SK159999A3 - Alternatively targeted adenovirus - Google Patents

Abstract

The present invention provides a trimer comprising three monomers, each having an amino terminus of an adenoviral fiber protein and each having a trimerization domain. The trimer exhibits reduced affinity for a native substrate than a native adenoviral fiber trimer. The present invention further provides an adenovirus incorporating the trimer of the present invention. The present invention also provides a cell line expressing a non-native cell-surface receptor to which an adenovirus having a ligand for the receptor binds, and a method of propagating an adenovirus using the cell line. The present invention also provides a method of purifying an adenovirus having a ligand for a substrate from a composition comprising the adenovirus. The method involves exposing the composition to the substrate under conditions to promote the ligand to selectively bind the substrate. Subsequently, the composition not bound to the substrate is separated from the substrate, after which the bound adenovirus is eluted from the substrate. The present invention further provides a method of inactivating an adenovirus having a ligand recognizing a blood- or lymph-borne substrate by exposing the virus to the substrate. Within the blood or lymph, the ligand binds its substrate, thereby adsorbing the free virus from the blood or lymph. Additionally, the present invention provides a chimeric blocking protein comprising a substrate for an adenovirus fiber, and a method of interfering with adenoviral receptor binding by incubating an adenovirus with such chimeric blocking protein in a solution such that the chimeric blocking protein binds the fiber.

Description

Alternatively, targeted adenovirus

Technical field

The invention relates to an alternatively targeted adenovirus and includes methods of producing and purifying said viruses as well as protein modifications that mediate alternative targeting.

BACKGROUND OF THE INVENTION

Adenoviral infection begins with the capture of the virion on the target cell. Adenovirus is captured on two cell surface proteins. Both proteins must be present in order to infect the target cell (Wickham et al., Cell, 73, 309-19 (1993)). The wild-type adenovirus first binds to the cell surface via the adenoviral receptor (AR). One such AR is the recently identified coxsackie virus and adenovirus (CAR) receptor (Bergelson et al., Science, 275, 1320-23 (1997); Tanako et al., Proc. Nat. Acad. Sci. USA, 94, 3352-56 (1997)). The MHC class I receptor is also AR (Hong et al., EMBO J., 16 (9), 2294-06 (1997)). Upon capture on AR, the virus attaches to and _in integrins, a family of heterodimeric cell surface receptors that mediate interaction with the extracellular matrix and play an important role in cellular signaling (Hynes, Cell, 69, 11-25 (1992)).

Once captured on the cell surface, infection proceeds by receptor-mediated internalization of the virus into endocytic vesicles (Svensson et al., J. Virol., 51, 687-94 (1984); Chardonnet et al., Virology, 40, 462 -77 (1970)). In the cell, virions are broken down (described in Greber et al., Cell, 75, 477-86 (1993)), the endosome is disrupted (Fitzerald et al., Cell, 32, 607-17 (1983)) and viral particles are transferred to the nucleus via a nuclear pore complex (Dales et al., Virology, 56, 465-83 (1973)).

The adenoviral virion is an uncoated icerader 65-80 nm in diameter (Horene et al., J. Mol. Biol., 1, 84-86 (1959)). The adenovirus Russian capsid contains 252 capsomers (240 hexons and 12 pentons) (Ginsberg et al., Virology, 28, 782-83 (1966)). Hexones and pentones were derived from three viral proteins (Maizel et al., Virology, 36, 11-15-25 (1968); Weber et al., Virology, 76, 709-24 (1977)). The hexon contains three identical proteins, each having 967 amino acids, namely polypeptide II (Roberts et al., Science, 232, 1148-51 (1986)). The penton comprises a base that binds to a capsid and a strand that noncovalently binds to and protrudes from the penton base. The IX, VI and IIIa proteins are also present in the adenovirus coat and stabilize the viral capsid (Stewart et al., Cell, 67, 145-54 (1991); Stewart et al., EMBO J., 12 (7) 2589-99 (1993)).

The penton base is highly conserved among the adenovirus serotypes and has five tripeptide RGD motifs (except for enteric adenovirus Ad40) (Neumann et al., Gene, 69, 153-57 (1988)). The adenovirus RGD tripeptides apparently mediates the binding of the adenovirus, and _the integrins and endocytosis of the virion (described in Wickham, et al., Wickham et al., Cell, 73, 309-19 (1993)).

The adenoviral strand is the homotrimer of the adenoviral polypeptide IV (Devaux et al., J. Molec. Biol., 215, 567-88 (1990)). The thread has three discrete domains. The amino terminal region is non-covalently attached to the penton base. The relatively long middle part domains containing a variable number of 15 repeating amino acid residues that form β-leaves protrude from the top of the viral particle (Yeh et al., Virus Res., 33, 179-98 (1991)). Approximately 200 amino acid residues at the carboxyl terminus form a nodal domain. The node mediates primary viral binding to cellular AR and trimerization of the fiber (Henry et al., J. Virol., 68 (8), 523946 (1994)). In order for the end of the strand to properly associate with the penton base, a trimerization region appears necessary (Novelli et al., Virology, 185, 365-76 (1991)). In addition to cell AR recognition and penton base binding, the fiber protein contributes to serotype integrity and mediates nuclear placement.

Fiber proteins from different adenoviral serotypes differ substantially. For example, the number of 15 amino acid β-leaf repeats varies among different adenoviral serotypes (Green et al., EMBO).

J., 2, 1357-65 (1983)). However, the node regions close to the related serotypes Ad2 and Ad5 show only 63% amino acid similarity (Chrobozcek et al., Virology, 186, 280-85 (1992)) and the fibrous nodes Ad2 and Ad3 show only 20% identity (Signas et al. J. Virol. 53, 672-78 (1985)). In contrast, the penton base sequences are 99% identical. Despite these differences in the node region, different conserved regions are demonstrated by aligning the Ad2 and Ad3 fiber gene sequences, most of which are also conserved among other human adenovirus fiber genes.

A number of factors present adenoviruses as attractive vectors for use in various gene transfer applications (e.g., cellular protein production, therapy, academic studies, etc.). For example, an adenovirus is a perfect expression vector. Recombinant adenovirus can be produced with high titers (for example, approximately 10 ¹³ viral particles / ml) and adenoviral vectors can transfer genetic material to a cell in which it either replicates or does not replicate (as opposed to retroviral vectors). The adenoviral genome can be manipulated to carry large amounts of exogenous DNA (up to about 7.5 kb), and the adenoviral capsid can potentiate even longer sequence transmission (Curiel et al., Hum. Gene Ther., 3, 147-54 (1992) )). In addition, some features suggest that adenoviruses represent a safe choice of gene transfer especially in therapeutic use. For example, adenoviruses do not integrate into the chromosome of the host cell, thereby minimizing the likelihood that the adenoviral vector will interfere with normal cell function. Adenoviral infection in humans does not correlate with malignancy and recombination of the adenoviral genome rarely occurs. Because of these benefits, doctors have been using these vectors as safe human vaccines for many years for gene therapy.

Due to the popularity of adenoviral vectors, efforts have been made to increase the ability of adenovirus to enter certain cells, for example, those that are not able to infect adenoviruses, by the so-called "adenovirus". "Targeting" (described, for example, in International Patent Application WO 95/26412 (Curiel et al.)), International Patent Application WO 94/10323 (Spooner et al.), In US Patent 5,543,328 (McClelland et al. ), in International Patent Application WO 94/24299 (Cotten et al.)). Of course, while the ability to target adenoviruses to certain cell types is an important task, it is far more important to prepare an adenovirus that infects only the desired cell type, which is called "exclusive targeting". In order to produce an exclusively targeted virus, its natural affinity for the host cell AR must first be eliminated, producing a recombinant adenovirus which is unable to productively infect a whole set of cells that are natural adenoviral targets. Efforts to abolish natural adenoviral cell affinity are logically concentrated to alter the fiber node. This effort was accompanied by disappointment because the suppression of important functions of the fiber protein in stable trimerization and penton base binding (Spooner et al., Supra) was unsuccessful. However, replacing a fiber node with a cell surface ligand (McClelland et al., Supra) produces a virus that is only suitable for infection of a cell type having said ligand. Such a strategy produces a virus that exhibits a number of the same targeting problems associated with wild-type adenoviruses (where fiber trimerization and cell trophism is mediated by the same protein domain), thus reducing the flexibility of the vector. Due to the lack of host cells and integration of fiber trimerization and targeting of functions, obtaining a mutant virus with substituted targeting is complicated. For example, removal of a fiber node and its replacement with a non-trimerizing ligand (e.g., McClelland et al., Supra) leads to the production of a virus lacking a suitable fiber protein. Therefore, it is necessary to create an adenoviral fiber that exhibits limited affinity for natural AR but still exhibits trimerization of the fiber and a function of binding to the penton base.

While exclusive adenoviral targeting requires a reduction in natural cellular trophism, abolishing the ability of natural targeting also reduces the ability of the virus to infect cell lines that are normally used to multiply (e.g., 293 cells) ((Curiel et al., Hum. Gene Ther. , 3, 147-54 (1992)) One published experiment overcomes this obstacle by using a cell line expressing a binding site on the surface of a relevant cell (McClelland et al., Supra) However, a number of cell lines do not express important cell lines. A number of available cell lines expressing potentially useful cell surface binding sites are inadequate for the production of recombinant adenoviruses, especially viruses useful in clinical applications (e.g., cell lines carrying and expressing essential adenoviral early genes from the E1, E2 and / or E4 regions). It is therefore necessary to provide a cell line and methods for producing a cell line in which a recombinant adenovirus that is substantially unable to productively infect cells via natural AR can be multiplied and packaged.

Typical protocols for purification of viral vectors from cell lysates include single or repeated centrifugation of viruses using a CsCl ₂ gradient. While such methods adequately isolate viruses, however, they need substantial material, which is CsCl ₂ , and are therefore relatively ineffective. Such protocols often cannot be used in many applications, which is a significant impediment to the commercial scale development of viral vectors. Other methods including column purification do not bind viruses specifically (Shabram et al., Hum. Gene Ther., 8, 453 (1997); Huyghe et al., Hum. Gene Ther., 6, 1403 (1995)). These methods often cause the presence of an unacceptable amount of contaminants compared to the form obtainable by affinity purification of other materials. There is a need for an efficient method of purifying and isolating recombinant viral vectors.

In many applications involving the in vivo introduction of viral vectors, it is necessary to infect the selected tissue (and introduce the gene). For example, the treatment of systemic infection and the introduction of a biologically active gene represent a major problem in gene therapy applications. However, ectopic expression of the transgene will damage many experimental applications. While theoretically, host blood cells can express proteins that facilitate the purification of foreign substances such as adenoviruses (News and Comment, Science, 275, 744-45 (1997)), the manipulation of such cells and their production in the host is difficult and intrusive. While anti-adenoviral hexon antibodies can inactivate the virus (Toogood et al., J, Gen. Virol., 73, 1429-35 (1992)), there are no effective protocols for introducing sufficient anti-hexon antiserum into the gene transfer recipient to occur. to reduce or prevent ectopic viral infection. This strategy may interfere with gene transfer protocols by blocking infection of the desired tissues.

It is therefore necessary to provide a method of inactivating recombinant viral vectors that retain the desired insertion attempt in the host animal.

SUMMARY OF THE INVENTION

The invention discloses a trimer comprising three monomers, each having an N-terminus of the adenoviral fiber protein and each having a trimerization domain. The trimmer exhibits limited affinity for the natural substrate compared to the natural trimer of the adenoviral fiber. The present invention further provides an adenovirus incorporating the trimer of the invention. The invention further provides a cell line expressing an unnatural cell surface receptor, wherein the adenovirus has a ligand by which it binds to it. Further described is a method of propagating adenovirus using a cell line.

The invention further provides a method of purifying an adenovirus having a substrate ligand from a composition comprising an adenovirus and a method for selectively binding a substrate. Subsequently, the composition that does not bind to the substrate is separated from the substrate and the bound adenovirus is eluted from the substrate.

The invention further provides a method of inactivating an adenovirus having a ligand recognizing a substrate derived from blood and lymph by contacting the substrate with the virus. In blood or lymph, the ligand binds to the substrate, adsorbing the free virus.

The invention provides a chimeric blocking protein comprising an adenoviral fiber substrate and a method of adenoviral receptor interference that binds to an adenovirus to a chimeric blocking protein in solution such that the chimeric blocking protein binds to the fiber.

The invention is further used in various in vivo and in vitro applications, such as therapy. It finds utility, for example, as a vector for introducing a therapeutic gene into a cell with minimal etopic infection. Specifically, the invention allows for more efficient production and construction of safer vectors for use in gene therapy. The invention can also be used as a research tool by preparing methods and reagents for studying adenoviral cell capture and infection and can be used to test receptor-ligand interaction. Similarly, recombinant filamentous protein trimmers can be used in receptor-ligand assays and as adhesive proteins in vitro and in vivo.

Further, the invention provides reagents and methods enabling biologists to examine the cellular biology of viral growth and infection. Such vectors of the invention are very useful in biological research

definition

The term 'adenovirus' is any virus of the genus Mastctdenoviridae or Aviaadenoviridae and may belong to any serotype of that genus. Adenoviral stocks that can be used as a source of adenovirus or adenoviral coat protein, such as penton base and / or fiber protein, can be amplified from adenoviral serotypes available from the American Type Culture Collection (ATCC, Rockville, MD) or from any other source.

A "ligand" can be any species selectively binding to an identifiable substrate.

The term "natural" means a protein or property of an unmodified virus or cell. Then, the non-natural protein may be a modified and mutated protein that differs from its natural homologue in the virus or in the cell. Alternatively, the non-natural protein may be a protein that does not have natural homology in the virus or in the cell.

The term "AR" refers to an adenoviral receptor. In particular, "AR" is a ligand binding to a mastadenoviral node.

The first species selectively binds to the substrate when it binds to the substrate with greater affinity than the second species. The first species does not bind selectively when it binds to a substrate with the same or less affinity than the second species, even if the first species binds with the same affinity.

trimers

The invention discloses a trimer comprising three monomers (for example, at least a portion of each of the three adenoviral fiber monomers) each having an N8 tail derived from the adenoviral fiber protein and each having a trimerization domain. The trimer of the invention exhibits limited affinity for a natural substrate, such as an antibody, a cell binding site (i.e., natural for the serotype from which the N-terminus is derived) compared to the natural adenoviral trimer of the fiber. The trimer may be the homotrimer or heterotrimer of the various fibrous monomers. The invention further describes any modification of the monomeric units reducing the affinity of the resulting trimer to its natural cell surface binding site (i.e., native AR). Affinity reduction is a substantial reduction in affinity (by at least one order, more orders of magnitude is preferred) relative to the untreated corresponding fiber.

When the trimerization domain is a ligand itself suitable for a natural cell surface binding site, such trimerization domains are present in the trimmers. Therefore, the trimerization domain of the monomer incorporated into the trimer of the invention is preferably not a ligand for CAR or MHC-1, a cell surface domain or a fiber recognition antibody. More preferably, the non-natural trimerization domain is not a ligand suitable for any natural binding site on the surface of a mammalian cell, wherein the binding site is an AR or other binding site on the cell surface. Adenoviruses incorporating such trimmers have a limited ability to infect their natural host cells and can serve as efficient vector sources for manipulating selectively targeted vectors. Therefore, while the trimerization domain is preferably not a ligand suitable for a cell-surface binding site, then the entire trimer may be such a ligand (a non-natural ligand as described herein). The trimerization domain may be a ligand suitable for a substrate other than a natural cell surface binding site, such as trimerization ligands. For example, the non-natural trimerization domain may be a ligand suitable for a substrate on an affinity column, on a molecule that is formed in blood or even on a cell surface, and is not a natural binding site on the cell surface (for example, a cell manipulated to express the substrate cell surface) protein that is not natural for the unmodified cell type).

The trimmer inclusion monomer may be all or part of the natural adenoviral monomer protein of the fiber. For example, the treated monomer may lack an adjustable number of residues or even identifiable domains as described herein. For example, the monomer need not include a natural node domain; it may lack one or more natural β-leaf repeats or be otherwise truncated. Thus, the monomer may have any other desired treatment, which may be as large as trimerization still occurs. Preferably, the monomer is not treated at the N-terminus (for example, the N-terminus of the monomer forms the nature of the N-terminus of the fiber) to ensure that the resulting trimer interacts correctly with the penton base. The invention further provides a composition comprising a trimer of the invention and an adenovirus penton base. It is preferred that the trimer and penton base be joined in the same manner as wild-type penton base fibers. Of course, while the trimmer contains modified fiber monomers, the pentone base may also be treated to include, for example, an unnatural ligand as described in U.S. Patent 5,559,099.

Mutant nodules

The fibrous monomer suitable for incorporation into the trimer of the invention has a trimerization domain that binds to the native mammalian AR (i.e., AR is natural for the adenovirus serotype) with less affinity than the natural adenoviral fiber. It is preferred that the trimmers incorporating such monomers are not ligands suitable for their natural cellular binding sites. The monomers may be treated in any manner suitable for reducing fiber affinity to natural AR while allowing monomer trimerization. For example, in one embodiment of the invention, the trimerization domain is a modified adenoviral fiber node domain that does not contain a naturally occurring receptor-binding amino acid. Any amino acid residue mediating or contributing to the interaction between the node and the native cellular AR is an amino acid suitable for mutation or deletion from the monomer. .

Natural amino acid residues may be selected for modification or deletion in any manner. For example, to deduce conserved residues that are likely to mediate binding to AR, sequences for different adenoviral serotypes can be compared. Alternatively, or in combination, the sequence may be mapped to produce a three-dimensional representation of the protein (such as a crystal structure) to deduce those residues most likely responsible for binding to AR. These assays can be performed using any conventional algorithm or program for deducing protein structural functional interaction. Alternatively, random mutations can be incorporated into the expression cassette of the cloned adenoviral strand. The method of introducing random mutations into a protein is carried out by Taq polymerase. For example, a knot encoding a fiber node (SEQ ID NO: 9; Roelvink et al., J. Virol., 70, 7614-21 (1996)) can serve as a template for PCR amplification of the adenoviral node of a fiber or a portion thereof. The frequency of transcript errors can be predetermined in most cases by varying the concentration of the two-valent cations in the PCR reaction (Weiss et al., J. Virol., 71, 4385-94 (1997); Zhou et al., Nucl. Acid. Res., 19 , 6052 (1991)). The PCR products can then be subcloned back into the template vector to replace the sequence in the strand coding sequence that serves as a source for the PCR reaction. This creates a fiber library, some of which will carry mutations that hinder natural binding to AR while retaining the ability to trimerize.

A monomer that lacks one or more amino acids as described herein may contain an unnatural residue (for example, several unnatural amino acids) in addition to or instead of the missing natural amino acid. Alternatively, natural amino acids can be detected from the node. It is preferred that the amino acid substitutes for another unnatural amino acid so as to maintain topology and, in particular, trimerization. The replacement of the amino acid preferably allows for a new monomer quality. For example, in order to maximize the binding to natural AR, the natural amino acid may be replaced by a residue (or a plurality of residues) with a different charge. Such substitution maximally interferes with the electrostatic interaction between the natural domains of the adenoviral node and the cellular AR In a similar case, where possible, the natural amino acid may be replaced by a larger molecular weight residue (or a plurality of residues). These residues have a longer side chain; maximally interferes with the spatial interaction between the natural adenoviral domains and cellular AR.

Unnatural trimerization domains

In another embodiment, the trimmer comprises engineered monomers that are chimeric adenoviral fiber polypeptides. A suitable chimeric monomer does not contain all or part of the trimerization domain that is natural to the source of the adenoviral serotype. The trimerization domain of such a monomer may be deleted from the virus or removed by insertion or substitution of unnatural amino acids. Of course, a monomer that does not contain a natural trimerization domain may also not contain the entire natural node. Since the natural trimerization domain is a ligand suitable for AR, the trimmer of the chimeric adenoviral fiber monomers lacking the natural trimerization domain binds to its natural AR with less affinity than the natural adenoviral fiber.

In order for the chimeric monomers to form the trimmer of the invention, replacement (i.e., unnatural) trimerization domains must be incorporated into the monomer. In order to maximize the targeting of the virus, it is preferred that the non-natural trimerization domain is not a ligand suitable for a mammalian cell surface receptor or other cell surface receptor. Any domain capable of forming homotrimers is suitable as a trimerization domain for inclusion in the trimers of the invention. For example, the chimeric monomer may comprise the trimerization domain of the heat shock factor (HSF) protein of K.lactis (Soreger and Nelson, Cell, 59, 807 (1989)), of the axonal dynein of trout (Garber et al., EMBO J , 8, 1727 (1989)), parainfluenza virus hemagglutinin protein (Coelingh et al., Virology, 162, 137 (1988)), type 1 reovirus sigma1 protein (Strong et al., Virology, 184, 12 (1991)) ) or other suitable trimmers. Alternatively, the chimeric monomer may include a modified leucine zipper motif Leucine zippers comprise a group of seven leucine repeats that mediate dimerization. Replacement of one or more leucines with isoleucine results in stable domain trimerization. An example of such a modified leucine zipper motif is the 32 amino acid GCN4p-II trimer (Harbury et al., Science, 262, 1401 (1993)).

Of these trimerization domains, the sigma 1 reovirus trimerization domain is preferred. This protein contains 17 alpha-helical groups of seven repeats resembling the double helix structure of the above mutant isoleucine zipper domains (Harbury et al., Nature, 371, 80-83 (1994)) . Thus, filamentous chimeras containing the sigma 1 domain may protrude from the virus to a greater distance than the corresponding chimeras containing shorter trimerization domains. An advantage of the sigma 1 reovirus trimerization domain over a mutant leucine zipper domain (e.g., GCN4) is that the sigma 1 domain is 22 nm long (Fraser et al., J. Virol., 64, 2990-3000 (1990)), whereas the GCN4 domain it is only 5 nm long (Harbury et al., Nature, 371, 80-83 (1994)). Another advantage with the use of the sigma 1 reovirus capture protein is that, unlike the central portion of the viral adenoviral protein, this protein tends to be intrinsic trimerized (Leone et al., Virology, 182, 336-45 (1991)). Fiber length appears to increase the efficiency and specificity of adenovirus cell attachment (Roelvink et al., J. Virol., 70, 7614-21 (1996)). For other chimeric fibers, other fibers with a possible sigma 1 domain are preferred.

Alternatively, the chimeric monomer may comprise a node domain from another adenovirus serotype. For example, the trimerization domain may be replaced by a mutant node of an adenoviral serotype that is capable of productively infecting a host (e.g., an Ad3 mutant node containing a mutation in loop H1). Alternatively, it may be replaced by a node of a serotype that is unable to productively infect the host. For example, the node of a mammalian adenovirus serotype fiber may be replaced by a node of avian serotype. While avian node mediates trimerization of fiber proteins, it is likely impossible for it to recognize mammalian AR. Therefore, these chimeric fibers do not contain the ability to bind to the natural AR of the host. Similarly, the fiber node of one mammalian adenovirus serotype may be replaced by a node from another mammalian serotype.

The treated and untreated nodule from porcine adenovirus NADC-1 fiber is the preferred domain and is well characterized. NADC-1 has defined ligands, for example galectin (which binds galactose) and LDZ and RGD peptides (which bind integrins) (see, for example, Hirabayashi et al., J Biol. Chem., 266, 13648-53 (1991)) . Thus, chimeric strands of human adenoviruses having NADC-1 nodes with such mutations may form trimmers and may associate with the penton base, but bind to natural cell surface receptors with limited affinity.

The unnatural trimerization domain can be ligated with the natural fiber monomer at any convenient location as long as the monomers can be correctly isolated (i.e., they are able to interact with the adenoviral penton base). For example, a domain can be incorporated into a natural node, disrupting its topology. Alternatively, the trimerization domain can be inserted beyond any repeat of 15 amino acids, preferably after the seventh, fifteenth or twenty-second repeats to mimic the natural size of the middle portion of the adenovirus. When an unnatural trimerization domain is incorporated into the central portion of an adenovirus, it may form the carboxyl terminus of the chimeric protein or be inserted into the middle of the amino acid sequence. Any number of trimerization domains can be incorporated into the fiber monomer as long as the resulting trimmer is properly coupled to the penton base.

Blocking domain

Another suitable chimeric monomer has a new ligand blocking domain that is suitable for the AR host. The blocking domain is any peptide that can tightly bind to a natural ligand (Hong et al., EMBO J., 16, 2294-2306 (1997)). In other words, the blocking domain is a substrate to which the (natural or engineered) fibrous monomeric ligand is selectively bound. It is necessary that the substrate-ligand interaction occurs at least immediately in the production of the virus and continues efficiently until the fibrous trimer is damaged. Because the natural ligand binds to the blocking domain, the ligand is unable to bind to its natural substrate on the cell surface. Since the natural trimerization do14 currency is a ligand suitable for natural AR, trimers of chimeric adenoviral fiber monomers having such blocking domains bind to natural AR with less affinity than natural adenoviral fiber. The blocking domain can be located at any position of the adenovirus to bind to the natural ligand without affecting trimerization or interaction with the penton base. For example, the blocking domain may be attached to the β-sheet described above or to loops either by fusion with a reading frame, by covalent post-translational modification. Alternatively, the blocking domain may be attached to another portion of the monomer. The blocking domain may also include a linker or spacer polypeptide that allows the blocking agent to interact with a natural ligand. If the blocking domain is attached via such a spacer, then the spacer may include protease-recognizing sites.

preparation

The monomers for inclusion in the trimers of the invention can be produced by any possible method. The mutant fiber protein can be produced by any suitable method. The mutant fiber protein can be synthesized using standard direct peptide synthesis (e.g., as described in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)), such as solid phase synthesis (e.g., Merrifiel, J. Am. Chem. Soc., 85, 2194-54 (1963); Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987) and US Patent 5,424,398). Alternatively, site-specific mutations (such as replacement of a node with an unnatural trimerization domain, removal, replacement, or mutation of residues that bind to AR, or addition of a blocking domain as described above) can be introduced into the monomer by ligation into an expression vector. that contains the edited location. Alternatively, a plasmid, oligonucleotide, or other vector encoding a desired mutation may be recombined with an adenoviral genome or expression vector that encodes a monomer to introduce the desired mutation. Oligonucleotide-directed site-specific mutagenesis is also suitable for this purpose (see, for example, Walder et al., Gene, 42, 133, (1986); Bauer et al, Gene, 37, 73 (1985), Craik, Biotechniques, 12). -19 (1995), US Patent 4,518,584 and 4,737,462). The manipulated DNA fragment encoding the engineered monomer can be subcloned into a suitable vector using a well known molecular genetics method. The fragment is then transcribed and the peptide subsequently translated in vitro in the host cell. Any suitable expression vector (described, for example, in Pouwels et al., Cloning Vectors; A Laboratory Manual (Elsevior, NY.1985)) and the corresponding suitable host cells can be used in the production of recombinant peptides. Expressive hosts include, but are not limited to, bacterial species, mammalian and insect host cell systems that include baculoviruses (e.g., Luckow et al., Bio / Technology, 6, 47 (1988)), and established cell lines, such as cell lines 293, COS-7, C127, 3T3, CHO, HeLa, BHK, etc. Especially preferred is an expression system suitable for preparing the engineered fibers of the invention, which is a baculovirus expression system (Wickham et al., J. Virol. 70: 6831-38 (1995)) This system allows for the production of large quantities of recombinant proteins, and the choice of an expression host reflects the type of peptide produced, which is primarily due to post-translational modifications.

When the monomers are prepared, they are tested for fiber protein activity. In particular, the ability of the monomers to form trimers, interact with the penton base and with the natural AR is tested. Any suitable test may be used to measure these parameters. Since improperly packed monomers are generally insoluble (Scopes: Protein Purification (3d Ed., 1994), Chapter 9, p. 270-82 (Springer-Verlag, New York)), one of the trimerization tests is the solubility of the recombinant fiber. The fiber solubility is determined by incorporating a certain amount of radioactive amino acid into the protein during synthesis. The lysate from the host cell that expresses the recombinant fiber protein can be centrifuged and the supernatant and pellet can be assayed by scintillation counting or Western analysis. Subsequently, proteins are separated in the pellet and supernatant (e.g., on an SDS-PAGE gel) to isolate the fiber protein for further testing. A comparison of the amount of radioactivity in the protein of the fiber isolated from the pellet versus the protein of the fiber isolated from the supernatant indicates whether the mutant protein is soluble. Alternatively, trimerization can be tested using monoclonal antibodies that recognize only the N-part of the trimeric form of the fiber (e.g., by immunoprecipitation, Western blotting, etc.). One such antibody is described in International Patent Application WO 95/26412. Another measure of trimerization is the ability of the recombinant fiber to complex with the penton base (Novelli and Boulanger, Virology, 185, 1189 (1995)), whereby only the fibrous trimmers can interact. This tendency can be tested by co-immunoprecipitation, gel mobility change assay, SDS-PAGE (samples boiled are monomers, otherwise the assay is performed with larger proteins), etc. Another measure of trimerization is the detection of the difference in molecular weight of the trimmer as opposed to the monomer . For example, the brewed and denatured trimmer will exhibit less molecular weight on the gel as compared to the undenatured stable trimmer (Hong and Angler, J. Virol., 70, 7071-78 (1996)).

The trimeric recombinant fiber must also be tested for its ability to bind to natural AR. Any suitable assay that can detect this property is suitable for use in the present invention. A preferred assay involves exposing cells expressing AR (e.g., 293 cells) with a recombinant fiber trimer under standard infection conditions. Subsequently, these cells are exposed to natural adenoviruses and the ability of the viruses to bind to the cells is monitored. Monitoring can be recorded by autoradiography (e.g., using radiolabeled viruses), immunocytochemistry or by measuring the degree of infection or by gene introduction (e.g., using a reporter gene). In contrast to the natural trimmers that reduce or substantially eliminate the subsequent binding of the virus to 293 cells, these trimmers insignificantly reducing the ability of the natural adenoviruses to subsequently bind to the cells are the trimmers of the invention. Limiting interference with substantial viral binding indicates that the trimer itself is not a ligand suitable for its native mammalian AR or at least binds with limited affinity.

Alternatively, a vector comprising a mutant strand encoding sequence (or a putative mutated strand library as described herein) can be introduced into a suitable host cell strain to express the strand 17 protein. Bacteria are preferably used as host cells for high efficiency monitoring. In this case, the mutants can be identified by testing the ability to bind soluble CAR protein. For example, a replica of a bacterial plate (e.g., on a nitrocellulose filter) can be cultured in a suitable culture medium to induce expression from the vector. The filter is then exposed to a solution suitable for the lysis of bacteria retained on the filters. Testing using radiolabeled CAR protein is then performed. It is preferred that the filter is first "blocked" by a solution with a large amount of protein, minimizing the non-specific adherence of the CAR probe to the filter. After hybridization, the filter is exposed to film, identifying colonies expressing fiber proteins that bind CAR. These colonies not hybridizing to the radiolabeled CAR probe (or bound with limited affinity, indicating a weaker signal) potentially express the fibrous monomers of the invention. Since the limitation of CAR binding can be due to either selective ligand removal or structural modification affecting trimerization in mutant fibers that do not exhibit CAR binding, the ability to trimerize must then be assessed by testing the bacterial library as described above.

Blocking proteins

As alternative methods of reducing natural viral trophism, the invention provides a chimeric blocking protein comprising an adenoviral fiber substrate. The chimeric blocking protein may comprise any suitable domain having a substrate that is recognized by a ligand on the adenoviral strand. For example, in the case of interference with the binding of wild-type adenovirus to the receptor, the chimeric blocking protein may comprise the extracellular domain of the cell surface protein CAR (Bergelson et al., Science, 275, 1320-23 (1997); Tomko et al, Proc. Nat. Acad). Sci. USA, 94, 3352-56 (1997)), an extracellular domain suitable for the MHC class 1 receptor (Hong et al., EMBO J., 16 (9), 2294-06 (1997)) or another similar extracellular domain substrate suitable for AR. However, in the case of interference by binding of recombinant adenoviruses to a substrate, adenoviruses having filamentous trimmers as described herein, the blocking protein may comprise a substrate that recognizes a ligand present on said trimer. While it is stated that the chimeric blocking protein may comprise domains from cell surface proteins, it is typically not the cell surface protein itself. Instead of a chimeric blocking protein, a freely soluble protein that is capable of interacting with the adenovirus in solution is preferred.

The chimeric blocking protein of the invention allows a method interfering with adenoviral receptor binding by incubating adenovirus with the chimeric blocking protein in solution such that the chimeric blocking protein binds to a ligand that is present on the adenoviral fiber. The virus and the chimeric blocking protein may be incubated for any length of time and under any suitable conditions to promote ligand binding to the fiber with the substrate on the chimeric blocking protein. Parameters, such as time, temperature and solution, which are chemically suitable to promote selective binding of the fiber ligand to the chimeric blocking protein substrate, may vary depending on the affinity with which the ligand selectively binds to the substrate. In general, when a known ligand-substrate system is used, these parameters are also known. When a new ligand-substrate system is used, then binding conditions can be largely predetermined as outlined herein (for example, using such conditions when testing a new ligand-substrate interaction in a protein library). However, it is preferred that the concentration of the chimeric blocking agent be sufficient to saturate the cell surface ligands that are present on the adenovirus fibers during incubation.

In addition to the included domain that exhibits a ligand-recognized substrate on the adenoviral strand, the chimeric blocking protein also has other domains. For example, the protein may include secretion promoting domains (e.g., Suter et al., EMBO, J., 10, 2395-2400 (1991); Beutler et al., J. Neurochem., 64, 475-81 (1995)) , which facilitates the collection of free chimeric blocking proteins from the protein producing cells. In addition, the chimeric blocking protein preferably further comprises a ligand domain (i.e., a ligand alongside a substrate suitable for a viral node), as well as those ligands described herein. The presence of a ligand on the chimeric blocking protein, notably the peptide tag and other similar sequences, allows purification and identification of the chimeric blocking protein after its production. A more preferred ligand is one that recognizes cell surface binding sites or other substrates as disclosed herein. Such blocking proteins function as "bi-specific" molecules suitable for altering adenoviral receptor binding. For example, when a chimeric blocking protein comprises a ligand for a cell surface binding site, then the blocking protein is capable of affecting the selective targeting of adenovirus by interfering with the binding of the fiber to the receptor by directing a new targeting with the ligand present on the chimeric blocking protein. Thus, the present invention provides a method of controlling adenovirus targeting by incubating an adenovirus with a chimeric blocking protein having a ligand that recognizes a substrate present at a binding site on the cell surface in solution, such that the chimeric blocking protein binds the adenovirus fiber to form a complex which comprises a substrate for the ligand.

In addition to a domain that exhibits a substrate recognized by a ligand on an adenoviral fiber (presumably on a non-adenovirus ligand domain), the chimeric blocking protein may also include a trimerization domain, such as the trimerization domains disclosed herein. The presence of the trimerization domains allows the chimeric blocking protein monomers to trimerize. While, like monomers, chimeric blocking proteins can saturate ligands present as fibers, such bonds are of course subject to dissociation and to some extent depend on the kinetics of the ligand-substrate interaction. Since the probability that all three ligand / substrate bonds between the trimer strand and the trimer blocking protein will be enhanced at the same time is substantially less than the probability that any such binding will be broken, the trimer blocking protein more easily saturates the available ligands that are present on the fiber. The trimeric structure effectively holds each substrate against the fiber node ligand, increasing the likelihood that each ligand is blocked.

Chimeric blocking proteins can be produced by any suitable method, such as direct protein synthesis, cell production, in vitro translation, or other methods known in the art. Described herein are a number of suitable methods that produce proteins and are known in the art.

viruses

The invention provides an adenovirus incorporated into the recombinant fiber trimers of the invention. The adenovirus of the invention does not infect its natural host cells via wild-type AR, as easily as the wild-type serotype, due to the aforementioned limitation of the affinity of filamentous trimmers present in the viral envelope (e.g. residues or by incorporating a blocking domain). Thus, the adenovirus preferably incorporates a non-adenovirus ligand, allowing propagation, isolation and / or targeting.

The virus may include any suitable ligand (e.g., a peptide that specifically binds to a substrate). For example, when targeting an adenovirus to a cell type other than a naturally infected cell (or a group of cell types other than a natural range or a set of host cells), the ligand may bind to a cell surface binding site (e.g., any site present on the cell surface with the adenovirus interacts, binding to the cell and thereby promoting cell entry) that is other than AR, or may even be any natural AR. The cell surface binding site may be any suitable type of molecule, but is typically a protein (including a modified protein), a saccharide, a glycoprotein, a proteoglycan, a lipid, a mucin molecule or a mucoprotein, or other similar molecule. Examples of potential cell surface binding sites include, but are not limited to, portions of heparin and chondroitin sulfate that are found in glycosaminoglycans; sialic acid portions found in glycoproteins and gangliosides, common carbohydrate molecules found in membrane glycoproteins including mannose, N-acetyl-galactosamine, N-acetyl-glucosamine, fucose and galactose; glycoproteins such as ICAM-1, VCAM, selectins (e.g., Eselectin, P-selectin, L-selectin, etc.) and integrin molecules and tumor-specific antigens present on carcinogenic cells, such as MUC-1 tumor-specific epitopes. The protein may then be expressed in a narrow group of cell types (e.g., heart muscle, skeletal muscle, smooth muscle, etc.) or expressed in a broad group comprising several cell types.

In other embodiments of the invention (for example, to allow purification and propagation in specifically engineered cell types), the non-natural ligand may bind to a compound other than the natural cell surface protein. Thus, the ligand may bind to proteins that arise in blood and / or lymph (e.g., albumin), to synthetic peptide sequences such as polyamino acids (e.g., polylysine, polyhistidine, etc.), to artificial peptide sequences (e.g., FLAG shown in SEQ. ID NO: 16) and RGD peptide fragments (Pasqualini et al., J. Cell. Biol. 130, 1189 (1995)). Alternatively, the ligand may bind to substrates that do not contain peptides, such as plastic (see, for example, Adey et al., Gene, 156, 27 (1995)), biotin (Saggio et al., Biochem. J., 293, 613 (1993)), DNA sequence (Cheng et al., Genen, 171, 1 (1996); Krook et al., Biochem. Biophys., Res. Commun., 204, 849 (1994)), streptavidin (Geibel et al., Biochemistry, 34, 15430 (1995), Katz, Biochemistry, 34, 15421 (1995)), nitrostreptavidin (Balas et al., Anal. Biochem., 243, 264 (1996)), heparin (Wickham et al., Nature Biotechnol., 14, 1570-73 (1996)), cationic substrates, metals such as nickel and zinc (see, for example, Rebar et al., Science, 263, 671 (1994); Qui et. al., Biochemistry, 33, 8319 (1994)) or other potential substrates. Examples of suitable ligands or substrates thereof for use in the method of the invention include, but are not limited to, a CR2 receptor that binds sequences to which amino acid residues are attached; CD4 receptor recognizing the HIV gp120 loop V3; transferrin receptor and its ligand (transferrin); low density lipoprotein receptor and ligand thereof; ICAM-1 receptor on the surface of lung epithelial and endothelial cells and its ligand; linear and cyclic peptide ligands for streptavidin or nitrostreptavidin (Katz, Biochemistry, 34, 15421 (1995)), galactin sequences that bind lactose, galactose and other galactose-containing compounds and asialoglycoproteins that recognize deglycosylated protein ligands. Other ligands and their binding sites preferably include (but are not limited to) short linear bands of amino acids (e.g., 6 amino acids or less) recognized by integrins, as well as polyamino acid sequences such as polylysine, polyarginine, and the like. Incorporation of a large number of lysines and / or arginines allows for differentiation of heparin and DNA. The ligand may also contain a commonly used peptide tag (for example, short amino acid sequences known to be recognized by available antisera), such as Shistosoma manosi glutathione-S-transferase (GST) sequences, thioredoxin beta-galactosidase or maltose binding protein (MPB). derived from E. coli, human alkaline phosphatase, octapeptide FLAG (SEQ ID NO: 16), hemaglutamine (HA) (Wickham et al., Nature Biotechnol., 14, 1570-73 (1996)), polyomavirus peptides, large peptide SV40 T antigen, BPV peptides, hepatitis C virus core, and E2 envelope peptides and single chain antibodies recognizing them (Chan, J. Gen. Virol., 77, 2531 (1996)), c-myc peptide, adenovirus penton base epitopes ( Stuart et al., EMBO J., 16, 1189-98 (1997)), epitopes present in the E2 envelope of hepatitis C virus SEQ ID NO: 17, SEQ ID NO: 18 (described, for example, in Khan, J. Gen. Virol., 77, 2531 (1996)) and others commonly used g. A preferred substrate suitable for the ligand tag is driven by antibodies, derivatives of such antibodies (such as a FAB fragment, single chain antibodies (ScAb), or a suitable substrate).

As mentioned, a suitable ligand may be specific for any desired substrate, such as that described herein or known in the art. Adenoviral vectors can also be manipulated to include novel ligands by first testing the ability of the peptides to react with the substrate. In general, a random or semi-random peptide library containing ligands can be prepared. This library is essentially a library in an expression vector system. This library can be tested by exposing the expressed proteins (i.e., putative ligands) to the desired substrate. Positive selective binding of sintering in the library to the substrate indicates a ligand suitable for said substrate, at least under assay conditions. Any assay capable of detecting the interaction between proteins and substrates can be used to test such a peptide library. Many of these assays are known in the art. One preferred assay useful for protein library testing is a phage system that utilizes a bacteriophage expression library (see, for example, Koivunen et al., Bio / Technology, 13, 265-70 (1995), Yanof23). et al., Proc Natl Acad Sci USA, 93, 7381-86 (1996), Barry et al., Nature Med., 2 (3), 299-305 (1996)) Phage binding to the substrate is tested. by subjecting the phage to a substrate attack, rinsing the substrate further, and selecting the phage that remains bound to the substrate. Then, limiting phage dilution can identify individual clones expressing the putative ligand. Of course, the insert present in such clones can be sequenced to determine ligand identity.

Phage is preferred for identifying potential ligands, since it best mimics viral interaction with the microenvironment. It should be noted that said phage expression is an extracellular system (such as the initial stage of viral infection). In addition, phage expression includes a virus (phage) that presents a potential ligand. Phage expression also offers considerably greater flexibility compared to other protein binding assays (especially intracellular assays). It should be noted that phage expression not only identifies the binding of proteins (ligands) to a particular substrate, but also identifies those ligands that bind under predetermined conditions. For phage expression, ligands useful for incorporation into adenovirus may be identified to allow purification under predetermined conditions. For example, a phage library can be tested by exposure to a particular plastic, resin, or other desired substrate that is used in an affinity column. Phage-expressed peptides that either bind to the substrate or elute from the substrate under specific conditions or within certain conditions (e.g., high or low salt content, pH, temperature, etc.) can be readily determined, but do not bind or are not eluting under other conditions. The adenovirus incorporating the ligand may be purified by exposure to the substrate under the conditions described herein.

Once identified, the ligand may be incorporated into any position of a virus that is capable of interacting with the substrate (i.e., the viral surface). For example, the ligand may be incorporated into a fiber, penton base, hexone or other suitable position. When the ligand is attached to the fibrous protein, it is preferable not to damage the interaction between viral proteins or monomers. It is preferred that the ligand itself is not an oligomerization domain. As such, it can interact adversely with the trimerization domain as discussed above. Preferably, the Li24 gand does not replace a portion of the fiber protein, which may adversely affect trimerization and interaction with pentone. Preferably, the ligand is added to the fibrous protein and incorporated in such a way that it can easily be exposed to the substrate (e.g., at the carboxyl terminus of the protein attached to the residue exposed to the substrate; located on the peptide spacer to come into contact with the substrate etc.). ), maximizing the presentation of Ugand to the substrate. When the ligand is attached to a portion of the pentone or the ligand replaces a portion thereof, it is preferred that this occurs in the hypervariable regions to ensure contact with the substrate. Further, when the ligand is attached to the pentone, the recombinant strand (e.g., 0-10 repeats) is truncated to maximize the presentation of the ligand to the substrate (see, for example, US Patent 5,559,099 (Wickham et al.)). When the ligand is attached to the hexone, it is preferred that this occurs in the hypervariable region (Miksza et al., J. Virol., 70 (3), 1836-44 (1996)).

When the ligand is incorporated into an adenoviral protein (or blocking protein), the ligand may comprise a portion of a natural sequence and a portion of a non-natural sequence. Similarly, sequences (either natural or unnatural) that contain a ligand in the protein do not necessarily adjacent to the amino acid chain that contains the protein. In other words, the ligand can be formed by some conformation of the protein, for example by packaging the protein in such a way that adjacent and / or non-adjacent sequences can be brought into proximity to each other. Of course, the adenovirus of the invention (or blocking protein) may contain large amounts of ligands, each binding to a different substrate. For example, the virus may comprise a first ligand allowing affinity purification as described herein and / or a third ligand suitable for virus inactivation as also described herein.

The protein comprising the ligand may also include other unnatural elements. For example, an unnatural, single protease recognition site may be incorporated into the amino acid sequence. Preferably, the protease site does not affect fiber trimerization or substrate specificity of the fiber ligand. A number of such protease sites are known in the art. For example, thrombin recognizes and cleaves a known amino acid sequence (Stenflo et al., J. Biol. Chem. 257, 12280-90 (1982)). The presence of protease recognition sequences allows virus purification according to some protocols as described above. The protein may be manipulated to include a ligand by any suitable method. These methods suitable for introducing mutations into proteins are described above.

In addition to the trimer and ligand, the virus of the invention may include one or more unnatural foreign genes. A "foreign gene" can be any suitable gene and is required to be either a therapeutic gene (i.e., a nucleic acid sequence encoding a product that produces a biological, preferably therapeutic response, either at the cell level or systemically) or a reporter gene (i.e., a nucleic acid sequence). acid, which encodes a product that can be detected in the cell). Preferably, the foreign gene is expressed in a cell into which the vector has been introduced. Preferably, the foreign gene exerts its effect at the RNA or protein level. For example, a protein encoded by a transferred therapeutic gene can be used to treat a hereditary disease. An example is the cDNA of the cystic fibrosis transmembrane conductivity regulator in the treatment of cystic fibrosis. Alternatively, a protein encoded by a therapeutic gene may exert its therapeutic effect by causing cell death. For example, expression of the gene itself may lead to cell death. An example is diphtheria toxin. Alternatively, the gene or gene expression may generate cells selectively sensitive to the undesired action of certain cells, which may result in cell death. An example is the expression of the HSV thymidine kinase gene, which causes the cell to be sensitive to antiviral compounds including aciclovir, ganciclovirus and FIAU (1- (2-deoxy-2-fluoro-β-Darabinofuranosyl) -5-iodouracil). However, the therapeutic gene may exert its effect at the RNA level. For example, by encoding an antisense message or ribozyme, a protein that affects 3-terminal splicing or processing (e.g., adenylation), or it may be a protein affecting the expression of another gene in a cell (i.e., if the expression of the gene involves all steps beginning with transcription up to production of the engineered protein), may further mediate a change in mRNA accumulation rate, may alter mRNA transport and / or may alter post-translational regulation. Of course, if use is required for the introduction of a foreign gene by the gene transfer method, then a gene whose sequence is known in the art is used.

The altered protein (e.g., a trimer or coat protein having a ligand) and the foreign gene (if present) can be incorporated into the adenovirus by any suitable method, many of which are known in the art. As stated herein, the protein is preferably identified by testing products that are produced in large volumes from genes in expression vectors (e.g., baculovirus vectors). Genes derived from vectors that carry the desired mutation can be simply subcloned into plasmids. These plasmids are then transfected into suitable cells for packaging (e.g. 293 cells). The transfected cells are then incubated with adenoviruses under conditions suitable for infection. At a certain frequency in cells, homologous recombination between the vector and the virus will produce an adenoviral genome carrying the desired mutation.

The adenoviruses of the invention may be either replication competent or insufficient. Preferably, the adenoviral vector comprises a genome with at least one modification that makes the replication deficient virus (described, for example, in International Patent Application WO 95/34671). Modifications of the adenoviral genome include, but are not limited to, adding a DNA segment, rearranging the DNA segment, deleting the DNA segment, replacing the DNA segment, or introducing a DNA lesion. The DNA segment may be as small as a single nucleotide and as large as an adenoviral genome (e.g., 36 kb), or alternatively may be equal to the maximum amount that can be packaged into an adenoviral virion (i.e., approximately 38 kb). Preferred modifications of the adenoviral genome include modifications in the E1, E2, E3, and / or E4 regions. Preferably, the adenovirus can also be co-integrated, which means ligation of the adenoviral genomic sequences with other sequences, such as those of other viruses, phages, or plasmids.

The adenovirus of the invention has a number of qualities which make it an attractive choice for use in gene transfer as well as in other applications. For example, an adenovirus does not infect its natural host cell more readily than wild-type adenovirus, causing mutant fiber trimmers (for example, selective mutations of residues responsible for binding to AR, replacement of the trimerization domain, or addition of a blocking domain as described herein). Further, the adenovirus has at least one unnatural substrate-specific ligand that allows virus propagation, targeting, purification and / or inactivation as described above. In the case of simple cloning, the ligands and trimerization domains are preferably separate domains, allowing the virus to be easily manipulated so that different ligands can be incorporated without disrupting the fiber trimerization. If a mutated fiber node has been incorporated into the fiber trimer, then the ligand can be incorporated into the node as described herein.

For incorporation into a host (such as an animal), it is possible to introduce the virus of the invention into a suitable carrier. As such, the invention provides a composition comprising the adenovirus of the invention and a pharmaceutically acceptable carrier. Any suitable preparation is within the scope of the invention, the exact formula of which of course depends on the nature of the desired application (e.g., cell type, application mode, etc.). A number of suitable formulations are described in US Patent 5,559,099 (Wickham et al.).

Cell line

As described herein, the adenovirus of the invention is not able to simply infect its natural host cell via AR because its ability to bind to AR is substantially attenuated (due to incorporation of the chimeric trimmers of the invention). The invention therefore provides a cell line capable of propagating said adenovirus. Preferably, the cell line promotes viral growth for at least about 10 passages (eg, about 15 passages), more preferably at least about 20 passages (eg, about 25 passages) or even 30 or more passages.

The adenovirus may be first cultured in a pacemaker cell line that expresses the native fiber protein gene. The resulting viral particles are therefore likely to contain both natural fibers encoded by the complementing cell line as well as unnatural fibers encoded by the adenoviral genome 28 mom (as described herein). Therefore, the population of such resulting viruses will contain both types of fibers. Such particles will be able to bind to, or enter, the packaging cell lines more efficiently than particles which do not contain natural fiber molecules. Thus, the use of such a fiber-encoding cell line makes it possible to cultivate and amplify the genomes of adenoviruses that encode chimeric, targeted adenoviral fibers with a suitable high titer. The resulting mixed stocks of adenoviruses produced from cell lines encoding a natural fiber molecule will contain natural and chimeric adenoviral fiber molecules. However, the particles contain genomes encoding only the chimeric adenoviral strand. In order to produce a pure stock solution of adenoviruses that exhibit only chimeric adenoviral fiber compounds, a mixed stock solution is used to infect a non-natural fiber producing cell line (such as 293 cells in the case of E1 deleted viruses). The resulting adenoviruses contain only the fiber molecules encoded by the genomes (i.e., the chimeric fiber molecules).

Similar cell lines are produced which complement the fiber. These cell lines are used to cultivate a mutant adenovirus lacking the fiber gene. However, the rate of production of these cell lines is generally not high enough to produce an adenovirus titer carrying a fiber deletion comparable to that of the adenoviral particles expressing the fiber. The lower titers produced by such mutants can be improved by temporarily regulating the expression of the natural strand to more complement the mutant adenoviral gene. One such strategy for producing an improved cell line employs an inducible promoter (e.g., a metallothionine promoter) to allow fiber production by a controlled and activated infection of adenovirus cells. Alternatively, the efficient mRNA splicing site introduced into the fiber gene in the complementing cell line enhances the strength of the production of the fiber protein in the cell line.

When the adenovirus is manipulated to contain a ligand specific for a cell surface binding site, any cell line that expresses the receptor and which is capable of promoting adenovirus growth can be used as the host cell line. Since a number of ligands do not bind to binding sites at the top of the cell (especially the novel ligands described herein), then the cell line can be manipulated to express the substrate for the ligand.

The invention describes a cell line expressing an unnatural cell surface binding site for which an adenovirus (or bi-specific blocking protein) has a ligand suitable for receptor binding. Any cell line capable of promoting the growth of adenovirus is suitable as a cell line which can be used according to the invention. If the adenovirus does not contain genes essential for viral replication, it is preferred that the cell line express a complementary amount of gene products. Since 293 cells promote the growth of adenoviruses, it is preferred that the cell line of the invention be derived from 293 cells.

An unnatural cell surface binding site is a substrate molecule as described herein. Adenovirus (or a bi-specific blocking protein) having a ligand is selectively bound thereto. The substrate may bind to the cell and thereby promote cell entry. When the ligand is on an adenovirus, the binding site may recognize an unnatural ligand incorporated into the adenovirus envelope or a ligand that is natural to the virus. For example, if the non-natural viral ligand is a peptide tag, then the binding site may be a single chain antibody receptor (ScAb) that recognizes the tag. Alternatively, the ScAb may recognize an epitope present in the mutated fibrous node region (if present) or even an epitope present on the natural adenoviral coat protein (e.g., fiber, penton, hexon, etc.). Alternatively, when the unnatural ligand recognizes a substrate on the cell surface (e.g., a membrane-bound protein), then the binding site may comprise the substrate. When the binding site is a natural receptor on the cell surface, the cell line can express a mutant receptor with reduced ability to interact with a cellular signaling cell pathway (e.g., a truncated receptor such as NMDA (Li, et al., Nat. Biotech., 14, 989 (1996)), with attenuated ion channel capability or other treatments Infection with such engineered proteins minimizes the secondary effect of viral infection on host cell metabolism by limiting activation of the intracellular message processing pathway and their various Briefly, the choice of binding site depends largely on the nature of the adenovirus, but in order to promote the specificity of the cell type with respect to adenovirus, then the binding site is preferably not a natural mammalian AR The binding site must be expressed on the cell surface to be for virus available In the case where the binding site is a protein, preferred are the leader sequences and sequences that assure attachment to the membrane (see, for example, Davitz et al., J. Exp. Med. 163, 1150 (1986)) which promote proper membrane integration.

The cell line can be formed in any standard manner. For example, a vector (e.g., an oligonucleotide, plasmid, viral, or other vector) containing a gene encoding a non-natural receptor can be introduced into the source cell line in a standard manner. Preferably, the vector also encodes an agent that allows the cells carrying it to be readily identifiable (for example, the vector may encode antibiotic resistance that kills non-plasmid-bearing cells). The vector will recombine with the cell genome at the same frequency to produce a transformed cell line expressing the binding site.

Method of propagation

Together with the cell surface of an unnatural adenoviral binding site on the cell surface, the invention describes a method of propagating the adenovirus of the invention. The method of the invention involves infecting a cell with an adenovirus that exhibits an unnatural ligand selectively binding to the receptor, incubating the cells, and recovering the adenoviruses that are produced in the cells. Cell-derived adenoviruses can be propagated again (for example, amplified) to form a very high titer virus stock. The ligand on the adenovirus may be any ligand described herein. Cells of the invention are infected with virus at any m.i.o. to promote effective infection of the cell line (e.g., about 1 to 10 m.o.i). Cell culture conditions depend on the nature of the host cell. However, one skilled in the art is able to select the culture conditions of a given cell type. Viruses are recovered from cells by standard methods, such as cell lysis. Thereafter, cells can be purified by standard procedures or other methods of the invention.

Method of cleaning

As mentioned herein, it is not necessary that the substrate suitable for the ligand that is introduced into the adenovirus be present on the cell surface. For example, the substrate may be on a support, such as an inorganic mass such as plastic, glass, metal, resins, or other materials commonly used in chromatographic or affinity separation. Examples of such supports include metals, natural polymeric carbohydrates, and synthetically modified, cross-linked or substituted derivatives thereof such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar polysaccharides, cellulose esters, especially nitric acid and carboxylic acid mixed cellulose esters. and cellulose ethers; natural nitrogen-containing polymers such as proteins and derivatives, including cross-linked or modified gelatins; natural polymers of hydrocarbons such as latexes and gums; synthetic polymers which can be prepared with a suitable porous structure, such as a vinyl polymer, including polyethylene, polypropylene, polystyrene, polyvinyl chloride, polyvinyl acetate and other partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the aforementioned polycondensates, such as polycondensates, polymers such as polyurethanes and polyepoxides; porous inorganic materials, such as alkali earth and magnesium metal sulphates or carbonates, including barium sulfate, calcium sulfate, calcium carbonate, alkali metal or alkaline earth metal, aluminum and magnesium silicates; silicon or aluminum oxides or hydrates such as clays, alumina, talc, kaolin, zeolite, silica gel or glass (these materials may be used as filters with the aforementioned polymeric materials) and mixtures or copolymers of the above classes, such as transplant copolymers, other materials commonly used in chromatographic and affinity separation Such supports may be prepared in the form of spheres, films, sheets, plates, etc. or they may be coated, laminated or bonded to, or otherwise bonded to, inert carriers. The carrier can be paper, glass, polymer film, fabric, etc.

The presence of a substrate suitable for the ligand on the surface of the adenovirus of the invention allows the adenoviruses to be easily purified with high affinity and taxonomic fidelity. The invention further provides a method of purifying an adenovirus carrying a ligand suitable for a substrate from an adenovirus-containing composition. The method includes exposing the substrate composition under conditions such as to promote selective binding of the ligand that is present on the adenovirus to the substrate. Thereafter, the composition (e.g., a substantial portion of the composition) that has not bound to it is removed from the substrate. This is followed by elution of the adenovirus bound to the substrate. Using this method, an adenovirus having a ligand can be isolated from various compositions (e.g., solutions, dispersions, suspensions, gels, and the like). A common adenovirus-containing composition is a cell lysate, which is produced, for example, from pacifying cells during adenovirus propagation.

Generally, the substrate binds to the substrate as described earlier herein. The fusion of the desired ligand-substrate complex with a suitable support material is well known in the art. The invention discloses any suitable method of producing a substrate having a substrate. The substrate itself may be plastic, glass, metal, resin, etc. Any means of exposing the composition comprising the adenovirus to the substrate is suitable for use in the invention. For example, the composition may pass through a column containing a substrate to which the substrate is bound. Of course, the composition may be mixed with a slurry of such a backing material (e.g., beads or other materials containing bound substrate), placed in a container (such as a test tube, plate depression, etc.) that is coated with the substrate, or otherwise exposed to the substrate.

Parameters such as time, temperature and solutions that are necessary for selective binding may vary depending on the affinity exhibited by the ligand for the substrate. When known ligand-substrate systems are used, the parameters are known. When new ligand-substrate systems are used, the binding conditions can be predetermined to a large extent as set forth herein (for example, using such conditions when ligand substrate substrates are tested in a protein library). It is preferred that the selective binding conditions do not allow selective binding of the other constituents of the composition to the substrate. Where other constituents selectively bind to a substrate, a substantial amount of adenovirus may be removed from the composition by association with the substrate.

After selective binding, the virus-free composition is removed from the substrate (for example, it is selectively eluted). Any suitable method of removing the adenovirus-depleted composition can be used, wherein the adenovirus remains selectively bound to the substrate. The conditions used to remove the adenovirus-depleted composition from the substrate are generally not sufficient to elute the adenovirus from the substrate. The method of removing the adenovirus-depleted composition is largely a function of the substrate and substrate type. For example, the adenovirus-depleted composition can be removed from the substrate-containing column by flushing the column with several volumes of a suitable solution. The adenovirus-depleted composition can be removed from the substrate slurry containing the substrate by repeated centrifugation, re-suspension in a suitable solution, and re-centrifugation. Alternatively, where the substrate is a magnetic material, it can be physically removed from the solution by exposing the solution containing particles to a magnet and washing the magnetic substrate. When the substrate is bound to the plate or to the indentation on the plate, the container may simply be washed with several volumes of a suitable solution.

When the adenovirus-depleted composition is removed from the substrate, the adenovirus is also released from the substrate. Any method can be used to remove the adenovirus from the substrate. In many applications, adenovirus can be released by exposing the adenovirus-substrate complex to an eluting agent that is incompatible with ligand-substrate binding. The time, temperature and solution parameters necessary to elicit selective elution of virus from the substrate may vary depending on the affinity by which the ligand selectively binds to the substrate. In general, when known ligand-substrate systems are used, these parameters are known. When new ligand-substrate systems are used, elution conditions can be predetermined to a large extent, for example, by adjusting the conditions under which the protein library is tested as described herein. When the ligand is incorporated into an adenovirus into a spacer or other peptide as described herein, the spacer may include a sequence that is recognized by a peptidase or other specific cleavage motif. Adenoviruses comprising this34 cleavage sequence can be released from the support by exposing the support to an agent that affects cleavage, such as an endoprotease or other agent. While the cleavage method damages the adenovirus ligand, it is preferred in many applications. For example, a virus purification ligand may interfere with a second ligand suitable for targeting the virus to a particular cell type. Removal of the purifying ligand thus allows the isolated adenovirus to more easily infect the cell type.

While any suitable binding or elution conditions can be used, the practical limitation is due to the ability of the adenovirus to survive said conditions. However, if the adenovirus is able to survive various environmental conditions, such as high salt concentration, high osmolality, and basic environment, the method can be used in a variety of conditions. Such conditions are even well known in the art.

In the process of purifying adenoviruses, it is not necessary to remove all or even a large portion of the viruses from the solution. In many applications, the amount of viruses present in the initial composition may saturate the amount of substrates present on the substrate. While the ligand on the substrate selectively binds to the substrate, such selective binding may not occur based on affinity. Such a substantial amount of substrate need not bind the available ligands in the separation step. Therefore, in order to recover as much adenovirus from the initial composition as possible, the adenovirus-depleted composition released from the substrate as described herein may be subjected to several cycles of purification. The virus thus obtained can be combined into a single stock solution. In a similar case, while other constituents of the initial composition preferably do not selectively bind to the resin, then there is no binding at least in the first purification steps. The presence of a baseline defect binding necessarily leads to contamination of the initial viral solution. To reduce or substantially reduce this base contamination, the viral stock solution can be subjected to successful purification when the base amount of contaminants reaches zero. The process of the invention is an economical, efficient and affordable method for purifying adenoviruses that exhibit ligands. The use of slurries and columns is common in industrial applications, allowing the process of the invention to be used commercially.

Method of cell infection

The unnatural ligand present on the virus of the invention (or on the virus / blocking protein complex) can recognize the substrate present at the binding site on the cell surface. The invention therefore provides a method of infecting a cell having a cell surface binding site comprising a substrate for an unnatural ligand. The method comprises contacting the cell with an adenovirus such that an unnatural ligand of the adenovirus (or virus / blocking protein complex) binds to a specific binding site on the cell surface, thereby causing adenovirus entry. Because the viruses of the invention incorporate filamentous trimmers exhibiting limited ability to bind natural mammalian AR, the adenovirus is incorporated into the cell primarily based on the unnatural ligand. Said method allows the selective targeting of a virus comprising a cell type ligand that expresses a binding site containing a substrate suitable for said ligand, without substantially infecting the cells with the native mammalian AR. In this case, when the virus is penton-based (such as a modified or unmodified penton base), the virus is incorporated by way of a ligand that is suitable for the pentone.

Any cell expressing on its surface a binding site comprising a substrate suitable for a ligand may be selectively targeted in accordance with the invention. The cell may be present as a single object or may be part of a larger collection of cells such as cell culture (either mixed or isolated), tumor, tissue (e.g. epithelial, muscle or other tissues), organ, organ system (e.g. circulatory, respiratory) , the gastrointestinal system, the urinary system, the nervous system, or the system of other organs) or even the whole organism (e.g., human). It is preferred that the target cells are selected from the group consisting of heart, blood vessels, smooth muscle, skeletal muscle, lung, liver, gall bladder, bladder, and ocular cells.

The method of infecting the cell ideally occurs when the adenovirus comprises a foreign gene, such as those described herein. When the adenovirus of the invention comprises a foreign gene, the method allows the adenovirus to serve as a vector introducing the gene into the target cell. The vectors and methods of the invention provide useful tools for introducing a foreign gene into a selected class of cells without significantly introducing the gene into cells ubiquitously or ectopically.

Method of virus inactivation

As noted, the unnatural ligand present on the virus of the invention can recognize the substrate present in blood and lymphatic fluid (such as the ligand present on the free protein that is produced in the blood, the protein present on the erythrocytes, etc.). Therefore, the invention describes a method of inactivating adenovirus having a ligand recognizing a substrate derived from blood or lymph by exposing the virus to the substrate. In blood or lymph, the ligand selectively binds to the substrate, whereby free virus is adsorbed from the liquid. It is preferred that the substrate be present in large molecules (such as albumin) or on the surface of erythrocytes (lacking the transcription tools required for virus propagation). Of course, the ligand suitable for virus inactivation may be present at any position on the viral envelope (Fender et al., Virology, 214, 110 (1995)). When antibodies recognizing and / or neutralizing adenoviruses primarily bind to epitopes on hexone (Gahery-Segard et al., Eur. J. Immunol.m 27, 653 (1997)), unnatural ligands suitable for virus inactivation are preferably incorporated into hexon as described herein.

A method that effectively inactivates adenoviruses has been implicated in limiting viral infection of the desired locus (tissue, cell type, etc.). In particular, the method effectively deactivates an individual virus by attaching it to a substrate while limiting its activity to contact the cell (and therefore enter the cell). Even when the virus so adsorbed comes into contact with the cell, it is substantially less likely that its incorporation is due to the presence of a substrate having particles. By combining these effects, the method effectively inactivates the multiple Russian stock solution (beyond the desired locus of infection) by dramatically reducing its effective free titer.

The method of virus inactivation complements another embodiment of the invention. For example, the viruses of the invention incorporate filamentous trimmers that exhibit reduced affinity for natural mammalian AR, substantially reducing the likelihood that the virus will infect cell types other than the desired cell type. The viruses of the invention may include substrate-specific ligands present at a cell-surface binding site, allowing the virus to target a predefined cell type. While these two advantages affect selective targeting and thereby substantially attenuate ectopic infection, viruses displaying a ligand recognizing a substrate derived from blood or lymph are less likely to contact ectopic tissue to effectively reduce viral titer.

While one skilled in the art is quite able to put the invention into practice after reading the description, the following examples further illustrate some of its features. The examples describe methods such as affinity chromatography, Southern blotting, PCR, DNA sequencing, vector construction (including DNA extraction, isolation, restriction enzyme digestion, ligation, etc.), cell culture (including antibiotic selection), cell transfection, screening proteins (Western blot, immunoprecipitation, immunofluorescence) etc. These are methods routinely used in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ^nd . Ed. (1989), Cold. Spring Harbor, New York).

BRIEF DESCRIPTION OF THE DRAWINGS

Figures no. 1A and 1B depict the three-dimensional node structure of the adenoviral protein (serotype 5). Picture no. 1A is a diagram representing β-sheet and loops internally connecting sheets. Picture no. 1B is a diagram that takes into account the relative size of amino acid residues.

Picture no. 2 shows sequence comparison between adenoviral serotypes.

Figures 3A to 3C show vectors forming recombinant adenoviral fiber trimmers that lack the natural domains of trimerization. 3A shows pAcT5S7GCNTS.PS.LS.X. Figure 3B shows pAcT5sigDel.TS.PS.LS. Picture no. 3C shows pAcT5S7sigDel.TS.PS LS.

Picture no. 4 shows pAcT5sigDel.GFP.TS.PS.LS which is a vector containing a gene encoding the fiber-sigDel-GFP chimera.

Figures no. Figures 5A-5D illustrate vectors useful in the construction of recombinant adenoviral vectors containing fiber trimers that do not have natural trimerization domains. Picture no. 5A shows pAS pGS HAAV. Picture no. 5B shows pAS pGS pK7. Picture no. 5C shows pAS T5S7sigDelpGS.HAAV.

Picture no. 5D shows pAST5S7sigDel.GFP.pGS.pK7.

Figures no. Figures 6A-6D illustrate vectors used in the construction of fiber trimers that do not have natural trimerization domains. Picture no. 6A represents pAcPig4KN. Picture no. 6B represents pAcPigKN D363E. Picture no. 6C represents pAcPigKN N437D. Picture no. 6D shows pAcPig4KN (FLAG).

Figures no. 7A to 7B represent vectors that are used in the preparation of a fiber trimmer that does not have a natural trimerization domain. Picture no. 7A shows PNS F5F2K. Picture no. 7B shows pNS Pig4.SS.

Figures no. Figures 8A to 8C represent vectors useful in the generation of an adenoviral vector having a chimeric fiber trimmer comprising a mutant NADC-1 node that does not have a natural receptor binding ability and contains a functionally unnatural ligand. Picture no. 8A shows pAcPig4KN D363E N437D. Picture no. 8B shows pAcPig4KN D363E N437D HAAV. Picture no. 8C shows pNS Pig D363E N437D HAAV SS.

Figures no. Figures 9A-9B represent vectors useful for generating chimeric blocking proteins of the invention that are capable of interfering with binding to the natural adenoviral receptor. Picture no. 9A shows pACSG2-sCAR. Picture no. 9B depicts pACSG2-sCAR-HAAV.

Figures no. Figures 10A to 10B represent vectors that can be used to generate chimeric blocking proteins capable of forming trimers interfering with binding to the natural adenoviral receptor. Picture no. 10A shows pAcSG2sCAR.sigDel. Picture no. 10B depicts pAcSG2-sCARsigDel (HAAV)

Figures no. Figures 11A-11E illustrate vectors useful for constructing adenoviral vectors that do not have specific unnatural ligands. 11A shows pBSSpGS. Picture no. 11B shows pBSS pGS (RKKK) 2. Picture no. 1 IC depicts pNSF5F2K (RKKK) 2. Picture no. 11D depicts pBSSpGS (FLAG). Picture no. 11E shows pNS F5F2K (FLAG).

Figures no. 12A to 12E represent vectors useful for generating a cell line expressing unnatural cell surface sites for a substrate. Picture no. 12A depicts pH00K3. Picture no. 12B depicts pRC / CMVβ-Puro. Picture no. 12C depicts pScHAHK. Picture no. 12D depicts pNSE4GLP.

Figures no. Figures 13A-13D represent vectors useful for generating a fiber-expressing cell line for the production of target adenoviral particles. Picture no. 13A denotes pCR2.1-TOPO + fiber. Picture no. 13B depicts pKSII Fiber. Picture no. 13C shows pSMTZeo-DBP. Figure 13D shows pSMTZeo-Fiber.

Picture no. 14 depicts pAdE1 (Z) E3 / E4 (B), a plasmid useful for the construction of target adenoviral particles having a genome encoding chimeric fibers.

Figures no. 15A to 15E illustrate the location of mutations in adenoviral nodes that interfere with ligand binding. Figure 15A is a top view; 15B is a side view and FIG. 15C is a bottom view of the node showing the position of the 3D9 mutation. Picture no. 15D is a top view, FIG. 15E is a side view, and FIG. 15F is a bottom node view illustrating CD loop mutation, FG loop mutation, and IJ mutation positions.

Picture no. 16 depicts a vector useful in the construction of a recombinant adenovirus comprising a short strand and a mutant node on a strand exhibiting limited affinity for the natural receptor.

Figures no. Figures 17A-17B depict vectors useful in constructing a cell line where it is possible to replicate adenoviruses lacking the natural function of cell binding (but targeting a pseudoreceptor). Figure 17A shows pCANTAB5E (HA). Picture no. 17B depicts pScFGHA.

DETAILED DESCRIPTION OF THE INVENTION

Example 1:

This example describes two fiber trimmers having two unnatural trimerization domains, each of which interacts with an adenoviral penton base. Fiber chimeras include the trimerization domains of the sigma reovirus

First

Two chimeras T5S7sigDel and TSsigDel were constructed. T5sigDel contained only the end of the Ad5 strand (T5) fused to sigDe with no mid-Ad sequence. The T5S7sigDel contains the end plus the first 7 repeats of the β-sheet mid-Ad (S7) fused to sigDel. The DNA and amino acid sequences of the two clones are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

The sigDel region of the sigma 1 reovirus gene was amplified by PCR and cloned into vector pAcT5S7GCNTS.PS A d.LS.X (Figure 3A) to produce the baculovirus transfer vector pAcT5sigDel.TS.PS.LS (Figure 3B). . This vector encodes the end of the Ad5 strand fused to the trimerization domain of the sigma 1 type 3 reovirus protein followed by the FLAG epitope near the C terminal. At the C-terminus of the gene, the vector also contains a large number of restriction sites, allowing cloning of the targeted and purified sequence into the gene.

The second vector pAcT5S7sigDel.TS.PS.LS (Figure 3C) was prepared by digesting the above PCR product with NheI and BamHI restriction enzymes and cloning the fragment into pAcT5S7GCNTS.PS.LS.X vector (Figure 3A), which was also it was digested with the restriction enzymes NheI and BamHI. The resulting vector encodes a protein containing the tail and the first seven repeats of the middle portion of the β-sheet of the Ad5 strand fused to sigDel followed by the FLAG epitope.

Recombinant baculovirus clones encoding each of the filamentous chimeras were then prepared by standard methods using each of the above plasmids. The resulting baculovirus clones were used to produce recombinant proteins in Tn5 insect cells. To compare the sigDel trimerization domain with the GCN domain, another baculovirus was constructed from the initial plasmid pAcT5S7GCNTS.PS.LS.X (FIG. 3A), which contained the GCN trimerization domain at the sigDel trimerization domain.

Cells infected with baculovirus were centrifuged three days after infection. The cell pellet was resuspended in PBS with protease inhibitors and freeze-thawed three times to release soluble intracellular proteins. The cell debris was then centrifuged at high speed to form a pellet and clean cell lysate was collected. The pellet was then resuspended in the same volume of PBS as described above.

The pellet and lysate samples were then run on a 12.5% polyacrylamide gel with 0.1% SDS and transferred to nitrocellulose by Western analysis using anti-FLAG M2 Mab (Kodak). These results demonstrate that more than 90% of each of the T5S7GCN.TS.PS.LS proteins is dissolved in the lysate,

T5sigDel.TS.PS.LS and T5S7sigDel.TS.PS.LS.

Proteins were further tested for their ability to form trimers. To test the trimerization of the chimera with lysates from each sample prior to electrophoresis on a 12.5% polyacrylic gel with 0.1% SDS, they were either boiled or not boiled. Western analysis of the cooked samples shows that the cooked samples migrate to a distance that corresponds to the molecular weights of the monomeric protein, whereas the uncooked proteins containing the sigDel trimerization domains migrated to a distance that corresponds to the molecular weights of the trimmers. Uncooked protein T5S7GCN.TS.PS.Ls also migrated as a trimer. A substantial part (more than half) of the uncooked sample migrated as monomer. Similar analyzes of wild-type fiber and sigma 1 protein show that these proteins, when not boiled, migrate completely as trimmers and migrate as monomers when cooked.

A large number of proteins were dissolved in the lysate (unlike the pellet), indicating that the proteins were correctly packaged. However, the migration of the uncooked samples shows that sigDel-containing chimeras are soluble trimmers and that the sigDel domain works better than GCN because they form more stable trimeric chimeras of the fiber.

To test the ability of the trimers to complex with the adenoviral penton base protein, the recombinant penton base is mixed in solution with the T5S7GCN.TS.PS.LS, T5sigDel.TS.PS.LS and T5S7sigDel.TS.PS.LS trimeric proteins. The resulting penton base / fiber chimera complex then immunoprecipitates with antibodies raised against the penton base associated with protein A-agarose. The precipitated sample is then loaded onto an SDS-PAGE gel and evaluated by Western analysis as described above using FLAG antibodies. Binding of FLAG antibodies shows that the filamentous chimera containing the FLAG epitope forms a complex with the penton base in solution.

Example 2:

This example describes the ability of filament-sigDel chimeras to incorporate an exogenous protein domain that is larger than a peptide tag.

The sequence coding for the modified version of the green fluorescent protein was amplified by PCR using primers containing restriction sites, allowing efficient cloning into the Fiber-sigDel chimeric plasmids described above in Example 1. Cloning of the GFP sequence in the correct orientation to the Spel plasmid restriction site pAcT5sigDel.TS.PS.LS (FIG. 3B) results in the plasmid pAcT5sigDel.GFP.TS.PS.LS (FIG. 4), which encodes the Fiber-sigDel-GFP chimera. The DNA sequence and amino acid sequence of this clone is shown as SEQ ID NO: 3.

This plasmid was then used to produce a recombinant protein using a baculovirus expression system as described above. The solubility of the fiber chimeric proteins (indicating proper packaging) and the ability of the resulting proteins to bind to the penton base (demonstrating trimerization) were confirmed. Production of a soluble trimeric protein containing GFP domains shows that large functional protein domains can break down into Fiber-sigDel chimeras as easily as a smaller peptide tag. The results suggest that such chimeras may also incorporate ligands such as ScAbs without substantially interfering with protein function.

Example 3.

This example describes the construction of recombinant adenoviral vectors containing fiber trimers that have unnatural trimerization domains.

The NdeI to BamHI fragment was cut from pAcT5S7sigDel.TS.PS.LS (Fig. 3C) to replace the corresponding fragments in pAS pGS HAAV (Fig. 5A) and pAS pGS pK7 (Fig. 5B), wherein the final transfer vector pAS T5S7sigDelGS.HAAV (Fig. 5C) and pAST5S7sigDelpGS.pK7 (Fig. 5D) are generated. The vectors encode a Fiber-sigDel chimera containing an RGD or pK7 binding domain at the C-terminus for binding to α _v integrin, and heparin sulfate-containing receptors are expressed in 293 cells.

These vectors are then linearized. Transfected 293 cells were incubated with E1, E3, and E4 deleted adenovirus AdCMVZ.1A prior to plasmid transfection (GenVec, Inc., Rockville, MD). Recombination of plasmid E4 + pNS with an E4 deleted vector results in the rescue of the E1-, E3-, E4 + vector, which is capable of replicating in 293 cells. Infected / transfected cells were harvested after five days and lysed to release virions. The lysate is then used to infect freshly plated cells and plaque-purified recombinant viruses. Plaques cross-contaminated with the original AdCMVZ.1A are stained blue when cultured on a culture medium containing the X-glu substrate. The white plaques (denoting the vector) are then amplified to form stock solutions of pure recombinant adenovirus.

Example 4

This example describes the production of targeted adenoviral particles having genomes encoding chimeric fibers. The chimeric strands represent the end of the Ad5 strand and the seven mid-repeats fused to the trimerization domain of sigovel from the reovirus followed by a high affinity RGD sequence that serves to bind to αv integrins.

Plasmid pAS T5S7sigDel.HAAV (FIG. 5C) was digested with the restriction enzyme DrdI, isolated and purified to contain a large fragment containing all adenoviral sequences. This fragment is then introduced, together with the linearized plasmid, by electroporesis into BJ5183 bacterial cells. Said plasmid contains most of the Ad genome located upstream of the filament gene with a small overlap of identical sequence to the plasmid pAS T5S7sigDel.HAAV. By recombination of the two pieces of DNA, a new plasmid is produced by homologous recombination in bacterial cells. This plasmid encodes an engineered adenoviral genome that is capable of replicating in a suitable complementary mammalian cell line (complementing E1 and strand). Plasmid DNA was isolated from selected colonies and found to be the correct plasmid by restriction analysis. This plasmid DNA is then used to transform bacterial DH5α cells in order to obtain an adequate amount of DNA for transfection into a fiber complementing cell line.

One microgram of plasmid is digested with the correct restriction enzyme and transfected with a fiber-complementing cell line, as described above. On days 0 to 4 after transfection, cells are induced with zinc and one to five days thereafter the cells are lysed. The lysate is transferred to fresh fiber complementing cells. This cycle of passage and lysis is repeated until the cytopathic effect occurs in the cells. LacZ cell lysate activity is also monitored throughout the cycle. This activity should grow along with the amplified vector. When an adequate titre of the "mixed" stock solution is obtained, the final passage is made into cells that are not complementary to the fiber and produces a targeted virus that lacks the natural fiber protein. The resulting virus was tested for its ability to bind to and enter cells by interacting with high affinity RGD sequence is _the integrins.

Example 5

This example describes four different fiber trimmers with unnatural trimerization domains. In particular, the fiber trimers mentioned in the examples are chimeras incorporating a portion of the NADC-1 fiber node, which is a porcine adenoid strain. Thus, the trimmers described in the examples contain known motifs suitable for receptor binding (i.e., galactin motif and RGD motif). Furthermore, the trimmers described in the examples incorporate mutations known to reduce the affinity of any motif suitable for receptor binding. Finally, this example describes the incorporation of unnatural ligand (FLAG) into the exposed loop of the unnatural trimer.

F

Using PCR, a node of the NADC-1 strand gene was amplified from a plasmid carrying the full-length gene. The PCR product was then cloned into a baculovirus expression plasmid to produce a plasmid that encodes a NADC-1 node plus an N-terminal polyhistidine tag (Pig4KN protein) for production and detection by Western blotting with antibodies against polyhistidine. The DNA and amino acid sequences of this clone are set forth in SEQ ID NO: 4.

The resulting plasmid pAcPig4KN (FIG. 6A) was mutated by site-directed mutagenesis using two pairs of oligonucleotide primers PigD363Es (SEQ ID NO: 10) and PigD363Ea (SEQ ID NO: 11) and PigN437Ds (SEQ ID NO: 12) and PigN437Da ( SEQ ID NO: 13). Said primers are used in the production of plasmid pAcPigKN D363E (FIG. 6B), in which the DNA sequence encoding the RGD integrin binding motif (a.k. 361-363 in the native fiber protein) has been mutated to a non-functional RGE sequence. A second pair of primers was used to produce the plasmid pAcPigKN N437D (FIG. 6C) in which the DNA sequence encoding the native amino acid N (if 437) was mutated to D. This mutation had previously been shown to break another galactin protein from its ligand, is galactose (Hirabayashi et al., J. Biol. Chem., 266, 23648-53 (1991)).

To demonstrate the possibility of incorporating a new binding motif into an exposed loop on a NADC-1 node, a final baculovirus plasmid was constructed. Analysis of the hydrophobicity of the NADC-1 loop protein showed that the protein sequence immediately before the RGD motif is likely to be an exposed loop capable of incorporating the amino acid sequence (e.g., polypeptide domains) for targeting or purification. Therefore, plasmid pAcPig4KN (FLAG) (Fig. 6D) was produced using complementary overlapping oligonucleotides that encode the FLAG binding domain. Oligonucleotides were pooled and cloned into plasmid pAcPig4KN (FIG. 11A), which contains a single natural restriction site AvrII, located just before the sequence coding for the RGD domain.

The four baculovirus transfer plasmids described above carrying the NADC-1 node genes were used to express the recombinant protein in insect cells using a baculovirus expression system. Tn5 insect cells were infected with recombinant baculovirus clones derived from plasmids. After three days, the cells were centrifuged to form a pellet and frozen three times and thawed in PBS containing protease inhibitors, releasing soluble intracellular protein. The cell debris was centrifuged to form a pellet and the clear lysate was decanted. The residual pellet was resuspended in PBS.

Lysate and pellet samples were then evaluated by SDS-PAGE and Western analysis to determine if the recombinant node proteins were soluble. Western analysis showed that most of the proteins of all four nodes are found in the cell lysate, suggesting that said proteins are soluble and properly folded. These results demonstrate that neither point mutations introduced into domains suitable for receptor binding nor FLAG binding sequences incorporated into the exposed loop affected the node negative packaging and its solubility.

To determine whether chimeric trimmers displaying the FLAG NADC-1 nodes can interact with FLAG antibodies, cell lysates were immunoprecipitated using anti-FLAG M2 antibodies and then blocked. Western analysis showed that the NADC-1 node containing the FLAG epitope is precipitated by antibodies prepared against FLAG. Thus, the fibrous trimmer NADC-1 is soluble and each is able to interact with monoclonal antibodies against FLAG M2.

Example 6:

This example describes the synthesis of a recombinant Ad5-based vector that contains the fibrous node NADC-1 (porcine adenovirus).

Using PCR, the NADC-1 strand gene gene was amplified from a plasmid containing the full-length gene. The PCR product was then cloned into the PNS F5F2K plasmid (FIG. 7A) to produce plasmid pNS Pig4.SS (FIG. 7B), which encodes the first seven β-repeats of the central portion of Ad5 fused to the NADC-1 node. The DNA and amino acid sequences of this clone are set forth in SEQ ID NO: 5.

Plasmid pNS Pig4.SS was then used to prepare a recombinant adenoviral vector. The plasmid was transfected into 293 cells that were infected with an adenoviral vector lacking the E4 region. Homologous recombination between the plasmid and the vector results in a replication competent vector containing E4 and having the chimeric strand NADC-1. The recombinant virus was then plaque purified on 293 cells. Pre-incubation of Ramos cells (which do not express and _in integrins but express adenovirus fiber receptor receptors) with the recombinant NADC-1 node blocked the transduction of these cells with the AdZ.PigSS vector, demonstrating that the vector contains a functional NADC node. The results show that the chimeric strand of NADC-1 can be correctly synthesized and incorporated into live virus particles.

Example 7:

This example describes an Ad5-based adenoviral vector having a chimeric filamentary trimer comprising a mutant NADC-1 node with attenuated receptor binding ability and comprising a functional unnatural ligand.

The ApaI to BamHI fragment containing the ND mutation in pAcPig4KN N437D (Fig. 6C) was cloned into plasmid pAcPig4KN D363E (Fig. 6B), which contains RGD-RGE mutations, to generate plasmid pAcPig4KN D363E N437D. 8A). This plasmid contains mutations in the NADC-1 node gene. Overlapping complementary oligonucleotide primers encoding the integrin binding domain and _in high affinity are therefore cloned into the natural site of AvrII, to form plasmid pAcPig4KN D363E N437D HAAV (FIG. 8B). The mutated EcoR1 to BamH1 fragment of the NADC-1 gene was then cloned into plasmid pNSPig4.SS (Figure 7B) to produce plasmid pNS Pig4 D363E N437D HAAV SS (Figure 8C). This plasmid was then used to prepare a recombinant adenovirus vector containing a mutated NADC-1 node that is targeted to α _v integrin as described above.

The ability of the double mutation in the NADC-1 node to block binding to a cell surface binding site (galactin and integrin) was confirmed by a competition assay. The ability of the resulting virus to target α _v integrin on the cell surface is confirmed using 293 cells as described above.

Example 8:

In this example, two chimeric blocking proteins are described which are capable of interfering with binding to the natural adenoviral receptor. Each blocking protein comprises a domain exhibiting a substrate suitable for the natural adenoviral fiber, namely the extracellular CAR domain.

The extracellular domain of CAR was amplified from the CAR gene (Bergelson et al Science, 275, 1320-23 (1997); Tomko et al., Proc. Nat. Acad. Sci. USA, 94, 3352-56 (1997)) by PCR. The PCR product was then cloned into a baculovirus expression vector to produce plasmid pACSG2-sCAR (FIG. 9A). Soluble CAR protein (sCAR) also contains a FLAG epitope suitable for purification and detection by Western analysis. The DNA and amino acid sequences of the sCAR clone are shown in SEQ ID NO: 6.

Western analysis of sCAR produced in insect cells using a baculovirus clone containing sCAR showed that the protein was secreted from the cell and that some of the secreted protein remained in the cell.

To assess whether the sCAR protein retained the function of natural CAR, radiolabeled type 2 adenovirus was preincubated in a solution containing various concentrations of sCAR and then contacted with 293 cells. Data show that increasing sCAR concentration blocked vi49 binding This result demonstrates that the soluble sCAR protein retained the structure and function of the natural extra-cellular CAR domain. These results demonstrate that pre-incubation with the sCAR protein can prevent binding to the natural adenoviral receptor via a CAR-binding ligand found on the adenoviral strand.

A second chimera containing sCAR was prepared in which the DNA sequence encoding the RGD targeting motif was cloned into the SpeI restriction site following the C-terminus of the sCAR using complementary overlapping primers. The chimeric gene retained the FLAG epitope at the C-terminus. The resulting plasmid SG2-sCAR-HAAV (Fig. 9B) was used in the production of recombinant sCAR protein. RGD as also performed for the sCAR protein described above. The DNA sequence of this clone is shown in SEQ ID NO: 7.

The sCAR.RGD protein was synthesized and secreted from insect cells similar to the sCAR protein. To determine whether the sCAR.RGD protein retained the function of the natural CAR protein, radiolabeled type 2 adenovirus was preincubated in a solution containing various concentrations of sCAR.RGD and contacted with Ramos cells that do not express integrins α _v , but express adenovirus fiber protein receptors. Pre-incubation of radiolabeled type 2 adenovirus with either sCAR or sCAR.RGD blocked the binding of the virus to Ramos cells. This result shows that the sCAR region present on the sCAR.RGD protein is functional.

A cell adhesion study was performed to determine if the sCAR.RGD protein retained the function of the natural RGD domain. Both the sCAR.RGD and sCAR proteins were immobilized on tissue culture plastic plates which subsequently came in contact with 293 cells (which express integrin α _v ). After the cells were incubated on the coated plates, the plates were washed and the number of cells remaining in contact with the plates was determined. The results show that cells adhere to sCAR.RGD coated plates while not adhering to sCAR coated plates or control plates, demonstrating that the RGD motif present on the sCAR.RGD protein is functional.

Example 9:

This example describes a method of directing adenoviral targeting using a chimeric blocking protein that contains a ligand suitable for a cell surface binding site.

The adenoviral vector carrying the lacZ reporter gene was pre-incubated with either the sCAR.RGD protein or sCAR as described in Example 8. The resulting complexes may then come into contact with either Ramos cells (which express the filament receptor (CAR) but lack them). integrins and _v ), or with HuVEC cells (which express both CAR and integrins and _v ), under conditions suitable for viral infection. The cells are then assayed for lacZ expression, and the level of expression will coincide with the degree of virus infection of the cells. The results show that both sCAR and sCAR.RGD effectively block the adenoviral transduction of Ramos cells, while sCAR, but not sCAR.RGD block the adenoviral transduction of HuVECs, indicating that the Ad / sCAR.RGD complex is targeted to integrins and and _to prevent adenovirus-mediated gene introduction into cells by means of sockets.

Example 10:

This example describes two chimeric blocking proteins that are capable of forming trimmers that interfere with binding to the natural adenoviral receptor. In particular, they are blocking proteins, each comprising a region exhibiting a substrate for the natural adenoviral fiber, namely the extracellular region of the CAR, and a trimerization region, namely the trimerization region of the sigDel protein of the Sigma-1 reovirus protein.

The sigDel trimerization region of Sigma-1 protein was amplified by PCR, and the resulting PCR product was cloned into plasmid pAcSG2-sCAR (Figure 9A). The resulting plasmid pAcSG2sCAR.sigDel (FIG. 10A) contains the chimera of the gene encoding the extracellular CAR region, the spacer region, the trimerization domain of the protein derived from the sigma 1 reovirus, and the FLAG binding region. The SpeI restriction site following the trimerization domain allows for easy cloning of regions suitable for targeting. An example is the RGD motif, which binds to integrins and _v . The DNA sequence and amino acid sequence of said clone is set forth in SEQ ID NO: 8.

PAcsCAR.sigDel is used to prepare baculovirus. Western analysis of cell lysates that have passed or not boiled and prepared from baculovirus-infected cells show that the uncooked chimeric sCAR.sigDel migrates as a trimer.

A second sCAR-containing chimera was prepared by cloning the DNA sequence encoding the RGD targeting motif into the SpeI restriction site following the C-terminus of sCAR.sigDel using a complementary overlapping primer. The resulting plasmid pAcSG2-sCARsigDel (HAAV) (Fig. No. 10 B) encodes a chimera consisting of the extracellular region of CAR, the spacer region, the trimerization region of the protein sigma 1 derived from reovirus, and the RGD motif, which binds with high affinity to integrins _and.

The plasmids pAcSG2sCAR.sigDel and pAcSG2-sCARsigDel.RGD (HAAV) were used in the production of recombinant baculoviruses which are suitable for the production of recombinant chimeric protein in insect cells using a standard method. Western analysis of cell lysates of baculovirus-infected cells that either passed or did not boil showed that the uncooked sCAR.sigDel protein migrated as a trimer.

To determine the ability of the sCAR.sigDel and sCARsigDel trimeric proteins to block adenoviral infection, an adenoviral vector carrying the lacZ gene indicating either the sCAR.sigDel or sCARsigDel.RGD trimer or sCAR monomer protein was pre-incubated. Several concentrations are used to obtain dose-dependent data. The resulting complexes are then contacted with 293 cells under conditions suitable for viral infection. The cells are then assayed for lacZ expression, the degree of which will correlate with the degree of virus infection of the cells. The results will be to the extent that the sCAR sigDel and sCARsigDel.RGD trimeric proteins are more effective at blocking adenovirus binding to cells by the sCAR protein than sCAR monomers.

Example 11:

This example describes a method for controlling the targeting of adenoviruses using a trimeric blocking protein that carries a ligand suitable for a cell surface binding site.

The adenoviral vector carrying the lacZ gene designation was pre-incubated with either the sCAR.sigDel, the sCARsigDel.RGD, or the sCAR described above. Similarly, adenovirus can be pre-incubated with a blocking protein isolated, for example, by the appearance of phage. The resulting complexes are then contacted with either Ramos or Hu VEC cells under conditions appropriate for viral infection. The cells are then assayed for lacZ expression, the degree of which then correlates with the degree of virus infection of the cells. The results show that while proteins will effectively block adenoviral transduction of Ramos cells, trimers are more effective at blocking adenovirus binding than sCAR monomers. Adenoviral transduction of HuVEC cells will be blocked by sCAR and sCAR.sigDel proteins. However, sCARsigDel.RGD will not effectively block adenoviral transduction of HuVEC cells. Such results indicate that the AdsCARsigDel.RGD complex is targeted to integrins α _v , while preventing the introduction of the gene into adenovirus-mediated cells by CAR.

Example 12:

The example describes the construction and evaluation of mutated fiber nodes, each having reduced affinity for natural substrates, particularly in the case of monoclonal antibodies raised against the natural fiber node.

Using site-directed mutagenesis, single mutations were introduced into the full-length Ad5 strand gene in a baculovirus vector. The resulting plasmids were then used to prepare recombinant baculovirus clones.

Each of the mutants plus the natural control of the Ad5 strand was used to produce protein in infected insect cells. Three days after infection, cells were harvested and lysed. Western analysis using a polyclonal antiserum, which recognizes the Ad5 strand, revealed the presence of a large amount of the filament protein in the lysate formed from cells infected with each of the vectors.

In cells infected with the five mutant clones (shown in Table 1) (as well as the natural fiber gene), the signal was predominantly found in a soluble portion of the lysates, indicating that the protein encoded by such a mutant was correctly packaged. The wild-type Ad5 strand sequences are set forth in SEQ ID NO: 9. The amino acids of SEQ ID NO: 9 altered by each of these mutations are set forth in Table 2. First

Table no. 1:

mutations Monoclonal antibodies ky 2C9 4B8 3d9 2E5 CD Loop (449 SGTVQ-GSGSG) + + Loop IJ (559 GSHN-GSGS) + + Loop FG (507 SHGKTA-GSGSGS) + + T533S / T353 (535 TIT-SIS) + + -t- K506 (506 K-R) + + + Addition at the C-terminus + + + + Natural fiber Ad5 + + + + Ad5 strand passed through boiling - - - -

Using Western blot analysis, each of the five soluble mutant fiber proteins, the native Ad5 strand, and the denatured Ad5 strand were tested with four monoclonal antibodies generated against the fiber node. The signals were detected by chemoluminescence and the signal strength of each band was compared. The results of the test are shown in Table 2. First

If no antibodies recognize the denatured strand, this means that each antibody binds only to properly packaged trimer strands. If none of the mutants shows limited affinity for 2E5 antibodies, this means that each mutant strand was indeed a trimer.

The K506R mutation substantially limits the affinity of the resulting strand for 3D9 antibodies without affecting the affinity for any other antibody. The position of this mutation in the fiber node is shown in FIG. 15A to 15C

Mutations in the CD, IJ, or FG loops, where 4-6 amino acids have been replaced by serines or glycines, substantially limit the affinity of the resulting mutant trimer for 2C9 antibodies. The double mutant T533S / T535S also reduced the affinity of the mutant loop for 4B8 antibodies. The placement of each of these mutations in the fiber node is shown in Figures 15D to 15F.

These results indicate that nodules on the trimer strand may be formed which exhibit reduced affinity for natural substrates. A similar assay protocol can be used to identify mutants that have limited affinity for cellular receptors. For example, a soluble form of sCAR that exhibits an FLAG epitope (or other tag) as described above may be used as a probe instead of the monoclonal antibodies described above. The blots are then screened for anti-FLAG monoclonal antibodies to detect mutations interfering with fiber-CAR binding.

Example 13:

This example describes the construction of a recombinant adenovirus comprising a mid-truncated strand (e.g., eight mid-repeats) and a mutant strand node (5) exhibiting reduced affinity for its natural receptor (i.e., CAR). Such a strand allows targeting through a ligand expressed in the penton base.

Using a standard recombinant procedure, a deletion was introduced into the mid-strand coding sequence. From the sequence shown in SEQ ID NO: 8, a portion of the mutant strand node was amplified by PCR from the middle repeat 22 to the end of the coding sequence. This section contains the K506R mutation (described in Example 12). The resulting product is used in the preparation of plasmid pAS T5S7F5K (R506K) (Fig. 16). Thus, the plasmid contains a gene encoding a truncated middle strand with reduced affinity for the natural substrate (3D9 antibodies). The adenovirus containing such a truncated middle portion fiber exhibits limited affinity for the natural substrate (3D9 antibodies). An adenovirus containing such a truncated middle portion will be able to bind to cells via a penton-based RGD ligand. A similar strategy can be used, for example, to prepare adenoviral vectors having truncated middle-portion fibers with reduced affinity for CAR.

Example 14:

This example describes the construction of adenoviral vectors having specific unnatural ligands that can be used to purify the vector using affinity chromatography. The base vector pNSF5F2K (Figure 8A) contains a gene that encodes a chimeric strand that contains the middle portion of the Ad5 strand and the Ad2 strand protein node. The Ad2 fiber gene contains a SpeI restriction site in the node region that encodes the flexible node loop H1 of the fiber node. This SpeI restriction site was used to incorporate sequences that encode the FLAG peptide of SEQ ID NO: 16 or a DNA / heparin binding ligand (SEQ ID NO: 15).

The base vector pBSSpGS (FIG. 11A) encodes an extension of 12 amino acids at the C-terminus (SEQ ID NO: 14). The codons encoding TS are also the only SpeI restriction sites that have been used to incorporate sequences that encode a FLAG peptide (SEQ ID NO: 16) or a DNA / heparin binding polypeptide (SEQ ID NO: 15), as described below.

Transfer plasmids (pBSSpGS (RKKK) 2 (FIG. 11B) and pNSF5F2K (RKKK) 2 (FIG. 11C)) suitable for introduction of a DNA / heparin-binding ligand into the adenoviral genome were generated using overlapping oligonucleotides. The sense and anti-sense oligonucleotides were mixed in equimolar ratios and cloned into the SpeI site of pBSSpGS (FIG. 11A) or pNS F5F2K (FIG. 8A) to form transfer plasmids. Sequencing in both directions across the insert region verified that the clones contained the correct sequence.

In a similar case, transfer plasmids pBSSpGS (FLAG) (FIG. 11D) and pNSF5F2K (FLAG) (FIG. 11E) suitable for introduction of the FLAG ligand (SEQ ID NO: 16) into the adenoviral genome were prepared. Sequencing in both directions across the insert region verified that the clones contained the correct sequence.

Plasmid DNA from the four transfer vectors was linearized with the restriction enzyme SalI, purified and transfected using calcium phosphate into 293 cells, which were pre-incubated for one hour with the adenovirus AdCMVZ.IIA (GenVec, Inc., Rockville, MD) with deletion. E1, E3 and E4 with a multiplicity of 1 ffu per cell. Recombination of plasmid E4 + pNS with an E4 deletion vector resulted in the rescue of vector E1-, E3-, E4 + capable of replicating in cells 293. Resulting vectors AdZ.F2K (RKKK) 2, AdZ.F2K (FLAG), Ad.F (RKKK) 12 and AdZ.F (FLAG) were isolated in two rounds of plaque formation on 293 cells.

For each vector, sequencing of PCR products obtained from viral template DNA was verified using primers that span the DNA insert region for the correct insert. Restriction analysis of Ad DNA from each virus showed that the viruses were pure and that the properly engineered virus contained a single BamHI restriction site.

Example 15:

This example describes that an adenoviral vector having an unnatural ligand can bind to a support associated with a substrate for said ligand.

The AdZ.PK vector was constructed similarly to the vectors described above. The virus has a fiber protein that contains polylysines. AdZ.PK was tested to see if the virus could bind to a substrate that contains a substrate for polylysine, which is heparin. 50 ml of heparin-agarose beads (SIGMA) were added to 1.0 ml phosphate buffers containing 150, 300, 500 and 1000 mM NaCl. 6,600 cpm of either AdZ or AdZ.PK was then added to saline solutions containing heparin and agarose beads and mixed by rocking for 60 minutes. The beads were then washed three times in buffer with the same salt content as the incubation buffer (150, 300, 500 and 1000 mM NaCl). The cpm value associated with the beads was then measured and it was shown that the binding of AdZ.PK was preferential to that of AdZ at NaCl concentrations of 150, 300 and 500 mM. However, at a NaCl concentration of 1000 mM, the binding of AdZ.PK to the beads was much lower and approximately equal to the background observed with AdZ.

These results indicate that the AdZ.PK vector binds to heparin bound to the support material and thus binding does not occur due to high salt concentration. Such a substrate can be used to purify the engineered vector by first attaching the virus to the substrate at a low salt concentration and then eluting the vector at a high salt concentration.

Example 16:

This example shows that an adenoviral vector exhibiting an unnatural ligand can be purified on a column containing a substrate for the ligand.

A 20 175 cm ² tissue culture flask containing a packer cell line 293 was infected at moi 5 with one of three vectors. AdZ.PK, AdZ.F2K (RKKK) 2, or AdZ.F (RKKK) 2, as described above. The cells are then incubated for two days and then the remaining adherent cells are separated from the plastic. The removed cells are then centrifuged at 3000 g to form a pellet. The culture medium is removed and the pellet is washed twice with PBS. The cells were then resuspended in a total volume of 5 ml PBS containing 10 mM MgCl ₂ .

The resuspended cells are then frozen and thawed three times to release the virus, and the cell debris is then centrifuged at 15,000 g for 15 minutes. The supernatant was passed through a column with a volume greater than 3 ml containing heparin bound to agarose beads. The column is then washed with 30 ml PBS followed by elution of the virus from the column using a gradual salt gradient. To elute the virus, 3 ml of a buffer containing a substantially higher concentration of NaCl (in 100 mM steps) is passed through the column. A 1 ml eluate (3 ml 200 mM, 3 ml 300 mM NaCl, 3 ml 400 mM NaCl to 2000 mM NaCl) is collected.

The fractions comprising the passage and wash fractions are then evaluated for adenoviral envelope proteins by Western blotting, the active virus particles are then evaluated using the A549 lacZ transduction step or plaque formation assay, and the overall purity by analytical58 high performance liquid chromatography (HPLC) as is described in Shabram et al., 1997, Hum. Genen Ther. 8, 453-46; Huyghe et al., 1995, Hum. Gene Ther., 6: 1403-1416; Shabram et al., WO 96/27677). The overall purity of the fractions showed that the peak of the adenovirus concentrations was assessed by passing the fractions through HPLC and comparing the profile with the column inlet fraction and the highly pure adenovirus preparation (prepared in three rounds of CsCl gradient purification).

Example 17:

This example describes the production of a pseudo-receptor for the construction of a cell line capable of replicating adenoviruses lacking the natural function of binding to a cell (but targeting the pseudo-receptor). In a specific case, an example of a pseudo-receptor comprises a single chain antibody (ScFv) binding domain.

First, a vector expressing ScFv from pHOOK3 (Fig. 12A) (Invitrogen), which encodes a ScFv, is synthesized with the murine Ig signal peptide. ScFv has a non-terminal epitope of the HA tag and its C-terminus is linked to a pair of myc epitopes followed by a PDGF receptor transmembrane anchor. The expression cassette comprising this construct was cloned into plasmid pRC / CMVβ-Puro (Figure 12B) to generate plasmid pScHAHK (Figure 12C). This plasmid has cloning sites for the insertion of genes following the CMV promoter and has single Agel and XbaI restriction sites at the C-terminus of the gene for addition of cytoplasmic sequences.

To show that the pseudo-receptor ScFv is expressed on the cell surface, 293 cells were transfected with either the plasmid pNSE4GLP (Figure 12D) carrying the green fluorescent protein gene for the detection of transfectants, or in combination with pScHAHK. One day after transfection, the surface immunofluorescence exhibited by pScHAHK using antibodies directed against the HA epitope demonstrates the correct surface expression of the pseudoreceptor.

To demonstrate that the expressed pseudo-receptor is functional, the transfected cells were contacted with magnetic particles of CAPTURE-TEC associated with antigens that are recognized by ScFv. After incubation, the particles were collected at the bottom of the tube using a magnet, washed and transferred to a culture plate. Culture plates were examined to identify cells expressing GFP under a fluorescence microscope. No staining was observed in cells transfected with pNSE4GLP alone, indicating that these cells did not bind to the particles. However, cells transfected with pNSE4GLP and pScHAHK were visible in the wells. This result shows that the double transfected cells bind to the particles.

Example 18:

This example describes the production of a pseudo-receptor for the construction of a cell line capable of replicating adenoviruses lacking the natural function of binding to a cell (but targeting the pseudo-receptor). An example of a pseudoreceptor comprises a binding region derived from single chain antibodies recognizing HA.

Anti-HA ScFvs were constructed as an N-Term-VL-VH fusion protein. RTPCR was converted to RNA to obtain hybridomas producing HA antibodies using primers specific for the k- or γ2β- and C-terminus of the VL and VH genes (Gillilan et al., Tissue Antigens, 47, 1-20 ( 1996)). After sequencing the resulting PCR products, specific oligonucleotides were labeled to amplify the VL-VH fusion in the second round of PCR. The final PCR product was cloned to give plasmid pCANTAB5E (HA) (FIG. 17A), which is suitable for the production of anti HA ScFv in E. coli. The expressed protein has a C terminalminal E peptide suitable for detecting the binding of HA penton base by Western analysis ELISA. After transformation of bacterial cells with plasmid pCANTAB5E (HA), Western analysis was performed using antibodies that recognize the E peptide. In this way, the peptide of the expected size was demonstrated.

To determine whether the anti-HA ScFv is functional, these antibodies were used in the protein A immunoprecipitation assay using adenoviral coat proteins (recombinant penton base) containing the HA epitope

Anti-HA ScFvs are able to precipitate HA containing penton base proteins These results show that the extracellular portion of the pseudo-receptor suitable for binding to an adenovirus having an unnatural ligand (i.e. HA) has been successfully constructed.

To generate the entire anti-HA pseudo-receptor, the anti-HA ScFv was cloned into plasmid pSCHAHK, in which HA was removed to produce plasmid pScFGHA (FIG. 17B). This plasmid will produce an anti-HA pseudo-receptor capable of binding recombinant adenoviruses that exhibit an epitope of HA similar to the adenoviruses described above having FLAG epitopes.

Example 19:

This example describes the generation of a fiber-expressing cell line that is suitable for the production of targeted adenoviral particles. The complementing cell line produces a fiber protein that either contains or does not contain other complementary genes from the adenoviral genome.

The entire adenovirus type 2 fiber gene was amplified from adenovirus type 2 DNA by PCR. The resulting product was cloned into plasmid pCR2.1-TOPO (Invitrogen) to produce plasmid pCR2.1-TOPO + fiber (Fig. 13A). The fiber2 gene was excised from plasmid pCR2.1-TOPO + Fiber with BamHI and Eagl restriction enzymes and then subcloned into plasmid pKSII (Stratagene) to construct plasmid pKSII Fiber (FIG. 13B). The fiber2 gene was then excised from the plasmid pKSII Fiber using the restriction enzymes KpnI and Eagl and then cloned into plasmid pSMTZeo-DBP (Fig. 13C). The resulting plasmid pSMTZeo-Fiber (FIG. 13D) encodes the entire fiber2 gene, which is under the control of the metallothionine promoter. In this construction, an efficient mRNA splicing site was placed upstream of the fiber gene, enhancing synthesis of the fiber protein following induction. Plasmid pSMTZeo-Fiber also contains a Zeo resistance marker, which allows the selection of cell lines for the antibiotic zeocin.

To produce a cell line, the plasmid pSMTZeo-Fiber was transfected into 293 cells (or some other cell line) whether or not adenovirus-complementing functions were added. Individual colonies of zeocin-resistant cells are then amplified in a standard manner and tested for fiber2 production (e.g., by Western analysis using anti-fiber2 antibodies) before and after induction with zinc, which activates the metallothionine promoter. The selected fiber-expressing clones were then tested for their ability to form plaques and / or complement the growth of adenoviruses containing mutated fibers. Clones that adequately complement mutated strands are useful for amplifying and culturing adenoviral particles that contain genomes encoding mutant strand genes.

SEQUENCE PROTOCOL

SEQUENCE NUMBER: 18 (2) Information about SEQ ID NO: 1:

(I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 960 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown

(ii) MOLECULAR TYPE: DNA (ix) FEATURES:

(A) Name / Key: CDS (B) Position: 1 ..957 (Xi) Sequence description: SEQ ID NO: 1: ATG AAG CGC GCA AGA CCG TCT GAA GAT ACC TTC AAC CCC GTG TAT CCA Met Lys 1 Arg Ala Arg Pro Ser 4 Asp Thr 10 Phé Asn Pro TAT GAC ACG GAA ACC GGT CCT CCA ACT GTG CCT TTT ACT CCT CCC Tyr Asp Thr Glu For Thr Val 25 Pro Phe Leu Thr Pro 30 TTT GTA TCC CCC AAT GGG TTT CAA GAG AGT CCC CCC GGG GTA CTC TCT Phe Val Ser Pro Asn Gly Phe 36 Gin Glu Ser 40 Pro Gly Val Leu Ser 45 TTG CGC CTA TCC GAA CCT CTA GTT ACC TCC AAT GGC ATG CTT GCG CTC Leu Arg Leu Ser Glu Pro Leu 56 Val Thr Asn Gly Met Leu Ala Leu AAA ATG GGC CTC TCT CTG GAC GAG GCC GGC AAC CTT ACC TCC Lys Met Gly Asn Gly Leu Ser 70 Leu Asp Glu Al Gly Asn Leu Thr Ser 75 80 CAA AAT GTA ACC ACT GTG CCA CCT CTC AAA AAA ACC AAG Gin Asn Val Thr Thr Val Ser For Pro Leu 90 Lys Lys Thr Lys Ser Asn AT AND AAC CTG GAA ATA TCT GCA CCC CTC ACA GTT ACC TCA GAA GCC Asn Leu Glu White Ser Ala 100 For Leu Thr 105 Val Thr Ser Glu Ala Leu ACT GTG GCT GCC GCC GCA CCT CTA ATG GTC GCG GACC AAC ACA CTC ACC Thr Val Ala Ala Ala Ala Pro 115 Leu Met Val Ala Gly Asn Thr Leu Thr 126 ATG CAA TCA CAG GCC CCG CTA ACC GTG CAC GAC TCC AAA ATT Met Gin Ser Gin Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser IJ.e 140

144

192

240

288

336

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432

GCČ ACC CAA GGA CCC GTC Pro Leu 150 ACA Thr GTG TCA GAA GGA 480 Ala 145 Thr gin wall Ser Glu Gly 155 Lys Leu Ala Ser Arg 160 GTC TCG GCG CTC GAG AAG ACG TCT CAA ATA CAC TCT GAT ACT ATC CTC 528 wall Ser Ala Leu Glu Lys Thr Ser gin Ile His Ser Asp Thr Ile Leu 165 170 175 CGG ATC ACC CAG GGA CTC GAT GAT GCA AAC AAA CGA ATC ATC GCT CTT 576 Arg Ile Thr gin Gly Leu Asp Asp Ala same time Lys Arg Ile Ile Ala Leu 180 185 190 GAG CAA AGT CGG GAT GAC TTG GTT GCA TCA GTC AGT GAT GCT CAA CTT 624 Glu gin Ser Arg Asp Asp Leu wall Ala Ser wall Ser Asp Ala gin Leu 195 200 205 GCA ATC TCC AGA TTG GAA AGC TCT ATC GGA GCC CTC CAA ACA GTT GTC 672 Ala Ile Ser Arg Leu Glu Ser Ser Ile Gly Ala Leu gin Thr wall wall 210 215 220 AAT GGA CTT GAT TCG AGT GTT ACC CAG TTG GGT GCT CGA GTG GGA CAA 720 same time Gly Leu Asp Ser Ser wall Thr gin Leu Gly Ala Arg wall Gly gin 225 230 235 240 CTT GAG ACA GGA CTT GCA GAC GTA CGC GTT GAT CAC GAC AAT CTC GTT 768 Leu Glu Thr Gly Leu Ala Asp wall Arg wall Asp His Asp same time Leu wall 245 250 255 GCG AGA GTG GAT ACT GCA GAA CGT AAC ATT GGA TCA TTG ACC ACT GAG 816 Ala Arg wall Asp Thr Ala Glu Arg same time Ile Gly Ser Leu Thr Thr Glu 260 265 270 CTA TCA ACT CTG ACG TTA CGA GTA ACA TCC ATA CAA GCG GAT TTC GAA 864 Leu Ser Thr Leu Thr Leu Arg wall Thr Ser Ile gin Ala Asp Phe Glu 275 280 285 TCT AGG GGA TCC GGC GGC ACT AGT GGC GGC GAC TAC AAG GAC GAC GAC 912 Ser Arg Gly Ser Gly Gly Thr Ser Gly Gly Asp Tyr Lys Asp Asp Asp 290 295 300 GAC AAG GGC CCT AGG GGC GCC CGC CGC GCC TCC CTT GGC TCT AGA TAA 960 Asp Lys Gly for Arg Gly Ala Arg Arg Ala Ser Leu Gly Ser Arg 305 310 315

(2) Information about SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 633 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (ix) FEATURES:

(A) Name / Key: CDS (B) Position: 1.630 (xi) Sequence Description · SEQ ID NO: 2:

ATG AAG CGC GCA AGA CCG TCT GAA GAT ACC TTC AAC CCC GTG TAT CCA 48 Met Lys Arg Ala Arg for Ser Glu Asp Thr Phe same time for wall Tyr for 325 330 33S TAT GAC ACG GAA ACC GGT CCT CCA ACT GTG CCT TTT CTT ACT CCT CCC 96

Tyr Asp Thr Glu 340 Thr Gly for for Thr 345 wall for Phe Leu Thr 350 for for TTT GTA TCC CCC AAT GGG TTT CAA GAG AGT CCC CCC GGG GGA GGG CTA 144 Phe wall Ser 355 for same time Gly Phe gin 360 Glu Ser for for Gly 365 Gly Gly Leu GCA TCA AGG GTC TCG GCG CTC GAG AAG ACG TCT CAA ATA CAC TCT GAT 192 Ala Ser 370 Arg wall Ser Ala Leu 375 Glu Lys Thr Ser gin 380 Ile His Ser Asp ACT ATC CTC CGG ATC ACC CAG GGA CTC GAT GAT GCA AAC AAA CGA ATC 240 Thr 385 Ile Leu Arg Ile Thr 390 gin Gly Leu Asp 395 Ala same time Lys Arg Ile 400 ATC GCT CTT GAG CAA AGT CGG GAT GAC TTG GTT GCA TCA GTC AGT GAT 288 Ile Ala Leu Glu gin 405 Ser Arg Asp Asp Leu 410 wall Ala Ser wall Ser 415 Asp GCT CAA CTT GCA ATC TCC AGA TTG GAA AGC TCT ATC GGA GCC CTC CAA 336 Ala gin Leu Ala 420 Ile Ser Arg Leu Glu 425 Ser Ser Ile Gly Ala 430 Leu gin ACA GTT GTC AAT GGA CTT GAT TCG AGT GTT ACC CAG TTG GGT GCT CGA 384 Thr wall wall 435 same time Gly Leu Asp Ser 440 Ser wall Thr gin Leu 445 Gly Ala Arg GTG GGA CAA CTT GAG ACA GGA CTT GCA GAC GTA CGC GTT GAT CAC GAC 432 wall Gly 450 gin Leu Glu Thr Gly 455 Leu Ala Asp wall Arg 460 wall Asp His Asp AAT CTC GTT GCG AGA GTG GAT ACT GCA GAA CGT AAC ATT GGA TCA TTG 480 same time 465 Leu wall Ala Arg wall 470 Asp Thr Ala Glu Arg 475 same time Ile Gly Ser Leu 480 ACC ACT GAG CTA TCA ACT CTG ACG TTA CGA GTA ACA TCC ATA CAA GCG 528 Thr Thr Glu Leu Ser 485 Thr Leu Thr Leu Arg 490 wall Thr Ser Ile gin 495 Ala GAT TTC GAA TCT AGG GGA TCC GGC GGC ACT AGT GGC GGC GAC TAC AAG 576 Asp Phe Glu Ser 500 Arg Gly Ser Gly Gly Thr Ser Gly Gly Asp 510 Tyr Lys GAC GAC GAC GAC AAG GGC CCT AGG GGC GCC CGC CGC GCC TCC CTT GGC 624 Asp Asp Asp 515 Asp Lys Gly for Arg Gly 520 Ala Arg Arg Ala Ser Leu Gly

TCT AGA TAA 633

Ser Arg

530 (2) Information about SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1704 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (ix) FEATURES:

(A) (B)

Name / Key: CDS Position-1 ..1701 (xi) Sequence Description: SEQ ID NO: 3:

ATG Met AAG Lys CGC GCA AGA CCG TCT GAA GAT ACC TTC Arg Ala 215 Arg for Ser Glu Asp 220 Thr Phe Asn For Val 225 Tyr for TAT GAC ACG GAA ACC GGT CCT CCA ACT GTG CCT TTT CTT ACT CCT CCC Tyr Asp Thr 230 Glu Thr Gly for for 235 Thr wall Pro Phe Leu Thr 240 for for TTT GTA TCC CCC AAT GGG TTT CAA GAG AGT CCC CCC GGG GTA CTC TCT Phe wall 245 Ser for same time Gly Phe 250 gin Glu Ser For Pro 255 Gly Val Leu Ser TTG CGC CTA TCC GAA CCT CTA GTT ACC TCC AAT GGC ATG CTT GCG CTC Leu 260 Arg Leu Ser Glu for 265 Leu wall Thr Ser Asn Gly Met Leu Ala Leu 275 AAA ATG GGC AAC GGC CTC TCT CTG GAC GAG GCC GGC AAC CTT ACC TCC Lys Met Gly same time Gly 280 Leu. Ser Leu Asp Glu 285 Ala Gly Asn Leu Thr 290 Ser CAA AAT GTA ACC ACT GTG AGC CCA CCT CTC AAA AAA ACC AAG TCA AAC gin same time wall Thr 295 Thr wall Ser for for 300 Leu Lys Lys Thr Lys Ser same time ATA AAC CTG GAA ATA TCT GCA CCC CTC ACA GTT ACC TCA GAA GCC CTA Ile same time Leu 310 Glu Ile Ser Ala for 315 Leu Thr Val Thr Ser Glu 320 Ala Leu ACT GTG GCT GCC GCC GCA CCT CTA ATG GTC GCG GGC AAC ACA CTC ACC Thr wall 325 Ala Ala Ala Ala for 330 Leu Met wall Ala Gly 335 Asn Thr Leu Thr ATG CAA TCA CAG GCC CCG CTA ACC GTG CAC GAC TCC AAA CTT AGC ATT Met 340 gin Ser gin Ala for 345 Leu Thr wall His Asp Ser 350 Lys Leu Ser Ile 355 GCC ACC CAA GGA CCC CTC ACA GTG TCA GAA GGA AAG CTA GCA TCA AGG Ala Thr gin Gly for 360 Leu Thr wall Ser Glu 365 Gly Lys Leu Ala Ser 370 Arg GTC TCG GCG CTC GAG AAG ACG TCT CAA ATA CAC TCT GAT ACT ATC CTC wall Ser Ala Leu 375 Glu Lys Thr Ser gin 380 Ile His Ser Asp Thr 385 Ile Leu CGG ATC ACC CAG GGA CTC GAT GAT GCA AAC AAA CGA ATC ATC GCT CTT Arg Ile Thr 390 gin Gly Leu Asp Asp 395 Ala same time Lys Arg 400 Ala Leu GAG CAA AGT CGG GAT GAC TTG GTT GCA TCA GTC AGT GAT GCT CAA CTT Glu gin 405 Ser Arg Asp Asp Leu 410 wall Ala Ser Val Ser Asp Ala gin Leu GCA ATC TCC AGA TTG GAA AGC TCT ATC GGA GCC CTC CAA ACA GTT GTC Ala 420 Ile Ser Arg Leu Glu 425 Ser Ser Ile Gly Ala Leu 4 31 Gin Thr wall wall 435 AAT GGA CTT GAT TCG AGT GTT ACC CAG TTG GGT GCT CGA GTG GGA CAA same time Gly Leu Asp Ser 440 Ser wall Thr gin Leu 445 Gly Ala Arg Val Gly 450 gin CTT GAG ACA GGA CTT GCA GAC GTA . CGC GTT GAT CAC GAC AAT CTC GTT Leu Glu Thr Gly 455 Leu Ala Asp wall Arg 460 wall 1 Asp His Asp Asn 464 Leu wall GCG AGA . GTG GAT ACT GCA . GAA CGT 'AAC : att GGA TCA TTG ACC : act ¹ GAG

144

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336

384

432

480

528

576

624

672

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768

816

Ala Arg wall Asp Thr Ala Glu Arg same time Ile Gly Ser Leu Thr Thr Glu 470 475 480 CTA TCA ACT CTG ACG TTA CGA GTA ACA TCC ATA CAA GCG GAT TTC GAA Leu Ser Thr Leu Thr Leu Arg wall Thr Ser Ile gin Ala Asp Phe Glu 485 490 495 TCT AGG GGA TCC GGC GGC ACT AGA GGA GGT GGA ATG AGC AAG GGC GAG Ser Arg Gly Ser Gly Gly Thr Arg Gly Gly Gly Met Ser Lys Gly Glu 500 505 510 515 GAA CTG TTC ACT GGC GTG GTC CCA ATT CTC GTG GAA CTG GAT GGC GAT Glu Leu Phe Thr Gly wall wall for Ile Leu wall Glu Leu Asp Gly Asp 520 525 530 GTG AAT GGG CAC AAA TTT TCT GTC AGC GGA GAG GGT GAA GGT GAT GCC wall same time Gly His Lys Phe Ser wall Ser Gly Glu Gly Glu Gly Asp Ala 535 540 545 ACA TAC GGA AAG CTC ACC CTG AAA TTC ATC TGC ACC ACT GGA AAG CTC Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu 550 555 560 CCT GTG CCA TGG CCA ACA CTG GTC ACT ACC TTC ACC TAT GGC GTG CAG for wall for Trp for Thr Leu wall Thr Thr Phe Thr Tyr Gly wall gin 565 570 575 TGC TTT TCC AGA TAC CCA GAC CAT ATG AAG CAG CAT GAC TTT TTC AAG Cys Phe Ser Arg Tyr for Asp His Met Lys gin His Asp Phe Phe Lys 580 585 590 595 AGC GCC ATG CCC GAG GGC TAT GTG CAG GAG AGA ACC ATC TTT TTC AAA Ser Ala Met for Glu Gly Tyr wall gin Glu Arg Thr Ile Phe Phe Lys 600 605 610 GAT GAC GGG AAC TAC AAG ACC CGC GCT GAA GTC AAG TTC GAA GGT GAC Asp Asp Gly same time Tyr Lys Thr Arg Ala Glu wall Lys Phe Glu Gly Asp 615 620 625 ACC CTG GTG AAT AGA ATC GAG TTG AAG GGC ATT GAC TTT AAG GAA GAT Thr Leu wall same time Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp 630 635 640 GGA AAC ATT CTC GGC CAC AAG CTG GAA TAC AAC TAT AAC TCC CAC AAT Gly same time Ile Leu Gly His Lys Leu Glu Tyr same time Tyr same time Ser His same time 645 650 655 GTG TAC ATC ATG GCC GAC AAG CAA AAG AAT GGC ATC AAG GTC AAC TTC wall Tyr Ile Met Ala Asp Lys gin Lys same time Gly Ile Lys wall same time Phe 660 665 670 675 AAG ATC AGA CAC AAC ATT GAG GAT GGA TCC GTG CAG CTG GCC GAC CAT Lys Ile Arg His same time Ile Glu Asp Gly Ser wall gin Leu Ala Asp His 680 685 690 TAT CAA CAG AAC ACT CCA ATC GGC GAC GGC CCT GTG CTC CTC CCA GAC Tyr gin gin same time Thr for Ile Gly Asp Gly for wall Leu Leu for Asp 695 700 705 AAC CAT TAC CTG TCC ACC CAG TCT GCC CTG TCT AAA GAT CCC AAC GAA same time His Tyr Leu Ser Thr gin Ser Ala Leu Ser Lys Asp for same time Glu

710

715

720

864

912

960

1008

1056

1104

1152

1200

1248

1296

1344

1392

1440

1488

1536

1584

AAG AGA GAC CAC ATG GTC CTG GG TTT GTG ACC GCT GCT GGG ATC Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly White

(+420) 725 730 735

ACA CAT GGC Gly ATG Met GAC GAG CTG TAC AAG GGT GGA GGT AGA TCT ACT Thr 740 His Asp Glu 745 Leu Tyr Lys Gly Gly 750 Gly Arg Ser Thr GGC GGC GAC TAC AAG GAC GAC GAC GAC AAG GGC CCT AGG GGC GCC Gly Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly for Arg Gly Ala 760 765 770 CGC GCC TCC CTT GGC TCT AGA TAA Arg Ala Ser Leu Gly Ser Arg 775

AGT 1632

Ser

755

CGC 1680

Arg

1704 (2) Information about SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1830 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (ix) FEATURES:

(A) Name / Key: CDS (B) Position: 1 ..1827 (xi) Sequence Description: SEQ ID NO: 4:

ATG AGA GGA TCT CAC CAT GGC GAT GGA GCT TTG 48 Met Arg Gly Ser His His His His His His Gly Glu 580 Asp Gly Ala Leu 570 575 TCC CTG ACA AAA ACC TTA GTC TAT CCC ACC CTG TGG ACG GGG CCT GCT 96 Ser Leu Thr Lys Thr Leu wall Tyr for Thr Leu Trp Thr Gly for Ala 585 590 595 600 CCC GAG GCC AAC GTC ACC TTC TCG GGG GAG AAT TCC CCA TCT GGC ATT 144 for GIU Ala same time wall Thr Phe Ser Gly Glu same time Ser for Ser Gly Ile 605 610 615 CTC AGA CTG TGT CTC AGC AGA ACC GGG GGC ACG GTC ATT GGC ACC CTG 192 Leu Arg Leu Cys Leu Ser Arg Thr Gly Gly Thr wall Ile Gly Thr Leu 620 625 630 TCT GTA CAA GGT AGC CTC ACG AAC CCC AGT ACC GGT CAG ACC CTG GGC 240 Ser wall gin Gly Ser Leu Thr same time for Ser Thr Gly gin Thr Leu Gly 635 640 645 ATG AAC CTT TAC TTT GAC GCA GAC GGC AAT GTG CTG TCT GAG AGC AAC 288 Met same time Leu Tyr Phe Asp Ala Asp Gly same time wall Leu Ser Glu Ser same time 650 655 660 CTC GTC CGA GGG TCC TGG GGA ATG AAA GAC CAA GAT ACC CTG GTG ACT 336 Leu wall Arg Gly Ser Trp Gly Met Lys Asp gin Asp Thr Leu wall Thr 665 670 675 680 CCC ATT GCC AAT GGG CAG TAC CTG ATG CCC AAC CTC ACT GCA TAC CCT 384 for Ile Ala same time Gly gin Tyr Leu Met for same time Leu Thr Ala Tyr for 685 690 695 CGC CTC ATA CAG ACC CTA ACT TCC AGC TAC ATT TAC ACA CAA GCG CAC 432 Arg Leu Ile gin Thr Leu Thr Ser Ser Tyr Ile Tyr Thr gin Ala His 700 705 710 CTT GAC CAC AAT AAC AGT GTG GTG GAC : atc : AAG ATA , GGG CTC AAC ; ACA 480

Leu Asp His 715 same time same time Ser wall wall 720 Asp Ile Lys Ile Gly 725 Leu same time Thr GAC CTG AGG CCC ACT GCG GCC TAC GGC CTA AGC TTT ACC ATG ACC TTC 528 Asp Leu 730 Arg for Thr Ala Ala 735 Tyr Gly Leu Ser Phe 740 Thr Met Thr Phe ACT AAC TCT CCC CCC ACC TCA TTT GGT ACC GAC CTG GTG CAA TTT GGC 576 Thr 745 same time Ser for for Thr 750 Ser Phe Gly Thr Asp 755 Leu wall gin Phe Gly 760 TAC CTG GGT CAG GAT AGC TCC CCC TCC TTC CTG AGA GAA CTT CCC CTT 624 Tyr Leu Gly gin Asp 765 Ser Ser for Ser Phe 770 Leu Arg Glu Leu for 775 Leu GCA TCC GAG GCG GGC TAC TTT GGC Aaa CTG GCA GCT GCC TCT GAG GAA 672 Ala Ser Glu Ala 780 Gly Tyr Phe Gly Lys 785 Leu Ala Ala Ala Ser 790 Glu Glu ATG CCA GCC CCT CCT GAG GCC CAG ACG CAG GAC CAA GCA GCT GAG GAG 720 Met for Ala 795 for for Glu Ala gin 800 Thr gin Asp gin Ala 805 Ala Glu Glu CCC CCG GCT CCT GCT GAG GCT GAG GCC CCC GCT CCT GCT GAG GCT GAG 768 for for 810 Ala for Ala Glu Ala 815 Glu Ala for Ala for 820 Ala Glu Ala Glu GCT GAG GCT GAA CCG CCC CGA AAA CCC CCT AGG GGT GAC CTG GCC GCC 816 Ala 825 Glu Ala Glu for for 830 Arg Lys for for Arg 835 Gly Asp Leu Ala Ala 840 CTA TAC AAT AGG GTC CAC AGC GAC ACC CGC GCA GAG GAC ACA CCA ACC 864 Leu Tyr same time Arg wall 845 His Ser Asp Thr Arg 850 Ala Glu Asp Thr for 855 Thr AGC CCC GAG TTG GTC ACA ACC TTG CCA GAC CCC TTT GTC CTC CCC CTA 912 Ser for Glu Leu 860 wall Thr Thr Leu for 865 Asp for Phe wall Leu 870 for Leu CCC GAC GGA GTC CCA ACC GGT GCG AGC ATT GTG TTG GAA GGT ACC CTC 960 for Asp Gly 875 wall for Thr Gly Ala 880 Ser Ile wall Leu Glu 885 Gly Thr Leu ACA CCC TCC GCT GTG TTT TTT ACC CTG GAT CTG GTG ACC GGG CCC GCC 1008 Thr for 890 Ser Ala wall Phe Phe 895 Thr Leu Asp Leu wall 900 Thr Gly for Ala AGT CTG GCG CTG CAC TTT AAC GTG CGC CTC CCA CTG GAA GGC GAA AAG 1056 Ser 905 Leu Ala Leu His Phe 910 same time wall Arg Leu for 915 Leu Glu Gly Glu Lys 920 CAC ATT GTG TGC AAC TCC AGA GAG GGT AGC AGC AAC TGG GGC GAA GAA 1104 His Ile wall Cys same time 925 Ser Arg Glu Gly Ser 930 Ser same time Trp Gly Glu 935 Glu GTA AGA CCG CAG GAG TTC CCC TTT GAA AGG GAA AAG CCA TTC GTC CTG 1152 wall Arg for gin 940 Glu Phe for Phe Glu 945 Arg Glu Lys for Phe 950 wall Leu GTC ATT GTC ATC CAA AGT GAC ACA TAC CAG ATC ACT GTG : AAC : GGG AAG 1200 wall Ile wall 955 Ile gin Ser Asp Thr 960 Tyr gin Ile Thr wall 965 same time ., Gly Lys CCT CTG GTG : GAT TTT CCA . CAG AGA ! CTA . CAG ; GGC : att 'ACC : CGT GCC TCC 1248 for Leu 970 wall Asp , Phe ; for , Gin 975 i Arg 1 Leu i Gin i Gly Thr Arg Ala Ser 980

CTA TC GGA GAC CTT GTG TTT ACC CGG Phe Thr Arg TTG ACA ATG TCA CCA CCC GGA 1296 Leu 985 Ser Gly Asp Leu Val Leu Thr 995 Met Tyr for for Gly 1000 GAC CCC CGT CCC ACA ACC TTG TTA CCA CCC CCC GCA GCT CCC CTG GAC 1344 Asp for Arg for 1005 Leu Leu Pro For Pro 1010 Ala Ala for Leu Asp 1015 GTA ATC CCA GAT GCC TAT GTG CTC AAT CTG CCC ACC GGA CTG ACG CCT 1392 wall Ile for Asp Ala Tyr 1020 Val Leu Asn Leu for Thr Gly Leu Thr 1030 for AGA ACA CTC CTC ACC GTC ACG GGA ACC CCC ACG CCC CTC GCC GAA TTT 1440 Arg Thr Leu Leu Thr Val Thr Gly Thr 1040 for Thr for Leu Ala Glu Phe TTT ATT GTG AAT CTG GTC TAT GAT TTA CAC TAT GAT TCC AAA AAT GTG 1488 Phe Val 1050 same time Leu Val Tyr Asp Leu 1055 His Tyr Asp Ser 1060 Lys same time wall GCC CTC CAC TTT AAT GTC GGC TTC ACC TCT GAC AGC AAA GGC CAC ATC 1536 Ala Leu His Phe Asn Val Gly Phe Thr 1070 Ser Asp Ser 1075 Lys Gly His Ile 1080 GCC TGC AAT GCC AGA ATG AAT GGC ACA TGG GGA AGT GAA ATC ACA GTG 1584 Ala Cys same time Ala Arg Met 1085 Asn Gly Thr Trp Gly 1090 Ser Glu Ile Thr Val TCT GAT TTC CCC TTT CAA AGG GGA AAA CCC TTC ACT CTG CAG ATT CTC 1632 Ser Asp Phe For Phe Gin 1100 Arg Gly Lys Pro 1104 Phe Thr Leu Gin ile 1110 Leu ACC AGA GAG GCA GAC TTC CAA GTC CTC GTA GAT AAA CAA CCT TTA ACC 1680 Thr Arg Glu Ala 1115 Asp Phe Gin Val Leu wall Asp Lys Gin Pro 1125 Leu Thr CAG TTT CAA TAC AGG CTG AAG GAA CTG GAC CAA ATC AAA TAT GTA CAC 1728 gin Phe Gin 1130 Tyr Arg Leu Lys Glu Leu 1135 · Asp gin Lys 1140 Tyr wall His ATG TTT GGC CAT GTT GTG CAA ACC CAC CTG GAA CAC CAA GTG CCA GAT 1776 Met Phe 1145 Gly His Val Gin Thr His 1150 Leu Glu His 1155 gin wall for Asp 1160 ACT CCA GTT TTT TCT ACT GCG GGA GTT TCG AAA GTT TAC CCT CAG ATA 1824 Thr for wall Phe Ser Thr Ala Gly Val Ser Lys wall Tyr for gin Ile

1165 1170 1175

CTG TAG 1830

Leu (2) Information about SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) 2253 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (ix) CHARACTERS (A) Name / Key: CDS (B) Position: 1. 2250

(xi) Sequence description: SEQ ID NO: 5.

ATG AAG CGC GCA AGA CCG TCT GAA GAT ACC TTC AAC CCC GTG TAT CCA Met Lys Arg Ala Arg 615 for Ser Glu Asp Thr 620 Phe same time for wall Tyr 625 for TAT GAC ACG GAA ACC GGT CCT CCA ACT GTG CCT TTT CTT ACT CCT CCC Tyr Asp Thr Glu 630 Thr Gly for for Thr 635 wall for Phe Leu Thr 640 for for TTT GTA TCC CCC AAT GGG TTT CAA GAG AGT CCC CCT GGG GTA CTC TCT Phe wall Ser 645 for same time Gly Phe gin 650 Glu Ser for for Gly 655 wall Leu Ser TTG CGC CTA TCC GAA CCT CTA GTT ACC TCC AAT GGC ATG CTT GCG CTC Leu Arg 660 Leu Ser Glu for Leu 665 wall Thr Ser same time Gly 670 Met Leu Ala Leu AAA ATG GGC AAC GGC CTC TCT CTG GAC GAG GCC GGC AAC CTT ACC TCC Lys 675 Met Gly same time Gly Leu 680 Ser Leu Asp Glu Ala 685 Gly same time Leu Thr Ser 690 CAA AAT GTA ACC ACT GTG AGC CCA CCT CTC AAA AAA ACC AAG TCA AAC gin same time wall Thr Thr 695 wall Ser for for Leu 700 Lys Lys Thr Lys Ser 705 same time ATA AAC CTG GAA ATA TCT GCA CCC CTC ACA GTT ACC TCA GAA GCC CTA Ile same time Leu Glu 710 Ile Ser Ala for Leu 715 Thr wall Thr Ser Glu 720 Ala Leu ACT GTG GCT GCC GCC GCA CCT CTA ATG GTC GCG GGC AAC ACA CTC ACC Thr wall Ala 725 Ala Ala Ala for Leu 730 Met wall Ala Gly same time 735 Thr Leu Thr ATG CAA TCA CAG GCC CCG CTA ACC GTG CAC GAC TCC AAA CTT AGC ATT Met gin 740 Ser gin Ala for Leu 745 Thr wall His Asp Ser 750 Lys Leu Ser Ile GCC ACC CAA GGA CCC CTC ACA GTG TCA GAA ' GGA AAG CTA GCC CTG ACA Ala 755 Thr gin Gly for Leu 760 Thr wall Ser Glu Gly 765 Lys Leu Ala Leu Thr 770 AAA ACC TTA GTC TAT CCC ACC CTG TGG ACG GGG CCT GCT CCC GAG GCC Lys Thr Leu wall Tyr 775 for Thr Leu Trp Thr 780 Gly for Ala for Glu 785 Ala AAC GTC ACC TTC TCG GGG GAG AAT TCC CCA TCT GGC ATT CTC AGA CTG same time wall Thr Phe 790 Ser Gly Glu same time Ser 795 for Ser Gly Ile Leu 800 Arg Leu TGT CTC AGC AGA ACC GGG GGC ACG GTC ATT GGC ACC CTG TCT GTA CAA Cys Leu Ser 805 Arg Thr Gly Gly Thr 810 wall Ile Gly Thr Leu 815 Ser wall gin GGT AGC CTC ACG AAC CCC AGT ACC GGT CAG ACC CTG GGC ATG AAC CTT Gly Ser 820 Leu Thr same time for Ser 825 Thr Gly gin Thr Leu 830 Gly Met same time Leu TAC TTT GAC GCA GAC GGC AAT GTG CTG TCT GAG AGC AAC CTC GTC CGA Tyr 835 Phe Asp Ala Asp Gly 840 same time wall Leu Ser Glu 845 Ser same time Leu wall Arg 850 GGG TCC TGG GGA ATG AAA GAC CAA GAT ACC CTG GTG ACT CCC ATT GCC Gly Ser Trp Gly Met Lys Asp gin Asp Thr Leu wall Thr for Ile Ala

855 860 865

144

192

240

288

336

384

432

480

528

576

624

672

720

768

AAT GGG Cag Tac Gin Tyr 870 CTG ATG CCC AAC CTC ACT 816 same time Gly Leu Met for same time Leu Thr Ala ' Tyr for Arg 880 Leu Ile CAG ACC CTA ACT TCC AGC TAC ATT TAC ACA CAA GCG CAC CTT GAC CAC 864 gin Thr Leu Thr Ser Ser Tyr Ile 890 Tyr Thr gin Ala His 895 Leu Asp His AAT AAC AGT GTG GTG GAC ATC AAG ATA GGG CTC AAC ACA GAC CTG AGG 912 same time same time 900 Ser Val Val Asp Ile 905 Lys Gly Leu same time 910 Thr Asp Leu Arg CCC ACT GCG GCC TAC GGC CTA AGC TTT ACC ATG ACC TTC ACT AAC TCT 960 for 915 Thr Ala Ala Tyr Gly 920 Leu Ser Phe Thr Met 925 Thr Phe Thr same time Ser 930 CCC CCC ACC TCA TTT GGT ACC GAC CTG GTG CAA TTT GGC TAC CTG GGT 1008 for for Thr Ser Phe Gly Thr Asp Leu Val 940 gin Phe Gly Tyr Leu 945 Gly CAG GAT AGC TCC CCC TCC TTC CTG AGA GAA CTT CCC CTT GCA TCC GAG 1056 gin Asp Ser Ser 950 Pro Ser Phe Leu Arg Glu 955 Leu for Leu Ala 960 Ser Glu GCG GGC TAC TTT GGC AAA CTG GCA GCT GCC TCT GAG GAA ATG CCA GCC 1104 Ala Gly Tyr Phe Gly Lys Leu Ala 970 Ala Ala Ser Glu Glu 975 Met for Ala CCT CCT GAG GCC CAG ACG CAG GAC CAA GCA GCT GAG GAG CCC CCG GCT 1152 for for 980 Glu Ala Gin Thr gin 985 Asp Gin Ala Ala Glu 990 Glu for for Ala CCT GCT GAG GCT GAG GCC CCC GCT CCT GCT GAG GCT GAG GCT GAG GCT 1200 for 995 Ala Glu Ala Glu Ala Pro 1000 Ala Pro Äla Glu Ala Glu Ala Glu Ala 1010 GAA CCG CCC CGA AAA CCC CCT AGG GGT GAC CTG GCC GCC CTA TAC AAT 1248 Glu for For Arg Lys Pro 1015 for Arg Gly Asp Leu 1020 Ala Ala Leu Tyr Asn AGG GTC CAC AGC GAC ACC CGC GCA GAG GAC ACA CCA ACC AGC CCC GAG 1296 Arg wall His Ser Asp Thr 1030 Arg Ala Asp 1035 Thr for Thr Ser Pro 1040 Glu TTG GTC ACA ACC TTG CCA GAC CCC TTT GTC CTC CCC CTA CCC GAC GGA 1344 Leu wall Thr Thr 1045 Leu Pro Asp For Phe Val 1050 Leu for Leu Pro 1055 Asp Gly GTC CCA ACC GGT GCG AGC ATT GTG TTG GAA GGT ACC CTC ACA CCC TCC 1392 wall For Thr Gly 1060 Ala Ser Val 1065 Leu Glu Gly Thr Leu 1070 Thr for Ser GCT GTG TTT TTT ACC CTG GAT CTG GTG ACC GGG CCC GCC AGT CTG GCG 1440 Ala Val Phe Phe Thr Leu Asp 1080 Leu Val Thr Gly Pro 1085 Ala Ser Leu Ala 1090 CTG CAC TTT AAC GTG CGC CTC CCA CTG GAA GGC GAA AAG CAC ATT GTG 1488 Leu His Phe Asn Val Arg Leu for Leu Glu Gly 1100 Glu Lys His Val 1105 TGC AAC TCC AGA GAG GGT AGC AGC AAC TGG GGC GAA GAA GTA AGA CCG 1536 Cys same time Ser Arg Glu Gly 1111 Ser Ser Asn Trp 1115 Gly Glu Glu Val Arg 1120 for CAG GAG TTC CCC TTT GAA AGG GAA AAG CCA TTC GTC CTG í GTC ATT 'GTC 1584 gin Glu Phe Pro Phe Glu Arg Glu Lys Pro Phe wall Leu wall Ile i Val

1125 1130 1135

ATC Ile CAA AGT Gin Ser 1140 GAC Asp ACA Thr TAC Tyr CAG ATC Gin White 1145 ACT Thr GTG wall AAC same time GGG AAG Gly Lys 1150 CCT for CTG Leu GTG wall 1632 GAT TTT CCA CAG AGA CTA CAG GGC ATT ACC CGT GCC TCC CTA TCC GGA 1680 Asp Phe Pro 1155 gin Arg Leu Gin Gly 1160 Ile Thr Arg Ala Ser 1166 Leu Ser Gly 1170 GAC CTT GTG TTT ACC CGG TTG ACA ATG TAC CCA CCC GGA GAC CCC CGT 1728 Asp Leu Val Phe Thr Arg Leu Thr Met Tyr Pro 1180 For Gly Asp for 1185 Arg CCC ACA ACC TTG TTA CCA CCC CCC GCA GCT CCC CTG GAC GTA ATC CCA 1776 for Thr Thr Leu Leu for Pro Pro Ala Ala for Leu Asp Val ile 1200 for GAT GCC TAT GTG CTC AAT CTG CCC ACC GGA CTG ACG CCT AGA ACA CTC 1824 Asp Ala Tyr Val Leu same time Leu Pro Thr 1210 Gly Leu Thr Pro Arg 1215 Thr Leu CTC ACC GTG ACG GGA ACC CCC ACG CCC CTC GCC GAA TTT TTT ATT GTG 1872 Leu Thr Val 1220 Thr Gly Thr For Thr 1225 for Leu Ala Glu Phe 1230 Phe Ile wall AAT CTG GTC TAC GAT TTA CAC TAT GAT TCC AAA AAT GTG GCC CTC CAC 1920 Asn Leu Val 123S Tyr Asp Leu His Tyr Asp Ser Lys Asn Val 1245 Ala Leu His 1250 TTT AAT GTC GGC TTC ACC TCT GAC AGC AAA GGC CAC ATC GCC TGC AAT 1968 Phe Asn Val Gly Phe Thr 1255 Ser Asp Ser Lys Gly 1260 His ile Ala Cys Asn 1265 GCC AGA ATG AAT GGC ACA TGG GGA AGT GAA ATC ACA GTG TCT GAT TTC 2016 Ala Arg Met Asn Gly 1270 Thr Trp Gly Ser Glu 1275 Ile Thr Val Ser Asp 1280 Phe CCC TTT CAA AGG GGA AAA CCC TTC ACT CTG CAG ATT CTC ACC AGA GAG 2064 for Phe Gin Arg 1285 Gly Lys For Phe Thr 1290 Leu gin Leu Thr 1295 Arg Glu GCA GAC TTC CAA GTC CTC GTA GAT AAA CAA CCT TŤA ACC CAG TTT CAA 2112 Ala Asp Phe 1300 gin wall Leu Val Asp 1305 Lys gin for Leu Thr 1310 gin Phe gin TAC AGG CTG AAG GAA CTG GAC CAA ATC AAA TAT GTA CAC ATG TTT GGC 2160 Tyr Arg Leu 1316 Lys Glu Leu Asp Gin 1320 Ile Lys Tyr Val His Met Phe Gly 1330 CAT GTT GTG CAA ACC CAC CTG GAA CAC CAA GTG CCA GAT ACT CCA GTT 2208 His Val Val gin Thr His 1335 Leu Glu His Gin Val 1340 For Asp Thr For Val 1345 TTT Phe TCT ACT Ser Thr GCG GGA GTT Ala Gly Val 1350 TCG AAA Ser Lys GTT TAC Val Tyr 1355 CCT for CAG ΆΤΑ Gin White CTG TAG Leu 1360 2253

(2) Information about SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 795 base pairs (B) TYPE: nucleic acid (C) STRING TYPE, unknown (θ) TOPOLOGY: unknown (ii) MOLECULAR TYPE ^- DNA (genomic) (ix) FEATURES:

(A) CDS name / key (B) Position. 1 ..792 (xi) Sequence description: SEQ ID NO: 6.

ATG GCG CTC by Met Ala Leu CTG CTG Leu Leu 755 TGC Cys TTC GTG CTC CTG TGC GGA Phe wall Leu Leu Cys Val Val Asp Phe 760 765 GCC AGA AGT TTG AGT ATC ACT ACT CCT GAA GAG ATG ATT GAA AAA GCC Ala Arg Ser Leu Ser Ile Thr Thr for Glu Glu Met Ile Glu Lys Ala 770 775 780 AAA GGG GAA ACT GCC TAT CTG CCG TGC AAA TTT ACG CTT AGT CCC GAA Lys Gly Glu Thr Ala Tyr Leu for Cys Lys Phe Thr Leu Ser for Glu 785 790 795 GAC CAG GGA CCG CTG GAC ATC GAG TGG CTG ATA TCA CCA GCT ' GAT AAT Asp gin Gly for Leu Asp Ile Glu Trp Leu Ile Ser for Ala Asp same time 800 805 810 815 CAG AAG GTG GAT CAA GTG ATT ATT TTA TAT TCT GGA GAC AAA ATT TAT gin Lys wall Asp gin wall Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr 820 625 830 GAT GAC TAC TAT CCA GAT CTG AAA GGC CGA GTA CAT TTT ACG AGT AAT Asp Asp Tyr Tyr for AS'POL Leu Lys Gly Arg wall His Phe Thr Ser same time 835 840 845 GAT CTC AAA TCT GGT GAT GCA TCA ATA AAT GTA ACG AAT TTA CAA CTG Asp Leu Lys Ser Gly Asp Ala Ser Ile · temporarily wall Thr same time Leu gin Leu 850 855 860 TCA GAT ATT GGC ACA TAT CAG TGC AAA GTG AAA AAA GCT CCT GGT GTT Ser Asp Ile Gly Thr Tyr gin Cys Lys wall Lys Lys Ala for Gly wall 865 870 875 GCA AAT AAG AAG ATT CAT CTG GTA GTT CTT GTT AAG CCT TCA GGT GCG Ala same time Lys Lys Ile His Leu wall wall Leu wall Lys for Ser Gly Ala 880 885 890 895 AGA TGT TAC GTT GAT GGA TCT GAA GAA ATT GGA AGT GAC TTT AAG ATA Arg Cys Tyr wall Asp Gly Ser Glu Glu Ile Gly Ser Asp Phe Lys Ile 900 905 910 AAA TGT GAA CCA AAA GAA GGT TCA CTT CCA TTA CAG TAT GAG TGG CAA Lys Cys Glu for Lys Glu Gly Ser Leu for Leu gin Tyr Glu Trp gin 915 920 925 AAA TTG TCT GAC TCA CAG AAA ATG CCC ACT TCA TGG TTA GCA GAA ATG Lys Leu Ser Asp Ser gin Lys Met for Thr Ser Trp Leu Ala Glu Met 930 935 940 ACT TCA TCT GTT ATA TCT GTA AAA AAT GCC TCT TCT GAG TAC TCT GGG Thr Ser Ser wall Ile Ser wall Lys same time Ala Ser Ser Glu Tyr Ser Gly 945 950 955 ACA TAC AGC TGT ACA GTC AGA AAC AGA GTG GGC TCT GAT CAG TGC CTG Thr Tyr Ser Cys Thr wall Arg same time Arg wall Gly Ser Asp gin Cys Leu 960 965 970 975 TTG CGT CTA AAC GTT GTC CCT CCT TCA AAT AAA GCT GGA , TCT GGA . TCC Leu Arg Leu same time wall wall for for Ser same time Lys Ala Gly Ser Gly Ser

144

192

240

288

336

384

432

480

528

576

624

672

720

980 985 990

GGC TCA GGG Gly Ser Gly TCT ACT AGT GGG GCC CAG CCG GCC 768 Ser 995 Thr Ser Gly Ala Gin Pro 1000 Ala Leu Gin Ala Ala Ala GAC TAT AAA GAT GAC GAC GAT AAG TGA 795 Asp Tyr Lys Asp Asp Asp Asp Lys 1010 1015

(2) SEQ ID NO: 7 Information:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 834 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (ix) FEATURES:

(A) Name / Key: CDS (B) Position: 1 ..831 (xi) Sequence Description: SEQ ID NO: 7:

ATG GCG CTC CTG CTG TGC TTC GTG CTC 48 Met Ala Leu Leu Leu 270 Cys Phe wall Leu Leu Cys Val Val Asp Phe 275 280 GCC AGA AGT TTG AGT ATC ACT ACT CCT GAA GAG ATG ATT GAA AAA GCC 96 Ala Arg Ser Leu Ser Ile Thr Thr for Glu Glu Met Ile Glu Lys Ala 285 290 295 AAA GGG GAA ACT GCC TAT CTG CCG TGC AAA TTT ACG CTT AGT CCC GAA 144 Lys Gly Glu Thr Ala Tyr Leu for Cys Lys Phe Thr Leu Ser for Glu 300 305 310 GAC CAG GGA CCG CTG GAC ATC GAG TGG CTG ATA TCA CCA GCT GAT AAT 192 Asp gin Gly for Leu Asp Ile Glu Trp Leu Ile Ser for Ala Asp same time 315 320 325 CAG AAG GTG GAT CAA GTG ATT ATT TTA TAT TCT GGA GAC AAA ATT TAT • 240 gin Lys wall Asp gin wall Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr 330 335 340 345 GAT GAC TAC TAT CCA GAT CTG AAA GGC CGA GTA CAT TTT ACG AGT AAT 288 Asp Asp Tyr Tyr for Asp Leu Lys Gly Arg wall His Phe Thr Ser same time 350 355 360 GAT CTC AAA TCT GGT GAT GCA TCA ATA AAT GTA ACG AAT TTA CAA CTG 336 Asp Leu Lys Ser Gly Asp Ala Ser Ile same time wall Thr same time Leu gin Leu 365 370 375 TCA GAT ATT GGC ACA TAT CAG TGC AAA GTG AAA AAA GCT CCT GGT GTT 384 Ser Asp Ile Gly Thr Tyr gin cys Lys wall Lys Lys Ala for Gly wall 380 385 390 GCA AAT AAG AAG ATT CAT CTG GTA GTT CTT GTT AAG CCT TCA GGT GCG 432 Ala same time Lys Lys Ile His Leu wall wall Leu wall Lys for Ser Gly Ala 395 400 405 AGA TGT TAC GTT GAT GGA TCT GAA GAA ATT GGA AGT GAC TTT AAG ATA 480 Arg Cys Tyr wall Asp Gly Ser Glu Glu Ile Gly Ser Asp Phe Lys Ile 410 415 420 425

AAA TGT GAA CCA AAA GAA GGT TCA CTT CCA TTA CAG TAT GAG TGG CAA Lys Cys Glu for Lys Glu Gly Ser Leu for Leu gin Tyr Glu Trp gin 430 435 440 AAA TTG TCT GAC TCA CAG AAA ATG CCC ACT TCA TGG TTA GCA GAA ATG Lys Leu Ser Asp Ser gin Lys Met for Thr Ser Trp Leu Ala Glu Met 445 450 455 ACT TCA TCT GTT ATA TCT GTA AAA AAT GCC TCT TCT GAG TAC TCT GGG Thr Ser Ser wall Ile Ser wall Lys same time Ala Ser Ser Glu Tyr Ser Gly 4 60 465 470 ACA TAC AGC TGT ACA GTC AGA AAC AGA GTG GGC TCT GAT CAG TGC CTG Thr Tyr Ser Cys Thr wall Arg same time Arg wall Gly Ser Asp gin Cys Leu 475 480 485 TTG CGT CTA AAC GTT GTC CCT CCT TCA AAT AAA GCT GGA TCT GGA TCC Leu Arg Leu same time wall wall for for Ser same time Lys Ala Gly Ser Gly Ser 490 495 500 505 GGC TCA GGG TCT ACT AGA GCC TGC GAC TGT CGC GGC GAT TGT TTT TGC Gly Ser Gly Ser Thr Arg Ala Cys Asp Cys Arg Gly Asp Cys Phe Cys 510 515 520 GGT ACT AGT GGG GCC CAG CCG GCC CTG CAG GCG GCC GCA GAC TAT AAA Gly Thr Ser Gly Ala gin for Ala Leu gin Ala Ala Ala Asp Tyr Lys 525 530 535 GAT GAC GAC GAT AAG TGA Asp Asp Asp Asp Lys

52B

576

624

672

720

768

816

834

540 (2) Information about SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1194 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: unknown (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (ix) FEATURES:

(A) Name / Key: CDS (B) Position: 1 ..1191 (xi) Sequence Description: SEQ ID NO: 8:

ATG GCG CTC CTG CTG TGC TTC GTG CTC CTG TGC GGA GTA GTG GAT TTC 48 Met Ala Leu Leu Leu Cys Phe wall Leu Leu Cys Gly val · wall Asp Phe 280 285 290 GCC AGA AGT TTG AGT ATC ACT ACT CCT GAA GAG ATG ATT GAA AAA GCC 96 Ala Arg Ser Leu Ser Ile Thr Thr for Glu Glu Met Ile Glu Lys Ala 295 300 305 310 AAA GGG GAA ACT GCC TAT CTG CCG TGC AAA TTT ACG CTT AGT CCC GAA 144 Lys Gly Glu Thr Ala Tyr Leu for Cys Lys Phe Thr Leu Ser for Glu 315 320 325 GAC CAG GGA CCG CTG GAC ATC GAG TGG CTG ATA TCA CCA GCT GAT AAT 192 Asp gin Gly for Leu Asp Ile Glu Trp Leu Ile Ser for Ala Asp same time 330 335 340 CAG AAG GTG GAT CAA GTG ATT ATT TTA TAT TCT GGA GAC AAA ATT TAT 240 gin Lys wall Asp gin wall Ile Ile Leu Tyr Ser Gly Asp Lys Ile Tyr

345 350 355 GAT GAC TAC TAT CCA GAT CTG AAA GGC CGA GTA CAT TTT ACG AGT AAT 288 Asp Asp Tyr Tyr for Asp Leu Lys Gly Arg wall His Phe Thr Ser same time 360 365 370 GAT CTC AAA TCT GGT GAT GCA TCA ATA AAT GTA ACG AAT TTA CAA CTG 336 Asp Leu Lys Ser Gly Asp Ala Ser Ile same time wall Thr same time Leu gin Leu 375 380 385 390 TCA GAT ATT GGC ACA TAT CAG TGC AAA GTG AAA AAA GCT CCT GGT GTT 384 Ser Asp Ile Gly Thr Tyr gin Cys Lys wall Lys Lys Ala for Gly wall 395 400 405 GCA AAT AAG AAG ATT CAT CTG GTA GTT CTT GTT AAG CCT TCA GGT GCG 432 Ala same time Lys Lys Ile His Leu wall wall Leu wall Lys for Ser Gly Ala 410 415 420 AGA TGT TAC GTT GAT GGA TCT GAA GAA ATT GGA AGT GAC TTT AAG ATA 480 Arg Cys Tyr wall Asp Gly Ser Glu Glu Ile Gly Ser Asp Phe Lys Ile 425 430 435 AAA TGT GAA CCA AAA GAA GGT TCA CTT CCA TTA CAG TAT GAG TGG CAA 528 Lys Cys Glu for Lys Glu Gly Ser Leu for Leu gin Tyr Glu Trp gin 440 445 450 AAA TTG TCT GAC TCA CAG AAA ATG CCC ACT TCA TGG TTA GCA GAA ATG 576 Lys Leu Ser Asp Ser gin Lys Met for Thr Ser Trp Leu Ala Glu Met 455 460 465 470 ACT TCA TCT GTT ATA TCT GTA AAA AAT GCC TCT TCT GAG TAC TCT GGG 624 Thr Ser Ser wall Ile Ser wall Lys same time Ala Ser Ser Glu Tyr Ser Gly 475 480 485 ACA TAC AGC TGT ACA GTC AGA AAC AGA GTG GGC TCT GAT CAG TGC CTG 672 Thr Tyr Ser Cys Thr wall Arg same time Arg wall Gly Ser Asp gin Cys Leu 490 495 500 TTG CGT CTA AAC GTT GTC CCT CCT TCA AAT AAA GCT GGA TCT GGA TCC 720 Leu Arg Leu same time wall wall for for Ser same time Lys Ala Gly Ser Gly Ser 505 510 515 GGC TCA GGG TCT ACT AGA GGA GGT GGT GCA TCA AGG GTC TCG GCG CTC 768 Gly Ser Gly Ser Thr Arg Gly Gly Gly Ala Ser Arg wall Ser Ala Leu 520 525 530 GAG AAG ACG TCT CAA ATA CAC TCT GAT ACT ATC CTC CGG ATC ACC CAG 816 Glu Lys Thr Ser gin Ile His Ser Asp Thr Ile Leu Arg Ile Thr gin 535 540 545 550 GGA CTC GAT GAT GCA AAC AAA CGA ATC ATC GCT CTT GAG CAA AGT CGG 864 Gly Leu Asp Asp Ala same time Lys Arg Ile Ile Ala Leu Glu gin Ser Arg 555 560 565 GAT GAC TTG GTT GCA TCA GTC AGT GAT GCT CAA CTT GCA ATC TCC AGA 912 Asp Asp Leu wall Ala Ser wall Ser Asp Ala gin Leu Ala Ile Ser Arg 570 575 580 TTG GAA AGC TCT ATC GGA GCC CTC CAA ACA GTT GTC AAT GGA CTT GAT 960 Leu Glu Ser Ser Ile Gly Ala Leu gin Thr wall wall same time Gly Leu Asp 585 590 595 TCG AGT GTT ACC CAG TTG GGT GCT CGA GTG GGA CAA CTT GAG ACA . GGA 1008 Ser Ser wall Thr gin Leu Gly Ala Arg wall Gly gin Leu Glu Thr Gly

600 605 610

CTT GCA Leu Ala 615 GAC GTA CGC GTT GAT CAC GAC AAT CTC GTT GCG AGA Val Ala Arg 630 1056 Asp Val Arg Val Asp His Asp Asn Leu 620 625 ACT GCA GAA CGT AAC ATT GGA TCA TTG ACC ACT GAG CTA TCA ACT CTG 1104 Thr Ala Glu Arg same time Gly Ser Leu Thr Thr Glu Leu 635 640 645 ACG TTA CGA GTA ACA TCC ATA CAA GCG GAT TTC GAA TCT AGG 1152 Thr Leu Arg Val Thr Ser ile Gin Ala Asp Phe Glu Ser Arg Thr Ser 650 655 660 ATG CAG GCG GCC GCA GAC TAT AAA GAT GAC GAT AAG TGA 1194 Met Gin Ala Ala Ala Asp Tyr Lys Asp Asp Asp Asp Lys 665 670 675 Information about SEQ ID NO: 9: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1743 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: unknown (D) TOPOLOGY: unknown

(ii) MOLECULAR TYPE: DNA (genomic) (ix) FEATURES:

(A) Name / Key: CDS (B) Position: 1 ..1743 (xi) Sequence Description: SEQ ID NO: 9:

ATG Met 1 AAG CGC LCA Arg Ala AGA Arg 5 CCG TCT GAA Pro Glu GAT ACC TTC AAC 48 Asp Thr 10 Phe Asn for wall Tyr 15 for TAT GAC ACG GAA ACC GGT CCT CCA ACT GTG CCT TTT CTT ACT CCT CCC 96 Tyr Asp Thr Glu Thr Gly for for Thr wall for Phe Leu Thr for for 20 25 30 TTT GTA TCC CCC AAT GGG TTT CAA GAG AGT CCC CCT GGG GTA CTC TCT 144 Phe wall Ser for same time Gly Phe gin Glu Ser for for Gly wall Leu Ser 35 40 45 TTG CGC CTA TCC GAA CCT CTA GTT ACC TCC AAT GGC ATG CTT GCG CTC 192 Leu Arg Leu Ser Glu for Leu wall Thr Ser same time Gly Met Leu Ala Leu 50 55 60 AAA ATG GGC AAC GGC CTC TCT CTG GAC GAG GCC GGC AAC CTT ACC TCC 240 Lys Met Gly same time Gly Leu Ser Leu Asp Glu Ala Gly same time Leu Thr Ser 65 70 75 80 CAA AAT GTA ACC ACT GTG AGC CCA CCT CTC AAA AAA ACC AAG TCA AAC 288 gin same time wall Thr Thr wall Ser for for Leu Lys Lys Thr Lys Ser same time 85 90 95 ATA AAC CTG GAA ATA TCT GCA CCC CTC ACA GTT ACC TCA GAA GCC CTA 336 Ile same time Leu Glu Ile Ser Ala for Leu Thr wall Thr Ser Glu Ala Leu 100 105 110 ACT GTG GCT GCC GCC GCA CCT CTA ATG GTC GCG GGC AAC ACA CTC ACC 384 Thr wall Ala Ala Ala Ala for Leu Met wall Ala Gly same time Thr Leu Thr 115 120 125 ATG CAA TCA CAG GCC CCG CTA ACC GTG CAC GAC TCC AAA CTT AGC ATT 4 32 Met gin Ser gin Ala for Leu Thr wall His Asp Ser Lys Leu Ser Ile

129 135 140

GCC ACC CAA GGA CCC CTC ACA GTG TCA GAA GGA AAG CTA GCC CTG CAA 480 Ala Thr gin Gly for Leu Thr Val Ser Glu Gly Lys Leu Ala Leu gin 145 150 155 160 ACA TCA GGC CCC CTC ACC ACC ACC GAT AGC AGT ACC CTT ACT ATC ACT 528 Thr Ser Gly for Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr 165 170 175 GCC TCA CCC CCT CTA ACT ACT GCC ACT GGT AGC TTG GGC ATT GAC TTG 576 Ala Ser for for Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu 180 185 190 AAA GAG CCC ATT TAT ACA CAA AAT GGA AAA CTA GGA CTA AAG TAC GGG 624 Lys Glu for Ile Tyr Thr Gin Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195 200 205 GCT CCT TTG CAT GTA ACA GAC GAC CTA AAC ACT TTG ACC GTA GCA ACT 672 Ala for Leu His wall Thr Asp Asp Leu same time Thr Leu Thr wall Ala Thr 210 215 220 GGT CCA GGT GTG ACT ATT AAT AAT ACT TCC TTG CAA ACT AAA GTT ACT 720 Gly for Gly wall Thr Ile Asn Asn Thr Ser Leu gin Thr Lys wall Thr 225 230 235 240 GGA GCC TTG GGT TTT GAT TCA CAA GGC AAT ATG CAA CTT AAT GTA GCA 768 Gly Ala Leu Gly Phe Asp Ser Gin Gly same time Met gin Leu same time wall Ala 245 250 255 GGA GGA CTA AGG ATT GAT TCT CAA AAC AGA CGC CTT ATA CTT GAT GTT 816 Gly Gly Leu Arg Ile Asp Ser Gin same time Arg Arg Leu Ile Leu Asp wall 260 265 270 AGT TAT CCG TTT GAT GCT CAA AAC CAA CTA AAT CTA AGA CTA GGA CAG 864 Ser Tyr for Phe Asp Ala Gin Asn gin Leu same time Leu Arg Leu Gly gin 275 280 285 GGC CCT CTT TTT ATA AAC TCA GCC CAC AAC TTG GAT ATT AAC TAC AAC 912 Gly for Leu Phe Ile same time Ser Ala His same time Leu Asp Ile same time Tyr same time 290 295 300 AAA GGC CTT TAC TTG TTT ACA GCT TCA AAC AAT TCC AAA AAG CTT GAG 960 Lys Gly Leu Tyr Leu Phe Thr Ala Ser same time same time Ser Lys Lys Leu Glu 305 310 315 320 GTT AAC CTA AGC ACT GCC AAG GGG TTG ATG TTT GAC GCT ACA GCC ATA 1008 wall same time Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile 325 330 335 GCC ATT AAT GCA GGA GAT GGG CTT GAA TTT GGT TCA CCT AAT GCA CCA 1056 Ala Ile same time Ala Gly Asp Gly Leu Glu Phe Gly Ser for same time Ala for 340 345 350 AAC ACA AAT CCC CTC AAA ACA AAA ATT GGC CAT GGC CTA GAA TTT GAT 1104 same time Thr same time for Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp 355 360 365 TCA AAC AAG GCT ATG GTT CCT AAA CTA GGA ACT GGC CTT AGT TTT GAC 1152 Ser same time Lys Ala Met wall Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp 370 375 380 AGC ACA GGT GCC ATT ACA GTA GGA AAC AAA AAT AAT GAT AAG CTA . ACT 1200 Ser Thr Gly Ala Ile Thr Val Gly same time Lys same time same time Asp Lys Leu Thr 385 390 395 400

TTG Leu TGG ACC ACA Trp Thr Thr CCA GCT CCA TCT CCT AAC GTA GAT 1248 for 405 Ala for Ser for Asn Cys 410 Arg Leu Asn Ala 416 Glu AAA GAT GCT AAA CTC ACT TTG GTC TTA ACA AAA TGT GGC AGT CAA ATA 1296 Lys Asp Ala Lys Leu Thr Leu wall Leu Thr Lys Cys Gly Ser Gin Ile 420 425 430 CTT GCT ACA GTT TCA GTT TTG GCT GTT AAA GGC AGT TTG GCT CCA ATA 1344 Leu Ala Thr Val Ser wall Leu Ala wall Lys Gly Ser Leu Ala Pro Ile 435 440 445 TCT GGA ACA GTT CAA AGT GCT CAT CTT ATT ATA AGA TTT GAC GAA AAT 1392 Ser Gly Thr Val gin Ser Ala His Leu ile ile Arg Phe Asp Glu same time 450 455 460 GGA GTG CTA AAC AAT TCC TTC CTG GAC CCA GAA TAT TGG AAC TTT 1440 Gly Val Leu same time same time Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe 465 470 475 480 AGA AAT GGA GAT CTT ACT GAA GGC ACA GCC TAT ACA AAC GCT GTT GGA 1488 Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly 485 490 495 TTT ATG CCT AAC CTA TCA GCT TAT CCA AAA TCT CAC GGT AAA ACT GCC 1536 Phe Met Pro Asn Leu Ser Ala Tyr for Lys Ser His Gly Lys Thr Ala 500 505 510 AAA AGT AAC ATT GTC AGT CAA GTT TAC TTA AAC GGA GAC AAA ACT AAA 1584 Lys Ser Asn White wall Ser gin wall Tyr Leu Asn Gly Asp Lys Thr Lys 515 520 525 CCT GTA ACA CTA ACC ATT ACA CTA AAC GGT ACA CAG GAA ACA GGA GAC 1632 for Le Thr Thr Ile Thr Leu same time Gly Thr gin Glu Thr Gly Asp 530 535 540 ACA ACT CCA AGT GCA TAC TCT ATG TCA TTT TCA TGG GAC TGG TCT GGC 1680 Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly 545 550 555 560 CAC AAC TAC ATT AAT GAA ATA TTT GCC ACA TCC TCT TAC ACT TTT TCA 1728 His Asn Tyr White same time Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser 565 570 575 TAC ATT GCC CAA GAA 1743 Tyr Ala Gin Glu

580 (2) Information about SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs (θ) TYPE: nucleic acid (C) STRING TYPE: single stranded (θ) TOPOLOGY: unknown (* ·) MOLECULAR TYPE: DNA (genomic) (xi) Sequence description SEQ ID NO: 10:

TGCATGCATA CTAGTCCTAG ATTCGAAATC CGCTTG (2) Information about SEQ ID NO: 1 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs (B) TYPE: nucleic acid (C) STRING TYPE: single-stranded (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (xi) Sequence description: SEQ ID NO: 11:

GCTCTAGAGG AGGTGGTGCA TCAAGGGTCT CGGCGCTC 38 (2) Information about SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (xi) Sequence description: SEQ ID NO: 12:

CCGGATCCCT ACAGTATCTG AGGGTAAAC (2) Information about SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: single-stranded (D) TOPOLOGY: unknown (ii) MOLECULAR TYPE: DNA (genomic) (xi) Sequence description: SEQ ID NO: 13:

GGGCACCATG GCGAAGATGG AGCTTTGTCC C (2) · SEQ ID NO: 14 '(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 14:

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Thr Ser 1 5 10 (2) Information about SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (ii) Sequence description: SEQ ID NO: 15:

Arg Lys Lys Lys Arg Lys Lys Lys 1 5 (2) Information about SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 16:

Asp Tyr Lys Asp Asp Asp Lys 1 5 (2) Information about SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 17:

For Lys Ala Arg Arg For Ala Gly Arg Thr Trp Ala Gin For 1 5 10 (2) Information about SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) CHAIN TYPE: not relevant (D) TOPOLOGY: not relevant (ii) MOLECULAR TYPE: peptide (xi) Sequence description: SEQ ID NO: 18:

Arg Pro Asp Asp Phe Asp Gin Gly Trp Gly Pro Thr Tyr 1 5

A | / Aif? - 77

Claims (25)

A trimer comprising three monomers, wherein each of said monomers has an N-terminus of an adenoviral fiber protein and each of said monomers has a trimerization region comprising an adenovirus fiber node lacking a natural amino acid binding substrate, and further having an unnatural amino acid that differs from the natural amino acid by charge or molecular weight. The trimer of claim 1, wherein said natural amino acid is replaced by a non-natural amino acid. The trimer of claim 1 or 2, wherein said natural amino acid binding substrate is in a β-sheet, The trimer of claim 1 or 2, wherein said natural amino acid binding substrate is in a loop that joins two β-sheets. A trimer comprising three monomers, each of said monomers having an N-terminus of the adenoviral fiber protein and each of said monomers having a trimeric axonal dynein trimerization region, parainfluenza virus hemaglutanin trimerization region, or a reovirus sigma-1 protein trimerization region. 6. A trimer comprising three monomers, wherein each of said monomers has an N-terminus of the adenoviral fiber protein and each of said monomers has a trimerization region comprising a modified leucine zipper motif. The trimmer of claim 6, wherein said leucine zipper motif is modified by replacing one or more leucine residues with isoleucine. The trimer of claim 6 or 7, wherein said trimerization region is derived from the yeast trimmer GCN4β-II. The trimer according to any one of claims 1 to 8, which is not a ligand for natural binding sites on the surface of a mammalian cell. The trimer of any one of claims 1 to 9, wherein at least one of the three said monomers comprises an unnatural polypeptide that interferes with the binding of said trimer to its natural cell surface binding site. A composition of matter comprising a trimer according to any one of claims 1 to 10 and an adenoviral penton base. The composition of claim 11, wherein said penton base comprises an unnatural ligand. An adenovirus comprising a trimer according to any one of claims 1 to 12. The adenovirus according to claim 13, which does not productively infect 293 cells. Adenovirus according to claim 13 or 14, comprising a non-adenovirus ligand. The adenovirus of claim 15, wherein said ligand binds to a substrate other than the natural mammalian adenovirus receptor. The adenovirus according to claim 15 or 16, wherein said ligand binds to a substrate other than the natural cell surface protein. The adenovirus according to claim 16 or 17, wherein said substrate is present on the cell surface. The adenovirus according to claim 16 or 17, wherein said substrate is present in an affinity column. The adenovirus according to claim 16 or 17, wherein said substrate is present on a blood-producing molecule. 21. A cell line expressing an unnatural cell surface receptor to which an adenovirus having a ligand for said receptor binds, wherein said cell surface receptor is an antibody molecule. The cell line of claim 21, wherein said antibody molecule comprises single chain antibodies. The cell line of claim 21 or 22, wherein said antibody molecule recognizes hemagglutinin. The cell line according to any one of claims 21 to 23, which can promote virus growth at least 10 passages. 25. A method of multiplying adenovirus comprising infecting a cell line according to any one of claims 21 to 24 with adenovirus, maintaining said cell line, and recovering adenoviruses produced by said cell line.