FR2842823A1 - MODIFIED ADENOVIRUSES FOR TARGETING B-LYMPHOCYTES - Google Patents

Abstract

The present invention relates to recombinant adenoviruses whose tropism has been modified to allow them to infect B lymphocytes. It also relates to methods for the preparation of such adenoviruses, as well as their uses, in particular for the transfer of nucleic acids into the cells. B lymphocytes in vitro, ex vivo or in vivo, for experimental, industrial, vaccine or therapeutic purposes. The invention more specifically describes the modification of the adenovirus fiber gene with the aim of targeting B lymphocytes, as well as other modifications and / or constructions making it possible to confer on the viruses a specific character for these cells. The invention is characterized in particular by the introduction into the fiber of the sequence of a targeting peptide recognizing the CD21 receptor expressed on the surface of B lymphocytes.

Description

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Modified adenoviruses for targeting B cells
The present invention relates to recombinant adenoviruses whose tropism has been modified to enable them to infect B lymphocytes. It also relates to methods for the preparation of such adenoviruses, as well as their uses, in particular for the transfer of DNA sequences into B lymphocytes in vitro, ex vivo or in vivo, for an experimental, industrial, vaccine or therapeutic purpose.

The invention more specifically describes the modification of the adenovirus fiber gene in order to target B lymphocytes, as well as other modifications and / or constructs which make it possible to give viruses a specific character for these cells. The invention is characterized in particular by the introduction into the fiber of the sequence of a peptide recognizing a receptor expressed on the surface of B lymphocytes.

 Adenoviruses are widely used as gene vectors. These viruses indeed have many qualities such as easy production at high titer, their genome does not integrate into the genome of the host cell, large inserts can be introduced therein and they can infect quiescent cells and dividing cells . These advantages make these viruses interesting candidates in the context of gene transfer for gene therapy or for experimental studies (gene transfer in animals, production of recombinant cells, analysis of gene expression, etc.).

 There are multiple serotypes of adenoviruses, infecting humans or animals. The adenoviruses currently used are mainly from subgroup C and from serotype 2 and 5. Mention may also be made of canine or bovine adenoviruses, as well as human adenoviruses of serotypes 7 or 12, for example.

 A limitation of adenoviral vectors, however, lies in the fact that they hardly infect hematological cells. However, within the framework of a gene therapy protocol against a pathology involving B lymphocytes, for example tumor pathologies such as leukemias and lymphomas, it is necessary to allow their infection to be effective. This as well in the case of a protocol

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 in-vivo or ex-vivo gene therapy, immunotherapy or a suicide gene strategy. In addition, in a gene therapy approach, it is important to infect the target cells most effectively while limiting the spread of the virus in other (non-target) cells. In addition, such a gene vector can be used in experimental approaches, for example to efficiently transfect B lymphocytes in order to test the action of various genes or promoters.

 The part of the virus which is responsible for the recognition of the natural receptor, CAR (Coxsackie-Adenovirus Receptor) (Bergelson et al., 1997) is a capsid protein, the fiber, and more precisely, its C-terminal part called "the button" (Louis et al., 1994). This part of the fiber is also responsible for initiating the trimerization of the protein (Hong and Engler 1996). This conformation is essential for its interaction with the base of the penton and its incorporation into the capsid.

To modify the tropism of the adenovirus, several strategies have been developed in the prior art. The first consists in the use of bispecific compounds. They consist of a part recognizing a domain of an adenovirus capsid protein (the fiber, the base of the penton or the hexon) and of a part which can interact with a target receptor. In this way cells were infected via the EGF receptor (Epidermal Growth Factor) (Watkins et al., 1997) or via the FGF receptor (Fibroblast Growth Factor) (Goldman et al., 1997). Many other membrane components have thus been targeted (Haisma et al., 1999; Kelly et al., 2000;
Schneider et al., 2000; Yoon et al., 2000). This technique is effective but it is difficult to envisage it in a gene therapy protocol. Indeed, there are two major problems which are (i) the stability of the bispecific compound and (ii) the use of a two-component system which can only be produced separately, which makes the notion of essential reproducibility difficult. for therapeutic trial protocols.

 The second strategy used to modify the tropism of adenoviruses is to graft on the fiber, in a genetic way, a peptide allowing the recognition of a cellular receptor. The addition of this peptide sequence should not interfere with the

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 trimerization in order to obtain a correct assembly of the virus. Numerous studies have been carried out in this direction, with in particular the use of a poly-lysine sequence (K7) or of an RGD sequence at the C-terminal end of the fiber (Wickham et al., 1997).

These modifications allow correct assembly of the virus and infection of the cells via the interaction poly-lysine / heparan sulfates or RGD / integrins of the [alpha] vss3-5 type. Other work has been done on the insertion of RGD sequence in the HI loop, which is presented on the surface of the button. Such viruses can thus infect a large number of cell types into which it naturally enters with low efficiency (Biermann et al., 2001; Cripe et al., 2001; Grill et al., 2001; Kasono et al., 1999; Nakamura et al., 2002; Vanderkwaak et al., 1999, W096 / 26281; W094 / 10323; W095 / 05201; W095 / 26412). These studies therefore show that the addition of peptides in the fiber button does not hinder its conformation and makes it possible to direct the infection to a predetermined receptor.

 The genetic modifications of the fiber described to date allow the infection of cell types which are not natural targets. However, the fact that the peptides used (poly-lysine and RGD) recognize receptors which are strongly expressed in many cell types only increases the tropism of adenoviruses. Furthermore, no adenovirus having a preferential tropism for B lymphocytes has been described in the prior art.

 The present invention describes the possibility of modifying the natural tropism of the adenovirus to allow good infection of B lymphocytes, preferably without increasing the natural spectrum of host cells of this virus. This is already very important because the natural adenovirus receptor is present on the surface of a large number of cell types.

 The present application now describes the construction and production of adenoviral vectors capable of infecting these cells, via a well-defined peptide recognizing a specific B receptor. The present application also describes recombinant adenoviral vectors exhibiting a modified and restricted tropism for B lymphocytes. B lymphocytes not being natural targets for adenoviruses, the

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 The present invention shows that it is possible to develop a tool for targeting these cells.

 A first object of the invention lies more particularly in a recombinant adenovirus, characterized in that it comprises a fiber whose sequence comprises a targeting peptide specific for a CD21 receptor.

 Another aspect of the invention resides in a process for the preparation or production of a recombinant adenovirus as defined above, as well as in the tools and constructions which can be used for this purpose (vectors, chimeric genes, cells, etc.) .

 Another aspect of the invention resides in a pharmaceutical composition comprising at least one recombinant adenovirus as defined above, and in its use for the transfer of genes into B lymphocytes, in particular for the treatment of pathologies associated with dysfunction of these cells or affecting these cells.

 Another object of the invention lies in the use of a recombinant adenovirus as defined above for the preparation of a composition intended for the transfer of genes into B lymphocytes in vivo, ex vivo or in vitro.

 Another aspect of the invention relates to an adenovirus fiber protein modified as defined above, any nucleic acid encoding such a protein, as well as their uses.

 The invention therefore relates, in general, to recombinant adenoviruses having a tropism for B lymphocytes.

 The term recombinant adenovirus designates, within the meaning of the invention, a modified viral particle comprising a viral genome contained in a capsid.

 The adenoviral genomes are double stranded linear DNA of approximately 36kb.

 They have two terminal regions, the ITR (Inverted Terminal Repeat) which are the

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 origins of viral genome replication and an encapsidation sequence (#). In addition, several regions (E1 to E4) called early encode elements necessary for the transcription and replication of the viral genome, and regions called late (L1 to L5) code the structural proteins of the virus.

 The recombinant adenoviral genomes constructed for gene therapy are deleted from (or defective for) Ela and / or Elb from the El region, the products of these genes being essential for viral production. This deletion (or this defective character) prevents possible dissemination of the vector in the organism. The sequence of interest is generally introduced in place of this region.

 Given the strong immunogenicity of the adenovirus and to prevent the risk of the emergence of RCA (Competent Adenovirus Replication) forms, other viral genomes deleted from other genes have been described. Thus defective recombinant adenoviruses for the following regions:: ElA; ElAetElB E1 and E3; E1 and E4 (in particular ORF3 or ORF6); Elet E2; Elet E2 and E3; Elet E2 and E4; Elet E3 and E4 were built. In the same vein, recombinant genomes lacking all the viral genes and comprising only the ITRs, the packaging sequence and the DNA sequence of interest have also been described. In a particular variant, the recombinant adenoviruses of the invention comprise a recombinant adenoviral genome comprising two ITR terminal regions, an packaging sequence (y) and a DNA sequence of interest, the transfer or expression of which in a determined host is desired. Preferably, they comprise an adenoviral genome defective for the El region at least.

 An essential characteristic of the recombinant adenoviruses of the invention lies in the presence of a modified fiber, which gives these viruses a particular tropism, and in particular the capacity to infect B lymphocytes. In this context, the invention now describes the construction of the gene encoding a chimeric fiber comprising a peptide specifically recognizing the CD21 receptor.

 The CD21 receptor is a surface marker for B lymphocytes (Nemerow et al., 1989). The CD21 (or CR2) glycoprotein is the EBV (Epstein Barr) receptor

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 Virus), which is also recognized by the complement C3dg. In order to allow a preferential infection of B lymphocytes, the inventors have developed adenoviruses possessing, in their fiber, a peptide specifically recognizing the CD21 receptor.

 The peptide chosen was defined on the basis of the sequence of the part of the glycoprotein GP350 / 220 (EBV envelope protein) responsible for the recognition of CD21. In 1987, the glycoprotein GP350 / 220 of the EBV envelope was defined as being responsible for the recognition of the CD21 receptor (Nemerow et al., 1987; Tanner et al., 1987). In addition, the comparison of the peptide sequence of this protein to that of the complement C3dg revealed two regions of strong homology: EDPGFFNVE for GP350 / 220 and EDPGKQLYNVE for C3dg, the latter being responsible for the recognition of C3dg to CD21 (Paneling et al., 1985).

 This is how the EDPGFFNVE region was defined as being responsible for EBV / CD21 recognition. In addition, experiments have demonstrated that this peptide sequence is capable of inhibiting the infection of B lymphocytes by this virus (Nemerow et al., 1989).

 Different peptides and different locations in the fiber were selected in order to have an optimal conformation of the sequence of interest on the surface of the fiber, and to allow a good interaction with the receptor.

 More particularly, the modified fiber according to the invention comprises a ligand peptide of a CD21 receptor comprising the sequence GEDPGFFNVE or a functional part thereof. Other targeting peptides are the peptides of sequences GEDPGFFNVEIP and GEDPGFFNVEIPEFP, which have been used in order to have an optimal conformation for the recognition of CD21. Targeting peptides according to the invention include any peptide comprising between 7 and 20 amino acids, preferably between 10 and 18, even more preferably between 11 and 17, of synthetic, natural or mixed origin, capable of binding the CD21 receptor to the surface of a B cell, preferably selectively. The term selectively indicates that the

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 binding to the CD21 receptor is more affine than binding to other cellular markers, although such non-specific binding cannot be completely excluded. The term functional part designates any fragment of a sequence defined above, or any derivative of such a sequence, which retains the ability to interact selectively with a CD21 receptor.

 The targeting peptide can be introduced into different positions in the adenovirus fiber, either as a replacement for existing sequences, or as a supplement. Preferably, the sequence encoding the targeting peptide is introduced at the 3 ′ end of the fiber gene (and the targeting peptide is therefore found at the C-terminal end of the fiber protein) or in the HI loop sequence (the HI loop being between the 535th and 547th residue in the fiber sequence of the adenovirus serotype 5).

 Furthermore, the targeting peptide can be linked directly to the fiber protein, or via a spacer sequence. Such a sequence is used essentially when the targeting peptide is introduced at the terminal end of the fiber. The spacing sequence does not affect the functionality of the targeting peptide and allows for greater flexibility thereof. By way of example, there may be mentioned in particular a poly-lysine sequence, (SG) i, in which i is an integer between 1 and 7, etc. A preferred spacer sequence is the sequence (Ser-Gly) 5-Ser.

Specific constructs according to the invention are fibers modified at the C-terminal end by adding a targeting peptide via a spacer sequence. The sequence of the C-terminus of the fiber is for example as follows: --- AQE- (SG) 5-S-targeting peptide.

 In a particular embodiment, the fiber further comprises one (or more) additional alteration reducing or blocking its interaction with the CAR receptor. Such alterations may include, for example, mutations

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 in the DG loop of the fiber button, which does not alter the trimerization but significantly reduces the interaction of the virus with its CAR receptor (Kirby et al., 1999). Alternatively, the adenovirus fiber button can be replaced by the domain that allows trimerization of the bacteriophage T4 fibritin (Krasnykh et al., 2001). Another possible alteration consists in replacing the fiber button of serotypes 2 and 5 (Had2, Had5) by the fiber button of a non-Mastadenovirus adenovirus.

 Such an alteration makes it possible to restrict the tropism of the recombinant adenovirus, and therefore to increase its specificity for B lymphocytes. Such specificity is of course particularly advantageous for use in gene therapy because it makes it possible to reduce the doses to be administered. This is an important advantage because the adenovirus is very immunogenic and can induce a strong undesirable immune reaction.

 Such an exchange was carried out using the button of the adenovirus BAV4 (Bovine AdenoVirus 4). The viruses were constructed, amplified and purified and initial tests made it possible to observe that the adenovirus thus modified infects human epithelial cells in a reduced manner (by a factor of 10-50).

 In this regard, another object of the invention resides in an adenovirus fiber protein comprising a targeting peptide specific for a CD21 receptor. In a preferred mode, the fiber further comprises a modification reducing the interaction with the CAR receptor.

 Another object of the invention resides in a nucleic acid encoding a fiber protein as defined above. It may be DNA or RNA, preferably DNA. Another object of the invention resides in a vector comprising such a nucleic acid, this vector possibly being a plasmid, virus, phage, cosmid, etc.

 The modified fibers can be constructed by genetic engineering, according to techniques known to those skilled in the art, such as for example by artificial synthesis,

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 cloning, ligation, enzymatic cleavage, amplification, site-directed mutagenesis, junction of oligonucleotides, etc.

 In a particular embodiment, the adenovirus according to the invention is produced from an adenovirus of serotype 2 or 5.

 The recombinant adenovirus comprising the modified fiber can be produced by various methods. Thus, the gene encoding the modified fiber can be introduced into the adenoviral genome, or introduced, in integrated or extra-chromosomal form, into the genome of the virus-producing cell.

 In this context, another subject of the invention relates to a process for the production of a recombinant adenovirus as defined above, comprising (i) the construction of a gene encoding the modified fiber, in particular encoding the fiber whose sequence comprises a targeting peptide specific for a CD21 receptor, (ii) the introduction of this gene into an adenoviral genome, (iii) the introduction of the adenoviral genome into an packaging cell, (iv) the amplification of the viruses and (v) purification of the viruses produced.

 The packaging cell used can be any permissive cell comprising or expressing the defective adenoviral proteins of the recombinant genome. In the case of an adenoviral vector defective for the E1 region, any cell expressing the E1 region of an adenovirus is used, such as cells 293, PER. C6, etc. It is also possible to use cells co-transfected with a plasmid or a helper virus providing the necessary functions.

 Another subject of the invention relates to a viral particle comprising an adenovirus fiber as defined above.

 Another subject of the invention relates to a method for the transfer of a nucleic acid into a B lymphocyte in vitro, ex vivo or in vivo, comprising bringing an organism, tissue, biological sample or cells comprising lymphocytes into contact B with a recombinant adenovirus, said recombinant adenovirus comprising said nucleic acid and comprising a modified fiber comprising a targeting peptide specific for a CD21 receptor.

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 Another subject of the invention relates to a pharmaceutical composition comprising a recombinant adenovirus as defined above and a pharmacologically acceptable vehicle. The vehicle or excipient can be any solution, suspension, gel, powder, etc., compatible with pharmaceutical use, such as in particular an isotonic, buffered, saline solution, etc.

 The invention can be used for the transfer and expression of nucleic acids of interest in B lymphocytes, in experimental or therapeutic approaches.

The nucleic acid can be a suicide gene, that is to say any nucleic acid encoding a protein toxic to the cell, either directly or conditionally (eg, TK, cytosine deaminase, etc.), a nucleic acid encoding an anti-tumor factor, a cytokine, an antisense, an antigen, a marker, etc. (such as, for example, I12, 1112, anti-bcr / abl anti-sense RNA). The invention can be used in the therapeutic field, for the preventive or curative treatment of pathologies involving or affecting B lymphocytes, such as tumor pathologies (leukemias, lymphomas, etc.), immune, inflammatory diseases, etc.

 Other aspects and advantages of the invention will appear on reading the examples which follow, which should be considered as illustrative and not limiting.

Caption of Figures Figure 1: of the last stage of construction of viral genomes having a fiber gene modified by homologous recombination in E. coli (after Renaut, L et al, 2002).

 Figure 2: shuttle allowing the insertion of fibers modified by molecular cloning between the MluI and Sali sites and homologous recombination with the vector pRECad.

 Figure 3: pRECad, its digestion with SwaI releases the viral genome.

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Example 1. Construction of chimeric fibers comprising a peptide targeting B cells.

1. Selection of targeting peptides
The targeting peptides chosen include the sequence GEDPGFFNVE or a functional part thereof. In order to verify that the peptide produced is capable of binding and then being internalized in B lymphocytes, its internalization in different cell types was tested, after fluorescent labeling of the peptides by confocal microscopy. The results obtained demonstrate that the peptide is capable of binding and being internalized in B lymphocytes.

2. Construction of genes encoding chimeric fibers
In order to effect the modifications to the fiber, the gene encoding the adenovirus 2 fiber (Had2) was cloned into pBluescript-II-SK (Stratagene) between the NotI and SalI restriction sites. PCR was performed on the Had2 genome with the primers 5'-AggAAAAAAgCggCCgCATgAAACgCgCCAgAC-3 'in 5' and 5'AggAAAAAAggTCgACTTATATCgATTCCTgggCAATgTAg-3 'in 3'. The second primer contains a ClaI site immediately before the Stop codon.

The oligonucleotides 5'- CgggTTCAggATCCggAgAggATCCCgggTTTTTCAACgTTgAATgTTAACg-3 'and 5'TCgACGTTAACATTCAACGTTGAAAAACCCGGGATCCTCTCCGGATCCTGAA end of the gene coding for the peptide EB is the gene peptide EB-3. A spacer sequence was also introduced between the sequence encoding the fiber and that encoding the EBV peptide. This sequence makes it possible to have no bulk and thus to keep an optimal conformation of the fiber and of the peptide. The C-terminus of the fiber has the sequence AQE (SG) 6EDPGFFNVEC.

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For the long peptide, the primers 5'AggAAAAAACgTCgACgTTAgAACgggAATTCgggAATTTCAACgTTgAAAAACC Cggg-3 'and 5'-CCCCCTggggTACTCTCTTTACgCgTATCCgAACCTCTAgTTACC-3' were used. The first hybridizes with the 3 'end of the fiber gene with the short peptide and makes it possible to lengthen the latter and the second hybridizes at the MluI site located 147 bp from the 5' end of the gene. the fiber. This PCR fragment was cloned in place of the MluI / SalI fragment of wild fiber cloned in pBs-sK.

The C-terminus of the resulting fiber has the sequence AQE (SG) 6EDPGFFNVEIPEFP.

With regard to the insertion of the peptides at the level of the HI loop, oligonucleotides were inserted into a SpeI site located at 1615 bp from the 5 ′ end of the fiber gene cloned in pBluescript-II-SK. For the short peptide, the 5'-CTAgCggCgAAgATCCCgggTTTTTCAACgTTgAAATTC-3 'oligonucleotides and 5'CTAggAATTTCAACgTTgAAAAACCCgggATCTTCgCCg-3' were used and for the long peptide, oligonucleotides 5'CTAgCggCgAAgATCCCgggTTTTTCAACgTTgAAATTCCCgAATTCC-3 'and 5'CTAgggAATTCgggAATTTCAACgTTgAAAAACCCgggATCTTCgCCg-3' were used.

 3. Expression of Chimeric Fibers and Functionality The genes encoding the modified fibers were transferred into a plasmid which contains, at 5 'to the gene of interest, the element of bacteriophage T7 allowing transcription in vitro. Thus, in order to demonstrate that the modified fibers are capable of trimerizing, in vitro syntheses were carried out with the in vitro transcription / translation kit from Promega and their products separated on non-denaturing polyacrylamide gel.

Example 2. Construction and Production of Recombinant Adenovirus Comprising a Chimeric Fiber Comprising a B Cell Targeting Peptide, and Functionality

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 The genes encoding the modified fibers were then cloned into a shuttle vector to allow their introduction into a viral genome previously constructed (Renaut et al., 2002b), in place of the gene encoding the wild fiber. This step was carried out by homologous recombination in E. coli (Chartier et al., 1996). The shuttle vector used contains 5502 bp from the right end of the Had5 genome which were amplified with the primers 5'-TTgTATAAAAAgCgCggCCgCAACTACCTTGCCTACCACTC-3 'which hybridizes to 25690 bp in the viral genome and primer 5'TTgTATAAAAAgCgTCgACTTCgTCgTCgTCgTCgTCgTCgTCgTCgTCgTCgTCgTCgTCgTCtTCg -3 'which hybridizes to 34322 bp in this same genome. This fragment was amplified from pBHGl 1 (Graham et al., 1989) which is a plasmid containing the entire Had5 genome, in the part which interests us, a fragment of 3130 bp containing the E3 region between 27865 bp and 30,995 bp had been deleted.

This fragment was cloned between the NotI and XhoI sites in pBS-SK, and a MluI site, absent in the Had5 fiber, was added with the complementary primers 5'CCCCCTggggTACTCTCTTTACgCgTATCCgAACCTCTAgTTACC-3 'and 5'ggTAACTAgAgGAggggggtgggg . Similarly, a SalI site was inserted by mutagenesis at the 3 'end of the fiber gene, upstream of the polyA addition site of the L5 region using the primers 5'- CTTACACTTTTTCATACATTgTCgACgAATAAAgAATCgTTTgTgTTATg-3' and 5 'CATAACACAAACgATTCTTTATTCgTCgACAATgTATgAAAAAgTgTAAg-3'.

At this stage, the different genes coding for the modified fibers have been cloned into the shuttle vector in place of the gene coding for the wild fiber by MluI / SalI. After a homologous recombination step in E. coli, we obtained the recombinant genomes carrying the genes encoding the modified fibers (figurel).

After digestion with SwaI, thus releasing the viral genomes from plasmids, these were introduced into HEK293 cells (Graham et al., 1977) by lipofection. These cells allow the production of recombinant viruses because they express E1 which allows transcomplementation since this gene is deleted in the genomes of the viruses previously obtained.

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A recombinant adenovirus was thus constructed and produced. The apical proteins of the capsid are Had2 fibers with, at their C-terminal end, the targeting peptide. This virus was produced with a titer equivalent to that of the virus carrying wild fiber (control virus), and it infects epithelial cells with the same efficiency as the control virus. These results demonstrate that the fact of having added this peptide does not hinder either the assembly of the virus or its intracellular traffic which is important for the transport of viral DNA and therefore of the gene of interest to the nucleus.

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Claims (18)

1. Recombinant adenovirus characterized in that it comprises a fiber whose sequence comprises a targeting peptide specific for a CD21 receptor.  2. Adenovirus according to claim 1, characterized in that the targeting peptide comprises the sequence GEDPGFFNVE, GEDPGFFNVEIP or GEDPGFFNVEIPEFP, or a functional part thereof.  3. Adenovirus according to claim 1 or 2, characterized in that the targeting peptide is introduced at the C-terminal end of the fiber or in the sequence of the HI loop.  4. Adenovirus according to any one of the preceding claims, characterized in that the targeting peptide is linked directly to the protein of the fiber, or via a spacer sequence.  5. Adenovirus according to claim 4, characterized in that the spacer sequence comprises an amino acid sequence (SG) j in which i is an integer between 1 and 7.  6. Adenovirus according to any one of the preceding claims, characterized in that the fiber further comprises one (or more) additional alteration of the button of the fiber reducing or blocking its interaction with the CAR receptor.  7. Adenovirus according to claim 6, characterized in that the additional alteration is chosen from a mutation in the DG loop of the fiber button, the replacement of the fiber button by the domain which allows the trimerization of the bacteriophage fibritin T4 and replacement of the fiber button with the fiber button of a non-Mastadenovirus adenovirus. <Desc / Clms Page number 19>  8. Adenovirus according to any one of the preceding claims, characterized in that it comprises a recombinant adenoviral genome comprising two ITR terminal regions, an packaging sequence (#) and a DNA sequence of interest, the transfer of which or expression in a specific host is desired.  9. Adenovirus according to claim 6, characterized in that it comprises an adenoviral genome defective for the El region.  10. Adenovirus according to any one of the preceding claims, characterized in that it is produced from an adenovirus of serotype 2 or 5.  11. Method for producing a recombinant adenovirus according to one of claims 1 to 10, comprising (i) the construction of a gene encoding the fiber whose sequence comprises a targeting peptide specific for a CD21 receptor, (ii ) the introduction of this gene into an adenoviral genome, (iii) the introduction of the adenoviral genome into an packaging cell, (iv) the amplification of the virus and (v) the purification of the viruses produced.  12. Adenovirus fiber protein, characterized in that it comprises a specific targeting peptide for a CD21 receptor.  13. Protein according to claim 12, characterized in that it further comprises a modification of the button of the fiber reducing the interaction with the CAR receptor.  14. Nucleic acid encoding a fiber protein according to claim 12 or 13.  15. Vector comprising a nucleic acid according to claim 14.  16. Method for the transfer of a nucleic acid into a B lymphocyte in vitro or ex vivo, comprising bringing an organism, tissue, biological sample or cells comprising B lymphocytes into contact with a recombinant adenovirus, <Desc / Clms Page number 20>  said recombinant adenovirus comprising said nucleic acid and comprising a modified fiber comprising a targeting peptide specific for a CD21 receptor.  17. Pharmaceutical composition comprising a recombinant adenovirus according to one of claims 1 to 10 and a pharmacologically acceptable vehicle.  18. Use of a recombinant adenovirus according to one of claims 1 to 10 for the preparation of a composition intended for the transfer of genes into B lymphocytes.