Peptides and combinations thereof for use in immunotherapy for acute myeloid leukemia (AML) and other hematological malignancies

Abstract

The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapeutic methods. Specifically, the present invention relates to the immunotherapy of cancer, particularly hematological tumors such as acute myeloid leukemia (AML). Furthermore, the present invention relates to tumor-associated T cell peptide epitopes that can be used, for example, as active pharmaceutical ingredients in vaccine compositions to stimulate anti-tumor immune responses or that can stimulate ex vivo T cells for transfer into patients. Peptides bound to major histocompatibility complex (MHC) molecules, and even the peptides themselves, may be targets for antibodies, soluble T cell receptors, and other binding molecules.

Description

[Technical Field]

[0001] The present invention relates to peptides, proteins, nucleic acids, and cells for use in immunotherapeutic methods. Specifically, the present invention relates to the immunotherapy of cancer, particularly hematological tumors such as acute myeloid leukemia (AML). Furthermore, the present invention relates to tumor-associated T cell peptide epitopes that can be used, for example, as active pharmaceutical ingredients in vaccine compositions to stimulate anti-tumor immune responses or that can stimulate ex vivo T cells for transfer into patients. Peptides bound to major histocompatibility complex (MHC) molecules, and even the peptides themselves, may be targets for antibodies, soluble T cell receptors, and other binding molecules.

[0002] The present invention relates to several novel peptide sequences and their variants that can be used in vaccine compositions for the induction of anti-tumor immune responses, especially against hematological tumors such as acute myeloid leukemia (AML), or targeted to the development of pharmaceutically / immunologically active compounds and cells.

[0003] The present invention relates to the field of molecular biology or medicine, more particularly to the field of molecular immunology. [Background technology]

[0004] Hematologic tumors include various malignant tumor diseases originating from myeloid or lymphoid hematopoietic cells. Therefore, the classification of these diseases is mainly based on the division of hematopoietic cell systems into lymphoid tumors and myeloid tumors. Among these, leukemia is the most important representative of hematologic tumors. Among leukemias, acute myeloid leukemia (AML) in particular is of great clinical importance.

[0005] Acute myeloid leukemia (AML) is a cancer of the myeloid blood cells characterized by the rapid proliferation of abnormal cells that make up the bone marrow and blood, interfering with the development of normal blood cells. Symptoms include fatigue, shortness of breath, a tendency to bruise or bleed, and an increased risk of infection. Symptoms may also affect the brain, skin, or gums. AML progresses rapidly as an acute leukemia and, if left untreated, is usually fatal within weeks or months.

[0006] Risk factors for AML include smoking, previous chemotherapy or radiation therapy, myelodysplastic syndrome, and exposure to benzene-based chemicals. The underlying mechanism is the replacement of normal bone marrow with leukemic cells, resulting in a reduction in red blood cells, platelets, and normal white blood cells. Diagnosis is usually based on bone marrow aspirate and specific blood tests. There are several subtypes of AML, each with varying treatment and outcomes.

[0007] AML is a rare disease with an annual incidence of approximately 3 cases per 100,000 people. In Germany, approximately 3,600 new cases are reported each year. AML is primarily a disease of older adults, with the median age of diagnosis being 63 years. AML accounts for approximately 80% of all acute leukemias in adults. Men are slightly more likely to develop the disease than women (ratio 1.4:1). In childhood, AML accounts for only 15-20% of all acute leukemia cases. However, rare acute leukemias in the neonatal period are usually AML.

[0008] First-line treatment for AML is usually chemotherapy, with the aim of inducing remission. Subsequent treatment may include additional chemotherapy, radiation therapy, or stem cell transplant.

[0009] A major challenge in curing AML is the elimination of leukemic stem and progenitor cells (LPCs), which are resistant to treatment and persist even after standard treatment. These cells are believed to be the main cause of leukemia relapse. Therefore, the majority of AML patients relapse despite achieving remission, resulting in a very low long-term survival rate for AML patients.

[0010] In recent years, the concept of T cell immunotherapy, such as checkpoint inhibitors, CAR T cells, adoptive T cell transfer, and vaccination strategies, has become increasingly important in the treatment of hematological tumors. The main prerequisite for developing the concept of antigen-specific immunotherapy is the identification of suitable targets that are naturally and frequently presented on the surface of tumor cells only in tumors and recognized by the patient's immune system. Such targets can be presented on the surface of tumor cells by HLA-independent molecules or HLA class I or HLA class II molecules.

[0011] Few target antigens for immunotherapy development have been reported in AML. Among HLA-independent surface antigens, only CD33, CD123, and FLT3 have been shown to be clinically relevant for antibody and CAR-T cell development. These surface antigens, which are not presented by HLA molecules, have not been shown to be clinically relevant for vaccination and adoptive TCR-mediated T cell transfer. Furthermore, only a few immunogenic AML-associated antigens have been reported to date as HLA-presented targets (WT1, PRAME, NY-ESO-1, and hTERT). Initial clinical trials have shown promising results in terms of in vivo immunogenicity and clinical responses in individual patients, but many of these antigens are restricted to a single HLA allotype, and their therapeutic value has not been demonstrated in more advanced clinical trials.

[0012] A widely used approach for identifying tumor antigens based on gene expression analysis and in silico prediction of gene-derived HLA ligands is extremely challenging due to the biased correlation between gene expression and HLA-restricted antigen presentation: gene expression cannot predict whether a given antigen will actually be presented on the cell surface of tumor cells.

[0013] In this context, there is a need to identify new tumor antigens that are pathophysiologically important for hematological malignancies in general, and AML in particular.

[0014] Therefore, the present invention aims to provide tumor-associated T-cell peptide epitopes that can be used to develop improved pharmaceuticals and methods for the diagnosis, prevention, and treatment of hematological tumors, such as acute myeloid leukemia (AML). More specifically, the present invention provides pharmaceutical compositions, such as peptide-based vaccines, that can be used for the diagnosis, prevention, and / or more effective long-term treatment of hematological tumors. Summary of the Invention [Means for solving the problem]

[0015] The present invention relates to a peptide and its pharmaceutically acceptable salts, The amino acid sequence is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32, and variant sequences thereof having at least 88% homology to SEQ ID NO: 1 to SEQ ID NO: 32, the variant sequence binds to a major histocompatibility complex (MHC) molecule and / or induces T cell cross-reactivity; the peptide is not a full-length polypeptide; Peptides and pharmaceutically acceptable salts thereof are provided.

[0016] For the first time, the inventors have been able to satisfy the need to provide new target antigens for the development of immunotherapies against hematological malignancies such as acute myeloid leukemia (AML) by adopting an approach that deviates from the common conventional approach of identifying tumor antigens based on gene expression analysis and in silico prediction of gene-derived HLA ligands.

[0017] Furthermore, the present inventors have realized that an important reason for the poor results of immunotherapy for the treatment of AML and other hematological tumors to date is that known treatments have focused on AML blasts and not on LPC-specific antigens, while taking into account the characteristics of AML and other hematological tumors (i.e., LPCs) that are resistant to standard treatments and often undergo relapse.

[0018] Therefore, in developing the peptides of the present invention, we identified and used target structures that are expressed to some extent not only in AML blasts but also in AML progenitor / stem cells and LPCs. Therefore, a novel approach was adopted. All peptides are frequently and exclusively found in the immunopeptidomes of AML patients but not in the immunopeptidomes of normal reference subjects. Therefore, the peptides of the present invention are particularly effective and have few side effects.

[0019] Using this method, the present inventors were able to identify 15 MHC / HLA class II peptides specific to hematological tumors and AML, each containing any of the amino acid sequences of SEQ ID NOs: 1 to 15. These MHC / HLA class II peptides can be used for any patient with a hematological tumor, regardless of their MHC / HLA allotype.

[0020] Furthermore, the present inventors were able to identify 17 types of MHC / HLA class I peptides specific to hematological tumors and AML, each containing any of the amino acid sequences of SEQ ID NO: 16 to SEQ ID NO: 32. These peptides include a peptide presented by MHC / HLA class I with an HLA allotype of A*11 (SEQ ID NO: 16), a peptide presented by MHC / HLA class I with an HLA allotype of A*03 (SEQ ID NO: 17), a peptide presented by MHC / HLA class I with an HLA allotype of A*02 (SEQ ID NO: 18 to SEQ ID NO: 20), a peptide presented by MHC / HLA class I with an HLA allotype of A*01 (SEQ ID NO: 21 to SEQ ID NO: 23), and a peptide presented by MHC / HLA class I with an HLA allotype of A*04 (SEQ ID NO: 24 to SEQ ID NO: 25). The peptides are peptides presented by MHC / HLA class I of type B*07 (SEQ ID NO: 24 to SEQ ID NO: 26), peptides presented by MHC / HLA class I of HLA allotype C*07 (SEQ ID NO: 24 to SEQ ID NO: 26), peptides presented by MHC / HLA class I of HLA allotype C*07 (SEQ ID NO: 27 to SEQ ID NO: 29), or peptides presented by MHC / HLA class I of HLA allotype B*08 (SEQ ID NO: 30 to SEQ ID NO: 32).

[0021] All selected peptides were frequently identified only in the immunopeptidome of the AML cohort, but not in the healthy controls.

[0022] MHC / HLA class II peptides according to the present invention are shown in Table 1 below. [Table 1]

[0023] MHC / HLA class I peptides according to the present invention are shown in Table 2 below. [Table 2]

[0024] According to the inventors' findings, the peptides or neoantigens KLKKMWKSPNGTIQNILGGTVF (SEQ ID NO: 1), AVEEVSLRK (SEQ ID NO: 16), and LAVEEVSLR (SEQ ID NO: 17) are of particular interest and therefore are particularly preferred. Furthermore, the (non-mutated) peptides DRVKLGTDYRLHLSPV (SEQ ID NO: 2), ETLHKFASKPASEFVK (SEQ ID NO: 3), PHRKKPFIEKKKAVSFHLVHR (SEQ ID NO: 4), SPGPFPFIQDNISFYA (SEQ ID NO: 5), IGSYIERDVTPAIM (SEQ ID NO: 6), SKPGVIFLTKKGRRF (SEQ ID NO: 7), DRQQMEALTRYLRAAL (SEQ ID NO: 8), SLLEADPFL (SEQ ID NO: 18), DIDTRSEFY (SEQ ID NO: 21), APESKHKSSL (SEQ ID NO: 24), APGLHLEL (SEQ ID NO: 25), and AYHELAQVY (SEQ ID NO: 27) are also of particular interest and are preferred. These peptides are of particular interest because they are naturally presented, found only in tumors, ie, not in normal tissues, and are recognized by T cells.

[0025] In the event of any discrepancy between the sequences set out in Tables 1 and 2 and those set out in the Sequence Listing, the information in these tables shall take precedence and apply.

[0026] As used herein, the term "peptide" generally refers to a series of amino acid residues linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. Peptides are preferably 7 to 12 amino acids long, more preferably 8 to 11 amino acids long, but may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or longer.

[0027] Furthermore, the term "peptide" generally includes salts of a series of amino acid residues linked together by a peptide bond between the α-amino and carbonyl groups of adjacent amino acids. The salts are preferably pharmaceutically acceptable salts of the peptide, such as chloride salts or acetate salts (trifluoroacetates). It should be noted that the peptides of the present invention cannot exist in vivo as salts, and therefore salts of the peptides of the present invention are in a state significantly different from the state of the peptides of the present invention in vivo.

[0028] Furthermore, the term "peptide" also includes "oligopeptides." As used herein, "oligopeptide" generally refers to a series of amino acid residues linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the oligopeptide is not critical to the present invention, so long as the correct epitope or epitopes are retained. The length of an oligopeptide is generally less than about 30 amino acid residues and greater than about 15 amino acids.

[0029] Furthermore, the term "peptide" also includes "polypeptides." A "polypeptide" generally refers to a series of amino acid residues linked together by peptide bonds between the α-amino and carbonyl groups of adjacent amino acids. The length of the polypeptide is not critical to the present invention, so long as the correct epitope is retained. Unlike the terms "peptide" and "oligopeptide," a "polypeptide" refers to a molecule containing more than about 30 amino acid residues.

[0030] By "variant" of an amino acid sequence, we mean, for example, that the side chains of one or two amino acid residues have been modified (e.g., by substitution with the side chains of another naturally occurring amino acid residue or other side chains), but that the peptide is still able to bind to an MHC molecule in substantially the same manner as a peptide consisting of the amino acid sequence of SEQ ID NOs: 1 to 32. For example, the peptide may be engineered to at least maintain (if not improve) its ability to interact with and bind to the binding groove of the appropriate MHC molecule and to at least maintain (if not improve) its ability to bind to the TCR of activated T cells.

[0031] The original (unmodified) peptides disclosed herein can be modified by substituting one or more residues at various positions within the peptide chain, unless otherwise specified, and may involve the modification of one or more residues selected from various positions. The substitutions are preferably located at the termini of the amino acid chain. Such substitutions can be conservative, e.g., substituting one amino acid for another of similar structure and properties, e.g., substituting a hydrophobic amino acid for another hydrophobic amino acid. Further conservative substitutions can be made between amino acids of the same size and chemical properties or between amino acids of similar size and chemical properties, e.g., substituting isoleucine for leucine. Studies of sequence variation within naturally occurring homologous protein families have shown that certain amino acid substitutions are often better tolerated than others, and such substitutions often correlate with similarities in size, charge, polarity, and hydrophobicity between the original and substituted amino acids, thereby defining them as "conservative substitutions." As used herein, a "conservative substitution" is defined as an exchange of amino acids within one of five groups: Group 1—nonpolar or slightly polar, small aliphatic residues (Ala, Ser, Thr, Pro, GlY); Group 2—polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3—polar, positively charged residues (His, Arg, Lys); Group 4—large, nonpolar, aliphatic residues (Met, Leu, Ile, Val, Cys); and Group 5—large, aromatic residues (Phe, Tyr, Trp). Less conservative substitutions may involve the replacement of one amino acid with another amino acid of similar properties but slightly different size, such as the replacement of an isoleucine residue with an alanine. Very less conservative substitutions may involve the replacement of a polar amino acid with an acidic amino acid, or even a basic amino acid with an acidic amino acid.However, such "extreme" substitutions cannot be excluded as potentially ineffective, because chemical effects cannot be fully predicted and extreme substitutions may lead to serendipitous new discoveries that cannot be predicted from simple chemical principles. It goes without saying that such substitutions may include structures other than common L-amino acids. That is, L-amino acids typically found in the antigenic peptides of the present invention may be substituted with D-amino acids, and such amino acids are also encompassed by the present disclosure. Furthermore, non-standard amino acids (i.e., amino acids other than the common amino acids that constitute natural proteins) may be used as substitutions to produce immunogens and immunogenic polypeptides of the present invention.

[0032] If substitutions at two or more positions result in a peptide with substantially equal or greater antigenic activity, as defined below, the combination of substitutions will be tested to determine whether it has an additive or synergistic effect on the antigenicity of the peptide. No more than four positions within a peptide will be substituted simultaneously.

[0033] Amino acid residues that do not substantially contribute to interaction with the T cell receptor can be engineered by substituting them with other amino acids that do not substantially affect T cell responsiveness and do not abolish binding to the relevant MHC.

[0034] Longer (extended) peptides may be preferred. MHC class I epitopes are typically 8-11 amino acids in length, but can also be generated by peptide processing from longer peptides or proteins containing the actual epitope. Residues flanking the actual epitope are preferably those that do not substantially affect the proteolytic cleavage required to expose the epitope during processing.

[0035] The peptides of the present invention can be extended by up to four amino acid residues, i.e., one, two, three, or four amino acids can be added to one or both termini, with the number of amino acids added to each termini being any combination ranging from 4:0 to 0:4. Extension combinations according to the present invention are shown in Table 3. [Table 3]

[0036] The amino acids used for the extension may be those derived from the peptide of the original sequence of the protein disclosed herein, or may be other amino acids, which may improve the stability or solubility of the peptide of the invention.

[0037] Thus, the epitopes of the present invention may be the same as naturally occurring tumor-associated or naturally occurring tumor-specific epitopes, or may include epitopes that differ by no more than four residues compared to the reference peptide, so long as they have substantially the same antigenic activity.

[0038] In another embodiment, the peptides of the present invention are extended by adding five or more amino acids to one or both termini, preferably up to a total length of 30 amino acids. This extension may result in a peptide that binds to MHC class II molecules. MHC class II binding can be tested by methods known in the art.

[0039] Thus, the present invention provides MHC class I epitope peptides and variants thereof, wherein the total length of the peptide or variant is 8 to 100 amino acids, preferably 8 to 30 amino acids, and most preferably 8 to 14 amino acids, i.e., 8, 9, 10, 11, 12, 13, or 14 amino acids; when the peptide is an extended class II molecule-binding peptide, the total length may be 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids.

[0040] It goes without saying that the peptides or variants of the present invention have the ability to bind to human major histocompatibility complex (MHC / HLA) class I or MHC class II molecules. Binding of the peptides or variants to MHC complexes may be tested by methods known in the art.

[0041] When testing T cells specific for a peptide of the invention against a substituted peptide, the substituted peptide achieves a half-maximal increase in lysis over background at a peptide concentration of about 1 mM or less, preferably about 1 μM or less, more preferably about 1 nM or less, even more preferably about 100 pM or less, and most preferably about 10 pM or less. The substituted peptide is preferably recognized by T cells from two or more individuals, preferably by T cells from at least two individuals, and more preferably by T cells from three individuals.

[0042] Those skilled in the art can assess whether T cells induced by a particular peptide variant can cross-react with the original peptide itself (Appay et al., 2006; Colombetti et al., Eur. J. Immunol. 36: 1805-1814 (2006); Fong et al., Proc. Natl. Acad. Sci. USA 98: 8809-8814 (2001); Zaremba et al., Cancer Res. 57: 4570-4577 (1997)).

[0043] These T cells can then substantially cross-react with and kill cells expressing polypeptides containing the native amino acid sequence of the cognate peptides defined in embodiments of the present invention. As evidenced by scientific literature and database information (Rammensee et al., Immunogenetics 50: 213-219 (1999); Godkin et al., Int. Immunol 9: 905-911 (1997)), specific positions in HLA-binding peptides typically contain anchor residues that form core sequences that fit into the binding motif of HLA receptors, determined by the polarity, electrophysical properties, hydrophobicity, and spatial properties of the polypeptide chain that constitutes the binding groove. Therefore, those skilled in the art can modify the amino acid sequences set forth in SEQ ID NOS: 1-32 while maintaining the known anchor residues, and determine whether such variants retain the ability to bind to MHC class I or MHC class II molecules. The variants of the present invention retain the ability to bind to the TCR of activated T cells and are therefore capable of substantially cross-reacting with and killing cells expressing a polypeptide comprising the native amino acid sequence of the cognate peptide defined in this embodiment of the present invention.

[0044] In the present invention, "homology" refers to the degree of identity between two amino acid sequences (i.e., peptide or polypeptide sequences). The aforementioned "homology" is determined by aligning and comparing the two sequences under optimal conditions for the two sequences to be compared. Such sequence homology can be calculated by creating an alignment using, for example, the ClustalW algorithm. Publicly available sequence analysis software, more specifically, Vector NTI, GENETYX, or other tools, are provided by public databases.

[0045] Thus, when the terms "% identity" or "percent identity" are used in reference to a sequence, these terms refer to aligning the sequence being compared ("comparison sequence") with a sequence described or claimed herein ("reference sequence"), and then comparing the comparison sequence to the sequence described or claimed herein. The percent identity is then determined according to the following formula: Identity (%)=100×[1-(C / R)] where C is the number of differences between the reference and compare sequences in the alignment length between the reference and compare sequences, and this difference is (i) each base or amino acid of the reference sequence that does not have a corresponding base or amino acid aligned to the compared sequence; (ii) each gap in the reference sequence; and (iii) each aligned base or amino acid of the reference sequence that differs from each aligned base or amino acid of the compared sequence; (iv) The alignment must start from the first position of the sequence being aligned. In the formula, R is the number of bases or amino acids of the reference sequence in the length of the alignment with the sequence to be compared, and gaps made in the reference sequence are also counted as bases or amino acids.

[0046] According to the present invention, "full-length polypeptide" refers to a protein source from which the peptide of the present invention is derived. "Full-length polypeptide" is also referred to as the "source gene / protein" from which the peptide of the present invention is derived. The protein or full-length polypeptide source of the peptide of the present invention may be highly overexpressed in cancer compared to normal tissue, or may not be highly overexpressed in cancer compared to normal tissue. In the context of the present invention, "normal tissue" refers to healthy peripheral blood mononuclear cells (PBMCs) or other normal tissue cells in which the gene source of the peptide of the present invention exhibits high tumor association. Furthermore, the peptide of the present invention itself is presented in tumor tissue. In the context of the present invention, "tumor tissue" refers to a sample obtained from a patient suffering from a cancer such as a blood tumor or acute myeloid leukemia (AML). Exemplary "full-length polypeptides" or their source proteins are listed in the "source protein" column of Table 6.

[0047] In one embodiment of the present invention, the peptide of the present invention is isolated. "Isolated" means that a material is removed from its original environment (e.g., if it is a natural material, it means that it is removed from its natural environment). For example, a natural polynucleotide or polypeptide present in the body of a living animal is not isolated, but if this polynucleotide or polypeptide is separated from some or all of the materials that coexist with it in nature, this polynucleotide or polypeptide is isolated. Such a polynucleotide may be part of a vector, and / or such a polynucleotide or polypeptide may be part of a composition, and such a vector or composition is isolated in that it is not part of the natural environment.

[0048] The peptides and / or nucleotides disclosed by the present invention may also be in a "purified" form. "Purified" does not necessarily imply absolute purity; it is intended as a relative definition and may include highly purified or only partially purified preparations, as the term is understood by those skilled in the relevant art. For example, it is customary to purify individual clones isolated from a cDNA library to ensure electrophoretic homogeneity. Purification of starting or natural materials by at least 10-fold, preferably 100-fold or 1,000-fold, and more preferably 10,000-fold or 100,000-fold is expressly contemplated. Furthermore, claimed polypeptides having a purity of preferably 99.999% by weight, or at least 99.99% or 99.9% by weight, and even more desirably 99% or greater by weight, are expressly encompassed by the present invention.

[0049] The peptides and nucleic acids of the present invention may be in a "concentrated form." As used herein, "concentrated" means that the concentration of a material is (for example) at least about 2-fold, about 5-fold, about 10-fold, about 100-fold, or about 1000-fold its natural concentration, advantageously 0.01% by weight, and preferably at least about 0.1% by weight. Furthermore, concentrated preparations of about 0.5%, about 1%, about 5%, about 10%, or about 20% by weight are contemplated. The sequences, constructs, vectors, clones, and other materials comprising the present invention may advantageously be in a concentrated or isolated form.

[0050] Against this background, another subject of the present invention is a pharmaceutical composition for the diagnosis, prevention and / or treatment of hematological tumors, in particular acute myeloid leukemia (AML), comprising: at least one peptide that binds to a major histocompatibility complex class II molecule (MHC class II molecule) and / or induces T cell cross-reactivity; and a pharmaceutically acceptable carrier; the at least one peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15 and variant sequences thereof having at least 88% homology to SEQ ID NO: 1 to SEQ ID NO: 15; the peptide is not a full-length polypeptide; It is a pharmaceutical composition.

[0051] Such pharmaceutical compositions comprise at least one MHC class II peptide according to the present invention and can therefore be used regardless of the patient's allotype.

[0052] The features, advantages and embodiments of the peptides of the present invention apply mutatis mutandis to the pharmaceutical compositions of the present invention.

[0053] A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Pharmaceutical compositions are preferably sterile and preferably manufactured according to GMP guidelines.

[0054] The pharmaceutical compositions of the present invention include the peptides of the present invention in free form or in the form of a pharmaceutically acceptable salt (see also above). As used herein, the term "pharmaceutically acceptable salt" refers to a derivative of the peptide of the present disclosure that has been modified by forming a salt with an acid or base. For example, an acid salt can be prepared by reacting the peptide of the present invention as a free base (the neutral form of the peptide of the present invention typically has a neutral -NH group) with a suitable acid. Suitable acids for preparing acid salts include organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. On the other hand, salts of bases with acid moieties that may be present on the peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

[0055] The pharmaceutical composition of the present invention is preferably an immunotherapeutic agent such as a vaccine. The pharmaceutical composition of the present invention may be administered directly to a patient's affected organ or the whole body via intradermal, intramuscular, subcutaneous, intraperitoneal, or intravenous routes, or may be added ex vivo to cells or human cell lines obtained from the patient and then administered to the patient. Alternatively, the composition may be used in vitro to select immune cell subpopulations obtained from the patient, and the selected immune cell subpopulations may then be readministered to the patient. When administering the nucleic acid of the present invention to cells in vitro, it may be useful to transfect the cells to co-express an immunostimulatory cytokine, such as interleukin-2.

[0056] The peptides of the present invention may be substantially pure. The peptides of the present invention may be combined with an immunostimulatory adjuvant, such as the TLR1 / 2 ligand XS15. In preliminary studies, XS15 was added to mutated or non-mutated viral peptides in the form of a water-in-oil emulsion and injected subcutaneously into healthy donors and tumor patients, resulting in potent CD8+ / CD8+ responses in vivo. + T cell responses and strong Th1CD4 +It has been shown that the peptides of the present invention can induce T cell responses. The peptides of the present invention may also be used in combination with immunostimulatory cytokines or administered using an appropriate delivery system (e.g., liposomes). The peptides of the present invention may also be conjugated to a suitable carrier, such as keyhole limpet hemocyanin (KLH) or mannan (see WO95 / 18145 and Longenecker et al., Ann. NY Acad. Sci. 690:276-291 (1993)). Furthermore, the peptides of the present invention may be tagged, fused proteins, or hybrid molecules. Peptides having the sequences described in the present invention are expected to stimulate CD4 T cells or CD8 T cells. However, stimulation of CD8 T cells is more efficient in the presence of help from CD4 helper T cells. Therefore, for MHC class I epitopes that stimulate CD8 T cells, a fusion partner or part of a hybrid molecule may be used to target CD4 T cells. + Epitopes that stimulate T cells are preferably provided. Epitopes that stimulate CD4 T cells or CD8 T cells are well known in the art and include those identified in the present invention.

[0057] According to the present invention, "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, etc. This allows to ensure very high coverage of the world population.

[0058] The pharmaceutical composition of the present invention may comprise the peptide according to the present invention as the only active ingredient, however, in another embodiment, the pharmaceutical composition of the present invention may comprise additional active ingredients.

[0059] The problems of the present invention can be completely solved by the disclosure of this specification.

[0060] In one embodiment of the present invention, the pharmaceutical composition of the present invention comprises: at least two peptides, preferably at least three peptides, more preferably at least four peptides, even more preferably at least five peptides, even more preferably at least six peptides, even more preferably at least seven peptides, even more preferably at least eight peptides, even more preferably at least nine peptides, and very preferably at least ten peptides; each peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15 and variant sequences thereof having at least 88% homology to SEQ ID NO: 1 to SEQ ID NO: 15; The peptide is not a full-length polypeptide.

[0061] This approach has the advantage that by increasing the number of MHC class II peptides contained in the pharmaceutical composition of the present invention, it is possible to immunize a larger number of people, regardless of their specific allotypes. By using 10 different peptides, it is possible to cover members of all populations.

[0062] In another embodiment, the pharmaceutical composition of the present invention comprises: KLKKMWKSPNGTIQNILGGTVF (SEQ ID NO: 1), DRVKLGTDYRLHLSPV (SEQ ID NO: 2), ETLHKFASKPASEFVK (SEQ ID NO: 3), PHRKKKPFIEKKKAVSFHLVHR (SEQ ID NO: 4), SPGPFPFIQDNISFYA (SEQ ID NO: 5), IGSYIERDVTPAIM (SEQ ID NO: 6), SKPGVIFLTKKGRRF (SEQ ID NO: 7), DRQQMEALTRYLRAAL (SEQ ID NO: 8), GNQLFRINEANQLMQ (SEQ ID NO: 9), LGQEVALNANTKNQKIR (SEQ ID NO: 10), NGRTFHLTRTLTVK (SEQ ID NO: 11), LDTMRQIQVFEDEPAR (SEQ ID NO: 12), VVGYALDYNEYFRDL (SEQ ID NO: 13), KHLHYWFVESQKDPEN (SEQ ID NO: 14), and ERPEWIHVDSRPF (SEQ ID NO: 15) The present invention includes different types of peptides containing any of the amino acid sequences listed above.

[0063] In yet another embodiment of the present invention, the pharmaceutical composition of the present invention comprises: at least one additional peptide that binds to a major histocompatibility complex class I molecule (MHC class I molecule) and / or induces T cell cross-reactivity; the at least one additional peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 32 and variant sequences thereof having at least 88% homology to SEQ ID NO: 16 to SEQ ID NO: 32; The peptide is not a full-length polypeptide.

[0064] This approach has the advantage that by adding an MHC / HLA class I peptide to the pharmaceutical composition of the invention, a (semi-)individualized or personalized composition can be obtained, this additional peptide being selected depending on the patient's MHC / HLA allotype.

[0065] In another embodiment of the invention, the at least one additional peptide is selected according to the MHC class I allotype of the individual to be treated, preferably selected according to Table 4 below, wherein the amino acid sequences of each MHC class I allotype group include variant sequences with at least 88% homology. [Table 4]

[0066] This approach provides pharmaceutical compositions comprising one or more MHC class II peptides (SEQ ID NOS: 1-15) of the present invention, as well as one or more additional peptides (SEQ ID NOS: 16-32) that specifically target the MHC allotype of the individual to be treated. This provides a (semi-)personalized pharmaceutical composition that can induce an optimized and maximally enhanced immune response in the individual. As a result, the pharmaceutical compositions of the present invention are particularly effective in / against hematological oncology diseases, particularly acute myeloid leukemia (AML).

[0067] In view of this, in one embodiment, the peptide of the present invention has the ability to bind to an MHC class I molecule or an MHC class II molecule, and upon binding to these MHC molecules, it can be recognized by CD4 T cells and / or CD8 T cells. The amino acid sequence of the peptide of the present invention preferably comprises a consecutive amino acid sequence set forth in any one of SEQ ID NOs: 1 to 32.

[0068] In yet another embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition of the present invention comprises: at least two additional peptides, preferably at least three additional peptides, more preferably at least four additional peptides, even more preferably at least five additional peptides, and most preferably at least six additional peptides; each additional peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 32, and variant sequences thereof having at least 88% homology to SEQ ID NO: 15 to SEQ ID NO: 32; The additional peptide is not a full-length polypeptide.

[0069] The more types of additional peptides are added, the more the immunostimulatory activity of the pharmaceutical composition of the present invention is enhanced in an allotype-specific manner.

[0070] In a preferred embodiment of the pharmaceutical composition of the present invention, the pharmaceutical composition contains a total of up to 10 peptides of the present invention. Of these, approximately 5 to 7 peptides are MHC class II peptides (SEQ ID NOS: 1 to 15) and approximately 3 to 5 peptides are MHC class I peptides (SEQ ID NOS: 16 to 32). This allows for the provision of a personalized "vaccine cocktail" for each patient. The specific vaccine peptides for each cocktail are selected based on the molecular phenotype of the disease and the patient's individual HLA allotype.

[0071] In yet another embodiment of the present invention, the pharmaceutical composition of the present invention is a vaccine, preferably a vaccine against a hematological tumor, more preferably a vaccine against acute myeloid leukemia (AML).

[0072] In another embodiment of the present invention, the pharmaceutical composition of the present invention further comprises an adjuvant, which is preferably XS15, more preferably XS15 dissolved in montanide.

[0073] XS15 is a TLR1 / 2 ligand and functions as an immune-stimulating adjuvant. Previous studies have shown that XS15 potently upregulates CD8 in vivo. + T cell responses and Th1CD4 +It has been shown to be able to induce T cell responses. See Rammensee et al. (2019), A new synthetic toll-like receptor 1 / 2 ligand is an efficient adjuvant for peptide vaccination in a human volunteer. J. Immunother. Cancer. 7(1):307, and Heitmann et al. (2022), A COVID-19 peptide vaccine for the induction of SARS-CoV-2 T cell immunity. Nature 601, 617-622. XS15 is preferably dissolved in montanide ISA51. Addition of this adjuvant optimizes the use of the pharmaceutical composition of the present invention as a vaccine.

[0074] Another subject of the present invention is an antibody that specifically recognizes the peptide of the present invention or a variant thereof, in particular a soluble antibody or a membrane-bound antibody, more preferably a monoclonal antibody or a fragment thereof, preferably an antibody that specifically recognizes the peptide of the present invention or a variant thereof when said peptide or variant thereof is bound to an MHC molecule.

[0075] As used herein, the term "antibody" is used broadly and includes both polyclonal and monoclonal antibodies. The term "antibody" encompasses intact or "full-length" immunoglobulin molecules, as well as fragments (e.g., CDRs, Fv fragments, Fab fragments, and Fc fragments) or polymers of such immunoglobulin molecules, as well as humanized forms of such immunoglobulin molecules, so long as they exhibit any of the desired properties, i.e., specifically recognize the peptides of the present invention or their variants. Antibodies of the present invention may be purchased from commercial vendors, if available. Alternatively, antibodies of the present invention may be produced using known methods.

[0076] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the antibodies and fragments thereof.

[0077] Another subject of the present invention relates to a T cell receptor or a fragment thereof that reacts with an HLA ligand that is a peptide of the present invention or a variant thereof, preferably a soluble T cell receptor or a membrane-bound T cell receptor, and preferably a T cell receptor that reacts with the peptide of the present invention or a variant thereof when the peptide or variant thereof is bound to an MHC molecule.

[0078] The term "T cell receptor" (abbreviated as "TCR") of the present invention refers to a heterodimeric molecule comprising an α polypeptide chain (α chain) and a β polypeptide chain (β chain), and this heterodimeric receptor can bind to peptide antigens presented by HLA molecules. "T cell receptor" also includes so-called γδ TCRs.

[0079] The features, properties, advantages and embodiments disclosed for the peptides and antibodies or fragments thereof of the present invention apply equally to the T cell receptors mentioned above.

[0080] A further subject of the present invention relates to a nucleic acid, optionally linked to a heterologous promoter sequence, which encodes a peptide of the invention or a variant thereof, an antibody of the invention or a fragment thereof, or a T-cell receptor of the invention or a fragment thereof; or an expression vector for expressing said nucleic acid.

[0081] The nucleic acid encoding a particular peptide, oligopeptide, or polypeptide may be a natural nucleic acid or a synthetically constructed nucleic acid. The nucleic acid (e.g., polynucleotide) may be, for example, DNA, cDNA, PNA, RNA, or a combination thereof. It may be single-stranded and / or double-stranded. It may be a natural polynucleotide or a stabilized polynucleotide, for example, a polynucleotide having a phosphorothioate backbone, and may contain or not contain introns, so long as it encodes a peptide of the present invention. Of course, the polynucleotide may only encode peptides containing natural amino acid residues linked by natural peptide bonds. Yet another aspect of the present invention provides expression vectors capable of expressing the above-mentioned (poly)peptides of the present invention or other (poly)peptides.

[0082] As used herein, the term "nucleic acid encoding a peptide" refers to a nucleotide sequence that encodes a peptide containing artificial (man-made) initiation and termination codons compatible with a biological system, and that is expressed by, for example, dendritic cells or other cell lines useful for producing TCRs. The term "promoter" refers to a DNA region that binds RNA polymerase and is involved in the initiation of transcription.

[0083] In one embodiment of the present invention, the nucleic acids of the present invention are isolated. The term "isolated" means that a material has been removed from its original environment (e.g., in the case of a naturally occurring material, it means that it has been removed from its natural environment). For example, a polynucleotide or (poly)peptide naturally occurring in the body of a living animal is not isolated, but a polynucleotide or (poly)peptide is isolated if it has been separated from some or all of the materials with which it coexists in nature. Such a polynucleotide may be part of a vector and / or such a polynucleotide or (poly)peptide may be part of a composition, and such a polynucleotide or (poly)peptide may be part of a vector or composition, but may be isolated in that such a vector or composition is not part of its natural environment.

[0084] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the nucleic acids and expression vectors described above.

[0085] Another subject of the present invention relates to a recombinant host cell, which comprises a peptide of the invention, an antibody or a fragment thereof of the invention, a T cell receptor or a fragment thereof of the invention, or a nucleic acid or an expression vector of the invention, and which is preferably chosen from mammalian and human cells, more preferably from antigen-presenting cells such as dendritic cells, T cells, NK cells, etc.

[0086] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the host cells described above.

[0087] Yet another subject of the present invention is a method for producing activated T lymphocytes in vitro, comprising the steps of: contacting T cells in vitro with antigen-bearing human MHC class I or II molecules expressed on the surface of suitable antigen-presenting cells or on the surface of an artificial structure that mimics an antigen-presenting cell for a time sufficient to antigen-specifically activate the T cells; The antigen is a peptide of the present invention.

[0088] Activated T cells that target the peptides of the present invention are useful for therapy. Accordingly, a further aspect of the present invention provides activated T cells obtained by the above-described method of the present invention.

[0089] The activated T cells produced by the above method selectively recognize cells that abnormally express a polypeptide comprising any of the amino acid sequences of SEQ ID NOs: 1 to 32.

[0090] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the above methods.

[0091] Another subject of the present invention relates to a pharmaceutical composition comprising at least one active ingredient or labeled active ingredient selected from the group consisting of a peptide of the invention, an antibody or fragment thereof of the invention, a T cell receptor or fragment thereof of the invention, a nucleic acid or expression vector of the invention, a recombinant host cell of the invention, and an activated T lymphocyte of the invention, and a pharmaceutically acceptable carrier, and optionally containing pharmaceutically acceptable additives and / or stabilizers.

[0092] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the invention mentioned earlier in this specification apply equally to the pharmaceutical compositions described above.

[0093] Another subject of the present invention relates to the peptide of the invention, the antibody of the invention or a fragment thereof, the T-cell receptor of the invention or a fragment thereof, the nucleic acid or expression vector of the invention, the recombinant host cell of the invention or the activated T-lymphocyte of the invention for use in medicine, preferably for use in the diagnosis and / or prevention and / or treatment of cancer or for use in the manufacture of a medicine, preferably a vaccine, against cancer, including hematological tumors, wherein said cancer or hematological tumor is further preferably acute myeloid leukemia (AML) or other neoplasms or tumors showing overexpression of the proteins from which the peptides shown in SEQ ID NO: 1 to SEQ ID NO: 32 are derived.

[0094] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the above uses.

[0095] Yet another subject of the present invention is a kit comprising: a) a container containing the pharmaceutical composition of the present invention, the peptide of the present invention or a variant thereof, the antibody of the present invention or a fragment thereof, the T cell receptor of the present invention or a fragment thereof, the nucleic acid or expression vector of the present invention, the recombinant host cell of the present invention, or the activated T lymphocyte of the present invention in a solution or a lyophilized formulation, b) a second container containing a diluent or reconstitution solution for the lyophilized formulation; c) optionally including instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation; Regarding the kit.

[0096] The kit of the present invention may further comprise one or more of (iii) a buffer, (iv) a diluent, (v) a filter, (vi) a needle, and (v) a syringe. The container is preferably a bottle, a vial, a syringe, or a test tube, and may be a multi-purpose container. The pharmaceutical composition is preferably lyophilized.

[0097] The kit of the present invention preferably includes a lyophilized formulation of the present invention in a suitable container and instructions for its reconstitution and / or use. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., dual-chamber syringes), and test tubes. The containers may be made of various materials, such as glass or plastic. The kit of the present invention and / or its container preferably include instructions for the reconstitution and / or use of the lyophilized formulation of the present invention, which may be affixed to or associated with the container. For example, a label affixed to the container may include instructions for reconstituting the lyophilized formulation to the aforementioned peptide concentration. The label may further include information that the lyophilized formulation is useful for subcutaneous administration or that the lyophilized formulation is intended for subcutaneous administration.

[0098] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the kits described above.

[0099] Another subject of the present invention is a method for producing a personalized anti-cancer pharmaceutical composition, preferably a vaccine, comprising the steps of: a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; b) comparing the peptides identified in step a) with a warehouse of peptides that have been pre-screened by comparing immunogenicity and / or over-representation in tumors with normal tissues; c) selecting from said warehouse at least one peptide that matches a TUMAP identified in said patient; and d) formulating a personalized vaccine based on step c). Including, wherein the warehouse comprises a plurality of peptides and / or a plurality of variant sequences of the invention.

[0100] "Personalized pharmaceutical composition" means a tailored treatment specific to a single patient that is used exclusively for the treatment of that patient, and includes actively personalized cancer vaccines and adoptive cell therapy using autologous patient tissue.

[0101] As used herein, the term "warehouse" refers to peptides of the present invention that have been pre-screened for immunogenicity and / or over-presentation in specific types of tumors, including hematological tumors, preferably acute myeloid leukemia (AML). This warehouse (e.g., in the form of a database) is composed of tumor-associated peptides that are highly overexpressed in cancer cells from multiple patients with various HLA-A, HLA-B, and HLA-C alleles. This warehouse includes MHC class I peptides and MHC class II peptides. More specifically, this warehouse includes the MHC class I A*11, A*03, A*02, A*01, B*07, C*07, and B*08 marker peptides. These peptides allow for quantitative comparison of the degree of T cell immunity induced by tumor-associated peptides (TUMAPs), thereby enabling important conclusions to be drawn regarding the ability of vaccines to induce anti-tumor responses.

[0102] In an exemplary embodiment, the identification of a peptide of the invention for inclusion in a vaccine of the invention is carried out by: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients by the aforementioned method; (b) comparing the peptides identified in step (a) with a warehouse of peptides that have been pre-screened by comparing immunogenicity and / or over-representation in tumors with corresponding normal tissues; and (c) selecting from said warehouse at least one peptide that correlates with a tumor-associated peptide identified in said patient; carried out by (d) At least one newly identified peptide from step (a) may be selected and subjected to a further step of confirming its immunogenicity.

[0103] Once peptides for a personalized peptide-based pharmaceutical composition (e.g., vaccine) have been selected, the pharmaceutical composition is manufactured as a liquid formulation in which individual peptides are dissolved in 20-40% DMSO, preferably about 30-35% DMSO (e.g., about 33% DMSO).

[0104] The features, properties, advantages and embodiments disclosed for the pharmaceutical compositions and / or peptides of the present invention apply equally to the above methods. DETAILED DESCRIPTION OF THE INVENTION

[0105] The present invention will be further described in detail by reference to the following embodiments, which describe further features, characteristics, and advantages of the present invention. Furthermore, the following embodiments are for illustrative purposes only and are not intended to limit the spirit or scope of the present invention. Features described in a particular embodiment are features of the present invention and may be considered general features that can be applied singly to all embodiments of the present invention, not just the particular embodiment. [Brief explanation of the drawings]

[0106] The present invention will be described and explained in more detail with reference to the following examples and figures, but the present invention is not limited to these examples and figures. [Figure 1]Immunopeptidome analysis of enriched primary CD34+CD38- LPCs is shown. (A) Representative flow cytometry analysis of LPC frequency before and after CD34+CD38- enrichment. (B) CD34+CD38- LPC frequency in primary AML patient samples (n=11) before and after sorting. Each data point represents an individual sample. Boxplots show the median and 25th-75th percentiles, with whiskers representing minimum to maximum values. Data were analyzed using a paired Wilcoxon signed-rank test. (C) In vivo leukemia engraftment of LPCs in NOD / SCID / IL2Rγnull mice (n=4) is shown. The frequencies of human CD33+ and CD33+CD117+ leukemia cells in the bone marrow, peripheral blood, spleen, and liver of NOD / SCID / IL2Rγnull mice 31 weeks after intrafemoral transplantation of 6 x 105 human CD34+CD38- LPCs are shown. (D, E) Cell surface expression of (D) HLA class I molecules and (E) HLA-DR molecules was measured by flow cytometry in CD34+ HPCs (n = 18), AML blasts (n = 11), and LPCs (n = 11) from healthy volunteers. Each data point represents an individual sample. Boxplots show the median and 25th-75th percentiles, with whiskers indicating minimum to maximum values. Data were analyzed using the Kruskal-Wallis test. (F, G) The number of HLA class I-presented peptides (F) and the number of HLA class II-presented peptides (G) are shown in LPC samples and autologous blast samples (HLA class I, n = 10; HLA class II, n = 11) identified by mass spectrometry. (H) Overlap analysis of HLA class I ligand IDs (left panel) and HLA class II peptide IDs (right panel) in LPC samples and autologous blast samples at the individual patient and entire cohort levels. (I) Peptide length distribution of HLA class I ligands (upper panel) and HLA class II peptides (lower panel) in the immunopeptidomes of LPC and autologous blasts. (J) Amino acid distribution in the HLA class I immunopeptidomes (left panel) and HLA class II immunopeptidomes (right panel) derived from LPC or AML blasts based on unique peptide IDs in each cohort.Abbreviations: BM, bone marrow; PB, peripheral blood; HPCs, hematopoietic progenitor cells; LPCs, leukemic progenitor cells; ID, identifier; UPN, unified patient number; aa, amino acid. [Figure 2] The amino acid distribution in the LPC-derived immunopeptidome and blast-derived immunopeptidome is shown. Position-specific amino acid distribution in (A) HLA class I immunopeptidome and (B) HLA class II immunopeptidome derived from LPC or AML blasts is shown. Specifically, the abundance of each amino acid in the 9-mer HLA class I ligand and 15-mer HLA class II peptide was calculated for each position within each peptide for each sample, and then the average amino acid frequency was calculated for each LPC cohort and blast cohort. [Figure 3]Figure 1 shows the identification of AML-associated and LPC-associated HLA class I target antigens by comparative immunopeptidome profiling. (A) Saturation analysis of proteins as sources of HLA class I-restricted peptides in the AML cohort. The number of protein IDs as sources of unique peptides is shown as a function of cumulative immunopeptidome analysis of AML samples (n = 47). Exponential regression allowed robust calculation of the maximum number of different protein IDs as sources of peptides (dotted line). The dashed line indicates the immunoproteome coverage obtained in the AML cohort as sources of peptides. (B) Overlap analysis of HLA class I ligand IDs in primary AML samples (n = 47) and benign samples (n = 332). (C) Comparative profiling of the HLA class I immunopeptidome based on the frequency of HLA-restricted presentation in the AML immunopeptidome (n = 47) and benign immunopeptidome (n = 332). The frequency of immunopeptidomes positive for each HLA ligand (X-axis) is shown on the Y-axis. The box and enlarged view on the left highlight the subset of AML-associated antigens that are frequently presented only in AML. (D) The population coverage of HLA class I allotypes in the AML cohort is compared with the global population. The frequency of individuals with up to six HLA allotypes in the AML dataset (X-axis) in the global population is shown as gray bars on the left Y-axis. The cumulative population coverage is shown as black dots on the right Y-axis. (E) A representative example of allotype-specific comparative profiling of the previously described HLA-A*01-positive samples (AML: n = 14, benign: n = 93) is shown. (F) HLA class I ligand ID overlap analysis of LPCenr samples (n = 10), AML samples (n = 47), and benign samples (n = 332). Overlap and comparative profiling were performed excluding one-hit wonder peptides.(G) Analysis of the proportion of AML-associated HLA class I-presented antigen peptides (all HLA-A*01, HLA-A*02, HLA-B*07, HLA-B*08, or HLA-C*07-restricted targets) presented by both AML blasts and LPCs (i.e., common antigens in LPC / AML). Abbreviations: IPep, immunopeptidome; n, number. [Figure 4]HLA class I immunopeptidomics are shown. (A) The frequencies of HLA-A allotypes (upper panel), HLA-B allotypes (middle panel), and HLA-C allotypes (lower panel) are shown in the AML immunopeptidomics dataset (n = 47) and the benign immunopeptidomics dataset (n = 332). P values ​​were calculated using a chi-square test. (B) Comparative profile analysis of allotype-specific immunopeptidomes based on the frequency of HLA-restricted presentation in AML immunopeptidomes and benign immunopeptidomes positive for HLA-A*02 (upper left panel), HLA-B*07 (upper right panel), HLA-B*08 (lower left panel), or HLA-C*07 (lower right panel). The frequency of immunopeptidomes (IPeps) positive for each HLA ligand (X axis) is shown on the Y axis. Comparative profile analysis was performed excluding one-hit wonder peptides. (C) Statistical analysis of the percentage of false-positive AML-associated antigen IDs at various presentation frequencies. The number of HLA-A*01, HLA-A*02, HLA-B*07, HLA-B*08, or HLA-C*07-restricted peptides identified based on immunopeptidome analysis of the AML cohort (n = 47) and benign cohort (n = 332) was compared against random virtual (HLA-matched) AML-associated peptides (left Y-axis). Virtual immunopeptidomes for AML and benign samples were generated in silico by weighted random sampling of the entire peptide ID pool contained in each original cohort. These randomized virtual immunopeptidomes were used to define AML-associated antigens by comparing simulated cohorts of AML samples and benign tissue samples. The process of peptide randomization, cohort construction, and AML-associated antigen identification was repeated 1,000 times, and the average values ​​of the resulting virtual AML-associated antigens were calculated and plotted against various thresholds. The false positive rate (right Y-axis) corresponding to the selected threshold (X-axis) was calculated, and the 1% and 5% false positive rates are indicated in the plot (dotted lines and arrows). Abbreviations: ID, identifier; FDR, false positive rate. [Figure 5]Identification of AML-associated and LPC-associated HLA class II target antigens by immunopeptidome comparative profiling. (A) Saturation analysis of proteins as sources of HLA class II-restricted peptides in the AML cohort. The number of protein IDs as sources of unique peptides is shown as a function of cumulative immunopeptidome analysis of AML samples (n = 47). Exponential regression allowed robust calculation of the maximum number of different protein IDs as sources of peptides (dotted line). The dashed line indicates the immunoproteome coverage obtained in the AML cohort as sources of peptides. (B) Overlap analysis of HLA class II peptide IDs in primary AML samples (n = 47) and benign samples (n = 312). (C) Comparative profiling of the HLA class II immunopeptidome based on the frequency of HLA-restricted presentation in the AML and benign immunopeptidomes. The frequency of immunopeptidomes positive for each HLA peptide (x-axis) is shown on the y-axis. The boxes on the left and their enlarged versions highlight the subset of AML-associated antigens frequently presented only in AML. (D) Hotspot analysis of KIT and FLT3 proteins by clustering HLA class II-presented peptides. The y-axis shows the frequency of presentation of amino acid counts within each cohort for each amino acid position (x-axis). Each box and its size highlights the hotspot identified for each amino acid at the position indicated on the x-axis. The ITD and TKD mutation regions within FLT3 are boxed. (E, F) Overlap analysis of (E) HLA class II peptide IDs and (F) their source protein IDs for LPCenr samples (n = 11), AML samples (n = 47), and benign samples (n = 312). Overlap analysis, comparative profile analysis, and hotspot analysis were performed excluding one-hit wonder peptides. (G) Analysis of the percentage of target peptides, target proteins, and target hotspots presented by AML-associated HLA class II that are presented on both AML blasts and LPCs (a common antigen in LPC / AML).(H) Comparative profile analysis of HLA class II peptide IDs found exclusively in AML (not identified in benign tissue samples) and LPC (not identified in benign tissue samples) based on the frequency of HLA-restricted presentation in LPC immunopeptidome samples (n = 11) and AML immunopeptidome samples (n = 47). The frequency of immunopeptidomes positive for each HLA peptide (X-axis) is shown on the Y-axis. Each box represents a subset of antigens found exclusively in LPC, a subset of LPC-associated antigens presented by both LPC and AML blasts, and a subset of antigens found exclusively in AML. (I) Mass spectrometry-based neoantigen validation using isotopically labeled synthetic peptides. The experimentally eluted P16A*11_mut peptide ("identification" on the X-axis) was validated with isotopically labeled synthetic peptides ("validation" on the X-axis). In the fragment ion spectrum (left), identified b-, y-, and internal ions are shown in red, blue, and orange, respectively. Ions containing the isotope-labeled amino acid (alanine) are indicated with an asterisk. It was confirmed that the isotope-labeled synthetic peptide and the experimentally verified peptide co-eluted at the same retention time (fragment ion chromatogram on the right). Abbreviations: AML, acute myeloid leukemia; IPep, immunopeptidome; n, number; AA, amino acid; npep, number of peptides; LPC, leukemia progenitor cell. [Figure 6]Identification of AML-associated HLA class II target antigens and LPC-associated HLA class II target antigens by immunopeptidome comparative profiling is shown. (A, B) Statistical analysis of the rate of false-positive AML-associated antigen IDs at various presentation frequencies. The number of (A) HLA class II-restricted peptides and (B) their source proteins identified based on immunopeptidome analysis of the AML cohort (n = 47) and benign cohort (n = 332) was compared against random hypothetical AML-associated peptides and their source proteins (left Y-axis), respectively. Virtual immunopeptidomes for AML and benign samples were generated in silico by weighted random sampling of the entire set of IDs contained in each original cohort. These randomized virtual immunopeptidomes were used to define AML-associated antigens by comparing mock cohorts of AML samples with mock cohorts of benign tissue samples. The process of peptide randomization, cohort construction, and AML-associated antigen identification was repeated 1,000 times, and the resulting mean values ​​of virtual AML-associated antigens were calculated and plotted against various thresholds. The false-positive rate (right Y-axis) corresponding to the selected threshold (X-axis) was calculated, and the 1% and 5% false-positive rates are indicated within the plot (dotted lines and arrows). (C) Overlap analysis of protein IDs as sources of HLA class II peptides in primary AML samples (n=47) and benign samples (n=312) is shown. (D) Comparative profiling of HLA class II immunopeptidomes at the protein level as sources of HLA peptides based on the frequency of proteins presented as sources of HLA-restricted peptides in AML and benign immunopeptidomes. The frequency of immunopeptidomes that were positive for each protein as a source of HLA peptide (X-axis) is shown on the Y-axis. The box on the left and its enlarged view highlight the subset of AML-associated antigens that are frequently presented exclusively in AML. (E) Hotspot analysis of AP2B1, HPRT, and IL1AP proteins by clustering of HLA class II-presented peptides.The frequency of presentation of the amino acid counts within each cohort for each amino acid position (x-axis) is shown on the y-axis. Each box and its size highlights the hotspots identified for each amino acid at the position indicated on the x-axis. (F) Comparative profile analysis of protein IDs as sources of HLA class II peptides found exclusively in AML (not identified in benign tissue samples) and protein IDs as sources of HLA class II peptides found exclusively in LPC (n = 11) based on the frequency of protein presentation as sources of HLA-restricted peptides in LPC immunopeptidome samples (n = 47). The frequency of immunopeptidomes that were positive for each protein as a source of HLA peptide (x-axis) is shown on the y-axis. Each box represents a subset of antigens found exclusively in LPC, a subset of LPC-associated antigens presented by both LPC and AML blasts, and a subset of antigens found exclusively in AML. Overlap analysis, comparative profile analysis, and hotspot analysis were performed excluding one-hit wonder peptides. Abbreviations: AML, acute myeloid leukemia; IPep, immunopeptidome; n, number; AA, amino acid; npep, number of peptides; LPC, leukemia progenitor cell; ID, identifier; FDR, false positive rate. [Figure 7] Validation of the spectra of HLA class I-restricted AML- and LPC-associated peptides is shown. A comparison of the fragment spectra (m / z on the x-axis) of HLA ligands eluted from primary samples ("identification") and their corresponding isotopically labeled synthetic peptides ("validation," shown inverted on the x-axis) is shown. Identified b and y ions are shown in red and blue, respectively. Ions containing isotopically labeled amino acids are indicated with an asterisk. [Figure 8]Validation of the spectra of HLA class II-restricted AML- and LPC-associated peptides is shown. A comparison of the fragment spectra (m / z on the x-axis) of HLA ligands eluted from primary samples ("identification") and their corresponding isotopically labeled synthetic peptides ("validation," shown inverted on the x-axis) is shown. Identified b and y ions are shown in red and blue, respectively. Ions containing isotopically labeled amino acids are indicated with an asterisk. [Figure 9] Cryptic peptides are represented in AML- and LPC-derived immunopeptidomes. (A) Genomic distribution of identified AML-associated cryptic peptides (n = 623). AML-associated cryptic peptides were not identified in any benign tissue samples. (B) Overlap analysis of cryptic HLA class I ligand IDs in LPCenr samples (n = 10) and AML samples (n = 47). (C) Genomic distribution of cryptic peptides found exclusively in AML (n = 514, upper panel) and LPC (n = 26, lower panel). (D) Examples of cryptic HLA peptides derived from the 5'UTR (upper panel, P1_cryA*02) and off-frame regions (lower panel, P2_cryB*07) are shown. Abbreviations refer to the CHRFAM7A and TSPAN2 transcripts on opposite strands. The enlarged view highlights three reading frames containing cryptic peptides in the 5'UTR or exon 3. (E) Validation of the spectra of the two cryptic peptides mentioned above, P1_cryA*02 (left panel) and P2_cryB*07 (right panel). The fragment spectra (m / z on the x-axis) of the cryptic peptides eluted from the primary sample ("identification") are compared with those of their corresponding isotopically labeled synthetic peptides ("validation," shown inverted on the x-axis). Identified b and y ions are shown in red and blue, respectively. Ions containing isotopically labeled amino acids are indicated with an asterisk. Abbreviations: ncRNA, non-coding RNA; UTR, untranslated region; n, number; LPC, leukemia progenitor cell; AML, acute myeloid leukemia. [Figure 10]Immunogenicity analysis of HLA class I-restricted targets. (A, B) Detection of pre-existing peptide-specific T cell responses by (A) IFN-γ ELISpot assay and (B) intracellular cytokine staining after 12 days of in vitro expansion of PBMC samples from a healthy volunteer (left panel in A) or an AML patient (middle and right panels in A and B). Representative examples are shown. (A) Data are shown as bar graphs with mean and SD. (B) Each graph shows live single cells stained for CD8 (left panel) and CD4 (right panel) and for IFN-γ and TNF as cytokines. (C) De novo induction of peptide-specific CD8+ T cells after in vitro priming of PBMCs from a healthy volunteer using aAPCs. Graphs show live single cells stained with PE-labeled multimers and CD8 for the specificities indicated in the graph. The negative control shows target antigen tetramer staining of T cells from the same donor primed with an HLA-matched control peptide. (D) Functionality of P12B*08-specific CD8+ T cells induced after in vitro priming using aAPCs was assessed by staining for intracellular cytokines (IFN-γ, TNF) and degranulation markers (CD107a). Representative examples are shown showing IFN-γ and TNF production and CD107a expression (upper panel) after stimulation with peptide P12B*08 compared with HLA-matched control peptide (lower panel). Graphs show live CD8+ single cells stained for IFN-γ and TNF (left panel) and CD107a (right panel). (E) De novo induction of peptide-specific CD8+ T cells using PBMCs from an AML patient. (F) Cytotoxicity of P16A*11_mut-specific effector T cells was analyzed by the VITAL cytotoxicity assay after in vitro priming of CD8+ T cells from a healthy volunteer.Prior to performing the VITAL assay, the abundance of P16A*11_mut-specific effector cells was measured by tetramer staining of polyclonal effector cells in a CD8+ T cell population primed with P16A*11_mut (top panel) and a control cell population from the same donor primed with an HLA-matched control peptide (bottom panel). P16A*11_mut-specific lysis of autologous target cells bearing P16A*11_mut, depending on the effector-to-target ratio, is shown compared to target cells bearing an HLA-matched control peptide (right panel). Nonspecific effectors did not show P16A*11_mut-specific lysis of the same target. Nonspecific effectors were assessed in three independent replicate experiments, and results are shown as the mean and SD of the three replicates. (G) Pie charts show the frequency of HLA class I-restricted antigenic peptides that induced detectable pre-existing T cell responses in an IFN-γ ELISpot assay (n = 17) (top panel), newly induced T cell responses after in vitro priming with aAPCs (middle panel), and the type of immunogenicity (bottom panel). Abbreviations: neg, HLA-matched negative control peptide; FSC, forward scatter; na, not applicable. [Figure 11]Immunogenicity analysis of HLA class II-restricted targets and the impact of immunopeptidome diversity and peptide-specific immune responses on patient survival are shown. (A, B) Detection of pre-existing peptide-specific T cell responses by (A) IFN-γ ELISpot assay and (B) intracellular cytokine staining after 12 days of in vitro expansion of PBMC samples from healthy volunteers (left panel in A and B) or AML patients (right panel in A). Representative examples are shown. (A) Data are shown as bar graphs with mean and SD. (B) Each graph shows live single cells stained for CD4 (left panel) and CD8 (right panel) and for IFN-γ and TNF as cytokines. (C) The intensity of T cell responses, assessed as the number of spots calculated in the IFN-γ ELISpot assay, after 12 days of stimulation of PBMCs from AML patients or healthy volunteers with each HLA class II-restricted antigen peptide associated with AML or LPC. Each dot represents data from an individual donor. Box plots show the median and 25th-75th percentiles, with whiskers indicating minimum to maximum. (D) Pie charts showing the frequency of recognition of AML- or LPC-associated HLA class II-restricted antigenic peptides (number of T cell responses / number tested) in PBMC samples from AML patients or healthy volunteers, assessed by IFN-γ ELISpot assay after 12 days of stimulation. A maximum of 37 AML patient samples and 25 healthy volunteer samples were tested per peptide. (E) The impact of HLA class II-restricted immunopeptidome diversity on overall survival in AML patients (n = 25) was assessed by presentation of unique HLA class II-restricted peptides found exclusively in AML. (F) The impact of antigen-specific immune responses to HLA class II-restricted AML-associated peptides and LPC-associated peptides on overall survival (OS, left panel) and failure-to-treat survival (FFS, right panel) in AML patients (n = 56) is shown. Kaplan-Meier analysis and log-rank tests were performed. Abbreviations: CI, confidence interval; HR, hazard ratio; HV, healthy volunteer; neg, negative peptide; AML, acute myeloid leukemia; LPC, leukemia progenitor cell. [Figure 12]Further characterization of HLA class II-restricted antigens is shown. (A) Structural comparison of CCL23_HUMAN, the protein that serves as the source of the AML-associated target P5II, and VMI2_HHV8P, a viral protein of human herpesvirus type 8 (HHV8). (B) Sequence alignment of CCL23_HUMAN and VMI2_HHV8P. The HLA-presented peptide P5II and its corresponding viral peptide are highlighted in bold. Dissimilar amino acids are shown in red. The symbols below each abbreviation indicate the degree of conservation. Asterisks indicate completely similar amino acids, colons indicate amino acids with very similar characteristics, and periods indicate amino acids with less similar characteristics. (C) Alignment of the physiochemical properties of P5II and its corresponding viral peptide P5II_HHV8P. Physiochemical properties were calculated using PepCalc software. The column direction (up and down) indicates hydrophilicity according to the Hopp-Woods scale. (D) Detection of pre-existing P5II-specific T cell responses, but not specificity for the corresponding viral peptide P5II_HHV8P, assessed by IFN-γ ELISpot assay using PBMC samples from healthy volunteers after 12 days of in vitro expansion. Representative examples are shown. Data are presented as bar graphs with mean and SD. (E) P6II-specific T cell responses assessed by IFN-γ ELISpot assay using PBMC samples from AML patients after 12 days of stimulation. (F) Functionality of P6II-specific T cells was assessed by intracellular cytokine staining using flow cytometry, revealing that P6II-specific responses were induced by CD8+ T cells. (G) In silico peptide prediction identified four hidden HLA class I-restricted peptide candidates for various patient HLA allotypes (A*01, A*02, B*08, B*13, C*06, and C*07). Abbreviations: neg., negative. [Figure 13]Longitudinal analysis of peptide-specific T cell responses after allogeneic stem cell transplantation (SCCT) is shown. (A) Schematic diagram of the treatment course of AML patient UPN23. After initial diagnosis (FD), the patient received induction therapy (idarubicin / cytarabine) followed by allogeneic stem cell transplantation (alloTx) 2 months after FD. After alloTx and respiratory syncytial virus (RSV) pneumonia testing, the patient received various immunosuppressive therapies (mycophenolate mofetil, cyclosporine (CSA), and prednisolone). AML-associated peptide-specific T cell responses were assessed by IFN-γ ELISpot assay 12, 17, and 32 months after FD. (B, C) T cell responses specific to P16A*11_mut are shown at various time points after allogeneic stem cell transplantation in patient UPN23. The calculated spot counts were calculated by normalizing the mean number of duplicate spots to 5 × 10 cells and then subtracting the normalized mean spot count for each negative control. [Figure 14] The impact of immunopeptidome diversity and peptide-specific immune responses on patient survival is shown. (A-C) The impact of HLA class I-restricted immunopeptidome diversity (A, B) and HLA class II-restricted immunopeptidome diversity (C) on overall survival (OS, A) and failed-failure survival (FFS, B and C) of AML patients (HLA class I: n = 26, HLA class II: n = 25) was assessed in terms of the presentation of unique peptides found exclusively in AML. (D) The impact of antigen-specific immune responses to HLA class II-restricted AML-associated peptides and LPC-associated peptides on failed-failure survival (FFS) in AML patients (n = 45) after allogeneic stem cell transplantation is shown. Kaplan-Meier analysis and log-rank test were performed. Abbreviations: HR, hazard ratio; CI, confidence interval. [Example]

[0107] 1. Materials and Methods Patients and blood samples For immunopeptidome analysis, PBMCs or bone marrow mononuclear cells (BMNCs) were collected from AML patients at diagnosis, relapse, or molecular remission at the Departments of Hematology and Oncology at the University of Tübingen and University of Dresden (Germany) and the Department of Hematology and Oncology at the University of California, San Francisco School of Medicine (California, USA). For T cell-based assays, PBMCs were collected from AML patients and healthy volunteers at various time points after allogeneic stem cell transplantation or complete remission. Cells were isolated by density gradient centrifugation and stored at -80°C. Clinical and survival data were collected during follow-up periods up to 48 months after diagnosis. Informed consent was obtained in accordance with the Declaration of Helsinki. This study was conducted in accordance with the guidelines of the local ethical committee. HLA typing was performed at the Department of Hematology and Oncology at the University of Tübingen (Germany).

[0108] HLA surface quantification HLA cell surface expression was measured using the QIFIKIT bead-based quantitative flow cytometry assay (Dako) according to the manufacturer's instructions. Briefly, cells were stained with a pan-HLA class I-specific W6 / 32 monoclonal antibody (produced in-house), an HLA-DR-specific L243 monoclonal antibody (produced in-house), or an IgG isotype control (BioLegend). A polyclonal goat FITC anti-mouse antibody (Dako) was used as the secondary antibody. After washing with normal mouse serum (affymetrix eBioscience), cell surface marker staining was performed using PE / Cy7 anti-human CD38 antibody (BioLegend), APC anti-human CD34 antibody (BD), and Pacific Blue anti-human CD45 antibody (BD). Aqua fluorescent-reactive dye (Invitrogen) was used as a viability marker. Analysis was performed on a FACS Canto II cytometer (BD). Only cell populations consisting of 100 or more cells were analyzed for HLA cell surface expression.

[0109] LPC enrichment Enrichment of LPCs from AML samples was performed by fluorescence-activated cell sorting (FACS) at the Institute for Stem Cell Biology and Regenerative Medicine, Stanford University (CA, USA) (UPN3–8, UPN11) or by magnetic-activated cell sorting (MACS) at the Institute for Cell Biology, Department of Immunology, University of Tübingen (Tübingen, Germany) (UPN01, UPN02, UPN09, UPN10). For FACS, PBMCs were stained with PE / Cy7 anti-human CD38 monoclonal antibody, APC anti-human CD34 monoclonal antibody, PerCP / Cy5.5 anti-human CD3 monoclonal antibody, PerCP / Cy5.5 anti-human CD19 monoclonal antibody, PerCP / Cy5.5 anti-human CD20 monoclonal antibody, or PerCP / Cy5.5 anti-human CD56 monoclonal antibody and sorted on a Becton Dickinson FACSAria II or FACSAria III. MACS was performed using the human CD34 MultiSort Kit and CD38 MicroBead Kit (both Miltenyi). Purity of sorted cells was determined by staining with PE / Cy7 anti-human CD38 monoclonal antibody (BioLegend), APC anti-human CD34 monoclonal antibody (BD), or Pacific Blue anti-human CD45 monoclonal antibody (BD). Aqua fluorescent reactive dye (Invitrogen) was used as a viability marker. Analysis was performed using a FACS Canto II cytometer (BD).

[0110] CD34 + HPCs were magnetically enriched (CD34 MicroBead Kit, human, Miltenyi) from hematopoietic stem cell apheresis samples mobilized with G-CSF from patients with non-hematologic malignancies (e.g., germ cell tumors) and healthy volunteers.

[0111] Mice and xenograft assay Xenotransplantation assays were performed at the Department of Biomedicine at the University of Basel and University Hospital of Basel, Switzerland. scid IL2rg tmWjl / Sz mice (NSG, Jackson Laboratory) were housed under pathogen-free conditions in accordance with Swiss federal and cantonal regulations. All animal experiments were approved by the Veterinary Medicine of the Canton of Basel-Stadt. Xenograft assays were performed as previously described. Briefly, presorted 6 × 10 5 Primary human CD34 + CD38 - AML cells or CD34 + CD38 + AML cells were transplanted into 8-week-old female NSG mice (n = 4–5 per group) by intrafemoral injection. Graft survival was monitored by conventional bone marrow aspirates or peripheral blood analysis as previously described. Graft survival was defined as a percentage of human leukemia cells of 1% or greater in the mouse peripheral blood or bone marrow, as analyzed by multicolor flow cytometry using antibodies against human leukemia antigens. The antibody panel included fluorescent antibodies against human CD33, human CD34, human CD133, human CD117, and human CD45 (BD Biosciences), fluorescent antibodies against human CD14 and human CD13 (eBiosciences), and fluorescent antibodies against human CD3 and human CD19 (BioLegend). All mice underwent final bone marrow, peripheral blood, and organ analysis using multicolor flow cytometry.

[0112] Isolation of HLA ligands Standard immunoaffinity purification was performed using the pan-HLA class I-specific W6 / 32 monoclonal antibody, the pan-HLA class II-specific Tu-39 monoclonal antibody, and the HLA-DR-specific L243 monoclonal antibody (all produced in-house, Department of Immunology, University of Tübingen). 32 HLA class I and II molecules were isolated and HLA ligands were extracted.

[0113] Mass spectrometry data acquisition Mass spectrometry data analysis was performed as previously described. Peptides were separated by nanoflow high-performance liquid chromatography (RSLCnano, Thermo Fisher) using a 50 μm x 25 cm PepMap Rapid Separation column (Thermo Fisher) with a 90-minute gradient elution from 2.4% to 32.0% acetonitrile. Eluted peptides were analyzed on an online Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher) equipped with a nanoelectrospray ion source, using top-speed collision-induced dissociation (CID) fragmentation (HLA class I peptides) or high-energy collision-induced dissociation (HCD) fragmentation (normalized collision energy: 35%) (HLA class II peptides) in data-dependent acquisition mode. The mass range for HLA class I peptide analysis was set to 400–650 m / z, and the charge states of the fragmented peptides were 2+ and 3+. For the analysis of HLA class II peptides, the mass range was limited to 400–1,000 m / z, and the charge state of the fragmented peptides was selected to be 2+–5+.

[0114] Data Processing Data processing was performed as previously described. Briefly, the SEQUEST HT search engine (University of Washington) was used to search the human proteome in the Swiss-Prot database (20,279 examined protein sequences, as of September 27, 2013) without enzyme restrictions. The precursor mass tolerance was set to 5 ppm, and the fragment mass tolerance was set to 0.02 Da. Oxidized methionine was allowed as a dynamic modification. The false discovery rate (FDR) was estimated using the Percolator algorithm and was limited to 5% for HLA class I and 1% for HLA class II. Peptide length was limited to 8–12 amino acids for HLA class I and 8–25 amino acids for HLA class II. Protein inference was disabled to allow peptides to have multiple protein annotations. HLA class I annotation was performed using NetMHCpan 4.0 and SYFPEITHI. Peptides were annotated with a percentile rank of <2% and a maximum score of ≥60%. For comparative profile analysis, peptides with ≤3 PSMs and presented in only one sample ("one hit wonders") were excluded.

[0115] Neoepitope screening To screen for neoepitopes, we used a non-patient mutFASTA database containing the top 100 recurrent AML-associated missense mutations listed in the COSMIC database (www.cancer.sanger.ac.uk; Forbes, SA et al. COSMIC: somatic cancer genetics at high-resolution. Nucleic Acids Res 45, D777-D783 (2017)), including the most common NPM1 frameshift mutations (types A, B, C, D, and E; Falini, B. et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 352, 254-266 (2005)) and FLT3-ITD mutations (Smith, CC et al. Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukemia. Nature 485, 260-263 (2012)) and FLT3-TKD mutations (Opatz, S. et al. Exome sequencing identifies recurring FLT3 N676K mutations in core-binding factor leukemia. Blood 122, 1761-1769 (2013); Bacher et al. Prognostic relevance of FLT3-TKD mutations in AML: the combination matters--an analysis of 3082 patients. Blood 111, 2527-2537 (2008); Thiede, C. et al.Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB subtypes and identification of subgroups with poor prognosis. Blood 99, 4326-4335 (2002); Vempati, S. et al. Arginine 595 is duplicated in patients with acute leukemias carrying internal tandem duplications of FLT3 and modulates its transforming potential. Blood 110, 686-694 (2007); Yamamoto, Y. et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood 97, 2434-2439 (2001)). Data processing of AML immunopeptidomic data using mutFASTA was performed as described above. To minimize false-positive identification, more stringent filtering criteria were applied: FDR for HLA class I was set to 5%, FDR for HLA class II was set to 1%, XCorr ≥ 1, and ΔScore ≥ 0.2. After manual spectral validation, candidate neoepitopes were produced as isotope-labeled synthetic peptides and used for spectral comparison and validation.

[0116] Peptide synthesis and spectral validation Peptides were synthesized using the 9-fluorenylmethyloxycarbonyl / tert-butyl method on a Liberty Blue peptide synthesizer (CEM). Experimental validation of eluted peptide spectra was performed by calculating the spectral similarity with the corresponding isotopically labeled synthetic peptides measured in a complex matrix. Synthetic peptide fragments containing isotopically labeled fragments had a mass shift compared to unlabeled fragments, and thus were penalized in the spectral similarity score. To minimize such effects, the synthetic peptide spectra were preprocessed by shifting the isotopically labeled synthetic peptides containing singly or doubly charged b- or y-ion peaks to the expected m / z positions of the corresponding unlabeled fragments. The intensities of the annotated b- and y-ion peaks were then used to calculate the spectral correlation between the eluted peptide spectrum and the preprocessed synthetic peptide spectrum.

[0117] Validation of mutation-derived HLA ligands by targeted PRM Using a highly sensitive targeted PRM MS method, we identified the HLA-A*11-restricted peptide AVEEVSLRK (P16 A*11_MUT To generate a PRM assay library, a complex biological matrix (HLA ligands isolated from JY cells) was spiked with a heavy isotope-labeled synthetic reference peptide and analyzed in MS2 mode using PRM on an LTQ Orbitrap Fusion Lumos mass spectrometer. For the validation of mutation-derived HLA ligands, each sample was spiked with the reference peptide and analyzed using the PRM MS method. The mass spectrometer was operated in PRM mode, with a resolution of 120,000 (AGC target: 1.5 × 10) on the Orbitrap mass spectrometer. 5 After acquiring the full mass spectrum with a maximum injection time of 50 ms, quadrupole and MS2 scans were performed on an Orbitrap mass spectrometer at a resolution of 60,000 (AGC target: 7.0 × 10) based on a scheduled inclusion list containing the target masses of mutant-derived peptides with a 2+ or 3+ charge state and the masses of heavy isotope-labeled reference peptides. 4Precursor ions were isolated using a 118 ms injection time (maximum injection time). Ion activation was performed by CID at 35% normalized collision energy. A spectral library was created from the msf file using Skyline software (version 3.7.0). The transition settings were precursor charge 2 and 3, ion charge 1 and 2, ion types y, b, a, and p, and product ions from ion 2 to the last ion -2. The ion match tolerance for the library was set to 0.02 m / z, and eight product ions were acquired. The method match tolerance was set to 0.02 m / z, and the MS / MS filter acquisition method was set to target.

[0118] Identification of cryptic peptides Cryptic HLA class I peptides were identified using the recently reported Peptide-PRISM. De novo peptide sequencing was performed using PEAKS X (Bioinformatics Solutions, Canada). Raw data were refined with the following settings: (1) Merge Option: No Merge; (2) Precursor Option: Correction; (3) Charge Option: No Correction; (4) Filter Option: No Filter; (5) Process: True; (6) Default: True; and (7) Assembled Chimeras: Yes. De novo sequencing was performed with a parent mass tolerance of 10 ppm. The fragment mass tolerance was set to 0.15 Da, and the enzyme was set to None. Possible modifications were defined as oxidation (M), pyroglutamate oxidation from Q (N-terminal Q), and carbamidomethylation (C). A maximum of three post-translational modifications were allowed per peptide. For each identified fragment ion mass spectrum, up to 10 new sequencing candidates and their corresponding average local confidence scores were reported. By applying the Chimera Spectrum option in PEAKS X, more than one top-10 candidate list could be assigned to a single fragment ion spectrum. Two tables ("All de novo candidates" and "de novo peptides") were exported from PEAKS for further analysis.

[0119] Using Peptide-PRISM, all de novo sequence candidates were matched to the six-frame translated human genome (hg38) and three-frame translated human transcriptome (ENSEMBL 90). The results were filtered for each classification (CDS, UTR5, off-frame, ncRNA, UTR3, intron, and intergenic regions) at a 10% FDR. NetMHCpan 4.0 was used to predict binding affinities for all identified HLA class I peptides across all HLA alleles in the corresponding samples.

[0120] Peptide-specific T cell proliferation and IFN-γ ELISpot assay PBMCs from AML patients and healthy volunteers were pulsed with 1 μg / mL (HLA class I) or 5 μg / mL (HLA class II) of each peptide and cultured for 12 days. 20 U / mL of IL-2 (Novartis) was added on days 3, 5, and 7. On day 12, PBMCs stimulated with each peptide were analyzed by ELISpot assay. Spots were counted using an ImmunoSpot S5 analyzer (CTL). T cell responses were considered positive if the number of counted spots per 500,000 cells was greater than 10 and the mean number of spots was at least three times the mean number of spots in the negative control.

[0121] Refolding Biotinylated HLA-peptide complexes were prepared as previously reported and tetramerized with PE-labeled streptavidin (Invitrogen Life Technologies) at a molar ratio of 4:1.

[0122] Peptide-specific CD8 by aAPC + T cell induction Peptide-specific cytotoxic T lymphocytes (CTLs) were primed using artificial antigen-presenting cells (aAPCs) as previously described. Specifically, 800,000 streptavidin-coated microspheres (5.6 μm diameter, Bangs Laboratories) were loaded with 200 ng of biotinylated peptide-HLA complexes and 600 ng of biotinylated anti-human CD28 antibody (clone 9.3, produced in-house). MACS-sorted CD8 CTLs were primed with 4.8 U / μl IL-2 (R&D Systems) and 1.25 ng / ml IL-7 (PromoKine). + T cells (CD8 microbeads, human, Miltenyi) were cultured. aAPC (1 × 10 6 CD8 + T cells were stimulated weekly with 200,000 aAPCs per T cell and 5 ng / ml IL-12 (PromoKine) for a total of four cycles. The induction of peptide-specific T cells was analyzed by tetramer staining.

[0123] Cytokine and tetramer staining Peptide-specific CD8 +T cell frequency and functionality were analyzed by tetramer staining and intracellular cytokine staining (ICS) as previously described. For ICS, cells were pulsed with 10 μg / ml of each peptide and then incubated with 10 μg / ml Brefeldin A (Sigma-Aldrich) and 10 μg / ml GolgiStop (BD) for 12–16 hours. Staining was performed using Cytofix / Cytoperm (BD), PerCP anti-human CD8 monoclonal antibody, Pacific Blue anti-human TNF monoclonal antibody, and FITC anti-human CD107a monoclonal antibody (all BioLegend), as well as PE anti-human IFN-γ monoclonal antibody (BD). PMA and ionomycin (Sigma-Aldrich) were used as positive controls. Negative control peptides used were GSEELRSLY (SEQ ID NO: 33), POL_HV1BR, HLA-A*01; YLLPAIVHI (SEQ ID NO: 34), DDX5_HUMAN, HLA-A*02; RLRPGGKKK (SEQ ID NO: 35), GAG_HV1BR, HLA-A*03; TPGPGVRYPL (SEQ ID NO: 36), NEF_HV1BR, HLA-B*07; DIAARNVL (SEQ ID NO: 37), FAK1_HUMAN, HLA-B*08; ASEDYVAPPK (SEQ ID NO: 38), MKX_HUMAN, HLA-A*11; and ETVITVDTKAAGKGK (SEQ ID NO: 39), FLNA_HUMAN, HLA class II. Peptide-specific CD8+ cells were expressed after priming with aAPCs. + T cell frequencies were measured by staining with PE-Cy7 anti-human CD8 monoclonal antibody (Biolegend) and HLA:peptide tetramer-PE. Tetramers of the same HLA allotype containing an irrelevant control peptide were used as negative controls. Peptide-specific CD8 + T cell frequency is determined by the CD8 + More than 0.1% of total T cells and peptide-specific CD8 +Priming was considered successful when the T cell frequency was at least three times higher. The same criteria were applied to the results of intracellular cytokine staining. Samples were analyzed using a FACS Canto II cytometer (BD).

[0124] Cytotoxicity assay Peptide-specific CD8 + The cytolytic potential of T cells was analyzed by a flow cytometry-based VITAL assay as previously described. CD8 cell-depleted autologous PBMCs were loaded with test peptides or HLA-matched control peptides and labeled with CFSE or FarRed. Effector cells were added at the effector-to-target ratios indicated in each graph. Specific lysis of target cells loaded with each peptide was calculated relative to control target cells.

[0125] Quantitation and statistical analysis Overlap analysis was performed using BioVenn. Population coverage of HLA allotypes was calculated using the IEDB population coverage tool (www.iedb.org). The FDR of AML-associated peptides with various presentation frequencies was calculated using a Python script developed in-house. Hotspot analysis of the HLA class II immunopeptidome (hotspot length ≥ 8 amino acids) was performed using an R script developed in-house. The R script mapped identified peptides to their source proteins according to their sequence and calculated the presentation frequency of single amino acid positions within each cohort. Flow cytometry data were analyzed using FlowJo 10.0.8 (Treestar). For survival analysis to examine the impact of immunopeptidome diversity, peptide yields of peptides found exclusively in AML were normalized by cell number and applied to immunopeptidome analysis. Overall survival (OS) and event-free survival (EFS) were calculated using the Kaplan-Meier method for the low- and high-diversity immunopeptidomes, categorized according to median peptide yield. Log-rank tests were performed to examine differences in survival between groups. Survival analysis examined the influence of preexisting antigen-specific immune responses to HLA class II-restricted AML-associated and LPC-associated peptides detected by IFN-γ ELISpot assay. Patients were divided into two groups: responders with peptide-specific T cell responses and non-responders without detectable peptide-specific T cell responses. All figures and statistical analyses were performed using GraphPad Prism 9.4.1 (GraphPad Software). Data are presented as mean ± SD. Box plots show the 25th or 75th percentile quartile and median values, with whiskers indicating the minimum and maximum values. Continuous data were tested for distribution, and individual groups were tested using two-tailed chi-squared tests, unpaired t-tests, unpaired Mann-Whitney U tests, Kruskal-Wallis tests, or paired Wilcoxon signed-rank tests, all performed two-tailed, with adjustments for multiple testing where appropriate.A P value <0.05 was considered to indicate statistical significance.

[0126] 2.Results Elucidating the antigenic landscape of primary LPCs by mass spectrometry-based immunopeptidomics To investigate the immunopeptidome landscape of primary leukemia stem and progenitor cells (LPCs), we first analyzed CD34 expression in PBMCs from a cohort of 26 AML patients. + CD38 - Screening for the presence of LPCs yielded a low median frequency of 0.18% (range 0.00-40.6%). Next, LPCs were enriched in the sample panel (n = 11) that showed adequate LPC frequencies. As a result, the frequency of LPCs was significantly enriched from 5.27% (range 0.02-40.6%) in the unsorted samples to 92.1% (range 40.0-99.7%) in the sorted samples (LPCs). enr population, Figure 1A and Figure 1B). UPN01-derived CD34 + CD38 - LPC enr The population was NOD / SCID / IL2Rγ null When xenotransplanted into (NSG) mice, human CD33 + Leukemia cells and CD33 + CD117 + We demonstrated that leukemia cells engrafted in vivo in the bone marrow, peripheral blood, spleen, and liver of NSG mice (n = 4, Figure 1C). T cell-mediated tumor immunosurveillance requires sufficient expression of HLA on tumor cells, particularly LPCs. Based on this, we quantified the cell surface expression of HLA on LPCs (n = 11) and determined the expression of CD34. + CD38 + CD34 from AML blasts (n=11) and healthy volunteers +We compared HLA class I and HLA-DR molecules in LPCs with those in HPCs (n = 18) (Figure 1D, Figure 1E). Cell surface expression of HLA class I and HLA-DR molecules in LPCs was heterogeneous among patients, with the number of HLA class I molecules per cell ranging from 31,054 to 310,084 (median: 81,874) and the number of HLA-DR molecules per cell ranging from 1,198 to 48,454 (median: 10,007). HLA class I cell surface expression was similar in AML blasts and HPCs, with the median number of HLA class I molecules per cell being 76,333 and 123,567, respectively (Figure 1D). Cell surface expression of HLA-DR molecules in LPCs was lower than that in AML blasts (median: 20,540) and HPCs (median: 28,349), but this difference was not significant (Figure 1E). LPCs enr Mass spectrometry-based analysis of the immunopeptidome naturally presented in the samples and autologous blast samples (n=10 each) revealed that LPC enr A total of 16,342 (range 127–7,603, median: 1,930) unique HLA class I ligands were identified from the samples, and a total of 32,961 (range 1,151–10,489, median: 5,588) unique HLA class I ligands were identified from the autologous blast samples (Figure 1F). enr HLA class II immunopeptidome mapping of the samples and autologous blast samples revealed that LPC enr A total of 16,638 (range 450-6,218, median: 1,458) different HLA class II-presented peptides were identified from the samples, and 25,128 (range 468-10,217, median: 2,238) different HLA class II-presented peptides were identified from the autologous blast samples.

[0127] Comparison of the LPC-derived immunopeptidome with that of AML blasts revealed substantial overlap in HLA-presented peptides between the LPC and autologous blast samples, with 39.4% of HLA class I peptides and 35.1% of HLA class II peptides shared in common. However, 6.8% of the identified HLA class I peptides and 18.7% of the identified HLA class II peptides were presented exclusively by LPC (Figure 1H). Length characterization of the identified HLA-presented peptides revealed that the peptide length distributions for HLA class I- and HLA class II-presented peptides were consistent with the expected peptide length distributions, and the distributions in the LPC and blast samples were similar (Figure 1I). Analysis of the overall or position-specific amino acid composition of HLA-presented peptides revealed no significant differences between the LPC-derived immunopeptidome and the blast-derived immunopeptidome (Figure 1J, Figure 2A, Figure 2B).

[0128] Taken together, these data indicate that presentation of HLA class I- and HLA class II-restricted antigens by LPCs was similar, but not identical, to that of AML blasts and emphasize the importance of selecting AML-associated target antigens for immunotherapy based on combined approaches, including LPCs.

[0129] Identification of LPC-associated HLA class I-restricted peptides using comparative immunopeptidomic profiling To identify novel AML-associated targets that target not only AML blasts but also LPCs, we comprehensively mapped the HLA class I immunopeptidome of 47 primary AML samples, including the above-mentioned samples sorted for LPCs or autologous blasts (Table 5). [Table 5]

[0130] A total of 72,042 unique HLA class I ligands were identified, mapping to 10,609 proteins as peptide sources, and it was estimated that up to 97% protein coverage could be achieved as peptide sources (Figure 3A). The number of peptides identified per patient ranged from 542 to 11,240 (median: 3,143). To identify AML-associated target antigens, PBMC samples and CD34 + We performed a comparative immunopeptidome profile analysis using a benign dataset (n = 332) including enriched HPC samples. This benign dataset contained 72,129 unique HLA class I ligands. Furthermore, overlap analysis revealed 13,019 HLA class I ligands found exclusively in AML (Figure 3B). Because the AML dataset contained a wide variety of HLA class I allotypes, at least one of these HLA class I ligands found exclusively in AML was identified in at least 20% of all analyzed samples (Figure 3C). A total of 48 HLA class I allotypes were covered, achieving a coverage rate of 99.9% of the global population with at least one allotype (Figure 3D). The allotype frequencies ranged from 2.1% to 55.3%, and no significant differences were observed for the majority of allotypes (72%, 39 of 54) compared to the benign dataset (Figure 4A).

[0131] To identify frequently occurring AML-associated HLA class I antigens, we performed HLA allotype-specific comparative profiling of the most common HLA class I allotypes: HLA-A*01 (frequency of 29.8% in the AML cohort), HLA-A*02 (frequency of 48.9% in the AML cohort), HLA-B*07 (frequency of 25.5% in the AML cohort), HLA-B*08 (frequency of 23.4% in the AML cohort), and HLA-C*07 (frequency of 55.3% in the AML cohort) (Figures 3E and 4B). This resulted in a global population coverage of 71.1%. This analysis revealed that 48 HLA-A*01-restricted AML-associated target antigens, 28 HLA-A*02-restricted AML-associated target antigens, 185 HLA-B*07-restricted AML-associated target antigens, 161 HLA-B*08-restricted AML-associated target antigens, and 11 HLA-C*07-restricted AML-associated target antigens were frequently presented in allotype-matched samples, with frequencies ranging from at least 20% to a maximum of 58% (Figures 3E, 4B, and 4C). As an additional validation step, AML-associated target antigens derived from PBMC samples obtained from AML patients (n = 8) who had achieved molecular remission were also presented. MR Using immunopeptidomic datasets, we established target antigens that are exclusively presented on malignant tumor cells. The majority of AML-associated targets defined by the above analysis (85% of FLT3 and 64% of NPM1) were identified regardless of the FLT3 and NPM1 mutation status of the analyzed patient samples. Furthermore, to identify AML-associated antigens that are also presented on LPCs, we analyzed all AML and benign datasets using the LPCs. enr Comparison of the immunopeptidomic data of the samples identified 2,322 LPC / AML common antigens presented by AML blasts and LPCs, but CD34 +These antigens were not observed in benign tissues containing enriched HPC (Figure 3F). Of the previously identified AML-associated target antigens, 41.9% (179 of 434) were common antigens between LPC and AML (Figure 3G), proving them to be the most important targets for dual-targeting T cells targeting AML blasts and LPC.

[0132] Identification of HLA class II-restricted antigens associated with AML and LPC For HLA class II, we identified a total of 61,205 unique peptides (590-10,733 per patient, median: 2,168) derived from 5,922 proteins. By analyzing 47 primary AML samples, we estimated that up to 85% protein coverage of peptide sources could be achieved (Table 5, Figure 5A). Using a previously established immunopeptidome profiling platform (Bilich, T. et al., The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy. Regular Article IMMUNOBIOLOGY AND IMMUNOTHERAPY (2019)), we analyzed three antigen groups: target peptides, target proteins, and target hotspots. First, we performed overlap and comparative profiling analysis at the peptide level using the benign data set (n = 312). We identified 10,931 HLA class II-restricted peptides exclusively present in AML (Figure 5B). After FDR correction, five of these peptides were present in at least 15% of the samples, confirming that they did not have length variants in benign tissue samples (target peptides, Figure 5C, Figure 6A). Next, we performed profiling analysis of proteins as sources of HLA peptides. We identified 311 proteins exclusively present in AML, including CCL23 and RRS1, which were frequently present. After FDR correction, we found that seven proteotypic peptides in CCL23 were presented in an HLA-restricted manner in association with AML, and two proteotypic peptides in RRS1 were presented in an HLA-restricted manner in association with AML (Figure 6B-D). As a third group, we analyzed hotspots found only in AML by clustering peptides. Five AML-associated hotspots were found in the FLT3, IL1AP, HPRT, KIT, and AP2B1 proteins with a frequency of at least 15% (Figures 5D and 6E).In the next step, the newly identified AML-associated target peptides, target proteins, and target hotspots are presented in the LPC. enr We screened the HLA class II immunopeptidome dataset for target peptides and protein antigens frequently found exclusively in LPCs and frequently associated with LPCs (Figures 5H and 6F). Similar to the results for HLA class I, the majority of identified HLA class II-restricted targets (64% for FLT3 and 50% for NPM1) were independent of the FLT3 and NPM1 mutation status of the analyzed patient samples. Among the AML-associated HLA class II target peptides, target proteins, and target hotspots, 66.7% (8 of 12) were antigens shared by LPCs and AMLs (Figure 5G), proving them to be the most important targets for targeting AML blasts and LPCs by dual-targeting T cells. Furthermore, we screened the HLA class II immunopeptidome dataset for target peptide and protein antigens frequently found exclusively in LPCs and frequently associated with LPCs (Figures 5H and 6F), identifying several interesting candidates for specific LPC immunotargeting.

[0133] The role of neoantigens and cryptic peptides in AML In addition to defining novel AML- and LPC-associated antigens, we screened the AML immunopeptidomic cohort for neoepitopes derived from naturally presented common mutations. Two peptides derived from NPM1 mutations were identified: the HLA-A*11-restricted peptide P16 A*11_mut (AVEEVSLRK; SEQ ID NO: 16) and HLA-A*03 restricted peptide P17 A*03_mut (LAVEEVSLR; SEQ ID NO: 17) and P15, an HLA class II-presented neoepitope derived from the R140Q mutation in IDH2 II_mutWe identified and validated the HLA class I-presented peptides (KLKKMWKSPNGTIQNILGGTVF; SEQ ID NO: 1) presented in the AML-associated HLA class I-targeted AML protein (Figure 5I, Figure 7, Figure 8). Furthermore, in addition to classical neoepitopes derived from mutations, we also focused on cryptic peptides derived from non-coding regions within the coding regions (off-frame) of annotated proteins, such as 5' and 3' untranslated regions (UTRs), non-coding RNAs (ncRNAs), intronic and intergenic regions, and frameshifted reading frames, as novel potential sources of tumor-associated antigens. Analysis of the AML and benign immunopeptidomics cohorts using Peptide-PRISM identified 623 AML-associated HLA class I-presented cryptic peptides, primarily derived from the UTR5 region and off-frame regions, none of which were identified in benign tissues (Figure 9A). Of these, a total of 109 peptides were identified in the LPC enr Among the peptides identified in the samples, 26 were peptides found exclusively in the LPC (Fig. 9B), and these were mainly derived from off-frame regions (Fig. 9C). P1_cry peptides were presented with high frequency (26% and 42% allotype specificity, respectively). A*02 (ILLSPPLLTI; SEQ ID NO: 40) and P2_cry B*07 (GPDDGRGVL; SEQ ID NO: 41) was selected for further validation and characterization. These peptides were derived from the UTR5 region of CHRFAM7A and an off-frame region of TSPAN2 (Figure 9D). Isotopically labeled synthetic peptides were used to verify their identification by mass spectrometry (Figure 9E). The identified neoepitopes and cryptic peptides further expanded the panel of novel AML-associated target antigens.

[0134] Novel AML / LPC-associated antigens reveal pre-existing and de novo inducible immune responses in AML patients and healthy volunteers A panel of peptides presented on LPC and frequently presented by HLA class I or HLA class II molecules, including neoepitopes, cryptic peptides, and AML-associated tumor antigens, restricted to common HLA allotypes, was selected for further characterization and immunogenicity analysis (Table 6). [Table 6]

[0135] Experimental spectra of selected peptide IDs were validated by comparison with fragment mass spectra using isotope-labeled synthetic peptides (Figure 9E, Figure 7, Figure 8). To detect pre-existing memory T cell responses to selected HLA class I-restricted peptides, IFN-γ ELISpot assays were performed using HLA-matched PBMCs obtained from healthy volunteers and AML patients (Figure 10A). IFN-γ secretion was observed in up to 30% of AML patient samples to five of 19 HLA class I peptides, and in up to 8% of healthy volunteer samples to one of 19 peptides (Table 2). These immune responses were primarily CD4+. + T cell-mediated expression was observed in vitro using artificial antigen-presenting cells (aAPCs). + T cell priming resulted in antigen-specific CD8 T cells expressing 12 of 13 HLA class I peptides investigated in at least 66% of the healthy volunteer samples analyzed. + It was confirmed that T cells were newly induced and effectively proliferated (Fig. 10C and Table 2). When stimulated with peptide, the induced T cells produced IFN-γ and TNF as cytokines and the degranulation marker CD107a (Fig. 10D). Notably, peptide-specific T cell responses were newly induced even in AML patient samples that did not show a pre-existing immune response (Fig. 10E). Furthermore, 4.0% of P16 A*11_mutPolyclonal effector cells containing specific T cells lysed 82% of peptide-bearing autologous cells, whereas nonspecific effector cell populations showed only a 3.4% relative lysis rate against the same target cells. This specific lysis was dependent on the effector-to-target ratio; decreasing the effector-to-target ratio reduced specific lysis (Figure 10F). Overall, 89.5% (17 of 19) of the selected HLA class I-restricted peptides were found to be immunogenic, and 26.3% (5 of 19) of the selected HLA class I-restricted peptides demonstrated the presence of pre-existing memory T cells in AML patients or healthy volunteers (Figure 10G).

[0136] Evaluation of the functionality of HLA class II-restricted peptides using IFN-γ ELISpot assay (Fig. 11A) and intracellular cytokine staining (Fig. 11B) revealed that 86.7% (13 of 15) of the peptides stimulated CD4 T cells at frequencies up to 33% in AML patients or healthy volunteers. + It was revealed that there was a strong pre-existing immune response mediated by T cells (Fig. 11C, Fig. 11D, Table 2). Interestingly, peptide P5 II Protein CCL23 as a source of (SKPGVIFLTKKGRRF) and this peptide P5 II showed high similarity to the structural, sequence, and physiochemical properties of the human herpesvirus 8 viral protein VMI2 (Fig. 12A, 12B, 12C). However, P5, detected in PBMC samples from healthy volunteers and AML patients, II Specific T cell response is P5 II It is specific and does not show cross-reactivity with this viral peptide (Fig. 12D). II (SPGPFPFIQDNISFYA) in one patient, CD8 +The peptides induced T cell-mediated immune responses (Figures 12E and 12F). In silico prediction using various patient HLA allotypes identified four HLA class I-restricted peptide candidates embedded in a long sequence (Figure 13G). Longitudinal evaluation of peptide-specific T cell responses using an IFN-γ ELISpot assay after allogeneic stem cell transplantation in one patient (Figure 13A) revealed an increase in responses over time, beginning 17 months after transplantation (Figures 13B and 13C). At 12 months after transplantation, no peptide-specific T cell responses were detectable, likely due to systemic immunosuppression caused by prednisolone treatment at that time. Diversity in antigen presentation and immune recognition of AML-associated antigens is associated with improved survival in AML patients As a final step, we investigated how antigen presentation and peptide-specific immune recognition exclusive to AML correlate with prognosis. Regarding the presentation of diverse peptides, known as immunopeptidome diversity, the presentation of antigens exclusive to AML did not differ significantly across patient backgrounds or tumor characteristics, such as age, sex, ELN, karyotype, FLT3-ITD mutation, or NPM1 mutation. Survival analysis revealed no effect of HLA class I immunopeptidome diversity on failure-to-treat (FFS) or overall survival (OS) (Figures 14A and 14B). While HLA class II-restricted peptide diversity significantly improved OS (Figure 11E), FFS was not improved in patients presenting a large number of diverse peptides (Figure 14C), suggesting the role of CD4 in tumor immune surveillance. +We demonstrated the importance of immune responses. We found that immune recognition of AML-associated antigens and LPC-associated antigens in AML patients (n = 56), i.e., pre-existing immune responses to HLA class II-restricted AML-associated antigens and LPC-associated antigens detected by IFN-γ ELISpot assay, significantly impacted FFS prognosis but not OS (Figure 11F). However, this cohort was selected to include only patients with sufficiently long survival times and did not take into account differences in cytogenetic therapy, such as stem cell transplantation, or previous treatments or disease stage. Therefore, a subgroup analysis of patients after allogeneic stem cell transplantation suggested the importance of antigen-specific T cell responses for immune surveillance, although statistical significance was not observed (Figure 14D).

Claims

1. A pharmaceutical composition for the diagnosis, prevention and / or treatment of hematological tumors, comprising: at least one peptide that binds to a major histocompatibility complex class II molecule (MHC class II molecule) and / or induces T cell cross-reactivity; and a pharmaceutically acceptable carrier; the at least one peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, and variant sequences thereof having at least 88% homology to SEQ ID NO: 1 to SEQ ID NO: 15; the peptide is not a full-length polypeptide; Pharmaceutical compositions.

2. at least two peptides, preferably at least three peptides, more preferably at least four peptides, even more preferably at least five peptides, even more preferably at least six peptides, even more preferably at least seven peptides, even more preferably at least eight peptides, even more preferably at least nine peptides, and very preferably at least ten peptides; each peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 15, and variant sequences thereof having at least 88% homology to SEQ ID NO: 1 to SEQ ID NO: 15; the peptide is not a full-length polypeptide; The pharmaceutical composition of claim 1.

3. KLKKMWKSPNGTIQNILGGTVF (SEQ ID NO: 1), DRVKLGTDYRLHLSPV (SEQ ID NO: 2), ETLHKFASKPASEFVK (SEQ ID NO: 3), PHRKKKPFIEKKKAVSFHLVHR (SEQ ID NO: 4), SPGPFPFIQDNISFYA (SEQ ID NO: 5), IGSYIERDVTPAIM (SEQ ID NO: 6), SKPGVIFLTKKGRRF (SEQ ID NO: 7), DRQQMEALTRYLRAAL (SEQ ID NO: 8), GNQLFRINEANQLMQ (SEQ ID NO: 9), LGQEVALNANTKNQKIR (SEQ ID NO: 10), NGRTFHLTRTLTVK (SEQ ID NO: 11), LDTMRQIQVFEDEPAR (SEQ ID NO: 12), VVGYALDYNEYFRDL (SEQ ID NO: 13), KHLHYWFVESQKDPEN (SEQ ID NO: 14), and ERPEWIHVDSRPF (SEQ ID NO: 15) 3. The pharmaceutical composition according to claim 1, comprising different types of peptides comprising any one of the amino acid sequences:

4. at least one additional peptide that binds to a major histocompatibility complex class I molecule (MHC class I molecule) and / or induces T cell cross-reactivity; the at least one additional peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 32, and variant sequences thereof having at least 88% homology to SEQ ID NO: 16 to SEQ ID NO: 32; the peptide is not a full-length polypeptide; 10. A pharmaceutical composition according to any one of the preceding claims.

5. The at least one additional peptide is selected according to the MHC class I allotype of the individual to be treated, preferably the at least one additional peptide is selected according to the MHC class I allotype of the individual to be treated, Table 1 are selected according to The pharmaceutical composition of claim 4 , wherein the amino acid sequences of each MHC class I allotype group comprise variant sequences having at least 88% homology.

6. at least two additional peptides, preferably at least three additional peptides, more preferably at least four additional peptides, even more preferably at least five additional peptides, and most preferably at least six additional peptides; each additional peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16 to SEQ ID NO: 32, and variant sequences thereof having at least 88% homology to SEQ ID NO: 16 to SEQ ID NO: 32; the additional peptide is not a full-length polypeptide; The pharmaceutical composition according to claim 4 or 5.

7. 10. The pharmaceutical composition according to any one of the preceding claims, which is a vaccine, preferably a vaccine against hematological malignancies, more preferably a vaccine against acute myeloid leukemia (AML).

8. 10. A pharmaceutical composition according to any one of the preceding claims, further comprising an adjuvant, preferably XS15, more preferably XS15 dissolved in montanide.

9. Peptides and pharmaceutically acceptable salts thereof, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 32, and variant sequences thereof having at least 88% homology to SEQ ID NO: 1 to SEQ ID NO: 32; the variant sequence binds to a major histocompatibility complex (MHC) molecule and / or induces T cell cross-reactivity; the peptide is not a full-length polypeptide, Preferably, the peptide has the ability to bind to an MHC class I molecule or an MHC class II molecule, and when bound to an MHC class I molecule or an MHC class II molecule, can be recognized by CD4 T cells and / or CD8 T cells; More preferably, the amino acid sequence comprises a continuous amino acid sequence shown in any one of SEQ ID NOs: 1 to 32. Peptides and pharmaceutically acceptable salts thereof.

10. A nucleic acid encoding the peptide or a variant thereof according to claim 9, optionally linked to a heterologous promoter sequence, or an expression vector for expressing said nucleic acid.

11. 11. A recombinant host cell comprising a peptide according to claim 9 or a nucleic acid or expression vector according to claim 10, preferably selected from antigen presenting cells such as, for example, dendritic cells, T cells, NK cells.

12. 1. A method for producing activated T lymphocytes in vitro, comprising: contacting T cells in vitro with antigen-bearing human MHC class I or II molecules expressed on the surface of suitable antigen-presenting cells or on the surface of an artificial structure that mimics an antigen-presenting cell for a time sufficient to antigen-specifically activate the T cells; The method of claim 9, wherein the antigen is a peptide according to claim 9.

13. Activated T lymphocytes that selectively recognize cells presenting a polypeptide comprising the amino acid sequence of claim 9, produced by the method of claim 16.

14. A kit comprising: (a) a container containing the pharmaceutical composition according to any one of claims 1 to 8, the peptide according to claim 9, the nucleic acid or expression vector according to claim 10, the recombinant host cell according to claim 11, or the activated T lymphocyte according to claim 13 in a solution or a lyophilized formulation; (b) said freezing; and optionally a second container containing a diluent or reconstitution solution for the dry formulation; (c) optionally including instructions for (i) use of the solution or (ii) reconstitution and / or use of the lyophilized formulation; kit.

15. 1. A method for producing a personalized anti-cancer vaccine, comprising: a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; b) comparing the peptides identified in step a) with a warehouse of peptides that have been pre-screened for immunogenicity and / or over-representation in tumors compared to normal tissues; c) selecting from said warehouse at least one peptide that matches a TUMAP identified in said patient; and d) formulating a personalized vaccine based on step c). Including, 10. The method of claim 9, wherein the warehouse comprises a plurality of peptide and / or variant sequences according to claim 9.

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