METHOD FOR MANUFACTURING A TUMOR VACCINE

Abstract

A method of identifying an effective composition of antigens for a tumor vaccine includes determination of antibodies produced by at least 10 patients after vaccination with an autologous vaccine obtained by gathering tumor material from the patients, separating the tumor cells from accompanying tissue, inactivating the tumor cells and providing them in a form suitable for administration as autologous vaccine, application of the autologous vaccine to the patients, isolation and determination of the antibodies produced by the patients' immune response that have been found to be shared by at least 80% of the patients, and identifying the antigens corresponding to the determined antibodies. A method of manufacturing a tumor vaccine is performed from the identified antigens and danger signals, and a tumor vaccine is obtained thereby.

Claims

1. A method of identifying an effective composition of antigens for a tumor vaccine, comprising the steps of determination of antibodies produced by at least 10 patients after vaccination with an autologous vaccine obtained by gathering tumor material from the patients, separating the tumor cells from accompanying tissue, inactivating the tumor cells and providing them in a form suitable for administration as autologous vaccine, application of the autologous vaccine to the patients, isolation and determination of the tumor specific antibodies produced by the patients that have been found to be shared by at least 80% of the patients, and identifying the antigens corresponding to the determined antibodies.

2. The method according to claim 1, wherein tumor specific antibodies produced by the patients that have been found to be shared by at least 50% of the patients are isolated and determined.

3. The method according to claim 1 or 2, wherein antibodies produced after vaccination with an autologous vaccine by at least 50 patients are determined.

4. The method according to claim 1, wherein the tumor is of epithelial or endothelial or mesothelial origin or a leukemia.

5. The method for manufacturing a tumor vaccine comprising identification of antigens according to claim 1 and assembling of the tumor vaccine from synthetic antigens thus identified and danger signals.

6. The method according to claim 5, wherein the danger signals are selected from the group consisting of HSP-27, HSP-47, HSP-60, HSP-70, HSP-72, HSP-90, HSP-105, HSP-110, multiple triacyl lipopeptides, glycolipids, lipopeptides, lipoproteins, lipoteichoic acid, zymosan—a Beta-glucan-, double-stranded RNA, poly I:C, lipopolysaccharide, fibrinogen, heparan sulfate and it's fragments, hyaluronic acid and it's fragments, nickel, Various opioid drugs, Bacterial flagellin, Profilin, diacyl lipopeptides, imidazoquinoline, loxoribine—a guanosine analogue-, bropirimine, single-stranded RNA, single-stranded Viral RNA, phagocytized bacterial RNA(24), unmethylated CpG Oligodeoxynucleotide DNA, bacterial ribosomal RNA sequence “CGGAAAGACC”, urea, and any mixture of two or more of them, which are substances able to activate Toll-like receptors.

7. The method according to claim 5, wherein the antigens are chosen from synthetic antigens, recombinant antigens, antigens of natural origin, peptides, proteins, glycans, branched polysaccharides and mixtures of two or more of those.

8. The method according to claim 5, wherein adjuvants enhancing the immune response chosen from the group consisting of Al(OH).sub.3, Aluminium salts, oil-in-water emulsions, liposomes, tocopherol acetate, saponine, detox. LPS from salmonellae, CpG oligo nucleotides—bacterial DNA—and combinations thereof, are added.

9. The method according to claim 5, wherein tumour tissue, blood, plasma or serum of the patient is analysed after treatment with the vaccine to determine additional antibodies, antigens corresponding to the determined antibodies are selected and added to the vaccine for further treatment.

10. A tumor vaccine obtained by assembling of the vaccine from danger signals and antigens according to the method of claim 5.

11. The tumor vaccine according to claim 10 adapted to intramuscular, intravenous, intracutaneous, intradermally, or subcutaneous application to the patient.

12. The tumor vaccine according to claim 10, wherein antigen presenting cells are contained.

13. The method according to claim 1, wherein tumor specific antibodies produced by the patients that have been found to be shared by at least 30% of the patients, are isolated and determined.

14. The method according to claim 1, wherein antibodies produced after vaccination with an autologous vaccine by at least 100 patients are determined.

15. The method according to claim 14, wherein tumor specific antibodies produced by the patients that have been found to be shared by at least 30% of the patients, are isolated and determined.

16. The method according to claim 5, wherein for identifying the effective composition antibodies produced after vaccination with an autologous vaccine by at least 100 patients are determined.

17. The method according to claim 16, wherein for identifying the effective composition tumor specific antibodies produced by the patients that have been found to be shared by at least 30% of the patients, are isolated and determined.

18. The method according to claim 17, wherein adjuvants enhancing the immune response selected from the group consisting of Al(OH).sub.3, Aluminium salts, oil-in-water emulsions, liposomes, tocopherol acetate, saponine, detox. LPS from salmonellae, CpG oligo nucleotides—bacterial DNA—and combinations thereof, are added.

19. The method according to claim 17, wherein tumour tissue, blood, plasma or serum of the patient is analysed after treatment with the vaccine to determine additional antibodies, antigens corresponding to the determined antibodies are selected and added to the vaccine for further treatment.

20. The tumor vaccine according to claim 10, wherein for identifying the effective composition antibodies produced after vaccination with an autologous vaccine by at least 100 patients are determined.

21. The tumor vaccine according to claim 20, wherein for identifying the effective composition tumor specific antibodies produced by the patients that have been found to be shared by at least 30% of the patients, are isolated and determined.

22. The tumor vaccine according to claim 10, wherein tumour tissue, blood, plasma or serum of the patient is analysed after treatment with the vaccine to determine additional antibodies, antigens corresponding to the determined antibodies are selected and contained in the vaccine for further treatment.

23. The tumor vaccine according to claim 10, wherein adjuvants enhancing the immune response selected from the group consisting of Al(OH).sub.3, Aluminium salts, oil-in-water emulsions, liposomes, tocopherol acetate, saponine, detox. LPS from salmonellae, CpG oligo nucleotides—bacterial DNA—and combinations thereof, are contained.

Description

EXAMPLE 1: METHOD OF IDENTIFICATION

[0037] Tumor material was received from 133 human patients who underwent radical nephrectomy, donating the material on a voluntary base. All patients gave informed consent. The tumor material (about 1 g) was placed in sterile RMPI 1640 transport medium (Invitrogen; Karlsruhe, Germany) and shipped at 2-8° C. within 24 h to the central laboratory. The median age of patients at primary diagnosis is 60 years and the male to female ratio is 2 to 1.

[0038] Preparation of the tumor cell lysate (TCL) started with removal of macroscopically visible non-tumor tissue. In order to obtain a single cell suspension, the remaining tumor tissue was cut into small pieces (˜1 mm.sup.3) and then passed through a sieve with a defined grid size (50 mesh). Cells are purified by Percoll density gradient centrifugation. Subsequently, the cells were incubated with interferon-γ and α-tocopherol acetate. The tumor cell suspension was aliquoted in single doses of 1 mL. Threefold rapid freezing at −82±5° C. and thawing was used to devitalize the cells.

[0039] For detection of antigens ELISA test kits were used. Sample dilutions were adjusted according to pre-experiments. Free hemoglobin (fHb) was measured to make sure, that no contamination from residual red blood cells of residual plasma influenced the results.

[0040] The concentrations of the danger signals HSP-60 and HSP-70 were detected by ELISA test kits. SDS-PAGE and Western Blotting were carried out according to standard procedures in 4-20% gradient mini-gels (SERVA, Heidelberg, Germany) utilizing a molecular weight marker (BioRad, Hercules, Calif., USA). Protein bands were insolubilized onto PVDF-membrane. Incubations with primary or secondary antibodies/protein-A-HRP were done in TBS 0.1% Tween 20, 1% w/v BSA. The substrate reaction (insoluble TMB+H.sub.2O.sub.2) [Seramun, Wolzig, Germany] was carried out for 20 min. The reaction was stopped by substrate removal (washing). Statistical analysis was carried out by descriptive methods using Microsoft Excel. Topological proteomics to demonstrate the heterogeneity of tumor tissue and cell suspensions were performed.

[0041] The results can be summarized as follows: There is a huge antigenic heterogeneity between the patients tumors. The antigenic composition of the tumors is highly individual on the found antigen pattern and concentrations. A few antigens are present on most tumors: TPS (Tissue polypeptide specific antigen [van Poppel H et al.: Serum tissue polypeptide antigen (TPA) as tumor marker for bladder cancer. Anticancer Res. 1996 July-August; 16(4B):2205-7.]), TPA (Tissue polypeptide antigen [Weber K.: Tissue polypeptide antigen (TPA) is related to the non-epidermal keratins 8, 18 and 19 typical of simple and non-squamous epithelia: re-evaluation of a human tumor marker. EMBO J. 1984 November; 3(11): 2707-2714.]), NSE (Neurone specific enolase [Lorenz J et al.: Neuron-specific enolase (NSE) and squamous cell carcinoma antigen (SCC) as serum markers in the diagnosis of bronchial carcinoma. Pneumologie. 1990 November; 44(11):1259-63.], CA-IX (carbonic anhydrase IX [Lam J S, Pantuck A J, Belldegrun A S, Figlin R A. G250: a carbonic anhydrase IX monoclonal antibody. Curr Oncol Rep. 2005; 7:109-115. Said J. Biomarker discovery in urogenital cancer. Biomarkers. 2005; 10 Suppl 1:S83-S86.], and CYFRA (CYFRA 21-1 is a fragment of cytokeratin 19 [Cytokeratin Fragment] [Pujol J L et al.: CYFRA 21-1: a new marker of epidermoid cancer of the bronchi. Comparison with 3 other markers. Presse Med. 1993 Jun. 19; 22(22):1039-42.]) are present in most tumors (>90%), in composition with at least 2 of this 5 in 100% of all RCC. TPA, TOPA and CYFRA are cytokeratins, which are represented by a peptide with a sequence present in all three antigens: Peptide sequence: QRGELAIKDANAKLSELEAALQRAKQ (Johansson A, Sandström P, Ullén A, et al. Epitope specificity of the monoclonal anticytokeratin antibody TS1. Cancer Res. 1999; 59(1): 48-51.). This peptide can be used for solid phase coating either for analytical techniques to detect specific antibodies against cytokeratins or as well for the affinity chromatographic purification of human antibodies of antibody expression systems like phage libraries.

[0042] The last step of the identification method according to the invention is the identification of the antigens recognized by the antibodies induced by the tumor vaccine. For this the gene-expression library Protagen: http://protagen.com/tests/autoimmunebiomarkers is used. The more than 7000 human proteins expressed are searched for the binding of the identified antibodies. Slides containing this spotted library are incubated with the patients serum (preferably serum drawn 4 weeks after the last vaccination with the tumor vaccine) at a dilution of 1:50 in PBS, 0.1% v/v Tween 20 (PBST) for 45 min. The slides are washed 3 times in (PBST) and now incubated with a fluorescence labelled anti-human-IgG for another 45 min (concentration: 1 μg/mL in PBST) before the slides are again washed 3 times in PBST. The evaluation is carried out by a fluorescence scan. Recognized antigens appear fluorescent.

EXAMPLE 2: VACCINE

[0043] For manufacturing a vaccine, the identified antigens are either obtained commercially or made in a known manner, adjusted to 10.sup.6 cells/mL and combined with the danger signals HSP-60 and/or HSP-70 in a concentration of 3 ng/mL up to 1000 μg/mL in cell NaCl/Glucose medium. The solution is filled into 1 mL sterile vials and can be stored below −40° C. until use. The vaccine can be used for the vaccination of patients. The composition of the vaccine can be done based on the statistical evaluation of the most present antigens or on an individual level, based on the preesixting anti-tumor response to get those patients boostered against their tumor.

[0044] Some of the treated human patients already show an immune response to their tumor. This existing immune response can be evaluated analogously to example 1. In those cases the vaccine composition is adjusted according to the antigen pattern recognized by the patients immune system.

[0045] It is contemplated that the method of identification and the methods of making vaccines can be applied to non-human mammals analogously.