Patent Application
20070212779
The present invention relates generally to targeting adenoviral vectors to dendritic cells. More specifically, the present invention relates to methods of targeting dendritic cells via a newly discovered dendritic cell marker, DC-SIGN.
An expanding body of evidence suggests that dendritic cells (DC) play a pivotal role in the immune system. Dendritic cells are recognized to serve as a key mediator of T cell based immunity. Stemming from their important function, dendritic cells have been proposed for utility in a number of clinical strategies, especially vaccinations. Immunotherapy approaches involving the use of genetically modified dendritic cells can promote immunity against pathogenic entities, both infectious and tumorigenic.
One key to practically realizing these immunotherapy approaches involving the use of genetically modified dendritic cells is the ability to deliver genes effectively to the dendritic cells. Delivery of genes to dendritic cells has been attempted using viral as well as non-viral vectors.
One candidate vector for gene delivery has been replication-defective adenoviral vector (Ad) of serotype 2 or 5. This vector has been suggested to be well suited for clinical applications by virtue of its high titer, efficient gene delivery and exuberant gene expression.
In spite of these theoretical advantages, the relative resistance of dendritic cells to adenoviral vector invention has hindered the full development of gene based immunotherapy strategies. The phenomenon of dendritic cell resistance to adenoviral mediated gene transfer has been shown to derive from a paucity of the primary adenoviral entry receptors coxsackie-adenovirus receptor (CAR).
In permissive cells, the projecting adenoviral fiber-knob protein mediates binding to the cell surface coxsackie-adenovirus receptor (CAR) followed by interaction with and internalization of the virion by either of the αv integrins αvβ3 or αvβ5. In this regard, adenovirus can be targeted to non-native receptors via a variety of trophism modification strategies. Re-routing adenoviral vectors to dendritic cells via CAR-independent infection has been shown to allow enhanced transduction efficiency. Such re-targeting approaches have included the use of chimerism for the fiber capsid protein such that the adenovirus is routed to the receptor for human adenovirus subgroup B. In addition, genetic incorporation of the targeting ligand CD40L, as well as the use of retargeting adapters incorporating anti-CD40-scFv or CD40L, have allowed retargeted gene delivery via the CD40 pathway with the achievement of enhanced adenovirus-mediated dendritic cell transduction.
These results have validated the concept that trophism modified adenovirus represents a useful means to achieve effective gene delivery to dendritic cell target cells for immunotherapy applications. Of note, the utility of any such retargeting approaches is linked to the cell specificity of the cell surface molecule targeted via the trophism modified adenovirus. This is especially relevant for in vivo delivery schemes whereby precise dendritic cell-specific gene delivery is a key technical goal. On this basis, the exploitation of highly specific dendritic cell makers for targeting via trophism modified adenovirus represents a logical and desirable means to further improve dendritic cell-based genetic immunotherapy approaches.
DC-SIGN (Dendritic Cell-Specific Intercellular adhesion molecule 3-Grabbing Nonintegrin, Genbank accession number AF209479) is a type II membrane protein. DC-SIGN is expressed at high levels on immature dendritic cells and is expressed on endometrium and placenta. Of note, whereas there are many described markers of activated/mature dendritic cells, there have been relatively few markers for immature dendritic cells. This is an important issue because vaccine approaches based on in vivo transduction must address immature dendritic cells as target cells.
Thus, there is a need for targeting immature dendritic cells via dendritic cell-specific marker, e.g., with an adenoviral vector. The present invention fulfills this need and desire in the art and provides a retargeting approach utilizing a recently identified dendritic cell-specific marker, DC-SIGN.
Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.
The invention is based, in part, on the identification of the dendritic cell marker DC-SIGN. A noteworthy aspect of this cell surface receptor is its high degree of selective expression on immature dendritic cells. The present invention provides for the identification and targeting of immature dendritic cells via DC-SIGN.
The utility of DC-SIGN as a target for tropism modified adenovirus remains unknown. The present invention demonstrates the feasibility of retargeting adenovirus to dendritic cells via DC-SIGN. Adenoviral vectors, which are genetically modified to incorporate the Fc-binding domain of Staphylococcus aureus Protein A into the adenovirus fiber protein, are targeted to DC-SIGN-expressing cells by binding to anti-DC-SIGN antibody. The results indicate that anti-DC-SIGN monoclonal antibody, not isotype matched control monoclonal antibody, significantly augmented gene transfer to DC-SIGN-expressing target cells. Of further note, the level of gene transfer achieved via the DC-SIGN targeted adenovirus exceed that achieved by un-modified Ad5 vector. Blocking experiments of the trophism modified adenovirus with excess anti-DC-SIGN monoclonal antibody confirmed that the augmented gene transfer achieved via the re-targeted adenovirus occurred exclusively via the DC-SIGN pathway. These studies clearly establish that one can modify adenovirus trophism such that gene transfer via the DC-SIGN pathway can be achieved. Of note, such DC-SIGN-mediated gene transfer allows enhanced transduction of DC-SIGN positive target cells.
The present invention is directed to a targeted recombinant adenovirus vector which may comprise a modified fiber protein with an immunoglobulin-binding domain and an anti-DC-SIGN antibody as targeting ligand. The invention provides for a targeted recombinant adenovirus vector, which may comprise: (i) a gene encoding a heterologous protein; (ii) a modified fiber protein comprising an immunoglobulin-binding domain; and (iii) an anti-DC-SIGN antibody, wherein binding of the immunoglobulin-binding domain to the antibody connects the antibody to the modified fiber protein, thereby targeting the adenovirus vector to a DC-SIGN positive cell. In one embodiment, the DC-SIGN positive cell may be an immature dendritic cell.
In one embodiment, the immunoglobulin-binding domain may be inserted at the HI loop or the carboxy terminal of the fiber protein. In another embodiment, immunoglobulin-binding domain inserted at the HI loop may be flanked by flexible linkers. In yet another embodiment, immunoglobulin-binding domain may be the Fc-binding domain of Staphylococcus aureus Protein A.
In one embodiment, the heterologous protein may be a tumor associated antigen. In another embodiment, the gene encoding the heterologous protein may be operably linked to a dendritic cell-specific promoter.
The invention also provides for a gene delivery system for the genetic manipulation of immune system cells with a vector encoding a DC-SIGN ligand, such as an anti DC-SIGN antibody. In one embodiment, the gene delivery system may comprise a targeted recombinant adenoviral vector. In another embodiment, the genetic manipulation may be selected from the group consisting of transduction, immunomodulation and maturation. In yet another embodiment, the immune system cells may be dendritic cells. The dendritic cells may include, but are not limited to, monocyte-derived dendritic cells, bone marrow-derived dendritic cells and cutaneous dendritic cells.
The present invention also encompasses methods of employing these DC-SIGN-targeted adenoviral vectors to deliver genes to immature dendritic cells. The invention provides for a method of gene transfer to immature dendritic cells which may comprise the step of contacting the cells with a vector which may comprise (i) a gene encoding a heterologous protein and (ii) a DC-SIGN targeting ligand, wherein the DC-SIGN targeting ligand targets the vector to a DC-SIGN positive cell and the vector mediates transfer of the gene encoding the heterologous protein to the dendritic cells. In one embodiment, the vector may be a targeted adenovirus vector, such as the targeted adenovirus vector described above. In another embodiment, the DC-SIGN targeting ligand may be an anti DC-SIGN antibody. In yet another embodiment, the heterologous protein may be a tumor associated antigen. In yet another embodiment, the gene encoding the heterologous protein may be operably linked to a dendritic cell-specific promoter.
The invention also provides for a method for modulating immunological status of dendritic cells which may comprise administering a composition comprising a DC-SIGN targeting ligand. In one embodiment, the DC-SIGN targeting ligand may be an anti-DC-SIGN antibody. In another embodiment, the composition may comprise a targeted recombinant adenoviral vector. The dendritic cells may include, but are not limited to, monocyte-derived dendritic cells, bone marrow-derived dendritic cells and cutaneous dendritic cells.
It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises”, “comprised”, “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes”, “included”, “including”, and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.
These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which:
A number of studies have highlighted the important consequences of genetically modified dendritic cells. A vector to achieve efficient gene transfer to this cell type becomes paramount to many immunomodulatory strategies and yet current vector systems have struggled with low efficiency of gene transfer. Adenovirus has been used in the context of dendritic cell transduction, but its efficiency of gene delivery has proven suboptimal. By means of targeting by anti-DC-SIGN antibody, the present invention successfully demonstrates enhanced gene transfer to dendritic cells by retargeting adenovirus to the dendritic cell-specific marker DC-SIGN.
The present invention describes an adenoviral vector targeting approach that combines the advantages of the previously established protein bridge-mediated and genetic modification of virus tropism. It is an object of the present invention to develop an adenoviral vector system in which genetic modifications done to both the adenoviral vector capsid and targeting ligand would allow them to self-associate into a stable complex.
The present invention is directed to a targeted recombinant adenovirus vector comprising a modified fiber protein with an immunoglobulin-binding domain and an anti-DC-SIGN antibody as targeting ligand. Binding of the immunoglobulin-binding domain to the Fc domain of the modified fiber protein would connect the antibody of the modified fiber protein, thereby targeting the adenovirus vector to DC-SIGN positive cells such as immature dendritic cells. The immunoglobulin-binding domain (for example, the Fc-binding domain of Staphylococcus aureus Protein A) can be inserted at the HI loop or the carboxy terminal of the modified fiber protein.
The present invention is directed to a targeted recombinant adenovirus vector comprising a modified fiber protein with an immunoglobulin-binding domain and an anti-DC-SIGN antibody as targeting ligand. The invention provides for a targeted recombinant adenovirus vector, comprising: (i) a gene encoding a heterologous protein; (ii) a modified fiber protein comprising an immunoglobulin-binding domain; and (iii) an anti-DC-SIGN antibody, wherein binding of the immunoglobulin-binding domain to the antibody connects the antibody to the modified fiber protein, thereby targeting the adenovirus vector to a DC-SIGN positive cell. In one embodiment, the DC-SIGN positive cell is an immature dendritic cell. In another embodiment, the immunoglobulin-binding domain is inserted at the HI loop or the carboxy terminal of the fiber protein. In yet another embodiment, immunoglobulin-binding domain inserted at the HI loop is flanked by flexible linkers. In another embodiment, immunoglobulin-binding domain is the Fc-binding domain of Staphylococcus aureus Protein A.
The 59 amino acids long domain C of Protein A can be incorporated into either the HI loop or the carboxy terminus of Ad5 fiber to create a docking site for the Fc domain of immunoglobulin or a Fc-modified targeting ligand. None of the modifications affected the yield or the growth dynamics of the resultant adenoviral vectors. The engineered fibers could be incorporated into mature Ad virions very efficiently. Apparently, none of these modifications caused any significant changes in the folding of the fiber, as its binding to natural adenoviral receptor, CAR, which requires the involvement of amino acid residues localized in two knob subunits, was not affected. The high degree of structural similarity of adenoviral fiber knob domains from different serotypes predicts the compatibility of Protein A domain C with the frameworks of fiber knobs other than that of Ad5.
The Fc domain of Ig functions as an element of the two-component mechanism mediating the association of the targeting ligand (e.g. anti-DC-SIGN antibody) with the virus. When mixed together, the Protein A-modified Ad and the targeting antibody undergo self-assembly into a targeting complex that can be purified from unincorporated ligand and then stored as a ready-to-use reagent while retaining its gene delivery properties.
The present invention is a new version of the protein bridge-based targeting approach that offers significant advantages over previously described methods. For instance, the targeting approach disclosed herein favorably compares to previously used strategy employing chemical cross-linking of antibodies to form targeting conjugate. Generation of those chemical cross-linked conjugates was proved to be inefficient and thus required large amounts of starting components. Reproducibility in the yields of the cross-linked conjugates is also an issue.
In accordance with the present invention, there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, “Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: A Practical Approach,” Volumes I and II (D. N. Glover ed. 1985); “Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic Acid Hybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcription and Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal Cell Culture” [R. I. Freshney, ed. (1986)]; “Imobilized Cells and Enzymes” [IRL Press, (1986)]; B. Perbal, “A Practical Guide to Molecular Cloning” (1984). Therefore, if appearing herein, the following terms shall have the definitions set out below.
The term used herein is intended to encompass both polyclonal and monoclonal antibodies. The term antibody is also intended to encompass whole antibodies, biologically functional fragments thereof, chimeric and humanized antibodies comprising portions from more than one species. Also encompassed in the term antibody are antibodies and biologically functional fragments thereof with alterations in glycosylation or with alterations in complement binding function.
Biologically functional antibody fragments are those fragments sufficient for binding to DC-SIGN, such as Fab, Fv, F(ab′)2, and sFv (single-chain antigen-binding protein) fragments. Antibody fragments can be generated by methods known to those skilled in the art, e.g. by enzymatic digestion of naturally occurring or recombinant antibodies; by recombinant DNA techniques using an expression vector that encodes a defined fragment of an antibody; by chemical synthesis; or by using bacteriophage to display and select polypeptide chains expressed from a V-gene library. One can choose among these or whole antibodies for the properties appropriate to a particular method.
Chimeric antibodies can comprise proteins derived from two different species. The portions derived from two different species can be joined together chemically by conventional techniques or can be prepared as a single contiguous protein using genetic engineering techniques (See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567, Neuberger et al., WO 86/01533 and Winter, EP 0,239,400). Such engineered antibodies can be, for instance, complementarity determining regions (CDR)-grafted antibodies (Tempest et al, Biotechnology 9:266-271 (1991)) or “hyperchimeric” CDR-grated antibodies which employ a human-mouse framework sequence chosen by computer modeling (Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989)).
Single chain V region fragments (“scFv”) can also be produced. Single chain V region fragments are made by linking L (light) and/or H (heavy) chain V (variable) regions by using a short linking peptide (Bird et al. (1988) Science 242:423). Any peptide having sufficient flexibility and length can be used as a linker in a scFv. Usually the linker is selected to have little to no immunogenicity. An example of a linking peptide is (GGGGS)3 (SEQ ID NO. 1) which bridges approximately 3.5 nm between the carboxy terminus of one V region and the amino terminus of another V region. Other linker sequences can also be used, and can provide additional functions, such as for attaching a drug or a solid support.
All or any portion of the H or L chain can be used in any combination. Typically, the entire V regions are included in the scFv. For instance, the L chain V region can be linked to the H chain V region. Alternatively, a portion of the L chain V region can be linked to the H chain V region or a portion thereof. Also contemplated are scFvs in which the H chain V region is from H 11, and the L chain V region is from another immunoglobulin. It is also possible to construct a biphasic, scFv in which one component is any target polypeptide and another component is a different polypeptide, such as a T cell epitope.
The scFvs can be assembled in any order, for example, VH-(linker)-VL or VL-(linker)-VH. There may be a difference in the level of expression of these two configurations in particular expression systems, in which case one of these forms may be preferred. Tandem scFvs can also be made, such as (X)-(linker)-(X)-(linker)-(X), in which X are target polypeptides, or combinations of the target polypeptides with other polypeptides. In another embodiment, single chain antibody polypeptides have no linker polypeptide, or just a short, inflexible linker. Exemplary configurations include VL-VH and VH-VL. The linkage is too short to permit interaction between VL and VH within the chain, and the chains form homodimers with a VL/VH antigen-binding site at each end. Such molecules are referred to in the art as “diabodies”.
ScFvs can be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as Escherichia coli, and the protein expressed by the polynucleotide can be isolated using standard protein purification techniques.
A particularly useful system for the production of scFvs is plasmid pET-22b(+) (Novagen, Madison, Wis.) in E. coli. pET-22b(+) contains a nickel ion binding domain consisting of 6 sequential histidine residues, which allows the expressed protein to be purified on a suitable affinity resin. Another example of a suitable vector is pcDNA3 (Invitrogen, San Diego, Calif.).
Humanized antibodies can also be used for methods of the invention. Humanized forms of non-human (e.g. murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab′, F(ab′).sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin.
A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. An “origin of replication” refers to those DNA sequences that participate in DNA synthesis. An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “operably linked” and “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
In general, expression vectors containing promoter sequences which facilitate the efficient transcription and translation of the inserted DNA fragment are used in connection with the host. The expression vector typically contains an origin of replication, promoter(s), terminator(s), as well as specific genes which are capable of providing phenotypic selection in transformed cells. The transformed hosts can be fermented and cultured according to means known in the art to achieve optimal cell growth.
A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence. A “cDNA” is defined as copy-DNA or complementary-DNA, and is a product of a reverse transcription reaction from an mRNA transcript.
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. A “cis-element” is a nucleotide sequence, also termed a “consensus sequence” or “motif”, that interacts with other proteins which can upregulate or downregulate expression of a specific gene locus. A “signal sequence” can also be included with the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell and directs the polypeptide to the appropriate cellular location. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes.
A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
The term “oligonucleotide” is defined as a molecule comprised of two or more deoxyribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use for the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence to hybridize therewith and thereby form the template for the synthesis of the extension product.
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to enzymes which cut double-stranded DNA at or near a specific nucleotide sequence.
“Recombinant DNA technology” refers to techniques for uniting two heterologous DNA molecules, usually as a result of in vitro ligation of DNAs from different organisms. Recombinant DNA molecules are commonly produced by experiments in genetic engineering. Synonymous terms include “gene splicing”, “molecular cloning” and “genetic engineering”. The product of these manipulations results in a “recombinant” or “recombinant molecule”.
A cell has been “transformed” or “transfected” with exogenous or heterologous DNA when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a vector or plasmid. With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA. A “clone” is a population of cells derived from a single cell or ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. An organism, such as a plant or animal, that has been transformed with exogenous DNA is termed “transgenic”.
As used herein, the term “host” is meant to include not only prokaryotes but also eukaryotes such as yeast, plant and animal cells. Prokaryotic hosts may include E. coli, S. tymphimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris, mammalian cells and insect cells and plant cells, such as Arabidopsis thaliana and Tobaccum nicotiana.
Two DNA sequences are “substantially homologous” when at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, preferably at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, most preferably at least about 90% , at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% at least about 98%, at least about 99%, at least about 99.5%, at least about 99.9% of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. Hybridization reactions can be performed under conditions of different “stringency.” Conditions that increase stringency of a hybridization reaction are well known. See for example, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C., and 68° C.; buffer concentrations of 10 x SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2 or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water.
For the purposes of the present invention, sequence identity or homology is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps. In particular, sequence identity may be determined using any of a number of mathematical algorithms. A nonlimiting example of a mathematical algorithm used for comparison of two sequences is the algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993;90: 5873-5877.
Another example of a mathematical algorithm used for comparison of sequences is the algorithm of Myers & Miller, CABIOS 1988;4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85: 2444-2448.
Advantageous for use according to the present invention is the WU-BLAST (Washington University BLAST) version 2.0 software. WU-BLAST version 2.0 executable programs for several UNIX platforms can be downloaded from ftp ://blast.wustl.edu/blast/executables. This program is based on WU-BLAST version 1.4, which in turn is based on the public domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Local alignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480; Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish & States, 1993;Nature Genetics 3: 266-272; Karlin & Altschul, 1993;Proc. Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated by reference herein).
In general, comparison of amino acid sequences is accomplished by aligning an amino acid sequence of a polypeptide of a known structure with the amino acid sequence of a the polypeptide of unknown structure. Amino acids in the sequences are then compared and groups of amino acids that are homologous are grouped together. This method detects conserved regions of the polypeptides and accounts for amino acid insertions and deletions. Homology between amino acid sequences can be determined by using commercially available algorithms (see also the description of homology above). In addition to those otherwise mentioned herein, mention is made too of the programs BLAST, gapped BLAST, BLASTN, BLASTP, and PSI-BLAST, provided by the National Center for Biotechnology Information. These programs are widely used in the art for this purpose and can align homologous regions of two amino acid sequences.
In all search programs in the suite the gapped alignment routines are integral to the database search itself. Gapping can be turned off if desired. The default penalty (Q) for a gap of length one is Q=9 for proteins and BLASTP, and Q=10 for BLASTN, but may be changed to any integer. The default per-residue penalty for extending a gap (R) is R=2 for proteins and BLASTP, and R=10 for BLASTN, but may be changed to any integer. Any combination of values for Q and R can be used in order to align sequences so as to maximize overlap and identity while minimizing sequence gaps. The default amino acid comparison matrix is BLOSUM62, but other amino acid comparison matrices such as PAM can be utilized.
Alternatively or additionally, the term “homology” or “identity”, for instance, with respect to a nucleotide or amino acid sequence, can indicate a quantitative measure of homology between two sequences. The percent sequence homology can be calculated as (Nref−Ndif)*100/Nref, wherein Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Nref=8; Ndif=2).
Alternatively or additionally, “homology” or “identity” with respect to sequences can refer to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the two sequences wherein alignment of the two sequences can be determined in accordance with the Wilbur and Lipman algorithm (Wilbur & Lipman, Proc Natl Acad Sci USA 1983; 80:726, incorporated herein by reference), for instance, using a window size of 20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4, and computer-assisted analysis and interpretation of the sequence data including alignment can be conveniently performed using commercially available programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc. CA). When RNA sequences are the to be similar, or have a degree of sequence identity or homology with DNA sequences, thymidine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence. Thus, RNA sequences are within the scope of the invention and can be derived from DNA sequences, by thymidine (T) in the DNA sequence being considered equal to uracil (U) in RNA sequences.
And, without undue experimentation, the skilled artisan can consult with many other programs or references for determining percent homology.
A “heterologous” region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. In another example, the coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. For example, a polynucleotide, may be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
In addition, the invention may includes portions or fragments of the fiber or fibritin genes. As used herein, “fragment” or “portion” as applied to a gene or a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments of these genes can be generated by methods known to those skilled in the art, e.g., by restriction digestion of naturally occurring or recombinant fiber or fibritin genes, by recombinant DNA techniques using a vector that encodes a defined fragment of the fiber or fibritin gene, or by chemical synthesis.
As used herein, “chimera” or “chimeric” refers to a single transcription unit possessing multiple components, often but not necessarily from different organisms. As used herein, “chimeric” is used to refer to tandemly arranged coding sequence (in this case, that which usually codes for the adenovirus fiber gene) that have been genetically engineered to result in a protein possessing region corresponding to the functions or activities of the individual coding sequences.
The “native biosynthesis profile” of the chimeric fiber protein as used herein is defined as exhibiting correct trimerization, proper association with the adenovirus capsid, ability of the ligand to bind its target, etc. The ability of a candidate chimeric fiber-fibritin-ligand protein fragment to exhibit the “native biosynthesis profile” can be assessed by methods described herein.
A standard Northern blot assay can be used to ascertain the relative amounts of mRNA in a cell or tissue in accordance with conventional Northern hybridization techniques known to those persons of ordinary skill in the art. Alternatively, a standard Southern blot assay may be used to confirm the presence and the copy number of the gene of interest in accordance with conventional Southern hybridization techniques known to those of ordinary skill in the art. Both the Northern blot and Southern blot use a hybridization probe, e.g. radiolabelled cDNA or oligonucleotide of at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, preferably at least 30, , at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, at least 41, at least 42, at least 43, at least 44, at least 45, at least 46, at least 47, at least 48, at least 49, more preferably at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95 and most preferably at least 100 consecutive nucleotides in length. The DNA hybridization probe can be labelled by any of the many different methods known to those skilled in this art.
Hybridization reactions can be performed under conditions of different “stringency.” Conditions that increase stringency of a hybridization reaction are well known. See for examples, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. 1989). Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25° C., 37° C., 50° C., and 68° C.; buffer concentrations of 10×SSC, 6×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2 or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionized water.
The labels most commonly employed for these studies are radioactive elements, enzymes, chemicals which fluoresce when exposed to untraviolet light, and others. A number of fluorescent materials are known and can be utilized as labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate. Proteins can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures. The preferred isotope may be selected from 3H, 4C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 25I, 131I, and 186Re.
Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques. The enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like. Many enzymes which can be used in these procedures are known and can be utilized. The preferred are peroxidase, β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090, 3,850,752, and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
The invention also encompasses viral vectors, preferably an adenoviral vector comprising the adenovirus of described herein. In one embodiment, adenovirus is operatively linked to a non-viral promoter.
Methods for making and/or administering a vector or recombinants or plasmid for expression of gene products of genes of the invention either in vivo or in vitro can be any desired method, e.g., a method which is by or analogous to the methods disclosed in, or disclosed in documents cited in: U.S. Pat. Nos. 4,603,112; 4,769,330; 4,394,448; 4,722,848; 4,745,051; 4,769,331; 4,945,050; 5,494,807; 5,514,375; 5,744,140; 5,744,141; 5,756,103; 5,762,938; 5,766,599; 5,990,091; 5,174,993; 5,505,941; 5,338,683; 5,494,807; 5,591,639; 5,589,466; 5,677,178; 5,591,439; 5,552,143; 5,580,859; 6,130,066; 6,004,777; 6,130,066; 6,497,883; 6,464,984; 6,451,770; 6,391,314; 6,387,376; 6,376,473; 6,368,603; 6,348,196; 6,306,400; 6,228,846; 6,221,362; 6,217,883; 6,207,166; 6,207,165; 6,159,477; 6,153,199; 6,090,393; 6,074,649; 6,045,803; 6,033,670; 6,485,729; 6,103,526; 6,224,882; 6,312,682; 6,348,450 and 6;312,683; U.S. patent application Ser. No. 920,197, filed Oct. 16,1986; WO 90/01543; WO91/11525; WO 94/16716; WO 96/39491; WO 98/33510; EP 265785; EP 0 370 573; Andreansky et al., Proc. Natl. Acad. Sci. USA 1996;93:11313-11318; Ballay et al., EMBO J. 1993;4:3861-65; Feigner et al., J. Biol. Chem. 1994;269:2550-2561; Frolov et al., Proc. Natl. Acad. Sci. USA 1996;93: 11371-11377; Graham, Tibtech 1990;8:85-87; Grunhaus et al., Sem. Virol. 1992;3:237-52; Ju et al., Diabetologia 1998;41:736-739; Kitson et al., J. Virol. 1991;65:3068-3075; McClements et al., Proc. Natl. Acad. Sci. USA 1996;93:11414-11420; Moss, Proc. Natl. Acad. Sci. USA 1996;93: 11341-11348; Paoletti, Proc. Nati. Acad. Sci. USA 1996;93: 11349-11353; Pennock et al., Mol. Cell. Biol. 1984;4:399-406; Richardson (Ed), Methods in Molecular Biology 1995;39, “Baculovirus Expression Protocols,” Humana Press Inc.; Smith et al. (1983) Mol. Cell. Biol. 1983;3:2156-2165; Robertson et al., Proc. Natl. Acad. Sci. USA 1996;93:11334-11340; Robinson et al., Sem. Immunol. 1997;9:271; and Roizman, Proc. Natl. Acad. Sci. USA 1996;93:11307-11312.
According to one embodiment of the invention, the expression vector is a viral vector, in particular an in vivo expression vector. In an advantageous embodiment, the expression vector is an adenovirus vector, such as a human adenovirus (HAV) or a canine adenovirus (CAV). Advantageously, the adenovirus is a human Ad5 vector, an El-deleted adenovirus or an E3-deleted adenovirus.
In one embodiment the viral vector is a human adenovirus, in particular a serotype 5 adenovirus, rendered incompetent for replication by a deletion in the E1 region of the viral genome. The deleted adenovirus is propagated in E1-expressing 293 cells or PER cells, in particular PER.C6 (F. Falloux et al Human Gene Therapy 1998, 9, 1909-1917). The human adenovirus can be deleted in the E3 region eventually in combination with a deletion in the E1 region (see, e.g. J. Shriver et al. Nature, 2002, 415, 331-335, F. Graham et al Methods in Molecular Biology Vol. 7: Gene Transfer and Expression Protocols Edited by E. Murray, The Human Press Inc, 1991, p 109-128; Y. ilan et al Proc. Natl. Acad. Sci. 1997, 94, 2587-2592; S. Tripathy et al Proc. Natl. Acad. Sci. 1994, 91, 11557-11561; B. Tapnell Adv. Drug Deliv. Rev.1993, 12, 185-199;X. Danthinne et al Gene Thrapy 2000, 7, 1707-1714; K. Berkner Bio Techniques 1988, 6, 616-629; K. Berkner et al Nucl. Acid Res. 1983, 11, 6003-6020; C. Chavier et al J. Virol. 1996, 70, 4805-4810). The insertion sites can be the El and/or E3 loci eventually after a partial or complete deletion of the E1 and/or E3 regions. Advantageously, when the expression vector is an adenovirus, the polynucleotide to be expressed is inserted under the control of a promoter functional in eukaryotic cells, such as a strong promoter, preferably a cytomegalovirus immediate-early gene promoter (CMV-IE promoter). The CMV-IE promoter is advantageously of murine or human origin. The promoter of the elongation factor la can also be used. In one particular embodiment a promoter regulated by hypoxia, e.g. the promoter HRE described in K. Boast et al Human Gene Therapy 1999, 13, 2197-2208), can be used. A muscle specific promoter can also be used (X. Li et al Nat. Biotechnol. 1999, 17, 241-245). Strong promoters are also discussed herein in relation to plasmid vectors. A poly(A) sequence and terminator sequence can be inserted downstream the polynucleotide to be expressed, e.g. a bovine growth hormone gene or a rabbit β-globin gene polyadenylation signal.
In another embodiment the viral vector is a canine adenovirus, in particular a CAV-2 (see, e.g. L. Fischer et al. Vaccine, 2002, 20, 3485-3497; U.S. Pat. No. 5,529,780; U.S. Pat. No. 5,688,920; PCT Application No. WO95/14102). For CAV, the insertion sites can be in the E3 region and /or in the region located between the E4 region and the right ITR region (see U.S. Pat. No. 6,090,393; U.S. Pat. No. 6,156,567). In one embodiment the insert is under the control of a promoter, such as a cytomegalovirus immediate-early gene promoter (CMV-IE promoter) or a promoter already described for a human adenovirus vector. A poly(A) sequence and terminator sequence can be inserted downstream the polynucleotide to be expressed, e.g. a bovine growth hormone gene or a rabbit β-globin gene polyadenylation signal.
The invention also provides for transformed host cells comprising such vectors. In one embodiment, the vector is introduced into the cell by transfection, electroporation or transformation. The invention also provides for a method for preparing a transformed cell expressing the adenovirus of the present invention comprising transfecting, electroporating or transforming a cell with the adenovirus to produce a transformed host cell and maintaining the transformed host cell under biological conditions sufficient for expression of the adenovirus in the host cell.
According to another embodiment of the invention, the expression vectors are expression vectors used for the in vitro expression of proteins in an appropriate cell system. The expressed proteins can be harvested in or from the culture supernatant after, or not after secretion (if there is no secretion a cell lysis typically occurs or is performed), optionally concentrated by concentration methods such as ultrafiltration and/or purified by purification means, such as affinity, ion exchange or gel filtration-type chromatography methods.
It is understood to one of skill in the art that conditions for culturing a host cell varies according to the particular gene and that routine experimentation is necessary at times to determine the optimal conditions for culturing the vector depending on the host cell. A “host cell” denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector. When referring to genetically altered cells, the term refers both to the originally altered cell and to the progeny thereof.
Polynucleotides comprising a desired sequence can be inserted into a suitable cloning or expression vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides can be introduced into host cells by any means known in the art. The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including direct uptake, endocytosis, transfection, f-mating, electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is infectious, for instance, a retroviral vector). The choice of introducing vectors or polynucleotides will often depend on features of the host cell.
As used herein, “chimera” or “chimeric” refers to a single polypeptide possessing multiple components, often but not necessarily from different organisms. As used herein, “chimeric” is used to refer to tandemly arranged protein moieties that have been genetically engineered to result in a fusion protein possessing regions corresponding to the functions or activities of the individual protein moieties.
As used herein the term “physiologic ligand” refers to a ligand for a cell surface receptor.
As used herein, “fragment” or “portion” as applied to a protein or a polypeptide, will ordinarily be at least 10 residues, more typically at least 20 residues, and preferably at least 30 (e.g., 50) residues in length, but less than the entire, intact sequence. Fragments of these genes can be generated by methods known to those skilled in the art, e.g., by restriction digestion of naturally occurring or recombinant genes, by recombinant DNA techniques using a vector that encodes a defined fragment of the gene, or by chemical synthesis.
The invention provides for the expression of a heterologous protein. In one embodiment, the heterologous protein is a tumor associated antigen. In another embodiment, the gene encoding the heterologous protein is operably linked to a dendritic cell-specific promoter.
The present invention is also directed to a method of gene transfer to immature dendritic cells using the DC-SIGN-targeted adenoviral vector disclosed herein. The invention also provides for a gene delivery system for the genetic manipulation of immune system cells with a vector encoding a DC-SIGN ligand, such as an anti DC-SIGN antibody. The anti DC-SIGN antibody may be a chimeric, humanized or single chain antibody.
In another embodiment, the DC-SIGN ligand is any ligand that binds DC-SIGN. In a preferred embodiment, the DC-SIGN targeting ligand is a peptide. Methods of identifying specific targeting ligands are well known to one of skill in the art (see, e.g., U.S. Pat. Nos. 6,632,621; 6,593,108; 5,939,322; 5,679,518 and 5,607,967; the disclosures of which are incorporated by reference).
In one embodiment, the gene delivery system comprises a targeted recombinant adenoviral vector. In another embodiment, the genetic manipulation is selected from the group consisting of transduction, immunomodulation and maturation. In yet another embodiment, the immune system cells are dendritic cells. The dendritic cells include, but are not limited to, monocyte-derived dendritic cells, bone marrow-derived dendritic cells and cutaneous dendritic cells.
The present invention also encompasses methods of employing these DC-SIGN-targeted adenoviral vectors to deliver genes to immature dendritic cells. The invention provides for a method of gene transfer to immature dendritic cells comprising the step of contacting the cells with a vector comprising (i) a gene encoding a heterologous protein and (ii) a DC-SIGN targeting ligand, wherein the DC-SIGN targeting ligand targets the vector to a DC-SIGN positive cell and the vector mediates transfer of the gene encoding the heterologous protein to the dendritic cells. In one embodiment, the vector is a targeted adenovirus vector, such as the targeted adenovirus vector described above. In another embodiment, the DC-SIGN targeting ligand is an anti DC-SIGN antibody. In yet another embodiment, the heterologous protein is a tumor associated antigen. In yet another embodiment, the gene encoding the heterologous protein is operably linked to a dendritic cell-specific promoter.
The present invention also provides for modulating the immunological status of dendritic cells by using the methods described herein. Antibody-based targeting resulted in modulation of the immunological status of dendritic cells by inducing their maturation. This was demonstrated phenotypically by increased expression of CD83, MHC, and costimulatory molecules, as well as functionally by production of IL-12 and an enhanced allostimulatory capacity in a mixed lymphocyte reaction (MLR). It has been reported that activation of dendritic cells to maturity renders them resistant to the effects of dendritic cell inhibitory cytokines like IL-10 as well as to direct tumor-induced apoptosis. The capacity with which murine dendritic cells can generate an immune response in vivo has been shown to correlate with the degree of their maturation.
The invention also provides for a method for modulating immunological status of dendritic cells comprising administering a composition comprising a DC-SIGN targeting ligand. In one embodiment, the DC-SIGN targeting ligand is an anti-DC-SIGN antibody. In another embodiment, the composition comprises a targeted recombinant adenoviral vector. The dendritic cells include, but are not limited to, monocyte-derived dendritic cells, bone marrow-derived dendritic cells and cutaneous dendritic cells.
It is specifically contemplated that pharmaceutical compositions may be prepared using the novel adenoviral vector of the present invention. In such a case, the pharmaceutical composition comprises the novel adenoviral vector of the present invention and a pharmaceutically acceptable carrier. A person having ordinary skill in this art would readily be able to determine, without undue experimentation, the appropriate dosages and routes of administration of this adenoviral vector of the present invention. It will normally be administered intradermally or parenterally, preferably intravenously, but other routes of administration will be used as appropriate. See Remington's Pharmaceutical Science, 17th Ed. (1990) Mark Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon Press.
The invention will now be further described by way of the following non-limiting example.
293/DC-SIGN or THP/DC-SIGN cells were washed with growth medium and then incubated on ice with either plain medium, or medium containing purified protein. In the latter instance, recombinant Ad5 fiber knob at the concentration 100 μg/ml and/or anti-DC-SIGN monoclonal antibody (clone #612) or isotype monoclonal antibody at the concentration 10 μg/ml were added to the medium. One hour later, the cells were washed and infected at a multiplicity of infection of 10 (293/DC-SIGN) or 500 (THP/DC-SIGN) virus particles per cell. After incubation on ice for 1 h, the medium containing the virus was removed, and the cells were washed with medium containing 10% FCS. Fresh medium was added and the incubation continued at 37° C. for twenty hours to allow for reporter expression. Luciferase activity in the cell lysates was measured according to the manufacturer's protocol (Promega). Each data point was set in triplicate and calculated as the mean of three determinations.
As shown in
Having thus described in detail advantageous embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.
This application is a continuation-in-part application of international patent application Serial No. PCT[US2004/028671 filed Sep. 3, 2004, which claims benefit of U.S. provisional application Ser. No. 60/499,843 filed Sep. 3, 2003. The foregoing applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer's instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention.
This invention was supported in part using federal funds from the Department of Defense. Accordingly, the Federal Government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60499843 | Sep 2003 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US04/28671 | Sep 2004 | US |
| Child | 11365203 | Mar 2006 | US |
