EP1124854A2 - Anticorps se liant a la capside de virus associes aux adenovirus (aav), modifiant le tropisme cellulaire et procede de transfert de genes cible - Google Patents

Anticorps se liant a la capside de virus associes aux adenovirus (aav), modifiant le tropisme cellulaire et procede de transfert de genes cible

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Publication number
EP1124854A2
EP1124854A2 EP99963196A EP99963196A EP1124854A2 EP 1124854 A2 EP1124854 A2 EP 1124854A2 EP 99963196 A EP99963196 A EP 99963196A EP 99963196 A EP99963196 A EP 99963196A EP 1124854 A2 EP1124854 A2 EP 1124854A2
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European Patent Office
Prior art keywords
aav
antibody
fragment
antibodies
receptor
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EP99963196A
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English (en)
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Jürgen KLEINSCHMIDT
Christiane Wobus
Andrea Kern
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Deutsches Krebsforschungszentrum DKFZ
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Deutsches Krebsforschungszentrum DKFZ
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—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
    • C12N2810/50—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

Definitions

  • Antibodies to the AAV capsid that change cell tropism and method for directed gene transfer are provided.
  • the present invention relates to antibodies or fragments thereof that bind to the capsid of adeno-associated viruses (AAV), thereby preventing the virus from binding to the viral receptor of the original target cell.
  • AAV adeno-associated viruses
  • These antibodies or the fragment (s) thereof can also serve as adapters for the fusion with a desired receptor ligand. After binding such an antibody to the AAV capsid, an AAV is obtained which has an altered tropism, that is to say can - depending on the fused ligand - bind to a new target cell.
  • the invention thus further relates to AAV vectors, to the capsid of which the antibody according to the invention or the fragment thereof are bound. These vectors can be used for directed gene transfer. Finally, the present invention relates to a method for directed gene transfer using this AAV vector.
  • the first event in a viral infection is the binding of the virus to the host cell surface.
  • the cell surface molecules to which the viruses bind are called virus receptors, while the viral proteins involved in this binding are called viral attachment proteins
  • VAP viral cell attachment protein
  • Virus receptors and viral ligands have been of increasing interest in recent years because knowledge of this interaction not only provides important information for the prevention of viral infections, but such knowledge may can also be used for the targeted manipulation of the infection with viral vectors.
  • the erythrocyte P antigen was described as a receptor for the autonomous parvovirus B19 (Brown et al., Science 262, pp. 114-117, 1993) and for the helper-dependent AAV-2, heparan sulfate proteoglycan was determined as a cellular receptor molecule (Summerford and Samulski, J. Virol. 72 (2), pp. 1438-1445, 1998).
  • viral vectors for example retroviral vectors, adenoviral vectors or vectors which are derived from adeno-associated viruses, are preferably used for gene transfer.
  • a disadvantage of the previous methods is that it has hardly been possible to modify the virus so that (only) the desired target cell is transduced (directed gene transfer) and the undesired transduction of non-target cells is avoided.
  • An expansion or change in the target cell spectrum ie tropism
  • the development of gene transfer methods that are as selective as possible is therefore becoming increasingly important.
  • AAV is a human parvovirus consisting of an uncoated icosahedral capsid with a single-stranded DNA genome (approx. 4.6 Kb). It has a broad host range and is able to integrate its genome into a preferred site of the host cell genome when no helper virus is present (Kotin et al., PNAS USA 87 (6), pp. 2211-2215, 1990).
  • helper virus eg adenovirus or herpes virus
  • a helper virus mobilizes the latent AAV and induces amplification of the AAV genome.
  • About 70% of the population have antibodies against AAV, for which no pathogenic properties are known.
  • the vectors derived from AAV consist only of the two 145 bp long terminal repeats that carry the signals in ice for replication, packaging and integration. Up to approx. 4.5 Kb foreign DNA can be inserted between these two elements.
  • the rep and cap genes and a helper virus are required in trans for packaging in recombinant viral vectors (rAAV).
  • rAAV recombinant viral vectors
  • AAV vectors combine a number of advantages: they contain no viral genes, have stable capsids, have a broad host range and are able to infect both proliferating cells and resting cells. In particular, they allow long-term expression of genes introduced into the differentiated tissues, for example muscle, brain and retina, without any significant immune response from the host.
  • the disadvantages of using AAV vectors for gene transfer are, inter alia, that although AAV has a broad host range, some cell types, for example haematopoietic stem cells and dendritic cells, can only be unsatisfactorily transduced.
  • the possibility of selective gene transfer with AAV would of course in principle be desirable for in vivo applications. However, little is known about the determinants of cell and tissue tropism in AAV, such as viral attachment proteins or attachment sequences, and so there is no way to manipulate AAV with regard to selective gene transfer.
  • the present invention relates to a monoclonal antibody which is characterized in that it binds to the capsid of an adeno-associated virus (AAV) and prevents the virus from binding to the virus receptor of the original target cell.
  • AAV adeno-associated virus
  • capsid used here denotes the icosahedral protein envelope which surrounds the AAV genome and is typically composed of the structural proteins VP1, VP2 and VP3.
  • original target cell means any cell to which the unmodified AAV binds.
  • the AAV includes the following types: AAV-1, AAV-2, AAV-3, AAV-4, AAV-5 and AAV-6.
  • the following groups are particularly suitable for the purposes of gene transfer: AAV-2, AAV-3, AAV-4, AAV-5 and AAV-6.
  • the prevention of the binding of the AAV to the viral receptor of the original target cell can be determined by several methods.
  • Various radioactive and non-radioactive binding tests have been developed in recent years to determine the binding of a virus to its target cell. These methods are applicable in the context of the present invention.
  • Radioactive binding tests are based on blot assays, in which cell extracts are dotted onto a nitrocellulose membrane or blotted according to SDS-PAGE and these are then incubated with radioactively labeled viruses. Bound viruses can be detected on an X-ray film after exposure (Bass, DM et al.
  • the monoclonal antibodies described in the present patent application were preferably isolated using a further non-radioactive approach. This is described below using AAV-2, which, however, should not be interpreted as being limited to this.
  • the non-radioactive binding assay was specially developed for the isolation of monoclonal antibodies that inhibit the binding of AAV-2 to the cell.
  • AAV-2 viruses were preincubated with hybridoma supernatants in order to then be incubated with cells. These cells with the viruses bound to them were fixed and non-specific binding sites were blocked in order to then detect the bound viruses by means of capsid ELISAs.
  • a biotinylated monoclonal antibody that binds to AAV-2 is used, which is isolated by streptavidin Peroxidase can be detected.
  • streptavidin Peroxidase When screening the hybridoma supernatants, a negative ELISA result was sought. If the sought monoclonal antibody is able to bind to AAV-2 and to prevent the virus from binding to the cell, no signal is obtained in the ELISA, since only AAV-2 particles that bind to cells are detected.
  • Non-binding of a virus results in non-infection.
  • these two processes are not identical since a virus can bind cells, but does not have to be absorbed into the cell at the same time, which would result in their infection.
  • the production of monoclonal antibodies comprises, for example, as a first step the production of polyclonal antibodies using AAV capsid proteins or fragments thereof (for example synthetic peptides) with suitable ligand sequences, for example the peptides or fragments thereof described in the examples, as an immunogen for the immunization of suitable animals and the production of cells which produce antibodies against the defined antigen, for example sensitized B lymphocytes.
  • AAV capsid proteins or fragments thereof for example synthetic peptides
  • suitable ligand sequences for example the peptides or fragments thereof described in the examples
  • Examples of cells that produce antibodies are spleen cells, lymph node cells, B-lymphocytes, etc.
  • animals that can be immunized for this purpose are mice, rats, horses, goats and rabbits.
  • the myeloma cells can be obtained from mice, rats, humans or other sources.
  • Cell fusion can be carried out, for example, by the well-known Köhler and Milstein method.
  • the hybridomas obtained by cell fusion are by means of the antigen according to the enzyme-antibody method or searched by a similar procedure. For example, clones are obtained using the limit dilution method.
  • the clones obtained are, for example, implanted intraperitoneally in BALB / c mice, the ascites are removed from the mouse after 10 to 14 days, and the monoclonal antibody is purified by known methods (for example ammonium sulfate fractionation, PEG fractionation, ion exchange chromatography, gel chromatography or affinity chromatography).
  • fragment means all parts of the monoclonal antibody (e.g. Fab, Fv or "single chain Fv” fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art.
  • the monoclonal antibody mentioned is an antibody derived from an animal (for example a mouse), a humanized antibody, a chimeric antibody, a human antibody or a fragment thereof.
  • Chimeric, human antibody-like or humanized antibodies have a reduced potential antigenicity, but their affinity for the target is not reduced.
  • the general production of chimeric and humanized antibodies or of antibodies similar to human antibodies has been described in detail (see for example Queen et al., PNAS USA 86, p. 10029, 1989; Verhoeyan et al., Science 239, p. 1534, 1988) .
  • Humanized immunoglobulins have variable scaffold areas, which essentially originate from a human immunoglobulin (with the name acceptor immunoglobulin) and the complementary nature of the determining regions, which essentially originate from a non-human immunoglobulin (e.g. from the mouse) (with the designation donor immunoglobulin).
  • the constant region (s), if any, also originate essentially from a human immunoglobulin.
  • humanized (as well as human) antibodies offer a number of advantages over antibodies from mice or other species: (a) the human immune system should not recognize the framework or the constant region of the humanized antibody as foreign and therefore the antibody should Response against such an injected antibody is lower than against a completely foreign mouse antibody or a partially foreign chimeric antibody; (b) since the effector area of the humanized antibody is human, it should interact better with other parts of the human immune system, and (c) injected humanized antibodies have a half-life that is essentially equivalent to that of naturally occurring human antibodies, which it is allows smaller and less frequent doses to be administered compared to antibodies from other species. Of course, these advantages also apply to human antibodies.
  • the antibody according to the invention is an antibody or a fragment thereof which binds to the capsid of AAV-2, AAV-3, AAV-4, AAV-5 or AAV-6 and the binding of Virus to the viral receptor of the original target cell prevented.
  • antibodies or fragments thereof described above are those which bind to the capsid proteins VP1, VP2 and VP3, in particular in the region of the amino acids 449-475, 545-556 and 585-598 (based on VPI of AAV-2) .
  • Antibodies according to the invention can be obtained from the hybridoma cell lines deposited on August 19, 1998 at DSMZ, Mascheroder Weg, Braunschweig under numbers ACC 2369 (results in C24-B) and ACC 2370 (results in C37-B). These are directed against AAV-2.
  • the present invention further relates to the monoclonal antibodies described above, which are furthermore characterized in that they are fused with a desired receptor ligand and can therefore serve to produce AAV vectors with an expanded or reduced host range, depending on the introduced new ligand sequence. to construct.
  • Suitable as receptor ligands are all ligands which bring about an expansion or reduction and thus a targeted change in the host range.
  • Ligands which bind primarily to receptors of malignant cells are also suitable for this purpose. Here are an example of the following ligands:
  • FGF Fibroblast Growth Factor
  • Tumors can be found.
  • EGF Epidermal Growth Factor
  • ligands are also of great importance for those receptors which allow the infection of target cells which enable the treatment of genetic diseases.
  • ligands are also of great importance for those receptors which allow the infection of target cells which enable the treatment of genetic diseases.
  • ligands are also of great importance for those receptors which allow the infection of target cells which enable the treatment of genetic diseases.
  • anti-human secretory component Fab fragments They bind to the "polymeric immunoglobulin receptor"
  • pIGR asialoglycoprotein
  • Erythropoietin for "targeting" hematopoietic cells that carry the erythropoietin receptor, for example for the treatment of sickle cell anemia.
  • an antibody fragment for example a Fab fragment
  • Fab fragment can be directly coupled to the ligand sequences which bind to the receptor of the desired target cell.
  • cross-linkers make groups available during the coupling, subdivide into homo- and heterobifunctional "cross-linkers".
  • the former have at least two identical reactive groups and allow one-step coupling, while the latter have at least two different reactive groups and allow sequential conjugation of proteins.
  • the most commonly used "cross-linkers” are homobifunctional and react with the primary amino groups of proteins. These include imido esters and NHS esters (N-hydroxysuccinimides). NHS esters are more stable and efficient than imido esters and react with primary and secondary amines to form an amide bond.
  • crosslinkers are sulfhydryl reagents containing thiol groups react, as well as other conjugation reagents that interact with other reactive groups (arginine-specific or carbonyl-specific "cross-linker”) or show no selectivity (eg photoaffinity reagents).
  • the "cross-linkers” mentioned above connect proteins via bridges, which allow different removal of the proteins.
  • the carbodiimide method is known as a method in which no bridges are formed, in which carboxyl groups are linked to primary amines via an amide bond.
  • the binding sequence of the monoclonal antibody can be cloned as a single chain antibody.
  • the approach developed for the cloning of scFv consisted in the direct cloning of the antibody genes from hybridoma cell lines.
  • the variable regions of the light and heavy chain are amplified by the polymerase chain reaction (PCR) with the help of antibody-specific oligonucleotide primers. (F. Breitling & S.
  • the cloned single chain antibodies can be expressed, for example, on a phage surface within a phage bank in order to investigate their binding to AAV capsids, for example by means of ELISA. Only those single-chain antibodies that show good binding are used further, for example to express them in E. coli. With the expressed single-chain antibodies, for example, purified via "His tags", their ability to compete for the binding of the virus to the receptor of the original target cell can then be checked again. The single-chain antibodies obtained in this way can then be fused with the desired ligand sequences or with a second single-chain antibody.
  • scFv with multivalent and multifunctional properties
  • Some scFv show a natural multimerization potential, whereby the bind variable domains of one scFv with the complementary domains of another scFv.
  • Other scFv can be fused together by fusion with a "Leucine Zipper" or an amphiphatic helix at the C-terminus.
  • a multifunctional approach concerns the fusion of a scFv with streptavidin. Biotinylated ligands, monoclonal antibodies or other scFv can now be easily conjugated to the scFv-streptavidin.
  • the present invention thus further relates to an AAV vector, preferably based on AAV-2, AAV-3, AAV-4, AAV-5 or AAV-6, which is characterized in that the monoclonal antibodies according to the invention described above or fragments thereof its capsid is bound and can no longer bind to the virus receptor of the original target cell, but possibly to the virus receptor of a desired target cell.
  • AAV vector preferably based on AAV-2, AAV-3, AAV-4, AAV-5 or AAV-6, which is characterized in that the monoclonal antibodies according to the invention described above or fragments thereof its capsid is bound and can no longer bind to the virus receptor of the original target cell, but possibly to the virus receptor of a desired target cell.
  • Such a vector allows the introduction of Fre d-DNA into a desired target cell.
  • the present invention thus also relates to the vector described above, which additionally contains a foreign DNA.
  • AAV vector plasmid which carries a foreign DNA to be expressed between the "inverted terminal repeats" (ITR) of AAV
  • AAV helper plasmid into a production cell line (for example 293T cells) and overinfection with a helper virus (e.g. Ad2, Ad5 or HSV-1) can be obtained ( Figure 1A).
  • helper virus e.g. Ad2, Ad5 or HSV-1
  • FIG. 1B The foreign DNA to be expressed can contain a reporter gene or a therapeutically interesting gene.
  • This foreign DNA should not exceed a maximum size of approximately 4.7 kilobases and can be selected by a specialist according to his wishes.
  • the recombinant AAV viruses can be released from the transfected cells by cell lysis.
  • a modification of the capsids with the antibodies described can be carried out in the cell lysate or after purification of the vectors.
  • the cleaning can be carried out using various methods known to the person skilled in the art.
  • the AAV vector is preferably modified so that it can bind target cells via different receptors.
  • the target cells are e.g. Tumor cells with receptors such as the folate receptor, the "epidermal growth factor” receptor and “fibroblast growth factor” receptor or receptors of hematopoietic cells, such as the erythropoietin receptor, SCF receptor and CD 34 in question.
  • the present invention further relates to a method for producing the AAV vector according to the invention, which comprises the steps described in the preceding sections.
  • the present invention also relates to a method for directed gene transfer, which is characterized in that the AAV vector according to the invention is used as a vehicle for the nucleic acid sequence to be introduced into the desired target cell.
  • the nucleic acid sequence to be introduced can be under the control of an inducible and / or repressible cell type-specific promoter, for example.
  • the AAV vectors according to the invention can be introduced into a cell, a tissue, organ, a patient or an animal by a number of methods, for example by ex vivo incubation of the purified AAV vectors with the desired target cells (e.g. Maas et al. Human Gene Therapy 9, 1049-1059 (1998), Zhou et al.
  • transgenic animals i.e. mammals
  • transgenic animals which are transgenic with respect to the nucleic acid sequence introduced by means of the AAV vector according to the invention
  • Methods for producing such transgenic animals can be found, for example, in WO 91/08216.
  • FIG. 2 Schematic representation of the test for the isolation of antibodies that block the binding of the virus to the cell.
  • Fig. 3 "Retargeting" of AAV-2
  • Example 1 Production of antibodies directed against the ligand sequences.
  • Balb-c mice were repeatedly immunized with synthetic peptides and with empty AAV-2 capsids.
  • the following four peptides were synthesized based on AAV-2 capsid gene sequences in which AAV-2 and AAV-3 differ.
  • the peptides had the following sequence:
  • AAV-2-1 GPPPPKPAERHKDDSC AAV-2-2 SRTNTPSGTTTQSRLQFSQAGASDIRDQSC AAV-2-3 QSGVLIFGKQGSEKTNVDIEKC AAV-2-4 SVSTNLQRGNRQAATADVNTQC
  • the two viruses AAV-2 and AAV-3 have a different cell tropism, although their capsid gene sequences differ slightly from each other in only four domains. Since peptides are only linear epitopes, but the ligand sequence on the viral capsid can also be a conformational epitope, an additional boost was made with empty AAV-2 capsids.
  • the spleen was removed from a mouse and the spleen cells isolated from it were fused with Ag8 cells.
  • the resulting clones secrete antibodies into the medium, which has now been tested for its properties with various essays.
  • all hybrid supernatants were tested for their ability to neutralize. In this neutralization test based on GFP (green fluorescent protein), a "screening" of the monoclonal was also found
  • Hybridoma supernatants which had a neutralizing effect, that is to say those which prevent the expression of GFP, could now be tested for their ability to prevent the binding of AAV-2 to the cellular receptor, to be examined.
  • This non-radioactive binding test was specially developed for this task (see Fig. 2).
  • hybridoma supernatants were first incubated with AAV-2 before these were applied to cells, for example Heia cells. The AA V-2 particles binding to the cell were fixed.
  • Hybridoma supernatants which bind to AA V-2 and prevent the capsid from binding to the cellular receptor, prevent binding to the cells.
  • the viruses bound to the cells are detected with the monoclonal A20 antibody, which recognizes assembled AAV-2 capsids (W istuba et al., Journal of Virology 71, pp. 1341-1352, 1997 ) .
  • the thus characterized clones which produce neutralizing un d t he binding inhibitory antibodies were then isolated to obtain monoclonal antibodies (Mab).
  • T he Maks thus obtained were again investigated for their properties. In addition to the just described A ssays they were still ⁇ fluorescence on their behavior in immune A d-5 (negative control) and Ad-5 / AAV-2 infected cells, Western blots of Ad / AAV-2 HeLa extracts and A AV 2 ELIS A examined. After analyzing this data and determining the subclass, the two Hybrido e C 24-B and C37-B were selected. The following table shows their e eatures:
  • Single chain antibodies were produced from both hybridomas, C24-B and C37-B.
  • oligonucleotide primers the sequence of which in Breitling et al. , Methods in Molecular Medicine, pp. 581-592, Vol. 13: Molecular Diagnosis of Infectious Diseases, Humana Press Inc. Totowa NJ, in the polymerase chain reaction (PCR) the variable domains of the light (VL) and the heavy Chain (VH) can be isolated.
  • PCR polymerase chain reaction
  • VL variable domains of the light
  • VH heavy Chain
  • diabodies are bivalent and bispecific because they consist of two scFv fused together.
  • the VH domain of one antibody (C24-B or C37-B) is replaced by a short linker with the sequence "AKTTPKLGG” (peptide linker, which is the approx. 3.5 nm between the C-terminus of the one
  • Example 3 Chemical coupling of Fab fragments Fab fragments of C24-B and C37-B were isolated according to standard methods. These are now chemically coupled with IgGs, which are directed against EGF and FGF receptors, or with folate, the ligand of the folate receptor. The conjugation takes place with the help of SPDP (3- (2-pyridildithio) propionic acid N-hydroxysuccinimide ester). The resulting complexes are purified by HPLC and can now be used to change the cell tropism of AAV vectors. For this purpose, AAV vectors are incubated with the purified Fab-IgG / ligand complexes. The complexes not bound to the AAV vectors are separated (e.g.) by centrifugation through a sugar cushion.
  • SPDP 3- (2-pyridildithio) propionic acid N-hydroxysuccinimide ester
  • AAV-2 vectors are used with the luciferase or LacZ reporter genes in order to obtain a quantitative and qualitative statement about the expression efficiency achieved.
  • all available AAV vectors can be used. These vectors are then incubated with the Fab-IgG / ligand complexes.
  • Example 4 Change in the Tropism of rAAV-2
  • the "diabodies" described in Example 2 and Fab-IgG / ligand conjugates described in Example 3 can now be used to change the tropism of rAAV-2.
  • the Fab-IgG / ligand conjugates are used to test the principle that rAAV-2 cells can be infected via a new receptor. After coupling Fab fragments to the ligand folate, for example, and then incubating with rAAV-2, it is shown that cells (eg HeLa, KB) which overexpress the folate receptor are infectible. This Infection cannot be prevented by an excess of heparin, which inhibits the natural AAV-2 infection, but it can be prevented by the presence of free Fab fragments or an excess of folate in the medium.
  • the Fab-anti-EGFR-IgG conjugate can be used to demonstrate "retargeting" on the EGFR analogously to the example described above with the folate ligand . (See Fig. 3).
  • a change in tropism is also possible via the "diabodies” described. These are used to make cells difficult to infect for AAV-2 (e.g. the cell lines Raji [human Burkitt lymphoma cell line], 9023 and 9050 [human lymphoblastoid cell lines]; Maass, G. et al. Human Gene Therapy 9, 1049-1059 , 1998) to make AAV-2 infection more accessible.
  • AAV-2 e.g. the cell lines Raji [human Burkitt lymphoma cell line], 9023 and 9050 [human lymphoblastoid cell lines]; Maass, G. et al. Human Gene Therapy 9, 1049-1059 , 1998) to make AAV-2 infection more accessible.
  • the "diabodies” are incubated with rAAV-2, the “diabodies” that are not bound to the AAV capsids are separated and then added to the cells mentioned above. This means that an efficient infection of the lymphoma cell lines that are barely infectable from un

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Abstract

L'invention concerne des anticorps monoclonaux ou des fragments de ces derniers qui se lient à la capside de virus associés aux adénovirus (AAV), empêchant ainsi la liaison du virus au récepteur viral de la cellule cible initiale. Cet anticorps ou un fragment de ce dernier peuvent en outre être fusionnés avec un ligand récepteur souhaité. Après liaison d'un tel anticorps à la capside de AAV, on obtient un AAV présentant un tropisme modifié, c'est-à-dire pouvant, en fonction du ligand fusionné, se lier à une nouvelle cellule cible et pouvant être utilisé pour la construction d'un vecteur AAV en vue d'un transfert de gènes.
EP99963196A 1998-10-29 1999-10-29 Anticorps se liant a la capside de virus associes aux adenovirus (aav), modifiant le tropisme cellulaire et procede de transfert de genes cible Withdrawn EP1124854A2 (fr)

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DE19849643 1998-10-29
DE1998149643 DE19849643A1 (de) 1998-10-29 1998-10-29 An das AAV-Kapsid bindender, den Zelltropismus verändernder Antikörper und Verfahren zum gerichteten Gentransfer
PCT/DE1999/003517 WO2000026254A2 (fr) 1998-10-29 1999-10-29 Anticorps se liant a la capside de virus associes aux adenovirus (aav), modifiant le tropisme cellulaire et procede de transfert de genes cible

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CA2265460A1 (fr) 1996-09-11 1998-03-19 The Government Of The United States Of America, Represented By The Secre Tary, Department Of Health And Human Services Vecteur de vaa4 et ses utilisations
CA2745131C (fr) 1998-05-28 2016-08-09 John A. Chiorini Vecteurs d'aav5 et leurs utilisation
AU2001277531A1 (en) * 2000-07-10 2002-01-21 Novartis Ag Bifunctional molecules and vectors complexed therewith for targeted gene delivery
US7419817B2 (en) 2002-05-17 2008-09-02 The United States Of America As Represented By The Secretary Department Of Health And Human Services, Nih. Scalable purification of AAV2, AAV4 or AAV5 using ion-exchange chromatography
WO2005017101A2 (fr) 2003-05-19 2005-02-24 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH & HUMAN SERVICES, NATIONAL INSTITUTES OF HEALTH Virus aviaire adenoassocie (aaav) et ses utilisations
WO2005056807A2 (fr) * 2003-12-04 2005-06-23 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services, National Institutes Of Health Vecteur viral associe aux adenovirus bovins et leurs utilisations
US8283151B2 (en) 2005-04-29 2012-10-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Isolation, cloning and characterization of new adeno-associated virus (AAV) serotypes
CN110799524A (zh) * 2017-06-27 2020-02-14 瑞泽恩制药公司 向性修饰的重组病毒载体及其用于将遗传材料靶向引入人细胞内的用途
CN112159467A (zh) * 2020-09-14 2021-01-01 和元生物技术（上海）股份有限公司 能够与aav1-13结合的抗体
CN113583112B (zh) * 2021-07-30 2022-07-19 上海勉亦生物科技有限公司 Aav特异性抗体及其应用
WO2023196892A1 (fr) 2022-04-06 2023-10-12 The Trustees Of The University Of Pennsylvania Immunisation passive avec des anticorps neutralisants anti-aav pour empêcher la transduction hors cible de vecteurs aav administrés par voie intrathécale
CN117285620B (zh) * 2023-11-27 2024-02-13 恺佧生物科技(上海)有限公司 抗aav9抗体及aav9滴度测定elisa试剂盒

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CA2175256A1 (fr) * 1993-10-28 1995-05-04 Andrea Kern Virus adeno-associe et son utilisation pour diagnostiquer un avortement precoce
DE19827457C1 (de) * 1998-06-19 2000-03-02 Medigene Ag Strukturprotein von AAV, seine Herstellung und Verwendung

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