI .7: Gene Transfer and Expression Protocols Edited by E. Murray, The Human Press Inc, 1991, p 109- 128; Y. Ilan et al Proc. Natl. Acad. Sci. 1997, 94, 2587-2592; S. Tripathy et al Proc. Natl. Acad. Sci. 1994, 91, 11557-11561; B. Tapnell Adv. Drug Deliv. Rev.1993, 12, 185-199;X. Danthinne et al Gene Thrapy 2000, 7, 1707-1714; K. Berkner Bio Techniques 1988, 6, 616- 629; K. Berkner et al Nucl. Acid Res. 1983, 11, 6003-6020; C. Chavier et al J. Virol. 1996, 70, 4805-4810). The insertion sites can be the El and/or E3 loci eventually after a partial or complete deletion of the El and/or E3 regions. Advantageously, when the expression vector is an adenovirus, the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells, such as a strong promoter, preferably a cytomegalovirus immediate-early gene promoter (CMV-IE promoter). The CMV-IE promoter is advantageously of murine or human origin. The promoter of the elongation factor lα can also be used. In one particular embodiment a promoter regulated by hypoxia, e.g. the promoter HRE described in K. Boast et al Human Gene Therapy 1999, 13, 2197-2208), can be used. A muscle specific promoter can also be used (X. Li et al Nat. Biotechnol. 1999, 17, 241-245). Strong promoters are also discussed herein in relation to plasmid vectors. A poly(A) sequence and terminator sequence can be inserted downstream the polynucleotide to be expressed, e.g. a bovine growth hormone gene or a rabbit β-globin gene polyadenylation signal. In another embodiment the viral vector is a canine adenovirus, in particular a CAV-2 (see, e.g. L. Fischer et al. Vaccine, 2002, 20, 3485-3497; U.S. Patent No. 5,529,780; U.S. Patent No. 5,688,920; PCT Application No. WO95/14102). For CAV, the insertion sites can be in the E3 region and /or in the region located between the E4 region and the right ITR region (see U.S. Patent No. 6,090,393; U.S. Patent No. 6,156,567). In one embodiment the insert is under the control of a promoter, such as a cytomegalovirus immediate-early gene promoter (CMV-IE promoter) or a promoter already described for a human adenovirus vector. A poly(A) sequence and terminator sequence can be inserted downstream the polynucleotide to be expressed, e.g. a bovine growth hormone gene or a rabbit β-globin gene polyadenylation signal.

The invention also provides for transformed host cells comprising such vectors, hi one embodiment, the vector is introduced into the cell by transfection, electroporation or transformation. The invention also provides for a method for preparing a transformed cell expressing the adenovirus of the present invention comprising transfecting, electroporating or transforming a cell with the adenovirus to produce a transformed host cell and maintaining the transformed host cell under biological conditions sufficient for expression of the adenovirus in the host cell.

According to another embodiment of the invention, the expression vectors are expression vectors used for the in vitro expression of proteins in an appropriate cell system. The expressed proteins can be harvested in or from the culture supernatant after, or not after secretion (if there is no secretion a cell lysis typically occurs or is performed), optionally concentrated by concentration methods such as ultrafiltration and/or purified by purification means, such as affinity, ion exchange or gel filtration-type chromatography methods.

It is understood to one of skill in the art that conditions for culturing a host cell varies according to the particular gene and that routine experimentation is necessary at times to determine the optimal conditions for culturing the vector depending on the host cell. A "host cell" denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.

Polynucleotides comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides can be introduced into host cells by any means known in the art. The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including direct uptake, endocytosis, transfection, f-mating, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is infectious, for instance, a retroviral vector). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.

A "fiber replacement protein" is a protein that substitutes for fiber and provides three essential features: trimerizes like fiber, lacks adenoviral tropism and has novel tropism. As used herein, "chimera" or "chimeric" refers to a single polypeptide possessing multiple components, often but not necessarily from different organisms. As used herein, "chimeric" is used to refer to tandemly arranged protein moieties that have been genetically engineered to result in a fusion protein possessing regions corresponding to the functions or activities of the individual protein moieties.

As used herein, the terms "fiber gene" refer to the gene encoding the adenovirus fiber protein. As used herein, "chimeric fiber protein" refers to a modified fiber as defined above.

As used herein the term "physiologic ligand" refers to a ligand for a cell surface receptor.

In addition, the invention may include portions or fragments of the fiber or fibritin proteins. As used herein, "fragment" or "portion" as applied to a protein or a polypeptide, will ordinarily be at least 10 residues, at least 11 residues, at least 12 residues, at least 13 residues, at Ieastl4 residues, at least 15 residues, at least 16 residues, at least 17 residues, at least 18 residues, at least 19 residues, more typically at least 20 residues, at least 21 residues, at least 22 residues, at least 23 residues, at least 24 residues, at least 25 residues, at least 26 residues, at least 27 residues, at least 28 residues, at least 29 residues and preferably at least 30 residues in length, at least 31 residues, at least 32 residues, at least 33 residues, at least 34 residues, at least 35 residues, at least 36 residues, at least 37 residues, at least 38 residues, at least 39 residues, at least 40 residues, at least 41 residues, at least 42 residues, at least 43 residues, at least 44 residues, at least 45 residues, at least 46 residues, at least 47 residues, at least 48 residues, at least 49 residues or at least 50 residues but less than the entire, intact sequence. Fragments of these genes can be generated by methods known to those skilled in the art, e.g., by restriction digestion of naturally occurring or recombinant fiber or fibritin genes, by recombinant DNA techniques using a vector that encodes a defined fragment of the fiber or fibritin gene, or by chemical synthesis. The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. EXAMPLES Example 1: Ad vectors Construction of 1 lF-scFv fiber containing Ad vectors. 1 IF was a composite fiber composed of the entire Ad5 fiber shaft connected to 12^th coiled-coil segment of fibritin. The predominant central region of fibritin has 13 consecutive α-helical coiled-coil segments linked by loops, while the predominant central region of the Ad5 fiber (shaft) consists of 22 β-repeats. pKanl 1F-Bael (FIGS. IA and IB) was the starting plasmid used to insert the cDNA of either a scFv or a scFv scaffold next to the 1 IF fiber. The insertion was by a PCR procedure to create Bael ends on the scFv cDNA and ligate the resulting fragment into the Bael site in this vector. An EcoRl digest opened up the plasmids to allow for recombination into the Ad rescue backbone, pVK700, a modified Ad5 genome, which allows for modified fibers to be recombined into the genome. Construction of pIX-scFv containing Ad vectors. pSILucIXNhe (FIGS. 2 A and 2B) was the starting plasmid used to insert the cDNA of either a scFv or a scFv scaffold at the C terminus of pIX. There was a short amino acid sequence following the C terminus of pIX, including a FLAG tag, which precedes the Nhel cloning site. PCR procedures created TSfhel ends on the scFv cDNA and the resulting fragment was ligated into the Nhel site in this vector. Standard pAdEasy techniques generated the viruses, including digesting the plasmids with Pmel to allow for recombination with commercially available vector pAdEasy. Example 3: Analysis of CsCl purified virions binding to ALK recombinant antigen

ALK scFv Ta-ALK) fused to 1 IF chimeric fiber. Three variants of 1 lF-a-ALK fiber containing adenoviral virions (and the control negative scFv, FW4.4) were prepared on (1) 28 cells, virions described as mosaic as they contain both 1 lF-a-ALK and Ad5 fibers, (2) 293 cells, described as non-mosaic as they contain only 1 lF-a-ALK fibers, and (3) 293-TAYT cells, described as TAYT-mosaic as they contain both 1 lF-a-ALK and TAYT Ad5 fibers. Binding analysis of these virion variants to the'ALK recombinant protein are shown in FIG. 3A, and demonstrate that all three versions of Ad.l lF-a-ALK could recognize recombinant ALK protein (naturally Ad.11F-FW4.4 does not, as this is the non-binder version of the single chain). There are differences in level of signal as the 1 lF-a-ALK fibers incorporate into virions at different levels depending on the cells the viruses are propagated upon.

ALK scFv Ca-ALK) fused to pDC capsid protein. An adenovirus with a- ALK fused or the negative scFv and an adenovirus with FW4.4 fused to the minor capsid protein, pIX were prepared. Binding analysis of this pIX-a-ALK virus to the ALK recombinant protein is shown in FIG. 3B and demonstrates that this virus recognizes the ALK recombinant protein. Example 4: Targeted gene transfer with Ad containing fibers fused with stabilized scFv.

Parental or clonal (Al 6) cells known to express GST-ALK in an artificial cell surface receptor were transduced with either lOOOvp/cell of either Ad5.1ucl, Ad.luc.l 1F-FW4.4 or Ad.luc.l lF-a-ALK for 1 hour on ice. Prior to virus transduction cells virus was coated with. GST-ALK (2ug per 1 x 1O⁹Vp) (for 15 minutes at RT). Cells were then cultured for a further 24 hours.

There is 10 fold increase in gene transfer with Ad.luc.l lF-a- ALK, when comparing parental and clonal cells as shown in FIG. 4. This increase is clearly blocked when the virus is precoated with the GST-ALK. Example 5: Stabilized antibodies

One method of directed evolution to increase the stability of a scFv is described by Graff et al. in Protein Eng Des SeI. 2004 Apr;17(4):293-304, the disclosure of which is incorporated by reference in its entirety. This method may be utilized for the selection of stabilized antibodies, such as stabilized scFv, for the invention described herein.

A random scFv library of humanized MFE-23 scFv (hMFE) is created by adapting the nucleotide analogue method of Zaccolo & Gerardi, 1999, J. MoI. Biol. 285:775-783. MFE- 23 scFV is an antibody fragment that targets colon cancer effectively for radioimmunodetection in vivo (see, e.g., Chester et al., 1994, Lancet 343:455-456 and Begent et al., 1996, Nat. Med. 2:979-984). The expression cassette of hMFE is amplified by PCR with the T3 and T7 promoter standard primers. Five mutagenic PCR conditions are used: 250 mM dPTP and 8-oxo-dGTP/five cycles; 25 mM dPTP and 8-oxo-dGTP/10 cycles; 25 mM mM dPTP and 8-oxo-dGTP/20 cycles. Other PCR components are 1 ng of pCThMFE, 250 mM each dNTP, 0.5 mM each primer, 3 units Taq polymerase (Gibco), 1 X Gibco PCR buffer supplemented with 2 mM MgCl₂. The reaction is cycled as follows: 94 C 1 min, 50 C 1 min, 72 C 3.5 min. PCR products from 10 and 20 cycles are isolated on a 1% agarose gel. Four 2200 bp fragments are excised from the gel and purified with a gel purification kit (Qiagen). PCR products from five cycles are diluted 1 : 10 for further amplification. The PCR products are then amplified in the absence of the nucleotide analogues for 25 cycles using Taq polymerase. The PCR conditions are identical with those listed above, with the exception of extension time at 72 C, which is changed to 3 min and 10 s. PCR products are purified using a PCR purification kit (Qiagen). Purified PCR fragments are digested with MeI and BamRl, gel purified and ligated overnight at 16 C into pCThMFE. Ligation reactions are transformed into 10 aliquots of DH5α-FT cells (Life Technologies). Transformants ar epooled and aliquots are plated to determine the library size. The library is amplified in LB/Amp50/Carb50 medium at 37 C and plasmid DNA is purified with a Qiagen Mazi-prep kit. Ten clones are selected from sequencing from the library and the mutagenesis rate is calculated. Library DNA is transformed into yeast strain EBYlOO (Boder & Wittrup, 1997, Nat. Biotechnol. 15:553-557) by the lithium acetate method of Gietz and co-workers (tto.trends.com). Transformants are pooled in SD-CAA (2% dextrose, 0.67% yeast nitrogen base, 1% casamino acids) and aliquots are placed to determine library diversity. The library is passaged twice in SD-CAA to reduce the concentration of untransformed EBYlOO. The library is grown to OD₆₀₀ = 10.0 in SD-CAA. Cells are transferred to SG-CAA to

OD₆₀₀ = 1.0 and grown for 18 h at 37 C. The hMFE library is screened by the equilibrium method as described in Boder & Wittrup, 1998, Biotechnol. Prog., 14:55-62. The optimal biotinylated CEA concentration is calculated from the mathematical model. Concentrations are selected to screen for 3- and 10-fold improvements. Yeast cells displaying mutated hMFE scFv are incubated in 0.35 or 0.2 nM biotinylated CEA at 37 C. CEA purified from primary human tumor samples (Calbiochem) are biotinylated by the succinimidyl eseter method on primary amines (Molecular Probes). Cells are washed with cold PBS-BSA (8 g/1 NaCl, 0.2 g/1 KCl, 1.44 g/1 Na₂HPO₄, 0.24 g/1 KH₂PO₄, 1 mg/ml BSA) and labeled with a 1 : 100 dilution of the monoclonal antibody 9El 0 on ice (Covance). Cells are washed with cold PSB-BSA and labeled with secondary reagents streptavidin-phycoerythrin (1:100)

(Pharmingen) and goat anti-mouse-FITC (1 :50) (Signa) on ice. Cells are washed with PBS- BSA and resuspended at a concentration of 10⁷ cells/ml and sorted on a Becton Dickinson FACStar flow cytometer (Flow Cytometry Center, MIT Cancer Research Center) with a sort rate of ~ 4000 cells/s. Cells are collected with gate settings designed to collect the cells displaying the highest PE fluorescent signal (carcinoembryonic antigen (CEA) binding) per FITC fluorescent signal (scFvs on surface). Four rounds of sorting and regrowth are performed to isolate a highly enriched (100%) population of improved mutants. Mutants are analyzed from both the third- and fourth-round sorts.

The random hMFE library is also screened from improved stability of the scFv. Cells are induced at 37 C. Yeast cells are incubated in 10 nM biotinylated CEA at 37 C. Cells are transferred to microcentrifuge tubes, washed with ice cold PBS-BSA and incubated in a 1:100 dilution of 9E10 (Covance) on ice. Cells are labeled with secondary reagents as described previously. Cells are sorted at a concentration of 10 cells/ml on a Becton Dickinson FACStar flow cytometer. Cells are sorted with gate settings designed to collect the highest FITC signal for the CEA binding population. Three rounds of sorting and regrowth are performed to isolate an enriched population of better displayed mutants. Sixteen mutants are analyzed from the third sort. For each clone, cells are labeled with 10 nM bio-CEA and 9E10. Six clones with higher display levels are selected for sequence analysis. Plasmids are rescued and sequenced as described previously. A single common stabilizing mutations, V_L W47L, is identified.

Another method for selecting for aggregation-resistant domain antibodies on phage is by heat denaturation, as described by Jespers et al. in Nat Biotechnol. 2004 Sep;22(9):l 161-5, the disclosure of which is incorporated by reference. This method may be utilized for the selection of stabilized antibodies, such as stabilized dABs, for the invention described herein. A human V_H dAb (HEL4) with biophysical properties similar to those of camels and llamas was identified (Jespers et al., 2004, J. MoI. Biol. 337:893-903). For example, the HEL4 dAb unfolds reversibly above 62.1 C (T_m, midpoint transition temperature) at concentrations as high as 56 μM. In contrast, heating a 5.0 μM solution of the DP47d dAb (a typical human V_H dAb encoded by the same germline gene as the HEL4 dAb) above 55 C led to irreversible unfolding and formation of aggregates (Jespers et al., 2004, J. MoI. Biol. 337:893-903). The human BElA dAb is devoid of mutations in the β-sheet scaffold and differs from the DP47d dAb only by mutation sin the loops comprising the complementarity- determining region^'s (CDRs). This suggests that the property of reversible unfolding among human VH dAbs may be more common than expected. The HEL4 and DP47d dAbs are therefore used to develop a method for the selection of human VH dAbs that unfold reversibly from those that aggregate irreversibly.

For phage display of a human V_H dAb library, two dAb repertoires are used and both are created in two steps by oligonucleotide-mediated diversification of several positions in the sequence of the DP47d dAb. The PCR-amplified DNA inserts are ligated into a fd bacteriophage vector with a tetracycline resistance gene that contains a c-myc tag between the leader sequence and gene III. The ligation products are transformed by electroporation into E. coli TGl or TBl cells and then plated on TYE plates (Trypton Yeast Extract) supplemented with 15 μg/1 of tetracycline (TYE-Tet), yielding library sizes of about J.6 x 10⁹ to 1.6 x 10¹⁰ clones. For monovalent display, dAb genes are ligated into a phagemid vector that contain the (His)₈ and VSV tags and gene III. Phage are prepared, purified and stores as described in McCafferty et al., 1990, Nature 348:552-554. For the analysis of phage proteins, 10¹⁰ are subjected to SDS PAGE (4% to 12% Bis-Tris gel; Invitrogen), and transferred to a PVDF Immobilon-P membrane (Millipore) for detection, the blocked membrane is incubated with murine anti-pill antibody (MoBiTec), then anti-murine horseradish peroxidase conjugate (Sigma-Aldrich) and electro-chemiluminscence reagents (Amersham Biosciences).

The HEL4 and DP47d dAbs are displayed in a multivalent state on the surface of filamentous bacteriophage, thereby providing a link between antibody phenotype and genotype and a means of selection (McCafferty et al., 1990, Nature 348:552-554). A selection wherein phage displaying the HEL4 and DP47d dAbs are mixed in 1 : 10 ratio respectively, incubated at 80 C for 10 min, cooled and selected on immobilized protein A. Bound phage are eluted with trypsin and used to reinfect bacteria. After two rounds of such selection, supernatants from infected colonies are tested by ELISA on immobilized hen egg lysozyme (HEL) to distinguish the HEL4 phage from the DP47d phage. By two rounds of heat denaturation and biopaniiing on protein A, phage dAbs that resist thermal aggregation are selected from those that do not. Next, a reportoire of human V_H dAbs is prepared by diversification of the loops comprising the CDRs in the DP47d dAb, and displayed multivalently on phage. After three rounds of heat denaturation followed by selection on protein A, most of the colonies secreted dAb phage that retain more than 80% of protein A- binding activity after heating.

* Φ *

Having thus described in detail advantageous embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

WHAT IS CLAIMED IS:

1. A modified adenovirus (Ad) comprising:

(a) a composite fiber comprising an adenovirus fiber shaft connected to twelvth coiled-coil segment of fibritin and

(b) a stabilized antibody.

2. The adenovirus of claim 1, wherein the adenovirus fiber shaft is an Ad5 fiber shaft.

3. The adenovirus of claim 1 or 2, wherein the stabilized antibody is a single chain antibody (scFv).

4. The adenovirus of any one of claims 1-3, wherein the composite fiber comprises nucleotides 584 to 2107 of pKanl lF-Bael (SEQ ID NO: 1).

5. A modified adenovirus (Ad) comprising a stabilized antibody inserted at the C terminus of pIX.

6. The adenovirus of claim 5, wherein the stabilized antibody is a single chain antibody (scFv).

7. The adenovirus of claim 5 or 6, wherein the composite pIX protein comprises nucleotides 3114 to 3569 of pSILucIXNhe (SEQ ID NO: 4).

8. A modified adenovirus (Ad) comprising a stabilized antibody inserted at the N terminus of pΙII(a).

9. The adenovirus of claim 8, wherein the stabilized antibody is a single chain antibody (scFv).

10. The adenovirus of any one of claims 1 to 9, wherein the adenovirus further comprises a transgene.

11. An adenoviral vector comprising the adenovirus of any one of claims 1 to 10.

12. A transformed host cell comprising the vector of claim 11.

13. The transformed host cell of claim 12, wherein the vector is introduced into the cell by infection, transfection, electroporation or transformation.

14. A method for preparing a transformed cell expressing the adenovirus of any of claims 1 to 10 comprising:

(a) infecting, transfecting, electroporating or transforming a cell with the adenovirus of any of claims 1 to 10 to produce a transformed host cell and

(b) maintaining the transformed host cell under biological conditions sufficient for expression of the adenovirus in the host cell.

15. A method for inhibiting tumor cell growth in a subject in need thereof comprising administering to the subject in need thereof a therapeutically effective amount of the adenovirus of any of claims 1 to 10 wherein the stabilized antibody targets the tumor cell such that the adenovirus infects the tumor cells and thereby inhibits tumor cell growth in the subject.

16. A method of increasing the ability of an adenovirus to transduce a specific cell type relative to an unmodified adenovirus, comprising the step of: modifying a composite fiber comprising an adenovirus fiber shaft connected to twelvth coiled-coil segment of fibritin by introducing a DNA sequence encoding a stabilized antibody into the 3' end of the composite fiber gene, wherein said modification increases the ability of said adenovirus to transduce a specific cell type relative to an unmodified adenovirus.

17. The method of claim 16 wherein the adenovirus fiber shaft is an Ad5 fiber shaft.

18. A method of increasing the ability of an adenovirus to transduce a specific cell type relative to an unmodified adenovirus, comprising the step of: modifying a gene encoding an adenoviral capsid protein by introducing a DNA sequence encoding a stabilized antibody into the 3' end of the minor capsid protein pIX gene, wherein said modification increases the ability of said adenovirus to transduce a specific cell type relative to an unmodified adenovirus.

19. • The method of any one of claims 16-18, wherein said stabilized antibody is directed towards a protein, wherein said protein is specific to a cell type.

20. The method of claim 19, wherein the cell type is a dendritic cell, fibroblast, immune cell, keratinocyte or tumor cell.

21. The method of claim 19, wherein said protein is a cell-surface protein.

22. The method of any one of claims 15 to 21 , wherein the stabilized antibody is a single chain antibody (scFv).