Class i and class ii hla tumor antigen peptides for the treatment of breast cancer

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prevention of breast cancer / mammary carcinoma, in particular locally recurrent or metastatic breast cancer, in a patient or patient group suffering from or suspected of suffering from breast cancer, 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 specific 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 class I and / or class II HLA peptides; a method for the manufacture of a pharmaceutical composition according to the invention; and the use of a pharmaceutical composition according to the invention for the manufacture of a pharmaceutical composition for the treatment of malignancies, leukemias and tumors.

Description

TECHNICAL FIELD

[0001] The present invention relates to a pharmaceutical composition for the treatment or prevention of breast cancer / mammary carcinoma, in particular locally recurrent or metastatic breast cancer / mammary carcinoma, in a patient or a group of patients suffering from or suspected to suffer from breast cancer / mammary carcinoma, to a method for the preparation of a pharmaceutical composition according to the present invention, and to a method for determining the regression, progression or occurrence of a breast cancer / mammary carcinoma disease, said pharmaceutical composition comprising at least 4 to 8 human leukocyte antigen (HLA)-A tumor antigen peptides corresponding to MHC class I complexes (herein also referred to as "HLA-A restricted tumor antigen peptides") and at least 2 tumor antigen peptides corresponding to MHC class II complexes (herein also referred to as "HLA restricted tumor antigen peptides", "HLA", "amino acid sequences of the invention", "compounds of the invention" and "polypeptides of the invention", respectively), wherein the HLA tumor antigen peptides are tumor-specific or tumor-associated HLA antigen peptides, and to the use of the same, including combinations thereof (i.e. binding to or having an affinity for these complexes), combination preparations (or parts thereof), for determining / identifying at least one HLA antigen peptide corresponding to MHC class I complexes and / or MHC class II complexes for use in a pharmaceutical composition. BACKGROUND

[0002] Despite the exhaustion of interdisciplinary approaches and traditional therapies, cancer remains one of the leading causes of death. Generally, cancer is treated by established methods, such as surgical tumor removal (resection), chemotherapy and / or radiotherapy.

[0003] In particular, drug resistance developed by cancer cells during chemotherapy and / or radiotherapy largely prevents the complete removal of all cancer cells from the body of a subject.

[0004] Newer treatment concepts aim at the integration of the patient's own immune system into the overall treatment concept by using specific measures, such as antibody therapy against immunosuppressive agents and recombinant immunoinformatics (i.e. therapy using information carriers). A prerequisite for the success of this strategy is that the immune system of the subject recognizes tumor-specific or tumor-associated antigens or epitopes, the effector function (by immune cells) of which is to be enhanced. Tumor cells differ biologically significantly from non-malignant cells of their origin. These differences are due to genetic alterations acquired during tumor development, which lead, among other things, to the formation of qualitatively or quantitatively altered molecular structures in tumor cells. If such tumor-associated structures are recognized by the specific immune system of the tumor-bearing host, they are referred to as tumor-associated epitopes.

[0005] Cancer / testis antigens (CTAs) refer to a group of tumor-associated proteins which are e.g. expressed by tumors of various histological origin (Fratta et al., Molecular Oncology, 5(2), April 2011, 164-182). In healthy adult vertebrates, the expression of these proteins is restricted to male germ cells. However, in cancer, the expression of these developmental antigens is frequently reactivated, thus can serve as a site for immune activation. Many CTAs are oncogenes, involved in cellular processes such as cell growth and division, inhibition of apoptosis and metastasis. They are usually responsible for tumorigenesis and malignant transformation, thus are classified as tumor antigens. The expression of CTAs in different malignancies is heterogeneous, often associated with tumor progression, which is why they can also serve as biomarkers for tumor disease progression. HLA antigen peptides of such CTAs are usually presented as degradation products on the tumor cell surface. It is known that this presentation of CTA-derived HLA antigen peptides can be used to develop new therapeutic approaches in combination with anti-CTA targeted immunotherapy.

[0006] Human leukocyte antigens (also known as HLA system, HL antigens, histocompatibility antigens, human leukocyte antigens) are a group of human genes that are essential for immune system function. The HLA system is known as the major histocompatibility complex (MHC) and is present in all vertebrates.

[0007] There are two types of MHC, i.e. class I MHC molecules (also referred to herein as “class I MHC complex” or “class I HLA complex”) and class II MHC molecules (also referred to herein as “class II MHC complex” or “class II HLA complex”). Both are present on the cell surface of all nucleated cells in the body of a vertebrate. MHC molecules consist of two polypeptide chains, one heavy a chain and one light b chain (see also Figure 1 and 2 ).

[0008] Class I MHC molecules are expressed on the cell surface of all nucleated cells and are recognized by CD8+ T cells (also known as T killer cells or cytotoxic T cells). Class II MHC molecules are mainly exposed on the cell surface of antigen-presenting cells and are recognized by CD4+ T cells (also known as T helper cells).

[0009] In blood cells, class I MHC molecules are exposed on the cell surface of platelets (also thrombocytes), but not on red blood cells. Their function is to present peptide fragments of non-self proteins on their cell surface and to transfer them from the cell into killer T cells (also cytotoxic T cells) to trigger an immediate immune system response against the specific non-self antigen presented by means of the MHC class I proteins. Since the class I MHC molecules present peptides derived from cytosolic proteins, the presentation pathway of class I MHC molecules is often referred to as the cytosolic or endogenous pathway.

[0010] Here, the HLA antigen peptides act as mediators between the corresponding MHC complex presented on the cell surface and the T cell receptor.

[0011] Class I HLA complexes (HLA-A, B and C) present intracellular antigen peptides (herein "HLA antigen peptides corresponding to MHC class I complexes" or "type 1 HLA antigen peptides" comprising 7 to 11, mainly 9 amino acids in their sequence, so-called nonamers) to T killer cells (also cytotoxic T cells), whereas HLA class II complexes (HLA-DR, DQ and DP) present exogenous antigen peptides (here "HLA antigen peptides corresponding to MHC class II complexes" or "type 2 HLA antigen peptides" comprising more than 11 amino acids, preferably 12 to 17 amino acids in their sequence) to T helper cells. In humans, class I HLA antigen peptides corresponding to MHC class I are subdivided into HLA-A, HLA-B and HLA-C antigen peptides.

[0012] It is known from the prior art that the binding of HLA antigen peptides corresponding to MHC class II complexes to the peptide binding pocket of the corresponding class II HLA complex takes place mainly via discrete anchor residues of the amino acids 1, 4, 6 / 7 and 9 of the HLA antigen peptide (see, for example, Sinigaglia and Hammer (1995), J. Exp. Med., 181, 449-451). According to the prior art, the interaction between the peptide binding pocket motif of the class II HLA complex and the discrete anchor residues of the amino acids 6 and 9 of the HLA antigen peptide corresponding to MHC class II complexes is particularly preferred. The amino acid residues of the amino acids 2, 3, 5, 7 and 8 of the respective HLA antigen peptide can be used for the interaction with the T cell receptor (Sant'Angelo et al. (2002), Recognition of core and flanking amino acids of MHC class II-bound peptides by the T-cell receptor, Eur J Immunol, 32(9), 2510-20).

[0013] In contrast, for HLA antigen peptides corresponding to MHC class I complexes, amino acids positions 1, 2 and 9 have been postulated as the main anchor residues associated with the corresponding class I HLA complex (see, e.g., Binkowski et al. (2012), PLoS ONE, 7(8), e41710; Yamada (1999), Tissue Antigens, 54(4), 325-32).

[0014] A new study in the USA is currently investigating the specific efficacy of only one HLA antigen peptide in the treatment of cancer (see WO 2013 / 135266, Inderberg-Suso et al. (2012), Oncoimmunology., 1(5), 670-686 and Slingluff (2011), Cancer J., 17(5), 343-350). Thus, the range of treatment is very limited and only one HLA antigen peptide is applied, the efficacy of which is questionable.

[0015] This is particularly true in tumor entities such as breast cancer / breast carcinoma, which is characterized by low homogeneity (i.e. high heterogeneity) and a lack of initial immunogenicity compared to other tumor entities. Thus, a clinically effective targeted immunological information and stimulation therapy relies on a multi-factorial information approach.

[0016] Thus, a major disadvantage of cancer immunotherapies is that they are based on the fact that the individual mutational pattern (profile) of the tumor of each cancer patient has to be decoded. A synthetic vaccine, e.g. an RNA-based vaccine, is then produced to match the determined profile of the mutational pattern of each individual patient (so-called vaccine production). The vaccine obtained in this way can only be used for the individual treatment of this particular patient.

[0017] Thus, in principle, these new vaccines for cancer immunotherapy are not applicable to other patients with the same tumor, but only to the single patient whose mutational panel has been previously analyzed for vaccine production. SUMMARY

[0018] Thus, in contrast to a fully individualized cancer immunotherapy, it is the task of the present application to provide a pharmaceutical composition for different subjects or a specific predetermined patient group (i.e. for a specific patient group having at least one identical HLA allele), which patients have an overlap in the mutational panel of the malignant or neoplastic tissue (breast cancer / breast carcinoma) (derivative patient group).

[0019] It is therefore an object of the present application to provide pharmacologically active agents and pharmaceutical compositions comprising such active agents, which are useful for the diagnosis, prevention and / or treatment of breast cancer and other diseases and conditions listed herein; and to provide methods of diagnosing, preventing and / or treating such cancers, comprising administering and / or using such agents and compositions.

[0020] In particular, it is an object of the present application to provide such pharmacologically active agents, pharmaceutical compositions and / or methods, which have certain advantages compared to the active agents, compositions and / or methods currently used and / or known in the state of the art. These advantages will be further described below.

[0021] In particular, it is an object of the present application to provide therapeutically active HLA antigen peptides which can be used as pharmacologically active HLA antigen peptides or as pharmacologically active agents, and to provide pharmaceutical compositions containing said HLA antigen peptides for the diagnosis, prevention and / or treatment of breast cancer and other diseases and conditions, in particular cancers as described herein, and to provide methods of diagnosing, preventing and / or treating such diseases and conditions, comprising administering and / or using such therapeutically active HLA antigen peptides and compositions.

[0022] In particular, it is a specific task of the present application to provide such HLA antigen peptides which are suitable for prophylactic, therapeutic and / or diagnostic use in warm-blooded animals, in particular mammals, most particularly humans.

[0023] According to the present application, these tasks are accomplished by a pharmaceutical composition according to claim 1 for the treatment or prevention of breast cancer, in particular locally recurrent or metastatic breast cancer, in a patient or group of patients suffering from or suspected of suffering from breast cancer, comprising a pharmacologically effective amount of 4 to 8, preferably 4, 5, 6, 7 or 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and at least 2, preferably 1, 2, 3 or 4 tumor antigen peptides corresponding to MHC class II complexes, characterized in that the HLA tumor antigen peptides are tumor-specific or tumor-associated HLA antigen peptides, wherein the HLA tumor antigen peptides in particular comprise the sequence in SEQ ID NO. 13 to SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 32 to SEQ ID No. 48.

[0024] Further advantageous embodiments are given in the dependent claims.

[0025] According to a particularly preferred embodiment of the present application, the pharmaceutical composition consists only of the following components: a carrier liquid (preferably water, a pharmaceutically acceptable saline solution and / or pharmaceutically DMSO, such carrier liquid compositions are known to the skilled person and are, for example, in the range of about 30% DMSO and 70% water), a pharmacologically effective amount of 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and 2 tumor antigen peptides corresponding to MHC class II complexes dissolved or suspended in the carrier liquid, and an adjuvant.

[0026] The pharmaceutical compositions and suitable dosage forms for applying the HLA tumor antigen peptides having a pharmaceutical activity are prepared according to standard procedures known in the prior art and are readily adaptable to any new or improved method of their preparation.

[0027] It is particularly advantageous that the above combination of pharmacologically effective amounts of HLA tumor antigen peptides administered to a patient or patient group suffering from breast cancer / mammary carcinoma effectively reduces the CA 15-3 level. CA 15-3 (Cancer Antigen 15-3) is a so-called glycoprotein used as a specific tumor marker in breast cancer. The CA 15-3 value is a laboratory value which is significantly above a threshold value in certain cancers, especially breast cancer / mammary carcinoma. In healthy individuals, the CA 15-3 threshold value is below 31 enzyme units per milliliter (< 31 U / ml). Preferably, the administration of pharmacologically effective amounts of tumor antigen peptides to a patient or patient group suffering from breast cancer / mammary carcinoma reduces the CA 15-3 value to below 60 U / ml, more preferably to below 50 U / ml, most preferably to below 40 U / ml, and to the normal value (< 31 U / ml).

[0028] It is particularly preferred that the pharmacologically effective amounts of HLA tumor antigen peptides are administered to a patient or patient group so as to effectively prolong the progression-free survival of the individual, preferably for at least 2 to 5 years.

[0029] In contrast to conventional methods, a surprising finding of the present application is that K D Tumor antigen peptides having a value in the range of 50 to 500 nM trigger effector cells (i.e. cytotoxic T cells) contrary to what is assumed in conventional in silico binding prediction models, which assume a low binding. In contrast, it is particularly advantageous for the efficacy of the tumor antigen peptides for treatment in a patient or patient group having at least one identical HLA allele that the HLA tumor antigen peptides used are immunogenic, which can be determined in advance using immunogenicity tests (for example, by Western blot, ELISA techniques, in particular by ELISPOT, preferably by interferon-gamma, interferon-alpha or interleukin (IL-2), or immunodetection using microscopic analysis).

[0030] As described herein, but not limited to any explanation, mechanism of action or assumption in the present application, two different classes of amino acid sequences of the present application were determined (in particular in the detection method described in Example 3 below) based on the ability to enhance the interaction of class I MHC complex and class II MHC complex, respectively, with at least one T cell receptor. These two classes of amino acid sequences of the present application are (as described below):

[0031] - "HLA tumor antigen peptide corresponding to MHC class I complex": (see in particular preferred examples in Tables 1-3).

[0032] - "HLA tumor antigen peptide corresponding to MHC class II complex": (see in particular preferred examples in Table 4).

[0033] Advantageously, the use / application of the pharmaceutical composition according to the present application or the specific combination of tumor antigen peptides corresponding to MHC class I complex and MHC class II complex contained therein to a patient or to a group of patients having at least one identical HLA allele is not only passive immunization (as in the case of treatment with antibodies, e.g. Herceptin), but active immunization (i.e. specific activation of T cells or B helper cells by the information carrier). Due to the specific activation of class II MHC molecules, CD4+ T cells (also called T helper cells) and B cells, which are mainly exposed at the cell surface of antigen-presenting cells, by tumor antigen peptides corresponding to MHC class II complex, the CD4+ T cells (also called T helper cells) and B cells are specifically activated.

[0034] In order to bind to a T cell receptor, the HLA tumor antigen peptides of the present application usually have one or more amino acid residues or one or more fragments of amino acid residues (i.e. each "fragment" comprises two or more amino acid residues adjacent to or very close to each other, i.e. in the primary or tertiary structure of the amino acid sequence) in their amino acid sequence, by which the amino acid sequences of the present application can bind to a T cell receptor (in particular to its binding pocket), the amino acid residues or the parts of amino acid residues thus form an "anchor" for binding to the T cell receptor (also referred to herein as "anchor amino acid").

[0035] The determination of this "anchor" can be assessed, for example, by computer simulation methods (e.g. artificial neural network NNAlign used in the publicly available NetMHC-4.0) or by targeted mutations (insertions or substitutions in the amino acid sequence of the HLA tumor antigen peptide).

[0036] The HLA tumor antigen peptides provided by the present application are preferably in substantially isolated form (as defined herein) or form part of a protein or polypeptide which can comprise or essentially consist of one or more HLA tumor antigen peptides of the present application and which can optionally further comprise one or more pharmacologically active HLA tumor antigen peptides (all optionally linked in a so-called oligopeptide by one or more linkers). For example, but not limited to, the tumor antigen peptides of the present application can be used as binding moieties in such proteins or polypeptides which can optionally include one or more additional amino acid sequences which can be used as binding moieties (i.e. against one or more targets other than T cell receptors) to provide monovalent, multivalent or multispecific polypeptides of the present application, respectively, as described herein. Such proteins or polypeptides can also be in substantially isolated form (as defined herein).

[0037] As disclosed herein, a pharmaceutical composition comprising a specific combination of HLA tumor antigen peptides corresponding to MHC class I complexes and HLA tumor antigen peptides corresponding to MHC class II complexes has proven to be particularly advantageous as it is able to specifically activate T cells as well as specifically activate B cells.

[0038] In other embodiments, the present application also relates to a pharmaceutical composition, a kit (or parts thereof), a method for determining / identifying pharmacologically active HLA antigen peptides corresponding to class I and / or class II MHC complexes, a method for the preparation of a preparation according to the present application, and the use of a preparation according to the present application for the manufacture of a pharmaceutical composition for the treatment of cancer, in particular breast cancer.

[0039] In particular, the polypeptides and pharmaceutical compositions of the present application can be used for the prevention and treatment of cancer, in particular breast cancer, which is characterized by mutated (herein referred to as "amino acid substitutions") altered wild-type HLA antigen peptides corresponding to MHC class I complexes and / or corresponding to MHC class II complexes (so-called HLA tumor antigen peptides, as defined herein).

[0040] Generally, prior to the present application, the cancer of a patient or patient group was treated by conventional methods such as chemotherapy, radiotherapy and / or cancer immunotherapy.

[0041] The traditional methods for treating the cancer of the present invention are, for example, surgical tumor removal (resection), chemotherapy and / or radiotherapy, wherein two or even all three treatment methods are often applied to the subject at the same time. In cancer immunotherapy methods, active and passive immunization are distinguished. In active immunization, the subject is given a substance that is intended to trigger an immune response in its immune system. In passive immunization, antibodies or antibody fragments are used. In adoptive immunotherapy (i.e. passive immunotherapy, compared to antibody treatment without direct immune modulation), white blood cells are taken from the subject, cultured ex vivo and then re-injected into the subject. If the treatment does not destroy all cells of the tumor and its metastases, the development of drug resistance can seriously hamper the further treatment of the cancer.

[0042] The present invention therefore also comprises a pharmaceutical composition for the treatment of cancer, as described above, which is performed after the traditional cancer treatment methods, i.e. after surgical resection (resection), chemotherapy and / or radiotherapy have been unsuccessful.

[0043] A particular achievement of the inventors is the finding that the HLA-A antigen peptides used according to the present invention are actually presented on the cell surface of the cells of the malignant tissue to be treated, in particular breast cancer, of the individual to be treated, in order to be effective in the treatment. Thus, the pharmaceutical composition applied and assembled before application comprises the HLA antigen peptides presented on the tumor cell surface of the breast cancer of the patient or patient group, as determined by ultra-high performance liquid chromatography (UHPCL) coupled with ESI mass spectrometry (MS). By such ligand analysis, the binding capacity of the HLA-A antigen peptides of the present invention to the corresponding MHC class I or class II complex has been proven in advance, unlike conventional cancer immunotherapy.

[0044] According to a preferred embodiment of the present invention, at least 60%, preferably at least 80%, most preferably 90%, ideally all of the HLA tumor antigen peptides comprised in the pharmaceutical composition to be administered are presented on the tumor cell surface of the breast cancer of the patient or patient group, as determined by a method as described herein.

[0045] According to a particularly preferred embodiment of the present invention, the HLA antigen peptides corresponding to MHC class I complexes are selected from the amino acid sequences given in SEQ ID No. 1 to 35 or have an amino acid identity of at least 85% to these amino acid sequences.

[0046] According to a particularly preferred embodiment of the present invention, the HLA antigen peptides corresponding to MHC class II complexes are selected from the amino acid sequences shown in SEQ ID No. 36 to 48.

[0047] The inventors of the present application have found that the HLA-A antigen peptides corresponding to MHC class I explicitly disclosed herein are particularly suitable for the treatment of breast cancer / mammary carcinoma in a subject or patient having at least one identical HLA allele exhibiting the A*01 and / or A*02 subtype. That is, the HLA-A tumor antigen peptides used herein preferably bind to the corresponding MHC class I complex of the A*01 and / or A*02 subtype.

[0048] According to one particularly preferred embodiment, the individual (subject or patient group) has a haplotype of the subgroup A*01 :01 and / or A*02:01.

[0049] Alternatively, the individual (subject or patient group) particularly preferably has a haplotype of the subgroup B*44:01 and / or A*18:01.

[0050] Indeed, the pharmaceutical composition according to the present application can be used for a tissue-independent treatment of a malignant tumor, a tumor and / or a leukemia (i.e. unlike conventional preparations, the cancer subtype does not initially play a role), so that the pharmaceutical composition can be administered across tissues for use as an anticancer drug. However, it was found that the administration / application of the specific combination of 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and at least 2 HLA tumor antigen peptides according to the present application can particularly advantageously be used for the treatment of breast cancer.

[0051] A further clinical benefit can be achieved by using the pharmaceutical composition for the treatment of a patient or a patient group having at least one identical HLA allele by further adding to the pharmaceutical composition a pharmacologically effective amount of at least one HLA-B tumor antigen peptide, preferably 1, 2, 3, 4 or 5 HLA-B tumor antigen peptides corresponding to MHC class I complexes and / or at least one HLA-C tumor antigen peptide, preferably 1, 2, 3, 4 or 5 HLA-C tumor antigen peptides corresponding to MHC class I complexes. It is understood that also the HLA-B tumor antigen peptide or the HLA-C tumor antigen peptide corresponding to the specific haplotype of the patient or patient group having at least one identical HLA allele is selected for the treatment or prevention.

[0052] According to a preferred embodiment of the application, the pharmaceutical composition according to the application comprises at least 6, 7, 8, 9, 10, 11 or 12 HLA antigen peptides corresponding to MHC class I complexes and / or MHC class II complexes, and / or at least one HLA antigen peptide which is analogous to at least one HLA antigen peptide exposed on the cell surface of cells of a malignant and / or neoplastic tissue (in particular breast cancer) from the individual to be treated, has at least one amino acid exchange in its amino acid sequence relative to the wild type of this HLA antigen peptide (so-called neoantigen peptide), and whose specific affinity to the T cell receptor of an endogenous T cell is increased by at least a factor of 3 (measured and / or expressed accordingly as K D Values). Pharmaceutical compositions with at least 10, 11 or 12 HLA antigen peptides corresponding to MHC class I complexes and / or MHC class II complexes have achieved particularly good success.

[0053] In this regard, it has been shown that pharmaceutical compositions consisting of a pharmacologically effective amount of 4 to 8, preferably 4, 5, 6, 7 or 8 HLA-A and / or HLA-B tumor antigen peptides corresponding to MHC class I complexes (in particular neoantigen peptides thereof) and 1, 2, 3 or 4 tumor antigen peptides corresponding to MHC class II complexes (in particular neoantigen peptides thereof) are particularly preferred.

[0054] According to a particularly preferred embodiment of the application, at least one of the HLA-A antigen peptides in the composition is a tumor-specific HLA-A antigen peptide (i.e. a cancer-testis antigen (CTA) which does not (no longer) occur in addition to the immunoprivileged spermatocytes of the patient / patient group's healthy / normal tissue, or a so-called neoantigen peptide), and wherein the specific dissociation (K D ) of the specific binding of the tumor-specific HLA-A antigen peptide is in the range of 10 to 50 nM, as determined by surface plasmon resonance.

[0055] The inventors have found that pharmaceutical compositions based on HLA antigen peptides are particularly effective when both T lymphocytes (short: T cells) and B lymphocytes (short: B cells) are activated by them, which is also an outstanding achievement of the present inventors.

[0056] T cells belong to the group of lymphocyte cells and play an important role in the human immune system. T cells recognize antigens via specific receptors, so-called T cell receptors (TCR). However, for this to happen, the antigen must be provided by an antigen-presenting cell (APC).

[0057] The stable binding of T cells to antigen-presenting cells requires the involvement of so-called co-stimulatory proteins. These include CD4 and CD8 (CD = "cluster of differentiation").

[0058] T cells carrying the CD4 signature are also referred to as CD4-positive T cells or T helper cells. In normal adult human blood, CD4+ T cells represent 27-57% of lymphocytes, or approximately 310-1570 cells per microliter.

[0059] The CD8-positive (CD8+) T cell group (also including regulatory T cells) comprises cytotoxic T cells or T killer cells. They play a special role in killing human body's own cells that are infected by viruses.

[0060] This feature of cellular immune defense is crucial in the present application, because the molecular biology and genetic alterations of tumor cells can be recognized and lysed by this T cell group.

[0061] In contrast, B cells are the only cells that can produce antibodies and, together with T cells, form a key component of the adaptive immune system. While T cells are involved in cell-mediated immune reactions, B cells are the carriers of humoral immune reactions (and are responsible for the formation of antibodies).

[0062] A pharmaceutical composition has been shown to comprise tumor-associated HLA antigen peptides, the expression level of which in tumor cells is at least three times higher than in healthy cells of a patient or a particularly determined patient group having at least one identical HLA allele, and wherein the tumor-associated HLA antigen peptides are associated with an increase in proliferation, invasion, angiogenesis and cytokeratin production of breast cancer / mammary carcinoma and have a particularly effective pharmacological effect in the treatment of breast cancer / mammary carcinoma.

[0063] It is particularly preferred that each individual HLA antigen peptide used herein for the treatment of breast cancer is contained in the pharmaceutical composition in an absolute concentration (i.e. dose of administration) of at least 100 to 600 pg, preferably 300 to 600 pg.

[0064] At the same time, it has been shown in further experiments that it is particularly preferred to have a pharmaceutical composition with an absolute concentration of > 600 pg / HLA tumor antigen peptide, i.e. at least 700 to 1,200 pg, preferably 800 to 1,200 pg, as this greatly intensifies the information (activation and / or training of the immune system). This is particularly advantageous if the immune system of the subject to be treated has been weakened by a standard treatment program (e.g. at least one surgery, radiation, chemotherapy and / or hormone therapy). In addition, an absolute concentration of each HLA tumor antigen peptide is preferred, as this significantly reduces the influence of HLA tumor antigen peptide degradation (e.g. by ligases) after the application of the HLA tumor antigen peptide to the subject.

[0065] It is very convenient when the composition is administered to the patient, that the pharmaceutical composition comprises an adjuvant which is able to form a granuloma at the site of administration. The advantage of the granuloma formation is that a depot effect can thus be achieved, whereby the HLA tumor antigen peptides are advantageously stored at the site of administration in a depot manner and can be delivered to the organism of the subject over a longer period of time. Thus, in particular advantageously, the weekly administration of the pharmaceutical composition according to the application is omitted. Preferably, the administration of the pharmaceutical composition for the treatment of a cancer disease within the meaning of the present application thus only needs to be carried out every 2 weeks, particularly preferably only every month, when used over a longer period of time.

[0066] In this regard, the pharmaceutical composition is preferably administered subcutaneously or intradermally, preferably substantially simultaneously at at least 2, more preferably at least 3, usually 3 to 4 administration sites which are remote from the tumor lesion and / or the area of the cancerous lymph node.

[0067] The advantage of the substantially simultaneous (consecutive) administration at multiple administration sites is, in particular in the case of high absolute concentrations of individual HLA antigen peptides (i.e. the dosing dose) in the range of > 600 pg, which require larger application volumes (> 1 mL) in order to completely dissolve the individual HLA antigen peptides, the application solution is administered as far as possible simultaneously, since this application solution has only a limited shelf life after opening.

[0068] Preferably, the pharmaceutical composition comprising each individual HLA antigen peptide in an absolute concentration (i.e. the dosing dose) of 300 to 600 pg only needs to be administered intradermally or subcutaneously every 2 weeks, preferably every 4 weeks, to the patient or to a specifically identified patient group with at least one identical HLA allele for at least one year.

[0069] It has been found that the pharmaceutical composition, wherein at least one HLA tumor antigen peptide has at least one mutation (as described in detail below) in relation to the wild-type HLA tumor antigen peptide, leads to an increased specific binding affinity (K D value) of the T-cell receptor of the individual treated with this HLA tumor antigen peptide in comparison to the wild-type HLA antigen peptide, preferably < 500 nM, more preferably < 50 nM, which shows a particularly beneficial effect in the treatment of breast cancer.

[0070] According to a preferred embodiment of the present application, the pharmaceutical composition is used for the treatment of breast cancer as monotherapy or in combination with other known therapies and / or compounds for the treatment of breast cancer. In this regard, the pharmaceutical composition will be administered to the patient or to a patient group (so-called adjuvant monotherapy) with at least one identical HLA allele as first-line therapy.

[0071] Alternatively, it can be provided that the patient to be treated with the pharmaceutical composition or the group of patients having at least one identical HLA allele has previously received at least one standard treatment procedure (e.g. at least one surgery, radiotherapy, chemotherapy and / or hormone therapy).

[0072] Particularly preferably, the breast cancer / breast adenocarcinoma to be treated is a hormone-positive, HER2 / neu or triple-negative breast adenocarcinoma.

[0073] HLA tumor antigen peptides

[0074] The present application also comprises HLA tumor antigen peptides corresponding to MHC class I complexes or MHC class II complexes, in particular for use in the treatment or prevention of breast cancer / breast adenocarcinoma in a patient or a group of patients suffering from or suspected to suffer from breast cancer / breast adenocarcinoma, respectively for use in a pharmaceutical composition according to the present application, wherein the HLA tumor antigen peptide comprises an amino acid sequence selected from the group consisting of the amino acid sequences given in SEQ ID No. 13 to 35 and SEQ ID No. 36 to 48, in particular the amino acid sequences given in SEQ ID No. 13 to SEQ ID No. 26, SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 32 to SEQ ID No. 48, or the HLA tumor antigen peptide has at least one mutation, preferably an amino acid substitution, relative to one of these amino acid sequences.

[0075] Particularly preferred in this respect is the use of the aforementioned HLA tumor antigen peptides in a method of treating breast cancer, in particular locally recurrent or metastatic breast cancer, in a patient or a group of patients having at least one identical HLA allele, which method comprises administering / applying to the patient or the group of patients a treatment regimen comprising a pharmacologically effective amount of at least one of the aforementioned HLA tumor antigen peptides.

[0076] In order to keep the immune system of the individual to be administered with the HLA tumor antigen peptide or the pharmaceutical composition according to the present application still fully functional and thus more easily trainable, it is advantageous if the individual has not yet received radiation, chemotherapy and / or hormone therapy against breast cancer, in particular locally recurrent or metastatic breast cancer, and / or has not received previous adjuvant chemotherapy in the recurrence 12 months or less since the last dose of chemotherapy drug was used.

[0077] Particularly preferably, the treatment regimen described herein, in particular the use of at least one of the aforementioned HLA tumor antigen peptides according to the present application, is effective in prolonging the progression-free survival of the individual.

[0078] A) HLA-A antigen peptides and HLA-A neoantigen peptides

[0079] An "HLA-A tumor antigen peptide corresponding to an MHC class I complex" is defined herein as an "HLA antigen peptide of the invention" or "amino acid sequence of the invention" (as defined herein), comprising:

[0080] a) an amino acid sequence consisting of 7 to 11 amino acids; and / or

[0081] b) a neo-antigen peptide having an amino acid sequence consisting of 7 to 11 amino acids, which

[0082] i) is similar to at least one HLA-A antigen peptide exposed on the cell surface of cells from a malignant and / or neoplastic tissue (in particular breast cancer) of the individual to be treated, and

[0083] ii) has at least one amino acid exchange in its amino acid sequence compared to the wild type of this HLA-A antigen peptide (so-called "HLA-A neo-antigen peptide"), and

[0084] iii) has an at least 3-fold increased specific affinity to the T cell receptor of an endogenous T cell

[0085] It was an outstanding achievement of the present inventors to recognize that an HLA-A neo-antigen peptide, which has at least one amino acid exchange in its amino acid sequence compared to the wild type of this HLA-A antigen peptide at amino acid position 1 (N-terminal), 2, 7 / 8 and / or C-terminal, has an at least 4-fold, particularly preferred at least 5-fold, most preferred at least 8-fold increased specific affinity (K D ) to the T cell receptor of an endogenous T cell, and is therefore particularly preferred for use in a composition according to the present invention.

[0086] Particularly preferred, the amino acid exchange in the amino acid sequence of the HLA-A antigen peptide is a single amino acid exchange at amino acid position 1 (N-terminal), 2, 7 / 8 or C-terminal.

[0087] Preferably, the amino acid exchange in the amino acid sequence of the HLA-A neo-antigen relative to the wild type of this HLA-A antigen peptide is a C / Y, A / V, D / Y, E / K, P / L, N / D or T / M exchange.

[0088] Preferably, the specific activity (KD) of the HLA-A peptide or HLA-A neo-antigen to the T cell receptor is less than 100 nM, more preferred less than 75 nM or most preferred less than 50 nM, such as less than 40 nM, 35 nM, 30 nM, 25 nM or 20 nM, determined by any suitable detection method known to the skilled person.

[0089] Preferably, the HLA-A tumor antigen peptide or HLA-A neoantigen is present in the pharmaceutical composition according to the application in a concentration of at least 100 to 600 pg as defined above, or preferably in an absolute concentration of > 600 pg relative to the volume of the pharmaceutical composition to be administered.

[0090] Preferably, the HLA-A tumor antigen peptide or HLA-A neoantigen is selected as defined in point (b) above.

[0091] According to one particularly preferred embodiment, the "HLA-A neoantigen peptide" is preferred, which comprises the following scaffold sequence:

[0092] (a) an amino acid sequence selected from the group consisting of SEQ ID No: 13 to 19 and 27 to 34; and / or

[0093] (b) a neoantigen having an amino acid sequence selected from the group consisting of SEQ ID No: 13 to 19 and 27 to 34;

[0094] and / or

[0095] (c) an amino acid sequence having an amino acid sequence having less than 100% sequence identity or similarity to a native HLA-A antigen peptide, for example at least 85%, more preferably at least 90% sequence identity (as defined herein) to an amino acid sequence selected from the group consisting of SEQ ID No.: 1 to 5, 13 to 19 and 27 to 34; and / or

[0096] (d) an amino acid sequence having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 5,

[0097] 13 to 19 and 27 to 34; and / or

[0098] (e) a compound, construct, protein or polypeptide consisting of at least two identical or different peptide sequences of at least one HLA-A tumor antigen peptide and one HLA-A, HLA-B and / or HLA-C tumor antigen peptide and / or its corresponding neoantigen, wherein the HLA antigen peptides are optionally connected to each other by a suitable linker (so-called oligopeptide).

[0099] Very preferably, the HLA-A tumor antigen peptide is as defined in (b), (d) and (e), respectively. Also in embodiments according to (e), the HLA-A tumor antigen peptide is preferably defined as described in (a) or (d), respectively. In case the HLA-A tumor antigen peptide and / or neoantigen defined in (e) are connected to each other by a linker, suitable linkers are known to the person skilled in the art.

[0100] The use of a compound, construct, protein or polypeptide according to the invention as defined under (e) consisting of at least two identical or different peptide sequences of HLA tumor antigen peptides and / or HLA neoantigens has the further advantage that the longer amino acid sequence of the compound, construct, protein or polypeptide is retained in the tissue of the subject for a longer time after administration, whereby the compound, construct, protein or polypeptide after being administered to the subject can be broken down, for example by the body's own enzymes, into smaller fragments (containing at least 7 to 11 amino acids in the pharmaceutically active form), which in the sense of the present invention have the biologically required functionality. That is, the individual fragments of the oligopeptide exhibit activity as HLA-A, HLA-B or HLA-C tumor antigen peptides, thereby contributing to the activation of T cells.

[0101] In a particular embodiment, any HLA-A peptide sequence can be a humanized and / or sequence-optimized sequence as further described herein.

[0102] B) HLA-B antigen peptides and HLA-B neoantigen peptides

[0103] "HLA-B antigen peptides" are defined herein as "HLA-B antigen peptides of the invention" or "amino acid sequences of the invention" (as defined herein), comprising:

[0104] a) an amino acid sequence consisting of 12 to 17 amino acids; and / or

[0105] b) a neoantigen peptide having an amino acid sequence consisting of 12 to 17 amino acids, which

[0106] i) is similar to at least one HLA-B antigen peptide exposed on the cell surface of cells from malignant and / or neoplastic tissue (in particular breast cancer / mammary carcinoma) of the individual to be treated, and

[0107] ii) has at least one amino acid exchange in its amino acid sequence compared to the wild type of this HLA-B antigen peptide (so-called "HLA-B neoantigen peptide"), and

[0108] iii) has at least a 3-fold increased specific affinity to the T cell receptor of endogenous T cells.

[0109] Furthermore, the inventors have recognized that an HLA-B neoantigen peptide having at least one amino acid exchange in its amino acid sequence in the 3rd, 8thand / or 10thamino acid has at least a 4-fold, particularly preferably at least a 7-fold, increased specific affinity to the T cell receptor of the body's own T cells compared to the wild type of this HLA-B antigen peptide, and is therefore particularly preferred for use in a preparation according to the invention.

[0110] It is particularly preferred that the amino acid exchange in the amino acid sequence of the HLA-B antigen peptide is a single amino acid exchange at amino acid position 3, 8 or 10.

[0111] Preferably, the amino acid exchange in the amino acid sequence of the HLA-B neoantigen is an E / A, E / K, R / W or D / A exchange relative to the wild type of the HLA-B antigen peptide.

[0112] Preferably, the specific activity of the HLA-B antigen peptide or the HLA-B neoantigen peptide for a T cell receptor as determined by any suitable detection method known to the person skilled in the art (K D ) is less than 100 nM, more preferably less than 75 nM or most preferably less than 50 nM, for example less than 40 nM, 35 nM, 30 nM, 25 nM or 20 nM.

[0113] Preferably, the HLA-B tumor antigen peptide or the HLA-B neoantigen is present in the pharmaceutical composition according to the application in a concentration of at least 100 to 600 pg as defined above, or preferably in an absolute concentration of > 600 pg relative to the volume of the pharmaceutical composition to be administered.

[0114] Preferably, the HLA-B peptide or the HLA-B neoantigen is selected as defined under point b) above.

[0115] According to one particularly preferred embodiment, the “HLA-B neoantigen” is preferred which comprises the following scaffold sequence:

[0116] (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 20, 21, 22 and 35; and / or

[0117] (b) an amino acid sequence having the following amino acid sequence: an amino acid sequence having at least 80%, for example at least 85%, more preferably at least 90% sequence identity (as defined herein) to an amino acid sequence selected from the group consisting of SEQ ID No: 6, 7, 8, 20, 21, 22 and 35; and / or

[0118] (c) an amino acid sequence having the following amino acid sequence: an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 6, 7, 8, 20, 21, 22 and 35; and / or

[0119] (d) a compound, construct, protein or polypeptide consisting of at least two identical or different peptide sequences of an HLA-B tumor antigen peptide and one HLA-A, HLA-B and / or HLA-C tumor antigen peptide and / or its corresponding neoantigen, wherein the HLA antigen peptides are optionally connected to each other by a suitable linker (so-called oligopeptide).

[0120] Very preferably, the HLA-B peptide is defined as in (a), (c) and (d), respectively. Also in embodiments according to (d), the HLA-B peptide is preferably defined as in (a) or (c). In case the HLA-B tumor antigen peptide and / or the HLA-B neoantigen as defined in (e) are connected to each other by a linker, suitable linkers are known to the person skilled in the art from the prior art.

[0121] The use of a compound, construct, protein or polypeptide according to the present application as defined in (d) consisting of at least two identical or different peptide sequences of an HLA tumor antigen peptide and / or HLA neoantigen has the further advantage that the longer amino acid sequence of the compound, construct, protein or polypeptide is retained in the tissue of the subject for a longer time after administration, whereby the compound, construct, protein or polypeptide after being administered to the subject can be broken down, for example by the body's own enzymes, into smaller fragments (containing at least 7 to 11 amino acids in the pharmaceutically active form), which in the sense of the present application have the biologically required functionality, that is to say the individual fragments of the oligopeptide exhibit activity as HLA-A, HLA-B or HLA-C tumor antigen peptide, thereby contributing to the activation of T cells.

[0122] In a particular embodiment, any HLA-B peptide sequence can be a humanized and / or sequence-optimized sequence as further described herein.

[0123] C) HLA-C antigen peptides and HLA-C neoantigen peptides

[0124] An "HLA-C antigen peptide" is defined herein as an "HLA antigen peptide of the present application" or "amino acid sequence of the present application" (as defined herein), comprising:

[0125] a) an amino acid sequence consisting of 8 to 11 amino acids; and / or

[0126] b) a neoantigen peptide having an amino acid sequence consisting of 8 to 11 amino acids, which

[0127] i) is similar to at least one HLA-C antigen peptide exposed on the cell surface of cells from a malignant and / or neoplastic tissue (in particular breast cancer / mammary carcinoma) of the individual to be treated, and

[0128] ii) at least one amino acid exchange in its amino acid sequence relative to the wild type of the HLA-C peptide (so-called "HLA-C neoantigen peptide"), and

[0129] iii) at least a 3-fold increase in the specific affinity to the T cell receptor of the endogenous T cell

[0130] Furthermore, the present inventors have recognized that HLA-C neoantigen peptides having at least one amino acid exchange in its amino acid sequence at amino acid position 1 (N- terminal), 4 and / or C-terminal compared to the wild type of the HLA-C antigen peptide have at least a 4-fold, particularly preferably at least a 5-fold, very particularly preferably at least a 7-fold increase in the specific affinity to the T cell receptor of the body's own T cells and are therefore particularly preferred for use in the formulation according to the application.

[0131] Particularly preferably, the amino acid exchange in the amino acid sequence of the HLA-C antigen peptide is a single amino acid exchange at amino acid position 1 (N-terminal), 4 or C-terminal.

[0132] Preferably, the amino acid exchange in the amino acid sequence of the HLA-C neoantigen peptide relative to the wild type of the HLA-C antigen peptide is a C / Y, A / P, L / F or T / M exchange.

[0133] Preferably, the specific activity of the HLA-C antigen peptide or the HLA-C neoantigen peptide to the T cell receptor (K D ) is less than 100 nM, more preferably less than 75 nM or most preferably less than 50 nM, for example less than 40 nM, 35 nM, 30 nM, 25 nM or 20 nM, as determined by any suitable detection method known to the person skilled in the art.

[0134] Preferably, the HLA-C tumor antigen peptide or the HLA-C neoantigen is present in the pharmaceutical composition according to the application in a concentration of at least 100 to 600 pg as defined above, or preferably in an absolute concentration of > 600 pg relative to the volume of the pharmaceutical composition to be administered.

[0135] Preferably, the HLA-C antigen peptide or the HLA-C neoantigen peptide is selected as defined in point b) above.

[0136] According to one particularly preferred embodiment, the "HLA-C neoantigen peptide" is preferred which comprises the following scaffold sequence:

[0137] (a) an amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26; and / or

[0138] (b) an amino acid sequence (as defined herein) having at least 80%, e.g. at least 85%, more preferably at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID No: 9 to 12 and 23 to 26; and / or

[0139] (c) an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to an amino acid sequence selected from the group consisting of SEQ ID No: 9 to 12 and 23 to 26; and / or

[0140] (d) a compound, construct, protein or polypeptide consisting of at least two identical or different peptide sequences of an HLA-C tumor antigen peptide and one HLA-A, HLA-B and / or HLA-C tumor antigen peptide and / or its corresponding neoantigen, wherein the HLA antigen peptides are optionally linked to each other by a suitable linker (so-called oligopeptide).

[0141] Very preferably, the HLA-C peptides are defined as in (a), (c) and (d), respectively. Also in embodiments according to (d), the HLA-C antigen peptides are preferably defined as in (a) and (c), respectively. In case the HLA-C tumor antigen peptides and / or HLA-C neoantigens defined in (d) are linked to each other by a linker, suitable linkers are known to the person skilled in the art from the prior art.

[0142] The use of a compound, construct, protein or polypeptide according to the application as defined in (d) consisting of at least two identical or different peptide sequences of an HLA tumor antigen peptide and / or HLA neoantigen has the further advantage that the longer amino acid sequence of the compound, construct, protein or polypeptide is retained in the tissue of the subject for a longer time after administration, whereby the compound, construct, protein or polypeptide after being administered to the subject can be broken down, e.g. by the body's own enzymes, into smaller fragments (containing at least 7 to 11 amino acids in the pharmaceutically active form), which in the sense of the present application have the biologically required functionality, that is to say the individual fragments of the oligopeptide exhibit activity as HLA-A, HLA-B or HLA-C tumor antigen peptides, thereby contributing to the activation of T cells.

[0143] In a particular embodiment, any HLA-C antigen peptide sequence can be a humanized and / or sequence-optimized sequence as further described herein.

[0144] D) HLA class II antigen peptides and HLA-II neoantigen peptides

[0145] An "HLA class II antigen peptide" is defined herein as an "HLA peptide of the invention" or "amino acid sequence of the invention" (as defined herein) comprising:

[0146] a) an amino acid sequence consisting of 13 to 20 amino acids, particularly preferably 13 to 17 amino acids, and / or

[0147] b) a neoantigen having an amino acid sequence consisting of 13 to 20 amino acids, particularly preferably 13 to 17 amino acids, which

[0148] i) resembles at least one HLA class II peptide exposed on the cell surface of cells from malignant and / or neoplastic tissue, particularly breast cancer, from the individual to be treated, and

[0149] ii) has at least one amino acid exchange in its amino acid sequence compared to the wild type of this HLA class II peptide (so-called "HLA class II neoantigen"), and

[0150] iii) has an at least 3-fold increased specific affinity to the T cell receptor of an endogenous T cell

[0151] Furthermore, the inventors have recognized that an HLA class II neoantigen having at least one amino acid exchange in its amino acid sequence compared to the wild type of this HLA class II peptide at amino acid position 3, 6, 10, 12, 13 and / or 14, particularly preferably at amino acid position 12 and / or 14, has an at least 4-fold increased specific affinity to the T cell receptor of an endogenous T cell and is therefore particularly preferred for use in a formulation according to the application.

[0152] Particularly preferably, the amino acid substitution in the amino acid sequence of the HLA class II peptide is a single amino acid substitution at amino acid position 12 or 14.

[0153] Preferably, the amino acid exchange in the amino acid sequence of the HLA class II neoantigen is an E / K, E / A, D / Y or T / M exchange relative to the wild type of this HLA class II peptide.

[0154] Preferably, the specific activity of the HLA class II peptide or the HLA class II neoantigen to the T cell receptor (K D ) is less than 100 nM, more preferably less than 75 nM, or most preferably less than 50 nM, for example less than 40 nM, 35 nM, 30 nM, 25 nM or 20 nM, as determined by any suitable detection method known to the person skilled in the art.

[0155] Preferably, the HLA tumor antigen peptide corresponding to an HLA class II MHC complex or the HLA neoantigen corresponding to an HLA class II MHC complex is present in the pharmaceutical composition according to the application in a concentration of at least 100 to 600 pg as defined above, or, preferably, in an absolute concentration of > 600 pg per volume of the pharmaceutical composition to be administered.

[0156] Preferably, the HLA class II peptide or the HLA class II neoantigen is selected as defined under b) above.

[0157] According to a particularly preferred embodiment, the "HLA class II neoantigen" is preferred comprising the following scaffold sequences:

[0158] (a) an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 37, 38, 41, 42, 43, 44, 45, 46, 47, 48; and / or

[0159] (b) a neoantigen having an amino acid sequence having at least 80%, such as at least 85%, more preferably at least 90% sequence identity (as defined herein) with an amino acid sequence selected from the group consisting of SEQ ID NO: 36 to 48; and / or

[0160] (c) a neoantigen having an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to an amino acid sequence selected from the group consisting of SEQ ID NO: 36 to 48; and / or

[0161] (d) a compound, construct, protein or polypeptide consisting of at least two identical or different HLA class II peptide sequences, wherein the HLA class II antigen peptides are linked together by a suitable linker (so-called oligopeptide).

[0162] (e) a compound, construct, protein or polypeptide consisting of at least one HLA class II antigen peptide sequence and one HLA class II antigen peptide sequence, HLA-A, HLA-B and / or HLA-C antigen peptide sequence and / or at least two identical or different peptide sequences of the respective neoantigen thereof, wherein the HLA antigen peptides are optionally linked to each other by a suitable linker (so-called oligopeptide).

[0163] Very preferably, the HLA class II antigen peptides are defined as in (a), (c) and (d), respectively. Also in embodiments according to (d), the HLA class II antigen peptides are preferably defined as in (a) or (c), respectively. In case the HLA class II tumor antigen peptides and / or the respective neoantigens as defined in (d) are linked to each other by a linker, suitable linkers are known to the person skilled in the art from the prior art.

[0164] The use of a compound, construct, protein or polypeptide according to the definition of (d) of the present application consisting of at least two identical or different peptide sequences of HLA tumor antigen peptides and / or HLA neoantigens has the further advantage that the longer amino acid sequence of the compound, construct, protein or polypeptide is retained in the tissue of the subject for a longer time after administration, whereby the compound, construct, protein or polypeptide after being administered to the subject can be broken down, for example by the body's own enzymes, into smaller fragments (containing at least 7 to 11 amino acids in the pharmaceutically active form), which in the sense of the present application have the biologically required functionality, that is to say the individual fragments of the oligopeptide exhibit activity as HLA-A, HLA-B or HLA-C tumor antigen peptides, thus contributing to the activation of T cells.

[0165] In a particular embodiment, any HLA class II antigen peptide sequence can be a humanized and / or sequence-optimized sequence as further described herein.

[0166] According to the present application, the treatment of breast cancer using at least 2 HLA class II tumor antigen peptides has the significant advantage that B cells are also activated in a targeted manner in the subject to be treated.

[0167] The use of HLA class II tumor antigen peptides according to the present application has the further advantage that the HLA antigen peptides having a longer amino acid sequence than HLA class I tumor antigen peptides can be broken down after application to the test subject, for example by the body's own enzymes, into smaller fragments (containing at least 7 to 11 amino acids), which have the activity of HLA-A, HLA-B or HLA-C antigen peptides, thus contributing to the activation of T cells.

[0168] "Fragment" means a portion of a polypeptide, which preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the full length of the reference polypeptide. A fragment can comprise 7, 8, 9, 10, 11 or more amino acids.

[0169] In particular, the amino acid sequences and polypeptides of the present application are preferred as defined in the claims, wherein:

[0170] The at least one HLA tumor antigen peptide is analogous to at least one HLA tumor antigen peptide exposed on the cell surface of cells from malignant and / or neoplastic tissue of the individual to be treated, which peptide has at least one amino acid exchange in its amino acid sequence compared to the wild type of said HLA tumor antigen peptide (i.e. HLA neoantigen peptide), which increases the specific affinity for the T cell receptor of the body's own T cells by at least a factor of 3.

[0171] The inventors' outstanding achievement is the finding that pharmaceutical compositions comprising more than 3 such HLA tumor antigen peptides are particularly effective. Thus, the pharmaceutical compositions according to the present application preferably comprise at least six, very preferably at least eight, very particularly preferably ten of the above HLA tumor antigen peptides corresponding to MHC class I and / or class II complexes.

[0172] Preferably, the monovalent amino acid sequence used in the present application (or a polypeptide comprising only one amino acid sequence of the present application) is a sequence which has an increased specific affinity of 3-fold to the T cell receptor of endogenous T cells compared to the corresponding wild-type HLA peptide sequence.

[0173] It is noted that "can specifically bind" and "specifically bind to" are used interchangeably herein to refer to the ability to specifically bind to the respective specified entity.

[0174] For the present application, it is important that the diagnosis of cancer is made in advance by the attending physician.

[0175] It is possible to combine amino acid sequences belonging to different categories used in the present application in a single polypeptide of the present application. In particular, it has been shown that the combination of HLA-A, HLA-B and / or HLA-C tumor antigen peptides and / or the corresponding neoantigen peptides in a single polypeptide of the present application has unique binding properties (see Figure 3 ).

[0176] The specific activity (KD) of the polypeptides of the present application comprising more than one component of an amino acid sequence corresponding to an HLA-A, HLA-B and / or HLA-C tumor antigen peptide and / or a neoantigen peptide of MHC class I or class II complexes can be determined according to one of the detection methods described above / below, as known to the person skilled in the art, wherein the compounds, constructs, proteins or polypeptides of the present application preferably have a specific activity similar to the specific activity of each of their components, i.e. a specific activity similar to the specific activity of (the individual component of) each of the class I or class II amino acid sequences comprised in the compounds, constructs, proteins or polypeptides of the present application. Some specific but non-limiting examples of the above preferred compounds, constructs, proteins or polypeptides are compounds, constructs, proteins or polypeptides comprising one of the following:

[0177] i) a wild-type HLA tumor antigen peptide corresponding to a MHC class I complex (respectively HLA-A, HLA-B or HLA-C peptide) and / or a MHC class II complex; or

[0178] ii) a neoantigen corresponding to a MHC class I complex (respectively HLA-A, HLA-B and HLA-C neoantigen) and / or a MHC class II complex, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 48; or

[0179] iii) neoantigens corresponding to MHC class I complexes (HLA-A, HLA-B and HLA-C neoantigens, respectively) and / or MHC class II complexes, the amino acid sequence of which has at least 80% sequence identity (as defined herein) to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 48; or

[0180] iv) an amino acid sequence comprising or consisting essentially of only one amino acid substitution relative to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 48; or

[0181] v) a compound, construct, protein or polypeptide consisting of at least two identical or different HLA antigenic peptide sequences, wherein the HLA peptides are optionally connected to each other by a suitable linker;

[0182] or suitable combinations thereof.

[0183] It is noted that the last preceding paragraph generally also applies to all amino acid sequences of the present application comprising one or more amino acid sequences of HLA tumor antigen peptides and / or HLA neoantigen peptides according to A), B), C) and D), respectively.

[0184] Of course, all of the above HLA tumor antigen peptides corresponding to MHC class I and class II complexes can be used and are effective in the treatment of the herein disclosed cancers, in particular in the treatment of breast cancer.

[0185] Some specific but non-limiting examples of such compounds, constructs, proteins or polypeptides of the present application are set forth, for example, in Tables 1-4, or are obvious to the person skilled in the art based on the present disclosure.

[0186] According to another preferred embodiment of the present application, the HLA tumor antigen peptides (in each case) are present as single member amino acid sequences, i.e. not as members of a compound, construct, protein or polypeptide consisting of at least two identical or different amino acid sequences of HLA tumor antigen peptides / neoantigens, wherein the HLA tumor antigen peptides / neoantigens are connected to each other by a suitable linker.

[0187] Preferably, the HLA tumor antigen peptides of the present application exhibit specific activity towards T cell receptors (T cells for this purpose), which can be determined using any suitable detection method known to the person skilled in the art, such as the EliSpot AlphaScreen detection method (as described herein) or a cell-based detection method (as described herein). Preferably, the blocking activity is determined using a cell-based detection method, such as described in Examples 3 and 4.

[0188] In particular, the compounds, constructs, proteins or polypeptides of the application comprising the amino acid sequence of the HLA antigen peptide of the application and belonging to MHC class I (as defined herein) have a specific affinity to the respective T cell receptor preferably between 10 and 50 nM.

[0189] It is also within the scope of the present application that the amino acid sequences of the present application can bind to two or more class I or class II MHC complexes, epitopes, components, domains or subunits of class I or class II MHC complexes. In this case, the MHC complex, epitope, component, domain or subunit of the MHC complex to which the amino acid sequences and / or polypeptides of the present application bind can be essentially the same or different (in the latter case, the amino acid sequences and polypeptides of the present application can bind to such different complexes, epitopes, components, domains or subunits of class I or class II MHC complexes with the same or different affinity and / or specificity, including combinations thereof).

[0190] It is also contemplated that the polypeptides of the present application will generally bind to all naturally occurring or synthetic analogs, variants, mutants, components and fragments of class I or class II MHC complexes, respectively. Also in this case, the amino acid sequences and polypeptides of the present application can bind to such analogs, variants, mutants, alleles, components and fragments with an affinity and / or specificity that is equal to or different from the affinity and specificity with which the amino acid sequences of the present application bind to the wild type of class I or class II MHC complexes, respectively.

[0191] It is also within the scope of the present application that the amino acid sequences and polypeptides of the present application bind to some analogs, variants or mutants of class I or class II MHC complexes, respectively, but not to others.

[0192] In a particular but non-limiting embodiment of the present application, the compounds, constructs, proteins or polypeptides comprising the HLA tumor antigen peptide of the present application can have an increased serum half-life compared to the amino acid sequence from which they are derived. For example, the amino acid sequences of the present application can be (chemically or otherwise) linked to one or more groups or moieties that prolong the half-life (such as PEG), such that they are half-life increased derivatives of the amino acid sequences of the present application.

[0193] Generally, the half-life of the compounds or polypeptides of the present application having an increased half-life is at least 1.5-fold, preferably at least 2-fold, such as at least 5-fold, for example at least 10-fold or more than 20-fold longer than the half-life of the respective amino acid sequence of the present application per se. For example, the half-life of the compounds or polypeptides of the present application having an increased half-life is more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours or even more than 24, 48 or 72 hours longer than the half-life of the respective amino acid sequence of the present application per se.

[0194] Generally, when the HLA tumor antigen peptides of the application (or compounds, constructs or polypeptides comprising the same) are administered to a patient (e.g. for therapeutic and / or diagnostic purposes, as defined herein), preferably, they are not an amino acid sequence naturally occurring in the patient; or, if naturally occurring in the patient, they are in a substantially isolated and at the same time concentrated form (as defined herein).

[0195] Furthermore, it will also be apparent to the skilled person that for pharmaceutical use, the HLA tumor antigen peptides of the application (as well as the compounds, constructs and polypeptides comprising the same) will be directed against human T cell receptors including combinations thereof as defined in the claims; wherein for veterinary purposes, the polypeptides of the application are preferentially directed against T cell receptors from the species to be treated including combinations thereof (as defined in the claims), or at least have cross-reactivity to T cell receptors from the species to be treated including combinations thereof.

[0196] The breast cancer / mammary carcinoma to be treated is preferably a hormone positive, HER2 / neu or triple negative breast cancer.

[0197] Method for determination / identification of HLA antigen peptides

[0198] The present application also comprises a method for the determination of a pharmaceutically active HLA tumor antigen peptide for the treatment or prevention of breast cancer or in a pharmaceutical composition according to the present application, said method comprising monitoring a tissue resection of a patient or a group of patients suffering from or suspected to suffer from breast cancer, said method comprising the following steps:

[0199] (a) providing a tissue sample from the tissue resection (preferably from a breast cancer / mammary carcinoma of the patient or group of patients), wherein the cells of the tissue sample express MHC class I and / or class II complexes and these complexes are presented on the cell surface, wherein said method step (a) of providing the tissue sample itself does not comprise a surgical intervention on one patient of the patient or group of patients, respectively;

[0200] (b) determining the following parameters using the tissue sample from the tissue resection provided in step (a):

[0201] i) the transcriptome of the provided tissue sample to determine all DNA sequences transcribed into mRNA sequences and to quantify the mRNA sequences, and

[0202] comparing the determined transcriptome to the transcriptome of a healthy tissue sample of the patient or group of patients to determine mRNA sequences that are up- and / or down-regulated by a factor of 3 from the threshold value in the healthy tissue sample;

[0203] ii) specific HLA haplotypes, preferably comprising the HLA haplotype of the patient or patient group, most preferably with respect to MHC class I complexes, and

[0204] if applicable, deriving preferred anchor positions (as defined herein) of MHC class I complexes;

[0205] iii) providing exome sequences of the provided tissue sample for identifying all possible protein-coding DNA sequences, and

[0206] comparing the exome of the provided tissue sample with the exome of a healthy tissue sample or with a gene database (library) of the patient or patient group to determine somatic mutations,

[0207] thereby advantageously excluding germline mutations in healthy tissue, thereby preventing an autoimmune reaction when / after applying the determined HLA antigen peptides to the patient or patient group (e.g. by taking a blood sample of nucleated cells, a healthy tissue sample can be obtained from the patient or patient group); and determining HLA tumor antigen peptides associated with increased proliferation, invasion, angiogenesis and keratin production of breast cancer / breast carcinoma; and

[0208] determining HLA tumor antigen peptides associated with increased proliferation, invasion, angiogenesis and keratin production of breast cancer / breast carcinoma;

[0209] iv) determining a ligand set for the HLA tumor antigen peptides determined in step (iii) for presentation at the cell surface of breast cancer / breast carcinoma,

[0210] optionally also determining HLA antigen peptides presented at the cell surface of a healthy tissue sample of the patient or patient group;

[0211] v) determining the specific binding affinity of the HLA tumor antigen peptides determined in step (iv) to the corresponding class I and / or class II MHC complexes expressed in the cells of the tissue sample, preferably breast cancer / breast carcinoma, and presented at the surface of these cells by database and / or ranking algorithms;

[0212] and / or

[0213] vi) optionally, determining the immunogenicity of the HLA tumor antigen peptides determined in step (iv) by an immunogenicity test, in particular by Western Blot, ELISA techniques, in particular by ELISPOT with microscopic analysis, AFM or immunoassay;

[0214] (c) selection of HLA tumor antigen peptides, preferably at least 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and at least 2 antigen peptides corresponding to MHC class II complexes, which fulfill the criteria of the parameters defined according to step (b) and which are expressed in the cells of the tissue sample provided and are presented onto the surface of these cells.

[0215] According to a preferred embodiment of the method for determining pharmaceutically active HLA tumor antigen peptides, it is checked immediately after providing the tissue sample of the patient or patient group in step (a) whether the BRCA1 and BRCA2 genes are mutated.

[0216] It is particularly preferred that the presentation of HLA tumor antigen peptides onto the surface of the cells of the tissue sample of the patient or patient group provided in step a) is determined by ultra-high performance liquid chromatography (UHPCL) in combination with ESI mass spectrometry (MC).

[0217] According to a preferred embodiment, the method for determining class I and / or class II HLA peptides in step (b) comprises generating a transcriptome of the tissue sample provided in step (a).

[0218] Preferably, the transcriptome is generated by RT-PCR followed by DNA microarray or DNA sequencing.

[0219] It is particularly preferred that the method for determining class I and / or class II HLA antigen peptides in step (b) comprises generating an exome sequencing of said tissue sample provided in step (a).

[0220] It is also desirable that the method for determining pharmaceutically active class I and / or class II HLA tumor antigen peptides in step (b) comprises matching the determined amino acid sequence of the HLA antigen peptide corresponding to the class I and / or class II MHC complex to a set of amino acid sequences, a collection of amino acid sequences or a library of amino acid sequences to rank the number of proteins (i.e. factors associated with breast cancer / breast carcinoma proliferation, invasion, angiogenesis and / or increased cytokeratin production) and the specific affinity to T cell receptors of endogenous T cells (content factors of tumor progression such as invasiveness, angiogenesis, and evasion mechanisms of the tumor against immune attacks).

[0221] The method further comprises matching the determined parameters of the amino acid sequence of the HLA tumor antigen peptide to a series, collection or library of amino acid sequences of healthy expression data of amino acid sequences which can bind to or have an affinity for MHC class I complexes or MHC class II complexes, including combinations thereof, as determined in step (i).

[0222] In such a method, the series, set or library of amino acid sequences can be a suitable series, set or library of amino acid sequences. For example, the series, set or library of amino acid sequences can be a series, set or library of class I and class II HLA tumor antigen peptides and / or MHC complexes (as described herein), e.g. a natural series, set or library of class I and class II HLA tumor antigen peptides and / or MHC complexes; a synthetic or semi-synthetic series, set or library of class I and class II HLA tumor antigen peptides and / or MHC complexes and / or series; a set or library of class I and class II HLA tumor antigen peptides and / or MHC complexes that have been affinity matured.

[0223] In order to specify the pharmacological efficacy of the HLA tumor antigen peptides determined by the resulting ranking, and in order to prevent or minimize the risk of an autoimmune reaction in the subject upon administration of the determined HLA tumor antigen peptides, the HLA tumor antigen peptides determined (pre)determined by the method according to the application are subjected to immunogenicity testing, in particular by Western blot, ELISA techniques, in particular with the aid of ELISPOT with microscopic analysis, AFM or immunoassay. Advantageously, in this aspect of the method of determining at least one pharmaceutically active HLA tumor antigen peptide, the time- and cost-intensive preparation of transcriptome and exome sequencing for the tissue sample can be dispensed with.

[0224] The present application also comprises a method for determining the regression, progression or occurrence of a breast cancer disease, comprising monitoring a tissue resection, in particular a breast tissue, of a patient suffering from or suspected of suffering from breast cancer, the method comprising the following steps:

[0225] (a) providing a tissue sample from a subject, wherein the cells of the tissue sample express HLA class I and / or class II complexes and present them to their cell surface;

[0226] (b) determining from the provided tissue sample a plurality of parameters selected from the group consisting of:

[0227] i) the concentration (expression level) of at least 4 to 8 HLA-A antigen peptides corresponding to MHC class I complexes and at least 2 antigen peptides corresponding to MHC class II complexes expressed in the cells of the provided tissue sample and presented on the cell surface of these cells; and

[0228] ii) the amino acid sequences of at least the 4 to 8 HLA-A antigen peptides and at least the 2 class II antigen peptides expressed in the cells of the provided tissue sample and presented on the cell surface of said cells; and

[0229] iii) the specific binding affinity of the HLA-A antigen peptides and class II antigen peptides determined in step ii) to T cell receptors of endogenous T cells; and

[0230] iv) the optional immunogenicity of the determined HLA-A antigen peptides and class II antigen peptides is determined by an immunogenicity test (e.g. by Western Blot, ELISA techniques, in particular by ELISPOT or immunodetection with microscopic analysis);

[0231] (c) the amino acid sequence of the determined HLA antigen peptides is matched to a series, set or library of amino acid sequences of amino acid sequences capable of binding to or having an affinity for the MHC class I complex or MHC class II complex determined in step (i) (including combinations thereof), respectively.

[0232] Further, in particular cost-reducing embodiments of the method for determining at least one HLA tumor antigen peptide corresponding to a class I and / or class II MHC complex for use in a pharmaceutical composition according to the present application, it is provided that the class I and / or class II HLA tumor antigen peptides exposed on the cell surface of cells of the malignant and / or neoplastic tissue from the individual to be treated (so-called ligandome determination) are determined exclusively (i.e. without prior preparation of a transcriptome, exome sequencing).

[0233] Here, the process comprises the following steps:

[0234] a) providing a tissue sample from a tissue resection of a subject, preferably suffering from or suspected to suffer from a locally recurrent breast cancer / breast adenocarcinoma; and

[0235] b) determining / selecting the HLA tumor antigen peptides exposed on the cell surface of cells of the sampled tissue according to the determination based on parameters from the provided tissue sample of step (a) according to step (b) of the above described method for determining a pharmaceutically active HLA tumor antigen peptide (as defined above); and

[0236] c) determining the affinity of the HLA tumor antigen peptides exposed on the cell surface of the harvested tissue cells to T cell receptors of endogenous T cells; and

[0237] d) ranking according to the number of proteins (as described herein) and the specific affinity (K D ) to T cell receptors of endogenous T cells.

[0238] wherein the sequences and / or combinations of sequences are HLA tumor antigen peptides corresponding to MHC class I complexes and / or MHC class II complexes or combinations of HLA tumor antigen peptides thereof, and wherein the individual sequences are selected from sequences of a nucleic acid database.

[0239] Detailed description and definitions

[0240] In the present specification and claims, the following terms are defined as follows:

[0241] In the context of the present application, features of the present application designated as "comprising" are intended to be understood as including the more limited description of the same features of the present application "consisting of or "consisting essentially of.

[0242] The term "and / or" is used to together or individually specify two features or components. Thus, for example, the term "and / or" used in the phrase "I and / or II" of the present disclosure includes "I and II", "I or II", "I", and "II".

[0243] A pharmaceutical composition is understood herein as a so-called informatic, which can be administered to a patient or a group of patients having at least one identical HLA allele and comprises a combination of HLA tumor antigen peptides according to the present application in the concentrations disclosed herein, wherein the HLA tumor antigen peptides represent the information carrier. This means that the arrangement of the amino acids in the amino acid sequence of the HLA tumor antigen peptides ("code") induces a sequence-specific activation of the immune system, in particular of T cells (via HLA tumor antigen peptides corresponding to MHC class I complexes) and / or B cells (via HLA tumor antigen peptides corresponding to MHC class II complexes). Thus, the in vitro or in vivo loading of MHC class I complexes or MHC class II complexes of tumor cells with HLA tumor antigen peptides having the same or slightly modified amino acid sequence as the HLA tumor antigen peptides of the pharmaceutical composition of the present application, or the contacting of T cells with said HLA tumor antigen peptides, leads to a sensitivity of the tumor cells to tissue or tissue-excision of the tumor cells to lysis due to specific cytotoxic or specifically activated T lymphocytes.

[0244] However, the main objective of the present application is not to load tumor cells with HLA tumor antigen peptides in vitro or in vivo, but to induce a specific activation and training of the immune system, in particular of T cells and B cells directed against tumor cells. For this purpose, the pharmaceutical composition according to the present application is preferably administered subcutaneously or intradermally, preferably substantially simultaneously (i.e. sequentially) at at least 2, particularly preferably at least 3 administration sites which are remote from the area of the tumor lesion and / or the cancerous lymph node. This has the particular advantage that the immune system or T cells recognizing the applied HLA tumor antigen peptides process the information applied in the form of the tumor antigen peptides, so that tumor cells presenting these HLA tumor antigen peptides on their surface are specifically recognized and lysed.

[0245] Thus, the method according to the present application has the advantage that in case the signal emitted by the tumor to the immune system is too weak (passive) or the tumor actively secretes substances that promote its growth (stimulate macrophages) (active, e.g. TREX or BD1 up), the immune system, in particular T cells, can be trained to the presence of the tumor even if the signal emitted by the tumor is too weak. The signal emitted by the tumor is too weak when the HLA antigen peptide is presented on the cell surface of the tumor cell low or reduced in response to an ongoing cytotoxic T cell attack (evasion mechanism).

[0246] For the purposes of the present application, the HLA tumor antigen peptide is an HLA tumor antigen peptide if it is a tumor-specific HLA antigen peptide (i.e. expressed and / or exposed only by tumor cells; a cancer testis antigen (CTA) does not occur in healthy / normal tissue of the adult patient / patient group anymore, or a so-called neoantigen peptide) or a tumor-associated HLA antigen peptide.

[0247] A "tumor-specific HLA antigen peptide" is a mutated HLA antigen peptide, e.g. resulting from a genetic mutation, wherein the genetic mutation is the cause of tumor growth and / or is associated with tumor development. The mutated gene product in the tumor is specific for the individual patient or a certain group of patients having at least one identical HLA allele.

[0248] A "tumor-associated HLA antigen peptide" includes non-mutated HLA antigen peptides that are expressed in some tissues of the patient or a certain group of patients having at least one identical HLA allele and in tumor cells in adulthood. Tumor-associated HLA antigen peptides are usually less immunogenic, as they are present in healthy cells can lead to immune tolerance (immunological tolerance). However, there is a risk of autoimmune reactions in inducing a strong immune response against HLA antigen peptides that are only associated with the tumor.

[0249] The immunogenic HLA tumor antigen peptide is also referred to herein as "epitope".

[0250] According to a preferred embodiment of the present application, the MHC complex corresponding to the tumor-associated HLA antigen peptide is associated with an increase in proliferation, invasion, angiogenesis and cytokeratin production of breast cancer / mammary carcinoma. The skilled person is familiar with the relevant databases and literature listing these effects (e.g. National Center for Biotechnology Information (NCBI) database).

[0251] The at least one HLA tumor antigen peptide within the scope of the present application is in particular formulated for subcutaneous administration. Thus, the expression / term "composition" refers to providing the at least one HLA tumor antigen peptide and an adjuvant in a pharmaceutical preparation having good applicability, including solutions, in particular injection solutions and infusion solutions, concentrates for the manufacture of injection and infusion preparations, powders for the manufacture of injection and infusion preparations and subcutaneous implants.

[0252] The pharmaceutical composition is prepared by dissolving or suspending the defined HLA tumor antigen peptide in a carrier fluid, i.e. a pharmacologically acceptable carrier, optionally adding further excipients, such as wetting agents, dyes, penetration enhancers, resorption enhancers, preservatives, antioxidants, light stabilizers.

[0253] The carrier fluid is preferably selected from sodium chloride injection for local injection, Ringer's injection, isotonic glucose, sterile water, glucose solution, lactated Ringer's injection, distilled water or mixtures thereof.

[0254] The good solubility is particularly advantageous if the amino acid sequence of the defined HLA tumor antigen peptide has as few hydrophobic amino acids as possible.

[0255] Preferably, dimethyl sulfoxide (DMSO), ethoxy glycol, ethanol, phosphatidylcholine, propylene glycol dipelargonate (DPPG) or glycolysis ethoxylated glycerol esters are suitable penetration enhancers.

[0256] According to a preferred embodiment of the present application, the pharmaceutical composition comprises water, a pharmaceutically acceptable aqueous salt solution and / or DMSO. For example, a pharmaceutical composition suitable for use comprises a mixture of about 30% DMSO and 70% water.

[0257] The term "class I HLA tumor antigen peptide (corresponding to MHC complex)" as used herein refers to a peptide sequence that binds to or is immunogenic for a class I MHC complex (HLA complex in humans). The class I HLA protein complex is used for the presentation of antigens at the cell surface and comprises a heavy chain with 3 domains (al, a2 and a3) and a beta2-microglobulin (b2M).

[0258] The term "class II HLA tumor antigen peptide (corresponding to MHC complex)" refers to a polypeptide sequence that binds to or is immunogenic for a class II MHC complex (HLA complex in humans). The class II HLA protein complex is used for the presentation of antigens at the cell surface and consists of two chains of almost equal size, an alpha chain and a non-covalently bound beta chain, each with two extracellular domains (al and a2 and bl and b2).

[0259] Thus, the polypeptides and pharmaceutical compositions of the present application (as defined herein) can be used for the prevention and treatment of breast cancer (also referred to herein as "cancer of the present application"). In general terms, "cancer of the present application" can be defined as diseases and conditions which can be suitably prevented and / or treated by suitably administering a tumor antigen peptide or a pharmaceutical composition of the present application (and, more specifically, a pharmaceutically effective amount thereof) to a subject, i.e. a person suffering from the disease or condition or at least one symptom thereof, and / or a person at risk of infection or development of such a disease or condition.

[0260] For the purposes of the present application, a "peptide sequence" (e.g., an HLA antigen peptide) having a "native sequence" includes a peptide sequence having the same (i.e., unmodified) amino acid sequence as a naturally occurring peptide sequence in a patient. Such a peptide sequence having a "native sequence" can be isolated from nature or produced recombinantly or synthetically. In particular, the term a peptide sequence having a "native sequence" includes naturally occurring truncated or secreted forms of a peptide sequence (e.g., an extracellular domain sequence), naturally occurring variants of a peptide sequence (e.g., alternative splice forms), and naturally occurring allelic variants.

[0261] As further described herein, the amino acid sequences used in the present application are individual variable HLA antigen peptide domains ("HLA" or "HLA complex"). An individual variable HLA antigen peptide domain is a region within a protein amino acid sequence that can be distinguished from the surrounding sequence according to defined properties (as further defined herein).

[0262] Amino acid sequences or regions within the amino acid sequence of the proteins of the present application that are HLA are also referred to herein as "HLA of the present application". Some preferred examples of individual variable HLA antigen peptide domains suitable for use in the present application become apparent from the further description herein and include, in particular, HLA-A, HLA-B, and HLA-C antigen peptides corresponding to MHC class I complexes and HLA-DR, DQ, and DP antigen peptides corresponding to MHC class II complexes.

[0263] Preferably, such neoantigen peptides (i.e., HLA antigen peptides that are only expressed and / or exposed by tumor cells) according to the present application that have less than 100% sequence identity or similarity to native HLA antigen peptides are characterized by amino acid substitutions in the amino acid sequence.

[0264] The following terms are used to describe sequence relationships between two or more amino acid sequences or polypeptide sequences: "reference sequence", "amino acid exchange", "sequence identity", "percent sequence identity", and "substantial identity".

[0265] In the context of the present application, the term "amino acid substitution" refers to the substitution of one amino acid for another in the amino acid sequence of an HLA antigen peptide to be synthesized relative to the wild type (i.e., native HLA antigen peptide) of that HLA antigen peptide to be synthesized. "Sequence identity", "percent sequence identity", or identity or similarity with respect to such amino acid sequences is defined herein as the percentage of amino acid residues in the amino acid sequence of a polypeptide that are identical (i.e., identical residues) or similar (i.e., amino acid residues that are from the same group based on common side chain features, see below) to the amino acid sequence of the wild type.

[0266] According to one preferred embodiment of the present application, the amino acid exchange of the HLA antigen peptide corresponding to an MHC class I and / or class II complex comprises at least one D / Y, C / Y, A / V, T / M, E / A or D / A exchange in the amino acid sequence at any position relative to the wild type of the HLA peptide.

[0267] As used herein, amino acids are abbreviated according to the universally accepted one-letter code of the IUPAC Nomenclature Commission. When two amino acids are separated by a hyphen ( / ), this indicates that the wild type amino acid (left side of the hyphen) has been replaced by another amino acid (right side of the hyphen) at the particular amino acid position in the relevant amino acid sequence.

[0268] In order to determine / obtain preferred anchor positions and preferred specific amino acid exchanges in the amino acid sequence of the HLA tumor antigen peptide, computer modeling methods are particularly suitable, for example based on the publications of Andreatta and Nielsen (Bioinformatics (2016) Feb 15; 32(4):511-7) and Nielsen et al. (Protein Sci., (2003) 12: 1007-17) by the algorithm NetMHC 4.0 (http: / / www.cbs.dtu.dk / services / NetMHC / ).

[0269] For the purpose of comparing two or more amino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and a second amino acid sequence can be calculated or determined by dividing the number of amino acids in the first amino acid sequence that are identical to the amino acids at the corresponding positions in the second amino acid sequence by [the total number of amino acids in the first amino acid sequence] and multiplying by [100%], wherein each deletion, insertion, substitution or addition of an amino acid in the second amino acid sequence compared to the first amino acid sequence is considered as a difference of a single amino acid (position).

[0270] An HLA tumor antigen peptide is "immunogenic" if the tumor cell exposes at its cell surface at least one corresponding MHC class I complex and / or at least one corresponding MHC class II complex recognizing and binding the HLA tumor antigen peptide, i.e. the HLA tumor antigen peptide exhibits a high specific activity for this MHC class I complex and / or MHC class II complex. The immunogenicity of an HLA tumor antigen peptide can be determined by Western blot, ELISA techniques, in particular by ELISPOT or immunodetection with microscopic analysis.

[0271] The HLA tumor antigen peptides according to the present application are preferentially immunogenic and are therefore also referred to as immunogenic HLA tumor antigen peptides ("epitopes"). The immunogenicity of the HLA tumor antigen peptides can be determined by suitable detection methods. Such methods are known to the skilled person and / or as described herein.

[0272] The term "specific affinity" or "specific binding affinity" of an HLA antigen peptide to a T cell receptor (TCR) refers to the specific and reversible binding of the HLA peptide to the TCR of an endogenous T cell. This "specific affinity" according to the present application is expressed by the dissociation constant (K D ) determined by a ligand binding assay, in units of molar.

[0273] Alternatively, the "specific affinity" of an HLA antigen peptide can also be determined by computer methods.

[0274] According to a preferred embodiment of the present application, the at least one HLA-A tumor antigen peptide is a tumor-specific HLA-A tumor antigen peptide (i.e. a cancer testis antigen (CTA) or a so-called neo-antigen peptide which is no longer present in healthy / normal tissue of the patient / patient group) and wherein the specific binding affinity of the tumor-specific HLA-A tumor antigen peptide is determined for the corresponding MHC class I complex in the range of K D determined as surface plasmon resonance, in the range of K D 10 to 50 nM.

[0275] Based on conventional computer-based binding prediction models (modelling), K D <50 nM are generally considered to be strongly binding, while those K D between 50 and 500 nM are considered to be weakly binding tumor antigen peptides. However, in the in vivo assays according to the present application, it has now been shown that K D values are not necessarily important. Surprisingly, it was found that tumor antigen peptides with K D values in the range of 50 to 500 nM trigger effector cells (i.e. cytotoxic T cells) contrary to the conventional computer-simulated binding prediction models which consider them to be of low binding. Thus, for the efficacy of the tumor antigen peptides, it is essential that the tumor antigen peptides are immunogenic.

[0276] The term "active substance enhancer / adjuvant" refers to an adjuvant which triggers and / or enhances the action of the HLA peptides at the first time. In principle, all common adjuvants known to the skilled person are suitable for the production of the formulations according to the present application.

[0277] However, the use of Montanide ISA 51 VG has proven to be particularly suitable. This forms so-called granulomas after the (pharmaceutical) formulation has been applied to the subject human, which advantageously store the HLA peptides in the form of a depot at the site of application and release them into the body of the subject human over a longer period of time. Thus, particularly advantageously, the (pharmaceutical) formulation according to the application does not need to be applied weekly. Preferably, the (pharmaceutical) formulation for the treatment of a cancer disease in the sense of the present application is used over a longer period of time, thus only needs to be applied every 2 weeks, particularly preferably only once a month.

[0278] The term "individual" (also referred to herein as "subject" or "patient") is used interchangeably with the term "subject" and means any mammal that is receiving treatment or has been diagnosed with a disease due to an abnormal physiological condition.

[0279] The terms "individual" and "subject" used in the present application include mammals, such as rodents, felines, ungulates, odd-toed ungulates or primates. In a particularly preferred embodiment, the subject is a human.

[0280] When reference is made herein to a "patient group", always reference is made to a group of individuals, preferably humans, who all have at least one, preferably at least two, most preferably at least three identical HLA alleles.

[0281] The patient allowed to be treated with the pharmaceutical composition has received standard treatment procedures (e.g. at least one surgery, radiation, chemotherapy and / or hormone therapy).

[0282] However, the pharmaceutical composition can also be given to a patient as a first line therapy or to a specifically identified patient group having at least one identical HLA allele.

[0283] According to a preferred embodiment of the present application, the individual has not received a chemotherapy for locally recurrent or metastatic breast cancer and / or has not received a prior adjuvant chemotherapy within 12 months or less since the last administration.

[0284] Particularly preferably, the individual has a haplotype of the subgroup A*01 or A*02.

[0285] A "haplotype" (abbreviation of "haploid genotype") is the sum of all specific alleles (= specific fingerprints) of a subject, which represents a variant of nucleotide sequences on one and the same chromosome in the genome of a living being. A specific haplotype can be individual-, population- or even species-specific.

[0286] For the purposes of the present invention, the transcriptome comprises the sum of all genes transcribed (i.e. transcribed from DNA sequences into mRNA sequences) in a cell at a given time, i.e. the total and quantified sum of all individual mRNA molecules produced in the cell. However, the creation of a transcriptome does not allow any statement about the "correctness" of the transcribed mRNA sequences.

[0287] In this context, the term "construction of a transcriptome" as used in the present disclosure means the analysis of the sum of all genes transcribed in a cell at a given point in time, preferably by quantitative real-time (RT)-PCR followed by DNA microarray or subsequent DNA sequencing. Typically, the construction of the transcriptome of a tissue sample provided in step (a) of the assay procedure of the present invention comprises the acquisition of more than 40,000 coding DNA sequences (raw data).

[0288] In genetics, the exome is the sum of the exons of an organism, i.e. all parts that can code for proteins. In humans, the exome comprises approximately 23,000 genes, approximately 50 million nucleobases. Whole exome sequencing (WES) examines all exons, i.e. the parts of the genome of a tissue section (i.e. the healthy tissue or tumor tissue of a test person) that code for proteins. Genetic diagnostics focuses on 1-2% of the human genome, in which 85% of known pathogenic mutations are found.

[0289] Thus, exome analysis involves sequencing the exome of a patient (and other relatives, if applicable), evaluating the sequence data and summarizing the results in a medical report. This diagnostic procedure is the method of choice for finding the cause of a disease, especially for patients with complex or non-specific symptoms and for whom a diagnosis has often not been explained for years.

[0290] In contrast to whole exome sequencing (WES), in which all protein-coding regions of approximately 23,000 known genes are enriched and sequenced, clinical exome sequencing (CES) enriches a subset of the exome. In WES, the focus is on determining disease-relevant genes described in the Human Gene Mutation Database (HGMD).

[0291] For the purposes of the present invention, the proteome refers to the sum of all proteins of at least one cell in a malignant or neoplastic tissue / tissue section or a cellular compartment thereof at a precisely defined condition and at a specific time. The proteome of a cell can be determined by proteome sequencing and is correlated to the genome of this cell by the transcriptome.

[0292] Immunotherapy is based on deciphering the individual mutational pattern (profile) of each cancer patient's tumor. Based on the profile of the mutational pattern, a synthetic vaccine, e.g. an RNA-based vaccine, is produced for each individual patient according to conventional therapeutic methods (i.e. vaccine production or production of a formulation according to the information of the present invention). These are subsequently used for the individualized treatment of the patient.

[0293] Basically, these new vaccines are not applicable to other patients with the same tumor, but only to the corresponding patient whose mutated tumor has previously been analyzed for vaccine production or information preparation of the present application. Thus, the recognition that different patients have overlaps in their malignant or neoplastic tissue / tumor excisions is a distinguished achievement of the present inventors. Advantageously, different patients can be divided into uniform patient groups, so that at least 50% of the HLA peptides of the preparation according to the present application are compatible with the mutated tumor of the patient group.

[0294] For the purposes of the present application, the sum of all HLA antigen peptides presented by MHC molecules at the cell surface is referred to as the (HLA) ligandome. It is believed that more than 105different HLA molecules are expressed at the cell surface and the number of identical HLA peptides presented can vary from a few copies per cell to up to 10,000 copies. Thus, approximately 10,000 different HLA peptides are presented on cells in different proportions.

[0295] The ligandome is influenced by various physiological, intrinsic and pathological (e.g. cancer or necrosis) factors, such as cell type or tissue type, infection or transformation of the cell, or simply the current state of the cell, which depends on the nutritional situation or external stress factors, leading to a change in the presented HLA peptides.

[0296] For example, at the beginning of the analysis of the HLA ligandome, Edman degradation can be used to initially understand the presented peptides. In one aspect, the general peptide motif of an allele can be determined by pool sequencing in this way, on the other hand, individual peptide sequences can already be determined by analyzing individual reverse phase high performance liquid chromatography (RP-HPLC) fractions.

[0297] Alternatively or in addition, in proteomics, the analysis of the ligandome can be carried out by modern mass spectrometers used, by which the sequence of many individual ligands can be unambiguously determined. There are two methods for ionization of the peptides or proteins required for this purpose: electrospray ionization (ESI) and matrix-assisted laser desorption / ionization (MALDI). In ESI, a coupling to an RP-HPLC system is very common. However, with increasing sensitivity of the mass spectrometer, capillary electrophoresis (CE) is also used as an analytical separation method.

[0298] In order to achieve higher sample throughput and higher sensitivity in the analysis of the ligandome, so-called UHPLC systems (ultra-high performance liquid chromatography) can be used. These HPLC systems use only 2 pm diameter material as packing of the separation column, thereby increasing speed, efficiency and chromatographic separation.

[0299] In ESI mass spectrometry, the direct coupling of HPLC and ESI interfaces allows for online separation of the sample, combined with an autosampler, enabling fully automated measurement procedures. Due to the continuous solvent flow from the HPLC, the sample can be measured in relatively short time. ESI mass spectrometry uses a wide range of instruments for analysis, such as quadrupole time-of-flight mass spectrometers, linear quadrupole ion traps, triple quadrupole or ion trap-orbitrap hybrid systems. This advantageously allows the identification of hundreds of HLA peptides in one measurement.

[0300] In the context of the present application, the term "deriving a ranking" means determining / selecting the number and affinity of HLA peptides exposed on the cell surface of cells of the harvested tissue (or tissue sections thereof).

[0301] Using the previous analysis, a cumulative ranking of HLA antigen peptides is derived in terms of protein mass and specific affinity (K D ) to T cell receptors of endogenous T cells. Thus, with regard to protein mass, factors are assessed which are in particular relevant to the content of tumor progression, such as invasiveness, angiogenesis, and evasion mechanisms of the tumor against an immune attack.

[0302] The algorithm uses a cumulative ranking system.

[0303] The cumulative (predicted) ranking is used to eliminate sequences.

[0304] In another embodiment, the highest ranked sequence likelihoods can be further confirmed by predicting their presence in a database of possible HLA antigen peptides with high specific affinity to T cell receptors of endogenous T cells (as defined herein) from sequence data, in particular limited to the organism / test person from which the HLA antigen peptides were obtained. In another embodiment, the highest ranked sequence likelihoods can be further confirmed by isolated coordinates of the HLA antigen peptides and / or their monomeric constituents (e.g. isoelectric point and molecular weight of the protein).

[0305] For the provision of (tumor) antigen peptides, the synthesized or isolated HLA tumor antigen peptides from the cumulative ranking can in principle be used for the preparation of (pharmaceutical) formulations of the present application and for the preparation of pharmaceutical compositions (such as so-called information formulations).

[0306] However, the use of synthetic HLA peptides is a good option. Production methods for synthetic peptides are known to the skilled person. Examples of such production methods are the Merrifield solid-phase peptide synthesis, the Bailey peptide synthesis and the N-carboxyanhydride method.

[0307] Another object of the present application is also (pharmaceutical) formulations of different dosage forms, which comprise a combination of the active ingredients according to the present application and optionally further active ingredients and / or excipients.

[0308] Preferred pharmaceutical preparations are tablets, chewable tablets, chewing gums, coated tablets, capsules, drops, fruit juices, syrups, suppositories, transmucosal therapeutic systems, transdermal therapeutic systems, solutions, injections, emulsions, suspensions, dry preparations ready for reconstitution, powders or sprays. Particularly preferred pharmaceutical preparations are injections or solutions.

[0309] Alternatively, the pharmaceutical preparation is present in a suitable administration device, preferably as a lyophilizate in a syringe, which allows reconstitution in situ with a pharmaceutically acceptable solution, such as saline.

[0310] Preferably, the (pharmaceutical) preparation according to the application is suitable for oral, intravenous, intramuscular, subcutaneous, intrathecal, epidural, buccal, sublingual, pulmonary, rectal, transdermal, nasal or intracerebroventricular administration, (pharmaceutical) preparations for subcutaneous or intravenous administration being particularly preferred.

[0311] Methods known in the prior art for preparing pharmaceutical compositions or dosage forms can be found, for example, in "Remington's Pharmaceutical Sciences". Pharmaceutical compositions for parenteral administration can comprise, for example, excipients, sterile water or saline, polyalkylene glycols, such as polyethylene glycol, oils of vegetable origin or hydrogenated naphthalenes. Biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compounds. Other potentially useful parenteral delivery systems for therapeutic anti-prion compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems and liposomes.

[0312] Contributing factors to tumor progression

[0313] Another important factor in determining the ranking is the identification of factors that play an important role in tumor progression (i.e. increase in tumor size and / or metastasis). Tumor progression is characterized by an increased growth rate, as well as increased invasiveness of the tumor.

[0314] Invasiveness refers to the degree of tissue penetration of a malignant tumor from its site of origin into adjacent tissue structures.

[0315] Angiogenesis describes the appearance of new blood vessels from an already existing vascular system and is an integral part of physiological processes (e.g. embryogenesis, wound healing) and pathological processes (e.g. diabetic retinopathy, chronic polyarthritis, tumor growth). It has long been known that in cancer new blood vessels form (angiogenesis), which is called tumor angiogenesis. Tumors consist of cells, which, like all other cells in the body, need nutrients and oxygen. In fact, due to the frequent division of cancer cells, their needs are particularly high. This is why tumors need their own blood vessels.

[0316] When a tumor develops, it initially has no blood vessels of its own. Thus, its growth is severely limited. Without its own blood vessels, a tumor will not grow beyond 1 to 2 mm. Metastasis formation also interacts with tumor angiogenesis, as tumor cells must reach a blood vessel in the periphery to metastasize. Only then can they be transported to a distant region of the body and form a metastasis there.

[0317] For example, a mutual dependency between class I HLA and integrin beta is known to stimulate signal transduction and cell proliferation. In this regard, integrin beta-mediated cell migration depends on its interaction with class I HLA molecules (Zhang and Reed, Hum Immunol. 2012 Dec, 73(12), 1239-1244).

[0318] The HLA peptide of the present application or a composition or formulation comprising the same can be used to modulate class I or class II HLA complexes, including combinations thereof, in vitro (e.g. in an in vitro or cellular assay) or in vivo (e.g. in a unicellular organism or a multicellular organism, in particular in a mammal, more particularly in a human, e.g. a human at risk of developing or suffering from a cancer of the present application).

[0319] In the context of the present application, "modulating" essentially means increasing the specific affinity of T cells for tumor-specific or tumor-associated MHC class I complexes or MHC class II complexes of malignant and / or neoplastic tissue, respectively, as measured by appropriate in vitro, cellular or in vivo assay methods, such as those mentioned herein. In particular, "modulating" means increasing the specific affinity of endogenous T cells of a patient or patient group for tumor-specific or tumor-associated MHC class I complexes or MHC class II complexes of malignant and / or neoplastic tissue by at least 1 %, preferably by at least 5%, such as 10% or by at least 25%, e.g. by at least 50%, at least 60%, at least 70%, at least 80% or 90% or more, compared to the affinity of T cells for tumor-specific or tumor-associated MHC class I complexes or MHC class II complexes of malignant and / or neoplastic tissue in the absence or prior to application of a pharmaceutical composition of the present application (as defined herein), under the same conditions and with the same assay methods.

[0320] In one embodiment, the present application comprises the above-mentioned method, wherein the expression profile of at least 5, preferably at least 6, most preferably at least 6 marker genes as shown in SEQ ID NO: 1 to SEQ ID NO: 48 is determined. As mentioned above, the marker genes are also defined by variants, again shown in Tables 2 to 4. Preferably, the expression profile is compared to the expression profile of a "reference". For example, the reference can be the expression profile of a healthy tissue (e.g. intestinal tissue or tissue of the liver, lung, etc.). In this context, the tissue of the affected individual (proband) can be used as "healthy tissue", from which it is known that no proliferative changes or even no metastases occur. Suitable examples are shown in the experimental part of the present application. However, data from tissue of external individuals (preferably healthy individuals) can also be used as "reference" or "reference value".

[0321] According to the present application, in the method for detecting cancer, at least 6, but preferably at least 8, most preferably at least 10 HLA antigen peptides corresponding to MHC class I complexes or MHC class I complexes (selected from the group of polypeptides / polypeptide fragments as shown in SEQ ID No: 1 to 48) will be determined, as described herein. For further embodiments, please refer to the experimental part.

[0322] The present application also comprises a method for preparing a pharmaceutical composition or (pharmaceutical) preparation according to the present application, wherein the method comprises the following steps:

[0323] (a) 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, which are exposed on the cell surface of the cells of the breast cancer / mammary carcinoma to be treated or of a patient group with the same haplotype, are determined by the determination method according to the present application as described above;

[0324] (b) the 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes and the at least 2 tumor antigen peptides corresponding to MHC class II complexes determined in step (a) are synthesized, wherein the definition of each HLA tumor antigen peptide is as defined above; and

[0325] (c) a pharmaceutical composition according to the present application is prepared, which comprises the at least 4 to 8 HLA-A tumor antigen peptides corresponding to MHC class I complexes, the at least 2 tumor antigen peptides corresponding to MHC class II complexes synthesized in step (b), and an adjuvant as defined herein.

[0326] The present application also comprises the use of a pharmaceutical composition or (pharmaceutical) preparation according to the present application for the preparation of a medicament or combination preparation for the treatment of malignant tumors, leukemias and tumors, in particular breast cancer.

[0327] The present application also comprises a pharmaceutical composition comprising an HLA peptide as defined in the present application and a pharmaceutically acceptable excipient.

[0328] The present application also relates to the HLA antigen peptides or neoantigen peptides of the present application, whether in vitro (for example in an in vitro or cellular assay) or in vivo (for example in a unicellular or multicellular organism, in particular in a mammal, more particularly in a human being, for example a human being at risk of developing or suffering from a cancer of the present application), for the manufacture of a preparation for the treatment of a cancer (for example, but not limited to, a pharmaceutical preparation as further described herein).

[0329] The present application also comprises a combination preparation for the treatment of a breast cancer, which is administered simultaneously, separately or sequentially,

[0330] The combination preparation comprises the following two separate preparations (a) and (b):

[0331] (a) a first preparation, together with a pharmaceutically acceptable carrier or diluent, comprising 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, as described herein and optionally determined by the method of the present application, and

[0332] (b) a second preparation, together with a pharmaceutically acceptable carrier or diluent, comprising an anticancer agent selected from the group consisting of anticancer alkylating agents, anticancer antimetabolites, anticancer antibiotics, herbal anticancer drugs, platinum coordination anticancer complexes, anticancer camptothecin derivatives, anticancer tyrosine kinase inhibitors, monoclonal antibodies, interferons, interleukins, biological response modifiers and other anticancer agents, or a pharmaceutically acceptable salt thereof.

[0333] The combination preparation according to the present application is particularly suitable for the treatment or prevention of breast cancer, in particular locally recurrent or metastatic breast cancer, in a patient or group of patients suffering from or suspected of suffering from breast cancer.

[0334] It is particularly preferred that the first preparation of the combination preparation according to the present application is thereby administered subcutaneously.

[0335] Particularly good experience has been made with the administration of the pharmaceutical composition according to the present application (first preparation) in combination with the administration of an herbal anticancer drug as second preparation, in particular artemether and / or curcumin.

[0336] However, it is preferred that the pharmaceutical composition according to the present application (first preparation) is administered in combination with a monoclonal antibody, in particular a monoclonal antibody directed against a target selected from the group consisting of CTLA4 (herein for example immunosuppressive proteins of EpCam, IDO, MIC, Fas and TRAIL; in particular: estrogen inhibitors from the aromatase inhibitor group for hormone-positive patients - (here e.g. letrozole and / or the estrogen blocker fulvestrant); antibodies against HER2-positive patients: Herceptin (TDM-1) as a second agent.

[0337] Also disclosed is a business method comprising selling 4 to 7 HLA-A antigen peptides and at least 2 antigen peptides corresponding to MHC class II complexes for the treatment of breast cancer in a human in order to effectively reduce the CA 15-3 score of the patient or a group of patients having at least one identical HLA allele, in particular to increase the progression-free survival or to reduce the likelihood of cancer recurrence or to increase the survival rate of the patient. In some embodiments, the selling is followed by treating the patient with the combination of HLA antigen peptides. BRIEF DESCRIPTION OF DRAWINGS

[0338] Herein is shown:

[0339] Figure 1 : Schematic representation of the HLA-A mediated binding of the T cell receptor to the MHC class I molecule, showing the anchor (amino acid) residues of the HLA-A antigen peptides (length 7-11 amino acids).

[0340] Figure 2 : Schematic representation of the HLA-B mediated binding of the T cell receptor to the MHC molecule, showing the anchor (amino acid) residues of the HLA-B antigen peptides (length 12-17 amino acids).

[0341] Figure 3 : Development of the specific tumor marker CA 15-3 after specific immunization with the composition of the application according to embodiment 3 of the tumor antigen peptide which represents an epitope of the primary tumor MC-HER2 / Neu (INC 14 / 1713; asterisk). The described course is 26 weeks.

[0342] Figure 4 : Development of the specific tumor marker CA 15-3 in combination with the liver marker gamma GT after specific immunization with the composition according to embodiment 3 of the tumor antigen peptide which represents a specific metastatic epitope of the disseminated MC-HER2 / Neu liver metastases (INC 14 / 1713; vertical line). The course shown is 34 weeks.

[0343] Figure 5 : Clinical efficacy of the pharmaceutical composition applied in embodiment 4. DETAILED DESCRIPTION

[0344] The application will be explained in more detail with the help of the following figures and examples, without limiting the application thereto.

[0345] The following table lists HLA tumor antigen peptides, all of which have been tested and are immunogenic. SEQ ID Nos: 13 to 48 list some preferred, but non-limiting, examples of amino acid sequences of tumor antigen peptides of the application, each of which is another embodiment of the application.

[0346] Table 1 [comparative example]

[0347]

[0348]

[0349]

[0350] Table 2

[0351]

[0352]

[0353]

[0354] Table 3

[0355]

[0356]

[0357] Table 4

[0358]

[0359]

[0360]

[0361] Example 1 : Transcriptomic analysis [mRNA expression]

[0362] All patients were subjected to transcriptomic analysis (sequencing) using the PANTHER chip analysis of Agilent (44K chip). The 44K chip used contains 44,000 gene probes, so in each case, each patient can be analyzed using this chip for the activity of more than 34,000 gene expression markers. In each case, 1000 genes with a 10-fold increase in relevance were determined.

[0363] The evaluation was based on published gene signatures known to the experts.

[0364] Example 2: EXOM sequencing

[0365] Exome sequencing was performed on standard frozen specimens of patients from which tumor DNA was extracted. Next generation sequencing of the exome was performed from tumor samples covering more than 95% of the human coding exonic regions. For this, more than 290,000 DNA related portions were selectively amplified and sequenced by means of semiconductor sequencing technology, among others.

[0366] The aim of the analysis was to determine possible mutations in order to design individual tumor antigen peptide immunization. In order to exclude germline, non-tumor related variations (SNP, single nucleotide polymorphisms), DNA was also isolated from the patient's nucleated blood cells and compared sequencing using the same method. Differentiation of potential neoantigen candidates was performed by the following six steps:

[0367] 1) Reduction of tumor / somatic mutations by excluding non-tumor specific polymorphisms.

[0368] 2) Further restriction of somatic mutations to predefined selection criteria of quality parameters, read coverage and impact on protein sequence. Mutations more complex than single amino acid substitution SNPs, as well as mutations in non-protein coding regions were excluded. Silent mutations were also excluded.

[0369] 3) Extension of mutations defined in this way to flanking 20s oligopeptides using known protein sequences (RefGene database). From these, 12x9 oligopeptides were formed in turn and the affinity to pre-known, patient-specific hypervariable HLA-I paratopes was determined (program NetMHC-4.0). Nine peptides with high affinity to the paratope were selected, which, due to the mutation (SNP), had a further increased affinity compared to the wild-type sequence.

[0370] 4) Exclusion of candidates carrying this mutation in the same position as a germline mutation in the pool of exons (NIH-NHLBI 6500 Exome Database Version 2 program ANNOVAR-Tool2.)

[0371] 5) Based on the results of the expression analysis (see findings of Example 1), peptides with low expression relevance in the tumor can be excluded.

[0372] 6) Finally, we will examine in detail whether further polymorphisms can exist in the vicinity of the single base exchange, thus further representing patient-specific deviations from the genome sequence defined from hg19.

[0373] The following is an example of the evaluation of the results of the described mutation analysis and filtering steps for one patient:

[0374] 1) Comparison of tumor DNA with normal DNA

[0375] a. "Exome_single_sample_Somatic" was generated using the Ion Reporter:

[0376] - 37494 variants in normal DNA, and

[0377] - 37299 variants in tumor DNA.

[0378] b. Using statistical analysis in Ion AmpliSeq Exome Panel (Ion Reporter software 4.6. Workflow version: 1.0) the "AmpliSeq Exome tumor-normal pair" detects and annotates rare somatic variants (SNP, InDels, CNV) in pairs. This results in: 1477 mutations in the tumor, accompanied by wildtype classification in normal DNA.

[0379] These 1477 variants are the starting point for further restriction.

[0380] 2) Restrict variants to protein coding SNPs (Single Nucleotide Polymorphisms) with amino acid substitution.

[0381] - Restriction to SNPs

[0382] - Tumor sequencing of at least 50x and min. 20x blood sequencing

[0383] - Must not occur in blood in any one run

[0384] - Must be located in an exon

[0385] - Must result in an amino acid exchange (missense mutation)

[0386] 3) From these variants, define 20s peptides and feed them into HLA paratope affinity analysis NetMHC.

[0387] Affinity analysis was performed on about 25*12 nonapeptide pairs (each mutant and wildtype) to analyze each of the 9 HLA loci. Out of these, 5814 affinities (2907 pairs) were analyzed. In 40 pairs, the affinity of the mutant peptide was at least 2-fold higher than the wildtype peptide.

[0388] Summary of variants with increased HLA affinity

[0389] 4) Exclude candidates carrying this mutation as germline mutations in the same position in the Exon Pool (NIH-NHLBI 6500 exome database version-2) (Programm ANNOVAR - Tool3.)

[0390] Database filter used: hg19_esp6500siv2_all

[0391] 5) Selection from the remaining 25 variants with significant mRNA expression ( > 3 fold compared to normal tissue) aligned to the transcriptome

[0392] Example 3: Patient with mMC (metastatic breast cancer), type_HER2-neu

[0393] 1) Clinical history of the patient

[0394] Patient, female

[0395] Preliminary clinical findings : Invasive ductal dual focus breast cancer (breast CA), right side, tumor max 2 cm to 5 cm (pT2), pN1 sn pN1 (3 / 5) G2, NO, ER-, PR-, HER-2 neu: 3+, Ki-67: 55%.

[0396] Treatment procedure

[0397] ■ After 2 weeks: Neoadjuvant chemotherapy (TCH w3) 6 cycles, trastuzumab;

[0398] ■ After 6 months: Irradiation of the right breast + lymphatic drainage area (LAG).

[0399] The tumor was completely destroyed by tumor radiotherapy or chemotherapy (CR = complete remission), result: patient cured and discharged.

[0400] Relapse : Local recurrence occurred 24 months after initial diagnosis with additional liver metastases (4 foci); result: ER-, PR+ (60%), HER-2 neu: 3+, Ki-67: 80%.

[0401] This recurrent breast CA with liver metastases is of significantly greater invasiveness (Ki-67: 80% vs. 55% (initial finding)).

[0402] Prognosis by the clinician : Patient discharged, prognosis / average OS (overall survival) of 6 months expected.

[0403] It is known that in breast CA, the relapse of a second primary malignancy (SPM) is associated with a worse prognosis and a worse OS (see "Breast cancer survivors face excess risk for second primary cancer in SEER analysis," Wei JL & al, Int J Clin Oncol, Mar 19, 2019), possibly triggered by the previous chemotherapy,

[0404] Treatment procedure :

[0405] 2 months after diagnosis : due to the occurrence of a local recurrence, the breast of the right breast and the adjacent tissue were surgically removed (mastectomy).

[0406] 4 months after diagnosis : bone metastases (bone metastases (spine)) and bronchial carcinoma (suspected LK hilar) occurred;

[0407] 9 months after diagnosis : the breast of the left breast and the adjacent tissue were surgically removed.

[0408] Despite the continued treatment with trastuzumab or T-DM1, the tumor progressed

[0409] 2) Individualized immunoinformatic therapy: analysis

[0410] After the treatment / handling procedure under point 1 was carried out without success, an immunotherapeutic method using the information of the patient's own immune system by applying tumor-associated and tumor-specific HLA antigen peptides in the form of synthetic peptides was used as part of a therapeutic trial.

[0411] The focus is on cellular immune defense, i.e. the activation of endogenous cytotoxic CD8+ T cells, which, as initial T cells, are able to recognize almost all possible pathogenic and malignant amino acid sequences (so-called "targets") through a highly differentiated receptor system and thus develop into effector cells. These effector T cells can destroy tumor cells recognized by HLA-presented antigens by secreting granzymes and perforin.

[0412] For this purpose, data were collected from the patient's biopsy tumor tissue using the following three steps by "next generation sequencing" (NGS) and "liquid chromatography coupled to tandem mass spectrometry" (LC-MS / MS)

[0413] ■ all mRNA expressions of the coding genetic regions (transcriptome - step (a)),

[0414] ■ tumor-specific somatic missense mutations as well as by (exome sequencing - step (b)) ​​​

[0415] ■ Tumor cell presented HLA class I+II HLA restricted ligands (HLA ligand set - step (c))

[0416] Step (a): mRNA expression data (about 40,000) are compared to expression levels of healthy tissue and filtered for

[0417] ■ significant (> 3-fold) deviation of expression values, and further

[0418] ■ importance regarding genes related to tumor development

[0419] ■ according to the criterion that these genes can not be expressed or only slightly expressed in other tissue types

[0420] ■ In addition, regarding the cancer-testis antigen set which is usually not expressed in healthy adult breast tissue of adults, significant expression in tumors is documented, as these can have the properties of tumor-specific antigens

[0421] This results in a separate data set of possible HLA tumor antigen peptides (number 86) which are weighted according to their expression abnormality / deviation from normal and their known importance for tumor proliferation -> factor 3 vs. normal; importance for tumor development: growth factors, angiogenic factors, metastatic factors.

[0422] Step (b): combinations of Somatic missense mutations single nucleotide variants (SNV) with one amino acid exchange and frameshift mutations (number: 57) are combined into another data set, as they can be important (new) antigens in their characteristic and thus have a high degree of tumor specificity.

[0423] Step (c): for the following, information from specialized databases is used to screen HLA restricted ligands (9-10 AS amino acid sequences (corresponding to MHC class I complexes) (number: 1100) and 12-15 AS sequences (corresponding to MHC class II complexes) (number: 730)

[0424] ■ which have already occurred in healthy tissue (= negative)

[0425] ■ protein matching with the desired sequences from the transcriptome and exome, whether the sequence of the determined tumor-related HLA antigen peptide is related to the proliferation, invasiveness, angiogenesis and / or increased production of cytokeratins of the breast cancer / breast carcinoma.

[0426] At the same time as these described tumor tissue examinations, the genetic haplotype and alleles of the HLA antigen peptides corresponding to the MHC class I complexes of the patients are determined. The patient individual results lead to the following assignment:

[0427] 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.

[0428] (In support of HLA-A*02:01 associated with B*18:01, this patient represents about 40% of the European (Caucasian) population).

[0429] 3) Individualized immunoinformative therapy: target selection

[0430] Step (a): From the dataset of the transcriptome, the amino acid sequences of the HLA tumor antigen peptides (nonamers) corresponding to the MHC complex with the highest allelic affinity (specific activity [nM] to T cell receptors) are first selected using the amino acid sequence of each protein. This selection criterion serves to determine by algorithm the probability of the corresponding tumor antigen peptide in the MHC complex being presented in vivo on the corresponding MHC complex (first prerequisite for a possible cellular immune response).

[0431] Step (b): From the dataset of the mutation test, the nonamers involving amino acid exchanges are determined with respect to the highest affinity (specific activity [nM] to T cell receptors) according to the alleles of the patient. In addition, for this purpose, polymers (oligopeptides) of 17 amino acids are determined under the affinity criterion. This selection criterion serves to determine by algorithm the probability of the corresponding tumor antigen peptide in the MHC complex being presented in vivo on the corresponding MHC complex (second prerequisite for a possible cellular immune response).

[0432] Step (c): Based on the new dataset determined in steps (a) and (b), the HLA-restricted ligands (dataset of the ligand panel) are added, the HLA tumor antigen peptides corresponding to the MHC class I complex and to the MHC class II complex are selected, which are (both individually and in particular in combination) the most promising epitope candidates to trigger a cellular immune response.

[0433] 4) Individualized immunoinformative therapy: peptide synthesis and delivery protocol

[0434] Step (a): The peptide concept selected for the customized application solution is synthesized as a chemical peptide.

[0435] Step (b): The following 8 HLA-A tumor antigen peptides corresponding to the MHC class I complex and 4 HLA-A tumor antigen peptides corresponding to the MHC class II complex are mixed in a 33% DMSO / H2O application solution and divided into 24 vial units of 1 ml each.

[0436] Tumor antigen peptide sequences

[0437]

[0438]

[0439] Dose of each tumor antigen peptide : 500 pg

[0440] 5) Individualized immunoinformative therapy: application protocol

[0441] Pre-treatment: 300 mg / m 2 Cyclophosphamide (single infusion); 3 days before first injection

[0442] Mode of administration: intradermal (i.d.)

[0443] Site of administration: left and right upper arm

[0444] Schedule of administration: 23 vaccinations on days 1, 2, 3, 8, 15, 22, 36, 50, 71, then every 3 weeks until day 365.

[0445] Adjuvant added at each application: 250 mg cream (Aldara) with 12.5 mg imiquimod locally at the injection site; 200 pg ipilimumab (Yervoy) because of CTLA4 elevation, alternated with 300 pg nivolumab (Opdivo) because of PD1 or PD-L1 elevation, administered directly subcutaneously (s.c.) in the proximity of the application site.

[0446] 6) Individualized immunoinformative therapy: 2 proofs of clinical efficacy.

[0447] Based on two important examples, the clinical efficacy of the application of immunotherapy in this case 1 is shown as follows:

[0448] Figure 3 The development of the specific tumor marker CA 15-3 and the liver marker gGT is shown after the specific pharmaceutical composition of the immunoinformative BITAP peptide (specific metastatic epitope for the disseminated mmC-HER2 / Neu (INC 14 / 1713) liver metastasis) in the specific application (id).

[0449] The course shown is 34 weeks.

[0450] The development of the specific tumor marker CA 15-3 is plotted after the specific immunoinformative composition of the tumor antigen peptide (epitope of the primary tumor MC-HER2 / Neu (INC 14 / 1713)) according to the application (star). The course shown is 26 weeks.

[0451] Example 4: mIBC with carcinomatous lymphangiomatosis (metastatic inflammatory breast cancer) patient

[0452] 1) Clinical history of the patient

[0453] Patient, female (46 years old)

[0454] Initial clinical finding: inflammatory breast cancer right with extensive carcinomatous lymphangiomatosis; initial tumor stage pT4, pn3a (24 / 29) (Level II: 14 / 19, Level III: 10 / 10), M0, LI, V0, G2. HR+, PR+, Androgen r.+, HER2 / neu-

[0455] Recurrence: First recurrence after 3 months post-surgery: progression found in the lower quadrant of the right breast, with a significant increase in the consistency of the breast tissue. Skin infiltration across the midline of the chest wall area and local regional tumor infiltration of the right axillary fat tissue and the chest lateral wall along the right pectoralis major muscle.

[0456] 5 months later, local regional tumor recurrence with extension into the axilla.

[0457] Chemotherapy from month 6 to month 11, with the possibility of stopping after 5 cycles if there is tumor progression during chemotherapy. Local regional tumor recurrence along the lateral chest wall with 7x11 cm, extensive infiltration and thickening of the skin and subcutaneous tissue.

[0458] Hormone therapy with aromatase inhibitors and irradiation of the right breast, chest wall, and supraclavicular / right cervical spine.

[0459] Prognosis of the clinician: total survival of 3-4 months is expected. (Inflammatory breast cancer is a relatively rare, particularly aggressive form of breast cancer, which in this case 2 has already spread to the lymphatic vessels (24 of 29 lymph nodes were already affected at the time of the initial diagnosis)).

[0460] Therapeutic procedure according to the application: After all therapeutic interventions available in the standard and conventional specifications of oncology medicine have been unsuccessful, the patient will be informed about the immunotherapeutic approach of the immune system using tumor-associated and tumor-specific antigens in the form of synthetic peptides for therapeutic testing.

[0461] The focus is on cellular immune defense, i.e. the activation of endogenous cytotoxic CD8+ T cells, which, as initial T cells, are able to recognize almost all possible pathogenic and malignant amino acid sequences (hereinafter referred to as "targets") via a highly differentiated receptor system and, in turn, develop into effector cells. These effector T cells can destroy tumor cells recognized by HLA-presented antigens by secreting granzymes and perforin.

[0462] Activation of these immune cells occurs via HLA class I ligands, i.e. antigens presented in the form of 8-10 amino acid sequences restrictively on class I HLA molecules.

[0463] Within 14-84 months after the start of the individualized immuno-informatic therapy, additional local recurrences (lymph nodes) occurred, each of which was surgically removed. However, distant metastases were prevented.

[0464] 2) Individualized immuno-informatic therapy: analysis

[0465] To identify tumor-associated and tumor-specific antigen peptides, data were determined from the patient's biopsy tumor tissue using next-generation sequencing (NGS) and liquid chromatography coupled to tandem mass spectrometry (LC-MS / MS) in the following three steps:

[0466] ■ mRNA expression of all coding gene regions (transcriptome - step (a)),

[0467] ■ tumor-specific somatic missense mutations as well as by (exome sequencing - step (b))

[0468] ■ HLA class I+II HLA-restricted ligands presented by tumor cells (HLA ligandome - step (c))

[0469] Step (a): mRNA expression data (about 40,000) were compared to expression levels of healthy tissue and filtered for:

[0470] ■ significant (> 3-fold) deviation of expression values, and further

[0471] ■ importance of genes in relation to tumor development

[0472] ■ criteria that these genes can not be expressed or only slightly expressed in other tissue types

[0473] ■ In addition, regarding the cancer-testis antigen group, which is usually not expressed in healthy adult breast tissue, significant expression in the tumor was documented, as they can have the properties of tumor-specific antigens

[0474] This resulted in a separate data set of possible HLA tumor antigen peptides (number 86), which were weighted according to their expression abnormalities / deviations from normal values and their known importance in tumor proliferation.

[0475] Step (b): The Somatic missense mutations Single nucleotide variants (SNVs) with one amino acid exchange as well as frameshift mutations (number: 41) were combined into another data set, as they can be important (neo)antigens in nature, thus being highly tumor-specific.

[0476] Step (c): For the following, the information from specialized databases was used to screen the HLA-restricted ligands (amino acid sequence of 9-10AS (corresponding to MHC class I complex) (number: 1321) and 12-15AS (corresponding to MHC class II complex) (number: 863)

[0477] ■that have already occurred in healthy tissue (= negative)

[0478] ■Matching with the proteins from the desired sequences from the transcriptome and exome, whether the sequence of the identified tumor-associated HLA antigen peptide is associated with an increase in proliferation, invasiveness, angiogenesis and / or cytokeratin production of the breast cancer / breast carcinoma.

[0479] In parallel to these described tumor tissue examinations, the genetic haplotype and alleles of the HLA antigen peptides corresponding to the MHC class I complex of the patient were determined. The patient individual results led to the following allocation:

[0480] 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

[0481] (In support of HLA-A*01:01 associated with B*08:01, this patient represents approximately 25% of the European (Caucasian) population).

[0482] 3) Individualized immunoinformative therapy: target selection

[0483] Step (a): From the data set of the transcriptome, the amino acid sequence of the HLA tumor antigen peptide (nonamer) corresponding to the MHC complex with the highest allelic affinity (specific activity [nM] to the T cell receptor) was first selected using the amino acid sequence of the respective protein. This selection criterion is used to determine by algorithm the probability of the corresponding HLA tumor antigen peptide being presented in vivo on the corresponding MHC complex (first prerequisite for a possible cellular immune response).

[0484] Step (b): From the data set of the mutation test, the nonamer variants involving the amino acid exchange are determined according to the alleles of the patients, relative to the highest affinity (specific activity of the T cell receptor [nM]). In addition, for this purpose, 17 amino acid polymers were determined under the affinity criterion. This selection criterion determines the probability of the corresponding tumor antigen peptide of the MHC complex being presented in vivo on the corresponding MHC complex (second prerequisite for a possible cellular immune response) by means of an algorithm.

[0485] Step (c): Based on the new data sets determined in steps (a) and (b), the HLA-restricted ligands (data set of the ligand panel) are added, the HLA tumor antigen peptides corresponding to MHC class I complexes and to MHC class II complexes are selected, which are (both individually and in particular in combination) the most promising epitope candidates for triggering a cellular immune response.

[0486] 4) Individualized immunoinformative therapy: Peptide synthesis and delivery protocol

[0487] Step (a): Synthesis of the peptide concept selected for the customized application solution as a chemical peptide.

[0488] Step (b): The following 9 HLA-A tumor antigen peptides corresponding to MHC class I complexes and 3 HLA tumor antigen peptides corresponding to MHC class II complexes are mixed in a 33% DMSO / H2O application solution and divided into 6 vial units, 1.5 ml per vial.

[0489] Tumor antigen peptide sequences

[0490]

[0491]

[0492] Dose of each peptide : 300 μg

[0493] Total peptide content per application dose (vial): 3.3 mg

[0494] 5) Individualized immunoinformative therapy: Application protocol

[0495] Pre-treatment: 3 million units of IFN alpha 2b (Roferon), 4 days before the first injection

[0496] Mode of administration: Subcutaneous (s.c.)

[0497] Site of administration: Left and right upper arm, left and right buttocks

[0498] Administration plan: 1st, 8th, 22nd, 50th, 180th and 360th day, 6 injections

[0499] Adjuvant added per administration: Montanide ISA 51 VG (1.5 ml) mixed 1 : 1 with peptide cocktail (1.5 ml); 300 pg nivolumab (Opdivo) injected s.c. directly adjacent to the injection site 30 min prior to peptide / Montanide injection when PD1 or PD-L1 is elevated.

[0500] 6) Individualized immunoinformative therapy: evidence of clinical efficacy

[0501] The clinical efficacy of the immunotherapy applied in Example 4 is shown as Figure 5 indicated.

[0502] The graph represents the development of the tumor markers CEA (column 1) and CA15-3 (column 2) as well as the liver values y-GT (column 3) and leukocyte count (column 4) during the 8 months after application of the pharmaceutical composition according to Example 4. Prior to this was an aggressive tumor progression - essentially based on inflammatory lymphangiomatosis cancer disease. The administration was carried out using a 1 : 1 mixture of Montanide ISA 51 VG with a peptide cocktail. The volume was 3 ml and was administered s.c. at 4 different administration sites. (See Section 5). In this peptide composition, is the initial administration (followed by 4 further administrations; see arrows in Figure 5

[0503] The 10 peptides contained in the pharmaceutical composition of Example 4 are tumor antigen peptides (or neoantigens). According to the information, antigens 1-4 and 10 have become epitopes, i.e. successfully activated the corresponding T cell receptors (TCRs) and produced effector cells and memory cells. SEQUENCE LISTING <110> PMCR LLC <120> Class I and class II HLA tumor antigen peptides for the treatment of breast cancer <130> PMC-0001-P-DE <140> DE 10 2019 114 735.2 <141> 2019-06-02 <160> 48 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> AA <213> Synthetic construct <220> <223> HLA-A antigen peptide​ <400> 1 CVGRRNYRFF Y 11 <210> 2 <211> 11 <212> AA <213> Synthetic construct <220> <223> HLA-A antigenic peptide <400> 2 HAVFVQSYY 9 <210> 3 <211> 8 <212> AA <213> Synthetic construct <220> <223> HLA-A antigenic peptide <400> 3 LLDPEDVD 8 <210> 4 <211> 8 <212> AA <213> Synthetic construct <220> <223> HLA-A antigenic peptide <400> 4 HTDIYANY 8 <210> 5 <211> 8 <212> AA <213> Synthetic construct <220> <223> HLA-A antigenic peptide <400> 5 AVFVQSYY 8 <210> 6 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-B antigenic peptide <400> 6 EEEAAAAAAY 10 <210> 7 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-B antigen peptide <400> 7 MEVLSQEIVR 10 <210> 8 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-B antigen peptide <400> 8 HMKKMMKDL 9 <210> 9 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-C antigen peptide <400> 9 SSTALHPCPF 10 <210> 10 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-C antigen peptide <400> 10 LSYLHVHTA 9 <210> 11 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-C antigen peptide <400> 11 YSLLSLLHT 9 <210> 12 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-C antigen peptide <400> 12 CPFTHGSSPM 10 <210> 13 <211> 11 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptide <400> 13 YVGRRNYRFF Y 11 <210> 14 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptide <400> 14 HVVFVQSYY 9 <210> 15 <211> 8 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptide <400> 15 LLDPEDVY 8 <210> 16 <211> 8 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptide <400> 16 HMDIYANY 8 <210> 17 <211> 8 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptide <400> 17 VVFVQSYY 8 <210> 18 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptides <400> 18 DEDEIKWWW 9 <210> 19 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-A neoantigen peptides <400> 19 FVNDKFMPL 9 <210> 20 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-B neoantigen peptides <400> 20 EEAAAAAAAY 10 <210> 21 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-B neoantigen peptides <400> 21 MEVLSQEIVW 10 <210> 22 <211> 9 <212> AA <213> Synthetic construct <220> <223> HLA-B neoantigen peptides <400> 22 HMKKMMKAL 9 <210> 23 <211> 10 <212> AA <213> Synthetic construct <220> <223> HLA-C neoantigen peptides <400> 23 SSTPLHPYPF 10 <210> 24 <211> 9 <212> AA <213> synthetic construct <220> <223> HLA-C neoantigen peptide <400> 24 FSYLHVHTA 9 <210> 25 <211> 9 <212> AA <213> synthetic construct <220> <223> HLA-C neoantigen peptide <400> 25 YSLLSLLHM 9 <210> 26 <211> 10 <212> AA <213> synthetic construct <220> <223> HLA-C neoantigen peptide <400> 26 YPFTHGSSPM 10 <210> 27 <211> 11 <212> AA <213> synthetic construct <220> <223> HLA-A neoantigen peptide <400> 27 TYLPTNASLS F 11 <210> 28 <211> 9 <212> AA <213> synthetic construct <220> <223> Class I neoantigen peptide <400> 28 DAVIVKLEI 9 <210> 29 <211> 9 <212> AA <213> synthetic construct <220> <223> Class I neoantigen peptide <400> 29 YYLDLSITR 9 <210> 30 <211> 10 <212> AA <213> Synthetic construct <220> <223> Class I HLA-A neoantigen peptide <400> 30 ILFGISLREV 10 <210> 31 <211> 9 <212> AA <213> Synthetic construct <220> <223> Class I HLA-A neoantigen peptide <400> 31 KVVEFLAML 9 <210> 32 <211> 9 <212> AA <213> Synthetic construct <220> <223> Class I HLA-A neoantigen peptide <400> 32 FVNDKFMPL 9 <210> 33 <211> 9 <212> AA <213> Synthetic construct <220> <223> Class I HLA-A neoantigen peptide <400> 33 FLLILKRDS 9 <210> 34 <211> 9 <212> AA <213> Synthetic construct <220> <223> Class I HLA-A neoantigen peptide <400> 34 RTPLSALCV 9 <210> 35 <211> 9 <212> AA <213> synthetic constructs <220> <223> HLA class I-B neoantigen peptides <400> 35 DEDEIKWWW 9 <210> 36 <211> 17 <212> AA <213> synthetic constructs <220> <223> HLA class II neoantigen peptides <400> 36 EDKKIDFSEF LSLLGDI 17 <210> 37 <211> 17 <212> AA <213> synthetic constructs <220> <223> HLA class II neoantigen peptides <400> 37 IHREDEDEIK WWWARLN 17 <210> 38 <211> 17 <212> AA <213> synthetic constructs <220> <223> HLA class II neoantigen peptides <400> 38 STKYSHKSPQ LSVHVTD 17 <210> 39 <211> 30 <212> AA <213> synthetic constructs <220> <223> HLA class II neoantigen peptides <400> 39 KYIQESQALA KRSCGLFQKL GEYYLQNAFL 30 <210> 40 <211> 14 <212> AA <213> synthetic constructs <220> <223> Class II HLA neoantigen peptides <400> 40 VDLIVEYEAF PKPE 14 <210> 41 <211> 15 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptides <400> 41 ATYSGAGYYL DLSIT 15 <210> 42 <211> 17 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptides <400> 42 HGSSFFLLIL KRDSAFI 17 <210> 43 <211> 17 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptides <400> 43 YSMKCKNVVP LNDLLLE 17 <210> 44 <211> 15 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptides <400> 44 PLQIILMPQV QPGLP 15 <210> 45 <211> 15 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptides <400> 45 LLHTEYSLLS LLHMQ 15 <210> 46 <211> 15 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptide <400> 46 NYLAEEITVD VRDEF 15 <210> 47 <211> 15 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptide <400> 47 REIDVLEREL NVLIF 15 <210> 48 <211> 15 <212> AA <213> Synthetic construct <220> <223> Class II HLA neoantigen peptide <400> 48 PSNYQPHQAC RITFL 15

Claims

1. A pharmaceutical composition for use in the treatment or prevention of breast cancer / mammary carcinoma in a patient or group of patients suffering from or suspected of suffering from breast cancer / mammary carcinoma, wherein, The pharmaceutical composition comprises a pharmacologically effective amount of: i. HLA tumor antigen peptides consisting of SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 30- SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 42; or ii. HLA tumor antigen peptides consisting of SEQ ID No. 13- SEQ ID No. 17, SEQ ID No. 20- SEQ ID No. 23, SEQ ID No. 43, SEQ ID No. 45 and SEQ ID No. 46, wherein SEQ ID No. 13-17, 20-23, 25, 28 and 30-35 are HLA tumor antigen peptides corresponding to MHC class I complexes, SEQ ID No. 36, 38, 39, 42, 43, 45 and 46 are HLA tumor antigen peptides corresponding to MHC class II complexes, and the HLA tumor antigen peptides are HLA antigen peptides associated with breast cancer.

2. The pharmaceutical composition according to claim 1, wherein, The breast cancer is locally recurrent or metastatic breast cancer.

3. The pharmaceutical composition according to claim 1 or 2, wherein, A pharmacologically effective amount of the tumor antigen peptides is administered to a patient or a group of patients having breast cancer to effectively reduce CA 15-3 levels.

4. The pharmaceutical composition according to claim 1 or 2, wherein, The immunogenic HLA tumor antigen peptides corresponding to MHC class I and II complexes, respectively, are determined by immunogenicity analysis selected from Western Blot, ELISA techniques, ELISPOT with microscopic analysis or immunodetection.

5. The pharmaceutical composition according to claim 1 or 2, wherein, The HLA tumor antigen peptides comprise HLA-A tumor antigen peptides which bind to the corresponding MHC class I complexes of the subtypes A*01 and / or A*02.

6. The pharmaceutical composition according to claim 1 or 2, wherein, The HLA tumor antigen peptides are presented on the surface of the breast cancer tumor cells of the patient or group of patients as determined by ultra-high performance liquid chromatography (UHPCL) combined with ESI mass spectrometry (MS).

7. The pharmaceutical composition according to claim 1 or 2, wherein The expression level of the tumor-associated HLA tumor antigen peptides in the tumor cells is at least three times higher than in healthy cells of the patient or a group of patients having at least one identical HLA allele as determined by qPCR, and wherein the tumor-associated HLA antigen peptides are associated with increased proliferation, invasiveness, angiogenesis and cytokeratin production of the breast cancer.

8. The pharmaceutical composition according to claim 1 or 2, wherein: at least one HLA tumor antigen peptide is a tumor-specific HLA-A tumor antigen peptide, and - the specific binding of the determined tumor-specific HLA-A tumor antigen peptide to the corresponding MHC class I complex determined by surface plasmon resonance K D in the range of 10 to 50 nM.

9. The pharmaceutical composition according to claim 1 or 2, wherein, the pharmacologically effective amount of each individual HLA antigen peptide in the composition, i.e. the dose administered, is in the range of 100 to 600 pg.

10. The pharmaceutical composition according to claim 1 or 2, wherein, The composition comprises an adjuvant capable of forming a granuloma at the site where the composition is administered to the patient.

11. The pharmaceutical composition according to claim 1 or 2, wherein, The pharmaceutical composition is administered subcutaneously or intradermally.

12. The pharmaceutical composition of claim 11, wherein, The pharmaceutical composition is administered simultaneously at at least 2 administration sites away from the tumor lesion and / or the area of cancerous lymph nodes. The pharmaceutical composition comprises a pharmacologically effective amount of: i. HLA tumor antigen peptides consisting of SEQ ID No. 25, SEQ ID No. 28, SEQ ID No. 30- SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 39, SEQ ID No. 42; or ii. HLA tumor antigen peptides consisting of SEQ ID No. 13- SEQ ID No. 17, SEQ ID No. 20- SEQ ID No. 23, SEQ ID No. 43, SEQ ID No. 45 and SEQ ID No. 46, wherein SEQ ID No. 13-17, 20-23, 25, 28 and 30-35 are HLA tumor antigen peptides corresponding to MHC class I complexes, SEQ ID No. 36, 38, 39, 42, 43, 45 and 46 are HLA tumor antigen peptides corresponding to MHC class II complexes, and the HLA tumor antigen peptides are HLA antigen peptides associated with breast cancer. The breast cancer is locally recurrent or metastatic breast cancer. A pharmacologically effective amount of the tumor antigen peptides is administered to a patient or a group of patients having breast cancer to effectively reduce CA 15-3 levels. The immunogenic HLA tumor antigen peptides corresponding to MHC class I and II complexes, respectively, are determined by immunogenicity analysis selected from Western Blot, ELISA techniques, ELISPOT with microscopic analysis or immunodetection. The HLA tumor antigen peptides comprise HLA-A tumor antigen peptides which bind to the corresponding MHC class I complexes of the subtypes A*01 and / or A*02. The HLA tumor antigen peptides are presented on the surface of the breast cancer tumor cells of the patient or group of patients as determined by ultra-high performance liquid chromatography (UHPCL) combined with ESI mass spectrometry (MS).

7. The pharmaceutical composition according to claim 1 or 2, The expression level of the tumor-associated HLA tumor antigen peptides in the tumor cells is at least three times higher than in healthy cells of the patient or a group of patients having at least one identical HLA allele as determined by qPCR, and wherein the tumor-associated HLA antigen peptides are associated with increased proliferation, invasiveness, angiogenesis and cytokeratin production of the breast cancer.

8. The pharmaceutical composition according to claim 1 or 2, wherein: at least one HLA tumor antigen peptide is a tumor-specific HLA-A tumor antigen peptide, and the pharmacologically effective amount of each individual HLA antigen peptide in the composition, i.e. the dose administered, is in the range of 100 to 600 pg. The composition comprises an adjuvant capable of forming a granuloma at the site where the composition is administered to the patient. The pharmaceutical composition is administered subcutaneously or intradermally. The pharmaceutical composition is administered simultaneously at at least 2 administration sites away from the tumor lesion and / or the area of cancerous lymph nodes.

13. The pharmaceutical composition of claim 11, wherein, The pharmaceutical composition is administered simultaneously at at least 4 administration sites away from the tumor lesion and / or the cancerous lymph node area.

14. The pharmaceutical composition according to claim 1 or 2, wherein, The at least one HLA tumor antigen peptide has at least one mutation relative to the wild type HLA tumor antigen peptide, which mutation results in an increased affinity of the individual treated with the HLA tumor antigen peptide to the T cell receptor compared to the wild type HLA antigen peptide.

15. The pharmaceutical composition according to claim 1 or 2, wherein, The pharmaceutical composition comprising each individual HLA tumor antigen peptide in an absolute concentration of 100-600 μg, i.e. the administered dose, is injected intradermally or subcutaneously to the patient or a group of patients having at least one identical HLA allele with an administration frequency of once every 2 weeks for at least one year.

16. Use of the pharmaceutical composition according to any one of claims 1 to 15 for the manufacture of a medicament for the treatment or prevention of breast cancer in a patient or group of patients suffering from or suspected to suffer from breast cancer.

17. A combined preparation for use in the treatment of breast cancer, which is for simultaneous, separate or sequential administration, The combined preparation comprises the following two separate preparations (a) and (b): (a) a first preparation comprising the pharmaceutical composition according to any one of claims 1 to 15 together with a pharmaceutically acceptable carrier or diluent, and (b) a second preparation comprising an anti-cancer agent selected from the group consisting of anti-cancer alkylating agents, anti-cancer antimetabolites, anti-cancer antibiotics, herbal anti-cancer agents, platinum coordination anti-cancer complexes, anti-cancer camptothecin derivatives, anti-cancer tyrosine kinase inhibitors, monoclonal antibodies, interferons, interleukins, biological response modifiers, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or diluent.

18. The combination preparation according to claim 17, wherein, The second preparation is a monoclonal antibody against an immunosuppressive protein selected from the group consisting of CTLA4, GM-CFS, TReg, EpCam, IDO, MIC, PDL1, Fas and PD1-L, TRAIL.

19. The combined preparation according to claim 17 or 18, wherein the second preparation is an estrogen inhibitor for hormone positive patients.

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