TW202229312A - Amidated peptides and their deamidated counterparts displayed by non-hla-a*02 for use in immunotherapy against different types of cancers - Google Patents

Abstract

The invention relates to a peptide comprising an amino acid sequence selected from the group consisting of (i) SEQ ID NO: 1 to SEQ ID NO: 113, and (ii) a variant sequence thereof which maintains capacity to bind to MHC molecule(s) and/or induce T cells cross-reacting with said variant peptide, or a pharmaceutically acceptable salt thereof.

Description

Aminated peptides and their deamidated counterparts revealed by non-HLA-A*02 for immunotherapy of different types of cancer

Refer to the Sequence Listing submitted as a compliant ASCII text file (.txt)

In accordance with the EFS-Web Legal Framework and 37 CFR §§1.821-825 (see MPEP §2442.03(a)), Rule 30 EPC, and §11 PatV, filed concurrently with this application in an ASCII-compliant document file (titled "" Sequence_listing_2912919-105001_ST25", created on July 19, 2021 and having a size of 43,501 bytes), an electronic sequence listing compliant with WIPO Standard ST.25, the entire contents of which are incorporated herein by reference. For the avoidance of doubt, if there is a discrepancy between the sequences mentioned in the specification and the electronic sequence listing, the sequence in the specification should be considered the correct sequence.

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapy methods. In particular, the present invention relates to immunotherapy of cancer. Furthermore, the present invention relates to tumor-associated T-cell peptide epitopes alone or in combination with other tumor-associated peptides, which peptides may, for example, serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor Tumor immune response, or ex vivo stimulation of T cells and their transfer into patients. Peptides or such peptides that bind to molecules of the major histocompatibility complex (MHC) can also be targets for antibodies, soluble T cell receptors, and other binding molecules.

The present invention relates to several novel peptide sequences of HLA class I molecules derived from human tumor cells and their variants, which can be used in vaccine compositions for eliciting anti-tumor immune responses, or for developing pharmaceutically active/immunoactive compounds and cellular targets.

According to the World Health Organization (WHO), in 2012 the four leading non-communicable diseases causing death worldwide involved cancer. In the same year, colorectal, breast and respiratory cancers were included in the top 10 causes of death in high-income countries.

Considering the serious side effects and costs associated with treating cancer, there is a need to identify factors that can be used to treat cancer.

There is also a need to identify biomarkers indicative of cancer, factors that lead to better cancer diagnosis, prognostic assessment, and prediction of treatment success. Cancer Immunotherapy

Immunotherapy of cancer represents an option for specific targeting of cancer cells while minimizing side effects. Cancer immunotherapy exploits the presence of tumor-associated antigens.

The current classification of tumor associated antigens (TAAs) includes the following main groups:

a) Cancer-testis antigens: The first TAAs ever identified that can be recognized by T cells belong to this category and were originally called cancer-testis (CT) antigens. Because cells of the testis do not express class I and class II HLA molecules, these antigens are not recognized by T cells in normal tissues and can therefore be considered immunologically tumor specific. Well-known examples of CT antigens are MAGE family members and NY-ESO-1.

b) Differentiation antigens: These TAA lines are shared between the tumor and the normal tissue in which the tumor appears. Most of the known differentiation antigen lines are found in melanoma and normal melanocytes. Examples include, but are not limited to, tyrosinase for melanoma and Melan-A/MART-1 or PSA for prostate cancer.

c) Overexpressed TAAs: Genes encoding widely expressed TAAs have been detected in histologically different types of tumors as well as in many normal tissues, often with lower expression levels. It is possible that, by normal tissue processing and possibly presenting many epitopes below threshold levels for T cell recognition, their overexpression in tumor cells can trigger anticancer by disrupting previously established tolerance reaction. Prominent examples of TAAs of this class are Her-2/neu, survivin, telomerase, or WT1.

d) Tumor specific antigens: These unique TAAs result from mutations in normal genes (such as beta-catenin, CDK4, etc.). Some of these molecular changes are associated with neoplastic transformation and/or progression. Tumor-specific antigens are often able to induce strong immune responses without the risk of normal tissue autoimmune responses. On the other hand, these TAAs are in most cases only associated with the exact tumor in which they were identified and are not shared among many individual tumors. Tumor specificity (or association) of a peptide may also occur if, in the case of a protein with a tumor-specific (association) isoform, the peptide is derived from a tumor-specific (association) exon.

e) Oncogenic viral proteins: These TAA-based viral proteins can play a key role in the carcinogenesis process and because they are foreign (not of human origin), they can evoke a T cell response. Examples of such proteins are the human papilloma type 16 virus proteins, E6 and E7, all expressed in cervical carcinomas. Human endogenous retroviruses (HERVs) make up a significant portion (about 8%) of the human genome. These viral elements were integrated into the genome millions of years ago and have been passed vertically through generations since then. The vast majority of HERVs have lost functional activity through mutation or truncation, while some endogenous retroviruses such as members of the HERV-K clade still encode functional genes and have been shown to form retroviral-like particles. Transcription of the HERV provirus is epigenetically controlled and silent under normal physiological conditions. However, reactivation and overexpression leading to active translation of viral proteins have been described in certain diseases and especially for different types of cancer. This tumor-specific expression of HERV-derived proteins can be manipulated for different types of cancer immunotherapy.

f) TAAs produced by aberrant post-translational modifications: Such TAAs can be produced by proteins that are neither specific nor overexpressed in tumors, but become tumor-associated by post-translational processing that is primarily active in tumors. Examples of this class arise from altered glycosylation patterns that in tumors result in novel epitopes for MUCl or events like protein splicing during degradation, which may or may not be tumor specific.

T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins, which are presented by MHC molecules. Antigens recognized by tumor-specific T lymphocytes, i.e., their epitopes may be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc., which are expressed in the cells of the corresponding tumor and are related to each other. Typically upregulated in cells of the corresponding tumor compared to unchanged cells of the same origin.

There are two types of MHC molecules, MHC class I and MHC class II. MHC class I molecules consist of alpha heavy chains and beta-2-microglobulin, and MHC class II molecules consist of alpha and beta chains. Its three-dimensional configuration results in a binding groove for non-covalent interactions with the peptide.

MHC class I molecules can be found on most nucleated cells. These molecules represent peptides resulting from proteolytic cleavage of major endogenous proteins, defective ribosomal products (DRIPs), and larger peptides. However, peptides derived from the endosome compartment or from exogenous sources are also frequently found on MHC class I molecules. This non-classical way of class I presentation is referred to in the literature as cross-presentation (Brossart and Bevan, 1997; Rock et al., 1990). MHC class II molecules can be found primarily on specialized antigen presenting cells (APCs) and present primarily peptides of exogenous or transmembrane proteins, such as those taken up by APCs during endocytosis and subsequently processed.

The complex of peptide and MHC class I is recognized by CD8 positive T cells with appropriate T cell receptor (TCR), while the complex of peptide and MHC class II molecule is recognized by CD4 positive with appropriate TCR Helper T cell recognition. It is well known that TCR, peptide and MHC are thereby present in stoichiometric amounts of 1:1:1.

CD4-positive helper T cells play an important role in inducing and sustaining effective responses by CD8-positive cytotoxic T cells. The identification of CD4-positive T cell epitopes derived from tumor associated antigens (TAAs) is of great importance for the development of medicinal products for triggering anti-tumor immune responses. At tumor sites, T helper cells support a cytotoxic T cell (CTL)-friendly interferon environment and attract effector cells such as CTLs, natural killer (NK) cells, macrophages, and granular leukocytes.

In the absence of inflammation, the expression of MHC class II molecules is primarily restricted to cells of the immune system, especially specialized antigen-presenting cells (APCs), e.g., monocytes, monocyte-derived cells, macrophages, dendritic cells. In cancer patients, tumor cells have been found to express MHC class II molecules (Dengjel et al., 2006).

The elongated (longer) peptides of the invention can serve as MHC class II active epitopes.

T helper cells activated by MHC class II epitopes play an important role in setting the effector function of CTLs in antitumor immunity. T helper epitopes that trigger TH1-type T helper cell responses support effector functions of CD8-positive killer T cells, including cytotoxic function against tumor cells that display tumor-associated peptide-MHC complexes on their cell surfaces. In this way, tumor-associated T helper peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses.

Because the constitutive expression of HLA class II molecules is usually restricted to immune cells, the possibility of directly isolating class II peptides from primary tumors was previously considered impossible. However, Dengjel et al. succeeded in identifying a large number of MHC class II epitopes directly from tumors (WO 2007/028574, EP 1 760 088 B1, the contents of which are hereby incorporated by reference in their entirety).

For an MHC class I peptide to trigger (elicit) a cellular immune response, it must also bind to an MHC molecule. This process depends on the alleles of the MHC molecule and the specific homopolymorphism of the amino acid sequence of the peptide. MHC class I binding peptides are typically 8-12 amino acid residues in length and typically contain two conserved residues ("anchors") in their sequence that interact with the corresponding binding grooves of the MHC molecule. In this way, each MHC allele has a "binding motif" that determines which peptides can specifically bind to the binding groove.

In an MHC class I-dependent immune response, not only must the peptide be able to bind to certain MHC class I molecules expressed by tumor cells, but it must also subsequently be recognized by T cells bearing specific TCRs.

For proteins to be recognized by T lymphocytes as tumor-specific or associated antigens and to be used in therapy, certain prerequisites must be met. Antigens should be expressed primarily by tumor cells rather than by normal healthy tissue, or by normal healthy tissue in relatively small amounts. It may be advantageous that the peptide is overrepresented by tumor cells compared to normal healthy tissue. Furthermore, it is desirable that the corresponding antigens are present not only in one type of tumor, but also in high concentrations. Tumor-specific and tumor-associated antigens are often derived from proteins that are directly involved in the transition of normal cells to tumor cells due to functions of the cells, such as the control or inhibition of apoptosis in the cell cycle. In addition, downstream targets of the protein directly caused by the transition can be up-regulated and thus can be tumor-associated indirectly. Such indirect tumor-associated antigens can also be targets for vaccination approaches (Singh-Jasuja et al., 2004, the contents of which are incorporated herein by reference in their entirety). Essentially, the epitope is present in the amino acid sequence of the antigen in order to ensure that such peptides ("immune peptides") derived from tumor-associated antigens elicit T cell responses in vitro or in vivo.

TAA can be a starting point for the development of T cell-based immunotherapy. Methods for identifying and characterizing TAAs are typically based on the use of T cells that can be isolated from patients or healthy subjects, or they are based on generating differential transcriptional profiles or differential peptide expression patterns between tumor and normal tissue. However, identifying genes that are overexpressed in tumor tissues or human tumor cell lines or selectively expressed in such tissues or cell lines will not provide precise information on the use of antigens transcribed from these genes in immunotherapy. This is because only individual subsets of epitopes of these antigens are suitable for such applications, since T cells with the corresponding TCR must be present and immune tolerance to this particular epitope must be absent or minimal.

In the case of targeting peptide-MHCs by specific TCRs (eg soluble TCRs) and antibodies or other binding molecules (scaffolds) according to the invention, the immunogenicity of the underlying peptide is secondary. In these cases, presentation is the determining factor. Deglycosylation and subsequent deamidation to produce tumor-associated peptides

TAA may not only be produced by proteins that are specific or overexpressed for tumors. It can also result from aberrant post-translational modification (PTM) of proteins that are neither specific nor overrepresented in tumors: however, such TAAs can be produced by post-translational processing that is primarily active in tumors. become tumor-associated.

While post-translational modifications alter and prolong the immune profile of the immunopeptidome, they have diverse functions and consequences. Some PTMs hinder the binding of modified peptides to HLA complexes (Andersen et al., 1999), which may contribute to the immune avoidance strategy of tumor cells. Other modifications lead to higher HLA affinity or increased immunogenicity, which has been associated with autoimmune diseases (Arentz-Hansen et al, 2000; McGinty et al, 2015; Sidney et al, 2018; Raposo et al, 2018 ), but could be developed for immunotherapy in malignancies (Zarling et al., 2006; Purcell et al., 2007; Petersen et al., 2009; Cobbold et al., 2013; Marcilla et al., 2014; Lin et al., 2019; Brentville et al., 2020).

Previous PTM analyses identified deamidation as a fairly common modification of HLA I-presented immune peptides (Han et al., 2011; Mei et al., 2020). This chemical reaction results in an amino acid conversion from aspartic acid (N) to aspartic acid (D) (Knorre, Kudryashova and Godovikova 2009; Mei et al., 2020).

N deamidation is enriched in peptides derived from membrane-associated proteins and is associated with the sequence motif N[X^P][ST], where X is any amino acid except proline and This is followed by serine (S) or threonine (T) (Han et al., 2011; Cao et al., 2017; Mei et al., 2020). This motif is an established recognition motif for N-glycosyltransferases (Yan and Lennarz 2005; Petersen, Purcell and Rossjohn 2009), which links N-deamidation to nascent in the endoplasmic reticulum (ER) Glycosylation. Mechanistically, a protein or polypeptide becomes glycosylated during its translation in the ER. After its export to the cytoplasm, it becomes deglycosylated by peptide-N-glycanase (PNGase). During this hydrolytic degradation process, the N residue is also deaminated to D, resulting in a terminal change in the amino acid sequence. After further degradation of the protein or polypeptide in the proteasome, the peptide is transported again to the ER. At this point, it binds to the HLA complex, translocates to the cell membrane and presents a deamidated peptide on the cell surface (see Figure 1) (Misaghi et al., 2004; Petersen, Purcell and Rossjohn 2009; Mei et al., 2020 ). This mechanism potentially allows T cells to recognize and eliminate cells with perturbed glycosylation and produced by altered tumor cell metabolism during infection.

Previous PTM analyses identified deamidation as a fairly common modification of HLA I-presented immune peptides (Han et al., 2011; Mei et al., 2020). This chemical reaction results in an amino acid conversion from aspartic acid (N) to aspartic acid (D) (see Figure 2) (Knorre, Kudryashova, and Godovikova 2009; Mei et al., 2020).

Although aberrant glycosylation in cancer was initially associated with immunosuppressive effects (Liu and Rabinovich 2005; Rodriguez et al, 2018; De Bousser et al, 2020), accumulating evidence suggests that it could also be developed for T cell-based therapy . Thus, glycoproteins themselves (Posey et al., 2016; Maher et al., 2016; RodrIguez, Schetters and van Kooyk 2018; De Bousser et al., 2020) or glycosylation-dependent deamidation peptides act as neoantigens and can be targeted by target. There are some well-described examples of the immunogenic role of deamidated peptides in the literature examining the pathogen human immunodeficiency virus type 1 envelope glycoprotein (GP) (Behrens et al., 2017; Ferris et al, 1999), hepatitis C GP E1 (Selby et al, 1999) and lymphocytic choriomeningitis virus GP 1 (Hudrisier et al, 1999) peptides, and tyrosinase peptides in melanoma (Mosse et al, 1998; Schaed et al, 2002; Altrich-VanLith et al, 2006; Ostankovitch et al, 2009).

Thus, the atypical pathways of deamidation peptides and antigen presentation described above serve as interesting targets for T cell-based tumor immunotherapy.

In a first aspect, the present invention relates to a peptide, or a pharmaceutically acceptable salt thereof, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO : 113; and variant sequences thereof that bind to MHC molecules and/or induce T cells to cross-react with the variant peptide.

The following tables (Table 1A and Table 2) show the peptides according to the invention, their corresponding SEQ ID NOs, and the expected source (basic) genes of these peptides.

Table 1A: Peptides according to the invention. Note that the two adjacent peptides in each row are the wild-type sequence containing a possible N-glycosylation motif (right column), and the mutant version lacking this N-glycosylation motif (left column), where the N(Asn) residue has been replaced by its deamidated variant D(Asp). SEQ ID NO Deamidation sequence SEQ ID NO WT sequence 1 VHDFTLPSW 114 VHNFTLPSW 2 FFQDSTFSF 115 FFQNSTFSF 3 IVRDLSCRK 116 IVRNLSCRK 4 YIDDVTLI 117 YIDNVTLI 5 GYIDDVTLI 118 GYIDNVTLI 6 ISDITEKNSGLY 119 ISNITEKNSGLY 7 VTRDDTASY 120 VTRNDTASY 8 AQDTTYLWW 121 AQNTTYLWW 9 IFDETGRF 122 IFNETGRF 10 QVDGSLLVI 123 QVNGSLLVI 11 NHITDTSLNLF 124 NHITNTSLNLF 12 ITDTSLNLF 125 ITNTSLNLF 13 TANYDTSHY 126 TANYNTSHY 14 WSDWSNPAY 127 WSNWSNPAY 15 TEGDFTKEASTY 128 TEGNFTKEASTY 16 VTQDDTGFY 129 VTQNDTGFY 17 DILDRTGHQL 130 DILNRTGHQL 18 GTDKQDSTLRY 131 GTDKQNSTLRY 19 MTDVDRDGTTAY 132 MTDVDRNGTTAY 20 TSDTSQYDTY 133 TSNTSQYDTY twenty one APFKDVTEY 134 APFKNVTEY twenty two NLYDWSASY 135 NLYNWSASY twenty three SYDETKIKF 136 SYNETKIKF twenty four FYDNSVIIF 137 FYNNSVIIF 25 FTDLITDESINY 138 FTDLITNESINY 26 IYPDASLLIQNI 139 IYPNASLLIQNI 27 DEAVRDITW 140 DEAVRNITW 28 PSDLSVFTSY 141 PSNLSVFTSY 29 RLWDFTMNAK 142 RLWNFTMNAK 30 IYNFRLWDF 143 IYNFRLWNF 31 VQPDSSYTY 144 VQPNSSYTY 32 RDATASLW 145 RNATASLW 33 ISDGMDSSAHY 146 ISDGMNSSAHY 34 LSDLSLADI 147 LSNLSLADI 35 VFHDHTYHL 148 VFHNHTYHL 36 YWDETLKEF 149 YWNETLKEF 37 RSLDCTVKTY 150 RSLNCTVKTY 38 KLTDNSNQF 151 KLTNNSNQF 39 LPFFTDKTLSF 152 LPFFTNKTLSF 40 LSDLTCNNY 153 LSNLTCNNY 41 NYLLYVSDF 154 NYLLYVSNF 42 AERDLDVTI 155 AERDLNVTI 43 FFTDKTLSF 156 FFTNKTLSF 44 KENQDHSYSL 157 KENQNHSYSL 45 YFVDVTTRI 158 YFVNVTTRI 46 KEVDDTLLVNEL 159 KEVNDTLLVNEL 47 RLPAADFTRY 160 RLPAANFTRY 48 FPYYLKIDY 161 FPYYLKINY 49 PSDGSMHNY 162 PSNGSMHNY 50 RSIDVTGQGF 163 RSINVTGQGF 51 RVDDITDQF 164 RVDNITDQF 52 LTEVEKDATALY 165 LTEVEKNATALY 53 SLIDITHGF 166 SLNITHGF 54 QYQDTTVSF 167 QYQNTTVSF 55 VYTDISHHF 168 VYTNISHHF 56 IYLDRTLLTTI 169 IYLNRTLLTTI 57 FYDLSIQSF 170 FYNLSIQSF 58 GTDQTGKGLEY 171 GTNQTGKGLEY 59 ILFSDSTRLSF 172 ILFSNSTRLSF 60 HVKDATMGY 173 HVKNATMGY 61 KAYDQTHLY 174 KAYNQTHLY 62 HPDLTSMTF 175 HPNLTSMTF 63 HLYYDVTEK 176 HLYYNVTEK 64 FHYDDTAGYF 177 FHYNDTAGYF 65 IYQFARLDY 178 IYQFARLNY 66 YHDQTISF 179 YHNQTISF 67 QAIDLSLNF 180 QAINLSLNF 68 VFDETKNLL 181 VFNETKNLL 69 KSYHDQTISF 182 KSYHNQTISF 70 HPFGYDLTL 183 HPFGYNLTL 71 VPRNQDESV 184 VPRNQNESV 72 DVSDKITFM 185 NVSDKITFM 73 DESKYTWSW 186 NESKYTWSW 74 LENMYDLTF 187 LENMYNLTF 75 QEVDISLHY 188 QEVNISLHY 76 TYLPTDASLSF 189 TYLPTNASLSF 77 KPREEQYDSTY 190 KPREEQYNSTY 78 DETIWYVRF 191 NETIWYVRF 79 FTDTSSYEY 192 FTNTSSYEY 80 LTNDQTLRL 193 LTNNQTLRL 81 VTETMGIDGSAY 194 VTETMGINGSAY 82 VTDVTEEHY 195 VTNVTEEHY 83 TFVDASRTLY 196 TFVNASRTLY 84 EVEGVIDGTYDY 197 EVEGVINGTYDY 85 EVQDHSTSSY 198 EVQNHSTSSY 86 ILPDITTTY 199 ILPNITTTY 87 ITDDTVQTY 200 ITNDTVQTY 88 WLDRSTILY 201 WLNRSTILY 89 HVSDVTVNY 202 HVSNVTVNY 90 HVDNSNLNY 203 HVNNSNLNY 91 GVDDTSLLY 204 GVNDTSLLY 92 GQYDDSLQAY 205 GQYNDSLQAY 93 HADLTTLTF 206 HANLTTLTF 94 YSIDVTNVM 207 YSINVTNVM 95 VYIDDSVEL 208 VYINDSVEL 96 IFVPTDRSL 209 IFVPTNRSL 97 RYVNDYTNSF 210 RYVNNYTNSF 98 AFDKTIVKL 211 AFNKTIVKL 99 VYVDTTELAL 212 VYVNTTELAL 100 EYQDFSTLF 213 EYQNFSTLF 101 VYLDASKVPGF 214 VYLNASKVPGF 102 IYPDGTLLI 215 IYPNGTLLI 103 RQDESYLNF 216 RQNESYLNF 104 HLFYDVTVF 217 HLFYNVTVF 105 NPADISVAL 218 NPANISVAL 106 SPKIFDSSW 219 SPKIFNSSW 107 GEPTSDITLL 220 GEPTSNITLL 108 DETHTLQF 221 NETHTLQF 109 DETAAYKIM 222 NETAAYKIM 110 AESLAVHDI 223 AESLAVHNI 111 GEYRCQTDL 224 GEYRCQTNL 112 AEFFDYTVRTL 225 AEFFNYTVRTL 113 TDFTKIASF 226 TNFTKIASF

Table IB: Peptides according to the invention. SEQ ID NO sequence 227 ELAGIGILTV 228 YLLPAIVHI

Table 2A: Non-deamidated peptides according to the invention and an exemplary source transcript ID from which the peptides were derived (ENST ID obtained from the ensemble database https://www.ensembl.org/), however , peptides may otherwise originate from other additional or alternative transcripts not listed herein. It is important to understand that while the source is indicated for the wild-type variant, it applies equally to the deaminated counterpart as shown in the summary of Table 1A. SEQ ID NO WT sequence Transcript ID 114 VHNFTLPSW ENST00000336375p218 115 FFQNSTFSF ENST00000377028p339 116 IVRNLSCRK ENST00000357639p423 117 YIDNVTLI ENST00000264144p359 118 GYIDNVTLI ENST00000264144p358 119 ISNITEKNSGLY ENST00000221992p464 120 VTRNDTASY ENST00000221992p205 121 AQNTTYLWW ENST00000221992p527 122 IFNETGRF ENST00000261875p303 123 QVNGSLLVI ENST00000296980p169 124 NHITNTSLNLF ENST00000261465p119 125 ITNTSLNLF ENST00000261465p121 126 TANYNTSHY ENST00000282903p539 127 WSNWSNPAY ENST00000344610p622 128 TEGNFTKEASTY ENST00000397910p136 129 VTQNDTGFY ENST00000161559p112 130 DILNRTGHQL ENST00000347842p291 131 GTDKQNSTLRY ENST00000271588p688 132 MTDVDRNGTTAY ENST00000415148p301 133 TSNTSQYDTY ENST00000263398p108 134 APFKNVTEY ENST00000268035p530 135 NLYNWSASY ENST00000233242p1365 136 SYNETKIKF ENST00000233242p3222 137 FYNNSVIIF ENST00000328668p286 138 FTDLITNESINY ENST00000260128p191 139 IYPNASLLIQNI ENST00000221992p101 140 DEAVRNITW ENST00000328668p271 141 PSNLSVFTSY ENST00000266674p61 142 RLWNFTMNAK ENST00000230173p311 143 IYNFRLWNF ENST00000230173p307 144 VQPNSSYTY ENST00000367796p1705 145 RNATASLW ENST00000356082p603 146 ISDGMNSSAHY ENST00000056233p291 147 LSNLSLADI ENST00000456448p81 148 VFHNHTYHL ENST00000056233p494 149 YWNETLKEF ENST00000377905p88 150 RSLNCTVKTY ENST00000344644p68 151 KLTNNSNQF ENST00000302347p209 152 LPFFTNKTLSF ENST00000217939p1730 153 LSNLTCNNY ENST00000349496p424 154 NYLLYVSNF ENST00000296474p481 155 AERDLNVTI ENST00000259056p212 156 FFTNKTLSF ENST00000217939p1732 157 KENQNHSYSL ENST00000261023p941 158 YFVNVTTRI ENST00000296585p1071 159 KEVNDTLLVNEL ENST00000379742p596 160 RLPAANFTRY ENST00000296795p65 161 FPYYLKINY ENST00000312265p95 162 PSNGSMHNY ENST00000324300p60 163 RSINVTGQGF ENST00000359337p999 164 RVDNITDQF ENST00000269228p958 165 LTEVEKNATALY ENST00000371994p236 166 SLNITHGF ENST00000375001p804 167 QYQNTTVSF ENST00000343028p72 168 VYTNISHHF ENST00000005995p270 169 IYLNRTLLTTI ENST00000262738p1284 170 FYNLSIQSF ENST00000260118p114 171 GTNQTGKGLEY ENST00000295550p114 172 ILFSNSTRLSF ENST00000287097p114 173 HVKNATMGY ENST00000296641p79 174 KAYNQTHLY ENST00000265362p122 175 HPNLTSMTF ENST00000231368p182 176 HLYYNVTEK ENST00000236040p536 177 FHYNDTAGYF ENST00000264868p356 178 IYQFARLNY ENST00000269217p886 179 YHNQTISF ENST00000264868p345 180 QAINLSLNF ENST00000256447p198 181 VFNETKNLL ENST00000329608p152 182 KSYHNQTISF ENST00000264868p343 183 HPFGYNLTL ENST00000441802p328 184 VPRNQNESV ENST00000264868p487 185 NVSDKITFM ENST00000243611p98 186 NESKYTWSW ENST00000357637p128 187 LENMYNLTF ENST00000339381p427 188 QEVNISLHY ENST00000281834p111 189 TYLPTNASLSF ENST00000269571p63 190 KPREEQYNSTY ENST00000390548p173 191 NETIWYVRF ENST00000425642p70 192 FTNTSSYEY ENST00000264833p397 193 LTNNQTLRL ENST00000372838p150 194 VTETMGINGSAY ENST00000269228p1065 195 VTNVTEEHY ENST00000333617p261 196 TFVNASRTLY ENST00000278407p235 197 EVEGVINGTYDY ENST00000397676p77 198 EVQNHSTSSY ENST00000440542p86 199 ILPNITTTY ENST00000337233p205 200 ITNDTVQTY ENST00000323853p993 201 WLNRSTILY ENST00000331898p68 202 HVSNVTVNY ENST00000262304p1001 203 HVNNSNLNY ENST00000265993p181 204 GVNDTSLLY ENST00000366794p978 205 GQYNDSLQAY ENST00000280800p107 206 HANLTTLTF ENST00000296754p68 207 YSINVTNVM ENST00000369939p573 208 VYINDSVEL ENST00000310325p375 209 IFVPTNRSL ENST00000321725p1165 210 RYVNNYTNSF ENST00000341846p449 211 AFNKTIVKL ENST00000263688p953 212 VYVNTTELAL ENST00000233838p602 213 EYQNFSTLF ENST00000379442p5194 214 VYLNASKVPGF ENST00000393203p410 215 IYPNGTLLI ENST00000006724p102 216 RQNESYLNF ENST00000378588p147 217 HLFYNVTVF ENST00000251582p108 218 NPANISVAL ENST00000227880p353 219 SPKIFNSSW ENST00000449174p135 220 GEPTSNITLL ENST00000361127p116 221 NETHTLQF ENST00000263087p954 222 NETAAYKIM ENST00000295770p641 223 AESLAVHNI ENST00000378588p233 224 GEYRCQTNL ENST00000294800p85 225 AEFFNYTVRTL ENST00000223127p59 226 TNFTKIASF ENST00000240487p203

Table 2B: Peptides according to the invention and one exemplary source transcript ID from which the peptides are derived (ENST ID obtained from the ensemble database https://www.ensembl.org/), however, the peptides may be of additional origin Additional or alternative transcripts not listed herein. SEQ ID NO sequence Transcript ID 227 ELAGIGILTV ENST00000381471p26 228 YLLPAIVHI ENST00000225792p148

The present invention furthermore generally relates to the peptides according to the invention for use in the treatment of proliferative diseases. Proliferative diseases in this context are, for example, acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastro-esophageal junction cancer, hepatocellular carcinoma, Head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and uterine cancer Endometrial cancer.

Especially preferred are peptides, alone or in combination, selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 113.

Therefore, another aspect of the present invention relates to the use of the peptides according to the present invention for the treatment (preferably combined treatment) of proliferative diseases.

The present invention furthermore relates to peptides according to the invention, which have the ability to bind to MHC molecule class I or - in elongated form, such as length variants - MHC class II.

The present invention further relates to elongated peptides which, after administration (eg, as a vaccine), can be processed intracellularly to produce shorter peptides, which consist or consist essentially of according to SEQ ID NO: 1 to SEQ ID NO: 113 Consisting of the amino acid sequences of these shorter peptides, these shorter peptides are then presented by HLA on the cell surface.

The present invention further relates to peptides according to the invention, wherein the peptides (each) consist or consist essentially of the amino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 113.

The present invention further relates to a peptide according to the invention, wherein the peptide comprises a non-peptide bond.

The invention further relates to a peptide according to the invention, wherein the peptide is part of a fusion protein, in particular fused to the N-terminal amino acid of the invariant chain (Ii) associated with the HLA-DR antigen or to an antibody (or to in the sequence of an antibody) such as, for example, an antibody specific for dendritic cells.

The present invention further relates to nucleic acids encoding the peptides according to the invention. The present invention further relates to a nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA or a combination thereof.

The present invention further relates to expression vectors capable of expressing and/or expressing nucleic acids according to the invention.

The present invention further relates to peptides according to the invention, nucleic acids according to the invention or expression vectors according to the invention for use in the treatment of diseases and in medicine, in particular for the treatment of cancer.

The present invention further relates to antibodies specific for peptides according to the invention or complexes of such peptides and MHC according to the invention and methods of making such antibodies.

The present disclosure may also relate to methods of producing antibodies that specifically bind to MHC class I molecules complexed with a peptide comprising, consisting of, or consisting essentially of the amino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 113, The method comprises: using a soluble form of the MHC class I molecule complexed with a peptide consisting of, or consisting essentially of, the amino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 113, causing cells containing the MHC class I molecule to express the MHC class I molecule immunization of a genetically engineered non-human mammal; isolation of mRNA molecules from antibody-producing cells of the non-human mammal; production of a phage-revealed library that exposes protein molecules encoded by the mRNA molecules; and isolation from the phage-revealed library of at least A bacteriophage, wherein the at least one bacteriophage exhibits the antibody that specifically binds to the MHC class I molecule complexed with, consisting of, or consisting essentially of a peptide according to the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 113 . In another aspect, the antibody can be a monoclonal antibody.

In one aspect, the antibody may have a binding affinity ( _Kd ) Binds to the MHC class I molecule complexed with an antigen comprising, consisting of, or consisting essentially of the amino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 113.

In another aspect, the method of producing an antibody can further comprise humanizing the antibody. In aspects, the method of producing an antibody can further comprise conjugating the antibody to a toxin. In another aspect, the method of producing an antibody can further comprise conjugating the antibody to an immunostimulatory domain.

In one aspect, the method of producing an antibody may further comprise modifying the antibody in the form of a bispecific antibody. In another aspect, the method of producing an antibody can further comprise modifying the antibody in the form of a chimeric antibody. In another aspect, the method of producing an antibody can further comprise modifying the antibody in Fv form. In one aspect, the method of producing an antibody can further comprise modifying the antibody in Fab form. In another aspect, the method of producing an antibody can further comprise modifying the antibody in Fab' form. In another aspect, the method of producing an antibody can further comprise labeling the antibody with a radionucleotide selected from the group consisting of: ^111In , ^99Tc , ^14C , ^131I , ^3H , ^32P , and ^35S . In another aspect, the non-human mammal can be a mouse.

The present invention further relates to TCRs engineered into autologous or allogeneic T cells, especially soluble TCRs and cloned TCRs, and methods of making these TCRs, and carrying or cross-reacting with the TCRs NK cells or other cells. The soluble TCR may have a binding affinity ( _Kd ) of <100 nM, more preferably <50 nM, more preferably <10 nM, more preferably <1 nM, more preferably <0.1 nM, more preferably <0.01 nM . Whereas the colonized cell based TCR may have a binding affinity ( _Kd ) of <50 μM, more preferably <25 μM, more preferably <10 μM, more preferably <1 μM, more preferably <0.1 μM.

Antibodies and TCRs are further examples of current immunotherapeutic uses of peptides according to the invention.

The present invention further relates to host cells comprising a nucleic acid according to the invention or an expression vector as hereinbefore described. The invention further relates to host cells according to the invention, which are antigen presenting cells and preferably dendritic cells.

The invention further relates to the method according to the invention, wherein the antigen is loaded into class I or class II expressed on the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell by contacting a sufficient amount of the antigen with the antigen-presenting cell on MHC molecules.

The invention further relates to the method according to the invention, wherein the antigen presenting cell comprises an expression vector capable of expressing the peptide comprising SEQ ID NO: 1 to SEQ ID NO: 113.

The invention further relates to activated T cells produced by the method according to the invention, wherein the T cells selectively recognize cells expressing a polypeptide comprising an amino acid sequence according to the invention.

The present invention further relates to a method of killing target cells in a patient that abnormally expresses a polypeptide comprising any amino acid sequence according to the present invention, the method comprising adding an effective amount of T cells as produced according to the present invention administered to the patient.

The invention further relates to any peptides as described, nucleic acids according to the invention, expression vectors according to the invention, cells according to the invention, activated T lymphocytes according to the invention, TCRs or antibodies or other peptides and/or peptides - Use of an MHC binding molecule as a medicament or in the manufacture of a medicament. Preferably, the agent is active against cancer.

Preferably, the agent is a soluble TCR or antibody-based cell therapy, vaccine or protein.

The invention further relates to the use according to the invention, wherein the cancers are acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastric- Esophageal junctional carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small Lung cancer, bladder cancer, and endometrial cancer, and preferably acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastric cancer -Esophageal junctional carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, Small cell lung cancer, bladder cancer, and endometrial cancer cells.

The present invention further relates to biomarkers based on the peptides according to the invention, referred to herein as "targets", which can be used to diagnose cancer, preferably acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colon Rectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovary cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer. A marker can be over-representation of the peptide itself, or over-representation of the corresponding gene. Markers can also be used to predict the probability of success of treatment, preferably immunotherapy, and immunotherapy that optimally targets the same target identified by the biomarker. For example, antibodies or soluble TCRs can be used to stain sections of tumors to detect the presence of peptides of interest in complexes with MHC.

Optionally, the antibody carries another effector function such as an immunostimulatory domain or toxin.

The present invention also relates to the use of these novel targets in the context of cancer therapy.

Stimulation of an immune response depends on the presence of antigens recognized as foreign by the host immune system. The discovery of the presence of tumor-associated antigens raises the possibility of using the host's immune system to interfere with tumor growth. Various mechanisms that control both the humoral and cellular arms of the immune system are now being explored for cancer immunotherapy.

Specific elements of cellular immune responses can specifically recognize and destroy tumor cells. Isolation of T cells from tumor-infiltrating cell populations or from peripheral blood suggests that such cells play an important role in innate immune defense against cancer. In particular, CD8-positive T cells play an important role in this response, and these CD8-positive T cells recognize MHC class I molecules with proteins derived from proteins located in the cytosol or defective ribosomal products (DRIPS). Peptides typically have 8 to 12 amino acid residues. Human MHC molecules are also known as human leukocyte-antigen (human leukocyte-antigen; HLA).

As used herein and unless otherwise stated, all terms are defined as given below.

The term "T cell response" means the specific proliferation and activation of effector functions induced by peptides in vitro or in vivo. For MHC class I-restricted cytotoxic T cells, the effector function can be peptide-pulsed target cells, peptide precursor-pulsed target cells, or lysis of native peptide-presenting target cells, by peptide-induced interferon (preferably interferon) - secretion of gamma, TNF-alpha, or IL-2), secretion by peptide-induced effector molecules (preferably granzyme or perforin), or degranulation.

The term "peptide" is used herein to designate a series of amino acid residues, typically one amino acid is linked to another by a peptide bond between the alpha amino group and the carboxyl group of adjacent amino acids .

In addition, the term "peptide" shall include salts of a series of amino acid residues, which are typically linked to one amino acid by a peptide bond between the alpha amino group and the carboxyl group of adjacent amino acids. linked to another amino acid. Preferably, the salt is a pharmaceutically acceptable salt of the peptide, such as, for example, the chloride or acetate (trifluoroacetate). It must be noted that the salts of the peptides according to the invention are substantially different from the peptides in their in vivo state, since the peptides are not in the form of salts or associated with relative ions in vivo.

The term "peptide" shall also include "oligopeptide". The term "oligopeptide" is used herein to designate a series of amino acid residues, typically one amino acid is linked to another by a peptide bond between the alpha amino group and the carboxyl group of adjacent amino acids acid. The length of the oligopeptide is not critical to the present invention as long as the correct epitope or epitopes are maintained in the oligopeptide.

The term "polypeptide" designates a series of amino acid residues, typically one amino acid is linked to another by a peptide bond between the alpha amino group and the carboxyl group of adjacent amino acids. The length of the polypeptide is not critical to the present invention as long as the correct epitope is maintained. In contrast to the terms peptide or oligopeptide, the term polypeptide is meant to refer to molecules containing more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide encoding such a molecule is "immune" (and therefore "immunogen" within the present invention) if it is capable of inducing an immune response. In the context of the present invention, immunogenicity is more specifically defined as the ability to induce a T cell response. Thus, an "immunogen" will be a molecule capable of inducing an immune response, and in the context of the present invention, a molecule capable of inducing a T cell response. In another aspect, the immunogen can be a peptide, a complex of a peptide and MHC, an oligopeptide, and/or a protein used to generate antibodies or TCRs specific thereto.

Class I T cell "epitopes" require short peptides that bind to MHC class I molecules to form a ternary complex (MHC class I alpha chain, beta-2-microglobulin, and a peptide), which The complex can be recognized by T cells with a matched T cell receptor that binds to the MHC-peptide complex with appropriate affinity. Peptides bound to MHC class I molecules are typically 8-12 amino acids in length, and most typically 9 amino acids in length.

In humans, there are three distinct loci encoding MHC class I molecules (MHC molecules in humans are also known as human leukocyte antigen (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02, and HLA-B*07 are examples of different MHC class I alleles that can be expressed from these loci.

The peptides of the invention, preferably when included in the vaccines of the invention as described herein, bind to at least one selected from the group consisting of: HLA-A*01:01, HLA-A*03 :01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01 and HLA-B*44:02, plus other HLA atypia as appropriate. Due to similarities in binding patterns, such as similarity in relative anchoring positions, some peptides bind to more than one allele, and this overlap is likely, but not limited to, HLA-A* which also binds to HLA-B*15 01 binding peptide, HLA-A*03 binding peptide also bound to HLA-A*11, HLA-B*07 binding peptide also bound to HLA-B*35 and HLA-B*51.

Vaccines may also include pan-binding MHC class II peptides. Thus, the vaccines of the present invention can be used to treat cancers in patients that are positive for the corresponding HLA allele without the need for selection against MHC class II alleles due to the pan-binding properties of these peptides.

If the peptides of the invention are combined with a peptide that binds to another allele, a higher percentage of any patient population can be treated than if directed against either MHC class I allele alone.

In a preferred embodiment, the term "nucleotide sequence" refers to a heteropolymer of deoxyribonucleotides.

Nucleotide sequences encoding particular peptides, oligopeptides, or polypeptides can be naturally occurring or they can be constructed synthetically. Typically, DNA segments encoding the peptides, polypeptides, and proteins of the invention are assembled from cDNA fragments and short oligonucleotide linkers, or from a series of oligonucleotides, to provide Synthetic genes expressed in recombinant transcription units of the regulatory elements of viral operons.

As used herein, the term "coding (or nucleotide encoding a peptide)" refers to a peptide-encoding core that includes an artificial (artificial) start codon and stop codon that are compatible with biological systems A nucleotide sequence that will be expressed by, for example, a dendritic cell or another cellular system suitable for TCR production.

As used herein, reference to a nucleic acid sequence includes both single-stranded and double-stranded nucleic acids. Thus, for example, with respect to DNA, unless the context dictates otherwise, a particular sequence refers to single-stranded DNA of such sequences, duplexes of such sequences and their complements (double-stranded DNA), and complements of such sequences.

The term "coding region" refers to the portion of a gene that naturally or normally encodes the expressed product of that gene in its natural genomic environment, ie, the region that encodes the initially expressed product of the gene in vivo.

The coding region can be derived from a non-mutated ("normal") gene, a mutated gene or an altered gene, or can even be derived from a DNA sequence or gene synthesized in its entirety in the laboratory using methods well known to those skilled in the art of DNA synthesis.

The term "expression product" means a polypeptide or protein that is the natural translation product of a gene and any nucleic acid sequence that results from the degeneracy of the genetic code and thus encodes equivalents of the same amino acid.

When referring to a coding sequence, the term "fragment" means a portion of DNA comprising a less than complete coding region, the expressed product of which retains substantially the same biological function or activity as the expressed product of the complete coding region.

The term "DNA segment" refers to a polymer of DNA in the form of individual fragments or as a component of a larger DNA construct, which has been derived in substantially pure form, that is, without contamination of endogenous material DNA isolated at least once and in an amount or concentration that enables the segment and its components to be identified, manipulated, and recovered by standard biochemical methods, eg, by use of a cloning vector. Such segments are provided in open reading frames not interrupted by internal non-translated sequences or introns typically found in eukaryotic genes. Sequences of non-translated DNA may be present downstream of the open reading frame, wherein the sequences do not interfere with the manipulation or representation of the coding region.

The term "pharmaceutically acceptable salts" refers to derivatives of the disclosed peptides wherein the peptides are modified by making acid or base salts of the reagents. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral _-NH2 group) involving reaction 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, salicylic acid, and the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid , nitric acid, phosphoric acid, and the like. Conversely, base salts of acid moieties that may be present on peptides are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like preparation. In a preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate), trifluoroacetate or hydrochloric acid (chloride).

The peptides of the present disclosure in the form of pharmaceutically acceptable salts are not found in nature. The peptides of the present disclosure bind to MHC molecules. MHC peptides sent to the endoplasmic reticulum (ER) for association with MHC molecules are longer precursors to final peptides that are transported to MHC molecules (peptide-MHC complexes) cell surface. The peptide presented by the MHC molecule never floats freely and is corrected to the appropriate size from the longer precursor in the ER even after association with the MHC molecule. At no time are the peptides of the present disclosure (with or without longer precursor peptides) in a pharmaceutically acceptable salt form, as seen in nature. The presence of the salt form means that the pendant and/or N-terminal and/or C-terminal binding to the salt ion is a significantly different structural/chemical change. Pharmaceutically acceptable salt forms of the peptides of the present disclosure require acids and/or bases that are capable of donating or accepting protons to the peptides at stoichiometrically defined molar concentrations, such salts only when not under cytoplasmic control formed in the environment.

The term "promoter" means a region of DNA involved in binding RNA polymerase to initiate transcription.

The term "isolated" means the removal of a substance from its original environment (eg, the natural environment if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living organism is not isolated, but is isolated from the same polynucleotide or polypeptide that is isolated from some or all of the coexisting species in the natural system. Such polynucleotides may be part of a vector and/or such polynucleotides or polypeptides may be part of a composition and still be isolated in that such a vector or composition is not part of its natural environment.

The polynucleotides and recombinant or immune polypeptides disclosed in accordance with the present invention may also be in "purified" form. The term "purified" does not require absolute purity, rather, it is intended as a relative definition, and can include highly purified preparations or only partially purified preparations, as those terms are understood by those skilled in the relevant art. For example, individual clones isolated from cDNA libraries have been conventionally purified to electrophoretic homogeneity. Purification of the starting material or natural material to at least one order of magnitude, preferably two or three orders of magnitude, and more preferably four or five orders of magnitude is expressly contemplated. Furthermore, claimed polypeptides having a purity by weight of preferably 99.999% or at least 99.99% or 99.9%; and even desirably 99% or greater are expressly encompassed.

Nucleic acid and polypeptide expression products disclosed in accordance with the present invention, as well as expression vectors containing such nucleic acids and/or such polypeptides, may be in "enriched form." As used herein, the term "enriched" means that the concentration of a substance is, for example, at least about 2, 5, 10, 100, or 1000 times its natural concentration, advantageously 0.01% by weight, preferably at At least about 0.1% by weight. Also encompassed are enriched formulations of about 0.5%, 1%, 5%, 10%, and 20% by weight. The sequences, constructs, vectors, clones, and other materials comprising the present invention may advantageously be in enriched or isolated form. The term "active fragment" means a fragment, usually a peptide, polypeptide or nucleic acid sequence, which produces an immune response (ie, is immunologically active) when administered to an animal alone or, as appropriate, with a suitable adjuvant or in a carrier, In animals such as mammals, eg, mice, rats, llamas, sheep, goats, dogs, or horses, and including humans, such immune responses take the form of stimulating T cell responses in recipient animals such as humans. Alternatively, "active fragments" can also be used to induce T cell responses in vitro.

As used herein, the terms "portion," "segment," and "fragment," when used in relation to a polypeptide, refer to a contiguous sequence of residues, such as amino acid residues, that form a subset of a larger sequence. For example, if the polypeptide is subjected to treatment with any common endopeptidase, such as trypsin or chymotrypsin, the oligopeptides resulting from such treatment will represent a portion, segment or fragment of the starting polypeptide. When used in relation to polynucleotides, these terms refer to the products produced by treating such polynucleotides with any endonuclease.

According to the present invention, when referring to sequences, the term "percent identity" or "percent identity" means the alignment of the sequence to be compared ("compared sequence") with the sequence described or claimed ("reference sequence"). Thereafter, the sequences are compared to the claimed or described sequences. The percent agreement is then determined according to the following formula: % Concordance = 100[1-(C/R)] where C is the number of differences between the reference and comparison sequences over the length of the alignment between the reference and comparison sequences, where (i) each base or amino acid in the reference sequence that does not have a corresponding aligned base or amino acid in the compared sequence, and (ii) each gap in the reference sequence, and (iii) each aligned base or amino acid in the reference sequence constitutes a difference that differs from the aligned base or amino acid in the compared sequence, and (iii) the alignment must begin at position 1 of the aligned sequences; And R is the number of bases or amino acids in the reference sequence over the length of the alignment to the comparison sequence, where any gaps created in the reference sequence are also counted as bases or amino acids.

If there is an alignment between the compared sequence and the reference sequence whose percent identity, as calculated above, is approximately equal to or greater than the specified minimum percent identity, then the compared sequence has the specified minimum percent identity to the reference sequence, even if There may be alignments where the above calculated percent identity is less than the specified percent identity.

As mentioned above, the present invention thus provides a peptide comprising a sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 113 or a variant thereof, which variant will induce T cells to cross with the peptide reaction. The peptides of the invention have the ability to bind to class I of MHC molecules or to bind extended versions of these peptides to class II.

In the present invention, the term "homology" refers to the degree of identity between the sequences (ie, peptide or polypeptide sequences) of two amino acid sequences (see above for percent identity). The above "homology" is determined by comparing two aligned sequences under optimal conditions at the sequences to be compared. This sequence homology can be calculated by generating alignments using, for example, the ClustalW algorithm. Commonly available sequence analysis software, more specifically Vector NTI, GENETYX or other tools, are provided through public databases.

Those skilled in the art will be able to assess whether T cells induced by variants of a particular peptide will be able to cross-react with the peptide itself (see, e.g., Appay et al., 2006; Colombetti et al., 2006; Fong et al., 2001; Zaremba et al., 1997, the contents of which are hereby incorporated by reference in their entirety).

By "variant" of a given amino acid sequence, the inventors mean that the side chains of, for example, one or both of the amino acid residues have been altered (for example, by substitution with another naturally occurring amino acid residue). side chain or some other side chain to replace it) so that the peptide can still bind to the HLA molecule in substantially the same way as the peptide consisting of the given amino acid sequence in SEQ ID NO: 1 to SEQ ID NO: 113 . For example, a peptide can be modified so that it at least maintains, if not modified, at least the ability to interact with and bind to the binding groove of a suitable MHC molecule, and in that way, it at least maintains, if not modified, to bind to activated T The ability of a cell's TCR to be suitable for an MHC molecule such as one selected from the group consisting of: HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA -B*07:02, HLA-B*08:01 and HLA-B*44:02, plus other HLA isoforms as appropriate.

These T cells can then cross-react with cells and kill cells expressing a polypeptide containing the native amino acid sequence of a cognate peptide as defined in aspects of the invention. As can be derived from scientific literature and databases (Rammensee et al., 1999; Godkin et al., 1997, which are incorporated herein by reference in their entirety), certain positions of HLA-binding peptides are typically used to form bindings that assemble into HLA molecules. Anchor residues of the motif's core sequence, which is defined by the polar, electrophysical, hydrophobic, and steric properties of the polypeptide chains that make up the binding groove. Thus, one skilled in the art will be able to modify the amino acid sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 113 by maintaining known anchor residues, and will be able to determine such variants Whether the ability to bind MHC class I or class II molecules is maintained. The variants of the present invention retain the ability to bind to the TCR of activated T cells, which can then cross-react with and kill cells expressing polypeptides containing cognate peptides as defined in aspects of the present invention. Natural amino acid sequence.

If not stated otherwise, the original (unmodified) peptides as disclosed herein can be modified by substituting one or more residues at different, possibly alternative sites within the peptide chain. Preferably, these substitutions are located at the ends of the amino acid chain. Such substitutions may have conservative properties, eg, where one amino acid is replaced by an amino acid of similar structure and properties, such as where a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be to replace amino acids of the same or similar size or chemical properties, such as where leucine is replaced by isoleucine. In studies of sequence variation in naturally occurring families of homologous proteins, certain amino acid substitutions are often more tolerated than others, and these substitutions often exhibit size, charge, and size between the original amino acid and its replacement , polarity, and hydrophobicity, and these are the basis for the definition of "conservative substitutions."

Conservative substitutions are interpreted herein as interchanges within one of the following five groups: Group 1 - small aliphatic non-polar or slightly polar 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 aliphatic Non-polar residues (Met, Leu, Ile, Val, Cys); and Group 5-large aromatic residues (Phe, Tyr, Trp).

In one aspect, conservative substitutions may include those described by Dayhoff in "The Atlas of Protein Sequence and Structure. Vol. 5", Natl. Biomedical Research , the contents of which are incorporated herein by reference in their entirety . For example, in one aspect, amino acids belonging to one of the following groups are interchangeable with each other, thus constituting a conservative interchange: Group 1: Alanine (A), Proline (P), Glycine (G ), aspartic acid (N), serine (S), threonine (T); group 2: cysteine (C), serine (S), tyrosine (Y) , Threonine (T); Group 3: Valine (V), Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Phenylalanine (F); group 4: lysine (K), arginine (R), histidine (H); group 5: phenylalanine (F), tyrosine (Y), tryptophan ( W), histidine (H); and group 6: aspartic acid (D), glutamic acid (E). In one aspect, conservative amino acid substitutions may be selected from the following: T→A, G→A, A→I, T→V, A→M, T→I, A→V, T→G, and/or or T→S.

In one aspect, conservative amino acid substitutions can include substitution of an amino acid by another amino acid of the same class, eg (1) Non-polar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp ; (2) Uncharged, polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) Acidic: Asp, Glu; and (4) Basic: Lys, Arg, His. Other conservative amino acid substitutions can also be made as follows: (1) Aromatics: Phe, Tyr, His; (2) Proton Donors: Asn, Gln, Lys, Arg, His, Trp; and (3) Proton Acceptors : Glu, Asp, Thr, Ser, Tyr, Asn, GIn (see, eg, US Pat. No. 10,106,805, the contents of which are incorporated herein by reference in their entirety).

In another aspect, conservative substitutions can be made according to Table 3. Methods for predicting resistance to protein modifications can be found in the literature (Guo et al., 2004, which is incorporated by reference in its entirety).

Table 3: List of conservative amino acid substitutions. original residue Conservative substitutions (others are known in the art) Ala Ser, Gly, Cys Arg Lys, Gln, His Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn, Lys, Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro, Ala, Ser His Asn, Gln, Lys Ile Leu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, His Met Leu, Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His Ser Thr, Cys, Ala Thr Ser, Val, Ala Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met, Ala, Thr

In another aspect, conservative substitutions may be those shown in Table 4 under the heading "Conservative Substitutions." If such substitutions result in a change in biological activity, more substantial changes, designated "exemplary substitutions" in Table 4, can be introduced and products screened if desired.

Table 4: Exemplary amino acid substitutions. original residue Exemplary substitutions (others are known in the art) Ala Val, Leu, Ile Arg Lys, Gln, Asn Asn Gln, His, Asp, Lys, Arg Asp Glu, Asn Cys Ser, Ala Gln Asn, Glu Glu Asp, Gln Gly Ala His Asn, Gln, Lys, Arg Ile Leu, Val, Met, Ala, Phe, Norleucin Leu Norleucin, Ile, Val, Met, Ala, Phe Lys Arg, Gln, Asn Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr Pro Ala Ser Thr Thr Ser, Ala Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser Val Ile, Leu, Met, Phe, Ala, Norleucin

Of course, such substitutions may involve structures other than common L-amino acids. Thus, D-amino acids can replace L-amino acids commonly found in antigenic peptides of the present invention and still be encompassed by the disclosure herein. In addition, non-standard amino acids (ie, amino acids other than the ubiquitous naturally occurring proteinogenic amino acids) may also be used for substitution purposes to generate immunogens and immunogenic polypeptides according to the present invention.

If substitutions at more than one position are found to result in a peptide having substantially equivalent or greater antigenic activity as defined below, then combinations of those substitutions will be tested to determine whether the combined substitutions result in a reduction in the antigenicity of the peptides additive or synergistic effects.

Peptides consisting primarily of amino acid sequences as indicated herein may have one or two non-anchor amino acids (see below for anchor motifs) interchangeable, while binding when compared to non-modified peptides The ability to MHC molecule class I or class II is not substantially changed or negatively affected. In another example, in a peptide consisting essentially of an amino acid sequence as indicated herein, one or two amino acids can be interchanged with its conservative exchange partner (see below), while when compared to a non-modified peptide Its ability to bind to MHC class I or class II molecules is not substantially altered or negatively affected.

Amino acid residues that do not substantially contribute to interaction with T cell receptors can be modified by replacement with other amino acids whose incorporation does not substantially affect T cell reactivity and does not eliminate Binding to the relevant MHC. Thus, regardless of the conditions given, the peptides of the invention may be any peptide (by which the inventors use this term to include oligopeptides or polypeptides) including amino acid sequences or a portion or variation thereof as given body.

Longer (elongated) peptides may also be suitable. It is possible that, although MHC class I epitopes are typically between 8 and 12 amino acids in length, they are processed by peptides from longer peptides or proteins that include the actual epitope. produce. Preferably, the residues flanking the actual epitope are residues that do not substantially affect the proteolytic breakdown necessary to expose the actual epitope during processing.

The peptides of the invention can be elongated by up to four amino acids, that is, 1, 2, 3 or 4 amino acids can be added to either end in any combination between 4:0 and 0:4 department. The combinations of elongations according to the invention can be found in Table 5.

Table 5: Combinations of peptide stretches of the invention C-terminal N-terminal 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4 N-terminal C-terminal 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acid used for elongation/extension can be a peptide of the original sequence of a protein or any other amino acid. Elongation can be used to enhance peptide stability or solubility.

Thus, epitopes of the invention may be identical to naturally occurring tumor-associated epitopes or tumor-specific epitopes, or may include epitopes that differ by no more than four residues from the reference peptide, so long as they have substantial The same antigenic activity is sufficient.

In an alternative embodiment, the peptide is elongated by more than 4 amino acids on either or both sides, preferably up to a total length of up to 30 amino acids. This action results in MHC class II-binding peptides. Binding to MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHC class I epitopes, wherein the peptides or variants have between 8 and 100, preferably between 8 and 30, and most preferably between 8 and 12 between, that is, the total length of 8, 9, 10, 11, 12 amino acids, and in the case of elongated class II binding peptides, the length can also be 13, 14, 15, 16 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptides or variants according to the invention will have the ability to bind to MHC class I or class II molecules. Binding of peptides or variants to MHC complexes can be tested by methods known in the art.

Preferably, when T cells specific for a peptide according to the invention are tested against the substituted peptide, the peptide concentration at which the substituted peptide achieves a half-maximal increase in lysis relative to background does not exceed about 1 mM, preferably preferably no more than about 1 μM, more preferably no more than about 1 nM, and still more preferably no more than about 100 pM, and most preferably no more than about 10 pM. Also preferably, the substituted peptides are recognized by T cells from more than one individual, at least two individuals, and more preferably three individuals.

In a particularly preferred embodiment of the present invention, the peptide consists or consists essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 113.

"Consisting essentially of" shall mean that, in addition to the sequence according to any one of SEQ ID NO: 1 to SEQ ID NO: 113 or a variant thereof, the peptide according to the invention contains an additional N-terminus and /or C-terminally positioned stretches of amino acids that do not necessarily form part of the peptides that function as epitopes of the MHC molecule.

However, these stretched chains are important to provide efficient introduction of the peptides according to the invention into cells. In one embodiment of the invention, the peptide is part of a fusion protein comprising, for example, an HLA-DR antigen-associated invariant chain (p33, hereinafter "Ii") derived from NCBI (GenBank Accession No. X00497) 80 N-terminal amino acids. In other fusions, the peptides of the invention may be fused to an antibody or a functional part thereof as described herein, in particular to a sequence of an antibody for specific targeting by the antibody, or eg to an antibody as described herein of dendritic cells for specific antibodies.

Additionally, the peptides or variants can be further modified to improve stability and/or binding to MHC molecules in order to elicit a stronger immune response. Methods for such optimization of peptide sequences are well known in the art and include, for example, the introduction of retro-peptide or non-peptide bonds.

In reverse peptide bonds, the amino acid residues are not linked by peptide (-CO-NH-) linkages but the peptide bonds are reversed. Such retro peptidomimetics can be prepared using methods known in the art, such as, for example, those described by Meziere and colleagues (Meziere et al., 1997, which are incorporated herein by reference). This method involves making pseudopeptides that contain changes involving the orientation of the main chain and not the side chains. They demonstrated that these pseudopeptides are useful for MHC binding and T helper cell responses. Retro-inverse peptides containing NH-CO bonds rather than CO-NH peptide bonds are much more resistant to proteolysis (Meziere et al., 1997).

Examples of non-peptide bonds are _{-CH2 -} NH, _-CH2S- , _-CH2CH2- , -CH= _CH- , _-COCH2- , -CH(OH)CH2-, and _-CH2SO- . US 4,897,445 provides a method for solid phase synthesis of non-peptide bonds ( _-CH2 -NH) in polypeptide chains involving polypeptides synthesized by standard procedures and by subjecting aminoaldehydes and amino acids in the presence of _NaCNBH3 Non-peptide bonds synthesized by reaction.

Peptides comprising the linkages described above can be synthesized with additional chemical groups present at their amine and/or carboxyl termini to enhance peptide stability, bioavailability, and/or affinity. For example, a hydrophobic group such as benzyloxycarbonyl, dansyl, or tertiary butoxycarbonyl can be added to the amine terminus of the peptide. Likewise, an acetyl or 9-intenylmethoxy-carbonyl group can be placed at the amine terminus of the peptide. Additionally, a hydrophobic group, a tertiary butoxycarbonyl group, or an amido group can be added to the carboxy terminus of the peptide.

Additionally, the peptides of the present invention can be synthesized to alter their stereoconfiguration. For example, the D-isomer of one or more amino acid residues of the peptide can be used instead of the common L-isomer. Additionally, at least one amino acid residue of the peptides of the invention can be substituted with one of the well-known non-naturally occurring amino acid residues. Changes such as these substitutions can be used to increase the stability, bioavailability and/or binding of the peptides of the invention.

Similarly, the peptides or variants of the invention can be chemically modified by reacting specific amino acids before or after synthesis of the peptide. Examples of such modifications are well known in the art (Lundblad, 2004, which is incorporated herein by reference). Chemical modification of amino acids includes, but is not limited to, modification by acylation, amidinoylation, pyridoxylation of lysine, reductive alkylation, amino group from 2,4,6-tris Trinitrobenzylation of Nitrobenzenesulfonic Acid (TNBS), Amide and Sulfhydryl Modification of Carboxyl by Peroxyformic Acid Oxidation of Cystine to Sulfalanine, Formation of Mercury Derivatives , formation of mixed disulfides with other thiol compounds, reaction with maleimide, carboxymethylation with iodoacetic acid or iodoacetamide, and amine groups with cyanate at alkaline pH Forsylation, but not limited to these (Coligan et al., 1995, the contents of which are incorporated herein by reference in their entirety).

Briefly, modifications of eg sperminyl residues in proteins are often based on the reaction of adjacent dicarbonyl compounds such as phenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione. to form adducts. Another example is the reaction of methylglyoxal with spermidine residues. Cystine can be modified without concomitant modification of other nucleophilic sites such as lysine and histidine. Therefore, a large number of reagents are available for the modification of cystine. The websites of companies such as Sigma-Aldrich (www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common. Disulfide bonds can be formed and oxidized during thermal treatment of biopharmaceuticals. Woodward's reagent K can be used to modify specific glutamic acid residues. N-(3-(dimethylamino)propyl)-N'-ethylcarbodiimide can be used to form intramolecular crosslinks between lysine residues and glutamic acid residues. For example, diethylpyrocarbonate is an agent used to modify histamine residues in proteins. Histidine can also be modified with 4-hydroxy-2-nonenal. The reaction of lysine residues and other alpha-amine groups is suitable, for example, for binding peptides to surfaces or cross-linking of proteins/peptides. Lysine is the attachment site of poly(ethylene) glycol and the major modification site of protein glycosylation. Methionine residues in proteins can be modified with, for example, iodoacetamide, bromoethylamine, and chloramine T.

Tetranitromethane and N-acetylimidazole can be used to modify tyrosinyl residues. Crosslinking via the formation of bistyrosine can be accomplished with hydrogen peroxide/copper ions.

Recent studies on modified tryptophan have used N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or 3-bromo-3-methyl-2-(2-nitrophenylmercapto) -3H-Indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG is often associated with increased circulation half-life, while proteins are cross-linked with glutaraldehyde, polyethylene glycol diacrylate, and formaldehyde for the preparation of hydrogels. Chemical modification of allergens for immunotherapy is often accomplished by aminoformylation with potassium cyanate.

Another embodiment of the present invention relates to a non-naturally occurring peptide, wherein the peptide consists or consists essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 113, and has been treated as a pharmaceutically acceptable Salts are produced synthetically (eg, synthetically). Methods of synthetically producing peptides are well known in the art. The salts of the peptides according to the invention differ substantially from the peptides in their in vivo state, since the peptides produced in vivo are free of salts. The non-natural salt forms of the peptides mediate the solubility of the peptides, especially in the context of pharmaceutical compositions comprising the peptides (eg, peptide vaccines as disclosed herein). Sufficient and at least substantial solubility of the peptide is required in order to effectively deliver the peptide to the subject to be treated. Preferably, the salt is a pharmaceutically acceptable salt of the peptide. The salts according to the invention include alkali metal salts and alkaline earth metal salts, such as salts _of the ^Hofmeister ionic sequence comprising the anions _PO43 , _SO42- ^{, CH3COO-} , Cl ^- , Br ^- , NO ₃ ^- , ClO ₄ ^- , I ^- , SCN ^- and cations NH ₄ ⁺ , Rb ⁺ , K ⁺ , Na ⁺ , Cs ⁺ , Li ⁺ , Zn ²⁺ , Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Cu ²⁺ and Ba ²⁺ . _In particular, the salt is selected from ( _NH4 ) _3PO4 , ( _{NH4 ) 2HPO4} , ( _{NH4 ) H2PO4} , _{( NH4 ) 2SO4} , _NH4CH3COO , _NH4Cl , NH ₄ Br, NH ₄ NO ₃ , NH ₄ CIO ₄ , NH ₄ I, NH ₄ SCN, Rb ₃ PO ₄ , Rb ₂ HPO ₄ , RbH ₂ PO ₄ , Rb ₂ SO ₄ , Rb ₄ CH ₃ COO, Rb ₄ Cl, Rb ₄ Br, Rb ₄ NO ₃ , Rb ₄ CIO ₄ , Rb ₄ I, Rb ₄ SCN, K ₃ PO ₄ , K ₂ HPO ₄ , KH ₂ PO ₄ , K ₂ SO ₄ , KCH ₃ COO, KCl , KBr, KNO ₃ , KClO ₄ , KI, KSCN, Na ₃ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ , Na ₂ SO ₄ , NaCH ₃ COO, NaCl, NaBr, NaNO ₃ , NaCIO ₄ , NaI, NaSCN , ZnCI ₂ Cs ₃ PO ₄ , Cs ₂ HPO ₄ , CsH ₂ PO ₄ , Cs ₂ SO ₄ , CsCH ₃ COO, CsCl, CsBr, CsNO ₃ , CsCIO ₄ , CsI, CsSCN, Li ₃ PO ₄ , Li ₂ HPO ₄ , LiH ₂ PO ₄ , Li ₂ SO ₄ , LiCH ₃ COO, LiCl, LiBr, LiNO ₃ , LiClO ₄ , LiI, LiSCN, Cu ₂ SO ₄ , Mg ₃ (PO ₄ ) ₂ , Mg ₂ HPO ₄ , Mg(H ₂ PO ₄ ) ₂ , Mg ₂ SO ₄ , Mg(CH ₃ COO) ₂ , MgCl ₂ , MgBr ₂ , Mg(NO ₃ ) ₂ , Mg(ClO ₄ ) ₂ , MgI ₂ , Mg(SCN) ₂ , MnCl ₂ , Ca ₃ (PO ₄ ), Ca ₂ HPO ₄ , Ca(H ₂ PO ₄ ) ₂ , CaSO ₄ , Ca(CH ₃ COO) ₂ , CaCl ₂ , CaBr ₂ , Ca(NO ₃ ) ₂ , Ca(ClO ₄ ) ₂ , CaI ₂ , Ca(SCN) ₂ , Ba ₃ (PO ₄ ) ₂ , Ba ₂ HPO ₄ , Ba(H ₂ PO ₄ ) ₂ , BaSO ₄ , Ba(CH ₃ COO) ₂ , BaCl ₂ , BaBr _2. Ba(N O ₃ ) ₂ , Ba(ClO ₄ ) ₂ , BaI ₂ , and Ba(SCN) ₂ . In particular, preferred are NH acetate, MgCl ₂ , KH ₂ PO ₄ , Na ₂ SO ₄ , KCl, NaCl, and CaCl ₂ , such as, for example, chloride or acetate (trifluoroacetate) (eg Berge et al., 1977, the contents of which are hereby incorporated by reference in their entirety).

In general, peptides and variants (at least those that contain peptide bonds between amino acid residues) can be synthesized by the Fmoc-polyamide mode of solid-phase peptide synthesis, such as by Lukas et al. (Lukas et al. et al., 1981, the contents of which are incorporated herein by reference in their entirety) and the references cited therein. Temporary N-amino protection is provided by a 9-intenylmethoxycarbonyl (Fmoc) group. Repeated decomposition of this highly base-labile protecting group was carried out using 20% piperidine in N,N-dimethylformamide. The pendant functionality can be protected as its butyl ether (in the case of serine, threonine and tyrosine), butyl ester (in the case of glutamic and aspartic acids), butoxy Carbonyl derivatives (in the case of lysine and histidine), trityl derivatives (in the case of cystine) and 4-methoxy-2,3,6-trimethylbenzenesulfonic acid Acyl derivatives (in the case of arginine). In the case of glutamic acid or aspartic acid as the C-terminal residue, a 4,4'-dimethoxybenzhydryl group was used to protect the side chain amido functionality. The solid support is based on three monomers dimethylacrylamide (main chain monomer), bisacryloylethylenediamine (crosslinking agent) and methylacryloyl sarcosinate (functionalizing agent) of polydimethyl-acrylamide polymers. The peptide-to-resin cleavable linker used was an acid labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their pre-formed symmetrical anhydride derivatives, with the exception of aspartic acid and glutamic acid, which use the reverse N,N-dicyclohexyl-carbodiimide /1 hydroxybenzotriazole-mediated coupling procedure to add. All coupling and deprotection reactions were monitored using ninhydrin, trinitrobenzenesulfonic acid or isotin test procedures. After the synthesis was complete, the peptide was cleaved from the resin support and side chain protecting groups were concomitantly removed by treatment with 95% trifluoroacetic acid containing a 50% scavenger mixture. Commonly used scavengers include ethanedithiol, phenol, anisole, and water, the exact choice depending on the constituent amino acids of the peptide being synthesized. In addition, a combination of solid-phase and liquid-phase methods for synthesizing peptides is possible (see, eg, Bruckdorfer et al., 2004 and references cited therein, the contents of which are incorporated herein by reference in their entirety).

Trifluoroacetic acid was removed by evaporation in vacuo, followed by trituration with diethyl ether to give the crude peptide. Any scavengers present were removed by a simple extraction procedure followed by lyophilization of the aqueous phase to yield the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from eg Calbiochem-Novabiochem (Nottingham, UK).

Purification can be performed by any one technique or combination of techniques, such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed phase separation using, for example, an acetonitrile/water gradient High performance liquid chromatography.

Analysis of peptides can be performed using thin layer chromatography, electrophoresis, especially capillary electrophoresis, solid phase extraction (CSPE), reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis And fast atom bombardment (fast atom bombardment; FAB) mass spectrometry, and MALDI and ESI-Q-TOF mass spectrometry.

To select overrepresented peptides, a representation distribution was calculated, which showed median sample representation as well as replicate variation. This distribution juxtaposes samples of tumor entities of interest with a baseline of normal tissue samples. Multiple tests can then be adjusted by calculating p-values for linear mixed-effects models (Pinheiro et al., 2015), by false discovery rates (Benjamini and Hochberg, 1995, the contents of which are hereby incorporated by reference in their entirety) to combine each of these distributions into an over-presentation score.

For identification and relative quantification of HLA ligands by mass spectrometry, HLA molecules from shake-frozen tissue samples were purified and HLA-associated peptides isolated. The isolated peptides were isolated and sequences were identified by on-line nano-electrospray-ionization (nanoESI) liquid chromatography-mass spectrometry (LC-MS) experiments . By combining acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma Squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer samples The resulting peptide sequences were validated by comparing the fragmentation pattern of the recorded natural tumor-associated peptide (TUMAP) with the fragmentation pattern of the corresponding synthetic reference peptide of the consensus sequence. Because peptides are directly identified as ligands for HLA molecules of the primary tumor, these results provide insights into acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glial cancer blastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, Direct evidence of native processing and presentation of identified peptides on primary cancer tissues obtained from prostate, renal cell, small cell lung, bladder, and endometrial cancers (Reference Example 1, Figures 3A-3D ).

Exploratory pipeline XPRESIDENT® v2.1 allows identification and selection of relevant transitions as potential targets for immunotherapy based on direct relative quantification of HLA-restricted peptide levels on cancer tissues compared to several different non-cancerous tissues and organs Rendered peptides. See, eg, US Patent Application Publication No. 2013/0096016, the contents of which are incorporated herein by reference in their entirety. This was done by developing label-free differential quantification, using acquired LC-MS data processed through a dedicated data analysis pipeline, which will be used for sequence identification, spectral clustering, ion calculation, residence time calibration, charge state deconvolution and Regularized algorithm combination to achieve.

Integration of additional sequence information from public sources (Olexiouk et al., 2016; Subramanian et al., 2011, the contents of which are incorporated by reference in their entirety) into the XPRESIDENT® discovery pipeline to enable identification of TUMAPs from atypical origins . A presentation level including error estimates is established for each peptide and sample. Peptides that are exclusively presented on tumor tissue relative to non-cancerous tissues and organs and peptides that are over-presented in tumors have been identified.

will be derived from acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cells Carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer tissue samples HLA-peptide complexes were purified and HLA-associated peptides were separated and analyzed by LC-MS (see Example 1). All TUMAPs contained in this application were developed using this method in acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction Cancer, Hepatocellular Carcinoma, Head and Neck Squamous Cell Carcinoma, Melanoma, Non-Hodgkin's Lymphoma, Non-Small Cell Lung Cancer, Ovarian Cancer, Esophageal Cancer, Pancreatic Cancer, Prostate Cancer, Renal Cell Carcinoma, Small Cell Lung Cancer , bladder cancer, and endometrial cancer samples, thereby confirming its role in acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastric cancer -Esophageal junctional carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, Presentation on small cell lung cancer, bladder cancer, endometrial cancer, and combinations thereof.

Quantification of ion counts using label-free LC-MS data in multiple acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastro-esophageal cancer Junctional carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell Lung, bladder, and endometrial cancers and TUMAPs identified on normal tissues. The method assumes that the LC-MS signal region of a peptide correlates with its abundance in the sample. All quantitative signals for peptides in various LC-MS experiments were normalized based on central tendency, averaged by sample and combined into a histogram, which is called a presentation distribution. The presented distribution incorporates different analytical methods such as protein database searching, spectral clustering, charge state deconvolution (de-charge), and residence time calibration and normalization.

In addition to overrepresentation of peptides, cryptic genes were tested for mRNA expression. mRNA data were obtained via RNASeq analysis of normal and cancer tissues (see Example 2, Figures 4A-4D). An additional source of normal tissue data is the database of publicly available RNA expression data from approximately 3000 normal tissue samples (Lonsdale, 2013, the contents of which are incorporated herein by reference in their entirety). Peptides derived from proteins whose mRNAs are highly expressed in cancer tissues but very low or absent in vital normal tissues are preferably included in the present invention.

The present invention provides peptides that are suitable for the treatment of cancers/tumors that over- or exclusively present the peptides of the invention, preferably acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, nerve Glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer , prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer. The peptides were shown by mass spectrometry to be detected by primary human acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastric- Esophageal junctional carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small HLA molecules are naturally present on lung, bladder, and endometrial cancer samples.

Many source genes/proteins from which the peptides are derived (also known as "full-length proteins" or "essential proteins" have been shown to be highly overrepresented in cancer compared to normal tissue - "normal tissue" in relation to the present invention will mean Refers to healthy blood, brain, heart, liver, lung, adipose tissue, adrenal gland, bile duct, bladder, bone, bone marrow, esophagus, eye, gallbladder, head and neck, large intestine, small intestine, kidney, lymph nodes, central nervous system, peripheral nerves, pancreas , parathyroid, peritoneal, pituitary, pleura, skeletal muscle, skin, spinal cord, spleen, stomach, thyroid, trachea, and ureter cells or other normal tissue cells such as breast, ovary, placenta, prostate, testis, thymus, uterus, which Both demonstrated a high tumor association of the source gene (see, Example 2). In addition, the peptide itself was strongly over-presented on tumor tissue but not on normal tissue - "tumor tissue" with respect to the present invention shall mean derived from patients suffering from Samples from patients: acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck cancer Squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer (See Example 1).

HLA-binding peptides can be recognized by the immune system, in particular T lymphocytes. T cells can destroy cells presenting the identified HLA-peptide complexes, such as acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, Gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, kidney cancer cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer cells.

The peptides of the invention are overrepresented in cancer tissues compared to normal tissues and thus can be used to generate antibodies and/or TCRs according to the invention, such as soluble TCRs (see Table 10). Furthermore, the peptides can also be used to generate antibodies and/or TCRs, especially soluble TCRs, according to the invention when complexed with the corresponding MHCs. Corresponding methods are well known to the skilled person and can also be found in the corresponding literature (see below). Therefore, the peptides of the present invention are suitable for generating an immune response in a patient whereby tumor cells can be destroyed. An immune response in a patient can be induced by direct administration to the patient of the described peptides or suitable precursor substances, such as elongated peptides, proteins, or nucleic acids encoding these peptides, ideally with an agent that enhances immunogenicity (also That is, adjuvant) is administered in combination to induce induction. Immune responses derived from such therapeutic vaccinations can be expected to be highly specific to tumor cells, as the target peptides of the present invention are not present on normal tissues in comparable amounts, thereby preventing the targeting of normal cells in the patient. Risk of unwanted autoimmune reactions.

The present specification further relates to TCRs comprising alpha chains and beta chains ("alpha/beta TCRs"). Also provided are peptides according to the invention which are capable of binding to TCRs and antibodies when presented by MHC molecules.

The present specification also relates to fragments of TCRs according to the invention, which are capable of binding to peptide antigens according to the invention when presented by HLA molecules. The term refers in particular to soluble TCR fragments, such as TCRs lacking the transmembrane portion and/or constant region, single-chain TCRs, and fusions thereof, eg, with immunoglobulins.

This specification also pertains to nucleic acids, vectors, and host cells expressing the TCRs and peptides of this specification; and methods of use thereof.

The term "T cell receptor" (abbreviated as TCR) refers to a heterodimeric molecule comprising an alpha polypeptide chain (alpha chain) and a beta polypeptide chain (beta chain), wherein the heterodimeric receptor is capable of binding to HLA via HLA Molecularly presented peptide antigens. The term also includes so-called gamma/delta TCRs.

In one embodiment, the present specification provides a method of producing a TCR as described herein, the method comprising culturing a host cell capable of expressing the TCR under conditions suitable for promoting the expression of the TCR.

In another aspect, the specification relates to a method according to the specification, wherein the antigen is loaded onto the surface of a suitable antigen-presenting cell or artificial antigen-presenting cell for expression by contacting a sufficient amount of the antigen with the antigen-presenting cell. class I or class II MHC molecules, or loading antigen onto class I or class II MHC tetramers by tetramerizing antigen/class I or class II MHC complex monomers.

The alpha and beta chains of the alpha/beta TCR and the gamma and delta chains of the gamma/delta TCR are generally considered to each have two "domains", namely a variable domain and a constant domain. A variable domain consists of the concatenation of a variable region (V) and a linker region (J). Variable domains may also include a leader (L). Beta and delta chains may also include regions of diversity (D). The alpha and beta constant domains may also include a C-terminal transmembrane (TM) domain, which anchors the alpha and beta chains to the cell membrane.

With respect to γ/δ TCRs, the term "TCRγ variable domain" as used herein refers to the concatenation of the TCRγV (TRGV) and TCRγJ (TRGJ) regions without a leader (L), and the term TCRγ constant domain refers to Extracellular TRGC region, or C-terminal truncated TRGC sequence. Likewise, the term "TCRδ variable domain" refers to the concatenation of the TCRδV (TRDV) and TCRδD/J (TRDD/TRDJ) regions without a leader (L), and the term "TCRδ constant domain" refers to the extracellular TRDC region, or C-terminal truncated TRDC sequence.

The alpha/beta heterodimeric TCRs of the present specification may have disulfide bonds introduced between their constant domains. Preferred TCRs of this type include those with a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, except that Thr 48 of TRAC and Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, these Cystine forms a disulfide bond between the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence.

An alpha/beta heterodimeric TCR of the present specification may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, with or without the above-introduced intrachain linkage, and the TRAC constant domain sequence and TRBC1 of the TCR Or the TRBC2 constant domain sequence can be linked by an initial disulfide bond between Cys4 of TRAC exon 2 and TRBC1 or Cys2 of TRBC2 exon 2.

The TCR of this specification may comprise a detectable label selected from the group consisting of radionuclides, fluorophores and biotin. The TCRs of this specification can be conjugated to therapeutic activators, such as radionuclides, chemotherapeutic agents, or toxins. detectable marker

Detectable labels can be, for example, fluorescent dyes, enzymes, substrates, bioluminescent substances, radioactive substances, and chemiluminescent labels. Exemplary enzyme labels include, but are not limited to, horseradish peroxidase, acetylcholinease, alkaline phosphatase, b-galactosidase, and luciferase. Exemplary fluorophores (fluorescent species) include, but are not limited to, rose bengal, luciferin, fluorescent isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Exemplary chemiluminescent labels include, but are not limited to, luminescent amines. Exemplary bioluminescent substances include, but are not limited to, luciferin and aequorin. Exemplary radioactive materials include, but are not limited to, bismuth-213 ( ²¹³ Bs), carbon-14 ( ¹⁴ C), carbon-11 ( ¹¹ C), chlorine-18 ( ¹⁸ Cl), chromium-51 ( ⁵¹ Cr), cobalt- 57 ( ⁵⁷ Co), cobalt-60 ( ⁶⁰ Co), copper-64 ( ⁶⁴ Cu), copper-67 ( ⁶⁷ Cu), dysprosium-165 ( ¹⁶⁵ Dy), erbium-169 ( ¹⁶⁹ Er), fluorine-18 ( ¹⁸ F), gallium-67 ( ⁶⁷ Ga), gallium-68 ( ⁶⁸ Ga), germanium-68 ( ⁶⁸ Ge), -166 ( ¹⁶⁶ Ho), indium-111 ( ¹¹¹ In), iodine-123 ( ¹²³ I ), iodine-124 ( ¹²⁴ I), iodine-125 ( ¹²⁵ I), iodine-131 ( ¹³¹ I), iridium-192 ( ¹⁹² Ir), iron-59 ( ⁵⁹ Fe), krypton-81 ( ⁸¹ Kr), Lead-212 ( ²¹² Pb), Li-177 ( ¹⁷⁷ Lu), Molybdenum-99 ( ⁹⁹ Mo), Nitrogen-13 ( ¹³ N), Oxygen-15 ( ¹⁵ O), Palladium-103 ( ¹⁰³ Pd), Phosphorus- 32 ( ³² P), potassium-42 ( ⁴² K), rhenium-186 ( ¹⁸⁶ Re), rhenium-188 ( ¹⁸⁸ Re), rubidium-81 ( ⁸¹ Rb), rubidium-82 ( ⁸² Rb), samarium-153 ( ¹⁵³ Sm), selenium-75 ( ⁷⁵ Se), sodium-24 ( ²⁴ Na), strontium-82 ( ⁸² Sr), strontium-89 ( ⁸⁹ Sr), sulfur 35 ( ³⁵ S), strontium-99m ( ⁹⁹ Tc) , thallium-201 ( ²⁰¹ Tl), tritium ( ³ H), xenon-133 ( ¹³³ Xe), ytterbium-169 ( ¹⁶⁹ Yb), ytterbium-177 ( ¹⁷⁷ Yb), and yttrium-90 ( ⁹⁰ Y). radionuclide

Radionuclides emit alpha or beta particles (eg, radioimmunoconjugates). Such radioisotopes include, but are not limited to: beta emitters such as phosphorus-32 ( ³² P), scandium-47 ( ⁴⁷ Sc), copper-67 ( ⁶⁷ Cu), gallium-67 ( ⁶⁷ Ga), yttrium-88 ( ⁸⁸ Y), yttrium-90 ( ⁹⁰ Y), iodine-125 ( ¹²⁵ I), iodine-131 ( ¹³¹ I), samarium-153 ( ¹⁵³ Sm), ruthenium-177 ( ¹⁷⁷ Lu), rhenium-186 ( ¹⁸⁶ Re ), rhenium-188 ( ¹⁸⁸ Re); and alpha emitters such as astatine-211 ( ²¹¹ At), lead-212 ( ²¹² Pb), bismuth-212 ( ²¹² Bi), bismuth-213 ( ²¹³ Bi), or actinium- ²²⁵ (225Ac). toxin

Toxins include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarben; alkylating agents, such as methylbis(chloroethyl) ) amine, thietipazide, melphalan, bischloroethyl nitrosourea (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, Cyclophosphamide, Methyldi(chloroethyl)amine, Busulfan, Dibromomannitol, Streptozotocin, Mitomycin C, Cisdichlorodiamineplatinum (II) (DDP) Cisplatin and carboplatin (paraplatin); anthracyclines including daunorubicin (previously known as daunorubicin), doxorubicin (adreomycin), detorubicin, carcinomycin, idarubicin, rubicin, mitoxantrone, and bisantrene; antibiotics including actinomycin (actinomycin D), bleomycin, calicheamicin, fucomycin, and anthramycin (AMC); and Splitting agents such as vinca alkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (TAXOL®), ricin, Pseudomonas exotoxin, gemcitabine, cytokinin B, gramicidin D, ethidium bromide, epecacin, etoposide, Paraside, colchicine, dihydroxyanthraxdione, 1-dehydrotestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, Hydroxyurea, asparaginase, corticosteroids, mitotane (O,P'-(DDD)), interferon, and mixtures of these cytotoxic agents.

Therapeutic agents include, but are not limited to, carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, Bleomycin, VEGF antagonists, EGFR antagonists, platinums, paclitaxel, irinotecan, 5-fluorouracil, genzecitabine, leucovorin, steroids, cyclophosphamide, melphalan, periwinkle Alkaloids, nitrogen mustards, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 Antagonists, interleukins (eg IL-12 or IL-2), IL-12R antagonists, toxin-conjugated monoclonal antibodies, tumor antigen-specific monoclonal antibodies, Erbitux®, Avastin®, Pertuzumab, Anti-CD20 antibody, Rituxan, RTM, ocrelizumab, ofatumumab, DXL625, Herceptin®, or any combination thereof. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin, and Pseudomonas toxins can be used to generate cell-type specific killing agents (Youle et al., 1980, Gilliland et al., 1980, Krolick et al., 1980 ). Other cytotoxic agents include cytotoxic ribonucleases (see US Pat. No. 6,653,104, the contents of each reference incorporated herein by reference in its entirety).

In one embodiment, a TCR of the present specification having at least one mutation in the alpha chain and/or at least one mutation in the beta chain has modified glycosylation compared to an unmutated TCR.

In one embodiment, a TCR comprising at least one mutation in the TCR alpha chain and/or TCR beta chain has a binding affinity and/or binding half-life for the peptide-HLA molecule complex comprising an unmutated TCR alpha chain and/or an unmutated TCR alpha chain At least twice the binding affinity and/or binding half-life of the TCR of the TCR beta chain. Affinity enhancement of tumor-specific TCRs and their developments relies on the existence of an optimal TCR affinity window. The existence of this window is based on the observation that it is specific for eg HLA-A*02-restricted pathogens when compared to TCRs that are specific for eg HLA-A*02-restricted tumor-associated autoantigens The TCR has a K _d value that is typically about 10 times lower. Although tumor antigens have the potential to be immunogenic, it is now known that because tumors are produced by an individual's own cells, only proteins that are mutated or processed using altered translation will be considered foreign by the immune system. Antigens that are upregulated or overexpressed (so-called self-antigens) will not necessarily induce a functional immune response against the tumor: T cells expressing TCRs that are highly reactive to these antigens will undergo a process called central tolerance Negative selection in the thymus means that only T cells with low affinity TCRs for self-antigens are retained. Thus, the affinity of a TCR or variant of the present specification for a peptide can be enhanced by methods well known in the art.

The present specification is further directed to a method of identifying and isolating TCRs according to the present specification, the method comprising incubating HLA-peptide monomers with PBMCs from a healthy donor that are negative for the corresponding HLA allele, with tetramer-phycoerythrin (phycoerythrin; PE) was incubated with PBMC, and high-binding T cells were isolated by fluorescence activated cell sorting (FACS) Calibur analysis.

The present specification is further directed to a method of identifying and isolating TCRs according to the present specification, the method comprising obtaining transgenic mice with all human TCRαβ loci (1.1 and 0.7 Mb) whose T cells express a diversity of TCRs compensating for mouse TCR deficiency Sexual human TCR immunoprofiling, immunization of mice with peptides, incubation of PBMCs obtained from transgenic mice with tetramer-phycoerythrin (PE), and isolation by fluorescence-activated cell sorting (FACS) Calibur analysis High binding T cells.

In one aspect, to obtain T cells expressing the TCRs of the present disclosure, nucleic acids encoding the TCR-alpha and/or TCR-beta chains of the present disclosure are cloned into expression vectors such as gamma retroviruses or lentiviruses. Recombinant viruses are generated and then tested for functionality, such as antigen specificity and functional binding. An aliquot of the final product is then used to transduce the target T cell population (usually purified from patient PBMC), allowing it to expand prior to infusion into the patient.

In another aspect, to obtain T cells expressing the TCR of the present specification, TCR RNA is synthesized by techniques known in the art, such as in vitro transcription systems. The in vitro synthesized TCR RNA was subsequently introduced into primary CD8 positive T cells obtained from healthy donors by electroporation to re-express tumor-specific TCR-alpha and/or TCR-beta chains.

To increase performance, the nucleic acid encoding the TCR of the present specification can be operably linked to a strong promoter, such as a retroviral long terminal repeat (LTR), cytomegalovirus (CMV), murine Murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin, ubiquitin, simian virus 40 (SV40)/CD4 composite promoter, elongation factor ; EF)-1a and the spleen focus-forming virus (SFFV) promoter. In a preferred embodiment, the promoter is heterologous to the expressed nucleic acid.

In addition to strong promoters, the TCR expression cassettes of this specification may contain additional elements that enhance expression of the transfected gene, including a central polypurine tract (cPPT) that facilitates nuclear translocation of lentiviral constructs (Follenzi et al., 2000), and woodchuck hepatitis virus post-transcriptional regulatory element (wPRE) that increases the level of expression of transgenic genes by increasing RNA stability (Zufferey et al. , 1999, the contents of which are incorporated herein by reference in their entirety).

The alpha and beta chains of the TCRs of the present invention may be encoded by nucleic acids located in separate vectors, or may be encoded by polynucleotides located in the same vector.

Achieving high-level TCR surface representation requires high-level transcription of the TCR-α and TCR-β chains of the introduced TCR. To do this, the TCR-alpha and TCR-beta chains of this specification can be cloned into bicis-trans constructs of a single vector that has been shown to overcome this obstacle. The use of an internal ribosomal entry site (IRES) between the TCR-α chain and the TCR-β chain results in a synergistic representation of the two chains since the TCR-α and TCR-β chains are transcribed from a single is produced from a single transcript that splits into two proteins during translation, ensuring the production of equimolar ratios of TCR-alpha and TCR-beta chains (Schmitt et al., 2009, the contents of which are incorporated by reference in their entirety) .

Nucleic acids encoding the TCRs of the present specification can be codon-optimized to increase expression from host cells. The redundancy of the genetic code allows some amino acids to be encoded by more than one codon, but some codons are sub-optimal compared to others due to the relative availability of matching tRNAs and other factors (Gustafsson et al. , 2004). Modifying the TCR-alpha and TCR-beta gene sequences so that each amino acid is encoded by optimal codons for mammalian gene expression, as well as eliminating mRNA instability motifs or cryptic splice sites has been shown to be significantly Enhancement of TCR-alpha and TCR-beta gene expression (Scholten et al., 2006, the contents of these references are incorporated herein by reference in their entirety).

Furthermore, mispairing between the introduced TCR chains and the endogenous TCR chains can lead to the acquisition of specificity, which poses a significant risk for autoimmunity. For example, the formation of mixed TCR dimers can reduce the number of CD3 molecules available to form properly paired TCR complexes, and thus can significantly reduce the functional binding capacity of cells expressing the introduced TCR (Kuball et al., 2007, cit. The contents are incorporated herein by reference in their entirety).

To reduce mispairing, the C-terminal domains of the introduced TCR chains of this specification can be modified to promote interchain affinity while reducing the ability of the introduced chains to pair with endogenous TCRs. These strategies may include replacement of the human TCR-alpha and TCR-beta C-terminal domains with their murine counterparts (murinized C-terminal domains); TCR-alpha by introducing a second cystine residue into the introduced TCR A second interchain disulfide bond is created in the C-terminal domain of both the TCR-α and TCR-β chains (cystine modification); the interacting residues in the C-terminal domains of the TCR-α and TCR-β chains are exchanged (“ "knob-in-hole"; and the variable domains of the TCR-alpha and TCR-beta chains were fused directly to CD3ζ (CD3ζ fusions) (Schmitt et al., 2009).

In one embodiment, the host cell is engineered to express the TCR of the present specification. In preferred embodiments, the host cells are human T cells or T cell precursor cells. In some embodiments, T cells or T cell precursor cell lines are obtained from cancer patients. In other embodiments, T cells or T cell precursor cell lines are obtained from healthy donors. With respect to the patient to be treated, the host cells of this specification may be allogeneic or autologous. In one embodiment, the host is a gamma/delta T cell transformed to express an alpha/beta TCR.

A "pharmaceutical composition" is a composition suitable for administration to humans in a medical setting. Preferably, the pharmaceutical composition is sterile and produced according to GMP guidelines.

The pharmaceutical composition comprises the peptide in free form or in the form of a pharmaceutically acceptable salt (see above). As used herein, "pharmaceutically acceptable salts" refer to derivatives of the disclosed peptides wherein the peptides are modified by making acid or base salts of the reagents. For example, acid salts are prepared from the free base (typically wherein the neutral form of the drug has a neutral _-NH2 group) involving reaction 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, salicylic acid, and the like; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid , nitric acid, phosphoric acid, and the like. Conversely, base salts of acid moieties that may be present on peptides are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, or the like preparation.

In a particularly preferred embodiment, the pharmaceutical composition comprises the peptide as a salt of acetic acid (acetate), trifluoroacetate or hydrochloric acid (chloride).

Preferably, the agent of the present invention is an immunotherapeutic agent such as a vaccine. The agent can be administered directly into the patient, into the affected organ, or systemically intravenous (i.v.), subcutaneous (s.c.), intradermal (i.d.), intraperitoneal (i.p.), intramuscular (i.m.) Administered or administered ex vivo to cells derived from a patient or human cell line and subsequently administered to the patient, or used in vitro to select a subset of patient-derived immune cells prior to administration to the patient. If the nucleic acid line is administered to cells in vitro, it can be applied to the cells to be transfected to co-express an immunostimulatory interleukin, such as interleukin-2. The peptides can be substantially pure or in combination with immunostimulatory adjuvants (see below) or used in combination with immunostimulatory interferons, or administered with a suitable delivery system such as liposomes. The peptides may also be conjugated to suitable carriers such as keyhole limpet hemocyanin (KLH) or mannan (see WO 95/18145 and Longenecker et al., 1993, both incorporated herein by reference in their entirety). Peptides can also be labeled, can be fusion proteins, or can be hybrid molecules. Peptides whose sequences are given in the present invention are expected to stimulate CD4 or CD8 T cells. However, stimulation of CD8 T cells is more effective in the presence of help provided by CD4 T helper cells. Thus, for MHC class I epitopes that stimulate CD8 T cells, fusion partners or slices of hybrid molecules are suitable to provide epitopes that stimulate CD4 positive T cells. CD4 and CD8 stimulating epitopes are well known in the art and include those epitopes identified in the present invention.

In one aspect, the vaccine comprises at least one peptide having the amino acid sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 113, and at least one additional peptide, preferably two to 50, more preferably two to 25, even better two to 20 and most preferably two, three, four, five, six, seven, eight, nine, ten, eleven , twelve, thirteen, fourteen, fifteen, sixteen, seventeen or eighteen peptides. Peptides can be derived from one or more specific TAAs and can bind to MHC class I molecules.

Another aspect of the invention provides nucleic acids (eg, polynucleotides) encoding the peptides or peptide variants of the invention. The polynucleotide can be, for example, DNA, cDNA, PNA, RNA, or a combination thereof, single- and/or double-stranded, or a polynucleotide (such as, for example, a polynucleotide having a phosphorothioate backbone) in its original or stabilized form, and it may or may not contain introns, so long as it encodes a peptide. Of course, only peptides containing naturally-occurring amino acid residues linked by naturally-occurring peptide bonds can be encoded by polynucleotides. Yet another aspect of the present invention provides an expression vector capable of expressing a polypeptide according to the present invention.

Various methods have been developed to ligate polynucleotides, especially DNA, to vectors, eg, via complementary sticky ends. For example, complementary homopolymer regions can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segments are then linked by hydrogen bonding between complementary homopolymer tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of linking DNA segments to vectors. Synthetic linkers containing various restriction enzymes are commercially available from a number of sources, including International Biotechnologies Inc. New Haven, CN, USA.

A desirable method of modifying DNA encoding the polypeptides of the invention uses the polymerase chain reaction as disclosed by Saiki et al. (Saiki et al., 1988, the contents of which are incorporated herein by reference in their entirety). This method can be used to introduce DNA into a suitable vector, eg, by engineering in suitable restriction sites, or can be used to modify DNA in other useful ways as known in the art. If a viral vector is used, a poxvirus or adenovirus vector system is preferred.

DNA (or RNA in the case of retroviral vectors) can then be expressed in a suitable host to produce polypeptides comprising the peptides or variants of the invention. Thus, DNA encoding the peptides or variants of the invention can be used according to known techniques, suitably modified in view of the teachings contained herein, to construct expression vectors which are then used to transform appropriate host cells for use in Expression and production of the polypeptides of the invention. Such techniques include, for example, those disclosed in US Pat.

DNA (or RNA, in the case of retroviral vectors) encoding the polypeptides constituting the compounds of the present invention can be linked to a variety of other DNA sequences for introduction into appropriate hosts. The companion DNA will depend on the identity of the host, the manner in which the DNA was introduced into the host, and whether episomal maintenance or integration is required.

Typically, the DNA is inserted into an expression vector, such as a plasmid, in the proper orientation and correct reading frame for expression. If necessary, the DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control sequences are commonly used in expression vectors. The vector is then introduced into the host via standard techniques. Typically, not all hosts will be converted by the vector. Therefore, selection will have to be made against the transformed host cells. One selection technique involves incorporating DNA sequences into an expression vector using any necessary control elements encoding selectable traits in transformed cells, such as antibiotic resistance.

Alternatively, the genes for such selectable traits can be on another vector that is co-transformed with the desired host cell.

The polypeptides can then be recovered in view of the classroom teachings disclosed herein by culturing host cells transformed with the recombinant DNA of the present invention under appropriate conditions known to those skilled in the art for a period of time sufficient to allow expression of the polypeptides.

Many expression systems are known, including bacteria (eg E. coli and Bacillus subtilis ), yeast (eg Saccharomyces cerevisiae ), filamentous fungi (eg Aspergillus spec . )), plant cells, animal cells and insect cells. Preferably, the system may be a mammalian cell such as CHO cells commercially available from the ATCC Cell Biology Collection.

Typical mammalian cell vector plasmids for constitutive expression contain a CMV or SV40 promoter with a suitable polyA tail and a resistance marker such as neomycin. An example is pSVL available from Pharmacia, Piscataway, NJ, USA. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia. Useful yeast plastid vectors are pRS403-406 and pRS413-416 and are commonly available from Stratagene Cloning Systems, La Jolla, 92037, USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integrating plasmids (YIps) and integrate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycp). CMV promoter-based vectors (eg, from Sigma-Aldrich) provide transient or stable expression, cytoplasmic expression or secretion, and N-terminal or C-terminal labeling in various combinations of FLAG, 3xFLAG, c-myc, or MAT. These fusion proteins allow the detection, purification and analysis of recombinant proteins. Double labeled fusions provide detection flexibility.

Strong human cytomegalovirus (CMV) promoter regulatory regions drive constitutive protein expression levels up to 1 mg/L in COS cells. For less intense cell lines, the protein level is typically about 0.1 mg/L. The presence of the SV40 origin of replication will result in high-level DNA replication in COS cells that allow SV40 replication. A CMV vector may contain, for example, a pMB1 (a derivative of pBR322) origin for replication in bacterial cells, b-lactamase, hGH polyA, and fl origin for ampicillin resistance selection in bacteria. Vectors containing the pre-pro-trypsin (PPT) leader sequence can direct secretion of FLAG fusion proteins into culture medium for purification using ANTI-FLAG antibodies, resins, and plates. Other vectors and expression systems are well known in the art for use with various host cells.

In another embodiment, two or more peptides or peptide variants of the invention are encoded in sequential order and behave accordingly (similar to a "beaded" construct). To do so, peptides or peptide variants can be linked or fused together by extending chains of linker amino acids, such as for example LLLLLL (SEQ ID NO: 229), or can be between peptides or peptide variants Linked without using any additional peptides. These constructs can also be used in cancer therapy and can induce immune responses involving both MHC I and MHC II.

The present invention also relates to host cells transformed with the polynucleotide vector constructs of the present invention. Host cells can be prokaryotic or eukaryotic. In some cases the bacterial cell may preferably be a prokaryotic host cell and is typically a strain of E. coli, eg, E. coli strain DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and commercially available from Rockville, RR1 (Accession ATCC 31343) of the American Type Culture Collection (ATCC), MD, USA. Preferred eukaryotic host cells include yeast, insect and mammalian mammalian cells, preferably vertebrate cells such as fibroblasts and colon cell lines from mouse, rat, monkey or human. Yeast host cells include YPH499, YPH500 and YPH501, which are commonly available from Stratagene Cloning Systems, La Jolla, CA 92037, USA. Preferred mammalian host cells include Chinese hamster ovary (CHO) cells which can be purchased from ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 which can be purchased from ATCC as CRL 1658, monkeys which can be purchased from ATCC as CRL 1650. Kidney-derived COS-1 cells and 293 cells, which are human embryonic kidney cells. Preferred insect cells are Sf9 cells, which can be transfected with a baculovirus expression vector. An overview of the selection of suitable host cells for expression can be found in the literature (Balbás and Lorence, 2004).

Transformation of an appropriate cellular host with the DNA constructs of the invention is accomplished by well-known methods, which typically depend on the type of vector used. For transformation of prokaryotic host cells, see, eg, Cohen et al. or Green and Sambrook (Cohen et al., 1972; Green and Sambrook, 2012). The transformation of yeast cells is described in Sherman et al. (Sherman et al., 1986). Beggs' method (Beggs, 1978) is also useful. With regard to vertebrate cells, reagents suitable for transfection of such cells, such as calcium phosphate and DEAE-polydextrose or liposomal preparations, are commercially available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. Electroporation is also suitable for transforming and/or transfecting cells and is well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells. The contents of each of these references are incorporated herein by reference in their entirety.

Successfully transformed cells, ie, cells containing the DNA constructs of the invention, can be identified by well-known techniques such as PCR. Alternatively, antibodies can be used to detect the presence of protein in the supernatant.

It will be appreciated that certain host cells of the present invention (eg, bacterial, yeast and insect cells) are suitable for use in preparing the peptides of the present invention. However, other host cells may be suitable for certain therapeutic approaches. For example, antigen presenting cells such as dendritic cells can be adapted to express the peptides of the invention so that they can be loaded into appropriate MHC molecules. Accordingly, the present invention provides host cells comprising a nucleic acid or expression vector according to the present invention.

In a preferred embodiment, the host cells are antigen-presenting cells, especially dendritic cells or antigen-presenting cells. APC containing a recombinant fusion protein containing prostatic acid phosphatase (PAP) was approved by the U.S. Food and Drug Administration (FDA) on April 29, 2010 for the treatment of asymptomatic or symptomatic Minimal metabolic HRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006, the contents of which are hereby incorporated by reference in their entirety).

Another aspect of the present invention provides a method of producing a peptide or variant thereof, the method comprising culturing a host cell and isolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, nucleic acid or expression vector of the invention is used in medicine. For example, peptides or variants thereof can be prepared for i.v., s.c., i.d., i.p., i.m. injection. Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v. Preferred methods of DNA injection include i.d., i.m., s.c., i.p., and i.v. Peptide or DNA can be administered, for example, in doses between 50 μg and 1.5 mg, preferably 125 μg to 500 μg and the dose will depend on the corresponding peptide or DNA. Doses in this range were successfully used in previous trials (Walter et al., 2012).

The polynucleotides used for live vaccination can be substantially pure or contained in a suitable carrier or delivery system. The nucleic acid can be DNA, cDNA, PNA, RNA, or a combination thereof. Methods for designing and introducing such nucleic acids are well known in the art. An overview is provided by Teufel et al. (Teufel et al., 2005, the contents of which are incorporated herein by reference in their entirety). Polynucleotide vaccines are easy to prepare, but the mode of action of these vectors in inducing immune responses is not fully understood. Suitable vectors and delivery systems include viral DNA and/or RNA, such as systems based on adenovirus, pox virus, retrovirus, herpes virus, adeno-associated virus, or hybrids containing elements of more than one virus. Non-viral delivery systems include cationic lipids and cationic polymers and are well known in DNA delivery technology. Physical delivery such as via a "gene gun" may also be used. The one or more peptides encoded by the nucleic acid can be a fusion protein, eg, having an epitope that stimulates T cells with corresponding opposite CDRs as described above.

The agents of the present invention may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate immune responses, such as those mediated by CD8-positive T cells and helper T (TH) cells, to antigens, and are therefore considered suitable for use in this study. The drug of invention.

Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligand derived from flagellin, FLT3 ligand, GM- CSF, IC30, IC31, Imiquimod (ALDARA®), Resiquimod, ImuFact IMP321, Interleukins such as IL-2, IL-13, IL-21, Interferon-alpha or -beta, or polyethylene glycol thereof Alcoholated Derivatives, IS Patch, ISS, ISCOMATRIX, ISCOMs, JuvImmune®, LipoVac, MALP2, MF59, Monophosphoryl Lipid A, Montagnier IMS 1312, Montagnier ISA 206, Montagny ISA 50V, Montagny ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® carrier systems, based on poly(lactic acid copolyglycolic acid) [PLG] and Polyglucose microparticles, bovine lactoferrin SRL172, viroids and other viroids, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulator (derived from saponin), Mycobacterial sense extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's adjuvant GM-CSF are preferred. Several immune adjuvants (eg, MF59) specific to dendritic cells and their preparations have been described previously (Allison and Krummel, 1995, the contents of which are incorporated herein by reference in their entirety).

Cytokines can also be used. Several interferons have been implicated directly in affecting the migration of dendritic cells to lymphoid tissues (eg, TNF), promoting the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (US Pat. No. 5,849,589, which is incorporated in its entirety). incorporated herein by reference) (eg, GM-CSF, IL-1, and IL-4) and act as immune adjuvants (eg, IL-2, IL-7, IL-12, IL-15, IL-21, IL-4) -23, IFN-alpha, IFN-beta) (Gabrilovich et al., 1996, the contents of which are incorporated herein by reference in their entirety). In one aspect, interleukins and immune adjuvants can be used in vitro, such as for expansion or activation of T cells, or for ex vivo use.

CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in a vaccine setting. Without being bound by theory, CpG oligonucleotides activate the innate (non-adaptive) immune system by passing through Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, in both prophylactic and therapeutic vaccines , Autologous cell vaccines and polysaccharide conjugates. More importantly, it enhances dendritic cell maturation and differentiation, resulting in enhanced activation of TH1 cells and strong cytotoxic T-lymphocyte (CTL) passage, even in the absence of CD4 T cell help so below. TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote TH2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, especially when the antigen is relatively weak necessary to induce a strong response. It also boosted immune responses and enabled antigen dose reductions by about two orders of magnitude, while in some experiments had comparable antibody responses to full-dose vaccines without CpG (Krieg, 2006). US 6,406,705 B1, incorporated herein by reference in its entirety, describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. The CpG TLR9 antagonist is dSLIM (dual stem-loop immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules can also be used, such as RNA binding TLR 7, TLR 8 and/or TLR 9.

Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (eg, CpR, Idera), such as dsRNA analogs of poly(I:C) and derivatives thereof (eg, AmpliGen®, Hiltonol®, poly(ICLC) , poly(IC-R), poly(I:C12U)), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, immune checkpoint inhibitors including ipil mAb, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and mipilimumab, bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting key structures of the immune system (eg, anti-CD40, anti-TGF-beta, anti-TNF-alpha receptors) and SC58175, which may act therapeutically or act as adjuvants . The amounts and concentrations of adjuvants and additives suitable for use in the context of the present invention can be readily determined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, requimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides and derivatives compounds, poly(I:C) and derivatives, RNA, sildenafil, and microparticle formulations with PLG or viroids.

In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is selected from the group consisting of: such as Granulocyte Macrophage Colony Stimulating Factor; GM-CSF, sand glostim), cyclophosphamide, imiquimod, resiquimod, interferon-alpha, or a mixture thereof.

In a preferred embodiment of the pharmaceutical composition according to the present invention, the adjuvant is cyclophosphamide, imiquimod or requimod. Even better adjuvants are Montagnier IMS 1312, Montagnier ISA 206, Montagny ISA 50V, Montagny ISA-51, poly ICLC (Hiltonol®) and anti-CD40 mAbs, or combinations thereof.

This composition is for parenteral administration, such as subcutaneous, intradermal, intramuscular or oral administration. The compositions can be administered via subcutaneous, intramuscular, intravenous, intraperitoneal, intrapleural, intravesicular, intrathecal, topical, oral administration, or a combination route. For this purpose, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. Additionally, the compositions may contain excipients such as buffers, binders, blasting agents, diluents, flavors, lubricants, and the like. Pharmaceutically acceptable carriers include, but are not limited to, excipients, lubricants, emulsifiers, stabilizers, solvents, diluents, buffers, vehicles, or combinations thereof. Peptides or T cells that recognize the peptides of the present disclosure in complexes with MHC molecules can also be administered in conjunction with immunostimulatory substances such as the cytokines shown in Table 6.

Table 6: Immune Stimulatory Interleukins interleukin EOTAXIN IL-15 G-CSF IL-17 GM-CSF IP-10 INF-γ MIP-2 IL-1α KC M-CSF LIF IL-1β LIX IL-2 MCP-1 IL-3 MIP-1α IL-4 MIP-1β IL-5 MIG IL-6 RANTES IL-7 TNFα IL-10 IL-12(P70) IL-12(p40) VEGF IL-123 IL-9 IL-18 IL-21

Interkinins, such as IL-2, IL-7, IL-12, IL-15, IL-21, IFN-α, and IFN-β can also be used to activate and/or expand T cells, such as T cells of the peptides of the present disclosure in complexes of molecules.

An extensive list of excipients that can be used in such compositions can be obtained, for example, from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Additional examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (Gennaro, 1997; Banker and Rhodes, 2002, the contents of which are incorporated by reference in their entirety). The compositions can be used to prevent, prevent and/or treat adenomatous disease or cancerous disease. Exemplary formulations can be found, for example, in EP2112253.

It is important to achieve that the immune response triggered by the vaccine according to the invention attacks the cancer at different cellular stages and at different developmental stages. In addition, attacking different cancer-associated signaling pathways. This is an advantage over vaccines targeting only one or a few targets, which cause tumors to readily adapt to challenge (tumor escape). Furthermore, not all individual tumors exhibit the same pattern of antigens. Thus, the combination of several tumor-associated peptides ensures that each individual tumor carries at least some targets. The compositions were designed in such a way that each tumor was expected to express several antigens and cover several independent pathways necessary for tumor growth and maintenance. Thus, vaccines can easily be used "off the shelf" for larger patient populations. This means that the pre-selected patients to be treated with the vaccine can be limited to HLA types, without any additional biomarkers for antigen presentation, but still ensure that several targets are simultaneously attacked by the induced immune response, from which effects on efficacy Language is important (Banchereau et al., 2001; Walter et al., 2012, the contents of which are hereby incorporated by reference in their entirety).

As used herein, the term "scaffold" refers to a molecule that specifically binds to, eg, an antigenic determinant. In one embodiment, the scaffold is capable of directing an entity to which a (eg, a second) antigen binding moiety is attached to a target site, such as to a specific type of tumor cell or tumor stroma bearing an antigenic determinant ( For example complexes of peptides and MHC, according to the present application). In another embodiment, the scaffold is capable of activating signaling through its target antigen (eg, T cell receptor complex antigen). Scaffolds include, but are not limited to, antibodies and fragments thereof, antigen binding domains of antibodies (including antibody heavy chain variable regions and antibody light chain variable regions), including at least one ankyrin repeat motif, and single domain antigen binding ; SDAB) molecules binding proteins, aptamers, (soluble) TCRs and (modified) cells such as allogeneic or autologous T cells. To assess whether a molecule is a scaffold that binds to a target, a binding assay can be performed.

"Specific" binding means that the scaffold binds the peptide-MHC complex of interest better than other naturally occurring peptide-MHC complexes to the extent that it is equipped with an active molecule capable of killing cells with a specific target The scaffold did not kill another cell that could not have a specific target, but presented other peptide MHC complexes. Binding to other peptide-MHC complexes is irrelevant if the cross-reactive peptide-MHC peptide is not naturally occurring, ie, is not derived from human HLA peptidomics. Assays to assess target cell killing are well known in the art. Such tests should be performed using target cells (primary cells or cell lines) with unaltered peptide-MHC presentation, or cells loaded with peptides such that naturally occurring peptide-MHC levels are reached.

Each scaffold can include a marker that provides: The bound scaffold can be detected by determining the presence or absence of the signal provided by the marker. For example, scaffolds can be labeled with fluorescent dyes or any other applicable cell marker molecules. Such marker molecules are well known in the art. For example, fluorescent labels provided by, for example, fluorescent dyes can provide visualization of bound aptamers by fluorescence or laser scanning microscopy or flow cytometry.

Each scaffold can, for example, be associated with a second active molecule such as IL-21, anti-CD3, and anti-CD28.

For additional information on polypeptide scaffolds, see, eg, the Background section of WO 2014/071978 A1 and the references cited therein, the contents of which are incorporated herein by reference in their entirety.

The peptides of the present invention can be used to generate and develop specific antibodies against MHC-peptide complexes. These antibodies can be used in therapy, targeting toxins or radioactive substances to diseased tissue. Another use of these antibodies may be to target radionuclides to diseased tissue for imaging purposes such as PET. This use can help detect small metastases or determine the size and precise location of diseased tissue.

Accordingly, another aspect of the present invention provides a method for producing recombinant antibodies that specifically bind to MHC class I or II molecules complexed with an HLA-restricted antigen, preferably a peptide according to the invention, the method comprising: utilizing A soluble form of the MHC class I or II molecule complexed with the HLA-restricted antigen immunizes a genetically engineered non-human mammal comprising cells expressing the human MHC class I or II molecule; isolation from antibody producing cells of the non-human mammal mRNA molecules; generating a phage display library that exposes protein molecules encoded by the mRNA molecules; and isolating at least one bacteriophage from the phage display library, the at least one phage display specifically binding to the MHC class I complexed with the HLA-restricted antigen or the antibody of the II molecule.

Another aspect of the present invention therefore provides antibodies that specifically bind to MHC class I or II molecules complexed with HLA-restricted antigens, wherein the antibodies are preferably polyclonal, monoclonal, bispecific, chimeric An antibody, an antibody fragment thereof, or a combination thereof. bispecific antibody

In one aspect, bispecific antibodies include antibodies capable of selectively binding two or more epitopes. Bispecific antibodies can be made in various ways (Holliger & Winter, 1993, the contents of which are incorporated by reference in their entirety), for example, chemically or from hybrid fusion tumors, or can be any bispecific antibody as described above. specific antibody fragments. Instead of whole antibodies, scfv dimers or diabodies can be used. Diabodies and scFvs can be constructed without Fc regions, using only variable domains (usually including variable domain components from the light and heavy chains of the source antibody), potentially reducing the effects of anti-genotypic responses. Other forms of bispecific antibodies include the single-chain "Janusins" described by Traunecker and colleagues (Traunecker et al., 1991, the contents of which are incorporated herein by reference in their entirety).

Bispecific antibodies typically comprise two distinct binding domains, wherein each binding domain specifically binds to two different antigens or to different epitopes on the same antigen. If the bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the first binding domain will generally have a lower affinity for the first epitope than the first At least one to two or three or four orders of magnitude of the binding domain's affinity for the second epitope, and vice versa. The epitopes recognized by the bispecific antibodies can be on the same or different targets (eg, on the same or different proteins). Bispecific antibodies can be made by combining binding domains that recognize different epitopes of the same antigen.

Some exemplary bispecific antibodies have two heavy chains (each with three heavy chain CDRs, followed by (N-terminal to C-terminal) CH1 domain, hinge, CH2 domain, and CH3 domain), and two immune A globulin light chain that confers antigen-binding specificity through association with each heavy chain. However, alternative architectures are envisaged, including bispecific antibodies, in which a light chain is associated with each heavy chain but does not (or minimally) contribute to antigen-binding specificity, or the light chain can bind through the heavy chain antigen-binding region One or more of the epitopes, or light chains, can be associated with each heavy chain and enable one or both of the heavy chains to bind to one or both epitopes.

In certain embodiments, bispecific antibodies may include antibody arms that combine with arms that bind to trigger molecules, such as T cells, on leukocytes in order to focus and localize cellular defense mechanisms to targeted disease cells Receptor molecules (eg, CD3), or Fc receptors for IgG (FcyRs), such as FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD 16). Bispecific antibodies can also be used to localize cytotoxic agents to targeted disease cells.

Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibodies). See, eg, WO 1996/016673; US Patent No. 5,837,234; WO 1998/002463; US Patent No. 5,821,337, the contents of which are incorporated herein by reference in their entirety.

Bispecific antibodies can have extended half-lives. In particular embodiments, half-life delay of bispecific antibodies can be achieved by increasing the hydrodynamic bulk of the antibody by coupling to an inert polymer such as polyethylene glycol or other pseudomorphic hydrophilic polymers; fusion or Conjugation to large disordered peptides; or fusion or coupling of antibodies to ligands. These and numerous other variations are described elsewhere (US Patent No. 7,083,784, US Patent No. 7,670,600, US Patent Application Publication No. 2010/0234575, and Zwolak et al., 2017, the contents of which are in their entirety incorporated herein by reference). Bispecific antibodies with extended half-life are described, for example, in US Patent No. 8,921,528 and US Patent Application Publication No. 2014/0308285, the contents of which are incorporated herein by reference in their entirety.

Methods for making bispecific antibodies are known in the art. The production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., 1983, WO 1993/008829, Traunecker et al., 1991 ; the contents of which are incorporated herein by reference in their entirety). polyclonal antibody

Methods for making polyclonal antibodies are known in the art. A polyclonal antibody is a heterogeneous population of antibody molecules derived from the serum of an animal immunized with an antigen. Polyclonal antibodies that selectively bind to peptides according to SEQ ID NO: 1 to SEQ ID NO: 113 or variants or fragments thereof can be made by methods well known in the art (see, e.g., Howard & Kaser (2007), It is hereby incorporated by reference in its entirety). Chimeric antibody

Chimeric antibodies are molecules whose different parts are derived from different animal species, such as those with variable regions derived from murine antibodies and human immunoglobulin constant regions, which are primarily used to reduce immunogenicity and increased production yields, eg, where murine monoclonal antibodies have higher yields from fusion tumors but are more immunogenic in humans, allowing the use of human murine chimeric monoclonal antibodies. Chimeric antibodies and methods for their production are known in the art (Cabilly et al, 1984; Morrison et al, 1984; Boulianne et al, 1984; European Patent Application 173494 (1986); WO 86/01533 (1986) European Patent Application 184187 (1986); Sahagan et al, 1986; Liu et al, 1987; Sun et al, 1987; Better et al, 1988; Harlow & Lane, 1998; is incorporated herein by reference in its entirety). Antibody fragment

In addition to intact immunoglobulins (or their recombinant counterparts), immunoglobulins that contain epitope binding sites (eg, Fab, F(ab') ₂ , or other fragments) can be synthesized. "Fragments" or minimal immunoglobulins can be designed using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in the present invention can be produced by synthetically fusing variable light and variable heavy chain regions. Combinations of antibodies are also of interest, eg, diabodies, comprising two distinct Fv specificities. Antigen-binding fragments of immunoglobulins include, but are not limited to, SMIPs (small molecule immunopharmaceuticals), camelid antibodies, nanobodies, and IgNARs.

Corresponding methods for producing such antibodies and single-chain MHC class I complexes, as well as tools for producing such antibodies, are disclosed in WO 03/068201, WO 2004/084798, WO 01/72768, WO 03/070752, and publications (Cohen et al., 2003a; Cohen et al., 2003b; Denkberg et al., 2003, which are hereby incorporated by reference in their entirety for purposes of the present invention).

Preferably, the antibody binds with a binding affinity of <100 nM, more preferably <50 nM, more preferably <10 nM, more preferably <1 nM, more preferably <0.1 nM, more preferably <0.01 nM complexes, which are also considered "specific" in the context of the present invention.

The present invention relates to peptides comprising amino acid sequences selected from the group consisting of ● SEQ ID NO: 1 to SEQ ID NO: 113, ● and variant sequences thereof, which maintain the ability to bind to MHC molecules and/or induce cross-reactivity of T cells with the variant peptide, or a pharmaceutically acceptable salt thereof.

The disclosed peptides bind to at least one of: HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA-B*07:02, HLA-B*08:01 and HLA-B*44:02, plus other HLA atypia as appropriate. Likewise, the ability of peptide variants to bind to molecules of the major histocompatibility complex (MHC) is described for HLA-A*01:01, HLA-A*03:01, HLA-A*24: 02, HLA-B*07:02, HLA-B*08:01, and HLA-B*44:02, plus other HLA atypia as appropriate.

In one embodiment, variant sequences of the above claimed peptides are meant to have substitutions in their so-called "anchor positions".

It should be noted that these anchor positions in the MHC restricted peptide comprise amino acid residues that mediate the binding of the peptide to the peptide binding groove in the MHC.

It only plays a small role in the binding reaction between the binding polypeptide and the peptide-MHC complex, meaning that substitutions in these positions do not significantly affect the immunogenic TCR/antibody binding of peptides modified in this way.

The following table (Table 7) shows the anchoring positions of the peptides and binding HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA-B*07:02, Preferred/accepted amino acid residues for HLA-B*08:01 and HLA-B*44:02.

Table 7: Anchor positions and preferred amino acids in these positions for the above HLA alleles. HLA subtypes Anchor position 1 Accepted amino acid residues Anchor position 2 Accepted amino acid residues Anchor position 3 Accepted amino acid residues HLA-A*01:01 position 2 Threonine (T) Serine (S) position 3 Aspartic acid (D) Glutamic acid (E) C-terminal Tyrosine (Y) HLA-A*03:01 position 2 Leucine (L) Valine (V) Not applicable Not applicable C-terminal Lysine (K) HLA-A*24:02 position 2 Tyrosine (Y) Not applicable Not applicable C-terminal Phenylalanine (F) HLA-B*07:02 position 2 Proline (P) Not applicable Not applicable C-terminal Leucine (L) HLA-B*08:01 position 3 Lysine (K) position 5 Lysine (K) Arginine (R) C-terminal Leucine (L) HLA-B*44:02 position 2 Glutamic acid (E) Not applicable Not applicable C-terminal Tryptophan (W) Phenylalanine (F) Tyrosine (Y)

Due to similarities in binding patterns, such as similarity in relative anchoring positions, some peptides bind to more than one allele, and this overlap is likely, but not limited to, HLA-A* which also binds to HLA-B*15 01 binding peptide, HLA-A*03 binding peptide also bound to HLA-A*11, HLA-B*07 binding peptide also bound to HLA-B*35 and HLA-B*51.

Furthermore, the present invention relates to variants thereof that are at least 88% homologous to SEQ ID NO: 1 to SEQ ID NO: 113, provided that they bind to MHC molecules and/or induce T cells to cross-react with the variant peptide.

In one embodiment, variant sequences of peptides claimed above mean sequences modified by at least one conservative amino acid substitution. The definition and scope of the term "conservative amino acid substitutions" are disclosed in Tables 3 and 4 and the descriptions associated therewith.

In one embodiment, the peptide has the ability to bind to MHC class 1 molecules and, when bound to the MHC, is capable of being recognized by CD4 and/or CD8 T cells.

In one embodiment, the MHC class I molecule is at least one selected from the group consisting of: HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA -B*07:02, HLA-B*08:01 and HLA-B*44:02, plus other HLA isoforms as appropriate.

The present invention further relates to peptides comprising sequences selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 113 or variants thereof, which are at least 88% different from SEQ ID NO: 1 to SEQ ID NO: 113 Homologous (preferably identical), wherein the peptide or variant has between 8 and 30 amino acids, and preferably between 8 and 12 amino acids, or if selected The peptide has a length of 9 amino acids, then between 9 and 30 amino acids, and preferably between 9 and 12 amino acids, or if the selected peptide has 10 amino acids The length of the acid is then between 10 and 30, and preferably between 10 and 12 amino acids.

In one embodiment, the peptide or variant thereof comprises 1 to 4 additional amino acids at the C-terminus and/or the N-terminus of the corresponding sequence. See Table 5 for additional details.

In one embodiment, the peptide or variant thereof is up to 30 amino acids in length. In one embodiment, the peptide or variant thereof is up to 16 amino acids in length. In one embodiment, the peptide or variant thereof is up to 12 amino acids in length.

In one embodiment, the peptide or variant thereof has a total length of 8 to 30 amino acids. In one embodiment, the peptide or variant thereof has a total length of 8 to 16 amino acids. In one embodiment, the peptide or variant thereof has a total length of 8 to 12 amino acids.

In one embodiment, the peptide or variant thereof has a total length of 9 to 30 amino acids. In one embodiment, the peptide or variant thereof has a total length of 9 to 16 amino acids. In one embodiment, the peptide or variant thereof has a total length of 9 to 12 amino acids.

In one embodiment, the peptide or variant thereof has a total length of 10 to 30 amino acids. In one embodiment, the peptide or variant thereof has a total length of 10 to 16 amino acids. In one embodiment, the peptide or variant thereof has a total length of 10 to 12 amino acids.

In one embodiment, the peptide or variant thereof has a length according to corresponding SEQ ID NO: 1 to SEQ ID NO: 113. In one embodiment, the peptide consists or consists essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 113.

The present invention further relates to a peptide according to the invention, wherein the peptide comprises a non-peptide bond.

The invention further relates to a peptide according to the invention, wherein the peptide is part of a fusion protein, which in particular comprises the N-terminal amino acid of the invariant chain (Ii) associated with the HLA-DR antigen, or wherein the peptide is fused to an antibody ( or fused into an antibody) such as, for example, an antibody specific for dendritic cells.

The present invention further relates to antibodies or functional fragments thereof, which specifically recognize or bind to a peptide according to the invention or a variant thereof, or when bound to an MHC molecule, to a peptide according to the invention or a variant thereof.

In one embodiment, the MHC molecule is at least one selected from the group consisting of: HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA-B *07:02, HLA-B*08:01 and HLA-B*44:02 isotype MHC molecules, plus other HLA isotypes as appropriate.

In other embodiments, the antibody is soluble or membrane bound. In other embodiments, the antibody is a monoclonal antibody or fragment thereof. In other embodiments, the antibody carries another effector function such as an immunostimulatory domain or a toxin.

The present invention further relates to a T cell receptor or a functional fragment thereof, which reacts with or binds to an MHC ligand, wherein the ligand is a peptide according to the invention or a variant thereof, or is bound to an MHC ligand The molecule is a peptide according to the invention or a variant thereof.

In one embodiment, the MHC molecule is at least one selected from the group consisting of: HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA-B *07:02, HLA-B*08:01 and HLA-B*44:02, plus other HLA isoforms as appropriate.

In other embodiments, the T cell receptor system is provided as a soluble molecule. In other embodiments, the T cell receptor carries another effector function such as an immunostimulatory domain or toxin.

The present invention further relates to nucleic acids encoding peptides or variants thereof according to the invention, antibodies or fragments thereof according to the invention, or T cell receptors or fragments thereof according to the invention.

The present invention further relates to a nucleic acid according to the present invention, which is DNA, cDNA, PNA, RNA or a combination thereof.

In one embodiment, the nucleic acid is linked to a heterologous promoter sequence. In one embodiment, the nucleic acid is provided as an expression vector that expresses and/or comprises the nucleic acid.

The invention further relates to recombinant host cells comprising a peptide according to the invention or a variant thereof, an antibody or fragment thereof according to the invention, a T cell receptor or a fragment thereof according to the invention, or a nucleic acid or expression according to the invention vector.

The invention further relates to an in vitro method for generating activated T lymphocytes, the method comprising combining T cells with antigen-loaded human class I or II expressed on the surface of an artificial construct suitable for antigen-presenting cells or mimicking antigen-presenting cells The MHC-like molecules are contacted in vitro for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide or a variant thereof according to the corresponding description.

In one embodiment, the antigen presenting cell comprises an expression vector capable of expressing the peptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 113 or the variant.

The present invention further relates to an in vitro method for producing a peptide according to the present invention, the method comprising culturing a host cell according to the present invention, and isolating the peptide from the host cell or its culture medium.

The present invention further relates to activated T lymphocytes produced by the method according to the present invention, wherein the T lymphocytes selectively recognize cells presenting the peptides according to the present invention or variants thereof. The presentation may be abnormal presentation or abnormal performance.

The present invention further relates to pharmaceutical compositions comprising at least one active ingredient selected from the group consisting of ● a peptide according to the invention or a variant thereof, ● an antibody or fragment thereof according to the invention, ● a T cell receptor according to the invention or a fragment thereof, ● a nucleic acid or expression vector according to the invention, ● a host cell according to the invention, ● or activated T lymphocytes according to the invention, and a pharmaceutically acceptable carrier.

In one embodiment, the pharmaceutical composition is a personalized pharmaceutical composition for an individual patient. In one embodiment, the pharmaceutical composition is a vaccine. The methods may also be adapted to generate T cell clones for downstream applications such as TCR isolation, or soluble antibodies, and other therapeutic options.

"Personalized Pharmacy" will mean therapy specifically tailored to an individual patient, which will be used only for that individual patient's treatment, including actively personalized cancer vaccines and donor-receptor cell therapy using autologous patient tissue.

The present invention further relates to the production of peptides according to the invention or variants thereof, antibodies or fragments thereof according to the invention, or T cell receptors or fragments thereof according to the invention and isolation from the host cells and/or their culture medium Methods of peptides or variants thereof, antibodies or fragments thereof, or T cell receptors or fragments thereof.

The present invention further relates to peptides according to the invention or variants thereof, antibodies or fragments thereof according to the invention, T cell receptors or fragments thereof according to the invention, nucleic acids or expression vectors according to the invention, hosts according to the invention Cells, or activated T lymphocytes according to the present invention, which are used for medicaments or for the manufacture of medicaments.

The present invention further relates to a method of killing target cells in a patient which abnormally express a polypeptide comprising any amino acid sequence according to the present invention. The method comprises administering to the patient an effective amount of activated T lymphocytes as in accordance with the present invention.

Likewise, the present invention relates to activated T lymphocytes according to the present invention, which are used to kill target cells of a patient, which target cells present a polypeptide comprising any amino acid sequence according to the present invention, or for the manufacture of Agents that kill such target cells.

The present invention further relates to a method of treating a patient, the patient ● diagnosed with cancer, ● have cancer, or ● risk of developing cancer, The method comprises administering to a patient an effective amount of a peptide according to the invention or a variant thereof, an antibody or fragment thereof according to the invention, a T cell receptor or fragment thereof according to the invention, a nucleic acid or expression vector according to the invention, Host cells according to the invention, or activated T lymphocytes according to the invention.

Likewise, the present invention further relates to peptides according to the invention or variants thereof, antibodies or fragments thereof according to the invention, T cell receptors or fragments thereof according to the invention, nucleic acids or expression vectors according to the invention, Host cells of the invention, or activated T lymphocytes according to the invention, for use in the treatment of a patient, the patient ● diagnosed with cancer, ● have cancer, or ● risk of developing cancer, or for the manufacture of a medicament for the treatment of such a patient.

The present invention further relates to the use according to the invention, wherein the medicament is a vaccine.

The present invention further relates to methods or peptides, antibodies, T cell receptors, nucleic acids, host cells or activated T lymphocytes for use according to the present invention, wherein the cancer is selected from the group consisting of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer.

The present invention further relates to a kit comprising: (a) a container containing a pharmaceutical composition containing the pharmaceutical composition according to the invention in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; (c) Optionally, at least one or more peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 113.

In other embodiments, the kit includes one or more of buffers, diluents, filters, needles, or syringes.

The present invention further relates to specific marker proteins and biomarkers based on the peptides according to the present invention which are herein "targeted", which can be used for the diagnosis and/or prognosis of at least one of the following: acute myeloid leukemia, breast cancer, cholangiocytes Carcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's Lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer.

The terms "antibody" or "antibodies" are used broadly herein and include both polyclonal and monoclonal antibodies. In addition to intact or "complete" immunoglobulin molecules, the term "antibody" also includes fragments of such immunoglobulin molecules (eg, CDR, Fv, Fab and Fc fragments) or polymers and human origins of immunoglobulin molecules A modified version, so long as it exhibits any desired property according to the invention (eg, specific binding to acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma , gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, Renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer marker polypeptide).

Wherever possible, antibodies of the invention can be purchased from commercial sources. Antibodies of the present invention can also be produced using well-known methods. Skilled artisans will understand full-length acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and cervical squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and intrauterine cancer Membrane cancer marker polypeptides or fragments thereof can be used to generate the antibodies of the present invention. The polypeptides used to generate the antibodies of the invention can be partially or completely purified from natural sources or can be produced using recombinant DNA techniques.

For example, a cDNA encoding a peptide according to the invention, such as a peptide according to a polypeptide of SEQ ID NO: 1 to SEQ ID NO: 113 or a variant or fragment thereof, can be expressed in prokaryotic cells (eg, bacteria) or eukaryotic cells (eg, yeast , insect, or mammalian cells), after which the recombinant protein can be purified and used to generate monoclonal or polyclonal antibody preparations that specifically bind to acute myeloid leukemia, breast cancer, Cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodge King's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer, bladder cancer, and endometrial cancer marker polypeptides.

Those skilled in the art will achieve: maximizing the passage of two or more distinct sets of monoclonal or polyclonal antibodies for their intended use (eg, ELISA, immunohistochemistry, in vivo imaging, immunotoxin therapy) Possibility of antibodies with desired specificity and affinity. Antibodies are tested for the desired activity by known methods, depending on the purpose for which they are intended to be used (eg, ELISA, immunohistochemistry, immunotherapy, etc. (Greenfield, 2014, the contents of which are incorporated by reference in their entirety). For example, antibodies Can be tested in ELISA assay or Western blot, immunohistochemical staining of formalin-fixed cancer or frozen tissue sections. Following its initial in vitro characterization, antibodies intended for therapeutic or in vivo diagnostic use are tested according to known clinical test methods .

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, comprising the individual antibodies of the population that are identical except for possible naturally occurring mutations that may be present in small amounts . Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and The remainder of the chains are identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired antagonistic activity (US 4,816,567 , which is incorporated herein by reference in its entirety).

Monoclonal antibodies of the present invention can be prepared using the fusion tumor method. In the fusionoma approach, a mouse or other suitable host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

Monoclonal antibodies can also be made by recombinant DNA methods such as those described in US 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be easily isolated using conventional procedures (eg, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies) and Sequencing.

In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art. For example, digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 and US 4,342,566, the contents of which are incorporated herein by reference in their entirety. Papain digestion of an antibody typically yields two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site and residual Fc fragment. Pepsin treatment produces F(ab') ₂ fragments and pFc' fragments.

Antibody fragments, whether or not linked to other sequences, may also include insertions, deletions, substitutions, or other selected modifications of specific regions or specific amino acid residues, provided that the activity of the fragment is not comparable to that of the unmodified antibody or antibody fragment. Not significantly altered or impaired. These modifications may provide some additional properties to remove/add disulfide capable amino acids, increase their biological longevity, alter their secretory properties, and the like. In any event, the antibody fragment must possess biologically active properties, such as binding activity, modulation of binding at the binding domain, and the like. Functional or active regions of antibodies can be identified by mutagenesis of specific regions of the protein, followed by expression and testing of the expressed polypeptide. Such methods will be apparent to those skilled in the art and may include site-specific mutagenesis of nucleic acids encoding antibody fragments.

The antibodies of the present invention may further comprise humanized antibodies or human antibodies. Humanized forms of non-human (eg, murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab' or other antigen-binding subsequences of antibodies) that contain Minimal sequence of non-human immunoglobulins. Humanized antibodies include human immunoglobulins (acceptor antibodies) in which residues from a complementary determining region (CDR) of the acceptor are determined by CDRs from a non-human species (donor antibody) with the desired specificity , affinity, and ability to replace residues in non-human species such as mouse, rat or rabbit. In some instances, Fv framework (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues found neither in the recipient antibody nor in the introduced CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one and typically both variable domains, wherein all or substantially all of the CDR regions correspond to those CDR regions of a non-human immunoglobulin, and the FR regions All or substantially all of them are those FR regions of human immunoglobulin consensus sequences. A humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), which is typically that of a human immunoglobulin.

Methods for humanizing non-human antibodies are well known in the art. Typically, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "introduced" residues, which are typically obtained from an "introduced" variable domain. Humanization can be performed essentially by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (US 4,816,567, the contents of which are incorporated herein by reference in their entirety) in which substantially less than complete human variable domains have been identified by corresponding sequences from non-human species replace. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from analogous sites in rodent antibodies.

Genetically transgenic animals (eg, mice) can be used which, after immunization, are capable of producing a complete immune profile of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that isotype combined deletion of antibody heavy chain linking regions in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into this germline mutant mouse will result in the production of human antibodies following antigen challenge. Human antibodies can also be produced in phage-displayed libraries.

In one aspect, antibodies of the present disclosure may also be obtained via phage exposure, or ribosome exposure, or yeast exposure, or bacterial exposure, or baculovirus exposure, or mammalian cell exposure, or mRNA exposure. These methods are all known techniques in the art, and the specific operations can be found in corresponding textbooks or operation manuals (Mondon et al., 2008; the content of which is incorporated herein by reference in its entirety). Using, for example, phage display, individual antibody genes can be inserted into the phage's DNA so that the antigen-binding variable regions of the antibody molecule can be coupled to the phage coat protein. After phage infection of E. coli, single-stranded DNA can be replicated in E. coli, and the phage can be reassembled and secreted into the medium without E. coli being lysed. Phage can be co-incubated with the target antigen; and after isolation of binding phage, amplification and purification can then be performed so that large numbers of clones can be screened. Phage exposure techniques are available in the literature (Liu et al., 2004, and US20100021477; the contents of which are incorporated herein by reference in their entirety).

In another aspect, the present disclosure can include methods for producing monoclonal antibodies using phage display methods. Specifically, mRNA can be prepared from animals immunized by the methods used to immunize animals, such as rabbits, rats, mice, guinea pigs, hamsters, goats, horses, chickens, sheep, and camels (eg, llama), after immunizing the animal, mRNA can be used as a template to prepare cDNA so that single-chain antibody (scFv) genes encoding only antibody variable regions can be prepared. The gene can be cloned into a phagemid vector. E. coli into which the phagemid vector was transduced was infected with phage to express the scFV antibody on the phage capsid. Screening of scFvs expressed in this manner against antigenic proteins or against peptide-MHC complexes can generate monoclonal scFV antibodies specific for the antigenic proteins or peptide-MHC complexes. In this context, mRNA preparation, cDNA preparation, sub-colonization into phagemid or transduction into E. coli, phage infection, and screening for monoclonal scFV antibodies specific for antigenic protein or peptide-MHC complexes can each be performed by known methods. For example, sub-cloning of the scFV gene into a phagemid vector containing two elements consisting of a gene fragment encoding a leader sequence (signal sequence) and phage coat protein III and an origin of replication for M13 and using M13 phage as phage can be used in M13 scFV antibodies are expressed on phage. In addition, phages obtained by screening can be infected with specific bacteria and cultured so that monoclonal antibodies specific to antigenic proteins can also be collected in large quantities from the culture. According to the methods used to generate the monoclonal antibodies of the present disclosure, not only scFV antibodies but also antibody fragments without constant regions, such as Fab antibody fragments, can be prepared.

In another aspect, the present disclosure can include phage-displayed repertoires in which the heavy and light chain variable regions of antibodies can be synthesized such that they include nearly all possible specificities.

In another aspect, the present disclosure can include passage of phage-revealed pools containing phage other than M13. Other phages such as lambda phage can also be used in the methods of the present disclosure. A library of lambda phage exposures has been generated that exposes peptides encoded by heterologous DNA on their surface (Sternberg et al., 1995; the contents of which are incorporated herein by reference in their entirety). Furthermore, the methods of the present disclosure can be extended to include viruses other than bacteriophage, such as eukaryotic viruses. Eukaryotic viruses can be produced which encode genes suitable for delivery to mammals and which encode and display antibodies capable of targeting a specific cell type or tissue to which the gene is to be delivered. For example, retroviral vectors have been generated that reveal functional antibody fragments (Russell et al., 1993; the contents of which are incorporated herein by reference in their entirety).

In another aspect, the present disclosure provides methods for generating peptides that specifically bind to HLA-restricted antigens (preferably consisting or consisting essentially of the amino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 113) ) A method for a recombinant antibody to a complexed MHC class I or II molecule, the method may comprise: using a soluble form of the MHC class I or class II molecule complexed with the HLA-restricted antigen to elicit cells comprising cells expressing the MHC class I or class II molecule immunizing a genetically engineered non-human mammal; isolating mRNA molecules from antibody-producing cells of the non-human mammal; producing a phage-revealed library that exposes protein molecules encoded by the mRNA molecules; and isolating at least one phage-revealed library from the phage-revealed library A bacteriophage, the at least one bacteriophage exhibiting specific binding to the antibody of the MHC class CI or class II complexed with the HLA-restricted antigen.

The antibodies of the present invention are preferably administered to a subject in a pharmaceutically acceptable carrier. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to impart isotonicity to the formulation. Examples of pharmaceutically acceptable carriers include saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped objects, eg, membranes, liposomes, or microparticles. Those skilled in the art will appreciate that certain carriers may be better depending on, for example, the route of administration and concentration of the antibody being administered.

Antibodies can be administered to a subject, patient, or cell by injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular) or by other methods such as infusion that ensure delivery of the antibody in an effective form to blood flow. Antibodies can also be administered by intratumoral or peritumoral routes for local as well as systemic therapeutic effects. Topical or intravenous injections are preferred.

Effective doses and schedules for administration of the antibody can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will understand that the dose of antibody that must be administered will vary depending on, for example, the subject to which the antibody will be administered, the route of administration, the particular type of antibody used, and other drugs being administered. Typical daily doses of antibodies used alone may range from 1 μg/kg body weight per day up to 100 mg/kg body weight or more, depending on factors such as those described above. After administration of the antibody, it is preferably used for the treatment of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, Hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer After cancer, and endometrial cancer, the efficacy of therapeutic antibodies can be assessed in a variety of ways well known to the skilled practitioner. For example, the size, number, and/or distribution of cancer in a subject receiving treatment can be monitored using standard tumor imaging techniques. A therapeutically administered antibody system that prevents tumor growth, causes tumor shrinkage, and/or prevents the development of new tumors is an effective antibody for the treatment of cancer compared to the disease process that would occur in the absence of antibody administration.

Another aspect of the present invention provides methods for generating soluble T cell receptors that recognize specific peptide-MHC complexes. Such soluble T cell receptors can be generated from specific T cell clones, and their affinity can be increased by targeted mutagenesis of the complementarity determining region. For the purpose of T cell receptor selection, phage exposure can be used (US 2010/0113300, Liddy et al., 2012, the contents of which are incorporated herein by reference in their entirety). Stabilization of T cell receptors during phage exposure and in practical use as drugs can be achieved, for example, by non-initial disulfide bonds, other covalent bonds (single-chain T cell receptors), or by dimerization domain to link the alpha and beta chains (Boulter et al., 2003; Card et al., 2004; Willcox et al., 1999, the contents of which are hereby incorporated by reference in their entirety). T-cell receptors can be linked to toxins, drugs, cytokines (see, eg, US 2013/0115191, the contents of which are incorporated herein by reference in their entirety), and domains such as anti-CD3 domains that recruit effector cells, in order to respond to Target cells perform specific functions. Furthermore, it is expressed in T cells used for donor-receptor transfer. Further information can be found in WO 2004/033685A1 and WO 2004/074322A1. Combinations of soluble TCRs are described in WO 2012/056407A1. Additional methods for production are disclosed in WO 2013/057586A1, the contents of which are incorporated herein by reference in their entirety.

Additionally, the peptides and/or TCRs or antibodies or other binding molecules of the invention can be used to validate a pathologist's diagnosis of cancer based on a biopsy sample.

Antibodies or TCRs can also be used in in vivo diagnostic assays. Typically, antibodies are labeled with radionucleotides such ^as111In , ^99Tc , ^14C , ^131I , ^3H , ^32P , ^and35S so that tumors can be localized using immunoscintigraphy. In one embodiment, the antibody or fragment thereof binds to the extracellular domain of two or more targets of a protein selected from the group consisting of the proteins described above with a binding affinity (Kd) of _&lt; 100 nM, More preferably <50 nM, more preferably <10 nM, more preferably <1 nM, more preferably <0.1 nM, more preferably <0.01 nM.

Antibodies for diagnostic use can be labeled with probes suitable for detection by various imaging methods. Methods for detecting probes include, but are not limited to, fluorescence, light, confocal and electron microscopy; magnetic resonance imaging and spectroscopy; fluorescence microscopy, computed tomography, and positron emission tomography. Suitable probes include, but are not limited to, luciferin, rose bengal, eosin and other fluorophores, radioisotopes, gold, gadolinium and other lanthanides, paramagnets, fluorine-18 and other positron emitting radionuclides. Additionally, probes can be bifunctional or polyfunctional and can be detected by more than one of the methods listed. The antibodies can be labeled directly or indirectly with the probes. Attachment of probes to antibodies includes covalent attachment of probes, incorporation of probes into antibodies, and covalent attachment of chelating compounds used to bind probes, as well as others well established in the art. For immunohistochemistry, disease tissue samples can be fresh or frozen or can be embedded in paraffin and fixed with a preservative such as formalin. Fixed or embedded sections contain the sample in contact with labeled primary and secondary antibodies, where the antibodies are used to detect protein expression in situ.

Another aspect of the invention includes an in vitro method for generating activated T lymphocytes, the method comprising contacting T cells in vitro with a peptide-loaded human MHC molecule suitable for expression on the surface of an antigen presenting cell for a sufficient amount of antigen specificity A period of time that activates the T cells in a manner wherein the antigen is a peptide according to the invention. Preferably, a sufficient amount of antigenic line is used with antigen presenting cells.

Preferably, the mammalian cell lacks or has a reduced level or function of the TAP peptide transport protein. Suitable cells lacking the TAP peptide transporter include T2, RMA-S and Drosophila cells. TAP is a transport protein associated with antigen processing.

Human peptides carrying the defective cell line T2 are available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA under Catalog No. CRL 1992; Drosophila cell line Schneider Line 2 is available from ATCC under Catalog No. CRL 19863 The mouse RMA-S cell line is described in Ljunggren et al. (Ljunggren and Karre, 1985, the contents of which are incorporated herein by reference in their entirety).

Preferably, the host cells are substantially free of MHC class I molecules prior to transfection. It is also preferred that the stimulator cells express molecules such as B7.1, B7.2, ICAM-1 and LFA3 that are important for providing co-stimulatory signals for T cells. Nucleic acid sequences for numerous MHC class I molecules and co-stimulatory molecules are publicly available from GenBank and EMBL databases.

In the case where MHC class I epitopes are used as antigens, the T cells are CD8 positive T cells.

If an antigen-presenting cell is transfected to express this epitope, preferably the cell comprises an expression vector capable of expressing a peptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 113 or a variant thereof.

Numerous other methods are available for generating T cells in vitro. For example, autologous tumor-infiltrating lymphocytes can be used for CTL passaging. Plebanski et al. (Plebanski et al., 1995) utilized autologous peripheral blood lymphocytes (PLB) in the preparation of T cells. Furthermore, it is possible to generate autologous T cell lines by pulsing dendritic cells with peptides or polypeptides or by infection with recombinant viruses. In addition, B cells can be used to generate autologous T cells. Additionally, macrophages pulsed with peptides or polypeptides or infected with recombinant viruses can be used to generate autologous T cells. S. Walter et al. (Walter et al., 2003, the contents of which are hereby incorporated by reference in their entirety) describe the in vitro sensitization of cells by the use of artificial antigen presenting cells (aAPCs), which are also directed against all Appropriate ways to select peptides to generate T cells. In the present invention, aAPCs are generated by coupling preformed MHC-peptide complexes to the surface of polystyrene particles (microbeads) via biotin:streptavidin biochemistry. This system allows accurate control of MHC density on aAPCs, which allows selective elicitation of high or low binding antigen-specific T cell responses from blood samples with high efficiency. In addition to the MHC-peptide complex, the aAPC should be loaded with other proteins with costimulatory activity, such as an anti-CD28 antibody coupled to its surface. In addition, such aAPC-based systems often require the addition of appropriate soluble factors such as interleukins, such as interleukin-12.

Allogeneic cells can also be used to generate T cells and the methods are described in detail in WO 97/26328, which is incorporated herein by reference in its entirety. For example, in addition to Drosophila cells and T2 cells, other cells such as CHO cells, baculovirus-infected insect cells, bacteria, yeast, and pox-infected target cells can be used to present antigens. Additionally, plant viruses can be used, see eg Porta et al. (Porta et al., 1994, the contents of which are incorporated herein by reference in their entirety), which describe the development of cowpea mosaic virus as a high yield system for the presentation of foreign peptides.

Activated T cells directed against the peptides of the present invention are suitable for therapy. Accordingly, another aspect of the present invention provides activated T cells obtainable by the aforementioned method of the present invention.

Activated T cells generated by the above method will selectively recognize cells aberrantly expressing a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 113.

Preferably, T cells recognize cells by interacting (eg, binding) with the HLA-peptide complex via their TCR. T cells are useful in a method of killing target cells in a patient that abnormally expresses a polypeptide comprising an amino acid sequence of the invention, wherein an effective amount of activated T cells is administered to the patient. T cells administered to a patient can be derived from the patient and activated as described above (ie, they are autologous T cells). Alternatively, the T cells are not from the patient but from another individual. Of course, it is preferred if the individual is a healthy individual. By "healthy individual" the inventors mean that the individual is generally in good health, preferably has a healthy immune system and more preferably is free of any disease that can be easily tested and detected.

In vivo, target cells for CD8-positive T cells according to the present invention may be cells of a tumor (which sometimes express MHC class II) and/or tumor-surrounding stromal cells (which sometimes also express MHC class II) (Dengjel et al., 2006).

The T cells of the present invention can be used as active ingredients in therapeutic compositions. Accordingly, the present invention also provides a method of killing target cells in a patient, the target cells of which abnormally express a polypeptide comprising an amino acid sequence of the present invention, the method comprising administering to the patient an effective amount of T as defined above cell.

By "abnormally expressed", the inventors also mean that the polypeptide is overexpressed compared to the level of expression in normal tissue, or that the gene is silent in the tissue from which the tumor is derived but is expressed in the tumor. By "overrepresented", the inventors mean that the polypeptide is at least 1.2-fold; preferably at least 2-fold, and more preferably at least 5-fold the level found in normal tissue or 10 times the level exists.

T cells can be obtained by methods known in the art, such as those described above.

Protocols for this so-called give-and-take transfer of T cells are well known in the art. Several summaries can be found (Gattinoni et al., 2006; Morgan et al., 2006, the contents of which are hereby incorporated by reference in their entirety).

Another aspect of the invention includes the use of peptides complexed with MHC to generate T cell receptors whose nucleic acids are cloned and introduced into host cells, preferably T cells. The engineered T cells can then be transferred to patients for cancer treatment.

Any molecule of the invention, ie, peptide, nucleic acid, antibody, expression vector, cell, activated T cell, T cell receptor, or nucleic acid encoding the same, is useful in the treatment of disorders characterized by cells that evade immune responses. Accordingly, any molecule of the present invention can be used as a medicament or for the manufacture of a medicament. Molecules can be used by themselves or in combination with other molecules of the invention or (a) known molecules.

The kit of the present invention preferably comprises 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 (eg, dual-chamber vials), syringes (such as dual-chamber syringes), and test tubes. The container can be formed from various materials such as glass or plastic. Preferably, the kit and/or container contains instructions on or associated with the container indicating instructions for reconstitution and/or use. For example, the label may indicate that the lyophilized formulation will be reconstituted to the peptide concentration as described above. The label may further indicate that the formulation is suitable or intended for subcutaneous administration.

The container holding the formulation can be a multiple-use vial, allowing repeated administrations (eg, 2-6 administrations) of the reconstituted formulation. The kit may further comprise a second container comprising a suitable diluent (eg, sodium bicarbonate solution).

After mixing of the diluent and lyophilized formulation, the final peptide concentration in the reconstituted formulation is preferably at least 0.15 mg/mL/peptide (= 75 µg) and preferably not more than 3 mg/mL/peptide (= 1500 µg). The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The kits of the present invention may have a single container containing a formulation of a pharmaceutical composition according to the present invention, with or without other components (eg, other compounds or pharmaceutical compositions of those other compounds), or may have for Distinct containers for each component.

Preferably, the kit of the present invention includes the formulation of the present invention packaged for use with a second compound such as an adjuvant (eg, GM-CSF), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic agent agent or inhibitor, apoptosis inducer or chelator) or a co-administration combination of a pharmaceutical composition thereof. The components of the kit can be pre-compounded or each component can be in separate distinct containers prior to administration to the patient. The components of the kit can be provided in one or more liquid solutions, preferably aqueous solutions, more preferably sterile aqueous solutions. The components of the kit can also be provided as entities which can be converted into liquids by adding suitable solvents, which are preferably provided in another distinct container.

The container of the therapeutic kit can be a vial, test tube, flask, bottle, syringe, or any other means of encapsulating a solid or liquid. Typically, where more than one component is present, the kit will contain a second vial or other container that allows for separate dosing. The kit may also contain another container for a pharmaceutically acceptable liquid. Preferably, the therapeutic kit will contain a device (e.g., one or more needles, syringes, droppers, pipettes, etc.) that enables the administration of the agents of the present invention, such agents being the current kit the components.

Current formulations are those suitable for administration of the peptide by any acceptable route, such as oral (enteral), nasal, ocular, subcutaneous, intradermal, intramuscular, intravenous or transdermal. Preferably, the administration is s.c., and optimally i.d. by means of an infusion pump.

Because the peptides of the present invention are derived from acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and cervical squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and intrauterine cancer Membrane cancer is separated, so the medicament of the present invention is preferably used for the treatment of acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastro-esophagus Junctional carcinoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell Lung cancer, bladder cancer, and endometrial cancer.

In an exemplary embodiment, the peptides included in the vaccine are identified by: (a) identifying tumor-associated peptides (TUMAPs) presented by tumor samples from individual patients; and (b) ) Select at least one peptide re-identified in (a) and confirm its immunogenicity.

Once the peptides are selected for use in the personalized peptide-based vaccine, the vaccine is produced. The vaccine is preferably a liquid formulation consisting of the individual peptides dissolved in between 20-40% DMSO, preferably between about 30-35% DMSO, such as about 33% DMSO.

Each peptide included in the product was dissolved in DMSO. The concentration of the single peptide solution must be chosen depending on the number of peptides to be included in the product. The single peptide-DMSO solution was mixed in equal parts to achieve a solution containing all peptides to be included in the product at a concentration of about 2.5 mg/mL/peptide. The mixed solution was then diluted 1:3 with water for injection to achieve a concentration of 0.826 mg/mL/peptide in 33% DMSO. The diluted solution was filtered through a 0.22 μM sterile filter. The final bulk solution is obtained.

The final bulk solution was filled in vials and stored at -20°C until use. One vial contains 700 µL of solution containing 0.578 mg of each peptide. Of these, 500 μL (approximately 400 μg per peptide) will be used for intradermal injection.

In addition to being suitable for use in the treatment of cancer, the peptides of the present invention are also suitable as diagnostic agents. Because peptides are derived from acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma squamous cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer produced, and since these peptides have been determined to be absent or present at lower levels in normal tissue, these peptides are useful in diagnosing the presence of cancer.

The presence of the claimed peptide on tissue biopsies in blood samples can assist pathologists in diagnosing cancer. Detection of certain peptides by means of antibodies, mass spectrometry, or other methods known in the art may suggest to the pathologist that the tissue sample is malignant or inflamed or substantially diseased, or may be used as acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma, melanoma Biomarkers for tumor, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer. The presence of groups of peptides can enable classification or sub-classification of diseased tissue.

Detection of peptides on diseased tissue samples may confer benefit with respect to treatments involving the immune system, especially where T lymphocytes are known or expected to be involved in the mechanism of action. Loss of MHC expression is a well-described mechanism by which infected or malignant cells evade immune surveillance. Thus, the presence of the peptide confirms that this mechanism is not utilized by the cells analyzed.

The peptides of the present invention can be used to analyze lymphocyte responses, such as T cell responses, or antibody responses to the peptides or peptides complexed to MHC molecules, to the peptides. These lymphocyte responses can be used as prognostic markers for deciding further treatment steps. These responses can also be used as surrogate response markers in immunotherapy approaches, targeting the induction of lymphocyte responses by various means, such as vaccination proteins, nucleic acids, autologous materials, and recipient transfer of lymphocytes. In a gene therapy setting, lymphocyte responses to peptides can be considered in assessing side effects. Monitoring lymphocyte responses can also be a valuable tool for follow-up examinations of transplantation therapy, such as for the detection of graft-to-host and host-to-graft disease.

The invention will now be described in the following examples describing preferred embodiments of the invention and with reference to the accompanying drawings, however, the invention is not limited thereto. For the purposes of this disclosure, all references cited herein are incorporated by reference in their entirety.

Additionally, it should be noted that experimental data and schemas may be disclosed herein for only selected peptide collections as claimed. Although complete datasets have been generated and are available upon request for all peptides disclosed and claimed herein, Applicants have decided not to incorporate all such complete datasets herein as this would be beyond the body of this application the controllable area. EXAMPLES Example 1 Identification and Quantification of Tumor-Associated Peptides Presented on the Cell Surface Tissue Samples

The patient's tissue was obtained from: BioIVT (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc. (Brea, CA, USA); BioServe (Beltsville, MD, USA); Capital BioScience Inc. (Rockville, MD, USA); Conversant Bio (Huntsville, AL, USA); Cureline Inc. (Brisbane, CA, USA); DxBiosamples (San Diego, CA, USA); Geneticist Inc. (Glendale, CA, USA); Indivumed GmbH (Hamburg, Germany); Kyoto Prefectural University of Medicine (KPUM) (Kyoto, Japan); Osaka City University (OCU) (Osaka, Japan); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK); Universität Bonn (Bonn, Germany); Asklepios Clinic St. Georg (Hamburg, Germany); Wald Westbrunn University Hospital (Barcelona, Spain); Cancer Immunotherapy Center (CCIT); Herlev, Denmark; Leiden University Medical Center (LUMC) (Leiden, Netherlands); Istituto Nazionale Tumori "Pascale", Molecular Biology and Viral Oncology Unit (Naples, Italy); Stanford Cancer Center (Palo Alto, CA, USA); Geneva University Hospital (Geneva, Switzerland) ; Heidelberg University Hospital (Heidelberg, Germany); Munich University Hospital (Munich, Germany); Tübingen University Hospital (Tuebingen, Germany).

Written informed consent was given to all patients prior to surgery or autopsy. Tissue was shake-frozen immediately after excision and stored at -70°C or below until dissociation of TUMAP. Isolation of HLA peptides from tissue samples

HLA peptide pools from shake-frozen tissue samples were prepared according to slightly modified protocols (Falk et al., 1991; Seeger et al., 1999) using HLA-A*02 specific antibodies BB7.2, HLA-A, HLA-B , HLA-C-specific antibody W6/32, HLA-DR-specific antibody L243, and HLA-DP-specific antibody B7/21, CNBr-activated agarose gel, acid treatment, and ultrafiltration by obtained by immunoprecipitation.

Table 8 shows the peptides and the HLA isoforms to which they bind, derived from HLA-A*01:01, HLA-A*03:01, HLA-A*24:02, HLA-B*07:02 , HLA-B*08:01 and HLA-B*44:02. However, due to similarities in binding patterns, such as similarity in relative anchoring positions, some peptides bind to more than one allele, and this overlap is likely to be, but not limited to, HLA-B*15 that also binds A*01 binding peptide, HLA-A*03 binding peptide also bound to HLA-A*11, HLA-B*07 binding peptide also bound to HLA-B*35 and HLA-B*51.

Table 8: Deamidated peptides according to the invention and the HLA alleles to which they bind. SEQ ID NO sequence total compound 1 VHDFTLPSW HLA-A*24:02 2 FFQDSTFSF HLA-A*01:01, HLA-A*24:02 3 IVRDLSCRK HLA-A*03:01 4 YIDDVTLI HLA-A*01:01, HLA-A*24:02 5 GYIDDVTLI HLA-A*24:02 6 ISDITEKNSGLY HLA-A*01:01 7 VTRDDTASY HLA-A*01:01, HLA-A*03:01 8 AQDTTYLWW HLA-A*24:02, HLA-B*44:02 9 IFDETGRF HLA-A*24:02 10 QVDGSLLVI HLA-A*01:01 11 NHITDTSLNLF HLA-A*01:01 12 ITDTSLNLF HLA-A*01:01, HLA-A*24:02 13 TANYDTSHY HLA-A*01:01 14 WSDWSNPAY HLA-A*01:01 15 TEGDFTKEASTY HLA-B*44:02 16 VTQDDTGFY HLA-A*01:01 17 DILDRTGHQL HLA-B*08:01 18 GTDKQDSTLRY HLA-A*01:01 19 MTDVDRDGTTAY HLA-A*01:01 20 TSDTSQYDTY HLA-A*01:01 twenty one APFKDVTEY HLA-B*07:02, HLA-B*44:02 twenty two NLYDWSASY HLA-A*01:01, HLA-A*03:01, HLA-B*44:02 twenty three SYDETKIKF HLA-A*24:02 twenty four FYDNSVIIF HLA-A*01:01, HLA-A*24:02 25 FTDLITDESINY HLA-A*01:01 26 IYPDASLLIQNI HLA-A*24:02 27 DEAVRDITW HLA-B*44:02 28 PSDLSVFTSY HLA-A*01:01 29 RLWDFTMNAK HLA-A*03:01 30 IYNFRLWDF HLA-A*24:02 31 VQPDSSYTY HLA-A*01:01, HLA-A*24:02, HLA-B*44:02 32 RDATASLW HLA-B*44:02 33 ISDGMDSSAHY HLA-A*01:01 34 LSDLSLADI HLA-A*01:01 35 VFHDHTYHL HLA-A*24:02, HLA-B*08:01 36 YWDETLKEF HLA-A*24:02 37 RSLDCTVKTY HLA-A*01:01 38 KLTDNSNQF HLA-A*24:02 39 LPFFTDKTLSF HLA-A*24:02, HLA-B*07:02 40 LSDLTCNNY HLA-A*01:01 41 NYLLYVSDF HLA-A*24:02 42 AERDLDVTI HLA-B*44:02 43 FFTDKTLSF HLA-A*24:02, HLA-B*08:01 44 KENQDHSYSL HLA-B*44:02 45 YFVDVTTRI HLA-A*24:02 46 KEVDDTLLVNEL HLA-B*44:02 47 RLPAADFTRY HLA-A*01:01, HLA-A*03:01, HLA-B*07:02 48 FPYYLKIDY HLA-B*07:02 49 PSDGSMHNY HLA-A*01:01 50 RSIDVTGQGF HLA-A*01:01 51 RVDDITDQF HLA-A*01:01, HLA-A*24:02, HLA-B*07:02 52 LTEVEKDATALY HLA-A*01:01, HLA-B*44:02 53 SLIDITHGF HLA-A*02:01, HLA-A*24:02, HLA-B*44:02 54 QYQDTTVSF HLA-A*24:02 55 VYTDISHHF HLA-A*24:02 56 IYLDRTLLTTI HLA-A*24:02 57 FYDLSIQSF HLA-A*01:01, HLA-A*24:02 58 GTDQTGKGLEY HLA-A*01:01 59 ILFSDSTRLSF HLA-A*01:01 60 HVKDATMGY HLA-A*01:01, HLA-A*03:01 61 KAYDQTHLY HLA-A*01:01, HLA-A*03:01, HLA-B*44:02 62 HPDLTSMTF HLA-A*01:01, HLA-B*07:02, HLA-B*08:01 63 HLYYDVTEK HLA-A*03:01 64 FHYDDTAGYF HLA-A*24:02 65 IYQFARLDY HLA-A*24:02 66 YHDQTISF HLA-A*24:02 67 QAIDLSLNF HLA-A*24:02 68 VFDETKNLL HLA-A*24:02 69 KSYHDQTISF HLA-A*24:02 70 HPFGYDLTL HLA-B*07:02, HLA-B*08:01 71 VPRNQDESV HLA-B*07:02, HLA-B*08:01 72 DVSDKITFM HLA-B*08:01 73 DESKYTWSW HLA-B*44:02 74 LENMYDLTF HLA-B*44:02 75 QEVDISLHY HLA-A*01:01, HLA-B*44:02 76 TYLPTDASLSF HLA-A*24:02, HLA-B*07:02 77 KPREEQYDSTY HLA-B*07:02, HLA-B*44:02 78 DETIWYVRF HLA-B*44:02 79 FTDTSSYEY HLA-A*01:01 80 LTNDQTLRL HLA-A*01:01 81 VTETMGIDGSAY HLA-A*01:01 82 VTDVTEEHY HLA-A*01:01 83 TFVDASRTLY HLA-A*01:01 84 EVEGVIDGTYDY HLA-A*01:01 85 EVQDHSTSSY HLA-A*01:01 86 ILPDITTTY HLA-A*01:01, HLA-A*03:01, HLA-A*24:02 87 ITDDTVQTY HLA-A*01:01, HLA-A*03:01 88 WLDRSTILY HLA-A*01:01, HLA-A*03:01 89 HVSDVTVNY HLA-A*01:01, HLA-A*03:01 90 HVDNSNLNY HLA-A*01:01, HLA-A*03:01 91 GVDDTSLLY HLA-A*01:01, HLA-A*03:01 92 GQYDDSLQAY HLA-A*01:01, HLA-A*03:01, HLA-B*44:02 93 HADLTTLTF HLA-A*01:01, HLA-A*24:02, HLA-B*07:02 94 YSIDVTNVM HLA-A*01:01, HLA-B*07:02 95 VYIDDSVEL HLA-A*24:02 96 IFVPTDRSL HLA-A*24:02 97 RYVNDYTNSF HLA-A*24:02 98 AFDKTIVKL HLA-A*24:02 99 VYVDTTELAL HLA-A*24:02 100 EYQDFSTLF HLA-A*24:02 101 VYLDASKVPGF HLA-A*24:02 102 IYPDGTLLI HLA-A*24:02 103 RQDESYLNF HLA-A*01:01, HLA-A*24:02, HLA-B*44:02 104 HLFYDVTVF HLA-A*24:02, HLA-B*08:01 105 NPADISVAL HLA-B*07:02, HLA-B*08:01 106 SPKIFDSSW HLA-B*07:02, HLA-B*08:01 107 GEPTSDITLL HLA-B*07:02, HLA-B*44:02 108 DETHTLQF HLA-B*44:02 109 DETAAYKIM HLA-B*44:02 110 AESLAVHDI HLA-B*44:02 111 GEYRCQTDL HLA-B*44:02 112 AEFFDYTVRTL HLA-B*44:02 113 TDFTKIASF HLA-B*08:01, HLA-B*44:02 Mass spectrometry

The obtained HLA peptide pools were separated according to their hydrophobicity by reverse phase chromatography (nanoAcquity UPLC system, Waters) and elution was analyzed in an LTQ velos equipped with an ESI source and a fusion hybrid mass spectrometer (ThermoElectron). peptides. The peptide cell was loaded directly onto an analytical fused silica microcapillary column (75 µm i.d. x 250 mm) packed with 1.7 µm C18 reverse phase material (Waters) with a flow rate of 400 nL per minute applied. Subsequently, peptides were separated using a two-step 180-minute binary gradient from 10% to 33% B at a flow rate of 300 nL per minute. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Gold-coated glass capillaries (PicoTip, New Objective) were used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in a data-dependent mode using the TOP5 strategy. Briefly, scan cycles were initiated with full scans with mass accuracy in orbitrap (R = 30000), followed by MS//MS/MS of the 5 most abundant precursor ions also in orbitrap (R = 7500). MS scans with kinetic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST at a fixed false discovery rate (q≤0.05) and additional manual control. In cases where the sequence of the identified peptide was uncertain, it was additionally verified by comparison of the fragmentation pattern of the native peptide generated to that of a synthetic reference peptide of sequence identity.

Label-free relative LC-MS quantification is performed by ion counting, ie by extraction and analysis of LC-MS features (Mueller et al., 2007). This method assumes that the LC-MS signal area of a peptide is related to its abundance in the sample. The extracted features were further processed by charge state deconvolution and residence time calibration (Mueller et al., 2008; Sturm et al., 2008). Ultimately, all LC-MS features were cross-referenced with sequence identification results to combine quantitative data from different samples and tissues to obtain peptide presentation profiles. Quantitative data were normalized in a second-order manner according to central tendency to account for differences within technical and biological replicates. Thus, each identified peptide can be associated with quantitative data, allowing relative quantification between sample and tissue. Additionally, all quantitative data obtained for peptide candidates were manually checked to ensure data consistency and to verify the accuracy of the automated analysis. For each peptide, a presentation distribution was calculated showing the average sample presentation as well as the repeat test variation. These distributions would be AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glial carcinoma) blastoma); GC (gastric cancer); GEJC (gastric-esophageal junction carcinoma); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin's carcinoma) NSCLC); NSCLCadeno (non-small cell lung cancer); NSCLCother (NSCLC samples that cannot be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer); UEC (endometrial cancer) samples were juxtaposed with baselines of normal tissue samples. Presentation profiles of exemplary overrepresented peptides are shown in panels 3A-3D. The figures show only their identification of peptides as dots, obtained on tissue samples that were positive for the indicated HLA alleles and processed with antibodies specific for the corresponding allotype.

Peptide presentations for various indications are shown in Table 9 for all peptides (SEQ ID NO: 1 to SEQ ID NO: 113). This table lists all indications for which the corresponding peptide was identified at least once, independent of the HLA type of the sample or the antibody used to process the sample.

Table 9: Presentation of peptides according to the invention on various cancer entities, and thus specific relevance of peptides as mentioned for diagnosis and/or treatment of cancer as indicated. Cancer types: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma) cell tumor); GC (gastric cancer); GEJC (gastric-esophageal junction carcinoma); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin's) lymphoma); NSCLCadeno (non-small cell lung cancer); NSCLCother (NSCLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer); UEC (endometrial cancer). SEQ ID NO sequence About Peptide Presentation of Cancer Types 1 VHDFTLPSW PRCA 2 FFQDSTFSF MEL, SCLC 3 IVRDLSCRK RCC 4 YIDDVTLI PACA, UEC 5 GYIDDVTLI NSCLCsquam, GBC 6 ISDITEKNSGLY GC, NSCLCadeno, NSCLCother, HNSCC, GBC, CRC 7 VTRDDTASY GC, CRC 8 AQDTTYLWW NSCLCadeno, GC 9 IFDETGRF UBC, PRCA 10 QVDGSLLVI OSCAR, NHL 11 NHITDTSLNLF HCC, GBC, PRCA, OC, NSCLCsquam, GEJC, BRCA 12 ITDTSLNLF RCC, PACA, NHL, CCC 13 TANYDTSHY OC, NSCLCsquam, UEC, RCC, NSCLCother, NSCLCadeno, GC, GBC 14 WSDWSNPAY HCC 15 TEGDFTKEASTY OC, NSCLCadeno, UEC, HNSCC 16 VTQDDTGFY NSCLCadeno, PACA, GC 17 DILDRTGHQL GBM 18 GTDKQDSTLRY MEL, OC, NSCLCadeno 19 MTDVDRDGTTAY NSCLCsquam, HNSCC, OSCAR, NSCLCadeno, UBC, GBC, GC, BRCA 20 TSDTSQYDTY NSCLCadeno, OSCAR, NHL, GC, AML twenty one APFKDVTEY RCC, NSCLCadeno, GC twenty two NLYDWSASY HCC, NSCLCsquam twenty three SYDETKIKF HCC, GBC twenty four FYDNSVIIF NSCLCsquam, OSCAR, NSCLCadeno, HNSCC, MEL, CRC, UEC, RCC, PACA, NSCLCother, GC, GBC, CCC 25 FTDLITDESINY HNSCC, NSCLCadeno 26 IYPDASLLIQNI GC, CRC 27 DEAVRDITW NSCLCsquam, CRC 28 PSDLSVFTSY NSCLCadeno, GC, BRCA, UEC 29 RLWDFTMNAK CCC, RCC, OC, NSCLCadeno, HNSCC, HCC, GBC, CRC 30 IYNFRLWDF GC, UBC 31 VQPDSSYTY PRCA, HCC 32 RDATASLW UBC, HNSCC, CCC, BRCA 33 ISDGMDSSAHY NSCLCother, NSCLCadeno, GC, NSCLCsquam, NHL, HNSCC, GEJC 34 LSDLSLADI HNSCC, OSCAR, NSCLCsquam, MEL, GBC 35 VFHDHTYHL NSCLCadeno, NSCLCsquam, NHL, GC, GBC, UEC, NSCLCother, UBC, RCC, PRCA, PACA, MEL, HCC, CRC, CCC, BRCA 36 YWDETLKEF HCC, RCC, CRC 37 RSLDCTVKTY RCC 38 KLTDNSNQF NSCLCsquam, MEL, HCC, GBM, BRCA, AML 39 LPFFTDKTLSF NSCLCsquam, OSCAR, UEC, OC, NSCLCadeno, GC, CRC, BRCA, NSCLCother, NHL, HNSCC, GBC 40 LSDLTCNNY NSCLCadeno, MEL, NSCLCsquam, NSCLCother, NHL, HNSCC, GC 41 NYLLYVSDF NSCLCadeno, GC, UEC, HNSCC, CRC 42 AERDLDVTI PACA, NSCLCadeno 43 FFTDKTLSF OSCAR, GC, HNSCC, BRCA 44 KENQDHSYSL NSCLCsquam, NHL, HNSCC, GC, CRC 45 YFVDVTTRI OSCAR, NSCLCother, NSCLCadeno, HNSCC, GBC 46 KEVDDTLLVNEL NSCLCsquam, OSCAR, OC, BRCA 47 RLPAADFTRY RCC, OSCAR, NSCLCsquam 48 FPYYLKIDY PRCA, OC 49 PSDGSMHNY NSCLCadeno, HNSCC, OSCAR, NSCLCsquam, SCLC, OC, MEL, GBC, BRCA, UEC, PRCA, PACA, NSCLCother, NHL, HCC, GEJC, GC, GBM, CRC 50 RSIDVTGQGF NSCLCadeno, GBM, PRCA, PACA, UBC, RCC, GC, GBC, BRCA, UEC, OSCAR, NSCLCsquam, NHL, MEL, HCC, CCC, AML 51 RVDDITDQF GBC, GC, PACA, RCC, BRCA, OSCAR, OC, MEL, HCC, AML 52 LTEVEKDATALY NSCLCadeno, NSCLCsquam, NSCLCother, NHL, GC, BRCA, SCLC, OC, HNSCC, HCC, GBC, OSCAR, MEL, GEJC, CCC 53 SLIDITHGF GC, PACA, RCC, NSCLCadeno, BRCA, UEC, OSCAR, OC, HNSCC, HCC, GEJC, CRC, CCC 54 QYQDTTVSF NSCLCadeno, NSCLCsquam, GC, PRCA, NSCLCother, OC, GBM, GBC, UBC, SCLC, RCC, OSCAR, MEL, HCC, CCC, BRCA 55 VYTDISHHF NSCLCsquam, NSCLCadeno, OC, UEC, GC, PRCA, OSCAR, NSCLCother, HNSCC, GBC, BRCA 56 IYLDRTLLTTI NSCLCadeno, NSCLCsquam, HNSCC, GBC, RCC, NSCLCother, OSCAR, GC, UEC, SCLC, MEL, HCC, CCC 57 FYDLSIQSF HCC, CRC, NSCLCother, NSCLCadeno, MEL, HNSCC, UEC, UBC, SCLC, OSCAR, OC, GC, GBC, AML 58 GTDQTGKGLEY NSCLCsquam, NSCLCadeno, UBC, HNSCC, GBC 59 ILFSDSTRLSF HNSCC, OSCAR, MEL 60 HVKDATMGY GC, HNSCC, OSCAR, PACA, NHL, UEC, MEL, HCC, GBC, CRC, CCC, BRCA 61 KAYDQTHLY NSCLCother, NSCLCadeno, PACA, OC, NSCLCsquam, HCC, CRC, BRCA 62 HPDLTSMTF RCC, NSCLCsquam, AML 63 HLYYDVTEK AML, GBM, GBC, CRC 64 FHYDDTAGYF NSCLCsquam, NHL, OSCAR, NSCLCother, HCC, GBC 65 IYQFARLDY GC 66 YHDQTISF NHL, PRCA, NSCLCsquam, NSCLCadeno 67 QAIDLSLNF OSCAR, NSCLCadeno, NHL 68 VFDETKNLL UEC, NSCLCsquam 69 KSYHDQTISF PACA, CCC 70 HPFGYDLTL RCC, PRCA, GC, CRC, UBC, SCLC, OSCAR, NSCLCother, NSCLCadeno, HNSCC 71 VPRNQDESV CLL 72 DVSDKITFM GC, CRC 73 DESKYTWSW OC, NSCLCsquam, NHL, HNSCC, GBM, GBC 74 LENMYDLTF NSCLCsquam, NHL 75 QEVDISLHY OSCAR, OC, HNSCC, BRCA, AML 76 TYLPTDASLSF NSCLCadeno, GC, GBC, NSCLCother, NSCLCsquam, MEL, CRC, UEC, PRCA, OC, HCC 77 KPREEQYDSTY NSCLCsquam, NSCLCadeno, PACA, UEC, OSCAR, HNSCC, GC, NSCLCother, CCC, BRCA 78 DETIWYVRF NHL, GBM, HCC, UEC, PRCA, OSCAR, NSCLCsquam, CRC, CLL, OC, NSCLCadeno, GC, GBC, CCC, BRCA 79 FTDTSSYEY GBM, BRCA, HCC, UEC, SCLC, NSCLCsquam, NSCLCadeno, HNSCC, GC, GBC 80 LTNDQTLRL HCC, UEC, CCC, BRCA, AML, NSCLCsquam, NHL 81 VTETMGIDGSAY NSCLCadeno, RCC, OSCAR, OC, NSCLCother, NHL, MEL, HNSCC, GEJC, GC, BRCA 82 VTDVTEEHY GBM, HNSCC, PRCA, NSCLCsquam, NSCLCother, GC 83 TFVDASRTLY NHL, MEL, GBC, RCC, OSCAR, NSCLCsquam, HCC, GC 84 EVEGVIDGTYDY NSCLCadeno, AML, NSCLCother, MEL, HNSCC, HCC, GEJC, GC, BRCA 85 EVQDHSTSSY OC, HNSCC, UEC, SCLC, PACA, OSCAR, NSCLCsquam, GBC 86 ILPDITTTY NSCLCadeno, MEL, GBC, UEC, SCLC, RCC, OSCAR, OC, NSCLCsquam, CCC 87 ITDDTVQTY NSCLCadeno, OSCAR, NSCLCsquam, NSCLCother, NHL, GC, AML, OC 88 WLDRSTILY NSCLCsquam, NSCLCadeno, GBM, GBC, SCLC, OSCAR, OC, MEL, GC, BRCA, AML 89 HVSDVTVNY UEC, NSCLCsquam, PRCA, PACA, OSCAR, OC, NSCLCadeno, MEL, HNSCC, GC, GBC, BRCA 90 HVDNSNLNY NSCLCadeno, RCC, NSCLCsquam, HNSCC, HCC, GBC, CRC, BRCA, AML 91 GVDDTSLLY NSCLCadeno, MEL, AML, NSCLCsquam, NHL, HCC, GBC 92 GQYDDSLQAY NSCLCadeno, NSCLCsquam, BRCA, RCC, PRCA, NSCLCother, MEL, HNSCC, GC, GBM 93 HADLTTLTF NSCLCsquam, OC, RCC, NSCLCadeno, NHL, UBC, PACA, HNSCC, GC, CRC 94 YSIDVTNVM GC, UEC, BRCA, OC, GBC, CRC 95 VYIDDSVEL PRCA, NSCLCadeno, HCC, PACA, GC 96 IFVPTDRSL NSCLCsquam, MEL, HNSCC, HCC, GC, GBC, RCC, PACA, OSCAR, GEJC, BRCA 97 RYVNDYTNSF NSCLCsquam, HNSCC, OSCAR, UBC, PRCA, NSCLCadeno, GBC 98 AFDKTIVKL UEC, NSCLCsquam, CRC, UBC, PACA, NSCLCadeno, HCC, CCC, BRCA 99 VYVDTTELAL HCC, GBC, PRCA, NSCLCadeno, CRC, NSCLCother 100 EYQDFSTLF CRC, PRCA, GC, GBC 101 VYLDASKVPGF NSCLCother, NSCLCadeno, GBC, OC, NSCLCsquam, HNSCC, HCC, BRCA 102 IYPDGTLLI CRC, GBC, HCC, GC, CCC 103 RQDESYLNF GC, HCC, RCC, UBC, OSCAR, OC, NSCLCother, NSCLCadeno, NHL, GEJC 104 HLFYDVTVF NHL, HCC, MEL, GEJC, GC 105 NPADISVAL NSCLCadeno, NSCLCsquam, OC, HNSCC, PRCA, OSCAR, NHL, CCC 106 SPKIFDSSW SCLC, BRCA, UEC, RCC, MEL, GC, GBM, CLL, CCC 107 GEPTSDITLL BRCA, NSCLCsquam, NSCLCadeno, MEL, UEC, NHL, HNSCC, HCC, CRC, AML 108 DETHTLQF OSCAR, NSCLCsquam, PACA, OC, NSCLCadeno, SCLC, PRCA, NSCLCother, NHL, GBC, BRCA, AML 109 DETAAYKIM NHL, OC, NSCLCsquam, OSCAR, HNSCC, UEC, PRCA, NSCLCadeno 110 AESLAVHDI HCC, UEC, UBC, PACA, NSCLCsquam, NSCLCadeno, NHL, HNSCC, CRC, AML 111 GEYRCQTDL OSCAR, RCC, PACA, OC, NSCLCsquam, NSCLCother, NSCLCadeno, HNSCC, HCC, GBM, BRCA 112 AEFFDYTVRTL NSCLCsquam, MEL, BRCA, RCC, OC, NHL, HNSCC 113 TDFTKIASF SCLC, OSCAR, RCC, NSCLCother, NSCLCadeno, MEL, HNSCC, GC, GBM, BRCA Example 2 Expression distribution of genes encoding peptides of the present invention

The over- or specific presentation of peptides on tumor cells compared to normal cells is sufficient to make them suitable for immunotherapy, and some peptides are tumor-specific regardless of the protein of origin that is also present in normal tissues. Again, the mRNA expression profile adds an additional level of safety in the selection of peptide targets for immunotherapy. Especially for therapeutic options with high safety risks, such as affinity matured TCRs, ideal target peptides would be derived from proteins that are unique to the tumor and not found on normal tissues. RNA source and preparation

After written informed consent had been obtained from each patient, surgically removed tissue samples were provided as indicated above (see Example 1). Tumor tissue samples were snap frozen immediately after surgery and later homogenized with a mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI reagent (Ambion, Darmstadt, Germany) followed by clearing with RNeasy (QIAGEN, Hilden, Germany); both methods were performed according to the manufacturer's protocol.

Total RNA from healthy human tissue for RNASeq experiments was obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK); Bio-Options Inc. (Brea, CA, USA); Geneticist Inc. (Glendale) , CA, USA); ProteoGenex Inc. (Culver City, CA, USA); Tissue Solutions Ltd (Glasgow, UK).

Total RNA from tumor tissue for RNASeq experiments was obtained from: Asterand (Detroit, MI, USA & Royston, Herts, UK); BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, MD, USA); Geneticist Inc (Glendale, CA, USA); Istituto Nazionale Tumori “Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, CA, USA); Heidelberg University Hospital (Heidelberg, Germany).

The quality and quantity of all RNA samples were assessed on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 Pico LabChip Kit (Agilent). RNAseq experiments

Gene expression analysis of tumor and normal tissue RNA samples was performed by next-generation sequencing (RNAseq) with CeGaT (Tübingen, Germany). Briefly, sequencing libraries were prepared using the Illumina HiSeq v4 reagent kit according to the provider's protocol (Illumina Inc., San Diego, CA, USA), which included RNA fragmentation, cDNA transformation, and addition of sequencing adapter proteins. Pools derived from multiple samples were equimolar pooled and sequenced on an Illumina HiSeq 2500 sequencer according to the manufacturer's instructions, resulting in 50 bp single-end reads. The processed reads were mapped to the human genome (GRCh38) using STAR software. Based on annotations in the ensembl sequence database (Ensembl77), performance data are provided at the transcript level as RPKM (reads per kilobase per million mapped reads, generated by the software Cufflinks) and at the exon level (total reads). , produced by the software Bedtools). Exon reads were normalized to exon length and alignment size to obtain RPKM values.

Figures 4A to 4D show exemplary expression profiles of the source genes of the invention that are highly overexpressed or exclusively expressed in AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellular carcinoma) ; CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); cancer); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin's lymphoma); NSCLCadeno (non-small cell lung cancer); NSCLC samples); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer) ); UBC (bladder cancer); UEC (endometrial cancer). Performance scores for other exemplary genes are shown in Table 9.

Table 10: Performance Scores. The table lists peptides from genes that are highly overrepresented (++) in tumors compared to a panel of normal tissues or overrepresented (+) in tumors compared to a panel of normal tissues. The baseline for this score is calculated from measurements of the following relevant normal tissues: adipose tissue; adrenal gland; bile duct; bladder; blood cells; blood vessels; bone marrow; brain; breast; esophagus; eye; gallbladder; head and neck; heart; large intestine ; Small Intestine; Kidney; Liver; Lung; Lymph Node; Peripheral Nerve; Ovary; Pancreas; Parathyroid; Peritoneum; Pituitary; Placenta; Pleura; Prostate; Skeletal Muscle; Skin; Spinal Cord; Spleen; Stomach; Testis; Thymus; Thyroid; Trachea; ureter; uterus. Where performance data for several samples of the same tissue type were available, the arithmetic mean of all corresponding samples was used for the calculations. SEQ ID NO sequence Gene expression in tumors over performance (+) High over performance (++) 114 VHNFTLPSW PRCA 178 IYQFARLNY HNSCC, OSCAR 117 YIDNVTLI CRC, GC, NSCLCadeno, NSCLCother, PACA, UBC CCC, HNSCC, NSCLCsquam, OC 143 IYNFRLWNF CCC 120 VTRNDTASY CRC 121 AQNTTYLWW CRC 119 ISNITEKNSGLY CRC 123 QVNGSLLVI CCC, NHL, NSCLCadeno, NSCLCsquam, PACA NSCLCother 135 NLYNWSASY HCC 115 FFQNSTFSF MEL 134 APFKNVTEY BRCA 136 SYNETKIKF HCC 171 GTNQTGKGLEY BRCA, GBC 159 KEVNDTLLVNEL BRCA, GBC, HNSCC, PACA 140 DEAVRNITW CCC, HNSCC, OSCAR 141 PSNLSVFTSY CRC, OC, UEC 145 RNATASLW CCC, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA 144 VQPNSSYTY HCC 160 RLPAANFTRY RCC 154 NYLLYVSNF GBC, GC, PACA 155 AERDLNVTI CRC, GC, NSCLCother, NSCLCadeno, PACA 170 FYNLSIQSF HCC 187 LENMYNLTF NHL 158 YFVNVTTRI CCC, PACA, UBC 152 LPFFTNKTLSF GC, PACA 142 RLWNFTMNAK CCC 220 GEPTSNITLL CLL 168 VYTNISHHF AML 174 KAYNQTHLY NSCLCother 131 GTDKQNSTLRY MEL 137 FYNNSVIIF CCC, HNSCC, OSCAR 180 QAINLSLNF AML NHL 148 VFHNHTYHL CCC, CRC, GBC 156 FFTNKTLSF GC, PACA 188 QEVNISLHY BRCA, CCC, HNSCC, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA AML, MEL 189 TYLPTNASLSF BRCA 116 IVRNLSCRK RCC 139 IYPNASLLIQNI CRC 146 ISDGMNSSAHY CCC, CRC, GBC, NSCLCother 183 HPFGYNLTL CCC, CRC, PACA 138 FTDLITNESINY PACA 198 EVQNHSTSSY OSCAR 118 GYIDNVTLI CRC, GC, NSCLCadeno, NSCLCother, PACA, UBC CCC, HNSCC, NSCLCsquam, OSCAR 225 AEFFNYTVRTL MEL 128 TEGNFTKEASTY OC Example 3 Validation of peptides by IdentControl and CoElution

To validate the peptides according to the invention, all peptides were synthesized using standard and well established solid phase peptide synthesis using the Fmoc strategy. When necessary, stable isotope labeled (SIL-) amino acids are used to introduce differential mass shifts and allow the use of these labeled peptides as internal standards (e.g., if the peptides are selected for identity confirmation in CoElution experiments). ). The identity and purity of each individual peptide was determined by mass spectrometry and analytical RP-HPLC. The peptide was obtained as a white to off-white lyophilisate (trifluoroacetate salt) in >50% purity. All TUMAPs are preferably administered as trifluoroacetate or acetate salts, other salt forms are also possible.

Initial validation of the peptides was achieved by spectral comparison by IdentControl. To this end, synthetic peptides were used to verify peptide identification results by obtaining high resolution reference MS2 spectra using matching fragmentation modes and collision energies as used to obtain native spectra. Automated spectral comparisons were performed using a sensitivity measure of spectral correlation and a cutoff score determined to yield 90% sensitivity at <1% FDR based on a benchmark dataset containing >10,000 manually validated spectra. The fuzzy identification results were further verified in the CoElution experiment.

Table 11: IdentControl results. Spectral correlation indicates the similarity of MS/MS spectra from endogenous detection peptides compared to synthetic peptides, with higher values indicating approximately similar spectra. Peptides were validated when they met a threshold of 0.75, or spectra were considered consistent according to manual checks. SEQ ID NO sequence Spectral correlation 1 VHDFTLPSW 0.957 2 FFQDSTFSF 0.953 3 IVRDLSCRK 0.977 4 YIDDVTLI 0.957 5 GYIDDVTLI 0.871 6 ISDITEKNSGLY 0.918 7 VTRDDTASY 0.957 8 AQDTTYLWW 0.791 9 IFDETGRF 0.953 10 QVDGSLLVI 0.800 11 NHITDTSLNLF 0.955 12 ITDTSLNLF 0.961 13 TANYDTSHY 0.887 14 WSDWSNPAY 0.968 15 TEGDFTKEASTY 0.921 16 VTQDDTGFY 0.917 17 DILDRTGHQL 0.961 18 GTDKQDSTLRY 0.851 19 MTDVDRDGTTAY 0.898 20 TSDTSQYDTY 0.895 twenty one APFKDVTEY 0.769 twenty two NLYDWSASY 0.943 twenty three SYDETKIKF 0.955 twenty four FYDNSVIIF 0.963 25 FTDLITDESINY 0.930 26 IYPDASLLIQNI 0.971 27 DEAVRDITW 0.904 28 PSDLSVFTSY 0.870 29 RLWDFTMNAK 0.962 30 IYNFRLWDF 0.931 31 VQPDSSYTY 0.860 32 RDATASLW 0.952 33 ISDGMDSSAHY 0.920 34 LSDLSLADI 0.811 35 VFHDHTYHL 0.862 36 YWDETLKEF 0.921 37 RSLDCTVKTY 0.816 38 KLTDNSNQF 0.898 39 LPFFTDKTLSF 0.907 40 LSDLTCNNY 0.855 41 NYLLYVSDF 0.994 42 AERDLDVTI 0.961 43 FFTDKTLSF 0.899 44 KENQDHSYSL 0.949 45 YFVDVTTRI 0.962 46 KEVDDTLLVNEL 0.981 47 RLPAADFTRY 0.943 48 FPYYLKIDY 0.84 49 PSDGSMHNY 0.946 50 RSIDVTGQGF 0.910 51 RVDDITDQF 0.965 52 LTEVEKDATALY 0.947 53 SLIDITHGF 0.968 54 QYQDTTVSF 0.959 55 VYTDISHHF 0.998 56 IYLDRTLLTTI 0.889 57 FYDLSIQSF 0.950 58 GTDQTGKGLEY 0.940 59 ILFSDSTRLSF 0.916 60 HVKDATMGY 0.922 61 KAYDQTHLY 0.890 62 HPDLTSMTF 0.873 63 HLYYDVTEK 0.863 64 FHYDDTAGYF 0.970 65 IYQFARLDY 0.944 66 YHDQTISF 0.966 67 QAIDLSLNF 0.941 68 VFDETKNLL 0.903 69 KSYHDQTISF 0.888 70 HPFGYDLTL 0.895 71 VPRNQDESV 0.927 72 DVSDKITFM 0.815 73 DESKYTWSW 0.784 74 LENMYDLTF 0.949 75 QEVDISLHY 0.895 76 TYLPTDASLSF 0.963 77 KPREEQYDSTY 0.897 78 DETIWYVRF 0.953 79 FTDTSSYEY 0.932 80 LTNDQTLRL 0.916 81 VTETMGIDGSAY 0.890 82 VTDVTEEHY 0.899 83 TFVDASRTLY 0.968 84 EVEGVIDGTYDY 0.948 85 EVQDHSTSSY 0.764 86 ILPDITTTY 0.947 87 ITDDTVQTY 0.768 88 WLDRSTILY 0.993 89 HVSDVTVNY 0.889 90 HVDNSNLNY 0.908 91 GVDDTSLLY 0.935 92 GQYDDSLQAY 0.961 93 HADLTTLTF 0.843 94 YSIDVTNVM 0.974 95 VYIDDSVEL 0.985 96 IFVPTDRSL 0.984 97 RYVNDYTNSF 0.853 98 AFDKTIVKL 0.871 99 VYVDTTELAL 0.948 100 EYQDFSTLF 0.946 101 VYLDASKVPGF 0.861 102 IYPDGTLLI 0.968 103 RQDESYLNF 0.941 104 HLFYDVTVF 0.913 105 NPADISVAL 0.945 106 SPKIFDSSW 0.758 107 GEPTSDITLL 0.948 108 DETHTLQF 0.942 109 DETAAYKIM 0.919 110 AESLAVHDI 0.950 111 GEYRCQTDL 0.957 112 AEFFDYTVRTL 0.890 113 TDFTKIASF 0.855

For further validation, the peptides were subjected to CoElution experiments using SIL internal standard peptides. To this end, SIL peptides were spiked into HLA peptidomics extracts from samples and subjected to liquid chromatography-targeted mass spectrometry (LC-MS) based on spectral similarity and CoElution in the residence time dimension to confirm peptide identity. If necessary, the amount of SIL-peptide incorporated was adjusted to the peptide ratio ionization factor (determined in the calibration curve). LC-MS was performed using predefined targeted MS2 scan events that utilized non-overlapping separation windows for SIL-peptide and native peptide species to avoid co-fragmentation. To confirm isotopic purity and the absence of co-fragmentation of the SIL-peptide and native peptide, a control experiment was performed in a non-HLA peptide containing a trypsin matrix, which must be confirmed to be free of any unlabeled signal. Peptide detection and validation by CoElution is determined by expert manual verification based on a number of predefined objective criteria, including the dot product (dot product; dotP) of SIL peptides compared to unlabeled peptide MS2 traces , The peak apex with the strongest transition and alignment exists in multiple consecutive scans. A list of peptides validated by CoElution can be found in Table 12.

Table 12: Peptides for positive CoElution experiments SEQ ID NO sequence SEQ ID NO sequence 3 IVRDLSCRK 63 HLYYDVTEK 4 YIDDVTLI 68 VFDETKNLL 5 GYIDDVTLI 70 HPFGYDLTL 6 ISDITEKNSGLY 73 DESKYTWSW 13 TANYDTSHY 75 QEVDISLHY 14 WSDWSNPAY 77 KPREEQYDSTY 15 TEGDFTKEASTY 79 FTDTSSYEY 16 VTQDDTGFY 80 LTNDQTLRL 18 GTDKQDSTLRY 82 VTDVTEEHY 20 TSDTSQYDTY 83 TFVDASRTLY twenty one APFKDVTEY 84 EVEGVIDGTYDY twenty two NLYDWSASY 85 EVQDHSTSSY 25 FTDLITDESINY 86 ILPDITTTY 27 DEAVRDITW 87 ITDDTVQTY 28 PSDLSVFTSY 88 WLDRSTILY 31 VQPDSSYTY 90 HVDNSNLNY 32 RDATASLW 91 GVDDTSLLY 35 VFHDHTYHL 92 GQYDDSLQAY 40 LSDLTCNNY 95 VYIDDSVEL 42 AERDLDVTI 96 IFVPTDRSL 44 KENQDHSYSL 98 AFDKTIVKL 45 YFVDVTTRI 99 VYVDTTELAL 46 KEVDDTLLVNEL 102 IYPDGTLLI 47 RLPAADFTRY 105 NPADISVAL 48 FPYYLKIDY 107 GEPTSDITLL 52 LTEVEKDATALY 110 AESLAVHDI 56 IYLDRTLLTTI 111 GEYRCQTDL 58 GTDQTGKGLEY 112 AEFFDYTVRTL 61 KAYDQTHLY Example 4 In vitro immunogenicity of MHC class I presenting peptides

To obtain information on the immunogenicity of the TUMAPs of the present invention, the inventors used in vitro experiments based on repeated stimulation of CD8+ T cells with artificial antigen presenting cells (aAPCs) loaded with peptide/MHC complexes and anti-CD28 antibodies. T cell sensitization assays to perform studies. Thus, the inventors can demonstrate the immunogenicity of the MHC class I-restricted TUMAPs of the invention, thereby demonstrating that these peptides are T cell epitopes against which CD8+ precursor T cell lines exist in humans (Tables 13A-13D) . In vitro sensitization of CD8+ T cells

To perform in vitro stimulation by artificial antigen-presenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28 antibody, the inventors first used healthy samples obtained from the University Clinic of Mannheim, Germany, after informed consent. Donor CD8 beads (Miltenyi Biotec, Bergisch-Gladbach, Germany), via positive selection from fresh HLA-A*01, HLA-A*24, HLA-B*08 or HLA-B*44 leukapheresis products CD8+ T cells were isolated.

PBMCs and isolated CD8+ lymphocytes were incubated until use in T cell medium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe, Germany) supplemented with 10% heat Passivated human AB serum (PAN-Biotech, Aidenbach, Germany), 100 U/ml penicillin/100 μg/ml streptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro, Oberdorla, Germany), 20 μg /ml Gentamycin (Cambrex). 2.5 ng/mL IL-7 (PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma, Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28-coated beads, T cell stimulation and readout were performed in a well-defined in vitro system using four different pMHC molecules per stimulation condition and eight different pMHC molecules per readout condition out.

Purified co-stimulatory mouse IgG2a anti-human CD28 Ab 9.3 (Jung et al., 1987) was chemically treated with sulfonated N-hydroxysuccinimidyl biotin as recommended by the manufacturer (Perbio, Bonn, Germany) Biotinylated. The beads used were 5.6 μM diameter streptavidin-coated polystyrene particles (Bangs Laboratories, Illinois, USA).

The pMHCs used for positive and negative control stimulation were A*02:01/MLA-001 (peptide ELAGIGILTV (SEQ ID NO:227) from modified Melan-A/MART-1) and A*02:01/DDX5, respectively -001 (YLLPAIVHI from DDX5, SEQ ID NO: 228).

800.000 beads/200 μl were plated in 96 well plates in the presence of 4 x 12.5 ng different biotin-pMHC, washed and then 600 ng biotin anti-CD28 was added in a volume of 200 μl. in 96-well plates by co-incubating ^1x106 CD8+ T cells with ^2x105 washed coated beads in 200 μl TCM supplemented with 5 ng/mL IL-12 (PromoCell) at 37°C medium initial stimulus. Half of the medium was then replaced by fresh TCM supplemented with 80 U/ml IL-2 and incubation was continued for 4 days at 37°C. Perform this stimulation cycle a total of three times. For pMHC multimer readouts using 8 different pMHC molecules per condition, a two-dimensional combinatorial encoding approach was used as previously described (Andersen et al., 2012), with minor modifications encompassing coupling to 5 different fluorophores. light dyes. Finally, multimer analysis was performed by staining cells with live/dead near-IR dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD, Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BD LSRII SORP hemocytometer equipped with appropriate lasers and filters was used. Peptide-specific cell lines were calculated as a percentage of total CD8+ cells. Evaluation of polymer analysis was performed using FlowJo software (Tree Star, Oregon, USA). In vitro sensitization of specific polymer+CD8+ lymphocytes was detected by comparison to negative control stimulation. If at least one evaluable in vitro stimulation well of a healthy donor is found to contain a specific CD8+ T cell line after in vitro stimulation (i.e., this well contains at least 1% of the CD8+ T cells specific multimer+, and Immunogenicity is detected for a given antigen if the percentage of specific polymer+cells is at least 10 times the median value of the negative control stimulation). In vitro immunogenicity of cancer peptides

For the HLA class I peptides tested, in vitro immunogenicity was demonstrated by passage of peptide-specific T cell lines. Exemplary flow cytometry results following staining for TUMAP-specific polymers of the five peptides of the invention are shown in Figure 7 along with the corresponding negative controls. Results from the 12 peptides of the present invention are summarized in Tables 13A to 13D.

Table 13A: In vitro immunogenicity of HLA-A*01 peptides of the invention

Exemplary results of in vitro immunogenicity experiments performed by applicants on HLA-A*01 restricted peptides of the invention. The results of in vitro immunogenicity experiments are indicated. Percentages of positive wells and donors (among evaluable persons) were as indicated <20%=+;20%-49%=++;50%-69%=+++;>=70%=+ +++ for an overview. SEQ ID NO sequence Positive holes [%] 14 WSDWSNPAY "+++" 28 PSDLSVFTSY "++" 40 LSDLTCNNY "+"

Table 13B: In vitro immunogenicity of HLA-A*24 peptides of the invention

Exemplary results of in vitro immunogenicity experiments performed by applicants on HLA-A*24 restricted peptides of the invention. Results of in vitro immunogenicity experiments are indicated. Percentages of positive wells and donors (among evaluable persons) were as indicated <20%=+;20%-49%=++;50%-69%=+++;>=70%=+ +++ to overview. SEQ ID NO sequence Positive holes [%] 30 IYNFRLWDF + 35 VFHDHTYHL + 41 NYLLYVSDF + 56 IYLDRTLLTTI +

Table 13C: In vitro immunogenicity of HLA-B*08 peptides of the invention

Exemplary results of in vitro immunogenicity experiments performed by applicants on HLA-B*08 restricted peptides of the invention. Results of in vitro immunogenicity experiments are indicated. Percentages of positive wells and donors (among evaluable persons) were as indicated <20%=+;20%-49%=++;50%-69%=+++;>=70%=+ +++ to overview. SEQ ID NO sequence Positive holes [%] 17 DILDRTGHQL +

Table 13D: In vitro immunogenicity of HLA-B*44 peptides of the invention

Exemplary results of in vitro immunogenicity experiments performed by applicants on HLA-B*44 restricted peptides of the invention. Results of in vitro immunogenicity experiments are indicated. Percentages of positive wells and donors (among evaluable persons) were as indicated <20%=+;20%-49%=++;50%-69%=+++;>=70%=+ +++ to overview. SEQ ID NO sequence Positive holes [%] 8 AQDTTYLWW + 27 DEAVRDITW + 32 RDATASLW + 42 AERDLDVTI + Example 5 MHC binding assay

Candidate peptides for T cell based therapy according to the invention were further tested for their MHC binding capacity (affinity). Individual peptide-MHC complexes are generated by UV-ligand exchange, wherein UV-sensitive peptides are cleaved after UV irradiation and exchanged for peptides of interest as analyzed. Only peptide candidates that effectively bind and stabilize peptide-accepting MHC molecules prevent dissociation of the MHC complex. To determine the yield of the exchange reaction, an ELISA based on the detection of the light chain (β2m) of the stabilized MHC complex was performed. Assays were performed as generally described by Rodenko et al. (Rodenko et al., 2006).

96-well MAXISorp plates (NUNC) were coated with 2 μg/ml streptavidin in PBS overnight at room temperature, washed 4 times and blocked in blocking buffer containing 2% BSA for 1 h at 37°C. The refolded HLA-A*02:01/MLA-001 monomer was used as a standard, which covers the range of 15-500 ng/mL. The peptide-MHC monomer of the UV exchange reaction was diluted 100-fold in blocking buffer. Samples were incubated at 37°C for 1 h, washed 4 times, incubated with 2 μg/ml HRP-conjugated anti-β2m for ₁ h at 37°C, washed again and probed with TMB solution quenched with _NH2SO4 . Absorption was measured at 450 nm. Candidate peptides exhibiting high exchange yields (preferably higher than 50%, optimally higher than 75%) are generally better for passage and production of antibodies or fragments thereof and/or T cell receptors or fragments thereof. Preferred because it exhibits sufficient binding to MHC molecules and prevents dissociation of MHC complexes.

The MHC-peptide binding results for the 112 peptides from the invention are summarized in Tables 14A-G.

Table 14A: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-A*01:01 was delineated by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange 4 YIDDVTLI ++ 6 ISDITEKNSGLY ++++ 7 VTRDDTASY ++ 10 QVDGSLLVI ++ 11 NHITDTSLNLF ++ 12 ITDTSLNLF ++++ 13 TANYDTSHY ++ 14 WSDWSNPAY ++++ 16 VTQDDTGFY ++++ 18 GTDKQDSTLRY ++++ 19 MTDVDRDGTTAY ++++ 20 TSDTSQYDTY +++ 25 FTDLITDESINY ++++ 28 PSDLSVFTSY ++++ 33 ISDGMDSSAHY ++++ 34 LSDLSLADI ++ 37 RSLDCTVKTY +++ 40 LSDLTCNNY ++++ 49 PSDGSMHNY ++++ 50 RSIDVTGQGF ++ 51 RVDDITDQF ++ 52 LTEVEKDATALY ++++ 58 GTDQTGKGLEY +++ 59 ILFSDSTRLSF +++ 60 HVKDATMGY +++ 79 FTDTSSYEY ++++ 80 LTNDQTLRL +++ 81 VTETMGIDGSAY ++++ 82 VTDVTEEHY ++++ 83 TFVDASRTLY ++++ 84 EVEGVIDGTYDY ++++ 85 EVQDHSTSSY ++ 86 ILPDITTTY ++ 87 ITDDTVQTY ++++ 88 WLDRSTILY ++++ 89 HVSDVTVNY ++ 90 HVDNSNLNY ++++ 91 GVDDTSLLY ++++ 92 GQYDDSLQAY ++ 93 HADLTTLTF ++ 94 YSIDVTNVM +++

Table 14B: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-A*02:01 was delineated by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange 53 SLIDITHGF ++++

Table 14C: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-A*03:01 was delineated by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange 3 IVRDLSCRK +++ twenty two NLYDWSASY ++ 29 RLWDFTMNAK +++ 47 RLPAADFTRY ++ 61 KAYDQTHLY ++++ 63 HLYYDVTEK +++

Table 14D: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-A*24:02 was delineated by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange 1 VHDFTLPSW ++ 2 FFQDSTFSF ++++ 5 GYIDDVTLI +++ 9 IFDETGRF ++ twenty three SYDETKIKF + twenty four FYDNSVIIF ++ 26 IYPDASLLIQNI ++++ 30 IYNFRLWDF ++++ 35 VFHDHTYHL ++++ 36 YWDETLKEF +++ 38 KLTDNSNQF ++ 41 NYLLYVSDF ++++ 43 FFTDKTLSF ++++ 45 YFVDVTTRI +++ 54 QYQDTTVSF +++ 55 VYTDISHHF ++++ 56 IYLDRTLLTTI ++++ 57 FYDLSIQSF ++++ 64 FHYDDTAGYF + 65 IYQFARLDY ++ 66 YHDQTISF + 67 QAIDLSLNF ++ 68 VFDETKNLL ++ 69 KSYHDQTISF ++++ 76 TYLPTDASLSF ++++ 95 VYIDDSVEL ++++ 96 IFVPTDRSL ++ 97 RYVNDYTNSF ++++ 98 AFDKTIVKL ++ 99 VYVDTTELAL ++++ 100 EYQDFSTLF ++++ 101 VYLDASKVPGF ++++ 102 IYPDGTLLI ++++ 103 RQDESYLNF ++ 104 HLFYDVTVF ++

Table 14E: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-B*07:02 was ranged by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange twenty one APFKDVTEY +++ 39 LPFFTDKTLSF ++++ 48 FPYYLKIDY +++ 62 HPDLTSMTF ++++ 70 HPFGYDLTL ++++ 71 VPRNQDESV ++++ 105 NPADISVAL ++++ 106 SPKIFDSSW +++ 107 GEPTSDITLL +++

Table 14F: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-B*08:01 was ranged by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange 17 DILDRTGHQL +++ 72 DVSDKITFM +++

Table 14G: MHC class I binding scores. Binding of HLA class I restricted peptides to HLA-B*44:05 was ranged by peptide exchange yields: ≧10% = +; ≧20% = ++; ≧50% = +++; ≧75 % = ++++ SEQ ID NO sequence Peptide exchange 8 AQDTTYLWW ++++ 15 TEGDFTKEASTY +++ 27 DEAVRDITW ++++ 32 RDATASLW ++++ 42 AERDLDVTI ++++ 44 KENQDHSYSL ++++ 46 KEVDDTLLVNEL ++++ 73 DESKYTWSW ++++ 74 LENMYDLTF ++++ 75 QEVDISLHY ++++ 77 KPREEQYDSTY ++++ 78 DETIWYVRF ++++ 108 DETHTLQF ++++ 109 DETAAYKIM +++ 110 AESLAVHDI +++ 111 GEYRCQTDL ++++ 112 AEFFDYTVRTL ++++ 113 TDFTKIASF ++++ List of references Y. et al, BMC. Biotechnol. 11(2011): 124. Allison, JP et al., Science 270(1995): 932-933. Altrich-VanLith, ML et al, J Immunol, 177(2006): 5440-50. Andersen, MH et al, J Immunol, 163(1999): 3812-18. Anderson, NL et al, J Proteome. Res 11(2012): 1868-1878. Andersen, RS et al., Nat Protoc. 7(2012): 891-902. Appay, V. et al, Eur. J Immunol. 36(2006): 1805-1814. Arentz-Hansen, H. et al, J Exp Med, 191: 603-12. 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Figure 1 shows the antigen processing pathway of deamidated peptides and their presentation by HLA class I. During translation, proteins with the glycosylation motif N[X^P][ST] become glycosylated at their N residues in the ER (1). Following its export, the cytosolic amidase PNGase removes N-linked oligosaccharides, resulting in deamidation to aspartate (D) (2). After further degradation in the proteasome (3) and re-delivery of the peptide into the ER (4), it binds to the HLA I complex (5). The peptide-HLA I complex then follows the classical antigen presentation pathway, translocating to the cell membrane and presenting the deamidated peptide on the cell surface (6).

Figure 2 shows the deamidation reaction from aspartic acid (N) to aspartic acid (D) (Figure 2).

Figures 3A-3D show the overrepresentation of various peptides in different cancer tissues compared to normal tissues. Upper panel: median MS signal intensity from technical replicate measurements is plotted as dots for normal samples (grey dots, left panel of the graph) and tumor samples with peptide detected (black dots, right panel of the graph) . Boxes show median, 25th percentile and 75th percentile of normalized signal strength, and whiskers extend to 1.5 interquartile range (interquartile range; IQR) still in the lower quartile ), and the highest data point still within 1.5 IQR of the upper quartile. Bottom part: The relative peptide detection frequency in each organ is shown as a spine plot. Numbers at the bottom of the graph indicate from N > 200 for HLA-A*01 positive normal samples; N > 210 for HLA-A*03 positive normal samples; N > 210 for HLA-A*24 normal samples > 180; for HLA-B*07 normal samples, N > 200; for HLA-B*08 positive normal samples, N > 90 and for HLA-B*44 normal samples, N > 210) or tumor indications (for HLA-B*44 normal samples) -A*01 positive cancer samples, N > 190; for HLA-A*03 positive cancer samples, N > 180; for HLA-A*24 positive cancer samples, N > 330; for HLA-B*07 positive cancer samples, N > 190; N > 100 for HLA-B*08 positive cancer samples and N > 210 for HLA-B*44 positive cancer samples) the number of samples with detected peptides in the total number of samples analyzed. If a peptide has been detected on a sample but cannot be quantified for technical reasons, the sample is included in this detection frequency representation, but no points are shown in the upper part of the figure. Tissues (left to right): normal samples: fat (adipose tissue); adrenal gland gl (adrenal gland); bile duct; bladder; blood cells; bloodvess (blood vessel); bone marrow; brain; breast; esoph (esophagus) ); eye; gallbladder bl (gall bladder); head and neck; heart; intest. la (large intestine); intest. sm (small intestine); kidney; liver; lung; ); ovary; pancreas; parathyr (parathyroid); perit (peritoneum); pituit (pituitary); placenta; pleura; prostate; skel. mus (skeletal muscle); skin; spinal cord; spleen; stomach; testis; thymus; thyroid; trachea; ureter; uterus. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma) cell tumor); GC (gastric cancer); GEJC (gastric-esophageal junction carcinoma); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin's) lymphoma); NSCLCadeno (non-small cell lung cancer); NSCLCother (NSCLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer); UEC (endometrial cancer). Panel 3A) Peptide: ISDITEKNSGLY (SEQ ID NO: 6), Panel 3B) Peptide: MTDVDRDGTTAY (SEQ ID NO: 19), Panel 3C) Peptide: IYLDRTLLTTI (SEQ ID NO: 56), Panel 3D) Peptide : TYLPTDASLSF (SEQ ID NO: 76).

Figures 4A-4D show exemplary expression profiles of the source genes of the present invention, which are overexpressed in different cancer samples. Tumor (black dots) and normal (grey dots) samples were grouped according to organ origin. The box and box-whisker plot represents the median FPKM value, i.e. the 25th percentile and the 75th percentile (box) plus box-whiskers that extend to still 1.5% of the lower quartile The lowest data point within the interquartile range (IQR) and the highest data point still within 1.5 IQR of the upper quartile. FPKM: Fragments per kilobase per million degrees of mapping. Tissues (left to right): normal samples: fat (adipose tissue); adrenal gland gl (adrenal gland); bile duct; bladder; blood cells; bloodvess (blood vessel); bone marrow; brain; breast; esoph (esophagus) ); eyes; gallbladder bl (gall bladder); head and neck; heart; intest. la (large intestine); intest. sm (small intestine); kidneys; liver; lungs; lymph nodes; (parathyroid); perit (peritoneum); pituit (pituitary); placenta; pleura; prostate; skel. mus (skeletal muscle); skin; spinal cord; spleen; stomach; testes; thymus; thyroid; trachea; ureter; uterus. Tumor samples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma) cell tumor); GC (gastric cancer); GEJC (gastric-esophageal junction carcinoma); HCC (hepatocellular carcinoma); HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin's) lymphoma); NSCLCadeno (non-small cell lung cancer); NSCLCother (NSCLC samples that could not be clearly assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR (esophageal cancer); PACA ( pancreatic cancer); PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (bladder cancer); UEC (endometrial cancer). Figure 4A) Ensembl ID: ENST00000336375p218, Peptide: VHNFTLPSW (SEQ ID NO: 114); Figure 4B) Ensembl ID: ENST00000264144p359, Peptide: FFQNSTFSF (SEQ ID NO: 115); Figure 4C) Ensembl ID: ENST00000254508p924, Peptide : IVRNLSCRK (SEQ ID NO: 116); Figure 4D) Ensembl ID: ENST00000314191p3305, Peptide: YIDNVTLI (SEQ ID NO: 117).

Figure 5 shows the results of an IdentControl experiment on an exemplary peptide, VYTDISHHF (SEQ ID NO: 55). Peptides were identified by IdentControl in data-dependent acquisition (DDA) mode comparing stable isotope labeled (SIL) standard fragments. Identity was confirmed using an indoor measured spectral correlation threshold.

Figure 6 shows an exemplary result of one of the CoElution experiments for the peptide ISDITEKNSGLY (SEQ ID NO: 6). Peptides were confirmed by CoElution using stable isotope labeled (SIL) internal standard and target MS (sPRM or IS-PRM). Non-overlapping MS2 separation windows using SIL-peptides and native peptides. Control experiments using non-HLA peptidomics samples (eg, trypsin digestion or 5% FA) as matrix were performed to confirm the isotopic purity of the SIL internal standard. Peptide identities are confirmed based on objective, predefined criteria in expert manual verification.

Figures 7A-7E show exemplary results of peptide-specific in vitro CD8+ T cell responses of healthy HLA-A*01+, A*24+ or B*44+ donors. Using artificial APCs coated with anti-CD28 mAb and HLA-A*01, A*24, or B*44 with SEQ ID NO: 56 (A, left panel), SEQ ID NO: 28 (B, left panel) panel), SEQ ID NO: 30 (C, left panel), SEQ ID NO: 32 (D, left panel) or complexes of SEQ ID NO: 14 (E, left panel) sensitive CD8+ T cells. After three stimulation cycles, by using A*24/SEQ ID NO:56(A), A*01/SEQ ID NO:28(B), A*24/SEQ ID NO:30(C),B *44/SEQ ID NO: 32 (D) or A*01/SEQ ID NO: 14 (E) 2D multimer staining was performed to perform detection of peptide reactive cells. The right panels (A, B, C, D and E) show control staining of cells stimulated with unrelated HLA/peptide complexes of the same isotype as the complex of interest. Live single cells are gated against CD8+ lymphocytes. The Brin gate helps exclude false positive events detected with polymers specific for different peptides. The frequency of specific polymer+ cells in CD8+ lymphocytes is indicated.

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Claims (21)

A peptide comprising an amino acid sequence selected from the group consisting of: ● SEQ ID NO: 1 to SEQ ID NO: 113, ● and variant sequences thereof, which maintain the ability to bind to MHC molecules and/or induce cross-reactivity of T cells with the variant peptide, or a pharmaceutically acceptable salt thereof. The peptide of claim 1, wherein ● The peptide has the ability to bind to MHC class I molecules, and/or ● wherein the peptide can be recognized by CD4 and/or CD8 T cells when bound to the MHC. The peptide or variant thereof of any one of claims 1 to 2, wherein the peptide or variant thereof has a total length of 8 to 30 amino acids. The peptide or variant thereof of any one of claims 1 to 3, wherein the peptide comprises a non-peptide bond. The peptide or variant thereof of any one of claims 1 to 4, wherein the peptide is part of a fusion protein. An antibody or a functional fragment thereof that specifically recognizes or binds to a peptide or variant thereof as claimed in any one of claims 1 to 5, or when bound to an MHC molecule as in claims 1 to 5 Any one of the peptides or variants thereof. A T cell receptor or a functional fragment thereof that reacts with or binds to an MHC ligand, wherein the ligand is a peptide or a variant thereof as claimed in any one of claims 1 to 5, or a peptide as claimed in any one of claims 1 to 5 or a variant thereof when bound to an MHC molecule. A nucleic acid encoding the peptide or its variant according to any one of claims 1 to 5, the antibody or its fragment according to claim 6, or the T cell receptor according to claim 7 or its Fragment. A recombinant host cell comprising the peptide or variant thereof according to any one of claims 1 to 5, the antibody or fragment thereof according to claim 6, the T cell receptor according to claim 7 or A fragment thereof or a nucleic acid or expression vector as claimed in claim 8. An in vitro method for the generation of activated T lymphocytes, the method comprising in vitro combining T cells with antigen-loaded human class I or class II MHC molecules expressed on the surface suitable for antigen-presenting cells or artificial constructs that mimic antigen-presenting cells is contacted for a period of time sufficient to activate the T cells in an antigen-specific manner, wherein the antigen is a peptide as claimed in any one of claims 1 to 5 or a variant thereof. An activated T lymphocyte produced by the method according to claim 11, which selectively recognizes cells presenting the peptide according to any one of claims 1 to 5 or a variant thereof. A pharmaceutical composition comprising at least one active ingredient selected from the group consisting of: ● A peptide or variant thereof as claimed in any one of claims 1 to 5, ● An antibody or fragment thereof as described in claim 6, ● a T cell receptor or fragment thereof as claimed in claim 7, ● The nucleic acid or expression vector as described in claim 8, ● a host cell as claimed in claim 9, ● or activated T lymphocytes as described in claim 10, and a pharmaceutically acceptable carrier. A method for producing a peptide or a variant thereof according to any one of claims 1 to 5, an antibody or a fragment thereof according to claim 6, or a T cell receptor or a fragment thereof according to claim 7 The method of , the method comprising culturing the host cell as claimed in claim 9, and isolating the peptide or variant thereof, the antibody or fragment thereof or the T cell receptor or fragment thereof from the host cell and/or its culture medium. The peptide or its variant as claimed in any one of claims 1 to 5, the antibody or its fragment as claimed in claim 6, the T cell receptor or its fragment as claimed in claim 7, or as claimed in claim 8 The nucleic acid or expression vector, the host cell according to claim 9, or the activated T lymphocyte according to claim 11, are used for medicine or for manufacturing medicine. A method of killing target cells in a patient, the target cells presenting a polypeptide comprising an amino acid sequence as given in any one of claims 1 to 5, the method comprising administering to the patient an effective amount of The activated T lymphocyte according to claim 11. The activated T lymphocyte according to claim 11, which is a) for use in the killing of target cells in a patient, the target cells presenting a polypeptide comprising an amino acid sequence as given in any one of claims 1 to 5, or b) Use in the manufacture of the agent for killing such target cells. A method of treating a patient, the patient ● Diagnosed with cancer, ● have cancer, or ● risk of developing cancer, The method comprises administering to the patient an effective amount of a peptide or variant thereof as claimed in any one of claims 1 to 5, an antibody or fragment thereof as claimed in claim 6, T as claimed in claim 7 A cell receptor or a fragment thereof, a nucleic acid or expression vector as claimed in claim 8, a host cell as claimed in claim 9, or an activated T lymphocyte as claimed in claim 11. The peptide or its variant as claimed in any one of claims 1 to 5, the antibody or its fragment as claimed in claim 6, the T cell receptor or its fragment as claimed in claim 7, or as claimed in claim 8 The nucleic acid or expression vector, the host cell as claimed in claim 9, or the activated T lymphocyte as claimed in claim 11 are used to treat a patient, the patient ● Diagnosed with cancer, ● have cancer, or ● risk of developing cancer, or for the manufacture of a medicament for the treatment of the patient. The method of claim 17 or the peptide, antibody, T cell receptor, nucleic acid, host cell or activated T lymphocyte for use as claimed in claim 18, wherein the cancer is selected from the group consisting of Cohorts: acute myeloid leukemia, breast cancer, cholangiocarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer, gastroesophageal junction cancer, hepatocellular carcinoma, head and neck squamous cell carcinoma cell carcinoma, melanoma, non-Hodgkin's lymphoma, non-small cell lung cancer, ovarian cancer, esophageal cancer, pancreatic cancer, prostate cancer, renal cell cancer, small cell lung cancer, bladder cancer, and endometrial cancer. A kit comprising: (a) a container containing a pharmaceutical composition containing the pharmaceutical composition as described in claim 12 in solution or in lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; (c) Optionally, at least one or more peptides selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 113. The kit of claim 20, further comprising one or more of a buffer, a diluent, a filter, a needle, or a syringe.

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