CN101068933A - Fiber-modified adenoviral vectors for enhanced transduction of tumor cells - Google Patents

Abstract

Adenoviral vectors which effectively transduce primary tumor cells are provided. The adenoviral vectors comprise a chimeric adenovirus fiber protein which includes at least a portion of a Subgroup C adenovirus fiber shaft and at least a portion of a Subgroup B adenovirus or serotype 37 adenovirus head, wherein the head region binds CD46.

Description

The fiber-modified adenovirus carrier that is used for enhanced transduction of tumor cells

The cross reference of related application

The application advocates to enjoy the U.S. Provisional Patent Application No.60/604 that submitted on August 25th, 2004, and 009 rights and interests are for reference during this patent application full content is incorporated herein.

Technical field:

The present invention relates to adenovirus carrier, it comprises chimeric fiber albumen and compares with non-chimeric adenoviral vectors, presents the especially enhanced transduction of primary tumo(u)r cell of tumour cell.

Background technology:

Gene therapy is as providing the means of extraneous nucleotide sequence to cell, and is some the most novel and bases of the most potential disease-resistant instrument.This method not only has great prospect in the treatment cancer but also in many other diseases of treatment comprise the cancer of gall-bladder fibrous lesions, anaemia, hemophilia, diabetes, Huntington's disease, acquired immune deficiency syndrome (AIDS), unusual high anteserum cholesterol, some immune deficiency and many forms.Gene therapy relies on launch vehicle usually, as the virus vector of exogenous array is provided to cell.Recombinant adenoviral vector has shown some anti-these treatment of diseases usefulness.In order to look back, referring to (1996) Gene Therapy 3:496-502 such as (1996) Mol.Med.Today 12:519-527 such as Kim and Smith.

The adenovirus carrier that two kinds of main types are arranged: incompetence rf (replication defective) and capable rf (replication-competent).Replication-defective vector lacks one or more necessary genes that duplicate usually.These incompetence duplicating virus are bred in the cell that replenishes its essential gene that lacks.Incompetence replication type adenovirus carrier has extensively been applied in the external and body transducer cell and has been expressed various transgenosiss.Have the ability the replication type adenovirus carrier do not lack usually any duplicate necessary adenoviral gene and can be in some cell copy choice.

Immunity system is played the part of pivotal player in the pathogeny of multiple cancer.When oncogenesis, there's a widespread conviction that immunity system fails fully to reply or fail suitably to reply for people, allows growth of cancer cells.At present, comprise the cancer standard medical treatment of chemotherapy, operation, radiotherapy and cell therapy, all have clear and definite boundary with respect to effect and toxicity.Stadium when up to now, these methods depend on type, the patient's of cancer basic health, diagnosis etc. obtains success in various degree.Can provide with combining of standard medical treatment in conjunction with improvement countermeasure and to strengthen effect and to reduce toxic method the concrete processing of the immunne response of cancer.

Strengthen antineoplastic immune from the body cancer cells as vaccine and studied for some time (Oettgen etc. using, " The History of Cancer Immunotherapy ",: BiologicTherapy of Cancer, Devita etc. (writing) J.Lippincot Co., the 87-199 page or leaf, 1991; Armstrong TD and Jaffee EM, Surg Oncol Clin N Am.11 (3): the 681-96 page or leaf, 2002 and Bodey B etc., Anticancer Res 20 (4): 2665-76 page or leaf, 2000).

Show: many cytokines work in the adjusting to the immunne response of tumour.For example, U.S. Pat 5,098, the tumour that 702 compositions of having described the TNF, the IL-2 that use cooperative effective quantity and IFN-β exist with opposing.U.S. Pat 5,078,996, US5,637,483 and US5,904,920 have described and have utilized GM-CSF treatment tumour.Yet directly taking the cytokine therapy cancer may not be practical, because they often are system toxicities.(referring to, for example, Asher etc., J.Immunol.146:3227-3234 page or leaf, 1991 and Havell etc., J.Exp.Med.167:1067-1085 page or leaf, 1988).

The expansion of this method relates to the use at the tumour cell of the genetic modification of vaccine site local expression cytokine.Various immunomodulating cytokines are verified an activity by using in tumor model, and these immunomodulating cytokines comprise as Golumbeck PT etc., Science 254:13-716 page or leaf, 1991; Gansbacher B etc., J.Exp.Med.172:1217-1224 page or leaf, 1990; Fearon ER etc., Cell 60:397-403 page or leaf, 1990; Gansbacher B etc., Cancer Res.50:7820-25 page or leaf, 1990; Teng M etc., PNAS 88:3535-3539 page or leaf, 1991; Columbo MP etc., J.Exp.Med.174:1291-1298 page or leaf, 1991; Aoki etc., ProcNatl Acad Sci USA.89 (9): 3850-4 page or leaf, 1992; Porgador A etc., Nat Immun.13 (2-3): 113-30 page or leaf, 1994; Dranoff G etc., PNAS 90:3539-3543 page or leaf, 1993; Lee CT etc., Human Gene Therapy 8:187-193 page or leaf, 1997; Nagai E etc., Cancer Immunol.Immonther.47:2-80,1998 and Chang A etc., HumanGene Therapy 11:839-850 page or leaf, IL-4, the IL-2, TNF-α, G-CSF IL-7, IL-6 and the GM-CSF that describe respectively in 2000.

The GM-CSF homology that use is expressed or the clinical trial of allos cell vaccine (GVAX ) have begun to be used for treatment (the Dummer R. of prostate cancer, melanoma, lung cancer, carcinoma of the pancreas, kidney and multiple myeloma, Curr Opin Investig Drugs 2 (6): 844-8 page or leaf, 2001; Simons J etc., Cancer Res.15; 59 (20): 5160-8 page or leaf, 1999; Soiffer R etc., PNAS 95:13141-13146,1998; Simons J etc., Cancer Res.15; The 57:1537-1546 page or leaf, 1997; Jaffee E etc., J.Clin Oncol.19:145-156 page or leaf, 2001; With Salgia R etc., J.Clin Oncol.21:624-630 page or leaf, 2003).

Although using adenoviral vectors transduction tumour cell, the common efficient of the transduction of primary tumo(u)r cell is low.Therefore, need to improve the especially transduction efficiency of primary tumo(u)r cell of tumour cell.

Summary of the invention:

The invention provides by using and have composition and the method for the proteic adenovirus transduction of chimeric fiber tumour cell with expressing heterologous Nucleotide in tumour cell, wherein, this chimeric fiber albumen comprises at least a portion of hypotype B adenovirus or serotype 37 adenovirus heads and at least a portion of subtype C adenovirus or cow adenovirus fibre axis.In one embodiment, tumour cell is external, body is interior or exsomatize transduction.In a preferred embodiment, tumour cell is the primary tumo(u)r cell.

In practice of the present invention, adenovirus can be have the ability rf (just tumour disappears molten) or incompetence rf and can the encode transgenosis or the transgenosis of can not encoding.

Correspondingly, selecting cell dissolved method is provided on the one hand, promptly, neoplastic cell among the cell killing group, this method is included in virus vector and/or particle optionally duplicates in neoplastic cell and kills under the condition of neoplastic cell, and the capable duplicating virus carrier and/or the virion of significant quantity of the present invention contacted with cell mass.Cell mass can be in external, direct body or in the indirect intravital device.

On the one hand, the invention provides in tumour cell for example method of cytokine (as GM-CSF) of express transgenic.In aspect preferred, tumour cell is the primary tumo(u)r cell.In addition, provide the method for producing tumor vaccine.Also provide tumor vaccine of the present invention is transported to mammiferous method.

The present invention further provides adenovirus particles, wherein, target ligands is contained in the capsid protein of virion.In further embodiment, capsid protein is that scleroproein and part are positioned at fibrinous C-end or HI ring.

Description of drawings:

Fig. 1 provides the representative of wild-type and the chimeric fiber structure of example.All adorned fibers can be sneaked into adenovirus carrier (for example having the Ad5 base carrier of leaving out in the E1 district).Under the control of RSV promotor, based on the vector expression beta-galactosidase enzymes of Av1nBg.Before CMV under the domination of early promoter, based on the vector expression green fluorescent protein of Ad5-CMV5-GFP.The axle of each carrier and source, interface are as shown in the figure.

Fig. 2 is the figure of the antitumous effect of the OV1991 in diagram FaDu human tau and the neck tumour xenotransplantation tumor model.

Fig. 3 is the figure of the antitumous effect of the chimeric oncolytic carrier of fiber in the human melanoma xenotransplantation of the diagram A375-luc tumor model.

Embodiment:

The invention provides by expressing heterologous nucleic acid is (for example in tumour cell with having the proteic adenovirus of chimeric fiber transduction tumour cell, the composition of adenovirus transgenosis) and method, wherein, this chimeric fiber albumen comprises at least a portion of hypotype B adenovirus or serotype 37 adenovirus heads and at least a portion of subtype C adenovirus or cow adenovirus fibre axis.In one embodiment, chimeric fiber albumen comprises at least a portion of Ad5 or Ad2 axle and at least a portion of Ad35 head.Usually tumour cell is the primary tumo(u)r cell.

Do not wish to accept the constraint of opinion or mechanism, the present invention with comprise with those chimeric fibers with Ad2, Ad5 or Ad35 natural fiber mutually specific energy more effectively transduce some cancer cells the adenovirus carrier with chimeric fiber that Ad5 or Ad2 axle (or afterbody) district and Ad35 head (knot) distinguish be viewed as basis.What is interesting is especially, compare with the non-chimeric adenoviral of single serotype, the chimeric adenoviral (adenovirus that for example has Ad5 or Ad2 axle (afterbody) and subgroup B adenovirus or serotype 37 adenovirus heads) with fiber head of the fibre axis of first serotype and second serotype has the ability of more effective transduction primary tumo(u)r cell.Comprised lung tumor cell, prostate tumor cells, head and neck tumour cell, tumor of bladder cell and kidney tumor cell by the non-restrictive example of the tumour cell type of the more effective transduction of chimeric adenoviral vectors of the present invention.The transduction of tumour cell is measured in the expression that can pass through marker gene (for example, beta-galactosidase enzymes or GFP), yet any transgenosis all can be included in the adenovirus of the present invention and by gland virus expression of the present invention.

Ad5 is a subtype C adenovirus and Ad35 is a hypotype B virus.With adenoviral serotype 3 (Sirena etc., J Virol.2004 May; The 4454-62 page or leaf), 11,14,16,21,3537 the same with 50 78 (9):, the Head Section of Ad35 fiber is in conjunction with CD46.Ad37 belongs to adenovirus hypotype D.Preferred implementation of the present invention have subtype C adenovirus such as Ad2 or Ad5 fibre axis and with the fiber head of CD46 bonded adenovirus.

Unless otherwise stated, practice of the present invention uses conventional chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cytobiology, cell cultures and genetically modified organism to learn a skill, and these all drop in the prior art.Referring to, for example, Maniatis etc., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York); Sambrook etc., 1989, Molecular Cloning, second edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Sambrook and Russell, 2001, Molecular Cloning, the third edition (Cold SpringHarbor Laboratory Press, Cold Spring, New York); Ausubel etc., 1992, Current Protocols in Molecular Biology (John Wiley ﹠amp; Sons comprises the regular update content); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992, Techniques for the Analysis of Complex Genomes, Academic Press, NewYork; Guthrie and Fink, 1991, Guide to east Genetics and MolecularBiology, Academic Press, New York; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B.D.Hames ﹠amp; S.J.Higgins writes, and 1984); Transcription And Translation (B.D.Hames and S.J.Higgins write, 1984); Culture Of Animal Cells (R.I.Freshney, Alan R.Liss, Inc., 1987); Immobilized Cells And Enzymes (IRLPress, 1986); B.Perbal, A Practical Guide to Molecular Cloning (1984); The treatise, and Methods In Enzymology (Academic Press, Inc., N.Y.); GeneTransfer Vectors For Mammalian Cells (J.H.Miller and M.P.Calos write, and 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In CellAnd Molecular Biology (Mayer and Walker write, Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M.Weir and C.C.Blackwell write, 1986); Riott, Essential Immunology, sixth version, Blackwell Scientific Publications, Oxford, 1988; Hogan etc., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

Definition

Unless indication is arranged in addition, the same meaning known to all terms used herein and those skilled in the art, and practice of the present invention can be used conventional microbiological technique and recombinant DNA technology known to those skilled in the art.

In this article, utilization comprises all patents, patent application, publication (comprising disclosed patent application), the publication of relevant with this specification sheets herein database login number and other content, illustrate background of the present invention and especially, provide and the case of putting into practice other relevant details.Comprise all patents, patent application, publication (comprising disclosed patent application), publication and other content with relevant with this specification sheets herein database login number all are incorporated herein for reference.

In description of the invention, use following term and be defined as follows.

On behalf of plaque, abbreviation " pfu " form the unit.

The infectious viral particle that term " virus ", " virion ", " carrier granule ", " vector particles " and " virion " form when being used interchangeably and can being interpreted as widely and being used to produce infectious particles into suitable cell or clone when for example virus vector of the present invention being transduceed.Can utilize that transfer DNA enters cell in the external or body of virion of the present invention.For purpose of the present invention, these terms relate to adenovirus, comprise the recombinant adenovirus that forms when adenovirus carrier of the present invention is encapsulated in the adenovirus capsid.

" adenovirus carrier " mentioned herein or " carrier of adenovirus " (being used interchangeably) are for wrapping into the polynucleotide structure of adenovirus virion.In some embodiments, adenovirus carrier of the present invention comprises the therapeutic gene sequence, as, the cytokine gene sequence.Exemplary adenovirus carrier of the present invention comprises, but be not limited to, DNA, be encapsulated in DNA in the adenovirus shell, be packaged in (as herpes simplex and AAV) in another virus or the viral shape form adenovirus DNA, be encapsulated in liposome adenovirus DNA, with polylysine compound adenovirus DNA, with synthetic polycation molecule compound, with the transferrin conjugated or with compound such as PEG compound with immunity ground " covering " antigenicity and/or increase half life, inclusive NAND viral protein conjugated adenovirus DNA.Therefore, term used herein " adenovirus carrier " or " carrier of adenovirus " comprise adenovirus or adenovirus particles.

The relevant term of adenovirus carrier used herein and of the present invention " rf of having the ability " is meant that adenovirus carrier of the present invention and particle are preferably in some rather than degree or all duplicate in the cell or tissue of other types still less.In an embodiment of the invention, adenovirus carrier and/or particle optionally duplicate in tumour cell and/or abnormality proliferation tissue such as solid tumor and other vegetation.These are included in U.S. Pat 5,677, and 178, US5,698,443, US5,871,726, US5,801,029, US5,998,205 and US6,432,700 and PCT open WO95/19434, WO 98/39465, WO 98/39467, WO 98/39466, WO 99/06576, WO 98/39464 and WO 00/15820 in disclosed virus.Such virus can be described as " oncolytic virus " or " oncolytic vectors " and can think " cytolytic " or " to cytopathic " and " the selecting cell dissolving " of thinking to influence target cell.

As used herein, term " virus vector " uses according to the generally acknowledged meaning of its technical field.The nucleic acid carrier structure that it refers to comprise at least one composition of viral source and can be packaged in vector particles.Utilize this virus vector and/or particle be used for DNA, RNA other nucleic acid is external or body in shift cell.The many virus vector forms that comprise adenovirus carrier are known in the prior art.

When term " derives from " adenovirus carrier that is used for context, be meant and adenovirus carrier genome homologous adenoviral serotype.In most cases, most of adenoviral gene groups come from a kind of serotype and it is said that in this case this adenovirus carrier derives from this kind serotype.When the adenovirus carrier genome only has a kind of adenovirus encoding sequence from second serotype, it is said that this adenovirus carrier only derives from first adenoviral serotype.

Term " chimeric fiber albumen " refers to comprise alpha-non-natural amino acid order, except or replace the adenoviral fiber protein of the natural fiber amino-acid sequence of a part.The alpha-non-natural amino acid order can be from the adenoviral fiber protein of different serotypes.The alpha-non-natural amino acid order can be any suitable length (for example 3 to about 200 amino acid).Exemplary " chimeric fiber albumen " has the axle and the head that derives from different adenoviral serotypes that derives from a kind of adenoviral serotype.

Term " carrier ", " polynucleotide carrier " " polynucleotide carrier structure ", " nucleic acid carrier structure " and " carrier structure " are used interchangeably in this article, be meant any nucleic acid construct that is used for transgenosis, as understood by one of ordinary skill in the art.

Term " duplicates necessary gene " and refers to nucleotide sequence, and it is transcribed is that virus vector is needed, to duplicate in target cell.For example, in adenovirus carrier of the present invention, duplicate necessary gene and can be selected from E1a, E1b, E2a, E2b and E4 gene.

As used in this article, " packing cell " be can the encapsidated adenovirus viral genome or the modifying factor group to produce the cell of virion.It can provide gene product or its Equivalent of losing.Therefore, the gene that can remove in adenovirus of packing cell provides supplementary functions and the adenoviral gene group can be packaged into adenovirus particles.These particulate produce the protein that needs replicator group and production and assembly infectious virus necessity.Particle also needs ripe necessary some protein of virion.This protein can provide by carrier or by packing cell.

Term " HeLa-S3 " is meant that (the human cervix neoplasms source cell that VA) provides is American Type Culture Collection, and called after ATCC CCL-2.2 for ATCC, Manassas.HeLa-S3 is clone's derivative of parental generation HeLa system (ATCC CCL-2).HeLa-S3 was cloned by (J.Exp.Med.103:273-284 page or leaf (1956)) such as T.T.Puck in nineteen fifty-five.

In this article, " process cleavage site certainly " or " processing cutting sequence " certainly is meant DNA or amino-acid sequence, wherein when translating or being translated, comprise (cis) cutting in the rapid molecular of the polypeptide of processing cleavage site, thereby cause the expression of discrete maturation protein or polypeptide product.This " processing cleavage site certainly " also can be described as the back translation or is total to the translation process cleavage site, for example, and 2A site, sequence or structural domain.2A site, sequence or structural domain discharge polypeptide in the mode of allowing the translation product generation of discrete downstream from the translation mixture thus and show translation effect (Donnelly, 2001) by modifying ribosomal activity to promote the hydrolysis of ester bond.In other words, 2A site, sequence or structural domain cut it by up time the C-end to produce elementary cleaved products (Furler; Palmenberg, Ann.Rev.Microbiol.44:603-623 page or leaf (1990)) demonstration " automatic protein enzymolysis " or " cutting ".

Term " nucleic acid " refer to or thymus nucleic acid or the Yeast Nucleic Acid and the polymkeric substance (" polynucleotide ") thereof of strand or double chain form.Unless special qualification the, this term contain comprise with reference to the similar bonding properties of nucleic acid and with the nucleic acid of the known analogue of the metabolic natural nucleotide of mode like the naturally occurring ucleotides.Unless otherwise stated, special nucleic acid molecule/polynucleotide also mean its conservative modification of modifying (for example degenerate code displacement) and complementary sequence and the sequence that spells out.Particularly, the 3rd mixed base of (or all) codons that the sub-displacement of degenerate code can be by generating its one or more selections and/or Hypoxanthine deoxyriboside residue (Batzer etc., Nucleic Acid Res.19:5081 page or leaf (1991); Ohtsuka etc., J.Biol.Chem.260:2605-2608 page or leaf (1985); Rossolini etc., MoI.Cell.Probes 8:91-98 page or leaf (1994)) institute's alternate sequence obtains.Nucleotide is represented by following standardized abbreviations by its base: VITAMIN B4 (A), cytosine(Cyt) (C), thymus pyrimidine (T) and guanine (G).

When being configured to function and concerning with another nucleic acid, nucleic acid is " effectively connecting ".For example, if two nucleotides sequences are classified effective connection as, or the state of the expression level of promotor or adjusting dna sequence dna influence coding or structural DNA sequence, it is " effective connection " that promotor or adjusting dna sequence dna are called as with coding RNA and/or proteic DNA sequence.Effectively the dna sequence dna that connects usually (but be not must) be successive.

Term " encoding sequence " and " coding region " are meant the nucleotide sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, meaningful RNA or sense-rna.In one embodiment, RNA is translated in cell then to produce protein.

Term " ORF " is meant open reading frame (Open Reading Frame).

Term " gene " is meant the qualification district that is positioned at genome and except foregoing encoding sequence, comprises other, mainly is to regulate, with the relevant nucleotide sequence of control of expressing, that is, and the transcribing and translating of encoding part.Gene also can comprise other 5 ' and 3 ' untranslated sequence and terminator sequence.According to the source of gene, provide further component to be, for example, intron.

Reference nucleic acid molecule such as promotor and gene coded sequence, term used herein " allogenic " and " external source " be meant derive to special virus or host cell be external source nucleotide sequence or, if derive from identical source, then this sequence is modified by its prototype and is formed.Therefore, it is endogenous but by for example codon optimized that the heterologous gene in virus or the cell comprises special virus or cell, the gene of modified.This term also comprises the multiple copy that the non-natural of naturally occurring nucleotide sequence exists.Therefore, this term is meant that to virus or cell be external or allogenic nucleic acid fragment, or is homologous but position in host's virus or cellular genome is undiscovered usually to virus or cell.

Reference nucleic acid molecule, term used herein " homologous " is meant the nucleotide sequence that natural link is arranged with host's virus or cell.

Term " replenishes " and " complementation " is meant between the complementary base residue that comprises in the antiparallel nucleotide sequence when forming hydrogen bond two nucleotide sequences of paired mutually.

Term " natural " is meant gene or the protein that is present in wild-type virus or the cellular genome.

Term " natural existence " or " wild-type " be used for describing can occurring in nature that find with different object artificial production.For example, be present in can separate from its source and the laboratory, have a mind to protein or the nucleotide sequence modified in the organism (comprising virus), for naturally occurring at occurring in nature through human.

Term used herein " target ligands " is meant the chemical part that preferably adenovirus particles is incorporated into required cellular type and/or tissue.The kind of this part includes, but are not limited to, peptide, polypeptide, single-chain antibody and polymer protein.Special part comprises the part of TNF superfamily, these parts comprise tumour necrosis factor (or TNF) as, for example, TNF-α and TNF-β, lymphotoxin (LT) is as LT-α and LT-β, with Fas antigen bonded Fas part; The CD40 part, the lymphocytic CD40 receptors bind of this part and B-; The CD30 part, the CD30 receptors bind of the neoplastic cell of this part and Hodgkin lymphomas; The CD27 part, NGF part and OX-40 part; Transferrin, this albumen combines with the transferrin receptor that is positioned at tumour cell, activated T-cell and nervous tissue cell; ApoB, it combines with hepatocellular ldl receptor; α-2-macroglobulin, this albumen and hepatocellular LRP receptors bind; α-I acid glycoprotein, the asialoglycoprotein receptors bind of this albumen and liver; Contain mannosazone, it combines with the mannose receptor of scavenger cell; Contain sialic acid-Lewis-X antigen peptide, the ELAM-I receptors bind of itself and activated endotheliocyte; The CD34 part, the CD34 receptors bind of itself and hemopoietic stem cell; ICAM-I, itself and LFA-I (CD11b/CD18) acceptor of thymus dependent cells or Mac-I (CD11a/CD18) receptors bind of scavenger cell; M-CSF, the c-fms receptors bind of itself and spleen and bone marrow macrophage; Circumsporozoite protein, itself and hepatocellular liver plasmodium falciparum receptors bind; VLA-4, the VCAM-I receptors bind of itself and activated endotheliocyte; HIV gpl20 and class II MHC antigen, the CD4 receptors bind of itself and T-helper; The ldl receptor land of apolipoprotein E (ApoE) molecule; Clone stimulating factor or CSF, itself and CSF receptors bind; Insulin-like growth factor, as IGF-I and IGF-II, they respectively with IGF-I and IGF-II receptors bind; Interleukin 1-14, they respectively with interleukin 1-14 receptors bind; Fv antigen-the binding domains of immunoglobulin (Ig); Gelatinase (MMP) inhibitor; Bombesin, gastrin releasing peptide; Substance P; Somatostatin; Luteinizing hormone releasing hormone (LHRH); Vasoactive peptide (VIP); Gastrin; Melanotropin (MSH); Ring RGD peptide and any other part or cell surface protein are in conjunction with (or target) molecule.In an embodiment of the invention, the peptide target ligands is inserted in the capsid protein of adenovirus carrier, be generally the scleroproein of adenovirus carrier.

With reference to nucleic acid molecule, term used herein " recon " is meant the nucleic acid molecule combination that utilizes recombinant DNA technology nucleic acid molecule to be combined together to form the filial generation nucleic acid molecule.With reference to virus, cell and organism, term used herein " recon ", " conversion " and " transgenosis " are meant host's virus, cell or the organism of introducing the heterologous nucleic acids molecule.Genome or nucleic acid molecule that nucleic acid molecule can be integrated into the host with being stabilized can also exist as extrachromosomal molecule.This extrachromosomal molecule can self-replacation.Should be appreciated that recombinant virus, cell and organism not only comprise the final product of conversion process but also comprise its recombinant progeny." non-conversion ", " non-transgenic " or " nonrecombinant " host are meant wild-type virus, cell or the organism that does not contain the heterologous nucleic acids molecule.

" controlling element " relates to control the nucleotide sequence of expression of nucleic acid.Controlling element comprises promotor, enhanser and termination signal.They also comprise the required nucleotide sequence of suitable translation of nucleotide sequence usually.

Term " promotor " is meant the untranslated dna sequence dna that is usually located at upstream of coding region, and it comprises the rna plymerase ii combining site and starts transcribing of DNA.Promoter region also can comprise other element that serves as the genetic expression setter.Term " minimal promoter " is meant promoter element, especially refers to inactive or lacks the TATA element that the promoter activity of upstream activation elements greatly lowers.

The meaning of term " enhanser " in the present invention can be any genetic constitution, for example, nucleotide sequence, it can strengthen with promotor and has transcribing of effective encoding sequence that is connected, make to a certain extent greater than the transcription activating that influenced by promotor itself when having effective the connection with this encoding sequence, just it strengthens from promotor and transcribes.

Term " expression " is the transcribing and/or translating of native gene, transgenosis or coding region in the phalangeal cell.With regard to antisense construct, expression can only refer to transcribing of antisense DNA.

Term used herein " up-regulated " is meant: compare with other cell, can find a large amount of RNA of specific gene in target cell.For example, compare with non-tumor cell, if tumour cell produces more multiterminal grain terminal enzyme (DNA) RNA, then this tumour cell has the just adjusting expression of Telomere terminal transferase.The amount of special RNA in target cell (for example tumour cell) be at least 3 times of other cell (non-tumor cell) for a long time, express and be considered to up-regulated.In another embodiment, the amount of special RNA is at least more than 5 times.In another embodiment, the amount of special RNA is at least more than 10 times.Those skilled in the art will know that and how to measure special RNA sequence the rna level of (for example Northern analyzes).

Term used herein " tumor-selective promoter activity " is meant that the promoter activity of promoter fragment of the present invention is higher than the non-tumor cell type in the tumour cell.

As using herein, " internal ribosome entry site " or " IRES " is meant that starting direct internal ribosome enters the element of initiator codon, as the ATG of cistron (protein coding region), cause the translation that does not rely on cap (cap-independent) of gene thus.(Jackson R J, Howell M T, Kaminski A (1990) Trends Biochem Sci 15 (12): 477-83 page or leaf and Jackson R J and Kaminski, A. (1995) RNA 1 (10): the 985-1000 page or leaf).The present invention includes the use of any IRES element, it can start the initiator codon that direct internal ribosome enters cistron." under the translation of IRES control " used herein is meant that translation is relevant with IRES and carries out in the mode that does not rely on cap.The example of " IRES " known in the prior art includes, but are not limited to IRES (Jackson etc., 1990, the Trends Biochem Sci15 (12): the 477-483 page or leaf) that can obtain from picornavirus; With the IRES that can obtain from virus or cell mRNA source, example for example, immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez etc. (1998) Mol.Cell.Biol.18 (11): the 6178-6190 page or leaf), fibroblast growth factor 2 and insulin-like growth factor, translation initiation factor eIF4G, yeast transcription factor TFIID and HAP4.Report has IRES too in various viruses such as Cardioviruses, rhinovirus, foot and mouth disease virus, HCV, Fu Luode muroid leukosis virus (FrMLV) and Moloney muroid leukosis virus (MoMLV).As used herein, " IRES " comprises the changes of function of IRES sequence, as long as this variation can start the initiator codon that direct internal ribosome enters cistron.In a preferred embodiment, IRES is a Mammals.In other embodiment, IRES is virus or protozoon.In illustrated embodiment disclosed herein, (can buy Duke etc. (1992) J.Virol 66 (3): can obtain IRES 1602-1609) from Novogen from encephalomyocarditis virus (ECMV).In another illustrated embodiment disclosed herein, IRES is from VEGF.In U.S. Pat 6,692, the example of IRES sequence has been described in 736.

Term " identical " or " identity " percentage before and after two or more Nucleotide or protein sequence, be meant and utilize for example Smith-Waterman algorithm or measure of a kind of sequence comparison algorithm of describing herein by visual inspection, when contrasting and be arranged in maximum unanimity, two or more sequences or subsequence are identical or the appointment percentage ratio of amino-acid residue or Nucleotide is identical.

" normal cell state " or " normal physiological state " is to be under the normal physiological conditions and not divide or the state of splitted cell with regulative mode, that is, cell is in the normal physiological state." abnormal cells state " is defined as relevant with the cell of same type, and this cell is in and does not divide/regulate splitting status and be under the normal physiological conditions.This shows that the cell with " abnormal cells state " shows unadjusted cell fission.

Term " cancer ", " cancer cells ", " neoplastic cell ", " tumorigenesis ", " tumour " and " tumour cell " (being used interchangeably) are meant the relative cell that independently increases of demonstration as used in this article, and they show misgrowth phenotype or the abnormal cells state that is characterized by remarkable cell proliferation control.Tumour cell can be proliferative cell, it is external to present or body in growth the cell that lacks contact inhibition, the cell that moves in can not body or can body in the cell that moves.Neoplastic cell can be a virulent or benign.This shows that cancer cells is considered to have the abnormal cells state.

Aggregatio mentium in the use of term " primary tumo(u)r cell " and the prior art.The primary tumo(u)r cell be separate from mammiferous tumour and the extensive cancer cells of vitro culture not.

Relevant with the special nucleus nucleotide sequence as used herein term " essentially consist " or " basic comprising " are meant: special nucleotides sequence is listed in or 5 ' or 3 ' end or two ends can have extra residue, wherein extra residue does not influence the fundamental sum novel feature of cited sequence in fact.

Term " cytokine " in this article " or phraseological Equivalent be meant and the common grade of immune system cell hormone comprise lymphocyte factor, the unicellular factor and other.Definition comprises, is not limited to, and works and round-robin hormone in blood not in the part, and, when used according to the invention, can produce the change of individual immunne response.

Term " from the antigen of tumour cell " or its phraseological Equivalent are meant self energy to cause any protein, carbohydrate or other component of the tumour cell of immunne response in this article.This definition is meant and comprises, but be not limited to, use the relevant antigen of whole tumour cell and institute thereof as antigen, and from cell paste isolated any component, as plasma membrane, cytoplasmic protein, transmembrane protein, from the carbohydrate part of the protein of cell surface or film purifying or the uniqueness relevant with cell surface.This definition also comprises from the cell surface of the special processing that needs cell so that those antigens that use.

Be meant the combination that comprises cell mass as the term " genetic modification tumour cell " that uses in this article, this combination by genetic modification with express transgenic, and give patient as the part method of therapy for cancer.The genetic modification tumour-cell vaccine comprise for the patient who treats be " from body " or " allogenic " tumour cell or with " bystander cell line " of taking from the patient's tumor cytomixis.In this article, the genetic modification tumour-cell vaccine of expression of GM-CSF can be meant " GVAX ".

Term " systemic immunity is replied " or phraseological Equivalent are meant immunne response herein, and this is replied and does not locate still influence individuality generally, therefore allow special the replying subsequently of identical stimulation generation.

Term " reverse of formation tumour " or phraseological Equivalent are meant inhibition, the degeneration of original tumour or partly or entirely disappear in this article.This definition is meant any minimizing on size, potentiality or the speed of growth that is included in original tumour.

Term " retardance growth of tumor " is meant the tumor growth rate that slows down, and suppressing tumour size or tumour cell number increases, and perhaps reduces tumour cell number, tumour size or tumour number.

Term " transduction " is meant by physical means to be introduced exogenous nucleic acid in the cell.For example, transduction comprises in the nucleic acid introducing cell of use virion of the present invention with external source.For the various technology that are used to operate mammalian cell, referring to Keown etc., Methods of Enzymology 185:527-537 page or leaf (1990).

As term " treatment ", " therapeutics uses of using in this article " or " medical science uses ", treat morbid state or symptom by any way with referring to or stop, obstruction, block or reverse disease or other do not wish that development any of symptom and all require the use of composition.

In this article, term " treatment significant quantity " or phraseological Equivalent are meant enough by stimulating or suppress to regulate the amount of the preparation that individual systemic immunity replys.Under Theratope of the present invention ground situation, it stimulates the mammiferous immunne response to cancer cells.Amount for Different Individual, different tumor type and different preparations can be different." treatment significant quantity " uses routine operation to cause " improving result of treatment " to determine by those skilled in the art.

Such as in this article use, be meant slow with Cancer-Related term " treatment result of improvement " and " enhanced result of treatment " or reduce the growth of cancer cells or solid tumor, or reduce sum or total dose,tumor of cancer cells.Therefore, " treatment result of improvement " or " enhanced therapeutics effect " is meant that according to any acceptable clinically standard, the improvement of patient condition comprises the increase of predicted life or the improvement of quality of life.In this article, term " inactivation cell " and " propagation deficient cells " or phraseological Equivalent are to cause that by the processing that makes their propagation defectives the treatment cell inactivation causes.This treatment causes that cell can not carry out the mitotic division of many wheels, but still keeps the ability of marking protein such as cytokine and/or tumour antigen.All can finish by the whole bag of tricks known to those skilled in the art.

" radiating cell " is an example of this inactivation cell.This radiating cell has been subjected to enough radiation so that their propagation defectives.

Term " individuality ", " main body " or its phraseological Equivalent are meant any individual Mammals.

Target cell

The invention provides a kind of in target cell such as primary tumo(u)r cell the method for expressing heterologous nucleic acid.Target cell is with having external, the stripped or interior transduction of body of the proteic adenovirus carrier of chimeric fiber, and wherein this chimeric fiber albumen comprises at least a portion of Ad5 or Ad2 axle and at least a portion of Ad35 head.

Target cell can be any cell or tissue type.In a preferred method, target cell is a tumour cell, is generally former target cell.In one embodiment, the primary tumo(u)r cell is selected from a kind of cell in lung tumor cell (for example non-small cell lung tumor cell), prostate tumor cells, head and neck tumour cell, tumor of bladder cell, melanoma tumor cell, lymphoma cell and the kidney tumor cell.

In some preferred implementation, this primary tumo(u)r cell is selected from bladder cancer, mammary cancer, colorectal carcinoma, kidney, liver cancer, lung cancer (for example nonsmall-cell lung cancer), ovarian cancer, cervical cancer, carcinoma of the pancreas, the rectum cancer, prostate cancer, cancer of the stomach, epidermal carcinoma; The hematopoiesis cancer of lymph or marrow pedigree; Between cancer such as the fibrosarcoma or the rhabdosarcoma in matter source; A kind of cancer cells in other tumor type such as melanoma, teratocarcinoma, neuroblastoma, neurospongioma and the gland cancer.

The advantage that chimeric adenoviral vectors of the present invention provides is the especially enhancing transduction of primary tumo(u)r cell of tumour cell.The enhancing transduction efficiency is meant needs less adenovirus carrier.This causes the minimizing of necessary adenovirus carrier amount of producing.Equally, because the adenovirus carrier transduction efficiency is more effective, can use virus to obtain same transduction efficiency than common non-chimeric adenoviral vectors less amount.

Adenovirus carrier of the present invention and method have several purposes.For example, used adenovirus carrier in cell, to introduce and the expressing heterologous encoding sequence in decades.The invention provides the more effectively adenovirus carrier and the method for introducing and expressing heterologous encoding sequence in the primary tumo(u)r cell.This provides a kind of effective tool of various transgenosiss to the primary tumo(u)r impact cell that be used to study.Also available carrier of the present invention is tested different therapeutics transgenosiss with method in tumour cell especially primary tumo(u)r cell.Adenovirus carrier of the present invention and method are provided for the carrier and the method for therapeutic applications, for example, and the cancer in the treatment Mammals.That method of the present invention can be used for is external, exsomatize or body in the transduction tumour cell.

Adenovirus carrier of the present invention

Such as in this article use, claim to classify as any and whole virus of adenovirus with term " adenovirus " and " adenovirus particles ", comprise any adenovirus of the infection mankind or animal, comprise whole groups, hypotype and serotype.Therefore, such as in this article use, " adenovirus " and " adenovirus particles " be meant viral or derivatives thereof own and cover all serotypes and hypotype and natural type and the recon form of existing, unless otherwise noted outside.Such adenovirus can be that wild-type maybe can be with well known in the prior art or as disclosed in this article variety of way modification.This modification comprises the modification that is packaged in the adenoviral gene group in the particle in order to produce infectious virus.This modification comprises disappearance well known in the prior art, as the one or more disappearance in E1a, E1b, E2a, E2b, E3 or the E4 coding region.This term also comprises and duplicating-the specificity adenovirus; Just, preferably at the cell or tissue of some type but in the less degree or the virus of in other type, not duplicating.Such virus is thought " cytolytic " or " cytopathogenic " virus (or carrier) sometimes, and, if they have this influence to neoplastic cell, think " oncolytic " virus (or carrier).

Adenovirus carrier coding chimeric fiber albumen of the present invention and adenovirus particles of the present invention contain chimeric fiber albumen, and wherein said chimeric fiber albumen comprises at least a portion of Ad35 head and at least a portion of Ad5 or Ad2 axle.The Ad35 head that keeps and the part of Ad5 or Ad2 axle provide effective transduction of cancer cells.Further describe chimeric adenoviral scleroproein useful in putting into practice the present invention below.

Adenovirus monoid that can be used according to the invention comprises any adenoviral serotype.At present can (ATCC, Manassas VA) obtain adenoviral serotype 1 to 51, and the present invention includes any other serotype of the adenovirus that obtains from any source from American Type Culture Collection.Adenovirus that can be used according to the invention can be the mankind or non-human source, as ox, pig, dog, monkey, birds source.For example, adenovirus can be hypotype A (for example, serotype 12,18,31), hypotype B (for example, serotype 3,7,11,14,16,21,34,35,50), subtype C (for example, serotype 1,2,5,6), hypotype D (for example, serotype 8,9,10,13,15,17,19,20,22-30,32,33,36-39,42-47,49,51), hypotype E (serotype 4), hypotype F (serotype 40,41) or any other adenoviral serotype.By whole specification sheets, the specific nucleotide in the adenovirus type 5 is compared.Those skilled in the art can determine the Nucleotide of the correspondence in other serotype, and therefore make up similar adenovirus carrier in other adenoviral serotype.In a preferred implementation, the adenoviral nucleic acid main chain derives from adenoviral serotype 2 (Ad2), 5 (Ad5) or 35 (Ad35) or comprises part adenoviral serotype 2 (Ad2) or the chimeric adenoviral main chain of the combination of 5 (Ad5) and part adenoviral serotype 35 (Ad35).Many examples of human and animal's adenovirus can obtain from American Type Culture Collection, can network address for example http://www.atcc.org/SearchCatalogs/CellBiology.cfm find.

Can find accession number to be respectively the adenoviral serotype 2,5,35 (bacterial strain Holden) of NC 001405, AY339865, AY128640 and AY271307 and the DNA and the protein sequence of 35 (bacterial strain 35p) in GenBank, its full content is incorporated herein by reference.In GenBank, can find accession number to be respectively the adenoviral serotype 11 of BAD11205 and BAB83691 and the protein sequence of 14 fibers, can find accession number in GenBank is the dna sequence dna of adenoviral serotype 3 fibers of X01998 and M12411, and its full content is incorporated herein by reference.Can find accession number to be respectively the adenoviral serotype 16 of AB073632 and AB073222 and 21 dna sequence dna in GenBank, its full content is incorporated herein by reference.Together with this sequence information, the details that the GenBank clauses and subclauses include usefulness is as beginning and terminator codon, protein sequence, the cDNA of each gene and the sequence mutation in a series of document with reference to the position of, cutoff signal, polyadenylation site, TATA signal, intron, each identification gene.

The genomic size restriction of adenovirus carrier adenovirus carrier (Bett etc., J Virol 67:5911-5921 page or leaf, 1993).This limits the quantity that can be inserted into the allogeneic dna sequence DNA in the carrier again, and therefore limits the quantity and/or the length that can be attached to the allogeneic coding sequence in the adenoviral gene group.Therefore, the incompetence duplicating virus is allowed the insertion of maximum allogeneic dna sequence DNA usually, but still is subjected to the size of adenovirus genomic dna and the quantitative limitation of the adenovirus DNA of removing.

In the scope of adenovirus carrier, term " 5 ' " is oppositely held the direction that repeats (ITR) on the left of can using with " upstream " exchange and be meant.In the scope of adenovirus carrier, term " 3 ' " can exchange the direction of using and being meant right side ITR with " downstream ".

Adenovirus carrier of the present invention comprises incompetence rf and capable replicating vector.The incompetence replicating vector does not duplicate in target cell, or duplicates with very low level.In one aspect, by removing or disappearance, the incompetence replicating vector has at least one coding region in the part or all of coding region of E1a, E1b, E2a, E2b or E4 deactivation usually.The method that is used to breed these carriers is known in the prior art.

In another aspect, adenovirus carrier is the replicating vector of having the ability.Capable replicating vector can duplicate in target cell.Capable duplicating virus comprise wild-type virus and in target cell through transforming so that the virus of duplicating.These comprise and duplicate specificity virus.Compare with other type, design is duplicated specificity virus with special in a type of cell or preferably duplicate.

Incompetence rf and capable replication type adenovirus carrier develop as the therapeutical agent that is used for cancer therapy.These carriers can have the disappearance of at least one E3 coding region or keep a natural E3 district sometimes.(referring to, for example, WO 02/067861, and WO 01/02540).

In target cell, in the situation of the adenovirus carrier of copy choice, developed the capable duplicating virus carrier of special weakening and be used for preferably destroying those cells at the cancer cells copy choice.For example, WO 95/19434, WO 96/17053, WO 98/39464, WO98/39465, WO 98/39467, WO 98/39466, WO 99/06576, WO 99/25860, WO 00/15820, WO 00/46355, WO 02/067861, WO 02/06862, U.S. Patent Application Publication US20010053352 and U.S. Pat 5,698,443, US5,871,726, US5,998,205 and US6, in 432,700, the capable replication type adenovirus structure of various cell-specifics of preferably duplicating (and therefore destroying) in some cell type has been described.Designed the replication type adenovirus carrier of having the ability so that in tumour cell, optionally duplicate.

Target cell can be a certain cell type, types of organization or have a certain cell state.At U.S. Patent number US5,998,205, US5,846,945, US5,801,029 and PCT open WO95/19434, WO 98/39465, WO 98/39467, WO 98/39466, WO 99/06576, WO 98/39464 and WO 00/15820 in, further described in the compositions and methods of the invention, find practicality duplicate Idiotype virus example.

Term " copy condition virus ", " preferred replication-competent virus ", " special replication-competent virus " and " copy choice virus " are the terms that is used interchangeably, and be preferred in some type cell or tissue but at less degree or the capable duplicating virus carrier and the particle that in other type, do not duplicate.In an embodiment of the invention, virus vector and/or in tumour cell and/or abnormality proliferation tissue such as solid tumor and other vegetation the grain copy choice.It is " oncolytic virus " or " oncolytic vectors " and can think " cytolytic " or " to cytopathic " and influence target cell " selecting cell dissolving " that such virus can be described as.

" preferably duplicate " and " copy choice " and " special duplicating " be used interchangeably and be meant virus duplicating in target cell more than in non-target cell, duplicating.The speed of virus replication is higher than in non-target cell in target cell, and is for example high at least about 3 times high, high at least about 10 times, high at least about 50 times and high at least about 100 times, 400 times, 500 times, 1000 times or even 1 * 10 in some cases ⁶Doubly.In one embodiment, virus is only duplicated (just, do not duplicate or duplicate with very low level) in non-target cell in target cell.

In another embodiment of the present invention, adenovirus carrier comprises the part or all of disappearance in E1B 19-kDa district.In one embodiment, adenovirus carrier comprises the whole E1 encoding sequences except that all or part of disappearance of E1b 19kDa encoding sequence.This disappearance causes E1b 19kDa protein not expressed or this protein is NOT-function.Adenovirus E 1 B 19-kDa district is meant the genome district of the adenovirus E 1 B gene of coding E1B 19kDa product.According to wild-type Ad5, E1B 19-kDa district is a 261bp zone that is located between Nucleotide (nt) 1714 and the nt 2244.At for example Rao etc., Proc.Natl.Acad.Sci.USA has described E1B 19-kDa district in the 89:7742-7746 page or leaf.The present invention includes the part or all of disappearance in E1B 19-kDa district and the embodiment of E1B 19-kDa region mutation, as long as the inhibition that this disappearance or sudden change reduce or remove the programmed cell death relevant with E1B 19-kDa.

In an embodiment of the invention, chimeric fiber albumen is included in described and CD46 bonded adenovirus, for example the allogeneic amino acid sequence at least a portion of Ad35 head (for example part).In some embodiments of the present invention, the allogeneic amino acid sequence is arranged in and CD46 bonded adenovirus for example the HI ring or the C-terminal of at least a portion of Ad35 head.

In an embodiment of the invention, adenovirus comprises the disappearance of duplicating necessary adenovirus encoding sequence.In one embodiment, replication defective adenoviral have with among inferior segment: E1a, E1b, E2a, E2b and the E4 at least one the district at least one disappearance.In one embodiment, adenovirus is the incompetence rf in the primary tumo(u)r cell.

In some embodiments of the present invention, adenovirus carrier comprises the disappearance of at least one adenovirus E3 encoding sequence.In one embodiment, this at least one adenovirus E3 encoding sequence is selected from 19K, 14.7K, 14.5K, 12.5K, 11.6K, 10.4K and the proteinic encoding sequence of 6.7K E3.In one embodiment, all the E3 encoding sequences are removed and/or suddenly change so that the respective egg white matter is not expressed or for non-functional.In one embodiment, adenovirus comprises the E3 encoding sequence of 10.4K, 14.5K and 14.7K.In one embodiment, all natural E3 encoding sequence is retained in the carrier.

In embodiments of the present invention; adenovirus carrier preferably duplicates in cancer cells; by the levels of replication (for example, cell killing and/or titre) in the cancer target cell relatively with at non-target cell as the levels of replication in normal or the contrast cell, show that it duplicates preference.Adenovirus titre in the target cancer cells and in the comparison of the titre of non-target cell type provides that total preference of duplicating is an enhanced and/or to duplicate be the key index that reduces in target cell in non-target cell.

In one aspect of the invention, adenovirus carrier comprises IRES element between gene, and it connects the translation of two or more encoding sequences.Connect the coding region and can be two adenovirus coding regions, two transgenes encoding districts or an adenovirus coding region and a transgenes encoding district.

The adenovirus carrier that comprises the IRES that connects two adenovirus coding regions is stable and provides in some embodiments than the carrier better specificity that does not contain IRES.Another advantage that contains the adenovirus carrier of IRES between gene is: the use IRES rather than the second allos transcriptional regulatory element (TRE) can be provided for the additional space in extra gene such as the therapeutic gene carrier.At US6, the example of the adenovirus carrier that contains IRES has been described in 692,736.In one aspect of the invention, disclosed in this article virus vector comprises at least one IRES in the polycistronic transcription thing usually, and wherein the output of this polycistronic transcription thing is regulated by the special TRE of allogenic target cell.For the adenovirus carrier that under IRES control, comprises the second adenovirus coding region, preferably will remove at the endogenesis promoter of second coding region under the IRES translation control, make endogenesis promoter not hinder transcribing of second coding region.If IRES comprises initiator codon, preferred second coding region with IRES in frame (frame).If initiator codon, as ATG, in IRES, preferably the initiator codon of second encoding sequence is removed and this IRES and second encoding sequence in frame.Perhaps, if, use the initiator codon of second coding region from IRES if IRES does not contain initiator codon or initiator codon is removed.In one embodiment, the IRES between allos TRE and introducing E1A and E1B transcribes under the control, and adenovirus carrier comprises gene, E1A and the E1B gene of adenovirus necessity.Therefore, E1A and E1B all transcribe under the control common, and because the existence of IRES can obtain the translation of E1B coding region.In one embodiment, the endogenesis promoter of E1A is removed.In another embodiment, the endogenous enhanser of E1A is removed and in also having other embodiment, endogenesis promoter and the E1A enhanser of E1A is removed.In another embodiment, the endogenesis promoter of E1B is removed.In further embodiment, E1B has the part or all of disappearance in the 19-kDa district of E1B.

In the preferred implementation that is used for the oncolytic adenovirus platform, comprise from the bicistronic mRNA of processing cutting sequence such as 2A or 2A class sequence or polycistron box and comprise early stage virogene of adenovirus (E1A, E1B, E2, E3 and/and or E4) or the gene (fiber, penton and six adjacent bodies) of expressing in the later stage of virus life cycle.

In some cases, owing to for example attribute of the suitable refractory of cancer target cell or special aggressiveness, the degree and/or the speed that perhaps strengthen the cytotoxin vigor are desirable.The example of the contributive virogene of pair cell toxicity includes, but are not limited to, dead albumen (ADP) gene of adenovirus.In another embodiment disclosed herein, adenovirus is included in has disappearance or the E1B gene that lacks of its endogenesis promoter in its endogenesis promoter.In other embodiment disclosed herein, the 19kDa district of E1B is removed.

For enhanced cell toxicity is provided to target cell, can there be one or more transgenosiss in the carrier with cytotoxin effect.In addition, perhaps, at random under the control of the alternative transcription of allos TRE and at random at IRES or certainly under the translation control of processing cutting sequence such as 2A or 2A class sequence, can be with pair cell toxicity and/or the contributive adenoviral gene of necrocytosis, as dead albumen (ADP) gene of adenovirus, be contained in the carrier.This can finish by the active expression with heterologous gene or genetically modified cell-specific of coupling target cell specific cell toxin.

Example adenovirus carrier of the present invention comprises, but be not limited to, DNA, be encapsulated in DNA in the adenovirus shell, be packaged in another virus or viral shape form (as herpes simplex and AAV) adenovirus DNA, be encapsulated in the intravital adenovirus DNA of lipid, with polylysine compound adenovirus DNA, with synthetic polycation molecule compound and with transferrin conjugation or compound with immunity ground " covering " antigenicity and/or increase the inclusive NAND viral protein conjugated adenovirus DNA of half life with compound such as PEG.

Adenoviral vector particle also can comprise as described below to fibrinous further modification.In one embodiment, adenovirus carrier of the present invention further comprises the target ligands that is contained in the particle capsid protein.For the example of target gland virus, referring to example WO 00/67576, WO 99/39734, US6,683,170, US6,555,368, US5,922,315, US5,543,328, US5,770,442 and US5,846,782.

In addition, adenovirus carrier of the present invention can also comprise the modification to other viral capsid proteins.The example of these sudden changes includes, but are not limited in U.S. Pat 5,731, those that describe in 190, US6,127,525 and US5,922,315.In U.S. Pat 6,057, other modification adenovirus has been described in 155, US5,543,328 and US5,756,086.

" E3 district " (can exchange with " E3 " and use) is a term of understanding easily in the prior art and the adenoviral gene group district that is meant coding E3 gene product.In various publications, for example comprise the E3 district being described in (1995) Curr.Topics Microbiol.Immunol.199:237-274 page or leaf such as Wold." part " in E3 district is meant the district less than whole E3, and comprises the polynucleotide of one or more polypeptide products in polynucleotide disappearance and coding E3 district.

Can generate the adenovirus structure that contains the E3 district, wherein contain the E3 adenoviral plasmid for example BHGE3 (Microbix Biosystems Inc. Toronto) and not contains the homologous recombination between the E3 adenoviral plasmid.

Perhaps, the adenovirus carrier that contains the E3 district can be introduced in the cell (for example 293 cells) with adenovirus structure or adenoviral plasmid structure, at this, they can carry out homologous recombination contains the E3 district with generation adenovirus.In this case, contain E3 adenovirus carrier and adenovirus structure or plasmid structure and comprise having the overlapping complementation district with the adenovirus that allows homologous recombination of enough sequences, for example, one contains the left hand district and another contains right hand region.

Perhaps, the E3 of containing adenovirus carrier of the present invention can utilize other ordinary method to comprise that any combination of standard weight group of methods (for example, utilizing restriction nuclease enzyme and/or PCR), chemosynthesis or these methods makes up.Further, can use molecular biological standard technique to form the excalation in E3 district.

In some embodiments, in adenovirus carrier, preserved the dead albumen (ADP) of adenovirus of E3 district in-line coding.As if under the control of main late promoter (MLP), the ADP gene is encoded to important protein matter (ADP) in the dissolving of acceleration host cell.Tollefson etc. (1996) J.Virol.70 (4): 2296 pages; Tollefson etc. (1992) J.Virol.66 (6): 3633 pages.Therefore, the adenovirus carrier that contains the ADP gene can make adenovirus carrier become more effective, can more effectively treat and/or reduce the dosage requirement.

In one embodiment, capable replicating vector is included in the gene that necessity is duplicated in control down of transcribing of selectivity TRE.In one embodiment, adenovirus carrier is the capable rf cancer idiosyncratic carrier that contains E1B, and wherein E1B has the disappearance in part or all of 19-kDa district.In embodiments of the present invention, duplicating necessary adenoviral gene is early gene, for example, and one or more among E1A, E1B, E2a, E2b and the E4.In further embodiment, one or more additional TRE can duplicate necessary adenoviral gene or transgenosiss with one or more, and for example, therapeutic gene forms effective connection.

Adenovirus carrier may further include additional allos TRE, and it can or cannot effectively connect by the gene identical with the special TRE of target cell.For example, TRE (as cellular type TRE special or that cell state is special) can be arranged side by side with second type of target cell specificity T RE." side by side " is meant that target cell specificity T RE and the 2nd TRE transcribe the identical gene of control.For these embodiments, special TRE of target cell and the 2nd TRE can be any of many arrangements, including, but not limited to, (a) adjacent each other (adjacency just); (b) 5 of two TRE ' towards the gene of being transcribed control (just can have intervening sequence between them); (c) 5 of a TRE ' and another TRE 3 ' towards gene.

Transcriptional regulatory element

Transcriptional regulatory element (TRE), and the discriminating, separation, sign, the method for genetic manipulation and the purposes of regulating effective encoding sequence that is connected that are used for them are known in the prior art.TRE can derive from monogenic transcriptional regulatory sequences, can function TRE be arranged in conjunction with producing from heterogeneic sequence, maybe can synthesize to generate TRE (for example CTP4 promotor).

According to the cell type that is present in tissue or the tumour, TRE can be tissue-specific, tumour-specific, the etap is specific, cell state is specific or the like.In this article, this TRE is called tissue-specific or the specific TRE of target cell together.Following more detailed description, target cell specificity T RE can comprise many structures, including, but not limited to, target cell specificity promoter and target cell specific enhancer; Allogeneic promoter and target cell specific enhancer; Target cell specificity promoter and allos enhanser; Allogeneic promoter and allos enhanser; With above-mentioned polymer.The promotor of target cell specificity T RE and enhanser component can be in any direction and/or any distances of the encoding sequence of being concerned about, as long as obtain required target cell specific transcriptional activity.

Many in the prior art known modes can be measured transcriptional activation, but (just, effectively connect) detection of the protein product by mRNA or encoding sequence usually and/or quantitatively measure under target cell specificity T RE control.

As further describing herein, target cell specificity T RE can be variation length and change sequence combination.Target cell specificity T RE preferably works in the cell that limits population (or type), for example, and prostatic cell, liver cell, melanoma cells etc.Correspondingly, in some embodiments, work in the preferred how undertissue in office of the TRE of the use type: prostate gland; Liver; Breast; Urethra (bladder); Colon; Lung; Ovary pancreas; Stomach and uterus.

Such as understood by a person skilled in the art, TRE is a polynucleotide sequence, and similarly, can be in various sequences displacements Presentation Function.The method of nucleotide substitution, interpolation and disappearance is known in the prior art, and the functional analysis that obtains easily (as CAT or the plain enzyme intelligencer's of fluorescent genetic analysis) allows those skilled in the art to measure the cell-specific transcripting regulating function whether sequence variations shows necessity.Therefore, can use the function conservative variations of TRE in the carrier disclosed herein, comprise that nucleic acid substitutes, adds and/or disappearance.Correspondingly, but the TRE of variation has kept the function in the target cell has not needed to show maximum function.In fact, it is necessary that the maximum transcriptional activation activity of TRE can always not obtain required result, and be enough by the level of inducing that the TRE fragment provides to some application.For example, if be used for the treatment of morbid state or slow down, it is enough being less than maximum replying, if for example target cell is not that the degree of fatal especially and/or disease is restriction relatively.

Some base modification can cause enhanced expression level and/or cell-specific.For example, as is known in the art, the nucleotide sequence of TRE inside disappearance or add removable transcription regulatory protein combining site make they than normal configuration state each other from nearer or farther, or rotate the offside that they are located at the DNA spiral, change TRE thus in conjunction with the spatial relation in the transcription factor, cause the minimizing or the increase of transcribing.Therefore, though be not wishing to be bound by theory, some modification of present disclosure expection TRE can cause as the adjusting expression level of TRE guiding, comprises the specific possibility of enhanced cell.Just superfluous when giving birth to the stronger intrusion form that increases, and/or when the faster and/or intrusion pattern (for example, in the non-responsiveness main body) of proof cell-lethal and opinion, strengthening finishing of expression level may cater to the need especially.

The TRE that is used for carrier of the present invention can comprise or not comprise silencer.The existence of silencer (negative regulatory element just well known in the prior art) can help to cut off transcribing in the non-target cell (with duplicating therefore).Therefore, by more effectively stoping duplicating in the non-target cell, the existence of silencer can be given the enhanced cell idiosyncratic carrier and be duplicated.Perhaps, lacking of silencer can promote duplicating in the target cell, therefore gives enhanced target cell specificity.

The transcriptional activity of TRE guiding (comprise and suppressing and enhancing) available several different methods well known in the prior art (and describing in more detail hereinafter) is measured, but the detection of the protein product of the sequence encoding that (just, effectively connects) down by mRNA and/or by TRE control and/or quantitatively measure usually.

As described in this article, TRE can variation length and change sequence combination.The big small portion of allos TRE determines that by the capacity of virus vector it depends on the expection form of carrier again.For the usually preferred minimum size of TRE, because this provides latent space to be used for the insertion of needed other sequence (as transgenosis and/or additional regulating and controlling sequence).In one embodiment, so additional regulating and controlling sequence is from processing cutting sequence such as 2A or 2A class sequence.

For instance, adenovirus carrier can be used and amount to up to 105% of about genome size, or about 1.8kb, does not need the appended sequence packing of virus sequence disappearance.If inessential sequence is removed, can allow the insertion (total insertion amount of about 6.4kb just) of extra 4.6kb from the adenoviral gene group.

With regard to the replication type adenovirus carrier of having the ability, reduce to minimum in order to make non-specific duplicating, preferably endogenous (adenovirus) TRE (just, natural E1A and/or E1B promotor) is removed from carrier.Except promoting that the target cell specificity is duplicated, endogenous TRE removes also to carrier provides bigger insertion amount, and it has special influence in the virion if adenovirus carrier is packed in.Even more importantly, the disappearance of endogenous TRE stoped so as to the possibility of the recombination event of removing allos TRE and endogenous TRE born its separately the adenovirus encoding sequence transcribe control (therefore allowing nonspecific duplicating).In one embodiment, adenovirus carrier is so constructed so that the endogenous transcriptional control sequence of one or more adenoviral genes is deleted and substituted by one or more allos TRE.Yet,, endogenous TRE can be kept in the adenovirus carrier as long as kept enough cell-specifics to duplicate preferably.By at endogenous TRE with duplicate and insert allos TRE between the genes encoding fragment that needs and make up these embodiments.Adenovirus carrier in the cell of the function by allos TRE relatively is provided duplicate the conduction analysis of duplicating in the cell with the function that allos TRE is not provided, determine that necessary cell-specific duplicates preference.

In some embodiments, compare with the base level of under the situation of no TRE, duplicating, in target cell, TRE increase at least about 2 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 400 to about 500 times, duplicate at least about 1000 times carriers.Acceptable difference can be measured (by use, for example level of rna blot analysis, rnase protection analysis or other assay determination mRNA well known in the prior art) by rule of thumb and depend on the desired use and/or the required result of carrier.

Can utilize the TRE that preferably works in the target cell to be created in the adenovirus carrier that specificity target cell is introduced.In an embodiment of the invention, target cell specificity or cell state specificity, allos TRE are tumor cell specific.Carrier can comprise single tumor cell specific TRE or tumor cell specific and many times of allos TRE that work in same cell.In another embodiment, carrier comprises the allos TRE of one or more tumor cell specifics and comprises one or more tissue-specific allos TRE in addition that all whereby TRE work in same cell.

In one embodiment, for the oncolytic adenovirus platform, the bicistronic mRNA or the polycistron box that contain from processing cutting sequence such as 2A or 2A class sequence comprise early stage virogene of adenovirus (E1A, E1B, E2, E3 and/or E4) or the gene (fiber, penton and six adjacent bodies) of expressing in the later stage of viral life cycle.

In some cases, owing to attribute or special aggressiveness that for example relative refractory of cancer target cell is healed, the degree and/or the speed that strengthen cytotoxic activity can cater to the need.The example of the contributive virogene of pair cell toxicity includes, but are not limited to, adenovirus death protein (ADP) gene.In another embodiment disclosed herein, adenovirus is included in the adenovirus E 1 B gene that has disappearance or endogenesis promoter disappearance in its endogenesis promoter.In other embodiment disclosed herein, the 19kDa coding region of E1B comprises part or all of disappearance, makes this 19kDa protein do not expressed or non-functional.

For enhanced cell toxicity is provided to target cell, the one or more transgenosiss with cytotoxin effect can be present in the carrier.In addition, perhaps, at random under the control of the alternative transcription of allos TRE and at random at IRES or certainly under the translation control of processing cutting sequence such as 2A or 2A class sequence, the contributive adenoviral gene of pair cell toxicity and/or necrocytosis, as adenovirus death protein (ADP) gene, can be contained in the carrier.This can finish by the activity of coupling target cell specific cell toxin and the expression of heterologous gene or genetically modified cell-specific.

As further described below, can be with many allos therapeutic genes or genetically modified any being contained in the capable duplicating virus carrier of the present invention.

Usually, foregoing bicistronic mRNA or polycistron box are placed under the control of transcriptional response element, cellular type or cell state are relevant with preferential transcriptional regulatory element of expressing in cancer or tumour cell usually.Correspondingly, the therapeutic gene that is contained in specified structure can change according to the type of the cancer in the treatment.

As known in the prior art, the activity of TRE is derivable.Derivable TRE usually shows low activity under the situation of no inductor, and under the situation that inductor exists for just regulating.Inductor comprises, for example, and nucleic acid, polypeptide, small molecules, organic compound and/or envrionment conditions such as temperature, pressure or hypoxemia.When only expressing when some suits the requirements constantly or in some position, maybe when utilizing inductor titration expression level to cater to the need, can preferred derivable TRE.For example, the transcriptional activity of PSE-TRE, PB-TRE and hKLK2-TRE is induced by male sex hormone, as describing with PCT/US98/04080 herein, specially is incorporated herein by reference.Correspondingly, in an embodiment of the invention, adenovirus carrier comprises derivable allos TRE.

Can there be various configurations as employed TRE among the present invention.TRE can comprise polymer.For example, TRE can comprise the series connection series of at least two, at least three, at least four or at least five target cell specificity T RE.These polymers also can comprise allogeneic promoter and/or enhancer sequence.Perhaps, TRE can comprise the one or more promoter regions together with one or more enhancings subarea.The TRE polymer can also comprise from heterogeneic promotor and/or enhancer sequence.The promotor of TRE and enhanser component can be positioned at relevant each other any direction with in any direction and/or in any distance from relevant encoding sequence, as long as obtain required target cell specific transcriptional activity.

In one embodiment, adenovirus is preferentially duplicated in tumour cell.In one embodiment, adenovirus comprises and duplicates the TRE that at least one necessary adenovirus encoding sequence effectively is connected.TRE includes, but are not limited to cell-specific TRE, cell state specificity T RE and tissue specificity TRE.The example that can be used for specificity T RE of the present invention comprises, but be not limited to PSA TRE, E2F TRE, Telomerase (TERT) TRE, urokinase plasminogen activator (uPA) TRE, upar (uPAR) TRE, PRL-3 Protein-tyrosine-phosphatase TRE.The present invention also expects to utilize the combination of TRE.Duplicating necessary adenovirus encoding sequence can effectively be connected with more than one allos TRE.Perhaps, in addition, the more than one adenovirus encoding sequence that duplicates necessity can effectively be connected with one or more allos TRE.The example that duplicates necessary adenovirus encoding sequence is the encoding sequence that is positioned at E1a, E1b, E2a, E2b and E4 district.Duplicating necessary odd encoder district can for example effectively be connected with IRES with the combination of a TRE or TRE.In one embodiment, at least one allos TRE with duplicate the first necessary adenovirus encoding sequence and effectively be connected, wherein the first adenovirus encoding sequence also effectively is connected with IRES and described IRES is same is connected with the second adenovirus encoding sequence that duplicates necessity.For example, at least one allos TRE effectively is connected with the encoding sequence of E1a coding region, and these E1a encoding sequences also effectively are connected with the IRES in downstream, and wherein this IRES also effectively is connected with the encoding sequence in E1b district.

As employed in this article, the TRE that derives from specific gene is equivalent to the gene in its source and is to regulate the polynucleotide sequence that the polynucleotide sequence of the effective connection in the host cell of expressing this gene is transcribed.For example, as employed in this article, " human gland kallikrein transcriptional regulatory element ", or " hKLK2-TRE " is polynucleotide sequence, preferred dna sequence dna, it is increased in transcribing of the polynucleotide sequence that allows the effective connection in the host cell that hKLK2-TRE works, as expressing cell (the preferred mammal cell of androgen receptor, more preferably human cell), as prostatic cell.Therefore, hKLK2-TRE replys and comprises at least a portion of hKLK2 promotor and/or hKLK2 enhanser (just, ARE or androgen receptor combining site) to the combination of androgen receptor.In WO99/06576, described human gland kallikrein enhanser and comprised the adenovirus carrier of enhanser, in specially being incorporated herein as a reference.

As employed in this article, " probasin (PB) transcriptional regulatory element " or " PB-TRE " are polynucleotide sequences, preferred dna sequence dna, its selectivity is increased in transcribing of the polynucleotide sequence that effectively connects in the host cell that allows PB-TRE to work, as express cell (the preferred mammal cell of androgen receptor, more preferably human cell, even more preferably prostatic cell).Therefore PB-TRE replys and comprises at least a portion of PB promotor and/or PB enhanser (just, ARE or androgen receptor combining site) to the combination of androgen receptor.In WO98/39466, described expressing the adenovirus carrier of the special cell of male sex hormone, in specially being incorporated herein as a reference.

As employed in this article, " prostate specific antigen (PSA) transcriptional regulatory element ", or " PSA-TRE ", or " PSE-TRE " is polynucleotide sequence, preferred dna sequence dna, its selectivity are increased in transcribing of the polynucleotide sequence that allows the effective connection in the host cell that PSA-TRE works, as expressing cell (the preferred mammal cell of androgen receptor, more preferably human cell, even more preferably prostatic cell).Therefore, PSA-TRE replys and comprises at least a portion of PSA promotor and/or PSA enhanser (just, ARE or androgen receptor combining site) to the combination of androgen receptor.The tissue-specific enhancer that activity is arranged in prostate gland and use in adenovirus carrier has been described in WO95/19434 and WO97/01358, in wherein all specially being incorporated herein as a reference.

As employed in this article, " carcinomebryonic antigen (CEA) transcriptional regulatory element " or " CEA-TRE " are polynucleotide sequences, preferred dna sequence dna, its selectivity increases transcribing of effective polynucleotide sequence that connects in the host cell that allows CEA-TRE to work, as express the cell (preferred mammal cell, even more preferably human cell) of CEA.CEA-TRE replys and comprises at least a portion of CEA promotor and/or enhanser to transcription factor and/or the cofactor relevant with producing the CEA cell.In WO98/39467, described expressing the adenovirus carrier of the special cell of carcinomebryonic antigen, in specially being incorporated herein as a reference.

As employed in this article, " α-Jia Taidanbai (AFP) transcriptional regulatory element " or " AFP-TRE " are polynucleotide sequences, preferred dna sequence dna, its selectivity is increased in and allows transcribing of (the effectively polynucleotide sequence sequence that connects) in the host cell that AFP-TRE works, as express the cell (preferred mammal cell, even more preferably human cell) of AFP.AFP-TRE replys and comprises at least a portion of AFP promotor and/or enhanser to transcription factor and/or the cofactor relevant with producing the AFP cell.The adenovirus carrier of expressing the special cell of α-Jia Taidanbai has been described in WO98/39465, in specially being incorporated herein as a reference.

As used herein, " mucin gene (MUC) transcriptional regulatory element " or " MUCl-TRE " are polynucleotide sequences, preferred dna sequence dna, its selectivity is increased in transcribe (the effectively polynucleotide sequence that connects) that allows in the host cell that MUCl-TRE works, as express the cell (preferred mammal cell, even more preferably human cell) of MUCl.MUCl-TRE replys and comprises at least a portion of MUC1 promotor and/or enhanser to transcription factor and/or the cofactor relevant with producing the MUCl cell.

As employed in this article, " urothelial cell specific transcriptional response element " or " urothelial cell specificity T RE " is polynucleotide sequence, preferred dna sequence dna, it is increased in transcribing of the polynucleotide sequence that allows the effective connection in the host cell that urothelial specificity T RE works, i.e. target cell.Known various urothelial cell specificity T RE to replying with urothelial cell cells involved protein (transcription factor and/or cofactor), and comprises at least a portion of urothelial specificity promoter and/or urothelial specific enhancer.The urothelial cell specific transcriptional regulating and controlling sequence of exemplary comprises human or rodentine uroplakin (UP), for example, and UPI, UPII, UPIII etc.In WO 01/72994, described human urine tract epithelial cell specificity uroplakin transcription regulating nucleotide sequence and comprised the adenovirus carrier of identical sequence, in specially being incorporated herein as a reference.

As employed in this article, " melanophore specific transcriptional response element " or " melanophore specificity T RE " is polynucleotide sequence, preferred dna sequence dna, it is increased in transcribing of the polynucleotide sequence that allows the effective connection in the host cell that melanocyte specificity T RE works, i.e. target cell.Known various melanophore specificity T RE replys the cell protein relevant with melanophore (transcription factor and/or cofactor), and comprises at least a portion of melanophore specificity promoter and/or melanophore specific enhancer.Described in this article and be used to measure melanophore specificity T RE activity and the method that is used for so whether definite designated cell allows melanophore specificity T RE to work.The example of melanophore specificity T RE that is used for the present invention practice is including, but not limited to the TRE of 5 ' flanking region of deriving from tyrosinase cdna, tyrosine oxidase related protein-1 gene, derive from 5 of tyrosine oxidase related protein-2 gene '-TRE of flanking region, derive from the MART-1 gene 5 ' flanking region TRE or derive from unconventionality expression in the melanoma gene 5 '-TRE of flanking region.

In one aspect of the method, the invention provides the adenovirus carrier that comprises the transitivity colorectal carcinoma specificity T RE that derives from the PRL-3 gene that effectively is connected with the gene or the transgenosis of adenoviral replication necessity.As employed in this article, " derive from the transitivity colorectal carcinoma specificity T RE of PRL-3 gene " or " PRL-3 TRE " is polynucleotide sequence, preferred dna sequence dna, its selectivity is increased in transcribing of the polynucleotide sequence that allows the effective connection in the host cell that PRL-3 TRE works, as cell (preferred mammal cell, more preferably human cell, even more preferably metastatic colon cancer cell).Transitivity colorectal carcinoma specificity T RE can comprise one or more regulating and controlling sequences, for example enhanser, promotor, transcription factor combining site etc., and it can derive from identical or different gene.One preferred aspect in, PRL-3 TRE comprises the PRL-3 promotor.In WO04/009790, a preferred PRL-3TRE of the 0.6kb sequence upstream that derives from the translation initiation codon that is used for the PRL-3 gene has been described, in specially being incorporated herein as a reference.

In one aspect of the method, the invention provides the adenovirus carrier that comprises the liver cancer-specific TRE that derives from the CRG-L2 gene that effectively is connected with the gene or the transgenosis of adenoviral replication necessity.As employed in this article, " derive from the liver cancer-specific TRE of CRG-L2 gene " or " CRG-L2TRE " is polynucleotide sequence, preferred dna sequence dna, its selectivity is increased in transcribing of the polynucleotide sequence that allows the effective connection in the host cell that CRG-L2 works, as cell (preferred mammal cell, more preferably human cell, even more preferably hepatocellular carcinoma cells).Hepatocellular carcinoma specificity T RE can comprise one or more regulating and controlling sequences, for example enhanser, promotor, transcription factor combining site etc., and it can derive from identical or different gene.One preferred aspect in, CRG-L2 TRE can derive from the 0.8kb sequence upstream of the translation initiation codon that is used for the CRG-L2 gene, or from the 0.7kb sequence that is contained in the 0.8kb sequence (residue 119-803); Or from the EcoRI that derives from the 0.8kb sequence to the NcoI fragment, as at U.S. Provisional Application US60/511, the description in 812, in specially being incorporated herein as a reference.

In yet another aspect, the invention provides the adenovirus carrier that comprises the EBV specific transcriptional controlling element (TRE) that effectively is connected with the gene or the transgenosis of adenoviral replication necessity.In one aspect, EBV specificity T RE derives from the sequence upstream of the translation initiation codon of LMP1, LMP2A or LMP2B gene, as at U.S. Provisional Application US60/423, further describing in 203, in specially being incorporated herein as a reference.EBV specificity T RE can comprise one or more regulating and controlling sequences, for example enhanser, promotor, transcription factor combining site etc., and it can derive from identical or different gene.

Yet in another aspect, the invention provides the adenovirus carrier that comprises the hypoxia response element (" HRE ") that effectively is connected with the gene or the transgenosis of adenoviral replication necessity.But HRE is the transcriptional regulatory element that comprises the combining site of transcription complex HIF-I or hypoxemia inducible factor-1, several genes in itself and the regulatory region, comprise that vascular endothelial growth factor and several encoding glycolytic enzyme comprise the gene of ENO1, interact.Correspondingly, in one embodiment, transcribing under the control of cell state specificity T RE such as HRE, adenovirus carrier comprises adenoviral gene, preferably duplicate necessary adenoviral gene, as in WO 00/15820, further describing, in specially being incorporated herein as a reference.

In also having on the other hand, the invention provides the adenovirus carrier that comprises " telomerase promoter " or " TERT promotor " that effectively be connected with the gene or the transgenosis of adenoviral replication necessity.Be meant natural TERT promotor and function fragment, sudden change and derivative thereof as term " telomerase promoter " or " the TERT promotor of using in this article ".That the TERT promotor needs not to be total length or wild-type promotor.Those skilled in the art will know that how to obtain fragment and optionally to test them from the TERT promotor.TERT promoter fragment of the present invention has the promoter activity of selecting for tumour cell, and the tumor-selective of the encoding sequence of effective connection is expressed.In one embodiment, TERT promotor of the present invention is mammiferous TERT promotor.In another embodiment, mammiferous TERT promotor is human TERT (hTERT) promotor.Referring to, WO98/14593 and WO 00/46355, for example, exemplary TERT promotor is found practicality in the compositions and methods of the invention.

Yet in another aspect, the invention provides the adenovirus carrier that comprises " the E2F promotor " that effectively be connected with the gene or the transgenosis of adenoviral replication necessity.Be meant natural E2F promotor and function fragment, sudden change and derivative thereof as the term " E2F promotor " that uses in this article.That the E2F promotor needs not to be total length or wild-type promotor.Those skilled in the art will know that how to obtain fragment and optionally to test them from the E2F promotor.E2F promoter fragment of the present invention has the promoter activity of tumour cell to be selected, and the tumor-selective of the encoding sequence of effective selection is expressed.The example of known in the prior art many E2F promotors.Referring to, for example, Nature Medicine 1997:3 (10) 1145-1149 pages or leaves such as Parr, WO 02/067861, US20010053352 and WO 98/13508.

Key protein in the metastasis of cancer is expressed and appeared to represent to albumen urokinase plasminogen activator (uPA) and cell surface receptor thereof, upar (uPAR) in many ever-present vegetations.In breast, colon, prostate gland, liver, kidney, lung and ovarian cancer, contain this two kinds of protein.The sequence composition that adjusting uPA and uPAR transcribe is widely studied.(Riccio etc. (1985) Nucleic Acids Res.13:2759-2771 page or leaf; Cannio etc. (1991) Nucleic Acids Res.19:2303-2308 page or leaf; Equally referring to WO 98/39464).

The preparation of adenovirus carrier and generation

It is known in the prior art and can obtain from commercial source that generation is used for the standards system of the adenovirus carrier that insertion sequence expresses, for example from Clontech (Palo Alto, CA) Adeno-X of (Clontechniques (in January, 2000) 10-12 page or leaf) ^TMExpression system is from Qbiogene (Carlsbad, Adenovator CA) ^TMAdenovirus system and AdEasy ^TM

For convenience's sake, it is available providing the plasmid of the necessary part of adenovirus.Plasmid pXC.l (McKinnon (1982) Gene 19:33-42) comprises the wild-type left hand end of Ad5.PBHG10 (Bett etc. (1994); Microbix Biosystems Inc. Toronto) is provided at a right hand extreme that has the Ad5 of disappearance at E3.PBHG11 provides bigger E3 disappearance, extra 0.3kb deleted (Bett etc. 1994).Perhaps, (Microbix Biosystems, use Inc.) provides the right hand extreme of the Ad5 with E3 total length to pBHGE3.

In order to handle early gene, the transcription initiation site of Ad5E1A at the ATG initiation site of virus genomic 498 places and E1A encode fragment at virus genomic 560 places.This district can be used for the insertion of allos TRE.

Can introduce restriction site by using polymerase chain reaction (PCR), the primer that uses in this place can be confined to the Ad5 genome, can comprise that maybe a part carries the plasmid of Ad5 genomic dna.For example, using under the situation of pBR322, primer can use in the pBR322 main chain the EcoRI site and in the XbaI site at nt 1339 places of Ad5.By carrying out PCR two steps, introduce the nucleotide sequence that causes unique restriction site at this zone center eclipsed primer and change, the operator can be that the insertion of allos TRE is prepared in this site.

Also available similar strategy is used for the insertion of allos TRE composition so that effectively be connected with E1B.The E1B promotor of Ad5 is made up of single high affinity recognition site and the TATA box of Sp1.This district extends to 1701 from Ad5nt 1636.By cell-specific allos TRE being inserted this district, the cell-specific that the operator can be the E1B gene is transcribed and is prepared.By using the left hand district of modifying by the cell-specific response element of regulating E1A, introduce allos TRE to regulate the template of E1B as being used to, the adenovirus carrier of generation depends on the cell-specific transcription factor that is used for E1A and E1B expression.In some embodiments, the 19kDa district of E1B partly or entirely is removed.

Similarly, cell-specific allos TRE can be inserted the upstream of raq gene so that its express cell specificity.The E2 early promoter, draw from about 27050-27150 at Ad5, by main and an accessory transcription initiation site, the latter accounts for 5% of this E2 transcript, two unconventional TATA boxes, two E2F transcription factor combining sites and ATF transcription factor combining site form (for the detailed commentary of E2 promoter structure referring to Swaminathan etc., Curr.Topicsin Micro.and Immunol. (1995) 199 (third part): the 177-194 page or leaf).

The E2 late promoter with by and therefore not receptor gene operation overlapping to the encoding sequence of the gene of chain encoding.Yet, the E2 early promoter only with to the proteic sequence of coding 33kD on the chain overlapping several base pairs.Merit attention, SpeI restriction site (Ad5 position 27082) is the proteic part terminator codon of above-mentioned 33kD and easily main E2 early transcription initiation site and the conjugated protein site of TATA is opened with UPSTREAM BINDING FACTOR combining site E2F and ATF branch.Therefore, the allos TRE with SpeI end inserts the cell specific expression that the SpeI site can be interrupted the endogenous E2 early promoter of Ad5 and the E2 transcript can be provided.

For E4, must utilize the right hand portion of adenoviral gene group.The E4 transcription initiation site is mainly at the about nt of Ad5 35605 places, the TATA box at the AUG/CUG of about nt 35631 places and ORF I at about nt 35532 places (Virtanen etc. (1984) J.Virol.51:822-831).With any other gene that is used for of above-mentioned strategy, allos TRE can be introduced the upstream of transcription initiation site.For the structure of the total length adenovirus that has allos TRE insertion in the E4 district, in these proteinic synthesizing, provide in the W162 cell (Weinberg etc. (1983) Proc.Natl.Acad.Sci.80:5383-5386 page or leaf) of the trans protein of E4 with additional defective and can carry out cotransfection and homologous recombination.

In one embodiment, the invention provides adenoviral gene and transcribe under the control and the adenovirus carrier of polynucleotide sequence under the control of the 2nd TRE element of coding ADP, and wherein preferably duplicate necessary adenoviral gene at a TRE.The dna sequence dna of coding ADP and the aminoacid sequence of ADP can openly obtain.Briefly, utilize technology well known in the prior art,, from Ad, obtain the ADP encoding sequence as PCR.Preferably, also can obtain Y leader sequence (it is the important sequence that is used to correct late gene expression) and it is connected with the ADP encoding sequence.Then, ADP encoding sequence (have or do not have Y leader sequence) is introduced the adenoviral gene group, for example, (drive by MLP) at the ADP of this place encoding sequence in the E3 district.The ADP encoding sequence can also insert in other position of adenoviral gene group, as the E4 district.Perhaps, the ADP encoding sequence can with dissimilar TRE, including, but not limited to, other viral TRE effectively connects.In one embodiment, carrier of the present invention has the ADP that effectively is connected with its natural TRE.

Can utilize the recon technology of prior art standard to prepare virus vector of the present invention.In the prior art, the modifying method that is modified with ability rf or incompetence duplicating virus carrier is well known and describes in this article with in the publication of originally quoting.Be used to modify transcribing that the whole bag of tricks that element enters adenovirus is described in this article and being standard of the prior art and well-known of adenovirus carrier and cloned, transgenic and expectation.The plasmid of various gland-containing virus genome different pieces is arranged in the prior art, comprise the plasmid that contains whole adenoviral gene group.(for example US20030104625) in the prior art also fully described the structure of these plasmids.In case select the site that is used to modify, available suitable plasmid carries out this modification.Then, should modify introducing total length adenovirus carrier genome by for example homologous recombination or external connection.Homologous recombination for example, can occur in mammalian cell (for example PerC6) or the bacterial cell (for example E.CoIi, referring to WO9617070).The genomic operation of virus vector can or comprise well-known molecular biology method in addition, includes, but are not limited to polymerase chain reaction (PCR), PCR-SOEing and restriction digest.If it is overlapping that use homologous recombination, two plasmids can be divided equally at least about the sequence of 500bp, although the lower efficient of tool usually can be recombinated in less overlap.If desired, but each plasmid of independent operation, succeeded by cotransfection in competent host, the complementary gene that provide suitable adenovirus carrier to breed.Usually utilize suitable transduction media,, plasmid is introduced proper host cell (for example 293, PerC.6, Hela-S3 cell) as cationic-liposome or calcium phosphate.Perhaps, can also utilize the right side of adenoviral gene group and the external all recombinant adenovirus derivatives that duplicates necessary part of structure gland-containing virus genome that are connected of left part.Berkner etc. (1983) Nucleic Acid Research 11:6003-6020 page or leaf; Bridge etc. (1989) J.Virol.63:631-638.

In the prior art, " the production cell " that is used for virus vector be well known (for example PCT/US98/04080).Producing cell is that adenovirus carrier is transferred the cell that is replicated and is packaged into virion with adenovirus carrier.Preferred packing cell is the cell that those qualifications that have been designed to can cause wild-type adenovirus particulate homologous recombination.If virus vector has the indispensable gene of removal or inactivation, then should produce cell and replenish this inactivation gene.The cell that can be used to produce adenovirus particles of the present invention comprises that human embryonic kidney cells is 293 (Graham etc., J Gen.Virol.36:59-72 page or leaf (1977)), the human embryos retinoblast is PER.C6 U.S. Pat 5,994,128 and US6,033,908; Fallaux etc., Hum.Gene Ther.9:1909-1917 (1998)) and human uterus's neck tumour source cell be HeLa-S3 (PCT applies for US04/11855).Perhaps or in addition, produce the gene that cell can be expressed in selective control in the virus vector or inactivation.An embodiment of the invention comprise the production cell that contains adenovirus carrier of the present invention.

The chimeric adenoviral scleroproein

Adenoviral fiber protein is attached in the cell receptor at virion and plays an important role.In the prior art, the fiber gene sequence from several different adenoviral serotypes is known.Scleroproein is divided into three structural domains.Conservative N-terminal comprises with the penton base gets in touch relevant sequence and nuclear localization signal.Shaft-like " axle " of variable-length comprises the repetition of a 15-amino acid beta structure, multiple number range from Ad3 6 in the Ad5 22.Comprise boundary between the repeating unit of beta structure in the amino acid whose conservative range flags axle of sequence TLWT and the ball heads structural domain.188 residues of the magnitude range in C-terminal header structure territory from 157 amino-acid residues of the staple fibre that is used for Ad41 to the Ad5 fiber.The fiber furcella is a homotrimer, and each virion has 12 by getting in touch the furcella that adheres to penton base mixture.Scleroproein must can trimerizing so that have function.The trimerization structural domain can be natural structural domain or allogenic trimerization structural domain.

Useful chimeric adenoviral fiber comprises at least a portion in subtype C adenovirus (for example Ad1,2,5 and 6 serotypes) axle district and Head Section at least a portion in conjunction with the hypotype B adenovirus (for example Ad3,7,11,14,16,21,35 and 50) of CD46 among the present invention.In one embodiment, all the axle district from Ad2 or Ad5 and all Head Section from Ad35.In another embodiment, chimeric fiber comprise subtype C adenovirus axle district a part and from the Head Section of CD46 bonded adenovirus.Fiber Head Section in conjunction with CD46 includes, but are not limited to, and those derive from fiber Head Section (Sirena etc., the J Virol.20045 month of Ad3; The 4454-62 page or leaf), 11,14,16,21,35,37 and 50 78 (9):.In another embodiment, chimeric fiber albumen comprises that cow adenovirus axle construction territory and deriving from is selected from Ad3,7,11,14,16,21,35,37,50 and the part in the header structure territory of the adenoviral serotype of hypotype D adenovirus.In another embodiment, chimeric fiber albumen comprises the part in subtype C adenovirus axle district and the part of hypotype D adenovirus Head Section.In following example, verified have the Ad2 that contains Ad2 or the Ad5 axle and the chimeric fiber of Ad35 Head Section or Ad5 and derive adenovirus carrier than the fibrinous identical adenovirus carrier that has natural Ad5 or Ad35 some primary tumo(u)r cell of more effectively transduceing.Those skilled in the art recognize that in the enhancing transduction that keeps the primary tumo(u)r cell, can further modify chimeric fiber albumen.

GenBank AAA75331 discloses the sequence of Ad35 fiber.This is an exemplary sequence and has many genome modification (Flomenberg etc., J.Infec.Dis., 155 (6) 1127-1134 (1987)).In the embodiment of this invention, deriving from the part of the adenovirus protein of Ad35 Head Section can be from any Ad35 genome modification.

This paper provides Ad2 well known in the prior art, Ad5 with the Ad35 sequence information and relevant indication is provided, and those skilled in the art can combine the part of proper A d2 or the Ad5 axle part with the Ad35 head to obtain the especially enhancing transduction of primary tumo(u)r cell of tumour cell.For example, those skilled in the art can lack with substitution analysis to determine how to combine various sequence parts.

In one embodiment, chimeric fiber albumen comprises complete adenoviral serotype 5 (Ad5) fibre axis (the 47-399 amino acid of SEQ ID NO:2) or complete Ad2 fibre axis (the 47-399 amino acid of SEQ IDNO:4).In another embodiment, chimeric fiber albumen comprises the Head Section from adenoviral serotype 35 scleroproeins (the 46-132 amino acid of SEQ ID NO:6).In another embodiment, chimeric fiber albumen comprises complete adenoviral serotype 5 (Ad5) fibre axis (the 47-399 amino acid of SEQID NO:2) or complete Ad2 fibre axis (the 47-399 amino acid of SEQ ID NO:4) and from the Head Section of adenoviral serotype 35 scleroproeins (the 46-132 amino acid of SEQ ID NO:6).In another embodiment, chimeric fiber albumen comprises sequence part that derives from Ad2 or Ad5 fibre axis and the sequence part that derives from the Ad35 Head Section.

In one embodiment, Ad5 or the block reservation of Ad2 axle KKTK sequence (the 91-94 amino acid of SEQ IDNO:2 or SEQ ID NO:4).In another embodiment, KKTK (the 91-94 residue of SEQ ID NO:2 or the SEQ ID NO:4) sequence in the natural axle sequence is removed or suddenlys change.In one embodiment, the Ad5 axle keeps KLGTGLSFD sequence (the 376-384 amino acid of SEQ IDNO:2) (Wu etc., the J Virol.20037 month; 77 (13): the 7225-35 page or leaf), the Ad2 axle keeps KLGAGLSFD sequence (the 376-384 amino acid of SEQ ID NO:4) or this axle comprises consensus motif KLGXGLXFD/N (SEQ ID NO:7; Wu etc., 2003).In one embodiment, the Ad5 axle keeps and comprises that having the 3rd of whippy structure territory repeats the GNLTSQNVTTVSPPLKKTK (the 76-94 amino acid of SEQ ID NO:2) in axle district.In another embodiment, the Ad35 axle comprises that this 3rd repeats (GTLQENIRATAPITKNN), and it lacks the sequence relevant with the elasticity of fiber (the 76-92 amino acid of SEQ ID NO:6).

Chimeric fiber protein can comprise further modification, including, but not limited to reducing vector particles and a special cellular type or surpassing the combining an of cellular type, enhanced virus carrier granule and a specific cellular type or combining and/or reduce modification to the immunne response of the adenovirus carrier in the beasts above a cellular type.The example of these modifications includes, but are not limited at U. S. application US10/403, and 337, WO 98/07877, WO 01/92299, WO 2003/62400 and U.S. Pat 5,962,311, US6,153,435, US6,455,314 and (J Virol.2003 July 1 such as Wu; 77 (13): those that describe the 7225-7235 page or leaf).

The non-natural part can be contained in the HI ring or at the proteic carboxyl terminal of chimeric fiber.

From processing cleavage site and 2A class sequence

In another aspect of the present invention, (for example 2A class sequence) is used for expressing two polypeptide that come from a mRNA " to process cleavage site certainly "." process cleavage site certainly " or " processing cutting sequence " certainly is meant DNA or aminoacid sequence, when wherein translating, comprise the cutting of in the rapid molecular of the polypeptide of processing cleavage site (cis), thereby cause the expression of discrete maturation protein or polypeptide product.This " processing cleavage site certainly " also can be described as the back and translates or with the translation process cleavage site, illustrate by 2A site, sequence or structural domain in this article.As used in this article, " from processed peptide " is defined as coding in this article from the peptide expression product of processing the dna sequence dna of cleavage site or sequence, it is when translation, mediate albumen or contain cutting in the rapid molecular of the albumen of processing cleavage site or polypeptide, to produce discrete maturation protein or polypeptide product.It is reported that 2A site, sequence or structural domain show translation effect by modifying ribosomal activity with the hydrolysis that promotes ester bond, discharge polypeptide (Donnelly etc. in the synthetic mode of carrying out of the downstream translation product that allows to disperse thus from the translation mixture.J Gen Virol.2001; May; 82 (Pt 5): the 1013-25 page or leaf).Perhaps, it is reported also that 2A site, sequence or structural domain cut its C-end by up time and show " autoproteolytic cleavage " or " cutting " (Furler to produce elementary depolymerization product; Palmenberg, Ann.Rev.Microbiol.44:603-623 page or leaf (1990)).

Because the ability of effective processing of its mediation polyprotein that has, after deliberation the mutation of 2A sequence (Donnelly etc., J.Gen.Virol.82:1027-1041 (2001)).Homologue and mutation 2A sequence are contained in the scope of the present invention.

Comprise the sequence of coding from processing cutting sequence, carrier structure as 2A between the open reading frame or 2A class sequence, can further comprise contiguous additional proteolysis cleavage site, after removing cutting, comprise the amino acid of processing cutting sequence certainly from processing cutting sequence.Term " additional proteolysis cleavage site " is meant and inserts the contiguous sequence of processing cleavage site certainly of adenovirus carrier of the present invention, as 2A or 2A class sequence, and provides by processing cutting sequence certainly and removes the remaining additional amino acid whose method in cutting back.Described exemplary " additional proteolysis cleavage site " in this article, and included, but are not limited to, had the furin cleavage site of consensus sequence RXK (R) R.Comprise the sequence of coding from processing cutting sequence, as the 2A between the open reading frame or 2A class sequence and can further comprise the carrier structure of additional proteolysis cleavage site, at U.S. Patent application US10/831, further describe in 302, in specially being incorporated herein as a reference.

In one embodiment, the invention provides and be used to remove residual amino acid whose method and the composition that is used for identical expression.Many new textures have been designed to remove additional amino acid from this PROTEIN C end.The furin cutting occurs in the C-terminal of cleavage site, and it has consensus sequence RXR (K) R, and wherein X is any amino acid.In one aspect, the invention provides by utilization and be selected from a kind of enzyme in the enzyme that is called as carboxypeptidase (CPs), it includes, but are not limited to carboxypeptidase D, E and H (CPD, CPE, CPH) and removes the alkaline amino acid residue R or the K that newly expose from the PROTEIN C end.Because CPs can remove the alkaline amino acid residue of PROTEIN C end, can remove the amino-acid residue that all derive from the furin cleavage site by CP, comprise basic aminoacids R or K specially, as RKKR (SEQ ID NO:25), RKRR (SEQ ID NO:26), RRRR (SEQ ID NO:27) etc.

In an embodiment of the invention, processing cutting sequence (for example 2A or 2A class sequence) effectively is connected with transgenosis with the adenovirus protein coding region certainly.Adenovirus protein CDS can process the upstream of cleavage site certainly at this, and genetically modified downstream.Perhaps, transgenosis CDS can process the upstream of cleavage site certainly at this, and adenovirus protein CDS is in the downstream.

Many CDS can with process cleavage site certainly and be connected.In one embodiment, Ad CDS is by processing cleavage site certainly and effectively be connected to first transgenosis and described first transgenosis effectively being connected to second transgenosis by processing cleavage site certainly.In one embodiment, the identical or different protein of first and second transgenes encodings.

Transgenosis

Carrier of the present invention can comprise one or more transgenosiss.In this way various heritabilities can be introduced in the target cell.

" cytokine " or phraseological Equivalent comprise, and are not limited to, and work and those hormones of round-robin in blood not in the part, and, when used according to the invention, produce the change of single immunne response.Also comprise the adhesion or the accessory molecule that cause that single immunne response changes in the definition of cytokine.Therefore, the example of cytokine comprises, but be not limited to IL-1 (a or P), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, GM-CSF, M-CSF, G-CSF, LIF, LT, TGF-P, y-IFN, a-IFN, P-IFN, TNF-a, BCGF, CD2 or ICAM.The description of foregoing cytokine and other applicable immunomodulator can be at " Cytokines and Cytokine Receptors " A.S.Hamblin, D.Male (writing), the Oxford University Press, New York, NY (1993)), or " Guidebook toCytokines and Their Receptors " N.A.Nicola (writing), the Oxford University Press, New York, NY (1995)) finds in.Under the situation of expectation human treatment purposes, cytokine is can preferred albumen with human form remarkable similar or derive from human sequence's (human origin just).In a preferred implementation, transgenosis is cytokine such as GM-CSF.

In addition, immunity system is had similar when active when it is proved to be demonstration, having the homologue of the basic structure identical with IL-2, the GM-CSF of human form, TNF-a etc. and/or other mammiferous cytokine of amino-acid sequence, is useful in the present invention.Similarly, find remarkable similar but have the protein of the conservative change of protein sequence, useful equally in the present invention with any special cytokine.Therefore, can be in protein sequence conservative substitution and do not hinder the functional of protein molecule, and therefore can produce as playing effect of cytokines among the present invention and but have protein with the slightly different amino-acid sequence of known array at present.This conservative substitution generally includes following displacement: glycine, L-Ala, Xie Ansuan, leucine, Isoleucine, aspartic acid, L-glutamic acid, asparagine, glutamine; Serine, Threonine; Methionin, arginine; And phenylalanine, tyrosine.

At last, the use of the odd number of the word among the application " cytokine " or plural form without limits and should not limit the explanation of the present invention and claim.Except cytokine, adhesion or auxilliary molecule or its combination can be used separately or be used in combination with cytokine.

RHuGM-CSF (GM-CSF) is the cytokine that is produced by inoblast, endotheliocyte, T cell and scavenger cell.Shown the growth of the hematopoietic cell of this cytokine induction granulocyte and macrophage system.In addition, it also stimulates the antigen processing of dendritic cell and presents function (presenting), and it is that immune major antigen is delivery cell (APC).The result of animal model experiment fully shows: generation GM-CSF tumour cell (GVAX just) can be induced the immunne response to parental generation, non-transduction tumour cell.

GM-CSF has increased antigen presentation ability (Gasson, Blood on March 15th, 1991 of dendritic cell (DC) subclass that can promote that powerful antitumor is replied; 77 (6): 1131-45; Mach etc., Cancer Res.2000 June 15; 60 (12): 3239-46; The summary of Mach and Dranoff, Curr Opin Immunol.2000 October; 12 (5): 571-5).Referring to, for example, Boon and Old, Curr Opin Immunol.1997 October 1; 9 (5): 681-3).Expectation tumour antigen mark is replied the systemic immunity that causes metastases of presenting of the T cell in the draining lymph node.Equally, the radiation tumour cell of expression of GM-CSF has shown the effect (as at the called after that further describes with the lower section " GVAX ") of the antitumor effective vaccine that excites.The high local concentrations of some cytokine of being carried by genetically modified cell has been found to cause tumour regression.(Abe etc., J.Cane.Res.Clin.Oncol.121:587-592 page or leaf (1995); Gansbacher etc., Cancer Res.50:7820-7825 page or leaf (1990); Fomi etc., Cancer and Met.Reviews 7:289-309 page or leaf (1988)).The open WO200072686 of PCT has described the tumour cell of expressing various cytokines.

In an embodiment of the invention, adenovirus comprises the GM-CSF encoding sequence that effectively is connected with the controlling element of the expression that is used for the primary tumo(u)r cell.In one embodiment, the GM-CSF encoding sequence human GM-CSF that encodes.Perhaps, GM-CSF encoding sequence codified murine GM-CSF.In some embodiments, the GM-CSF encoding sequence is a cDNA sequence (for example SEQ ID NO:16).In other words, the GM-CSF encoding sequence does not comprise the intron sequences that splices outside before the translation.In another embodiment, GM-CSF coding sign indicating number sequence is genome encoding sequence (for example SEQ ID NO:15).In other words, this encoding sequence comprises at least one the natural GM-CSF intron that splices outside before the translation.In one embodiment, GM-CSF encoding sequence coding SEQ ID NO:14.In the Genbank number of registering on the books: AF373868, AC034228, AC034216, M10663 and NM000758, can find other example of GM-CSF encoding sequence.

In one embodiment, mark of transgenes encoding.In another embodiment, cytotoxic protein of transgenes encoding.The proteic carrier of available code cytotoxin can be used to strengthen the degree of therapeutic efficacy by the speed that improves cytotoxic activity.The virus replication that this can regulate by coupling and the expression of corresponding selecting cell toxicity and one or more metabolic enzymes realize, these enzymes for example HSV-tk, nitroreductase, Cytochrome P450 maybe to make 5-flurocytosine (5-FC) metabolism be Isocytosine deaminase (CD), Procaine esterase (CA), deoxycytidine kinase (dCK), purine nucleoside phosphorylase (PNP), the carboxypeptidase G 2 (CPG2 of chemotherapeutic 5 FU 5 fluorouracil (5-FU); J Med Chem.2004 such as Niculescu-Duvaz May 6; 47 (10): 2651-2658), thymus gland glycosides Starch phosphorylase (TP), thymidine kinase (TK) or xanthine-guanine phosphoribosyl transferase (XGPRT).Also can utilize this class transgenosis to give bystander effect.

Genetically modified other examples that can be contained in the adenovirus carrier of the present invention comprise the factor that can start programmed cell death, antisense or ribozyme, it is together with the protein of propagation necessity of bootable mRNAs coding pair cell of other performances or pathogenic agent, as structural protein, transcription factor, polysaccharase etc., virus or other pathogenic protein, hyperplasia in the pathogen cells wherein, cytotoxin albumen, for example, diphtheria series, ricin, toxalbumin etc., coding nucleic acid enzyme (for example ribonuclease A) or proteolytic enzyme (trypsinase for example, papoid, Proteinase K, the gene of genetically engineered kytoplasm modification carboxypeptidase etc.), chemokine, as MCP3 α or MIP-1, derive from virus, the pore-forming protein of bacterium or mammalian cell, fusion gene, chemotherapy sensitization gene and radiation sensitization gene.Other relevant gene comprises cytokine, antigen, transmembrane protein etc., as IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 or flt3, GM-CSF, G-CSF, M-CSF, IFN-α ,-β ,-γ, TNF-α ,-β, TGF-α ,-β, NGF, MDA-7 (melanoma differentiation associated gene-7, mda-7/ interleukin II 4) etc.Further example comprises, short apoptosis gene such as Fas, Bax, Caspase, TRAIL, Fas part, nitric oxide synthase (NOS) etc.; Can cause fusion gene such as V22, the VSV etc. of cytogamy or promotion cytogamy; Tumor suppressor gene such as p53, RB, p16, p17, W9 etc.; Gene such as pQE30/en, the angiostatin etc. of gene relevant and coding anti-angiogenic proteins with the cell cycle.

Although can use any relevant gene or encoding sequence in practice of the present invention, some gene or its fragment are especially suitable.For example, the coding region of coding immunogen polypeptide, toxin, immunotoxin and cytokine is useful in practice of the present invention.These coding regions comprise those coding regions and the coding region that comprises the materials that those codings are following, other coding regions above: promote and the immunocyte interacting proteins, as B7, CD28, MHC class I, MHC class II, TAPs, tumor associated antigen is as from MART-1, the immunogen sequence of gp100 (pmel-17), tyrosine oxidase, tyrosine oxidase related protein 1, tyrosine oxidase related protein 2, the melanotropin acceptor, MAGE1, MAGE2, MAGE3, MAGE12, BAGE, GAGE, NY-ESO-1, beta-catenin is white, MUM-1, CDK-4, cysteine aspartase 8, KIA 0205, HLA-A2R1701, α-Jia Taidanbai, the protein of telomerase catalytic, G-250, MUC-1, oncofetal protein, p53, Her2/neu, triosephosphate isomerase, CDC-27, LDLR-FUT, telomerase reverse transcriptase, PSMA, blocking-up suppresses the cDNA of the antibody of signal (CTLA4 blocking-up), chemokine (MIP1 α, MIP3 α, CCR7 part and calprotectin), the angiogenesis inhibitor gene comprises, but be not limited to coding METH-1, METH-2, the TrpRS fragment, the proliferin related protein, the prolactin fragment, PEDF, angiostatin, the various segmental gene of extracellular matrix protein matter and somatomedin/cytokine inhibitor; The various fragments of extracellular matrix protein matter comprise, but be not limited to, angiostatin, pQE30/en, the prokinin statin, Fibrinogen-E fragment, thrombospondin, TUMSTATIN (tumstatin), CANSTATIN (canstatin), restin (restin), somatomedin/cytokine inhibitor comprises, but be not limited to the VEGF/VEGFR antagonist, sFlt-1, sF1k, sNRP1, angiogenin/tie antagonist, sTie-2, chemokine (IP-10, PF-4, Gro-β, IFN-γ (Mig), IFN α, FGF/FGFR antagonist (sFGFR), Ephrin/Eph antagonist (sEphB4 and sephrinB2), PDGF, TGF β and IGF-1.Be suitable for the gene codified enzyme that practice of the present invention utilizes (as for example, urease, renin, zymoplasm, metalloprotease, nitric oxide synthase, superoxide dismutase, other enzymes known to catalase and those skilled in the art), enzyme inhibitors (alpha1-antitrypsin for example, Antithrombin III, cell or hiv protease inhibitor, Type 1 plasminogen activator inhibitor-1, the tissue depressants of metalloprotease etc.), cystic fibrosis is striden the film conduction and is regulated (CFTR) albumen, Regular Insulin, dystrophin, or the major histocompatibility complex of class I or II (MHC) antigen.Equally usefully the corresponding gene polypeptide expressed can be adjusted/be regulated to coding, can suppress bacterium, the polypeptide of parasite or virus infection or its growth (for example, antigenic peptide, epitope and trans-dominant (transdominant) the albumen modification of passing through to compete the effect of inhibition natural protein), programmed cell death inductor or inhibitor are (for example, Bax, Bcl2, other materials known to BclX and those skilled in the art), cytostatic agent (for example, p21, p16, Rb etc.), apolipoprotein (for example, ApoAI, ApoAIV, ApoE etc.), oxygen free radical scavenger, polypeptide with antitumous effect, antibody, toxin, immunotoxin, mark (for example, beta-galactosidase enzymes, luciferase etc.) or in the prior art approved to clinical state treatment or prevent useful valuable any other gene.Further transgenosis comprises that those codings suppress the polypeptide of cell fission or signal transduction, tumor suppressor protein (for example, p53, Rb, p73), activate the polypeptide of host immune system, tumour (is for example closed associated antigen, early stage or the late antigen of MUC-1, BRCA-1, HPV such as E6, E7, L1, L2 etc.) gene, optionally combine with cytokine.

The present invention further comprises the adenovirus carrier that contains two or more transgenosis combinations.In some cases, two or more transgenosiss have the mode of collaborative, complementary and/or nonoverlapping toxicity and effect.

When design adenovirus carrier of the present invention, consider genetically modified biological activity, for example, in some situation, advantageously in carrier, insert the transgenosis render transgenic and only express or main the expression in the early stage or late period of adenovirus infection.For some transgenosiss, preferably in the virus early expression transgenosis of life cycle.In the case, this transgenosis can be inserted in any early stage district (for example, E3) or insert L1 district, upstream.

GVAX

The invention still further relates to tumour cell, change method target antigen or antigenic single immunne response by the proteic adenovirus carrier transduction of the chimeric fiber that at least a portion of containing Ad5 or Ad2 axle and at least a portion Ad35 head are provided.Usually tumour cell is the primary tumo(u)r cell.Exemplary tumour cell comprises from same individual (from body) or from the tumour cell of Different Individual (allogenic).In another embodiment, tumour cell be from the tumor cell line of tumour of just treating or cancer same type.

The present invention further comprises by introducing Mammals and improves method to the immunne response of tumour cell, tumour cell is wherein replied raising to the mammalian immune of tumour cell with the proteic adenovirus carrier transduction of the chimeric fiber of at least a portion with at least a portion of containing Ad5 or Ad2 axle and Ad35 head.In one embodiment, the immunne response of raising is a humoral immunoresponse(HI).In another embodiment, the immunne response of raising is a cellullar immunologic response.In another embodiment, the immunne response of raising is cell and humoral immunoresponse(HI).

In relevant embodiment, this method comprises that further the tumour cell of deactivation transduction and transduction tumour cell give Mammals.The tumour cell that gives can be the transduction tumour cell from body, allogenic or bystander cell line (below further explanation).Usually, before introducing, make the tumour cell of the transduction propagation defective that becomes.In one embodiment, this Mammals is the mankind that have with the tumour cell of transduction tumour cell same type.In a preferred embodiment, after the transduction tumour cell was introduced, this mammiferous tumour cell showed growth-inhibiting or necrocytosis.

In another aspect, the present invention contains the propagation defective tumour cell of the genetic modification that the chimeric recombinant adenovirus of the nucleic acid of at least a human cell's factor of encode transduces by the usefulness of introducing the treatment significant quantity, be provided for promoting to tumour in the Mammals, or its antigenic systemic immunity method of replying, wherein this transduction tumour cell is identical with this tumor type and express at least a common antigen.

In some embodiments, the systemic immunity of tumour is replied cause tumour regression or suppress growth of tumor.

In one embodiment, tumour cell derives from the Mammals with tumour, as the mankind.In some embodiments, before introducing, tumour cell refrigerates.The tumor cell type for the treatment of in some embodiments, is selected from tumor of prostate, melanoma, nonsmall-cell lung cancer, mammary cancer, epidermal carcinoma, bladder cancer, prostate cancer, head and neck cancer, the preceding pathology of tumour, carcinopolypus, ovarian cancer, cervical cancer, leukemia and kidney.When the tumour cell class for the treatment of was prostate cancer, prostate tumor cells system can be selected from DU1 45, PC-3 and LnCaP.

In a preferred embodiment of the present invention, utilize adenovirus carrier of the present invention that human GM-CSF transgenosis is exsomatized and import former human tumor cell.After transduction, this cell of radiation makes their become incompetence propagation.Then, the expression of GM-CSF cell of propagation defective is introduced patient's (for example, by intracutaneous or subcutaneous route) again and played Theratope thus.

Tumour cell is selected from the tumour cell from body, allogenic tumour cell and tumor cell line.Can be by transduction tumour cell in external, stripped or the body.In U.S. Pat 5,637,483, US5,904, in 920 and US6,350,445, described by genetic modification with the express cell factor, for example, GM-CSF, introduce again subsequently the treatment that is used for cancer in the patient body from body and allogenic cancer cells, in specially being incorporated herein as a reference.In U.S. Pat 6,033, in 674 and US5,985,290, the expression of GM-CSF genetic modification tumour cell that is used for treatment of pancreatic cancer or the form of " express cell factor cell vaccine " (" GVAX ") have been described, in specially being incorporated herein as a reference.In U.S. Pat 6,464, in 973, a bystander cell line system that general immunomodulatory gene is modified has been described, in specially being incorporated herein as a reference.

Use the clinical trial of expression of GM-CSF cell vaccine (GVAX) to begin to be used for the treatment of prostate cancer, melanoma, lung cancer, carcinoma of the pancreas, kidney and multiple myeloma.Described the many clinical trials that utilize the GVAX cell vaccine, it should be noted that at melanoma and prostate gland, kidney and carcinoma of the pancreas (Cancer Res.1999 such as Simons JW; 59:5160-5168; Cancer Res.1997 such as SimonsJW; 57:1537-1546; Proc.Natl.Acad.SciUSA 1998 such as Soiffer R; 95:13141-13146; Jaffee waits J Clin Oncol 2001; 19:145-156; J Clin Oncol 200321:624-30 such as Salgia; J Clin Oncol 200321:3343-50 such as Soiffer; 18 days 96 (4) February of J Natl Cancer Inst.2004 such as Nemunaitis: 326-31).

The invention provides promotion to mammalian subject, the method for the raising immunne response of the cancer of preferred human patient.Neededly be, this method affect is replied the systemic immunity of cancer, that is, and and t cell response and/or B cell response.This improvement comprises to patient introduces the expressed at least a antigenic genetic modification tumour cell of tumour cell that patient has, and utilization has the proteic adenovirus carrier of chimeric fiber and finishes this genetic modification, wherein chimeric fiber albumen comprises at least a portion in subtype C adenovirus (for example Ad1,2,5 and 6 serotypes) axle district and Head Section at least a portion in conjunction with hypotype B adenovirus (for example Ad3,7,11,14,16,21, the 35 and 50) Head Section of CD46, for example, at least a portion of Ad5 or Ad2 axle and Ad35 head.The use of this adenovirus carrier promotes transduction and genetically modified expression.Although can use any known or method that makes the cell proliferation defective of finding afterwards,, the genetic modification tumour cell incompetence that becomes is bred usually by radiation.When the genetic modification tumour cell is introduced the experimenter, excited the antigenic immunne response of cancer.

In one approach, the genetic modification tumour cell comprises the cell of modified single population with the express cell factor and gives the experimenter as the part methods of treatment.In other method, modify two or more genetic modification tumour cell population one by one to express different transgenosiss and to give the experimenter.Therapeutic modality can comprise one or more other cancer therapeutical agent or therapy.

Usually, the genetic modification tumour cell that uses in practice of the present invention comprises one or more tumour cells from body, allogenic tumour cell and tumor cell line (bystander cell line just).For instance, in one approach, the genetic modification tumour cell (just, GVAX) provides as allogenic or bystander cell line system and one or more other cancer therapeutical agent is expressed by autogenous cell.In other method, when (just, in the time of GM-CSF), one or more other transgenosiss are expressed by allogenic or bystander cell line system by the autogenous cell express cell factor.Directly relatively the mouse tumour cell by various cytokines transductions proves, the secrete GM-CSF tumor cell induction the most comprehensive antitumor protection.In a preferred implementation, the cytokine of genetic modification tumor cells expression of the present invention is GM-CSF (" GVAX ").Utilize the improvement adenovirus carrier that the GM-CSF encoding sequence is introduced this carrier, this carrier has at least a portion and its Head Section at least a portion in conjunction with the hypotype B adenovirus Head Section of CD46, for example the chimeric fiber albumen of at least a portion of Ad5 or Ad2 axle and Ad35 head that comprises subtype C adenovirus axle district.The optimized encoding sequence of GM-CSF is at Science 230 (4731): 1282-5 such as Huebner K., the genome sequence of describing in 1985, yet, sometimes in practice of the present invention, find the practicality (Cantrell etc. of the GM-CSF of cDNA form, Proc.Natl.Acad.Sci., 82,6250-6254,1985).

In some embodiments, the tumour cell of genetic modification refrigerates before introducing.In a preferred embodiment, the genetic modification tumour cell is introduced into the same individual from its origin.In another preferred embodiment, genetic modification tumour cell and tumour derive from Different Individual.In some preferred implementation, the tumor type for the treatment of is selected from the cancer of bladder, breast, colon, kidney, liver, lung, ovary, uterine cervix, pancreas, rectum, prostate gland, stomach, epidermis; The hematopoiesis tumour of lymph or marrow pedigree; Between tumour such as the fibrosarcoma or the rhabdosarcoma in matter source; Other tumor type such as melanoma, teratocarcinoma, neuroblastoma, neurospongioma, gland cancer and non-little pneumonocyte cancer.

Before introducing patient, the genetic modification tumour cell preferably from about 50-200rads/min, more preferably shines under the dosage of about 120-140rads/ branch.Cell is preferably with being enough to significantly to suppress the further total dose irradiation of propagation of 100% cell.Therefore, expectation is used from about 10,000-20, and 000rads, best with about 15, the total dose of 000rads is shone this cell.

From body

Use from the transgene expression tumour cell of body genetic modification has advantage, because each patient's tumor is expressed one group of unique tumour antigen, it is different from from the similar MHC-coupling of other patient's tissue tumour cell.Referring to, for example, Kawakami etc., J.Immunol., 148,638-643 (1992); Darrow etc., J.Immunol., 142,3329-3335 (1989); With Hom etc., J.Immunother., 10,153-164 (1991).On the contrary, MHC-coupling tumour cell has advantage: patient does not need to perform the operation and just can obtain to be used for its tumor sample that the genetic modification tumour cell produces.

One preferred aspect in, the present invention includes method: (a) obtain tumour cell from mammalian subject with tumour by the treatment cancer of carrying out following steps; (b) utilize and to have at least a portion of containing subtype C adenovirus axle district and Head Section at least a portion in conjunction with the hypotype B adenovirus Head Section of CD46, the proteic adenovirus carrier of chimeric fiber of at least a portion of Ad5 or Ad2 axle and Ad35 head for example, modify this tumour cell, make them can produce one or more genetically modified growth levels relevant with the unmodified tumour cell; (c) make and modify the tumour cell incompetence propagation that becomes; (d) to the mammalian subject that obtains tumour cell or have with the Mammals of the identical MHC type of Mammals of acquisition tumour cell and introduce this modification tumour cell again.The tumour cell of introducing be from body and be the MHC-coupling to the host.Be incorporated into mammalian subject in preferably that said composition is subcutaneous, intracutaneous or the tumour.

Single autologous tumor cell can express more than a kind of transgenosis or each transgenosis can be passed through the different tumor cells expressions from body.In one aspect of the invention, contain proteic adenovirus carrier of chimeric fiber and the genetically modified nucleotide sequence of coding as described herein by introducing, for example, cytokine, as GM-CSF, modify the allos tumour cell, its expression/control sequence with promotor and expression necessity thereof effectively is connected.In another aspect, by contain coding at least one with promotor and express the introducing of the carrier of effective other the genetically modified nucleotide sequences that are connected of necessary expression/control sequence, modify the identical autologous tumor cell or second autologous tumor cell.Utilize identical or different carrier, the one or more genetically modified nucleotide sequences of coding are introduced identical or different autologous tumor cell.The genetically modified nucleotide sequence of encoding can or cannot further comprise the selected marker sequence that effectively is connected with promotor.

Allosome

As Jaffee etc., Seminars in Oncology, 22,81-91 (1995) is described, and the researchist has sought surrogate from body and MHC-coupling cell as tumor vaccine.Early stage tumor vaccine countermeasure is the understanding that plays antigen presenting cell (APCs) effect according to the preventive vaccination tumour cell, and antigen presenting cell makes tumour antigen present its MHC class I and II molecularity, and the immune T cell of direct activation arm.The result of Huang etc. (Science, 264,961-965,1994) shows by secrete cytokines such as GM-CSF to make host's special APCs rather than preventive vaccination tumour cell load immune T cell arm, so that bone marrow APCs is added to tumor area.Bone marrow APCs occupies whole cell proteins of the tumour that is used to process, and antigen-presenting peptide on its MHC class I and II molecule is filled immune CD4+ and CD8+T cell arm thus then, causes the tumour-specific anti-tumor immune response of system.These result's hints be not necessary or best in order to cause that antitumor immune response uses from body or MHC coupling tumour cell, and the transfer of allogenic mhc gene (the different heredity from same species are individual) can strengthen immunogenicity of tumor.More particularly, in some cases, the repulsion of the MHC class I molecule of tumour expressing heterologous cause to follow-up competition unmodified parental generation tumour excite the enhanced system immunne response, such as above-mentioned Jaffee etc. and above-mentioned Huang etc. description.

Such as in this article description, " tumor cell line " comprises the cell that derives from tumour at first.Such cell is transformant (just, showing indeterminate growth in cultivation) normally.One preferred aspect in, the invention provides by carrying out the method for following steps treatment cancer: (a) obtain tumor cell line; (b) utilization contains the proteic adenovirus carrier modification of chimeric fiber tumor cell line, so that these cells can produce the one or more genetically modified growth levels relevant with the unmodified tumour cell, wherein this albumen comprises at least a portion in subtype C adenovirus axle district and Head Section at least a portion in conjunction with the Head Section of the hypotype B adenovirus of CD46, for example at least a portion of Ad5 or Ad2 axle and Ad35 head; (c) make this modification tumor cell line incompetence breed and (d) this tumor cell line be introduced at least a tumour mammalian subject (host) of the tumour of the tumor cell line same type that has and obtained.This introducing tumor cell line is allogenic and non-MHC coupling to the host.Such allos clone has advantage: they can prepare in advance, characterize, five equilibrium and storage (being frozen) are beneficial to introduce patient with characterizing good cell in the tubule that contains known express transgenic cell.The method that is used for the allos cell that producer gene modifies has been described in WO 00/72686A1 for example, in specially being incorporated herein as a reference.

In a method for preparing genetic modification express transgenic allos cell, with in the clone of an allos tumor cell line of one or more coding therapeutic agent core acid sequences (transgenosis) introducing (just, deriving from an individuality except the individuality for the treatment of).In other method, one or more transgenosiss are introduced isolating allos tumor cell line.Usually, cell or cell population be from the tumor cell line of tumour for the treatment of or cancer same type.Tumour and/or tumor cell line can be any type of cancers, including, but not limited to, the cancer of bladder, breast, colon, kidney, liver, lung, ovary, uterine cervix, pancreas, rectum, prostate gland, stomach, epidermis; The hematopoiesis tumour of lymph or marrow pedigree; Between tumour such as the fibrosarcoma or the rhabdosarcoma in matter source; Or other tumour, comprise melanoma, teratocarcinoma, neuroblastoma, neurospongioma, gland cancer and non-little pneumonocyte cancer.

In one aspect of the invention, have the proteic adenovirus carrier of chimeric fiber by introducing and modify allogenic tumour cell, wherein this chimeric fiber albumen comprises at least a portion in subtype C adenovirus axle district and Head Section at least a portion in conjunction with the hypotype B adenovirus Head Section of CD46, at least a portion of Ad5 or Ad2 axle and Ad35 head for example, it comprises coding and promotor and the necessary effective genetically modified nucleotide sequence that is connected of expression control sequenc of expression, for example cytokine such as GM-CSF.In another aspect, have by introducing and aforesaidly to contain with promotor and express proteic another adenovirus carrier of other genetically modified chimeric fiber of effective at least one that is connected of necessary expression control sequenc, modify the identical allos tumour cell or the second allos tumour cell.Utilize identical or different carrier, the genetically modified nucleotide sequence of coding can be introduced in the identical or different allos tumour cell.The genetically modified nucleotide sequence of encoding can or cannot further comprise the selected marker sequence that effectively is connected with promotor.Make us desirably the high-caliber cytokine of allos expression of cell lines, for example GM-CSF.

In practice of the present invention, with one or more allos clones and from body cancer antigen, for example autologous tumor cell (it comprises allos clone composition jointly) is cultivated together, gives patient with this allos clone composition then.Cancer antigen promptly, provides from the body cancer cells normally by the cancer cells that will treat.In the case, make become incompetence propagation of said composition by irradiation usually, wherein, as mentioned above, this allos cell and cancer cells are cultivated at tissue culturing plate's upper flat plate, and utilized the caesium source at room temperature to shine.In specified introducing, the allos cell with change according to its combination from the ratio of body cancer cells.

Can use any suitable processing approach that allos clone composition is introduced patient, said composition is preferably subcutaneous, introduce in intracutaneous or the tumour.

In practice of the present invention, the use of allos clone has the treatment advantage: suffer from the carninomatosis people by the express transgenic clone of genetic modification is introduced, with from body cancer antigen, the paracrine of treatment transgenosis such as cytokine produces the effective immunne response that causes tumour.This has been avoided cultivating and autologous tumor cell being transduceed into each patient's needs.

The onlooker

One further aspect in, the invention provides and express at least one genetically modified general immunomodulatory gene and modify transgene expression bystander cell line.Identical universal bystander cell line system can express and surpass a transgenosis, or individual transgenosis can be expressed by different universal bystander cell line systems.General bystander cell line system comprises and innately lacks main tissue affinity class I (MHC-I) antigen and main histocompatibility class II (MHC-II) antigen or modified so that they lack MHC-I antigen and the antigenic cell of MHC-II.In one aspect of the invention, have the proteic carrier adenovirus carrier of chimeric fiber by introducing and modify general bystander cell line system, wherein this chimeric fiber albumen comprises at least a portion in subtype C adenovirus axle district and Head Section at least a portion in conjunction with the carrier hypotype B adenovirus Head Section of CD46, for example at least a portion of Ad5 or Ad2 axle and Ad35 head wherein this carrier comprise coding and promotor and express for example nucleotide sequence of cytokine such as GM-CSF of transgenosis that necessary expression control sequenc effectively is connected.In another aspect, by introducing comprise coding at least one with promotor and express the introducing that necessary expression control sequenc effectively is connected the carrier of other genetically modified nucleotide sequence, modifies identical universal bystander cell line and is or second universal bystander cell line is.Utilize identical or different carrier, the genetically modified nucleotide sequence of coding is introduced identical or different universal bystander cell line system.The genetically modified nucleotide sequence of encoding can or cannot further comprise the selected marker sequence that effectively is connected with promotor.Excite any transgenosis combination of anti-tumor immune response in practice of the present invention, to find practicality.Universal bystander cell line system is the serum-free medium what stipulate preferably, grows in the preferred suspension.

The example of preferred generic bystander cell line system is K562 (ATCC CCL-243; Lozzio etc., Blood 45 (3): 321-334 (1975); Klein etc., Int.J.Cancer 18:421-431 (1976)).In U.S. Pat 6,464, in 973 case description the detailed description from generation to generation of human bystander cell line system, in specially being incorporated herein as a reference.

Make us desirably, high-caliber cytokine is expressed by universal bystander cell line system, for example, and GM-CSF.

In practice of the present invention, with one or more universal bystander cell line systems and from body cancer antigen, for example, autologous tumor cell (it comprises general bystander cell line system: compositions jointly) is cultivated together, gives patient with this universal bystander cell line system: compositions then.Can use any suitable processing approach that the universal bystander cell line system: compositions is introduced patient.Introduce in preferably that said composition is subcutaneous, intracutaneous or the tumour.

Cancer antigen is normally promptly provided from the body cancer cells by the cancer cells that will treat.In the case, make become incompetence propagation of said composition, wherein, as mentioned above this allos cell and cancer cells are cultivated at tissue culturing plate's upper flat plate, and utilized the caesium source at room temperature to shine by irradiation.

In given processing, bystander cell line with change according to composition from the ratio of body cancer cells.For the bystander cell line that produces GM-CSF, in given introducing, bystander cell line should make the GM-CSF that produces level of significance in treatment with ratio from the body cancer cells.Except the GM-CSF threshold value, bystander cell line with should be from the ratio of body cancer cells greater than 1: 1.Utilize common method well known in the prior art, can determine the adequate rate of bystander cell line and tumour cell or tumour antigen.

In practice of the present invention, the use of bystander cell line system has the treatment advantage: by with express cell factor bystander cell line system and at least a other cancer therapeutical agent (by identical or different cell expressing) with from body cancer antigen, introducing has the patient of cancer, the paracrine of immunomodulating cytokines produces, and causes the effective immunne response to tumour.This has been avoided cultivating and autologous tumor cell being transduceed into each patient's needs.

Usually the minimum dose of about 3500Rads is enough to as killed cells and makes its propagation defective that becomes, although about 30, the dosage of 000Rads is acceptable.In some embodiments, before introducing Mammals,, or time shine this cell from the dosage of about 120-140rads/min preferably from about 50-200rads/min.Usually, when using irradiation, the illumination levels that needs is 2,500rads, 5,000rads, 10,000rads, 15,000rads or 20,000rads.In one embodiment, use about 10, the dosage as killed cells of 000Rads and make its propagation defective that becomes.Be appreciated that irradiation just makes cell become and breeds a method of defective, and comprise in the present invention cause cell repeatedly the splitted deactivation but keep express transgenic (for example cytokine) ability other method (for example, with ametycin, cycloheximide, with similar reagents on the principle, or the bonded of suicide gene is handled in the cell).

Be used to put into practice the compositions and methods of the invention

The invention provides and comprise recombinant adenoviral vector of the present invention and/or particle and/or utilize recombinant adenoviral vector of the present invention and/or particle by the pharmaceutical composition of the tumour cell of genetic modification and medicine acceptable carrier, and the method for using said composition.

In one aspect, the invention provides the composition of the adenovirus carrier that comprises the treatment significant quantity of the present invention in the medicine acceptable carrier, be fit to introduce individual with unit dosage form part or system, aseptic parenteral solution or suspension, the outer solution of aseptic non-enteron aisle or oral liquid or suspension, oil-in-water or water-in-oil emulsion etc.It is known in the prior art to be used for the outer dispenser prescription of parenteral and parenteral.Composition also comprises freeze-drying and/or the recombinant forms and the particle of the present invention of adenovirus carrier.The acceptable drug carrier is, for example salts solution, protamine sulfate (Elkins Sinn, Inc., Cherry Hill, N.J.), water, water-containing buffering liquid, as phosphate buffered saline buffer and Tris damping fluid, polybrene (polybrene) (Sigma Chemical, St.Louis MO) and phosphate buffered saline(PBS) and sucrose.From the instruction that is comprised herein, the selection of suitable pharmaceutical carrier will be apparent to those skilled in the art.These solution are aseptic and usually are no particulate matter except required adenovirus virion.Said composition can comprise the approximate as required physiological condition of the acceptable auxiliary substance of medicine such as pH calibration and buffer reagent, toxicity calibration agent etc., for example sodium-acetate, sodium-chlor, Repone K, calcium chloride, Sodium.alpha.-hydroxypropionate etc.Can comprise the vehicle that strengthens the adenovirus infection cell.

In one embodiment, with effective inhibition, prevent or the amount of tumoricidal growth (treatment significant quantity just) is introduced the host with adenovirus carrier of the present invention.This can be for example by virus vector in the tumour cell duplicate and/or the tumour cell transduction after genetically modified expression finish.Such introducing can be carried out to any amount of tumour target cell there to be effective conveying adenovirus carrier, as carrier system being introduced or directly being injected tumour.In a method, to be at least 5 * 10 ⁹The amount of virion/kg body weight carrier system is introduced, and generally speaking, such amount is no more than 1 * 10 ¹³Virion/kg body weight.In other method, with at least 2 * 10 ¹⁰The amount of individual virion is introduced carrier in the tumour, and this amount is no more than 1 * 10 usually ¹³Virion/kg body weight.In another method, carrier is instilled into experimenter's bladder.In the case, can be with as the bladder enhanser pre-treatment bladder described in the US20040131590.The definite dosage of being introduced depends on the size of the tumour that comprises age, body weight and patient's sex and treating and the various factors of severity.Can introduce the carrier one or many.The single or multiple of composition is introduced and can be undertaken by treatment selected dosage level of doctor and pattern.If necessary, can reduce immunne response, make to allow to repeat to introduce and/or strengthen and duplicate by reducing immunne response to virus by the antibody that uses various immunosuppressor or remove preexist.The introducing of adenovirus carrier of the present invention can combine with other antitumor flow process, and many examples wherein are known in the prior art.Antitumor flow process like this changes according to the type of the cancer of treatment.The example of antitumor flow process is composed as follows to be further described.

Available several different methods uses liposome, direct injection, systemic injection, conduit, topical application, suction etc. to finish conveying.

Then, the invention provides treatment and have superfluous method of giving birth to state (patient who has cancer usually) experimenter, comprise adenovirus carrier of the present invention from treatment significant quantity of the present invention to the experimenter that introduce.Adenovirus carrier can be have the ability rf or incompetence rf.One aspect of the present invention relates to the incompetence propagation of becoming (just, by irradiation) and gives the purposes of tumour cell of express transgenic of experimenter's genetic modification.With the genetic modification of treatment significant quantity, propagation defective type tumour cell is introduced the experimenter, is generally the human carcinomas patient.

Modify tumour cell by transduction with aforesaid adenovirus carrier of the present invention.In one embodiment, the present invention carries out the single transduction of tumour cell population with the one or more genetically modified a kind of recombinant adenoviral vectors of coding.In other embodiments, the present invention relies on many transductions (infection) of the tumor cell group with identical recombinant adenoviral vector.In other embodiment, the present invention carries out many or single transduction by the different genetically modified recombinant adenovirus of encoding.

Transducer cell is incorporated into Mammals to be caused with stimulation mode, the adjusting of individual system immunne response.In others, the present invention relates to the purposes of the one or more genetically modified modification tumour cells of expression (as the primary tumo(u)r cell) of the reverse of original tumour or inhibition.Also transducible tumour cell is to express many resistances protein (MDR).Can identical adenovirus carrier, different adenovirus carrier or even non-adenovirus carrier on the MDR that encodes.Other carrier can be used in combination with the transduction tumour cell with adenovirus carrier of the present invention.Different carriers can to desired result suitable basically simultaneously or other the time be transported to tumour cell.

In yet another aspect, the invention provides the method for enhancing to the tumour antigen immunne response.In this method, modify the primary tumo(u)r cell with express transgenic and introducing experimenter, as the human carcinomas patient with adenovirus carrier of the present invention.In the significant quantity and the mode (for example intracutaneous or subcutaneous) of treatment, should modify tumour cell and introduce to obtain the mammalian immune of antigenic raising is replied.

In related embodiment, genetic modification tumour cell and adenovirus carrier of the present invention are all introduced the experimenter.Such genetic modification tumour cell can and be introduced from body, allogenic or bystander cell line and can take place simultaneously or can take place successively.

The transducer cell of express transgenic needs not to be identical tumor type with the tumour cell that Mammals is had.For example, bystander cell line express transgenic and be tumour cell or the tumour inequality that is had with Mammals in vivo.In another example, transduction is not identical tumor type with the cell of expression of GM-CSF, but can be with mammiferous tumour cell expressing tumor antigen.Therefore, improved immunne response to mammal tumor.

Tumour and the transduction tumour cell (being the primary tumo(u)r cell) normally same type cancer and have common antigen.This cancer can be the cancer of any kind, including, but not limited to, bladder cancer, osteocarcinoma, brain and spinal cord cancer, brain and spinal cord cancer, cervical cancer, colorectal carcinoma, uterine endometrium and other uterus carcinoma, esophagus cancer, gall-bladder and cholangiocarcinoma, stomach (stomach) cancer, head and neck cancer, kidney, leukemia, liver cancer, lung cancer, lymphoma, melanoma, multiple myeloma, neuroblastoma, ovarian cancer, carcinoma of the pancreas, prostate cancer, hemopathy, retinoblastoma, skin carcinoma, soft tissue sarcoma, carcinoma of testis, thyroid carcinoma, uterus carcinoma, nephroblastoma, original neoplastic lesion, carcinopolypus etc.

In one embodiment, this method comprises propagation defective tumour cell and the auxiliary agent adenovirus carrier for example of the present invention that introducing is not transduceed, and auxiliary agent that is introduced into and cytokine-expressing tumour cell are introduced simultaneously or successively.In a method, adenovirus carrier of the present invention for example comprises transgenosis, the encoding sequence of cytokine such as GM-CSF, and introduce with propagation defective tumour cell, wherein this tumour cell is from body or allogenic.

The experimenter is human patient normally.For human patient, if allogeneic coding sequence is contained in the carrier, this allogeneic coding sequence can be the human origin, in the mankind, show the high homology and the gene of the species that are closely related identical or equivalent functions biologically although can use, if the product of allogeneic coding sequence does not produce/cause reverse immune response in container.In one embodiment, the protein of allogeneic coding sequence coding treatment or the RNA of treatment.The therapeutic activity amount of nucleotide sequence or therapeutic gene is the significant quantity that reaches required necessity as a result in dosage and for some time.This amount can change according to the various factors including, but not limited to experimenter's sex, age, body weight etc.

Those of ordinary skill is appreciated that from doctor or patient's angle, in fact anyly alleviates or prevents from not wish that symptom (for example, the symptom relevant with disease, to the susceptibility of environmental factors, weather aging etc.) wishes.

Give pharmaceutical composition of the present invention give can in individuality, produce immunne response or treat Cancer-Related other treatment after, before, replacement or combination.For example, other form by chemotherapy, radiotherapy and immunotherapy and adoptive transfer can prior to or treat Mammals simultaneously.For example, composition of the present invention may use with chemotherapeutics such as cisplatin, compound cisplatin/cyclophaphamide, taxol or cisplatin/endoxan/Zorubicin.In the situation of using such therapy, can use them in one way or when not hindering the immunogenicity of composition of the present invention.Mammals also can be introduced other vaccine or other composition so that excite immunne response.Such alternate sets compound can comprise tumour antigen vaccine, the antigenic nucleic acid vaccine of codes for tumor, antiidiotype vaccine and comprise the cell vaccine of other type of cytokine-expressing tumor cell line.

In one embodiment, the processing transducer cell is to remove other components of major part of using in the preparation cell.Especially, remove other biological additive in foetal calf serum, bovine serum component or the substratum.In one embodiment, as centrifugal by the multiple tenderness, washed cell becomes the vehicle of suitable pharmacology affinity.The vehicle of affinity comprises etc. and to ooze (isotonic) salt solution, there are or do not have the ion or the amino acid of the similar phosphoric acid salt of damping fluid of physiology affinity or Hepes and nutrient such as glucose, physiology affinity, with various substratum, especially those lack the substratum of other immunogen component.Can use equally and carry reagent, as white protein and blood plasma cut and nonactive enriching agent.In one embodiment, nonactive biological components on they are present in degree in the pharmacology preparation, derive from the mankind, and even can obtain in advance from the experimenter that will treat.

Yet others of the present invention relate to the test kit that is used to regulate to the mammiferous immunne response of tumour antigen.These test kits comprise having the proteic recombinant adenovirus of the present invention of chimeric fiber and can or cannot further comprise for example nucleic acid of cytokine such as GM-CSF of coding transgenosis, wherein this chimeric fiber albumen comprises at least a portion in subtype C adenovirus axle district and Head Section at least a portion in conjunction with the hypotype B adenovirus Head Section of CD46, for example at least a portion of the Ad35 of Ad5 or Ad2 axle and Ad35 head.The storer that this test kit further includes usefulness for example is used for placing and storage tumour cell or tumor fragment, and within it with the recombinant virus tumour cell of transduceing.In some embodiments, test kit further comprises the digestive ferment that tumor tissues is converted into the single cell suspension.In some embodiments, this enzyme is a collagenase.In some embodiments, test kit further comprises the auxiliary agent of the propagation defective type tumour cell of the recombinant virus transduction that comprises the genetic material that is not contained the Codocyte factor.

Embodiment

Following by reference case description the present invention, it provides by mode of explaining and does not think to limit by any way the present invention.Utilize standard technique known in the prior art or following concrete described technology.Only use routine test, those skilled in the art considered that maybe can be clear: the many specific embodiment of the invention that specifically described in this article equivalence.Such scheme is meant and is included in following claims scope.

Embodiment 1: human tumour cell line and cell cultures

Human tau that uses among the embodiment of Miao Shuing and neck cancer system and human melanoma cell series are listed in table 1 in this article.

Table 1. tumor cell line

Clone	Describe	Source/class-mark
			Head and neck cancerous cell line

A-253	The mankind, epidermal carcinoma	ATCC，HTB-41
			A431	The mankind, epidermal carcinoma	ATCC，CRL-2592
FaDU	The mankind, squamous cell carcinoma (SQCC)	ATCC，HTB-43
			SCC-9	The mankind, tongue, SQCC	ATCC，CRL-1629
SCC-15	The mankind, tongue, SQCC	ATCC，CRL-1623
			Detroit 562	The mankind, the pharynx cancer	ATCC，CCL-138
CAL 27	The mankind, tongue SQCC	ATCC，CRL-2095
			RPMI 2650	The mankind, nasal septum, SQCC	ATCC，CCL-30
Melanoma cell series
			A375-luc	The mankind, skin, malignant melanoma	CRL-1619 (modification) with expressing luciferase
A2058	The mankind, skin, malignant melanoma	ATCC，CRL-11147
			C32	The mankind, skin, malignant melanoma	ATCC，CRL-1585
SK-Mel-28	The mankind, skin, malignant melanoma	ATCC，HTB-72
			WM-266-4	The mankind, skin, malignant melanoma	ATCC，CRL-1676
G-361	The mankind, skin, malignant melanoma	ATCC，CRL-1424

In RPMI 1640 substratum that contain 10%FBS, cultivate human tau and the neck cancer system and the human melanoma cell series of listing in the table 1.In RPMI 1640 substratum that contain 10%FBS, cultivate human prostate cancer cell line, PC3M-2AC6.In the RPMI that contains 10%FBS, L-glutaminate (2mM), non-essential amino acid (0.1mM), yellow soda ash (0.075%) and Sodium.alpha.-ketopropionate (1mM), cultivate human prostate cancer cell line, and LNCaP (ATCC, CRL-10995).In the RPMI that is supplemented with 10%FBS and L-glutaminate (2mM), cultivate human prostate cancer cell line, and PC-3 (ATCC, CRL-1435).

The structure of embodiment 2:E1 defective, the chimeric Ad5 carrier of expression GFP fiber

As described below, in order to produce, at first make up shuttle plasmid, pAd5LtRt-SmaI based on the chimeric Ad5 carrier of fiber.At first, with the left hand end (bp 1-1009bp) of two primers by pcr amplification Ad5DNA;

Primer 1 (5 '-GAATTCTAGGGATAACAGGGTAATCATCATCAATAATATACCTT-3 '; SEQ ID NO:17) and primer 2 (5 '-CCCGGGGTGCTCCACATAAATCT-3 '; SEQ ID NO:18).Use be called primer 3 (5 '-AAGCTTTAGGGATAACAGGGTAATCATCATCAATAATATACCTT-3 '; SEQ ID NO:19) and primer 4 (5 '-CCCGGGGGAATACATACCCGCAGG-3 '; SEQ ID NO:20) the 580bp sequence is not held on the right side of the second cover primer amplification Ad5DNA.The recognition sequence of I-SceI is merged in primer 1 and 3.Digest a PCR product and digest the 2nd PCR product with EcoRI and Smal with HindIII and SmaI.Connect the fragment cloning that is generated to be advanced pBlueScript (EcoRI CA) and HindIII site are to produce pAd5LtRt-SmaI for Stratagene, La Jolla to the fragment gel-purified that generated and by three-dimensional.By (genomic dna CA) produces that to comprise with the I-SceI site be the plasmid pFLAd5.CMV5-GFP of the Ad5GFP vector gene group on boundary for Qbiogene, Carlsbad in conjunction with Ad5.CMV5-GFP among linearizing pAd5LtRt SmaI of Smal-and the E.coli.

Produce the fiber chimeric vector with several steps.At first use the SmaI digested plasmid, pFLAd5.CMV5-GFP, and gel-purified is connected the not fragment of end fragment (being respectively nt.1 to 3047 and 32652 to 33231) of the left side that contains Ad5.CMV5-GFP and right side with the oneself, with generation pAd5Lt ﹠amp; RtSmaI-CMV5-GFP.By respectively in conjunction with the linearizing pAd5Lt ﹠amp of SmaI-; RtSmaI-CMV5-GFP and AvlnBg5T35H, AvlnBg35F AvlnBg-5T3H (Smith etc., 2003) genomic dna, and CRAd:hUPII-E1a-IRES-E1b/Fb5/3LL-RGD, produce plasmid pFLAd5.CMV5-GFP5T35H, the pFLAd5.CMV5-GFP-35F, pFLAd5.CMV5-GFP-5T3H and the pFLAd5.CMV5-GFP-5T3H-RGD that contain and total length Ad5DNA that contain chimeric fiber the coding region coding region with GFP coding region replacement E1.The gene of the shaft portion of the celloglobulin in the capable replication type adenovirus of encoding condition, CRAd:hUPII-E1a-IRES-E1b/Fb5/3 LL-RGD derives from Ad5, and the knot of coding region obtains from Ad3.By at proteinic C-terminal in conjunction with the RGD part, further modify this viral chimeric fiber albumen.Advance in the PER.C6 cell with I-SceI digestion total length plasmid and transfection, to produce the fiber chimeric vector.

The structure of embodiment 3:E1 defective and human expression of GM-chimeric Ad5 carrier of CSF fiber

In order to produce these carriers, at first, structure shuttle plasmid as described below, pAd5LtRtSma-CAGhGM.With primer PSR3 (5-CTTCGAGGAATTCAGGATGTGGCTGCAGAGCCTGCTG-3; SEQ ID NO:21) and PSR4 (5-CTTCGAGAAGCTTACTCCTGGACTGGCTCCCAG-3; SEQ ID NO:22) carries out the gene of pcr amplification coding hGMCSF from pDR20hGMF.Incorporate restriction endonuclease recognition sequence into primer PSR3 (EcoRI) and PSR 4 (HindIII) to promote the clone.With EcoRI and HindIII digestion PCR product (435bp) and fragment that gel-purified produced.With primer PSR6 (5-CTTCGAGAAGCTTCAGACATGATAAGATACATTG-3; SEQ ID NO:23) and PSR7 (5-CTTCGAGGGATCCTACCACATTTGTAGAGGTTTAC-3; SEQID NO:24) carries out poly-(A) signal in pcr amplification SV40 late period from the pCAT-base.The restriction endonuclease recognition sequence transformation is advanced among primer PSR6 (HindIII) and the PSR7 (BamHI).With HindIII and BamHI digestion PCR product (222bp) and fragment that gel-purified produced.Two gel-purified fragment clonings are gone into pGEM-7Z, and (EcoRI WI) and BamHI site are to produce pGMCSF-SV40pA-7Z for Promega Corp, Madison.

Digest the plasmid that is produced and utilize Klenow fragment to fill terminal with BamHI with the dNTP mixture.Carry out phenol-chloroform extraction and ethanol sedimentation subsequently, digest DNA and the gel-purified 670bp fragment that is produced with EcoRI.Secondly, digest pAd5Lt﹠amp with BgIII; RtSmaI-CMV5-GFP utilizes Klenow fragment to fill the end that is produced with the dNTP mixture.Gel-purified contains the left distal end (357bp) of Ad5DNA and the 4.3KB fragment of right end (583bp) and plasmid main chain.By HindIII and BamHI digestion and gel-purified from pRRLsinhCAG.gammapptpre (Shoji etc., 1997.J.Gen Virol.78:2657-2664; 1989 Gene79:269-277.urce such as Miyazaki) obtain to contain the 1.6KB fragment of CAG promotor (cmv enhancer, chicken β actin promoter and intron and rabbit globulin intron) in.All three gel-purified fragments are connected to produce shuttle plasmid, pAd5LtRtSma-CAGhGM.In E.coli, SmaI linearizing pAd5LtRtSma-CAGhGM is combined generation total length plasmid, pFLAd5-CAG-hGM-5F with the genomic dna (referring to SEQ ID NO:1) of Ad5-CMV5-GFP.In order to produce the similar full-length clone that contains the knot coding region that derives from Ad35 and Ad3, by with StuI digestion succeeded by contain left side and right side not end fragment the segmental gel-purified of restriction enzyme and from ligation, make up new shuttle plasmid, pAd5Lt ﹠amp from pFLAd5-CAG-hGM-5F; RtStu-CAG-hGM.With the plasmid that the StuI linearizing is produced, the Ad5-CMV5-GFP-5T35H of combining source in E.coli and the genomic dna of Ad5-CMV5-GFP-5T3H-RGD produce pFLAd5.CAG-hGM-5T35 and pFLAd5.CAG-hGM-5T3H-RGD respectively.With I-SceI digestion total length plasmid and be transfected in the PER.C6 cell to produce Ad5.CAG-hGM, Ad5.CAG-hGM-5T35H and Ad5.CAG-hGM-5T3H-RGD.

Embodiment 4: the method that is used to prepare the primary tumo(u)r cell.

The tumor tissues (for example lung) that surgery obtains is presented pathology assessment to determine its identity as tumor tissues (being nonsmall-cell lung cancer).At transhipment substratum (Td substratum; Contain among the α of 1% human serum albumin-MEM), under 40 ℃, the laboratory is transported/be transported to remaining tumour.In most cases, less than 24 hours with this tumour of interior transhipment.The transhipment substratum of sucking-off immediately and wash tumour and utilize Stomacher laboratory stirrer (Brinkmann Westbury NY) to be processed into the single cell suspension in 30 minutes after arriving by mechanical digestion with the Td substratum of 50ml.Remove cell debris and group by filtration and Ficol density gradient centrifugation (Amersham Pharmacia, Uppsala, Sweden).The cell flat board is cultivated in 6-well tissue culture ware.

Embodiment 5: utilize step isolated cells system or the infection of primary tumo(u)r cell and the mensuration of transduction percentage ratio among the embodiment 3

In the substratum that contains 2% foetal calf serum (FBS), under 37 ℃, primary tumo(u)r cell that flat board is cultivated or clone are exposed to Ad5 base GFP carrier (for example the using chimeric fiber) 2hr of or different infection multiplicity (MOIs).Serum-concentration is increased to 4-10% and this cell is further cultivated 24hr think that GFP expresses and prepare.With the PBS washed cell and utilize flow cytometry to measure transduction percentage ratio.

In order to measure and comparison Ad5 fiber mosaic and the transduction efficiency that carries wild-type fiber Ad5, the total amount that contains 2%FBS be in the substratum of 0.5ml under 37 ℃, be transduction two hours with the carrier of 50 particle/cells (ppc) with the human cell who selects.The suitable medium that contains 4%FBS with 2ml is replaced the infection substratum.With cell cultures 24 hours so that be that transgenosis (GFP) express and to prepare by beta-galactosidase enzymes or green fluorescence egg matter.Express in order to monitor beta-galactosidase enzymes, cell monolayer is fixed and with 5-bromo-4-chloro-3-indoles-β-D-semi-lactosi pyranoside (X-GaI) 24h that dyes.Under high power field, measure the per-cent of transduction with opticmicroscope by adding up blue cell number that every all transit cell leads; Every well is added up three visuals field and is determined the transduction average percentage.Express in order to monitor GFP,, use the PBS washed cell then with trypsin treatment isolated cell individual layer.Measure the per-cent of transduction by flow cytometry.In triple wells, carry out each carrier transduction experiment and measure the average percentage of transduceing.

Embodiment 6: the human cell's that external Ad5 and chimeric fiber are carrier mediated transduction.

As described in Example 5, with 50 particles/cell transduction cell.In infection back 24 hours, the beta-galactosidase enzymes of monitoring infected cell was expressed.The result is as shown in table 2 and reflected the per-cent of transducer cell.

The human cell's of external Ad5 of table 2. and the mediation of Ad5 fiber chimeric vector transduction.

		Avlnbg	-5T35H	-35F	-5T3H
						Lung	MRC-5	2	31	14	16
H1299	63	85	55	83
					H460		16	60	15	17
DMS 114	22	44	31	6
					H446		72	95	54	15
A549	22	74	42	43
					Prostate gland		PC3	1	35	13	17
DU145	16	43	28	27
						LNCaP	4	7	5	1
Head and neck	SCC9		5	22							10	9
					Fadu	0.5	15	7	9
	Detroit 562	0.25	6	2						3
					A253	0.3	3	1	1
	Bladder	SW780	9	36						18	17
RT4					4	24	13	10
		UC14	4	22					6	7

Kidney	SW839	3	21	8	4
						786-0	1	28	12	14

Embodiment 7: the transduction of former human non-small cell lung cancer's cell that external Ad5 and chimeric fiber are carrier mediated.

Express mensuration cell transduction percentage ratio with fiber chimeric vector transducer cell and with the 24hr after infection of the FACS described in embodiment 5 by monitoring GFP.The result is as shown in table 3.

The transduction of former human non-small cell lung cancer's cell of external Ad5 of table 3. and the mediation of Ad5 fiber chimeric vector.

Carrier	The % transduction
		Ad5.CMV5-GFP	5.8
Ad5.CMV5-GFP-35F	8.4
		Ad5.CMV5-GFP-5T35H	10.6
Ad5.CMV5-GFP-5T3H	7.9
		Ad5.CMV5-GFP-5T3H-RGD	8.05

Embodiment 8: the density of selecting cell surface receptor in the human tumour cell line.

As mentioned above, compare with the fiber chimeric adenoviral vectors, melanoma and head are relative little to the sensitivity that Ad5 infects with neck cancer (HNC) clone.In order to study the Basic of Biology of these clones, measure the cell levels of the used acceptor of adenovirus carrier to the dull and stereotyped relevant antagonism of Ad5 rather than fiber chimeric vector.The tumour cell of cultivating with PBS washing and use 0.025% trypsinase is with itself and plate isolation, with PBS (pH7.4) washing once and suspension (pH 7.4) in PBS.With the anti-COxsackie-adenovirus receptor of murine antibody guiding (CAR, Rmcb, Upstate biotechnology, Lake placid, NY), CD46 (Clone E4.3, BD Biosciences, Pharmingen, SanDiego, CA) α _vβ ₃(Chemicon International, Temecula, CA) or α _vβ ₅(ChemiconInternational, Temecula CA), under 4 ℃ with cell cultures 30min.Subsequently, (San Diego CA) cultivates 30min down at 4 ℃ for BD Biosciences, Pharmingen with PBS washed cell three times and with the secondary anti-mouse IgG of FITC-conjugated.With after the PBS washing, with cell suspension in PBS and with the flow cytometry analysis to measure positive cell percentage.Table 4 and 5 provides the data of human tau and neck cancerous cell line and melanoma cell series respectively.

The cell surface receptor of selecting in table 4. human tau and the neck cancerous cell line is expressed (% positive cell)

Virus	A-253	A431	FaDu	SCC-9	Detroit562
						CAR	16	22	2	6	3
CD46	79	64	94	95	93

The cell surface receptor of selecting in the human melanoma cell series of table 5. is expressed (% positive cell)

Virus	WM-266-4	A375-luc	SK-MEL-28	G361	A2058
						CAR	0.3	2	9	2	39
CD46	14	69	5	4	39
						αvβ3	62	44	32	1	39
αvβ5	2	28	2	1	3

These studies have shown that: melanoma and the low-level CAR of HNC expression of cell lines.On the contrary, especially for head and neck cancerous cell line, the height of being on close level of the CD46 of mensuration.In addition, the head and the neck cancerous cell line of all five tests have very low-level α _vβ ₃And α _vβ ₅Integrin and therefore can not being detected by flow cytometry; Yet, α in most of melanoma cells _vβ ₃The expression level of integrin is high.Therefore, CD46, Ad3 by high expression level and former the acceptor of Ad35 and low-level CAR express, former the acceptor of the Ad5 on melanoma and HNC cell, the relative sensitivity of fiber chimeric vector are described and to the resistance of Ad5.

Embodiment 9: the structure that contains E1 and human expression of GM-chimeric Ad5 carrier of CSF fiber

The E1 oncolytic vectors that contains that contains chimeric fiber produces by several steps.At first, by in conjunction with Smal-linearizing pAd5LtRtSmaI among the E.coli and the genomic dna of Ad5, make up the total length plasmid, pFLAd5.The shuttle plasmid pFLAd5 that produces comprises with the I-SceI site being the Ad5 genome on boundary.Secondly, with XhoI digestion pFLAd5 and gel-purified with is connected certainly the left side that contains Ad5 and right side not the fragment of end fragment with generation pAd5-LtRtXhoI.Utilize PCR whole fiber coding region to be deleted and inserted the recognition sequence of SwaI to produce pAd5-LtRtXhodelfiber.Genomic dna in conjunction with XhoI-linearizing pAd5LtRtXhodelfiber and CG0070 (describing as the embodiment among the WO2005030261) produces the plasmid pFLAr20pAE2fhGmdelfiber that contains total length CG0070DNA O-fiber coding region.Digest from Genetic TherapyInc. with XbaI and EcoRV, the recombinant plasmid that (GTI) obtains and contain the gene of coding Ad5 fibre axis and Ad35 fiber knot, pFBSE5T35H, and utilize the standard technique gel-purified to contain the fragment of chimeric fiber coding region.By in conjunction with the gel-purified fragment among SwaI linearizing pFLAr20pAE2fhGmdelfiber and the E.coli, produce the plasmid that wherein Ad5 axle and Ad35 knot replace Ad5 fiber coding region, pFLAr20pAE2fGm-5T35H.Produce the chimeric oncolytic adenovirus carrier of fiber, OV1191 by digesting pFLAr20pAE2fGm-5T35H with I-SceI and being transfected into the PER.C6 cell.

For generation contains E1 oncolytic vectors OV1192, from CRAd:hUPII-Ela-IRES-Eib/Fb5/3 _LLObtain to contain the 3.6kb EcoRI and the KpnI restriction enzyme enzyme fragment of coding chimeric fiber albumen (Ad5 axle and Ad3 knot) gene in the genomic dna of-RGD, and be cloned into pBlueScript to produce pBlue-5T3H-RGD.Secondly, by obtain the 3.16kb restriction enzyme enzyme fragment of cross-fiber coding region with EagI and KpnI digestion pBlue-5T3H-RGD.In E.coli, gel-purified fragment and SwaI-linearizing pFLAr20pAE2fhGmdelfiber combination are to produce pFLAr20pAE2fhGM-5T3H-RGD.Advance in the PER.C6 cell to produce OV1192 with plasmid and transfection that I-SceI digestion is produced.

By several steps, produced three other to its be positioned at the extra ATG of intrinsic E1A ATG upstream deleted CG0070, OV 1191 similar with 1192 contain the E1 oncolytic vectors.At first, the left side that will obtain from pFLAr21pAe2fF does not hold KpnI restriction enzyme enzyme fragment from being connected to produce pAr21Lt with the right side; RtKpn-E2f.From the total length plasmid, obtain 1.3-kb NheI-KpnI fragment among the pAr21pAE2fe (GTI).In this total length plasmid, deleted the extra ATG upstream of E1A ATG.Replace respective segments from pAr21LtRt Kpn-E2f to produce pAr21LtRtKpn-E2fe with the restriction enzyme enzyme fragment.In conjunction with the genomic dna of KpnI linearizing pAr21LtRtKpn-E2fe and CG0070, OV1191 and OV1192, produce three total length plasmids respectively, pFLAr20pAE2fe-5fiber, pFLAr20pAE2fe-5T35H and pFLAr20pAE2fe-5T3H-RGD.Advance the PER.C6 cell with the linearizing of I-SceI digestion and with pFLAr20pAE2fe-5fiber, pFLAr20pAE2fe-5T35H and pFLAr20pAE2fe-5T3H-RGD transfection, produce OV1193, OV1194 and OV1195 respectively.

In addition, it places oncolytic adenovirus carrier that E1A expresses under hTERT promotor domination in having produced.At first, in order to replace the E2F-1 promotor with the hTERT promotor, the 1293bp Nhe-KpnI fragment that derives from pAr6pATrtexE3F of the NheI-KpnI restriction enzyme enzyme fragment quilt of pAr21LtRtKpn-E2f substitutes.Plasmid with Kpnl linearizing generation, pAr21LtRtKpn-Trtex, and combining source in the genomic dna of CG0070, OV1191, OV1192 to produce pFLAr20pATrtex-5fiber, pFLAr20pATrtex-5T35H and pFLAr20pATrtex-5T3H-RGD.Advance the PER.C6 cell with the linearizing of I-SceI enzymic digestion and with pFLAr20pATrtex-5fiber, pFLAr20pATrtex-5T35H and pFLAr20pATrtex-5T3H-RGD transfection, produce OV1196, OV1197 and OV1198 respectively.

The adenovirus of other of Shi Yonging comprises CV802 in this article, and it is to contain the wild-type Ad5 of whole wild-type dna sequence dnas and be used for positive regulation.Addl312 is the incompetence replicating vector that has a disappearance in the E1a gene, and is used as the negative control carrier.

Utilize two-wheeled cesium chloride density gradient centrifugation purifying all to contain the E1 carrier.As previously described, utilize spectrophotometry virion titre (for example,, waiting 1996) referring to Mittereder.

Embodiment 10: the human cell's that external Ad5 and chimeric fiber are carrier mediated transduction and cytotoxicity.

By tumour and normal cell series being exposed, measure the vitro cytotoxicity of Ad5 of the present invention and Ad5 chimeric fiber carrier to the serial dilution thing of virus seven days.Explanation (CellTiter 96  A according to producer _QueousNon-Radioactive Cell Proliferation Assay, Promega, Madison WI) utilizes the MTS cytotoxicity analysis to measure cell viability.Do not feel the ratio mapping that absorption value may be interpreted as the percentage ratio of not infection control and itself and carrier dosage.The dose-response curve of S shape is suitable for these data and utilizes GraphPad Prism software version 3.0 to calculate each EC that duplicates ₅₀Value.EC ₅₀Value is the carrier amount in every cell (PPC) particle, 50% of the maximum light absorption that its minimizing exposed cell is cultivated.

In four typical case's heads and neck cancer and melanoma cell series, tested the external molten cell potential of chimeric fiber oncolytic adenovirus carrier.These data are compiled in table 6 and 7.

Table 6: the EC of typical head and neck cancerous cell line ₅₀Value

Virus	A-253	SCC-9	FaDu	A431
					CV802	16	12	72	31
OV1193	205	59	323	291
					OV1194	20	6	23	55
OV1195	7	4	34	20
					OV1191	20	39	23	49
OV1192	14	24	60	30
					OV1196	189	40	206	302
OV1197	13	5	9	28
					OV1198	11	1	25	38

Table 7: the EC50 value of typical melanoma cell series

Virus	WM-266-4	A375-luc	G-361	A2058
					CV802	667	45	52	16
OV1193	882	377	177	29
					OV1194	13	58	161	30
OV1195	9	19	40	23
					OV1191	17	27	101	21
OV1192	25	14	83	34
					OV1196	1253	102	88	34
OV1197	8	13	23	15
					OV1198	9	7	18	23

These digital proofs: with the low EC50 value of fiber chimeric vector OV1194, OV1195, OV1197 and OV1198 infection, and can be by the CD46 of high expression level, former the acceptor of Ad3 and Ad35, with former acceptor of Ad5 and HNC cell on low-level CAR expression, the melanoma, explain corresponding resistant to Ad5.

Embodiment 11: the mensuration of the human GM-CSF level by chimeric fiber Ad vector expression.

Express for assessing human GM-CSF, the tumour cell of cultivating is infected under 50 virions/cell, after infection, collected supernatant liquor in 24 and 72 hours and pass through commercially available elisa assay (R﹠amp; D Systems, Minneapolis MN) expresses to measure total GM-CSF.In analysis buffer, with 10 times-1000 times of culturing cell supernatant liquor dilutions.Obtain data at spectrophotometer 490nm place and utilize the SoftMax software package to analyze this data.Human GM-CSF typical curve has usually＞0.995 R ²The sensitivity of value and analysis is 7.8pg/mL normally.The human GM-CSF expression amount of the head and the typical chimeric fiber carrier of neck cancerous cell line shown in table 8A (24 hours) and table 8B (72 hours), and melanoma cell series as showing 9A (24 hours) and showing shown in the 9B (72 hours).

Table 8A. infects the human GM-CSF expression (ng/10 in back 24 hours typical heads and the neck cancerous cell line ⁶Cell/sky).

Virus	A-253	A431	Detroit 562	FaDu	SCC-9
						OV1193	5	10	2	5	7
OV1194	132	212	135	809	561
						OV1195	147	212	107	398	476

Table 8B. infects the human GM-CSF expression (ng/10 in back 72 hours typical heads and the neck cancerous cell line ⁶Cell/sky).

Virus	A-253	A431	Detroit 562	FaDu	SCC-9
						OV1193	285	81	84	131	154
OV1194	659	384	360	1073	2011
						OV1195	468	376	323	1163	1469

Table 9A. infects the back 24 hours human GM-CSF in the typical melanoma cell series and expresses (ng/10 ⁶Cell/sky).

Virus	A-375	WM-266-4	A2058	G-361	SK-MEL-28
						CG0070	4	0.4	7	0.3	1.3
OV1194	370	17	68	11	15
						OV1195	312	21	63	8	14

Table 9B. infects the back 72 hours human GM-CSF in the typical melanoma cell series and expresses (ng/10 ⁶Cell/sky).

Virus	A-375	WM-266-4	A2058	G-361	SK-MEL-28
						CG0070	76	14	145	25	70
OV1194	2074	188	672	130	146
						OV1195	1347	246	302	177	158

The result shows: when infecting typical case's head and neck cancer and melanoma cell series with fiber chimeric vector OV1194 and OV1195, obtained the high-caliber expression of human GM-CSF in 24 and 72 hours after infection.

Embodiment 12: usefulness in the body of the chimeric fiber Ad carrier in the xenotransplantation tumor model.

Assessment contains the usefulness of E1 chimeric fiber Ad carrier in containing FaDu (head and neck cancer) or the heteroplastic nude mice of A375-luc (melanoma).With FaDu (in the Hanks buffering salt (HBSS) of 100 μ l 5 * 10 ⁶Cell) or A375-luc (in 100 μ l HBSS 2 * 10 ⁶) injection nude mice (Hsd:Athymic Nude-nu; Simonsen laboratories, Gilroy CA) in the abdomen of right side.When tumour reaches 50-150mm ³The time, with mice group (n=10) and with 1 * 10 ¹⁰Handle four times in the PBS tumour of individual particulate viral agent or 50 μ l volume doses.In two-dimensional space, measure the size of tumour weekly for twice, and to calculate gross tumor volume be W * (L) ²Mean tumour volume ± the SE of the every treatment group of * II/6. average with vector injection after fate map.

Embodiment 13: a kind of separation, with adenovirus of the present invention transduction with heavily introduce the method for mammiferous primary tumo(u)r cell.

From skin and subcutaneous pathology, lymphoglandula and lung, liver and soft tissue metastasis tumor resection cell.After the excision, transfer to sterile chamber and be transported to the laboratory sample is aseptic.

All down-streams are preferably carried out under aseptic condition.Perhaps handle by collagenase or by the machinery disassociation with the tumor tissues depolymerization, and growth and/or freezing in DMSO in cultivating subsequently with 50%FCS (collagenase processings) or human albumin (the mechanical disassociation).The number of cultivating nascent and secondary autologous tumor cell can enlarge, and utilizes the recombinant adenovirus of the present invention of coding hGM-CSF to transduce some or all of cell to produce hGM-CSF subsequently.

Preferably after the confirmation transducer cell is aseptic, great-hearted and also can produces cytokine, irradiation transduction tumour cell, be suspended in the physiological saline, and the volume of injection (with 0.25-1.0ml) is reaching five different sites intracutaneous and subcutaneous injection) return in the former donor.The cell concentration of per injection is usually 5 * 10 ⁵-5 * 10 ⁸Between and in one embodiment 4 * 10 ⁶-5 * 10 ⁷Between the cell.Can award many injections.In one embodiment, carry out many injections at different times (for example 7 days, 14 days, 21 days, January at interval or its combination).In one embodiment, carry out injection up to three times.Wherein, the injection plan can be depending on transducer cell output.

Embodiment 14: be used to separate, with adenovirus transduction of the present invention with heavily introduce other method of mammiferous primary tumo(u)r cell.

Utilize operation technique to obtain to be used for the tumor tissues (for example from body) of vaccine production.The preparation of vaccine bebcell relates to 48 hours manufacturing processed.Vaccine is made by the metastatic tumo(u)r that comprises lymphoglandula, lung, pleural effusion, suprarenal gland, Tiroidina subcutaneous nodule and the acquisition of unusual sour jujube lump position.

Briefly, tumor sample is removed from individuality, washing and be cut into preferred weight with the HBSS damping fluid is 3 gram or following parts.In each part Petri dish of organizing of 5ml collagenase/gram that dropped into an independent interpolation.Tumor tissues is chopped into 1-3mm ³Section and put into the bacterium separator bag.

This volume is preferably less than 20ml.With bag sealing and be placed in mechanical homogenizer or the Stomacher laboratory stirrer and homogenize until complete digestion (common 30 to 40 minutes, that is, the content until bag is to have the indigested fragment of tissue of the minority dense cream of (if having indigested fragment of tissue)).Usefulness 20ml growth medium dilutes homogenate then, and being settled to final volume is 40ml, and descends 2, centrifugal 10 minutes of 000RPM at 4 ℃.Removing supernatant liquor and cell pellet is suspended on ice in the 10ml growth medium.Repeat these steps until with whole treatments of the sample and can wash essentially no enzyme (for example collagenase) and be suspended in the growth medium.Available then Trypan measures the number and the vitality of cell in the cell suspension.

Then by 4 ℃ 2, centrifugal 10min under the 000rpm is concentrated to cell suspension the suitable concn that is used to transduce.Then bead is re-suspended into the final cell density that is used to transduce (for example 10 ⁷Cell/ml).Some cells are reserved be used for delayed type hypersensitivity (Delayed TypeHypersensitivity) and (DTH) test, establish clone and low temperature storage.Recombinant adenovirus transduction remaining cell with the human GM-CSF that for example encodes.

The plaque-forming unit (pfu) that need transduce by total cellular score (dead+great-hearted, as after tumour digestion, the to calculate) mensuration that increases or the number of virion.For example, if require 10 infection multiplicities, the sum of cell multiply by 10.Then in substratum with viral dilution to 10 times of ultimate density.Then, the adenovirus GM-CSF suspension with 0.1 milliliter of every milliliter of tumour suspension adds cell suspension.Recombinant adenovirus can be for example, to have the E1 and the E3 replication defective virus of the standard first-generation gene elmination of the human GM-CSF cDNA that introduces E1 deletion district.Certainly, cell factor cDNA can be introduced virus genomic other district.

Tenderness was once mixed in per 10 minutes, and at room temperature (being about 23-25 ℃) was with cell cultures 1 hour.Add tumor cell suspension by the growth medium with proper volume, the volume of tumor cell suspension doubles then.Transfer to cell suspension in the suitable tissue culture vessel and at 37 ℃ of following culturing cells.

Under 37 ℃, with the recombinant adenovirus tumour cell of transduceing whole night.After transduceing whole night, with the careful washed cell of HBSS, and use gamma emitter 10 then, 000Rads shone 24 hours down.The cell of small portion (reach sum 10%) may be put into substratum cultivated 48 hours.

If GM-CSF is a transgenosis, can utilize ELISA (for example to utilize commercially available test kit, for example from R﹠amp from the GM-CSF secretory product of transducer cell; The D laboratory, Minneapolis MN) measures to prove the suitable goal gradient of GM-CSF output.In some embodiments of the present invention, goal gradient is 20ng GM-CSF/10 at least ⁶Cell/24 hour are 40ngGM-CSF/10 at least ⁶Cell/24 hour, 100ng GM-CSF/10 at least ⁶Cell/24 hour or surpass 1000ng GM-CSF/10 ⁶Cell/24 hour.

In addition, the routine that can be used for bacterium and fungi is cultivated to guarantee the sterile state of manipulating cells.Then, the cell that is used for vaccination and immunologic evaluation is freezing and preserve in liquid nitrogen at the 10%DMSO/90% foetal calf serum.

The example of vaccination operation is as follows.With HBSS solution with cell thawing and washed twice.For introducing, the cell resuspending that is no more than 1 hour of will thawing before introducing is in the cumulative volume with aseptic salt at the most in the damping fluid of 1ml.Preferably give to be no more than altogether the volume of 1ml, single injection is formed in vaccination, injects the alternately upper arm or the meropodium of base with 23 or 25 gauge needle intracutaneous.The cumulative volume of each injection depends on the dosage level of introducing.

The description of sequence in the sequence table

Therefore, with relevant sequence table and simultaneously disclosed content are disclosed simultaneously in conjunction with being incorporated herein by reference.Below be the description that is contained in the sequence in the sequence table:

SEQ ID NO:1 is coding Ad5 scleroproein (Ad5-CMV5-GFP; Bp position: nucleotide sequence 28338-30083)

SEQ ID NO:2 is an Ad5 fiber aminoacid sequence, and length is 581 amino acid.

SEQ ID NO:3 is the fibrinous nucleotide sequence of coding Ad2

SEQ ID NO:4 is an Ad2 fiber aminoacid sequence

SEQ ID NO:5 is coding Ad35 scleroproein (Ad5-CMV5-GFP-35F; Bp position 28337-29308) nucleotide sequence

SEQ ID NO:6 is an Ad35 fiber aminoacid sequence, and length is 323 amino acid.

SEQ ID NO:7 is the adenovirus fiber consensus motif, KLGXGLXFD/N

SEQ ID NO:8 is coding 5T35H scleroproein (Ad5-CMV5-GFP-5T35H; The nucleotide sequence of gene (ORF) Bp position 28338-30110)

SEQ ID NO:9 is the amino-acid sequence (afterbody and axle derive from Ad5, and the interface obtains from Ad35) of 5T35H fiber: length is 590 amino acid.

SEQ ID NO:10 is coding 5T3H scleroproein (Ad5-CMV5-GFP-5T3H; The nucleotide sequence of gene (ORF) Bp position 28338-30097)

SEQ ID NO:11 is the aminoacid sequence (afterbody and axle derive from Ad5, and the interface obtains from Ad3) of 5T3H fiber: length is 587 amino acid

SEQ ID NO:12 is coding 5T3H-RGD scleroproein (Ad5-CMV5-GFP-5T3H; The nucleotide sequence of gene (ORF) Bp position 30217-32052)

SEQ ID NO:13 is the aminoacid sequence (afterbody and axle derive from Ad5, and the interface obtains from Ad3) of 5T3H-RGD fiber: length is 611 amino acid.The RGD sequence is positioned at fibrinous C-terminal.

SEQ ID NO:14 is the aminoacid sequence of hGM-CSF

SEQ ID NO:15 is the nucleotide sequence of coding hGM-CSF

SEQ ID NO:16 is the nucleotide sequence of the cDNA of coding hGM-CSF

SEQ ID NO:17 is an adenovirus 5PCR primer 1

SEQ ID NO:18 is an adenovirus 5PCR primer 2

SEQ ID NO:19 is an adenovirus 5PCR primer 3

SEQ ID NO:20 is an adenovirus 5PCR primer 4

SEQ ID NO:21 is human GM-CSF PCR primer PSR3

SEQ ID NO:22 is human GM-CSF PCR primer PSR4

SEQ ID NO:23 is poly-(A) signal PCR primer PSR6 in SV40 late period

SEQ ID NO:24 is poly-(A) signal PCR primer PSR7 in SV40 late period

SEQ ID NO:25 is the amino-acid residue RKKR that derives from the furin cleavage site, and it can be removed by carboxypeptidase.

SEQ ID NO:26 is the amino-acid residue RKRR that derives from the furin cleavage site, and it can be removed by carboxypeptidase.

SEQ ID NO:27 is the amino-acid residue RRRR that derives from the furin cleavage site, and it can be removed by carboxypeptidase.

Sequence table

＜110〉Cell Genesys Inc.

＜120〉be used for the fiber-modified adenovirus carrier of the enhancing transduction of tumour cell

<130>3802-105-11 PCT

<140>

<141>

<150>60/604,009

<151>2004-08-25

<160>27

<170>PatentIn version 3.3

<210>1

<211>1746

<212>DNA

＜213〉adenovirus type 5

<400>1

atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata tgacacggaa 60

accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa tgggtttcaa 120

gagagtcccc ctggggtact ctctttgcgc ctatccgaac ctctagttac ctccaatggc 180

atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa ccttacctcc 240

caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat aaacctggaa 300

atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc cgcacctcta 360

atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt gcacgactcc 420

aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct agccctgcaa 480

acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc ctcaccccct 540

ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta tacacaaaat 600

ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct aaacactttg 660

accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac taaagttact 720

ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg aggactaagg 780

attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga tgctcaaaac 840

caactaaatc taagactagg acagggccct ctttttataa actcagccca caacttggat 900

attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa aaagcttgag 960

gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc cattaatgca 1020

ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct caaaacaaaa 1080

attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact aggaactggc 1140

cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga taagctaact 1200

ttgtggacca caccagctcc atctcctaac tgtagactaa atgcagagaa agatgctaaa 1260

ctcactttgg tcttaacaaa atgtggcagt caaatacttg ctacagtttc agttttggct 1320

gttaaaggca gtttggctcc aatatctgga acagttcaaa gtgctcatct tattataaga 1380

tttgacgaaa atggagtgct actaaacaat tccttcctgg acccagaata ttggaacttt 1440

agaaatggag atcttactga aggcacagcc tatacaaacg ctgttggatt tatgcctaac 1500

ctatcagctt atccaaaatc tcacggtaaa actgccaaaa gtaacattgt cagtcaagtt 1560

tacttaaacg gagacaaaac taaacctgta acactaacca ttacactaaa cggtacacag 1620

gaaacaggag acacaactcc aagtgcatac tctatgtcat tttcatggga ctggtctggc 1680

cacaactaca ttaatgaaat atttgccaca tcctcttaca ctttttcata cattgcccaa 1740

gaataa 1746

<210>2

<211>581

<212>PRT

＜213〉adenovirus type 5

<400>2

Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro

1 5 10 15

Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro

20 25 30

Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser

35 40 45

Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu

50 55 60

Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser

65 70 75 80

Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn

85 90 95

Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu

100 105 110

Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr

115 120 125

Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile

130 135 140

Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln

145 150 155 160

Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr

165 170 175

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu

180 185 190

Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly

195 200 205

Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr

210 215 220

Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr

225 230 235 240

Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala

245 250 255

Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val

260 265 270

Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln

275 280 285

Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn

290 295 300

Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu

305 310 315 320

Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile

325 330 335

Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro

340 345 350

Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp

355 360 365

Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp

370 375 380

Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr

385 390 395 400

Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu

405 410 415

Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile

420 425 430

Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile

435 440 445

Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn

450 455 460

Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe

465 470 475 480

Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Ala Val Gly

485 490 495

Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala

500 505 510

Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys

515 520 525

Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp

530 535 540

Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly

545 550 555 560

His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser

565 570 575

Tyr Ile Ala Gln Glu

580

<210>3

<211>1749

<212>DNA

＜213〉adenovirus type 2

<400>3

atgaaacgcg ccagaccgtc tgaagacacc ttcaaccccg tgtatccata tgacacagaa 60

accgggcctc caactgtgcc ctttcttacc cctccatttg tttcacccaa tggtttccaa 120

gaaagtcccc ctggagttct ctctctacgc gtctccgaac ctttggacac ctcccacggc 180

atgcttgcgc ttaaaatggg cagcggtctt accctagaca aggccggaaa cctcacctcc 240

caaaatgtaa ccactgttac tcagccactt aaaaaaacaa agtcaaacat aagtttggac 300

acctccgcac cacttacaat tacctcaggc gccctaacag tggcaaccac cgctcctctg 360

atagttacta gcggcgctct tagcgtacag tcacaagccc cactgaccgt gcaagactcc 420

aaactaagca ttgctactaa agggcccatt acagtgtcag atggaaagct agccctgcaa 480

acatcagccc ccctctctgg cagtgacagc gacaccctta ctgtaactgc atcacccccg 540

ctaactactg ccacgggtag cttgggcatt aacatggaag atcctattta tgtaaataat 600

ggaaaaatag gaattaaaat aagcggtcct ttgcaagtag cacaaaactc cgatacacta 660

acagtagtta ctggaccagg tgtcaccgtt gaacaaaact cccttagaac caaagttgca 720

ggagctattg gttatgattc atcaaacaac atggaaatta aaacgggcgg tggcatgcgt 780

ataaataaca acttgttaat tctagatgtg gattacccat ttgatgctca aacaaaacta 840

cgtcttaaac tggggcaggg acccctgtat attaatgcat ctcataactt ggacataaac 900

tataacagag gcctatacct ttttaatgca tcaaacaata ctaaaaaact ggaagttagc 960

ataaaaaaat ccagtggact aaactttgat aatactgcca tagctataaa tgcaggaaag 1020

ggtctggagt ttgatacaaa cacatctgag tctccagata tcaacccaat aaaaactaaa 1080

attggctctg gcattgatta caatgaaaac ggtgccatga ttactaaact tggagcgggt 1140

ttaagctttg acaactcagg ggccattaca ataggaaaca aaaatgatga caaacttacc 1200

ctgtggacaa ccccagaccc atctcctaac tgcagaattc attcagataa tgactgcaaa 1260

tttactttgg ttcttacaaa atgtgggagt caagtactag ctactgtagc tgctttggct 1320

gtatctggag atctttcatc catgacaggc accgttgcaa gtgttagtat attccttaga 1380

tttgaccaaa acggtgttct aatggagaac tcctcactta aaaaacatta ctggaacttt 1440

agaaatggga actcaactaa tgcaaatcca tacacaaatg cagttggatt tatgcctaac 1500

cttctagcct atccaaaaac ccaaagtcaa actgctaaaa ataacattgt cagtcaagtt 1560

tacttgcatg gtgataaaac taaacctatg atacttacca ttacacttaa tggcactagt 1620

gaatccacag aaactagcga ggtaagcact tactctatgt cttttacatg gtcctgggaa 1680

agtggaaaat acaccactga aacttttgct accaactctt acaccttctc ctacattgcc 1740

caggaataa 1749

<210>4

<211>582

<212>PRT

＜213〉adenovirus type 2

<400>4

Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro

1 5 10 15

Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro

20 25 30

Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser

35 40 45

Leu Arg Val Ser Glu Pro Leu Asp Thr Ser His Gly Met Leu Ala Leu

50 55 60

Lys Met Gly Ser Gly Leu Thr Leu Asp Lys Ala Gly Asn Leu Thr Ser

65 70 75 80

Gln Asn Val Thr Thr Val Thr Gln Pro Leu Lys Lys Thr Lys Ser Asn

85 90 95

Ile Ser Leu Asp Thr Ser Ala Pro Leu Thr Ile Thr Ser Gly Ala Leu

100 105 110

Thr Val Ala Thr Thr Ala Pro Leu Ile Val Thr Ser Gly Ala Leu Ser

115 120 125

Val Gln Ser Gln Ala Pro Leu Thr Val Gln Asp Ser Lys Leu Ser Ile

130 135 140

Ala Thr Lys Gly Pro Ile Thr Val Ser Asp Gly Lys Leu Ala Leu Gln

145 150 155 160

Thr Ser Ala Pro Leu Ser Gly Ser Asp Ser Asp Thr Leu Thr Val Thr

165 170 175

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asn Met

180 185 190

Glu Asp Pro Ile Tyr Val Asn Asn Gly Lys Ile Gly Ile Lys Ile Ser

195 200 205

Gly Pro Leu Gln Val Ala Gln Asn Ser Asp Thr Leu Thr Val Val Thr

210 215 220

Gly Pro Gly Val Thr Val Glu Gln Asn Ser Leu Arg Thr Lys Val Ala

225 230 235 240

Gly Ala Ile Gly Tyr Asp Ser Ser Asn Asn Met Glu Ile Lys Thr Gly

245 250 255

Gly Gly Met Arg Ile Asn Asn Asn Leu Leu Ile Leu Asp Val Asp Tyr

260 265 270

Pro Phe Asp Ala Gln Thr Lys Leu Arg Leu Lys Leu Gly Gln Gly Pro

275 280 285

Leu Tyr Ile Asn Ala Ser His Asn Leu Asp Ile Asn Tyr Asn Arg Gly

290 295 300

Leu Tyr Leu Phe Asn Ala Ser Asn Asn Thr Lys Lys Leu Glu Val Ser

305 310 315 320

Ile Lys Lys Ser Ser Gly Leu Asn Phe Asp Asn Thr Ala Ile Ala Ile

325 330 335

Asn Ala Gly Lys Gly Leu Glu Phe Asp Thr Asn Thr Ser Glu Ser Pro

340 345 350

Asp Ile Asn Pro Ile Lys Thr Lys Ile Gly Ser Gly Ile Asp Tyr Asn

355 360 365

Glu Asn Gly Ala Met Ile Thr Lys Leu Gly Ala Gly Leu Ser Phe Asp

370 375 380

Asn Ser Gly Ala Ile Thr Ile Gly Asn Lys Asn Asp Asp Lys Leu Thr

385 390 395 400

Leu Trp Thr Thr Pro Asp Pro Ser Pro Asn Cys Arg Ile His Ser Asp

405 410 415

Asn Asp Cys Lys Phe Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Val

420 425 430

Leu Ala Thr Val Ala Ala Leu Ala Val Ser Gly Asp Leu Ser Ser Met

435 440 445

Thr Gly Thr Val Ala Ser Val Ser Ile Phe Leu Arg Phe Asp Gln Asn

450 455 460

Gly Val Leu Met Glu Asn Ser Ser Leu Lys Lys His Tyr Trp Asn Phe

465 470 475 480

Arg Asn Gly Asn Ser Thr Asn Ala Asn Pro Tyr Thr Asn Ala Val Gly

485 490 495

Phe Met Pro Asn Leu Leu Ala Tyr Pro Lys Thr Gln Ser Gln Thr Ala

500 505 510

Lys Asn Asn Ile Val Ser Gln Val Tyr Leu His Gly Asp Lys Thr Lys

515 520 525

Pro Met Ile Leu Thr Ile Thr Leu Asn Gly Thr Ser Glu Ser Thr Glu

530 535 540

Thr Ser Glu Val Ser Thr Tyr Ser Met Ser Phe Thr Trp Ser Trp Glu

545 550 555 560

Ser Gly Lys Tyr Thr Thr Glu Thr Phe Ala Thr Asn Ser Tyr Thr Phe

565 570 575

Ser Tyr Ile Ala Gln Glu

580

<210>5

<211>972

<212>DNA

＜213〉adenovirus type 35

<400>5

atgaccaaga gagtccggct cagtgactcc ttcaaccctg tctaccccta tgaagatgaa 60

agcacctccc aacacccctt tataaaccca gggtttattt ccccaaatgg cttcacacaa 120

agcccagacg gagttcttac tttaaaatgt ttaaccccac taacaaccac aggcggatct 180

ctacagctaa aagtgggagg gggacttaca gtggatgaca ctgatggtac cttacaagaa 240

aacatacgtg ctacagcacc cattactaaa aataatcact ctgtagaact atccattgga 300

aatggattag aaactcaaaa caataaacta tgtgccaaat tgggaaatgg gttaaaattt 360

aacaacggtg acatttgtat aaaggatagt attaacacct tatggactgg aataaaccct 420

ccacctaact gtcaaattgt ggaaaacact aatacaaatg atggcaaact tactttagta 480

ttagtaaaaa atggagggct tgttaatggc tacgtgtctc tagttggtgt atcagacact 540

gtgaaccaaa tgttcacaca aaagacagca aacatccaat taagattata ttttgactct 600

tctggaaatc tattaactga ggaatcagac ttaaaaattc cacttaaaaa taaatcttct 660

acagcgacca gtgaaactgt agccagcagc aaagccttta tgccaagtac tacagcttat 720

cccttcaaca ccactactag ggatagtgaa aactacattc atggaatatg ttactacetg 780

actagttatg atagaagtct atttcccttg aacatttcta taatgctaaa cagccgtatg 840

atttcttcca atgttgccta tgccatacaa tttgaatgga atctaaatgc aagtgaatct 900

ccagaaagca acatagctac gctgaccaca tccccctttt tcttttctta cattacagaa 960

gacgacgaat aa 972

<210>6

<211>323

<212>PRT

＜213〉adenovirus type 35

<400>6

Met Thr Lys Arg Val Arg Leu Ser Asp Ser Phe Asn Pro Val Tyr Pro

1 5 10 15

Tyr Glu Asp Glu Ser Thr Ser Gln His Pro Phe Ile Asn Pro Gly Phe

20 25 30

Ile Ser Pro Asn Gly Phe Thr Gln Ser Pro Asp Gly Val Leu Thr Leu

35 40 45

Lys Cys Leu Thr Pro Leu Thr Thr Thr Gly Gly Ser Leu Gln Leu Lys

50 55 60

Val Gly Gly Gly Leu Thr Val Asp Asp Thr Asp Gly Thr Leu Gln Glu

65 70 75 80

Asn Ile Arg Ala Thr Ala Pro Ile Thr Lys Asn Asn His Ser Val Glu

85 90 95

Leu Ser Ile Gly Asn Gly Leu Glu Thr Gln Asn Asn Lys Leu Cys Ala

100 105 110

Lys Leu Gly Asn Gly Leu Lys Phe Asn Asn Gly Asp Ile Cys Ile Lys

115 120 125

Asp Ser Ile Asn Thr Leu Trp Thr Gly Ile Asn Pro Pro Pro Asn Cys

130 135 140

Gln Ile Val Glu Asn Thr Asn Thr Asn Asp Gly Lys Leu Thr Leu Val

145 150 155 160

Leu Val Lys Asn Gly Gly Leu Val Asn Gly Tyr Val Ser Leu Val Gly

165 170 175

Val Ser Asp Thr Val Asn Gln Met Phe Thr Gln Lys Thr Ala Asn Ile

180 185 190

Gln Leu Arg Leu Tyr Phe Asp Ser Ser Gly Asn Leu Leu Thr Glu Glu

195 200 205

Ser Asp Leu Lys Ile Pro Leu Lys Asn Lys Ser Ser Thr Ala Thr Ser

210 215 220

Glu Thr Val Ala Ser Ser Lys Ala Phe Met Pro Ser Thr Thr Ala Tyr

225 230 235 240

Pro Phe Asn Thr Thr Thr Arg Asp Ser Glu Asn Tyr Ile His Gly Ile

245 250 255

Cys Tyr Tyr Met Thr Ser Tyr Asp Arg Ser Leu Phe Pro Leu Asn Ile

260 265 270

Ser Ile Met Leu Asn Ser Arg Met Ile Ser Ser Asn Val Ala Tyr Ala

275 280 285

Ile Gln Phe Glu Trp Asn Leu Asn Ala Ser Glu Ser Pro Glu Ser Asn

290 295 300

Ile Ala Thr Leu Thr Thr Ser Pro Phe Phe Phe Ser Tyr Ile Thr Glu

305 310 315 320

Asp Asp Glu

<210>7

<211>9

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic adenovirus 2 fiber consensus motifs

<220>

<221>MOD_RES

<222>(4)

＜223〉variable amino acid

<220>

<221>MOD_RES

<222>(7)

＜223〉variable amino acid

<220>

<221>MOD_RES

<222>(9)

＜223〉Asn or Asp

<400>7

Lys Leu Gly Xaa Gly Leu Xaa Phe Xaa

1 5

<210>8

<211>1773

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: coding 5T35H scleroproein (Ad5-CMV5-GFP-5T35H; The Bp position

Gene ground synthesizing ribonucleotide sequence 28338-30110)

<400>8

atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata tgacacggaa 60

accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa tgggtttcaa 120

gagagtcccc ctggggtact ctctttgcgc ctatccgaac ctctagttac ctccaatggc 180

atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa ccttacctcc 240

caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat aaacctggaa 300

atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc cgcacctcta 360

atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt gcacgactcc 420

aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct agccctgcaa 480

acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc ctcaccccct 540

ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta tacacaaaat 600

ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct aaacactttg 660

accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac taaagttact 720

ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg aggactaagg 780

attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga tgctcaaaac 840

caactaaatc taagactagg acagggccct ctttttataa actcagccca caacttggat 900

attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa aaagcttgag 960

gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc cattaatgca 1020

ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct caaaacaaaa 1080

attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact aggaactggc 1140

cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga taagctaact 1200

ttgtggaccg gaataaaccc tccacctaac tgtcaaattg tggaaaacac taatacaaat 1260

gatggcaaac ttactttagt attagtaaaa aatggagggc ttgttaatgg ctacgtgtct 1320

ctagttggtg tatcagacac tgtgaaccaa atgttcacac aaaagacagc aaacatccaa 1380

ttaagattat attttgactc ttctggaaat ctattaactg aggaatcaga cttaaaaatt 1440

ccacttaaaa ataaatcttc tacagcgacc agtgaaactg tagccagcag caaagccttt 1500

atgccaagta ctacagctta tcccttcaac accactacta gggatagtga aaactacatt 1560

catggaatat gttactacat gactagttat gatagaagtc tatttccctt gaacatttct 1620

ataatgctaa acagccgtat gatttcttcc aatgttgcct atgccataca atttgaatgg 1680

aatctaaatg caagtgaatc tccagaaagc aacatagcta cgctgaccac atcccccttt 1740

ttcttttctt acattacaga agacgacgaa taa 1773

<210>9

<211>590

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the synthetic amino acid array of 5T35H fiber (derive from Ad5 tail and the axle and

From the interface that Ad35 obtains)

<400>9

Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro

1 5 10 15

Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro

20 25 30

Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser

35 40 45

Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu

50 55 60

Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser

65 70 75 80

Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn

85 90 95

Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu

100 105 110

Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr

115 120 125

Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile

130 135 140

Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln

145 150 155 160

Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr

165 170 175

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu

180 185 190

Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly

195 200 205

Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr

210 215 220

Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr

225 230 235 240

Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala

245 250 255

Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val

260 265 270

Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln

275 280 285

Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn

290 295 300

Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu

305 310 315 320

Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile

325 330 335

Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro

340 345 350

Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp

355 360 365

Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp

370 375 380

Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr

385 390 395 400

Leu Trp Thr Gly Ile Asn Pro Pro Pro Asn Cys Gln Ile Val Glu Asn

405 410 415

Thr Asn Thr Asn Asp Gly Lys Leu Thr Leu Val Leu Val Lys Asn Gly

420 425 430

Gly Leu Val Asn Gly Tyr Val Ser Leu Val Gly Val Ser Asp Thr Val

435 440 445

Asn Gln Met Phe Thr Gln Lys Thr Ala Asn Ile Gln Leu Arg Leu Tyr

450 455 460

Phe Asp Ser Ser Gly Asn Leu Leu Thr Glu Glu Ser Asp Leu Lys Ile

465 470 475 480

Pro Leu Lys Asn Lys Ser Ser Thr Ala Thr Ser Glu Thr Val Ala Ser

485 490 495

Ser Lys Ala Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Asn Thr Thr

500 505 510

Thr Arg Asp Ser Glu Asn Tyr Ile His Gly Ile Cys Tyr Tyr Met Thr

515 520 525

Ser Tyr Asp Arg Ser Leu Phe Pro Leu Asn Ile Ser Ile Met Leu Asn

530 535 540

Ser Arg Met Ile Ser Ser Asn Val Ala Tyr Ala Ile Gln Phe Glu Trp

545 550 555 560

Asn Leu Asn Ala Ser Glu Ser Pro Glu Ser Asn Ile Ala Thr Leu Thr

565 570 575

Thr Ser Pro Phe Phe Phe Ser Tyr Ile Thr Glu Asp Asp Glu

580 585 590

<210>10

<211>1764

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: 5T3H celloglobulin (Ad5-CMV5-GFP-5T3H; The Bp position

The synthesizing ribonucleotide sequence of gene 28338-30097)

<400>10

atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata tgacacggaa 60

accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa tgggtttcaa 120

gagagtcccc ctggggtact ctctttgcgc ctatccgaac ccctagttac ctccaatggc 180

atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa ccttacctcc 240

caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat aaacctggaa 300

atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc cgcacctcta 360

atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt gcacgactcc 420

aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct agccctgcaa 480

acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc ctcaccccct 540

ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta tacacaaaat 600

ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct aaacactttg 660

accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac taaagttact 720

ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg aggactaagg 780

attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga tgctcaaaac 840

caactaaatc taagactagg acagggccct ctttttataa actcagccca caacttggat 900

attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa aaagcttgag 960

gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc cattaatgca 1020

ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct caaaacaaaa 1080

attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact aggaactggc 1140

cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga taagctaact 1200

ttgtggaccg gtccaaaacc agaagccaac tgcataattg aatacgggaa acaaaaccca 1260

gatagcaaac taactttaat ccttgtaaaa aatggaggaa ttgttaatgg atatgtaacg 1320

ctaatgggag cctcagacta cgttaacacc ttatttaaaa acaaaaatgt ctccattaat 1380

gtagaactat actttgatgc cactggtcat atattaccag actcatcttc tcttaaaaca 1440

gatctagaac taaaatacaa gcaaaccgct gactttagtg caagaggttt tatgccaagt 1500

actacagcgt atccatttgt ccttcctaat gcgggaacac ataatgaaaa ttatattttt 1560

ggtcaatgct actacaaagc aagcgatggt gccctttttc cgttggaagt tactgttatg 1620

cttaataaac gcctgccaga tagtcgcaca tcctatgtta tgactttttt atggtccttg 1680

aatgctggtc tagctccaga aactactcag gcaaccctca taacctcccc atttaccttt 1740

tcctatatta gagaagatga ataa 1764

<210>11

<211>587

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the synthetic amino acid array of 5T3H fiber (derive from Ad5 tail and the axle and

From the interface that Ad3 obtains)

<400>11

Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro

1 5 10 15

Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro

20 25 30

Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser

35 40 45

Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu

50 55 60

Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser

65 70 75 80

Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn

85 90 95

Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu

100 105 110

Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr

115 120 125

Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile

130 135 140

Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln

145 150 155 160

Thr Ser G1y Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr

165 170 175

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu

180 185 190

Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly

195 200 205

Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr

210 215 220

Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr

225 230 235 240

Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala

245 250 255

Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val

260 265 270

Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln

275 280 285

Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn

290 295 300

Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu

305 310 315 320

Val Asn Leu Ser Thr Ala Lys G1y Len Met Phe Asp Ala Thr Ala Ile

325 330 335

Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro

340 345 350

Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp

355 360 365

Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp

370 375 380

Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr

385 390 395 400

Leu Trp Thr Gly Pro Lys Pro Glu Ala Asn Cys Ile Ile Glu Tyr Gly

405 410 415

Lys Gln Asn Pro Asp Ser Lys Leu Thr Leu Ile Leu Val Lys Asn Gly

420 425 430

Gly Ile Val Asn Gly Tyr Val Thr Leu Met Gly Ala Ser Asp Tyr Val

435 440 445

Asn Thr Leu Phe Lys Asn Lys Asn Val Ser Ile Asn Val Glu Leu Tyr

450 455 460

Phe Asp Ala Thr Gly His Ile Leu Pro Asp Ser Ser Ser Leu Lys Thr

465 470 475 480

Asp Leu Glu Leu Lys Tyr Lys Gln Thr Ala Asp Phe Ser Ala Arg Gly

485 490 495

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Val Leu Pro Asn Ala Gly

500 505 510

Thr His Asn Glu Asn Tyr Ile Phe Gly Gln Cys Tyr Tyr Lys Ala Ser

515 520 525

Asp Gly Ala Leu Phe Pro Leu Glu Val Thr Val Met Leu Asn Lys Arg

530 535 540

Leu Pro Asp Ser Arg Thr Ser Tyr Val Met Thr Phe Leu Trp Ser Leu

545 550 555 560

Asn Ala Gly Leu Ala Pro Glu Thr Thr Gln Ala Thr Leu Ile Thr Ser

565 570 575

Pro Phe Thr Phe Ser Tyr Ile Arg Glu Asp Glu

580 585

<210>12

<211>1836

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: 5T3H-RGD celloglobulin (Ad5-CMV5-GFP-5T3H-RGD;

The synthesizing ribonucleotide sequence of gene Bp position 30217-32052)

<400>12

atgaagcgcg caagaccgtc tgaagatacc ttcaaccccg tgtatccata tgacacggaa 60

accggtcctc caactgtgcc ttttcttact cctccctttg tatcccccaa tgggtttcaa 120

gagagtcccc ctggggtact ctctttgcgc ctatccgaac ctctagttac ctccaatggc 180

atgcttgcgc tcaaaatggg caacggcctc tctctggacg aggccggcaa ccttacctcc 240

caaaatgtaa ccactgtgag cccacctctc aaaaaaacca agtcaaacat aaacctggaa 300

atatctgcac ccctcacagt tacctcagaa gccctaactg tggctgccgc cgcacctcta 360

atggtcgcgg gcaacacact caccatgcaa tcacaggccc cgctaaccgt gcacgactcc 420

aaacttagca ttgccaccca aggacccctc acagtgtcag aaggaaagct agccctgcaa 480

acatcaggcc ccctcaccac caccgatagc agtaccctta ctatcactgc ctcaccccct 540

ctaactactg ccactggtag cttgggcatt gacttgaaag agcccattta tacacaaaat 600

ggaaaactag gactaaagta cggggctcct ttgcatgtaa cagacgacct aaacactttg 660

accgtagcaa ctggtccagg tgtgactatt aataatactt ccttgcaaac taaagttact 720

ggagccttgg gttttgattc acaaggcaat atgcaactta atgtagcagg aggactaagg 780

attgattctc aaaacagacg ccttatactt gatgttagtt atccgtttga tgctcaaaac 840

caactaaatc taagactagg acagggccct ctttttataa actcagccca caacttggat 900

attaactaca acaaaggcct ttacttgttt acagcttcaa acaattccaa aaagcttgag 960

gttaacctaa gcactgccaa ggggttgatg tttgacgcta cagccatagc cattaatgca 1020

ggagatgggc ttgaatttgg ttcacctaat gcaccaaaca caaatcccct caaaacaaaa 1080

attggccatg gcctagaatt tgattcaaac aaggctatgg ttcctaaact aggaactggc 1140

cttagttttg acagcacagg tgccattaca gtaggaaaca aaaataatga taagctaacc 1200

ctatggacag gtccaaaacc agaagccaac tgcataattg aatacgggaa acaaaaccca 1260

gatagcaaac taactttaat ccttgtaaaa aatggaggaa ttgttaatgg atatgtaacg 1320

ctaatgggag cctcagacta cgttaacacc ttatttaaaa acaaaaatgt ctccattaat 1380

gtagaactat actttgatgc cactggtcat atattaccag actcatcttc tcttaaaaca 1440

gatctagaac taaaatacaa gcaaaccgct gactttagtg caagaggttt tatgccaagt 1500

actacagcgt atccatttgt ccttcctaat gcgggaacac ataatgaaaa ttatattttt 1560

ggtcaatgct actacaaagc aagcgatggt gccctttttc cgttggaagt tactgttatg 1620

cttaataaac gcctgccaga tagtcgcaca tcctatgtta tgactttttt atggtccttg 1680

aatgctggtc tagctccaga aactactcag gcaaccctca taacctcccc atttaccttt 1740

tcctatatta gagaagatga cggtggaggc ggttcaggcg gaggtggctc tggcggtggc 1800

ggatcctgtg actgccgcgg agactgtttc tgctaa 1836

<210>13

<211>611

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the synthetic amino acid array of 5T3H-RGD fiber (derive from Ad5 tail and

Axle and the interface that obtains from Ad3)

<400>13

Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro

1 5 10 15

Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro

20 25 30

Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser

35 40 45

Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu

50 55 60

Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser

65 70 75 80

Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn

85 90 95

Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu

100 105 110

Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr

115 120 125

Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile

130 135 140

Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln

145 150 155 160

Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr

165 170 175

Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu

180 185 190

Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly

195 200 205

Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr

210 215 220

Gly Pro Gly Val Thr Ile Asn Aan Thr Ser Leu Gln Thr Lys Val Thr

225 230 235 240

Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala

245 250 255

Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val

260 265 270

Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln

275 280 285

Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn

290 295 300

Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu

305 310 315 320

Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile

325 330 335

Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro

340 345 350

Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp

355 360 365

Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp

370 375 380

Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr

385 390 395 400

Leu Trp Thr Gly Pro Lys Pro Glu Ala Asn Cys Ile Ile Glu Tyr Gly

405 410 415

Lys Gln Asn Pro Asp Ser Lys Leu Thr Leu Ile Leu Val Lys Asn Gly

420 425 430

Gly Ile Val Asn Gly Tyr Val Thr Leu Met Gly Ala Ser Asp Tyr Val

435 440 445

Asn Thr Leu Phe Lys Asn Lys Asn Val Ser Ile Asn Val Glu Leu Tyr

450 455 460

Phe Asp Ala Thr Gly His Ile Leu Pro Asp Ser Ser Ser Leu Lys Thr

465 470 475 480

Asp Leu Glu Leu Lys Tyr Lys Gln Thr Ala Asp Phe Ser Ala Arg Gly

485 490 495

Phe Met Pro Ser Thr Thr Ala Tyr Pro Phe Val Leu Pro Asn Ala Gly

500 505 510

Thr His Asn Glu Asn Tyr Ile Phe Gly Gln Cys Tyr Tyr Lys Ala Ser

515 520 525

Asp Gly Ala Leu Phe Pro Leu Glu Val Thr Val Met Leu Asn Lys Arg

530 535 540

Leu Pro Asp Ser Arg Thr Ser Tyr Val Met Thr Phe Leu Trp Ser Leu

545 550 555 560

Asn Ala Gly Leu Ala Pro Glu Thr Thr Gln Ala Thr Leu Ile Thr Ser

565 570 575

Pro Phe Thr Phe Ser Tyr Ile Arg Glu Asp Asp Gly Gly Gly Gly Ser

580 585 590

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Cys Asp Cys Arg Gly Asp

595 600 605

Cys Phe Cys

610

<210>14

<211>144

<212>PRT

＜213〉mankind

<400>14

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln Pro Trp Glu His

20 25 30

Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp

35 40 45

Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe

50 55 60

Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys

65 70 75 80

Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met

85 90 95

Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr Ser

100 105 110

Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys

115 120 125

Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu

130 135 140

<210>15

<211>2027

<212>DNA

＜213〉mankind

<400>15

tgtggctgca gagcctgctg ctcttgggca ctgtggcctg cagcatctct gcacccgccc 60

gctcgcccag ccccagcacg cagccctggg agcatgtgaa tgccatccag gaggcccggc 120

gtctcctgaa cctgagtaga gacactgctg ctgagatggt aagtgagaga atgtgggcct 180

gtgcctaggc cacccagctg gcccctgact ggccacgcct gtcagcttga taacatgaca 240

ttttcctttt ctacagaatg aaacagtaga agtcatctca gaaatgtttg acctccaggt 300

aagatgcttc tctctgacat agctttccag aagcccctgc cctggggtgg aggtggggac 360

tccattttag atggcaccac acagggttgt ccactttctc tccagtcagc tggctgcagg 420

aggagggggt agcaactggg tgctcaagag gctgctggcc gtgcccctat ggcagtcaca 480

tgagctcctt tatcagctga gcggccatgg gcagacctag cattcaatgg ccaggagtca 540

ccaggggaca ggtggtaaag tgggggtcac ttcatgagac aggagctgtg ggtttggggc 600

gctcactgtg ccccgagacc aagtcctgtt gagacagtgc tgactacaga gaggcacaga 660

ggggtttcag gaacaaccct tgcccaccca gcaggtccag gtgaggcccc acccccctct 720

ccctgaatga tggggtgaga gtcacctcct tccctaaggc tgggctcctc tccaggtgcc 780

gctgagggtg gcctgggcgg ggcagtgaga agggcaggtt cgtgcctgcc atggacaggg 840

cagggtctat gactggaccc agcctgtgcc cctcccaagc cctactcctg ggggctgggg 900

gcagcagcaa aaaggagtgg tggagagttc ttgtaccact gtgggcactt ggccactgct 960

caccgacgaa cgacattttc cacaggagcc gacctgccta cagacccgcc tggagctgta 1020

caagcagggc ctgcggggca gcctcaccaa gctcaagggc cccttgacca tgatggccag 1080

ccactacaag cagcactgcc ctccaacccc ggtgagtgcc tacggcaggg cctccagcag 1140

gaatgtctta atctaggggg tggggtcgac atggggagag atctatggct gtggctgttc 1200

aggaccccag ggggtttctg tgccaacagt tatgtaatga ttagccctcc agagaggagg 1260

cagacagccc atttcatccc aaggagtcag agccacagag cgctgaagcc cacagtgctc 1320

cccagcagga gctgctccta tcctggtcat tattgtcatt atggttaatg aggtcagagg 1380

tgagggcaaa cccaaggaaa cttggggcct gcccaaggcc cagaggaagt gcccaggccc 1440

aagtgccacc ttctggcagg actttcctct ggccccacat ggggtgcttg aattgcagag 1500

gatcaaggaa ggggggctac ttggaatgga caaggacctc aggcactcct tcctgcggga 1560

agggagcaaa gtttgtggcc ttgactccac tccttctggg tgcccagaga cgacctcagc 1620

ccagctgccc tgctctgccc tgggaccaaa aaggcaggcg tttgactgcc cagaaggcca 1680

acctcaggct ggcacttaag tcaggccctt gactctggct gccactggca gagctatgca 1740

ctccttgggg aacacgtggg tggcagcagc gtcacctgac ccaggtcagt gggtgtgtcc 1800

tggagtgggc ctcctggcct ctgagttcta agaggcagta gagaaacatg ctggtgcttc 1860

cttcccccac gttacccact tgcctggact caagtgtttt ttatttttct ttttttaaag 1920

gaaacttcct gtgcaaccca gattatcacc tttgaaagtt tcaaagagaa cctgaaggac 1980

tttctgcttg tcatcccctt tgactgctgg gagccagtcc aggagtg 2027

<210>16

<211>435

<212>DNA

＜213〉mankind

<400>16

atgtggctgc agagcctgct gctcttgggc actgtggcct gcagcatctc tgcacccgcc 60

cgctcgccca gccccagcac gcagccctgg gagcatgtga atgccatcca ggaggcccgg 120

cgtctcctga acctgagtag agacactgct gctgagatga atgaaacagt agaagtcatc 180

tcagaaatgt ttgacctcca ggagccgacc tgcctacaga cccgcctgga gctgtacaag 240

cagggcctgc ggggcagcct caccaagctc aagggcccct tgaccatgat ggccagccac 300

tacaagcagc actgccctcc aaccccggaa acttcctgtg caacccagac tatcaccttt 360

gaaagtttca aagagaacct gaaggacttt ctgcttgtca tcccctttga ctgctgggag 420

ccagtccagg agtaa 435

<210>17

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic adenovirus 5PCR primer 1

<400>17

gaattctagg gataacaggg taatcatcat caataatata cctt 44

<210>18

<211>23

<212>DNA

<213>

Artificial sequence

<220>

＜223〉description of artificial sequence: synthetic adenovirus 5PCR primer 2

<400>18

cccggggtgc tccacataaa tct 23

<210>19

<211>44

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic adenovirus 5PCR primer 3

<400>19

aagctttagg gataacaggg taatcatcat caataatata cctt 44

<210>20

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic adenovirus 5PCR primer 4

<400>20

cccgggggaa tacatacccg cagg 24

<210>21

<211>37

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the human GM-CSF PCR of synthetic primer PSR3

<400>21

cttcgaggaa ttcaggatgt ggctgcagag cctgctg 37

<210>22

<211>33

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: the human GM-CSF PCR of synthetic primer PSR4

<400>22

cttcgagaag cttactcctg gactggctcc cag 33

<210>23

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic SV40 polyadenylic acid in late period signal PCR primer PSR6

<400>23

cttcgagaag cttcagacat gataagatac attg 34

<210>24

<211>35

<212>DNA

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic SV40 polyadenylic acid in late period signal PCR primer PSR7

<400>24

cttcgaggga tcctaccaca tttgtagagg tttac 35

<210>25

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>25

Arg Lys Lys Arg

1

<210>26

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>26

Arg Lys Arg Arg

1

<210>27

<211>4

<212>PRT

＜213〉artificial sequence

<220>

＜223〉description of artificial sequence: synthetic peptide

<400>27

Arg Arg Arg Arg

1

Claims (30)

1. the method for an expressing heterologous nucleic acid in the primary tumo(u)r cell, comprise: with containing the proteic adenovirus carrier of the chimeric fiber described primary tumo(u)r cell of transduceing, wherein said chimeric fiber albumen comprise the subtype C adenovirus axle district at least a portion and at least a portion of Head Section of hypotype B adenovirus, wherein Head Section is combined with CD46.

2. method according to claim 1, wherein said chimeric fiber albumen comprise at least a portion of Ad5 or Ad2 axle and at least a portion of Ad35 head.

3. method according to claim 1, wherein cell is by external transduction.

4. method according to claim 1, wherein cell is transduceed in the body.

5. method according to claim 1, wherein chimeric fiber albumen comprises the amino acid of 46-136 among the SEQ ID NO:6.

6. method according to claim 1, wherein chimeric fiber albumen comprises the amino acid of 47-399 among the amino acid of 47-399 among the SEQ ID NO:2 or the SEQ ID NO:4.

7. method according to claim 5, wherein chimeric fiber albumen comprises the amino acid of 47-399 among the amino acid of 47-399 among the SEQ ID NO:2 or the SEQ ID NO:4.

8. method according to claim 1, wherein said primary tumo(u)r cell is selected from lung tumor cell, prostate tumor cells, head and neck tumour cell, tumor of bladder cell and kidney tumor cell.

9. method according to claim 1, wherein said primary tumo(u)r cell are non-small cell lung tumor cells.

10. method according to claim 1, wherein said adenovirus is preferentially duplicated in described primary tumo(u)r cell.

11. method according to claim 10, wherein said adenovirus comprise and duplicate the allos transcriptional regulatory element (TRE) that at least one necessary adenovirus encoding sequence effectively is connected.

12. method according to claim 11, wherein said TRE is selected from cell-specific TRE, cell state specificity T RE and tissue specificity TRE.

13. method according to claim 11, wherein said TRE is selected from PSA TRE, E2FTRE, Telomerase (TERT) TRE, urokinase plasminogen activator (uPA) TRE, probasinTRE, tyrosine oxidase related protein-2 TRE, MART-1 TRE, CRGL2 TRE and PRL-3 Protein-tyrosine-phosphatase TRE.

14. method according to claim 11 is in the adenovirus coding region of wherein said adenovirus encoding sequence in being selected from E1a, E1b, E2a, E2b and E4.

15. method according to claim 10, wherein said adenovirus comprise the some or all of disappearance in E1B 19-kDa district.

16. method according to claim 1, wherein said adenovirus comprise the disappearance of duplicating necessary adenovirus encoding sequence.

17. it is the incompetence replication type adenovirus that method according to claim 16, wherein said adenovirus make the adenovirus in the primary tumo(u)r cell.

18. method according to claim 17, the wherein said adenovirus encoding sequence that duplicates necessity is selected from E1a, E1b, E2a, E2b and E4.

19. method according to claim 1, wherein said adenovirus comprise the disappearance of at least one adenovirus E3 encoding sequence.

20. being selected from, method according to claim 19, wherein said adenovirus E3 encoding sequence be used for 19K, 14.7K, 14.5K, 12.5K, 11.6K, 10.4K and the proteic encoding sequence of 6.7K E3.

21. method according to claim 19, wherein all the E3 encoding sequence lacks.

22. method according to claim 19, wherein said adenovirus comprise 10.4K, 14.5K and 14.7K E3 encoding sequence.

23. method according to claim 1, wherein said adenovirus comprise the E3 encoding sequence that all are natural.

24. method according to claim 1, wherein said adenovirus comprise the GM-CSF encoding sequence that the controlling element of expressing with GM-CSF wherein effectively is connected in described primary tumo(u)r cell.

25. method according to claim 24, wherein said GM-CSF encoding sequence is the human sequence.

26. method according to claim 24 further comprises in the tumour cell and the tumour cell introducing Mammals with transduction of deactivation transduction.

27. method according to claim 26, wherein said tumour cell to described Mammals from body.

28. method according to claim 26, wherein said tumour cell is allogenic to described Mammals.

29. method according to claim 26, wherein said Mammals comprise the tumour cell with the tumour cell same type of transduceing.

30. method according to claim 29, wherein said Mammals are human.

Images