WO2008143936A1 - Methods of enhancing adenoviral delivery - Google Patents

Abstract

The present invention provides methods and compositions for enhancing delivery of adenoviral based vectors, in particular adenoviral based vectors for gene therapy comprising administering these vectors in conjunction with an anti-hexon antibody and/or an F(ab) fragment of an anti-hexon antibody and/or a Clg peptide or combinations thereof, and methods and compositions for enhancing delivery of a recombinant adenoviral based vaccine vectors. The present invention also provided compositions and/or kits for use in the methods.

Description

METHODS OF ENHANCING ADENOVIRAL DELIVERY

This invention claims the benefit of priority under 35 U.S. C. 119(e) of U.S.S.N.: 60/938,894 filed May 18, 2007, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods and compositions for enhancing the delivery of adenoviral vectors used in gene therapy and/or vaccines.

BACKGROUND

Dose dependent adverse effects have been associated with systemic administration recombinant adenoviruses for gene therapy. One aspect of the response to adenovirus is complement activation (Batshaw, M. L. et al (1999) Hum. Gene Ther. 10:2419-2437; Cichon, G. et al. (2001) Gene Ther. 8:1794-1800; Muruve, D. A. et al, (1999) Hum. Gene Ther. 10:965-976; Raper, S. E. et al (2003) MoI. Genet. Metab 80:148-158). The complement cascade can be activated through one or both of two major pathways; both pathways converge to generate the membrane attack complex. The classical pathway is antibody-dependent. It is activated by the binding of CIq, a subunit of the first complement protein Cl, to an immune complex formed from antigen specific antibodies and the microbes (Cooper, N. R. (1985) Adv. Immunol. 37:151-216). The alternative pathway is activated by C3, another complement protein which binds directly to the microbe and is antibody independent (Nilsson, B. and K. Nilsson Ekdahl (1997) "Components of the alternative pathway" In K. Rother, G. O. Till, and G. M. Hansen (eds.), The Complement System. Springer- Verlag, Berlin).

The present invention provides methods of utilizing the complement activation pathway to promote complement-mediated antibody-dependent adenovirus infection. SUMMARY OF THE INVENTION

The present invention provides methods and compositions for enhancing delivery of adenoviral based vectors to a cell, in particular adenoviral based vectors for gene therapy and methods and compositions for enhancing delivery of a recombinant adenoviral based vaccine vectors.

In one embodiment the invention provides a method of enhancing recombinant adenoviral vector delivery to a cell, the method comprising administering an adenoviral vector in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof.

In another embodiment, this invention relates to a method of enhancing recombinant adenoviral vector delivery to a cell, the method comprising administering an adenoviral vector in conjunction with an anti-hexon antibody and optionally CIq.

In yet another embodiment, this invention relates to a method of enhancing recombinant adenoviral vector delivery to a cell, the method comprising administering an adenoviral vector in conjunction with or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and optionally CIq .

In yet another embodiment, this invention relates to a method of enhancing recombinant adenoviral vector delivery to a cell, the method comprising administering an adenoviral vector in conjunction with an anti-hexon antibody or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and optionally a CIq peptide.

In one embodiment the invention provides a method of enhancing recombinant adenoviral vector delivery to a cell, the method comprising administering an adenoviral vector in conjunction with an anti-hexon antibody and a CIq peptide and optionally an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody, wherein the cell is ex vivo.

In yet another embodiment, this invention relates to compositions comprising an adenoviral vector and/or antihexon antibody and/or a CIq peptide and/or or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody or combinations thereof and kits comprising the same.

Brief Description of the Drawings

Figure 1. Fluorescent intensity measured in vitro as a result of Ad-GFP infection in HeLa and RD cells in the presence of human sera. GFP expression measured as relative fluorescent intensity (RFU) resulted from Ad-GFP infection of HeLa cells (A) and RD cells (B) in the presence of high titer serum (solid circles), heat inactivated high titer serum (open circles), low titer serum (solid diamonds), and heat inactivated low titer serum (open diamonds). Two controls were included: Baselines were cells without Ad-GFP infection (solid triangles), and maximum expression was cells infected with Ad-GFP in the absence of human serum (solid squares).

Figure 2. SDS-PAGE analysis of purified capsid proteins. The 4-20% acrylamide gradient gel was silver stained. Lanes are (1) purified hexon; (2) purified penton base (UI); (3) fiber; and (4) the column-purified rAd used for the preparation of the purified capsid components. Arrows represent the migration positions of the adenovirus protein components.

Figure 3. Removal ofanti-hexon antibodies with free purified hexons abrogated Ad- GFP infection of RD cells. RD cells were infected with Ad-GFP in the presence of free capsid proteins at two low titer serum dilutions: 1 :80 (panel A) and 1 :160 (panel B). Free capsid proteins are: Fibers (doted columns), hexons (black columns), free penton base (columns with diagonals), and mixture of all 3 capsid proteins (white columns).

Figure 4. CIq restored Ad-GFP infectability of RD cells in the presence of heat- inactivated low titer serum. RD cells were infected with Ad-GFP in the presence of heat- inactivated low titer serum. Purified CIq or C4 were serially diluted into the Ad-GFP and low titer serum mixture, starting at 1 : 10. Baseline is RD cells alone (horizontal bars), RD cells infected with Ad-GFP (diagonals bars), infected with Ad-GFP in the presence of heat- inactivated low titer serum and C4 protein (white columns) or CIq protein (black columns).

Figure 5. CIq mediate Anti-hexon antibody coated Ad-GPF infection of RD cells. Ad- GFP was co-cultured with native anti-hexon antibodies (anti-Hx columns), heated inactivated anti-hexon antibody (Heat anti-hx columns), heated inactivated anti-hexon antibody plus CIq, CIq alone, media alone (Ad-GFP columns), and RD cells alone serve as baseline. Three dilutions ofanti-hexon antibody were used in the co-cultures: 1 :40 (black columns), 1 :80 (white columns), and 1 :160 (diagonal columns).

DETAILED DESCRIPTION

The present invention provides methods and compositions for enhancing delivery of adenoviral based vectors, in particular adenoviral based vectors for gene therapy and methods and compositions for enhancing delivery of a recombinant adenoviral based vaccine vectors. The present invention is based, in part, on a discovery by the inventors that activation of the antibody dependent complement pathway, in particular the binding of the CIq protein to virus bound anti-hexon antibodies, can facilitate entry of a adenoviral vector into CAR negative cells. The present invention also provides compositions and/or kits for use in the methods.

General

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 in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook, et al, 1989"); 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 & S.J. 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); F.M. Ausubel, et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Lie. (1994).

5.1 Terminology

As used herein, the term "adenovirus" refers to viruses of the genus adenoviridiae. The term "recombinant adenovirus" refers to viruses of the genus adenoviridiae capable of infecting a cell whose viral genomes have been modified through conventional recombinant DNA techniques. The term recombinant adenovirus also includes chimeric (or even multimeric) vectors, i.e. vectors constructed using complementary coding sequences from more than one viral subtype.

As used herein, the term "recombinant adenovirus vector(s)" refers to a vector construct comprising adenoviral nucleotide sequences and optionally, one or more heterologous nucleotide sequences. In a preferred embodiment, the recombinant adenovirus vectors comprise adenoviral nucleotide sequences that have reduced homology to the helper adenovirus nucleic acid sequences. In another preferred embodiment, the recombinant adenovirus vector encodes a replication-defective adenovirus. In accordance with this embodiment, the recombinant adenovirus vector may be engineered to comprise a mutated adenovirus genome by, e.g., introducing one or more mutations in an adenovirus genome (e.g., introducing deletions in one or more coding regions for adenoviral proteins).

As used herein, the term "adenoviridae" refers collectively to animal adenoviruses of the genus mastadenovirus including but not limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera. In particular, human adenoviruses include the A-F subgenera as well as the individual serotypes thereof. A-F subgenera including but not limited to human adenovirus types 1 , 2, 3, 4, 4a, 5, 6, 7, 7a, 7d, 8, 9, 10, 11 (AdI IA and AdI IP), 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.

As used herein, the term "ElA gene" and "ElB region" refers to the immediate early genes of the adenovirus genome first transcribed following infection. For example, the El A coding region spans nucleotide 560-1542 and the ElB coding region spans 1714-2242. As used herein, the term "E2B gene" refers to the early gene of the adenovirus genome that encodes the 14OkD DNA polymerase. The E2 region also encodes the precursor to the terminal protein (8OkD) that is cleaved during viral assembly to 55kD while covalently bound to DNA. The E2B coding region spans nucleotide 8367-5197 of adenovirus type 5. GenBank^® deposits of the complete human adenovirus type 5 genome are available, see for example, AY339865 and AC000008.

As used herein, the term "expression cassette" is used herein to define a nucleotide sequence capable of directing the transcription and translation of a heterologous coding sequence and the heterologous coding sequence to be expressed. An expression cassette comprises a regulatory element operably linked to a heterologous coding sequence so as to achieve expression of the protein product encoded by said heterologous coding sequence in the cell.

As used herein, the term "heterologous" in the context of nucleic acid sequences, amino acid sequences and antigens refers to nucleic acid sequences, amino acid sequences and antigens that are foreign and are not naturally found associated with a particular adenovirus.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the nucleotide sequences being linked are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.

As used herein, the term "regulatory element" refers to promoters, enhancers, transcription terminators, insulator regions, silencing region, polyadenylation sites, intron sequences, post transcriptional regulatory elements and the like. The term "promoter" is used in its conventional sense to refer to a nucleotide sequence at which the initiation and rate of transcription of a coding sequence is controlled. The promoter contains the site at which RNA polymerase binds and also contains sites for the binding of regulatory factors (such as repressors or transcription factors). Promoters may be naturally occurring or synthetic. When the vector to be employed is a viral vector, the promoters may be endogenous to the virus or derived from other sources. The regulatory elements may be arranged so as to allow, enhance or facilitate expression of the transgene only in a particular cell type. For example, the expression cassette may be designed so that the transgene is under control of a promoter which is constitutively active, or temporally controlled (temporal promoters), activated in response to external stimuli (inducible), active in particular cell type or cell state (selective) constitutive promoters, temporal viral promoters or regulatable promoters.

As used herein, the term "infecting" means exposing the recombinant adenovirus to a complementing cell line under conditions so as to facilitate the infection of the producer cell with the recombinant adenovirus. In complementing cells which have been infected by multiple copies of a given virus, the activities necessary for viral replication and virion packaging are cooperative. Thus, it is preferred that conditions be adjusted such that there is a significant probability that the cells are multiply infected with the virus. An example of a condition which enhances the production of virus in the cell is an increased virus concentration in the infection phase. However, it is possible that the total number of viral infections per cell can be overdone, resulting in toxic effects to the cell. Consequently, one should strive to maintain the infections in the virus concentration in the range of 10⁶ to 10¹⁰, preferably about 10⁹, virions per ml. Chemical agents may also be employed to increase the infectivity of the cell line. For example, the present invention provides a method to increase the infectivity of cell lines for viral infectivity by the inclusion of a calpain inhibitor. Examples of calpain inhibitors useful in the practice of the present invention include, but are not limited to, calpain inhibitor 1 (also known as N-acetyl-leucyl-leucyl-norleucinal, commercially available from Boehringer Mannheim). Calpain inhibitor 1 has been observed to increase the infectivity of cell lines to recombinant adenovirus (see, e.g. U.S. Patent No. 7,001,770 herein incorporated by reference in its entirety).

As used herein, the term "culturing under conditions to permit replication of the viral genome" means maintaining the conditions for complementation so as to permit the recombinant adenovirus to propagate in the cell. It is desirable to control conditions so as to maximize the number of viral particles produced by each cell. Consequently it will be necessary to monitor and control reaction conditions such as temperature, dissolved oxygen, pH, etc. Commercially available bioreactors such as the CelliGen Plus Bioreactor (commercially available from New Brunswick Scientific, Inc. 44 Talmadge Road, Edison, NJ) have provisions for monitoring and maintaining such parameters. Optimization of infection, transfection and culture conditions will vary somewhat, however, conditions for the efficient replication and production of virus may be achieved by those of skill in the art taking into consideration, for example, the known properties of the producer cell line, properties of the virus and the type of bioreactor.

As used herein, the term "helper adenovirus nucleic acid sequence(s)" refers to a nucleic acid sequence(s) that: (i) provides viral functions for the replication of a recombinant adenovirus vector and/or its packaging into infectious virions; and (ii) is (are) not replicated or assembled into viral particles to a measurable degree.

As used herein, the terms, "recombinant adenovirus production cell line", "recombinant adenovirus complementation cells", and "recombinant adenovirus complementation cell lines" are synonyms and mean a cell able to propagate recombinant adenoviruses by providing viral functions for replication of a recombinant adenovirus and/or its packaging into infectious virions.

As used herein, the term "transfection" or "transformation" means the introduction of a nucleic acid into a cell. A host cell that receives the introduced DNA or RNA has been "transformed" and is a "transformant" or a "clone." Examples of transformation methods which are very well known in the art include liposome delivery, electroporation, CaPO₄ transformation, DEAE-Dextran transformation, microinjection and viral infection. Anti-hexon Antibodies

The anti-hexon antibodies of the present invention can be produced by any suitable methods known in the art. The antibodies may be polyclonal or monoclonal or combinations thereof. Capsid proteins to serve as antigens for generating the antibodies can be prepared by methods know in the art (e.g., Vellekamp, G., et al (2001) Hum. Gene Ther. 12:1923-1936). In a preferred embodiment whole virus may be used to generate the anti-hexon antibodies.

Neutralizing anti-hexon antibodies for use in the present invention can be selected for by methods known in the art. By way of example, and not limitation, anti -hexon antibodies can be selected for by exposing an adenoviral particle to the anti-hexon antibody and measuring the infectivity of CAR negative cells exposed to CIq and the adenoviral particle exposed to the anti- hexon antibody. Such a method is described herein below in the examples.

Antigen binding fragments of Anti-hexon Antibodies

The antigen binding fragments of anti-hexon antibodies of the present invention can be produced by any suitable methods known in the art (e.g., L. Presta (2003) Current Opinions in Structural Biology 13:519-525). Non-limiting examples of antigen body binding fragments include, but are not limited to, Fab, Fab', F(ab')₂, and Fv fragments. By way of example and not- limitation, an adenoviral vector can be administered in conjunction with an anti-hexon antibody, or an F(ab)' fragment.

In one embodiment, the amount of anti-hexon antibody administered in conjunction with the adenoviral based vectors is sufficient to bind between about 25% to about 75% of the 240 hexon proteins on an adenoviral particle. In another embodiment the amount of anti-hexon antibody and antigen binding fragment of an anti-hexon antibody (e.g., F(ab)' fragment ) administered in conjunction with the adenoviral based vectors is sufficient to bind to between about 90% to about 100% of the 240 hexon proteins on an adenoviral particle.

CIq protein

The CIq protein of the present invention may be produced by any suitable method known the art. Purified CIq protein is also commercially available (e.g., purified human CIq protein from Quidel, Santa Clara, CA). In one embodiment, the method of enhancing recombinant adenoviral vector delivery to a cell comprises administering an adenoviral vector to a cell in conjunction with an anti-hexon antibody and a CIq peptide and optionally an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody, wherein the cell is ex vivo. Preferably the amount of CIq protein administered is in excess of the anti-hexon antibody. Recombinant Adenovirus (rAd)

The recombinant adenovirus vectors to be administered in conjunction with an anti- hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof can comprise adenoviral nucleotide sequences and optionally, one or more heterologous nucleotide sequences. In a preferred embodiment, the recombinant adenovirus vectors comprise adenoviral nucleotide sequences having decreased homology to the adenovirus nucleic acid sequences of the complementing cell lines. The lack of homology between the adenoviral helper nucleic acid sequences and recombinant adenovirus vectors reduces the possibility of the viral genome recombining to produce replication competent adenovirus. In a preferred embodiment, the recombinant adenovirus vector encodes a replication-defective adenovirus. In accordance with this embodiment, the recombinant adenovirus vector may be engineered to comprise a mutated adenovirus genome by, e.g., introducing one or more mutations in an adenovirus genome {e.g., introducing deletions in one or more coding regions for adenoviral proteins). Preferably, the mutations in the adenovirus genome result in lower levels of expression of adenoviral proteins than wild-type adenovirus. The reduction in adenoviral protein expression reduces the immune response to the adenoviral proteins in a subject.

In one embodiment the recombinant adenovirus vector is derived from a human adenovirus serotype 5 and comprises deletions of the EIa, EIb and protein IX functions, and deletions in the E3 region (see, e.g., U.S. Patent Nos. 6,210,939 and 5,932,210, herein incorporated by reference in their entirety).

In another embodiment, the recombinant adenovirus vector encodes an El deleted replication-defective adenovirus and comprises a mutated genome with a partial or complete (preferably, a complete) deletion of the E2B polymerase function, and includes a heterologous nucleotide sequence. In one embodiment, the recombinant adenovirus vector encodes a replication-defective adenovirus and comprises a mutated genome with a partial or complete (preferably, a complete) deletion of the ElA coding region, ElB coding region, E2B polymerase coding region and includes a heterologous nucleotide sequence in the deleted El coding region.

In an embodiment of the invention, deletions in the E2B region include those sufficient to lead to the production of a non-functional DNA polymerase. In a preferred embodiment of the invention the deletion in the E2B region retains sequences that encode viral proteins on the opposite strand. Mutations, that may be used in the practice of the invention include, but are not limited to, the E2b deletion of nucleotides 7274 to about 7881 (see Amalfitano et al., 1998, herein incorporated by reference in its entirety). In yet another embodiment of the invention point mutations may be genetically engineered into the E2B coding region which result in a decrease in functional adenovirus polymerase expression. In a specific embodiment of the invention, the start codon of the E2B gene may be mutated to prevent translation of the E2B mRNA, thereby eliminating the function of E2B polymerase activity.

The heterologous nucleotide sequences can be introduced into any region of the genome (e.g., the amino or carboxy-termini). In a specific embodiment, a heterologous nucleotide sequence is introduced into one of the deleted adenoviral coding regions, such as the El , E2B or E3 coding region, of the mutated adenoviral genome. In a preferred embodiment of the invention, the heterologous nucleotide sequence is introduced into the deleted El coding region of the mutated adenoviral genome.

In accordance with the invention, the recombinant adenovirus vectors comprise an adenoviral genome or a portion thereof obtained and/or derived from any adenoviridae or a combination of adenoviridae. In a preferred embodiment, the recombinant adenovirus vectors comprise an adenoviral genome or portion thereof obtained and/or derived from a human adenoviridae. In another preferred embodiment, the recombinant adenovirus vectors comprise an adenoviral genome or portion thereof obtained and/or derived from the human adenovirus serotype 2 or 5.

In one embodiment the recombinant adenovirus vector is derived from a human adenovirus serotype 5 and comprises deletions of the EIa, EIb and protein DC functions, and deletions in the E3 region (see, e.g., U.S. Patent Nos. 6,210,939 and 5,932,210, herein incorporated by reference in their entirety) and the E2b region. By way of example, and not limitation, the recombinant adenovirus vector derived from a human adenovirus serotype 5 can comprise a deletion of base pairs 357 to about base pairs 4050, such as, for example, base pairs 360 to between about base pairs 4030, a deletion of base pairs 28,597 to between about base pairs 30,471 and a deletion in the E2b region as described in Amalfitano, A. et al (1998), herein incorporated by reference in its entirety.

In another embodiment, the recombinant adenovirus vector is derived from a human adenovirus serotype 5 and comprises deletions of the same adenoviral sequences as shown in the adenoviral vector in Figure 6. The present invention relates to recombinant adenovirus expression vectors comprising an "expression cassette" which is inserted into the mutated adenoviral genome to be administered in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof. As used herein, the term "expression cassette" is defined as a nucleotide sequence capable of directing the transcription and translation of a heterologous coding sequence and the heterologous coding sequence to be expressed. An expression cassette comprises a regulatory element operably linked to a heterologous coding sequence so as to achieve expression of the protein product encoded by said heterologous coding sequence in the cell.

In an embodiment of the invention, the heterologous nucleotide sequence is obtained and/or derived from a source other than the recombinant adenovirus vector. In accordance with the invention, the heterologous nucleotide sequence may encode a moiety, peptide, polypeptide or protein possessing a desired biological property or activity.

In certain embodiments, the heterologous nucleotide sequence encodes a biological response modifier such as a cytokine, cytokine receptor, hormone, growth factor or growth factor receptor. Non-limiting examples of such biological response modifiers include interferon (IFN)-alpha, IFN-beta, IFN gamma, interleukin (IL-I), JL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-IO, IL-12, IL-15, EL-18, IL-23, erythropoietin (EPO), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF), thymic stromal lymphopoietin (TSLP), GM-CSF, TNFR and TNFR ligand superfamily members including TNFRSF 18 and TNFSFl 8. In a preferred embodiment the nucleotide sequence encodes an interferon, such as Interferon alpha 2b. (see, e.g. U.S. Patent No.: 6,835,557, herein incorporated by refrence in its entirety).

In other embodiments, the heterologous nucleotide sequence encodes an antibody. In yet other embodiments, the heterologous nucleotide sequence encodes a chimeric or fusion protein.

In certain embodiments, the heterologous nucleotide sequence encodes an antigenic protein, a polypeptide or peptide of a virus belonging to a different species, subgroup or variant of adenovirus other than the species, subgroup or variant from which the recombinant adenovirus vector is derived. In certain embodiments, the heterologous nucleotide sequence encodes an antigenic protein, polypeptide or peptide obtained and/or derived from a pathogenic microorganism.

In yet another embodiment of the invention, the heterologous nucleotide sequence is a cancer therapeutic gene. Such genes include those that enhance the antitumor activity of lymphocytes, genes whose expression product enhances the immunogenicity of tumor cells, tumor suppressor genes, toxin genes, suicide genes, multiple-drug resistance genes, antisense sequences, and the like. Thus, for example, the adenoviral vector of this invention can contain a foreign gene for the expression of a protein effective in regulating the cell cycle, such as p53, Rb, or mitosin, or in inducing cell death, such as the conditional suicide gene thymidine kinase.

According to the invention, if the heterologous nucleotide sequence of the recombinant adenovirus vector is to be expressed in host cells, a transcriptional control element, also called a promoter/enhancer sequence, should be provided. The promoter/enhancer sequence may be widely active or may, alternatively, be tissue specific. The promoter/enhancer sequence may be derived from a non-adenovirus source or may be an adenovirus promoter. In a preferred embodiment, the promoter/enhancer sequences used to regulate the expression of the heterologous nucleotide sequence are not shared with those promoter/enhancer sequences that regulate the expression of the helper adenovirus nucleic acid sequences, hi accordance with this embodiment, a promoter can be any promoter known to the skilled artisan. For example, the promoter can be a constitutive promoter, a tissue-specific promoter or an inducible promoter. Examples of promoters that may be used in accordance with the invention include: the SV40 early promoter (Benoist and Chambon, 1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980), the herpes thymidine kinase promoter (Wagner et al., 1981), the regulatory sequences of the metallothionein gene (Brinster et al., 1982), the beta-actin promoter, the CMV promoter, the SR-alpha promoter, the hFer/SV40 promoter, the EIf-I promoter, the Tet promoter, the Ecdysone promoter and a rapamycin promoter.

In a specific embodiment, a native promoter is utilized to regulate the expression of a nucleotide sequence encoding an adenoviral protein, hi alternative embodiment, a promoter that is not native to the adenoviral gene encoding the protein being expressed (i.e., a heterologous promoter) is utilized to regulate the expression of the protein. In certain embodiments, the promoter is a constitutive promoter (e.g. , a viral, cellular or hybrid constitutive promoter). In other embodiments, the promoter is an inducible promoter. In yet other embodiments, the promoter is a tissue-specific promoter.

In certain embodiments, it is desirable to use a constitutive promoter, such as a CMV promoter, β-actin promoter, SR-alpha promoter or hFer/S V40 promoter, to regulate the expression of the heterologous nucleotide sequence, hi certain other embodiments, it is desirable to use a constitutive promoter, such as a RSV promoter, SV40 promoter or EIf-I promoter, to regulate the expression of the heterologous nucleotide sequence. In yet other embodiments, it is desirable to use an inducible promoter, such as a Tet promoter or Ecdysone promoter, to regulate the expression of the heterologous nucleotide sequence of the adenovirus vector.

In yet another embodiment of the invention, an inducible promoter can be used in the adenoviral vector of the invention. These promoters will initiate transcription only in the presence of an additional molecule. Examples of inducible promoters include those obtainable from a β-interferon gene, a heat shock gene, a metallothionine gene or those obtainable from steroid hormone-responsive genes. Tissue specific expression has been well characterized in the field of gene expression and tissue specific and inducible promoters such as these are very well known in the art. These genes are used to regulate the expression of the foreign gene after it has been introduced into the target cell.

The desirable size of inserted non-adenovirus or heterologous nucleotide sequence is limited to that which permits packaging of the recombinant adenovirus vector into virions, and depends on the size of retained adenovirus sequences. The genome of a human adenovirus is approximately 36 kilobase pairs in length (measured to be 35938 nucleotides in length by (Davison et al., 2003). The total size of the recombinant adenovirus to be packaged into virions should be about 37735 nucleotides in length (about 105% of the normal genome length). Therefore, it may be desirable to exclude additional portions of the adenovirus genome, such as the E3 region, in the recombinant adenovirus vector in order to maximize expression of the inserted heterologous nucleotide sequence.

Insertion of a foreign gene sequence into a recombinant adenovirus vector can be accomplished by either a complete replacement of a viral coding region with a heterologous nucleotide sequence or by a partial replacement or by adding the heterologous nucleotide sequence to the viral genome. Complete replacement would probably best be accomplished through the use of PCR-directed mutagenesis. Briefly, PCR-primer A would contain, from the 5' to 3' end: a unique restriction enzyme site, such as a class IIS restriction enzyme site (i.e., a "shifter" enzyme; that recognizes a specific sequence but cleaves the DNA either upstream or downstream of that sequence); a stretch of nucleotides complementary to a region of the gene that is to be replaced; and a stretch of nucleotides complementary to the carboxy-terminus coding portion of the heterologous nucleotide sequence. PCR-primer B would contain from the 5' to 3' end: a unique restriction enzyme site; a stretch of nucleotides complementary to the gene that is to be replaced; and a stretch of nucleotides corresponding to the 5' coding portion of the heterologous or non-native gene. After a PCR reaction using these primers with a cloned copy of the heterologous or non-native gene, the product may be excised and cloned using the unique restriction sites. Digestion with the class HS enzyme and transcription with the purified phage polymerase would generate a RNA molecule containing the exact untranslated ends of the viral gene that carries now a heterologous or non-native gene insertion. In an alternate embodiment, PCR-primed reactions could be used to prepare double- stranded DNA containing the bacteriophage promoter sequence, and the hybrid gene sequence so that RNA templates can be transcribed directly without cloning.

When inserting a heterologous nucleotide sequence into the recombinant adenovirus vector of the invention, the intergenic region between the end of the coding sequence of the heterologous nucleotide sequence and the start of the coding sequence of the downstream gene can be altered to achieve a desired effect. As used herein, the term "intergenic region" refers to nucleotide sequence between the stop signal of one gene and the start codon (e.g., AUG) of the coding sequence of the next downstream open reading frame. An intergenic region may comprise a non-coding region of a gene, i.e., between the transcription start site and the start of the coding sequence (AUG) of the gene. This non-coding region occurs naturally in some viral genes.

In an embodiment of the invention, sequences referred to as "insulators" may be inserted into the expression cassette, in the intergenic region downstream of the heterologous nucleotide sequence (Di Simone et al., 2001 ; Martin-Duque et al., 2004a; Pluta et al., 2005; Puthenveetil et al., 2004; Qu et al., 2004; Rincon-Arano and Recillas-Targa, 2004; Takada et al., 2000) The insertion of such insulators can result in decreased expression of adenoviral proteins, as compared to wild type, which is useful in reducing the immunogenity and toxicity of the adenovirus vectors. Insulator sequences that may be used in the practice of the invention are well known to those of skill in the art and include, for example, hypersensitive site 4 (HS4) of the β-globin gene locus. The HS4 locus has been used in retroviruses (Emery et al., 2002; Jakobsson et al., 2004; Pannell and Ellis, 2001; Yannaki et al., 2002; Yao et al., 2003) and also adenovirus vectors (Cheng et al., 2004; Martin-Duque et al., 2004b; Steinwaerder and Lieber, 2000; Ye et al., 2003). The region of the HS4 locus being responsible for the control of gene expression through chromatin rearrangement and blocking activities has been attributed to the transcriptional modulator CTCF (Bell et al., 1999; Dunn and Davie, 2003; Dunn et al., 2003; Emery et al., 2002; Farrell et al., 2002; Jakobsson et al., 2004; Kanduri et al., 2002; Lewis and Murrell, 2004; Lutz et al., 2000; Mukhopadhyay et al., 2004; Pannell and Ellis, 2001; Recillas- Targa et al., 2002; Saitoh et al., 2000; Szabo et al., 2002; Thorvaldsen et al., 2002; Valadez- Graham et al., 2004; Yannaki et al., 2002; Yao et al., 2003; Yusufzai and Felsenfeld, 2004; Yusufzai et al., 2004; Zhang et al., 2004; Zhao and Dean, 2004). In an embodiment of the invention, an insulator comprising four head to tail copies of the CTCF binding site from the hypersensitive site 4 of the β-globin gene locus may be used as an insulator. In another embodiment, other synthetic insulator sequences (Bell et al., 2001 ; Brasset and Vaury, 2005; Zhao and Dean, 2004) may also be used.

In yet another embodiment of the invention, the recombinant adenoviruses of the invention may include post-transcriptional regulatory element (PRE) that function to increase transgene expression. Such elements including, for example, the woodchuck hepatitis PRE (Donello et al., 1998), the hepatitis B virus PRE (Huang and Yen, 1994) or the herpes simplex PRE (Liu and Mertz, 1995) are inserted into the expression cassette at a location downstream of the heterologous gene (Appleby et al., 2003; Breckpot et al., 2003; Brun et al., 2003; Glover et al., 2002; Glover et al., 2003; Gropp et al., 2003; Mangeot et al., 2002; Robert et al., 2003; Schwenter et al., 2003; Werner et al., 2004; Xu et al., 2003; Yam et al., 2002; Zufferey et al., 1999).

The present invention also provides a recombinant adenovirus wherein the expression cassette is engineered to contain an intron sequence engineered into the 5' untranslated region of the heterologous gene (Choi et al., 1991; Hermening et al., 2004; Lee et al., 1997; Xu et al., 2002; Xu et al., 2003). The intron sequences to be used in the practice of the invention can be generated from know consensus splicing sequences using, for example, PCR with primers that incorporate the necessary consensus splicing signals. Intron sequences include a 5' splice donor site and a 3' splice region that includes a branch point sequence and a 3' splice acceptor AG site. The 3' splice region may further comprise a polypyrimidine tract. Consensus sequences for the 5' splice donor site and the 3' splice region used in RNA splicing are well known in the art (See, Moore, et al., 1993, The RNA World, Cold Spring Harbor Laboratory Press, pp. 303-358). In addition, modified consensus sequences that maintain the ability to function as 5' donor splice sites and 3' splice regions may be used in the practice of the invention. Briefly, the 5' splice site consensus sequence is AG/GURAGU (where A=adenosine, U=uracil, G=guanine, C=cytosine,

and/=the splice site). The 3' splice site consists of three separate sequence elements: the branch point or branch site, a polypyrimidine tract and the 3' consensus sequence (YAG). The branch point consensus sequence in mammals is YNYURAC (Y=pyrimidine; N=any nucleotide). The underlined A is the site of branch formation. A polypyrimidine tract is located between the branch point and the splice site acceptor and is important for efficient branch point utilization and 3' splice site recognition. Other pre-messenger RNA introns beginning with the dinucleotide AU and ending with the dinucleotide AC have been identified and referred to as U12 introns. U12 intron sequences as well as any additional sequences that function as splice acceptor/donor sequences may also be used to generate the expression cassette of the invention.

In yet another embodiment of the invention the 5' untranslated region of the expression cassette comprises the adenovirus tripartite leader .

In one embodiment the expression vector comprises one or more heterologous nucleotide sequences, CMV promoters, a tripartite leader sequences, synthetic introns, WPRE sequences, polyA regions and CTCF binding sites. By way of example, and not limitation, the recombinant adenovirus vectors of the invention can comprise the expression cassette shown in Figure 5.

The expression of the inserted heterologous nucleotide sequence can be determined by various indexes including, but not limited to, protein or mRNA expression levels, measured by following non-limiting examples of assays: immunostaining, immunoprecipitation and immunoblotting, enzyme-linked immunosorbent assay, nucleic acid detection {e.g. , Southern blot analysis, Northern blot analysis, Western blot analysis), employment of a reporter gene {e.g. , using a reporter gene, such as Green Fluorescence Protein (GFP) or enhanced Green Fluorescence Protein (eGFP), integrated to the viral genome the same fashion as the interested heterologous gene to observe the protein expression), or a combination thereof. Procedures of performing these assays are well known in the art (see, e.g. Flint et al., PRINCIPLES OF VIROLOGY, MOLECULAR BIOLOGY, PATHOGENESIS, AND CONTROL, 2000, ASM Press pp 25-56, the entire text is incorporated herein by reference).

For example, expression levels can be determined by infecting cells in culture with a recombinant adenovirus of the invention and subsequently measuring the level of protein expression by, e.g., Western blot analysis or ELISA using antibodies specific to the gene product of the heterologous nucleotide sequence, or measuring the level of RNA expression by, e.g. , Northern blot analysis using probes specific to the heterologous sequence. Similarly, expression levels of the heterologous sequence can be determined by infecting an animal model and measuring the level of protein expressed from the heterologous nucleotide sequence of the recombinant virus of the invention in the animal model. The protein level can be measured by obtaining a tissue sample from the infected animal and then subjecting the tissue sample to Western blot analysis or ELISA, using antibodies specific to the gene product of the heterologous sequence. Further, if an animal model is used, the titer of antibodies produced by the animal against the gene product of the heterologous sequence can be determined by any technique known to the skilled artisan, including but not limited to, ELISA.

According to the invention, a recombinant adenovirus vector may be propagated in microorganisms, for example, as part of a bacterial plasmid or bacteriophage, in order to obtain large quantities of recombinant adenovirus vector.

Production of Recombinant Adenovirus

In accordance with the invention, recombinant adenovirus (preferably, recombinant replication-defective adenovirus) may be produced by co-transfecting an appropriate cell type with recombinant adenovirus vector and helper adenovirus nucleic acid sequences. Co- transfection may be performed by the DEAE dextran method (McCutchan and Pagano, 1968), the calcium phosphate procedure (Graham and van der Eb, 1973) or by any other method known in the art, including but not limited to microinjection, lipofection, and electroporation. Amounts of recombinant adenovirus vector and helper adenovirus nucleic acid sequences used in transfection are approximately 0.2 to 10 μg of DNA per 10 cells, but may vary among different DNA constructs and cell types. Cells suitable for transfection include any cell line permissive for adenvirus infection, including, but not limited to HeLa cells, 293-D22 cells, A549 cells, HCT-15 cells, IGROV-I cells, U87 cells and W162 cells.

Alternatively, a recombinant adenovirus complementing cell line may be transfected with recombinant adenovirus vector to produce of recombinant adenovirus (preferably, recombinant replication-defective adenovirus). See, e.g., US patent application 60/674,488 and U.S. Publication No.: 2006/0270041 (the disclosures of which are herein incorporated by reference),

Recombinant adenovirus of the present invention may be produced by any suitable method, many of which are known in the art (see, e.g., (Berkner and Sharp, 1983; Berkner and Sharp, 1984; Brough et al., 1992). In the preferred practice of the invention, the recombinant adenoviruses are derived from the human adenoviridae. In a preferred embodiment of the invention, the recombinant adenovirus is derived from the human adenovirus serotype 2 or 5.

In a preferred practice of the invention, the produced recombinant adenovirus is a replication-defective adenovirus comprising a mutated genome with a partial or complete (preferably, complete) deletion of the ElA coding region, ElB coding region, and/or E2B polymerase coding region, and includes one or more heterologous nucleotide sequences in the El region.

In another embodiment of the invention, the recombinant adenovirus is a replication- defective adenovirus and comprises a mutated genome with a partial or complete (preferably, complete) deletion of the ElA coding region, ElB coding region, E2B polymerase coding region, and E3 coding region, and includes one or more heterologous nucleotide sequences in the deleted El coding region.

In another embodiment of the invention, the recombinant adenovirus is a replication- defective adenovirus and comprises a mutated genome with a partial or complete (preferably, complete) deletion of the ElA coding region, ElB coding region, E2B polymerase coding region, and E4 coding region, and includes one or more heterologous nucleotide sequences in the deleted El coding region.

In another embodiment of the invention, the recombinant adenovirus is a replication- defective adenovirus and comprises a mutated genome with a partial or complete (preferably, complete) deletion of the ElA coding region, ElB coding region, E2B polymerase coding region, E3 coding region, and E4 coding region and includes one or more heterologous nucleotide sequences in the deleted El coding region.

The preferred recombinant adenoviruses of the present invention comprise viral DNA sequences that have reduced homology with the adenoviral DNA sequences in the recombinant adenovirus production cell, which reduces the possibility of the viral genome recombining with the cellular DNA to produce RCAs. In certain embodiments, the quantity of recombinant adenovirus is titrated. Titrating the quantity of the adenovirus in the culture may be performed by techniques known in the art. In a particular embodiment, the concentration of viral particles is determined by the Resource Q assay as described by (Shabram et al., 1997b). As used herein, the term "lysis" refers to the rupture of the virus-containing cells. Lysis may be achieved by a variety of means well known in the art. For example, mammalian cells may be lysed under low pressure (100-200 psi differential pressure) conditions, by homogenization, by microfluidization, or by conventional freeze-thaw methods. Exogenous free DNA/RNA may be removed by degrecombinant adenovirusation with DNAse/RNAse.

Virus-containing cells may be frozen. Virus may be harvested from the virus- containing cells and the medium. In one embodiment, the virus is harvested from both the virus-containing cells and the medium simultaneously. In a particular embodiment, the virus producing cells and medium are subjected to cross-flow microfiltration, for example, as described in U.S. Patent Number 6,146,891, under conditions to both simultaneously lyse virus-containing cells and clarify the medium of cell debris which would otherwise interfere with virus purification.

As used herein, the term "harvesting" means the collection of the cells containing the recombinant adenovirus from the media and may include collection of the recombinant adenovirus from the media. This may be achieved by conventional methods such as differential centrifugation or chromatographic means. At this stage, the harvested cells may be stored or further processed by lysis and purification to isolate the recombinant virus. For storage, the harvested cells should be buffered at or about physiological pH and frozen at - 7O⁰C.

Virus may also be harvested from the virus-containing cells and medium separately. The virus-containing cells may be collected separately from the medium by conventional methods such as differential centrifugation. Harvested cells may be stored frozen or further processed by lysis to liberate the virus. Virus may be harvested from the medium by chromatographic means. Exogenase free DNA/RNA may be removed by degrecombinant adenovirusation with DNAse/RNAse, such as BENZONASE (American International Chemicals, Inc.). The virus harvest may be further processed to concentrate the virus by methods such as ultrafiltration or tangential flow filtration, for example, as described in U.S. Patent Numbers 6,146,891; 6,544,769 and 6,783,983.

As used herein, the term "recovering" means the isolation of a substantially pure population of recombinant virus particles from the lysed producer cells and optionally from the supernatant medium. Viral particles produced in the cell cultures of the present invention may be isolated and purified by any method which is commonly known in the art. Conventional purification techniques such as chromatographic or differential density grecombinant adenovirusient centrifugation methods may be employed. For example, the viral particles may be purified by cesium chloride grecombinant adenovirusient purification, column or batch chromatography, diethylaminoethyl (DEAE) chromatography (Haruna et al., 1961 ; Klemperer and Pereira, 1959; Philipson, 1960), hydroxyapatite chromatography (U.S. Patent Application Publication Number US2002/0064860) and chromatography using other resins such as homogeneous cross-linked polysaccharides, which include soft gels (e.g., agarose), macroporous polymers based on synthetic polymers, which include perfusion chromatography resins with large "throughpores", "tentacular" sorbents, which have tentacles that were designed for faster interactions with proteins (e.g., fractogel) and materials based on a soft gel in a rigid shell, which exploit the high capacity of soft gels and the rigidity of composite materials (e.g., Ceramic HyperD® F) (Broschetti, 1994; Rodrigues, 1997). In the preferred practice of the invention, the virus is purified by column chromatography in substantial accordance with the process of (Huyghe et al., 1995b) as described in Shabram, et al., United States Patent 5,837,520 issued November 17, 1998; see also U.S. Patent No 6,2661,823, the disclosures of which are herein incorporated by reference.

The recombinant adenovirus production cell lines producing virus may be cultured in any suitable vessel which is known in the art. For example, cells may be grown and the infected cells may be cultured in a biogenerator or a bioreactor. Generally, "biogenerator" or "bioreactor" means a culture tank, generally made of stainless steel or glass, with a volume of 0.5 liter or greater, comprising an agitation system, a device for injecting a stream of CO₂ gas and an oxygenation device. Typically, it is equipped with probes measuring the internal parameters of the biogenerator, such as the pH, the dissolved oxygen, the temperature, the tank pressure or certain physicochemical parameters of the culture (for instance the consumption of glucose or of glutamine or the production of lactate and ammonium ions). The pH, oxygen, and temperature probes are connected to a bioprocessor which permanently regulates these parameters. In other embodiments, the vessel is a spinner flask, a roller bottle, a shaker flask or in a flask with a stir bar providing mechanical agitation. In another embodiment, a the vessel is a WAVE Bioreactor (WAVE Biotech, Bridgewater, NJ, U.S.A.).

Recombinant adenoviruses may be propagated in the recombinant adenovirus production cell lines of the invention. Virus may be produced by culturing the cells; optionally adding fresh growth medium to the cells; inoculating the cells with the virus; incubating the inoculated cells; optionally adding fresh growth medium to the inoculated cells; and optionally harvesting the virus from the cells and the medium. Typically, when the concentration of viral particles, as determined by conventional methods, such as high performance liquid chromatography using a Resource Q column, as described in (Shabram et al., 1997b), begins to plateau, the harvest is performed.

Proteins produced by recombinant adenoviruses grown in the recombinant adenovirus production cell lines (e.g., adenovirus comprising a deletion of the ElA and ElB coding regions and comprising a heterologous nucleotide sequence, or adenovirus comprising a deletion of El A, ElB and E2B polymerase coding regions and comprising a heterologous nucleotide sequence, adenovirus comprising a deletion of the ElA, ElB, E2B and E3 coding regions and comprising a heterologous nucleotide sequence, or adenovirus comprising a deletion of El A, ElB, E2B polymerase coding regions, E3 and E4 coding regions and comprising a heterologous nucleotide sequence) may also be isolated and purified. Proteins, polypeptides and peptides may be purified by standard methods, including, but not limited to, salt or alcohol precipitation, affinity, preparative disc-gel electrophoresis, isoelectric focusing, high pressure liquid chromatography (HPLC), reversed-phase HPLC, gel filtration, cation and anion exchange and partition chromatography, and countercurrent distribution. Such purification methods are well known in the art and are disclosed, e.g., in "Guide to Protein Purification ", Methods in Enzvmologv, Vol. 182, M. Deutscher, Ed., 1990, Academic Press, New York, NY.

Therapeutic Use

The recombinant adenoviruses can be administered in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof for gene therapy. The recombinant adenoviruses can be used for in vivo or ex vivo gene therapy. For in vivo gene therapy, recombinant adenovirus is directly administered to a subject in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof. For ex vivo gene therapy, cells are infected with the recombinant adenovirus in conjunction with an anti-hexon antibody and CIq and optionally an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody in vitro and then the infected cells are transplanted into the subject. In a specific embodiment, the recombinant adenovirus is directly administered in vivo, where a protein of interest is expressed. hi one embodiment, the present invention comprises a method for the treatment of cancer comprising administering a therapeutically effective amount of a recombinant adenovirus vector comprising one or more nucleotide sequences encoding a therapeutic protein in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof to a subject. The recombinant adenovirus vectors can comprise one or more nucleotide sequences encoding a therapeutic protein may be delivered to any cancerous tissue or organ using any delivery method known in the art, including, but not limited to intratumoral or intravesical administration. Examples of cancers that may be treated by the methods include, but are not limited to, carcinoma of the bladder and upper respiratory tract, vulva, cervix, vagina or bronchi; local metastatic tumors of the peritoneum; broncho-alveolar carcinoma; pleural metastatic carcinoma; carcinoma of the mouth and tonsils; carcinoma of the nasopharynx, nose, larynx, oesophagus, stomach, ovary, prostate colon and rectum, gallbladder, or skin; or melanoma or hematological cancers such as leukemia. By way of example, and not limitation, a recombinant adenovirus of the present invention comprising an expression cassette encoding interferon alpha 2b can be used in the treatment of bladder cancer. In one embodiment the recombinant adenovirus vector shown in Figure 6 is used in the methods described herein to treat bladder cancer.

Non-limiting examples of therapeutically effective amounts of the recombinant adenovirus vectors of the invention comprising one or more nucleotide sequences encoding a therapeutic protein are in the range of between about 1 xlO⁸ particles/ml to about 1 xlO¹² particles/ml or between about 1 xlO⁹ particles/ml to about 1 xlO¹¹ particles/ml. In one embodiment, the recombinant adenovirus vector shown in Figure 6 is administered to a subject with bladder cancer in the range of between about 1 xlO⁸ particles/ml to about 1 xlO¹² particles/ml or between about 1 xlO⁹ particles/ml to about 1 xlO¹¹ particles/ml. In another embodiment, a cell is infected ex vivo with a recombinant adenovirus. Th cell is exposed ex vivo to the recombinant adenovirus in conjunction with an anti-hexon antibody and CIq and optionally an antigen binding fragment (e.g., F(ab)' fragment) of an anti- hexon antibody and/or a CIq peptide or combinations thereof. The resulting recombinant cell is administered to a subject. The resulting recombinant cells can be delivered to a subject by various methods known in the art. Recombinant blood cells {e.g., hematopoietic stem or progenitor cells) are preferably administered intravenously. The amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art. In accordance with the invention, any cells which can be infected with a recombinant adenovirus can be for purposes of gene therapy. Non-limiting examples include epithelial cells (e.g., respiratory epithelial cells), endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes, blood cells (such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes), and various stem or progenitor cells (in particular, hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.). In a preferred embodiment, the cell used for gene therapy is autologous to the subject. In an embodiment in which recombinant cells are used in gene therapy, the proteins encoded by the genome of the recombinant adenovirus are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.

The recombinant adenovirus recombinant adenovirus can be administered in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof to immunize a subject. For example, the recombinant adenovirus may be used to generate antibodies against a heterologous antigen encoded by the recombinant adenovirus. The amount of recombinant adenovirus to be used to immunize a subject and the immunization schedule will be determined by a physician skilled in the art and will be administered by reference to the immune response and antibody titers of the subject.

The antibodies generated against an antigen by immunization with a recombinant adenovirus may used in diagnostic immunoassays, passive immunotherapy, and generation of anti-idiotypic antibodies. The generated antibodies may be isolated by standard techniques known in the art (e.g. , immunoaffinity chromatography, centrifugation, precipitation, etc.) and used in diagnostic immunoassays. The antibodies may also be used to monitor treatment and/or disease progression. Any immunoassay system known in the art may be used for this purpose including, but not limited to, competitive and noncompetitive assay systems using techniques such as recombinant adenovirusioimmunoassays, ELISA (enzyme-linked immunosorbent assays), "sandwich" immunoassays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunorecombinant adenovirusiometric assays, fluorescent immunoassays, protein A immunoassays and immunoelectrophoresis assays, to name but a few.

The recombinant adenoviruses can be administered in conjunction with an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof to produce antibodies for use in passive immunotherapy, in which short-term protection of a subject is achieved by the administration of pre-formed antibody directed against a heterologous antigen. The antibodies generated by the recombinant adenovirus of the present invention can also be used in the production of anti- idiotypic antibody. The anti-idiotypic antibody can then in turn be used for immunization, in order to produce a subpopulation of antibodies that bind the initial antigen (Jerne, 1974; Jerne et al., 1982).

In certain embodiments, the antibody produced by immunization with a recombinant adenovirus is modified prior to administration to a subject. For example, the antibody may be humanized and/or affinity matured.

Compositions and Methods of Administering Recombinant Adenovirus

The invention encompasses compositions comprising a recombinant adenovirus (preferably, replication-defective recombinant adenovirus) generated by the methods of the invention and compositions comprising an anti-hexon antibody and/or an antigen binding fragment (e.g., F(ab)' fragment) of an anti-hexon antibody and/or a CIq peptide or combinations thereof. In a preferred embodiment, the compositions are pharmaceutical compositions suitable for administration to a subject.

The pharmaceutical compositions of the present invention comprise an effective amount of recombinant adenovirus, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeiae for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the pharmaceutical composition is administered. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. These compositions can be formulated as a suppository. Oral formulation can include standard carriers such as pharmaceutical grecombinant adenoviruses of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain an effective amount of recombinant adenovirus, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

The amount of the pharmaceutical composition of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose- response curves derived from in vitro or animal model test systems.

Non-limiting examples of therapeutically effective amounts of the recombinant adenovirus vectors of the invention comprising one or more nucleotide sequences encoding a therapeutic protein are in the range of between about 1 x10 particles/ml to about 1 xlO particles/ml or between about 1 xlO⁹ particles/ml to about 1 xlO¹¹ particles/ml.

By way of example, and not limitation for the treatment of superficial bladder cancer in a subject, course of treatment comprising a dose of from 1 x xlO^lo.particles/ml to about 1 x 10¹².particles/ml, most preferably approximately 1 xlO¹¹ particles/ml encoding interferon alpha.2b in a volume of approximately 100 ml is instilled intravesically for a period of approximately one hour. By way of example, and not limitation, an alternate course of treatment may comprise a dose of from 1 x xlθ^lo.particles/ml to about 1 x 10¹².particles/ml most preferably approximately 1 xlθ^π particles/ml encoding interferon alpha2b in a volume of approximately 100 ml is instilled intravesically for a period of approximately one hour followed by a second substantially equivalent dose within 7 days, 5 days, 4 days, 3 days, 2 days or on consecutive days following the first dose. Each course of treatment is repeatable, depending on the course of disease progression. In the case of intravesically administered recombinant vectors for the treatment of bladder cancer, optimal interferon gene expression is generally observed when the courses of treatment are distanced by at least 14 days, more preferably about 30 days, and most preferably about 90 days.

Methods of administration of the compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The pharmaceutical compositions of the present invention may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g. , oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the lungs by any suitable route. Pulmonary administration can be employed, e.g. , by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non- porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue. In another embodiment the administration can be intravesicular administration.

In another embodiment, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used (Buchwald et al., 1980; Langer, 1983; Saudek et al., 1989; Sefton, 1987). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); (Langer and Peppas, 1983); (During et al., 1989; Howard et al., 1989; Levy et al., 1985). In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, i.e., the lung, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by (Langer, 1990).

In a specific embodiment, a composition of the invention is a vaccine or immunizing composition comprising a recombinant adenovirus (preferably, replication-defective recombinant adenovirus) generated by the methods of the invention, and a suitable excipient. Many methods may be used to introduce the vaccine compositions, these include but are not limited to intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. It may be preferable to introduce the recombinant adenovirus vaccine composition via the natural route of infection of adenovirus.

Non-limiting examples of therapeutically effective amounts of the recombinant adenovirus vectors of the invention comprising one or more nucleotide sequences encoding a therapeutic protein are in the range of between about 1 xlO particles/ml to about 1 xlO particles/ml or between about 1 xlO⁹ particles/ml to about 1 xlO¹¹ particles/ml.

In some embodiments it may be desirable to administer the recombinant adenovirus vector in conjunction with enhancing agents that facilitate the transfer of the nucleic acid encoding a therapeutic protein, for example interferon, to a target cell, such as , for example, a cancer cell. Examples of such delivery enhancing agents include detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates. Alcohols include for example the aliphatic alcohols such as ethanol, N- propanol, isopropanol, butyl alcohol, acetyl alcohol. Glycols include glycerine, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol. Acetates such as acetic acid, gluconic acid, and sodium acetate are further examples of delivery-enhancing agents. Hypertonic salt solutions like IM NaCl are also examples of delivery-enhancing agents. Bile salts such as taurocholate, sodium tauro- deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other astringents such as silver nitrate may be used. Heparin-antagonists like quaternary amines such as protamine sulfate may also be used. Anionic, cationic, zwitterionic, and nonionic detergents may also be employed to enhance gene transfer. Exemplary detergents include but are not limited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride, Zwittergent 3-14 detergent, CHAPS (3-[(3-

Cholamidopropyl)dimethylammoniol]-l-propanesulfon- ate hydrate), Big CHAP, Deoxy Big CHAP, Triton-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC-F68 detergent, Tween 20 detergent, and TWEEN 80 detergent (CalBiochem Biochemicals). Particularly preferred enhancing agents and methods are described in Engler et al., U.S. Pat. No. 6,312,681, issued Nov. 6, 2001, Engler et al., U.S. Pat. No. 6,165,779, issued Dec. 26, 2000, and Engler et al., U.S. Pat. No. 6,392,069, issued May 21, 2002, the entire teachings of which are herein incorporated by reference. A particularly preferred enhancing agent useful in the practice of the present invention is a compound termed Syn3 of the Formula I in U.S. Pat. No. 6,392,069. Additional enhancing agents useful in the practice of the present invention include, but are not limited to, the compounds of the Formulas II, III, IV, and V and their pharmaceutically acceptable salts in WO2004/ 108088. By way of example, and not limitation, the enhancing agents may be administered concomitant with the vector or prior to the administration of the vector.

The compositions and methods of the present invention may be practiced alone or in combination with conventional chemotherapeutic agents or treatment regimens. Examples of such chemotherapeutic agents include inhibitors of purine synthesis (e.g., pentostatin, 6- mercaptopurine, 6-thioguanine, methotrexate) or pyrimidine synthesis (e.g., PaIa, azarbine), the conversion of ribonucleotides to deoxyribonucleotides (e.g., hydroxyurea), inhibitors of dTMP synthesis (5-fluorouracil), DNA damaging agents (e.g., radiation, bleomycines, etoposide, teniposide, dactinomycine, daunorubicin, doxorubicin, mitoxantrone, alkylating agents, mitomycin, cisplatin, procarbazine) as well as inhibitors of microtubule function (e.g., vinca alkaloids and colchicine). Chemotherapeutic treatment regimens refers primarily to nonchemical procedures designed to ablate neoplastic cells such as radiation therapy. These chemotherapeutic agents may be administered separately or may be included with the formulations of the present invention for co-administration. The present invention may also be practiced in combination with conventional immunotherapeutic treatment regiments such as BCG in the case of superficial bladder cancer.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

Patents, patent applications, publications, product descriptions, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties.

EXAMPLES

Materials and Methods

Cell lines and sera used for studies.

HeLa and RD cell lines were from ATCC, designated as CCL-2 and CCL- 136, respectively. They were maintained in cultures according to ATCC protocols. Human sera were derived from blood samples from healthy normal blood donors from the San Diego Blood Bank (San Diego, California) in accordance with the San Diego Blood Bank informed consent guidelines. Where indicated human sera were heat inactivated in water bath for 30 minutes at 56⁰C.

Neutralizing antibody (NAb) assay.

A previously described assay was used to determine the titer of anti-adeno virus neutralizing antibodies in human serum (Rahman, A. et al. (2001) MoI. Ther. 3:768-778). The NAb titer for each serum sample was reported as the reciprocal dilution that inhibited Ad-GFP (adenovirus vector expressing a green fluorescent protein transgene) transduction by 50%, defined as ID_5O. Sera with titers greater than 320 were considered to be high titer sera and sera with titers 20-320 were considered to be low titer sera (Tsai, V. et al (2004). Clin. Cancer Res. 10:7199-7206). Sera with titers less than 20 were not used for the study.

Purification of capsid proteins from adenovirus.

Capsid proteins were prepared from E 1/E3 -deleted rAd with the transgene for p53 grown in HEK 293 cells and first purified by anion exchange chromatography followed by gel filtration (Vellekamp, G., et al (2001) Hum. Gene Ther. 12:1923-1936). This rAd lacks protein DC. The virus was further purified to remove any residual free proteins by centrifugation against a high glycerol pad as follows. The virus (4 x 10¹⁴ particles at 1.2 x 10¹² particles/ml) in Buffer A (14 mM Tris, 11 mM sodium phosphate, 2 mM MgCl₂, 2% sucrose, 10% glycerol, (w/v), pH 8.1 at 4⁰C) was placed in multiple 38.5 ml polyallomer centrifuge tubes on top of 2 ml and 4 ml pads, of 25% and 70% glycerol, respectively, in Buffer A. The virus was centrifuged for 30 minutes at 26,000 RPM in a Beckman SW28 swinging bucket rotor at 1O⁰C. The supernatant was removed and the concentrated virus was dialyzed against Buffer A at 4⁰C.

The concentrated virus was heat-disrupted at 57⁰C for 1 hour, then cooled and loaded on a 20 ml DEAE-Fractogel column at 4⁰C equilibrated with Buffer A. After washing with column with additional buffer, the fiber was eluted with 50 mM NaCl in the same buffer. This was followed by a NaCl gradient from 50-600 mM with penton base eluting as a small peak at approximately 200 mM and the hexon eluting as a large peak at approximately 300 mM NaCl. The fiber-, penton-, and hexon-containing fractions were separately pooled, concentrated with Millipore Ultrafree-15 centrifugal filtration devices, and run on a Superdex-200 gel filtration column equilibrated with Buffer A. Peak fractions were pooled, 0.22 μm filtered, analyzed, and stored at -8O⁰C until used. The proteins were analyzed by RP-HPLC or by western blot for fiber (Vellekamp, G., F. et al. (2001) Hum. Gene Ther. 12:1923-1936). The protein concentrations were determined by Bio-RAd protein assay.

Blocking of anti-adenovirus antibodies with purified capsid proteins.

RD cells were plated at 5x10⁴ cells per well in 96 well flat-bottom plate over night at 37⁰C 5% CO₂. The next day, specific dilution of human low titer serum was mixed with serial 10 folds dilutions of individual capsid protein. The starting dilution of the capsid protein was 100 fold excess calculated based on the capsid proteins on each virion. The mixtures were cultured with Ad-GFP (1/1 v/v) containing 8xlO⁸ Ad-GFP particles/ml for 1 hour at 37⁰C 5% CO₂. This serum, capsid protein, adenovirus mixture was transferred to cultured RD cells in 96 well flat-bottom plates. The relative fluorescent intensity in each well was measured the following day on a CytoFluor 4000 Fluorescence Multi-well Plate Reader (PerSeptive Biosystems, Foster City CA). Data were expressed as percent transduction as described previously (Rahman, A. et al 2001. MoI. Ther. 3:768-778) or as RFU (relative fluorescent unit) reported by the instrument. All samples were done in quadruplicates. Five different experiments were done with low titer serum isolated from different blood donors. Enhancement of transduction with complement protein, CIq.

RD cells were cultured as described above. Experiment conditions were similar to above except low titer human serum was heat inactivated before use. Purified human CIq protein (Quidel, Santa Clara, CA) was serially diluted in 10 folds increment starting at lOOμg/ml. The heated inactivated serum, CIq, and Ad-GPF were then added to the cultured RD cells. Human C4 complement protein (Quidel, Santa Clara, CA) was used as controls. Data were expressed as RFU as reported by CytoFluor plate reader. All samples were done in quadruplicates. Five different experiments were done with low titer serum isolated from five different blood donors.

Purification of anti-hexon antibodies from human sera.

Hexon protein was conjugated to Affigel-15 (Bio-Rad). Affigel-15 was supplied as a suspension in ispropanol. 100 ul of the matrix was resuspended and rinsed with 400 ul of deionized water on ice. The matrix was briefly centrifuged to pellet the matrix and the excess liquid removed. The hexon protein was diluted with 50 mM HEPES buffer to a final volume of 450 ul ~ (180 ul of hexon at 0.91 ug/ul). Conjugation was allowed to proceed overnight at 4⁰C with continuous inversion. The Affigel 15-hexon matrix was rinsed with two 500 ul volumes of PBS buffer changes. Between each wash the matrix was spun down and the wash material aspirated.

Sera samples were diluted 1:1 with dPBS to 1 ml. Diluted sera were mixed with the hexon conjugated Affigel-15 matrix. The tubes were mixed by inversion for 3 hr at 4⁰C. The sera-matrix suspension was briefly centrifuged and the supernatant transferred to a fresh tube labeled anti-hexon minus sera. The column was washed twice with 400 ul of PBS. The anti- hexon antibodies were eluted with 100 ul of 0.2M glycine (pH 2.5) and rapidly resuspended, then spun down. The supernatant was added to a fresh tube containing 80 ul of IM Tris, pH 7.5. The matrix was extracted again with 200 ul and 620 ul of water sequentially and pooled with the anti-hexon fraction.

For the neutralization assay the sera was used neat at 6 ul while the anti-hexon and hexon minus samples and buffer controls were used at 24 ul to account for their dilution. Eluted fractions were stored at -2O⁰C until required.

RESULTS

Characterization of neutralizing antibodies against adenovirus in human sera using HeLa and RD cell lines. Previously it was ashown that neutralization of adenovirus by human sera can be determined in vitro by a serum titration assay that measured the reduction of GFP expression after Ad-GFP infection in HeLa cells and that sera isolated from normal donors have various neutralizing antibodies titers. Titers were defined as the reciprocal dilution that gave a 50% reduction of GFP expression. We classified neutralizing titer greater than 320 as high titer, titer less than 320 as low titer (Rahman, A. et al (2001). MoI. Ther. 3:768-778; Tsai, V. et al (2004). Clin. Cancer Res. 10:7199-7206). Here using the same assay system, we infected HeLa cells with Ad-GFP in the presence of native or heat inactivated sera from high titer or low titer human sera. As shown in Figure IA, HeLa cells were readily infected with Ad-GFP, and preincubation of Ad-GFP with varying amounts of human serum reduced GFP expression in a dose-dependent manner. Heat-inactivated low titer serum became slightly more neutralizing, shifting the midpoint of GFP expression from 60 to 120. There was no change in neutralization for high titer serum under similar conditions.

RD cells were reported by others not easily infected with adenovirus (Polacek, C. et al (2005) Virus Res. 113:107-115). As seen in Figure IB, under identical experimental conditions as with HeLa cells, there was no measurable GFP expression when RD cells were infected with Ad-GFP. However in the presence of low titer serum, there was a robust GFP expression. This expression was highly dependent on serum concentration. Inactivation of the low titer serum with heat completely prevented this GFP expression.

Purification of adenovirus capsid proteins. To determine the potential role of antibody specificity from the low titer serum responsible for the observed Ad-GFP infectivity in RD cells, purified Ad capsid proteins were required. These were prepared from column-purified recombinant adenovirus. After concentration of the virus by centrifugation against a glycerol pad to remove any residual free proteins and high molecular weight contaminants (Vellekamp, G.et al. (2001) Hum. Gene Ther. 12:1923-1936) the virus was heat disrupted, cooled, and chromatographed on an anion exchange column. The peak fractions containing the purified capsid protein components were separately pooled, concentrated, and purified by gel filtration chromatography. The purified fiber (17 μg/ml), penton (30 μg/ml) and hexon (910 μg/ml) were >90% pure as determined by SDS-PAGE (Fig. 2) and their identities were confirmed by RP-HPLC for penton and hexon or by western blot for fiber. Anti-hexon antibodies in low titer serum mediate adenovirus infection in RD cells. Low titer serum was diluted at 1 : 80 and 1 :160 for the capsid protein competition assay. These dilutions corresponded to peak GFP expression, and low GFP expression respectively (Fig. IB). As shown in Figure 3 A, at the 1 :80 serum dilution, the presence of 100-fold excess of purifed hexon protein completely abrogated GFP fluorescence in RD cells. This inhibition was dose dependent, i.e., the inhibitory effect was reversed as hexon protein concentrations were reduced. This suggested that the removal of anti-hexon antibodies present in the low titer serum with free hexon proteins abrogate Ad-GFP infection of RD cells. The addition of free fiber or penton base proteins to block anti-fiber or anti-penton base antibodies in low titer serum, did not reduce GFP expression in RD cells. Similarly, at the 1 :160 dilution of low titer serum, the addition of purifed hexon protein abrogated Ad-GFP infection of RD cells in a dose dependent manner. Interestingly, removal of anti-fiber antibodies in the serum by addition of purifed fiber protein slightly enhanced Ad-GFP infection of RD cells (Fig. 3B), suggesting that the GFP signal reflected the net balance of inhibitor and enhancing components present in the serum.

Heat sensitive CIq in low titer human serum facilitated Ad-GFP infection in RD cells. RD cells were exposed to Ad-GFP in the presence of heat-inactivated low titer serum. As shown on Figure 4, RD cells alone or RD cells infected with Ad-GFP gave similar minimum fluorescent intensity. In the presence of heat-inactivated low titer human serum, there was no change in fluorescent intensity at 24 hours post Ad-GFP exposure (data not shown). However, in the presence of a replacement concentration of CIq normally found in human serum (51-125 μg/ml) to the heat inactivated low titer serum, there was a dose dependent increase of fluorescent intensity in RD cells. C4, another heat labile complement protein had no effect on the infection of Ad-GFP in RD cells co-cultured with heat inactivated low titer serum (Fig. 4).

Anti-hexon antibodies and CIq proteins found in low titer human sera were responsible for the infectability of adenovirus in RD cells. As shown above, in the absence of low titer human serum, RD cells were not permissive for adenovirus infection. Also from the above results two components found in low titer human sera were responsible for adenovirus infection of RD cells, they were CIq and anti-hexon antibodies. We then purified anti-hexon antibodies from the low titer human sera, and co-cultured the purified antibodies with Ad-GFP in the RD cells, GFP expression in RD cells went up in a dose dependent manner (Fig. 5). At 1 :40, there was a significant increase of fluorescent intensity from baseline of 3000 RFU to above 16000 RFU. This increase of fluorescence was abrogated if the purified anti-hexon antibodies were subjected to heat inactivation at 56⁰C for 30 minutes. However if CIq protein was added to the heat-inactivated anti-hexon antibodies, the fluorescent intensity was restored in RD cells. CIq or heat inactivated anti-hexon antibodies individually had no effect on the infectability of adenovirus in RD cells.

Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. All references cited herein are incorporated by reference to the same extent as if each individual publication, patent application, or patent, was specifically and individually indicated to be incorporated by reference.

Claims

1. A method of enhancing recombinant adenoviral vector delivery to a cell, the method comprising administering an adenoviral vector in conjunction with an anti-hexon antibody and/or an F(ab)' fragment of an anti-hexon antibody and/or a CIq peptide or combinations thereof.

2. The method of claim 1, wherein an anti-hexon antibody is administered in conjunction with the adenoviral vector.

3. The method of claim 1, wherein an an F(ab)' fragment of an anti-hexon antibody is administered in conjunction with the adenoviral vector.

4. The method of claim 1 , wherein an anti-hexon antibody and an

F(ab)' fragment of an anti-hexon antibody is administered in conjunction with the adenoviral vector.

5. The method of claim 3 , further comprising the administration of a C 1 q peptide.

6. The method of claim 4, further comprising the administration of a C 1 q peptide.

7. The methods of claims 1-6, wherein the cell is ex vivo.