EP1664099A2 - Epitopes adenoviraux - Google Patents

Epitopes adenoviraux

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Publication number
EP1664099A2
EP1664099A2 EP20040768408 EP04768408A EP1664099A2 EP 1664099 A2 EP1664099 A2 EP 1664099A2 EP 20040768408 EP20040768408 EP 20040768408 EP 04768408 A EP04768408 A EP 04768408A EP 1664099 A2 EP1664099 A2 EP 1664099A2
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EP
European Patent Office
Prior art keywords
peptide
epitopes
antibodies
antibody
epitope
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EP20040768408
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German (de)
English (en)
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Niels Rudi Pedersen
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Individual
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Individual
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Priority claimed from GB0321063A external-priority patent/GB0321063D0/en
Priority claimed from GB0321179A external-priority patent/GB0321179D0/en
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Publication of EP1664099A2 publication Critical patent/EP1664099A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/235—Adenoviridae
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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
    • C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2710/10334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2710/14011—Baculoviridae
    • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
    • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to Adenoviral epitopes, in particular to epitopes of the penton base (Pb) of Adenovirus (Ad) which are recognised by neutralising antibodies .
  • Peptides incorporating these epitopes have utility in methods of treatment and evaluation/diagnosis of a patient.
  • Ads adenoviruses
  • Ads are easy to grow to high titers and in a suitable clinical grade.
  • Ads are biologically safe compared to other vectors such as retroviruses or lentiviruses since they are mostly responsible for benign infections in immunocompetent individuals, and their lack of genomic integration reduces the risk of genomic recombination and oncogenic transformation.
  • Ads are able to transduce both dividing and nondividing cells and are particularly appropriate in clinical applications such as cancer therapy due to their transient expression.
  • Ad capsid epitopes in hexon and fiber proteins that were responsible for the immune response.
  • the immunogenicity in humans and the characterization of neutralizing and non-neutralizing epitopes in the third major Ad capsid component, the Pb has not been systemically studied.
  • Ad antibodies that recognise linear and conformational epitopes in the penton base protein of Adenovirus .
  • the present inventors have further been able to identify the Pb antigenic epitopes and to evaluate which are epitopes for neutralising antibodies (NA) that can interfere with effective gene therapy transfer. It has also been shown that Pb antibodies make a significant contribution toward anti-Ad neutralisation.
  • one or more peptides which are able to mimic the Pb neutralising antibody epitopes in simultaneous administration with an Ad vector (e.g. a recombinant Ad vector in gene therapy) .
  • Ad vector e.g. a recombinant Ad vector in gene therapy
  • These peptides can block the neutralising antibodies and help the Ad vector itself to escape the neutralising effect of the antibodies, thus enabling more effective gene delivery.
  • the invention provides a peptide comprising an adenoviral penton base epitope for a (human) neutralising antibody.
  • the 'epitope 1 either has the sequence of a naturally occurring penton base epitope for NA or is a related sequence which is immunologically equivalent.
  • said epitope is from Ad5.
  • Three major immunodominant domains have been identified and the epitope is preferably a fragment from within region 50-120, 191-233 (preferably 193-230) or
  • Ad5 or an equivalent region within another Ad serotype, or an immunologically equivalent variant of such a fragment .
  • a further group of suitable epitopes are immunologically equivalent variants or fragments of these 10 epitopes.
  • the numbers correspond to the a ino acid in the Ad5 penton base protein.
  • Preferred variants are the corresponding regions in other Ad serotypes, particularly Ad3.
  • the epitope will preferably comprise at least 5 amino acids, e.g. at least 6 or 7 amino acids .
  • Further suitable variants include the hexamers of Table 2.
  • a preferred group of epitope fragments have an amino acid sequence selected from the group comprising
  • NSIRYSE LGFDPVTGL, AIRGDTF, LDDRSHWG, SGSGAE, FTQYRS YL, YLVDNKS, LKVGRQ, PYVYK and GLVMPGV, any of the underlined sequences of Table 2, or immunologically equivalent variants or fragments thereof.
  • Particularly preferred peptides are those which consist or comprise the following sequences NSIRYS, VTGL, SGAE, and FTQ.
  • Immunologically equivalent variants will be able to generate or are immunologically reactive with neutralising antibodies and will have sequence similarity with one of the sequences listed above, suitable immunologically equivalent epitopes/peptides are listed in Table 2.
  • Variants will typically have 3 or more, preferably 4 or more, e.g. 5 or more amino acids which are preferably identical to or possibly homologous with amino acids in the actual Pb sequence.
  • Amino acid pairs which can be considered homologous include leucine and isoleucine; cysteine and serine; asparagine and glutamine; and aspartic acid and glutamic acid.
  • Fragments and variants of the 10 epitopes defined above will typically be at least 6 or 7 amino acids in length, preferably 8 or 9 amino acids in length, more preferably 10 or 11, most preferably 12 amino acids in length.
  • Variants will typically have at least 50%, if not 60%, preferably at least 70 or 80%, more preferably at least 90% sequence homology with these naturally occurring Ad5 epitopes (or equivalent epitopes in other serotypes) . Sequence 'homology' allows for conservative substitution of individual amino acids, Arg for Lys, Phe for Tyr, Glu for Asp etc.
  • variants will have at least 40%, if not 50%, more preferably at least 60 or 70%, most preferably at least 80% sequence identity with these naturally occurring Ad5 epitopes (or equivalent epitopes in other serotypes) .
  • a method of determining immunological equivalence can be derived from the Examples which describe how bacteriophage expressing putative epitopes may be generated and their binding reactivity with (e.g. immobilised) Pb antibodies assessed.
  • Variants and fragments may be more or less able to block the NA than a named sequence and still be considered ' immunogenically equivalent'; typically fragments and variants will have at least 20%, preferably at least 35%, more preferably at least 50%, most preferably at least 65% of the immunogenic activity of a named epitope .
  • Each peptide of the invention incorporating an epitope will typically be no more than 25, preferably no more than 20 amino acids in length, e.g. 15 to 10 amino acids, most preferably 12 amino acids.
  • Preferred peptides for use in co-administration with an Ad vector will comprise 2 or more epitopes as defined above, e.g. two epitopes or more in tandem (these peptides may therefore exceed the preferred values given above and be of 30, 40 or even 50 amino acids in length) , or circularised peptides incorporating disulfide bridges .
  • the peptides may further be modified in a conventional manner to increase half life e.g. by N- or C-terminal modification but will typically be administered with the Ad vector without such modifications .
  • the peptide may comprise no other amino acids apart from the epitope or epitopes .
  • a 'neutralising antibody' is one which inhibits the ability of an Ad to replicatively infect a cell, typically at the step of cell uptake or endocytosis.
  • the invention provides a pharmaceutical composition comprising a peptide comprising an adenoviral penton base epitope for a human neutralising antibody and a pharmaceutically acceptable carrier, diluent or adjuvant.
  • the peptides of the invention may be administered together with an Ad vector in a combined preparation for gene delivery or the two components may be administered separately.
  • the invention provides a pharmaceutical composition comprising a peptide comprising an adenoviral penton base epitope for a human neutralising antibody and an (recombinant) adenoviral vector.
  • the invention provides a product containing (a) a peptide comprising an adenoviral penton base epitope for a human neutralising antibody, and (b) an adenoviral vector as a combined preparation for separate, simultaneous or sequential administration to a patient as part of gene therapy or cancer treatment .
  • an "adenoviral vector” being an adenovirus based particle which is capable of delivering genetic material, typically but not exclusively recombinant genetic material, into a cell.
  • Suitable therapeutic genes which may be introduced into a patient in an adenoviral vector are known in the art and the list of suitable candidates, particularly in cancer therapy, are increasing all the time.
  • the invention provides a peptide comprising an adenoviral penton base epitope for a human neutralising antibody for use in therapy, in particular for use in gene or cancer therapy as a masking or blocking agent for a co-administered Ad vector.
  • the invention provides a method of gene or cancer therapy using an Adenoviral vector wherein a peptide comprising an adenoviral penton base epitope for a human neutralising antibody is co-administered to the patient with said vector.
  • co-administration need not be, but may be, as part of a single product or 2 simultaneously administered products .
  • the peptides of the invention may be administered to a patient as a mini peptide or a mini gene encoding the peptide which is inserted in a plasmid or other vector, e.g. an Ad vector.
  • a plasmid or other vector e.g. an Ad vector.
  • nucleic acid molecules encoding the peptides constitute a further aspect of the invention and the various therapeutic methods or uses of the peptides described herein apply equally to nucleic acid molecules encoding the peptides and to expression vectors incorporating these regions of nucleic acid, e.g. plasmids or viral vectors.
  • the Adenoviral vector may be of any serotype .
  • the sequences referred to herein are based on the conditionally replicative mutant adenovirus AddI1520.
  • Epitopes in other Ad may vary slightly in position or sequence but equivalent epitopes can be readily identified by sequence comparison and tested according to the methods described in the Examples section herein.
  • a preferred group of epitope fragments are NSIRYS, VTGL, SGAE, FTQ, AVTGID, HSGAEF and NFTQVG and immunologically equivalent variants thereof.
  • Ads are the cause of common upper respiratory infections, and thus most people have preexisting immunity.
  • Adenoviral vector itself.
  • An alternative application of the present invention is the modification of these epitopes.
  • Site directed mutagenesis may be used to modify the Ad genome so that the produced capsid Pb protein incorporates one or more modified epitopes which are able to avoid the NA.
  • the present invention provides an adenovirus wherein one or more of the penton base epitopes for neutralising antibodies has been modified to reduce its reactivity with a corresponding neutralising antibody.
  • a reduction in 'reactivity' includes a reduced ability to be recognised by NA and a reduced ability to generate NA; a reduction in immunogenicity.
  • Pb capsomers may be swapped between Ad serotypes from different subgroups in order to improve the efficacy of Ad virions or Ad-derived vectors used as therapeutic agents in cancer or gene therapey.
  • the present invention provides a modified Adenovirus which incorporates one or more Pb capsomers from a non-native serotype. "Non-native" means that the capsomer is from a different serotype than the rest (or most of the rest) of the Ad.
  • the peptides can be incorporated in an ELISA test, for example, to check the presence and level of different penton base antibodies in the patient. This will give information as to the suitable dose of Ad vector which should be administered to the patient and also may be used to monitor the patient during the period of Ad administration, e.g. during the gene therapy trial. This is particularly useful as the type and quantity of NA varies significantly from patient to patient.
  • the present invention provides a method of evaluating a patient ' s serum, which method comprises contacting a ( in vi tro) sample of the serum with one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody.
  • a ( in vi tro) sample of the serum with one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody.
  • Preferred peptides are discussed above.
  • the sample is preferably contacted with three or more, preferably 5 or more peptides incorporating the neutralising epitopes .
  • These methods may conveniently be carried out on a solid support, e.g. the peptides described above or antibodies thereto may be immobilised on a solid support.
  • the invention provides a solid support having immobilised thereon one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody.
  • the solid support will have immobilised thereon 3 or more, e.g. 5 or more peptides incorporating the neutralising epitopes.
  • Methods of immobilising peptides on solid supports are well known in the art.
  • the peptides described above are of particular utility when Ad of serotype 5 is used as the adenoviral vector as the epitopes which have been characterised are from an Ad of this serotype .
  • Ad of serotype 5 is used as the adenoviral vector as the epitopes which have been characterised are from an Ad of this serotype .
  • the penton base sequence is highly conserved between Ad of the same subgroup and a 98.6% homology has been found between Ad2 and Ad5.
  • the peptides of the invention may also be used with an Ad2 or Ad3 vector.
  • the peptides described herein may also be used as a vaccine for preventing Ad infection.
  • the invention provides a vaccine comprising one or more peptides comprising an adenoviral penton base epitope for a human neutralising antibody.
  • the peptide would be used as an adjuvant, like tetanus toxoid or alum.
  • the peptides, in the form of lipopeptides can be injected with recombinant Ad hexon proteins (which may be a complete hexon protein or an active fragment thereof, activity in this case being in the context of immunogenicity and the ability to act as an adjuvant) .
  • Ad hexon proteins which may be a complete hexon protein or an active fragment thereof, activity in this case being in the context of immunogenicity and the ability to act as an adjuvant
  • the C-terminus of the peptide is preferably modified to enhance immunogenicity e.g. by a palmitoyl-lysylamide group (as in J. Virology 76 [2000] , pp 127-135) .
  • the invention also provides a method of preventing or ameliorating natural Adenoviral infection in a patient which comprises administering to said patient a vaccine as described above.
  • a further application of the present invention is the use of the peptides of the invention in a method of haemofiltration or dialysis where the blood of the patient is taken out of the body and passed across a filter that contains these peptides.
  • the peptides can chelate the NA and retain them in the filter, the blood can then be given back to the patient with much lower concentrations of NA.
  • the invention provides a method of filtering blood to remove neutralising antibodies to epitopes of the adenoviral penton base, which method comprises contacting the blood outside the body with the peptides of the invention described above, preferably these peptides will be immobilised on a solid support which filters the blood.
  • the blood is then preferably returned to the patient .
  • the blood is passed through an affinity column which incorporates the peptides of the invention.
  • An affinity column comprising the peptides of the invention is a further aspect of the present invention.
  • An alternative method of masking the neutralising ability of the NA is to use antibodies or antibody fragments directed against the neutralising antibodies. These antibody or Ab fragments can also attach to the NA so they cannot act on the adenovirus and as such can be considered immunoreactive with these NA.
  • Methods of raising antibodies to known epitopes is well known in the art.
  • the various therapeutic and diagnostic methods and uses discussed above which utilise the peptides of the invention apply, mutatis mutandis to the use of antibody or antibody fragments to the various epitopes described herein.
  • Ficfure 1 shows Western blot analysis of sera from patients 1, 2, 3 and 4 taken before (day 0 [DO]) or after (day 10 [D10] ) Addll520 administration. Aliquots of serum dilutions were reacted with membrane strips transferred with recombinant Ad Pb protein electrophoresed under ' native conditions (a) or a mixture of SDS-denatured hexon, Pb, and fiber proteins separated by conventional SDS-PAGE (b and c) .
  • Control samples (lane 9 in panel a; lanes 9 to 11 in panel b; lanes 5 and 6 in panel c) consisted of strips that were reacted with rabbit anti-fiber ( ⁇ Fi) , anti-Pb ( ⁇ Pb) , or anti-whole Ad virion ( ⁇ Ad) antibodies, respectively.
  • Ficrure 2 shows the virus NA activity of Pb antibodies from human sera.
  • Ad5Luc3 recombinant was preincubated without (control, no antibody [con] ) or with total serum samples from patients 1 (PI) , 2 (P2) , 3 (P3) , or 4 (Pr) (using the DO sera of patients 1 and 2 and the D10 sera of patients 3 and 4) and then incubated with HeLa cells.
  • Ad5Luc3 was preincubated with or without isolated Pb antibodies.
  • isolated Pb antibodies were added to virus-cell monolayers after pre-attachment of Ad5Luc3 to HeLa cells at 4°C for 30 min.
  • virus infection was allowed to proceed for 18 h at 37°C, and the cells were processed for luciferase assays.
  • the NA effect was indirectly assayed by the level of luciferase activity expressed as arbitrary units and then normalized to the percentage of the control samples .
  • Figure 3 demonstrates epitope mapping in Ad Pb protein.
  • the Pb sequence is represented linearly. Under the Pb line are shown the positions of the epitopes defined in Table 2. The patient sera reacting with the different epitopes are indicated in parentheses. Above the Pb line are shown the major IDRs in humans, as defined in the present study, and in mice, after immunization with recombinant Pb (11) . The probability of immunogenicity (as evaluated by the antigenic index of Jameson-Wolf) is presented at the top of the figure .
  • Figure 4 gives graphical representation of phage-mediated depletion of Pb epitope-specific antibodies .
  • Phages carrying the Pb epitopes indicated at the top of each panel were amplified and incubated with D10 serum samples from patients 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, and 16, as well as samples 18, 19, 20, and 21 from healthy donors. After removal of the phage-antibody complexes by centrifugation, the serum supernatants were assayed for their effect on Ad-mediated gene transfer by using the replication-competent Ad5Luc3 vector. Luciferase expression was compared in cells infected with Ad5Luc3 treated with depleted or nondepleted sera.
  • Addll520 was given an intravenous injection in the arm vein (vena mediana cubiti) for the first dose (day 1) and then by direct intratumoral injection under ultrasound guidance on days 2, 15, 16, 29, and 30 (Habib, N., 2002. supra). All given doses were 1 ml in volume and contained 3 x 10 11 PFU of AddJ.1520, which had been found to be well tolerated in a previous phase 1 study (Habib, N. et al., 2001 supra) .
  • Viruses and mammalian cells Viruses and mammalian cells.
  • the Ad mutant AddI1520 was donated by A.J. Berk
  • Ad recombinant Ad5Luc3 (Mittal, S.K., et al. 1993. Virus Res. 28: 67-90), a kind gift from F. Graham (McMaster University, Hamilton, Ontario, Canada) , is a replication-competent Ad that carries the luciferase reporter gene under the control of the simian immunodeficiency virus early promoter inserted into the E3 region of the Ad5 genome .
  • Ad5GFP vector (Adeno-CMV5-GFP) was obtained from Quantum Biotechnologies, Inc. (Montreal, Quebec, Canada) . HeLa and 293 cells were grown as monolayers in Dulbecco modified Eagle medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) , L-glutamine, and antibiotics.
  • Dulbecco modified Eagle medium Invitrogen
  • FCS fetal calf serum
  • Spodoptera frugiperda (Sf9) cells were maintained as monolayers at 28°C in Grace insect medium supplemented with 10% FCS and antibiotics.
  • the recombinant baculoviruses expressing Ad2 capsid proteins hexon, Pb, and fiber have been described previously (Karayan, L. et al., 1997. J. Virol. 71: 8678-8689 and Molinier- Frenkel, V. et al . , 2002. J. Virol. 76: 127-135).
  • Recombinant Ad capsid proteins were produced in Sf9 cells and purified as described in previous studies (Boulanger, P., and F. Puvion. 1973. Eur. J. Biochem. 39: 37-42 and Molinier-Frenkel, V. et al . , supra).
  • SDS-denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 10% acrylamide gels by using a discontinuous buffer system (Laemmli, U.K. 1970. Nature 227: 680-685). Native proteins were separated in 8% polyacrylamide gels in the same discontinuous buffer system but without SDS and twice the normal buffer concentration. Transfer of proteins onto nitrocellulose membranes (Hybond ECL; Amersham Biosciences) was carried out by using a semidry blotting system.
  • Blots were blocked with skimmed milk in TBS-T (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20) and then successively reacted with patient sera, followed by the addition of phosphatase-labelled anti-human immunoglobulin M (IgM) and IgG conjugate (Sigma-Aldrich) .
  • TBS-T 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.05% Tween 20
  • IgM phosphatase-labelled anti-human immunoglobulin M
  • IgG conjugate Sigma-Aldrich
  • Recombinant Pb protein was electrophoresed in a preparative SDS-PAGE gel and transferred onto a nitro-cellulose membrane. The protein band was then localized by Ponceau red staining and excised. Membrane strips with immobilized Pb protein were incubated with human sera overnight at 4°C. After several rinses with phosphate-buffered saline (PBS) , the antibodies were eluted with 5 mM glycine-HCl (pH 2.3) -500 mM NaCl-5% FCS for 30 s and immediately neutralized with 0.5 M Na 2 HP0 4 (pH 9.5) to a final concentration of 50 mM. Two successive elutions were performed to maximize the antibody recovery.
  • PBS phosphate-buffered saline
  • the quantity of Pb antibodies eluted from the nitrocellulose membranes were determined in comparison to their corresponding original sera by using dot blot analysis . Aliquots of Pb antibody samples and of the patients ' sera from which Pb antibodies were isolated (at dilutions of 1:500, 1:1,000, 1:2,000, and 1:3,000) were spotted onto the same nitrocellulose membrane. The membrane was then reacted with peroxidase-conjugated anti-human IgG antibody and developed by chemiluminescence by using the SuperSignal substrate (Pierce) . The amounts of antibodies recovered from the patients ' sera were quantitatively determined by densitometric analysis of the dots by using the VersaDoc image analyzer and the Quantity One program (Bio-Rad) . Circulating anti-Ad antibodies.
  • Ad5Luc3 virus was used according to the protocol described above, except that the cells were harvested at 18 h after infection and the cell lysates were analyzed for luciferase activity by using luciferin substrate and a Lumat LB 9501 luminometer (Berthold, Germany) .
  • the filamentous phage hexapeptide library was a kind gift of G. Smith (University of Columbia, Columbia, Mo.) .
  • Affinity selection of phages bound to immobilized Pb antibodies was carried out according to published protocols (Hong, S.S. et al., 2000, Viral Immunol. 13: 353-371; and Hong, S.S., and P. Boulanger. 1995. EMBO J. 14: 4714-4727; and Hong, S.S., 1997, EMBO J. 16:
  • the phagotopes carried by the phages were identified by DNA sequencing of the piII gene of the phage .
  • the principle of this method is based on the normal content of IgG in human serum (ca. 15 mg/ l) .
  • IgG molecule is 150 kDa
  • incubation of 0.1 ⁇ l of serum in 100 ⁇ l of PBS (1:1,000 dilution, 6 x 10 10 moles of IgG) with 10 11 phages (give pill proteins per phage, carrying one epitope each) would provide a 10-fold excess of epitope over their corresponding antibody molecules . Phage and serum were incubated overnight at
  • the whole-antibody titer against Ad5 which was assayed for six patients (patients 3 to 8 , DO and D10 samples; Table 1), ranged between 128 to 1,024 in the pretreatment samples, whereas by 10 days after Ad administration there was an overall augmentation in antibody titer for all six patients: either 4-fold (patient 6), 16-fold (patient 4), 32-fold (patients 3, 5, and 7), or 64-fold (patient 8).
  • the anti-Ad5 NA titers of the serum samples were analyzed, and 14 sera of 21 (67%) showed low titers of pre-existing NA (titers of ⁇ 256) , whereas 7 sera (33%) had high titers of >1,024 (Table 1) .
  • NA titers At 10 days after Ad treatment, there was an increase in NA titers for 12 patients (71%) , whereas there was no change for the five other patients .
  • the increase in NA titers ranged from 2- to 16-fold, with one patient (patient 8) showing a 512-fold increase.
  • the patients ' sera were assayed for IgM and IgG subclass antibodies against native or denatured hexon, Pb, and fiber proteins. However, the reactivity of IgM antibodies were found to be very low and negligible compared to IgG antibodies (data not shown) , and thus only IgG reactivity is described here.
  • the antibody reactivity profile of the patients ' sera are summarized in Table 1. In the samples taken before Ad injection, 13 samples (62%) had hexon antibodies, 17 samples (81%) had Pb antibodies, and 17 samples (81%) had fiber antibodies. At 10 days after virus injection, most of the patients ' sera showed either an increase in reactivity or acquired antibodies to hexon (4 of 17 patients [24%]), Pb (8 of 17 patients [45%]), or fiber (7 of 17 patients [41%]) proteins.
  • the isolated antibodies were tested for their reactivity against Pb protein by Western blot analysis (Fig. lc) .
  • the positive reactivity of all four different Pb antibodies suggested that the brief acid treatment during the elution process was apparently not detrimental to their antigen-binding capacity and to their functionality as antibodies.
  • Their nonreactivity against fiber protein that was present on the same blot showed that the antibodies recovered were specific toward Pb proteins (Fig. lc) .
  • the amounts of IgG antibodies recovered were determined by dot blotting, followed by quantitative densitometric analysis.
  • the Pb antibodies recovered from serum samples 1, 2, 3, and 4 contained 1/2,500, 1/2,000, 1/1,428, and 1/1,666 of the total IgG content of their original sera, respectively.
  • the Pb of the Ad virion has been shown to react with cell surface integrin molecules which subsequently result in the internalization of the virion.
  • Pb antibodies could therefore have a neutralizing effect on Ad infection by interfering with the step of virion endocytosis and internalization.
  • the Ad5Luc3 virus used was a replication-competent virus that carried the luciferase reporter gene in its deleted E3 region (Mittal, S.K., et al., 1993, Virus Res. 28: 67-90).
  • Ad5Luc3 was preincubated for 1 h at 37°C with (i) mock, serum, (ii) total serum, or (iii) the corresponding isolated Pb antibodies .
  • the total sera were diluted accordingly to normalize their IgG content to that of their corresponding Pb antibody preparations .
  • the virus-antibody samples were added to HeLa cells, and infection was allowed to occur for 18 h at 37°C.
  • the cells were harvested and assayed for luciferase activity as a measure of Ad infection efficiency.
  • the four serum samples from patients 1, 2, 3, and 4 blocked Ad-mediated gene delivery by 65, 83, 96, and 98%, respectively (Fig. 2a) .
  • Their corresponding Pb antibodies inhibited Ad-mediated gene transfer by 59, 56, 77, and 86%, respectively (Fig. 2b) . This result suggested that a significant proportion of the Ad Na activity from the serum samples could be attributed to Pb antibodies .
  • Ad5Luc3 virus was first allowed to attach to HeLa cells at 4°C for 30 in. Unattached viruses were then removed, Pb antibodies were added to the virus-cell monolayer for another 30 min at
  • phagotopes recovered from immobilized Pb-specific Pb antibodies were grouped according to conserved residues and peptide motifs and then aligned with homologous regions of the Ad Pb sequence (underlines, with residue numbers as indicated) . Homologous or identical residues at similar locations are in boldface. Patient numbers correspond to the patient identification numbers in Table 1. Note that motif LDD-WGV likely corresponded to a discontinuous epitope.
  • epitopes corresponded to regions of high antigenic index, according to the predictive method of Jameson-Wolf (Fig. 3) . This was the case for the three major epitopes of the first class, mapped to positions S3 NSIRYSE 59 , 217 LGFDPVTGL 225 , AND 338 AIRGDTF 344 in the Pb sequence. Interestingly, one of the epitopes contained the tripeptide sequence, RGD (underlined) , which is responsible for interacting with cell surface integrins .
  • the three epitopes of class III that mapped within the regions 113 LDDRSHWG 121 , 313 SGSGAE 318 , AND 400 FTQYRSWYL 408 showed a high probability of accessibility and antigenicity (Fig. 3) .
  • 70 YLVDNKS 7S , 193 LKVGRQ 198 , AND S53 PYVYK SS6 corresponded to regions of high antigenic index; however, the epitope 224 GLVMPGV 230 had a relatively low probability of antigenicity.
  • the unique phage carrying the epitope RGDVTF was amplified and incubated with 17 different serum samples which had Pb antibodies and moderate to high Ad NA titers (D10 samples from patients 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 14, and 16 and samples 18, 19, 20, and 21 from healthy donors) .
  • Ad NA Ad NA
  • the serum supernatants were tested for Ad NA, which were indirectly assayed by their effect on gene transduction by using the Ad5Luc3 vector.
  • the phage library carrying random peptides was used as shown in Fig.

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Abstract

L'invention concerne un peptide compreant un épitope à base de Penton adénoviral pour un humain neutralisant les anticorps ; ledit épitope peut être un fragment provenant de la région 50-120, 191-233 ou 310-408 d'adénovirus 5 ou une région équivalente à l'intérieur d'un autre sérotype d'adénovirus, ou un variant équivalent de manière immunologique dudit fragment. Les épitopes préférés comprennent GGRNSIRYSELA, TRVYLVDNKSTD, QTINLDDRSHWG, HYLKVGRQNGVL, FRLGFDPVTGLV, VTGLVMPGVYTN, SNSSGSGAEENS, DHAIRGDTFATR, DSTFTQYRSWYL, RRTCPYVYKALG. L'invention concerne également l'utilisation desdits peptides et des molécules d'acides nucléiques dans la thérapie et les formulations comprenant lesdites molécules, éventuellement associées à un vecteur adénoviral.
EP20040768408 2003-09-09 2004-09-09 Epitopes adenoviraux Withdrawn EP1664099A2 (fr)

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GB0321063A GB0321063D0 (en) 2003-09-09 2003-09-09 Adenoviral epitopes
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PCT/GB2004/003861 WO2005023848A2 (fr) 2003-09-09 2004-09-09 Epitopes adenoviraux

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EP2971008B1 (fr) 2013-03-14 2018-07-25 Salk Institute for Biological Studies Compositions d'adénovirus oncolytiques
CL2016000164A1 (es) * 2016-01-21 2016-07-29 Pontificia Universidad Católica De Chile Anticuerpos monoclonales específicos para el antígeno piii de adenovirus humano (adv), producidos y secretados por hibridomas celulares, útiles para la detección y el diagnóstico de la infección causada por adv.
JP7015551B2 (ja) 2016-02-23 2022-02-15 ソーク インスティテュート フォー バイオロジカル スタディーズ ウイルス動態への影響を最小限にするための治療用アデノウイルスにおける外因性遺伝子発現
JP7054527B2 (ja) 2016-02-23 2022-04-14 ソーク インスティテュート フォー バイオロジカル スタディーズ アデノウイルスの複製動態を測定するための高スループットアッセイ
CA3045892A1 (fr) 2016-12-12 2018-06-21 Salk Institute For Biological Studies Adenovirus synthetiques ciblant une tumeur et leurs utilisations
EP3715374A1 (fr) 2019-03-23 2020-09-30 Ablevia biotech GmbH Composé de séquestration d'anticorps indésirables chez un patient
US11986536B2 (en) 2019-03-23 2024-05-21 Ablevia Biotech Gmbh Compound for the sequestration of undesirable antibodies in a patient
JP2023542390A (ja) 2020-09-23 2023-10-06 アブレヴィア バイオテック ゲーエムベーハー ウイルスベクターの有効性を増強するための化合物
WO2023180502A1 (fr) 2022-03-24 2023-09-28 Ablevia Biotech Gmbh Composé pour augmentation de l'efficacité des virus oncolytiques

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