WO1996001429A1

WO1996001429A1 - Method of identifying and producing antigen peptides and use thereof as vaccines

Info

Publication number: WO1996001429A1
Application number: PCT/EP1995/002593
Authority: WO
Inventors: Gabriele Niedermann; Klaus Eichmann; Bernhard Maier
Original assignee: MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Priority date: 1994-07-04
Filing date: 1995-07-04
Publication date: 1996-01-18
Also published as: EP0769147A1; AU2981395A; DE4423392A1; JPH10505665A; CA2192565A1

Abstract

The invention concerns in vitro methods of identifying and producing antigen peptides and peptide mixtures which simulate in vivo trimming of foreign or the body's own proteins; and the use of the antigen peptides or peptide mixtures identified and produced by the claimed method, for example, for identifying vaccine components against pathogens or tumour cells, or competitive or antagonistic peptides in auto-immune diseases or in the production of vaccines.

Description

Methods for the identification and production of antigenic peptides and their use as vaccines

DESCRIPTION

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.

One of the most important basic principles of the body's own defense reactions is the presentation of processed foreign protein by polymorphic body's own structures, the so-called tissue tolerance antigens (major histocompatibility antigens = major histocompatibility complex antigens = MHC 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 (CTLs) 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.

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

(1992), 173; J.J. Monaco, S. Cho and M. Attaya, Transport protein genes in the murine MHC: possible implications for antigen processing, Science 259 (1990), 1723; M.J. Andrblewicz, K.S. Anderson and P. Cresswell, Evidence that transporters associated with antigen processing translocate a major histocompatibility complex class I-binding Peptide into the endoplasmic reticulum in an ATP-dependent manner., Proc. Natl. Acad. Be. USA 90

(1993), 9130; JC Shepherd, TN Schumacher, PG Ashton-Rickardt, S. I aeda, HL Ploegh, C. Janeway Jr. and S. Tonegawa, TAPl-dependent peptide translocation in vitro is ATP dependent and peptide selective, Cell 74 (1993), 577; JJNeetjes and F. Momburg, Cell biology of antigen presentation. Curr. Opin. Immunol. 5 (1993) 27).

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. Rammenee, Allele specific Motifs Revealed by Sequencing of Self-peptides Eluted from MHC Molecules, Nature 351 (1991), 290). With the help of these anchor motifs, antigenic peptides of proteins from viruses, bacteria, parasitic protozoa and tumor cells were predicted and identified (HJ Pamer, MJ Bevan, Precise Prediction of a Dominant Class I MHC-restricted Epitope of Listeria monocytogenes, Nature 353 (1991), 852 ; HJ Wallny DK, S. Faath, G. Jung, A. van Pel, T. Boon, HG. Rammenee, Identification and quantification of a Naturally Presented Peptide as Recognized by Cytotoxic T Lymphocytes Specific for an Immunogenic Tumor Variant, Int. Immunol 4: 1088 (1992).

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).

After assembly of the MHC-peptide complexes, these are transported to the cell surface, where they are presented to the immune system.

The number of "naturally processed and presented peptides" (NPP) that have been identified to date is relatively small and is limited to a small spectrum of different peptides of proteins expressed in large quantities. The identification of antigenic determinants, which are presented on the cell surface in only a very small amount due to the low expression rate, poor processing, slow transport into the ER or suboptimal binding to MHC molecules, is itself possible with the methods available to date difficult to impossible using highly sensitive methods such as tandem mass spectrometry. The general, experimentally empirically confirmed rule that all peptides generated in the cell compete for transport into the ER and for available free MHC molecules also applies to peptides that arise from proteins of pathogenic organisms or cancer cells. This fact makes it extremely difficult to isolate the very small amount of antigenic peptides from a large excess of endogenous peptides that occupy most of the MHC molecules.

However, the identification of these rare antigenic peptides is of great importance for the vaccine development and immunotherapy of many diseases. 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.

The solution to these problems is provided by the embodiments characterized in the claims.

Thus, 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:

(a) proteolytic degradation by cellular processing mechanisms from exogenously added proteins or polypeptides containing the antigenic peptides to a peptide size that allows association with MHC molecules;

(b) association of the antigenic peptides and / or peptide mixtures obtained in step (a) with suitable MHC molecules;

(c) isolation of the MHC molecule-peptide complexes;

(d) separation of the antigenic peptides and / or mixtures of peptides from the MHC molecules; and

(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:

(a) Proteolytic degradation by cellular processing mechanisms of exogenously added proteins or polypeptides, which contain the antigenic peptides into one. Peptide size that allows association with MHC molecules;

(b) fractionation of the antigenic peptides and / or peptide mixtures obtained in layer (a);

(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.

If desired, 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.

In order to facilitate the identification of antigenic peptides generated by the cellular processing machinery and presented by MHC molecules on the cell surface, 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.

Compared to the previously used methods and methods of peptide elution from cell lysates or from affinity-purified MHC molecules, the method described here can greatly enrich specific exogenous, e.g. peptide epitopes derived from a pathogen can be achieved. In addition to immunodominant peptide epitopes, 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.

As in principle all P ^* oteine of the cellular machinery processed and presented, in which the method according erfindungs¬ achieves high accumulation of peptide epitopes that are subdominant under normal conditions. If you can generate CTLs against eptitopes that represent peptide segments that are under such high functional pressure that they have a very limited genetic variability, such CTLs can be used to generate a very effective immune response. Once activated CTLs also recognize subdominant epitopes which are presented in the lowest concentrations on infected cells, whereas the limit concentration for the activation of the T cells and differentiation to CTLs is much higher.

Many viruses owe their persistence to this fact, since the dominant epitopes constantly mutate and thus the virus as a whole eludes immune elimination, despite the constant presence of immune reactions. 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.

In a preferred embodiment of the method according to the invention, 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.

In a further preferred embodiment of the method according to the invention, the proteolytic degradation of the proteins or polypeptides takes place by proteasomes from cultivated cells, in particular human cells. This embodiment is 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.

The isolated proteasomes can be used without further treatment for the proteolytic degradation of exogenously added proteins or polypeptides. In a preferred embodiment of the present invention, however, 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. Sei., 88 (1991) 10277-10281; CC Winter, BM Carreno, RV Turner, S. Koenig and WB Biddison. The 45 pocket of HLA.A2.1 plays a role in presentation of influenza virus matrix peptides and alloantigens. J. Immunol. 146 (1991) 3508-3512). On the other hand, 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).

If the MHC class I molecules originate from cellular microsomes, 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. In this case, 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. In this case, 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. As already mentioned above, 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). On the other hand, recombinant MHC class II molecules (Arimilli, S., Cardoso, C, Mukku, P., Baichwal, V. and Nag, B., Refolding and reconstitution of functionally active complexes of human leucocyte antigen DR2 and myelin basic protein from recombinant alpha and beta chains. J. Biol. Chem. 270 (1995) 971).

As far as is known in the prior art, 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. However, it must be assumed that 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.

In a further preferred embodiment of the method according to the invention, the antigenic peptides are associated with MHC class II molecules by joint incubation in CPLs.

With this embodiment of the in vitro method according to the invention, the in vivo situation of loading the MHC class II molecules with antigenic peptides is mimicked. Therefore, 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. For this embodiment, it is necessary to provide a source of natural protein or polypeptide which enables sufficient amounts for an effective loading and presentation by a sufficiently high number of MHC molecules. On the other hand, 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.

In a further preferred embodiment of the method according to the invention, the MHC molecules originate from TAP-negative cell lines. An example of such a cell line is 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). According to the technical teachings described there, the person skilled in the art can produce further TAP-negative cell lines.

The embodiments described above either increase the relative amount of protein of interest to be degraded or increase the number of free MHC molecules.

Thus, by adding 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. 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. Such a purification or fractionation step is particularly advantageous if a heterogeneous fraction of peptides is found. In particular, in the case of processed peptides which bind to MHC class II molecules, 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. In addition, 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. Surprisingly, it was found that the 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. For this purpose, 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. This is achieved through the use of a suitable detection system for antigenic peptides, for example polyclonal, antigen-specific CTL lines. These 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. Another method known in the prior art for generating epitope-specific CTLs is stimulation with peptide-pulsed EBV-B cells.

These CTLs can also be used to search for particularly subdominant peptides that are just naturally processed sufficiently to be able to be detected.

By using preferably recombinant ^* . 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.

In the case of peptides or peptide mixtures which bind to MHC class II molecules, the active fractions are preferably identified by a T cell proliferation test known in the prior art.

Finally, the 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.

Both Edman chemistry and tandem mass spectrometry e.g. as electrospray ionization tandem mass spectrometry in connection with microcapillary HPLC belong to the prior art (cf. e.g. Hunt et al., Science 255 (1992), 1261-1264).

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.

With this embodiment of the method according to the invention, clinically relevant pathogens, tumor antigens or autoimmune antigens can be examined for their antigenic peptide components after cellular processing.

In a further preferred embodiment of the method according to the invention, 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.

In a further preferred embodiment of the method according to the invention, 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.

The genes coding for 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:

(a) proteolytic degradation by cellular processing mechanisms from exogenously added proteins or polypeptides which contain the antigenic peptides to a peptide size which permits association with MHC molecules,

(b) Obtaining the peptide mixtures.

Surprisingly, it was found that the complex peptide mixtures which result from the digestion of antigenic proteins or polypeptides, in particular by proteasomes, as a peptide vaccine are able to induce CTLs without having to identify or isolate individual peptide sequences.

In addition, the use of the peptide mixtures as a vaccine has a considerable advantage over the individual peptides previously used. With single peptide vaccines known to date, 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. Thus, 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.

On the other hand, the use of attenuated viruses known from the prior art, with which cytotoxic T cells can be effectively induced, is dangerous since the viruses can revert to pathogenic viruses in the event of a mutation. Again, mostly killed cytotoxic T-lymphocytes cannot be induced with killed viruses or protein vaccines, since the antigenic proteins cannot get into the r.ytosol, the place where antigens are processed for presentation via MHC class I molecules.

By using complex peptide mixtures after proteolytic degradation of antigens, especially in proteasomes, as a peptide vaccine, an induction of cytotoxic T cells can surprisingly be achieved. The advantages of 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.

To increase the yield of the peptides and / or polypeptides formed during the proteolytic degradation of proteins or polypeptides, an enzyme reactor is preferably used, 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. However, 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. For this purpose, the outlet opening preferably comprises a filtration unit, e.g. an Amicon filter (molecular weight cut-off 3000 to 10000 daltons).

Furthermore, the present invention also relates to a new device for the proteolytic degradation of proteins or polypeptides, which has the features mentioned above. With this device, 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.

Finally, 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.

Competitive or antagonistic peptides 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.

As already stated above, 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.

It is further preferred that 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 μg, more preferably 50-500 μg, particularly preferably 50-200 μg and for example 100 μg. In a particularly preferred embodiment of the present invention, 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. By administering the combination of protein or polypeptide vaccine and peptide vaccine, simultaneous stimulation of helper T cells and cytotoxic T cells can be achieved, whereby an increased antibody formation and an increased cellular immune response is achieved.

In this connection it should be noted that the joint administration of 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. The term "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. It should also be noted here that the 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.

Finally, 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.

The invention is further illustrated by the following examples:

Example 1:

Isolation of proteasomes from cultured cells

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).

10 ml aliquots of the supernatant obtained after the last centrifugation step were mixed with polyethylene glycol 6000 to a final concentration of 5% (w / v). After incubation for 30 min at 4 ° C., precipitated material was removed by centrifugation at 20,000 × g. The supernatant was reduced to 12.5%

(W / V) polyethylene glycol 6000, centrifuged and the resulting pellet in 2 ml of cold Tris buffer (50 mM Tris-HC1, pH 7.2, 1 mM EGTA, 5 mM MgCl ₂ 2, 0.5 mM / 3- Mercaptoethanol) dissolved. The resuspended material was on a Mono-Q

(HR5 / 5) column (Pharmacia, Uppsala, Sweden) applied, which was connected to a Waters HPLC system (Millipore, Milford, Massachusetts, USA). The bound proteins were eluted with a NaCl gradient; Buffer A (20 mM Tris-HCl pH 7.2), Buffer B

(20 mM Tris-HCl pH 7.2, 1 M NaCl), 0-10 min at 100% for buffer A, 10-65 min with a linear increase to 34% for buffer B, 65-95 min with a linear increase to 36% for buffer B (Flow rate: 0.5 ml / min), 90-105 min with a linear increase to 100% for buffer B (flow rate: 1 ml / min). The size of the fractions collected was 0.5 ml each.

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.

Example 2:

Cleavage of synthetic polypeptides by proteasomes

The cleavage of synthetic polypeptides (20 μg) with 0.5 μg isolated proteasomes was carried out in a volume of 300 μl Tris buffer (50 mM Tris-HCl pH 7.8, 1 mM EGTA, 2 mM MgCl ₂ , 0.5 mM β - mercaptoethanol) for 36 h at 37 ° C. To check the cleavage reaction, 30 μl 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. eluted with an acetonitrile gradient; 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.

The resulting peptide mixtures were sequenced by Edman degradation using a Hewlett Packard device (model HP 1005A). To determine the proteosome cleavage sites, the data obtained from Edman sequencing was evaluated as described by Boes et al (J. Exp. Med. 179 (1994), 901-909). Example 3:

Test for stimulation of cytotoxic T lymphocytes

RMA (Ljunggren and Karre, J. Exp. Med. 162 (1985), 1745-1759; Karre et al, Nature 319 (1986), 675-678), 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 μCi 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).

Example 4:

Immunoprecipitation of proteasomes

The immunoprecipitations were carried out at 4 ° C. To reduce any undesirable protein interactions that may occur with protein G or A Sepharose during the actual immunoprecipitation, 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 μl of a 10% (w / v) solution of this protein G or A-Sepharose are mixed with 10 μg of a monoclonal anti-proteasome antibody (KB Hendil, P. Kristensen and W. Uerkvitz; Human proteasomes analyzed with monoclonal antibodies, Biochem. J. 305 (1995) 245-252) or with an antiserum for one hour. Excess unbound antibodies are removed by washing the Sepharose beads three times. The Sepharose is then incubated with cell lysate or cytosol. To produce cytosol, cells (eg Tl cells) were cultivated in roller bottle cultures to a density of approximately 1 × 10 ⁶ cells per ml. 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.

To carry out the immunoprecipitation, 30 μl of a 10% solution of protein G or A-Sepharose beads, which contain immobilized antiproteasome antibodies, were incubated with 400 μl of cytosol for one hour while rotating. The Sepharose beads were then washed three times. The presence of proteasomes could be determined by measurements of the proteasome activity and by Western blots of the cytosol and the Sepharose solution. The successful immunoprecipitation was shown by the disappearance of the proteasomes from the cytosol and their detection in the Sepharose solution.

Example 5:

Operation of an enzyme reactor

Proteasome (20S or 26S proteasome) were ert using the method described in Example 4 Immunoprecipitation isoli ^". Instead of proteasomes, however, other enzyme Antigenprozessierungs- 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. In addition, 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 . To activate the proteasomes and to improve the solubility of the substrates, a detergent such as the buffer SDS (0.003 to 0.04% w / v) or 0.4 M guanidinium hydrochloride was added if necessary. When using 26S proteasomes, 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.

Since the proteasomes are enzymatically active for several weeks, they could be used several times for the operation of an enzyme reactor. Example 6:

Identification of individual antigenic peptide sequences

Antigenic proteins or polypeptide parts thereof (e.g. viral, bacterial, parasitic, tumor or self-antigens) of natural, recombinant or synthetic origin have been reacted with 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). However, 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.

The antigenic peptide sequences were identified as follows.

(A) 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). In the T-cell assay, among others, 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. USA, 88, 10277-10281; CC Winter, BM Carreno, RV Turner, S. Koenig and WE Biddison, J. Immunology, 146, 3508-3512) used as target or stimulator cells. Polyclonal CTL lines or 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.

The 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.

(B) In order to identify the antigenic peptide sequences, 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. In addition, 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.

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.

When using MHC molecules that have not bound any antigenic, "endogenous" peptides (e.g. recombinant MHC molecules), the sequences of the eluted peptides were determined directly, without separation of the peptide mixtures by mass spectrometry.

Claims

Patent claims

1. A method for producing and identifying antigenic peptides and peptide mixtures, which comprises the following steps. *

(a) proteolytic degradation by cellular processing mechanisms of exogenously added proteins or polypeptides which contain the antigenic peptides to a peptide size which permits association with MHC molecules;

(c) isolation of the MHC molecule-peptide complexes;

(d) separation of the antigenic peptides and / or peptide mixtures from the MHC molecules; and

2. The method of claim 1, wherein the MHC molecules are MHC class I molecules.

3. The method according to claim 1 or 2, wherein the proteolytic degradation of the proteins or polypeptides takes place by proteasomes.

4. The method according to any one of claims 1 to 3, wherein the MHC class I molecules from cellular microsomes. Cells from a patient or modified cells with a specificity for a single MHC class I molecule.

5. The method according to any one of claims 1 to 3, wherein one uses recombinant MHC class I molecules.

6. The method of claim 1, wherein the MHC molecules are MHC class II molecules.

7. The method according to claim 1 or 6, wherein the proteolytic degradation of the proteins or polypeptides by endosomal and / or lysosomal enzymes, preferably in the presence of the MHC class II molecules.

8. The method according to any one of claims 1, 6 and 7, wherein the MHC class II molecules originate from trans-Golgi vesicles, endosomes, CPLs, cells of a patient or modified cells with a specificity for a single MHC class II molecule.

9. The method according to any one of claims 1, 6 and 7, wherein recombinant MHC class II molecules are used.

10. The method according to any one of claims 1 to 9, wherein the proteins or polypeptides are of natural origin.

11. The method according to any one of claims 1 to 9, wherein the proteins or polypeptides are of recombinant or synthetic origin.

12. The method according to any one of claims 1 to 11, wherein the isolation of the MHC molecule-peptide complexes is carried out by precipitation with comformation epitope-specific anti-MHC antibodies.

13. The method according to any one of claims 1 to 12, wherein the separation of the antigenic peptides and / or peptide mixtures from the MHC molecules by elution, preferably acid elution of the peptides and / or peptide mixtures from the MHC molecules.

14. The method according to claim 13, wherein the elution is followed by a peptide purification step using HPLC, gel filtration or capillary electrophoresis.

15. The method according to any one of claims 1 to 14, wherein the separation of the antigenic peptides from the MHC molecules comprises the identification of fractions containing antigenic peptide.

16. The method according to claim 15, wherein the identification of the fractions is carried out 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.

17. The method according to claim 16, characterized in that one uses cytotoxic cells from polyclonal CTL lines or T cell clones.

18. The method according to claim 15, wherein the identification of the fractions is carried out by a T cell proliferation test.

19. The method according to any one of claims 1 to 18, wherein the amino acid sequence of the antigenic peptides is determined by sequencing using preferably automatic Edman chemistry or mass spectrometry.

20. The method according to any one of claims 1 to 19, wherein the protein or polypeptide (a) originates from a pathogen, or corresponds to a protein or polypeptide or a part thereof which originates from a pathogen, (b) a tumor antigen or is part of it, or (c) is or is a part of an autoimmune antigen.

21. The method according to any one of claims 1 to 20, wherein the antigenic peptide is an immunodominant or subdominant peptide.

22. A method for producing and identifying antigenic peptides and mixtures of peptides, comprising the following steps:

(b) fractionation of the antigenic peptides and / or peptide mixtures obtained in step (a);

(c) testing the fractions obtained 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).

23. A method for producing antigenic peptide mixtures, comprising:

(b) Obtaining the peptide mixtures.

24. The method according to any one of claims 1 to 23, characterized in that the proteolytic degradation of proteins or polypeptides is carried out in a device which comprises: a container which contains cellular processing enzymes which bring about the degradation of the proteins or polypeptides to a peptide size which permits association with MHC molecules,

the container having at least one inlet opening for the supply of proteins and polypeptides and at least one outlet opening for removing the processed peptides.

25. The method according to claim 24, characterized in that the cellular processing enzymes are immobilized on a carrier material and / or on walls of the container.

26. The method according to claim 24 or 25, characterized in that the processing enzymes are immobilized by chemical coupling or by immunoadsorption.

27. The method according to any one of claims 24 to 26, characterized in that the outlet opening of the container is designed such that it permits the passage of the processed peptides, but not the passage of the proteins or polypeptides used as the starting material and the enzymes.

28. The method according to claim 27, characterized in that the outlet opening comprises a filtration unit.

29. The method according to any one of claims 24-28, characterized in that the container is thermostable.

30. The method according to any one of claims 24-29, characterized in that the method is carried out continuously.

31. Use of an antigenic peptide or peptide mixture prepared according to one of claims 1 to 30 for the identification of vaccine components against pathogens or tumor cells or of competitive or antagonistic peptides in autoimmune diseases.

32. Use of peptides or peptide mixtures as can be obtained by the method according to any one of claims 1-30 for the preparation of a vaccine.

33. Use according to claim 32, characterized in that one uses the peptide mixtures obtained by proteolytic degradation, optionally after size fractionation, without first separating them into individual peptides.

34. Use according to claim 32 or 33, characterized in that the vaccine additionally contains an adjuvant.

35. Use according to any one of claims 32-34, characterized in that the vaccine is administered several times at intervals.

36. Use according to any one of claims 32-35, characterized in that the peptide vaccine is administered in combination with a protein or polypeptide vaccine.

37. Use according to any one of claims 32-36, characterized in that the peptide vaccine is administered by injection, orally or topically.

38. Use according to claim 37, characterized in that the topical administration is carried out by applying a plaster containing the vaccine and optionally a penetration aid.

39. Pharmaceutical composition, characterized in that it contains, as the active ingredient, peptide mixtures as obtainable by the process according to one of claims 1 to 30, optionally together with pharmaceutically customary excipients and additives.

40. Device for proteolytic degradation of proteins or polypeptides, comprising:

a container which contains cellular processing enzymes which bring about the degradation of the proteins or polypeptides to a peptide size which permits association with MHC molecules, the container having at least one inlet opening for the supply of proteins and polypeptides and at least one outlet opening for removing the processed peptides.

41. Apparatus according to claim 40, characterized in that it further has the features of one of claims 25 to 29.