The subject of the present invention is the use of all or part of an antigen of the class I major histocompatibility complex and/or of a type III module of fibronectin to allow or facilitate the attachment of an adenovirus onto a host cell and/or its entry into the latter. The invention also relates to the use of a ligand capable of modulating the infectivity of an adenovirus toward a host cell, mediated by either of the polypeptides mentioned above. Finally, the invention relates to a biopanning method for identifying or selecting a cellular receptor for an adenovirus or one of these ligands, in particular of viral origin.
Adenoviruses are DNA viruses with a broad host spectrum. They have been detected in numerous animal species and can infect various cell types. Numerous serotypes have been characterized within each species which exhibit a genomic organization and an infectious cycle which are comparable. In general, the adenoviral genome consists of a double-stranded linear DNA molecule of about 36 kb containing the genes encoding the viral proteins and, at its ends, two inverted repeats (designated ITRs) which are involved in replication and the encapsidation region.
Adenoviruses replicate in the nuclei of the cells infected. The infectious cycle occurs in two stages. The early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and the transcription of the viral DNA. These stages are followed by the late phase during which the structural proteins which constitute the viral particles are synthesized. The assembling of the new virions takes place in the nucleus. In the first instance, the viral proteins assemble so as to form empty capsids having an icosahedral structure, into which the adenoviral DNA is encapsidated. The viral particles are released and are capable of infecting other permissive cells. In this regard, the fiber and the penton base which are present at the surface of the capsids play a critical role in the cellular attachment of the virions and their internalization.
The adenovirus binds to the surface of permissive cells through the intermediacy of the trimeric fiber and a cellular receptor which has so far not been identified. Next, the particle is internalized by endocytosis through the binding of the penton base to the cellular integrins xcex1vxcex23 and xcex1vxcex25 (Belin and Boulanger, 1993, J. Gen. Virol. 74, 1485-1497; Mathias et al., 1994, J. Virol. 68, 6811-6814; Nemerow et al., 1994, Trends Cell. Biol. 4, 52-55; Wickham et al., 1993, Cell 73, 309-319; Wickham et al., 1994, J. Cell Biol. 127, 257-264). The Ad2 fiber comprises 580 amino acids (aa) whose sequence is disclosed in Herissxc3xa9 et al. (1981, Nucleic Acid Res. 9, 4023-4042). That of Ad5 has 582 amino acids (Chroboczek and Jacrot, 1987, Virology 161, 549-554). Its molecular mass is 62 kDa, but the native fiber behaves like a 160-180 kDa molecule, confirming its assembly in the form of a trimer.
The fiber is composed of 3 domains (Chroboczek et al., 1995, Current Top. Microbiol. Immunol. 199, 165-200):
(1) At the N-terminus, the xe2x80x9ctailxe2x80x9d, which is highly conserved from one serotype to another, interacts with the penton base and ensures the anchorage of the molecule in the capsid.
(2) The xe2x80x9cstemxe2x80x9d is a structure in the form of a rod of variable length depending on the serotypes. For example, the stem of the Ad5 fiber contains 22 repeats of a motif of 15 residues which could adopt a xcex2 sheet conformation. The number of these repeats differs from one serotype to another, which explains the variations in length.
(3) Finally, at the distal end of the stem, the xe2x80x9cheadxe2x80x9d or terminal sphericle is a globular structure containing trimerization signals (Hong and Engler, 1996, J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J. Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991, Virology 185, 365-376). Most of the experimental data show that it is the head domain which is responsible for the binding to permissive cells (Krasnykh et al., 1996, J. Virol. 70, 6839-6846).
(4) The complexity of the adenoviral attachment suggests that it could be serotype-dependent and that several cellular proteins could participate in it. As regards Ad2, Hong and Boulanger (1995, EMBO J. 14, 4714-4727) have identified a number of peptide motifs found in several cellular surface proteins which are capable of interacting with the capsid proteins (penton base and fiber), in particular the type III 5 and 14 modules of human fibronectin. The authers proceeded by immobilizing, on an inert support, penton base or fiber (ligand) with which they reacted a library of phages expressing random hexapeptides (designated phagotopes). The phages adsorbed, which in theory express phagotopes interacting with a motif carried by the adenoviral protein, are then eluted either conventionally at acidic pH or by competition with the other nonimmobilized capsid partner (eluent). However, the cellular receptor for adenoviruses and the region of the head precisely involved in the binding to the receptor have so far not yet been clearly identified.
A new technique of xe2x80x9cbiopanningxe2x80x9d has now been carried out in which the immobilized ligand consists of the head domain of the Ad5 fiber and the eluent consists of a neutralizing antibody directed against the latter and two classes of phagotopes isolated depending on the antibody used. The first corresponds to a conserved sequence within the xcex1-2 domain of the antigens of the class I major histocompatibility complex (xcex1-2 MHC-I) and the second to a sequence found in the III modules of human fibronectin (FNIII). The data presented in the examples which follow support the hypothesis that the xcex1-2 MHC-I constitutes the primary receptor for the serotype C adenoviruses and confirm the participation of the FNIIIs as coreceptor or cofactor. The regions of these two receptors and of the fiber which interact with each other have also been identified. In addition, an antagonist peptide has been generated which reproduces the motif of the xcex1-2 MHC-I domain which neutralizes the attachment of adenoviruses and an agonist peptide reproducing the FNIII motifs which stimulates attachment.
Accordingly, the subject of the present invention is the use of a polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown:
(a) in SEQ ID NO: 1 starting with the leucine residue at position 1 and ending with the glutamine residue at position 25,
(b) in SEQ ID NO: 2 starting with the asparagine residue at position 1 and ending with the asparagine residue at position 26,
(c) in SEQ ID NO: 3 starting with the valine residue at position 1 and ending with the asparagine residue at position 25,
(d) in SEQ ID NO: 4 starting with the serine residue at position 1 and ending with the arginine residue at position 25, and/or
(e) in SEQ ID NO: 5 starting with the asparagine residue at position 1 and ending with the serine residue at position 25; to allow or facilitate the attachment of an adenovirus to a host cell and/or the entry of the said adenovirus into the said host cell.
For the purposes of the present invention, xe2x80x9cpolypeptidexe2x80x9d is understood to mean any molecule consisting of a succession of at least 6, and preferably of at least 8, amino acids. The term polypeptide comprises both peptide molecules of short length (from 6 to a few tens of residues) and molecules which are longer (up to several hundreds of residues), provided, however, the envisaged use is allowed. It is specified that a polypeptide in use within the framework of the present invention may be derived from a native polypeptide as found in nature, in particular in humans, or a portion thereof. It may also be a chimera and comprise additional residues of any origin fused at the N- and/or C-terminus and/or inserted so as to form an open reading frame. It is also possible to use a mutant obtained by mutation, deletion, insertion and/or substitution of one or more amino acids relative to the sequences disclosed in the sequence identifiers (SEQ ID).
A preferred polypeptide within the framework of the present invention comprises, in addition, appropriate elements to ensure its anchorage in a cell membrane or its presentation at the surface of a cell. Such elements are known to persons skilled in the art. As a guide, there may be mentioned the presence of a signal peptide generally associated at the N-terminal position and of a transmembrane region exhibiting a high degree of hydrophobicity. However, use may also be made of other techniques, for example chemical techniques, to anchor or bind a polypeptide to a membrane or a cell surface.
xe2x80x9cHomologous amino acid sequencexe2x80x9d is understood to mean a sequence having a degree of homology of at least 70%, advantageously of at least 80%, preferably of at least 90% with at least 6 continuous amino acids of one of the sequences mentioned. The term identical refers to 100% homology. Persons skilled in the art know the general rules which make it possible to calculate the degree of homology between two sequences. The procedure is generally carried out by aligning sequences possibly with the aid of specialist computer programs. It may be necessary to artificially introduce vacant positions. Once the optimum alignment has been achieved, the degree of homology is established by counting all the positions in which the amino acids of the two sequences are found to be identical, relative to the total number of positions.
xe2x80x9cAttachment of an adenovirus to a host cellxe2x80x9d is understood to mean the binding of the viral particle to the cell. xe2x80x9cEntry of an adenovirus into a host cellxe2x80x9d denotes the penetration of the virus into the host cell. The attachment and/or the entry are preferably mediated, at least in part, by the polypeptide(s) in use within the framework of the present invention by interaction with the adenoviral capsid. Of course other polypeptide or nonpolypeptide molecules may also participate in these processes which are recognized in the art to be complex and multifactorial. They can be evaluated by any prior art technique, such as those described below using a permissive cell line and particles which are radio-actively labeled or which express a reporter gene, for example the luciferase gene. At 0xc2x0 C., only the attachment can take place, the viral penetration requiring a temperature of 37xc2x0 C.
For the purposes of the present invention, an adenovirus may be of human or animal (canine, avian, bovine and the like) or hybrid origin comprising genome fragments. These viruses and their genome are described in the literature (see for example Graham and Prevec, Methods in Molecular Biology, Vol. 7; Gene Transfer and Expression Protocols; Ed: E. J. Murray, 1991, The Human Press Inc., Clinton, N.J.). A replication-defective recombinant adenovirus expressing in particular a gene of therapeutic interest is preferred. Advantageously, the adenoviral genome is modified by deletion or mutation of sequences essential for replication and, in particular, contained in the E1, E2, E4 and/or L1-L5 regions (see for example international application WO 94/28152).
According to a first variant, the subject of the present invention is the use of a polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 1 starting with the leucine residue at position 1 and ending with the glutamine residue at position 25.
Advantageously, a polypeptide in use within the framework of the present invention comprises an amino acid sequence homologous or identical to all or part of an antigen of the class I major histocompatibility complex (MHC-I) and, preferably, of the heavy chain of the latter.
All the cells of an organism have on their membrane molecules called histocompatibility antigens which define each individual. The corresponding genes, more than about ten, are located on chromosome 6 in humans and exhibit high polymorphism, which makes it possible to ensure a high variability of these identity markers. There are two different categories of these histocompatibility antigens, classes I and II respectively, whose structure and functions are distinct. The class I molecules, called HLA (for Human Leukocyte Antigen), are involved in presenting antigenic peptides at the cell surface and play an essential role in the antiviral immune responses exerted by the cytotoxic T lymphocytes.
The MHC-I molecules are heterodimers composed of a non-MHC light chain designated xcex22-microglobulin (xcex22m) and a heavy chain encoded by the MHC genes, which are noncovalently linked. The heavy chain is a membrane protein whose N-terminal part is oriented outside the cell whereas the C-terminal portion is cytoplasmic. The former comprises 3 domains designated xcex11, xcex12 and xcex13 having about 90 amino acids in each. It is followed by a transmembrane region of about 25 amino acids and then the C-terminal region of about thirty amino acids. Most of the variations between the products of the different alleles are located in the xcex11 and xcex12 domains, the xcex13 domain being relatively conserved and xcex22m being invariable (for a review and the sequence comparison between the members of the MHC-Is, see Bjorkman and Parham, 1990, Annu. Rev. Biochem. 59, 253-288).
Among the polypeptides suitable for the purposes of the present invention, there may be mentioned more particularly the HLA A, B, C, D, E and F antigens or polypeptides derived therefrom.
In a particularly advantageous manner, the polypeptide in use within the framework of the present invention comprises a sequence homologous or identical to all or part of the C-terminal region of the xcex12 domain of the MHC-I heavy chain and, more particularly, to the part centered on the tryptophan residue at position 167, in particular that extending from residues 156 to 180 (SEQ ID NO: 1). The numbering to which reference is made is in accordance with that used, for example, in Bjorkman and Parham (1990, supra).
According to another variant, a polypeptide in use within the framework of the present invention comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown.
in SEQ ID NO: 2 starting with the asparagine residue at position 1 and ending with the asparagine residue at position 26,
in SEQ ID NO: 3 starting with the valine residue at position 1 and ending with the asparagine residue at position 25,
in SEQ ID NO: 4 starting with the serine residue at position 1 and ending with the arginine residue at position 25, and/or
in SEQ ID NO: 5 starting with the asparagine residue at position 1 and ending with the serine residue at position 25.
A preferred polypeptide comprises an amino acid sequence homologous or identical to fibronectin and, in particular, to at least one of its type III modules and, in particular, to modules FNIII 1, 4, 5 and/or 14. Of course, it may comprise several of them. It is also possible to envisage using human fibronectin or a peptide derived therefrom, for example, by mutation or fragmentation. As a guide, the fibronectin encoded by a single gene is a molecule which is involved in adhesion and cell contact phenomena. Its sequence and its characteristics are described in the literature accessible to persons skilled in the art (see in particular Bork and Doolittle, 1992, Proc. Natl. Acad. Sci. USA 89, 8990-8994 and Dickinson et al., 1994, 236, 1079-1092). It is composed of 14 so-called type III modules (numbered from 1 to 14) whose primary sequence may vary, but whose xcex2-sheet conformation is conserved.
According to a particularly advantageous embodiment, a polypeptide as defined above is more particularly intended to allow or to facilitate the attachment of a serotype C adenovirus to a host cell and/or its entry into the latter. Among the adenoviruses which may be envisaged, there may be mentioned more particularly serotypes 2 and 5.
The present invention also relates to a host cell capable of expressing a polypeptide in use within the framework of the present invention and its use to allow or to facilitate the attachment of an adenovirus to its surface and/or the entry of the said adenovirus. Various types of host cells may be considered. They may be cells of any origin, for example of microorganisms, yeasts, insects, plants or animals. A mammalian cell and, in particular, a human cell of the primary or tumor type or derived from a line which can be cultured in vitro will be preferred in particular. It may be of a hematopoietic (totipotent stem cell, leukocyte, lymphocyte, monocyte, macrophage and the like), hepatic or renal origin, from the central nervous system, fibroblast, epithelial, pulmonary or muscular (myocyte, myoblast, satellite cell, cardiomyocyte and the like) origin. A particularly preferred cell is or is derived from the 293 line established from embryonic kidney cells by integration of the adenoviral E1 region (Graham et al., 1977, J. Gen. Virol. 36, 59-72). It is indicated that the expression of one or more polypeptides in use within the framework of the present invention at the surface of a host cell not usually expressing the MHC-Is and/or fibronectin should allow its infectivity by an adenovirus. It could be used as a new cell producing adenoviral vectors. It is also possible to envisage the case of an overexpression in a cell naturally expressing the said polypeptide. An overexpressing line derived from the 293 line should make it possible to improve the yields of production of an adenovirus of interest. Of course, the polypeptide in use within the framework of the present invention may be associated with the cell by chemical means or by means of a ligand recognizing a cell surface protein. However, it is also possible to envisage expression by recombinant DNA techniques. Such an embodiment is within the capability of persons skilled in the art. As a guide, the nucleotide sequence encoding the polypeptide in question may be isolated (by standard PCR or cloning techniques) or chemically synthesized before being inserted into a conventional expression vector under the control of appropriate regulatory elements, the vector being introduced into the host cell by any prior art technique. The host cell in use within the framework of the present invention may also be modified so as to complement a defective adenovirus by transfection of (an) appropriate fragment(s) of adenoviral genome.
The subject of the present invention is also the use of a ligand capable of influencing the attachment of an adenovirus to a host cell and/or its entry into the latter, which are mediated by a polypeptide as defined above. The ligand in use in the invention may be of any type. There may be mentioned, for example, the peptides, hormones, antibodies or derivatives thereof and, in particular, single-chain antibodies of the scFv (for single chain fragment variable) type and soluble receptors lacking their transmembrane region. In particular, such a ligand may be derived from a polypeptide in use in the present invention. In accordance with the aims pursued by the present invention, the ligand may have a negative (antagonist) or positive (agonist) influence. Preferably, a preferred ligand has a dissociation constant with respect to the adenovirus of between 0.01 and 100 nM, advantageously between 0.1 and 50 nM, and most preferably between 0.5 and 10 nM.
In the case of an antagonist, the interaction of the ligand with the fiber will make it possible to reduce or inhibit the process of attachment and/or of entry of an adenovirus. In this context, a particularly preferred ligand is based on a polypeptide as defined in SEQ ID NO: 1. By way of example, there may be mentioned a polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids contained in the sequence as shown in SEQ ID NO: 6 starting with the arginine residue at position 1 and ending with the arginine residue at position 20. The use of the peptide designated MH20 in the examples which follow will be preferred.
In the case of a positive influence, the ligand in use within the framework of the present invention is used to allow or to stimulate the attachment and/or the entry of adenoviruses. A ligand which is suitable for the purposes of the invention comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 7 starting with the arginine residue at position 1 and ending with the serine residue at position 20. A preferred example consists of the peptide designated below FN20.
The present invention also relates to a ligand comprising an amino acid sequence homogous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 6 or 7.
However, it may also be a ligand of adenoviral origin. According to this embodiment, a preferred ligand is derived from the fiber of an adenovirus, in particular from the part of the head which interacts with the abovementioned polypeptides. A peptide motif chosen in this region therefore ought to influence the infectivity of the adenoviruses with respect to a host cell expressing the polypeptide. Advantageously, a ligand covering residues 438 to 486 of the fiber of an adenovirus is used. More particularly, a ligand of a polypeptide as defined by SEQ ID NO: 1 is preferably derived from an Ad5 and comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 8, starting at the amino acid leucine at position 1 and ending at the amino acid aspartic acid at position 18. A ligand which may also be envisaged may be derived from the fiber of a serotype 2 adenovirus and may comprise an amino acid sequence homologous or identical to at least 6 amino acids of the sequence as shown in SEQ ID NO: 9 starting at the threonine residue at position 1 and ending at the valine residue at position 16.
The ligand of a polypeptide as defined by SEQ ID NO: 2 to 5 is more particularly characterized by an amino acid sequence homologous or identical to at least 6 amino acids of the sequence as shown in SEQ ID NO: 10 starting at the leucine residue at position 1 and ending at the threonine residue at position 14 (Ad5) or in SEQ ID NO: 11 starting at the asparagine residue at position 1 and ending at the asparagine residue at position 13 (Ad2).
The subject of the present invention is also the use of a ligand according to the invention for the preparation of a medicament intended to inhibit or reduce an infection by an adenovirus. In this context, the use of an antagonist ligand with a therapeutic or prophylactic objective will be preferred. The use of a ligand according to the invention, preferably an agonist ligand, is appropriate for the preparation of a medicament intended to promote or facilitate an infection by an adenovirus, and in particular a recombinant adenovirus carrying a therapeutic gene intended for gene (curative) or anti-viral (AIDS) or anti-cancer therapy. Such a medicament finds it usefulness, for example, in association with gene therapy treatments so as to improve viral infection in a patient treated with a recombinant adenovirus. It is possible to envisage a parenteral or oral administration or alternatively administration by aerosol. The administration may be made in a single dose or a dose repeated once or several times after a certain period of time. The appropriate dosage and formulation vary according to different parameters, for example the individual, the disease to be treated, the desired effect, the route of administration or alternatively the adenovirus in question.
The present invention also relates to a method for selecting or identifying a cellular receptor for a virus in an appropriate sample, comprising:
(a) the immobilization, onto an inert support, of a reagent of viral origin comprising all or part of a surface protein of the said virus which determines its attachment to the cellular receptor,
(b) the incubation with the sample for a determined time,
(c) the elution of the sample retained in step (b) with all or part of an antibody directed against the said reagent of viral origin, and
(d) the analysis of the sample eluted in step (c).
The inert support may be, without limitation, in any form (cone, tube, well, beads and the like) and may be made of any material (natural, synthetic, such as polymers, chemically modified or otherwise and the like). The attachment of the reagent to the inert support may be carried out in a direct or indirect manner. In a direct manner, the procedure will be preferably carried out by adsorption, that is to say noncovalently, although the establishment of covalent bonds may also be considered. In an indirect manner, an anti-reagent compound capable of interacting with the reagent may be attached beforehand so as to immobilize the whole onto the inert support. According to an advantageous embodiment, the sample consists of a so-called random library, and in particular an expression library (genomic fragments, cDNA) or a peptide library or, preferably, a phage library expressing peptide motifs (phagotopes). Such libraries are described in the literature or are commercially accessible. With the aim of selecting or identifying a cellular receptor for an adenovirus, there is preferably used as reagent of viral origin all or part of the fiber and, in particular, of the head of an adenovirus and, as eluent, an anti-fiber neutralizing antibody (inhibitor of the attachment of the virus to the surface of the host cell). The fiber or its fragments may be produced by the recombinant route and the antibodies by the hybridoma technique or by genetic engineering (production of single chain antibody scFv, Fab and the like). It is indicated that most anti-fiber antibodies are neutralizing. The analysis is carried out by comparing the sequence of the eluted sample with data banks. Such an analysis is within the capability of persons skilled in the art.
Finally, the present invention also relates to a method for selecting or identifying the part of a viral protein which determines the attachment of a virus to a cellular receptor in an appropriate sample, comprising:
(a) the immobilization, onto an inert support, of all or part of an antibody directed against the said viral protein,
(b) the incubation with the said sample for a determined time,
(c) the elution of the sample retained in step (b) with a reagent of viral origin comprising all or part of the said viral protein, and
(d) the analysis of the sample eluted in step (c).
The specific embodiments cited above may also apply in this context.
The subject of the present invention is also the use of a bifunctional ligand for targeting an adenovirus to a cell surface protein other than the natural cellular receptor for the said adenovirus, the said bifunctional ligand comprising a first ligand part capable of interacting with the fiber of the said adenovirus, a second ligand part capable of interacting with the said cell surface protein and, optionally, a spacer between the said first and second ligand parts.
For the purposes of the present invention, a bifunctional ligand is capable of interacting with two different species, one preferably situated at the surface of an adenovirus and the other at the surface of a host cell, at the level of a cell surface protein other than the natural cellular receptor for the said adenovirus. Moreover, the two ligand parts may be optionally separated by a spacer comprising from one to about fifteen amino acids which are preferably not charged. Of course, the order of the species is of no importance, it being possible for the domain interacting with the adenoviral protein to be at the N or at the C terminus of the bifunctional ligand, the C-terminal position being preferred. The use of such a bifunctional ligand makes it possible to target an adenovirus to a host cell of interest, for example a tumor cell, an infected cell, a particular cell type or a category of cells carrying a specific surface marker. After binding of the said bifunctional ligand to the cell surface protein, the species recognizing the adenoviral protein is exposed, which should create xe2x80x9cluresxe2x80x9d of viral receptors of the same type as the primary receptors (xcex12 domain of the MHC-Is) and create or increase the number of adenovirus primary receptors at the surface of the host cell.
Preferably, the ligand part interacting with the adenoviral fiber has the characteristics of the ligand defined above; it is in particular derived from the xcex12 domain of the MHC-I and preferably comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids contained in SEQ ID NO: 6. Still more preferably, it consists of the peptide MH20 (SEQ ID NO: 6).
As regards the ligand part interacting with the cell surface protein, it is adapted to the host cell which it is desired to target. In the case of a cell infected by the HIV virus (Human Immunodeficiency Virus), the ligand may be an antibody fragment against fusin, the CD4 receptor or against an exposed viral protein (envelope glycoprotein) or alternatively the part of the TAT protein of the HIV virus extending from residues 37 to 72; (Fawell et al., 1994, Proc. Natl., Acad. Sci. USA 91, 664-668). In the case of a tumor cell, the choice will be for a ligand recognizing a tumor-specific antigen (MUC-1 in the case of breast cancer, antigens of the papillomavirus HPV and the like) or one which is over-expressed (IL-2 receptor overexpressed in certain lymphoid tumors). If it is desired to target the T lymphocytes, it is possible to use a T cell receptor ligand. Moreover, transferrin is a good candidate for hepatic targeting. There may also be mentioned the peptide EGF (abbreviation for Epidermal Growth Factor) which allows targeting to cells expressing the EGF receptor or the GRP peptide (for Gastrin Releasing Peptide) having the sequence (SEQ ID NO: 24) GNHWAVGHLM (Michael et al., 1995, Gene Ther. 2, 660-668) which binds to the GRP cellular receptor. In general, the ligands which may be used in the context of the invention are widely described in the literature.
A bifunctional ligand in use within the framework of the present invention may be obtained by recombinant DNA techniques, by synthesis or by chemical coupling of the two parts of the ligands in question. Preferably, the adenovirus to be targeted is recombinant and carries a cytotoxic gene or is capable of inducing cellular apoptosis. Such genes are perfectly known. There may be mentioned, in particular, the gene encoding thymidine kinase of the HSV-1 virus (herpes simplex virus type 1).
By way of preferred examples, there may be mentioned a bifunctional ligand comprising the peptide MH20 and GRP. The MH20 and GRP peptide domains may be inversely oriented: respectively MH20-GRP when MH20 is at the N-terminal position and GRP-MH20 when MH20 is at the C-terminal position. Another embodiment uses a ligand having an MH20 species and an antibody species of the ScFv (Single Chain FV fragment) type. According to a particularly preferred embodiment, the said bifunctional ligand comprises an amino acid sequence homologous or identical to all or part of the sequence as shown:
(i) in SEQ ID NO: 22 starting at the arginine residue at position 1 and ending with the methionine residue at position 35, or
(ii) (ii) in SEQ ID NO: 23 starting at the lysine residue at position 1 and ending with the arginine residue at position 35.
The present invention also relates to a bifunctional ligand as defined above and, in particular the ligands GRP-MH20 or MH20-GRP. The ligand GRP-MH20 is particularly preferred.
The subject of the present invention is also a cell carrying at its surface such a bifunctional ligand. The advantage of such a cell is to increase the number of primary receptors of the MHCI-xcex12 type. The said cell is advantageously a mammalian cell of any origin (see above). It is preferably a cell for complementation of an adenovirus defective for the E1 function and optionally for another function (E2, E4, E2 and E4, and the like, see Application WO94/28152). A preferred example is the 293 line. It may be generated by recombinant DNA techniques (expression of the bifunctional ligand by means of an appropriate vector comprising the elements allowing expression at the cell surface, for example signal sequence and/or transmembrane region), by covalent or noncovalent chemical bonding or by simple interaction between the cell and the ligand.