HK1109421B - Chimeric adenoviruses for use in cancer treatment - Google Patents

Description

Chimeric adenoviruses for the treatment of cancer

This application claims priority from U.S. provisional application serial No. 60/574,851 filed on 26.5.2004, which is hereby incorporated by reference.

Technical Field

The present invention relates generally to the field of molecular biology, and more specifically to oncolytic adenoviruses having therapeutic applications.

Background

Cancer is a leading cause of death in the united states and other areas. Depending on the type of cancer, surgery, chemotherapy and/or radiation are typically used for treatment. These treatments often fail and it is clear that new treatments are needed, either alone or in combination with conventional techniques.

One approach is to use adenovirus, either alone or as a vehicle capable of delivering anti-cancer therapeutic proteins to tumor cells. Adenoviruses are nonenveloped icosahedral double-stranded DNA viruses with a genome of approximately 36 kilobase pairs. Each end of the viral genome has a short sequence known as Inverted Terminal Repeat (ITR), which is essential for viral replication. All human adenovirus genomes detected to date have the same overall structure; that is, the gene encoding a particular function is located at the same position in the viral genome. The viral genome contains 5 early transcription units (E1A, E1B, E2, E3 and E4), 2 delayed early units (IX and Iva2) and 1 late unit (major late) that is processed to produce 5 families of late mRNAs (L1-L5). The proteins encoded by the early genes are involved in replication, while the late genes encode viral structural proteins. A portion of the viral genome can be easily replaced with foreign DNA and recombinant adenoviruses are structurally stable, properties that make these viruses potentially useful for gene Therapy (see Jolly, D. (1994) cancer Gene Therapy 1: 51-64).

Currently, research efforts to produce clinically useful adenoviral therapy have focused on the adenoviral serotype, Ad 5. The genetics of this human adenovirus is well characterized and the system for its molecular manipulation is well described. Mass production methods have been developed to support clinical applications and some clinical experience with this formulation is available, see Jolly, d. (1994) Cancer Gene Therapy, 1: 51-64. Research involving the use of human adenoviruses (Ad) in cancer therapy has focused on developing Ad 5-based adenoviruses that are more potent in, or preferentially target, specific tumor cell types; and there is a need to produce more effective oncolytic viruses if it is desired to make adenoviral therapy clinically practical.

Ad5 is only one of 51 currently known adenoviral serotypes, which are classified into subgroups A through F based on various properties including erythrocyte coagulation properties (see, Shenk, "Adenovirdae: The Viruses and Their Replication," Fields Virology, Vol.2, Fourth Edition, Knipe, ea., Lippincott, Williams & Wilkins, pp.2265-2267 (2001)). These serotypes differ at several levels, such as pathology in humans and rodents, cell receptors for attachment, but these differences are largely ignored as potential methods for developing more effective oncolytic adenoviruses (see Stevenson et al (1997) J.Virol.71: 4782. 4790; Krasykh et al (1996) J.Virol.70: 6839. 6846; Wickhamamet. 1997) J.Virol.71: 8221. 8229; Legrand et al (2002) curr. Gene Ther.2: 323. Barnett et al (2002) Biochim. Biophys. acta.1-3: 1-14; U.S. patent application 2003/0017138), in addition to fibrin changes (fiber optics).

The use of differences between adenoviral serotypes may provide a source for more effective adenoviral-based therapies that utilize novel adenoviruses with enhanced selectivity and potency. There is a need for such improved adenovirus-based therapies.

Summary of The Invention

The present invention provides novel chimeric adenoviruses, or variants or derivatives thereof, useful for virus-based therapy. In particular, the invention provides a chimeric adenovirus, or a variant or derivative thereof, having a genome comprising the E2B region,

wherein said E2B region comprises a nucleic acid sequence derived from a first adenovirus serotype and a nucleic acid sequence derived from a second adenovirus serotype;

wherein said first and second adenovirus serotypes are each selected from adenovirus subgroup B, C, D, E or F, and are distinct from each other; and the number of the first and second groups,

wherein the chimeric adenovirus is oncolytic and exhibits an increased therapeutic index for a tumor cell.

In one embodiment, the chimeric adenovirus further comprises regions encoding a fiber protein, a penton protein, and a hexon protein, wherein the nucleic acids encoding the proteins are all from the same adenovirus serotype. In another embodiment, the chimeric adenoviruses of the invention comprise a modified E3 or E4 region.

In another embodiment, the chimeric adenovirus exhibits an increased therapeutic index in a colon tumor cell, a breast tumor cell, a pancreatic tumor cell, a lung tumor cell, a prostate tumor cell, an ovarian tumor cell, or a hematopoietic tumor cell. In a particularly preferred embodiment, the chimeric adenovirus exhibits an increased therapeutic index in colon tumor cells.

In a preferred embodiment, the E2B region of the chimeric adenovirus comprises SEQ ID NO: 3. in a particularly preferred embodiment, the chimeric adenovirus comprises SEQ ID NO: 1.

the present invention provides a recombinant chimeric adenovirus, or a variant or derivative thereof, having a genome comprising an E2B region,

wherein said E2B region comprises a nucleic acid sequence derived from a first adenovirus serotype and a nucleic acid sequence derived from a second adenovirus serotype;

wherein said first and second adenovirus serotypes are each selected from adenovirus subgroup B, C, D, E or F, and are distinct from each other;

wherein the chimeric adenovirus is oncolytic and exhibits an increased therapeutic index for a tumor cell; and

wherein the chimeric adenovirus has been made replication-deficient by deletion of one or more adenoviral regions encoding proteins involved in adenoviral replication selected from the group consisting of E1, E2, E3, and E4.

In one embodiment, the chimeric adenovirus of the invention further comprises a heterologous gene encoding a therapeutic protein, wherein said heterologous gene is expressed in a cell infected with said adenovirus. In a preferred embodiment, the therapeutic protein is selected from the group consisting of cytokines and chemokines, antibodies, prodrug converting enzymes, and immunomodulatory proteins.

The invention provides methods for using the chimeric adenoviruses of the invention for therapeutic purposes. In one embodiment, the chimeric adenovirus can be used to inhibit the growth of a cancer cell. In a particular embodiment, the polypeptide comprising SEQ ID NO: 1 is used to inhibit the growth of colon cancer cells.

In another embodiment, the chimeric adenoviruses of the invention are useful as vectors for delivering therapeutic proteins to cells.

The present invention provides a method for preparing the chimeric adenovirus of the present invention, wherein the method comprises:

h) aggregating adenovirus serotypes representing adenovirus subgroups B through F, thereby generating an adenovirus mixture;

i) passaging the pooled adenoviral mixture from step (a) in actively growing cultures of tumor cells at a particle/cell ratio high enough to promote recombination between serotypes, but not so high as to produce premature cell death;

j) harvesting the supernatant from step (b);

k) infecting a quiescent culture of tumor cells with the supernatant harvested in step (c);

l) harvesting cell culture supernatant from step (d) before any signs of cytopathic effect (CPE) appear;

m) infecting a quiescent culture of tumor cells with the supernatant harvested in step (e); and

n) isolating the chimeric adenovirus from the supernatant harvested in step (f) by plaque purification.

Drawings

FIG. 1: retention time profile of Ad on TMAE HPLC column. A) Retention profiles for each Ad serotype used to generate the original starting viral pool. B) Retention profiles of 20 generation passaged pools derived from HT-29, Panc-1, MDA-231, and PC-3 cell lines, respectively.

FIG. 2: cytolytic activity of each virus pool. A) HT-29, B) MDA-231, C) Panc-1 and D) PC-3 cells were infected with the respective virus pools at a VP/cell ratio of 100-0.01. MTS assays were performed on different days post infection (as indicated in the figure) for different cell lines. Each data point in the plot represents a quadruplicate test, with results expressed as mean +/-standard deviation. Each panel depicts a representative assay and all viral pools were tested at least 3 independent times for target tumor cell lines (legend: - ● -Ad 5-)Initial viral pool, - ■ -specific cells derived from the pool, passage 20).

FIG. 3: cytolytic activity of ColoAd1 and Ad5 on human tumor cell lines. MTS assays were performed on A) a large panel of human tumor cell lines, and B) a panel of human colon cancer cell lines to determine their potential potency specificity. The MTS assay was performed on different days according to different cell lines. Each panel is a representative experiment repeated at least 3 times. Each data point in the plot represents a quadruplicate test, with the results being averaged+/-Standard deviation representation (legend: - ● -Ad 5; -)—ColoAd1)。

FIG. 4: cytolytic activity of ColoAd1 and Ad5 on one panel indicated cells. HS-27, HUVEC and SAEC cells (primary fibroblasts, endothelial and epithelial cells, respectively) were infected with ColoAd1 and Ad5 at VP/cell ratios of 100-0.01. MTS assays were performed on different cells on different days post-infection, with each panel representing a representative experiment repeated at least 3 times. Each data point in the plot represents a quadruplicate test, and the results are expressed as mean +/-standard deviation (legend: - ● -Ad 5-)—ColoAd1)。

FIG. 5: cytolytic activity of ColoAd1, Ad5 and ONYX-015 against primitive normal endothelial cells (HUVEC) and a colon tumor cell line (HT-29). Each panel is a representative experiment repeated at least 3 times. Each data point in the plot represents a quadruplicate test, with results expressed as mean +/-standard deviation (legend: - ● -Ad 5-)—ColoAd1、—■—Onyx-015)。

FIG. 6: cytolytic activity of ColoAd1, Ad11p and Ad5 on normal epithelial cell lines (SAEC) and human colon cancer cell lines (HT-29). Each panel is a representative experiment repeated at least 3 times. Each data point in the plot represents a quadruplicate test, with results expressed as mean +/-standard deviation (legend: - ● -Ad 5-)—Ad11p、—■—ColoAd1)。

FIG. 7: cytolytic activity of the recombinant virus. Construction according to the description of example 6 represents 4Recombinant viruses of the viral population (Adp11, ColoAd1, left-hand Ad11 p/right-hand ColoAd1(ColoAd1.1) and left-hand ColoAd 1/right-hand Ad11p (ColoAd1.2)). The lytic activity of each population in HT29 cells was determined as described previously. (legend: - ● -Ad 5-)—Ad11p、—■—ColoAd1、—υ—ColoAd1.1、—▲—ColoAd1.2)。

Detailed Description

All publications, including patents and patent applications, mentioned in the specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication was specifically and individually indicated to be incorporated herein by reference in its entirety.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the test methods described below are those well known and commonly employed in the art.

The terms "adenovirus," "serotype," or "adenovirus serotype" as used herein refer to any of the 51 human adenovirus serotypes currently known or later isolated. See, for example, Strauss, "Adenoviruses infestations inhumans", The Adenoviruses, Ginsberg, ea., Plenum Press, New York, NY, pp.451-596 (1984). These serotypes are classified in subgroups A to F (see, Shenk, "Adenovirdae: The Viruses and The radiation," Fields virology, Vol.2, Fourth Edition, Knipe, ea., Lippincott Williams & Wilkins, pp.2265-2267, 2001), as shown in Table 1.

TABLE 1

Subgroup of	Adenovirus serotype
		A	12，18，31
B	3，7，11，14，16，21，34，35，51
		C	1，2，5，6
D	8-10，13，15，17，19，20，22-30，32，33，36-39，42-49，50
		E	4
F	40，41

The term "chimeric adenovirus" as used herein refers to an adenovirus whose nucleic acid sequence consists of at least two of the adenoviral serotypes described above.

The term "parental adenovirus serotype" as used herein refers to the adenovirus serotype from which most of the genome of the chimeric adenovirus is derived.

The term "homologous recombination" as used herein refers to the exchange or recombination of two nucleic acid molecules having homologous sequences in the region of homology.

As used herein, the term "titer" refers to the lytic potential of a virus, representing its ability to replicate, lyse and spread. For the purposes of the present invention, the titer is the ratio of the cytolytic activity of a given adenovirus according to the invention to the cytolytic activity of Ad5 in the same cell line, i.e.the titer is AdX IC₅₀IC of/Ad 5₅₀Wherein X is the particular adenovirus serotype being tested, and wherein the titer of Ad5 is assigned a value of 1.

The term "oncolytic virus" as used herein refers to a virus that preferentially kills cancer cells over normal cells.

The term "quality index" or "therapeutic window" as used herein refers to a number that indicates the oncolytic potential of a given adenovirus and is determined by dividing the potency of the adenovirus in a cancer cell line by the potency of the same adenovirus in a normal (i.e., non-cancer) cell line.

The term "modified" as used herein refers to a molecule having a nucleotide or amino acid sequence that is different from a naturally occurring nucleotide or amino acid sequence, i.e., a wild-type nucleotide or amino acid sequence. The modified molecule is capable of retaining the function or activity of the wild-type molecule, i.e., the modified adenovirus may retain its oncolytic activity. Modifications include mutations of the nucleotides described below.

The term "mutation" as used herein in relation to a polynucleotide or polypeptide refers to a naturally occurring, synthetic, recombinant, or chemical alteration or difference in the primary, secondary, or tertiary structure of the polynucleotide or polypeptide, respectively, as compared to a reference polynucleotide or polypeptide (e.g., as compared to the wild-type polynucleotide or polypeptide). Mutations include such changes as deletions, insertions or substitutions.

The term "deletion" as used herein is defined as a change in the polynucleotide or amino acid sequence in which one or more polynucleotide or amino acid residues, respectively, are absent.

The term "insertion" or "addition" as used herein refers to a change that occurs in a polynucleotide or amino acid sequence resulting in the addition of one or more polynucleotides or amino acid residues, respectively, as compared to the naturally occurring polynucleotide or amino acid sequence.

The term "substitution" as used herein results from the replacement of one or more polynucleotides or amino acids with a different polynucleotide or amino acid, respectively.

As used herein, the term "adenoviral derivative" refers to a modified adenovirus of the invention, to or in the viral genome of which additions, deletions or substitutions have been made, which exhibits a higher potency and/or therapeutic index, or is otherwise therapeutically more useful (i.e., lower immunogenicity, increased clearance) than the parental adenovirus. For example, derivatives of the adenoviruses of the invention may have a deletion in one of the early genes of the viral genome, including but not limited to the E1A or E2B region of the viral genome.

The term "variant" as used herein in relation to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that may have a change in primary, secondary or tertiary structure as compared to a reference polynucleotide or polypeptide, respectively (e.g., as compared to a wild-type polynucleotide or polypeptide). For example, the amino acid or nucleic acid sequence may contain mutations or modifications that differ from a reference amino acid or nucleic acid sequence. In some embodiments, the adenoviral variants can be of different isotypes or polymorphisms. Variants may be naturally occurring, synthetic, recombinant, or chemically modified polynucleotides or polypeptides isolated or produced using methods known in the art. The alteration in the polynucleotide sequence of the variant may be silent. That is, they may not alter the amino acids encoded by the polynucleotide. If the alteration is limited to this type of silent alteration, the variant will encode a polypeptide having the same amino acid sequence as the reference sequence. Alternatively, such changes in the polynucleotide sequence of the variant may alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide, which results in conservative or non-conservative amino acid changes as described below. Such polynucleotide alterations may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Various codon substitutions (e.g., silent changes that produce various restriction sites) can be introduced to optimize their cloning into plasmids or viruses, or their expression in particular prokaryotic or eukaryotic systems.

The term "adenoviral variant" as used herein refers to an adenovirus whose polynucleotide sequence differs from a reference polynucleotide (e.g., wild-type adenovirus) as described above. This difference is limited such that the polynucleotide sequences of the parent and variant are similar overall and identical in most regions. In this application, a first nucleotide or amino acid sequence is assumed to be "similar" to a second sequence when compared, meaning that they have little sequence difference (i.e., the first and second sequences are nearly identical). In the present application, the polynucleotide sequence differences present between the adenoviral variant and the reference adenovirus do not result in differences in potency and/or therapeutic index.

The term "conservative" as used herein refers to the replacement of an amino acid residue by a different amino acid residue having similar chemical properties. Conservative amino acid substitutions include the substitution of isoleucine or valine for leucine, glutamic for aspartic acids, or serine for threonine. Typically, insertions or deletions are in the range of about 1-5 amino acids.

The term "non-conservative" as used herein refers to the replacement of an amino acid residue with a different amino acid residue having different chemical properties. Non-conservative substitutions include, but are not limited to, substitution of glycine (G) for aspartic acid (D), lysine (K) for asparagine (N), or arginine (R) for alanine (A).

The one letter codes for amino acids include the following: a ═ alanine, R ═ arginine, N ═ asparagine, D ═ aspartic acid, C ═ cysteine, Q ═ glutamine, E ═ glutamic acid, G ═ glycine, H ═ histidine, I ═ isoleucine, L ═ leucine, K ═ lysine, M ═ methionine, F ═ phenylalanine, P ═ proline, S ═ serine, T ═ threonine, W ═ tryptophan, Y ═ tyrosine, V ═ valine.

It will be appreciated that polypeptides often contain amino acids other than these 20 amino acids (commonly referred to as the 20 naturally occurring amino acids), and that many amino acids, including the terminating amino acids, may be modified into a given polypeptide by natural processing (e.g., glycosylation and other post-translational modifications) or by chemical modification techniques known in the art. Even the common modifications that occur naturally in polypeptides are numerous and cannot be exhausted here; they are described in basic texts and in more detailed monographs, as well as in the long study literature, and are well known to those skilled in the art. Among the known modifications that may be present in the polypeptides of the invention, there are exemplified: acetylation, acylation, ADP-ribosylation, amination, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a polynucleotide or polypeptide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation (selenoylation), sulfation, transfer of RNA-mediated addition of amino acids to proteins such as arginylation and ubiquitination.

Such modifications are known to those skilled in the art and have been described in great detail in the scientific literature. For example, several particularly common modifications (glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation) are described in most basic texts, such as i.e. Creighton, Proteins-structures and molecular Properties, 2nd Ed., W.H.Freeman and Company, New York, 1993. There are many detailed reviews on this subject, such as those described by Wold, f., Posttranslational equivalent Modification of Proteins, b.c. johnson, ed., Academic Press, New York, pp 1-12, 1983; seifter et al, meth.enzymol.182: 626. 646, 1990 and Rattan et al, Protein Synthesis: posttranslation modifications and Aging, ann.n.y.acad.sci.663: 48-62, 1992.

As is known and mentioned above, those skilled in the art will recognize that polypeptides are not always perfectly linear. For example, a polypeptide may be branched due to ubiquitination; they may be circular, with or without branches, typically due to post-translational events, including natural processing events and events resulting from non-naturally occurring manual manipulations. Cyclic, branched and branched cyclic polypeptides can be synthesized by non-translational natural processes as well as by entirely synthetic methods.

Modifications can occur anywhere in the polypeptide, including the peptide backbone, the amino acid side chains, and the amino or carboxyl termini. Indeed, blocking amino or carboxyl groups of polypeptides by covalent modification is common in naturally occurring and synthetic polypeptides, and such modifications may also be present in the polypeptides of the invention. For example, the amino-terminal residue of a polypeptide prepared in e.coli (e.coli) will almost always be N-formylmethionine before being subjected to proteolytic processing.

The modifications that occur in a polypeptide are often related to how the polypeptide is produced. For example, for a polypeptide prepared by expressing a cloned gene in a host, the nature and extent of most modifications will depend on the host cell's posttranslational modification capability and the modification signals present in the amino acid sequence of the polypeptide. For example, as is well known, glycosylation does not generally occur in bacterial hosts such as E.coli. Thus, when glycosylation is desired, the polypeptide should be expressed in a glycosylation host (glycosylation host), typically a eukaryotic cell. Insect cells typically undergo the same post-translational glycosylation as mammalian cells, and thus, insect cell expression systems have been developed to efficiently express mammalian proteins in natural glycosylation patterns and the like.

One skilled in the art will recognize that the same type of modification may be present to the same or different extents at several sites in a given polypeptide. Likewise, a given polypeptide may contain multiple types of modifications.

In the present application, the following terms are used to describe the sequence relationship between two or more polynucleotide or amino acid sequences: "reference sequence", "comparison window", "sequence identity", "percentage of sequence identity", "substantial identity", "similarity", and "homology". A "reference sequence" is a defined sequence that is used as a basis for performing a sequence comparison, and a reference sequence may be a subset of a larger sequence, e.g., as a fragment of a full-length cDNA or gene sequence given in a sequence listing or may comprise the entire cDNA or gene sequence. Typically, the reference sequence is at least 18 nucleotides or 6 amino acids in length, often at least 24 nucleotides or 8 amino acids in length, and usually at least 48 nucleotides or 16 amino acids in length. Because two polynucleotide or amino acid sequences can each (1) comprise a sequence that is similar between two molecules (i.e., a portion of the complete polynucleotide or amino acid sequence), and (2) can further comprise a sequence that is sufficiently different from the two polynucleotide or amino acid sequences, sequence comparisons between two (or more) molecules are typically performed by comparing the sequences of the two molecules over a "comparison window" to identify and compare local regions of sequence similarity. In this application, a "comparison window" refers to a fragment of conceptually at least 18 contiguous nucleotide positions or 6 amino acids, wherein a polynucleotide sequence or amino acid sequence can be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences, and wherein the portion of the polynucleotide sequence in the comparison window can comprise 20% or less additions, deletions, substitutions, and the like (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The comparison window is determined, for example, by using smith Waterman, adv.appl.math.2: 482(1981) by using Needleman and Wunsch, j.mol.biol.48: 443(1970) by using Pearson and Lipman, proc.natl.acad.sci. (u.s.a.) 85: 2444(1988) Studies of the similarity methods by computer processing of GAP, BESTFIT, FASTA and TFASTA in these algorithms (Genetics computer group, 575Science Dr., Madison, Wis.), or by examining sequences that can be optimally aligned for comparison windows and selecting the optimal alignment produced by various methods (i.e., to obtain the highest percentage of homology over the comparison window).

In this application, the term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide-nucleotide or residue-residue basis) over the comparison window. The term "percent sequence identity" is the percent sequence identity obtained by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical nucleobase (e.g., A, T, C, G, U or I) or residue occurs on both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100. The term "substantial identity" as used herein indicates a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence having at least 85% sequence identity, preferably at least 90-95% sequence identity, more typically at least 99% sequence identity, to a reference sequence when compared over a comparison window of at least 18 nucleotide (6 amino acid) positions, more typically over a comparison window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage sequence identity is calculated by comparing the reference sequence to deleted or added sequences that may include a total of 20% or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence. The term "similarity", when used in reference to a polypeptide, can be determined by comparing the amino acid sequence and conservative amino acid substitutions of one polypeptide to another. The term "homology", when used to describe polynucleotides, means that two polynucleotides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions in at least 70% of the nucleotides (typically about 75-99%, and more preferably at least about 98-99%).

In the present application, "homology", when used to describe polynucleotides, means that two polynucleotides, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions in at least 70% of the nucleotides (typically about 75-99%, and more preferably at least about 98-99%).

In the present application, "polymerase chain reaction" or "PCR" refers to a procedure in which a specific DNA fragment is amplified according to the description in US4,683,195. Generally, sequence information or information beyond requirements from the ends of the polypeptide fragment of interest is available, allowing the design of oligonucleotide primers; these primers are opposite to each other and are identical or similar to the sequences of the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers are identical to the ends of the amplified material. PCR can be used to amplify specific DNA sequences from total genomic DNA, cDNA transcribed from total cellular RNA, plasmid sequences, and the like (see generally Mullis et al, Cold Spring Harbor Symp. Quant. biol., 51: 263, 1987; Erlich, ed., PCR Technology, Stockton Press, NY, 1989).

In the present application, "stringency" typically occurs at about T_m(melting temperature) -5 deg.C (specific probe T)_mLow 5 ℃) to about less than T_mIn the range of 20 ℃ to 25 ℃. As will be appreciated by those skilled in the art, stringent hybridization can be used to identify or detect identical polynucleotide sequences or to identify or detect similar or related polynucleotide sequences. In the present application, the term "stringent conditions" means that only those cases where there is at least 95% and preferably at least 97% sequence identityThe hybridization that takes place.

In this application, "hybridization" shall include "any method of joining together polynucleotide strands and complementary strands by base pairing" (Coombs, J., Dictionary of Biotechnology, Stockton Press, New York, N.Y., 1994).

In the present application, the term "therapeutically effective dose" or "effective amount" refers to an amount of adenovirus that is capable of ameliorating a disease symptom or condition. The dose at which the growth of the tumor or metastasis slows or stops, or the tumor or metastasis is found to be reduced in size, is considered a therapeutically effective dose for treating the cancer or its metastasis, resulting in an extension of the patient's life span.

Adenovirus of the invention

The present invention provides a chimeric adenovirus, or a variant or derivative thereof, in which the nucleotide sequence of the E2B region of said chimeric adenovirus in its genome comprises a nucleic acid sequence derived from at least two adenoviral serotypes, each serotype being selected from the adenoviral subgroups B, C, D, E and F, respectively, and being distinct from each other. The chimeric adenoviruses of the invention are oncolytic and exhibit an enhanced therapeutic index for tumor cells.

Isolation of chimeric adenoviruses

The chimeric adenoviruses of the invention, or variants or derivatives thereof, can be produced by modifying a technique known as "bioselection" in which an adenovirus with desired properties, such as enhanced tumorigenicity or cell type specificity, is produced by genetic selection under controlled conditions (Yanet al (2003) J.Virol.77: 2640-.

In the present invention, a mixture of adenoviruses of different serotypes is pooled and passaged, preferably for at least two generations, on a subconfluent culture of tumor cells at a particle/cell ratio that is high enough to promote recombination between serotypes, but not so high as to cause premature cell death. Preferably the particle/cell ratio is about 500 particles/cell and can be readily determined by one skilled in the art. In the present application, a "subconfluent culture" of cells refers to a monolayer or suspension culture in which the cells are actively growing. For cells grown as a monolayer, an example would be a culture in which about 50-80% of the area available for cell growth is covered by cells. Preferably, about 75% of the growth area is covered by cells.

In a preferred embodiment, said adenoviral mixture comprises adenoviral subgroups B, C, D, E and the adenoviral serotypes represented by F. Group a adenoviruses are not included in the mixture because they are associated with tumor formation in rodents. Preferably, tumor cell lines useful in the bioselection method include, but are not limited to, those derived from breast, colon, pancreas, lung, and prostate. Some solid tumor cell lines that can be used for "bioselective" passaging of the adenoviral mixture include, but are not limited to, MDA231, HT29, PAN-1, and PC-3 cells. Hematopoietic cell lines include, but are not limited to, Raji and Daudi B-lymphocytes, K562 erythroid primary cells (erythroblastoid cells), U937 myeloid cells, and HSB 2T-lymphocytes.

Adenoviruses produced in these initial generations can be used to infect quiescent tumor cells at a sufficiently low particle/cell ratio to allow infection of cells by only one adenovirus. After passage under such conditions up to 20 passages, supernatants were harvested from the last passage before the cytopathological effect seen (CPE, see Fields virology, Vol.2, Fourth Edition, Knipe, ea., Lippincott Williams & Wilkins, pp.135-136) to increase the selection of high titer viruses. The harvested supernatant may be concentrated using techniques well known to those skilled in the art. A preferred method for obtaining quiescent cells in monolayer culture (i.e., where active cell growth ceases) allows the culture to grow for a further 3 days after confluence, where confluence means that the entire available area for cell growth is occupied (covered by cells). Similarly, suspension cultures can be grown to a density characterized by the absence of active cell growth.

Detection of serotype distribution of concentrated supernatants from pools containing bioselected adenoviruses can be carried out by determining the retention time of the harvested virus pools on an anion exchange column, where different adenovirus serotypes are known to have characteristic retention times (Blanche et al (2000) Gene Therapy 7: 1055-; see example 3, fig. 1A and 1B. The adenoviruses of the invention can be isolated from the concentrated supernatant by dilution and plaque purification or other techniques well known in the art and grown for further characterization. Techniques well known in the art can also be used to determine the sequence of the isolated chimeric adenovirus (see example 5).

An example of a chimeric adenovirus of the invention is the chimeric adenovirus ColoAd1, which was isolated during bioselection using HT29 colon cells. ColoAd1 has the sequence of SEQ ID NO: 1. Most of the nucleotide sequence of ColoAd1 corresponds to the nucleic acid sequence of the Ad11 serotype (SEQ ID NO: 2) (Stone et al, (2003) Virology 309: 152-. Compared with Ad11, there were two deletions in the nucleotide sequence of ColoAd1, one of 2444 base pairs in length within the E3 transcription unit region of the genome (27979-30423 base pairs of SEQ ID NO: 2), and the other of smaller length, 25 base pairs in length within the E4orf4 gene (33164-33189 base pairs of SEQ ID NO: 2). E2B transcription unit region of ColoAd1(SEQ ID NO: 3), which encodes adenovirus protein DNA polymerase and terminal proteins, located in SEQ ID NO: between 5067 and 10354 base pairs of 1, is a homologous recombination region between Ad11 and Ad3 serotypes. Within this region of ColoAd1, there was a 198 base pair change compared to the sequence of Ad11 (SEQ ID NO: 1). These changes resulted in a more extended nucleotide range of the E2B region of ColoAd1, homologous to the sequence of the E2B region (SEQ ID NO: 8) portion of Ad3, and a longest extended range with homology of 414bp length between ColoAd1 and Ad 3. The E2B region of ColoAd1(SEQ ID NO: 3) has a stronger potency than ColoAd1 adenovirus compared to unmodified Ad11 adenovirus (see example 6, FIG. 7). In other embodiments, a chimeric adenovirus of the invention can comprise nucleic acid sequences from more than two adenovirus serotypes.

The selectivity of the chimeric adenoviruses of the invention, or variants or derivatives thereof, in a particular tumor type can be assessed by determination of the cytolytic capacity in tumor cells obtained from the same tissue as the pool of adenoviruses of the initial passage. For example, chimeric adenoviruses ColoAd1(SEQ ID NO: 1) originally derived from an adenovirus pool passaged from HT-29 colon tumor cell lines were retested in HT-29 cells and from other colon-derived tumor cell lines including DLD-1, LS174T, LS1034, SW403, HCT116, SW48, and Colo320DM (see FIG. 3B). Any available colon tumor cell line would have an equivalent effect on such an assessment. Similar assays can be performed on isolated adenovirus clones from pools of adenoviruses selected on other tumor cell types in appropriate tumor cells including, but not limited to, prostate cell lines (e.g., DU145 and PC-3 cell lines), pancreatic cell lines (e.g., Panc-1 cell line), breast tumor cell lines (e.g., MDA231 cell line), and ovarian cell lines (e.g., OVCAR-3 cell line). Other tumor cell lines available have the same effect on the isolation and identification of the adenoviruses of the invention.

The chimeric adenoviruses of the invention have an improved therapeutic index compared to the adenoviral serotype from which they were derived (see fig. 6, which compares the cytolytic activity of the chimeric adenoviruses ColoAd1 and Ad11 p).

The present invention also includes chimeric adenoviruses constructed using recombinant techniques well known to those skilled in the art. Such chimeric adenoviruses comprise a region of nucleotide sequence derived from an adenoviral serotype which is integrated by recombinant techniques into the genome of another adenoviral serotype. The integrated sequences confer such properties as tumor specificity or enhanced potency against the parental adenovirus serotype. For example, the E2B region of ColoAd1(SEQ ID NO: 3) may be integrated into the genome of Ad35 or Ad 9.

Adenovirus derivatives

The invention also includes chimeric adenoviruses of the invention modified to provide other therapeutically useful chimeric adenoviruses. Such modifications include, but are not limited to, those described below.

One modification is the generation of a derivative of the chimeric adenovirus of the invention that substantially lacks the ability to bind p53 as a result of a mutation in the adenovirus gene encoding the E1B-55K protein. These viruses typically include partial or complete deletions of the E1B-55K region (see U.S. Pat. No. 5,677,178). An Ad5 mutant with deletions in the region of the E1B-55K protein responsible for binding p53 is described in US 6,080,578. Other preferred modifications of the chimeric adenoviruses of the invention are mutations in the E1A region, as described in US5,801,029 and US5,972,706. These types of modifications provide chimeric adenoviruses of the invention with greater selectivity for tumor cells.

Other modification examples encompassed by the present invention are chimeric adenoviruses which exhibit a higher degree of tissue specificity due to the arrangement of viral replication under the control of a tissue-specific promoter, as described in US5,998,205. Replication of the chimeric adenoviruses of the invention can also be placed under the control of an E2F response element as described in U.S. patent application 09/714,409. Such modifications provide a viral replication control mechanism based on the presence of E2F, resulting in enhanced tumor tissue specificity, and are distinct from control achieved by tissue-specific promoters. In both embodiments, the tissue-specific promoter and the E2F response element are operably linked to an adenovirus gene essential for replication of the adenovirus.

Other modifications of the invention include the use of chimeric adenoviruses of the invention (e.g., ColoAd1) as scaffolds for the production of novel replication-defective adenovirus vectors. Replication-defective adenovirus vectors can be used to deliver and express therapeutic genes as described in Lai et al ((2002) DNA Cell Bio.21: 895-913). The present application provides first generation (in which the E1 and E3 regions are deleted) and second generation (in which the E4 region is also deleted) adenoviral vectors derived from the chimeric adenoviruses of the invention. These vectors can be readily prepared using techniques well known to those skilled in the art (see Imperial and Kochanek (2004) curr. Top. Microbiol. Immunol.273: 335-.

Another modification encompassed by the present invention is the insertion of a heterologous gene marker or reporter that serves to track the efficacy of viral infection. One embodiment of such a modification is the insertion of the Thymidine Kinase (TK) gene. Using radiolabeled TK-reactive substrates, expression of TK in infected cells can be used to track the level of residual virus in the cells following viral infection (Sangro et al (2002) mol. imaging biol. 4: 27-33).

Methods for constructing modified chimeric adenoviruses are well known in the art. See Mittal, S.K, (1993) Virus Res.28: 67-90 and Hermiston, T.et al (1999) methods in Molecular Medicine: adenovirus Methods and Protocols, W.S.M.Wold, ed, Humana Press. Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, liposome transformation). Generally, enzymatic reactions and purification steps are performed according to the manufacturer's instructions. These techniques and procedures are generally performed according to conventional methods in the art and various conventional references which are mentioned throughout the present specification (see, generally, Sambrook et al, Molecular Cloning: Alaboratory Manual, 2nd. edition (1989) Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y.). The nomenclature used herein and the laboratory procedures in analytical chemistry, organic synthetic chemistry, and pharmaceutical formulation described below are those well known and commonly employed by those skilled in the art.

Determination of therapeutic potential

The therapeutic utility of the chimeric adenoviruses of the invention, or variants or derivatives thereof, can be assessed by detecting their cytolytic potential in tumor cells derived from tissues of interest as therapeutic targets. Tumor cell lines that can be used to detect these adenoviruses include, but are not limited to, colon cell lines (including, but not limited to, DLD-1, HCT116, HT29, LS1034, and SW48 cell lines), prostate cell lines (including, but not limited to, DU145 and PC-3 cell lines), pancreatic cell lines (including, but not limited to, Panc-1 cell lines), breast tumor cell lines (including, but not limited to, MDA231 cell lines), and ovarian cell lines (including, but not limited to, OVCAR-3 cell lines). Hematopoietic cell lines include Raji and Daudi B-lymphocytes, K562 erythroid blasts, U937 myeloid cells and HSB 2T-lymphocytes. Any other tumor cell line available can be used to evaluate and identify the use of the adenoviruses of the invention to treat neoplasia.

The cytolytic activity of the adenoviruses of the invention can be determined in representative tumor cell lines and the resulting data converted into a measure of potency, which can be used as a standard (i.e., assuming a potency of 1) since adenoviruses belong to subgroup C, preferably Ad 5. The preferred method for determining cytolytic activity is the MTS assay (see example 4, figure 2).

The therapeutic index of the adenoviruses of the invention in a particular tumor cell line can be calculated by comparing the potency of a given adenovirus in the tumor cell line with the potency of the same adenovirus in a non-cancer cell line. Preferred non-cancer cell lines are SAEC cells derived from the epithelium, and HUVEC cells derived from the endothelium (see fig. 4). These two cell types represent normal cells from the organ and vasculature, respectively, and are available depending on the mode of delivery of the adenovirus and are representative of potential toxic sites in adenovirus therapy. However, practice of the invention is not limited to the use of these cells, and other non-cancer cell lines (e.g., B cells, T cells, macrophages, monocytes, fibroblasts) may also be used.

The ability of the chimeric adenoviruses of the invention to reduce tumorigenesis or tumor cell burden in nude mice possessing a tumor cell graft can also be compared to the corresponding ability in untreated mice possessing the same tumor cell burden to assess the ability of the chimeric adenoviruses of the invention to affect target tumor cell growth (i.e., cancer) (see example 7).

The adenoviruses of the invention can also be evaluated using primary human tumor explants (Lamet al (2003) Cancer Gene Therapy; Grill et al (2003) mol. Therapy 6: 609-614), which provide a detection environment present in tumors that would not normally be provided using tumor xenograft studies.

Therapeutic applications

The invention provides the use of the chimeric adenoviruses of the invention for inhibiting the growth of tumor cells, and the use of adenoviral vectors derived from these chimeric adenoviruses for the delivery of therapeutic proteins useful in the treatment of tumors or other disease states.

Pharmaceutical compositions and administration

The invention also relates to pharmaceutical compositions comprising the chimeric adenoviruses of the invention (including variants and derivatives thereof) formulated for therapeutic administration to a patient. For therapeutic use, a sterile composition containing a pharmacologically effective dose of an adenovirus is administered to a human patient or a non-human diseased animal for the treatment of, for example, a neoplastic condition. Typically, the composition will comprise about 10 of the aqueous suspension¹¹One or more adenovirus particles. Pharmaceutically acceptable carriers or excipients are often used in such sterile compositions. A variety of aqueous solutions can be used, such as water, buffer, 0.4% saline, 0.3% -glycine, and the like. These solutions are sterile and generally free of particulate matter other than the desired adenoviral vector. The composition may contain pharmaceutically acceptable auxiliary substances as necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents and the like (e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.). Excipients that enhance adenovirus infection of cells may be included (see US 6,392,069).

The adenoviruses of the invention can also be delivered to tumor cells by liposome or immunoliposome delivery, which can selectively target tumor cells based on cell surface properties presented by the tumor cell population (e.g., the presence of cell surface proteins that bind to immunoglobulins in immunoliposomes). Typically, aqueous suspensions containing virosomes are encapsulated in liposomes or immunoliposomes. For example, conventional methods (U.S. Pat. No. 5,043,164, U.S. Pat. No. 4,957,735, U.S. Pat. No. 4,925,661, Connor and Huang, (1985) J.Celbiol.101: 581, Lasic D.D, (1992) Nature 355: 279, Novel Drug Delivery (eds. Prescott and Nimmo, Wiley, New York-, 1989), Reddy et al, (1992) J.Immunol.148: 1585) can be provided to encapsulate the suspension of adenovirus virions in capsules to form immunoliposomes. Immunoliposomes comprising antigens that specifically bind to cancer cells (e.g., CALLA, CEA) present on cancer cells in an individual can be used to target the virions of those cells (Fisher (2001) Gene Therapy 8: 341-348).

To further enhance the efficacy of the adenoviruses of the invention, they may be modified to exhibit enhanced tropism for particular cancer cell types. For example, as shown in PCT/US98/04964, proteins on the outer envelope of an adenovirus can be modified to have a chemical agent, preferably a polypeptide, that binds to receptors present on tumor cells to a greater extent than normal cells (see also US5,770,442 and US5,712,136). The polypeptide may be an antibody, preferably a single chain antibody.

Adenovirus therapy

The adenoviruses of the invention or pharmaceutical compositions thereof may be administered for the treatment of neoplastic diseases or cancers. In therapeutic applications, the compositions are administered to a patient already affected by a particular neoplastic disease in an amount sufficient to cure or at least partially block the condition and its complications. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose" or "effective dose". The amount effective for this use depends on the severity of the condition, the general condition of the patient and the route of administration.

For example, but not by way of limitation, a human patient or non-human mammal having a solid tumor or a hematological neoplastic disease (e.g., pancreatic, colon, ovarian, lung or breast cancer, leukemia or multiple myeloma) can be treated by administering a therapeutically effective dose of the inventionAn adenovirus that exhibits an increased therapeutic index for a certain tissue type. For example, a preferred chimeric adenovirus for the treatment of colon cancer would be the adenovirus ColoAd1(SEQ ID NO: 1). Suspensions of infectious adenovirus particles can be delivered to tumor tissue by a variety of routes, including intravenous, intraperitoneal, intramuscular, subcutaneous, and topical. Administration may be by infusion (e.g., into the peritoneal cavity for treatment of ovarian cancer, into the portal vein for treatment of liver cancer or metastasis from other non-hepatic primary tumors to the liver) or other appropriate route containing about 10³-10¹²An adenovirus suspension of one or more viral particles, and other suitable routes include direct injection into a tumor mass (e.g., a breast tumor), an enema (e.g., colon cancer), or a catheter (e.g., bladder cancer). Other routes of administration may also be applicable to cancers of other origin, i.e., by nebulization (e.g., for pulmonary delivery to treat bronchial, small cell lung, non-small cell lung, lung adenocarcinoma, or laryngeal cancer) or direct administration to a tumor site (e.g., bronchial, nasopharyngeal, laryngeal, cervical cancer).

Adenoviral therapy using the adenoviruses of the invention may be used in combination with other anti-tumor regimens, such as conventional chemotherapy or x-ray therapy for the treatment of a particular cancer. The treatments may be simultaneous or sequential. One preferred chemotherapeutic agent is cisplatin, and the preferred dosage may be selected by the practitioner based on the characteristics of the cancer to be treated and other factors routinely considered when administering cisplatin. Preferably, it may be in the range of 50-120mg/m²The dose of (a) is intravenously administered cisplatin over a period of 3-6 hours. More preferably, at 80mg/m²The dose of (a) was intravenously administered cisplatin over a period of 4 hours. Another preferred chemotherapeutic agent is 5-fluorouracil, which is usually administered in combination with cisplatin. The preferred dosage of 5-fluorouracil is 800-1200mg/m per day²For 5 consecutive days.

Adenoviral therapy using the adenovirus of the invention as an adenoviral vector may also be used in combination with other genes known to be useful in virus-based therapy, see US5,648,478. In this case, the chimeric adenovirus further comprises a heterologous gene encoding a therapeutic protein incorporated into the genome of the virus, such that the heterologous gene is expressed in the infected cell. As used herein, a therapeutic protein refers to a protein that is expected to provide a therapeutic effect when expressed in a given cell.

In one embodiment, the heterologous gene is a prodrug activating gene, such as Cytosine Deaminase (CD) (see, US5,631,236, US5,358,866, and US5,677,178). In another embodiment, the heterologous Gene is a known inducer of cell death, such as apoptin (apotin) or Adenovirus Death Protein (ADP), or a fusion protein such as fusogenic transmembrane glycoprotein (Danen-Van Oorshot et al (1997) Proc. Nat. Acad. Sci.94: 5843. multidot. 5847; Tollefson et al (1996) J.Virol.70: 2296. multidot. 2306; Fu et al (2003) mol.Therpy 7: 48-754, 2003; Ahmed et al (2003) Gene Therapy 10: 1663. multidot. 1671; Galanet et al 2001) Human Therapy 12 (7): 811).

Other examples of heterologous genes or fragments thereof include those genes encoding immunomodulatory proteins (e.g., cytokines or chemokines). Examples include interleukin 2(US 4,738,927 or US5,641,665), interleukin 7(US 4,965,195 or US5,328,988) and interleukin 12(US 5,457,038), tumor necrosis factor alpha (US 4,677,063 or US5,773,582), interferon gamma (US 4,727,138 or US4,762,791) or GM CSF (US5,393,870 or US5,391,485, Mackensen et al (1997) Cytokine growth factor Rev.8: 119-128). Other immunomodulatory proteins also include macrophage inflammatory proteins including MIP-3. Monocyte chemotactic protein (MCP-3 alpha) may also be used, and an example of a preferred heterologous gene is a chimeric gene consisting of a gene encoding a protein that crosses the cell membrane (e.g., VP22 or TAT) fused to a gene encoding a protein that is preferentially toxic to cancer cells but not normal cells.

The chimeric adenoviruses of the invention can also be used as vectors for the delivery of genes encoding therapeutically useful RNA molecules, i.e., siRNAs (Dorsett and Tuschl (2004) Nature RevDrug Disc 3: 318-329).

In some cases, genes encoding for disruption of MHC class I antigen presenting molecules (Hewitt et al, (2003) Immunology 110: 163-169), blockade of complement, inhibition of IFN and IFN-inducing mechanisms, chemokines and cytokines, NK cell-based killing (Orange et al, (2002) Nature Immunol.3: 1006-1012; Mireile et al, (2002) Immunogenetics 54: 527-542; Alcami (2003) Nature Rev.Immunol.3: 36-50), down regulation of immune responses (e.g.IL-10, TGF-Beta, Khong and Res (2002) Nature. IL-3: 999; and Bosun J. Immunol.423; Bosun J. 2003) protein diffusing in tumor cells (2003) J. Immunol.428; 2000: 2000-428; and strengthening of tumor cell proliferation in tumor cells (2003) are incorporated into the chimeric adenoviruses of the invention to further enhance the ability of the oncolytic viruses to eradicate tumors 839).

Reagent kit

The invention also relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the above-described compositions of the invention. Such containers may be accompanied by a notice in a format prescribed by a governmental agency regulating the manufacture, use and sale of pharmaceuticals or biological products, which reflects that formal approval by the agency regulating the manufacture, use and sale of such products is available for human consumption.

The invention is further described by the following examples which illustrate specific embodiments of the invention, and various uses thereof. These examples, which illustrate certain specific aspects of the invention, should not be described as limiting the scope of the disclosure.

The practice of the present invention will be carried out using conventional techniques of cell culture, molecular biology, microbiology, recombinant DNA manipulation, and immunology sciences, which are well known to those skilled in the art, unless otherwise specified. Such techniques are explained in detail in the literature, see, for example, Cell Biology: a Laboratory Handbook: celis (Ed), Academic press n.y. (1996); graham, f.l.and Prevec, l.adonvirus-based expression vectors and recombinants vaccenes.in: vaccines: new applications to Immunological schemes R.W.Ellis (ed) butterworth PD 363-390; grahan and Prevec management of adonoviruses.in: methods in Molecular Biology, Vol.7: gene Transfer and expression techniques, E.J.Murray and J.M.Walker (eds) HumanaPress Inc., Clifton, N.J.pp 109-; sambrook et al (1989), Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring harbor Laboratory Press; sambrook et al (1989) and Ausubel et al (1995), short protocols in Molecular Biology, John Wiley and Sons.

Examples

Method of producing a composite material

Standard techniques are used for recombinant nucleic acid methods, polynucleotide synthesis, and microbial culture and transformation (e.g., electroporation, lipofection). Generally, enzymatic reactions and purification steps are performed according to the manufacturer's instructions. In general, the techniques and methods are performed according to conventional methods in the art and various general references (see, generally, Sambrook et al, molecular cloning: A Laboratory Manual, 2nd. edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) referred to throughout the specification. The terms and laboratory procedures used in this application encompass analytical chemistry, organic synthetic chemistry, pharmaceutical formulation and delivery, and treatment of patients. Methods for constructing adenoviral mutants are widely known in the art (see Mittal, S.K., Virus Res., 1993, vol: 28, pages67-90 and Hermiston, T.et. al., Methods in Molecular Medicine: Adenoviral Methods and Protocols, W.S.M.Wold, ed, Humana Press, 1999). In addition, the adenovirus 5 genome is registered with Genbank 10 accession # M73260 and the virus is available from the American type culture Collection, Rokville, Maryland, USA, accession number VR-5.

Viruses and cell lines

In addition to HUVEC (Vec Technologies, Rensselaer, NY) and SAEC (Clonetics, Walkersville, MD), Ad serotypes Ad3(GB strain), Ad4(RI-67 strain), Ad5(Adenoid 75 strain), Ad9(Hicks strain), Ad11p (Slobitski strain), Ad16(Ch.79 strain) and all cell lines were purchased from ATCC: MDA231-mt1 (a derivative isolated by doctor Deb Zajchowski from a fast-growing MDA231 cell subcutaneous xenograft) and Panc1-sct (derived by doctor Sandra Biroc from a fast-growing Panc1 cell subcutaneous xenograft). Ad40 was hewed by doctor William s.m. wold, st.

Example 1: virus purification and quantification

Viral stocks were propagated on 293 cells and purified on a CsCl gradient (Hawkins et al, 2001). The method for quantifying viral particles is based on the following Shabram et al (1997) Human Gene Therapy 8: 453-465, except that the anion exchanger TMAEfractogel was used instead of Resource Q. Briefly, a 1.25ml column was packed with Fractogel EMD TMAE-650(S) (Cat #116887-7EM Science, Gibbstown, NJ 08027). HPLC separation on Agilent HP 1100HPLC using the following conditions: buffer a 50mM HEPES, pH 7.5; buffer B1.0M NaCl in buffer a; the flow rate was 1 ml/min. After column equilibration in buffer A for not less than 30 minutes, about 10⁹-10¹¹Each sample virus particle was loaded onto the column in a volume of 10-100. mu.l, followed by 4 column volumes of buffer A. A linear gradient over 16 column volumes was then applied and ended in 100% buffer B.

The column eluate was monitored at A260 and A280nm, the peak regions calculated, and the ratio of 260nm to 280nm determined. The viral peaks were identified as those with a A260/A280 ratio close to 1.33. Each sample series included one standard virus. Standard number of virus particles per ml has been determined using Lehmberg et al (1999) j.chrom.b, 732: 411-423. The a260nm peak region for each sample directly corresponds to the number of virus particles in the sample, within the range of virus concentrations used. The number of viral particles per ml in each test sample was calculated by multiplying the number of viral particles per ml in the known standard times the ratio of the sample to the standard a260nm viral peak region.

After each sample gradient, the column was regenerated by washing the column with 2 column volumes of 0.5N NaOH followed by 2 column volumes of 100% buffer a, 3 column volumes of 100% buffer B, and then 4 column volumes of 100% buffer a.

Example 2: biological selection

The viral serotypes representing adenovirus subgroups B to F were pooled and subjected to 2 rounds of passage at high particle/cell ratios on pooled cultures of the target tumor cell line, allowing recombination to occur between serotypes. Supernatants (1.0, 0.1, 0.01, 0.001ml) from round 2 high virus particle/cell infection, subconfluent cultures were then used to infect OVER-confluent (over-confluent) T-75 tissue culture dishes of a panel of target tumor cell lines PC-3, HT-29, Panc-1 and MDA-231. To obtain over-confluence, each cell line was seeded at a seeding rate that allowed the cell lines to reach confluence between 24-40 hours after seeding, and the cells were allowed to grow for a total of 72 hours after seeding before infection. This step was performed to maximize the rate of confluence of the cells to mimic growth conditions in human solid tumors.

Cell culture supernatants were harvested from the first dishes in 10-fold serial dilutions that did not show any sign of CPE at day 3 or 4 post infection (for HT-29 and PC-3, 10-20 passages were modified to harvest the second dish, i.e., 100-fold dilutions were harvested, where CPE was detectable at day 3 of infection). Each harvest was used as starting material for selective passage of the virus. This process was repeated until the virus pool reached the 20-generation bioselective band.

Standard methods are used (Tollefson, A., Hermiston, T.W., and Wold, W.S.M.; "Preparation and recommendation of CsCl-banded Adenovirus Stock”inAdenovirus Methods and ProtocolsHuman Press, 1999, pp 1-10, W.S.M.Wold, Ed) isolated various viruses from each bioselective pool by 2 rounds of plaque purification on A549 cells. Briefly, dilutions of the supernatant harvested from passage 20 on each target tumor line were used to infect a549 cells in a standard plaque assay. Well-separated plaques were harvested and the same plaque detection method was used to generate round 2 independent plaques from these harvests. Well-separated plaques from round 2 plaque purification were considered pure and infected cultures were prepared using these purified plaques and the oncolytic titer of the culture supernatants was determined by MTS assay as described above.

Example 3: serotype characterization

The following reactions were performed using a DNA hybridization reaction with Shabram et al (1997) Human Gene Therapy 8: 453: 465 the parental adenovirus serotype comprising the virus pool or isolated ColoAd1 adenovirus was identified in a similar manner except that the resource q was replaced with TMAE Fractogel (EM Industries, Gibbstown, NJ) as described in example 1 (see, figure 1).

During the gradient, adenovirus type 5 eluted at approximately 60% buffer B. Other serotypes (3, 4,9, 11p, 16, 35 and 40) were identified in the same manner as Blanche et al (2000) Gene Therapy 7: the characteristic retention times consistent with the retention times on Q Sepharose XL as disclosed in 1055-.

Example 4: cytolysis assay

The lytic capacity of the virus was determined using a modified MTT assay (Shen et al, 2001). Briefly, MTS assay (Promega, CellTiter) was used because the conversion of MTS by cells into aqueous, soluble formazan reduced time and avoided the use of volatile organic solvents associated with MTT assaysAqueous Non-Radioactive CellProliReferration Assay) replaced the MTT Assay.

To perform the assay, cells were seeded at a defined density that each tumor cell line was able to produce a confluent monolayer within 24 hours. These densely seeded cells were allowed to grow for an additional 2 days before exposure to the test virus. Infection of tumor and primary normal cells was performed in quadruplicate with 3-fold serial dilutions of virus, starting with a particle/cell ratio of 100 and ending with a particle/cell ratio of 0.005. Infected cells were incubated at 37 ℃ and MTS assays were performed at the time points specified for each primary cell or tumor cell line. Uninfected cells were used as negative controls and were set to 100% survival in a given assay.

Example 5: DNA sequencing

DNA sequencing of the Ad11p (SEQ ID NO: 2) and ColoAd1(SEQ ID NO: 1) genomic DNA was performed as follows. Briefly, purified adenoviral DNA from ColoAd1 and Ad11p was partially digested with the restriction endonuclease Sau3A1, and then shotgun cloned into the plasmid vector pBluescript II (Stratagene, La Jolla, Calif.). Positive clones were propagated and sequenced using primers M13R and KS (Stratagene, La Jolla, CA). Using a Sequencher^tm(Gene Codes Corp., Ann Arbor, Michigan) modifies, edits and assembles the sequence reactions. The gaps covered were amplified with conventional oligonucleotide primers and sequenced. The ends of the viral genome were directly sequenced off the adenoviral DNA. In summary, each genome was sequenced at 3X + coverage and 431 bases at 2 Xcoverage.

To determine the origin of the ColoAd1E2B region, two primer pairs were designed, one pair for the E2B pTP gene (9115bp, 5 'GGGAGTTTCGCGCGGACACGG 3' (SEQ ID NO: 4) and (9350bp, 5 'GCGCCGCCGCCGCGGAGAGGT 3' (SEQ ID NO: 5), one pair for the DNA polymerase genes (7520bp, 5 'CGAGAGCCCATTCGTGCAGGTGAG 3' (SEQ ID NO: 6) and 7982bp, 5 'GCTGCGACTGCGGCCGTCTGT 3' (SEQ ID NO: 7)) and used for PCR isolate DNA fragments from each serotype (Ad3, 4, 5,9, 11p, 16 and 40) obtained by electrophoresis on a PTC-200thermocycler from MJ Research (Watertown, MA) using the Advant 2PCR kit (Clonetics, Walkersville, MD; Cat # K1910-Y) followed by sequencing on an ABI sequencer, sequence 3 together with the sequence of these fragments.

The E2B region of Ad3 was sequenced with isolated Ad3DNA and overlapping primers.

Sequence information was analyzed using the Vector NTI program (Informatix).

Example 6: construction of recombinant viruses

Genomic DNA of Ad11p (SEQ ID NO: 2) and ColoAd1(SEQ ID NO: 1) was purified from CsCl gradient-bound virus particles. Genomic DNA was digested with PacI, which only cleaves one of the two genomes once in the viral genome. On the ColoAd1 nucleotide sequence (SEQ ID NO: 1), the Pac1 cleavage occurred at base 18141; on the Ad11 nucleotide sequence (SEQ ID NO: 2), the Pac1 cleavage occurred at base 18140. The digested DNA was mixed in equal amounts and ligated in the presence of T4DNA ligase overnight at 16 ℃. CaPO by Invitrogen, Carlsbad, CA (Cat # K2780-01)₄Transfection kit this ligation mixture was transfected into a549 cells. Isolated plaques were selected and screened by restriction enzyme digestion and PCR analysis to distinguish between 4 virus populations (Ad11p, colorad 1, left-end Ad11 p/right-end colorad 1 (colorad 1.1) and left-end colorad 1/right-end Ad11p (colorad 1.2)).

The virus lytic capacity of each population was determined in several cell lines (including HT29 and HUVEC cell lines) as described in example 3. The results showed titers ranging from lowest to highest as Ad11p, colorad 1.2, colorad 1.1, colorad 1 (see results in HT29 cells shown in figure 7).

Also constructed are chimeric adenoviruses pCJ144 and pCJ146, which contain the full-length ColoAd1 genome in which the wild-type Ad11p E3 and E4 regions, respectively, have been reconstituted. These modifications were introduced into BJ5183E.Coli (Chartier et al (1996) J.Virol.70: 4805-4810) by homologous recombination. Both chimeric adenoviruses showed reduced lytic capacity in HT29 and HUVEC cells compared to ColoAd1 or ColoAd1.2.

Example 7: in vivo efficacy of adenovirus

In a classical nude mouse test with a human cancer graft, 5X 10 was used⁶One cell was injected subcutaneously into the posterior side of the mouse. When the tumor size reaches 100-¹⁰Individual particles of virus were injected continuously for 5 days (total 1X 10¹¹Individual particles). A reduction in tumor size will be observed relative to the PBS control in combination with other control viruses (Ad5, ONYX-015).

Example 8: selectivity of ColoAd1 on Primary human tissue grafts

Tissue samples from colon tumors and adjacent normal tissues removed during surgery were placed in culture and infected with the same number of ColoAd1 or Ad 5. Culture supernatants were collected 24 hours post infection and the number of virus particles produced was determined. Compared to Ad5, colorad 1 produced more virus particles per input particle on tumor tissue, but it produced fewer virus particles per input particle on normal tissue.

Claims (17)

1. A chimeric adenovirus having a genome comprising an E2B region, wherein

The nucleotide sequence of the E2B region encodes a polypeptide consisting of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof; and the number of the first and second groups,

the chimeric adenoviruses are oncolytic and exhibit an increased therapeutic index for tumor cells.

2. The adenovirus of claim 1, further comprising regions encoding a fiber protein, a penton protein, and a hexon protein, wherein the nucleic acids encoding the fiber protein, the penton protein, and the hexon protein of the adenovirus are from the same adenovirus serotype.

3. The adenovirus of claim 1, further comprising a modified E3 region.

4. The adenovirus of claim 1, further comprising a modified E4 region.

5. The adenovirus of claim 1, wherein said tumor cell is a colon tumor cell, a breast tumor cell, a pancreatic tumor cell, a lung tumor cell, a prostate tumor cell, an ovarian tumor cell, or a hematopoietic tumor cell.

6. The adenovirus of claim 5, wherein said tumor cell is a colon tumor cell.

7. The adenovirus of claim 1, wherein the nucleotide sequence of said adenovirus consists of seq id NO: 1.

8. A recombinant chimeric adenovirus having a genome comprising an E2B region, wherein

The nucleotide sequence of the E2B region consists of SEQ ID NO: 3, preparing a composition;

the chimeric adenoviruses are oncolytic and exhibit an increased therapeutic index for tumor cells; and

the chimeric adenovirus has been made replication-deficient by deletion of one or more adenoviral regions encoding proteins involved in adenoviral replication selected from the group consisting of E1, E2, E3, and E4.

9. The replication deficient adenovirus of claim 8, wherein the E1 and E3 regions are deleted.

10. The replication deficient adenovirus of claim 9, further comprising a deletion in the E4 region.

11. The adenovirus of claim 1 or 8, further comprising a heterologous gene, wherein said heterologous gene is expressed in a cell infected with said adenovirus.

12. The adenovirus of claim 11, wherein said heterologous gene is thymidine kinase.

13. The adenovirus of claim 11, wherein said heterologous gene encodes a therapeutic protein selected from the group consisting of: cytokines and chemokines, antibodies, prodrug converting enzymes, and immunomodulatory proteins.

14. Use of the adenovirus of claim 1 in the manufacture of a medicament for infecting a cancer cell to inhibit growth of the cancer cell.

15. The use of claim 14, wherein the cancer cell is a colon cancer cell.

16. The use of claim 15, wherein the nucleotide sequence of said adenovirus consists of seq id NO: 1.

17. Use of an adenovirus according to claim 13 in the manufacture of a medicament for infecting a cell for delivery of a therapeutic protein to said cell.