EP0900280A2 - Utilisation d'un polypeptide a titre de recepteur cellulaire des adenovirus - Google Patents

Utilisation d'un polypeptide a titre de recepteur cellulaire des adenovirus

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Publication number
EP0900280A2
EP0900280A2 EP98905474A EP98905474A EP0900280A2 EP 0900280 A2 EP0900280 A2 EP 0900280A2 EP 98905474 A EP98905474 A EP 98905474A EP 98905474 A EP98905474 A EP 98905474A EP 0900280 A2 EP0900280 A2 EP 0900280A2
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EP
European Patent Office
Prior art keywords
ligand
residue
adenovirus
seq
ending
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP98905474A
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German (de)
English (en)
French (fr)
Inventor
Pierre Boulanger
Saw See Hong
Lucie Karayan
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Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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Priority claimed from FR9701005A external-priority patent/FR2758821B1/fr
Application filed by Centre National de la Recherche Scientifique CNRS filed Critical Centre National de la Recherche Scientifique CNRS
Publication of EP0900280A2 publication Critical patent/EP0900280A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10345—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/859—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from immunoglobulins
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses

Definitions

  • the subject of the present invention is the use of all or part of an antigen of the major histocompatibility complex of class I and / or of a type III module of fibronectin to allow or facilitate the attachment of an adenovirus on a host cell and / or its entry therein.
  • the invention also relates to the use of a ligand capable of modulating the infectivity of an adenovirus with respect to a host cell, mediated by one or the other of the polypeptides mentioned above.
  • the invention relates to a bio-mulching 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 found in many animal species and can infect various cell types. Many serotypes have been characterized within each species which have a comparable genomic organization and infectious cycle.
  • the adenoviral genome consists of a linear, double-stranded DNA molecule of approximately 3 kb containing the genes coding for the viral proteins and at its ends two reverse repeats (designated ITR) involved in replication and the packaging region.
  • ITR reverse repeats
  • Adenoviruses replicate in the nuclei of infected cells. The infectious cycle takes place in 2 stages. The early phase precedes the initiation of replication and makes it possible to produce the early proteins regulating the replication and transcription of viral DNA.
  • the structural proteins which constitute the viral particles are synthesized.
  • the assembly of new virions takes place in the nucleus.
  • the viral proteins assemble so as to form empty capsids of icosahedral structure, in which the adenoviral DNA is packaged. Viral particles are released and can infect other permissive cells.
  • the fiber and the base penton present on the surface of the capsids play a critical role in the cellular attachment of virions and their internalization.
  • the adenovirus binds to the surface of permissive cells via the trimeric fiber and a hitherto unidentified cellular receptor. Then, the particle is internalized by endocytosis by binding of the penton base to the cellular integrins ⁇ v ⁇ 3 and ⁇ v ⁇ s (Belin and Boulanger, 1993, J. Gen. Virol. 74, 1485-1497; Mathias et al., 1994 , J. Virol. 68, 681 1-6814; 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.
  • the Ad2 fiber contains 580 amino acids (aa), the sequence of which is disclosed in Hérissé et al. (1981, Nucleic Acid Res. 9, 4023-4042). 0 That of Ad 5 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 molecule of 160-180 kDa confirming its assembly in the form of a trimer.
  • the fiber is made up of 3 domains (Chroboczek et al., 1995, Current Top. 5 Microbiol. Immunol. 199, 165-200):
  • the “stem” is a rod structure of variable length depending on the serotypes.
  • the stem of the Ad5 fiber contains 22 repeats of a pattern 0 of 15 residues which could adopt a ⁇ -sheet conformation. The number of these repetitions differs from one serotype to another, which explains the variations in length.
  • the "head” or terminal sphericle is a globular structure containing the trimerization signals (Hong and Engler, 1996, 5 J. Virol. 70, 7071-7078; Novelli and Boulanger, 1991, J. Biol. Chem. 266, 9299-9303; Novelli and Boulanger, 1991, Virology 755, 365-376). Most experimental data show that it is the head area which is responsible for binding to permissive cells (Krasnykh et al., 1996, J. Virol. 70, 6839-6846).
  • the adsorbed phages which in theory express phagotopes interacting with a motif carried by the adenoviral protein, are then eluted either conventionally at acid pH or by competition with the other non-immobilized capsid partner (eluent).
  • eluent non-immobilized capsid partner
  • the immobilized ligand is constituted by the domain of the head of the Ad5 fiber and the eluent by a neutralizing antibody directed against the latter and isolated two classes of phagotopes according to the antibody used.
  • the first corresponds to a sequence conserved within the ⁇ -2 domain of major class I histocompatibility complex antigens ( ⁇ -2 MHC-I) and the second to a sequence found in modules III of human fibronectin ( FNIII).
  • ⁇ -2 MHC-I constitutes the primary receptor for adenoviruses of serotypes C and confirm the participation of FNIII as co-receptor or co-factor.
  • the regions of these two receptors and of the fiber interacting with each other have also been highlighted.
  • an antagonistic peptide reproducing the motif of the ⁇ -2 MHC-I domain was generated which neutralizes the attachment of adenoviruses and an agonist peptide reproducing the FNIII motifs which stimulates attachment.
  • polypeptide comprising an amino acid sequence homologous or identical to at least 6 continuous amino acids of the sequence as shown:
  • polypeptide means any molecule constituted by a chain of at least 6 and, preferably at least 8, amino acids.
  • polypeptide includes both peptide molecules of short length (from 6 to a few tens of residues) as molecules of greater length (up to several hundred residues), on the condition, however, of allowing the intended use.
  • a polypeptide in use in the context of the present invention can be derived from a native polypeptide as found in nature, in particular in humans, or from a part thereof. It can also be chimeric and include additional residues of any origin fused in N and / or C-terminal 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 with respect to the sequences disclosed in the sequence identifiers (SEQ ID).
  • a preferred polypeptide in the context of the present invention comprises in in addition to suitable elements to ensure its anchoring in a cell membrane or its presentation on the surface of a cell.
  • suitable elements are known to those skilled in the art.
  • other techniques for example chemical techniques, can also be used to anchor or bind a polypeptide to a membrane or a cell surface.
  • homologous amino acid sequence is meant a sequence having a degree of homology of at least 70%, advantageously of at least 80%, preferably of at least 90% with at least minus 6 continuous amino acids from one of the sequences listed.
  • the identical term refers to 100% homology.
  • Those skilled in the art know the general rules which make it possible to calculate the degree of homology between two sequences. This is generally done by aligning the sequences, possibly using specialized computer programs. It may be necessary to artificially introduce vacant locations. Once the optimal alignment is achieved, the degree of homology is established by counting all the positions in which the amino acids of the two sequences are found identically, compared to the total number of positions.
  • attachment of an adenovirus to a host cell is meant the binding of the viral particle to the cell.
  • entity of an adenovirus into a host cell is meant the penetration of the virus into the host cell.
  • the attachment and / or entry are preferably mediated at least in part by the polypeptide (s) in use in the context of the present invention by interaction with the adenoviral capsid.
  • polypeptide polypeptide
  • other polypeptide molecules or not can also participate in these processes recognized in the art field as complex and multifactorial.
  • an adenovirus can be of human or animal origin (canine, avian, bovine, etc.) or a hybrid 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, NJ). It is preferred to use a recombinant adenovirus defective for replication and expressing in particular a gene of therapeutic interest.
  • the adenoviral genome is modified by deletion or mutation of sequences essential for replication and, in particular, included in the regions E1, E2, E4 and / or L1-L5 (see for example international application WO 94/28152).
  • 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 in position 1 and ending with the glutamine residue in position 25.
  • 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 major histocompatibility complex of class I (MHC-I) and, preferably , of the latter's heavy chain.
  • MHC-I major histocompatibility complex of class I
  • histocompatibility antigens All the cells of an organism present on their membrane molecules called histocompatibility antigens which define each individual. The corresponding genes, more than a dozen, are located on chromosome 6 in humans and exhibit significant polymorphism, which makes it possible to ensure great variability of these identity markers. There are two different categories of these histocompatibility antigens, respectively Class I and II, whose structure and functions are distinct. Class I molecules, called FILA (for Human Leukocyte Antigen in English) intervene to present antigenic peptides on the cell surface and play an essential role in immune responses 8/33929
  • antiviral drugs exerted by cytotoxic T lymphocytes.
  • the MHC-I molecules are heterodimers composed of a non-MHC light chain designated ⁇ 2-microglobulin ( ⁇ 2m) and a heavy chain coded by the MHC genes, linked noncovalently.
  • the heavy chain is a membrane protein whose N-terminal part is oriented outside the cell while the C-terminal part is cytoplasmic.
  • the first includes 3 domains designated ⁇ l, ⁇ 2 and ⁇ 3 of approximately 90 amino acids each. It is followed by a transmembrane region of approximately 25 amino acids and then by the C-terminal region of around thirty amino acids.
  • polypeptides suitable for the purposes of the present invention there may be mentioned more particularly the HLA antigens A, B, C, D, E and F or polypeptides derived therefrom.
  • the polypeptide used in the context of the present invention comprises a sequence homologous or identical to all or part of the C-terminal region of the ⁇ 2 domain of the heavy chain of MHC-I and, more particularly, to the part centered on the tryptophan residue in position 167, in particular that extending from residues 156 to 180 (SEQ ID NO: 1).
  • the numbering referred to is consistent with that used for example in Bjorkman and Parham (1990, supra).
  • a polypeptide in use in the context 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 residue asparagine in position 1 and ending with the asparagine residue in position 26, in SEQ ID NO: 3 starting with the valine residue in position 1 and ending with the asparagine residue in position 25, in SEQ ID NO: 4 starting with the serine residue in position 1 and ending with the arginine residue in position 25, and / or in SEQ ID NO: 5 starting with the asparagine residue in position 1 and ending with the serine residue in 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. Good heard, he can understand several. One can also consider the use of human fibronectin or a peptide derived therefrom, for example by mutation or fragmentation. For information, fibronectin encoded by a single gene is a molecule involved in the phenomena of cell adhesion and contact. Its sequence and its characteristics are described in the literature accessible to the skilled person (see in particular Bork and Doolittle, 1992, Proc. Natl. Acad. Sci.
  • a polypeptide as defined above is more particularly intended to allow or facilitate the attachment of an adenovirus of serotype C to a host cell and / or its entry therein.
  • adenoviruses mention may be made more particularly of serotypes 2 and 5.
  • the present invention also relates to a host cell capable of expressing a polypeptide in use in the context of the present invention and its use to allow or facilitate the attachment of an adenovirus to its surface and / or the entry of said adenovirus.
  • host cells can be considered. They can be cells of any origin, for example microorganisms, yeasts, insects, plants, animals. A mammalian cell and in particular a human cell of primary type, tumor or from a line will be preferred. cultivable / ' /; vitro.
  • a particularly preferred cell is or is derived from line 293 established from. of embryonic kidney cells by integration of the El adenoviral region (Graham et al., 1977, J. Gen. Virol. 36, 59-72).
  • polypeptides in use in the context of the present invention on the surface of a host cell not usually expressing MHC-I and / or fibronectin should allow its infectivity by an adenovirus . It could be used as a new cell producing adenoviral vectors.
  • an overexpression in a cell naturally expressing said polypeptide An overexpression line derived from line 293 should make it possible to improve the production yields of an adenovirus of interest.
  • the polypeptide used in the context of the present invention can be associated with the cell by chemical means or by means of a ligand recognizing a cell surface protein. However, one can also envisage expression by recombinant DNA techniques.
  • nucleotide sequence coding for the polypeptide in question can 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 technique of the art.
  • the host cell used in the context of the present invention can also be modified so as to complement a defective adenovirus by transfection of appropriate fragment (s) of adenoviral genome.
  • the present invention also relates to the use of a ligand capable of influencing the attachment of an adenovirus to a host cell and / or its entry into the latter mediated by a polypeptide as defined above.
  • the ligand used in the invention can be of any nature. We can cite for example the peptides, hormones, antibodies or their derivatives and, in particular, single chain antibodies of the scFv type (for single chain fragment variable in English) and soluble receptors lacking their transmembrane region.
  • a ligand can be derived from a polypeptide used in the present invention.
  • the ligand can have a negative (antagonist) or positive (agonist) influence.
  • 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.
  • a particularly preferred ligand is based on a polypeptide as defined in SEQ ID NO: 1.
  • polypeptide comprising an amino acid sequence homologous or identical to at least 6 amino acids Continues included in the sequence as shown in SEQ ID NO: 6 starting with the arginine residue in position 1 and ending with the arginine residue in position 20. It will be preferred to use the peptide designated MH20 in the examples which follow.
  • the ligand used in the context of the present invention is used to allow or stimulate the attachment and / or entry of adenoviruses.
  • a ligand 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 in 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 which is ho ogue or identical to at least 6 continuous amino acids of the sequence as shown in SEQ ID NO: 6 or 7.
  • a preferred ligand is derived from the fiber of an adenovirus, in particular from the part of the head interacting with the abovementioned polypeptides.
  • a peptide motif chosen in this region should therefore influence the infectivity of adenoviruses with respect to a host cell expressing the polypeptide.
  • a ligand of a polypeptide as defined by SEQ ID NO: 1 preferably derives from 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 with the amino acid leucine in position 1 and ending with the amino acid aspartic acid in position 18.
  • a ligand also conceivable can derive from the fiber of an adenovirus of serotype 2 and include a amino acid sequence homologous or identical to at least 6 amino acids of the sequence as shown in SEQ LD NO: 9 starting at the threonine residue in position 1 and ending at the valine residue in position 16.
  • the ligand of a polypeptide such 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 started both at the leucine residue in position 1 and ending at the threonine residue in position 14 (Ad5) or in SEQ ID NO: 11 starting at the asparagine residue in position 1 and ending at the asparagine residue in position 13 (Ad2).
  • the present invention also relates to 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.
  • an antagonist ligand will be preferred for a therapeutic or prophylactic purpose.
  • the use of a ligand according to the invention preferably an agonist, is suitable for the preparation of a medicament intended to promote or facilitate infection by an adenovirus, and, in particular, of a recombinant adenovirus carrying a gene therapeutic for gene therapy (curative) or anti-viral (AIDS) or anti-cancer.
  • Such a drug finds its usefulness for example in combination with gene therapy treatments in order to improve viral infection in a patient treated with a recombinant adenovirus.
  • a parenteral, oral or aerosol route of administration The administration can take place in single dose or repeated one or more times after a certain interval of interval.
  • 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 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) immobilization on an inert support of a reagent of viral origin comprising all or part of a surface protein of said virus determining its attachment to the cellular receptor,
  • step (c) eluting the sample retained in step (b) with all or part of an antibody directed against said reagent of viral origin
  • the inert support can be without limitation in any form (cone, tube, well, beads or the like) and of any material (natural, synthetic such as polymers, chemically modified or not ).
  • the fixing of the reagent on the inert support can be carried out directly or indirectly. Directly, the procedure is preferably by adsorption, that is to say in a non-covalent manner although the establishment of covalent bonds can also be considered. Indirectly, an anti-reagent compound capable of interacting with the reagent can be fixed beforehand so as to immobilize the assembly on the inert support.
  • the sample consists of a library called random and, in particular of expression (genomic fragments, cDNA) or peptide or, preferably, phages expressing peptide motifs (phagotopes).
  • libraries are described in the literature or commercially accessible.
  • an anti- neutralizing fiber in order to select or identify a receiver cell of an adenovirus, all or part of the fiber and, in particular, of the head of an adenovirus and, as eluent, an anti- neutralizing fiber (inhibitor of virus attachment to the surface of the host cell).
  • the fiber or its fragments can be produced recombinantly and the antibodies by the hybridoma technique or by genetic engineering (production of single chain antibodies scFv, Fab ).
  • the present invention also relates to a method for selecting or identifying the part of a viral protein determining the attachment of a virus to a cellular receptor in an appropriate sample, comprising: (a) immobilization on an inert support of all or part of an antibody directed against said viral protein, (b) incubation for a determined time with said sample,
  • step (c) eluting the sample retained in step (b) with a reagent of viral origin comprising all or part of said viral protein
  • the present invention also relates to the use of a bifunctional ligand for targeting an adenovirus to a cell surface protein other than the natural cellular receptor of said adenovirus, said bifunctional ligand comprising a first ligand part capable of interacting with the fiber of said adenovirus, a second ligand part capable of interacting with said cell surface protein and, optionally, a spacer between said first and second ligand parts.
  • a bifunctional ligand is capable of interacting with two different entities, one preferably located on the surface of an adenovirus. and the other on the surface of a host cell, at the level of a cell surface protein other than the natural cellular receptor of said adenovirus.
  • the two ligand parts can optionally be separated by a spacer comprising from 1 to fifteen amino acids, preferably uncharged.
  • the order of the entities does not matter, the domain interacting with the adenoviral protein being able to be in N or in C-terminal of the bifunctional ligand, the C-terminal position being preferred.
  • bifunctional ligand makes it possible to target an adenovirus towards 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.
  • 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.
  • the entity recognizing the adenoviral protein is exposed, which should create “decoys” of viral receptors of the same type as the primary receptors ( ⁇ 2 domain of MHC-I) and create or increase the number of primary adenovirus receptors on the surface of the host cell.
  • the ligand part interacting with the adenoviral fiber has the characteristics of the ligand defined above, it is in particular derived from the ⁇ 2 domain of MHCI and preferably comprises an amino acid sequence homologous or identical to at least 6 continuous amino acids included in the SEQ. ID NO: 6. Even more preferably, it consists of the peptide MH20 (SEQ ID NO: 6).
  • the ligand part interacting with the cell surface protein this is adapted to the host cell which it is desired to target.
  • the ligand may be a fragment of antibody against fusine, the CD4 receptor or against an exposed viral protein (envelope glycoprotein) or even the part HIV virus TAT protein extending from residues 37 to 72; (Fawell et al. 1994, Proc. Natl. Acad. Sci. USA 91, 664-668).
  • a tumor cell As it is a tumor cell, the choice will be made on a ligand recognizing a specific tumor antigen (MUC-1 in the case of breast cancer, HPV papilloma virus antigens, etc.) or overexpressed (IL-2 receptor overexpressed in certain lymphoid tumors). If it is desired to target T cells, a T cell receptor ligand can be used. In addition, transferrin is a good candidate for liver targeting.
  • EGF abbreviation of Epidermal Growth Factor
  • GRP for Gastrin Releasing Peptide
  • GNHWAVGHLM sequence GNHWAVGHLM
  • the adenovirus to be targeted is recombinant and carries a cytotoxic gene or capable of inducing cellular apoptosis.
  • genes are well known. Mention may in particular be made of the gene coding for the thymidine kinase of the HSV-1 virus (herpes simplex virus type 1).
  • a bifunctional ligand comprising the peptide MH20 and the GRP.
  • the MH20 and GRP peptide domains can be oriented inversely: MH20-GRP respectively when the MH20 is in the N-terminal position and GRP-MH20 when the MH20 is in the C-terminal position.
  • Another embodiment uses a ligand having an MH20 entity and an antibody entity of the ScFv (Single Chain Fv fragment) type.
  • said bifunctional ligand comprises an amino acid sequence homologous or identical to all or part of the sequence as shown.
  • the present invention also relates to a bifunctional ligand as defined above and, in particular, the GRP-MH20 or MH20-GRP ligands.
  • the GRP-MH20 ligand is particularly preferred.
  • the present invention also relates to a cell carrying on its surface such a bifunctional ligand.
  • the advantage of such a cell is to increase the number of primary receptors of the MHCl- ⁇ 2 type.
  • Said cell is advantageously a mammalian cell of any origin (see above). It is preferably a complement cell for an adenovirus defective in function
  • El and possibly another function (E2, E4, E2 and E4, etc., see application WO94 / 28152).
  • a preferred example is line 293. It can be generated by recombinant DNA techniques (expression of the bifunctional ligand by means of an appropriate vector comprising the elements allowing expression on the cell surface, for example signal sequence and / or transmembrane region), by covalent chemical bond or not or by simple interaction between the cell and the ligand.
  • the present invention is illustrated by reference to the following figures:
  • Figure 1 shows the phagotopes obtained after bio-mulching using the antibody 1D6.3 (a) or 7A2.7 (b) as a ligand.
  • the phagotope peptide motifs are aligned with respect to the sequence of the Ad5 head (the methionine residue initiating the fiber representing +1.
  • the regions forming ⁇ -sheet structures (Xia et al., 1994, supra) are underlined and indicated by (D), (E) and (F). Identical or conserved residues in the sequences are indicated in bold.
  • Figure 2 shows the phagotopes obtained after bio-mulching using (a) the antibody 7A2.7 or (c) 1D6.3 as eluent, (b) and (d) the consensus sequences determined from the phagotopes (a) and (c) respectively as well as the homologous sequences found by analysis of the SWISS PROT database. Residues stored at similar positions are shown in bold.
  • FIG 3 illustrates the expression of the luciferase gene in HeLa cells infected with the Ad5Luc3 virus at a constant MOI (0.16 f ⁇ u / 10 cells).
  • Ad5Luc3 is added to the cells cooled to 0 ° C in the presence of increasing molarities of peptide
  • a) FN20 (0 to 500 ⁇ M) or
  • MH20 (0 to 50 ⁇ M).
  • the controls correspond to the incubation of the peptides after the attachment of Ad5Luc3 ( ⁇ ) or after endocytosis (D).
  • Figure 4 illustrates the expression of the luciferase gene in Daudi-
  • the Ad5Luc3 is brought into contact with the cells pre-cooled to 0 ° C for 1 h to allow viral attachment but not entry.
  • the luciferase activity is evaluated after 18 h of culture at 37 ° C.
  • the RLU values represent the average of three separate experiments.
  • Figure 5 illustrates the principle of the bifunctional ligand method mimicking the primary adenovirus receptors.
  • Figure 6 shows the luciferase enzyme activity as a function of the MOI of Ad5Luc3 on NIH (a), Swiss 3T3 (b) and HeLa (c) cells.
  • HeLa cells are cultured in monolayers according to the techniques of the art.
  • a DMEM medium Dulbecco's Modified Eagle's Medium; Gibco
  • FCS heat-inactivated fetal calf serum
  • the Daudi HLA- (ATCC CCL213) and HLA + (Quille, et al., 1988, J. Immunol. 141, 17-20) cells are maintained in RPMI 1640 medium (Gibco) supplemented with 15% FCS.
  • Ad5Luc3 is an adenovirus competent for replication which contains the luciferase gene placed under the control of the SV40 virus early promoter (simian virus 40) inserted in the E3 region of the adenoviral genome (Mittal et al., 1993, Virus Research 28, 67-90).
  • the murine mAbs 1D6.3 and 7A2.7 were generated by conventional techniques by injecting the head of the Ad5 fiber produced in the bacteria recombinantly (Henry et al., 1994, J. Virol. 68, 5239- 5246; Douglas et al., 1996, Nature Biotech 14, 1574-1578) to Balb / C mice.
  • the fusion and obtaining of hybridoma clones are conventional techniques within the reach of those skilled in the art.
  • the secreting clones are selected by their recognition of the antigen which served for immunization in ELISA. They are said to have neutralizing activity against virions (Michael et al., In preparation).
  • the reactivity of the antibodies is tested with regard to the fiber head domain of 3 different serotypes (Ad2, Ad5 and Ad3) prepared by recombinant route.
  • the corresponding sequences are isolated by PCR (Polymerase Chain Reaction) from viral genomic DNA and then introduced into the AcNPV virus (Autographa californica Nuclear Polyhedrosis Virus) under the control of the polyhedrine promoter (Luckow and Summer, 1989, Virology 170, 31- 39).
  • Recombinant proteins are expressed in Sf9 insect cells (Spodoptera frugiperda).
  • the general technology is detailed in Karayan et al. (1994, Virology 202, 782-796) and Novelli and Boulanger (1991, Virology 185, 365-376).
  • the Ad5 sequences carrying the last repeated motif of the stem followed by the fiber head are cloned using the primers shown in SEQ ID NO: 12 and 13.
  • the sense primer corresponds to nucleotides 32164 to 32205 of the Ad5 genome (Chroboczek and Jacrot, 1987, Virology 161, 549-554), includes 4 mismatches so as to create a BamHI site and to replace threonine at position 388 of the native fiber with an initiating ATG codon.
  • the antisense primer corresponds to nucleotides 32919 to 32883 of the Ad5 genome and makes it possible to create a Kpnl site to facilitate the subsequent cloning steps.
  • the recombinant protein harvested from the supernatants of Sf9 cells is designated F5-AT386.
  • the Ad2 head is produced from the baculovirus vector described in Louis et al. (1994, J. Virol. 68, 4104-4106).
  • the expression product designated F2-AT388 begins at position 388 (by replacing the Ala of the native sequence with a Met) and carries, in addition to the domain of the head, the last repeated motif of the stem.
  • a sense primer (SEQ ID NO: 14) is used which is designed to introduce an Ncol cloning site and to replace the Asn and Ser codons at position 124 and 125 with Met and Ala codons respectively.
  • the antisense primer introduces a Kpnl site.
  • the expression product is designated F3-AT124.
  • the wells of an ELIS A plate are covered with the recombinant protein
  • F5-AT386, F2-AT388 or F3-AT124 on which the mAb 1D6.3 or 7A2.7 is reacted then a labeled anti-mouse antibody (for example with phosphatase or peroxidase).
  • a labeled anti-mouse antibody for example with phosphatase or peroxidase.
  • a positive reaction is observed with regard to the recombinant protein F5-AT386 native. No reaction is detected in the wells containing the F5-AT386 protein denatured in SDS or those containing the native or denatured products F2-AT388 and F3-AT124.
  • the cells in the semi-confluent state are put in the presence of a constant quantity of radioactive virions (10 ′ cpm for 5 ⁇ 10 cells) at a multiplicity of infection (MOI) of 1000 virions per cell for 1 h at 0 ° C in the presence of mAb 1D6.3 or 7 A2.7 (dilutions 1: 10, 1: 8, 1: 4 and 1: 2 of the supernatants of respective hybridomas whose concentration in mAb is estimated at 0 , 1- 0.2 ⁇ g / ml, which corresponds to an excess in mAb compared to the virions present in the inoculum of 100, 250, 500 and 1000 respectively).
  • MOI multiplicity of infection
  • the cells are then washed in the presence of PBS, fixed with 0.1% of paraformaldehyde in PBS, dried and covered with the K4 emulsion in the form of a gel (Ilford Nuclear Research). After a week-long exposure and development (development agent D19B, Kodak), the samples are stained briefly with 0.5% toluidine blue taken up in HO and examined under a microscope. The density of the reduced silver grains around the contour of the cells is representative of the number of virions [C] bound to the cell surface.
  • EXAMPLE 2 Identification of the epitopes of mAbs 1D6.3 and 7A2.7.
  • the immobilized antibodies are brought into contact with a phage library expressing hexapeptides (phages FUSES; Scott and Smith, 1990, Science 249, 386-390).
  • phages FUSES a phage library expressing hexapeptides
  • the phages retained are eluted either by a conventional acid elution buffer or, more selectively, by competition in the presence of an excess of recombinant protein F5-AT386.
  • the hexapeptide motifs (phagotopes) carried by the eluted phages are determined by sequencing the protein pIII fUSE5 by the method of Sanger et al. (1977, Proc. Natl. Acad. Sci. USA 74, 5463-5467).
  • mAb 1D6.3 retains different phagotopes with overlapping sequences, and are homologous to the head of Ad5 extending between the residues Val at position 438 and Asp at position 462.
  • a central motif of sequence LAPISGTVQSAHLIIRFD corresponding to the amino acids at positions 445 to 462 (SEQ ID NO: 8). This motif is centered on the His residue at position 456, which corroborates the presence of histidine in several independent phagotopes.
  • three phagotopes contain histidine close to a serine (GISHTG and GASHTV) and a homologous sequence is found in the head QSAHLi).
  • the LAPIS sequence is represented in several phagotopes in the form L-P, VAP-S and LIPFNS.
  • WALFRS are homologous to the YWNFR motif. Based on this data, the mAb 7A2.7 epitope was mapped between residues 473 and 486 of the fiber.
  • Ad5 (SEQ ID NO: 10).
  • mAbs 1D6.3 and 7A2.7 recognize adjacent segments of 15 to 20 aa in the linear sequence of the Ad5 head, the residues extending from positions 445 to 462 and 473 to 486 respectively.
  • the two epitopes occupy continuous regions from a spatial point of view.
  • the epitope of mAb 1D6.3 covers part of the CD loop and of the ⁇ D sheet, while the epitope of mAb 7A2.7 is located at the level of the adjacent segment DE and of the two sheets ⁇ E and F.
  • the epitope 1D6.3 is located inside a sheet R while the epitope 7A2.7 is oriented more laterally with respect to sheet R.
  • FIG. 2a The phagotopes produced by competition with mAb 7A2.7 are shown in Figure 2a. Their analysis makes it possible to derive a consensus sequence (FIG. 2b) which exhibits homology with motifs 1, 3, 5 and 14 of the type III module of human fibronectin (SEQ ID NO: 2 to 5) (Main et al., 1992, Cell 77, 671-678). The latter are situated at the level of the ⁇ B sheet and of the adjacent loop BC of the module FNIII (Dickinson et al., 1994, J. Mol. Biol. 236, 1079-1092).
  • the sequence of the fusion product comprising a copy of the pentadecapeptide (designated GST-FNxl) can be diagrammed as follows: GST- (thrombin cleavage site-PGIS-GGGGG-ILDSMGRLE-RHILWTPANTPAMGY (V) -ELKLNS- stop. Constructions GST-FNx2 and GST-FNx3 have 2 and 3 repeats of the pentadecapeptide between the LE and EL residues of the cloning cassette, respectively.
  • the sense and antisense oligonucleotides (SEQ ID NO: 18 and 19) coding for the consensus sequence obtained by competition with mAb 1D6.3 ( Figure 2d) are inserted according to the same strategy as above at the C-terminal end of GST to give the constructions GST-MHCxl, GST-MHCx2 and GST-MHCx3 depending on the number of patterns present.
  • the chimeric proteins GST-FN and GST-MHC are produced in E. coli, extracted and affinity-purified on agarose-glutathione beads (Sigma) according to conventional methods (Smith and Johnson, 1988, Gene 67, 31-40 ).
  • the complete fibers of serotypes 2, 3 and 5 are produced recombinantly according to the baculovirus / Sf9 cell technology already used.
  • the F2-FL582 construction carrying the Ad2 fiber gene is described in Novelli and Boulanger (1991, Virology 185, 365-376).
  • the sequences coding for the Ad5 and Ad3 fibers are isolated by PCR using the viral DNA as a template and appropriate sense and antisense primers such as those reported in SEQ ID NO: 20 and 13 and 21 and 15.
  • the amplified segment is introduced into a baculovirus vector under the control of the polyhedrin promoter and the expression product recovered from the culture supernatants.
  • F5-FL581 and F3-FL320 are obtained corresponding to the fibers Ad5 and Ad3 respectively.
  • the capacity of the FNIII module and of the recombinant ⁇ -2 MHC-I domain to bind the adenoviral fiber is evaluated by immunotransfer after incubation / ' // vitro of the fusion proteins GST-FN and GST-MHC and of the recombinant fibers F2-FL582, F5-FL581 and F3-FL320.
  • the complexes formed are isolated on glutathione agarose beads under the conditions described by Johnson et al. (1995, J. Biol. Chem.
  • the luminograms (Hyperfiim ⁇ - max, Amersham) are analyzed at 610 nm using an automatic densitometer (REP-EDC, Helena Laboratories, Beaumont, TX) .
  • REP-EDC automatic densitometer
  • the antibody 4D2.5 recognizes the FNPVYP epitope of the tail domain conserved in most mammalian adenoviruses.
  • GST-FNxl, GST-FNx2 and GST-FNx3 bind to F5-AT386 and F2-AT388 with great efficiency while F3-AT124 is retained at a lower level.
  • a similar behavior is observed with the chimeric proteins GST-MHCxl, GST-MHCx2 and GST-MHCx.3 which retain F5-AT386 and F2-AT388 with an efficiency greater than F3-AT 124.
  • the Ad5 fiber binds to the GST-FN and GST-MHC fusion proteins and this with an affinity 2 to 3 times greater compared to the Ad2 fiber and 10 to 15 times greater compared to the Ad3 fiber.
  • the binding efficiency is not dependent on the number of motifs present in the fusion protein, the intensity being comparable and even sometimes lower between GST-FNxl and GST-FNx3 and GST-MHCxl and GST-MHCx3 . This can be explained by the fact that the tandem patterns can adopt a conformation which harms the bond.
  • the FN20 and MH20 peptides are tested for the attachment of the reporter adenovirus Ad5Luc3 to HeLa cells cultured in vitro.
  • the test is carried out in part at 0 ° C, a temperature which allows the attachment of viruses to the surface of permissive cells but, on the other hand, blocks the entry of viruses and the recycling of receptors.
  • Ad5Luc3 (MOI 0.16 pfu / 10 cells) is previously incubated with increasing amounts of peptides (0.01 to 500 nM) at room temperature for 2 hours then the mixture is added to a cell culture placed on ice.
  • the non-adsorbed viruses and the peptides are eliminated by washing and the culture is continued for 18 h at 37 ° C. after addition of a preheated medium.
  • the cell lysates are prepared in a conventional manner and the luciferase activity expressed in RLU (for Relative Light Units in English) is determined (Promega, Madison, WI substrate; Lumat LB-9501 luminometer, Brethold Bioanalytical, Wildbad, Germany).
  • the results of competition with the FN20 peptide are presented in Figure 3a. No significant effect is obtained up to a molarity of 10 ⁇ M then a progressive increase in the luciferase activity appears beyond 25 ⁇ M. In particular, the activity increases by a factor of 100 between 25 and 100 ⁇ M.
  • the FN20 peptide therefore has a stimulating effect on viral attachment. It does not confer any apparent cytotoxicity and has no negative effect on the expression of the luciferase gene once the virus is pre-attached (peptide added to cell culture after the viral attachment step at 0 ° C) or pre-endocytosed ( peptide added to cell culture after the virus attachment and penetration step).
  • the MH20 peptide FIG.
  • the wild adenoviruses Ad5, Ad2 Ad3 are preincubated for 2 h at room temperature with the inhibitor peptide MH20 at a constant molarity (25 ⁇ M), the MOI varying from 0.2 to 2 pfu / cell.
  • the mixture is placed in the presence of the HeLa cells for 1 h at 0 ° C. and the culture continued at 37 ° C. after removal of the non-adsorbed viruses under the same conditions as those described above.
  • the level of synthesis of the hexon, of the 100k protein, of the penton base and of the fiber is estimated by immunotitration (Wohlfart, 1988, J. Virol. 62, 2321-2328) on the cell extracts collected 48 h after the infection.
  • EXAMPLE 7 Affinity of the Ad5 fiber for the peptides FN20 and MH20.
  • the fiber adsorbed on either peptide is eluted with an adequate solution (1M urea, 1 M NaOH and 1% SDS) and then precipitated in the presence of trichloroacetic acid.
  • the radioactivity contained in the precipitate recovered on a GF / C filter is counted using a liquid scintillation spectrometer (Beckman LS-6500) and the dissociation constants (Kd) determined according to Scatchard (1949, Annls NY Acad. Sci . 57, 660-672).
  • EXAMPLE 8 The infectivity of Ad5 is dependent on the expression of MHC-I on the surface of permissive cells.
  • the Daudi line of B lymphoblastoids established from a lymphoma of
  • Burkitt is naturally deficient in the expression of ⁇ -2 microglobulin and, therefore, does not have on its surface HLA class I molecules (Daudi HLA-).
  • the Daudi-derived E8.1 cell line was generated by transfection of a gene coding for ⁇ -2 microglobulin in order to restore the expression of HLA class I molecules on their surface (Daudi-HLA +; Quillet et al., 1988, J. Immunol. 141, 17-20).
  • the attachment experiments of Ad5Luc3 at 0 ° C. were carried out on Daudi HLA- and Daudi-HLA + cells with an MOI of three orders of magnitude higher than that used in the case of HeLa cells (0.3 to 150 pfu / 30 cells).
  • the luciferase activity measured in the cell lysates 18 h post-infection is represented in FIG. 4.
  • the infection concerns Daudi-HLA + cells
  • the luciferase activity increases regularly in a dependent MOI manner until reaching a plateau above 5 pfu / 10 cells.
  • the luminescent signal is very weak (3 to 4 orders of magnitude) compared to to that observed with cells provided with functional HLA molecules on their surface.
  • This example describes the construction of a bifunctional peptide which contains two domains, one recognizing the head of the adenoviral fiber and the other a cellular surface protein.
  • a peptide is used below in which the MH20 is fused to GRP (Gastrin Releasing Peptide).
  • GRP Neuron Releasing Peptide
  • Two constructions are possible, orienting the two peptide domains in an inverted manner, N- versus C-terminal, giving rise to the peptide MH20-GRP (SEQ ID No: 22) and to the peptide with opposite orientation GRP- MH20 (SEQ ID No: 23 ).
  • the binding of this peptide to GRP receptors on the cell surface should create “decoys” of viral receptors of the same type as the primary receptors, ie the ⁇ 2 domain of class I molecules of MHC.
  • the apparent result is to create or increase the number of primary viral adenovirus receptors on the surface of cells having GRP receptors.
  • HeLa Human (HeLa) or murine cells (Swiss-3T3 or NIH-3T3, ATCC CRL-1658) are rinsed and incubated between 0 and 4 ° C with an isotonic solution (PBS) at 500 ⁇ M in peptide. After 1 h, the solution is removed and replaced with the viral inoculum (Ad5Luc3) according to a protocol already described (see above or Hong et al., 1997, EMBO J. 16, 2294-2306).
  • PBS isotonic solution
  • the virus is incubated for one hour between 0 and 4 ° C to allow its attachment, then the excess of non-adsorbed virus is rinsed with culture medium pre-cooled to 4 ° C and the cells are replaced at 37 ° C in the presence of culture medium preheated to 37 ° C.
  • the virus is endocytosed at 37 ° C and the viral cycle then takes place for 18 to 20 h at 37 ° C.
  • FIG. 6 it can be seen that the curves obtained in the absence of any peptide (control curves indicated: - peptide) and those in the presence of peptide in the orientation MH20 on the N-terminal side - linked to the GRP in C-terminal ( MH20 - GRP) have an identical slope.
  • GRP on the N-terminal side and
  • the increase in the number of adsorbed viruses is significant, as shown by the increase in luciferase activity: 8 to 10 times for NIH-3T3, 5 to 6 times for HeLa and 2 cells 3 times for Swiss-3T3.
  • the binding of the GRP portion of the bifunctional peptide, a ligand for GRP receptors, has made it possible to increase the apparent number of receptors for the AdS fiber, of the alpha 2 domain type of MHC class I molecules (MHC I- ⁇ 2) .
  • ORGANISM Mastadenovirus
  • STRAIN serotype 5

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