WO2000066153A1

WO2000066153A1 - RAS ONCOGEN p21 PEPTIDE VACCINES

Info

Publication number: WO2000066153A1
Application number: PCT/NO2000/000142
Authority: WO
Inventors: Jon Amund Eriksen; Gustav Gaudernack; Marianne Klemp Gjertsen; Mona Møller
Original assignee: Norsk Hydro ASA
Current assignee: Norsk Hydro ASA
Priority date: 1999-04-30
Filing date: 2000-04-28
Publication date: 2000-11-09
Anticipated expiration: 2001-10-30
Also published as: NO992102D0; AU4438900A; CA2372187A1; NO992102L; NO309798B1; AR023806A1; JP2002543149A; EP1173199A1

Abstract

The invention relates to synthetic peptide mixtures which elicit T cellular immunity, for use in cancer vaccines and compositions for anti-cancer treatment. Preferred peptides are RAS p21 (5-21, 49-73 and 52-70) mutant peptides and in particular the mutants G12A, G12C, G12D, G12R, G12S, G12V, G13D, Q61H, Q61K, Q61L and Q61R.

Description

RAS oncogen p21 peptide vaccines

This invention relates to use of synthetic peptide mixtures for prophylaxis and/or treatment of cancers.

The genetic background for the onset of cancer is alterations in proto-oncogenes and oncogenes and tumour supressor genes. Proto-oncogenes are normal genes of the cell which have the potential of becoming oncogenes. All oncogenes code for and function through a protein. In the majority of cases they have been shown to be components of signal transduction pathways. Oncogenes arise in nature from proto-oncogenes through point mutations or translocations, thereby resulting in a transformed state of the cell harbouring the mutation. Cancer develops through a multistep process involving several mutational events in oncogenes and tumour supressor genes.

In its simplest form a single base substitution in a proto-oncogene may cause the resulting gene product to differ in one amino acid.

In experimental models involving murine tumours it has been shown that point mutations in intracellular "self-proteins may give rise to tumour rejection antigens, consisting of peptides differing in a single amino acid from the normal peptide. The T cells recognizing these peptides in the context of the major histocompatibility (MHC) molecules on the surface of the tumour cells are capable of killing the tumour cells and thus rejecting the tumour from the host. (Boon, T. et al, Cell, 1989, Vol. 58, p 293-303)

In the field of human cancer immunology the last two decades has seen intensive efforts to characterize genuine cancer specific antigens.

Initially, particular effort has been devoted to the analysis of antibodies to human tumour antigens. The prior art suggests that such antibodies could be used both for diagnostic and therapeutical purposes, for instance in connection with an anti-cancer agent. One problem is that antibodies can only bind to tumour antigens that are exposed on the surface of tumour cells. For this reason the efforts to produce a cancer treatment based on the immune system of the body has been less successful than expected.

Antibodies typically recognise free antigen in native conformation and can potentially recognize almost any site exposed on the antigen surface. In contrast to the antibodies produced by the B cells, T cells recognize antigens only in the context of MHC molecules, designated HLA (human leucocyte antigen) in humans, and only after appropriate antigen processing, usually consisting of proteolytic fragmentation of the protein, resulting in peptides that fit into the groove of the MHC molecules. This enables T cells to recognize also peptides derived from intracellular proteins. T cells can thus recognize aberrant peptides derived from anywhere in the tumour cell, in the context of MHC molecules on the surface of the tumour cell, and subsequently can be activated to eliminate the tumour cell harbouring the aberrant peptide.

The HLA molecules are encoded by the HLA region on the human chromosome No 6. The class I molecules are encoded by the HLA A, B and C subloci, and the class II molecules are encoded by the DR, DP and DQ subloci. All the gene products are highly polymorphic. Different individuals thus express distinct HLA molecules that differ from those of other individuals. This is the basis for the difficulties in finding HLA matched organ donors in transplantations. The significance of the genetic variation of the HLA molecules in immunobiology is reflected by their role as immune-response genes. Through their peptide binding capacity, the presence or absence of certain HLA molecules governs the capacity of an individual to respond to peptide epi topes. As a consequence, HLA molecules determine resistance or susceptibility to disease.

T cells may control the development and growth of cancer by a variety of mechanisms. Cytotoxic T cells, both HLA class I restricted CD8+ and HLA Class II restricted CD4+, may directly kill tumour cells carrying the appropriate tumour antigens. CD4+ helper T cells are needed for induction and maintenance of cytotoxic T cell responses as well as for antibody responses, and for inducing macrophage and LAK cell killing.

Although the prior art (i.e. the prior art referred to in WO 92/14756 belonging to the applicant of the present patent application) has identified many oncogenes and their protein products, and a recently published study has shown that the T cell repertoire of a healthy person includes T cells with a specificity against a synthetic peptide fragment derived from one p21 ras oncogene product, no studies prior to WO 92/14756 have defined the correct antigens or antigenic sites giving rise to tumour specific T cell immunity.

WO 92/14756 is based on the idea that another possible approach for combating cancer is by using the body's own immune system through an activation and strengthening of the immune response from specific T cells. In WO 92/14756 synthetic peptides and fragments of oncogene protein products which elicit T cellular immunity, and cancer vaccines and compositions for anti-cancer treatment comprising said peptides or peptide fragments, are disclosed. Despite this previous invention, there is still a need for more effective products for being able to combat cancer.

It has been great concerns about using peptide mixtures for vaccination of patients due to the overhanging danger that some of the peptides in the mixture are immunodominant and thus supress the HLA presentation of the other peptides. ( Pion S, Christianson GJ, Fontaine P, Roopenian DC, Perreault C, Blood 1999 Feb l;93(3):952-62; Shaping the repertoire of cytotoxic T-lymphocyte responses: explanation for the immunodominance effect whereby cytotoxic T lymphocytes specific for immunodominant antigens prevent recognition of nondominant antigens.)

From experiments performed in vitro, it is known that various mutated ras peptides may compete for binding to the HLA molecule responsible for presentation to the relevant T cells (WO 92/14756, Figures 4,5 and 7) and that peptides of the same length, but representing different mutations may inhibit the binding and recognition of a peptide representing another mutation with different degrees of efficacy (T. Gedde-Dahl III et al., Human Immunol. 33, 266-274, 1992 and B.H. Johanssen et al., Scand.J. Immunol, 33, 607-612, 1994). From these facts, the immunodominance issue has been regarded as a problem regarding mutated RAS peptide vaccines.

However, these observations were all made under the experimental conditions that can be obtained in vitro. In a vaccinated patient several other conditions may contribute to the operational immunodominance of a given peptide in a mixture of other peptides. Of particular interest may be resistance to degradation by proteolytic enzymes in the tissue where the antigen is injected, which may influence the half life of the peptides in vivo, and differences in hydrophobicity which may influence the rate of uptake of the peptides by the relevant antigen presenting cells in the tissue. Contrary to what would have been expected from the in vitro experiments the inventors surprisingly found that the immune response was significantly higher in groups of patients with colorectal cancer and pancreatic cancer treated with a cocktail of mutant ras peptides compared to single ras peptide treatment as described in WO 92/14756. These results demonstrate that rather than competing with the other peptides and thus creating immunodominance, a peptide may give rise to an immune response that augments the response against the other peptides in the mixture. This phenomenon can best be explained by a positive feedback loop generated by the cascade of T-helper related chemokines and cytokines that will be released initially following T cell activation by a peptide specific T cell. These factors will recruit more professional antigen presenting cells and T cells to the lymph node where activation takes place and subsequently also to the site of the vaccine, and will also provide an amplification of the production of the growth and maturation factors that are required for successful T cell proliferation. The rational basis for the feedback based augmentation effect is that the number of T cells that have the potential to recognize all the peptides in the mixture greatly outnumbers the number of T cells specific for a single peptide. In concert the activation of more T cells specific for individual peptides present in the peptide mixture will increase the magnitude of the response also against single peptides in the mixture.

Furthermore, it was surprisingly found that ras peptides in the administered cocktails that do not correspond to the ras mutation actually existing in the tumour of the patient, also did generate T cells that were clinically relevant due to cross-reactivity with peptides corresponding to the actual mutation. The effect of using a ras peptide mixture is therefore twofold, it creates an amplification of the T cells specific for a single peptide and adds a second component consisting of T cell clones that are specific for another peptide, but which cross reacts with the tumour relevant mutation. Such T cell clones are otherwise not elicited by single peptide vaccination, since they were not observed in patients treated with a single peptide vaccination.

Thus, it is a main object of the invention to provide a more effective therapeutic agent, vaccine and pharmaceutical composition then those disclosed in WO 92/14756 for prophylaxis and/or treatment of cancer.

Another object of the invention is to provide a therapeutic agent, vaccine and pharmaceutical composition that induces immune responses and activation of T cells.

These and other objects of the invention are achieved by the attached claims.

The peptides and peptide fragments disclosed in WO 92/14756 constitute the peptides used in the peptide mixtures in this invention. Thus, reference is made to the specification of WO/92/14756.

The invention is further explained by the examples and attached figures. Description of the figures

Fig. 1: In figure 1 the results from two clinical trials with vaccination of colorectal cancer patients with position 12 and/or position 13 mutant RAS peptides are compared.

Fig. 2:

In figure 2 the results from two clinical trials of vaccination of pancreatic cancer patients with position 12 mutant RAS peptides are compared.

Fig. 3:

Figure 3 shows different response patterns of four patients with colorectal adenocarcinoma that had received vaccination with a cocktail of five different position 12 and 13 mutant ras peptides, sequence id. no. 2, 3, 4, 6 and 7.

Fig. 4:

Figure 4 shows different T cell responses against individual mutant ras peptides for two responding patients.

Fig. 5:

Figure 5 shows the reactivity of peripheral T cells obtained after vaccination of a patient with colorectal adenocarcinoma.

Fig. 6:

Figure 6 shows the reactivity of tumour infiltrating lymphocytes (TILs) obtained after vaccination of a patient with advanced pancreatic cancer.

Fig. 7: The extensive cross reactivity with different ras mutations, of ras specific T cells generated by vaccination with a mixture of ras peptides, was also demonstrated at a clonal level. This is shown in figure 7. Fig. 8:

Figure 8 shows the specificity of T cell clones obtained from vaccination of patients with melanoma cancer with a cocktail of four different position 61 mutant RAS peptides, sequence id. no. 9,10, 11 and 10.

5

Examples

Synthesis of the peptides:

10 The peptides were synthesised by using solid phase peptide synthesis. N-a-Fmoc-amino acids with appropriate side chain protection ( Ser(tBu), Thr(tBu), Lys(Boc), His(Trt), Arg(Pmc), Cys(Trt), Asp(O-tBu), Glu(O-tBu) ) were used. The Fmoc-amino acids were activated by TBTU prior to coupling. 20% piperidine in DMF was used for selective removal of Fmoc after each coupling. Detachment from the resin and final removal of

15 side chain protection was performed by 95% TFA (aq.) containing appropriate scavengers. The peptides were purified and analysed by reversed phase. The peptides were analysed and characterised using standard methods for peptides.

The following RAS peptides were synthesised by this method: 20

Table 1. RAS peptides with mutation in position 12 and 13 and the corresponding normal p21 sequence.

12-A-p21 RAS (5-21) KLVVVGAAGVGKSALTI

25 12-C-p21 RAS (5-21) KLVVVGACGVGKSALTI

12-D-p21 RAS (5-21) KLVVVGADGVGKSALTI

12-R-p21 RAS (5-21) KLVVVGARGVGKSALTI

12-S-p21 RAS (5-21) : KLVVVGASGVGKSALTI

12-V-p21 RAS (5-21) : KLVVVGAVGVGKSALTI

30 13-D-p21 RAS (5-21) : KLNVVGAGDVGKSALTI Table 2. RAS peptides with mutation in position 61 and the corresponding normal p21 sequence.

61-H-p21 RAS (49-73) ETCLLDILDTAGHEEYSAMRDQYMR 5 61-K-p21 RAS (49-73) ETCLLDILDTAGKEEYSAMRDQYMR 61-L-p21 RAS (49-73) ETCLLDILDTAGLEEYSAMRDQYMR 61-R-p21 RAS (49-73) ETCLLDILDTAGREEYSAMRDQYMR 61-H-p21 RAS (52-70) LLDILDTAGHEEYSAMRDQ 61-K-p21 RAS (52-70) LLDILDTAGKEEYSAMRDQ 10 61-L-p21 RAS (52-70) LLDILDTAGLEEYSAMRDQ 61-R-p21 RAS (52-70) LLDILDTAGREEYSAMRDQ normal p21 RAS(52-70) LLDILDTAGQEEYSAMRDQ

15

Biological experiments:

Vaccination with a cocktail of ras peptides instead of single peptides increases the efficacy of vaccination. By comparing the two types of vaccination regimens in groups of cancer patient having pancreatic or colorectal cancer, we observed that the percentage of patients

20 obtaining an immune response is significantly higher in groups of cancer patients vaccinated with a cocktail of mutant RAS peptides as compared to single RAS peptide vaccination. This is demonstrated for patients with colorectal cancer and pancreatic cancer in figure 1 and 2 respectively.

25 In figure 1 the results from two clinical trials with vaccination of colorectal cancer patients with position 12 and/or position 13 mutant RAS peptides are compared. In one trial the patients were vaccinated with one single mutant RAS peptide, either sequence id. no. 2,3,4,6 or 7, and in the other trial a cocktail containing all five mutant RAS peptides was used for vaccination. In the patients vaccinated with a single peptide 52% (14 out of 27

30 vaccinated patients) obtained an immune response, and in the patients given the five peptide cocktail the immune response rate was 67 % (18 of 23 vaccinated patients). The single peptide was administered intradermally to Dukes B and C patients together with GM-CSF (30mg) in two series; first 4 injections of lOOmg peptide with one week between each dose was given, and then, four weeks after the last injection, 4 x 300mg was given, again one week between each 300mg dose. The five peptide cocktail was administered to Dukes C and D patients together with GM-CSF (30mg) as four injections of 500 mg cocktail (lOOmg of each peptide) with one week between each injection in 15 of the patients, and as four injections of 2000mg cocktail (400mg of each peptide) in 12 of the patients. The number of responding patients was 10 in the group receiving the low dose (67%) and 8 in the high dose group (67%). It is notable that the patient group receiving single peptide vaccinations predominantly contained patients that were in a better clinical condition (mainly resected cancer patients in Dukes stages B and C), whereas the group of patients receiving the vaccine cocktail mainly contained patients with advanced disease (Dukes stage D, with liver metastasis, that did not respond to chemotherapy/irradiation therapy). It is conceivable that the immune status of the patients in the latter group was lower than in the former group, since both prior treatment (cytostatics and irradiation) and large tumour burden may severely impair immune functions. It was therefore highly unexpected to us that the number of responders to the vaccine was higher in the group having advanced disease.

Increasing the dose did not result in an increased number of responders, as increasing the dose of single peptide vaccine in the same patients did not result in more responders, and in two groups with high and low dose peptide cocktail the number of responders were equal.

In figure 2 the results from two clinical trials with vaccination of pancreatic cancer patients with position 12 mutant RAS peptides are compared. In one trial patients with resected pancreatic cancer (Whippel operation) were vaccinated with one single mutant RAS peptide, either sequence id. no. 2,3,4 or 6, and in the other trial, involving patients with inoperable (terminal) pancreatic cancer, a cocktail containing all four mutant RAs peptides was used for vaccination. Again we observed that the patients with advanced disease given the vaccine cocktail were able to respond to the vaccine despite their deteriorating clinical condition and surprisingly that the number of responders were higher in this group of patients with non-resected tumour as compared to the tumour resected group that were given single peptide vaccine. In the patients vaccinated with a single peptide 42% (5 out of 12 vaccinated patients) obtained an immune response, and in the patients given the four peptide cocktail the immune response rate was 56 % (19 of 34 vaccinated patients). The single peptide was administered intradermally to tumour resected patients together with GM-CSF (30μg) in six injections of lOOμg peptide with one week between first four doses, two weeks between dose four and five and four weeks between dose five and six. The four peptide cocktail was administered to non resectable patients together with GM-CSF (30μg) in six injections of 400 μg (lOOμg of each peptide) following the same schedule as for the single peptide administration.

Again we observed that the patients with advanced disease given the vaccine cocktail were able to respond to the vaccine despite their deteriorating clinical condition and surprisingly, that the number of responders were higher in the group of patients with non-resected tumours that received vaccine cocktail.

The combined results shown in Figure 1 and 2 demonstrate that the vaccine cocktail is superior to single peptide vaccines and is highly efficient even under clinical conditions that do not favour an immune response.

Furthermore it is demonstrated in figures 3 and 4 that by vaccination with mixtures of RAS peptides it is possible to raise T cells against all the various peptides in the mixture.

Figure 3 shows different response patterns of four patients with colorectal adenocarcinoma that had received vaccination with a cocktail of five different position 12 and 13 mutant ras peptides, sequence id. no. 2, 3, 4, 6 and 7. The peptide cocktail was administered intradermally to patients with colorectal adenocarcinoma together with GM-CSF (30μg) in four weekly injections of 0,5 mg peptide cocktail. The T cell responses were tested in pre- and post-vaccination samples after one in vitro stimulation. All four patients also showed development of a positive DTH reaction to the vaccine cocktail. The results in figure 3 were obtained by using standard proliferation assays and peptides with sequence id. no. 2, 3, 4, 6, 7 and 8 (wild type). The results show that one patient generated an immune response against all five peptides in the cocktail whereas the three other patients generated an immune response against four of the five peptides used in the vaccine. Since reactivity with the wild type peptide was not observed in blood samples harvested before vaccination, but only in post vaccination samples (patient 2 and 3), and since the peptide with sequence id. no. 8 was not part of the vaccine mixture, this reactivity must be due to the stimulation of clones of T cells with the capacity of cross reaction between the wild type ras sequence and sequences containing some of the amino acid substitutions generated by mutation. In no instance was side reactions indicative of an autoimmune reaction observed in patients demonstrating such a cross reactivity. This may be related to the fact that the level of p21 ras expression in tumours is higher than in the corresponding non-transformed tissue.

In figure 4 the different T cell responses against individual mutant ras peptides for two non-resected pancreatic cancer patients are shown. The patients were vaccinated with a cocktail containing four mutant ras peptides with sequence id. no. 2, 3, 4 and 6. T cell responses were investigated in vitro in peripheral blood mononuclear cells (PBMC). Here, PBMCs from the two patients were tested against the 4 single mutant peptides (sequence id.no. 2, 3, 4 and 6) and the normal ras peptide (sequence id. no 8) after one stimulation in vitro. Briefly, PBMC were seeded 1 x 10⁶ per well in 24- well plates (Costar) with 25 μM of single mutant ras peptide or the mixture of mutant ras peptides in 1 ml of RPMI-1640 (Gibco, Paisley, UK) substituted with 15% heat-inactivated human serum. After three days of culture the media was supplemented with 10 U/ml IL-2 (Amersham). Cultured cells (5 x 10⁴ / well) were tested after 9 - 12 days for specific proliferating capacity against single mutant ras peptides/peptide mixture and normal ras peptide at 25 μM concentration, with or without IL-2 (1 U/ml), by using autologous, irradiated (30 Gy) PBMC (5 x 10⁴ cells /well) as APCs. Proliferation was measured at day 3 after overnight incubation with ³H-thymidine 3.7 x 10⁴ Bq/well (Amersham). Values are given as mean counts per minute (cpm) from triplicates. An antigen-specific response was considered positive when the stimulatory index (SI) (response with antigen divided by response without antigen) was above 2. Both patients mounted an immune response against all of the sub-components of the vaccine (sequence id. no 2, 3, 4 and 6).

Figure 5 shows the reactivity of peripheral T cells obtained after vaccination of a patient with colorectal adenocarcinoma. The patient received a vaccination with a cocktail consisting of seven different position 12 and 13 mutant ras peptides, sequence id. no.l, 2, 3, 4, 5, 6 and 7. The peptide cocktail was administered intradermally to a patient with advanced colorectal cancer (Dukes D) together with GM-CSF (30μg) in four weekly injections of 0,7 mg peptide cocktail at four different sites. The results show T cells that react with five of the different ras mutations are generated, but there is no cross-reactivity with wild type ras. T cell responses were investigated as mentioned above. Briefly, PBMC were tested after one in vitro stimulation in standard proliferation assays with peptides with sequence id. no. 1, 2, 3, 4, 5, 6, 7 and 8 (wild type).

Furthermore it is shown that vaccination with cocktails of RAS peptides generates T cells that are cross-reactive with the different ras mutations This is demonstrated in figures 6,7 and 8.

Figure 6 shows the reactivity of tumour infiltrating lymphocytes (TILs) obtained after vaccination of a patient with advanced pancreatic cancer. The patient received a vaccination with a cocktail consisting of four different position 12 mutant ras peptides, sequence id. no. 2, 3, 4 and 6. The peptide cocktail was administered intradermally to a patient with advanced pancreatic cancer together with GM-CSF (30μg) in six injections of 0,4 mg peptide cocktail with one week between the first four doses, two weeks dose four and five and four weeks between dose five and six. The T cells were obtained from a tumour biopsy taken after the patient was successfully immunised by the peptide vaccination (positive DTH test). The TIL's were expanded extensively in vitro by coculture in recombinant human interleukin 2 (rIL2) and were shown to be homogeneously expressing the same T cell receptor Vβ chain by analysis with monoclonal antibodies, indicating a monoclonal origin of the TIL cell line generated. The results in figure 6 show that the TIL cell line cross-react with all four of the different mutant ras peptides contained in the vaccine cocktail. TILs were tested in standard proliferation assays with peptides with sequence id. no. 2, 3, 4 and 6.

The extensive cross reactivity with different ras mutations, of ras specific T cells generated by vaccination with a mixture of ras peptides, was also demonstrated at a clonal level. Figure 7 summarises the specificity of five T cell clones obtained after vaccination of a patient with advanced pancreatic cancer with a cocktail consisting of four different position 12 mutant ras peptides, sequence id. no. 2, 3, 4 and 6. The results show that T cell clones can be either strictly specific for a single RAS mutation as described before (TLC1, TLC2 and TLC4) or cross-reactive with two or more different RAS mutations (TLC4 and TLC5). None of the TLC generated from this patient showed evidence of cross-reactive with wild type RAS (seq.id.no. 8). The peptide cocktail was administered as described for figure 6. The T cell clones were obtained from blood samples from patients showing an immune response to the vaccine by a positive delayed type hypersensitivity test (DTH), by cloning under limiting dilution conditions. The results in figure 7 were obtained using standard proliferation assays and peptides with sequence id. nos. 2,3,4,6 and 8 (wild type).

Figure 8 shows the specificity of T cell clones obtained from peripheral blood after vaccination of patients with melanoma with a cocktail of four different position 61 mutant RAS peptides, sequence id. no. 9,10, 11 and 10. The results show that both T cells that are strictly specific for single RAS mutations as well as T cells that are cross-reactive with two, three of four different RAS mutations RAS are generated by vaccination with this peptide cocktail. None of the T cell clones studied showed any evidence of cross-reactivity with wild type RAS. The peptide cocktail was administered intradermally to tumour resected patients together with GM-CSF (30μg) in six injections of 0.32 mg peptide cocktail with one week between the first four doses, two weeks between dose four and five and four weeks between dose five and six. The T cell clones were obtained from blood samples from patients showing a immune response to the vaccine by a positive delayed type hypersensitivity test (DTH), after culturing of T cells under limiting dilution conditions. The results in figure 8 were obtained using standard proliferation assays and peptides with sequence id. nos. 13, 14, 15, 16 and 17 (wild type).

The consequence of this is that also the RAS peptides contained in the administered cocktails that do not correspond to the ras mutation actually existing in the tumour of the patients, also generate T cells that are clinically relevant due to cross-reactivity. Cross-reactive T cells are never observed after single peptide vaccination and using RAS peptide cocktails is therefore a new way to further strengthen an immune response against cancer associated ras mutations. Our data show that in the majority of patients that have received a cocktail of mutant ras peptides, immune-responses to most of the sub-components contained in the vaccine preparation is observed. This is of great importance and shows that there is no immunodominance between the different mutant ras peptides and that the peptides are presented to T cells by most common HLA molecules. Thus, cancer vaccines consisting of different mutant ras peptides can be used broadly in the clinical setting without the need for HLA-typing and prior determination of the ras mutation. Actually, by using a cocktail of mutant peptides, cross-reactive T cells that are clinically relevant will be generated in the patients. The results in figure 6 showing anti mutant ras reactivity in tumour infiltrating lymphocytes that can be found in biopsy specimens post peptide vaccination, are of great importance because they demonstrates that cross-reactive T cells are capable of localising their target tumour area in situ.

Administration

The peptide mixture according to this invention can be administered to the patient as described in WO 92/14756.

The peptide mixtures of this invention can be administered in an amount in the range of 1 nanogram (1 ng) to 1 gram (lg) to an average human patient or individual to be vaccinated. It is preferred to use a dose in the rage of 1 microgram (1 mg) to 1 milligram (1 mg) for each administration.

Claims

1. Use of a peptide mixture comprising of at least two peptides defined as a) having a point of mutation or translocation as compared to a corresponding fragment of the oncogene protein; and b) corresponding to, completely cover or being a fragment of a processed oncogene protein fragment as presented by a cancer cell or other antigen presenting cells (APC); and c) inducing specific T cell responses to the actual oncogene protein fragment produced by the cell by processing and presented in the HLA molecule, for the manufacture of a therapeutical agent for the prophylaxis and/or treatment cancer.

2. Use of a peptide mixture comprising of at least two peptides defined as a) having a point of mutation or translocation as compared to a corresponding fragment of the oncogene protein; and b) corresponding to, completely cover or being a fragment of a processed oncogene protein fragment as presented by a cancer cell or other antigen presenting cells (APC); and c) inducing specific T cell responses to the actual oncogene protein fragment produced by the cell by processing and presented in the HLA molecule, for the manufacture of a vaccine for the prophylaxis and/or treatment cancer.

3. Use of a peptide mixture comprising of at least two peptides defined as a) having a point of mutation or translocation as compared to a corresponding fragment of the oncogene protein; and b) corresponding to, completely cover or being a fragment of a processed oncogene protein fragment as presented by a cancer cell or other antigen presenting cells (APC); and c) inducing specific T cell responses to the actual oncogene protein fragment produced by the cell by processing and presented in the HLA molecule, for the manufacture of a pharmaceutical composition for the prophylaxis and/or treatment cancer.

4. Use according to claim 1-3, wherein the peptides have an amino acid sequence as the natural p21 ras protein except that the residue Gly in the positions 12 and or 13 and/or Gin in position 61 are replaced by any other amino acid.

5. Use according to claim 1-3, wherein the peptides are selected from a group consisting of A and B:

A) the peptides shown in Table 1 (sequence identity nos. 1,2,3,4,5,6 and 7), or fragments thereof;

B) the peptides shown in Table 2 (sequence identity nos. 9,10,11, 12, 13, 14, 15, and 16), or fragments thereof.

6. A pharmaceutical composition comprising a peptide mixture as defined in claim 3-5, and a pharmaceutically acceptable carrier, diluent and/or excipient.

7. A process for manufacture of a pharmaceutical composition, which comprises the step of incorporating a peptide mixture as defined in claim3-5, together with a pharmaceutically acceptable carrier, diluent and/or excipient.

8. A cancer vaccine comprising a peptide mixture as defined in claim 2, 4 and 5, and a pharmaceutically acceptable carrier, diluent and/or excipient.

9. A process for manufacture of a cancer vaccine, which comprises the step of incorporating a peptide mixture as defined in claim 2, 4 and 5, together with a pharmaceutically acceptable carrier, diluent and/or excipient.

10. Method for treatment of a patient afflicted with cancer consisting of induction of T cell immunity to oncogene proteins by stimulating in vivo or in vitro with a peptide mixture as defined in claim 1-5.

11. Method according to claim 10 wherein the amount of the peptide mixture used is in the range of 1 nanogram (1 ng) to 1 gram (lg) and preferentially in the rage of 1 microgram (1 mg) to 1 milligram (1 mg) for each administration.

12. Method for vaccination of a person disposed for or afflicted with cancer consisting of induction of T cell immunity to oncogene proteins by stimulating in vivo with a peptide mixture as defined in claim 2, 4 and 5.

13. Method for vaccination of a person disposed for or afflicted with cancer, consisting of administering a peptide mixture according to the claims 2, 4 or 5, one or more times, in an amount sufficient for induction of specific T cell immunity oncogene proteins.

14. Method according to claim 13, wherein the amount of the peptide mixture is in the range of 1 nanogram (1 ng) to 1 gram (1 g) and preferentially in the range of 1 microgram (1 mg) to 1 milligram (1 mg) for each administration.