WO1996001429A1 - Techniques d'identification et de production de peptides antigeniques et leur utilisation en tant que vaccins - Google Patents

Techniques d'identification et de production de peptides antigeniques et leur utilisation en tant que vaccins Download PDF

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Publication number
WO1996001429A1
WO1996001429A1 PCT/EP1995/002593 EP9502593W WO9601429A1 WO 1996001429 A1 WO1996001429 A1 WO 1996001429A1 EP 9502593 W EP9502593 W EP 9502593W WO 9601429 A1 WO9601429 A1 WO 9601429A1
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Prior art keywords
peptide
peptides
molecules
mhc
proteins
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PCT/EP1995/002593
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German (de)
English (en)
Inventor
Gabriele Niedermann
Klaus Eichmann
Bernhard Maier
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to JP8503679A priority Critical patent/JPH10505665A/ja
Priority to EP95925827A priority patent/EP0769147A1/fr
Priority to AU29813/95A priority patent/AU2981395A/en
Publication of WO1996001429A1 publication Critical patent/WO1996001429A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56977—HLA or MHC typing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/12—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by hydrolysis, i.e. solvolysis in general
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

  • the invention relates to in vitro methods for identifying and producing antigenic peptides and peptide mixtures which simulate the in vivo processing of foreign or proprietary protein and the use of the antigenic peptides identified by the method according to the invention, for example for identifying vaccine components against pathogens or tumor cells or of competitive or antagonistic peptides in autoimmune diseases. Furthermore, the invention relates to the use of peptides and peptide mixtures as vaccines and an enzyme reactor for the production of peptide mixtures.
  • MHC antigens major histocompatibility complex antigens
  • the processed foreign or proprietary proteins presented as peptides by the MHC molecules are recognized by two different types of lymphocytes with different functions in the immune system, the so-called cytotoxic T-lymphocytes and the helper T-lymphocytes.
  • Cytotoxic T-lymphocytes play an important role in the defense against both intracellular viral and bacterial infections and against degenerate cancer cells. They recognize antigens in the form of small peptides that are bound to class I MHC antigens on the surface of infected or degenerate cells. The peptides recognized by the defending CTLs are in the cell while of the normal cell cycle through continuous, proteolytic degradation of the antigen proteins.
  • Helper T lymphocytes also play a central role in the body's defense. For example, they perform a function in the proliferation and differentiation of antigen-recognizing B cells. Helper T cells recognize peptides presented by MHC class II molecules located on antigen presenting cells. The peptides recognized by the T helper cells are mainly exogenous, i.e. non-body or extracellular origin.
  • proteaso A very important role in the generation of antigenic peptides in a cell, which are presented by MHC class I molecules, is played by multienzyme complexes called proteaso (AL Goldberg and KL Rock, Proteolysis, proteasomes and antigen presentation, Nature 357 (1992) , 375). These proteasomes can be isolated from the cytosol of cells.
  • the assembly of the antigenic peptides with MHC class I molecules takes place in the endoplasmic reticulum (ER) after the peptides have been transported through the ER membrane. Participants in the transport of the peptides into the ER and the assembly with MHC class I molecules are members of the "ABC transporter” protein family (Higgins, ABC transporters: from microorganisms to man. Annu. Rev. Cell Biol. 8 (1992), 67), called “Transporter for Antigen Presentation” (TAP) (JJ Monaco, A molecular model of MHC class-I-restricted antigen processing, Immunol. Today 13
  • the assembly of the peptides with the MHC molecules is a selective process and requires that the peptide is preferably of a certain size (9 ⁇ 1 amino acids) (HG Rammenee, K. Falk and O. Rotzschke, Peptides naturally presented by MHC class I molecules Annu. Rev. Immuol. 11 (1993), 213). Furthermore, certain "anchor amino acids" must bind molecules in MHC class I molecules. Individual, allele-specific anchor motifs were determined for various MHC molecules (HG Rammenee, K. Falk and 0. Rotzschke, MHC molecules as peptide receptors. Curr. Opin. Immunol. 5 (1993), 35; KRO Falk, S. Stevanovic, G. Jung, HG.
  • the processing route of the antigenic peptides presented by the MHC class II molecules is broadly similar to that of the molecules presented by MHC class I molecules. Particularities result particularly from the fact that the mostly exogenous proteins go through the endocytic degradation pathway, must have a length of about 12 amino acids for the presentation and in special compartments, the so-called compartments for peptide loading (CPL) with MHC Class II molecules associate after this early have passed through the endosomal processing route and the peptide loading-preventing invariant chain has been degraded (Schmid & Jackson, Nature 369 (1994), 103-104 and references cited therein; Germain and Margulies, Annu. Rev. Immunol. 11 (1993), 403 -450).
  • CPL compartments for peptide loading
  • NPP naturally processed and presented peptides
  • the object of the present invention was therefore to provide a method with which these rare antigenic peptides bound to MHC molecules can be identified.
  • Another object underlying the present invention was to produce antigenic peptides or peptide mixtures containing antigenic peptides in a simple manner and to use them for the production of peptide vaccines.
  • a first aspect of the present invention relates to a method for producing and identifying antigenic peptides and peptide mixtures, which comprises the following steps:
  • step (b) association of the antigenic peptides and / or peptide mixtures obtained in step (a) with suitable MHC molecules;
  • step (e) Determination of the amino acid sequence of the antigenic peptides and / or peptide mixtures obtained in step (d).
  • a second aspect of the present invention relates to a method for producing and identifying antigenic peptides and peptide mixtures, which comprises the following steps:
  • step (c) testing the fractions obtained for antigenic activity, preferably in a T cell assay, and (d) optionally determining the amino acid sequence of the antigenic peptides and / or peptide mixtures obtained in step (b).
  • Antigenic peptide is understood here to mean a peptide that is recognized by the immune system and is therefore in particular able to elicit a cellular immune response or an antibody reaction.
  • a peptide size that allows association with MHC molecules is preferably about 9 ⁇ 1 amino acids for MHC class I molecules, while preferably about 12 ⁇ 1 to 25 + 1 amino acids for MHC class II molecules.
  • Cellular processing mechanisms are understood here to be those which are exerted by cytosolic multienzyme complexes (proteases) and further, sometimes not yet shown, enzymes / enzyme complexes and, if appropriate, the subsequent processing ("trimming") in microsomes.
  • the MHC molecule-peptide complexes can preferably be isolated from microsome or CLP lysates. According to the second aspect of the present invention, however, an association of the proteolytically generated peptides with MHC molecules, isolation of the MHC molecule-peptide complexes and exclusive elution of the peptides is not necessary, since antigenic peptides or peptide fractions of the proteolytically generated peptide mixture are also in one T cell assay can be tested for their antigenic effectiveness.
  • the stoichiometric ratio between endogenous peptides and antigenic peptides must be manipulated so that the antigenic peptides are in a large excess for transport into the ER and for binding to MHC molecules. This will achieved by adding exogenous protein or polypeptide in step (a). At the same time, the amount of endogenous peptides that are already bound to MHC molecules in the ER should be small. This means that more free MHC molecules are available for the antigenic peptides.
  • the method described here can greatly enrich specific exogenous, e.g. peptide epitopes derived from a pathogen can be achieved.
  • this also makes it possible to identify subdominant peptide epitopes which, under normal physiological conditions, reach the cell surface in too low a concentration in MHC-bound form in order to induce specific CTL precursor cells for expansion and differentiation.
  • Dominant epitopes often represent proteins or protein segments which are formed in large quantities by the pathogen (e.g. envelope proteins of viruses), but which are not necessarily under such a high functional selection pressure that they cannot be changed within certain limits. This leads to e.g. in the case of infection with human immunodeficiency virus (HIV), most of the immune responses to protein segments are made, which are very variable.
  • the dominant epitopes cover the subdominant epitopes in quantity in the MHC-bound presentation. The concentration of the subdominant epitopes on the cell surface is so low that it is not sufficient as a signal to differentiate the T cells into CTLs.
  • the method according to the invention can thus advantageously be used to identify and produce viral antigenic peptides which can be used for prophylaxis and immunotherapy against such viruses.
  • the method according to the invention makes it possible to isolate antigenic peptides from cellular MHC molecules in amounts which are several orders of magnitude above the amounts which can be achieved using methods known in the prior art.
  • Antigenic peptides can be obtained directly from complete microsome lysates, cell lysates or from affinity-purified, loaded MHC molecules.
  • the MHC molecules are MHC class I molecules.
  • Suitable MHC molecules are those molecules which have a suitable conformation in order to be able to present the antigenic peptides.
  • the proteolytic degradation of the proteins or polypeptides takes place by proteasomes from cultivated cells, in particular human cells.
  • proteasomes are particularly preferred if the peptides from the degradative degradation of the proteins or polypeptides are to be presented by MHC class I molecules.
  • the proteasomes are preferably according to the Boes et al. , J. Exp. Med. 17 (1994), 901 isolated from cultured cells, especially human cells. Practically, the protein with the isolated proteasomes is incubated in microsome standard buffer (Levy et al., Cell 67 (1991), 265) for different periods (0.5 to 96 hours) at 37 ° C.
  • the human cell line K-562 (ATCC No. CCL 243) expressing this enzyme complex, for example, can also serve as a source of proteasomes.
  • proteasomes can be used without further treatment for the proteolytic degradation of exogenously added proteins or polypeptides.
  • proteasomes are used which are immobilized on a solid support. Immobilization can be achieved by chemical coupling e.g. using a bifunctional linker reagent. On the other hand, immobilization can also be carried out by immunoadsorption using immobilized antibodies directed against proteasomes.
  • the MHC class I molecules used according to the first aspect of the present invention for association with the antigenic peptides and / or peptide mixtures obtained by proteolytic degradation can consist of cellular microsomes, cells of a patient or modified cells with specificity for a single MHC class I molecule (K. Takahashi, LC Dai, TR Fuerst, WE Biddison, PL Earl, B. Moss and FA Ennis. Specific lysis of human immunodeficiency virus type 1-infected cells by a HLA-A3.1- restricted CD8 + cytotoxic T. lymphocyte clone that recognizes a conserved peptide sequence within the gp41 subunit of the envelope protein. Proc. Natl. Acad.
  • HLA.A2.1 plays a role in presentation of influenza virus matrix peptides and alloantigens. J. Immunol. 146 (1991) 3508-3512).
  • recombinant MHC class I molecules can also be used (Matsumura, M., Saito, Y., Jackson, MR, Song, ES and Peterson, PA. In vitro peptide binding to soluble empty class I MHC molecules isolated from transfected Drosophila melanogaster cells. J. Biol. Chem. 26 (1992) 23589).
  • the isolation of the microsomes is preferably carried out according to the method described by Saraste et al. , Proc. Natl. Acad. Be. USA 83 (1986), 6425.
  • the antigenic peptides can be associated with the MHC class I molecules by incubating the peptides with microsomes.
  • the unloaded MHC class I molecules are mainly present in the microsomal fraction (Levy et al., Op. Cit.). Accordingly, it is possible to directly incubate the antigenic peptides obtained after proteasome degradation with the microsomal fraction of the endoplasmic reticulum to obtain MHC class I molecule-antigenic peptide complexes. Incubation is preferably 1 minute to 36 hours at 37 ° C.
  • Another preferred embodiment of the invention relates to a method wherein the MHC molecules are MHC class II molecules.
  • the proteolytic degradation of the proteins or polypeptides can take place by endosomal and / or lysosomal enzymes, preferably in the presence of suitable MHC class II molecules.
  • This embodiment of the method according to the invention is particularly advantageous when the antigenic peptides are to be presented by MHC class II molecules.
  • the antigenic peptides to be loaded on MHC class II molecules are not primarily processed by degradation by means of a proteasome complex, but by proteolytic degradation in endosomal / lysosomal compartments.
  • the MHC class II molecules to be provided for the formation with antigenic peptide can be made from trans-Golgi vesicles, endosomes, CPLs, cells of a patient or modified cells with specificity for a single MHC class II molecule (Riberdy, JM, Awa, RR, Geuze, HJ and Cresswell, P., Transport and intracellular distribution of MHC class II-molecules and associated invariant chain in normal and antigen-processing mutant cell lines. J. Cell. Biol. 125 (1994), 1225-1237).
  • recombinant MHC class II molecules (Arimilli, S., Cardoso, C, Mukku, P., Baichwal, V.
  • MHC class II molecules are prevented in the case of CPLs from binding to a suitable antigenic peptide at least when entering the compartment by the invariant chain.
  • the proteolytic degradation of the invariant chain begins or is completed in these compartments, so that the MHC class II molecules can subsequently associate with the desired antigenic peptide.
  • the antigenic peptides are associated with MHC class II molecules by joint incubation in CPLs.
  • this preferred embodiment of the method according to the invention should particularly lead to the fact that the actually interesting antigenic peptides can be recognized by the MHC class II molecules and made available for further studies.
  • the proteins or polypeptides used for proteolytic degradation can be of natural origin.
  • the proteins or polypeptides can be of recombinant or synthetic origin. This embodiment is particularly preferred since recombinant or synthetic polypeptides are easier to produce by conventional methods than the isolation of these proteins or polypeptides from natural sources.
  • the processes developed in recent years make it particularly easy to provide recombinant proteins in sufficient quantities, which can then be broken down by the cellular processing machinery and taken up in large quantities by free MHC molecules.
  • Synthetic polypeptides or proteins can be produced by standard methods, for example using F-moc chemistry, using a peptide synthesis device from Applied Biosystems Inc. or a corresponding other device.
  • the number of suitable MHC class II molecules can optionally be increased by in vitro translations and / or translocation of MHC class II molecules in microsomes or trans-Golgi vesicles.
  • the MHC molecules originate from TAP-negative cell lines.
  • T2 T2
  • This cell line and its production is described in Levy et al. , op. cit., Salter et al. , EMBO J.5 (1986), 943-949, Salter et al. , Immunogenetics 21 (1985), 235-246 and De Mars et al. , Proc. Natl. Acad. Be. USA 82: 8183-8187 (1985).
  • the person skilled in the art can produce further TAP-negative cell lines.
  • exogenous natural, recombinant or synthetic polypeptides to the cellular processing and presentation machinery, an excess of exogenous antigens is created Peptides that compete with endogenous cellular peptides.
  • the amount of free MHC molecules in microsomes can be increased by in vitro translation and translocation of MHC molecules in microsomes according to methods known in the art and by using microsome preparations from TAP-negative cell lines. The latter embodiment prevents the MHC molecules from being loaded with endogenous peptides prior to contact with antigenic peptides originating from exogenous sources and thus being blocked.
  • Isolation of the MHC molecule-peptide complexes according to the first aspect of the present invention can e.g. by precipitation with conformation epitope-specific anti-MHC antibodies.
  • Such antibodies can be of polyclonal or monoclonal origin. Methods for immunoprecipitation with such antibodies are well known in the art (see e.g. Burgert & Kvist, Cell 41 (1985), 987).
  • the antigenic peptides can be separated from the MHC molecules by elution, preferably acid elution, of the peptides from the MHC molecules.
  • elution preferably acid elution
  • Methods of this type for elution, preferably for acid elution, have also been sufficiently described in the prior art (cf. Germain and Margulies; cited above and references cited therein).
  • the elution is preferably followed by a peptide purification step or peptide fractionation step, for example using HPLC, gel filtration or capillary electrophoresis.
  • a purification or fractionation step is particularly advantageous if a heterogeneous fraction of peptides is found.
  • a subsequent sequencing of the peptides without prior purification or fractionation of the relevant antigenic peptides is often difficult because of the offset NH 2 ends.
  • prior purification is also advantageous for peptides that bind to MHC class I molecules.
  • Reverse phase HPLC is preferably used as the HPLC method.
  • Fractions of peptides and / or peptide mixtures with a defined size are obtained by the above-mentioned fractionation procedure.
  • This fractionation step is carried out according to the second aspect of the present invention directly after proteolytic degradation, i.e. performed without prior association with MHC molecules.
  • proteolytic degradation found antigenic peptides in such a high yield that the step of association with MHC molecules can often be dispensed with.
  • a T cell assay is preferably carried out to identify antigenic peptide fractions.
  • peptides processed by proteasomes which can bind to MHC I molecules, are tested in particular by a standard lysis test with the aid of cytotoxic T cells, which are preferably specific for the protein or polypeptide or a pathogen containing the protein or polypeptide.
  • the cytotoxic cells can originate, for example, from polyclonal CTL lines or T cell clones.
  • Such a standard lysis test is, for example, the chromium freezing method known in the prior art; see. e.g. Boes et al. , op. cit.
  • the method according to the invention enables the production and identification of antigenic peptides which, of course, correspond largely to naturally processed and presented peptides.
  • a suitable detection system for antigenic peptides for example polyclonal, antigen-specific CTL lines.
  • CTLs can be generated in vitro from peripheral blood lymphocytes by contact with autologous Epstein-Barr virus transformed B cells (EBV-B cells) which express the recombinant antigen in excess.
  • EBV-B cells Epstein-Barr virus transformed B cells
  • Another method known in the prior art for generating epitope-specific CTLs is stimulation with peptide-pulsed EBV-B cells.
  • CTLs can also be used to search for particularly subdominant peptides that are just naturally processed sufficiently to be able to be detected.
  • Antigens from exogenous sources are also rarely presented on the EBV-B cells in combination with processed antigenic peptides in combination with MHC molecules, which are presented in vivo in insufficient numbers to activate CTLs.
  • the standard lysis set represents the control authority for the authenticity of the identified antigenic peptides with naturally processed and presented antigens.
  • the active fractions are preferably identified by a T cell proliferation test known in the prior art.
  • amino acid sequence of antigenic peptides and / or mixtures of peptides can be determined by sequencing using preferably automated Edman chemistry or mass spectrometry, e.g. Tandem mass spectroscopy.
  • the protein or polypeptide used for proteolytic degradation can (a) correspond to a pathogen or a protein or polypeptide or a part thereof that originates from a pathogen, (b) be a tumor antigen or a part thereof, or (c) be an autoimmune antigen or a part thereof.
  • the antigenic peptide is a subdominant peptide.
  • the advantages of the method according to the invention for identifying subdonminant antigenic peptides have already been discussed above.
  • This preferred embodiment of the method according to the invention can therefore be used in particular and is advantageous when such subdominant antigenic peptides are to be recognized by the immune system and its subsequent stimulation is to be used in order to effectively combat the pathogen / tumor antigen / autoimmune antigen.
  • the selection of presentable antigenic peptides is increased by transfection of a cell line in which the cellular processing mechanism takes place and can express the MHC molecules with genes coding for TAP molecules, preferably from different species.
  • TAP molecules are cloned (Levy et al., Loc. Cit., Germain & Margulies, loc. Cit.) Or can be isolated and cloned from other species by methods known in the art (eg hybridization with probes from conserved regions of the gene) become. Since it is assumed that peptide binding to TAP is a selective process, transfection with other TAP molecules from different ethnic groups (assuming a certain polymorphism between different ethnic groups), rats, mice, hamsters, to name just a few examples, the absolute number of different peptide-binding conformations on TAP molecules increase. This means that absolutely more different peptides can be loaded onto suitable MHC molecules, which in particular also increases the possibilities for identifying subdominant antigenic peptides.
  • the genes are preferably cloned into expression vectors.
  • the transfection of TAP-negative cell lines with such genes is particularly preferred.
  • a third aspect of the present invention relates to a method for producing antigenic peptide mixtures, comprising the steps:
  • cytotoxic T lymphocytes which are particularly important for the elimination of virus-infected cells or tumor cells, can only be induced to a limited extent, since a specific MHC molecule can only ever bind a specific set of peptides.
  • a peptide epitope that is effective for one patient can be completely ineffective for another patient. Due to the enormous MHC polymorphism in the human population, it would probably take years or decades to the epitopes of relevant viral or tumor antigens are identified for all MHC molecules. In addition, an MHC typing would have to be carried out by each person to be immunized and then an appropriate selection or mixture of peptides for the respective MHC constellation would be administered.
  • a vaccine containing the complex peptide mixtures are in particular that the identification of individual epitopes for different MHC molecules and an MHC typing of the person to be vaccinated is no longer necessary. Furthermore, the corresponding peptide vaccines no longer have to be produced synthetically by complex and expensive methods, but can be produced in a simple and efficient manner by enzymatic digestion of recombinant polypeptides or proteins.
  • an enzyme reactor comprising:
  • a container that contains cellular processing enzymes, especially proteasomes, that degrade the proteins or polypeptides cause a peptide size that allows association with MHC molecules, the container having at least one inlet opening for the supply of proteins and at least one outlet opening for the removal of the processed peptides.
  • the container is preferably thermostable.
  • the cellular processing enzymes are preferably immobilized by known methods - as discussed above - on a carrier material and / or on walls of the container.
  • the immobilization can take place by chemical coupling or preferably by immunoadsorption.
  • the enzymes can also be freely available in solution.
  • the outlet opening of the enzyme reactor is preferably designed such that it allows the processed peptides to pass through, but not the proteins or polypeptides used as starting material to pass through.
  • the outlet opening of the enzyme reactor preferably also does not allow the enzymes to pass through.
  • the outlet opening preferably comprises a filtration unit, e.g. an Amicon filter (molecular weight cut-off 3000 to 10000 daltons).
  • the present invention also relates to a new device for the proteolytic degradation of proteins or polypeptides, which has the features mentioned above.
  • the process can be carried out continuously, i.e. the proteins or polypeptides used as starting material can be fed continuously to the reactor and the processed peptides can be removed continuously.
  • the invention also relates to the use of the antigenic peptides produced by the method according to the invention for the identification of vaccine components against pathogens or tumor cells or of competitive or antagonistic peptides in autoimmune diseases.
  • the antigenic peptides identified and / or produced by the method according to the invention can be used, for example, alone or coupled to a carrier according to methods known in the art for immunization against pathogens or for prophylaxis against and / or for the therapy of tumor growth.
  • autoimmune diseases can be used in the prophylaxis and therapy of autoimmune diseases to displace previously identified autoimmune stimulating peptides from binding to MHC molecules. They can also be used for competitive binding to T cell receptors without stimulating the T cells.
  • the peptides or peptide mixtures which are obtainable by the process according to the invention are suitable for the production of a vaccine.
  • the complex peptide mixtures obtained by proteolytic degradation can optionally be used after size fractionation without being separated into individual peptides beforehand.
  • the vaccine preferably additionally contains an adjuvant, which, for example, consists of aluminum hydroxide, emulsions of mineral oils (cf. Freund's adjuvant), saponin, silicon compounds, thiourea, liposomes, quil-A-containing liposomes, ISCOMS, lipopeptides, endotoxins from gram-negative bacteria, exotoxi - can be selected from gram-positive bacteria and Haemophilus pertussis.
  • an adjuvant which, for example, consists of aluminum hydroxide, emulsions of mineral oils (cf. Freund's adjuvant), saponin, silicon compounds, thiourea, liposomes, quil-A-containing liposomes, ISCOMS, lipopeptides, endotoxins from gram-negative bacteria, exotoxi - can be selected from gram-positive bacteria and Haemophilus pertussis.
  • the peptide vaccine is administered several times, for example at weekly, monthly or half-yearly intervals.
  • the dosage of the peptide vaccine is preferably 10-1000 ⁇ g, more preferably 50-500 ⁇ g, particularly preferably 50-200 ⁇ g and for example 100 ⁇ g.
  • the peptide vaccine is administered in combination with a protein or polypeptide vaccine.
  • the protein or polypeptide vaccine can contain the protein or polypeptide or / and another protein or polypeptide, which preferably originates from the same pathogen, used as the starting material for the production of the peptide vaccine.
  • peptide vaccine and polypeptide or protein vaccine is not only limited to the peptides and / or peptide mixtures produced by the method according to the invention, but can be carried out with any peptide vaccines.
  • co-administration in this context means that the peptide vaccine and the protein or polypeptide vaccine can be administered both in a single dosage unit and in separate dosage units simultaneously or at different times during the treatment period.
  • Administration can be by injection, orally or topically, e.g. by applying a vaccine and possibly a penetration aid, e.g. a physiologically acceptable organic solvent, such as a patch containing DMSO.
  • a penetration aid e.g. a physiologically acceptable organic solvent, such as a patch containing DMSO.
  • a topical application of peptide vaccines is not limited to the peptide vaccines obtainable by the method according to the invention, but can be used with any peptide vaccines.
  • yet another object of the present invention is a pharmaceutical composition which, as an active ingredient, contains peptide mixtures which are produced by proteolytic degradation of exogenous polypeptides and proteins are obtainable by cellular processing enzymes, optionally together with pharmaceutically customary carriers, auxiliaries and additives.
  • EL-4 cells (ATCC TIB39) were cultivated in RPMI medium with 10% fetal calf serum, 3-mercaptoethanol, L-glutamine and antibiotics in roller bottle cultures to a density of approximately 1 ⁇ 10 6 cells / ml.
  • About 2 x 10 9 cells were washed 2 x with cold phosphate-buffered saline (PBS), in 100 ml imidazole buffer (20 mM imidazole-HCl pH 6.8, 100 mM KCl, 20 mM EGTA, 2 mM MgCl 2 , 10% Sucrose) and then exposed to N 2 cavitation. The resulting suspension was then centrifuged (15 min at 1500 xg, 15 min at 15000 xg, 90 min at 150000 xg).
  • PBS cold phosphate-buffered saline
  • the peptidase activity of the proteasomes was determined fluorometrically using the substrate Suc-LLVY-MCA as described by McGuire and Martino (Biochim. Biophys. Acta. 873 (1986), 279-289).
  • the column fractions with activity (about 35% B) were combined and the purity of the proteasomes was determined by electrophoresis in 12% polyacrylamide / SDS gels and 5% native polyacrylamide gels and staining of the proteins by silver staining.
  • the cleavage of synthetic polypeptides (20 ⁇ g) with 0.5 ⁇ g isolated proteasomes was carried out in a volume of 300 ⁇ l Tris buffer (50 mM Tris-HCl pH 7.8, 1 mM EGTA, 2 mM MgCl 2 , 0.5 mM ⁇ - mercaptoethanol) for 36 h at 37 ° C.
  • Tris buffer 50 mM Tris-HCl pH 7.8, 1 mM EGTA, 2 mM MgCl 2 , 0.5 mM ⁇ - mercaptoethanol
  • 30 ⁇ l of the mixture were separated by reverse phase HPLC (Sephasil, C18, 2.1 / 10 Smart System, Pharmacia, Uppsala, Sweden).
  • the material applied to the column was at a flow rate of 100 ul / min.
  • Solution A 0.1% (vol / vol), trifluoroacetic acid (TFA);
  • Solution B 0.081% (vol / vol), TFA in 80% acetonitrile-H 2 0; 0-10 min at 0% for solution B, 10-40 min with a linear increase to 75% for solution B.
  • EL-4 (ATCC TIB 39) IC-21 were used as target cells (ATCC TIB 186) cells or 48 h with ConA-activated spleen cells used in standard 51 Cr release assays.
  • the target cells were labeled with 100 ⁇ Ci 51 Cr (New England Nuclear Research Products, Boston, Mass.) For 90 min at 37 ° C. and washed twice with RPMI.
  • the labeled target cells were then sensitized to CTL-mediated lysis by preincubation with synthetic peptides or the reaction products of a proteasome cleavage for 2 h at room temperature, washed and added to the effector cells (CTLs).
  • CTLs effector cells
  • the immunoprecipitations were carried out at 4 ° C.
  • Protein G or A Sepharose beads (Pharmacia, Uppsala, Sweden) were used for 30 min with a detergent-containing buffer. pre-incubated with rotation. The Sepharose was then sedimented by centrifugation (3 min. At 1000 ⁇ g) and washed three times with a detergent-free buffer. 30 ⁇ l of a 10% (w / v) solution of this protein G or A-Sepharose are mixed with 10 ⁇ g of a monoclonal anti-proteasome antibody (KB Hendil, P. Kristensen and W.
  • a monoclonal anti-proteasome antibody KB Hendil, P. Kristensen and W.
  • the cells were centrifuged, washed twice with PBS and then at a density of 2 x 10 7 cells per ml in lysis buffer (20 mM imidazole, pH 6.8, 100 mM KC1, 20 mM EGTA, 2 mM MgCl 2 , 10% sucrose ) added. The cells were then destroyed by freeze / thaw steps. By centrifugation for 15 min. at 3000 xg, the cell nuclei and by centrifugation for 15 min. centrifuged the mytochondria and lysosomes at 10,000 xg. By ultracentrifuging the supernatant for 90 min. at 100,000 xg the microsomes were removed. The supernatant thus obtained was then used in immunoprecipitation.
  • proteasome (20S or 26S proteasome) were ert using the method described in Example 4 Immunoprecipitation isoli ". Instead of proteasomes, however, other enzyme Antigen processing ists- natural or recombinant origin can be used, which can also be isolated by conventional protein purification methods. The enzymes can be used in the enzyme reactor in soluble or immobilized form. Obtained or recombinant by conventional cleaning methods Enzymes can be immobilized on Sepharose or other known carrier materials using known chemical coupling methods.
  • the proteasomes were placed in an enzyme reactor.
  • polypeptides and proteins of recombinant, synthetic or natural origin in the denatured and reduced state were added to the enzyme reactor.
  • the reaction buffer contained 20 mM Hepes (pH 7.8), 0.5 mM 2-mercaptoethanol, 1 mM EGTA and 5 mM MgCl 2 .
  • a detergent such as the buffer SDS (0.003 to 0.04% w / v) or 0.4 M guanidinium hydrochloride was added if necessary.
  • ubiquitin and ATP as well as an ATP regenerating system were added.
  • the reaction was at 37 ° C for 10 min. carried out up to 24 hours. Subsequently, the peptide fragments obtained were separated from the proteasomes and from any proteins and other reactants still present by a filter limiting the outlet opening of the reaction vessel (cut-off size of the pores 3000 to 10,000 daltons). The separation was carried out by low-speed centrifugation or by pumping off the reaction solution.
  • the peptide mixtures were thus obtained in sterile form. They can be used either directly or after mixing with an adjuvant for in vitro and in vivo immunizations.
  • the peptide mixtures were also used to identify individual antigenic peptides, as described in Example 6.
  • Antigenic proteins or polypeptide parts thereof e.g. viral, bacterial, parasitic, tumor or self-antigens
  • isolated 20S or 26S proteasomes resulting in peptides of a size that allow association with MHC molecules.
  • the peptide mixtures were obtained using an enzyme reactor (see Example 5).
  • the proteins can also be reacted with soluble proteasomes in a suitable reaction vessel, the reaction after 10 min. is stopped up to 24 hours by adding 2% acetic acid.
  • antigenic peptide sequences were identified as follows.
  • the peptide mixture is separated using reverse-phase HPLC (for example on a C18 4.6 x 250 mm column) or using capillary zone electrophoresis. An aliquot of the fractions was then subjected to a T cell assay (eg chromium release assay, proliferation test).
  • a T cell assay eg chromium release assay, proliferation test.
  • PHA-stimulated blood lymphocytes (PBL) EBV-transformed B cells and cells that only express one HLA molecule
  • K. Takahashi, L.-C. Dai TR Fuerst , WE Biddison, PL Earl, B. Moss and FA Ennis, Proc. Natl. Acad. Sei.
  • T cell clones can be generated in vitro from peripheral blood lymphocytes through * contact with autologous Epstein-Bar virus transformed B cells (EBV-B cells). Furthermore, as T cells, non-specifically (for example PHA) stimulated PBL from virus-infected patients or from tumor patients or PBL or tumor-infiltrating lymphocytes (TILL) that were specifically with autologous virus-infected cells or tumor cells were stimulated.
  • PHA Epstein-Bar virus transformed B cells
  • sequence of the peptides therein was then determined in the positive fractions by Edman degradation or mass spectrometry. If necessary, positive fractions were subjected to a separation in a second dimension. The steps of separation of the peptide mixture, determination of the anti-gene peptide fractions in the T cell assay and sequence analysis were also carried out in a coupled manner.
  • the peptide mixture was introduced via a capillary HPLC column directly into a mass spectrometer (eg tandem electrospray mass spectrometer), the HPLC eluent being divided by a split in such a way that part of the mass spectrometer and another part of a suitable one Reaction vessel (eg 96-well plate) was introduced, in which a T-cell assay was then carried out.
  • a mass spectrometer eg tandem electrospray mass spectrometer
  • a suitable one Reaction vessel eg 96-well plate
  • the peptide mixture was brought into contact with MHC molecules.
  • the peptides of the peptide mixture are first associated with suitable MHC molecules.
  • the MHC molecules were used as MHC molecules bound to the cell surfaces of intact cells (e.g. PBL from patients, cells which only express a certain HLA molecule and other suitable cells) or as MHC molecules present in microsomes of such cells.
  • isolated MHC molecules were used, which were immobilized on a carrier material.
  • the MHC molecules were obtained from cell lysates by immunoprecipitation with MHC-specific antibodies, but recombinant MHC molecules can also be used.
  • peptides After the peptide mixture has been incubated with the MHC molecules, bound peptides are eluted from the binding pit of the MHC molecules by means of acid elution. The eluted peptides were then separated by reverse phase HPLC or capillary zone electrophoresis and aliquots of the fractions were tested in a T cell assay, the positive fractions, such as under (A), the sequences of the peptides were determined.
  • endogenous peptides e.g. recombinant MHC molecules
  • sequences of the eluted peptides were determined directly, without separation of the peptide mixtures by mass spectrometry.

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Abstract

L'invention concerne des techniques in vitro d'identification et de production de peptides ou de mélanges de peptides antigéniques, qui simulent la modification in vivo de protéines exogènes ou endogènes, ainsi que l'utilisation des peptides ou mélanges de peptides antigéniques identifiés et produits selon la technique de l'invention, par exemple pour identifier des composants de vaccins contre des pathogènes ou des cellules tumorales, ou bien des peptides compétitifs ou antagonistes dans le cas de maladies auto-immunes, ou pour produire des vaccins.
PCT/EP1995/002593 1994-07-04 1995-07-04 Techniques d'identification et de production de peptides antigeniques et leur utilisation en tant que vaccins WO1996001429A1 (fr)

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JP8503679A JPH10505665A (ja) 1994-07-04 1995-07-04 抗原ペプチドの同定及び製造法並びにワクチンとしてのその使用
EP95925827A EP0769147A1 (fr) 1994-07-04 1995-07-04 Techniques d'identification et de production de peptides antigeniques et leur utilisation en tant que vaccins
AU29813/95A AU2981395A (en) 1994-07-04 1995-07-04 Method of identifying and producing antigen peptides and use thereof as vaccines

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DE4423392A DE4423392A1 (de) 1994-07-04 1994-07-04 Verfahren zur Identifizierung antigener Peptide
DEP4423392.2 1994-07-04

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026538A1 (fr) * 1996-01-19 1997-07-24 Virginia Mason Research Center Strategie de determinant antigenique de peptide specifique d'un allele aux fins de la mise au point de vaccin
US6861234B1 (en) 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
US6977074B2 (en) 1997-07-10 2005-12-20 Mannkind Corporation Method of inducing a CTL response
US6994851B1 (en) 1997-07-10 2006-02-07 Mannkind Corporation Method of inducing a CTL response
US7232682B2 (en) 2001-11-07 2007-06-19 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
WO2011050344A2 (fr) 2009-10-23 2011-04-28 Mannkind Corporation Immunothérapie pour le cancer et procédé de traitement du cancer
EP2394655A2 (fr) * 2001-04-06 2011-12-14 Mannkind Corporation Séquences d'épitope
EP2481418A1 (fr) 2007-02-15 2012-08-01 MannKind Corporation Procédé d'amélioration de la réponse des cellules T

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EP2248910A1 (fr) * 2000-04-28 2010-11-10 Mannkind Corporation Synchronisation d'épitopes dans les cellules présentatrice d'antigène
KR101711458B1 (ko) * 2015-03-06 2017-03-02 인하대학교 산학협력단 올리고당류 또는 펩티드류 분리용 고분자 부착 실리카 모세관 및 이의 제조방법
CN113993991A (zh) * 2019-04-18 2022-01-28 智能干细胞科技公司 宿主细胞中免疫蛋白酶体的过表达用于产生抗原呈递细胞

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WO1992007952A1 (fr) * 1990-10-30 1992-05-14 Immulogic Pharmaceutical Corporation Methodes de dosage faisant appel a une liaison competitive entre un peptide et des antigenes de complexe majeur d'histocompatibilite
WO1992021033A1 (fr) * 1991-05-17 1992-11-26 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Determination de motifs de peptides sur des molecules du complexe majeur d'histocompatibilite
US5200320A (en) * 1987-12-07 1993-04-06 National Jewish Center For Immunology And Respiratory Medicine Method for identifying useful polypeptide vaccines

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US5200320A (en) * 1987-12-07 1993-04-06 National Jewish Center For Immunology And Respiratory Medicine Method for identifying useful polypeptide vaccines
WO1992007952A1 (fr) * 1990-10-30 1992-05-14 Immulogic Pharmaceutical Corporation Methodes de dosage faisant appel a une liaison competitive entre un peptide et des antigenes de complexe majeur d'histocompatibilite
WO1992021033A1 (fr) * 1991-05-17 1992-11-26 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Determination de motifs de peptides sur des molecules du complexe majeur d'histocompatibilite

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H. KROPSHOFER ET AL.: "Self-peptide released from class II HLA-DR1 exhibits a hydrophobic two-residue contact motif.", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 175, TOKYO, JP, pages 1799 - 1803 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997026538A1 (fr) * 1996-01-19 1997-07-24 Virginia Mason Research Center Strategie de determinant antigenique de peptide specifique d'un allele aux fins de la mise au point de vaccin
US6977074B2 (en) 1997-07-10 2005-12-20 Mannkind Corporation Method of inducing a CTL response
US6994851B1 (en) 1997-07-10 2006-02-07 Mannkind Corporation Method of inducing a CTL response
US7364729B2 (en) 1997-07-10 2008-04-29 Mannkind Corporation Method of inducing a CTL response
US6861234B1 (en) 2000-04-28 2005-03-01 Mannkind Corporation Method of epitope discovery
EP2394655A2 (fr) * 2001-04-06 2011-12-14 Mannkind Corporation Séquences d'épitope
EP2394655A3 (fr) * 2001-04-06 2012-05-02 Mannkind Corporation Séquences d'épitope
US7232682B2 (en) 2001-11-07 2007-06-19 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
US8637305B2 (en) 2001-11-07 2014-01-28 Mannkind Corporation Expression vectors encoding epitopes of target-associated antigens and methods for their design
EP2481418A1 (fr) 2007-02-15 2012-08-01 MannKind Corporation Procédé d'amélioration de la réponse des cellules T
WO2011050344A2 (fr) 2009-10-23 2011-04-28 Mannkind Corporation Immunothérapie pour le cancer et procédé de traitement du cancer

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AU2981395A (en) 1996-01-25

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