ANTI-TUMOR MEDICAMENT BASED ON ADENOVIRUS

Abstract

The invention provides manipulated adenovirus, i.e. a viral particle based on a manipulated adenovirus, for use as a medicament, especially for use in the treatment of tumours. The viral particle of the invention has the advantage of having a preference or specificity for tumour cells, yielding a preferred infection of tumour cells. The viral particle is based on adenovirus, especially type C, preferably serotype 2 (Ad2), more preferably serotype 5 (Ad5), in which the native entire fiber protein, and its coding sequence, respectively, is deleted and replaced by a fusion protein providing specificity for cell surface bound polysialic acid.

Claims

1. Adenoviral particle for use in the treatment of a tumour comprising a fusion protein comprising from N-terminus to C-terminus a tail domain of an adenoviral fiber protein and an amino acid sequence of an endosialidase of bacteriophage origin, lacking its bacteriophage attachment domain, wherein the fusion protein specifically binds to polysialic acid.

2. Adenoviral particle according to claim 1, wherein the endosialidase of bacteriophage origin, lacking its bacteriophage attachment domain is selected from amino acid sequences having an identity of at least 51% to one of SEQ ID NO: 5 (EndoK1E), SEQ ID NO: 6 (EndoK1F), SEQ ID NO: 7 (EndoNK1), and SEQ ID NO: 8 (Endo92).

3. Adenoviral particle according to claim 1, wherein at least 1 pseudo-repeat of SEQ ID NO: 14 is arranged between the tail domain and the endosialidase.

4. Adenoviral particle according to claim 1, wherein the tail domain an amino acid sequence of amino acids 1 to 44 of SEQ ID NO: 4.

5. Adenoviral particle according to claim 4, wherein a flexible linker peptide containing at least 2 repeats of one or more amino acid sequences selected from (PT).sub.XP, wherein X is 1 to 10, and S.sub.3N.sub.10 (SEQ ID NO: 17) is arranged N-terminally to the endosialidase and C-terminally to the at least one pseudo-repeat.

6. Adenoviral particle according to claim 1, wherein the fusion protein from N-terminus to C-terminus consists of the tail domain, at least one pseudo-repeat, a linker peptide, and one endosialidase of bacteriophage origin, lacking its bacteriophage attachment domain.

7. Adenoviral particle according to claim 2, wherein SEQ ID NO: 5 (Endo-K1E) contains the mutation K200A (SEQ ID NO: 41) and/or the mutation R386A and/or R437A (SEQ ID NO: 42), SEQ ID NO: 6 (Endo-K1F) contains the mutation K410A (SEQ NO: 9) and/or the mutation R596A and/or R647A (SEQ ID NO: 10), SEQ ID NO: 7 (Endo-NK1) contains the mutation R503A and/or R554A (SEQ ID NO: 43), and SEQ ID NO: 8 (Endo92) contains the mutation R436A and/or R437A (SEQ ID NO: 44).

8. Adenoviral particle according to claim 1, comprising wild-type adenoviral proteins with the exception of the fiber protein encoded by nucleotides No. 31037 to 32782 in the sequence accessible at GenBank at accession No. AY339865.1.

9. Adenoviral particle according to claim 8 containing the wild-type adenoviral proteins of adenovirus C serotype 5 and lacks the coding sequence for E3 protein.

10. Adenoviral particle according to claim 1, wherein the tumour is selected from glioblastoma, medulloblastoma, rhabdomyosarcoma, small cell carcinoma, and small cell lung carcinoma.

11. Adenoviral particle according to claim 1, containing DNA encoding the amino acid sequences.

12. Adenoviral particle according to claim 11, wherein the coding sequence for adenoviral early proteins, optionally additionally the coding sequence for E1A, is functionally arranged under the control of a polysialyltransferase promoter of mammalian origin or a telomerase promoter.

13. Adenoviral particle according to claim 1 wherein the fusion protein has a sequence of at least 90% identity to one of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4.

14. Adenoviral particle according to claim 1, wherein the endosialidase has a reduced enzymatic activity on polysialic acid.

15. Process for producing an adenoviral particle according to claim 1 by expressing a DNA in a mammalian cell, the DNA encoding a fusion protein comprising from N-terminus to C-terminus a tail domain of amino acids No. 1 to 44 of SEQ ID NO: 4 and an amino acid sequence of an endosialidase of bacteriophage origin, lacking its bacteriophage attachment domain.

16. Process according to claim 15, wherein the adenoviral particle is a fusion protein comprising from N-terminus to C-terminus a tail domain of an adenoviral fiber protein and an amino acid sequence of an endosialidase of bacteriophage lacking its bacteriophage attachment domain, wherein the fusion protein specifically binds to polysialic acid.

Description

[0051] The invention will now be described in greater detail by way of examples with reference to the figures, which show in

[0052] FIG. 1 a scheme of the domains of the wild-type adenovirus serotype 5 fiber protein,

[0053] FIG. 2 a scheme of an embodiment of the fusion protein having 1 pseudo-repeat,

[0054] FIG. 3 a scheme of an embodiment of the fusion protein having 8 pseudo-repeats,

[0055] FIG. 4 a scheme of an embodiment of the fusion protein having 14 pseudo-repeats,

[0056] FIG. 5 a scheme of an embodiment of the fusion protein having 18 pseudo-repeats,

[0057] FIG. 6 an amino acid sequence alignment of preferred endosialidase domains,

[0058] FIG. 7 a scheme for cloning the coding sequence for fusion protein to replace the natural fiber protein in adenovirus,

[0059] FIG. 8 a Western blot specific for the fusion protein of the invention on cell culture samples infected by viral particles of the invention,

[0060] FIG. 9 a graph on the titer of viral particles of the invention in different cell lines,

[0061] FIGS. 10A-10D micrographs of a cell line not expressing polysialic acid in the presence of an embodiment of the viral particle of the invention,

[0062] FIGS. 11A-11D micrographs of a cell line expressing polysialic acid in the presence of the embodiment of the viral particle of FIG. 10 a) to d),

[0063] FIGS. 12A-12D micrographs of a cell line not expressing polysialic acid in the presence of an embodiment of the viral particle of the invention,

[0064] FIGS. 13A-13D micrographs of a cell line expressing polysialic acid in the presence of the embodiment of the viral particle of FIG. 12 a) to d),

[0065] FIGS. 14A-14F FACS results from different cell line cultures infected by viral particles of the invention,

[0066] FIGS. 15A-15B electron micrographs of wild-type adenovirus and at c) and d) electron micrographs of a viral particle of the invention, and

[0067] FIG. 16 shows an amino acid sequence comparison of preferred endosialidase domains of the fusion protein.

[0068] FIG. 17 describes the effect of wild-type oncolytic adenovirus compared with a virus expressing polySia-binding fusion protein (307-Ad) on human tumour growth when applied to transplanted tumours on nude mice.

[0069] FIG. 1 for comparison shows a scheme of the domains of the wild-type fiber protein of adenovirus serotype 5, from N-terminus to C-terminus consisting of a tail domain of 44 amino acids (aa) that binds to the penton base, 22 pseudo-repeats at amino acids 45 to 407, and the knob domain at amino acids 408 to 581 that binds to CAR.

[0070] FIGS. 2 to 5 depict embodiments of the polySia-binder, which from N-terminus to C-terminus consist of the penton base binding tail domain (tail) that is represented by the wild-type tail domain of 44 aa, directly adjacent at least 1 pseudo-repeat, namely in FIG. 2 a polySia-binder containing 1 pseudo-repeat (designated C521), in FIG. 3 a polySia-binder containing 8 pseudo-repeats (designated C414), in FIG. 4 a polySia-binder containing 14 pseudo-repeats (designated C307), and in FIG. 5 a polySia-binder containing 19 pseudo-repeats (designated C235), in each embodiment directly adjacent a linker, which is represented by the (PT)8P linker (SEQ ID NO: 16), directly adjacent the endosialidase, which is represented by the endosialidase of bacteriophage K1F, in the K410A mutant form, in the wild-type form (Endosialidase NF K410A/wt) or in a completely inactive double mutant form (Endosialidase NF R596A/R647A).

[0071] The designations of these exemplary embodiments indicate the deletions that were made, counting from the C-terminus of the wild-type fiber protein. Optionally all embodiments (C235, C307, C414 and C521) contained the mutation K410A which resulted in a reduced endosialidase activity and did essentially not affect binding to cell surface bound polysialic acid. All of the aforementioned embodiments can optionally contain the double mutation R596A/R647A, abolishing the endosialidase activity but not affecting the binding to polysialic acid. Generally, a deletion can be indicated by a , D, d, or delta, with the letter N indicating a deletion from the N-terminus, the letter C indicating a deletion from the C-terminus.

[0072] The N-terminal section of 247 amino acids of the wild-type form is not contained in the polySia-binder in order to delete the wild-type bacteriophage attachment domain.

[0073] In the examples, in all viral genomes of the invention and in the wild-type adenovirus used, the coding sequence for E3 protein was deleted.

[0074] Exemplary DNA sequences in the form of a bacterial plasmid for cloning a viral particle containing the polySia-binder of the invention are shown in SEQ ID NO: 45, SEQ ID NO: 49 and SEQ ID NO: 51. Therein, the endosialidase is represented by EndoK1F (endoNF), and the expression cassette is under the control of the endosialidase promoter (SEQ ID NO: 45 and 49) or a telomerase promoter (SEQ ID NO: 51), respectively. In SEQ ID NO: 45, nucleotides 28582 to 32136 encode the polySia binder, SEQ ID NO: 48 shows the amino acid sequence of the polySia binder. The N-terminally truncated E3 14.7K encoded by SEQ ID NO: 45 is given as SEQ ID NO: 47, SEQ ID NO: 46 shows the amino acid sequence of the C-terminally truncated E3 14.5K encoded by SEQ ID NO: 45. In SEQ ID NO: 49, nucleotides 29611 to 33165 encode the polySia binder, its amino acid sequence is given as SEQ ID NO: 50. In SEQ ID NO: 51, nucleotides 28447 to 32001 encode the polySia binder, its amino acid sequence is given as SEQ ID NO: 52.

[0075] Exemplary complete virus genomes containing a coding sequence for the polySia binder of the invention are given as hTert-Ad-C235fiber-(PT8)P-N247EndoNF-K410A (SEQ ID NO: 53), wherein nucleotides 28447 to 32001 encode the polySia binder, its amino acid sequence is given as SEQ ID NO: 57. SEQ ID NO: 54 to SEQ ID NO: 56 and SEQ ID NO: 58 give the further amino acid sequences of the codons indicated in SEQ ID NO: 53. A complete virus genome containing a coding sequence for the polySia binder termed hTert-Ad-C307fiber-(PT8)P-N247EndoNF-K410A is shown at SEQ ID NO: 59 (hTERT-Ad5_fiberEndo), encoding the polySia binder at nucleotides 28447 to 31782 (Fiber-Endo), the amino acid sequence of which is given as SEQ ID NO: 63. SEQ ID NO: 60 to 62 give the amino acid sequences of the codons indicated in SEQ ID NO: 59 A complete virus genome termed hTert-Ad-C414fiber-(PT8)P-N247EndoNF-K410A is given at SEQ ID NO: 64, encoding the polySia binder at nucleotides 28447 to 31464 (C414fiber-(PT8)P-N247EndoNF-K410A), the amino acid sequence is given at SEQ ID NO: 68. SEQ ID NO: 65 to 67 give further amino acid sequences of the codons indicated in SEQ ID NO: 64. These viral genomes contain DNA sections which form homologous arms for integration of the virus genome into the genome of the target cell by homologous recombination and encode the endosialidase of EndoNF as an embodiment of the endosialidase of phage origin without its phage attachment domain.

[0076] FIG. 6 shows the amino acid sequence alignment (Clustal Omega 1.2.1, available from EMBL-EBI) of the preferred endosialidases N76-Endo92 (EndoPhi 92-dN76, SEQ ID NO: 8), N147-Endo-NK1(CUS3) (EndoNK1 dN147, SEQ ID NO: 7), N247-Endo-K1F (EndoK1F-dN247, SEQ ID NO: 6), and N40-Endo-K1E (EndoK1E-dN40, SEQ ID NO: 5) of the polySia-binder. In the alignment, * denotes an identical amino acid, .and :denote amino acids having homology. The alignment shows that these endosialidases of bacteriophage origin have an amino acid identity of at least 51%.

Example 1: Cloning of DNA Encoding an Adenovirus Containing the PolySia-binder

[0077] Generally, the DNA encoding the adenoviral particles are available by replacing the coding sequence for the fiber protein from the genome encoding a wild-type adenovirus by a sequence encoding the polySia-binder.

[0078] FIG. 7 schematically shows the cloning procedure for deleting the coding sequence for the wild-type fiber protein, contained in a bacterial vector containing the entire adenovirus serotype 5 genome, optionally lacking the coding sequence for E3 protein, by homologous recombination with a linear chloramphenicol resistance cassette (Chloramphenicol resistance), containing restriction sites (SpeI) flanked at both ends with sections homologous (homologous arms 5-HA, 56 bp (base pairs) and 3-HA, 52 bp) to the wild-type sequence. A subsequent digest with the specific restriction enzyme (SpeI) removed the chloramphenicol resistance cassette and linearized the plasmid for subsequent homologous recombination.

[0079] The coding sequence for the fusion protein was produced by PCR amplification of sections with primers having overlapping terminal sections, followed by a one-step fusion PCR (Szewczyk et al., Nature Protocols 2006, 3111-3120 and Erratum). The outermost primers of the fusion product contained homologous sections, and the fusion product was integrated into the previously prepared vector lacking the wild-type fiber encoding sequence using the Red/ET mediated recombination (available from Genebridges, USA). The primers used are indicated in FIG. 7, for PCRI (and PCR VI) prim-1 (SEQ ID NO: 30) and prim-2 (SEQ ID NO: 31), for PCRII prim-3 (SEQ ID NO: 32) and prim-4 (SEQ ID NO: 33), for PCRIII prim-5 (SEQ ID NO: 34) and prim-6 (SEQ ID NO: 35), for PCRIV prim-7 (SEQ ID NO: 36) and prim-8 (SEQ ID NO: 37), for PCRV prim-9 (SEQ ID NO: 38) and prim-10 (SEQ ID NO: 39) and for PCRVI prim-1 (SEQ ID NO: 40) and prim-11 (SEQ ID NO: 39).

[0080] The kanamycin resistance cassette was removed using its FRT sites by FLP/FRT recombination.

[0081] The plasmid containing the entire genome for the recombinant adenovirus having the polySia-linker in the place of the wild-type fiber protein was linearized by PacI and transfected into HEK293 cells for producing the adenovirus having the polySia binder, which construct is also referred to as Ad-polySia-binder herein. Ad-polySia-binder adenoviral particles were shown to contain the DNA encoding the genome having the polySia binder, both for the genome encoding all adenoviral proteins except the wild-type fiber protein, and for the embodiment, in which genome in addition the coding sequence for E3 was deleted.

[0082] In addition, the coding sequence contained an expression cassette for enhanced green fluorescent protein (EGFP) as a reporter, the coding sequence of which was functionally arranged under the control of the E1B promoter. The CDS (codons) of EGFP was linked to the CDS of E1B via an IRES motif.

[0083] FIG. 8 shows a Western blot analysis of culture supernatant and cell lysate of HEK293 cells infected with the DNA encoding Ad-polySia-binder using a primary antibody (Abcam Anti-Adenovirus Fiber monomer and trimer antibody [4D2] (ab3233)) directed against the pseudo-repeats. Lanes M show size marker proteins, lanes 1 contains the soluble fraction of lysate 72 h post infection (p.i.) and lane 2 contains the insoluble fraction of lysate 72 h p.i. of the wild-type Adenovirus 5 showing the fiber protein at 62 kDa (box) as a positive control, lane 3 contains culture medium 72 h p.i. lane 4 contains the soluble fraction of lysate 72 h p.i., lane 5 contains the insoluble fraction of lysate 72 h p.i. of the construct C307 showing the polySia-binder at 104 kDa (box) and 122 kDa, lane 6 contains culture medium 72 h post infection (p.i.) and lane 7 contains the soluble fraction of lysate 72 h p.i., lane 8 contains the insoluble fraction of lysate 72 h p.i. of the construct of C414 showing the polySia-binder at 93 kDa (box) and 111 kDa. The detection of the polySia-binder protein at two sizes corresponds to the result of the removal of the C-terminal chaperone domain from the endosialidase domain.

Example 2: Producing Adenovirus Containing the PolySia-binder

[0084] For producing Ad-polySia binder viral particles, HEK293-polySia+cells were cultivated in 60 T-75 culture flasks under cell culture conditions in Gibco DMEMGlutaMAX, containing 2% v/v FCS, Penicillin (100 U/mL) and Streptomycin (100 g/mL). Cells were grown to about 90% confluency and then infected with the C307 construct with an MOI of 5. The infected cells were incubated until a strong cytopathic effect was observable and nearly all cells were detached. Cells were then collected by centrifugation and lysed by three repetitions of freezing and thawing. Subsequently, particles were concentrated by CsCl gradient centrifugation (96,000g, 4 h), yielding 5,9410.sup.10 ifu (infectious particles)/mL in a total volume of 2 mL. The proportion of infectious particles in total particles was 1.25%.

[0085] These Ad-polySia binder viral particles were used to infect cell line cultures of HEK293 cells and of HEK293 cells that were genetically manipulated to express cell-surface bound polysialic acid (HEK293-polySia+) which in the example represented tumour cells bearing polysialic acid on their cell surface. HEK293-polySia+ were generated by retroviral transduction of HEK293 cells with a DNA encoding for the murine ST8SiaIVunder the control of the CMV promoter.

[0086] The results are depicted in FIG. 9, showing that HEK293 cells (hatched bars) were infected only to a very small extent, whereas HEK293 cells expressing cell-surface bound polysialic acid (HEK293-polySia+, dark bars) were infected to a significantly higher extent. In FIG. 8, the X-axis gives the volume in L of 10.sup.6 ifu/mL added to each cell culture, the Y-axis gives the amount of infectious particles produced after isolated from 310.sup.5 cells that were counted at the time of infection from the cell culture at 48 h post infection. Infection was for 30 min with subsequent change of the medium to remove unbound viral particles.

[0087] The predominant infection of polysialic acid bearing cells and the very small infection of cells having CAR but no polysialic acid is shown for different amounts of viral particle containing preparations used. This result is shown in FIG. 9 for the embodiments of Ad-polySia binder viral particles C307 (4) and C414 (3).

[0088] When using wild-type adenovirus as a positive control, both HEK293 cells and HEK293-polySia+ cells, production of virus was found approximately to the same extent for each cell line, indicating no preference for one of the cell lines, i.e. no preference for the polysialic acid bearing cells.

[0089] FIGS. 10 to 13 show microscope pictures at the magnification indicated of cultivated cells, with the left hand microscopic pictures showing fluorescence images and the right hand pictures showing light microscopic pictures of the identical subject area, i.e. without excitation of fluorescence. For excitation of fluorescence, light of 485 nm was irradiated onto the cell samples, detection was at 530 nm, indicating GFP. For these analyses, HEK cells and HEK293-polySia+ cells that express surface-bound polysialic acid following cultivation under standard cell culture conditions to 80% confluence were contacted for 30 min with 100 of viral particles prepared directly from cell lysate by freezing and thawing without concentration via a CsCl gradient. Unbound viral particles were then removed by washing and medium exchange, and the cells were incubated for a further 48 h under cell culture conditions.

[0090] FIG. 10 shows HEK293 cells following contact with viral particles C307 containing the K410A mutation. The fluorescence images of FIGS. 10A and 10C show that essentially no HEK293 cells present in the light microscopic pictures of FIGS. 10B and 10D, respectively, were infected by the viral particle of the invention.

[0091] In contrast, HEK293-polySia+ cells when contacted with the viral particles C307 containing the K410A mutation were effectively infected. Fluorescence images of FIGS. 11A and 11C show individual HEK293-polySia+ cells, marked up by the inserted arrowheads, fluoresce, indicating presence of the viral particle. The fluorescing cells are also marked up by inserted arrowheads in the light microscopic pictures of FIGS. 11B and 11D.

[0092] FIG. 12 shows HEK293 cells, which do not express polysialic acid, following contact with viral particles C414 containing the K410A mutation. The fluorescence images of FIGS. 12A and 12C show that essentially no HEK293 cells were infected by the viral particle. FIGS. 12B and 12D show the cells in light microscopy.

[0093] FIG. 13 shows that HEK293-polySia+ cells when contacted with the viral particles C307 containing the K410A mutation were effectively infected. Fluorescent cells are marked up by arrowheads in the fluorescence images of FIGS. 13A and 13B as well as in light microscopic images of FIGS. 13C and 13D.

[0094] The results of FIGS. 10 to 13 demonstrate that the viral particles of the invention infect mammalian cells bearing cell surface bound polysialic acid and also CAR (HEK293-polySia+), whereas cells which bear CAR but no cell surface bound polysialic acid (HEK293) are essentially not infected, showing the significant specificity of the viral particles for cells bearing cell surface bound polysialic acid. In addition, these results show that infection of cells bearing cell surface bound polysialic acid by the viral particles results in amplification of the viral particle within the infected cells.

[0095] The predominance of infection of cells for those cells bearing cell surface bound polysialic acid, represented by HEK293-polySia+, over cells not bearing cell surface bound polysialic acid by viral particles that were produced in infected cells shows that the specificity of the viral particles for cells bearing cell surface bound polysialic acid is stable, i.e. inheritable, and allows for secondary infection of such cells by viral particles that are generated within cells that were primarily infected by viral particles.

[0096] Therefore, these results indicate that cells bearing cell surface bound polysialic acid can specifically be infected by the viral particles of the invention, e.g. essentially without infecting cells bearing CAR but no cell surface bound polysialic acid. The viral particles used for initial infection can also be termed primary viral particles. Further, the results show that the viral particles multiply within infected cells to generate secondary viral particles having the same specificity for infecting cells bearing cell surface bound polysialic acid. The stable inheritance of the specificity in secondary viral particles for cells bearing cell surface bound polysialic acid allows re-targeting of the secondary viral particles to cells bearing cell surface bound polysialic acid.

[0097] For fluorescence-activated cell sorting (FACS) analysis, HEK293 cells and HEK293-polySia+ cells after contacting with viral particles as described above were incubated in fresh culture medium for 48 h under cell culture conditions and brought into suspension by the treatment with Cell Dissociation Buffer, enzyme-free, PBS (available from Gibco, USA) according to the manufacturer's guidelines. For detection of infected cells EGFP fluorescence was measured directly. For comparison, wild-type adenovirus containing the same EGFP cassette was used, and C307 and C414, both additionally bearing the K410A mutation. HEK293 cells were used for representing cells having the CAR receptor and essentially having no cell surface bound polysialic acid, and HEK293-polySia+ cells were used for representing tumour cells bearing cell surface bound polysialic acid.

[0098] The FACS results are shown in FIGS. 14A-14F, for wild-type adenovirus in A) HEK293 cells and B) in HEK293-polySia+ cells, both showing infection, for viral particles C307 in C) HEK293 cells and D) in HEK293-polySia+cells, of which essentially only HEK293-polySia+ cells were infected, and for viral particles C414 in E) HEK293 cells and F) in HEK293-polySia+ cells, of which essentially only HEK293-polySia+ cells were infected. These results confirm the specificity of the viral particles of the invention for mammalian cells bearing cell surface bound polysialic acid.

[0099] FIGS. 15C and 15D show transmission electron microscopic pictures of the viral particles C307 in comparison to wild-type adenovirus in FIGS. 15A and 15B. It can be seen that the viral particles of the invention have the overall shape of adenovirus and bear a ligand (indicated by arrows) attached to the penton protein, which ligand seems to locate at the penton protein in a position similar to the ligand visible on the wild-type adenovirus. In the wild-type, the knob domain and the shaft domain (pseudo-repeats) are indicated, in the viral particle of the invention, the shaft domain and the endosialidase domain (PBXD) are indicated. As the fiber protein of the wild-type adenovirus is replaced by the polySia-binder protein, it is assumed that the ligand seen in FIGS. 15C and 15D is the polySia-binder protein, which is present in the position of the fiber protein in the wild-type, and that the polySia-binder protein allows for formation of stable viral particles, e.g. stabilizing arrangement of penton proteins.

Example 3: Use of Adenoviral Particles Containing the PolySia-binder in the Treatment of Tumour

[0100] For establishment of a mouse model of polySia-expressing human xenograft tumours, 110.sup.7 human rhabdomyosarcoma cells (TE671) were injected s.c. into the flank of nude mice. Once the tumours had reached a palpable size of 100 mm.sup.3, virotherapy was applied by intratumoural infiltration of 510.sup.8 ifu of the polySia-specific 307-Ad, or hTert-Ad as control, respectively.

[0101] Treatment was repeated after 10 days and tumour size was monitored each 3-4 days using a digital caliper. Size was calculated using the formula V=(LB.sup.2)/2. The results are depicted in FIG. 16, wherein the polySia-binder (307-Ad) corresponding to SEQ ID NO: 3 was produced by the method steps described in Example 1 and viral particles were produced as described in Example 2. hTert-Ad, containing the wild-type fiber protein, was used as a control. The result shows that the viral particle containing the polySia-binder according to the invention (.box-tangle-solidup.) retarded tumour growth to a significantly higher degree than the control viral particle having no polysialic acid-specificity (.square-solid.).

[0102] This in vivo example shows that the polySia-binder of the invention when expressed on an adenoviral particle results in retardation of tumour growth of polysialic acid bearing tumour cells.