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Definitions

• 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.
• 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.
• gene therapy to treat e.g. diseases of the blood vessels, cancer, inflammatory diseases and infectious diseases such as HIV can be envisaged.
• gene therapy is not a useful method in human medicine.
• 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.
• vectors have so far not been available .
• Adenoviruses 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 and Cusack S: Adenovirus Fiber, Current
• Each shaft repeat has two three-amino acid regions which form ⁇ - 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 ⁇ -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).
• the FLAG tetra-amino acid motif 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. ) .
• 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.
• the objects of the invention are obtained by the recombinant adenovirus and the method for rescuing the virus fibers as claimed in the claims.
• a recombinant adenovirus with changed tropism 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 .
• 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:
• 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.
• 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 .
• re-targeting of Ad is achieved through the introduction of a new cell-binding ligand into the fiber (Fig. 1) .
• a new cell-binding ligand 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 HIA-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 EGF-derived neurotrophic factor (vd Bruggen P, Traversaari C, Chomez P, Lurquin D, De Plaen E, vd Eynde B, Knuth A and Boon T: A Gene encoding an
• Ad vectors can be made replication competent or incompetent for permissive cells.
• 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.
• 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.
• 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:
• 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.
• 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,
• Recombinant fibers are constructed using, methodology based on PCR (Clackson T, G ⁇ ssow 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, BioTechniqrues 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 :
• 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.
• trimerisation motifs are synthesized, cloned and sequenced in the project.
• a new cellbinding ligand is introduced into the fiber in addition to the external trimerisation amino acid motif.
• 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.
• sequences are e.g. sequences that are present in the wild type knob or in SEQ ID NO: 10 - 12.
• Type A where the trimerisation motif is fused to the fiber gene downstream of the fiber shaft after the TL T 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/ ⁇ 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 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.
• 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.
• 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
• C ⁇ Constant domain from ⁇ chain of T cell receptor against MAGEl/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
• RGD The amino acid sequence arginine-glycine-aspartic acid
• V ⁇ Variable domain from ⁇ chain of T ceil receptor against MAGEl/HLA Al .
• SEQ ID NO: 12 Example 2
• 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.
• 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.
• the fibers would be detected in the nucleus, due to the presence of the native nuclear localization signal in the fiber tail.
• 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) .
• NLS nuclear localization signal
• SEQ ID NO: 9 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. 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) .
• 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 , 70: 4805-4810, 1996) containing the entire Ad5 genome as a Pacl-Pacl fragment is used as starting material.
• 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 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 Muni sites and the wild type genome sequence between the fiber stop codon and the Muni site.
• 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.
• 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 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.
• Ad made re-targeted by the present invention
• tumor diseases the following options exist:

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