EP1181058A2 - Vaccins contre des antigenes dependant de la conformation ainsi que contre des antigenes qui ne sont pas ou pas exclusivement des proteines ou des peptides - Google Patents

Vaccins contre des antigenes dependant de la conformation ainsi que contre des antigenes qui ne sont pas ou pas exclusivement des proteines ou des peptides

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Publication number
EP1181058A2
EP1181058A2 EP00951201A EP00951201A EP1181058A2 EP 1181058 A2 EP1181058 A2 EP 1181058A2 EP 00951201 A EP00951201 A EP 00951201A EP 00951201 A EP00951201 A EP 00951201A EP 1181058 A2 EP1181058 A2 EP 1181058A2
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Prior art keywords
peptides
antigen
conformation
dna
sequences
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English (en)
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Steffen Goletz
Uwe Karsten
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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Max Delbrueck Centrum fuer Molekulare in der Helmholtz Gemeinschaft
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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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • Vaccine against conformation-dependent antigens as well as against antigens that are not or not exclusively proteins or peptides
  • the invention relates to naczines against conformation-dependent antigens and against antigens which are not or not exclusively proteins or peptides. Furthermore, the invention relates to methods for their preparation and their use and human anti-idiotypic antibody fragments against the MUCl conformation epitope and amino acid sequences of mimicry peptides against the MUCl conformation epitope and antiidiotypic antibody fragments against the TF antigen and amino acid sequences of mimicry peptide hydrides against the tep antigen .
  • Target structures of vaccines against pathogens of infectious diseases and non-infectious diseases, including tumors can be proteins or peptides, carbohydrates or lipids, and combinations of these.
  • the immunogenic determinant can be determined either by the sequence of the amino acids of a section of the molecule (sequence epitope) or by a certain spatial arrangement of binding forces that does not correspond to the linear arrangement of the amino acids (conformation epitope). Conformation epitopes are more common than sequence epitopes; Mixed forms also occur.
  • Conformation epitopes and antigens that are not or not exclusively proteins or peptides are difficult to convert into an effective and practicable vaccine. Conformation epitopes usually only develop in the native protein and not in shorter peptides. Antigens that are not or not exclusively proteins or peptides, such as glycostructures or lipids, are not very immunogenic. Their synthesis is often complex. A particularly serious circumstance is that in many cases these antigens are not properly presented to the immune system. An effective antigen presentation is, among other things, a prerequisite for the development of cytotoxic T lymphocytes, i.e. for an effective cellular defense. Finally, the very effective form of DNA vaccination is not applicable to these antigens.
  • DNA vaccination instead of a protein or peptide antigen, the coding DNA sequence is injected intramuscularly or intradermally as a vaccine, as such or packed into a vector.
  • a vaccine as such or packed into a vector.
  • an effective humoral response and cellular response can be achieved (Wolff J.A. et al., Science 247: 1465, 1990; Ulmer, JB et al, Vaccine 12: 1541, 1994; Raz, E. et al., Cancer Res. 52: 1954, 1992).
  • a particularly successful method is the so-called "Prime Boost Protocol” (Keystone Symposia: DNA-Vaccines, April 12-17, 1999, Snowbird, Utah, USA, conference proceedings), in which the intradermal, intramuscular or intrarectal injection of a DNA (Priming), followed by a booster with the corresponding antigen.
  • a corresponding recombinant virus vector particle for example fowlpox, adeno or alpha virus-derived constructs
  • the "prime boost” process is known to result in a strong cellular immune response with the activation of specific cytotoxic T cells, which is particularly desirable in the case of tumor vaccines.
  • the immune response can be significantly enhanced by additional administration of suitable cytokines, also in the form of DNA, of immunostimulatory CpG-DNA motifs (non-methylated cytosine guanine dinucleotides) or of suitable adjuvants (eg aluminum phosphates).
  • suitable cytokines also in the form of DNA
  • immunostimulatory CpG-DNA motifs non-methylated cytosine guanine dinucleotides
  • suitable adjuvants eg aluminum phosphates
  • the object of the invention is to circumvent the disadvantages mentioned above and to develop a vaccine, in particular a DNA vaccine, even for those cases which have not been accessible to a corresponding vaccination.
  • the invention is implemented according to the claims. It relates, on the one hand, to a method by which the field of application of vaccination, in particular DNA vaccination, to conformation-dependent antigens and mixed forms, these also fall within the scope of the invention under the concept of conformation epitopes, and antigens, the relevant epitopes of which, or not exclusively, proteins or Peptides are, for example carbohydrates, combined carbohydrate-peptide epitopes, lipids, glycolipids, expanded and the disadvantages listed above can thus be avoided. According to the invention, this takes place by means of a detour via a peptide (mimicry peptide) which immunologically depicts the original epitope (the antigen determinant) but which has a different amino acid sequence.
  • a detour via a peptide (mimicry peptide) which immunologically depicts the original epitope (the antigen determinant) but which has a different amino acid sequence.
  • the mimicry peptide is preferably prepared using the methods of the phage display or ribosome display known per se (Scott J.K. and Smith, GP Science, 249: 386, 1990; Winter, G. et al., Annu Rev Immunol, 12: 433, 1994; HanesJ. Et al, Proc Natl Acad Sei USA, 95: 14130, 1998), either as a shorter peptide from peptide libraries or in the form of an anti-idiotypic antibody fragment from corresponding libraries.
  • the third, but more complex method is the generation of anti-idiotypic antibodies using hybridoma technology.
  • the common goal of the three methodological variants mentioned is to "rewrite" the original conformational epitope or the epitope, which is not or not exclusively a protein or peptide, into an immunologically corresponding sequence epitope, which enables better immunological presentation and is suitable for DNA vaccination is.
  • the vaccines in particular the DNA vaccines, can be used not only in the form of the example described (Prime Boost protocol), but also in comparable variants and in the form of the DNA vaccine alone or the mimicry structures alone in appropriately suitable formulations .
  • the invention relates to vaccines against conformation-dependent antigens.
  • the relevant conformation epitopes are "rewritten" with the aid of the phage display or ribosome display method into an immunologically corresponding sequence epitope imitating the conformation epitope.
  • the primary reagents used are molecules that specifically bind the target antigen in its desired conformation, e.g. Antibodies, antibody fragments or receptors.
  • Antibody fragments (anti-idiotypic antibody fragments, Ab2) or linear or circular peptides are obtained from the various gene libraries, which specifically bind the primary reagents and imitate the antigen immunologically.
  • anti-idiotypic antibodies are obtained using hybridoma technology and fragments are isolated if necessary. These mimicry peptides are rewritten into a DNA and used as a DNA vaccine.
  • One method is the so-called "Prime Boost Protocol", in which the intradermal, intramuscular or intrarectal injection of DNA (priming), in the form of a plasmid DNA, linear DNA or a plasmid replicon vector, from a booster with the corresponding antigen, alone, in the form of a chemical coupling to proteins, in the form of bacteriophages as fusion proteins with phage coat proteins on their surface, in the form of a fusion protein on the surface of other viruses or attenuated biological carriers or in the form of dendritic cells loaded with the peptide, is followed.
  • both the DNA and the expressed mimicry peptide are required, which is possible without any problems when using the phage display or ribosome display technology.
  • a corresponding recombinant virus vector particle e.g. fowlpox, adeno or alpha virus-derived constructs
  • the immune response can be significantly enhanced by the additional administration of suitable cytokines, also in the form of DNA, of immunostimulatory CpG-DNA motifs (non-methylated cytosine guanine dinucleotides) or of suitable adjuvants (e.g. aluminum phosphates).
  • the invention also relates to vaccines against antigens which are not or not exclusively proteins or peptides, according to claim 3.
  • a target antigen type of the group antigens which are not or not exclusively proteins or peptides are glycostructures, further immunogenic ones Structures are combined carbohydrate-protein epitopes, lipids, glycolipids or synthetic structures.
  • a method is known with which a monoclonal anti-idiotypic antibody is obtained with the help of hybridoma technology, which immunologically imitates pure carbohydrate structures.
  • a vaccine preferably a DNA vaccine, of this antibody or a suitable fragment thereof is used for the vaccination.
  • the present invention extends this method from DE196 27 352 AI in several points.
  • Antiidiotypic antibody fragments can be obtained directly from antibody gene libraries using the phage display technique or the ribosome display technique. With this method, human antibody fragments can also be obtained directly. Combined carbohydrate-peptide epitopes can also be used.
  • the invention also relates to vaccines, to the full extent as described for conformation-dependent antigens, against the antigens glycopeptides, glycolipids, lipids, synthetic structures or other antigens which are no or only partially proteins or peptides, the relevant epitopes having improved immunogenic structures, processes their manufacture and their use.
  • the approach of immunotherapy for tumor diseases assumes that it is possible to strengthen or activate the natural immune response.
  • the rationale for vaccination is to combat the residual disease (metastasis prophylaxis) after conventional therapy (eg surgical removal of the majority of the tumor cells).
  • Mimicry peptides immunologically mimic the original antigen or epitope. They do this as far as possible, but not one hundred percent. For the application in the context of a vaccine (in particular in the case of a tumor vaccine), this is to be seen rather positively in the sense that specifically inhibiting processes, for example tolerance phenomena, are avoided.
  • a widespread carcinoma antigen is the epithelial mucin, MUC1, whose immunodominant epitope occurs repeatedly in the extracellular part of the molecule.
  • This epitope forms a type I ß turn in its native state, but only on synthetic peptides under certain conditions, e.g. if the threonine in the immunodominant region is glycosylated with GalNAc ⁇ l-0-Thr or Galßl-3GalNAc ⁇ l-0-Thr ( Karsten, U., et al, Cancer Res. 58: 2541-2549, 1998).
  • This epitope is usually perceived by the immune system as a typical conformation epitope, cf.
  • Example 1 A widespread carcinoma antigen is the epithelial mucin, MUC1, whose immunodominant epitope occurs repeatedly in the extracellular part of the molecule.
  • This epitope forms a type I ß turn in its native state, but only on synthetic peptides under certain conditions, e.g. if the
  • this conformation epitope is imitated by means of the phage display technique by immunologically identical (or almost identical) sequence epitopes which are part of a tumor vaccine in the form of a DNA in a DNA vaccination vector (Example 1).
  • the invention therefore also relates to human anti-idiotypic antibody fragments against the MUCl conformation epitope and all DNA sequences which encode these fragments and protein sequences or DNA or partial protein sequences which can be derived from them and which have the corresponding properties.
  • Fragments containing the desired DNA of the scFv and the peptides were amplified using the PCR and then sequenced.
  • the numbering, e.g. Q33, corresponds to a certain isolated clone; the sequences of the different scFv are aligned with each other (alignment); the complete sequence of a clone can be read continuously for each clone across the different blocks)
  • R6 SGGGGSGGGGSTDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI
  • the invention furthermore also relates to amino acid sequences of mimicry peptides against the MUCl conformation epitope and all DNA sequences which code for these amino acid sequences, DNA and peptide and partial peptide sequences which are derived from them and which have the same properties.
  • Antigens that are not or not exclusively proteins or peptides e.g. carbohydrate antigens
  • carbohydrate antigens like conformational epitopes of proteins
  • the selection of mimicry peptides according to the invention by means of the phage display technique can lead to a "rewriting" of the antigen into a peptide sequence, which in turn enables the use of the DNA vaccination technique, cf.
  • Example 2
  • the invention also relates to protein sequences of anti-idiotypic antibody fragments against TF and amino acid sequences of mimicry peptides against the TF carbohydrate epitope and all DNA sequences which code these amino acid sequences and DNA and protein or peptide and partial sequences which are derived from them and which have the same properties.
  • mice were immunized ip with a suspension of living human breast carcinoma cells from the T-47D cell line (Keydar., Et al, Eur J Cancer, 15: 659, 1979) after treatment with neuraminidase (V.cholerae) without adjuvant.
  • X63-Ag8.653 (Kearney, JF, et al, J Immunol 123: 1548, 1979) served as the fusion cell line.
  • the hybridoma technology itself was carried out according to standard methods (for example Peters, HH, et al, "Monoclonal Antibodies, Production and Characterization", Berlin 1985; Friemel, H., "Immunological Working Methods", 4th edition, Jena 1991).
  • the specificity analysis of the monoclonal antibodies (mAb) produced by the hydridome cell lines was based on enzyme immunoassays with natural glycoproteins and synthetic peptides and glycopeptides, immunofluorescence analyzes with various cell lines and immunohistochemical tests on tissue sections.
  • the epithelial mucin, MUC1 was clearly identified as a specific antigen.
  • IgGl, k with a small proportion of IgM of the same specificity was determined as the isotype using a commercial isotyping kit (Pharmingen, San Diego, USA).
  • An epitope Mapping as part of the ISOBM TD-4 International Workshop on Monoclonal Antibodies against MUC1 (Tumor Biol. 19, SupplJ, 1998) defined the epitope as APDTRPAP.
  • the antibody binds only slightly to a single unit (a repeat), although it contains the epitope sequence.
  • Binding to non-glycosylated peptides depends on the length of the peptide, more precisely on the number of repeats lined up (Fig. La). It is known from the literature that the native conformation of the PDTRP motif only forms when the peptide length is more than 3 repeats (FontenotJ.D., Et al., J Biomol Struct Dyn 13: 245, 1995). - The binding of mAb A76-A / C7 to a single MUC1 unit (1 repeat) is greatly increased if it is glycosylated in the region of the epitope on the thr with GalNAc- or Galßl-3GalNAc (Fig. Lb; see also Karsten , U., et al., Cancer Res, 58: 2541, 1998).
  • the antibody was followed by ammonium sulfate precipitation
  • the one antibody gene library (Griffin 1 Library; http://www.mrc-cpe.cam.ac.uk/ ⁇ phage/) consists of more than 10 9 phages, each with different combinations of the variable regions of the heavy and light chains human Antibodies with partially randomized hypervariable regions, which are connected to a peptide piece (linker) and are covalently bound to a phage coat protein (pIII). It is derived from another antibody gene library (Griffiths, A. et al., 1994, EMBO j., 13: 3245-3260).
  • the second, smaller gene library consists of scFv with the same framework (single framework library), which was pre-selected for active folding of the antibody fragments by binding to protein L and protein A (I. Tomlinson, 9th anniverary conference: "Antibody engineering”, IBC -Conferences, SanDiego 1998; I.Tomlinson, 10th anniverary conference: "Antibody engineering", IBC-Conferences, SanDiego 1999; speaker abstract).
  • the first library comes from the Dr.G. Winter laboratory and the second from the Dr.I.Tomlinson laboratory (each MRC Center for Protein Engineering, Cambridge, UK).
  • the specific phages were selected in 2-3 rounds (phage panning) under Use of the proteolytic selection method with the helper phage KM 13 (Kristensen, P. and Winter, G., Folding & Design, 3: 321, 1998).
  • the purified monoclonal antibody A76-A / C7 (35 ⁇ g / ml in 4 ml), which was immobilized in a test tube (Immunotube, Nunc, Wiesbaden) overnight at 4 ° C. in PBS, served as antigen.
  • A76-A / C7 was incubated with the phages; the phages bound to the antibodies were obtained by magnetic beads with immobilized anti-IgG antibodies (Deutsche Dynal, Hamburg).
  • the phages specifically bound in the selection rounds were, after stringent washing steps (up to 20 times PBS / 0.1% Tween20 and then 20 times PBS), replaced by the tandem repeat (100 ⁇ g / ml; Biosynthan, Berlin-Buch) and then treated with trypsin (proteolytic selection method). Between the rounds of selection, the eluted phages in the bacteria were multiplied with helper phages and selected again.
  • the selection and testing was carried out as described for the generation of the anti-idiotypic antibodies. Analogously to this, additional linear and circular mimicry peptides were obtained with further peptide libraries. These are peptide libraries which were produced analogously to the peptide library described above.
  • the expressed peptides are linear peptides with 7 amino acids and circular peptides with 7 randomized amino acids, flanked by two cysteines (CX7C), circular peptides with 10 randomized amino acids, flanked by two cysteines (CX10C), and circular peptides with a total of 9 randomized amino acids, with two internal and two flanking cysteines (CX3CX3CX3C).
  • the form of the peptides and antibody fragments coupled to phages was used for the ELISA tests.
  • the anti-idiotypic svFv and the mimicry peptides can be divided into groups that: - bind only to A76-A / C7 to A76-A / C7 and to other MUC1 -specific antibodies that either only bind to the conformation epitope (in the Bind PDTR region glycosylated MUCl tandem repeat (type A) or their binding is greatly increased by the PDTR glycosylation of the MUC1 tandem repeat (conformational induction) (type B) to MUC1 -specific antibodies, which in addition to type A and B also MUC1 -specific antibodies bind, which bind glycosylated and unglycosylated MUC1 tandem repeats to the same extent (type D) have a strong binding to MUC1 -specific antibodies which are concerned with the glycosylation of the PDTR region of the MUC1 repeats to
  • the mimicry peptides and anti-idiotypic antibody fragments were also examined in ELISA inhibition tests to determine whether, in the form of the synthesized peptides or purified scFv (alone or coupled to phages), the binding of the A76-A / C7 to the glycosylated MUC1 peptide (im Inhibit epitope PDTR with GalNAc glycosylated tandem repeat) and non-glycosylated oligomers of the 20-mer tandem repeat specifically and concentration-dependent.
  • These experiments were carried out with streptavidin-coated microtest plates (BioTeZ, Berlin-Buch) and biotinylated MUC1 peptides (Biosynthan, Berlin-Buch; Fig. Lc) as well as with normal ELISA test plates, on which the MUC1 peptides were immobilized by drying .
  • mice of the Balb / c strain were intraperitoneally immunized with mimicry peptides and anti-idiotypic antibody fragments in the form of the synthesized peptides or purified scFv alone, each coupled to the KLH protein or coupled to bacteriophages in PBS, mixed with incomplete Freund's adjuvant. Mixtures of anti-idiotypic scFv phages or mimicry peptide phages from the different groups (see above) were used. Three weeks later, the same approach was used to boost without an adjuvant. The booster was repeated after 3 weeks and blood was drawn from the mice 10 days later.
  • the serum was tested in ELISA tests for antibodies that specifically recognize the conformation-dependent epitope of MUC1 (experimental setup as above).
  • the Mixtures of the anti-idiotypic scFv and the mimicry peptides generate a strong reaction against the conformation-dependent epitope of the MUC 1.
  • the antiidiotypic scFv were directionally cloned into a DNA vaccination vector.
  • the scFv were cut out of the phagemid vector by Sfil and Notl and cloned directionally into different DNA vaccination vectors which had previously been cleaved with the same enzymes.
  • a suitable vector here is the vector pVAC2 (I.Harmer et al., Keystone Symposium “DNA-Vaccines", Snowbird, USA, 1999; poster and poster abstract), which, after the scFv has been inserted into the DNA vaccination vector, is a fusion protein encoded from the antiidiotypical scFv with a tetanus toxoid, which acts as an adjuvant and enhances the immune response against the fused protein portion (C. King et al., 1998, Nat.Medicine 4: 1281-86).
  • the mimicry peptides were also cloned into various DNA vaccine vectors, which were cloned using the known method of PCR cloning, in which the sequences coding for the mimicry peptides were inserted into the DNA vaccination vectors using synthetic primers DNA vaccination vectors based on the pVAC2 produced, each coding for a fusion protein of the mimicry peptide with the tetanus toxoid, the DNA of the vaccination vector was multiplied according to methods known per se, cleaned and then injected into mice.
  • DNA vaccination vectors which encode anti-idiotypic scFv or mimicry peptides as fusion proteins with the tetanus toxoid, which come from the different groups with different binding patterns for MUC 1 -specific antibodies (see above), were used for the immunization. 50 ⁇ g or 200 ⁇ g of total DNA were used as the dose and administered intra-muscularly. Four weeks later, the same approach was used for the booster and the booster was repeated after 4 weeks and blood was drawn from the mice 10 days later. The serum was tested in ELISA tests for antibodies that specifically recognize the conformation-dependent epitope of MUC1 (experimental setup as above).
  • the immunization with the mixtures of the DNA vaccine vectors resulted in a strong humoral immune reaction against the conformation-dependent epitope of the MUC1 and a strong reaction against the tetanus toxoid both in the antiidiotypic scFv and in the mimicry peptides.
  • mice were used in the mouse tumor challenge model, which stably transfected with the cDNA of the transmembrane form of the human MUCl.
  • the MUCl-positive mouse cell lines express the conformation epitope of the MUCl, which was tested by immunological studies with the A76-A / C7.
  • Several strains of mice were used in the studies (Balb / c, DBA 2 and C57BL / 6).
  • the mice were injected subcutaneously with 10 6 to 10 7 tumor cells in 200 ⁇ l PBS near the peritoneum and the tumor growth (tumor size in mm) was measured over 20-30 days.
  • Prime Boost vaccination scheme :
  • a combination of DNA vaccination vectors (coding for scFv-tetanus toxoid or mimicry peptide-tetanus toxoid fusion protein) with two candidates from each of the 4 different groups of the anti-idiotypic-scFv or mimicry peptides were used for the immunizations (priming) .
  • the same combinations of the antiidiotypic scFv or mimicry peptides were used in their protein form in an incomplete Freund's adjuvant.
  • the scFv were purified by methods known per se by nickel chelate chromatography and the mimicry peptides were chemically coupled to KLH by methods known per se.
  • the same vaccination when subsequently injected with the same tumor cells without transfected MUCl, has an average tumor size of over 200 mm 2 (after 20 days).
  • the injection of MUCl-positive mouse tumor cell lines into mice without prior vaccination also results in strong tumor growth (> 200 mm 2 after 20 days).
  • An immunization and booster with the proteins of the antiidiotypic scFv or the mimicry peptides coupled to KLH without DNA vaccination vectors results in an immune response against the MUC1 tumor cells, but the tumor protection is many times less than with the prime boost protocol with the DNA Vaccination vectors.
  • mice were mixed with 100 ⁇ g asialoglycophorin (Sigma, Deisenhofen) in PBS, mixed with Freund's adjuvant , immunized intraperitoneally. After 24 h, 100 ⁇ g / kg body weight of cyclophosphamide in PBS i.p. administered. The booster was carried out after 2 weeks with 100 ⁇ g asialoglycophorin.
  • X63-Ag8.653 was used as the fusion cell line (Kearney, J.F., et al., J Immunol 123: 1548, 1979).
  • the hybridoma technique was carried out according to standard methods (e.g. Peters, H.H., et al., "Monoclonal Antibodies, Production and Characterization", Berlin 1985; Friemel, H., “Immunological Working Methods", 4. AufL, Jena 1991).
  • the specificity analysis of the monoclonal antibodies produced by the hydridoma cell lines was based on enzyme immunoassays with natural glycoproteins, synthetic peptides and glycopeptides, glycolipids and neoglycolipids and synthetic polyacrylamide-carbohydrate conjugates, absorption analyzes on synthetic carbohydrate conjugates (Synsorb, Edmontonomensomenom, cellulite analysis, Chemondomedomensol, cellulite analysis, Chemondomedomedomone, Chemontomedomelizone, Chemondomedomensol, cellulite analysis, Synmont, cellulosomal enzyme analysis) as well as immunohistochemical studies on tissue sections.
  • the tumor-associated carbohydrate epitope Thomsen-Friedenreich (TF) was clearly identified as a specific antigen:
  • TFß which can occur on the glycan chains of glycolipids, as well as other carbohydrate structures, peptide or lipid components are not bound.
  • A78-G / A7 binds highly specifically to various carcinoma cell lines in immunofluorescence studies and to various carcinomas in histochemical Investigations.
  • the isotype IgM, k was determined for the A78-G / A7 using a commercial isotyping kit (Pharmingen, San Diego, USA).
  • A78-G / A7 was isolated from cell culture supernatants by means of ammonium sulfate precipitation, followed by affinity chromatography on a ProteinG affinity matrix to remove unwanted IgG antibodies from the calf serum and finally by affinity chromatography using a goat anti-mouse Ig affinity matrix (percellulose, BioTeZ , Berlin-Buch) cleaned (Dr.G.Butschak).
  • the one antibody gene library consists of more than 10 10 phages, each with different combinations of the variable regions of the heavy and light chains of human antibodies with partially randomized hypervariable regions, which are linked to a piece of peptide (linker) and covalently linked to a phage coat protein (pIII) are bound. It is derived from another antibody gene library (Griffiths, A. et al., 1994, EMBO J., 13: 3245-3260).
  • the second, smaller gene library consists of scFv, which were pre-selected for active folding of the antibody fragments.
  • the first library comes from the Dr.G.
  • the purified antibody was incubated with the phages; the phages bound to the antibody were obtained by magnetic beads with immobilized anti-IgM antibodies (Deutsche Dynal, Hamburg).
  • the phages specifically bound in the selection rounds (3 h at RT) became specific after stringent washing steps (up to 20 times PBS / 0.1% Tween20 and then 20 times PBS) by the TF-bearing glycoprotein asialoglycophorin (100-165 ⁇ g / ml) eluted and then partially treated with trypsin (proteolytic selection method).
  • the eluted phages in the bacteria were propagated with helper phages and selected again. Two to three rounds of selection were carried out. Identification of peptides using a peptide gene library that specifically mimic the Thomsen-Friedenreich antigen
  • Example for the generation of anti-idiotypic antibodies was in several rounds of selection from a peptide gene library (. Oligino, L, et al, J Biol Chem 272: 29046, 1997) has coupled to the phage coat protein pIII 10 7 different short peptides, specifically binding peptides obtained (in collaboration with Dr. H. Gollasch, Robert-Rössle-Klinik, Berlin-Buch).
  • the peptides expressed are randomized nonapeptides flanked by two cysteines and thus circularized, which increases stability and affinity.
  • the selection and testing was carried out as described in the generation of the anti-idiotypic antibodies.
  • the potential mimicry peptides and anti-idiotypic antibody fragments were examined in ELISA inhibition tests to determine whether they specifically inhibit the binding of the A78-G / A7 and / or other TF-recognizing antibodies and lectins to the disaccharide TF ⁇ .
  • the glycoprotein asialoglycophorin carrying the TF ⁇ was immobilized on ELISA plates by drying, and the binding of the monoclonal antibodies and lectins was inhibited by the mimicry peptides or anti-idiotypic antibody fragments in the form of the synthesized peptides or purified scFv alone or coupled to phages in a concentration-dependent manner (Fig. 2).
  • mice of the Balb / c strain and the NMRI strain were immunized intraperitoneally with mimicry peptides and anti-idiotypic antibody fragments in the form of the synthesized peptides or purified scFv alone, each coupled to the KLH protein or coupled to bacteriophages in PBS, mixed with complete Freund's adjuvant. Three weeks later, the same approach was used to boost without an adjuvant. The booster was repeated after 3 weeks and blood was drawn from the mice 10 days later. The serum was examined in ELISA tests for antibody bindings against the Thomsen-Friedenreich antigen. Vaccination with TF-mimicking peptides in a mouse tumor model
  • the mouse colon carcinoma cell line C-26 was in the medium RPMI 1640 with
  • Tumor model 10 cells of the syngeneic colon carcinoma cell line C-26 s.c. transplanted, in two variants: a) untreated and b) pretreated with neuraminidase from V.cholerae (Serva, Heidelberg) (TF positive). The tumor size was determined externally at weekly intervals. After 3 weeks, the animals were sacrificed and the liver was prepared to determine the number of metastases visible on the surface of the liver.
  • Vaccination The vaccination of the mice was started 6 weeks before the tumor transplant.
  • the phage preparation or the purified scFv (and corresponding controls) were emulsified 1: 1 with incomplete Freund's adjuvant and i.p. injected. Four weeks later there was a boost (without adjuvant). After a further 2 weeks, the tumor transplantation (tumor challenge) was carried out with untreated and neuraminidase-treated C-26 cells.
  • Culture supernatant of the A76-A / C7 (diluted 1:80) was preincubated with the scFv phages purified by polyethylene glycol precipitation in the indicated concentrations (volume percentage of adjusted phage solutions in PBS) for one hour and then added to the MUCl glycopeptide plate for 2 hours .
  • the detection was carried out using an anti-mouse POD antibody (Dako).
  • the scFv phages Q6, Q7 and Q8 are examples of anti-idiotypic scFv, while Q4 and Q10 are examples of control scFv that bind the A78-A / C7 but are not anti-idiotypic scFv.
  • A78-G / A7 binding to asialoglycophorin by scFv phages The asialoglycophorin (A-GP) was immobilized on the ELISA plate by drying (25ng / well) and then blocked with 30% FCS in RPMI. Culture supernatant of the A78-G / A7 (diluted 1:20) was pre-incubated for one hour with the scFv phages purified by polyethylene glycol precipitation in the stated concentrations (volume percentage of adjusted phage solutions in PBS) and then for 2 hours on the A-GP plate given. The detection was carried out using an anti-mouse POD antibody (Dako).
  • the scFv phages P9, P13, P16, P3 and K3 are examples of antiidiotypic scFv, while P8 and Ql are examples of control scFv, of which P8 binds the A78-G / A7 but is not an antiidiotypical scFv and Ql Is phage that does not bind the A78-G / A7.

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Abstract

Procédé qui permet d'appliquer la technologie extrêmement performante de la vaccination à l'aide d'acide désoxyribonucléique (ADN) non seulement sur des épitopes de séquence de protéines ou de peptides, mais aussi sur des épitopes de conformation. Ce procédé permet en outre l'utilisation de la vaccination à l'ADN pour des antigènes qui ne sont pas ou pas exclusivement des protéines ou des peptides. Le vaccin préféré selon la présente invention contient en tant que constituant essentiel un acide désoxyribonucléique (ADN) codant une séquence peptidique qui représente quant à elle l'imitation immunologique (mimétisme) d'un antigène dépendant de la conformation, y compris des épitopes de conformation protéiques, ou d'un antigène qui n'est pas ou qui n'est que partiellement une protéine ou un peptide. Le peptide d'imitation qui est ou peut être également une partie du vaccin selon la présente invention est soit un anticorps antiidiotype, un fragment d'anticorps, un peptide dérivé dudit fragment ou un peptide à liaison spécifique obtenu par sélection dans une génothèque peptidique. Les domaines d'utilisation de la présente invention sont l'immunologie médicale et vétérinaire, dont la thérapie d'accompagnement pour les pathologies tumorales.
EP00951201A 1999-05-27 2000-05-29 Vaccins contre des antigenes dependant de la conformation ainsi que contre des antigenes qui ne sont pas ou pas exclusivement des proteines ou des peptides Withdrawn EP1181058A2 (fr)

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US7109308B1 (en) 1994-03-08 2006-09-19 Human Genome Sciences, Inc. Antibodies to human vascular endothelial growth factor 2
US7186688B1 (en) 1994-03-08 2007-03-06 Human Genome Sciences, Inc. Methods of stimulating angiogenesis in a patient by administering vascular endothelial growth factor 2
US7153827B1 (en) 1994-03-08 2006-12-26 Human Genome Sciences, Inc. Vascular endothelial growth factor 2 and methods of use
US6608182B1 (en) 1994-03-08 2003-08-19 Human Genome Sciences, Inc. Human vascular endothelial growth factor 2
US5932540A (en) 1994-03-08 1999-08-03 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
US6040157A (en) 1994-03-08 2000-03-21 Human Genome Sciences, Inc. Vascular endothelial growth factor 2
ATE309360T1 (de) 1994-03-08 2005-11-15 Human Genome Sciences Inc Vaskularer endothelialer wachstumsfaktor 2
US7223724B1 (en) 1999-02-08 2007-05-29 Human Genome Sciences, Inc. Use of vascular endothelial growth factor to treat photoreceptor cells
NZ518077A (en) 2000-08-04 2003-11-28 Human Genome Sciences Inc Biologically active fragments, analogues and derivatives of VEGF-2 for the treatment of peripheral artery diseases such as critical limb ischemia and coronary disease
US7402312B2 (en) 2001-04-13 2008-07-22 Human Genome Sciences, Inc. Antibodies to vascular endothelial growth factor 2 (VEGF-2)
WO2002083704A1 (fr) 2001-04-13 2002-10-24 Human Genome Sciences, Inc. Facteur de croissance 2, endothelial, vasculaire
US7658924B2 (en) 2001-10-11 2010-02-09 Amgen Inc. Angiopoietin-2 specific binding agents
US7521053B2 (en) 2001-10-11 2009-04-21 Amgen Inc. Angiopoietin-2 specific binding agents
JP2005289809A (ja) 2001-10-24 2005-10-20 Vlaams Interuniversitair Inst Voor Biotechnologie Vzw (Vib Vzw) 突然変異重鎖抗体
EP1539235A2 (fr) 2002-07-01 2005-06-15 Human Genome Sciences, Inc. Anticorps qui se lient specifiquement a reg iv
WO2004009632A2 (fr) 2002-07-22 2004-01-29 Nemod Immuntherapie Ag Procede de production d'une mucine immunostimulatrice (muc1)
PT2312318E (pt) * 2002-12-03 2015-01-14 Univ North Carolina State Ligandos a proteína de prião e métodos de uso
GB0324265D0 (en) * 2003-10-16 2003-11-19 Medical Res Council Peptide
US7871782B2 (en) 2004-03-29 2011-01-18 The University Court Of The University Of Aberdeen Specific binding members against synaptophysin
WO2006100582A1 (fr) * 2005-03-25 2006-09-28 Glycart Biotechnology Ag Molecules de liaison d'antigene orientees mcsp et a fonction amelioree d'affinite de liaison au recepteur fc et effectrice
PL1888640T3 (pl) 2005-05-18 2012-08-31 Ablynx Nv Ulepszone nanociała skierowane przeciwko czynnikowi martwicy nowotworów typu alfa
DE102005023617A1 (de) 2005-05-21 2006-11-23 Aspre Ag Verfahren zum Mischen von Farben in einem Display
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NZ587506A (en) 2008-01-25 2012-09-28 Univ Aarhus Selective exosite inhibition of papp-a activity against igfbp-4
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