|Title of Invention||
"A COMPOSITION CONTAINING ATLEAST ONE PEPTIDE, PROTEIN OR PROTEIN FRAGMENT WITH AN IMMEDIATELY ACTIVITY"
|Abstract||Pharmaceutical composition containing at least one peptide, protein or protein fragment with an immunomodulatory activity together with an adjuvant, wherein said adjuvant is polyarginine.|
|Full Text||The invention relates to the field of immuncmodulation.
The invention is a development of a therapeutic vaccine based on tumour cells. It is essentially dependent on the following conditions: there are qualitative or quantitative differences between tumour cells and normal cells; the immune system is fundamentally capable of recognising these differences; the immune system can be stimulated - by active specific immunisation with vaccines - to recognise tumour cells by means of these differences and cause them to be rejected.
In order to achieve an anti-tumour response, at least two conditions must be satisfied: firstly, the tumour cells must express antigens which do not occur on normal cells, or occur only to the extent that the immune system can distinguish qualitatively between normal and tumour tissue. Secondly, the immune system must, be activated accordingly in order to react to these antigens. A serious obstacle in the immune therapy of rumours is their low immunogenicity, particularly in humans.
Recently, tumour-associated and tumour-specific antigens have been discovered which constitute such neo-epitopes and thus ought to constitute potential targets for an attack by tne immune system. The fact that the immune system nevertheless does not succeed in eliminating the tumours which express these neo-epitopes would then obviously not be due to the absence of neo-epitopes but due to the fact that the immunological response to these neo-antigens is inadequate.
For immunotherapy of cancer on a cellular basis, two general strategies have been developed: on the one hand, adoptive immunotherapy which makes use of the in vitro
expansion cf tumour-reactive T-lymphocytes and their
reintroduction into the patient; on the other hand, active immunotherapy which uses tumour cells in the expectation that this will give rise to either new or more powerful immune responses to tumour antigens, leading to a systemic tumour response.
Tumour vaccines based on active immunotherapy have been prepared in various ways; one example consists of irradiated tumour cells mixed with immuncstimulant adjuvants such as Corynebacterium parvum or Bacillus Calmette Guerin (BCG) in order to provoke immune reactions against tumour antigens (Oettgen and Old, 1991) .
In recent years, in particular, genetically modified tumour cells have been used for active immunotherapy against cancer. A survey of these different approaches in which tumour cells are alienised for more powerful immunogenicity by the incorporation cf various genes is provided by Zatloukal et al., 1993. One cf the strategies used hitherto uses tumour cells which are genetically modified in order to produce cytokines.
The identification and isolation of tumour antigens and tumour-associated antigens (TAs) or peptides derived therefrom, (e.g. as described by Wolfel et al. , 1994 a) and 12-94 b) ; Carrel et al . , 1993, Lehmann et al . , 1989, Tibbets et al., 1993, or in the published International Applications WO 92/20356, WO 94/05304, WO 94/23031, WO 95/00159) was the prerequisite for another strategy using tumour antigens as immunogens for tumour vaccines, both in the form of proteins and in the form of peptides. However, a tumour vaccine in the form of tumour antigens as such is not sufficiently immunogenic to trigger a cellular immune response which would be necessary to eliminate tumour cells carrying tumour anticen. To ensure that antigen-presenting cells (APCs'
have defined peptide antigens on their surface it was proposed to "pulse" the peptides, but this resulted in inefficient loading of the cells with peptides (Tykocinski et al., 1996); it was also shown that the co-administration of adjuvants had only limited success in intensifying the immune response (Oettgen and Old, 1991) .
A third strategy for active immunotherapy in order to increase the efficacy of tumour vaccines is based on xenogenised (alienised) autologous tumour cells. This concept is based on the assumption that the immune system reacts to tumour cells which express a foreign protein and that, in the course of this reaction, an immune response is also provoked against those tumour antigens which are presented by the tumour cells of the vaccine.
A central role is played in the regulation of the specific immune response by a trimolecular complex consisting of the components of T-cell-antigen receptor, MHC (Major Histocompatibility Complex) molecule and the ligand thereof which is a peptide fragment derived from a protein.
MHC molecules, (or the corresponding human molecules, the HLAs) are peptide receptors which allow the binding of numerous different ligands, with stringent specificity. The prerequisite for this is provided by allele-specific peptide motifs which have the following specificity criteria: the peptides have a defined length, depending on the MHC haplotype, this length generally being from eight to ten amino acid groups in the MHC-I haplotype. Typically, two of the amino acid positions are so-called "anchors" which can only be occupied by a single amino acid or by amino acid groups with closely related side chains. The exact position of the anchor amino acids in
the peptide and the requirements made on their properties vary with the MHC-haplotypes. The C-terminus of the peptide ligands is frequently an aliphatic or a charged group. Such MHC-I-peptide-ligand motifs have hitherto been known, inter alia, for H-2Kd, Kb; Kk, Kkml, Db, HLA-A*0201, A*0205 and B*2705 haplotypes.
Within the scope of the protein conversion inside the cell, regular, degenerate and foreign gene products, e.g. viral proteins or tumour antigens, are broken down into small peptides; some of them constitute potential ligands for MHC molecules. This provides the prerequisite for their presentation by MHC-molecules and, as a result, the triggering of a cellular immune response, although it has not yet been clearly explained how the peptides are produced as MHC ligands in the cell. Foreign or alienised proteins and the fragments thereof may also be recognised, bound and eliminated by immunoglobulins which constitute the humoral immune response. This is also true of all tumour antigens: using the example of tumour associated antigens MUC1, CEA and HER2/neu it has been shown that immunoglobulins which have specificity for these proteins are able to recognise and kill the protein-carrying cells. In order to trigger a tumour antigen-specific humoral immune response, therefore, various forms of MUC1 and CEA were tried out as immunogens (e.g. in recombinant poxvectors; Bronte et al., J. Immunol. 154:5282 1995; in animal models and preliminary clinical trials.
Within the scope of the invention, some ideas were pursued which had come up in the development of a cellular tumour vaccine: whereas non-malignant normal body cells are tolerated by the immune system, the body reacts to a normal cell by means of an immune response if this cell synthesises proteins foreign to the body, e.g. as the result of a viral infection. The reason for
this is that the MHC molecules present foreign peptides which originate from the foreign proteins. Consequently, the immune system registers that something undesirable and alien has happened to this cell. APCs (these include macrophages, dendritic cells, Langerhans cells, B-cells and possibly the recently discovered biphenotypic cells which have the properties of both 3-cells and also macrophages; Tykocinski et al., 1996) are activated, a new specific immunity is generated and the cell is eliminated.
Tumour cells admittedly contain the tumour-specific tumour antigens in question but are ineffective vaccines as such, because they are ignored by the immune system as the result of their low immuncgenicity. If a tumour cell were to be charged not with a foreign protein but with a foreign peptide, in addition to the foreign peptides the cell's own tumour antigens will be recognised as foreign by this cell. By alienisation with a peptide the intention is to direct the cellular immune response triggered by the foreign peptides against the tumour antigens.
The reason for the low immunogenicity of tumour cells may be a problem not of quality but of quantity. For a peptide derived from a tumour antigen, this may mean that it is indeed presented by MHC molecules but in a concentration which is too low to trigger a cellular tumour-specific immune response. An increase in the number of tumour-specific peptides on the tumour cell should thus also result in alienisation of the tumour cell, resulting in the triggering of a cellular immune response. It has been proposed to present the tumour antigen or the peptide derived from it on the cell surface by transfecting it with a DNA coding for the protein or peptide in question, as described in International Publications WO 92/20356, WO 94/05304,
WO 94/23031 and WO 95/00159.
German Patent Application P 195 43 649.0 discloses a cellular vaccine which contains as active component tumour cells charged with one or more peptides so that the tumour cells in context with the peptides are recognised as foreign by the patient's immune system and trigger a cellular immune response. An essential feature of the peptides is that they are ligands for the MHC-haplotype of the patient. The peptides are therefore recognised as foreign by the patient's immune system because they may be, on the one hand, "foreign peptides" or "xenopeptides", i.e. they are different from peptides derived from proteins which are expressed by the patient's tumour cells. Another category of peptides is derived from tumour antigens expressed by the patient's cells. These bring about an increase in immunogenicity by the fact, chat they are present on the tumour cells of the vaccine in a concentration which is greater than the concentration of the same peptide on the patient's tumour cells.
The aim of the present invention was to provide a new pharmaceutical composition having an immunomodulatory activity, particularly a vaccine.
In furtherance of the concept of the cellular vaccines disclosed in German Patent Application P 195 43 649.0, a pharmaceutical composition has been developed within the scope of the present invention which contains pepcides having an immunomodulatory effect not in context with cells but together with an adjuvant, in order to trigger or intensify a cellular and/or humoral, preferably systemic, immune response to pathogens or an anti-tumour response or to bring about tolerance to proteins with an autoimmune activity.
The invention relates to a pharmaceutical composition containing one or more peptides with an immunomodulatory effect together with an adjuvant. The composition is characterised in that the adjuvant has the ability to increase the binding of the peptide to cells of the individual to be treated or to promote the entry of the peptide into cells and bring about an increase in the immunomodulatory effect of the peptide.
The term "immunomodulatory effect" denotes, on the one hand, the triggering or intensification of a cellular and/or humoral, preferably systemic, immune reaction. In this embodiment, the pharmaceutical composition according to the invention acts as a vaccine.
In a preferred embodiment the peptides are ligands for at least one MHC molecule expressed by the individual to be treated.
The human MHC molecules are hereinafter also referred to as HLA (Human Leucocyte Antigen) in accordance with International Conventions.
The term "cellular immune response" denotes in particular the cytotoxic T-cell immunity which, as a result of the generation of cytotoxic CD8-positive T-cells and CD4-positive helper-T-cells, brings about destruction of the tumour cells or of the cells attacked by the pathogen.
The expression "humoral immune response" denotes the production of immunoglobulins which selectively recognise tumour cells or structures derived from pathogens and consequently, together with other systems such as, for example, complement, ADCC (antibody dependent cytotoxicity) or phagocytosis, bring about the destruction of these tumour cells or the pathogenic
agents or the cells attacked by them.
The peptide contained in the vaccine is derived from an antigen against which a cellular and/or humoral immune response is to be triggered. This ensures that T-cells or other cytotoxic effector cells which recognise the disease-causing agent or the tumour cells which contain the antigen, and/or antibodies are generated.
For immunisation against pathogenic agents of disease such as bacteria, viruses and parasites, peptides are used which are derived from a protein of the pathogen or pathogens in question. Particularly suitable are proteins which are unaffected by the high general mutation rate of these pathogens. Published examples include HPV1S/17 (Human Papilloma Virus; Feltkamp et al., 1995), Hepatitis 3 Virus Core Antigen (Vitiello et al., 1995), Plasmodium Berghell \Widmann et al., 1992), influenza virus nucleoprotein and hepatitis C virus.
In one embodiment of the invention, the peptide is derived from a tumour antigen with a view to triggering an anti-tumour response and the pharmaceutical composition is used as a tumour vaccine. In this case, when the vaccine is used therapeutically, the peptide is derived from a tumour antigen which is expressed by the patient's tumour cells. These tumour antigens are, for example, those which are expressed by the patient in a concentration which is too lew, with the result that the tumour cells are not recognised as foreign.
The tumour antigens of the patient can be determined in the course of drawing up the diagncsis and treatment plan by standard methods: tumour antigens can easily be detected by immunohistochemistry using antibodies. If the tumour antigens are enzymes, e.g. tyrosinases, they can be detected by enzyme assays. In the case cf tumour
antigens with a known sequence, the RT-PCR method can be used. Boon, T., et al. , 1994; Coulie, P.G., et al. , 1994; Weynants, P., et al. , 1994. Other methods of detection are assays based on CTLs with specificity for the tumour antigen which is to be detected. These assays have been described, for example, by Herin et al., 1987; Coulie et al. , 1993; Cox et al., 1994; Rivoltini et al., 1995; Kawakami et al., 1995; and have been described in WO 94/14459; these references also disclose various tumour antigens and peptide epitopes derived therefrom. A summary of known tumour antigens and peptides derived therefrom which may be used for the purposes of the invention is provided in the Table .
A tumour vaccine containing peptides derived from a tumour antigen may be used not only therapeutically but also prophylactically. For propylactic use, it is preferable to use a mixture of peptides derived from representatives of commonly occurring tumour antigens. When the tumour vaccines according to the invention are used therapeutically, one or more peptides are used, which can be expected to be contained in tumour antigens of the patient.
The tumour vaccine according to the invention has the advantage, over a cellular vaccine based on autologous tumour cells, that it is therapeutically useful even for patients at a relatively early stage (stage I and II) of the disease, who have insufficient tumour cells to produce a cell vaccine.
In a preferred embodiment of the invention the peptide is matched to the MHC-I- or MCH-II - subtype of the patient to be vaccinated, with a view to triggering a cellular immune response; the peptide thus has a sequence or contains a sequence which ensures that it binds to an MHC-molecule.
In another embodiment, the pharmaceutical composition in its form as a tumour vaccine also contains a polypeptide with an immunostimulant effect, particularly a cytokine. In a preferred embodiment of the invention the cytokine used is interleukin 2 (IL-2) or GM-CSF, e.g. in a dosage of about 1000 units; other examples of cytokines are IL-4, IL-12, IFN-α, IFN-β, ILN-γ, IFN-ω, TNF-α and combinations thereof, e.g. IL-2 + IFN-γ, IL-2 + IL-4, IL-2 + TNF-α or TNF-α + IFN-γ.
In one embodiment of the invention the pharmaceutical composition serves to confer tolerance to proteins or the fragments thereof which trigger autoimmune-induced diseases, i.e. for the treatment of autoimmune diseases. The peptides used in this embodiment of the invention are derived from proteins which cause autoimmune diseases.
In contrast to the application of the invention as a tumour vaccine or as a vaccine against pathogenic agents in which the peptides substantially match a portion of the original protein (tumour antigen or protein of the pathogen) to the extent that the peptide is recognised as the "original antigen", when the invention is applied to the treatment of autoimmune diseases, peptides are used, inter alia, which differ from the amino acid sequence of the original protein in some crucial respects. These peptides do indeed bind to the MHC-molecule as a result of their anchor positions but they have mutations in their sequence which cause these peptides to act as antagonists which switch off the activated specific T-cells again (Kersh and Allen, 1996) .
Suitable peptide antagonists are both "natural" antagonists which were discovered in viruses (Bertoletti
et al., 1994) and also antagonists found by systematic
searching, e.g. by screening peptide libraries. Examples of peptide antagonists are peptides which are able to switch off T-cells which are specific for myelin basic protein; these were tested for their effectiveness in animal experiments (Brocke et al., 1996) .
A peptide which is supposed to trigger a cellular immune response must be capable of binding to an MHC-molecule. In order that the immune response is triggered in the patient, the individual to be treated must therefore have a corresponding HLA-molecule in their repertoire. The determination of the HLA-subtype of the patient thus constitutes one of the most important prerequisites for effective administration of a peptide to this patient, in terms of obtaining a cellular immune response.
The HLA subtype of the patient can be detected using standard methods such as the micro-lymphotoxicity test (Practical Immunol., 1989). This test is based on the principle of mixing lymphocytes isolated from the patient's blood first with antiserum or a monoclonal antibody against a specific HLA molecule in the presence of rabbit complement (C). Positive cells are lysed and absorb an indicator dye, whereas undamaged cells remain unstained.
RT-PCR can also be used to determine the HLA-I or HLA-II-haplctype of a patient (Curr. Prot . Mol. Biol. Chapters 2 and 15). In order to do this, blood is taken, from the patient and RNA is isolated from it. This RNA is subjected first to reverse transcription, resulting in the formation of cDNA from the patient . The cDNA is used as a matrix for the polymerase chain reaction with primer pairs which specifically bring about the amplification of a DNA fragment which represents a certain HLA-haplotype. If after agarose gel electrophoresis a DNA band appears, the patient
expresses the corresponding HLA molecule. If the band does not appear, the patient is negative for it.
The definition of a peptide used according to the invention by means of an HLA-molecule defines them in terms of their anchor amino acids and their length; defined anchor positions and length ensure that the peptide fits into the peptide binding fork of the HLA molecule in question. This means that the immune system will be stimulated and a cellular immune reaction will be provoked against the tumours cells of the patient, if a peptide derived from a tumour antigen is used.
Peptides which are suitable for the purposes of the present invention are available in a wide range. Their sequence may be derived from naturally occurring immunogenic proteins or the cellular breakdown products thereof, e.g. viral or bacterial peptides, or from. tumour antigens, or they may be antagonists to peptides derived from proteins which induce autoimmune diseases.
Suitable peptides may be selected, for example, on the basis of peptide sequences known from the literature.
With a view to triggering a cellular immune response, the peptides may be defined, e.g. by means of the peptides described by Rammensee et al . , 1993, Rammensee et al., 1995, Falk et al., 1991, for the different HLA motifs, peptides derived from immunogenic proteins of various origins, which fit into the binding forks of the molecules of the various HLA-subtypes. For peptides which have a partial sequence of a protein with an immunogenic activity, it is possible to establish which peptides are suitable candidates by means of the polypeptide sequences already known or possibly still to be established, by sequence comparison taking account of the HLA-specific requirements. Examples of suitable
peptides are found, for example, in Rammensee et al . ,
1993, Falk et al., 1991, and Rammensee, 1995 and in
WO 91/09869 (HIV peptides); peptides derived from tumour
antigens are described, inter alia, in the published
International Patent Applications WO 95/00159 and
WO 94/05304. Reference is hereby made to the disclosure
of these references and the Articles cited therein in
connection with peptides. Preferred candidates are the
peptides whose immunogenicity has already been
demonstrated, i.e. peptides derived from known
immunogens such as viral or bacterial proteins.
Instead of using the original peptides which fit into the binding forks of MHC-I or MKC-II molecules, i.e. peptides which are derived unchanged from natural proteins, it is possible to carry cut variations as required, using the minimum requirements regarding anchor positions and lengchs, specified on the basis of the original peptide sequence, provided that these variations not only do not impair but preferably enhance the effective immunogenicity of the peptide made up of its binding affinity to the MKC molecule and its ability to stimulate T-cell receptors. In this case, therefore, synthetic peptides are used according to the invention which are designed in accordance with the requirements relating to an MHC-I molecule. Thus, for example, starting from the H2-Kd-ligand Leu Phe Glu Ala Ile Glu Gly Phe Ile (LFEAIEGFI) it is pcssicle to change the amino acids which are not anchor amino acids in such a way as to obtain the peptide of the sequence Phe Phe Ile Gly Ala Leu Glu Glu Ile (FFIGALEEI) ,- moreover, the anchor amino acid Ile at position 9 can be replaced by Leu. The determination of epitopes of MHC-I- or MHC-II-ligands or the variation thereof may be carried out, for example, using the principle described by Rammensee et al., 1995. The length of the peptide preferably corresponds to the minimum sequence of 8 to 10 amino
acids required for binding to the MHC-I molecule, together with the necessary anchor amino acids. The MHC-II-binding motif which extends over nine amino acids has a higher degree of degeneration in the anchor positions. Methods have recently been developed, starting from X-ray structural analysis of MHC-II molecules, which permit accurate analysis of the MHC-II binding motifs and, on the basis thereof, variations in the peptide sequence (Rammensee et al. , 1995, and the original literature cited therein).
If desired, the peptide may also be lengthened at the C-and/or N-terminus provided that this lengthening does not interfere with the binding capacity of the MHC molecule, i.e. that the extended peptide can be processed at cellular level down to the minimum sequence.
In one embodiment of the invention the peptide may be extended with negatively charged amino acids, or negatively charged amino acids may be incorporated in the peptide, at positions other than the anchor amino acids, in order to achieve electrostatic binding of the peptide tc a polycationic adjuvant such as polylysine.
The term "peptides" for the purposes of the present invention also includes by definition larger protein fragments or whole proteins which are guaranteed to be processed, after the application of the APCs, into peptides which fit the MHC-molecule.
In this embodiment of the invention the antigens used not in the form of a peptide but as a protein or protein fragment or as a mixture of proteins or protein fragments. The protein is an antigen or tumour antigen from which the fragments obtained after processing are derived. In this embodiment, the adjuvant serves to
enable or enhance the charging of cells, particularly APCs such as dendritic cells or macrophages, with the tumour antigen or fragments. Proteins or protein fragments thus absorbed are processed by the cells and can then be presented to the immune effector cells in the MHC context and thus trigger or intensify an immune response (Braciale and Braciale, 1991; Kovacsovics Bankowski and Rock, 1995; York and Rock, 1996).
The embodiment of the invention in which proteins or larger protein fragmencs are used as antigens has the advantage that there is less dependency on the HLA-type of the patient, as the protein is processed into a number of fragments and there is hence greater variability as to the "fitting form" of the peptides.
If proteins or protein fragments are administered, the identity of the processed end product can be demonstrated by chemical analysis (Edman degradation or mass spectrometry of processed fragments; cf . the summarising article by Rammensee et al. , 1995 and the original literature cited therein) or by biological assays (ability of the APCs to stimulate T-cells which are specific for the processed fragments).
In principle, peptide candidates which are suitable for producing a cellular immune response are selected in several stages: generally, the candidates are first tested in a peptide binding test for their binding capacity to an MHC molecule, preferably by series of tests.
One suitable method of investigation is based on the ability of peptides to stabilise empty MHC-molecules, as described for example by Stuber et al. , 1994 and McIntyre et al., 1996. The peptide is applied to cells
which are capable of expressing the MHC-molecule in
question but which do not bind any endogenous peptides in the MHC-context because of a genetic defect. Suitable cell lines of this type are RMA-S (mouse) and T2 (human) and the transfected variants thereof. Then only the MHC-molecules stabilised by the peptide in question are detectable, preferably by means of the FACS analysis based on flow cytometry (Flow Cytometry, 1989; FACS Vantage TM User's Guide, 1994; CELL Quest ™ User's Guide, 1394) . Stable MHC molecules are detected with a suitable anti-MHC antibody and with a second (e.g. polyclonal) antibody marked with a fluorescent dye, e.g. with FITC (fluorescein isothiocyanate). In the flow, individual cells are excited by a laser of a certain wavelength; the fluorescence emitted is measured and is dependent on the quantity of peptide bound to the cell.
Another method of determining the quantity of peptide bound is the Scatchard blot, as described by Sette et al . , 1994. Peptide labelled with I125, for example, is used for this and incubated overnight with isolated or recombinantly produced MHC molecules at 4°C with various defined concentrations of peptide. In order to determine non-specific interaction of the peptide, an excess of unlabelled peptide is added to some of the samples, preventing the non-specific interaction of the labelled peptide. Then the non-specificaily bound peptide is removed, e.g. by gel chromatography. The quantity of bound peptide is then determined in a scintillation counter using the radio-activity emitted. The data thus obtained are evaluated using standard methods.
A summary of methods of characterising the MHC/peptide interaction, the analysis of MHC-ligands and peptide-binding assays which may be used within the scope of the present invention is provided by Rammensee et al., 1995.
In a second step, peptide candidates with good binding qualities are tested for their immunogenicity:
The triggering of a cellular immune response can be confirmed by detecting peptide-specific CTLs (Current Protocols in Immunology, Chapter 3). Another indication of the presence of a cellular immune response is provided when, in the absence of T-cells, there is no immune response in an experimental animal (which is achieved by treating the animal with antibodies which deplete the CD4- or CD8-cells) (Current Protocols in Immunology, Chapter 3) .
A cellular immune response can also be demonstrated by detecting a "delayed-type hypersensitivity" (DTH) reaction in immunised animals. For this purpose, peptides are injected into the sole of the paw in mice and the swelling at the injected site is measured (Grohman et al., 1995; Puccetti et al., 1994).
The induction of a humoral immune response by peptides which are foreign antigens to the organism or antigens expressed in low concentrations by the organism to be treated, can be determined by detecting specific antibodies in the serum. A suitable method cf detecting the antibody level in the serum is enzyme-linked immuncassay (ELISA). The specific antibodies are detected, after binding to the peptide used for immunisation, by means of a staining reaction. An alternative method is Western blot. In this, specific serum antibodies bind to the peptide immobilised on a membrane. Bound antibodies are finally detected again with a staining reaction (reference for both methods: Current Protocols in Immunology. Editors: Coligan et al. , 1991) .
Particularly after vaccination with foreign antigens,
e.g. of viral origin, the formation of antibodies can be expected. However, it cannot be ruled out that specific antibodies may also be formed against mutated or over-expressed peptides derived from cellular tumour antigens. Tumour destruction by such antibodies might take place after antibody binding to tumour cells by other components of the immune system such as, for example, complement, antibody-dependent cytotoxicity (ADCC) or phagocytosis by macrophages (Roitt I.M., Brostoff J., Male D.K. Immunology, Churchill Livingstone).
The triggering of a cellular immune response by peptides which are derived from proteins whose immunogenic activity is unknown may be tested, for example, as described by Rivoltini et al., 1995 or Kawakami et al., 1994a. For this, T-cells are needed which are able to recognise the desired peptide when it is presented by MHC-molecules. In the case of peptides which originate from tumour cells, the corresponding T-cells are obtained from the tumour-infiltrating lymphocytes (TILs) as described by Kawakami et al., 1994b; in the case of foreign peptides, T-cells of this kind are obtained analogously from the peripheral blood. The T-cells are incubated with cell lines such as T2 (Alexander et al . , 1989) or RMA-S (Karre et al., 1986) which have been mixed with the peptide in question, and they lyse them if it is an immunogenic peptide.
Another possible way of testing MHC-binding peptide candidates for their immunogenicity consists in investigating the binding of the peptides to T2 cells. One such test is based on the peculiar nature of T2 cells (Alexander et al., 1989) or RMA-S-cells (Karre et al., 1986) that they are defective in the TAP peptide transporting mechanism and only present stable MHC-molecules when they are applied to peptides which are
presented in the MHC context. T2 cells or RMA-S cells stably transfected with an HLA gene, e.g. with HLA-A1 and/or HLA-A2 genes, are used for the test. If the cells are mixed with peptides which are good HLA ligands, by being presented in the HLA context in such a way as to be recognised as foreign by the immune system, these peptides cause the HLA molecules to appear in significant quantities on the cell surface. Detection of the HLAs on the cell surface, e.g. by means of monoclonal antibodies, makes it possible to identify suitable peptides (Malnati et al., 1995; Sykulev et al., 1994). Here again, a standard peptide known to have a good HLA-binding capacity is appropriately used.
With a view to the broadest possible application of the pharmaceutical composition according to the invention it is preferable to use a mixture of several peptides, each of which is able to bind to another MHC-molecule, preferably to one of two or three of the most commonly occurring MHC-subtypes. A vaccine based on a mixture of peptides which can bind to these haplotypes can be used to cover a wide population of patients.
In one embodiment of the invention, the vaccine may have a number of peptides with different sequences. In this case, the peptides used may differ from one another, on the one hand, in that they bind to different HLA subtypes. In this way, it is possible to detect several or all the HLA subtypes of a patient or of a larger group of patients.
Another, possibly additional, variability with regard to the peptides used may consist in the fact that peptides which bind to a certain HLA subtype differ in their sequence which is not crucial to HLA binding, being derived, for example, from different proteins of the same pathogenic agent or from different pathogens.
Variability of this kind can be expected to intensify the stimulation of the immune response or to confer immunity against a variety of pathogens.
The quantity of effective peptide in the composition according to the invention may vary over a wide range. The quantity of peptide depends, inter alia, on the method of administration and the particular formulation used. The quantity of peptide to be administered may be about 1.0 µg to about 5000 µg per dose, generally 1.0 µg to about 1000 µg, particularly about 10 µg to about 500 µg. It may be administered once or several times and, if administered several times, preferably at least three times. For therapeutic use, in particular, the peptide may be administered at intervals (e.g. once a week to once a month) over any desired length of time as determined by the specific immune status of the patient or the progress of the disease.
The pharmaceutical composition according to the invention may also be used ex vivo: the principle of possible ex vivo administration consists in cultivating APCs, e.g. dendritic cells, ex vivo, incubating the cell culture with the composition according to the invention and administering the APCs, which now present the peptide in an MHC-context, to the individual who is to be treated. Methods known from the literature may be used for this type of application, as described, for example, by Porgador and Gilbca, 1995; Young and Inabe, 1996 .
The adjuvant contained in the composition according to the invention has the property of aiding the entrance of the peptide into the cells or binding the peptide to the cells of the patient. The adjuvant may, for example, make the membranes of target cells into which the peptide is supposed to penetrate pervious for at least a
short time in order to allow the peptide to be conveyed into the cell in this way. It would be advantageous, but not absolutely necessary, for the peptide to be bound to the adjuvant, e.g. by electrostatic interaction between electronegative peptides and polycationic adjuvant. Entry of the peptide into the cell can also be achieved by the fact that the peptide is able to pass through the cell membrane on the grounds of its spatial closeness thereto, as soon as the adjuvant has made it permeable. The effect of the adjuvant may also be based on the fact that it increases the concentration of the peptide on the cell surface which is critical to its absorption into the cell or that it brings about phagocytosis or liquid transport (pinocytosis) of the peptide into the cell.
Surprisingly, the presence of the adjuvant not only increases the uptaKe of the peptide into the cell but also results in a potentiation of the immunomodulatory effect of the peptide, which would appear to be due to correct presentation of the peptide by MHC-molecules.
In one embodiment, adjuvants, inter alia, may theoretically all be the membrane-permeabilising substances which are used for transporting nucleic acids into the cell; in connection with this, reference is made to WO 93/13768, which mentions such substances.
In a preferred embodiment of the invention, the adjuvant is a basic polyamino acid or a mixture of basic polyamino acids.
The degree of polymerisation of the polyamino acids may vary over a wide range. It may be about 5 to about 1000, more particularly about 15 to 500.
Preferably, polyarginine is used as the adjuvant within
the scope of the present invention.
Another preferred adjuvant for the purposes of this invention is polylysine.
Examples of other suitable, particularly polycationic, organic compounds (basic polyamino acids) are polyornithine, histones, protamines, polyethyleneimines or mixtures thereof.
The adjuvant is optionally conjugated with a cellular ligand (e.g. with transferrin, gp120; LDL (Low Density Lipoprotein), α-fetuin, EGF (Epidermal Growth Factor) peptides or with a representative of other cellular ligands which have been described for the transportation of DNA by means of receptor-mediated endocytosis in WO 93/07283), carbohydrate residues such as mannose or fuccse (ligands for macrophages , or antibodies or antibody fragments against cell surface proteins.
Optionally, polycationic adjuvants such as polylysine or polyarginine, which are optionally conjugated with a cellular ligand, occur as the constituents of a complex with DNA, e.g. in the form of plasmid DNA which is free from sequences coding for functional peptides.
Without wishing to be tied to the theory, the effect of the pharmaceutical composition according to the invention would appear to consist in the fact that the peptide penetrates into the target cells with the help of the adjuvant or binds to cells which occur in the endodermal region of the skin. Target cells include, for example, antigen-presenting cells by which the peptide, optionally after processing, is presented to the B- and/or T-cells. Examples of target cells are macrophages, fibroblasts, keratinocytes, Langerhans cells, dendritic cells or B-cells.
Within the scope of the present invention, investigations were carried out to find out whether small peptides are absorbed by a greater degree by macrophage-like antigen-presenting cells (APCs) in the presence of basic polyamino acids or glycosylated forms of polycation. Regarding the sugar residues used, it is known that they are absorbed by macrophages using receptor mediated endocytosis. As for APCs, it is assumed that in vivo they constitute the type of cell which absorbs the peptides and presents other immune cells. Results of in vitro tests which show that APCs endocytose increased quantities of peptide antigens in the presence of the adjuvants tested are an indication that these adjuvants are also suitable, in vivo, for potentiating the presentation of the peptides to the cytotoxic effector cells and the activation thereof, leading to an overall more powerful immune response to the target contained in the vaccine.
The adjuvants used may also be components in particle form, optionally in addition to the adjuvants mentioned above. The particles may theoretically be any materials which are also used to produce column material for peptide synthesis, e.g. silica gel or synthetic resins, provided that they are physiologically acceptable and particles can be produced from them which are small enough to enter the cells. Using adjuvants in particle form it is possible to achieve high local concentrations of peptide, making it easier for it to be absorbed into the cells .
The type of adjuvant used, the suitability of modification thereof with a cellular ligand or the addition of DNA and the necessary quantity of adjuvant in relation to peptide may be determined empirically, e.g. the particular ratio of peptide to adjuvant chosen, which may theoretically vary over a wide range, can be
determined by titration.
Adjuvants may in theory be tested by the same methods as the peptides, optionally in a number of steps:
The ability of an adjuvant to increase the binding and/or internalisation of a peptide to APCs may be measured, for example, in a first step by incubating APCs with fluorescent-labelled peptides and adjuvant. An increased uptake or binding brought about by the adjuvant can be determined by throughflow cytometry by comparison with cells mixed with peptide on its own.
In a second step the adjuvants to be tested can be investigated in vitro to see whether and to what extent their presence results in presentation of a peptide to APCs, and the method used for testing peptides above may be used to measure the MHC-concentration on the cells.
Another possible way of testing the efficiency of an adjuvant is to use an in vitro model system. Here, APCs are incubated together with adjuvant and peptide and the relative activation of a T-cell clone which specifically recognises the peptide used is measured (Coligan et al . , 1991; Lopez et al., 1993).
The efficiency of the formulation may also be demonstrated by means of the cellular immune response by demonstrating a delayed-type hypersensitivity (DTH) reaction in immunised animals.
Finally, the immunomodulatory effect of the formulation is measured in animal trials. Established tumour models may be used, with peptide sequences recognised by immune cells. The vaccine containing peptide and adjuvant is administered in varying proportions with regard to the amount of peptide to adjuvant and the total amount. The
protection from tumour growth is a measurement of the effectiveness of a tumour vaccine.
The pharmaceutical composition may be administered parenterally, topically, orally or locally. It is preferably given by parenteral, e.g. subcutaneous, intradermal or intramuscular route, preferably by subcutaneous or intradermal route, in order to reach skin cells in particular (keratinocytes, fibroblasts), dendritic cells, Langerhans cells or macrophages as the target cells. Within the scope of tumour therapy the tumour vaccine may also be administered by intratumoral route.
The composition according to the invention for parenteral administration is generally in the form of a solution or suspension of the peptide and adjuvant in a pharmaceutically acceptable carrier preferably an aqueous carrier. Examples of aqueous carriers which may be used include water, buffered water, saline solution (0.4%), glycine solution (0.3%), hyaluronic acid and similar known carriers. Apart from aqueous carriers it is also possible to use solvents such as dimethylsulphoxide, propyleneglycol, dimethylformamide and mixtures thereof. The composition may also contain pharmaceutically acceptable excipients such as buffer substances and organic salts in order to achieve normal osmotic pressure and/or effective lyophilisation. Examples of such additives are sodium and potassium salts, e.g. chlorides and phosphates, sucrose, glucose, protein hydrolysates, dextran, polyvinylpyrrolidone or polyethyleneglycol. The compositions may be sterilised by conventional methods, e.g. by sterile filtration. The composition may be decanted directly in this form or lyophilised and mixed with a sterile solution before use.
In one embodiment, the pharmaceutical composition according to the invention is in the form of a topical formulation, e.g. for dermal or transdermal application. The pharmaceutical composition may, for example, take the form of hydrogel based on polyacrylic acid or polyacrylamide (such as Dolobene®, Merckle), as an ointment, e.g. with polyethyleneglycol (PEG) as the base, like the standard ointment DAB 8 (50% PEG 300, 50% PEG 1500), or as an emulsion, especially a microemulsion based on water-in-oil or oil-in-water, optionally with added liposomes. Suitable permeation accelerators (entraining agents) include sulphoxide derivatives such as dimethylsulphoxide (DMSO) or decylmethylsulphoxide (decyl-MSO) and transcutol (diethyleneglycolmonoethyl-ether) or cyclodextrin, as well as pyrrolidones, e.g. 2-pyrrolidone, N-methyl-2-pyrrolidcne, 2-pyrrolidcne-5-carboxylic acid or the biodegradable N-(2-hydrcxyethyl)-2-pyrrolidcne and the fattv acdd esters thereof, urea derivatives such as dodecylurea, 1,3-didodecylurea and 1,3-diphenylurea, terpenes, e.g. D-limonene, menthone, a-terpinol, carvol, limonene oxide or 1,8-cineol.
Other formulations are aerosols, e.g. for administering as a nasal spray or for inhalation.
The composition according to the invention may also be administered by means of liposomes which may take the form of emulsions, foams, micelles, insoluble monolayers, phospholipid dispersions, lamella layers and the like. These act as carriers for conveying the peptides to their target of a certain tissue, e.g. lymphoid tissue or tumour tissue or to increase the half-life of the peptides.
If the composition according to the invention is in the form of a topical formulation it may also contain UV-absorbers in order to act, for example, as a sun
protection cream, for example, when the formulation is used prophylactically against melanoma.
The person skilled in the art will find suitable formulations and adjuvants in standard works such as "Remington's Pharmaceutical Sciences", 1990.
Summary of Figures
Fig. 1: Vaccination of DBA/2 mice against mastocytoma
P815 Fig. 2: Vaccination of DBA/2 mice against mastocytoma
P815 using a single peptide vaccine Fig. 3: Vaccination of DBA/2 mice against melamona M-3
with a mixture of peptides Fig. 4: Vaccination of DBA/2 mice against melamona M-3
metastases Fig. 5 : Testing the potentiation of the binding of
peptides to APCs by basic polyamino acids Fig. 6: Testing the permeabilisation of the cell
membrane by basic polyamino acids Fig. 7: Testing the internalisation of peptides by
basic polyamino acids
In the Examples which follow, the following materials and methods were used unless otherwise stated:
A) Cell lines
The murine melanoma cell line Cloudman S91 (clone M-3; ATCC No. CCL 53.1), the mastocytoma cell line P815 (ATCC No. TIB 64)' and the monocyte macrophage cell line P388D1 (ATCC TIB 63) were obtained from ATCC.
B) Peptide synthesis
The peptides were synthesised in a peptide synthesiser
(Model 433 A with feedback monitor, Applied Biosystems, Foster City, Canada) using TentaGel S PHB (Rapp, Tubingen) as a solid phase using the Fmoc method (HBTU
activation, Fastmoc™, scale 0:25 mmol). The peptides were dissolved in 1 M TEAA, pH 7.3, and purified by reverse chromatography on a Vydac C 18 column. The sequences were confirmed by flight time mass spectrometry on an MAT Lasermat (Finnigan, San Jose, Canada).
C) List of the peptides used (Table Removed)
Peptide mixture I for M-3 melanoma vaccine: kpep!43, kpep!45, kpep!46, kpep!50.
Peptide mixture III for mastocytoma P815 vaccine kpepl!7, kpep!88, Kpepl62, Kpepl63, Kpepl64
Dl) Individual peptide vaccine
a) Individual peptide control vaccine without adjuvant
was prepared by taking up the peptide in a
concentration of 1 mg/ml in PBS. The incubation
period up to injection was 4 hours at ambient
b) Individual peptide vaccines with polylysine as
adjuvant were prepared by mixing peptide and
polylysine in the specified amounts in HBS. The
incubation time up to injection was 4 hours at
i) In order to obtain a vaccine containing 16 ug of
effective peptide, 11.3 µ9 of cclylysine were mixed with 160 µg of peptide kpepll7 in a total volume cf 1 ml of HBS.
ii) In order to obtain a vaccine containing 110 µg of effective peptide, 74 µg of polylysine were mixed with 1 mg of peptide kpepll7 in a total volume of 1 ml HBS.
c) Individual peptide control vaccines with incomplete
Freund's adjuvant (IFA) were prepared by
emulsifying peptide and IFA in the amounts
specified. The incubation time up to injection was
30 minutes at ambient temperature.
i) For a control vaccine containing 16 µg of active
peptide, 192 µg of peptide kpep117 were emulsified in 600 µl of HBS with 600 µ1 of IFA.
ii) For a control vaccine containing 100 µg of
effective peptide, 1.2 mg of peptide kpepll7 were emulsified in 600 µl of HBS with 600 µl of IFA.
D2) Peptide mixtures as vaccines
a) Peptide mixture I as a control vaccine without
adjuvant contained 250 µg of each of the peptides
kpep143, kpep145, kpep146 and kpepl50 in a total
volume of 1 ml PBS.
b) Peptide mixture III as a control vaccine without
adjuvant contained 250 µg of each of the peptides
kpepll7, kpepll8, Kpepl62, Kpepl63 and Kpepl64 in a
total volume of 1 ml of PBS.
c) Peptide mixture I as a vaccine with polylysine as
adjuvant was prepared by mixing 1 mg of peptide
mixture I (containing 250 µg of each peptide) with
74 µg of polylysine in HBS. The incubation period
up to injection was 4 hours at ambient temperature.
d) Peptide mixture III as a vaccine with polylysine as
adjuvant was prepared by mixing 1.25 mg of peptide
mixture III (containing 250 µg of each peptide)
with 93 µg of polylysine in HBS. The incubation period up to injection was 4 hours at ambient temperature.
e) Peptide mixture I as a control vaccine with
incomplete Freund's adjuvant was prepared by
emulsifying 1.2 me of peptide mixture I in 600 µl
of HBS (containing 300 µg of each peptide) with
600 µl of IFA. The incubation time up to injection
was 30 minutes at ambient temperature.
f) Peptide mixture III as control vaccine with
incomplete Freund's adjuvant was prepared by
emulsifying 1.5 mg of peptide mixture III in 600 µl
of HBS (containing 300 µg of each peptide) with
600 µl of IFA. The incubation time up to injection
was 30 minutes at ambient temperature.
g) For topical application with polylysine as
adjuvant, 1 mg of peptide mixture I (containing 250 µg of each peptide) was incubated with 74 µg of polylysine for 4 hours in a total volume of 400 µl of HBS. The mixture obtained was stirred into 1.6 g of the hydrogel DOLOBENE (Merckle).
h) For topical administration of a control vaccine without an adjuvant, 1 mg of peptide mixture I (containing 250 µg of each peptide) in a total volume of 200 µl of HBS was stirred into 1.8 g of the hydrogel DOLOBENE (Merckle) .
i) The preparation of fucose-coupled polylysine (chain length: 240) was carried out using the method described by MacBroom et al., 1992, achieving a substitution of about 40%.
j) When transferrin/polylysine conjugates (prepared as described in WO 93/07283) were used, the quantity was adjusted so that the absolute quantity of polylysine was 75 µg per mg of peptide. When plasmid DNA (empty plasmid pSP65, LPS-free, Boehringer Mannheim) was integrated in the complexes, the ratio was 37.5 µg of DNA/75 µg of polylysine/1 µg of peptide. When 160 µg were used instead of 1 mg of peptide, the quantities of the other components were reduced by the same factor (6.25) .
E) Injection of the vaccines
Before the subcutaneous injection the mice were anaesthetised in an isolated air chamber in groups of up to eight animals. After 3.5 minutes of
halothan treatment (4% in 02, flow rate 4) the mice were anaesthetised for about 1 minute; during this time the vaccines were injected subcutaneously.
The intraperitoneal injection was given without any-previous anaesthetic. The volume of the injection was 100 µl of each vaccine per animal, corresponding to 100 µg of individual peptide or peptide mixture I per animal. In the case of peptide mixture III, the total amount of peptide administered to each mouse was 125 µg.
F) Topical application of the vaccine
For each mouse, 200 mg of ointment containing 100 µg of peptide or peptide mixture I or 125 µg of peptide mixture I was rubbed into the skin of the shaved animals, all over their back and in their ears. The correct quantity was monitored using scales.
G) Use of the vaccine against tumour growth in the mouse model
The procedure for testing the efficacy of the cancer vaccine in the prophylactic or therapeutic mouse model corresponded to the principle described in WO S4/21808, unless otherwise specified, using the DBA/2 model as the mouse model.
The present invention relates to a Pharmaceutical composition containing at least one peptide, protein or protein fragment with an immunomodulatory activity together with an adjuvant, wherein said adjuvant is polyarginine.
Vaccination of DBA/2 mice against mastocytomas P815
160 µg of the peptide of sequence KYQAVTTTL (kpepll8) derived from the tumour antigen P815 described by Lethe et al . , 1992, a ligand of H2-Kd, was mixed with 11.8µg
- 34 -
of polylysine 300 in 500 µ1 of HBS and incubated for 4 hours at ambient temperature. Then 500 µl of BBSS (Earl's buffered saline solution) were added. 100 µ1
portions of the resulting mixture were administered subcutaneously to 8 mice at intervals of one week. After this pre-immunisation, tumours were set after another week, by injecting each mouse contralaterally with 5 x 104 cells of the mastocytoma cell line P815 (ATCC No. TIB 64; these cells express the tumour antigen from which the peptide P815 is derived) in 100 µl of EBSS. The results of these tests are shown in Fig. 1 (filled-in squares).
In a parallel trial, 200 µg of the peptide were mixed with 500 µl of HBS and then emulsified with 500 µl of Freund's adjuvant. 8 mice were pre- immunised with 100 µl of the resulting emulsion and then tumours were set with P815 cells as described above (Fig. 1: filled-in circles).
For another parallel experiment, a cellular tumour vaccine was prepared as fellows:
160 µg of peptide kpep 118 were mixed with 3 µg of transferrin-polylysine (TfpL) , 10 µg of pL and 6 µg of pSP65 (LPS free) in 500 µl of HBS buffer. After 30 minutes at ambient temperature the above solution was added to a T 75 cell culture flask with 1.5 x 106 cells of the allogenic fibroblast cell line NIH3T3 (ATCC No. CRL 1658) in 20 ml of DMEM medium (10% FCS, 20 mM glucose) and incubated at 37°C. After 3 hours, the cells were mixed with 15 ml of fresh medium and incubated overnight at 37°C with 5% C02. 4 hours before administration, the cells were irradiated with 20 Gy. The vaccine was prepared as described in WO 94/21808 . The pre-immunisation with this vaccine was carried out at intervals of one week with 10 5 cells; after another
week, the tumours were set as described above (Fig. 1: filled-in triangles). It was found that the vaccine containing the peptide combined with polylysine offered best protection for the mice against tumour formation.
Vaccination of DBA/2 mice against mastocytoma P815 with a single peptide vaccine
Three single peptide vaccines containing either peptide kpepll7 on its own in PBS (Fig. 2a), peptide kpepll7 emulsified in IFA (Fig. 2b) or peptide kpepll7 with polylysine (chain length: 240) as adjuvant (Fig. 2c) were tested for their protective effect against a P815 tumour challenge. The vaccines were prepared as described in Section D above. The injection volume was 100 µl in each case; the injection was given subcutaneously (sc) or intraperitoneally (ip). Naive mice were used as a negative control, a whole cell vaccine consisting of GM-CSF secreting P815 cells was the positive control (P815-GM-CSF; 105 cells in 100 µl were injected subcutanecusly into each animal). Each experimental group consisted of eight animals and three vaccinations were carried out at seven day intervals. One week after the last vaccination the animals were given a contralateral tumour challenge with 5 x 104 P815 cells. The animals were inspected daily and the appearance of any tumours was monitored at weekly intervals.
Peptide kpepll7 with polylysine as adjuvant produced the best antitumour effect when 100 µg were injected subcutaneously into each animal (three of the eight animals were protected). This effect was approximately as good as the one achieved with the whole cell vaccine (four out of eight animals protected) . 16 µg of peptide together with polylysine per animal was less effective
(two animals protected), but significantly better than 100 µg of peptide in PBS (Fig. 2a, no protective effect). Also when emulsified in IFA the peptide did not achieve the activity which it achieved together with polylysine (Fig. 2c).
Vaccination of DBA/2 mice against mastocytoma P815 with a tumour vaccine containing P815 single peptide or mixtures of P815 peptides
The following peptides were used to prepare the vaccine: kpepllS (100 µg per injection)
Peptide mixture III (kpepl17, kpepll8, kpep1627 kpep163, Kpep164) : this peptide mixture contains all the P815 peptides known hitherto; 25 µg of each peptide were administered in each injection).
GM-CSF secreting P815 cells were used as the positive control.
In preliminary tests, kpepll7 proved to be the peptide with the best protective effect against P815 tumour setting when 100 µg of peptide were used together with polylysine (7.5 µg of pciylysine/100 µg of peptide, corresponding to the standard ratio; polylysine: chain length = 200). A smaller amount (16 µg) of kpepll7 had been less effective. In this example, 100 µg of kpepll8 were injected into each animal, on one occasion only with pclylysine (group B), then with transferrin polylysine (group C) and again with transferrin polylysine/DNA (group D). kpepll8 with IFA was used as control. In this experiment, kpepll8 on its own showed no protective effect against tumour setting.
In the experiments carried out in Example 4, it was shown that a vaccine containing a peptide mixture of melanoma peptides had a protective effect against melanoma. Therefore, this example was used to test whether the concept of the peptide mixture is also suitable for P815.
The peptide mixture III was administered once with only
polylysine (group E), once with transferrin polylysine (group F) and once with transferrin-polylysine/DNA (group G). Peptide mixture III in IFA was used as the
control. Naive mice were used as the negative control;
GM-CSF-transfected P815 cells were used as the positive
control (105 ceils per mouse).
The experiments carried out in this Example proved tc be rather untypical, compared with the other experiments: in the positive control group (GM-CSF secreting cells) all the animals developed tumours shortly after tumour setting, and the majority of these tumours disappeared just as quickly as they had formed. One possible explanation for this is that the tumour grew for a while before it was destroyed. A second possible explanation would be that the swelling diagnosed as a tumour did net originate from tumour growth but was the result of a powerful immune cell infiltration (granulcma). Since the animals were not dissected the reason could not be established definitively; in any case, the tumours produced were finally destroyed. Another interesting result was obtained in group G in which the animals were treated with a combination of peptide mixture III and polylysine. All the animals developed tumours, but in two of the animals the size of the swelling of the tumour (or the immune cell infiltration) was relatively small, did not increase, and the mice did not look unhealthy. These two animals were not killed but were kept under observation. Surprisingly, the tumours were
undetectable nine weeks after tumour setting, a result which had not been observed before. Finally, two out of eight animals destroyed their tumours. The destruction of the tumours would appear to be a result of the content of kpepll7 in the peptide mixture, which would be analogous to Example 2, in which two out of eight animals were protected with 16 µg of kpepll7 and three out of eight animals were protected with 100 µg of kpepll7. However, the protective effect might also have been produced by more than one peptide in the mixture.
Protection of DBA/2 mice against melanoma M-3 by pre-immunisation with a tumour vaccine containing a mixture of peptides
A prophylactic vaccine was used containing a mixture of melanoma peptides (peptide mixture I, paragraph D2)
The procedure for pre-immunisation with the vaccine and the setting of the tumours corresponded to the procedure described in Example 2, except that the tumour setting was carried out with M3 cells (105 cells per animal) . The control vaccine used was whole cell vaccine from M-3 cells which secrete IL-2 and prepared as described by Schmidt et al. 1995. Under the test conditions used, this vaccine achieved 100% protection (Figs. 3a - c) .
Fig. 3 illustrates the protective effect of the peptide vaccines with polylysine as adjuvant; 50% of the treated mice were protected from the M-3 tumour challenge. This effect could be achieved if the peptide-polylysine vaccine was injected subcutaneously or applied to the skin as a hydrogel (Fig. 3a and b). No protection was obtained under the test conditions chosen if the peptide vaccine was administered by intraperitoneal route with
polylysine as adjuvant. All the mice developed tumours, although with a time delay, compared with animals in the naive control group which were not treated before the
tumour challenge. The same marginal effect was observed when the peptide mixture I was used as an IFA vaccine. There was merely a time delay in the appearance of the tumours, which had occurred in all the animals after six weeks, both in the group treated subcutaneously and in the group treated by intraperitoneal route (Fig. 3c).
Protection of DBA/2 mice against M-3 metastases
A therapeutic vaccine was used containing a mixture of melanoma peptides (peptide mixture 1 described in paragraph D2). Three vaccinations were given at intervals of one week. The first vaccination was given five days after the setting of the metastases and consequently vaccination was given against a five day metastasis. 1.2 x 104 M-3 cells were injected for the metastasis setting, using the procedure described in WO 94/21808 and by Schmidt et al., 1996.
The vaccine used was peptide mixture I: without the adjuvant (pepmixl PBS), with IFA as adjuvant (IFA pepmixl) or with fucose-modified polylysine (fpL pepmixl) . The control groups were giver, no vaccine (naive) or the M-3 whole cell vaccine producing IL-2 mentioned in Example 4, paragraph 2. Fig. 4 shows that the best protection was achieved in the group treated with peptide mixture 1 with fucose-modified polylysine as adjuvant (Fig. 4a). 50% of the mice were able to reject the metastases (4/8). This treatment was even more effective than the one with the whole cell vaccine which protected only 33% of the mice (3/9) . The peptide vaccines with IFA or without adjuvant only resulted in a
delay in the growth of the metastases into a tumour (Fig. 4b).
Testing of various basic polyamino acids for their ability to potentiate the internalising and/or binding of peptides to APCs
For these tests a fluorescence assay was used: a model peptide antigen of the sequence LFEAIEGFI (MHC Kd-restricted) was labelled with the fluorescent dye fluorescein isothiocyanate (FITC) in accordance with the manufacturer's instructions (Molecular Probes). The uptake or binding of FITC-labelled peptide on its own ("pulsed") or together with various concentrations of basic amino acids (polylysine with a chain length of 16 to 490, polyarginine with a chain length of 15 to 720) by the MHC Kd-restricted monocyte macrophage cell line P388D1 was measured by throughflow cytometry. In order to do this, 1 x 106 P388D1 cells were incubated in a final volume of 1 ml of medium (DMEM/10% FCS) in a centrifugal test tube with 5 µg of FITC-labelled peptide on its own or with a mixture of peptide and polyamino acid for 30 minutes at 37°C and then washed thoroughly to eliminate any free peptide. The polyamino acids were added in a concencration of 50, 25, 12, 6 and 3 µg per ml of medium, containing 5 µg of FITC-labelled peptide. The relative fluorescence intensity of the various samples was compared in order to assess the efficiency of uptake and/or binding of the peptide. The results of these tests are shown in Fig. 5; the tests were carried out using 25 µg of pL450 and pArg450, respectively. Under the conditions used, polyarginine was found to be about five times more efficient than polylysine.
Peptides can be absorbed by APCs by means of specific
mechanisms such as macropinocytosis or receptor-mediated endocytosis (Lanzavecchia, 1996) . An alternative mechanism may consist in the polyamino acids making the cell membrane permeable and in this way allowing peptides to diffuse from the medium into the cytoplasm. The possible internalisation of FITC-labelled peptides in the presence or absence of basic polyamino acids was investigated on the basis of the principle published by Avrameas et al., 1996: particles internalised by cells are transported in endosomes. Compared with the cytoplasm or cell culture medium which have neutral pH values, these organelles with a pH of about 5 are acidic . The fluorescence emitted by FITC is strongly pH-dependent. In an environment with pH conditions such as those found in endosomes, fluorescence is suppressed with a factor of about 3 to 5. Therefore, FITC-labelled peptides which are absorbed by the cells into the endosomes show reduced fluorescence. When monesin is added, the low pH of the endosomes is neutralised, leading to a measurably greater fluorescence of the internalised FITC-labelled peptides.
The possible permeabilisation of the cell membrane was tested by measuring the release of the cytoplasmic enzyme lactate dehydrogenase (LDH) after incubation of P388D1 cells with polyamino acids (polylysine or polyarginine) under isotcnic conditions, using the commercially obtainable kit (Cytotox 96, Promega, Madison, Wisconsin, USA) in accordance with the manufacturer's instructions. It was found that the incubation of APCs with certain basic polyamino acids such as polylysine (pLys) and polyarginine (pArg) increases the uptake or binding of the peptides to APCs. (In the test carried out, pArg was found to be about five times more efficient than pLys, under comparable conditions.) On the basis of the results obtained in Fig. 6a it can be assumed that the effect of pLys
consists in making the cell membranes pervious, which is expressed by high concentrations of cytoplasmic enzyme released under isotonic conditions. By contrast, after pArg treatment (Fig. 6b), virtually no LDH was detected. A significant increase in fluorescence after monensin treatment indicates that the loading of the peptides achieved with pArg causes them to be accumulated in vesicles inside the cell (Fig. 7).
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1. Pharmaceutical, composition containing at least one peptide, protein or protein fragment with an immunomodulatory activity together with an adjuvant, wherein said adjuvant is polyarginine.
2. Pharmaceutical composition as claimed in claim 1 wherein the peptide or the cellular breakdown product of the protein or protein fragment is a ligand for at least one MHC molecule.
3. Pharmaceutical composition as claimed in claim 2, wherein the peptide or the cellular breakdown product of the protein or protein fragment is a ligand for an MHC-I molecule.
4. Pharmaceutical composition as claimed in claim 2, wherein the peptide or the cellular breakdown product of the protein or protein fragment is a ligand for an MHC-II molecule.
5. Pharmaceutical composition as claimed in one of the preceding claims, wherein it contains a peptide which is derived from a protein of a pathogenic agent.
6. Pharmaceutical composition as claimed in claim 5, wherein the peptide is derived from a bacterial protein.
7. Pharmaceutical composition as claimed in claim 5, wherein the peptide is derived from a viral protein.
8. Pharmaceutical composition as claimed in one of Claims 1 to 4 for use as a tumour vaccine, wherein the protein is a tumour antigen or the protein fragment or the peptide or peptides is or are derived from a tumour antigen or antigens
9. Pharmaceutical composition as claimed in claim 8 for therapeutic use, wherein the tumour antigen or antigens is or are derived from tumour antigens.
10. Pharmaceutical composition as claimed in Claim 8 for prophylactic use, wherein the tumour antigens are derived from representatives of commonly occurring tumour antigens.
11. Pharmaceutical composition as claimed in one of Claims 8 to 10, wherein
the tumour antigen(s) is or are melanoma antigens.
12. Pharmaceutical composition as claimed in one of Claims 8 to 11, wherein it also contains a cytokine.
13. Pharmaceutical composition as claimed in claim 12, wherein, the cytokine is selected from the group IL-2, IL-4, IL-12, IFN-α, IFN-ß, IFN-γ, IFN- ω, TNF-α, GM-CSF or mixtures thereof.
14. Pharmaceutical composition as claimed in one of claims 2 to 11, wherein it contains a plurality of peptides which differ in that they bind to different MHC-subtypes of the individual to be treated.
15. Pharmaceutical composition as claimed in one of claims 2 to 14, wherein it contains one or more peptides which are derived from a naturally occurring Immunogenic protein or tumour antigen, or a cellular breakdown product thereof.
16. Pharmaceutical composition as claimed in one of claims 2 to 14, wherein it contains one or more peptides which are different from peptides derived from naturally occurring immunogenic protein(s) or tumour antigen(s) or cellular breakdown product(s) thereof.
17. Pharmaceutical composition as claimed in claim 1, wherein the peptide is an antagonist of a peptide which is derived from a protein which causes an autoimmune disease.
18. Pharmaceutical composition as claimed in claim 1, wherein polyarginine is conjugated with a cellular ligand.
19. Pharmaceutical composition as claimed in claim 18, wherein the ligand is a carbohydrate group.
20. Pharmaceutical composition as claimed in claim 19, wherein the ligand is fucose.
21. Pharmaceutical composition as claimed in claim 18, wherein the ligand is transferrin.
22. Pharmaceutical composition as claimed in one of claims 1 to 21 for parenteral administration.
23. Pharmaceutical composition as claimed in claim 22, wherein it takes the form of a solution or suspension of the peptide and the adjuvant in a pharmaceutically acceptable carrier.
24. Pharmaceutical composition as claimed in one of claims 1 to 21 for topical application.
25. Pharmaceutical composition as claimed in claim 24 in the form of a hydro gel.
26. Pharmaceutical composition substantially as hereinbefore described with reference to an as illustrated in the accompanying drawings.
|Indian Patent Application Number||0922/DEL/2000|
|PG Journal Number||37/2008|
|Date of Filing||10-Oct-2000|
|Name of Patentee||BOEHRINGER INGELHEIM INTERNATIONAL GMBH|
|Applicant Address||D-55216 INGELHEIM AM RHEIN, GERMANY.|
|PCT International Classification Number||A61K 48/00|
|PCT International Application Number||N/A|
|PCT International Filing date|