HLA TUMOR ANTIGEN PEPTIDES OF CLASS I AND II FOR TREATING MAMMARY/BREAST CARCINOMAS

Abstract

The present invention relates to a pharmaceutical composition for use in the treatment or prophylaxis of mammary/breast carcinomas, in particular locally recurring or metastasizing mammary carcinomas in a patient or group of patients, who has or is suspected of having a breast carcinoma, comprising at least 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and at least 2 tumor antigen peptides corresponding to MHC class II complexes, wherein the HLA tumor antigen peptides are tumor-exclusive or tumor-associated HLA antigen peptides and are directed against at least one MHC complex including combinations thereof; a pharmaceutical composition, a kit (or parts thereof); a method for determining an HLA peptide of class I and/or II; a method for preparing a pharmaceutical composition according to the invention; and the use of a pharmaceutical composition according to the invention for the preparation of a pharmaceutical composition for the treatment of malignancies, leukemias and neoplasms.

Claims

1. Pharmaceutical composition for use in the treatment or prophylaxis of mammary/breast carcinomas, in particular locally recurrent or metastatic breast carcinomas in a patient or group of patients suffering from or suspected of suffering from mammary/breast carcinoma, comprising a pharmacologically effective amount comprising 4 to 8 HLA tumor antigen peptides corresponding to MHC class I complexes and at least 2 tumor antigen peptides corresponding to MHC class II complexes, characterized in that the HLA tumor antigen peptides are tumor-exclusive or tumor-associated HLA antigen peptides, in particular those associated with mammary/breast carcinomas, wherein the HLA tumor antigen peptides comprise sequences contained in the sequences SEQ ID No. 13-SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 32-SEQ ID No. 48.

2. Pharmaceutical composition according to claim 1 for use as recited in claim 1, wherein administering the pharmacologically effective amount of the tumor antigen peptides to the patient or group of patients suffering from breast carcinoma is effective to reduce the CA 15-3 level.

3. Pharmaceutical composition according to claim 1 or 2 for the use as claimed in claim 1, wherein the HLA tumor antigen peptides corresponding to the MHC class I and class II complexes, respectively, are immunogenic HLA tumor antigen peptides determined by means of an immunogenicity assay, in particular by Western blot, ELISA techniques, ELISPOT or immunodetection with microscopic analysis.

4. Pharmaceutical composition according to one of claims 1 to 3 for the use as claimed in claim 1, wherein the HLA-A tumor antigen peptides bind to the corresponding MHC class I complex of subtype A*01 and/or A*02.

5. Pharmaceutical composition according to one of claims 1 to 4 for the use as claimed in claim 1, further comprising at least one HLA-B tumor antigen peptide corresponding to the MHC class I complexes and/or at least one HLA-C tumor antigen peptide corresponding to the MHC class I complexes.

6. Pharmaceutical composition according to one of claims 1 to 4 for the use as claimed in claim 1, wherein the HLA tumor antigen peptides corresponding to the respective subtype of the MHC class I complexes are selected from the group consisting of the amino acid sequences given in SEQ ID Nos. 13 to 35 or have at least one amino acid exchange with respect to these amino acid sequences.

7. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the HLA tumor antigen peptides corresponding to the MHC class II complexes are selected from the group consisting of the amino acid sequences set forth in SEQ ID Nos. 36 to 48.

8. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the HLA tumor antigen peptides are presented on the surface of the patient's or group of patients' mammary/breast carcinoma tumor cells, as determined by ultra-high performance liquid chromatography (UHPCL) in conjunction with ESI mass spectrometry (MS).

9. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the expression level of the tumor-associated HLA tumor antigen peptides in the tumor cells is at least three times higher than in the healthy cells of the patient or group of patients having at least one identical HLA allele as determined by qPCR, and wherein tumor-associated HLA antigen peptides are associated with proliferation, invasiveness, angiogenesis, and an increase in cytokeratin production of mammary/breast carcinoma.

10. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein: at least one HLA-A tumor antigen peptide is a tumor exclusive HLA-A tumor antigen peptide, and specific binding of the tumor-exclusive HLA-A tumor antigen peptide determined against the corresponding MHC class I complex with a K.sub.D in the range of 10 to 50 nM occurs as determined by surface plasmon resonance.

11. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the pharmacologically effective amount of each individual HLA antigen peptide in the composition is in an absolute concentration (i.e., administration dose) in the range of 100 to 600 μg.

12. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the composition comprises an adjuvant that is capable of forming a granuloma at the site of application of the composition to a patient.

13. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the pharmaceutical composition is applied subcutaneously or intradermally and, preferably substantially simultaneously at least 2 application sites, preferably at least 4 application sites, remote from a tumor lesion and/or the cancerous lymph node area.

14. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein at least one HLA tumor antigen peptide has at least one mutation with respect to the wild-type HLA tumor antigen peptide (the mutanome) that results in an increase in affinity for the T-cell receptor of the individual treated with HLA tumor antigen peptide as compared to the wild-type HLA antigen peptide.

15. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the pharmaceutical composition is used for the treatment of mammary/breast carcinoma as monotherapy or in combination with other known therapies and/or compounds for the treatment of mammary/breast carcinoma.

16. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the patients to be treated with the pharmaceutical composition have received a standard therapy procedure (e.g., at least one surgery, radiation, chemotherapy, and/or hormone therapy).

17. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the pharmaceutical composition is administered as a first-line therapy to the patient or group of patients having at least one identical HLA allele.

18. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the pharmaceutical composition comprising each individual HLA tumor antigen peptide in the pharmaceutical composition at an absolute concentration (i.e., administration dose) of 100 to 600 μg is administered intradermally or subcutaneously once every 2 weeks for a period of at least one year to a patient or group of patients having at least one identical HLA allele.

19. Pharmaceutical composition according to any preceding claims for the use as claimed in claim 1, wherein the mammary/breast cancer is a hormone positive, HER2/neu, or triple negative breast cancer.

20. HLA tumor antigen peptide corresponding to MHC class I complexes or MHC class II complexes, in particular for use in the treatment of mammary/breast carcinoma or for a pharmaceutical composition according to any one of claims 1 to 19 selected from the group consisting of the amino acid sequences given in Seq ID No. 13-Seq ID No. 26, Seq ID No. 28, Seq ID No. 29 and Seq ID No. 32-Seq ID No. 48, respectively.

21. HLA tumor antigen peptide corresponding to claim 20 for use in a method of treating mammary/breast carcinomas, particularly locally recurrent or metastatic mammary/breast carcinomas in an individual, the method comprising administering to the individual a treatment regimen comprising a pharmacologically effective amount of the HLA tumor antigen peptide.

22. HLA tumor antigen peptide corresponding to claim 20 or 21, wherein the individual has not yet received radiation, chemotherapy, and/or hormonal therapy for the breast cancer, particularly locally recurrent or metastatic breast cancer, and/or has not received prior adjuvant chemotherapy in recurrence for 12 months or less since the last dose of a chemotherapy agent.

23. HLA tumor antigen peptide according to one of claims 20 to 22, wherein the treatment regimen is effective to prolong progression-free survival of the individual by at least 2 to 5 years.

24. HLA tumor antigen peptide according to one of claims 20 to 23, that is immunogenic in the patient or group of patients, determined by means of an immunogenicity test, in particular by Western blot, ELISA techniques, in particular by ELISPOT, AFM or immunodetection with microscopic analysis.

25. HLA tumor antigen peptide to any preceding claims, wherein the individual has the haplotype with the subtype A*01:01 and/or A*02:01.

26. HLA tumor antigen peptide according to claim 25, wherein the individual has the haplotype with the subgroup B*44:01 and/or A*18:01.

27. Method for determining pharmaceutically active HLA tumor antigen peptides for use in the treatment or prophylaxis of mammary/breast carcinomas or in a composition according to any one of the preceding claims, comprising monitoring a tissue resection of a patient or group of patients suffering from or suspected of suffering from mammary/breast cancer, the method comprising the following steps: (a) providing a tissue sample of the patient or group of patients, wherein the cells of the tissue sample express class I and/or class II MHC complexes and present them on their surface, wherein said method step (a) of providing the tissue sample does not itself comprise any surgical intervention in the patient or one of the patients of the group of patients; (b) Determine the following parameters using the provided tissue sample from step (a): i) the transcriptome of the tissue sample provided, and Comparison with the transcriptome of a healthy tissue sample from the patient or group of patients to determine up- and/or down-regulated mRNA sequences that differ by a factor of 3 from the threshold in the healthy tissue sample; and ii) the specific HLA haplotype of the patient or patient group; and iii) the exome sequence of the tissue sample provided, and Comparing the exome of the provided tissue sample with the exome of a healthy tissue sample or with a gene database of the patient or patient group to determine somatic mutations, and Determine the HLA tumor antigen peptides that have somatic mutations and are up- or down-regulated relative to the healthy tissue sample, determined in step (i); and Determine the HLA tumor antigen peptides associated with proliferation, invasiveness, angiogenesis, and an increase in cytokeratin production of a mammary/breast carcinoma; and iv) of the ligandome to determine the tumor antigen peptides presented on the surface of the cells of the mammary/breast carcinoma determined in step (iii); and v) the specific binding affinity of the HLA tumor antigen peptides determined in step (iv) against the corresponding MHC complex of the cell of the breast cancer by means of a database and/or a ranking algorithm, and vi) the immunogenicity of the HLA tumor antigen peptides determined in step (iv) by means of an immunogenicity test, in particular by Western blot, ELISA techniques, in particular by ELISPOT, AFM or immunodetection with microscopic analysis; (c) Selection of HLA tumor antigen peptides that meet the criteria according to the parameters defined in step (b) and that are expressed in the cells of the provided tissue sample and presented on the surface of these cells.

28. Method according to claim 27, wherein, after providing the tissue sample from the patient or group of patients in step (a), the BRCA1 and BRCA2 genes are analyzed for the presence of mutations.

29. Method according to claim 27 or 28, wherein the tissue sample provided in step (a) is the tissue sample of a mammary/breast carcinoma.

30. Method according to one of claims 27 to 29, wherein determining whether the HLA antigen peptides are presented on the surface of the cells of the tissue sample of the patient or group of patients provided in step (a) is performed by ultra-high performance liquid chromatography (UHPCL) in conjunction with ESI mass spectrometry (MS).

31. Method according to one of claims 27 to 30, wherein determining a plurality of parameters according to step (b) comprises generating a transcriptome of the tissue sample provided in step (a).

32. Method according to one of claims 27 to 31, wherein determining a plurality of parameters according to step (b) comprises generating an exome sequencing for said tissue sample provided in step (a).

33. Method for producing a pharmaceutical composition according to any of claims 1 to 19, comprising the following steps: (a) Determining at least 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and at least 2 tumor antigen peptides corresponding to MHC class II complexes exposed on the cell surface of cells from a mammary/breast carcinoma of the patient or group of patients to be treated having at least one identical HLA allele, by the determination method of any one of claims 27 to 32; (b) synthesizing the 4 to 8 HLA-A tumor antigen peptides determined in step (a) corresponding to MHC class I complexes and at least 2 tumor antigen peptides corresponding to MHC class II complexes; and (c) Preparing the pharmaceutical composition according to the invention comprising at least the 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes synthesized in step (b), the at least 2 tumor antigen peptides corresponding to MHC class II complexes, and an adjuvant as defined herein.

34. Combination preparation for use in the treatment of breast/mammary carcinoma with simultaneous, separate, or sequential administration, comprising the following two separate preparations (a) and (b): (a) a first preparation comprising, together with a pharmaceutically acceptable carrier or diluent, 4 to 8 HLA-A tumor antigen peptides corresponding to the MHC class I complexes and at least 2 tumor antigen peptides corresponding to the MHC class II complexes of any one of claims 1 to 19, and optionally determined by the method of any one of claims 27 to 32, and (b) a second preparation comprising, together with a pharmaceutically acceptable carrier or diluent, an anticancer agent selected from the group consisting of anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, herbal anticancer agents, platinum-coordinated anticancer complex compounds, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, monoclonal antibodies, interferons, interleukins, biological response modifiers, and other anticancer agents, or a pharmaceutically acceptable salt thereof.

35. Combination preparation according to claim 34, wherein the second preparation is a monoclonal antibody, in particular against an immunosuppressive protein selected from the group consisting of CTLA4, GM-CFS, TReg, EpCam, IDO, MIC, PDL1, Fas and PD1-L, TRAIL.

36. Combination preparation according to claim 34 or 35, the second preparation being an estrogen inhibitor for hormone-positive patients.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0334] Herein shows:

[0335] FIG. 1: Schematic representation of HLA-A-mediated binding of a T cell receptor to a class I MHC molecule, showing the anchor (amino acid) residues of the HLA-A antigenic peptide (7-11 amino acids in length).

[0336] FIG. 2: Schematic representation of HLA-B-mediated binding of a T cell receptor to an MHC molecule, showing the anchor (amino acid) residues of the HLA-B antigenic peptide (12-17 amino acids in length).

[0337] FIG. 3: the development of the specific tumor marker CA 15-3 after specific immune information by the composition according to the invention of tumor antigen peptides according to embodiment 3 (INC 14/1713; star), which depict the epitopes of the primary tumor MC-HER2/Neu. The course depicted comprises 26 weeks.

[0338] FIG. 4: the development of the specific tumor marker CA 15-3 in combination with the liver marker Gamma GT after application of specific immune information by the composition of tumor antigen peptides according to embodiment 3 (INC 14/1713; vertical line), which represent the specific metastatic epitopes of the liver metastases of the scattered MC-HER2/Neu. The imaged course covers 34 weeks.

[0339] FIG. 5: The clinical efficacy of the applied pharmaceutical composition of embodiment 4.

EXEMPLARY EMBODIMENTS

[0340] With the aid of the following figures and embodiments, the present invention will be explained in more detail without limiting the invention to the same.

[0341] The following tables list HLA tumor antigen peptides, all of which have been tested and are immunogenic. SEQ ID Nos: 13 to 48 list some preferred, but not limiting, examples of amino acid sequences of tumor antigen peptides of the invention, each of which is another embodiment of the present invention.

TABLE-US-00001 TABLE 1 [Comparison example]: specific Target protein activity [Sequence Wild-type  towards  position in the HLA T cell overall sequence Tumor antigenic peptide peptide receptor [nM] of the protein] HLA-A Antigenic peptide  CVGRRNYRFFY 132.00 ZDHHC18  (Subtype: A29-02) p.C228Y (SEQ ID NO: 1) HLA-A Antigenic peptide HAVFVQSYY 141.00 SMAD 4 (SEQ ID NO: 2) p.A406V HLA-A Antigenic peptide LLDPEDVD 20986.09   PLEC (SEQ ID NO: 3) p.D220Y HLA-A Antigenic peptide HTDIYANY 142.87 PCSK1 (SEQ ID NO: 4) p.T128M HLA-A Antigenic peptide AVFVQSYY  65.00 SMAD4 (SEQ ID NO: 5) p.A406V HLA-B Antigenic peptide EEEAAAAAAY 147.31 FBX02 (SEQ ID NO: 6) p.E39A HLA-B Antigenic peptide MEVLSQEIVR 5407.15  GRIPAP1 (SEQ ID NO: 7) p.R822W HLA-B Antigenic peptide HMKKMMKDL 131.67 STIM1 (SEQ ID NO: 8) p.D247A HLA-C Antigenic peptide SSTALHPCPF 180.82 PRR21 (SEQ ID NO: 9) p.A63P HLA-C Antigenic peptide LSYLHVHTA 283.98 STS (SEQ ID NO: 10) p.L284F HLA-C Antigenic peptide YSLLSLLHT 1550.46  STK40 (SEQ ID NO: 11) p.T107M HLA-C Antigenic peptide CPFTHGSSPM 160.14 PRR21 (SEQ ID NO: 12) p.C67Y

TABLE-US-00002 TABLE 2 specific Target protein activity [Sequence Amino acid Wild-type Amino towards position sequence of HLA peptide acid T cell in the total Neoantigen peptide the neoantigen (comparative ex- receptor sequence of (Subtype) peptide example) change [nM] the protein] HLA-A Neoantigen peptide YVGRRNYRFFY CVGRRNYRFFY C/Y 16.00 ZDHHC18 (Subtype: A01.01; A29.02; [228-238] C16.01) p.C228Y (SEQ ID NO: 13) HLA-A Neoantigen peptide HVVFVQSYY HAVFVQSYY A/V 32.00 SMAD 4 (Subtype: A01.01; A29.02) [405-413] (SEQ ID NO: 14) p.A406V HLA-A Neoantigen peptide LLDPEDVY LLDPEDVD D/Y 45.67 PLE (Subtype: A01.01) [372-379] (SEQ ID NO: 15) p.D379Y HLA-A Neoantigen peptide HMDIYANY HTDIYANY T/M 24.97 PCSK1 (Subtype: A01.01; A29.02) [174-181] (SEQ ID NO: 16) p.T175M HLA-A Neoantigen peptide VVFVQSYY AVFVQSYY A/V 18.00 SMAD4 (Subtype: A01.01; A29.02) [406-413] (SEQ ID NO: 17) p.A406V HLA-A Neoantigen peptide (TSA) DEDEIKWWW DEDEIEWWW E/K  9.77 TP53BP2 (Subtype: A02.01, B18.01) [1091-1099] (SEQ ID NO: 18) p.E1096K HLA-A Neoantigen peptide (TSA) FVNDKFMPL FVNDKFMPP P/L 12.73 ZC3H12A (Subtype: A02.01) [269-277] (SEQ ID NO: 19) p.P277L HLA-B Neoantigen peptide EEAAAAAAAY EEEAAAAAAY E/A 41.45 FBXO2 (Subtype: A01.01; B44.03) [37-46] (SEQ ID NO: 20) p.E39A HLA-B Neoantigen peptide MEVLSQEIVW MEVLSQEIVR R/W 25.66 GRIPAP1 (Subtype: A01.01; B44.03) [813-822] (SEQ ID NO: 21) p.R822W HLA-B Neoantigen peptide HMKKMMKAL HMKKMMKDL D/A 16.48 STIM1 (Subtype: A01.01; B08.01) [240-248] (SEQ ID NO: 22) p.D247A HLA-C Neoantigen peptide SSTPLHPYPF SSTALHPCPF A/P 44.19 PRR21 (Subtype: A01.01; C16.01) [60-69] (SEQ ID NO: 23) p.A63P HLA-C Neoantigen peptide FSYLHVHTA LSYLHVHTA L/F 38.46 STS (Subtype: A01.01; C16.01) [284-292] (SEQ ID NO: 24) p.L284F HLA-C Neoantigen peptide YSLLSLLHM YSLLSLLHT T/M 42.42 STK40 (Subtype: A01.01; C16.01) [99-107] (SEQ ID NO: 25) p.T107M HLA-C Neoantigen peptide YPFTHGSSPM CPFTHGSSPM C/Y 30.96 PRR21 (Subtype: C16-01) [67-76] (SEQ ID NO: 26) (B08.01) p.C67Y

TABLE-US-00003 TABLE 3 Amino acid Target protein sequence of  Amino specific activity [Sequence position in the HLA tumor acid towards T cell the total sequence  antigen peptide exchange receptor [nM] of the protein] Klasse I HLA-A Antigen peptide TYLPTNASLSF —  137.50 ERB2 (Subtype: A24) [63-72] (SEQ ID NO: 27) Klasse I HLA-Antigen peptide DAVIVKLEI — 7376.79 PKD2 (Subtype: B51.01) [861-869] (SEQ ID NO: 28) Klasse I HLA-Antigen peptide YYLDLSITR R/I 3674.81 PKD2 (Subtype: A24.02) (p.R397I) [391-399] (SEQ ID NO: 29) Klasse I HLA-A Antigen peptide ILFGISLREV —   13.61 MAGEC1 (Subtype: A02) [26-35] (SEQ ID NO: 30) Klasse I HLA-A Antigen peptide KVVEFLAML —   58.22 MAGEC1 (Subtype: A02.01) [150-158] (SEQ ID NO: 31) Klasse I HLA-A Neoantigen peptide FVNDKFMPL P/L   12.73 ZC3H12A (Subtype: A02.01) p.P277L [269-277] (SEQ ID Nr.: 32) Klasse I HLA-A Neoantigen peptide FLLILKRDS N/D 10793.42  ENTHD2 (Subtype: A02.01) p.N104D [96-105] (SEQ ID Nr.: 33) Klasse I HLA-A Neoantigen peptide RTPLSALCV P/L 7172.33 ASIP (Subtype: A02.01)  p.P92L [86-94] (SEQ ID Nr.: 34) Klasse I HLA-B Neoantigen peptide DEDEIKWWW E/K    9.77 TP53BP2 (Subtype: B18.01  p.E1096K [1091-1099] (SEQ ID Nr.: 35)

TABLE-US-00004 TABLE 4 Target protein [Sequence Amino acid position in sequence of  Amino the total another  Immunogenic the HLA tumor acid sequence of target (+ . . . highly antigen peptide exchange the protein] haplotype immunogenic Class II HLA-Neoantigen peptide EDKKIDFSEFLSLLGDI — S100A7 + (SEQ ID Nr: 36) [66-82] Class II HLA-Neoantigen peptide IHREDEDEIKWWWARLN E/K TP53BP2 + (SEQ ID Nr: 37) p.E1096K [1087-2003] Class II HLA-Neoantigen peptide STKYSHKSPQLSVHVTD Y/H CD33 + (SEQ ID Nr: 38) p.Y129H [124-140] Class II HLA-Antigen peptide KYIQESQALAKRSCGLFQ — AFP + (SEQ ID Nr: 39) KLGEYYLQNAFL [403-432] Class II HLA-Antigen peptide VDLIVEYEAFPKPE — KIT + (SEQ ID Nr: 40) [331-344] Class II HLA-Neoantigen peptide ATYSGAGYYLDLSIT R/I PKD2 + DRB1.09*01 p.R3971 [384-398] (SEQ ID Nr: 41) Class II HLA-Neoantigen peptide HGSSFFLLILKRDSAFI N/D ENTHD2 + DRB1.16*01 p.N104D [92-108] (SEQ ID Nr: 42) Class II HLA-Neoantigen peptide YSMKCKNVVPLNDLLLE p.Y537N ESR1 auf + (Subtype: DRB1*03) [526-542] Subtype (SEQ ID Nr: 43) A01 Class 11 HLA-Neoantigen peptide PLQIILMPQVQPGLP p.P931L SMG8 auf + (Subtype: DRB1*03) [918-932] Subtype (SEQ ID Nr: 44) A01 Class II HLA-Neoantigen peptide LLHTEYSLLSLLHMQ p.T102M STK40 auf + (Subtype: DRB1*03) [89-103] Subtype (SEQ ID Nr: 45) A01 Class II HLA-Neoantigen peptide NYLAEEITVDVRDEF p.E625A STON2 auf + (Subtype: DRB1*03) [622-636] Subtype (SEQ ID Nr: 46) A01 Class II HLA-Neoantigen peptide REIDVLERELNVLIF KIAA1804 auf + (Subtype: DRB1*07) Subtype (SEQ ID Nr: 47) A01 Class II HLA-Antigen peptide PSNYQPHQACRITFL p.PPSMQNHIP ARID1A auf + (Subtype: DRB1*07) 1575-1583 [1569-1583] Subtype (SEQ ID Nr: 48) HQACRITFL A01

Example 1: Transcriptome Analysis [mRNA Expression]

[0342] Transcriptome analysis (sequencing) was performed in all patients using PANTHER chip analysis (44K chip) from Agilent Technologies. The 44K chip used contains 44,000 gene probes, so that the activity of >34,000 gene expression markers could be analyzed with this chip per patient in each case. In each case, 1,000 genes with increased relevance were determined 10-fold.

[0343] The evaluation is based on published gene signatures known to the expert.

Example 2: EXOM Sequencing

[0344] Exome sequencing is performed from the standard frozen specimen of the patients from whom the tumor DNA is extracted. Next-generation sequencing of the exome is performed from the tumor sample with coverage of more than 95% of the entire human coding exon region. For this purpose, among others, >290,000 relevant sections of DNA are selectively amplified and sequenced by semiconductor sequencing technology.

[0345] The aim of the analysis is to define possible mutations for the design of an individual tumor antigen peptide immunization. To exclude non-tumor associated variants (SNPs, Single Nucleotide Polymorphism) of the germline, DNA is simultaneously isolated from nucleated blood cells of the patients and comparatively sequenced using the same methodology. The differentiation of potential neoantigen candidates was performed in the following six steps: [0346] 1) Selection of tumor/somatic mutations is reduced by exclusion of non-tumor specific polymorphisms. [0347] 2) The predefined selection of somatic mutations is further restricted to quality parameters, read coverage and influence on the protein sequence. More complex mutations than single amino acid substitutions SNPs, and mutations in non-protein coding regions are excluded. Silent mutations are also excluded. [0348] 3) The mutations defined in this way are expanded to flanking 20s oligopeptides using the known protein sequences (RefGene database). From these, 12×9 oligopeptides each were formed sequentially and the affinity to the pre-known, patient-specific hypervariable HLA-I paratopes was determined (program NetMHC-4.0). Nonapeptides with high affinity to the paratopes, which show a further increase in affinity compared to the wild type sequence due to the mutation (SNP), were selected. [0349] 4) Candidates carrying this mutation as a germline mutation in an exome pool at the same position are excluded (NIH-NHLBI 6500 exome database version-2 program ANNOVAR-Tool2.) [0350] 5) Based on the results of the expression analysis (see findings under embodiment example 1), peptides with a low expression relevance in the tumor can be excluded. [0351] 6) Finally, we will examine in detail whether further polymorphisms may be present in the vicinity of the single base exchange and thus represent further, patient-specific deviations from the hg19 defined genome sequence.
The following is an example of the evaluation of the results of the described mutation analysis and filtration steps for one patient: [0352] 1) Comparison of tumor DNA versus normal DNA [0353] a. “Exome_single_sample_Somatic” using the IonReporter yields: [0354] 37494 variants in normal DNA, and [0355] 37299 variants in tumor DNA. [0356] The paired analysis “AmpliSeq Exome tumor-normal pair” detects and annotates rare somatic variants (SNPs, InDels, CNVs) using statistical analysis in the Ion AmpliSeq Exome Panel (Ion Reporter Software 4.6. Workflow Version: 1.0). This results in: 1477 mutations in the tumor with concurrent wild type categorization in the normal DNA. [0357] These 1477 variants are the starting point for further restrictions. [0358] 2) Restriction of variants to protein-coding SNP (single nucleotide polymorphisms) with amino acid substitution. [0359] Restriction to SNPs [0360] At least 50× sequenced in tumor and min. 20× sequenced in blood [0361] Must not have occurred in the blood in any single run [0362] Must be positioned in an exon [0363] Must result in amino acid exchange (missense mutation) [0364] 3) From these variants, 20s peptides are defined and fed to the HLA paratope affinity analysis NetMHC. [0365] ˜25*12 nonapeptide pairs (each mutant and wild type) are submitted to affinity analysis for analysis in 9 HLA loci each. Out of these 5814 affinities (2907 pairs) are analyzed. In 40 pairs, an affinity at least 2-fold higher in the mutated peptide than in the wild-type peptide is detected.

[0366] Summary of Variants with Increased HLA Affinity [0367] 4) Candidates that carry this mutation as a germline mutation at the same position in an exome pool (NIH-NHLBI 6500 exome database version-2) are excluded (Programm ANNOVAR-Tool3.) [0368] Database filters used: hg19_esp6500siv2_all [0369] 5) Selection from remaining 25 variants with significant mRNA expression (>3 fold versus normal tissue) in alignment with transcriptome

Example 3: Patients with mMC (Metastatic Breast Carcinoma), Type_HER2-Neu

[0370] 1) Clinical history of the patients

[0371] Patient, Female

[0372] Initial clinical findings: invasive ductal bifocal breast carcinoma (breast CA), right, tumor 2 cm to 5 cm in greatest extent (pT2), pN1sn pN1 (3/5) G2, NO, ER-, PR-, HER-2 new: 3+, Ki-67: 55%.

[0373] Treatment Procedure: [0374] 2 weeks later: Neoadjuvant chemotherapy (TCH w3) 6 cycles, trastuzumab; [0375] 6 months later: Irradiation of the right breast+lymphatic drainage area (LAG).

[0376] Tumor completely destroyed by radiotherapy or chemotherapy (CR=complete remission) of tumor, findings: patient discharged from hospital as cured.

[0377] Recurrence: Local recurrence with additional liver metastases (4 foci) occurs 24 months after initial diagnosis; findings: ER−, PR+(60%), HER-2 new: 3+, Ki-67: 80%.

[0378] This recurrent mammary/breast CA occurred with liver metastases and also had significantly greater aggressiveness (Ki-67: 80% versus 55% (initial finding)).

[0379] Prognosis of the clinicians: The patient was discharged from the hospital with a prognosis of OS (overall survival) expected/average of 6 months.

[0380] The well-known fact that in breast CA, second primary malignancy (SPM) flared up again is associated with worse prognosis and with worse OS (see “Breast cancer survivors face excess risk for second primary cancer in SEER analysis,” Wei J L & al,

[0381] Int J Clin Oncol, Mar. 19, 2019), likely triggered by prior chemotherapy,

[0382] Treatment Procedure: [0383] 2 months after diagnosis: surgical removal of the mammary gland and adjacent tissues of the right breast (mastectomy) because of a local recurrence that had occurred there. [0384] 4 months after diagnosis: Appearance of bone metastases (osseous metastases (spine)) and bronchial carcinoma (suspicious LK pulmonary hilus); [0385] 9 months after diagnosis: surgical removal of the mammary gland and adjacent tissues of the left breast.

[0386] Tumor progression despite trastuzumab or T-DM1 continuous therapy [0387] 2) Individualized Immune Information Therapy: Analytics

[0388] After therapy/treatment procedures under point 1 had been carried out without success, the immunological therapy approach of informing the patient's own immune system by means of application of tumor-associated and tumor-specific HLA antigen peptides in the form of synthesized peptides was used as part of a curative trial.

[0389] The focus was on the cellular immune defense, i.e. the activation of the endogenous cytotoxic CD8+ T cells, which as naive T cells are able to recognize virtually all conceivable pathogenic as well as malignant amino acid sequences (the so-called “targets”) by means of a highly differentiated receptor system and thus develop into effector cells. These effector T cells can destroy the tumor cells recognized via the HLA-present antigens by secreting granzyme and perforin.

[0390] For this purpose, data were collected from biopsied tumor tissue of the patients via “next generation sequencing” (NGS) and “liquid chromatography-coupled tandem mass spectrometry” (LC-MS/MS) using the following three steps [0391] the mRNA expression of all coding gene regions (transcriptome—step (a)), [0392] the tumor-specific somatic miss-sense mutations as well as via (exome sequencing—step (b)) [0393] the HLA-restricted ligands of the HLA classes I+II presented by the tumor cells (HLA ligandome—step (c))
to step (a): The mRNA expression data (approximately 40,000) were compared with the expression levels of healthy tissue and filtered with respect to [0394] significant (>3-fold) deviation of the expression values, and further [0395] with regard to the significance of the genes concerned for tumor development [0396] according to the criterion that these genes may not be expressed or only slightly expressed in other tissue types [0397] furthermore, with regard to the group of cancer-testis antigens, which are generally not expressed in healthy mammary tissues of adults, notable expressions in the tumor were recorded, since these may have the quality of a tumor-specific antigen

[0398] This resulted in a separate data set of possible HLA tumor antigen peptides (number 86), which were weighted with respect to their expression abnormalities/deviations from normal and their known importance for tumor proliferation->factor 3 vs. normal; importance for tumor development: growth factors, angiogenesis factors, metastasis factors.

to step (b): The somatic miss-sense mutations (single nucleotide variants (SNV) with an exchange of one amino acid as well as frame-shift mutations) (number: 57) were combined in another data set, since they are potentially significant (neo)-antigens in character and thus highly tumor-specific.
to step (c): The HLA-restricted ligands (amino acid sequences of 9-10 AS (corresponding to MHC class I complexes) (number:1100) and sequences of 12-15 AS (corresponding to MHC class II complexes) (number:730) were screened using information from specialized databases for [0399] already appeared in healthy tissue (=negative) [0400] In protein match with promising sequences from transcriptome and exome, whether sequences of the identified tumor-associated HLA antigen peptides are associated with proliferation, invasiveness, angiogenesis, and/or an increase in cytokeratin production of mammary/breast carcinoma.

[0401] In parallel to these described tumor tissue examinations, the genetic haplotype, the alleles of the HLA antigen peptides corresponding to the patient's MHC class I complexes were determined. The patient-individual result resulted in the following assignment:

HLA-A*02:01, A*24:02, B*18:01, B*51:01, C*07:01, C*15:02; HLA-DRB1*09:01; DRB1*16:01, DQB1*03:03, DQB1*05:02.

[0402] (With the support of HLA-A*02:01 in connection wirh B*18:01 this patient represents about 40% of the population living in Europe (Caucasian)). [0403] 3) individualized immune information therapy: target selection
to step (a): From the data sets of the transcriptome, amino acid sequences of the respective proteins were first used to select amino acid sequences of the HLA tumor antigen peptides corresponding to the MHC complexes (nonamers) with the highest allelic affinities (specific activity towards T cell receptor [nM]). This selection criterion is used to algorithmically determine the probability with which the respective HLA tumor antigen peptide is presented in vivo on the corresponding MHC complexes (a first prerequisite for a possible cellular immune response).
to step (b): From the data sets of the mutation tests, nonameric variants involving amino acid exchange were determined with respect to the highest affinities (specific activity towards T cell receptor [nM]) based on the alleles of the patient. Also, polymers of 17 amino acids (oligopeptides) were determined under the affinity criterion for this purpose. This selection criterion algorithmically determines the probability with which the respective tumor antigen peptide corresponding to the MHC complexes is presented in vivo on the corresponding MHC complexes (a second prerequisite for a possible cellular immune response).
step (c): Based on the new data sets identified in step (a) and (b), with the addition of the HLA-restricted ligands (the data set of the ligandome), a selection of HLA tumor antigen peptides corresponding to MHC class I complexes and corresponding to MHC class II complexes was made, which were the most promising epitope candidates for eliciting a cellular immune response, both individually and especially in their combination. [0404] 4) Individualized Immune Information Therapy: Peptide Synthesis and Delivery Solution
step (a): The peptide concepts selected for the custom application solution were synthesized as chemical peptides.
step (b): the following 7 HLA-A tumor antigen peptides corresponding to MHC class I complexes and 2 HLA tumor antigen peptides corresponding to MHC class II complexes were mixed in a 33% DMSO/H.sub.2O application solution and divided into 24 vial units of 1 ml each.

[0405] Sequences of the Nine Tumor Antigen Peptides

TABLE-US-00005 Identification Nr. Amino acid sequence number Class  1 ILFGISLREV SEQ-ID Nr.: 30 HLA-A  2 KVVEFLAML SEQ-ID Nr.: 31 HLA-A  3 DEDEIKWWW SEQ-ID Nr.: 35 HLA-B  4 FVNDKFMPL SEQ-ID Nr.: 32 HLA-A  5 TYLPTNASLSF SEQ-ID Nr.: 25 HLA-A  6 FLLILKRDS SEQ-ID Nr.: 33 HLA-A  7 DAVIVKLEI SEQ-ID Nr.: 28 HLA-A  8 RTPLSALCV SEQ-ID Nr.: 34 HLA-A  9 EDKKIDFSEFLSLLGDI SEQ-ID Nr.: 36 class II 10 STKYSHKSPQLSVHVTD SEQ-ID Nr.: 38 class II 11 HGSSFFLLILKRDSAFI SEQ-ID Nr.: 42 class II 12 KYIQESQALAKRSCGLFQKLGEYYLQNAFL SEQ-ID Nr.: 39 Klasse II (Oligopeptide)

[0406] Dose per tumor antigen peptide: 500 μg [0407] 5) Individualized Immune Information Therapy: Application Protocol

[0408] Pretreatment: 300 mg/m.sup.2 cyclophosphamide (single infusion); 3 days before first injection

[0409] Application: intra-dermal (i.d.)

[0410] Application site: left and right upper arm

[0411] Administration plan: 23 vaccinations on days 1, 2, 3, 8, 15, 22, 36, 50, 71 and on every 3 weeks until day 365.

[0412] Adjuvants added per application: 12.5 mg imiquimod in 250 mg cream (Aldara) topically at injection site; 200 μg ipilimumab (Yervoy), because CTLA4 elevated, alternating with 300 μg nivolumab (Opdivo), because PD1 or PD-L1 elevated, subcutaneously (s.c.) directly adjacent to application site [0413] 6) Individualized immune information therapy: 2 evidences of clinical effects.

[0414] Based on two significant examples, the clinical efficacy of the applied immunotherapy in the presented case 1 can be shown below:

[0415] FIG. 3 shows the evolution of the Specific Tumor Marker CA 15-3 in combination with the liver marker Gamma GT after specific application (i.d.) specific pharmaceutical composition of immune information by BITAP peptides (star line), which are the specific metastatic epitopes of liver metastases of the scattered mMC-HER2/New (INC 14/1713).

[0416] The course shown covers 34 weeks.

the development of the specific tumor marker CA 15-3 after specific immune information by the composition according to the invention of tumor antigen peptides (star) mapping the epitopes of the primary tumor MC-HER2/Neu (INC 14/1713). The course shown covers 26 weeks.

[0417] Beispiel 4: Patients with mIBC with lymphangosis carinomatosis (metastatic inflammatory breast cancer) [0418] 1) Clinical history of the patients

[0419] Patient, female (46 years)

[0420] The initial clinical findings: inflammatory breast carcinoma right with extensive lymphangiosis carcinomatose; Initial tumor stage pT4, pn3a (24/29)(Level 11:14/19, Level III: 10/10), MO, L1, VO, G2. HR+, PR+, Androgenr.+, HER2/neu−

[0421] Recurrence: Postoperatively, a first recurrence already after 3 months: Progression of findings intramammary in the area of the right lower quadrant with significant increase in consistency of the breast tissue. Skin infiltration crossing the midline in the area of the thoracic wall as well as locoregional tumor infiltration of the axillary adipose tissue and the lateral thoracic wall along the pectoralis major muscle on the right.

[0422] After 5 months locoregional tumor recurrence, with extension into the axilla.

[0423] Chemotherapy from month 6 to month 11, discontinued after 5 cycles if tumor progression during chemotherapy. Locoregional tumor recurrence, 7×11 cm along ventrolateral thoracic wall. Skin and subcutaneous tissue extensively infiltrated and thickened.

[0424] Hormone therapy with aromatase inhibitor and irradiation of the right breast, chest wall and supraclavicular/cervical right side.

[0425] Prognosis of the clinicians: Overall survival for an expected 3-4 months. (Inflammatory breast carcinoma is a relatively rare, particularly aggressive form of breast carcinoma, which in this case 2 had already spread to the lymphatics (24 of 29 lymph nodes were already affected at initial diagnosis)).

[0426] Treatment process according to the invention: After all therapies available in the standard and regular canon of oncological medicine had been carried out without success, the immunological therapy approach of informing the patient's own immune system by means of application of tumor-associated and tumor-specific antigens in the form of synthesized peptides was used in the context of a curative trial.

[0427] The focus was on the cellular immune defense, i.e. the activation of the endogenous cytotoxic CD8+ T cells, which as naive T cells are able to recognize virtually all conceivable pathogenic as well as malignant amino acid sequences (hereinafter the “targets”) by means of a highly differentiated receptor system and thus develop into effector cells. These effector T cells can destroy the tumor cells recognized via the HLA-present antigens by secretion of granzyme and perforin.

[0428] Activation of these immune cells occurs via HLA class I ligands, i.e. antigens presented as sequences of 8-10 amino acids restricted on class I HLA molecules.

[0429] In months 14-84 after initiation of individualized immunoinformatics therapy, additional local recurrences (lymph nodes) occurred, each of which was surgically removed. However, distant metastasis was prevented. [0430] 2) Individualized Immune Information Therapy: Analytics

[0431] To identify tumor-associated and tumor-specific antigen peptides, data were determined from biopsied tumor tissues of the patients using next generation sequencing (NGS) and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) in the following three steps: [0432] the mRNA expression of all coding gene regions (transcriptome—step (a)), [0433] the tumor-specific somatic miss-sense mutations as well as via (exome sequencing—step (b)) [0434] the HLA-restricted ligands of the HLA classes I+II presented by the tumor cells (HLA ligandome—step (c))
to step (a): The mRNA expression data (approximately 40,000) were compared with the expression levels of healthy tissue and filtered with respect to [0435] significant (>3-fold) deviation of the expression values, and further [0436] the significance of the genes concerned for tumor development [0437] the criterion that these genes may not be expressed or only slightly expressed in other tissue types [0438] furthermore, with regard to the group of cancer-testis antigens, which are generally not expressed in healthy mammary tissues of adults, notable expressions in the tumor were recorded, since these may have the quality of a tumor-specific antigen

[0439] This resulted in a separate data set of possible HLA tumor antigen peptides (number 86), which were weighted with respect to their expression abnormalities/deviations from normal and their known importance in tumor proliferation.

to step (b): The somatic miss-sense mutations (single nucleotid variants (SNV) with an exchange of one amino acid as well as frame-shift mutations) (number: 41) were combined in another data set, since they are potentially significant (neo)-antigens in character and thus highly tumor-specific.
to step (c): The HLA-restricted ligands (amino acid sequences of 9-10 AS (corresponding to MHC class I complexes) (number:1321) and sequences of 12-15 AS (corresponding to MHC class II complexes) (number:863) were screened using information from specialized databases for [0440] already appeared in healthy tissue (=negative) [0441] protein match with promising sequences from transcriptome and exome, whether sequences of the identified tumor-associated HLA antigen peptides are associated with proliferation, invasiveness, angiogenesis, and/or an increase in cytokeratin production of mammary/breast carcinoma.

[0442] In parallel to these described tumor tissue examinations, the genetic haplotype, the alleles of the HLA antigen peptides corresponding to the patient's MHC class I complexes were determined. The patient-individual result resulted in the following assignment:

HLA-A*01:01, A*29:02, B*08:01, B*44:03, C*07:01, C*16:01; HLA-DRB1*03:01; DRB1*07:01, DRB3*01:01, DRB1*03:01, DRB4*01:01, DQB1*02:01, DPB1*01:01

[0443] (With the support of HLA-A*01:01 in connection with B*08:01 this patient represents about 25% of the population living in Europe (Caucasian)). [0444] 3) Individualized Immune Information Therapy: Target Selection
to step (a): From the data sets of the transcriptome, amino acid sequences of the respective proteins were first used to select amino acid sequences of the HLA tumor antigen peptides corresponding to the MHC complexes (nonamers) with the highest allelic affinities (specific activity towards T cell receptor [nM]). This selection criterion is used to algorithmically determine the probability with which the respective HLA tumor antigen peptide is presented in vivo on the corresponding MHC complexes (a first prerequisite for a possible cellular immune response).
to step (b): From the data sets of the mutation tests, nonameric variants involving amino acid exchange were determined with respect to the highest affinities (specific activity towards T cell receptor [nM]) based on the alleles of the patient. Also, polymers of 17 amino acids were determined under the affinity criterion for this purpose. This selection criterion algorithmically determines the probability with which the respective tumor antigen peptide corresponding to the MHC complexes is presented in vivo on the corresponding MHC complexes (a second prerequisite for a possible cellular immune response).
step (c): ased on the new datasets identified in step (a) and (b), with the addition of the HLA-restricted ligands (the dataset of the ligandome), a selection was made of HLA tumor antigen peptides corresponding to MHC class I complexes and corresponding to MHC class II complexes that were the most promising epitope candidates for eliciting a cellular immune response, both individually and, most importantly, in combination. [0445] 4) Individualized Immune Information Therapy: Peptide Synthesis and Delivery Solution
step (a): The peptide concepts selected for the custom application solution were synthesized as chemical peptides.
step (b): the following 9 HLA-A tumor antigen peptides corresponding to MHC class I complexes and 2 HLA tumor antigen peptides corresponding to MHC class II complexes were mixed in a 33% DMSO/H.sub.2O application solution and divided into 6 vial units of 1.5 ml each.

[0446] Sequences of the Nine Tumor Antigen Peptides

TABLE-US-00006 Identification Nr. Amino acid sequence number Class  1 LLDPEDVY SEQ-ID Nr.: 15 HLA-A  2 HVVFVQSYY SEQ-ID Nr.: 14 HLA-A  3 HMDIYANY SEQ-ID Nr.: 16 HLA-A  4 YVGRRNYRFFY SEQ-ID Nr.: 13 HLA-A  5 VVFVQSYY SEQ-ID Nr.: 17 HLA-A  6 HMKKMMKAL SEQ-ID Nr.: 22 HLA-B  7 EEAAAAAAAY SEQ-ID Nr.: 20 HLA-B  8 SSTPLHPYPF SEQ-ID Nr.: 23 HLA-B  9 MEVLSQEIVW SEQ-ID Nr.: 21 HLA-B 10 YSMKCKNVVPLNDLLLE SEQ-ID Nr.: 43 class II 11 NYLAEETVDVRDEF SEQ-ID Nr.: 46 class II 12 LLHTEYSLLSLLHMQ SEQ-ID Nr.: 45 class II

[0447] Dose per peptide: 300 μg

[0448] Total peptide content per application dose (vial): 3.3 mg [0449] 5) Individualized Immune Information Therapy: Application Protocol

[0450] Pretreatment: 3 million units of IFN alpha 2b (Roferon), 4 days before first injection

[0451] Application: subcutaneous (s.c.)

[0452] Application site: left and right upper arm, left and right hip

[0453] Administration plan: 6 injections on days 1, 8, 22, 50, 180 and 360

[0454] Adjuvants added per application: Montanide ISA 51 VG (1.5 ml), mix 1:1 with peptide-vial (1.5 ml); 300 μg nivolumab (Opdivo), as PD1 or PD-L1 elevated, sub-cutaneous (s.c.) directly adjacent to injection site, 30 min before peptide/montanide injection. [0455] 6) Individualized immune information therapy: evidence of clinical effects

[0456] The clinical efficacy of the applied immunotherapy in the illustrated embodiment 4 is shown in FIG. 5.

[0457] The graph represents the development of the tumor markers CEA (bar 1) and CA15-3 (bar 2) as well as the liver value gamma-GT (bar 3) and the leukocyte count (bar 4) during a period of 8 months after application of the pharmaceutical composition according to embodiment 4. This was preceded by aggressive tumor progression—essentially based on inflammatory lymphangiosis carcinomatosis. The application was performed with a 1:1 mixture of Montanide ISA 51 VG with the peptide cocktail. The volume was 3 ml and was applied s.c. at the 4 different application loci (see pt 5). In this peptide composition, it is the initial application (followed by 4 further applications; see arrows in FIG. 5).

[0458] The 10 peptides contained in the pharmaceutical composition according to embodiment 4 are tumor antigen peptides (or neoantigens). By information, antigens Nos. 1-4 and 10 have become epitopes, i.e. succeeded in activating the corresponding T cell receptors (TCR) and developing effector and memory cells