CA2378324A1

CA2378324A1 - Recombinant adenovirus

Info

Publication number: CA2378324A1
Application number: CA002378324A
Authority: CA
Inventors: Leif Lindholm
Original assignee: Individual
Current assignee: Got-A-Gene AB
Priority date: 1999-07-06
Filing date: 2000-06-30
Publication date: 2001-01-11
Also published as: CN1359391A; AU763733B2; AU6040100A; KR20020092886A; JP2003531568A; WO2001002431A1; EP1196435A1

Abstract

Recombinant adenovirus with changed tropism. In the adenovirus the native pentone fibre, comprising 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 moti f have been introduced into the virus fiber. Further, the invention relates to the recombinant adenovirus for the treatment of human diseases, either in vi vo or by in vitro methods and also to a method for rescuing of recombinant adenovirus fibers into the adenovirus genome.

Description

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.
Backcxround 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 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 15 amino acid fiber shaft motif which is repeated six times in Ad3 and 22 times in Ad2 and Ad5 (Chrobozek J, Ruigrok RWH a'nd 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 Ad-receptor (See review in in the previous ref.). Each shaft repeat has two three-amino acid regions which form ~3-sheets 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 ~i-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, 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 C-terminally 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 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.

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 Sacl 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.

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. 1). 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 6250, 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 6250 recognizes a determinant present in renal-cell carcinoma and absent from normal kidney.
Int J Cancer 38: 489-94, 1986.). 6250 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 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.

~ 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, Gussow 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. PCR-ligation-PCR (Alvaro Ali S, Steinkasserer A: PCR-ligation-PCR Mutagenesis: A Protocol for Creating Gene Fusions and Mutations, BioTechniques 18: 746-750, 1995) and splicing by overlap extension (SOE) (Horton RM and Pease LR: Recombination and mutagenesis of DNA sequences using PCR, in McPherson MJ (ed), 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 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 triple-helix formation. FEBS Letters 344: 191-195 (1994).) and a 5 31 as "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 10 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 2). The sequence of the 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.

Type D/0 This is a variant of Type D where the fiber shaft downstream of the cellbinding ligand in Type D was removed. Type D and (D/~) 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 from Psedomonas exotoxin ~ SEQ ID NO: 4, derived from tissue prothrombin activator ~ SEQ ID NO: 5, 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 no 17 of human Ad5 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 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 cellbinding 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

A1 RGD + +

Al EGF + + +

Al 6250 HK + + + +

A1 6250 KH + + + +

A1 6250 KHJCH2 + + + +

A1 VaLV(3C~3 + +

A7 RGD + +

A7 EGF + + +

A7 6250 HK + + + +

A7 6250 KH . + + + +

A7 6250 KHJCH2 + + + +

3 A7 VaLV(3C(3 + +

A7 IgG3 EGF + + +

A7 (Gly4Ser)4 G250VKVH + + + +

A22 EGF + + +

A2 2 RGD + +

Type B

B (Gly4Ser) 4 RGD + +

Type C

C IgG3 EGF + + +

C (Gly4Ser)4-G250VKVH + + + +
Type D
N/D EGF + + +
N/D G250 HKCKy + + + +
F2/D EGF + + +
F3/D EGF + + +
Type D/0 F2 D/0 6250 HKCK + + +
F2 D/0 6250 HKCKy + + + +
F2 D/0 EGF + + +
F3 D/0 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 6250 a-Ig: antibody against mouse immunoglobulin C(3: Constant domain from (3 chain of T cell receptor against MAGE1/HLA Al. SEQ ID NO: 11.
CH2: immunoglobulin heavy chain constant domain 2 EGF: epidermal growth factor 6250: monoclonal antibody specific for renal carcinoma H: heavy chain variable sequence from 6250 (SEQ ID NO:

15) 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 6250 (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 A1. SEQ ID NO: 10 i : Variable domain from ~3 chain of T cell receptor against MAGE1/HLA A1. SEQ ID NO: 12 Example 2:
Nuclear localization of recombinant fibers (Tables 2 and 3) 5 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 10 localization in Sf9 cells despite the presence of the nuclear addressing signal in the fiber tail.
Table 2 15 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 A RGD App. 50 A7 RGD App. 100 A7 EGF App. 100 A7 scTCR App. 50 A7 6250 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 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 T-antigen 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 40 Oncoprotein Targeted to the Nucleus. Mo1 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 +
3 0 A VaLV(3C~3 +
A VaLV~iC~iCk +
A RGD -A NLS RGD +

A7 NLS RGD +

For abbreviations, se Table 1 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, ~T Virol, 70:
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 Pacl 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 Munl 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 into the fiber-deleted 3kb fragment (the 3 kb fiber shuttle) between Ndel and Xhol.
The 3 kb fiber shuttle with recombinant fiber is re-introduced 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 Pacl 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.

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 cell-binding 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 cell-binding 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.

In the case of tumor diseases, the following options exist:
I. Use of vectors to introduce transgenes into tumors, 5 such as ~ anti onco genes -~ "suicide" genes ~ genes for immune modulatory substances or tumor antigens 10 ~ 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 15 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 20 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.

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 Gln Gln Val Glu Asp Leu Gln Gly Gln Val Gln His Ley Gln Ala Ala Phe Ser Gln Tyr Lys Lys Val Glu Leu Phe Pro Asn Gly <210> 2 <211> 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 Gln Ile Gly Asp Lys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Gly Ile Ala Arg Ile Lys Lys Leu Ile Gly Glu <210> 3 <211> 6 <212> PRT

<213> Pseudomonas aeruginosa <301> Brinkmann U, Buchner J, Pastan I

<302> Independent domain folding of Pseudomonas exotoxin and single chain immunotoxins: Influence of interdomain connections <303> Proc Natl Acad Sci US

<304> 89 <306> 3075-3079 <307> 1992 <400> 3 Ala Ser Gly Gly Pro Glu <210> 4 <211> 7 <212> PRT

<213> Homo Sapiens <301> Brinkmann U, Buchner J, Pastan I

<302> Independent domain folding of Pseudomonas exotoxin and single chain immunotoxins: Influence of interdomain connections <303> Proc Natl Acad Sci US

<304> 89 <306> 3075-3079 <307> 1992 <400> Ala Ser Glu Gly Asn Ser Asp <210> 5 <211> 8 <212> PRT

<213> Mus musculus <301> Brinkmann U, Buchner J, Pastan I

<302> Independent domain folding of Pseudomonas exotoxin and single chain immunotoxins: Influence of interdomain connections <303> Proc Natl Acad Sci US

<304> 89 <306> 3075-3079 <307> 1992 <400> 5 Ala Ser Thr Pro Glu Pro Asp Pro <210> 6 <211> 13 <212> PRT
<213> Staphylococcus aureus <400> 6 Ala Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys Ser Asp <210> 7 <211> 11 <212> PRT

<213> Homo Sapiens <301> Dangl JL, Wensel TG, Mornson 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 <210> 8 <211> 11 <212> PRT

<213> Adenovirus type 5 <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 <210> 9 <211> 8 <212> PRT

<213> Simian virus 40 <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 <210> 10 <211> 119 <212> PRT
<213> Homo Sapiens <400> 10 Gln Lys Val Thr Gln Ala Gln Thr Glu Ile Ser Val Val Glu Lys Glu Asp Val Thr Leu Asp Cys Val Tyr Glu Thre Arg Asp Thr Thr Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Gly Glu Leu Val Phe Leu Ile Arg Arg Asn Ser Phe Asp Glu Gln Asn Glu Ile Ser Gly Arg Tyr Ser Trp Asn Phe Gln Lys Ser Thr Ser Ser Phe Asn Phe Thr Ile Thr Ala Ser Gln Val Val Asp Ser Ala Val Tyr Phe Cys Ala Leu Gly Gly Val Asn Asn Asn Ala Gly Asn Met Leu Thr Phe Gly Gly Gly Thr Arg Leu Met Val Lys Pro <210> 11 <211> 133 <212> PRT
<213> Homo sapiens <400> 11 Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thre Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Lys Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Set Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Ala Ala Ala <210> 12 <211> 114 <212> PRT
<213> Homo sapiens <400> 12 Asp Ser Gly Val Thr Gln Thr Pro Lys His Leu Ile Thr Ala Thr Gly Gln Arg Val Thr Leu Arg Cys Ser Pro Arg Ser Gly Asp Leu Ser Val Tyr Trp Tyr Gln Gln Ser Leu Asp Gln Gly Leu Gln Phe Leu Ile His Tyr Tyr Asn Gly Glu Glu Arg Ala Lys Gly Asn Ile Leu Glu Arg Phe Ser Ala Gln Gln Phe Pro Asp Leu His Ser Glu Leu Asn Leu Ser Ser Leu Glu Leu Gly Asp Ser Ala Leu Val Phe Cys Ala Ser Asn Ile Ala Gly Gly Ser Tyr Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 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 <210> 14 <211> 1746 <212> DNA
<213> Human adenovirus type S
<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 48 ccc gtg tat cca Met Lys Arg Ala Arg Pro Ser Glu Asp Thr Phe Asn Pro Val Tyr Pro tat gac acg gaa acc ggt cct cca act gtg cct ttt 96 ctt act cct ccc Tyr Asp Thr Glu Thr Gly Pro Pro Thr Val Pro Phe Leu Thr Pro Pro ttt gta tcc ccc aat ggg ttt caa gag agt ccc cct 144 ggg gta ctc tct Phe Val Ser Pro Asn Gly Phe Gln Glu Ser Pro Pro Gly Val Leu Ser ttg cgc cta tcc gaa cct cta gtt acc tcc aat ggc 192 atg ctt gcg ctc Leu Arg Leu Ser Glu Pro Leu Val Thr Ser Asn Gly Met Leu Ala Leu aaa atg ggc aac ggc ctc tct ctg gac gag gcc ggc 240 aac ctt acc tcc Lys Met Gly Asn Gly Leu Ser Leu Asp Glu Ala Gly Asn Leu Thr Ser caa aat gta acc act gtg agc cca cct ctc aaa aaa 288 acc aag tca aac Gln Asn Val Thr Thr Val Ser Pro Pro Leu Lys Lys Thr Lys Ser Asn ata aac ctg gaa ata tct gca ccc ctc aca gtt acc 336 tca gaa gcc cta Ile Asn Leu Glu Ile Ser Ala Pro Leu Thr Val Thr Ser Glu Ala Leu act gtg get gcc gcc gca cct cta atg gtc gcg ggc 384 aac aca ctc acc Thr Val Ala Ala Ala Ala Pro Leu Met Val Ala Gly Asn Thr Leu Thr atg caa tca cag gcc ccg cta acc gtg cac gac tcc 432 aaa ctt agc att Met Gln Ser Gln Ala Pro Leu Thr Val His Asp Ser Lys Leu Ser Ile gcc acc caa gga ccc ctc aca gtg tca gaa gga aag 480 cta gcc ctg caa Ala Thr Gln Gly Pro Leu Thr Val Ser Glu Gly Lys Leu Ala Leu Gln WO 01!02431 PCT/SE00/01390 aca tca ggc ccc ctc acc acc acc gat agc agt acc 528 ctt act atc act Thr Ser Gly Pro Leu Thr Thr Thr Asp Ser Ser Thr Leu Thr Ile Thr gcc tca ccc cct cta act act gcc act ggt agc ttg 576 ggc att gac ttg Ala Ser Pro Pro Leu Thr Thr Ala Thr Gly Ser Leu Gly Ile Asp Leu aaa gag ccc att tat aca caa aat gga aaa cta gga 624 cta aag tac ggg Lys Glu Pro Ile Tyr Thr Gln Asn Gly Lys Leu Gly Leu Lys Tyr Gly get cct ttg cat gta aca gac gac cta aac act ttg 672 acc gta gca act Ala Pro Leu His Val Thr Asp Asp Leu Asn Thr Leu Thr Val Ala Thr ggt cca ggt gtg act att aat aat act tcc ttg caa 720 act aaa gtt act Gly Pro Gly Val Thr Ile Asn Asn Thr Ser Leu Gln Thr Lys Val Thr gga gcc ttg ggt ttt gat tca caa ggc aat atg caa 768 ctt aat gta gca Gly Ala Leu Gly Phe Asp Ser Gln Gly Asn Met Gln Leu Asn Val Ala gga gga cta agg att gat tct caa aac aga cgc ctt 816 ata ctt gat gtt Gly Gly Leu Arg Ile Asp Ser Gln Asn Arg Arg Leu Ile Leu Asp Val agt tat ccg ttt gat get caa aac caa cta aat cta 864 aga cta gga cag Ser Tyr Pro Phe Asp Ala Gln Asn Gln Leu Asn Leu Arg Leu Gly Gln ggc cct ctt ttt ata aac tca gcc cac aac ttg gat 912 att aac tac aac Gly Pro Leu Phe Ile Asn Ser Ala His Asn Leu Asp Ile Asn Tyr Asn aaa ggc ctt tac ttg ttt aca get tca aac aat tcc 960 aaa aag ctt gag Lys Gly Leu Tyr Leu Phe Thr Ala Ser Asn Asn Ser Lys Lys Leu Glu gtt aac cta agc act gcc aag ggg ttg atg ttt gac 1008 get aca gcc ata Val Asn Leu Ser Thr Ala Lys Gly Leu Met Phe Asp Ala Thr Ala Ile gcc att aat gca gga gat ggg ctt gaa ttt ggt tca 1056 cct aat gca cca Ala Ile Asn Ala Gly Asp Gly Leu Glu Phe Gly Ser Pro Asn Ala Pro aac aca aat ccc ctc aaa aca aaa att ggc cat ggc 1104 cta gaa ttt gat Asn Thr Asn Pro Leu Lys Thr Lys Ile Gly His Gly Leu Glu Phe Asp tca aac aag get atg gtt cct aaa cta gga act ggc 1152 ctt agt ttt gac Ser Asn Lys Ala Met Val Pro Lys Leu Gly Thr Gly Leu Ser Phe Asp agc aca ggt gcc att aca gta gga aac aaa aat aat 1200 gat aag cta act Ser Thr Gly Ala Ile Thr Val Gly Asn Lys Asn Asn Asp Lys Leu Thr ttg tgg acc aca cca get cca tct cct aac tgt aga 1248 cta aat gca gag Leu Trp Thr Thr Pro Ala Pro Ser Pro Asn Cys Arg Leu Asn Ala Glu aaa gat get aaa ctc act ttg gtc tta aca aaa tgt 1296 ggc agt caa ata Lys Asp Ala Lys Leu Thr Leu Val Leu Thr Lys Cys Gly Ser Gln Ile ctt get aca gtt tca gtt ttg get gtt aaa ggc agt 1344 ttg get cca ata Leu Ala Thr Val Ser Val Leu Ala Val Lys Gly Ser Leu Ala Pro Ile tct gga aca gtt caa agt get cat ctt att ata aga 1392 ttt gac gaa aat Ser Gly Thr Val Gln Ser Ala His Leu Ile Ile Arg Phe Asp Glu Asn gga gtg cta cta aac aat tcc ttc ctg gac cca gaa 1440 tat tgg aac ttt Gly Val Leu Leu Asn Asn Ser Phe Leu Asp Pro Glu Tyr Trp Asn Phe aga aat gga gat ctt act gaa ggc aca gcc tat aca 1488 aac ggt gtt gga Arg Asn Gly Asp Leu Thr Glu Gly Thr Ala Tyr Thr Asn Gly Val Gly ttt atg cct aac cta tca get tat cca aaa tct cac 1536 ggt aaa act gcc Phe Met Pro Asn Leu Ser Ala Tyr Pro Lys Ser His Gly Lys Thr Ala aaa agt aac att gtc agt caa gtt tac tta aac gga 1584 gac aaa act aaa Lys Ser Asn Ile Val Ser Gln Val Tyr Leu Asn Gly Asp Lys Thr Lys cct gta aca cta acc att aca cta aac ggt aca cag 1632 gaa aca gga gac Pro Val Thr Leu Thr Ile Thr Leu Asn Gly Thr Gln Glu Thr Gly Asp aca act cca agt gca tac tct atg tca ttt tca tgg 1680 gac tgg tct ggc Thr Thr Pro Ser Ala Tyr Ser Met Ser Phe Ser Trp Asp Trp Ser Gly cac aac tac att aat gaa ata ttt gcc aca tcc tct 1728 tac act ttt tca His Asn Tyr Ile Asn Glu Ile Phe Ala Thr Ser Ser Tyr Thr Phe Ser tac att gcc caa gaa taa Tyr Ile Ala Gln Glu ***
<210> 15 <211> 120 <212> PRT
<213> Mus musculus <400> 15 Asp Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Leu Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr Tyr Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Leu Val Ala Ala Ile Asn Ser Asp Gly Gly Ile Thr Tyr Tyr Leu Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Leu Phe Tyr Cys Ala Arg His Arg Ser Gly Tyr Phe Ser Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Ser <210> 16 <211> 116 <212> PRT
<213> Mus musculus <400> 16 Asp Ile Val Met Thr Gln Ser Gln Arg Phe Met Ser Thr Thr Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Val Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Met Gln Ser Glu Asp Leu Ala Asp Phe Phe Cys Gln Gln Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro Thr V al S er

Claims

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.

5. 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, derived from tissue prothrombin activator; SEQ ID NO: 5, 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 Ad5.

10. 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.

13. An adenovirus according to claim 11, wherein the single chain fragment is a single chain fragment of the monoclonal antibody 6250 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 .alpha.-helical coiled coil motif, or any other peptide capable of rendering functio-nally trimerised fibers.

15. 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 anal 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.

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

19. An adenovirus according to claim 18, wherein the sequences are present in the wild type knob.

20. 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. A method of producing a recombinant adenovirus with changed tropism, comprising:
I. rescuing recombinant adenovirus fibers into the adeno-virus genome by the following steps:
a) subcloning of a 9 kb fragment (from Spe1 to the end of genome), b) further subcloning of a 3 kb fragment between Sac1 and Kpn1, c) deletion of the native fiber gene coding for the native penton fiber between Nde1 and Mun1 and replacing the missing sequence with the sequence SEQ ID NO: 13 containing an Xho1 site;
d) ligation of recombinant fiber gene coding for between Nde1 and Xho1 of construct under c) above;
e) re-introduction of construct under d) above into the 9 kb fragment cut with Nhe1 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 of the Spe1 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.