AU763733B2 - Recombinant adenovirus - Google Patents

Description

WO 01/02431 PCT/SE00/01390 1 RECOMBINANT ADENOVIRUS Field of the invention The present invention relates to new recombinant adenovirus with changed tropism. More particularly the recombinant adenovirus has been constructed by removing the native knob structure and replacing it with a new cell binding ligand and an external trimerisation motif.

The invention also relates to the new adenovirus for treatment of human diseases. Also included is a method for rescuing of recombinant adenovirus fibers into the adenovirus genome.

Background of the invention.

Clinical gene therapy was introduced in 1989. The aim at that time was to correct a gene defect in the immune system through the in vitro introduction of a healthy gene into the defect cells of the patient and transfusion of the treated cells back to the patient. Since that time, the possible indications for gene therapy have increased dramatically. Today, ten years after its introduction, the use of gene therapy to treat e.g.

diseases of the blood vessels, cancer, inflammatory diseases and infectious diseases such as HIV can be envisaged.

At present, however, gene therapy is not a useful method in human medicine. One main reason is that gene therapy demands the packaging of the genes to be delivered into gene-carriers, or vectors, which can be injected into WO 01/02431 PCT/SE00/01390 2 patients and which will target the genes only to the intended cells. Such vectors have so far not been available.

Adenoviruses (Ad) are DNA viruses without an envelope, shaped as regular icosahedrons with a diameter of 60-85 nm. Cell-binding takes place through fiber proteins, anchored to the virion at the corners of the icosahedron.

The fiber protein is not necessary for assembly and release of intact virions. Assembly of virions take place in the nucleus of infected cells.

The fiber protein, which is a homotrimer of a fiber polypeptide, contains three functionally different parts: an N-terminal tail anchoring the fiber non-covalently to the penton base in the virion and which furthermore contains the nuclear-localization signal; an approximate amino acid fiber shaft motif which is repeated six times in Ad3 and 22 times in Ad2 and Ad5 (Chrobozek J, Ruigrok RWH and Cusack S: Adenovirus Fiber, Current Topics in Microbiology and Immunology, 1995, p 163-200); and a C-terminal globular domain, the knob, which contains the ligand which binds to the cellular Adreceptor (See review in in the previous ref.). Each shaft repeat has two three-amino acid regions which form Psheets and two amino acid regions which constitute the turns of the native extended fiber shaft. The crystal structure of the trimerised, cell-binding domain has been determined and shows a unique topology different from other anti-parallel p-sandwiches (Di Xia, Henry LJ, Gerard RD and Deisenhofer J: Crystal structure of the receptor-binding domain of adenovirus type 5 fiber protein at 1.7 A resolution, Structure 2: 1259-1270, WO 01/02431 PCT/SE00/01390 3 1994.). Binding of the fiber to the penton base of the virion can take place also in a cell-free system, i.e.

the fiber can bind to fiberless virions (Boudin M-L and Boulanger P: Assembly of Adenovirus Penton Base and Fiber, Virology, 116: 589-604, 1982).

It seems possible that the fiber can tolerate structural modifications as long as the ability to bind to the penton base and to be transported to the nucleus is retained. Some attempts at modifying the Ad fiber in order to change the binding properties of the virus have been made. A short peptide ligand has been added Cterminally of the knob (Michael SI, Hoy JS, Curie DT and Engles JT: Addition of a short peptide ligand to the adenvorirus fiber protein. Gene Therapy 2: 660-8, 1995.) and an octapeptide has been introduced into one of the knob "loops". By introducing the FLAG tetra-amino acid motif into the Ad penton, it has been shown possible to target Ad to cells normally not infected by Ad. This was done by targeting with bi-specific antibodies where one specificity was directed against the FLAG peptide and the other against the new target cell (Wickham TJ, Segal DM, Roelvink PW, Carrion ME, Lizonova A, Lee GM and Kovesdi I: Targeted Adenovirus Gene Transfer to Endothelial and Smooth Muscle Cells by Using Bispecific Antibodies. J.

Virol., 70: 6831-6838, 1996.). It would therefore seem possible to target Ad to a broad range of human cells which would be very useful for the purpose of human gene therapy. For these reasons and for the reason that Ad have been used extensively for gene therapeutic applications (Trapnell BC and Gorziglia: Gene therapy using adenoviral vectors, Current Opinion in Biotechnology, 5: 617-625, 1994.) a method has now been WO 01/02431 PCT/SE00/01390 4 developed to create recombinant re-targeted Ad-virus which can be useful for human gene therapy.

Accordingly it is an object of the present invention to provide a recombinant adenovirus with changed tropism.

Another object of the invention is the recombinant adenovirus for treatment of human diseases.

A further object of the invention is a method for rescuing of recombinant adenovirus fibers into the adnovirus genome.

Summary of the invention The objects of the invention are obtained by the recombinant adenovirus and the method for rescuing the virus fibers as claimed in the claims.

According to the invention there is provided a recombinant adenovirus with changed tropism. The adenovirus is characterized in that the native pentone fibre, which comprises a fibre tail, a fibre shaft and a fibre knob including a trimerisation motif, has been changed in that the native knob containing the cell binding structure and the native trimerisation motif has been removed and a new cellbinding ligand and an external trimerisation motif have been introduced into the virus fiber.

The structural modification has been performed by DNA technology at the gene level or by chemical or immunological means at the virus level.

According to another aspect of the invention adenovirus, as identified above, is used for the treatment of human diseases, either in vivo or by in vitro methods.

WO 01/02431 PCT/SE00/01390 A further aspect of the invention is a method for rescuing of recombinant adenovirus fibers into the adenovirus genome comprising the following steps: a) subcloning of a 9kb fragment (from Spel to end of genome), b) further subcloning of a 3kb fragment between Sad and Kpnl, c) deletion of the fibergene between Ndel and Munl and replacing the missing sequence with SEQ ID NO: 13 in the Sequence listing containing an Xhol site; d) ligation of recombinant fiber between Ndel and Xhol of construct under c) above; e) re-introduction of construct under d) above into the 9 kb fragment cut with Nhel using homologous recombination in E. coli; f) isolation of the recombinant 9 kb fragment under e) and re-creation of the adenovirus genome by joining 9 kb fragment to the 27 kb fragment from the beginning of the genome to the Spel site by Cosmid cloning.

Detailed description of the invention Figure legends Fig. 1: Summary of modifications to native fiber carried out in the invention.

Fig. 2: Recombinant adenovirus fibers.

Fig. 3: Method for rescuing of recombinant fiber genes into the Ad genome.

Fig. 4a: Recombinant fibers rescued into Ad genomes which are capable of giving CPE/plaques on transfected cells and in secondary cultures.

WO 01102431 PCT/SE00/01390 6 Fig. 4b: Recombinant fibers rescued into Ad genomes which are capable of giving CPE/plaques on transfected cells and in secondary cultures.

In the present invention re-targeting of Ad is achieved through the introduction of a new cell-binding ligand into the fiber (Fig. Any cell binding peptide can be used, e.g. a monoclonal antibody or a fragment thereof whether as a single chain fragment or Fab, a T cell receptor or a fragment thereof, an integrin binding peptide such as RGD or a growth factor such as Epidermal Growth Factor.

Ligands which so far have been applied include Epidermal Growth Factor (EGF), the amino acid motif RGD, a single chain fragment of a cloned T-cell receptor (scTCR) reactive with MAGE-1 peptides associated with HLA-A1 (vd Bruggen P, Traversaari C, Chomez P, Lurquin D, De Plaen E, vd Eynde B, Knuth A and Boon T: A Gene encoding an Antigen Recognized by Cytolytic T Lymphocytes on a Human Melanoma, Science 13 December 1991, 1643-1647.), a single chain fragment (scFv) of the monoclonal antibody G250, which with high selectivity has been shown to react with a protein antigen on human renal carcinoma cells (Oosterwijk E, Ruiter DJ, Hoedemaeker PhJ, et al: Monoclonal antibody G250 recognizes a determinant present in renal-cell carcinoma and absent from normal kidney.

Int J Cancer 38: 489-94, 1986.). G250 has been extensively evaluated and has been applied in clinical trials (see the previous ref.).

Ad vectors can be made replication competent or incompetent for permissive cells. For tumor therapy, replication competent Ad has the potential advantage that it can replicate and spread within the tumor (Miller R and Curiel DT: Towards the use of replicative adenoviral WO 01/02431 PCT/SE00/01390 7 vectors for cancer gene therapy, Gene Therapy 3: 557- 559). This may theoretically result in an increase of the chosen effector mechanism over that obtainable with replication incompetent vectors. Furthermore, infectious virus may contribute to an anti tumor effect by cytopathogenic effects in infected cells as well as by evoking an anti viral immune response which may harm infected cells.

Construction, expression and evaluation of recombinant fibers The aim has been to develop a universal method for the construction of functional Ad fibers with changed binding-specificity to make possible the construction of re-targeted Ad.

The adenovirus fiber peptide carries several biological functions which are necessary to retain in order to produce active virus particles. The following fiber features are deemed to be of key importance in the construction of functional recombinant fiber peptides: The ability to form parallel homotrimers. This function is carried by the N-terminal amino acid sequence of the wild type fiber knob and is necessary for the fiber to be able to bind to penton base and to form the functional cell binding knob.

The ability to bind to penton base to form penton capsomeres. This function is carried by the wild type fiber tail.

The ability to express a cell-binding ligand allowing for attachment to target cells. This function is carried by the wild type fiber knob.

WO 01/02431 PCT/SE00/01390 8 Since adenovirus is assembled in the nucleus of infected cells, the ability to be transported into the nucleus of infected cells is vital to virus formation.

The nuclear localization signal is mainly, but perhaps not exclusively, carried by the wild type fiber tail.

In the first stage recombinant fibers are constructed and evaluated in vitro after cell-free expression in a coupled transcription/translation system. Analysis by SDS-PAGE and autoradiography is performed to reveal the presence of an open reading frame and give information on the size of the translated product. In the next stage recombinant fibers are cloned into Baculovirus and expressed in insect cells allowing for functional studies of the fibers. Such studies include ability to form trimers as evaluated by immunostaining with monoclonal antibody 2A6.36 which has been shown to react only with trimerised fibers (Shin Hong J and Engler JA: The amino terminus of the adenovirus fiber protein encodes the nuclear localization signal, Virology 185: 758-767, 1991), expression of functional ligand as evidenced by ability to bind to cells expressing the corresponding receptor and ability to bind to penton-base either in solution or on virions.

Recombinant fibers are constructed using,methodology based on PCR (Clackson T, Gqssow D and Jones PT: General application of PCR to gene cloning and manipulation, in PCR, A Practical Approach, Eds McPherson MJ, Quirke P and Taylor GR, IRL Press, Oxford, p 187, 1992), e.g. PCRligation-PCR (Alvaro Ali S, Steinkasserer A: PCRligation-PCR Mutagenesis: A Protocol for Creating Gene Fusions and Mutations, BioTechniques 18: 746-750, 1995) WO 01/02431 PCT/SE00/01390 9 and splicing by overlap extension (SOE) (Horton RM and Pease LR: Recombination and mutagenesis of DNA sequences using PCR, in McPherson MJ Directed Mutagenesis, IRL Press 1991, p 217.). Cloning is performed according to standard methods. Recombinant fibers are sequenced using Perkin Elmer ABI Prism and are expressed in mammalian cells and in insect cells and stained with monoclonal antibodies specific for fiber tail, trimeric fiber and the new cell-binding ligand. The following parameters are evaluated after immunostaining: trimerisation nuclear transportation expression of the new cell-binding ligand.

Finally, recombinant fibers are rescued into the Ad genome by a newly invented procedure described below and recombinant virus particles are produced.

The invention will be further illustrated with the following non-limiting examples: Example 1: Fiber peptides are made where the knob is replaced with an external trimerisation motif which is introduced after the TLWT motif which ends the shaft portion of the fiber.

The purpose behind the introduction of an external trimerisation motif is two-fold: a) to remove the knob containing the native trimerisation signal but also the cell-binding part of the fiber, and b) simultaneously to supply the necessary trimerisation signal. Two different amino acid motifs have been used, i.e. the 36 amino acid "Neck Region Peptide" NRP (SEQ ID NO: 1 in Sequence WO 01/02431 PCT/SE00/01390 listing) from human "Lung Surfactant Protein D" Hoppe H-J, Barlow PN and Reid KBM: A parallel three stranded helicalbundle at the nucleation site of collagen triplehelix formation. FEBS Letters 344: 191-195 (1994).) and a 31 aa "Zipper" motif where the leucine residues on positions 1 and 4 have been replaced with isoleucine residues pII (SEQ ID NO: 2 in Sequence listing) (Harbury PB, Tao Zhang, Kim PS and Alber T: A Switch Between Two-, Three-, and Four-Stranded Coiled Coils in GCN4 Leucine Zipper Mutants. Science 262: 1401-1407, 1993.).

The DNA sequences for these trimerisation motifs are synthesized, cloned and sequenced in the project.

To replace the cellbinding function of the knob a new cellbinding ligand is introduced into the fiber in addition to the external trimerisation amino acid motif.

To augment the efficiency of nuclear transportation of recombinant fibers an external nuclear localisation sequence is added to the fiber in some cases.

In further embodiments the fiber in addition contains sequences which increase the survival of the fiber in the cytosol of infected cells, thereby enhancing transportation into the nucleus and virus assembly. Such sequences are e.g. sequences that are present in the wild type knob or in SEQ ID NO: 10 12.

The following types of fibers are constructed using the methods mentioned above (see Fig The sequence of the WO 01/02431 PCTSE00/01390 11 wild type fiber is shown in the sequence listing as SEQ ID NO 14.

Type A where the trimerisation motif is fused to the fiber gene downstream of the fiber shaft after the TLWT motif which constitutes the four first amino acids of the fiber knob or downstream of the second turn (Turn b) of any shaft repeat, the remaining shaft repeats having been removed.

The new cellbinding ligand is introduced downstream of the trimerisation signal with an amino acid linker motif being added between the trimerisation signal and the cellbinding ligand.

Type B similar to type A but with a linker motif introduced immediately upstream of the trimerisation signal.

Type C where the trimerisation motif is introduced after the first shaft repeat and in turn followed the shaft repeats 17 through 21. The new cellbinding ligand is introduced downstream of the trimerisation signal with an amino acid linker motif being added between the trimerisation signal and the cellbinding ligand.

Type D where the cellbinding ligand is introduced between the restriction sites Nhel and Hpal in the fiber shaft, with an amino acid linker being added both upstream and downstream of the ligand.

WO 01/02431 PCT/SE00/01390 12 Type D/A This is a variant of Type D where the fiber shaft downstream of the cellbinding ligand in Type D was removed. Type D and are constructed with the normal knob and with the knob being replaced with an external trimerisation signal as in Types A and B.

Type E which are similar to Type A but with part of the knob being retained immediately upstream of the external trimerisation motif.

The following amino acid motifs are used as linkers in the above fiber constructs: SEQ ID NO: 3, derived SEQ ID NO: 4, derived SEQ ID NO: 5, derived immunoglobulin SEQ ID NO: 6, derived SEQ ID NO: 7, derived IgG3 SEQ ID NO: 8, derived from from from from from Psedomonas exotoxin tissue prothrombin activator the hinge region of mouse Staphylococcal protein A the hinge region of human from shaft repeat no 17 of human Recombinant fibers are cloned into Baculovirus and expressed in Sf9 cells and/or cloned into the vector pSecTag and expressed in COS cells as secreted proteins.

Expression is monitored by immunostaining with monoclonal antibodies 4D2.5 (anti Ad5 fiber) and 2A6.36 (anti trimerised Ad5 fiber). Expression and trimerisation is WO 01/02431 PCT/SEOO/01390 13 obvious in all recombinant fibers irrespective of length and trimerisation motif.

The various fibers which have been constructed and shown to be able to form trimers and express the new cell binding ligand are shown in Table 1. The results show that the invented technology is capable of generating trimerising fibers which express a new celibinding ligand. It should therefore be possible to make functional virus with such fibers.

Table I. Results from immunostaining of different recombinant fibers Detecting antibody Fiber 4D2 2A6 a-EGF a-Ig a-Id Type A Al RGD Al EGF Al G250 HK Al G250 KH Al G250 KHJCH2 Al VcLLVPCO A7 RGD A7 EGF A7 G250 HK A7 G250 KH A7 G250 KHJCH2 A7 VctLVP3Cf A7 IgG3 EGF A7 (Gly4Ser)4 G25OVKVH A22 EGF A22 RGD Type B B (Gly4Ser)4 RGD Type C C IgG3 EGF WO 01/02431 PCT/SE00/01390 14 C (Gly4Ser)4- G250VKVH Type D N/D EGF N/D G250 HKCKy F2/D EGF F3/D EGF Type D/A F2 D/A G250 HKCK F2 D/A G250 HKCKy F2 D/A EGF F3 D/A EGF Abbreviations used in Table 1: 2A6: antibody against trimerized fiber 4D2: antibody against fiber a-EGF: antibody against epidermal growth factor a-Id: anti idiotypic antibody specific for G250 a-Ig: antibody against mouse immunoglobulin Cp: Constant domain from p chain of T cell receptor against MAGE1/HLA Al. SEQ ID NO: 11.

CH2: immunoglobulin heavy chain constant domain 2 EGF: epidermal growth factor G250: monoclonal antibody specific for renal carcinoma H: heavy chain variable sequence from G250 (SEQ ID NO: IgG3:amino acid linker derived from hinge region of human IgG3, SEQ ID NO: 7 J: immunoglobulin joining chain sequence K: light chain variable sequence from monclonal antibody G250 (SEQ ID NO: 16) RGD: The amino acid sequence arginine-glycine-aspartic acid Va: Variable domain from a chain of T cell receptor against MAGE1/HLA Al. SEQ ID NO: Vp: Variable domain from P chain of T cell receptor against MAGE1/HLA Al. SEQ ID NO: 12 WO 01/02431 PCT/SE00/01390 Example 2: Nuclear localization of recombinant fibers (Tables 2 and 3) Nuclear localization is assessed by immunostaining of fibers in Sf9 cells 24 hours after infection with the relevant Baculovirus clone. Some results are shown in Table 2 below. It is clear from these experiments that some recombinant fibers show a grossly impaired nuclear localization in Sf9 cells despite the presence of the nuclear addressing signal in the fiber tail.

Table 2 Nuclear localization of native and selected recombinant fibers in Sf9 cells Fiber of fiber-expressing Sf9 cells showing nuclear localization after infection Wild type 100 N/D EGF 100 A RGD App. A7 RGD App. 100 A7 EGF App. 100 A7 scTCR App. A7 G250 scFvs 0 Recombinant and native fibers have also been expressed in COS cells, targeted for expression in the cytosol after cloning into the vector pcDNA 3.1. In this case it was expected that the fibers would be detected in the nucleus, due to the presence of the native nuclear localization signal in the fiber tail. However, nuclear localization has so far only been detected in the wild type fiber and in fibers with single-chain T-cell WO 01/02431 PCT/SE00/01390 16 receptors, i.e. the fibers which have produced the most efficient virus (se below).

Since nuclear localization of fibers are crucial to virus assembly, an attempt is made to improve the efficiency of nuclear addressing by adding an external nuclear localization signal (NLS), in this case the SV40 large Tantigen NLS having the amino acid sequence SEQ ID NO: 9 (Fisher-Fantuzzi L and Vesco C: Cell-Dependent Efficiency of Reiterated Nuclear Signals in a Mutant Simian Virus Oncoprotein Targeted to the Nucleus. Mol Cell Biol, 8:5495-5503, 1988). The external NLS sequence is added immediately up-stream of the RGD motif. It is found that the presence of the external NLS dramatically improved the nuclear localization in the cases where it has been investigated. In fact, as mentioned above the fiber constructs lacking the external NLS were undetectable in the transfected cells (Table 3).

Table 3 Nuclear localization of native and selected recombinant fibers in COS cells after targeting for expression in the cytosol Fiber Nuclear localization Wild type A VaLVPCP A VaLVCPCk A RGD A NLS RGD A7 RGD A7 NLS RGD A22 RGD For abbreviations, se Table 1 WO 01/02431 PCT/SE00/01390 17 The evidence given above support the hypothesis that recombinant fibers are poorly transported into the nucleus despite the presence of the intact tail region (see also below) and that this may possibly be corrected by the incorporation of an external NLS in the fiber construct.

Example 3: METHOD FOR RESCUING OF RECOMBINANT FIBERS INTO VIRIONS The wild type fiber in the Ad genome is exchanged for recombinant fibers by the following method (see Fig 3).

The plasmid pTG3602 (Chartier C, Degryse E, Gantzer M, Dieterle A, Pavirani A and Mehtali M: Efficient generation of Recombinant Adenovirus Vectors by Homologous Recombination i Escherichia Coli, J Virol, 4805-4810, 1996) containing the entire Ad5 genome as a Pacl-Pacl fragment is used as starting material. The approximate 9kb fragment of the genome between Spel and Pad and containing the wild type fiber gene is cloned separately in pBluescript. From this fragment an approximate 3kb fragment between Sacl.and Kpnl is further subcloned. A deletion of the native fiber gene with the exception of the N-terminal nucleotides upstream of the Ndel site of the fiber, between the Ndel site and the Muni site, which begins at base 38 after the stop codon of the fiber, is created in the 3kb fragment. The deleted sequence is replaced with SEQ ID NO: 13 which restores the Ndel and Munl sites and the wild type genome sequence between the fiber stop codon and the Munl site. In addition the added sequence, SEQ ID NO: 13, contains an Xhol site allowing for ligation of recombinant fibers WO 01/02431 PCT/SE00/01390 18 into the fiber-deleted 3kb fragment (the 3 kb fiber shuttle) between Ndel and Xhol.

The 3 kb fiber shuttle with recombinant fiber is reintroduced into the 9 kb fragment cut with Nhel using homologous recombination in E.coli (see ref. in previous passage). The resulting recombinant 9 kb fragment is finally excised from the vector with Spel and Pad and joined to the isolated 27 kb fragment by Cosmid cloning.

The presence of an insert of the expected properties is verified in all cosmid clones by PCR. Cosmid clones are also restricted with Hind III and the presence of restriction fragments of the expected size verified on gels.

Recombinant Ad genomes are isolated after restriction with Pac 1 and used to transfect suitable cells. The occurrence of plaques is determined by microscopic inspection of the transfected cell cultures.

Supernatants are harvested from primarily transfected cultures and used to infect secondary cultures. The occurrence of cytopathogenic effects and plaques are monitored by microscopy.

The particular fiber constructs that have been successfully rescued into virus are shown in figure 4a and 4b.

WO 01/02431 PCT/SE00/01390 19 Conclusion: For gene therapy to be useful for treatment of human diseases there is a need for injectable vectors with ability to target specific cells or a specific tissue (Miller N and Vile R: Targeted vectors for gene therapy.

FASEB J, 9: 190-199, 1995).

The present invention describes methods whereby knobless, trimerisation-competent fibers with new cellbinding ligands can been created and rescued into virus and have identified locations within the fiber-shaft which tolerates inserts of foreign ligands. The importance of intracellular trafficking of recombinant fibers has also been identified. Recombinant virus made using the invented technology should be highly useful in human medicine. Virtually unlimited opportunities for targeted gene-therapy may be developed by the combination of the technology described here and the identification of cellbinding ligands by phage-display.

So far trimerisation-competent fibers with a human scTCR have been and rescued into functional virus. Since single chain antibodies are large and highly complex peptides it seems highly likely that also other scAbs and cellbinding ligands, e.g. peptides identified from peptide libraries by means of phage-display, could be incorporated into Ad-fibers and rescued into virus using the same technology.

There are many ways in which Ad, made re-targeted by the present invention, may be applied to human gene therapy.

WO 01/02431 PCT/SE00/01390 In the case of tumor diseases, the following options exist: I. Use of vectors to introduce transgenes into tumors, such as anti onco genes "suicide" genes genes for immune modulatory substances or tumor antigens genes for anti angiogenetic factors II. Use of infectious virus. This has the added value over the use of non replicating vectors that virus can spread from cell to cell within a tumor, thereby multiplying the initial hit on the tumor..Tumor cell destruction may occur not only by the cell-destroying mechanism engineered into the vector but also by the cell destruction which is associated with the virus infection per se and by the attack of the body's immune response on the virus infected cells. This principle has already been tested in man through the direct intra-tumoral injection of an adenovirus which has been made gene manipulated to replicate only in p53 mutant tumor cells. The experience from these limited trials on large "head-and-neck" tumors are partially encouraging with a complete regress of 2/11 treated tumors which are otherwise resistant to any form of known treatment.

P:\OPERUEHU\491117 spa.doc-5 June. 2(X13 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

*oo* EDITORIAL NOTE APPLICATION NUMBER 60401/01 The following Sequence Listing pages 1 to 12 are part of the description. The claims pages follow on pages 21 to 23.

WO 01/02431 PCT/SE00/01390 Sequence listing <110> Got-A-Gene AB <120> Recombinant adenovirus <130> 2001575 <160> 16 <170> MS Word 97 <210> 1 <211> 36 <212> PRT <213> Homo sapiens <301> Hoppe HJ, Barlow PN, Reid KBM <302> A parallel three stranded a-helical bundle at the nucleation site of collagen triple-helix formation <303> FEBS Letters <304> 344 <306> 191-195 <307> 1994 <400> 1 Pro Asp Val Ala Ser Leu Arg Gin Gin Val Glu Asp Leu Gin Gly 1 5 10 Gin Val Gin His Ley Gin Ala Ala Phe Ser Gin Tyr Lys Lys Val 25 Glu Leu Phe Pro Asn Gly WO 01/02431 WO 0102431PCT/SEOO/01390 2 <210O> 2 <21 1> 31 <212> PRT <213> Homo sapiens <301> Harbury PB, Zhang T, Kim PS, Albert T <302> A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants <303> Science <304> 262 <306> 1401-1407 <307> 1993-11-26 <400> 2 Met Lys Gin Ile Gly Asp Lys Ile Gin Glu Ile Leu Ser Lys le Tyr His 1 5 10 le Glu Asn Gly Ile Ala Arg Ile Lys Lys Leu le Gly Glu 25 <210> 3 <21 1> 6 <212> PRT <213> Pseudomonas aeruginosa <301> Brinkmann U, Buchner J, Pastan 1 <302> Independent domain foiding of Pseudomonas exotoxin and single chain immunotoxins: Influence of interdomain connections <303> Proc Nat] Acad Sci US <304> 89 WO 01102431 WO 0102431PCTSEOOO139O 3 <306> 3075-3079 <307> 1992 <400> 3 Ala Ser Gly Gly Pro Glu 1 <210O> 4 <21 1> 7 <212> PRT <213> Homo sapiens 1> Brinkmnann U, Buchner 3, Pastan 1 <302> Independent domain folding of Pseudon-onas exotoxin and single chain immunotoxins: Influence of interdomain connections <303> Proc Nati Acad Sci US <304> 89 <306> 3075-3079 <307> 1992 <400> Ala Ser Glu Gly Asn Ser Asp 1 <210> <21 1> 8 <212> PRT <213> Mus musculus WO 01102431 WO 0102431PCT/SEOO/01390 4 <301> Brinkmann U, Buchner J, Pastan 1 <302> Independent domain folding of Pseudomonas exotoxin and single chain immunotoxins: Influence of interdomain connections <303> Proc Nat] Acad Sci US <304> 89 <306> 3075-3079 <307> 1992 <400> Ala Ser Thr Pro Glu Pro Asp Pro 1 <210O> 6 <21 1> 13 <212> PRT <213> Staphylococcus aureus <400> 6 Ala Lys Lys Leu Asn Asp Ala Gin Ala Pro Lys Ser Asp 1 5 <210> 7 <211> 11 <212> PRT <213> Homo sapiens WO 01/02431 PCT/SE00/01390 <301> Dangl JL, Wensel TG, Morrison SL, Streyer L, Herzenberg LA and Oi

T

<302> Segmental flexibility and complement fixation of genetically engineered chimeric human, rabbit and mouse antibodies <303> EMBO Journal <304> 7 <306> 1989 <307> 1988 <400> 7 Thr Pro Leu Gly Asp Thr Thr His Thr Ser Gly 1 5 <210> 8 <211> 11 <212> PRT <213> Adenovirus type <301> Stouten PFW, Sander C, Ruigrok WH, Cusack S <302> New triple-helical model for the shaft of the adenovirus fibre <303> Journal of molecular biology <304> 226 <306> 1073-1084 <307> 1992 <400> 8 Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu 1 5 WO 01/02431 PCT/SE00/01390 6 <210> 9 <211> 8 <212> PRT <213> Simian virus <301> Fisher-Fantuzzi L and Vesco C 8:5495-5503, 1988 <302> Cell-Dependent Efficiency of Reiterated Nuclear Signals in a Mutant Simian Virus 40 Oncoprotein Targeted to the Nucleus <303> Molecular Cell Biology <304> 8 <306> 5495-5503 <307> 1992 <400> 9 Asp Pro Lys Lys Lys Arg Lys Val 1 <210> <211> 119 <212> PRT <213> Homo sapiens <400> Gin Lys Val Thr Gin Ala Gin Thr Glu Ile Ser Val Val Glu Lys Glu 1 5 10 Asp Val Thr Leu Asp Cys Val Tyr Glu Thre Arg Asp Thr Thr Tyr 25 Tyr Leu Phe Trp Tyr Lys Gin Pro Pro Ser Gly Glu Leu Val Phe Leu Ile 40 WO 01/02431 PCT/SEOO/01390 7 Arg Arg Asn Scr Phe Asp Giu Gin Asn Glu Ile Ser Gly Arg Tyr Ser 55 60 Trp Asn Phe Gin Lys Scr Thr Scr Ser Phe Asn Phe Thr le Thr Ala 75 Ser Gin Val Val Asp Ser Ala Val Tyr Phe Cys Ala Lcu Gly Gly Val 90 Asn Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg 100 105 110 Leu Met Val Lys Pro 115 <210> 11 <211I> 133 <212> PRT <213> Homo sapiens <400> 11 Giu Asp Leu Asn Lys Val Phe Pro Pro Giu Val Ala Val Phe Giu 1 5 10 Pro 5cr Glu Ala Glu le Ser His Thr Gin Lys Ala Thre Leu Val Cys 25 Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Lys Ser Trp Trp 40 Val Asn Gly Lys Giu Val His Ser Giy Val Set Thr Asp Pro Gin Pro 55 Leu Lys Glu Gin Pro Ala Leu Asn Asp Ser Mrg Tyr Cys Leu Ser Ser 70 Mrg Lcu Mrg Val 5cr Ala Thr Phe Trp Gin Asn Pro Mrg Asn His Phe 85 Mrg Cys Gin Val Gin Phe Tyr Gly Leu Ser Glu Asn Asp Giu Trp Thr 100 105 110 Gin Asp Mrg Ala Lys Pro Val Thr Gin le Val 5cr Ala Glu Ala Trp Giy 115 120 125 Mrg Ala Asp Ala Ala Ala 130 <210> 12 <21 1> 114 WO 01/02431 PCT/SE00/01390 8 <212> PRT <213> Homo sapiens <400> 12 Asp Ser Gly Val Thr Gin Thr Pro Lys His Leu Ile Thr Ala Thr Gly 1 5 10 Gin Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val 25 Tyr Trp Tyr Gin Gin Ser Leu Asp Gin Gly Leu Gin Phe Leu Ile His 40 Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe 55 60 Ser Ala Gin Gin Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser 75 Leu Glu Leu Gly Asp Ser Ala Leu Val Phe Cys Ala Ser Asn lie Ala 90 Gly Gly Ser Tyr Thr Gin Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 100 105 110 Leu <210> 13 <211> 52 <212> DNA <213> Artificial sequence <223> Sequence replacing the fiber gene sequence which was deleted between the Ndel restriction site in the fiber tail and the Munl site which begins at base 38 after the stop codon in the fiber. The sequence restores the Ndel and Munl sites and the wild type genome sequence between the fiber stop codon and the Munl site. In addition the added sequence contains an Xhol site allowing for the ligation of recombinant fibers.

<400> 13 tatgcactcg agtaaagaat cgtttgtgtt atgtttcaac gtgtttatttt tc WO 01/02431 PCTSEOOO1390 9 <210> 14 <21 1> 1746 <212> DNA <213> Human adenovirus type <221> CDS <222> 1-1746 <223> 1- 129 Fiber tail 130-1200 Fiber shaft 1201-1746 Fiber knob <400> 14 atg aag cgc gca aga ccg tct gaa gat acc ttc aac ccc gtg tat cca. 48 Met Lys Mrg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro 1 5 10 tat gac acg gaa. acc ggt cct cca act gtg cct itt ctt act cct ccc 96 Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu. Thr Pro Pro 25 ttt gta tcc ccc aat ggg ttt caa. gag agt ccc cct ggg gta ctc tct 144 Phe Val Ser Pro Asn Gly Phe. Gin Glu Ser Pro Pro Gly Val Leu Ser 40 ttg cgc cta tcc gaa. cct cia gil acc tcc aat ggc atg ctt gcg ctc 192 Leu. Ag Leu. Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu, Ala Leu 55 aaa atg ggc aac ggc ctc ict ctg gac gag gcc ggc aac cit acc tcc 240 Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Lou Thr Scr 70 7S caa aat gta acc act gtg agc cca cct ctc aaa aaa ace aag tca aac 288 Gin Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn 90 ata aac cig gaa ala, Id gca ccc ctc aca gtt ace tea gaa gce cta 336 le Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu 100 105 110 act gtg got gcc gcc gca cct cta. atg gtc gcg ggc aac aca ctc acc 384 Thr Val Ala Ala Ala Ala Pro Leu. Met Val Ala Gly Asn Thr Leu Thr 115 120 125 atg caa, tca cag gcc ccg cia acc gig cac gac icc aaa ctt agc alt 432 Met Gin Ser Gin Ala Pro Leu Tbr Val His Asp Ser Lys Leu. Ser le 130 135 140 gcc acc caa gga. ccc ctc aca gig tca gaa, gga aag cta gcc ctg caa. 480 Ala Thr Gin Gly Pro Lou Thr Val Ser Glu Gly Lys Lou Ala Leu Gin 145 150 155 160 WO 01/02431 WO 0102431PCT/SEOOO 1390 aca tca ggc ccc ctc acc acc acc gat agc agt acc ctt act ate, act 528 Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr 165 170 175 gcc tea ccc cct cta act act gec act ggt agc ttg ggc att gac ttg 576 Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Scr Leu Gly Ile Asp Leu 180 185 190 aaa gag ccc att tat aca caa. aat gga aaa. cta gga cta aag tac ggg 624 Lys Glu Pro Ile Tyr Thr Gin Asn Gly Lys Leu Gly Leu Lys Tyr Gly 195 200 205 get cct ttg cat gta aca gac gac cta aac act ttg ae gta gca act 672 Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr 210 215 220 ggt cca ggt gtg act att aat aat act tee ttg eaa, act aaa gtt act 720 Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gin Thr Lys Vai Thr 225 230 235 240 gga gec tig ggt ttt gat tea caa gge aat atg caa. ctt aat gta gca 768 Gly Ala Leu Giy Phe Asp Ser Gin Giy Asn Met Gin Leu Asn Vai Ala 245 250 255 gga gga eta agg att gal tct caa. aac aga ege ctt ata ctt gal gtt 816 Gly Gly Leu Mrg Ie Asp Ser Gin Asn Arg Arg Leu le Leu Asp Vai 260 265 270 agt tat ceg ttt gal get caa aac caa cta aat eta aga eta gga cag 864 Ser Tyr Pro Phe Asp Ala Gin Asn Gin Leu Asn Leu Arg Leu Gly Gin 275 280 285 ggc cet ctt ttt ala aac tea gee cac aac ttg gat alt aac tac aac 912 Gly Pro Leu Phe Ile Asn Scr Ala His Asn Leu Asp Ile Asn Tyr Asn 290 295 300 aaa ggc cli tac ttg ttt aca get tea aac: aal tee aaa aag ctl gag 960 Lys Gly Leu Tyr Leu Phe Thr Ala Scr Asn Asn Ser Lys Lys Leu Glu 305 310 315 320 gtl aac eta age act gee aag ggg ttg atg lit gac gct aca gee ata 1008 Vai Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile 325 330 335 gee att aat gca gga gal ggg ctl gaa ttt ggt lea ccl aat gca eca 1056 Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro 340 345 350 aac aca aal ccc etc aaa aca aaa alt ggc cat ggc eta gaa Itt gat 1104 Asn Thr Asn Pro Leu Lys Thr Lys Ile Giy His Gly Leu Giu Phe Asp 355 360 365 tea aac aag get atg gtl eel aaa eta gga act ggc: ctt agt ttt gac 1152 Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp 370 375 380 age aca ggl gee alt aca. gta gga aac aaa aal aal gat aag eta act 1200 Ser Thr Gly Ala Ilie Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr 385 390 395 400 ttg tgg ace aca eca. gel eca tel ect aae lgt aga eta aat gca. gag 1248 Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Mrg Leu Asn Ala Giu 405 410 415 WO 01/02431 WO 0102431PCT/SE00101390 I1I aaa gat gct aaa ctc act ttg gtc tta aca aaa tgt ggc agt caa ata 1296 Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gin le 420 425 430 ctt gct aca gtt tca gtt ttg get git aaa ggc agt ttg gct cca ata 1344 Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro le 435 440 445 tct gga aca gtt caa, agt gct cat ctt att ata aga ttt gac gaa aat 1392 Ser Gly Thr Val Gin Ser Ala His Leu le le Arg Phe Asp Giu Asn 450 455 460 gga gtg cta cta aac aat t cc ttc ctg gac cca gaa tat tgg aac ttt 1440 Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp, Asn Phe 465 470 475 480 aga aat gga gat ctt act gaa. ggc aca gcc tat aca aac ggt gtt gga 1488 Arg Asn Gly Asp Leu Thr Giu Gly Thr Ala Tyr Thr Asn Gly Val Gly 485 490 495 itt atg cct aac cta tca gct tat cca aaa tct cac ggt aaa act gcc 1536 Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala 500 505 510 aaa agt aac all gtc agt caa gtt tac tia aac gga gac aaa act aaa 1584 Lys Ser Asn Ilie Val Ser Gin Vai Tyr Leu Asn Giy Asp Lys Thr Lys 515 520 525 cct gta, aca cta acc att aca cta aac ggt aca cag gaa, aca gga gac 1632 Pro Vai Thr Leu Thr le Tbr Leu Asn Giy Thr Gin Giu Thr Gly Asp 530 535 540 aca act cca agt gca tac tct atg tca. ttt tca tgg gac tgg tct ggc 1680 Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Giy 545 550 555 560 cac aac tac att aat gaa ata lit gcc aca tcc tct tac act ttt tca 1728 His Asn Tyr le Asn Glu le Phe Ala Thr Ser Ser Tyr Thr Phe Ser 565 570 575 tac att gcc caa gaa taa Tyr le Ala Gin Giu <210> <211> 120 <212> PRT <213> Mus musculus <400> Asp Val Lys Leu Vai Giu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly 1 5 10 WO 01/02431 WO 0102431PCT/SEOO/01390 12 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Scr Asn Tyr 25 Tyr Met Ser Trp Val Arg Gin Thr Pro Glu Lys Arg Leu Glu Leu Val 40 Ala Ala le Asn Ser Asp Gly Gly Ilie Thr Tyr Tyr Leu Asp Thr Val 55 Lys Gly Arg Phe Thr le Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 70 Leu Gin Met Ser Ser Leu Lys Ser Glu Asp Tin Ala Leu Phe Tyr Cys 85 90 Ala Arg His Arg Ser Gly Tyr Phe Ser Met Asp Tyr Trp Gly Gin Gly 100 105 110 Thr Ser Val Thr Val Ser Ser Gly Ser 115 <210> 16 <211> 116 <212> PRT <213> Mus musculus <400> 16 Asp le Val Met Tin Gin Ser Gin Arg Phe Met Ser Thr Tin Val Giy 1 5 10 Asp Mrg Val 5cr le Tin Cys Lys Ala Ser Gin Asn. Vai Val Ser Ala 25 Val Ala Trp Tyr Gin Gin Lys Pro Giy Gin Ser Pro Lys Leu Leu Ile 40 Tyr 5cr Ala Ser Asn Mrg Tyr Thr Gly Val Pro Asp Mrg Phe Tin Giy 55 Ser Giy 5cr Gly Thr Asp Phe Tin Leu Tr Ile Ser Asn Met Gin Ser 70 75 Glu Asp Leu Ala Asp Phe Phe Cys Gin Gin Tyr Scr Asn Tyr Pro Trp 90 Tin Phe Gly Gly Gly Tin Lys Leu Giu Ile Lys Mrg Ala Asp Ala Ala 100 105 110 Pro Tin Val Ser 115

Claims (20)

1. A recombinant adenovirus with changed tropism, wherein the native pentone fiber, comprising a fiber tail, a fiber shaft and a fiber knob including a tri- merisation motif, has been structurally modified in that the native knob containing the cellbinding structure and the native trimerisation motif has been removed and a new cell-binding ligand and an external trimerisation motif have been introduced into the virus fiber, c h a r a c t e r i z e d in that the new cell-binding ligand has been introduced into the fiber shaft.

2. An adenovirus according to claim 1, wherein the new cell binding ligand has been introduced downstream of the fiber shaft repeats.

3. An adenovirus according to claim 1, wherein the new cell binding ligand has been introduced between the restriction sites Nhel and Hpal in the fiber shaft.

4. An adenovirus according to claim 1, wherein said structural modification has been performed by DNA tech- nology at the gene level or by chemical or immunological means at the virus level. An adenovirus according to claim 1, which is either replication competent or replication incompetent.

6. An adenovirus according to claim 1, wherein amino acid linkers have been introduced upstream and downstream of the cell-binding ligand.

7. An adenovirus according to claim 1, wherein the shaft repeats downstream of the restriction site Hpal have been removed.

8. An adenovirus according to claim 1, wherein an amino acid linker motif has been added between the fiber shaft and the trimerisation motif and/or between the tri- merisation motif and the cellbinding ligand as a linker.

9. An adenovirus according to claim 8, wherein the amino acid linker motif is any of the following: SEQ ID NO: 3, derived from Pseudomonas exotoxin; SEQ ID NO: 4, AMENDED SHEET POT irternational ApiPici PCSE/1390 lPT ,ca J j PCT/SE00/01390 22 04-10-2001 derived from tissue prothrombin activator; SEQ ID NO: derived from the hinge region of mouse immunoglobulin; SEQ ID NO: 6, derived from Staphylococcal protein A; SEQ ID NO: 7, derived from the hinge region of human IgG3; SEQ ID NO: 8, derived from shaft repeat 17 of human An adenovirus according to any one of claims 1- 9, wherein the new cell-binding ligand is any cell- binding peptide.

11. An adenovirus according to claim 10, wherein the cellbinding ligand is a monoclonal antibody or a fragment thereof whether as a single chain fragment or Fab, a T cell receptor or a fragment thereof, an integrin binding peptide such as RGD or a growth factor such as Epidermal Growth Factor.

12. An-adenovirus according to claim 11, containing any of the sequences SEQ ID NO: 10-12. i

13. An adenovirus according to claim 11, wherein the single chain fragment is a single chain fragment of the monoclonal antibody G250 with heavy chain variable region with SEQ ID NO: 15 and light chain variable region with SEQ ID NO: 16.

14. An adenovirus according to claim 1, wherein the external trimerisation motif is an a-helical coiled coil motif, or any other peptide capable of rendering functio- nally trimerised fibers. An adenovirus according to claim 14, wherein the external trimerisation motif is the neck region peptide of human lung surfactant protein D, SEQ ID NO: 1 or a 13 amino acid "Zipper" motif where the leucine residues on positions 1 and 4 have been replaced with isoleucine re- sidues, SEQ ID NO: 2.

16. An adenovirus according to any one of the pro- ceeding claims, wherein an external nuclear localisation signal (NLS) has been introduced in the fiber.

17. An adenovirus according to claim 16, wherein the NLS is the SV40 large-T antigen NLS. AMENDED SHEET P:\OPERUEHU4 I1117 spa doc-5 Jlm.. 2(m3 -23-

18. An adenovirus according to any one of the proceeding claims, wherein the fiber in addition contains sequences which increase the survival of the fiber in the cytosol of the infected cells, thereby enhancing transportation into the nucleus and virus assembly.

19. An adenovirus according to claim 18, wherein the sequences are present in the wild type knob. An adenovirus according to claim 19, wherein the sequences are as presented in SEQ ID NO: 10-12.

21. An adenovirus according to any one of claims 1-20, for the treatment of human diseases, either in vivo or by in vitro methods.

22. The use of an adenovirus according to any one of claims 1-21 in the manufacture of a medicament for the treatment of human disease, either in vivo or by in vitro method.

23. A method of producing a recombinant adenovirus with changed tropism, 20 comprising: I. rescuing recombinant adenovirus fibers into the adenovirus genome by the following steps: a) subcloning of a 9 kb fragment (from Spel to the end of genome), b) further subcloning of a 3 kb fragment between Sad and Kpnl, c) deletion of the native fiber gene coding for the native penton fiber between Ndel and Munl and replacing the missing sequence with the sequence SEQ ID NO: 13 containing an Xhol site; d) ligation of recombinant fiber gene coding for between Ndel and Xhol of construct under c) above; e) re-introduction of construct under d) above into the 9 kb fragment cut with Nhel using homologous recombination in E. coli; P:\OPER\JEHU24') 117 spa do-5 June. 21X)3 -24- f) isolation of the recombinant 9 kb fragment under e) and re-creation of the adenovirus genome by joining 9 kb fragment to the 27 kb fragment from the beginning of the genome of the Spel site by Cosmid cloning; and II. transfecting a cell with the adenovirus obtained in step f) to enable said cell to express the recombinant adenovirus.

24. An adenovirus according to any one of claims 1 to 21, a use according to claim 22 or a method according to claim 23, substantially as herein disclosed with reference to the Figures and/or Examples. DATED this 5 th day of June, 2003 GOT-A-GENE AB by DAVIES COLLISON CAVE 15 Patent Attorneys for the Applicant(s)