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  • C12N2810/00—Vectors comprising a targeting moiety
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  • C12N2810/00—Vectors comprising a targeting moiety
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  • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
  • C12N2810/855—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from receptors; from cell surface antigens; from cell surface determinants

Definitions

• the present invention relates to an alternately targeted adenovirus and includes methods for producing and purifying such viruses as well as protein modifications mediating alternate targeting.
• Adenoviral infection begins with the attachment of the virion to the target cell.
• the adenovirus attaches to two cellular surface proteins, both of which must be present for the virus to infect the target cell (Wickham et al., Cell, 73, 309-19 (1993)).
• Wild-type adenovirus first binds the cell surface by means of a cellular adenoviral receptor (AR).
• AR cellular adenoviral receptor
• One such AR is the recently-identified coxsackievirus and adenovirus receptor (CAR) (Bergelson et al., Science, 275, 1320-23 (1997); Tanako et al., Proc. Nat. Acad.
• the MHC class I receptor also is an AR (Hong et al, EMBO J., 16(9), 2294-06 (1997)).
• the virus attaches to a v integrins, a family of a heterodimeric cell-surface receptors mediating interaction with the extracellular matrix and playing important roles in cell signaling (Hynes, Cell, 69, 11-25 (1992)).
• infection proceeds by receptor-mediated internalization of the virus into endocytotic vesicles (Svensson et al., J.
• the adenoviral virion is a non-enveloped icosahedron about 65-80 nm in diameter (Home et al., J. Mol. Biol, 1, 84-86 (1959)).
• the adenoviral capsid comprises 252 capsomeres ⁇ 240 hexons and 12 pentons (Ginsberg et al., Virology, 28, 782-83 (1966)).
• the hexons and pentons are derived from three viral proteins (Maizel et al., Virology, 36, 115-25 (1968); Weber et al., Virology, 76, 709-24 (1977)).
• the hexon comprises three identical proteins of 967 amino acids each, namely polypeptide II (Roberts et al., Science, 232, 1148-51 (1986)).
• the penton contains a base, which is bound to the capsid, and a fiber, which is non-covalently bound to and projects from, the penton base. Proteins IX, VI, and Ilia also are present in the adenoviral coat and are thought to 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 serotypes of adenovirus and (except for the enteric adenovirus Ad40) has five RGD tripeptide motifs (Neumann et al., Gene, 69, 153-57 (1988)).
• the RGD tripeptides apparently mediate adenoviral binding to a v integrins and endocytosis of the virion (Wickham et al. (1993), supra; Bai et al., J Virol, 67, 5198-3205 (1993)).
• the adenoviral fiber is a homotrimer of the adenoviral polypeptide IN (Devaux et al., J Molec. Biol, 215, 567-88 (1990)). Structurally, the fiber has three discrete domains. The amino-terminal tail domain attaches non-covalently to the penton base. A relatively long shaft domain comprising a variable number of repeating 15 amino acid residues forming ⁇ -sheets extends outward from the vertices of the viral particle (Yeh et al., Virus Res., 33, 179-98 (1991)). Lastly, roughly 200 amino-acid residues at the carboxy-terminal form the knob domain.
• the knob mediates primary viral binding to the cellular AR and fiber trimerization (Henry et al., J. Virol, 68(8), 5239-46 (1994)).
• the trimerization domain of a fiber is a ligand for a cell-surface receptor native for the adenoviral serotype.
• the trimerization domain also appears necessary for the tail of the fiber to properly associate with the penton base ( ⁇ ovelli et al., Virology, 185, 365-76 (1991)).
• the fiber protein contributes to serotype integrity and mediates nuclear localization.
• Fiber proteins from different adenoviral serotypes differ considerably. For example, the number of 15 amino-acid ⁇ -sheet repeats differs between adenoviral serotypes (Green et al., EMBO J, 2, 1357-65 (1983)). Moreover, the knob regions from the closely related Ad2 and Ad5 serotypes are only 63% similar at the amino acid level (Chroboczek et al., Virology, 186, 280-85 (1992)), and Ad2 and Ad3 fiber knobs are only 20% identical (Signas et al., J. Virol, 53, 672-78 (1985)). In contrast, the penton base sequences are 99% identical. Despite these differences in the knob region, a sequence comparison of even the Ad2 and Ad3 fiber genes demonstrates distinct regions of conservation, most of which are also conserved among the other human adenoviral fiber genes.
• the adenovirus is a superior expression vector.
• Recombinant adenovirus can be produced in high titers (e.g., about 10 13 viral particles/ml), and adenoviral vectors can transfer genetic material to non-replicating, as well as replicating, cells (in contrast with retroviral vectors).
• the adenoviral genome can be manipulated to carry a large amount of exogenous D ⁇ A (up to about 7.5 kb), and the adenoviral capsid can potentiate the transfer of even longer sequences (Curiel et al., Hum. Gene Ther., 3, 147-54 (1992)).
• adenoviruses represent a safe choice for gene transfer, a particular concern for therapeutic applications. For example, adenoviruses do not integrate into the host cell chromosome, thus minimizing the likelihood that an adenoviral vector will interfere with normal cell function.
• adenoviral infection does not correlate with human malignancy, and recombination of the adenoviral genome is rare. Due to these advantages, clinicians have employed adenoviral vectors safely as a human vaccine and for gene therapy for many years.
• adenoviral vectors Based on the popularity of adenoviral vectors, efforts have been made to increase the ability of adenovirus to enter certain cells, e.g., those few cells it does not infect, an approach referred to as "targeting" (see, e.g., International Patent Application WO
• Typical protocols for purifying viral vectors from packaging cell lysates involve centrifuging the viruses through a CsCl 2 gradient one or more times. While such methods adequately isolate viruses, they generally require considerable material (CsCl 2 ) and are therefore relatively inefficient. Moreover, such protocols are not readily amenable to high throughput application, presenting a significant barrier to economic development of viral vectors on a commercial scale. Other methods involving column purification do not bind the viruses specifically (Shabram et al., Hum. Gene Ther., 8, 453 (1997); Huyghe et al., Hum. Gene Ther., 6, 1403 (1995)), often resulting in an unacceptable amount of contaminants compared to the purity obtainable in affinity purification of other materials. Thus, there is a need for an efficient method of purifying and isolating recombinant viral vectors.
• 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 to selectively bind the substrate.
• 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.
• 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.
• the present invention is useful in a variety of applications, in vitro and in vivo, such as therapy, for example, as a vector for delivering a therapeutic gene to a cell with minimal ectopic infection. Specifically, the present invention permits more efficient production and construction of safer vectors for gene therapy applications.
• the present invention is also useful as a research tool by providing methods and reagents for the study of adenoviral attachment and infection of cells and in a method of assaying receptor- ligand interaction.
• the recombinant fiber protein trimers can be used in receptor-ligand assays and as adhesion proteins in vitro or in vivo.
• the present invention provides reagents and methods permitting biologists to investigate the cell biology of viral growth and infection.
• the vectors of the present invention are highly useful in biological research.
• Figures 1A and IB depict the three-dimensional structure of an adenoviral knob protein (serotype 5).
• Figure 1 A is a ribbon diagram representing ⁇ -sheets and the loops interconnecting the sheets.
• Figure IB is a filled-in diagram taking into account the relative sizes of the amino acid residues.
• Figure 2 is a sequence comparison between adenoviral serotypes.
• Figures 3A-3C depict vectors for creating recombinant adenoviral fiber trimers having non-native trimerization domains.
• Figure 3A depicts pAcT5S7GCNTS.PS.LS.X.
• Figure 3B depicts pAcT5sigDel.TS.PS.LS.
• Figure 3C depicts pAcT5S7sigDel.TS.PS.LS.
• Figure 4 depicts pAcT5sigDel.GFP.TS.PS.LS, a vector containing a gene encoding a fiber-sigDel-GFP chimera.
• Figures 5A-5D depict vectors useful for the construction of recombinant adenovirus vectors containing fiber trimers having non-native trimerization domains.
• Figure 5A depicts pAS pGS HAAV.
• Figure 5B depicts pAS pGS pK7.
• Figure 5C depicts pAS T5S7sigDelpGS.HAAV.
• Figure 5D depicts pAST5S7sigDel.GFP.pGS.pK7.
• Figures 6A-6D represent vectors used in the construction of fiber trimers having non-native trimerization domains.
• Figure 6A represents pAcPig4KN.
• Figure 6B represents pAcPigKN D363E.
• Figure 6C depicts pAcPigKN N437D.
• Figure 6D depicts pAcPig4KN(FLAG).
• Figures 7A-7B represent vectors employed in creating a fiber trimer having a non- native trimerization domain.
• Figure 7A depicts PNS F5F2K.
• Figure 7B depicts pNS Pig4.SS.
• Figures 8A-8C represent vectors useful for creating an adenoviral vector having a chimeric fiber trimer comprising a mutant NADC- 1 knob lacking native receptor-binding ability and containing a functional non-native ligand.
• Figure 8 A depicts pAcPig4KN D363E N437D.
• Figure 8B depicts pAcPig4KN D363E N437D HAAV.
• Figure 8C depicts pNS Pig4 D363E N437D HAAV SS.
• Figures 9A-9B represent vectors useful for creating chimeric blocking proteins of the present invention able to interfere with native adenoviral receptor binding.
• Figure 9A depicts pACSG2-sCAR.
• Figure 9B depicts pACSG2-sCAR-HAAV.
• Figures 10A-10B represent vectors useful for creating chimeric blocking proteins able to form trimers interfering with native adenoviral receptor binding.
• Figure 10A depicts pAcSG2sCAR.sigDel.
• Figure 10B depicts pAcSG2-sCARsigDel (HAAV).
• Figures 11A-1 IE depict vectors useful for creating construction of adenovirus vectors having specific non-native ligands.
• Figure 11 A depicts pBSSpGS.
• Figure 1 IB depicts pBSS pGS (RKKK)2.
• Figure 11C depicts pNSF5F2K(RKKK)2.
• Figure 11D depicts pBSSpGS (FLAG).
• Figure 1 IE depicts pNS F5F2K(FLAG).
• Figures 12A-12E represent vectors useful for creating a cell line expressing a non- native cell surface binding site substrate.
• Figure 12A depicts pHOOK3.
• Figure 12B depicts pRC/CMVp-Puro.
• Figure 12C pScHAHK.
• Figure 12D depicts pNSE4GLP.
• Figures 13A-13D represent vectors useful for creating a fiber-expressing cell line for the production of targeted adenovirus particles.
• Figure 13A depicts pCR2.1- TOPO+fiber.
• Figure 13B depicts pKSII Fiber.
• Figure 13C depicts pSMTZeo-DBP.
• Figure 13D depicts pSMTZeo-Fiber.
• Figure 14 depicts pAdEl(Z)E3/E4(B), a plasmid useful for the construction of targeted adenovirus particles having genomes encoding chimeric fibers.
• Figures 15A-15E illustrate the locations of mutations within adenoviral knobs which interfere with ligand binding.
• Figure 15A is a top view, Figure 15B a side view, and Figure 15C a bottom view of the knob illustrating the location of the 3D9 mutation.
• Figure 15D is a top view, Figure 15E a side view, and Figure 15F a bottom view of the knob illustrating the locations of the CD loop mutation, the FG loop mutation, and the IJ mutation.
• Figure 16 depicts a vector useful for the construction of a recombinant adenovirus containing a short-shafted fiber and a mutant fiber knob exhibiting reduced affinity for its native receptor.
• Figures 17A-17B depict vectors useful for constructing a cell line able to replicate adenoviruses lacking native cell-binding function (but targeted for a pseudo-receptor).
• Figure 17A depicts pCANTAB5E(HA).
• Figure 17B depicts pScFGHA.
• An adenovirus is any virus of the genera Mastadenoviridae or Aviaadenoviridae, and can be of any serotype within those genera.
• Adenoviral stocks that can be employed as a source of adenovirus or adenovirus coat protein such as penton base and/or fiber protein can be amplified from the adenovirus serotypes currently available from American Type Culture Collection (ATCC, Rockville, MD), or from any other source.
• a ligand is any species selectively binding an identifiable substrate.
• Native refers to a protein or property of an unmodified virus or cell.
• a non- native protein can be a modified or mutated protein differing from its native homologue within the virus or cell.
• a non-native protein can be a protein having no native homologue within the virus or cell.
• An AR refers to an adenoviral receptor.
• an AR is a ligand binding the mastadenoviral knob.
• a first species is selectively bound to a substrate if it binds the substrate with greater affinity than a second species.
• the first species is not selectively bound if binds the substrate with the same or lesser affinity than the second species, even if the first species binds with some affinity.
• the present invention provides a trimer comprising three monomers (e.g., at least a portion of each of three adenoviral fiber monomers), each having an amino terminus derived from an adenoviral fiber protein and each having a trimerization domain.
• the inventive trimer exhibits reduced affinity for a native substrate, such as an antibody, a cellular binding cite, etc. (i.e., native to the serotype from which the shaft, and particularly the amino-terminus, is drawn) as compared to a native adenoviral fiber trimer.
• the trimer can be a homotrimer or a heterotrimer of different fiber monomers.
• any modification of the monomeric units reducing the affinity of the resulting trimer for its native cell surface binding site is within the scope of the invention.
• the reduction in affinity is a substantial reduction in affinity (such as at least an order of magnitude, and preferably more) relative to the unmodified corresponding fiber.
• trimers possessing such trimerization domains present some of the same problems for targeting as native adenoviral fiber trimerization domains. Therefore, the trimerization domain of a monomer incorporated into the trimer of the invention preferably is not a ligand for the CAR or MHC-1 cell surface domains, or antibodies recognizing the fiber.
• the non-native trimerization domain is not a ligand for any native mammalian cell-surface binding site, whether the site is an AR or other cell surface binding site.
• adenoviruses incorporating such trimers exhibit reduced ability to appreciably infect their native host cells, and can serve as efficient source vectors for engineering selectively targeted vectors. Therefore, while the trimerization domain preferably is not a ligand for a cell surface binding site, the entire trimer can be such a ligand (by virtue of a non-native ligand as discussed herein).
• trimerization domain can be a ligand for a substrate other than a native cell surface binding site, as such trimerization-ligands do not present the same concern for cell targeting as do trimerization domains which are ligands for cell surface binding sites.
• the non-native trimerization domain can be a ligand for a substrate on an affinity column, on a blood-borne molecule, or even on a cell surface when it is not a native cell-surface binding site (e.g., on a cell engineered to express a substrate cell surface protein not native to the unmodified cell type).
• a monomer for inclusion into a trimer can be all or a part of a native adenoviral fiber monomeric protein.
• a modified monomer can lack a sizable number of residues, or even identifiable domains, as herein described.
• a monomer can lack the native knob domain; it can lack one or more native shaft ⁇ -sheet repeats, or it can be otherwise truncated.
• a monomer can have any desired modification so long as it trimerizes.
• a monomer preferably is not modified appreciably at the amino terminus (e.g., the amino-terminus of a monomer preferably consists essentially of the native fiber amino-terminus) to ensure that the resultant trimer interacts properly with the penton base.
• the present invention also provides a composition of matter comprising a trimer of the present invention and an adenoviral penton base.
• the trimer and the penton base associate much in the same manner as wild-type fibers and penton bases.
• the trimer comprises modified fiber monomers
• the penton base can also be modified, for example, to include a non-native ligand, for example as is described in U.S. Patent 5,559,099.
• a fiber monomer for incorporation into the trimer of the present invention has a trimerization domain which binds a native mammalian AR (i.e., an AR native for the adenoviral serotype of interest) with less affinity than a native adenoviral fiber.
• Trimers incorporating such monomers preferably are not ligands for their native cellular binding sites.
• the monomers can be modified in any manner suitable for reducing the affinity of the fiber for native AR while permitting the monomers to trimerize.
• the trimerization domain is a modified adenoviral fiber knob domain lacking a native receptor-binding amino acid.
• any native amino-acid residue mediating or assisting in the interaction between the knob and a native cellular AR is a suitable amino acid for mutation or deletion from the monomer.
• the knob domain can lack any number of such native receptor-binding amino acids, so long as, in the aggregate, the monomers associate to form a trimer of the present invention.
• Native amino acid residues for modification or deletion can be selected by any method. For example, the sequences from different adenoviral serotypes can be compared to deduce conserved residues likely to mediate AR-binding. Alternatively or in combination, the sequence can be mapped onto a three dimensional representation of the protein (such as the crystal structure) to deduce those residues most likely responsible for AR binding.
• random mutations can be introduced into a cloned adenoviral fiber expression cassette.
• One method of introducing random mutations into a protein is via the Taq polymerase.
• a clone encoding the fiber knob can serve as a template for PCR amplification of the adenoviral fiber knob, or a portion thereof.
• the error rate of the transcripts can be largely predetermined (see, e.g., Weiss et al., J. Virol, 71, 4385-94 (1997); Zhou et al., Nucl Acid. Res., 19, 6052 (1991)).
• the PCR products then can be subcloned back into the template vector to replace the sequence within the fiber coding sequence employed as a source for the PCR reaction, thus generating a library of fibers, some of which will harbor mutations which diminish native AR binding while retaining the ability to trimerize.
• a monomer lacking one or more amino acids, as herein described, can optionally comprise a non-native residue (e.g., several non-native amino acids) in addition to or in place of the missing native amino acid(s); of course, alternatively, the native amino acid(s) can simply be deleted from the knob.
• the amino-acid is substituted with another non-native amino acid to preserve topology and, especially, trimerization.
• the replacement amino acid preferably confers novel qualities to the monomer. For example, to maximally ablate binding to the native AR, a native amino acid can be substituted with a residue (or a plurality of residues) having a different charge.
• Such a substitution maximally interferes with the electrostatic interaction between native adenoviral knob domains and cellular ARs.
• a native amino acid can be substituted with a heavier residue (or a plurality of residues) where possible. Heavier residues have longer side-chains; hence, such a substitution maximally interferes with the steric interaction between native adenoviral knob domains and cellular ARs.
• the trimer includes modified monomers which are chimeric adenoviral fiber polypeptides.
• a suitable chimeric monomer lacks all or a portion of the trimerization domain native to the source adenoviral serotype.
• the trimerization domain of such a monomer can be deleted from the virus, or the trimerization domain can be ablated by inserting or substituting non-native amino acids into the domain.
• a monomer lacking the native trimerization domain can also lack the entire native knob.
• the native trimerization domain is a ligand for a native AR
• a trimer of chimeric adenoviral fiber monomers lacking the native trimerization domain binds its native AR with less affinity than the native adenoviral fiber.
• the chimeric monomers to form a trimer of the present invention they must incorporate a replacement (i.e., non-native) trimerization domain.
• the non-native trimerization domain is not a ligand for a mammalian cell-surface receptor, or any cell-surface receptor. Any domain able to form homotrimers is a suitable trimerization domain for inclusion into the trimers of the present invention, and several are known in the art.
• a chimeric monomer can include the trimerization domain from the heat shock factor (HSF) protein ofK. lactis (Sorger and Nelson, Cell, 59, 807 (1989)), trout axonal dynein (Garber et al., EMBO J, 8, 1727 (1989)), parainfluenza virus hemagglutanin protein (Coelingh et al., Virology, 162, 137 (1988)), the sigma 1 protein of reovirus type 1 (Strong et al., Virology, 184, 12 (1991)), or other suitable trimer.
• a chimeric monomer can include a modified leucine-zipper motif.
• Leucine zippers comprise heptad repeats of leucines, which mediate dimerization. However, replacement of one or more leucine with isoleucine results in stable trimerization of the domains.
• An example of such a modified leucine zipper motif is the 32 amino acid GCN4p-II trimer (Harbury et al., Science, 262, 1401 (1993)).
• the reovirus sigma 1 trimerization domain is preferred.
• This protein contains 17 alpha helical heptad repeats, reminiscent of the coiled-coil trimer structure of the aforementioned mutant isoleucine zipper domains (Harbury et al., Nature, 371, 80-83 (1994)). Fiber chimeras containing the sigma 1 domain can thus protrude farther from the virus than corresponding chimeras containing shorter trimerization domains.
• An advantage of the reovirus sigma 1 trimerization domain over a mutant leucine-zipper (e.g., GCN4) domain is that the sigma 1 domain is 22 nm long (Fraser et al., J.
• a chimeric monomer can alternatively include a knob domain from another adenoviral serotype.
• the trimerization domain can be replaced with a mutated knob from an adenoviral serotype capable of productive infection within the host species (e.g., a mutant knob of Ad3 containing a mutation in the HI loop).
• it can be replaced with a knob from a serotype not capable of productive infection within the host species.
• the fiber knob of a mammalian adenoviral serotype can be replaced with a knob from an avian serotype.
• the avian knob mediates trimerization of the fiber proteins, it is likely unable to recognize a mammalian AR; hence, such chimeric fibers lack the native ability to bind the native host AR.
• the fiber knob of one mammalian adenoviral serotype can be replaced with a knob from another mammalian serotype.
• a modified or unmodified knob from the porcine adenovirus NADC-1 fiber is a preferred domain, as the NADC-1 is well characterized.
• the NADC-1 knob has identifiable ligands, e.g., galectin (which binds galactose), and LDZ and RGD peptides, (which bind integrins) (see, e.g., Hirabayashi et al., J. Biol. Chem., 266, 13648-53 (1991)).
• galectin which binds galactose
• LDZ and RGD peptides which bind integrins
• the non-native trimerization domain can be ligated to the native fiber monomer at any suitable site, so long as the monomers can trimerize properly (i.e., be capable of interacting with an adenoviral penton base).
• the domain can be inserted into the native knob to disrupt knob topology.
• the trimerization domain can be inserted after any of the 15 amino acid shaft repeats, preferably after the 7 th , 15 th , or 22° repeats to mimic native adenoviral shaft size.
• the non-native trimerization domain is inserted into the adenoviral shaft, it can form the carboxy-terminus of the chimeric protein, or it can be inserted into the middle of the amino acid sequence.
• any number of trimerization domains can be so inserted into the fiber monomer, so long as the resulting trimer properly associates with the penton base.
• Another suitable chimeric monomer has a novel domain blocking the ligand for the native host AR.
• the blocking domain is any peptide which can be tightly bound to the native ligand. (See, e.g., Hong et al., EMBO J., 16, 2294-2306 (1997)).
• the blocking domain is a substrate to which the (native or modified) fiber monomer ligand selectively binds.
• the ligand-substrate interaction occurs at least immediately upon viral production and effectively continues until the fiber trimer is destroyed.
• the native ligand binds the blocking domain, the ligand is incapable of binding its native substrate on cell surfaces.
• trimers of chimeric adenoviral fiber monomers having such blocking domains bind the native AR with less affinity than a native adenoviral fiber.
• the blocking domain can be at any position on the adenovirus to bind the native ligand without appreciably affecting trimerization or penton base interaction.
• the blocking domain can be appended to the above-referenced ⁇ -sheets or loops, either by fusion within the reading frame, by covalent post-translational modification, etc.
• the blocking domain can be appended to another portion of the monomer, such as the shaft.
• the blocking domain can also include a linker or spacer polypeptide to afford an opportunity for the blocking domain to interact with the native ligand. If the blocking domain is attached via such a spacer, the spacer can include a protease recognition site for subsequent cleavage, as described herein.
• the monomers for inclusion into the trimers of the present invention can be produced by any suitable method.
• the mutant fiber protein can be synthesized using standard direct peptide synthesizing techniques (e.g., as summarized in Bodanszky, Principles of Peptide Synthesis (Springer- Verlag, Heidelberg: 1984)), such as via solid-phase synthesis (see, e.g., Merrifield, J. Am. Chem. Soc, 85, 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res., 30, 705-739 (1987); and U.S. Patent 5,424,398).
• site-specific mutations can be introduced into the monomer by ligating into an expression vector a synthesized oligonucleotide comprising the modified site.
• a plasmid, oligonucleotide, or other vector encoding the desired mutation can be recombined with the adenoviral genome or with an expression vector encoding the monomer to introduce the desired mutation. Oligonucleotide-directed site- specific mutagenesis procedures also are appropriate ⁇ .
• the DNA fragment encoding the modified monomer can be subcloned into an appropriate vector using well known molecular genetic techniques. The fragment is then transcribed and the peptide subsequently translated in vitro within a host cell.
• Any appropriate expression vector e.g., Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevior, NY: 1985)
• suitable host cells can be employed for production of recombinant peptides.
• Expression hosts include, but are not limited to, bacterial species, mammalian or insect host cell systems including baculovirus systems (e.g., Luckow et al., Bio/Technology, 6, 47 (1988)), and established cell lines such 293, COS-7, C127, 3T3, CHO, HeLa, BHK, etc.
• An especially preferred expression system for preparing modified fibers of the invention is a baculovirus expression system (Wickham et al., J. Virol, 70, 6831-38 (1995)) as it allows the production of high levels of recombinant proteins.
• the choice of expression host has ramifications for the type of peptide produced, primarily due to post-translational modification.
• the monomers are assayed for fiber protein activity. Specifically, the ability of the monomers to form trimers, interact with the penton base, and interact with native ARs is assayed. Any suitable assay can be employed to measure these parameters. For example, as improperly folded monomers are generally insoluble (Scopes, "Protein Purification” (3d Ed., 1994), Chapter 9, p. 270-82 (Springer-Verlag, New York)), one assay for trimerization is whether the recombinant fiber is soluble. Determining solubility of the fiber is aided if an amount of radioactive amino-acid is incorporated into the protein during synthesis.
• Lysate from the host cell expressing the recombinant fiber protein can be centrifuged, and the supernatant and pellet can be assayed via a scintillation counter or by Western analysis. Subsequently, the proteins within the pellet and the supernatant are separated (e.g., on an SDS-PAGE gel) to isolate the fiber protein for further assay. Comparison of the amount of radioactivity in the fiber protein isolated from the pellet vis-a-vis the fiber protein isolated from the supernatant indicates whether the mutant protein is soluble. Alternatively, trimerization can be assayed by using a monoclonal antibody recognizing only the amino portion of the trimeric form of the fiber (e.g., via immunoprecipitation, Western blotting, etc.).
• trimerization is the ability of the recombinant fiber to form a complex with the penton base (Novelli and Boulanger, Virology, 185, 1189 (1995)), as only fiber trimers can so interact. This propensity can be assayed by co- immunoprecipitation, gel mobility-shift assays, SDS-PAGE (boiled samples run as monomers, otherwise, they run as larger proteins), etc.
• a fourth measure of trimerization is to detect the difference in molecular weight of a trimer as opposed to a monomer.
• a boiled and denatured trimer will run as a lower molecular weight than a non- denatured stable trimer (Hong and Angler, J. Virol, 70, 7071-78 (1996)).
• a trimeric recombinant fiber must also be assayed for its ability to bind native
• a preferred assay involves exposing cells expressing a native AR (e.g., 293 cells) to the recombinant fiber trimers under standard conditions of infection. Subsequently, the cells are exposed to native adenoviruses, and the ability of the viruses to bind the cells is monitored. Monitoring can be by autoradiography (e.g., employing radioactive viruses), immunocytochemistry, or by measuring the level of infection or gene delivery (e.g., using a reporter gene). In contrast with native trimers which reduce or substantially eliminate subsequent viral binding to the 293 cells, those trimers not substantially reducing the ability of native adenoviruses to subsequently bind the cells are trimers of the present invention.
• a vector including a sequence encoding a mutated fiber can be introduced into a suitable host cell strain to express the fiber protein.
• the host cells are bacteria.
• mutants can be identified by assaying the ability to bind the soluble CAR protein.
• a replica of the bacterial plate e.g., on a nitrocellulose filter lift
• the filter is exposed to a solution suitable for lysing the bacteria adhering to it, and the probed with a radiolabled CAR protein.
• the filter is first "blocked" with a high protein solution to minimize nonspecific adherence of the CAR probe to the filter.
• the filter is exposed to film to identify colonies expressing fiber proteins that bind the CAR. Those colonies not hybridizing to the radiolabeled CAR probe (or binding with reduced affinity as indicated by weaker signal) potentially express fiber monomers of the present invention.
• mutant fibers identified as non-CAR binding by such a bacterial library screen must be assayed for the ability to trimerize, as described above.
• the present invention provides a chimeric blocking protein comprising a substrate for an adenovirus fiber.
• the chimeric blocking protein can include any suitable domain having a substrate recognized by the ligand on the adenoviral fiber.
• the chimeric blocking protein can comprise the extracellular domain of the CAR cell-surface protein (Bergelson et al., Science, 275, 1320-23 (1997); Tomko et al., Proc. Nat. Acad. Sci.
• the blocking protein can comprise a substrate recognized by a ligand present on the trimer. While, as mentioned, the chimeric blocking protein can comprise domains from cell-surface proteins, typically it is not itself a cell-surface protein. Instead, the chimeric blocking protein is preferably a free soluble protein able to interact with an adenovirus in solution.
• a chimeric blocking protein of the present invention affords a method of interfering with adenoviral receptor-binding by incubating an adenovirus with the chimeric blocking protein in a solution such that the chimeric blocking protein binds the ligand present on the adenoviral fiber.
• the virus and the chimeric blocking protein can be incubated for any length of time, and under any suitable conditions, to promote the ligand on the fiber to bind the substrate on the chimeric blocking protein.
• the parameters of time, temperature, and solution chemistry suitable for promoting selective binding between the fiber ligand and the chimeric blocking protein substrate can vary according to the affinity with which the ligand selectively binds the substrate.
• the binding conditions can, in large measure, be predetermined as discussed herein (e.g., by employing such conditions when screening the protein library for the novel ligand-substrate interaction).
• concentration of the chimeric blocking proteins is sufficient to saturate the cell-surface ligands present on the fibers of the adenovirus during the incubation.
• a chimeric blocking protein in addition to including a domain having a substrate recognized by the ligand on an adenoviral fiber, can have other domains.
• the protein can include domains to promote secretion (see, e.g., Suter et al., EMBO, J., 10, 2395-2400 (1991); Beutler et al, J. Neurochem., 64, 475-81 (1995)), thus aiding in the collection of free chimeric blocking proteins from cells producing the protein.
• the chimeric blocking protein preferably further includes a ligand domain (i.e., a ligand in addition to the substrate for the viral knob), such as those ligands described herein.
• a ligand on the chimeric blocking protein facilitates purification and identification of the chimeric blocking protein after production.
• a more preferred ligand is one recognizing a cell surface binding site or other substrate, as discussed herein.
• Such blocking proteins function as "bi-specific" molecules for altering adenoviral receptor binding.
• a chimeric blocking protein includes a ligand for a cell-surface binding site
• the blocking protein is able to effect selective targeting of the adenovirus by interfering with fiber-mediated receptor binding while directing novel targeting through the ligand present on the chimeric blocking protein.
• the present invention provides a method of directing adenoviral targeting by incubating an adenovirus with a chimeric blocking protein having a ligand recognizing a substrate present on a cell surface binding site in a solution such that the chimeric blocking protein binds the adenoviral fiber to form a complex, and thereafter exposing the complex to a cell having a substrate for the ligand.
• the chimeric blocking protein also can include a trimerization domain, such as those trimerization domains discussed herein.
• trimerization domains permits the chimeric blocking protein monomers to trimerize. While, as monomers, the chimeric blocking proteins can saturate the ligands present on the fibers, such bonds are, of course, subject to dissociation at a certain rate depending on the kinetics of the ligand-substrate interaction. However, because the probability that all three ligand/substrate bonds between a trimeric fiber and the trimeric blocking protein will be severed at the same time is significantly less than the probability that any one such bond will be broken, a trimeric blocking protein more easily saturates the available ligands present on the fiber. In effect, the trimeric structure effectively holds each substrate against the fiber knob ligand, thereby increasing the likelihood that each ligand is blocked.
• the chimeric blocking proteins can be produced by any suitable method, such as by direct protein synthesis, cellular production, in vitro translation or other method known in the art. Many suitable methods for producing proteins are described elsewhere herein and are otherwise known in the art.
• the present invention provides an adenovirus incorporating the recombinant fiber trimers of the present invention.
• the adenovirus of the present invention does not infect its native host cell via the native AR as readily as the wild-type serotype, due to the above-mentioned reduction in affinity of the fiber trimers present in the viral coat (e.g., via replacement of the trimerization domain with a non-ligand trimerization domain, selective mutation of the responsible residues, or incorporation of a blocking domain, as herein described).
• the adenovirus preferably incorporates a non-adenoviral ligand to facilitate its propagation, isolation and/or targeting.
• the virus can include any suitable ligand (e.g., a peptide specifically binding to a substrate).
• the ligand can bind a cell surface binding site (e.g., any site present on the surface of a cell with which the adenovirus can interact to bind the cell and thereby promote cell entry) other than its native AR or even any native AR.
• a cell surface binding site can be any suitable type of molecule, but typically is a protein (including a modified protein), a carbohydrate, a glycoprotein, a proteoglycan, a lipid, a mucin molecule or mucoprotein, or other similar molecule.
• potential cell surface binding sites include, but are not limited to: heparin and chondroitin sulfate moieties found on glycosaminoglycans; sialic acid moieties found on mucins, 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., E-selectin, P-selectin, L-selectin, etc.), and integrin molecules; and tumor-specific antigens present on cancerous cells, such as, for instance, MUC-1 tumor-specific epitopes.
• heparin and chondroitin sulfate moieties found on glycosaminoglycans sialic acid moieties found on mucins, glycoproteins, and gangli
• the protein can thus be expressed in a narrow class of cell types (e.g., cardiac muscle, skeletal muscle, smooth muscle, etc.) or expressed within a broader group encompassing several cell types.
• the non-native ligand can bind a compound other than a natural cell-surface protein.
• the ligand can bind blood- and/or lymph-borne proteins (e.g., albumin), synthetic peptide sequences such as polyamino acids (e.g., polylisine, polyhistadine, etc.), artificial peptide sequences (e.g., FLAG SEQ ID NO: 16), and RGD peptide fragments (Pasqualini et al., J. Cell. Biol , 130, 1189 ( 1995)).
• blood- and/or lymph-borne proteins e.g., albumin
• synthetic peptide sequences such as polyamino acids (e.g., polylisine, polyhistadine, etc.)
• artificial peptide sequences e.g., FLAG SEQ ID NO: 16
• RGD peptide fragments Pasqualini et al., J. Cell. Biol , 130, 1189 ( 1995)
• the ligand can bind non-peptide substrates, such as plastic (e.g., Adey et al., Gene, 156, 27 (1995)), biotin (Saggio et al, Biochem. J, 293, 613 (1993)), a DNA sequence (Cheng et al., Gene, 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 (Balass et al., Anal.
• plastic e.g., Adey et al., Gene, 156, 27 (1995)
• biotin Saggio et al, Biochem. J, 293, 613 (1993)
• a DNA sequence Choeng et al., Gene, 171, 1, (
• Suitable ligands and their substrates for use in the method of the invention include, but are not limited to: CR2 receptor binding the amino acid residue attachment sequences, CD4 receptor recognizing the V3 loop of HIV gpl20, transferrin receptor and its ligand (transferrin), low density lipoprotein receptor and its ligand, the ICAM-1 receptor on epithelial and endothehal cells in lung and its ligand, linear or 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.
• additional ligands and their binding sites preferably include (but are not limited to) short (e.g., 6 amino acid or less) linear stretches of amino acids recognized by integrins, as well as polyamino acid sequences such as polylysine, polyarginine, etc. Inserting multiple lysines and or arginines provides for recognition of heparin and DNA.
• a ligand can comprise a commonly employed peptide tag (e.g., short amino acid sequences known to be recognized by available antisera) such as sequences from glutathione-S-transferase (GST) from Shistosoma manosi, thioredoxin ⁇ -galactosidase, or maltose binding protein (MPB) from E. coli.
• GST glutathione-S-transferase
• MPB maltose binding protein
• a preferred substrate for a tag ligand is an antibody directed against it, a derivative of such an antibody (e.g., a FAB fragment, Single Chain antibody (ScAb)), or other suitable substrate.
• a suitable ligand can be specific for any desired substrate, such as those recited herein or otherwise known in the art.
• adenoviral vectors can also be engineered to include novel ligands by first assaying for the ability of a peptide to interact with a given substrate.
• a random or semirandom peptide library containing potential ligands can be produced, which is essentially a library within an expression vector system.
• Such a library can be screened by exposing the expressed proteins (i.e., the putative ligands) to a desired substrate. Positive selective binding of a species within the library to the substrate indicates a ligand for that substrate, at least under the conditions of the assay.
• any assay able to detect interactions between proteins and substrates is appropriate, and many are known in the art.
• one preferred assay for screening a protein library is the phage display system, which employs bacteriophage expressing the library (e.g., Koivunen et al., Bio/Technology, 13, 265-70 (1995); Yanofsky et al., Proc. Nat. Acad. Sci. U.S.A., 93, 7381-86 (1996); Barry et al., Nature Med, 2(3), 299-305 (1996)).
• Binding of the phage to the substrate is assayed by exposing the phage to the substrate, rinsing the substrate, and selecting for phage remaining bound to the substrate. Subsequently, limiting dilution of the phage can identify individual clones expressing the putative ligand. Of course, the insert present in such clones can be sequenced to determine the identity of the ligand.
• Phage display is preferred for identifying potential ligands because it best mimics viral interaction with the microenvironment.
• phage display is an extracellular system (as is the initial stage of viral infection); moreover, phage display incorporates an actual virus (phage) presenting the actual potential ligand.
• Phage display also offers significantly more flexibility than other protein binding assays (especially intracellular assays).
• phage display not only identifies proteins (ligands) binding to a particular substrate, but it identifies those which bind under predefined conditions. Thus, the use of phage display can identify ligands useful for incorporation into an adenovirus to facilitate purification under largely predefined conditions.
• the phage display library can be screened by exposure to a particular plastic, resin, or other desired substrate used in an affinity column.
• Phage expressing peptides that either bind the substrate or that are eluted from the substrate under a specific condition or range of conditions (e.g., high or low salt, pH, temperature, etc.), but do not so bind or elute under other conditions, can be readily identified.
• adenovirus incorporating the ligand can be purified by expositing it to the substrate under like conditions, as discussed herein.
• a given ligand can be incorporated into any location of the virus capable of interacting with a substrate (i.e., the viral surface).
• the ligand can be incorporated into the fiber, the penton base, the hexon, or other suitable location.
• the ligand is attached to the fiber protein, preferably it does not disturb the interaction between viral proteins or monomers.
• the ligand preferably is not itself an oligomerization domain, as such can adversely interact with the trimerization domain as discussed above.
• the ligand preferably does not replace a portion of the fiber protein, as such perturbance can adversely affect trimerization and interaction with the penton.
• the ligand preferably is added to the fiber protein, and is incorporated in such a manner as to be readily exposed to the substrate (e.g., at the carboxy-terminus of the protein, attached to a residue facing the substrate, positioned on a peptide spacer to contact the substrate, etc.) to maximally present the ligand to the substrate.
• the ligand is attached to or replaces a portion of the penton, preferably it is within the hypervariable regions to ensure that it contacts the substrate.
• the recombinant fiber is truncated or short (e.g., from 0 to about 10 shaft repeats) to maximally present the ligand to the substrate (see, e.g., U.S. Patent 5,559,099 (Wickham et al.)).
• the ligand is attached to the hexon, preferably it is within a hypervariable region (Miksza et al., J. Virol, 70(3), 1836-44 (1996)).
• the ligand When engineered into an adenoviral protein (or blocking protein), the ligand can comprise a portion of the native sequence in part and a portion of the non-native sequence in part. Similarly, the sequences (either native and/or normative) that comprise the ligand in the protein need not necessarily be contiguous in the chain of amino acids that comprise the protein. In other words, the ligand can be generated by the particular conformation of the protein, e.g., through folding of the protein in such a way as to bring contiguous and/or noncontiguous sequences into mutual proximity.
• an adenovirus of the present invention (or a blocking protein) can comprise multiple ligands, each binding to a different substrate.
• a virus can comprise a first ligand permitting affinity purification as described herein, a second ligand that selectively binds a cell-surface site as described herein, and/or a third ligand for inactivating the virus, also as described herein.
• the protein including the ligand can include other non-native elements as well.
• a non-native, unique protease site also can be inserted into the amino acid sequence.
• the protease site preferably does not affect fiber trimerization or substrate specificity of the fiber ligand. Many such protease sites are known in the art. For example, thrombin recognizes and cleaves at a known amino acid sequence (Stenflo et al., J. Biol.
• a virus of the present invention can include one or more non-native passenger genes as well.
• a “passenger gene”” can be any suitable gene, and desirably is either a therapeutic gene (i.e., a nucleic acid sequence encoding a product that effects a biological, preferably a therapeutic, response either at the cellular level or systemically), or a reporter gene (i.e., a nucleic acid sequence which encodes a product that, in some fashion, can be detected in a cell).
• a therapeutic gene i.e., a nucleic acid sequence encoding a product that effects a biological, preferably a therapeutic, response either at the cellular level or systemically
• a reporter gene i.e., a nucleic acid sequence which encodes a product that, in some fashion, can be detected in a cell.
• a passenger gene is capable of being expressed in a cell into which the vector has been internalized.
• the passenger gene exerts its effect at the level of RNA or protein.
• a protein encoded by a transferred therapeutic gene can be employed in the treatment of an inherited disease, such as, e.g., the cystic fibrosis transmembrane conductance regulator cDNA for the treatment of cystic fibrosis.
• the protein encoded by the therapeutic gene can exert its therapeutic effect by effecting cell death.
• expression of the gene in itself can lead to cell killing, as with expression of the diphtheria toxin.
• a gene, or the expression of the gene can render cells selectively sensitive to the killing action of certain drugs, e.g., expression of the HSV thymidine kinase gene renders cells sensitive to antiviral compounds including aciclovir, ganciclovir, and FIAU (l-(2-deoxy-2-fluoro- ⁇ -D-arabinofuranosil)-5- iodouracil).
• the therapeutic gene can exert its effect at the level of RNA, for instance, by encoding an antisense message or ribozyme, a protein which affects splicing or 3 ' processing (e.g., polyadenylation), or a protein affecting the level of expression of another gene within the cell (i.e., where gene expression is broadly considered to include all steps from initiation of transcription through production of a processed protein), perhaps, among other things, by mediating an altered rate of mRNA accumulation, an alteration of mRNA transport, and or a change in post-transcriptional regulation.
• gene transfer technology to deliver a given passenger gene, its sequence will be known in the art.
• the altered protein e.g., the trimer or the coat protein having the ligand
• the passenger gene can be incorporated into the adenovirus by any suitable method, many of which are known in the art.
• the protein is preferably identified by assaying products produced in high volume from genes within expression vectors (e.g., baculovirus vectors).
• the genes from the vectors harboring the desired mutation can be readily subcloned into plasmids, which are then transfected into suitable packaging cells (e.g., 293 cells). Transfected cells are then incubated with adenoviruses under conditions suitable for infection. At some frequency within the cells, homologous recombination between the vector and the virus will produce an adenoviral genome harboring the desired mutation.
• Adenoviruses of the present invention can be either replication competent or replication deficient.
• the adenoviral vector comprises a genome with at least one modification therein, rendering the virus replication deficient (see, e.g., International Patent Application WO 95/34671).
• the modification to the adenoviral genome includes, but is not limited to, addition of a DNA segment, rearrangement of a DNA segment, deletion of a DNA segment, replacement of a DNA segment, or introduction of a DNA lesion.
• a DNA segment can be as small as one nucleotide and as large as the adenoviral genome (e.g., about 36 kb) or, alternately, can equal the maximum amount which can be packaged into an adenoviral virion (i.e., about 38 kb).
• Preferred modifications to the adenoviral genome include modifications in the El, E2, E3, and/or E4 regions.
• An adenovirus also preferably can be a cointegrate, i.e., a ligation of adenoviral genomic sequences with other sequences, such as other virus, phage, or plasmid sequences.
• the adenovirus of the present invention has many qualities which render it an attractive choice for use in gene transfer, as well as other, applications.
• the adenovirus does not infect its native host cells as readily as does wild-type adenovirus, due to the mutant fiber trimers (e.g., selective mutation of residues responsible for AR binding, replacement of the trimerization domain, or addition of a blocking domain, as herein described).
• the adenovirus has at least one non-native ligand specific for a substrate which facilitates viral propagation, targeting, purification, and/or inactivation as discussed herein.
• the ligands and the trimerization domains preferably are separate domains, thus permitting the virus to be easily be reengineered to incorporate different ligands without perturbing fiber trimerization.
• the fiber trimer incorporates a mutated fiber knob
• the ligand can be incorporated into the knob, as herein described.
• a virus of the present invention can be incorporated into a suitable carrier.
• the present invention provides a composition comprising an adenovirus of the present invention and a pharmacologically acceptable carrier.
• Any suitable preparation is within the scope of the invention, the exact formulation, of course, depends on the nature of the desired application (e.g., cell type, mode of administration, etc.), many suitable preparations are set forth in U.S. Patent 5,559,099 (Wickham et al.).
• an adenovirus of the present invention does not readily infect its native host cell via the native AR because its ability to bind ARs is significantly attenuated (due to the incorporation of the chimeric trimers of the present invention). Therefore, the invention provides a cell line able to propagate the inventive adenovirus.
• the cell line can support viral growth for at least about 10 passages (e.g., about 15 passages), and more preferably for at least about 20 passages (e.g., about 25 passages), or even 30 or more passages.
• the adenoviruses can be first grown in a packaging cell line which expresses a native fiber protein gene.
• the resultant viral particles are therefore likely to contain both native fibers encoded by the complementing cell line and non-native fibers encoded by the adenoviral genome (such as those fibers described herein); hence a population of such resultant viruses will contain both fiber types. Such particles will be able to bind and enter packaging cell lines via the native fiber more efficiently than particles which lack native fiber molecules.
• the employment of such a fiber- encoding cell line permits adenovirus genomes encoding chimeric, targeted adenovirus fibers to be grown and amplified to suitably high titers.
• the resultant "mixed" stocks of adenovirus produced from the cell lines encoding the native fiber molecule will contain both native and chimeric adenovirus fiber molecules; however, the particles contain genomes encoding only the chimeric adenovirus fiber.
• the "mixed” stock is used to infect a packaging cell line which does not produce native fiber (such as 293 for El -deleted viruses).
• the resultant adenoviruses contain only the fiber molecules encoded by the genomes (i.e., the chimeric fiber molecules).
• Similar fiber-complementing cell lines have been produced and used to grow mutant adenovirus lacking the fiber gene.
• the production rates of these cell lines have generally not been great enough to produce adenovirus titers of the fiber- deleted adenovirus comparable to those of fiber-expressing adenovirus particles.
• the lower titers produced by such mutants can be improved by temporally regulating the expression of the native fiber to more fully complement the mutant adenovirus genome.
• One strategy to produce such an improved cell line is to use of an inducible promoter, (e.g., the metallothionine promoter), to permit fiber production to be controlled and activated once the cells are infected with adenovirus.
• an efficient mRNA splice site introduced into the fiber gene in the complementing cell line improves the level of fiber protein production in the cell line.
• any cell line expressing that receptor and capable of supporting adenoviral growth is a suitable host cell line.
• a cell line can be engineered to express the substrate for the ligand.
• the present invention provides a cell line expressing a non-native cell-surface biding site to which an adenovirus (or a bi-specific blocking protein) having a ligand for the receptor binds.
• an adenovirus or a bi-specific blocking protein having a ligand for the receptor binds.
• Any cell line capable of supporting adenoviral growth is a suitable cell line for use in the present invention.
• the adenovirus lacks genes essential for viral replication, preferably the cell line expresses complementing levels of the gene products.
• the cell line of the present invention is derived from 293 cells.
• the non-native cell surface binding site is a substrate molecule, such as those described herein, to which an adenovirus (or a bi-specific blocking protein) having a ligand selectively binding that substrate can bind the cell and thereby promote cell entry.
• an adenovirus or a bi-specific blocking protein
• the binding site can recognize a non-native ligand incorporated into the adenoviral coat or a ligand native to a virus.
• the binding site can be a single chain antibody (ScAb) receptor recognizing the tag.
• the ScAb can recognize an epitope present in a region of a mutated fiber knob (where present), or even an epitope present on a native adenoviral coat protein, (e.g., on the fiber, penton, hexon, etc.).
• the non-native ligand recognizes a cell-surface substrate (e.g., membrane-bound protein)
• the binding site can comprise that substrate.
• the substrate binding side is native to a cell-surface receptor
• the cell line can express a mutant receptor with decreased ability to interact with the cellular signal transduction pathway (e.g., a truncated receptor, such as NMD A, (Li, et al., Nat.
• the binding site preferably is not a native mammalian AR.
• the binding site must be expressed on the surface of the cell to be accessible to the virus.
• the binding site is a protein, it preferably has leader sequence and a membrane tethering sequence (see, e.g., Davitz et al., J. Exp. Med. 163, 1150 (1986)). to promote proper integration into the membrane.
• the cell line can be produced by any standard method. For example, a vector
• the vector e.g., an oligonucleotide, plasmid, viral, or other vector
• a gene encoding the non-native receptor can be introduced into source cell line by standard means.
• the vector also encodes an agent permitting the cells harboring it to be selected (e.g., the vector can encode resistance to antibiotics which kill cells not harboring the plasmid). At some frequency, the vector will recombine with the cell genome to produce a transformed cell line expressing the binding site.
• the present invention provides a method of propagating the inventive adenovirus.
• the inventive method involves infecting the cell with an adenovirus having a non-native ligand selectively binding to the receptor, incubating the cells, and recovering the adenoviruses produced within the cells.
• Adenoviruses recovered from the cells can be propagated again (e.g., amplified) to produce viral stocks of very high titer.
• the ligand on the adenovirus can be any ligand, such as those discussed herein.
• the cells of the present invention are infected by the virus at any suitable m.o.i.
• Viruses are recovered from the cells by standard means, such as by cell lysis. Thereafter they can be purified by standard methods or the method of the present invention.
• the substrate for the ligand engineered into the adenovirus need not be present on the surface of a cell.
• the substrate can be located on a support, e.g., an inanimate support such as plastic, glass, metal, resin, or other material commonly employed in chromatographic or affinity separation.
• Such supports include metals, natural polymeric carbohydrates and their synthetically modified, cross-linked or substituted derivatives, such as agar, agarose, cross-linked alginic acid, substituted and cross-linked guar gums, cellulose esters, especially with nitric acid and carboxylic acids, mixed cellulose esters, and cellulose ethers; natural polymers containing nitrogen, such as proteins and derivatives, including cross-linked or modified gelatins; natural hydrocarbon polymers, such as latex and rubber; synthetic polymers which may be prepared with suitably porous structures, such as vinyl polymers, including polyethylene, polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and its partially hydrolyzed derivatives, polyacrylamides, polymethacrylates, copolymers and terpolymers of the above polycondensates, such as polyesters, polyamides, and other polymers, such as polyurethanes or polyepoxides; porous inorganic materials such as sulf
• the present invention 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 present on the adenovirus to selectively bind the substrate.
• the composition e.g., at least a significant portion of the composition
• the composition is removed from the substrate, after which the adenovirus bound to the substrate is eluted from the substrate.
• an adenovirus having a ligand can be purified from a variety of compositions (e.g., solutions, dispersions, suspensions, gels, etc.). While adenoviruses can be present in a variety of compositions, a common composition containing adenoviruses is a cell lysate, such as produced from a packaging cell during adenoviral propagation.
• a common composition containing adenoviruses is a cell lysate, such as produced from a packaging cell during adenoviral propagation.
• the substrate is bound to a support, as previously described. Fusing desired ligand-substrates to a suitable support material is known in the art, and the present invention contemplates any suitable method for engineering a support having the substrate. Indeed, as mentioned, the substrate can itself be such a plastic, glass, metal, resin, etc.
• any method of exposing the composition containing the adenovirus to the substrate is suitable for use in the present inventive method.
• the composition can be passed through a column comprising the support onto which the substrate is bound.
• the composition also can be mixed with a slurry of such a support (e.g., beads or other preparation comprising the support-bound substrate), placed into a container (e.g., a tube, the well of a dish, etc.) which has been coated with the substrate, or otherwise exposed to the substrate.
• the parameters of time, temperature, and solution chemistry necessary to promote selective binding can vary according to the affinity with which the ligand selectively binds the substrate. Generally, where known ligand-substrate systems are employed, these parameters are also known. Where novel ligand-substrate systems are employed, however, the binding conditions can, in large measure, be predetermined as discussed herein (e.g., by employing such conditions when screening the protein library for the novel ligand-substrate interaction). Preferably, the conditions for selective binding do not permit selective binding of other constituents of the composition to the substrate. Where other constituents do not selectively bind the substrate, a significant amount of the adenovirus can be removed from the composition by association with the substrate.
• the adenoviral-deprived composition is removed from the presence of the substrate (e.g., selectively eluted).
• Any suitable method for so removing the adenoviral-deprived composition from the substrate can be employed, provided the adenovirus remains selectively bound to the substrate.
• the conditions employed for removing the adenoviral-deprived composition from the substrate generally are insufficient to elute the adenovirus from the substrate.
• the method of removing the adenoviral-deprived composition is largely a function of the type of substrate and support.
• the adenoviral-deprived composition can be removed from a column comprising the substrate by rinsing the column with several volumes of a suitable solution.
• the adenoviral-deprived composition can be removed from a slurry of the support containing the substrate by repeated centrifugation, resuspension in a suitable solution, and recentrifugation.
• the support is a magnetic material
• it can be physically removed from the solution by exposing the vessel containing the solution to a magnet and rinsing the magnetic support.
• the dish can simply be rinsed with several volumes of a suitable solution.
• the adenovirus is eluted from the substrate.
• Any method for separating the adenovirus from the substrate is suitable for use in the present inventive method.
• the adenovirus can be liberated by exposing the support-adenovirus complex to an elution solution incompatible with the ligand-substrate bond.
• the parameters of time, temperature, and solution chemistry necessary to promote selective elution of the virus from the support can vary according to the affinity with which the ligand selectively binds the substrate. Generally, where known ligand-substrate systems are employed, these parameters are also known.
• the elution conditions can, in large measure, be predetermined, for example, by adjusting the conditions when screening a protein library, as discussed herein.
• the spacer can include a peptidase recognition sequence or other specific cleavage motif.
• Adenoviruses containing such a cleavage sequence can be liberated from the support by exposing the support to an agent effecting the cleavage, such as an endoprotease or other agent. While the cleavage method severs the ligand from the adenovirus, in many applications this is preferred.
• the ligand for purifying the virus might interfere with a second ligand for targeting the virus to a particular cell type. Removal of the purifying ligand thus permits the isolated adenovirus to more readily infect the cell type of interest.
• any suitable binding or elution conditions can be employed, a practical limit is set by the ability of the adenovirus to survive the conditions.
• the present method can be employed under a wide range of conditions. In any event, such conditions are known to those of skill in the art.
• the inventive method for purifying adenoviruses need not remove all of the virus from the solution, or even a majority of the virus. Indeed, in many applications, the amount of virus present in the initial composition can saturate the amount of substrate present on the support. Moreover, while the ligand on the adenovirus selectively binds the substrate, such selective binding can be of any affinity. As such, a substantial amount of substrate can not bind available ligands in the separation step. Therefore, to obtain as much adenovirus from the initial composition as possible, the adenoviral-depleted composition removed from the support, as herein described, can be subjected to successive rounds of purification, and the viruses obtained from each round can be combined into a single stock.
• the complete absence of erroneous binding is not common, at least in early rounds of purification.
• the presence of background levels of erroneous binding necessarily results in some contamination of the initial viral stock obtained.
• the viral stock can be subjected to successive rounds of purification until the background level of contaminants approaches zero.
• the present inventive method provides an economical, efficient, and reliable means of purifying adenoviruses having known ligands.
• the use of slurries and columns is common in industrial applications, rendering the present method amenable to high throughput, or commercial- scale application.
• the non-native ligand present on the virus of the present invention can recognize a substrate present within a cell surface binding site. Therefore, the present invention provides a method of infecting a cell having a cell surface binding site including a substrate for the non-native ligand. The method involves contacting the cell with the adenovirus such that the non-native ligand of the adenovirus (or on the virus/blocking protein complex) binds the particular cell surface binding site and thereby effects entry of the adenovirus.
• the viruses of the present invention incorporate fiber trimers having reduced ability to bind native mammalian ARs
• the adenovirus is internalized into the cell primarily due to the non- native ligand.
• the present inventive method effects selective targeting of the virus comprising the ligand to a cell type expressing a binding site comprising the substrate for that ligand without significant infection of cells via native mammalian ARs.
• the ligand is on the penton base (such as a modified or unmodified penton base)
• the virus is internalized via the ligand on the penton.
• a cell can be present as a single entity, or can be part of a larger collection of cells, such as a cell culture (either mixed or pure), a tumor, a tissue (e.g., epithelial, muscle, or other tissue), an organ, an organ system (e.g., circulatory system, respiratory system, gastrointestinal system, urinary system, nervous system, integumentary system or other organ system), or even an entire organism (e.g., a human).
• the cells being targeted are selected from the group consisting of heart, blood vessel, smooth muscle, skeletal muscle, lung, liver, gallbladder, urinary bladder, and eye cells.
• the method for infecting a cell ideally is carried out wherein the adenovirus includes a passenger gene, such as those vectors herein described.
• the adenovirus of the present invention includes a passenger gene
• the method permits the adenovirus to serve as a vector for introducing that gene into a targeted cell. Once internalized, the passenger gene is expressed within the cell.
• the vectors and methods of the present invention provide useful tools for introducing a passenger gene into a selected class of cells without significantly providing the gene to cells ubiquitously or ectopicly.
• the non-native ligand present on the virus of the present invention can recognize substrate present within blood or lymphatic fluid (such as a ligand present on a free blood-borne protein, a protein present on erythrocytes, etc.). Therefore, the present invention 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 selectively binds its substrate, thereby adsorbing the free virus from the fluid.
• the substrate is present within a large macromolecule (e.g., albumin) or on the surface of erythrocytes (which lack transcription machinery required to propagate viruses).
• a ligand for inactivating the virus can be present at any location on the viral coat (Fender et al., Virology, 214, 110 (1995)).
• antibodies recognizing and/or neutralizing adenoviruses primarily bind epitopes on the hexon (Gahery-Segard et al., Eur. J. Immunol, 27, 653 (1997))
• non- native ligands for inactivation of the virus preferably are incorporated into the hexon, as herein described.
• the method assists in confining the viral infection to a desired locus (tissue, cell type, etc.). Specifically, the method effectively inactivates an individual virus by tethering it to the substrate, thereby reducing its ability to contact (and therefore enter) a cell. Even where a virus so adsorbed does contact a cell, it is significantly less likely to be internalized due to the presence of the particle having the substrate. Due to the aggregation of these effects, the inventive method effectively inactivates a viral stock (outside of the desired locus of infection) by dramatically reducing its effective free titer.
• the viruses of the present invention incorporate fiber trimers having reduced affinity for native mammalian ARs, thereby substantially reducing the likelihood that the virus will infect cell types other than the desired cell type.
• the viruses of the present invention can include ligands specific for a substrate present on a cell surface binding site, permitting the virus to be targeted to a predetermined cell type. While those two qualities effect selective targeting, and thereby significantly attenuate ectopic infection, viruses also having a ligand recognizing a blood- or lymph- borne substrate are much less likely to even contact an ectopic tissue by reason of the effective reduction of viral titer.
• EXAMPLE 1 This example describes two different fiber trimers having non-native trimerization domains, each of which interacts properly with the adenoviral penton base. Specifically, the fiber chimeras incorporate the reovirus sigma 1 trimerization domain.
• T5S7sigDel contained only the Ad5 fiber tail (T5) fused to sigDel without any Ad fiber shaft sequence.
• T5S7sigDel contained the tail plus the first 7 ⁇ -sheet repeats of the Ad shaft (S7) fused to sigDel.
• S7 fused to sigDel.
• the DNA and respective amino acid sequences of these two clones are set forth at SEQ ID NO: 1 and SEQ ID NO:2.
• the sigDel region of the reovirus sigma 1 gene was amplified via PCR and cloned into the vector, pAcT5S7GCNTS.PS.LS.X (Fig.
• pAcT5sigDel.TS.PS.LS (Fig. 3B)
• This vector encodes the Ad5 fiber tail fused to the N-terminal trimerization domain of reovirus type 3 sigma 1 protein followed by a FLAG epitope near the C-terminus.
• the vector also contains multiple restriction sites to facilitate the cloning of targeting and purification sequences into the gene.
• the second vector, pAcT5S7sigDel.TS.PS.LS (Fig. 3C) was created by cutting the above PCR product with the restriction enzymes Nhel and BamHI and cloning this fragment into the vector, pAcT5S7GCNTS.PS.LS.X (Fig. 3 A), also cut with Nhel and BamHI.
• the resultant vector encodes a protein containing the tail and first seven ⁇ -sheet shaft repeats of Ad5 fiber fused to sigDel, followed by a FLAG epitope.
• Recombinant baculovirus clones encoding each of the fiber chimeras were then generated by standard means using each of the above plasmids.
• the resultant baculovirus clones were used to produce recombinant proteins in Tn5 insect cells.
• another baculovirus was constructed from the initial plasmid, pAcT5S7GCNTS.PS.LS.X (Fig. 3A), which contained the GCN trimerization domain in place of the sigDel trimerization domain.
• the baculovirus-infected cells were pelleted at 3 days post infection.
• the cell pellet was resuspended in PBS plus protease inhibitors and freeze-thawed three times to release the soluble intracellular proteins.
• the cell debris were then pelleted by centrifugation at high speed and the cleared cell lysate was removed. The pellet was then resuspended in the same volume of PBS as previously.
• the proteins were further assayed for their ability to form trimers.
• the lysates from each sample were either boiled or not boiled prior to running the samples on a 0.1% SDS, 12.5% polyacrylamide gel.
• Western analysis of the boiled samples showed that the boiled samples migrated at molecular weights corresponding to the size of the monomeric protein, whereas the unboiled proteins containing the sigDel trimerization domains migrated at molecular weights commensurate with a trimer.
• the unboiled T5S7GCN.TS.PS.LS protein also migrated as a trimer; however, a significant portion (over half) of the unboiled sample migrated as a monomer.
• recombinant penton base is mixed in solution with the T5S7GCN.TS.PS.LS, T5sigDel.TS.PS.LS and T5S7sigDel.TS.PS.LS trimeric fiber proteins.
• the resultant penton base/fiber chimera complex is then immunoprecipitated with anti-penton base antibody coupled to protein A-agarose.
• the precipitated sample is then run on an SDS- PAGE gel and evaluated by Western analysis as described above using the FLAG antibody. Binding of the FLAG antibody indicates that the fiber chimera containing the FLAG epitope complexes with the penton base in solution.
• This example demonstrates the ability of the fiber-sigDel chimeras to incorporate exogenous protein domains larger than peptide tags.
• EXAMPLE 3 This example describes the construction of recombinant adenovirus vectors containing fiber trimers having non-native trimerization domains.
• the Ndel to BamHI fragment is excised from pAcT5S7sigDel.TS.PS.LS (Fig. 3C), to replace the corresponding fragments in pAS pGS HAAV (Fig. 5A), and pAS pGS pK7 (Fig. 5B), to produce the final transfer vectors pAS T5S7sigDelpGS.HAAV (Fig. 5C) and pAST5S7sigDelpGS.pK7 (Fig. 5D), respectively.
• the vectors encode the fiber- sigDel chimera containing either the RGD or pK7 binding domains at their C-terminus for binding to an a v integrin and heparin sulfate-containing receptors that are expressed by 293 cells.
• This example describes the production of targeted adenovirus particles having genomes encoding chimeric fibers.
• the chimeric fibers represent the Ad5 fiber tail and seven shaft repeats fused to the sigDel trimerization domain from reovirus followed by a high affinity RGD sequence for binding ay integrins.
• the plasmid, pAS T5S7sigDel.HAAV (Fig. 5C) is cut with the restriction enzyme Drdl, and the large fragment containing all the adenovirus sequences is isolated and purified. This fragment is then electroporated into B J5183 bacterial cells along with a linearized plasmid, containing the majority of Ad genome prior to the fiber gene with a small overlap of identical sequence with the pAS T5S7sigDel.HAAV plasmid. Upon recombination of the two pieces of DNA, a new plasmid is produced in the bacterial cells through homologous recombination.
• This plasmid encodes a modified adenovirus genome that is capable of replicating in the appropriate complementing mammalian cell line (El and fiber-complementing).
• the plasmid DNA from selected colonies is isolated and confirmed to be the correct plasmid by restriction analysis.
• This plasmid DNA is then used to transform DH5a bacterial cells in order to obtain adequate amounts of DNA for transfection into the fiber-complementing cell line.
• One microgram of the plasmid is cut with the appropriate restriction enzyme and transfected into a fiber-complementing cell line, such as the cell line described above.
• a fiber-complementing cell line such as the cell line described above.
• the cells are induced with zinc, and 1-5 days later the cells are lysed.
• the lysate is passaged onto fresh fiber-complementing cells. This passage and lysis cycle is repeated until a cytopathic effect develops in the cells.
• the LacZ activity of the cell lysate is also followed, as it should increase as the recombinant vector is amplified.
• a final passage onto non- fiber-complementing cells is made to produce a targeted virus lacking a native fiber protein.
• the resulting virus is then assayed for its ability to bind and enter cells via the interaction of its high affinity RGD sequence with a v integrins.
• the exemplified fiber trimers are chimeras incorporating the knob portion of the NADC-1 fiber, a porcine adenoidal strain.
• the exemplified trimers thus, contain known receptor-binding motifs (i.e., a galectin motif and an RGD motif).
• exemplified trimers incorporate mutations known to reduce the affinity of each of the receptor-binding motifs.
• this example describes the incorporation of a non-native ligand (FLAG) into an exposed loop of a non-native trimer.
• FLAG non-native ligand
• the PCR product was then cloned into a baculovirus expression plasmid to produce a plasmid which encoded the NADC-1 knob plus an N- terminal polyhistidine tag (the Pig4KN protein) for purification and detection by Western analysis using an anti-polyhistidine antibody.
• the DNA and amino acid sequences of this clone are set forth at SEQ ID NO:4.
• the resultant plasmid, pAcPig4KN (Fig. 6A), was then mutated by site-directed mutagenesis using the two oligonucleotide primer pairs PigD363Es (SEQ ID NO: 10) and PigD363Ea (SEQ ID NO:l 1), and PigN437Ds (SEQ ID NO: 12) and PigN437Da (SEQ ID NO: 13).
• the former pair of primers was used to produce the plasmid pAcPigKN D363E (Fig. 6B), in which the DNA sequence encoding the RGD integrin binding motif (a.a. 361-363 in the native fiber protein) was mutated to the non- functional sequence RGE.
• the 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 (a.a. 437) was mutated to a D.
• This mutation has been previously shown to abrogate the binding of another galectin protein to its ligand, galactose (Hirabayashi et al., J. Biol. Chem., 266, 23648-53 (1991)).
• a final baculovirus plasmid was constructed to demonstrate the feasibility of incorporating a novel binding motif into an exposed loop on the NADC-1 knob.
• Hydrophobicity analysis of the NADC-1 knob protein revealed that the protein sequence immediately prior to the RGD motif was likely to be an exposed loop that would be capable of incorporating additional amino acid sequences (e.g., polypeptide domains) for the purpose of targeting or purification. Therefore, the plasmid, pAcPig4KN(FLAG) (Fig. 6D), was produced using complementary overlapping oligonucleotides, which encoded the FLAG binding domain. The oligonucleotides were annealed and cloned into the plasmid pAcPig4KN (Fig. 11 A), which contained a unique, native restriction site, Avrll, just prior to sequence encoding the RGD domain.
• the four baculovirus transfer plasmids described above carrying NADC-1 knob genes were used to express recombinant protein in insect cells using the baculovirus expression system.
• Tn5 insect cells were infected with the recombinant baculovirus clones derived from the plasmids. After three days the cells were pelleted and freeze- thawed three times in PBS plus protease inhibitors to release the soluble intracellular protein. The debris were pelleted and the cleared lysate was decanted. The remaining pellet was resuspended in PBS .
• NADC-1 knob-FLAG domains can interact with the FLAG antibody
• cell lysates are immunoprecipitated using anti-FLAG M2 antibody and then blotted.
• Western analysis will demonstrate that the NADC-1 knob containing the FLAG epitope is precipitated by the anti-FLAG antibody.
• the NADC-1 -fiber trimers are soluble, and each is capable of interacting with the anti-FLAG M2 monoclonal antibody.
• This example describes the synthesis of recombinant Ad5-based vector containing an NADC-1 (porcine adenovirus) fiber knob.
• the knob of the NADC-1 fiber gene was amplified from a plasmid containing the full length gene.
• the PCR product was then cloned into the plasmid PNS F5F2K (Fig. 7A) to produce the plasmid, pNS Pig4.SS (Fig. 7B) which encodes the first 7 ⁇ -repeats of the Ad5 shaft fused to the NADC-1 knob.
• the DNA and amino-acid sequences of this clone are set forth at SEQ ID NO:5.
• the pNS Pig4.SS plasmid was then used to create a recombinant adenovirus vector.
• the plasmid was transfected into 293 cells which had been infected with an adenovirus vector lacking the E4 region. Homologous recombination between the plasmid and the vector produced an E4-containing, replication competent vector having the chimeric NADC-1 fiber. The recombinant virus was then plaque purified on 293 cells.
• EXAMPLE 7 This example describes an Ad5-based adenoviral vector having a chimeric fiber trimer comprising a mutant NADC-1 knob with attenuated receptor-binding ability and containing a functional non-native ligand.
• FIG. 6C is cloned into the plasmid pAcPig4KN D363E (Fig. 6B) containing the RGD- RGE mutation to create the plasmid pAcPig4KN D363E N437D (Fig. 8A) containing both mutations in the NADC-1 knob gene.
• Overlapping, complementary oligonucleotide primers encoding the high affinity a ⁇ integrin binding domain are thereafter cloned into the native Avrll site to produce the plasmid pAcPig4KN D363E N437D HAAV (Fig. 8B).
• the mutated NADC-1 gene fragment EcoRI to BamHI is then cloned into the plasmid pNSPig4.SS (Fig. 7B) to create the plasmid, pNS Pig4 D363E N437D HAAV SS (Fig. 8C).
• This plasmid is then used to create a recombinant adenovirus vector containing the mutated and ⁇ integrin-targeted NADC-1 knob as described above.
• the ability of the double mutation in the NADC-1 knob to block binding to the native cell surface binding sites (galectin and integrin) is confirmed via competition assays.
• the ability of the resultant virus to target cell-surface a,, integrin is confirmed using 293 cells, as discussed above.
• the blocking protein each include a domain having a substrate for the native adenovirus fiber, namely the extracellular domain of the CAR.
• the extracellular domain of CAR was amplified from the CAR gene (Bergelson et al., supra; Tomko et al., supra) via PCR.
• the PCR product was then cloned into a baculovirus expression vector to create the plasmid pACSG2-sCAR (Fig. 9A).
• the soluble CAR protein (sCAR) also contained a FLAG epitope for purification and for detection by Western analysis.
• the DNA and amino acid sequences of this sCAR clone are set forth at SEQ ID NO:6.
• a second sCAR-containing chimera was produced in which DNA sequence encoding an RGD targeting motif was cloned into an Spel site following the C-terminal end of sCAR using complementary, overlapping primers.
• the chimeric gene retained the FLAG epitope on the C-terminus.
• the resultant plasmid, SG2-sCAR-HAAV (Fig. 9B), was used to produce recombinant sCARRGD protein as was done for sCAR protein described above.
• the DNA sequence of this clone is set forth at SEQ ID NO:7.
• the sCARRGD protein was synthesized and secreted from insect cells similarly to the sCAR protein.
• radiolabeled adenovirus type 2 were preincubated in a solution containing various concentrations of sCAR.RGD and then exposed to Ramos cells, which do not express a v integrins but do express receptors for the fiber protein of adenovirus.
• Preincubation of radiolabeled adenovirus type 2 with either sC AR or sC AR.RGD blocked virus binding to Ramos cells. This result demonstrates that the sCAR domain present in the sCAR.RGD protein is functional.
• sCAR.RGD protein retains the function of the native RGD domain. Both sCAR.RGD, and sCAR were immobilized onto tissue culture plastic plates, which were subsequently contacted with 293 cells (which express a,, integrin). After the cells were incubated on the coated plates, the plates were rinsed, and the number of cells remaining in contact with the plates were assayed. The results showed that cells adhered to plates coated with sCAR.RGD, while they did not adhere to plates coated with sCAR or control plates, demonstrating that the RGD motif present in the sCAR.RGD protein is functional.
• This example demonstrates the inventive method of directing adenoviral targeting using a chimeric blocking protein having a ligand for a cell surface binding site.
• An adenovirus vector carrying a lacZ reporter gene is preincubated with either the sCAR.RGD protein or the sCAR protein, described above in Example 8.
• the resultant complexes are then exposed to either Ramos cells (which express fiber receptor (CAR) but lack & repeat integrins) or HuVEC cells (which express both CAR and a ⁇ integrins) under conditions suitable for viral infection. Subsequently, the cells are assayed for lacZ expression, the level of which will correlate to the degree to which the viruses infect the cells.
• EXAMPLE 10 This example describes two chimeric blocking proteins able to form trimers interfering with native adenoviral receptor binding.
• the blocking proteins each include a domain having a substrate for the native adenovirus fiber, namely the extracellular domain of the CAR, and a trimerization domain, namely the sigDel trimerization domain of the Sigma- 1 reovirus protein.
• the sigDel trimerization domain of the Sigma- 1 reovirus protein is amplified by
• the resultant plasmid, pAcSG2sCAR.sigDel contains a gene chimera encoding the extracellular domain of CAR, a spacer region, the trimerization domain from sigma 1 protein of reovirus, and a FLAG binding domain.
• An Spel restriction site following the trimerization domain allows for the convenient cloning of targeting domains, such as the high affinity RGD motif which binds a v integrins.
• the DNA and amino acid sequences of this clone are set forth at SEQ ID NO: 8.
• PAcsCAR.sigDel was used to make baculovirus.
• Western analysis of boiled and unboiled cell lysates from baculovirus-infected cells showed that the unboiled chimeric sCAR.sigDel migrated as a trimer.
• a second sCAR-containing chimera is produced in which DNA sequence encoding an RGD targeting motif is cloned into an Spel site following the C-terminal end of sCARsigDel using complementary, overlapping primers.
• the resultant plasmid, pAcSG2-sCARsigDel (HAAV) (Fig. 10B), encodes a chimera having the extracellular domain of CAR, a spacer region, the trimerization domain from sigma 1 protein of reovirus, and the high affinity RGD motif which binds a ⁇ integrins.
• pAcSG2sCAR.sigDel and pAcSG2-sCARsigDel.RGD (HAAV) plasmids were used to produce recombinant baculovirus which are used to produce the recombinant chimeric protein in insect cells by standard means.
• Western analysis of boiled and unboiled cell lysates from bacculovirus-infected cells demonstrated that the unboiled sCAR.sigDel protein migrated as a trimer.
• an adenovirus vector carrying a lacZ reporter gene is preincubated with either the sCAR.sigDel or the sCARsigDel.RGD trimer or the sCAR monomeric protein.
• concentrations are employed to generate dose-response data.
• the resultant complexes are then exposed to 293 cells under conditions suitable for viral infection. Subsequently, the cells are assayed for lacZ expression, the level of which will correlate to the degree to which the viruses infect the cells.
• the results will demonstrate that the trimeric sCAR.sigDel and sCARsigDel.RGD proteins are more potent in blocking adenovirus binding to via the sCAR protein cells than the sCAR monomers.
• EXAMPLE 11 This example demonstrates the inventive method of directing adenoviral targeting using a trimeric blocking protein having a ligand for a cell surface binding site.
• adenovirus vector carrying a lacZ reporter gene is preincubated with either sCAR.sigDel, sCARsigDel.RGD, or sCAR described above.
• the adenovirus can be preincubated with a blocking protein isolated, for example, by phage display.
• the resultant complexes are then exposed to either Ramos cells or HuVEC cells under conditions suitable for viral infection. Subsequently, the cells are assayed for lacZ expression, the level of which will correlate to the degree to which the viruses infect the cells. The results will demonstrate that, while such proteins will effectively block adenovirus transduction of Ramos cells, the trimers are more potent in blocking adenovirus binding than the sCAR monomers.
• both sCAR and sCAR.sigDel will block adenovirus transduction of HuVEC cells; however, sCARsigDel.RGD will not effectively block adenovirus transduction of HuVEC cells.
• Ad sCARsigDel.RGD complex is targeted to a ⁇ integrins while avoiding adenoviral-mediated gene delivery to cells via CAR.
• EXAMPLE 12 This example describes the construction and evaluation of mutated fiber knobs each having reduced affinities for native substrates, particularly monoclonal antibodies raised against the native fiber knob. Using site-directed mutagenesis, separate mutations were introduced into the full length Ad5 fiber gene in a baculoviral vector. The resultant plasmids were then used to generate recombinant baculoviral clones.
• each of the mutants plus a native Ad5 fiber control, were used to produce protein in infected insect cells. Three days post infection, the cells were harvested and lysed. Western analysis using polyclonal antisera recognizing the Ad5 fiber revealed the presence of high amounts of fiber protein in lysates from cells infected with each of the vectors. In cells infected with five of the mutant clones (see table 1) (as well as the native fiber gene), the signal was predominantly in the soluble portion of the lysates, indicating that the protein encoded by each mutant was correctly folded.
• the sequences of the wild- type Ad5 fiber is set forth a SEQ ID NO:9. The amino acids of SEQ ID NO:9 changed by each of these mutations is indicated in Table 1.
• EXAMPLE 13 This example describes the construction of a recombinant adenovirus containing a short-shafted fiber (e.g., 8 shaft repeats) and a mutant fiber(5) knob having reduced affinity for its native receptor (i.e., CAR). Such a fiber permits targeting via a ligand expressed in the penton base.
• a short-shafted fiber e.g., 8 shaft repeats
• a mutant fiber having reduced affinity for its native receptor (i.e., CAR).
• CAR native receptor
• a deletion is introduced into the sequence encoding the fiber shaft.
• a portion of the mutant fiber knob from the 22 d shaft repeat until the end of the coding sequence and containing the K506R mutation (see Example 12) is amplified by PCR from SEQ ID NO:8.
• the resultant product is used to create the pAS T5S7F5K(R506K) plasmid (Fig. 16).
• the plasmid thus, contains a gene encoding a short-shafted fiber with reduced affinity for a native substrate (the 3D9 antibody).
• An adenovirus having such a short-shafted fiber will be able to bind to cells via the RGD ligand on the penton base.
• a similar strategy can be used to create adenoviral vectors having short-shafted fibers with reduced affinity for the CAR.
• This example demonstrates the construction of adenovirus vectors having specific non-native ligands that can be used to purify the vector via affinity chromatography.
• the base vector pNSF5F2K ( Figure 8A) contains a gene which encodes a chimeric fiber having the shaft of the Ad5 fiber and the knob of the Ad2 fiber protein.
• the Ad2 fiber gene contains an Spel restriction site in the region of the knob which encodes the flexible, exposed HI loop of the fiber knob. This Spel restriction site was used to insert sequences which encode the FLAG peptide SEQ ID NO: 16 or a DNA/heparin-binding ligand (SEQ ID NO: 15).
• the base vector pBSSpGS ( Figure 11 A) encodes a C-terminal 12 amino acid extension (SEQ ID NO: 14).
• the codons encoding the TS also are a unique Spel site that was used to insert sequences which encode the FLAG peptide (SEQ ID NO: 16) or the DNA/heparin-binding polypeptide (SEQ ID NO: 15) as described below.
• Transfer plasmids (pBSS pGS (RKKK)2 ( Figure 1 IB) and pNSF5F2K(RKKK)2 ( Figure 11 C)) for introducing the DNA/heparin-binding ligand into the adenoviral genome were created using overlapping oligonucleotides.
• Sense and antisense oligonucleotides were mixed in equimolar ratios and cloned into the Spel site of pBSS pGS (Fig. 11 A) or pNS F5F2K (Fig. 8 A) to create the transfer plasmids. Sequencing in both directions across the region of the inserts verified that the clones contained the appropriate sequence.
• transfer plasmids pBSSpGS (FLAG) ( Figure 1 ID) and pNSF5F2K(FLAG) ( Figure 1 IE) for introducing the FLAG ligand (SEQ ID NO: 16) into the adenoviral genome were created. Sequencing in both directions across the region of the inserts verified that the clones contained the appropriate sequence.
• the plasmid DNA from the four transfer vectors were linearized with Sail, purified and transfected using calcium phosphate into 293 cells which had been preincubated for 1 h with the El, E3, E4-deleted adenovirus AdCMVZ.l 1A (GenVec, Inc., Rockville, MD) a multiplicity of 1 ffu per cell. Recombination of the E4+ pNS plasmid with the E4-deleted vector resulted in the rescue of an E1-, E3-, E4+ vector capable of replication in 293 cells.
• the resultant vectors, AdZ.F2K(RKKK)2 The resultant vectors, AdZ.F2K(RKKK)2,
• AdZ.F2K(FLAG), AdZ.F(RKKK)2 and AdZ.F(FLAG), were isolated in two successive rounds of plaquing on 293 cells. Each vector was verified to contain the correct insert by sequencing PCR products derived from virus DNA template using primers spanning the region of the insert DNA.
• This example demonstrates that an adenoviral vector having a non-native ligand can bind a support conjugated to a substrate for that ligand.
• AdZ.PK was constructed similarly to the vectors described above; the virus has a fiber protein containing polylysines.
• AdZ.PK was assayed to determine whether the virus could bind a support having a substrate for polylisine, heparin.
• 50 ml of heparin-agarose beads (SIGMA) were added to 1.0 ml of phosphate buffers containing 150, 300, 500 and 1000 mM NaCl, respectively. 6600 cpm of either AdZ or AdZ.PK were then added to the saline buffers containing the heparin-agarose beads and rocked for 60 min.
• AdZ.PK vector binds a heparin-linked support material and that binding is ablated by high salt concentration. Therefore, such a support can be used to purify the modified vector by first binding the virus to the support at low salt conditions and then eluting the vector at high salt conditions.
• an adenoviral vector having a non-native ligand can be purified on a column comprising substrate for that ligand.
• 20 175 cm2 tissue culture flasks containing 293 packaging cell lines are infected at an m.o.i. of 5 with one of the three vectors: AdZ.PK, AdZ.F2K(RKKK)2 or AdZ.F(RKKK)2 described above.
• the cells are then incubated for 2 days, after which any remaining adherent cells are then dislodged from the plastic.
• the removed cells are centrifuged at 3,000 g to form a pellet, the culture medium removed, and the pellet gently washed 2 times with PBS.
• the cells are then resuspended in a total volume of 5 ml PBS containing 10 mM MgCl 2 .
• the resuspended cells are then freeze-thawed 3 times to release the virus, and the cell debris is then centrifuged at 15,000 g for 15 min. The supernatant is passed over a 3 ml column containing heparin-linked agarose beads. The column is then washed with 30 ml of PBS followed by elution of the virus from the column by a salt step gradient.
• the overall purity of the fractions determined to contain peak adenovirus concentrations is evaluated by running the fractions on HPLC and comparing the profile to a pre-column fraction and a highly purified adenovirus preparation (prepared by 3 successive rounds of purification on CsCl gradients).
• the exemplary pseudo-receptor includes a binding domain from a single-chain antibody (ScFv).
• ScFv single-chain antibody
• Figure 12A Figure 12A
• Human a vector expressing the ScFv from pHOOK3
• Figure 12A Figure 12A
• the ScFv has an N- terminal HA epitope tag, and its C-terminus is linked to a pair of myc epitopes followed by the PDGF receptor transmembrane anchor.
• transfected cells were exposed to magnetic CAPTURE-TEC beads conjugated with antigens recognized by the ScFv. Following incubation, the beads were collected in the bottom of a tube using a magnet, washed, and transferred to a culture dish. The culture dishes were then viewed under a fluorescence microscope to identify GFP-expressing cells. No staining was observed from cells transfected only with pNSE4GLP alone, indicating that these cells did not bind the beads. However, cells transfected with pNSE4GLP and pScHAHK were observed in the wells. This result demonstrates that the doubly transfected cells bound to the beads.
• EXAMPLE 18 This example describes the production of a pseudo-receptor for constructing a cell line able to replicate adenoviruses lacking native cell-binding function (but targeted for the pseudo-receptor).
• the exemplary pseudo-receptor includes a binding domain from a single-chain antibody recognizing HA.
• Anti-HA ScFv was constructed as an N-Term- VL-VH fusion protein.
• RT-PCR was performed on RNA obtained from hybridomas producing HA antibodies using primers specific for K- or ⁇ 2 ⁇ - and C-terminus of the VL and VH genes (see Gilliland et al., Tissue Antigens, 47, 1-20 (1996)).
• specific oligonucleotides were designed to amplify the VL-VH fusion in a second round of PCR.
• the final PCR product was cloned to create the pCANTAB5E(HA) plasmid (Fig. 17A) for production of anti HA ScFv in E. coli.
• the expressed protein has a C- terminal E peptide for detection of binding to HA-tagged penton base via Western analysis of ELISA assay.
• Western analysis using an antibody recognizing the E peptide revealed a protein of the expected size.
• the anti-HA ScFv was used in protein A immunoprecipitation assays using adenoviral coat proteins (recombinant penton base) containing the HA epitope.
• the anti-HA ScFv was able to precipitate HA-containing penton base proteins.
• EXAMPLE 19 This example describes the creation of a fiber-expressing cell line for the production of targeted adenovirus particles.
• the complementing cell line produces a fiber protein with or without additional complementary genes from the adenovirus genome.
• the entire adenovirus type 2 fiber gene was amplified from adenovirus type 2
• the resultant product was cloned into the pCR2.1-TOPO plasmid (Invitrogen) to make the plasmid pCR2.1-TOPO+fiber (Fig. 13 A).
• the fiber2 gene was then excised from the pCR2.1-TOPO+fiber plasmid with the restriction enzymes BamHI and Eagl, and it was then subcloned into the plasmid, pKSII (Stratagene), to construct the plasmid pKSII Fiber (Fig. 13B).
• the fiber2 gene was then excised from the pKSII Fiber plasmid using the restriction enzymes Kpnl and Eagl, and it was then cloned into the plasmid, pSMTZeo-DBP (Fig. 13C).
• the resultant plasmid, pSMTZeo-Fiber (Fig. 13D) encoded the entire fiber2 gene under control of the metallothionine promoter.
• This construct also placed an efficient mRNA splice site before the fiber gene to enhance fiber protein synthesis following induction.
• the pSMTZeo-Fiber plasmid also contains a Zeo resistance marker to allow selection of cell lines on the antibiotic zeocin.
• the pSMTZeo-Fiber plasmid is transfected into 293 cells (or some other cell line) with or without additional adenovirus complementing functions.
• Individual zeocin-resistant cell colonies are then amplified by standard means and tested for fiber2 production (e.g., by Western analysis using an anti-fiber2 antibody) before and after induction with zinc, which activates the metallothionine promoter.
• Selected fiber- expressing clones are then tested for the ability to plaque and/or complement the growth of adenoviruses containing mutated fibers. Clones that adequately complement mutated fibers are suitable for amplifying and growing adenovirus particles having genomes encoding mutant fiber genes. All references cited herein are hereby inco ⁇ orated by reference to the same extent as if each reference were individually and specifically indicated to be inco ⁇ orated by reference and were set forth in its entirety herein.
• NAME WICKHAM, THOMAS J.
• NAME ROELVINK, PETRUS W.
• NAME LIZONOVA, ALENA
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• AGC GCC ATG CCC GAG GGC TAT GTG CAG GAG AGA ACC ATC TTT TTC AAA 1200 Ser Ala Met Pro Glu Gly Tyr Val Gin Glu Arg Thr He Phe Phe Lys 600 605 610
• AAG AGA GAC CAC ATG GTC CTG CTG GAG TTT GTG ACC GCT GCT GGG
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• CAG ACC CTA ACT TCC AGC TAC ATT TAC ACA CAA GCG CAC CTT GAC CAC 864 Gin Thr Leu Thr Ser Ser Tyr He Tyr Thr Gin Ala His Leu Asp His 885 890 895
• 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 Ala Ala Ser Glu Glu Met Pro Ala 965 970 975
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• MOLECULE TYPE DNA (genomic)
• SEQUENCE DESCRIPTION SEQ ID NO: 13: GGGCACCATG GCGAAGATGG AGCTTTGTCC C 31
• MOLECULE TYPE peptide
• SEQUENCE DESCRIPTION SEQ ID NO: 15: Arg Lys Lys Lys Arg Lys Lys Lys 1 5

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