Symptoms of anti-PRAME binding

Abstract

1. A method for treating a metastatic lesion that presents a peptide comprising SLLQHLIGL (SEQ ID NO: 310) on its cell surface, comprising the steps of selecting a patient having a metastatic lesion and administering to the patient a composition comprising recombinant or activated T lymphocytes that express a T cell receptor or a functional fragment thereof that reacts with or binds to an MHC ligand comprising SLLQHLIGL (SEQ ID NO: 310).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Reference to sequence listing submitted as an XML 1.0 format compliant file (.xml)

[0002] In accordance with the EFS-Web legal framework and 37 CFR § 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and PatV § 11, an electronic sequence listing in the form of an XML 1.0 format file conforming to WIPO Standard ST.26 has been submitted contemporaneously with the present application, the entire contents of which are incorporated herein by reference. For the avoidance of doubt, in case of discrepancies between the sequences set forth in the specification and the electronic sequence listing, the sequences in the specification shall be deemed correct. [Technical field]

[0003] The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention further relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides, which may act as active pharmaceutical ingredients of vaccine compositions, for example, to stimulate anti-tumor immune responses or to stimulate T-cells ex vivo and transfer to patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or the peptides themselves, can also be targets of antibodies, soluble T-cell receptors and other binding molecules.

[0004] The present invention relates to several novel peptide sequences and their variants derived from HLA class I molecules of human tumor cells that can be used to elicit anti-tumor immune responses in vaccine compositions or as targets for the development of pharmacologic / immunologically active compounds and cells. [Background technology]

[0005] According to the World Health Organization (WHO), cancer was ranked one of the world's four leading non-communicable fatal diseases in 2012. In the same year, colorectal, breast and respiratory tract cancers were among the top ten causes of death in high-income countries.

[0006] Cancer immunotherapy Cancer immunotherapy represents an option to specifically target cancer cells while minimizing side effects. Cancer immunotherapy exploits the presence of tumor-associated antigens.

[0007] The current classification of tumor associated antigens (TAA) includes the following major groups:

[0008] a) Cancer-testis antigens: The first ever identified TAAs capable of being recognized by T cells belong to this class, originally called cancer-testis (CT) antigens. Since cells of the testis do not express class I and II HLA molecules, these antigens cannot be recognized by T cells from normal tissues and can therefore be considered immunologically tumor-specific. Well-known examples of CT antigens are the MAGE family members, PRAME and NY-ESO-1.

[0009] b) Differentiation antigens: These TAAs are shared between tumors and the normal tissue from which they originate. Most of the known differentiation antigens are found in melanomas and normal melanocytes. Examples include, but are not limited to, tyrosinase and Melan-A / MART-1 in melanoma and PSA in prostate cancer.

[0010] c) Overexpressed TAAs: Genes encoding widely expressed TAAs have been detected not only in various types of tumors histologically, but also in many normal tissues, generally at lower expression levels. While many of the epitopes processed and potentially presented by normal tissues are below the threshold level for T cell recognition, their overexpression in tumor cells may trigger anticancer responses by breaking previously established immune tolerance. Prominent examples of this class of TAAs are Her-2 / neu, survivin, telomerase, or WT1.

[0011] d) Tumor-specific antigens: These unique TAAs arise from mutations in normal genes (such as β-catenin, CDK4, BCR-ABL, etc.). Some of these molecular alterations are associated with neoplastic transformation and / or tumor progression. Tumor-specific antigens can generally induce a strong immune response without the risk of an autoimmune response against normal tissues. On the other hand, these TAAs are most often only associated with the exact tumor in which they were identified and are not usually shared between many individual tumors. In the case of proteins with tumor-specific (associated) isoforms, tumor specificity (or association) of peptides can also arise if the peptides arise from tumor-specific (associated) exons.

[0012] e) Oncoviral proteins: These TAAs are viral proteins that may play an important role in the carcinogenesis process and, being foreign (not of human origin), can elicit T cell responses. An example of such a protein is the human papilloma type 16 virus proteins E6 and E7, which are expressed in cervical carcinomas.

[0013] Human endogenous retroviruses (HERVs) represent a significant portion of the human genome (approximately 8%). These viral elements integrated into the genome millions of years ago and have been vertically transmitted for many generations since then. Although the majority of HERVs have lost functional activity through mutations or truncations, some endogenous retroviruses, such as members of the HERV-K clade, have been shown to still encode functional genes and form retrovirus-like particles. Transcription of HERV proviruses is epigenetically regulated and remains silent under normal physiological conditions. However, in certain diseases and especially in various types of cancer, reactivation and overexpression leading to active translation of viral proteins have been described. This tumor-specific expression of HERV-derived proteins can be exploited for various types of cancer immunotherapy.

[0014] f) TAAs resulting from aberrant post-translational modifications: Such TAAs may arise from proteins that are neither tumor-specific nor overexpressed, but may still become tumor-associated due to post-translational processes that are primarily active in tumors. Examples of this class arise from events such as changes in glycosylation patterns that result in novel epitopes in tumors, as in the case of MUC1, or protein splicing during degradation, which may or may not be tumor-specific.

[0015] T cell-based immunotherapy targets peptide epitopes derived from tumor-associated or tumor-specific proteins presented by MHC molecules. The antigens, i.e., the epitopes, recognized by tumor-specific T lymphocytes can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc., that are expressed in the cells of each tumor and are usually upregulated compared to unmodified cells of the same origin.

[0016] There are two classes of MHC molecules: MHC class I and MHC class II. MHC class I molecules are composed of alpha (heavy) chains and beta 2 microglobulin (light chain, β2m), while MHC class II molecules are composed of alpha and beta chains. Their three-dimensional conformation results in a binding groove that is used for non-covalent interactions with peptides.

[0017] MHC class I molecules are found on most nucleated cells. They present mainly endogenous proteins, defective ribosomal products (DRIPs) and peptides resulting from proteolytic cleavage of larger peptides. However, peptides originating from endosomal compartments or exogenous sources are also frequently found on MHC class I molecules. This non-classical mode of class I presentation is referred to in the literature as cross-presentation (Rock, Gamble, and Rothstein, 1990; Brosart and Bevan, 1997). MHC class II molecules are found mainly on professional antigen presenting cells (APCs) and mainly present peptides of exogenous or transmembrane proteins that are taken up by the APCs during, for example, endocytosis and subsequently processed.

[0018] It is well known that peptide-MHC class I complexes are recognized by CD8+ T cells with the appropriate T cell receptor (TCR), whereas peptide-MHC class II complexes are recognized by CD4+ helper T cells with the appropriate TCR, resulting in a 1:1:1 stoichiometry of TCR, peptide, and MHC.

[0019] CD4+ helper T cells play an important role in inducing and maintaining effective responses by CD8+ cytotoxic T cells. Identification of CD4+ T cell epitopes derived from tumor-associated antigens (TAA) is of great importance for the development of pharmaceutical products to trigger antitumor immune responses. At the tumor site, helper T cells support a cytokine environment favorable to cytotoxic T cells (CTLs) and attract effector cells, such as CTLs, natural killer (NK) cells, macrophages, and granulocytes.

[0020] According to various sources, over 90% of cancer deaths are caused by lesions including metastases (Hanahan and Weinberg, 2000). To date, there are only a few therapeutic options to address such metastatic lesions.

[0021] Therefore, there is an urgent need for new and effective treatments for such conditions, as well as the identification of factors that represent biomarkers for such metastatic lesions and that would lead to better diagnosis, prognosis assessment, and prediction of therapeutic success of such metastatic lesions. Summary of the Invention [Means for solving the problem]

[0022] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific components of the described devices or process steps of the described methods, since the described devices and methods may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that the singular forms "a", "an" and "the" as used in this specification and the appended claims include singular and / or plural referents unless the context clearly dictates otherwise. Furthermore, when a parameter range bounded by numerical values ​​is given, it should be understood that the range is deemed to include these limits. It should further be understood that the embodiments disclosed herein are not intended to be understood as separate embodiments that are unrelated to each other. Features described in one embodiment are also intended to be disclosed in conjunction with other embodiments shown herein. In some cases, if a particular feature is not disclosed in one embodiment but is disclosed in another embodiment, a person skilled in the art will understand that this does not necessarily mean that the feature cannot be disclosed in the other embodiment. Those skilled in the art will appreciate that while it is the intent of the present application to disclose such features in other embodiments, this has not been done merely for the sake of clarity and to keep the specification to a manageable length.

[0023] Furthermore, the contents of the prior art documents referenced herein are incorporated by reference, particularly those prior art documents that disclose standard or routine methods, in which case incorporation by reference is primarily intended to provide a full and enabling disclosure and to avoid redundant repetition.

[0024] According to a first aspect of the present invention there is provided a peptide comprising the amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a pharma- ceutically acceptable salt thereof, said peptide for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with, suffering from, or at risk of developing, (i), a metastasis or metastatic lesion (ii).

[0025] This language is deemed to encompass both Swiss-style claim language permitted in some countries (in which case the brackets are deemed not to exist) and EPC 2000 language (in which case the brackets and any content within them are deemed not to exist).

[0026] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0027] The method includes administering to the patient one or more therapeutically effective doses of a peptide comprising the amino acid sequence of SEQ ID NO:310 (SLLQHLIGL), or a pharma- ceutically acceptable salt thereof.

[0028] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or a metastatic lesion, comprising as an active ingredient a peptide comprising the amino acid sequence of SEQ ID NO:310 (SLLQHLIGL) or a pharma- ceutically acceptable salt thereof.

[0029] In one embodiment, the treatment or composition does not include co-administration (simultaneous or sequential) with a peptide that is a fragment of Prostate specific Membrane antigen (PSMA), the amino acid sequence of which is disclosed in UniProt reference number Q04609.

[0030] In particular, the treatment is 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297It does not include co-administration (simultaneous or sequential) with 1297V (GLPSIPVHPV, SEQ ID NO: 377).

[0031] In one embodiment, the peptides used in the treatment do not include any N- or C-terminal residues beyond the sequence set forth in SEQ ID NO:1.

[0032] In one embodiment, the metastasis or metastatic lesion is PRAME positive. As used herein, the term "PRAME positive metastasis or metastatic lesion" refers to a metastasis or metastatic lesion comprising cells expressing PRAME. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells the amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0033] The term "metastasis" refers to the spread of cancer cells or tissues from a primary tumor. Cancer occurs after cells undergo genetic changes that allow them to grow rapidly and indefinitely. The cells eventually undergo metaplasia, followed by dysplasia, and then anaplasia, resulting in a malignant phenotype often referred to as the "primary tumor." This malignant tumor allows for invasion into the circulation and then invasion of a second site for tumor formation.

[0034] Some cells of the primary tumor acquire the ability to penetrate the walls of lymphatic or blood vessels and then circulate through the bloodstream to other parts and tissues of the body. This process is known as lymph node metastasis or hematogenous metastasis. After tumor cells are arrested at another site, they again penetrate the blood vessel or wall and continue to grow, eventually forming another clinically detectable tumor. This new tumor is known as a metastasis (the plural is "metastases" and both terms may be used interchangeably herein) and generally causes a metastatic lesion. Metastasis is one of the hallmarks of cancer that distinguishes it from benign tumors. Most cancers can metastasize, but some do not. For example, basal cell carcinoma rarely metastasizes.

[0035] Regarding nomenclature, the following rules apply: (i) The term "metastatic breast cancer" relates to breast cancer as a primary tumour that releases cancer cells throughout the body that may or may not colonise the same or other organs or tissues and form metastases. (ii) The term "breast cancer metastasis" refers to metastases in the breast or other organs or tissues that have spread from a breast cancer as a primary tumor.

[0036] This nomenclature is used, for example, (i) metastatic lung cancer; (ii) lung cancer metastasis; and / or (ii) metastatic liver cancer; It also relates to all other tumor or cancer types or metastases.

[0037] Thus, in diagnosis, a metastasis found anywhere in the body is often considered, for example, a lung cancer metastasis if the patient has been diagnosed with a primary lung tumor, or a colon cancer metastasis if the patient has been diagnosed with a primary colon tumor.

[0038] This nomenclature is used throughout this application.

[0039] In one embodiment, the metastases or metastatic lesions according to the present invention occur in one or more vital organs, preferably at least one selected from the group consisting of the brain, spinal cord, heart, lungs, liver, bone marrow, blood, trachea, skin, kidneys, pancreas and intestines.

[0040] In one embodiment, metastases or metastatic lesions according to the invention are 1 cm or greater in diameter, in one embodiment thereof, such metastases or metastatic lesions occur in vital organs.

[0041] In one embodiment, 10 or more metastases or metastatic lesions are found in the patient, preferably 10 or more metastases or metastatic lesions, in one embodiment of which such metastases or metastatic lesions occur in vital organs.

[0042] In one embodiment, the metastasis or metastatic lesion has progressed beyond the lymphatic system.

[0043] In one embodiment, the metastasis or metastatic lesions are not limited to the lymphatic system.

[0044] Metastases can, and often do, acquire further mutations and evolve independently of their original tumors at the metastatic site, so information obtained from observations of primary tumors does not necessarily apply to those metastases, and the independent growth of metastases may introduce some differences between primary tumors and the metastases derived from them that may affect the clinical outcome of cancer.

[0045] Some of these differences may affect the presentation levels of pHLA and may include, but are not limited to:

[0046] (a) Differences in antigen peptide-presenting complexes. An overview of the loss of MHC class I antigen presentation during cancer evolution can be found in (Dhatchinamoorthy, Colbert, & Rock, 2021). In particular, downregulation of antigen processing and presentation complexes in metastasis has been shown through reduced expression of TAP1 (Ling et al., 2017), HLA (McGranahan et al., 2017; Watkins et al., 2020), and b2M (Campo et al., 2014).

[0047] (b) Downregulation of specific genes and antigens Apart from the downregulation of the MHC presentation pathway in metastases, reduced expression of tumor antigens used in clinical trials, such as TRPM8 ( Fuessel et al., 2006 ), has also been reported ( Yao et al., 2019 ).

[0048] Both mechanisms of downregulation of antigen processing pathways and downregulation of specific antigens may contribute to the effects seen in Figure 42 showing presentation of peptide KRT5-004 (STASAITPSV, SEQ ID NO:312).

[0049] KRT5-004 is related to the parent protein keratin 5, also known as KRT5, K5, or CK5, a protein encoded by the KRT5 gene in humans. It dimerizes with keratin 14 to form intermediate filaments (IFs), which constitute the cytoskeleton of basal epithelial cells. This protein is involved in several diseases, including epidermolysis bullosa simplex and breast and lung cancer.

[0050] When comparing HNSCC (head and neck squamous cell carcinoma) primary tumors with HNSCC metastases, KRT5-004 expression is completely lost. SEQ ID NO: 312 is detected in nearly 50% of primary HNSCC tumor samples, but is completely absent from the metastatic HNSCC tumor samples analyzed.

[0051] Furthermore, differences in chemosensitivity to common chemotherapy drugs have been reported when comparing primary and metastatic tumor samples from the same patient (Furukawa et al., 2000).

[0052] FIG. 40 shows that peptide PRAME 004 (SLLQHLIGL, SEQ ID NO: 310) is presented on selected metastases but not on healthy tissues.

[0053] FIG. 43 shows that peptide PRAME 004 (SLLQHLIGL, SEQ ID NO: 310) is differentially presented on selected metastases and selected healthy tissues.

[0054] FIG. 45 shows that peptide PRAME 004 (SLLQHLIGL, SEQ ID NO: 310) is differentially presented on primary and metastatic triple-negative breast cancer (TNBC).

[0055] Figures 48, 49A, and 49B show experiments with patient-derived xenografts (PDXs), in which tumor metastases were xenografted in a preclinical mouse model with a tumor biology as close as possible to the in vivo situation in patients. The main genetic and histological characteristics of the patient metastases remained unchanged over time (mouse passage). For this reason, the PDX models used are superior to cell line-derived xenografts (CDXs), which do not preserve, let alone possess, the physiological characteristics of metastases, including the immunopeptidome.

[0056] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0057] Metastases or metastatic lesions can be analyzed by various means to determine whether they display, on the surface of at least one of their cells, the amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0058] In one embodiment, a tumor biopsy or another diagnostically relevant sample (such as a blood, lymph, cerebrospinal fluid, saliva or urine sample containing suspended cells, sHLA, exosomes, tumor-derived extracellular vesicles (EVs), etc.) is taken and subjected to immunoprecipitation of peptide-MHC complexes followed by analysis of the thus obtained peptidome by mass spectrometry. Respective methods are disclosed, for example, in Fritsche et al., 2018, the contents of which are incorporated herein by reference.

[0059] Another possibility is to use a labeled T cell receptor or TCR mimetic antibody specific for a peptide-MHC complex containing the peptide of SEQ ID NO: 310 (SLLQHLIGL). In one embodiment, a biopsy or sample of the metastasis is obtained, evaluated by routine immunological methods (slicing, homogenization, etc.), and then incubated with the T cell receptor of the TCR mimetic antibody. For methods, see, for example, Hoydahl et al., 2019, the contents of which are incorporated herein by reference.

[0060] In another embodiment, the mRNA encoding the parent protein giving rise to the peptide of interest or encoding its particular exon can be determined, for example, by qRT-PCR or any other mRNA detection technique. Such methods are within the routine of a skilled artisan. See, for example, (Wong and Medrano, 2005; Moon et al., 2020), the contents of which are incorporated herein by reference.

[0061] Another possibility is to apply RNA-Seq technology to metastasis. RNA-Seq (short for "RNA-Sequencing") is a sequencing technology that uses next-generation sequencing (NGS) to analyze the continuously changing cellular transcriptome to reveal the presence and amount of RNA in a biological sample at a given moment. In particular, RNA-Seq facilitates the ability to examine alternative gene splicing transcripts, post-transcriptional modifications, gene fusions, mutations / SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments. In addition to mRNA transcripts, RNA-Seq can interrogate various RNA populations to include total RNA, miRNA, small RNA such as tRNA, and ribosome profiling. RNA-Seq can also be used to determine exon / intron boundaries and to verify or revise previously annotated 5' and 3' gene boundaries. Recent advances in RNA-Seq include native RNA molecular sequencing by single-cell sequencing, in situ sequencing of fixed tissues, and single-molecule real-time sequencing.

[0062] Respective HLA status can be determined by routine methods of HLA serotyping and HLA haplotyping, for example as disclosed in (Zhang et al., 2014), the contents of which are incorporated herein by reference.

[0063] A2 is a human leukocyte antigen serotype within the HLA-A serogroup. The serotype is determined by antibody recognition of the α2 domain of the HLA-A α chain. In A2, the α chain is * The α-chain is encoded by the 02 gene, and the β-chain is encoded by the B2M locus.

[0064] HLA‐A * HLA-A is a specific set of class I major histocompatibility complex (MHC) alleles at the HLA-A locus. *The HLA-A2 allele group can code for a large number of proteins, and as of December 2013, there are 456 different HLA-A * The serotype typing was HLA-A. * HLA can be specified by up to 02, which is usually sufficient to prevent transplant rejection (the original motivation for HLA specification). Genes can be further divided by gene sequencing and analysis. HLA can be specified by as many as nine numbers and letters (e.g., HLA-A * 02:101:01:02N). HLA‐A * Although 02 is common worldwide, geographical regions may separate specific variants of the allele.

[0065] The term "peptide" as used herein is intended to include salts of a series of amino acid residues that are typically connected to one another by peptide bonds between the alpha amino and carbonyl groups of adjacent amino acids. Preferably, the salts are pharma- ceutically acceptable salts of peptides, such as chloride or acetate (trifluoroacetate). It should be noted that peptides are not salts in vivo, so that salts of peptides according to the present description are substantially different from their state(s) in vivo.

[0066] As used herein, "pharmaceutical acceptable salt" refers to a derivative of the peptide of the present disclosure, in which the peptide is modified by making an acid or base salt of the drug. For example, an acid salt is prepared from a free base (where the neutral form of the drug typically has a neutral -NH2 group) by reaction with a suitable acid. Suitable acids for preparing acid salts include both 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, as well as inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Conversely, preparations of base salts of acid moieties that may be present on the peptide are prepared using pharmaceutical acceptable bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine, and the like.

[0067] For example, the pharma- ceutically acceptable salt is selected from the chloride, acetate, trifluoroacetate, phosphate, nitrate, sulfate, bromide, propionate, glycolate, pyruvate, oxalate, malate, maleate, malonate, succinate, fumarate, tartrate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, salicylate, sodium salt, potassium salt, ammonium salt, calcium salt, or trimethylamine salt.

[0068] SEQ ID NO:310 (SLLQHLIGL, alias: PRAME-004) is a peptide related to PRAME, a protein encoded by the PRAME gene.

[0069] PRAME (Preferentially Expressed Antigen in Melanoma), also known as Opa-interacting protein 4, CT130 and MAPE, is a protein and tumor antigen of the cancer / testis antigen family. PRAME is 509 amino acids in length and has a mass of 57,890 Da. PRAME has the Entrez identifier number 23532 and the UniProt identifier number P78395.

[0070] PRAME is expressed at high levels in the majority of tumors, as well as in some types of leukemia. PRAME is the best-characterized member of the PRAME family of leucine-rich repeat (LRR) proteins. Mammalian genomes contain multiple members of the PRAME family, whereas only one PRAME-like LRR protein has been identified in other vertebrate genomes. PRAME is a cancer / testis antigen that is expressed at very low levels in normal adult tissues, except testis, but at high levels in a variety of cancer cells.

[0071] PRAME-004 is a nine amino acid peptide derived by degradation of PRAME by the ubiquitin-proteasome system (UPS). PRAME-004 is also called PRA425-433 because it contains AA residues 425-433 of the PRAME protein. PRAME-004 is then presented on the cell surface of the respective cells by major histocompatibility complex (MHC) class I molecules.

[0072] The inventors have found that PRAME-004 is displayed with high selectivity on MHC class I molecules of primary tumors (see, for example, International Publication No. 2018172533A2 and US Patent No. 20180273602, the contents of which are incorporated by reference in their entirety). Thus, the inventors describe that PRAME-004 can be used as a target for entities capable of binding to PRAME-004 for the treatment of various primary tumors.

[0073] However, we have surprisingly discovered that PRAME-004 is also presented by metastases and metastatic lesions, for which only very limited therapeutic options have been available until now.

[0074] As used herein, the term "metastasis" refers to the spread of cancer cells from where they first formed (i.e., initial or primary site) to another part of the host's body (i.e., a different or secondary site). In metastatic cancer, cancer cells break off from the original (primary) tumor, travel through the blood or lymphatic system, and form new (secondary) tumors in the same or other organs or tissues of the body. These newly formed pathological sites are called metastases or metastatic tumor(s). The new (or secondary) metastatic tumors are of the same type of cancer as the primary tumor. Because metastatic cancer cells share some characteristics with the primary cancer, they are generally referred to by the same name as the primary cancer. For example, breast cancer that spreads to the lungs is generally referred to as metastatic breast cancer (not lung cancer) and is therefore treated as breast cancer, not as lung cancer.

[0075] In some cases of metastatic cancer, the source of the cancer cannot be identified (for example, when the primary tumor cannot be located). This type of cancer is called cancer of unknown primary or occult primary.

[0076] Cancer that spreads from where it began to another part of the body is called metastatic cancer. The direct extension and penetration by cancer cells into adjacent tissues is called "cancer invasion" and is the first step in the process of metastasis (see below). In many types of cancer, metastatic cancer is also called advanced or stage IV (4) cancer. However, the terms stage IV (4) and advanced cancer can also refer to cancers that are large but have not spread to other parts of the body (e.g., locally advanced cancers).

[0077] The process by which cancer cells spread to other parts of the body is called metastasis. The term metastasis refers to the spread of a pathogen from its initial (primary) site to a different (secondary) site in the host's body. As used herein, the term metastasis refers to the spread of cancerous cells or tumors from its initial (primary) site to a different (secondary) site in the host's body. Thus, as used herein, metastatic cancer is cancer associated with metastasis, which is the spread of cancer from its primary site (where the cancer originated) to other places in the body.

[0078] Additionally, as used herein, the term metastasis is intended to mean the occurrence of secondary tumors in parts of the body that are different and / or distant from the original primary cancer (Fares et al., 2020).

[0079] Thus, as used herein, metastasis is the dissemination of tumor cells from a primary neoplasm to secondary sites in a multistep process that is often depicted as a simple series of sequential events: escape from the primary tumor and local invasion, intravasation and survival in the circulation, and extravasation and metastatic seeding (Riggio, Varley, and Welm, 2021).

[0080] Metastasis is divided into two main stages: the physical dissemination of cancer cells from the primary tumor to adjacent tissues, and the adaptation and thus successful colonization of these cells into the adjacent tissue microenvironment, i.e., the metastatic growth of the macroscopic tumor, including the metastatic lesion. In one embodiment, the terms "metastasis" and "metastatic lesion" are used synonymously.

[0081] Metastasis refers to the accumulation of cancer cells of the same type as the primary tumor, but localized and separated from the site of the primary tumor. This accumulation can be in the same or different organ or tissue, and can lead to neoplastic growth. Separation from the primary tumor can be confirmed, for example, by any of the following invasive or non-invasive methods or any combination thereof: Macroscopic assessment, e.g. through visual or instrument-guided (e.g. endoscopy) examination, of metastasis formation, e.g. during surgery or screening of cancer patients. Histopathological evaluation of tissues harvested from surgical procedures, including biopsies. For this evaluation, a skilled artisan (e.g., a trained pathologist) may wish to further utilize various types of physical or chemical treatments of the harvested tissue (e.g., FFPE preservation), staining with chemical reagents (including, e.g., dyes or antibodies that bind to molecular or genetic markers), or known additional analyses that may further facilitate the identification of cancer cells to confirm metastasis formation. Medical imaging techniques such as computed tomography (CT), magnetic resonance tomography (MRI), positron emission tomography (PET), ultrasound, X-ray or any combination of these (e.g. PET / MRI). Biomarker-based assays such as prostate serum antigen (PSA) screening or other assays that quantify biomolecules indicative of primary and / or metastatic cancer in clinical samples including, but not limited to, blood, urine, stool, etc.

[0082] Most cancer cells that spread die at some stage during the metastatic process. But if conditions are favorable for them at every step, some of them can form new tumors in other parts of the body. Metastatic cancer cells can also remain inactive in distant sites for years and then start growing again if they can.

[0083] Cancer can spread to almost any part of the body, but some types of cancer are more likely to metastasize to certain areas than others. Certain organ sites (sometimes called "fertile soil" or "metastatic niches") may be particularly permissive for metastatic seeding and colonization by certain types of cancer cells as a result of local properties that are inherent in normal tissues or induced remotely by the systemic effects of the primary tumor. Cancer stem cells may be variably involved in various stages or any or all of primary tumor formation and metastasis (Hanahan and Weinberg, 2011).

[0084] In further embodiments, metastatic cancer appears after a long period of undetectable disease following surgery or systemic therapy due to relapse or recurrence, e.g., in breast cancer, metastatic relapse can occur months to decades after initial diagnosis and treatment.

[0085] Thus, metastatic cancer can arise de novo, where metastases are present at the time of original diagnosis and the cancer has already spread before detection, but de novo occurrence is more often the result of relapse (recurrence), where metastases appear after definitive treatment (Riggio, Varley, & Welm, 2021).

[0086] Representative cancers prone to metastasis may include adrenocortical carcinoma, breast carcinoma, lung cancer, melanoma, colon cancer, renal cell carcinoma, prostate cancer, cervical cancer, cervical squamous cell carcinoma and adenocarcinoma, bile duct carcinoma, bladder cancer, bladder urothelial carcinoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, rectal cancer, esophageal cancer, esophageal carcinoma, liver cancer, liver hepatocellular carcinoma, oral and pharyngeal cancer, multiple myeloma, ovarian cancer, ovarian serous cystadenocarcinoma, sarcoma, gastric adenocarcinoma, testicular germ cell tumor, thymoma, uterine carcinosarcoma, endometrial carcinoma, and gastric cancer. In some embodiments, the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, endometrial carcinoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, gastric adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, embryonal sarcoma, The cancer may be from a cancer selected from the group consisting of: ovarian cancer, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, cholangiocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0087] (Liu and Cao, 2016), the contents of which are incorporated herein by reference in their entirety, show that primary tumors can generate favorable microenvironments in secondary organs and tissue sites for subsequent metastasis, i.e., pre‐metastatic niches (PMNs). The pre‐metastatic niche can be prepared and established through complex interactions between primary tumor‐derived factors, bone marrow‐derived cells recruited by the tumor, and local stromal components. Liu et al. proposed six features that may define the pre‐metastatic niche that allow tumor cell colonization and promote metastasis, including (1) immunosuppression, (2) inflammation, (3) angiogenesis / vascular permeability, (4) lymphangiogenesis, (5) organ tropism, and (6) reprogramming.

[0088] For example, primary tumor-derived components, bone marrow-derived cells (BMDCs) recruited by the tumor, and the local stromal microenvironment of the host (or future metastatic organ components) may be important factors in the formation of the premetastatic niche. Numerous molecular and cellular components that contribute to premetastatic niche formation have been identified in various tumor models. In addition to being secreted by tumor cells, these niche-promoting molecular components can also be produced by myeloid and stromal cells. They may work together with cellular components to initiate, polarize, and establish the premetastatic niche in the future metastatic organ.

[0089] Representative primary tumor determinants of organ-specific metastasis can be found, for example, in Table 1 of (Liu and Cao, 2016), the contents of which are incorporated by reference.

[0090] Tumor-derived extracellular vesicles (EVs) may travel far from their original site to act as potential mediators to educate the premetastatic niche. EVs can be classified into the categories of exosomes (30–100 nm in diameter), microvesicles (100–1,000 nm in diameter), and a newly identified cancer-derived EV population called “large oncosomes” (1–10 mm in diameter). Exosomes, containing proteins, mRNA, microRNA, small RNA, and / or DNA fragments, may promote premetastatic niche formation by mediating communication between tumor cells and surrounding components or by horizontally transferring their contents to recipient cells. Tumor-derived microvesicles may mediate crosstalk between tumor cells and host cells in the secondary microenvironment for premetastatic niche formation. Tumor-derived large oncosomes contain metalloproteinases, RNA, caveolin-1, and the GTPase ARF6, suggesting that metastatic tumor cells can program distant sites into premetastatic niches via secretion of large oncosomes.

[0091] Some embodiments of the present disclosure may include a method of inhibiting metastatic lesions in a subject comprising the steps of selecting a subject having cancer that presents a peptide consisting of SLLQHLIGL (SEQ ID NO: 310) on its cell surface at an increased exosomal level of one or more markers for metastatic lesions compared to a control exosomal level of the one or more markers for metastatic lesions, where the marker for metastatic lesions is at least one selected from the group consisting of PMN-promoting molecules listed in Table 1 of (Liu and Cao, 2016), and administering to the selected subject a T cell and / or bispecific molecule of the present disclosure in an amount effective to inhibit metastatic lesions in the subject.

[0092] In one embodiment, the treatment may be a treatment of a patient experiencing metastatic cancer. The treatment of the present disclosure may also be administered to a patient with cancer who has elevated exosomal levels of one or more markers of metastatic lesions, but prior to the identification of any metastasis, to prevent metastasis. Similarly, patients who may develop potentially malignant neoplasms may be treated by the methods described herein. Subjects in need of treatment may be identified by a diagnosis of a potentially malignant neoplasm. Treatment groups may include subjects who cannot undergo traditional cancer treatments, such as surgery, radiation therapy, or chemotherapy. Patients with metastatic cancer or at risk of cancer metastasis may not be able to undergo certain cancer treatments due to other diagnoses, physical conditions, or comorbidities. For example, debilitated patients, such as elderly patients or those experiencing cancer cachexia, may not be good candidates for surgery, as they are at risk of not being able to tolerate invasive procedures. Patients who are already immunocompromised or have chronic infections may not be able to undergo chemotherapy, as many chemotherapy drugs can harm the immune system.

[0093] Metastases can, and often do, acquire further mutations and evolve independently of their original tumors at the metastatic site, so information obtained from observations of primary tumors does not necessarily apply to those metastases, and the independent growth of metastases may introduce some differences between primary tumors and the metastases derived from them that may affect the clinical outcome of cancer.

[0094] Some of these differences may affect the presentation levels of pHLA and may include, but are not limited to:

[0095] (c) Differences in antigen peptide-presenting complexes. An overview of the loss of MHC class I antigen presentation in cancer evolution can be found in (Dhatchinamoorthy, Colbert, & Rock, 2021). In particular, downregulation of antigen processing and presentation complexes in metastasis has been shown through reduced expression of TAP1 (Ling et al., 2017), HLA (McGranahan et al., 2017; Watkins et al., 2020), and β2M (Campo et al., 2014).

[0096] (d) Downregulation of specific genes and antigens Apart from the downregulation of the MHC presentation pathway in metastases, reduced expression of tumor antigens used in clinical trials, such as TRPM8 ( Fuessel et al., 2006 ), has also been reported ( Yao et al., 2019 ).

[0097] Both mechanisms of downregulation of antigen processing pathways and downregulation of specific antigens may contribute to the effects seen in Figure 42 showing presentation of peptide KRT5-004 (STASAITPSV, SEQ ID NO:312).

[0098] KRT5-004 is related to the parent protein keratin 5, also known as KRT5, K5, or CK5, a protein encoded by the KRT5 gene in humans. It dimerizes with keratin 14 to form intermediate filaments (IFs), which constitute the cytoskeleton of basal epithelial cells. This protein is involved in several diseases, including epidermolysis bullosa simplex and breast and lung cancer.

[0099] When comparing HNSCC (head and neck squamous cell carcinoma) primary tumors with HNSCC metastases, KRT5-004 expression is completely lost. SEQ ID NO: 312 is detected in nearly 50% of primary HNSCC tumor samples, but is completely absent from the metastatic HNSCC tumor samples analyzed.

[0100] Furthermore, differences in chemosensitivity to common chemotherapy drugs have been reported when comparing primary and metastatic tumor samples from the same patient (Furukawa et al., 2000).

[0101] Those skilled in the art have at their disposal a variety of routine approaches to determine whether a cell, or a metastasis or metastatic lesion is PRAME positive. Based on Entrez Identification No. 23532 and UniProt Identification No. P78395, those skilled in the art can also use immunohistochemical methods (such as ELISA, RIA, etc.) in which an antibody or binding agent that binds to PRAME protein in a suitable tissue sample is used. Alternatively, those skilled in the art can detect the presence or absence of PRAME mRNA by RT-PCR or other routine methods.

[0102] In a preferred embodiment of the invention, the term metastasis or metastatic lesion excludes the primary tumor.

[0103] According to one embodiment of the invention, the peptide has the ability to bind to an MHC class I or class II molecule and / or the peptide, when bound to said MHC, is capable of being recognized by CD4 or CD8 T cells.

[0104] Peptide-MHC class I complexes are recognized by CD8+ T cells bearing the appropriate T cell receptor (TCR).

[0105] According to one embodiment of the invention, the pharma- ceutically acceptable salt is a chloride or acetate salt.

[0106] According to further embodiments, the peptide may also have a total length of 9-30 amino acids. Preferably, the peptide has 9-12 amino acids. In one embodiment, said peptide comprises 1-4 additional amino acids at the C-terminus and / or N-terminus of SEQ ID NO: 310. For further details, see Table 1.

[0107] [Table 1]

[0108] In one embodiment, said peptides have a respective length according to SEQ ID NO: 310. In one embodiment, the peptide consists or consists essentially of an amino acid sequence according to SEQ ID NO:310.

[0109] According to another aspect of the invention there is provided an antibody, or functional fragment thereof, which specifically recognises or binds to a peptide according to above, or to a peptide according to above when bound to an MHC molecule.

[0110] The antibody or functional fragment is provided for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with metastasis or metastatic lesion (i), suffering from (ii), or at risk of developing (iii).

[0111] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0112] The method includes administering to a patient one or more therapeutically effective doses of a peptide according to the above, or an antibody, or functional fragment thereof, that specifically recognizes or binds to a peptide according to the above when bound to an MHC molecule.

[0113] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or metastatic lesions, comprising as an active ingredient a peptide according to the above, or an antibody or functional fragment thereof that specifically recognizes or binds to the peptide according to the above when bound to an MHC molecule. In one embodiment, said treatment or composition does not include co-administration (simultaneous or sequential) with an antibody or functional fragment thereof that binds a peptide that is a fragment of prostate specific membrane antigen (PSMA).

[0114] In particular, the treatment is 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297 It does not include co-administration (simultaneous or sequential) with an antibody or functional fragment thereof that binds 1297V (GLPSIPVHPV, SEQ ID NO: 377).

[0115] The term "antibody" as used herein refers to an antibody composition consisting of a homogenous antibody population, i.e. a homogenous population of whole immunoglobulins, or fragments or derivatives thereof that retain target binding ability. It is particularly preferred that such antibodies are selected from the group consisting of IgG, IgD, IgE, IgA and / or IgM, or fragments or derivatives thereof that retain target binding ability.

[0116] As used herein, the term "functional fragment" refers to a fragment of such an antibody that retains target binding ability, e.g. ·CDR (complementarity determining region) Hypervariable region Variable domain (Fv) IgG or IgM heavy chain (consisting of VH, CH1, hinge, CH2 and CH3 regions) IgG or IgM light chains (consisting of the VL and CL regions), and / or Fab and / or F(ab)2 This refers to the following.

[0117] As used herein, the term "derivative" is intended to refer to protein constructs that are structurally distinct from the typical antibody concept, yet have some structural relationship and still retain target binding ability, such as scFv, Fab and / or F(ab)2, as well as bi-, tri- or higher specificity antibody constructs, all of which are described below.

[0118] Other antibody derivatives known to the skilled person are bispecific antibodies (diabodies), camelid antibodies, nanobodies, domain antibodies, scFvs, bivalent homodimers with two chains consisting of IgA (two IgG structures linked by a J chain and a secretory component), shark antibodies, antibodies consisting of a New World primate framework and non-New World primate CDRs, dimerization constructs containing CH3+VL+VH, and antibody conjugates (e.g. antibodies or fragments or derivatives linked to toxins, cytokines, radioisotopes or labels). These types are well described in the literature and can be used by the skilled person based on the present disclosure without further inventive activity.

[0119] Methods for producing hybridoma cells are disclosed in (Koehler and Milstein, 1975).

[0120] Methods for the production and / or selection of chimeric or humanized mAbs are known in the art, for example, U.S. Patent No. 6,331,415 by Genentech describes the production of chimeric antibodies, U.S. Patent No. 6,548,640 by the Medical Research Council describes CDR grafting technology, and U.S. Patent No. 5,859,205 by Celltech describes the production of humanized antibodies.

[0121] Methods for the production and / or selection of fully human mAbs are known in the art. These may include the use of transgenic animals immunized with the respective proteins or peptides, or the use of suitable display technologies such as yeast display, phage display, B-cell display or ribosome display, in which antibodies from libraries are screened against human iRhom2 in stationary phase.

[0122] In vitro antibody libraries are disclosed inter alia by MorphoSys in US Patent No. 6,300,064 and by MRC / Scripps / Stratagene in US Patent No. 6,248,516. Phage display technology is disclosed, for example, by Dyax in US Patent No. 5,223,409. Transgenic mammalian platforms are described, for example, by Taconic Artemis in EP Patent No. 1,480,515 A2.

[0123] IgG, IgM, scFv, Fab and / or F(ab)2 are antibody formats well known to those skilled in the art. The relevant enabling technologies are available from the respective textbooks.

[0124] As used herein, the term "Fab" refers to an IgG / IgM fragment containing the antigen-binding region, said fragment being composed of one constant domain and one variable domain from each heavy and light chain of the antibody.

[0125] The term "F(ab)2" as used herein refers to an IgG / IgM fragment consisting of two Fab fragments connected together by disulfide bonds.

[0126] As used herein, the term "scFv" refers to a single-chain variable fragment that is a fusion of the variable regions of the heavy and light chains of an immunoglobulin linked together with a short linker, usually serine (S) or glycine (G). This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of a linker peptide.

[0127] Modified antibody formats are, for example, bi- or trispecific antibody constructs, antibody-based fusion proteins, immunoconjugates, etc. These types are well described in the literature and can be used by the skilled artisan on the basis of the present disclosure with further inventive activity.

[0128] Antibodies capable of binding to peptides bound to MHC are sometimes referred to as "TCR mimetic antibodies" or "TCR-like antibodies." In general, such antibodies can be generated by the methods described above. Methods for generating TCR-like antibodies are disclosed, for example, in (He et al., 2019), the contents of which are incorporated herein by reference in their entirety.

[0129] TCR mimetic antibodies that bind to HLA-restricted peptides derived from PRAME are disclosed, for example, in (Chang et al., 2017), the contents of which are incorporated herein by reference in their entirety. See also U.S. Patent Application Publication No. 2018 / 0148503 (T Cell Receptor-Like Antibodies Specific for PRAME Peptides) (Eureka Therapeutics Inc), the contents of which are incorporated herein by reference in their entirety.

[0130] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0131] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0132] According to another aspect of the invention there is provided a T cell receptor or functional fragment thereof which reacts with or binds to an MHC ligand, said ligand being a peptide according to above, or a peptide according to above when bound to an MHC molecule. The T cell receptor is provided for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with metastasis or metastatic lesion (i), suffering from (ii) or at risk of developing (iii).

[0133] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0134] The method includes the step of administering to the patient a T cell receptor or functional fragment thereof that reacts with or binds to an MHC ligand, the ligand being one or more therapeutically effective doses of a peptide according to the above, or a peptide according to the above when bound to an MHC molecule.

[0135] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or metastatic lesions comprising a T cell receptor or a functional fragment thereof which reacts with or binds to an MHC ligand, said ligand being a peptide according to above as an active ingredient, or a peptide according to above when bound to an MHC molecule.

[0136] In one embodiment, the treatment does not include co-administration (simultaneous or sequential) of a T cell receptor or a functional fragment thereof that binds a peptide that is a fragment of prostate-specific membrane antigen (PSMA) and that is bound to an MHC molecule.

[0137] In particular, the treatment is 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297 1297V (GLPSIPVHPV, SEQ ID NO: 377) and does not include co-administration (simultaneous or sequential) with a T cell receptor or functional fragment thereof that binds to a peptide bound to an MHC molecule.

[0138] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0139] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0140] According to one embodiment, the T cell receptor is provided as a soluble molecule.

[0141] As used herein, a soluble T cell receptor refers to a heterodimeric truncated variant of a native TCR, e.g., comprising the extracellular portions of the TCR α and β chains linked by disulfide bonds, but lacking the transmembrane and cytoplasmic domains of the native protein. The term "soluble T cell receptor α and β chain sequences" refers to TCR α and β chain sequences lacking the transmembrane and cytoplasmic domains. The soluble TCR α and β chain sequences (amino acid or nucleic acid) may be identical to the corresponding sequences in a native TCR, or may comprise mutated soluble TCR α and β chain sequences compared to the corresponding native TCR sequences. As used herein, the term "soluble T cell receptor" encompasses soluble TCRs comprising mutated or non-mutated soluble TCR α and β chain sequences. Mutations may be in the variable or constant regions of the soluble TCR α and β chain sequences and may include, but are not limited to, amino acid deletions, insertions, substitution mutations, as well as modifications of the nucleic acid sequence that do not change the amino acid sequence. The soluble TCRs of the present invention in any case retain the binding function of their parent molecules.

[0142] PRAME‐004 specific TCR Complexes of peptides and MHC class I molecules are recognized by CD8+ T cells bearing the appropriate T cell receptor (TCR), whereas complexes of peptides and MHC class II molecules are recognized by CD4+ helper T cells bearing the appropriate TCR, thereby recognizing that TCR, peptide and MHC exist in a 1:1:1 stoichiometry.

[0143] This interaction is highly specific. For example, in an MHC class I-dependent immune response, the peptide must not only be able to bind to a specific MHC class I molecule expressed by tumor cells, but must also be subsequently recognized by T cells bearing a specific T cell receptor (TCR). Usually, when targeting peptide-MHC complexes with said specific TCRs (e.g., soluble TCRs) and antibodies according to the present invention, presentation is the determining factor for a successful response.

[0144] The invention further relates to T cell receptors (TCRs), in particular soluble TCRs (sTCRs) and cloned TCRs integrated into autologous or allogeneic T cells, and methods for making these as well as NK cells or other cells bearing or cross-reacting with said TCRs.

[0145] Structurally, these subgroups of T cell receptors (TCRs) include an alpha chain and a beta chain ("alpha / beta TCR"). These TCRs specifically bind to peptides according to the invention, such as SLLQHLIGL (PRAME-004) (SEQ ID NO: 310), when presented by MHC molecules. The present description also relates to fragments of such TCRs according to the invention that can still specifically bind to peptide antigens according to the invention, such as PRAME-004 (SEQ ID NO: 310), when presented by HLA molecules. This relates to soluble TCR fragments, such as TCRs lacking transmembrane portions and / or constant regions, single chain TCRs, and fusions thereof, such as with immunoglobulins (Igs). For example, TCRs and fragments thereof of the present disclosure may include those disclosed in US Patent Publication No. 20180273602, US Patent Application No. 10800832, and US Patent Publication No. 20200123221, the contents of which are incorporated herein by reference in their entirety.

[0146] The alpha and beta chains of alpha / beta TCRs, and the gamma and delta chains of gamma / delta TCRs, structurally have two "domains", a variable domain and a constant domain. The variable domain consists of a variable region (V) linked to a junction region (J). The variable domain may also contain a leader region (L). The beta and delta chains may also contain a diversity region (D). The alpha and beta constant domains may also contain a C-terminal transmembrane (TM) domain that anchors the alpha and beta chains to the cell membrane.

[0147] The majority of available TCR structures are αβTCRs, formed by TCRα and TCRβ chains. A minority of TCRs are γδTCRs, consisting of TCRγ and TCRδ chains. The TCRβ and TCRδ chains are thought to resemble antibody heavy chains, while the TCRα and TCRγ chains are thought to resemble antibody light chains (Rudolph, Stanfield, and Wilson, 2006).

[0148] As mentioned above, each TCR chain is characterized by two immunoglobulin domains: variable (V) and constant (C). Both the variable and constant domains have a conserved β-sandwich structure, allowing variable domains from different TCRs to be numbered and compared (Dunbar and Deane, 2016). The IMGT numbering has been used for structural analysis of TCRs (Glanville et al., 2017; Dunbar et al., 2014). In each variable domain, there are three hypervariable loops with the highest degree of sequence and structure variability, known as complementarity determining regions (CDR1, CDR2, and CDR3). The remainder of the TCR structure adjacent to the CDRs is collectively known as the TCR "framework".

[0149] CDRs may contain one or more "alterations" such as substitutions, additions or deletions from a given sequence, provided that the TCR retains the ability to bind to a peptide:MHC complex. The alterations may include substitutions of an amino acid with a similar amino acid, e.g., conservative substitutions. Similar amino acids are those that have side chain moieties with related properties summarized, for example, in: (i) basic side chains: lysine, arginine, histidine; (ii) acidic side chains: aspartic acid and glutamic acid; (iii) uncharged polar side chains: asparagine, glutamine, serine, threonine and tyrosine; and (iv) non-polar side chains: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

[0150] Outside the variable portion of the TCR, the TCR structure is highly conserved, and therefore only a small portion of the chains generate the actual specificity of the TCR repertoire. As mentioned above, TCRs are divided into a remarkably diverse TCR (10 15 ~10 61 They are generated by genomic rearrangements of the germline TCR loci, a process called V(D)J recombination, which has the potential to generate multiple TCR subunits (presumably spanning multiple possible receptors).

[0151] Despite this potential diversity, TCRs from T cells that recognize the same pMHC epitope often share conserved sequence features. Analyses demonstrate that each epitope-specific repertoire contains a collection of receptors that share core sequence similarity, along with a dispersed and diverse set of "outlier" sequences. Identifying shared motifs in the core sequence can highlight key conserved residues that drive essential elements of TCR recognition (Glanville et al., 2017; Dash et al., 2017, both specifically incorporated by reference herein). These analyses provide insight into epitope-specific repertoires and generalizable fundamental features of adaptive immune recognition.

[0152] Sequence analysis focusing exclusively on high probability contact sites in CDR3 appears to provide a means to cluster TCRs by shared specificity, since the majority of these possible contacts are in CDR3, and only short, typically linear stretches of amino acids contact antigenic peptide residues (IMGT positions 107-116), whereas stem positions in CDR3 (IMGT positions 104, 105, 106, 117, and 118) are never within 5 Å of the antigen (Glanville et al., 2017). While there is always at least one CDR3β contact, there are multiple cases where no CDR3α contact is made, suggesting that the former is necessary, although both are usually involved. Thus, now well-established features of TCR repertoire analysis include the length, charge, and hydrophobicity of the CDR3 region, clonal diversity (within an individual), and amino acid sequence sharing (between individuals). For example, by using the GLIPH algorithm, TCR sequences may be organized into distinct groups that share specificity within or across individuals.

[0153] Thus, the estimated number of specific T cell receptors, and thus the repertoire of amino acid sequences of the relevant variable regions, is relatively small, and the availability of even one antigenic receptor sequence readily enables one skilled in the art to generate and search for other related T cell receptors sharing the same specificity. Because the general methods for generating TCRs are known, and the specific interactions between peptide-MHC and receptors have been extensively studied, knowledge of peptide-MHC complexes alone should provide the skilled artisan with sufficient information to be able to generate specific subsets of the variable regions described herein for the T cell receptors of the present invention (or the specific fragments thereof as described), without being overly burdened by, for example, the lack of specific indications of the relevant locations of the receptor.

[0154] In one embodiment, to obtain T cells expressing the TCR of the present invention, nucleic acids encoding the TCR-alpha chain and / or TCR-beta chain of the present invention are cloned into an expression vector, such as a gammaretrovirus, lentivirus, or a non-viral vector, such as a transposon, nanoplasmid, CRISPR. Recombinant viruses or vectors are generated and then tested for functionality, such as antigen specificity and functional avidity. An aliquot of the final product is then used to transduce a target T cell population (usually purified from the patient's PBMCs) and expanded prior to infusion into the patient.

[0155] In another embodiment, to obtain T cells expressing the TCR of the present invention, TCR RNA is synthesized by techniques known in the art, such as in vitro transcription systems. The in vitro synthesized TCR RNA is then introduced by electroporation into primary CD8+ T cells obtained from healthy donors to re-express the tumor-specific TCR-alpha and / or TCR-beta chains.

[0156] In one embodiment, the TCRs herein having at least one mutation in the alpha chain and / or having at least one mutation in the beta chain have modified glycosylation compared to the non-mutated TCR.

[0157] The alpha / beta heterodimeric TCRs herein may have a disulfide bond introduced between their constant domains. Preferred TCRs of this type include those having a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, except that Thr48 of TRAC and Ser57 of TRBC1 or TRBC2 are replaced by cysteine ​​residues, which form a disulfide bond between the TRAC constant domain sequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

[0158] With or without the above-mentioned introduced interchain bond, the alpha / beta heterodimeric TCR of the present specification may have a TRAC constant domain sequence and a TRBC1 or TRBC2 constant domain sequence, and the TRAC constant domain sequence of the TCR and the TRBC1 or TRBC2 constant domain sequence may be linked by a native disulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 of TRBC1 or TRBC2.

[0159] Thus, in one further or alternative embodiment, the antigen recognition construct of the present invention comprises CDR1, CDR2, CDR2bis and CDR3 sequences in combination as provided in SEQ ID NOs: 12-128 displaying the respective variable chain alleles together with the CDR3 sequence. Thus, preferred are antigen recognition constructs of the present invention comprising at least one, and preferably all four CDR sequences CDR1, CDR2, CDR2bis and CDR3. Preferably, the antigen recognition construct of the present invention comprises each CDR1, CDR2bis and CDR3 of one individual TCR variable region of the present invention disclosed herein (see SEQ ID NOs: 12-128 and the Examples section).

[0160] In one embodiment, the TCR alpha variable domain has at least one mutation relative to the TCR alpha domain set forth in SEQ ID NOs: 12-128, and / or the TCR beta variable domain has at least one mutation relative to the TCR alpha domain set forth in SEQ ID NOs: 12-128. In one embodiment, a TCR comprising at least one mutation in the TCR alpha variable domain and / or the TCR beta variable domain has a binding affinity and / or binding half-life for a TAA peptide-HLA molecule complex that is at least twice as long as a TCR comprising an unmutated TCR alpha domain and / or an unmutated TCR beta variable domain.

[0161] The antigen recognition construct of the present invention may comprise a TCR alpha or gamma chain, and / or a TCR beta or delta chain, wherein the TCR alpha or gamma chain comprises a CDR3 having at least one, at least two, at least three, at least four, or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 14, 26, 38, 50, 62, 74, 86, and 110, and / or the TCR beta or delta chain comprises a CDR3 having at least one, at least two, at least three, at least four, or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 20, 32, 44, 56, 68, 80, 92, and 116.

[0162] Most preferably, in some additional embodiments where the disclosure refers to an antigen recognition construct comprising any one, two, three or all of the CDR1, CDR2, CDR2bis and CDR3 regions of the TCR chain disclosed herein (see Table 1), an antigen recognition construct comprising each CDR sequence of the present invention with no more than three, two, preferably only one modified amino acid residues may be preferred. The modified amino acid residues may be selected from amino acid insertion, deletion or substitution. Most preferably, the three, two, preferably only one modified amino acid residues are the first or last amino acid residues of each CDR sequence. When the modification is a substitution, in some embodiments, it is preferred that the substitution is a conservative amino acid substitution.

[0163] Such conservative substitutions may be when an amino acid is replaced by an amino acid of similar structure and characteristics, for example, when a hydrophobic amino acid is replaced by another hydrophobic amino acid. Even more conservative would be the replacement of an amino acid of the same or similar size and chemical properties, such as replacing leucine with isoleucine. In studies of sequence variants in families of naturally occurring homologous proteins, some amino acid substitutions are often tolerated better than others, and these are often correlated with similarity in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, which is the basis for defining "conservative substitutions".

[0164] Conservative substitutions are defined herein as exchanges 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).

[0165] Less conservative substitutions may involve the replacement of one amino acid with another having similar characteristics but slightly different size, such as the replacement of an alanine with an isoleucine residue. Highly non-conservative substitutions may involve the replacement of a polar amino acid, or a basic amino acid with an acidic amino acid. However, such "radical" substitutions cannot be dismissed as potentially ineffective, since chemical effects are not entirely predictable, and radical substitutions may produce fortuitous effects that are inherently unpredictable from simple chemical principles.

[0166] If substitutions at multiple positions are found to result in an antigen recognition construct of the present invention with substantially equivalent or greater antigen binding activity, combinations of the substitutions are tested to determine whether the combined substitutions result in additive or synergistic effects on antigen binding activity. For example, no more than four positions, no more than three positions, no more than two positions, or no more than one position in the CR3 region of the antigen recognition construct of the present invention will be substituted at the same time.

[0167] In the case where the antigen recognition construct of the invention is composed of at least two amino acid chains, such as a two-chain TCR, or an antigen-binding fragment thereof, the antigen recognition construct may comprise an amino acid sequence according to SEQ ID NO: 14 in a first polypeptide chain, an amino acid sequence according to SEQ ID NO: 20 in a second polypeptide chain, or an amino acid sequence according to SEQ ID NO: 26 in a first polypeptide chain, an amino acid sequence according to SEQ ID NO: 32 in a second polypeptide chain, or an amino acid sequence according to SEQ ID NO: 38 in a first polypeptide chain, an amino acid sequence according to SEQ ID NO: 44 in a second polypeptide chain, or an amino acid sequence according to SEQ ID NO: 50 in a first polypeptide chain. or an amino acid sequence according to SEQ ID NO: 56 in the first polypeptide chain, or an amino acid sequence according to SEQ ID NO: 62 in the first polypeptide chain, an amino acid sequence according to SEQ ID NO: 68 in the second polypeptide chain, or an amino acid sequence according to SEQ ID NO: 74 in the first polypeptide chain, an amino acid sequence according to SEQ ID NO: 80 in the second polypeptide chain, or an amino acid sequence according to SEQ ID NO: 86 in the first polypeptide chain, an amino acid sequence according to SEQ ID NO: 92 in the second polypeptide chain, or an amino acid sequence according to SEQ ID NO: 110 in the first polypeptide chain and an amino acid sequence according to SEQ ID NO: 116 in the second polypeptide chain.

[0168] Any one of the aforementioned dual chain TCRs, or antigen binding fragments thereof, is a preferred TCR of the invention. In some embodiments, the CDR3 of the dual chain TCR of the invention may be mutated. Mutations in the above CDR3 sequences preferably include substitution, deletion, addition or insertion of no more than three, preferably no more than two, most preferably no more than one amino acid residue. In some embodiments, the first polypeptide chain may be a TCR alpha or gamma chain and the second polypeptide chain may be a TCR beta or delta chain. A combination of alpha beta or gamma delta TCRs is preferred.

[0169] The TCR, or antigen-binding fragment thereof, in some embodiments is composed of TCR alpha and TCR beta chains, or gamma and delta chains. Such dual-chain TCRs comprise a variable region within each chain, each of which comprises one CDR1, one CDR2, or more preferably one CDR2bis, and one CDR3 sequence. The TCR comprises the CDR1, CDR2, CDR2bis, and CDR3 sequences as contained in the variable chain amino acid sequences of SEQ ID NOs: 15 and 21, or 27 and 33, or 39 and 45, or 51 and 57, or 63 and 69, or 75 and 81, or 87 and 93, or 111 and 117.

[0170] Some embodiments of the invention relate to a TCR, or fragment thereof, consisting of a TCR alpha and a TCR beta chain, said TCR comprising a variable region sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from the alpha and beta chains according to SEQ ID NOs: 15 and 21, or 27 and 33, or 39 and 45, or 51 and 57, or 63 and 69, or 75 and 81, or 87 and 93, or 111 and 117.

[0171] In a particularly preferred embodiment, the present invention provides an improved TCR, designated R11P3D3_KE, composed of a TCR alpha and a TCR beta chain, said TCR comprising a variable region sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity with an amino acid sequence selected from the alpha and beta chains according to SEQ ID NOs: 113 and 119. This TCR showed surprisingly improved functionality in terms of tumor cell recognition compared to its parent receptor, designated herein as R11P3D3.

[0172] The TCR of the present invention may further comprise a constant region derived from any suitable species, such as any mammal, for example, human, rat, monkey, rabbit, donkey, or mouse. In one embodiment of the present invention, the TCR of the present invention further comprises a human constant region. In some preferred embodiments, the constant region of the TCR of the present invention may be slightly modified, for example, by the introduction of a heterologous sequence, preferably a mouse sequence, which may increase TCR expression and stability. In some preferred embodiments, the variable region of the TCR of the present invention may be slightly modified, for example, by the introduction of a single point mutation, to optimize the stability of the TCR and / or to enhance TCR chain pairing.

[0173] Some embodiments of the invention relate to a TCR, or fragment thereof, consisting of a TCR alpha and a TCR beta chain, said TCR comprising a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or preferably 100% sequence identity to an amino acid sequence selected from the alpha and beta chains according to SEQ ID NOs: 16 and 22, or 28 and 34, or 40 and 46, or 52 and 58, or 64 and 70, or 76 and 82, or 88 and 94, or 112 and 118.

[0174] The TCR alpha or gamma chain of the present invention may further comprise a CDR1 having at least one, at least two, at least three, at least four, or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 12, 24, 36, 48, 60, 72, 84 and 108, and / or a CDR2 having at least one, at least two, at least three, at least four, or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 13, 25, 37, 49, 61, 73, 85, and 109, and / or a CDR2bis having at least one, at least two, at least three, at least four, or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 120, 121, 122, 123, 124, 125, 126, and 128.

[0175] According to the present invention, the TCR β or δ chain may further comprise a CDR1 having at least one, at least two, at least three, at least four or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 18, 30, 42, 54, 66, 78, 90 and 114, and / or a CDR2 having at least one, at least two, at least three, at least four or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 19, 31, 43, 55, 67, 79, 91 and 115, and / or a CDR2bis having at least one, at least two, at least three, at least four or at least five amino acid substitutions of an amino acid sequence selected from SEQ ID NOs: 19, 31, 43, 55, 67, 79, 91 and 115.

[0176] The antigen recognition construct may in a further embodiment comprise a binding fragment of a TCR, said binding fragment comprising on one chain the sequence of SEQ ID NO: 12, 13, 14, 120, 11, 18, 19, 20, or 24, 25, 26, 121, or 30, 31, 32, or 36, 37, 38, 122, or 42, 43, 44, or 48, 49, 50, 123, or 54, 55, 56, or 60, 61, 62, 124, or 66, 67, 68, or 72, 73, 74, 125, or 78, 79, 80, or 84, 85, 86, 126, or 90, 91, 92, or 108, 109, 110, 128, or 114, 115, 116 amino acid sequences.

[0177] In a further embodiment of the invention, the antigen recognition construct described elsewhere herein is a TCR, or a fragment thereof, composed of at least one TCR alpha and one TCR beta chain sequence, said TCR alpha chain sequence comprising CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 12-14 and 120, said TCR beta chain sequence comprising CDR1-CDR3 sequences having the amino acid sequences of SEQ ID NOs: 18-20, or said TCR alpha chain sequence comprising CDR1, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 24-26 and 121. the TCR β chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 30 to 32, or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 36 to 38 and 122, the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 42 to 44, or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 48 to 50 and 123. and CDR3 sequences, wherein the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 54 to 56, or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 60 to 62 and 124, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 66 to 68, or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 72 to 74 and 125, The TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 78 to 80, or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 84 to 86 and 126, and the TCR β chain sequence comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 90 to 92, or the TCR α chain sequence comprises CDR1, CDR2, CDR2bis, and CDR3 sequences having the amino acid sequences of SEQ ID NOs: 108 to 110 and 128, and the TCR β chain sequence comprisesIt comprises CDR1 to CDR3 sequences having the amino acid sequences of SEQ ID NOs: 114 to 116.

[0178] In a further embodiment of the invention, the antigen recognition construct as hereinbefore described is a TCR, or a fragment thereof, comprising at least one TCR alpha and one TCR beta chain sequence, wherein said TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 15, said TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 21, or said TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 27, said TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 33, or said TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 39, said TCR beta chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 45, or said TCR alpha chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO: 51, The TCR β chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:57, or the TCR α chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:63, the TCR β chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:69, or the TCR α chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:75, the TCR β chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:81, or the TCR α chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:87, the TCR β chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:93, or the TCR α chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:111, and the TCR β chain sequence comprises a variable region sequence having the amino acid sequence of SEQ ID NO:117.

[0179] In a further embodiment of the invention, the antigen recognition construct as hereinbefore described is a TCR or a fragment thereof further comprising a TCR constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76, 82, 88, 94, 112 and 118, preferably the TCR is composed of at least one TCR alpha and one TCR beta chain sequence, The alpha chain sequence comprises a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 16, 28, 40, 52, 64, 76, 88, and 112, and the TCR beta chain sequence comprises a constant region having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% sequence identity to an amino acid sequence selected from SEQ ID NOs: 22, 34, 46, 58, 70, 82, 94, and 118.

[0180] Also disclosed herein is an antigen recognition construct as previously described comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 17 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 23, and the invention relates to a TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 29. and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 35, and in a further embodiment the invention provides a TCR comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 41 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 47. or 100% sequence identity to the amino acid sequence of SEQ ID NO:53; and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO:59; and in a further embodiment the invention provides an antigen recognition construct which is a TCR and comprises a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% ...9. and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 71; in a further embodiment the invention provides an antigen recognition construct comprising a first TCR chain which is a TCR and has at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 77;or 100% sequence identity with the amino acid sequence of SEQ ID NO: 83 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 83, and in a further embodiment the invention provides an antigen recognition construct comprising a TCR, a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 89 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity with the amino acid sequence of SEQ ID NO: 95. In a further embodiment, the invention provides an antigen recognition construct comprising a first TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 113 and a second TCR chain having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 119.

[0181] As used herein, the terms "murine" or "human" when referring to an antigen recognition construct, or a TCR, or any component of a TCR described herein (e.g., the complementarity determining regions (CDRs), variable region, constant region, α chain, and / or β chain), refer to a TCR (or component thereof) derived from a mouse or human unrearranged TCR locus, respectively.

[0182] In one embodiment of the invention, a chimeric TCR is provided, where the TCR chain comprises sequences from multiple species. Preferably, the TCR of the invention may comprise an alpha chain comprising a human variable region of the alpha chain and a murine constant region, for example a murine TCR alpha chain.

[0183] According to another aspect of the present invention, there is provided a nucleic acid encoding the peptide according to the above, or the antibody or fragment thereof according to the above, or the T cell receptor or fragment thereof according to the above. In various embodiments, the nucleic acid is provided in the form of DNA or RNA. In one embodiment, the nucleic acid is provided in the form of a vector or plasmid. In one embodiment, the nucleic acid comprises two or more repeats (concatamers) of the coding sequence separated by a short nucleotide stretch ("spacer").

[0184] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0185] The method comprises the step of administering to the patient one or more therapeutically effective doses of a peptide according to above, or an antibody or fragment thereof according to above, or a nucleic acid encoding a T cell receptor or fragment thereof according to above.

[0186] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or a metastatic lesion comprising as an active ingredient a peptide according to the above, or an antibody or fragment thereof according to the above, or a nucleic acid encoding a T cell receptor or fragment thereof according to the above.

[0187] In one embodiment, the treatment or composition does not include co-administration (simultaneous or sequential) with a peptide that is a fragment of prostate-specific membrane antigen (PSMA), or a nucleic acid encoding an antibody or T cell receptor that binds such a peptide when bound to an MHC molecule.

[0188] In particular, the treatment is 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297 1297V (GLPSIPVHPV, SEQ ID NO: 377), which does not include co-administration (simultaneous or sequential) with a nucleic acid encoding an antibody or a T cell receptor or functional fragment thereof that binds to a peptide bound to MHC

[0189] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0190] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0191] Optionally, the nucleic acid is provided for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with metastasis or metastatic lesion (i), suffering from (ii), or at risk of developing (iii).

[0192] Such nucleic acids may be mRNA or DNA. Such nucleic acids may be delivered as plasmids or linear molecules. Such nucleic acids may be delivered by viral vectors or encapsulated in liposomes. Such mRNAs may contain modified nucleosides, such as pseudouridine or 1-methylpseudouridine, to reduce immunogenicity. Such mRNAs may be G / C codon optimized to reduce uridine content.

[0193] According to another aspect of the invention there is provided a recombinant host cell comprising a peptide according to above, an antibody or fragment thereof according to above, a T cell receptor or fragment thereof according to above, or a nucleic acid according to above.

[0194] According to another aspect of the invention there is provided a recombinant T lymphocyte expressing at least one vector encoding a T cell receptor according to above.

[0195] The T lymphocytes are provided for use in (the manufacture of a medicament for) the treatment of a patient who has been diagnosed with metastasis or a metastatic lesion (i), is suffering from (ii), or is at risk of developing (iii).

[0196] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0197] The method comprises administering to the patient one or more therapeutically effective doses of recombinant T lymphocytes expressing at least one vector encoding a T cell receptor according to above.

[0198] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or metastatic lesions comprising as an active ingredient a recombinant T lymphocyte expressing at least one vector encoding a T cell receptor according to above.

[0199] In one embodiment, the treatment or composition comprises a peptide that is a fragment of prostate specific membrane antigen (PSMA) and that is bound to an MHC molecule, in particular PSMA. 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297 1297V (GLPSIPVHPV, SEQ ID NO: 377) and does not include co-administration (simultaneous or sequential) with recombinant T lymphocytes expressing a vector encoding a T cell receptor or a functional fragment thereof that binds to a peptide bound to an MHC molecule.

[0200] In one embodiment, the recombinant T lymphocytes are produced by a method comprising isolating cells from a subject, transforming the cells with at least one vector encoding a T cell receptor to produce recombinant T lymphocytes, and expanding the recombinant T lymphocytes to produce a population of recombinant T lymphocytes.

[0201] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0202] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0203] In one embodiment, the recombinant T lymphocyte is a CD8+ (CD8 positive) T lymphocyte. CD8+ T lymphocytes (also called cytotoxic T cells CTL, T killer cells, cytolytic T cells, or killer T cells) are T lymphocytes that kill cancer cells, cells infected (especially by viruses), or cells that are otherwise damaged.

[0204] Most cytotoxic T cells express a T cell receptor (TCR) that can recognize a specific antigen. Antigens are molecules that can stimulate an immune response and are often produced by cancer cells or viruses. Antigens within a cell bind to class I MHC molecules and are carried to the cell surface by class I MHC molecules, where they can be recognized by T cells.

[0205] If the TCR is specific for that antigen, it binds to the complex of class I MHC molecule and antigen, and the T cell destroys the cell. For the TCR to bind to a class I MHC molecule, the former must be associated with a glycoprotein called CD8, which binds to the constant part of the class I MHC molecule. These T cells are therefore called CD8+ T cells.

[0206] According to some embodiments, the T cell receptor is (1) a CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 12, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 13, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 14, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 18, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 19, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 20, or (2) a CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 25, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 26, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 30, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 31, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 32, or (3) a CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 37, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 38, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 42, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 43, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 44, or (4) a CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 48, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 49, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 50, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 54, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 55, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 56; (5) a CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 60, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 61, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 62, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 66, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 67, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 68; (6) a CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 72, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 73, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 74, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 78, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 79, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 80; (7) A CDR1 α chain comprising the amino acid sequence of SEQ ID NO: 84, a CDR2 α chain comprising the amino acid sequence of SEQ ID NO: 85, a CDR3 α chain comprising the amino acid sequence of SEQ ID NO: 86, a CDR1 β chain comprising the amino acid sequence of SEQ ID NO: 90, a CDR2 β chain comprising the amino acid sequence of SEQ ID NO: 91, and a CDR3 β chain comprising the amino acid sequence of SEQ ID NO: 92 and the T cell receptor is HLA-A * It is capable of binding to a peptide consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 310) in a complex with .02.

[0207] According to some embodiments, the T cell receptor is (1) an alpha chain variable domain comprising SEQ ID NO: 15, and a beta chain variable domain comprising SEQ ID NO: 21, or (2) an α chain variable domain comprising SEQ ID NO: 27 and a β chain variable domain comprising SEQ ID NO: 33; or (3) an alpha chain variable domain comprising SEQ ID NO: 39 and a beta chain variable domain comprising SEQ ID NO: 45; or (4) an alpha chain variable domain comprising SEQ ID NO: 51 and a beta chain variable domain comprising SEQ ID NO: 57; or (5) an alpha chain variable domain comprising SEQ ID NO: 63 and a beta chain variable domain comprising SEQ ID NO: 69; or (6) an alpha chain variable domain comprising SEQ ID NO: 75 and a beta chain variable domain comprising SEQ ID NO: 81; or (7) an alpha chain variable domain comprising SEQ ID NO: 87 and a beta chain variable domain comprising SEQ ID NO: 93; or (8) an alpha chain variable domain comprising SEQ ID NO: 111, and a beta chain variable domain comprising SEQ ID NO: 117 and the T cell receptor is HLA-A * It is capable of binding to a peptide consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 310) in a complex with .02.

[0208] According to another aspect of the present invention, there is provided an in vitro method for producing activated T lymphocytes, comprising the step of contacting T cells in vitro with antigen-loaded human class I MHC molecules expressed on the surface of a suitable antigen-presenting cell or an artificial construct that mimics an antigen-presenting cell for a period of time sufficient to activate said T lymphocytes in an antigen-specific manner, said antigen being a peptide according to above.

[0209] According to another aspect of the present invention, there is provided an activated T lymphocyte produced by the method described above, which selectively recognizes cells presenting the peptide described above.

[0210] The T lymphocytes are provided for use in (the manufacture of a medicament for) the treatment of a patient who has been diagnosed with metastasis or a metastatic lesion (i), is suffering from (ii), or is at risk of developing (iii).

[0211] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0212] The method comprises the step of administering to the patient one or more therapeutically effective doses of activated T lymphocytes produced by the method described above that selectively recognize cells presenting a peptide according to the above.

[0213] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or metastatic lesions, comprising as an active ingredient activated T lymphocytes produced by the above-described method, which selectively recognize cells presenting the above-described peptide.

[0214] In one embodiment, the treatment presents a peptide that is a fragment of prostate specific membrane antigen (PSMA), in particular PSMA 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297 It does not encompass co-administration (simultaneous or sequential) with activated T lymphocytes that recognize cells that do not present 1297V (GLPSIPVHPV, SEQ ID NO: 377).

[0215] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0216] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is HLA-A*02:01 positive.

[0217] In one embodiment, the activated T lymphocytes are CD8+ (CD8 positive) T lymphocytes.

[0218] Adoptive cell therapy: manufacturing γδ T cells To isolate γδ T cells, in one embodiment, γδ T cells can be isolated from a subject or from a composite sample of a subject. In one embodiment, the composite sample can be a peripheral blood sample, a cord blood sample, a tumor, a stem cell precursor, a tumor biopsy, a tissue, lymph, or from an epithelial site of a subject that directly contacts the external environment, or derived from stem progenitor cells. γδ T cells can be directly isolated from a composite sample of a subject, for example, by sorting γδ T cells expressing one or more cell surface markers using flow cytometry techniques. Wild-type γδ T cells can exhibit a number of antigen recognition, antigen presentation, costimulation, and adhesion molecules that can be associated with γδ T cells. Wild-type γδ T cells can be isolated from a composite sample using one or more cell surface markers, such as specific γδ TCR, antigen recognition, antigen presentation, ligand, adhesion molecule, or costimulation molecule. Various molecules associated with or expressed by γδ T cells can be used to isolate γδ T cells from complex samples, for example a mixed population of Vδ1+, Vδ2+, Vδ3+ cells or any combination thereof can be isolated.

[0219] For example, peripheral blood mononuclear cells can be collected from a subject using an apheresis machine, including, for example, a Ficoll-Paque™ PLUS (GE Healthcare) system, or another suitable device / system. From the collected sample, γδ T cell(s), or a desired subpopulation of γδ T cell(s), can be purified, for example, using flow cytometry techniques. Cord blood cells can also be obtained from umbilical cord blood at the time of the subject's birth.

[0220] Using positive and / or negative selection of cell surface markers expressed on harvested γδ T cells, γδ T cells, or populations of γδ T cells expressing similar cell surface markers, can be isolated directly from peripheral blood samples, umbilical cord blood samples, tumors, tumor biopsies, tissues, lymphatic fluids, or from epithelial samples of subjects. For example, γδ T cells can be isolated from composite samples based on positive or negative expression of CD2, CD3, CD4, CD8, CD24, CD25, CD44, Kit, TCRα, TCRβ, TCRα, TCRδ, NKG2D, CD70, CD27, CD30, CD16, CD337 (NKp30), CD336 (NKp46), OX40, CD46, CCR7, and other suitable cell surface markers.

[0221] The process may include harvesting or obtaining leukocytes or PBMCs from a leukapheresis product. Leukapheresis may include drawing whole blood from a donor and separating the components using an apheresis machine. The apheresis machine separates the desired blood components and returns the remainder to the donor's circulation. For example, an apheresis machine may be used to harvest leukocytes, plasma, and platelets, while the red blood cells and neutrophils are returned to the donor's circulation. A commercially available leukapheresis product may be used in this process. Another way to obtain leukocytes is from the buffy coat. To isolate the buffy coat, anticoagulated whole blood is obtained from the donor and centrifuged. After centrifugation, the blood is separated into plasma, red blood cells, and buffy coat. The buffy coat is the layer located between the plasma layer and the red blood cell layer. Leukapheresis harvesting may result in a higher purity and a significantly increased mononuclear cell content than that achieved by harvesting the buffy coat. The mononuclear cell content possible in leukapheresis can typically be 20-fold higher than that obtained from buffy coats. To enrich for mononuclear cells, the use of a Ficoll gradient for further separation may be necessary.

[0222] To deplete αβT cells from PBMCs, αβTCR expressing cells can be separated from PBMCs, e.g. by magnetic separation using CliniMACS® magnetic beads coated with anti-αβTCR antibodies, followed by cryopreservation of αβTCR-T cell depleted PBMCs. To produce a "ready-made" T cell product, cryopreserved αβTCR-T cell depleted PBMCs can be thawed and activated in small / medium scale, e.g. 24 to 4-6 well plates or T75 / T175 flasks, or in larger scale, e.g. 50ml-100 liter bags, in the presence of aminobisphosphonates, e.g. zoledronate, and / or isopentenyl pyrophosphate (IPP) and / or cytokines, e.g. interleukin 2 (IL-2), interleukin 15 (IL-15), and / or interleukin 18 (IL-18), and / or other activators, e.g. Toll-like receptor 2 (TLR2) ligands, for 1-10 days, e.g. 2-7 days.

[0223] Engineering αβTCR and CD8αβ-expressing γδT cells The γδ T cells of the present disclosure may be engineered for use to treat a subject in need of treatment for a condition. αβTCR-expressing γ-retroviruses were generated to generate γδ T cells that specifically bind to αβTCR, e.g., PRAME-004-MHC complexes. Because γδ T cells may not express CD8, γδ T cells may require CD8α homodimers or CD8αβ heterodimers in addition to αβTCR to recognize PRAME-004 / MHC-I complexes displayed on the cell membrane of target cells, e.g., cancer cells. To that end, αβTCR / CD8-expressing γ-retroviruses were generated to transduce isolated γδ T cells using the methods described herein. The sequences of CD8α or variants thereof and CD8β or variants thereof may be selected from SEQ ID NOs: 1-11.

[0224] By transducing Vγ9δ2 T cells with αβTCR retrovirus and CD8αβ retrovirus, we generated αβTCR-expressing Vγ9δ2 T cells in which the αβTCR specifically binds to peptide-MHC complexes.

[0225] Autologous T cell manufacturing process The embodiments of the present disclosure may be used to generate 10 billion (10×10 9 The process may involve about a 7 to about 10 day process resulting in the production of more than 100,000 cells. In addition, the concentrations of some raw materials may be optimized to reduce the cost of goods by 30%.

[0226] The T cell manufacturing process of the present disclosure may include thawing PBMCs on day 0, followed by resting overnight, e.g., 24 hours, without cytokines, followed by activating the rested PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on non-tissue culture treated plates. IL-7 is a homeostatic cytokine that promotes T cell survival by preventing apoptosis. IL-7 may be added to the PBMCs during resting.

[0227] The T cell manufacturing process of the present disclosure may include thawing PBMCs on day 1, followed by resting for 4-6 hours in the presence of IL-7, or in the presence of IL-7+IL-15, or no cytokines, followed by activation of the rested PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on non-tissue culture treated plates.

[0228] The T cell manufacturing process of the present disclosure may include thawing PBMCs on day 1 (no settling, no cytokines), followed by activating the thawed PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on tissue culture plates. Cells may be harvested and counted on days 8-10, followed by activation panel analysis.

[0229] The T cell manufacturing process of the present disclosure may include resting PBMCs for a period of about 4 hours according to one embodiment of the present disclosure. For example, the T cell manufacturing process may include isolation and cryopreservation of PBMCs from leukapheresis, which may be tested for sterility; thawing, resting (e.g., for about 4 hours), and activation of the T cells; transduction with viral vectors; proliferation with cytokines; cell splitting / feeding, which may be tested for cell number and immunophenotyping; harvesting and cryopreservation of pharmaceutical cells, which may be tested for cell number and mycoplasma, and release after cryopreservation, which may be tested for viability, sterility, endotoxin, immunophenotyping, integrated vector copy number, and vesicular stomatitis virus glycoprotein G (VSV-g).

[0230] The T cell manufacturing process of the present disclosure may include resting the PBMCs overnight (about 16 hours). For example, the T cell manufacturing process may include isolation of PBMCs, which may be used fresh or frozen until ready to use, or may be used as starting material for T cell manufacturing, and may also allow selection of lymphocyte populations (e.g., CD8, CD4, or both); thawing and resting the lymphocytes overnight, e.g., about 16 hours, which may allow apoptotic cells to die and restore T cell function (this step may not be necessary when using fresh material); activation of lymphocytes, which may use anti-CD3 and anti-CD28 antibodies (soluble or bound to a surface, e.g., magnetic or biodegradable beads); transduction of TCR or bispecific molecules, which may use lentiviral or retroviral constructs encoding the TCR or bispecific molecules, or may use non-viral methods; and expansion, harvesting, and cryopreservation of lymphocytes, which may be performed in the presence of cytokine(s), serum (ABS or FBS), and / or cryopreservation medium.

[0231] Table 2a summarizes the characteristics of T cells produced according to one embodiment of the present disclosure after a short rest period of about 4 hours and after an overnight rest period of about 16 hours.

[0232] [Table 2a]

[0233] The T cell manufacturing process of the present disclosure may include using fresh PBMCs that are not obtained by thawing cryopreserved PBMCs, thereby minimizing cell loss due to freezing, thawing, and / or resting of PBMCs and maximizing cell numbers at the start of the manufacturing process. For example, the T cell manufacturing process may include isolation of fresh PBMCs on day 0, activation of fresh lymphocytes using, for example, anti-CD3 and anti-CD28 antibodies (soluble or bound to a surface, for example, magnetic or biodegradable beads) in a bag, for example, Saint-Gobain VueLife AC bags coated with anti-CD3 and anti-CD28 antibodies; transduction of TCR or bispecific molecules on day 1, for example, using lentiviral or retroviral constructs encoding the TCR or bispecific molecule, or non-viral methods, for example, liposomes; expansion of lymphocytes on day 2, harvesting on day 5 / 6, and cryopreservation in the presence of cytokine(s), serum (ABS or FBS), and / or cryopreservation medium.

[0234] Engineering αβ T cells to express αβ TCR and CD8αβ The engineered αβ T cells of the present disclosure may be used to treat a subject in need of treatment for a condition. αβTCR-expressing γ-retroviruses were generated to generate αβ T cells expressing αβ TCRs, e.g., as shown in the sequence listing below, that specifically bind to PRAME-004 / MHC complexes. Expression of exogenous CD8α homodimers or CD8αβ heterodimers in CD8+ and / or CD4 T cells may improve recognition of PRAME-004 / MHC-I complexes on the cell membrane of target cells, e.g., cancer cells, by αβ TCR. To that end, αβTCR / CD8-expressing γ-retroviruses were generated to transduce T cells using the methods described herein. The sequences of CD8α or variants thereof and CD8β or variants thereof may be selected from SEQ ID NOs: 1-11.

[0235] Treatment methods Compositions comprising engineered αβ T cells (e.g., CD4+ and CD8+ T cells) and / or γδ T cells expressing recombinant TCRs and / or bispecific molecules that bind PRAME-004 as described herein may be administered for prophylactic and / or therapeutic treatments. In therapeutic applications, the pharmaceutical compositions may be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. Engineered αβ T cells and / or γδ T cells may also be administered to reduce the likelihood of developing, contracting, or worsening a condition. An effective amount of therapeutic engineered αβ T cells and / or γδ T cell populations may vary based on the severity and course of the disease or condition, previous treatments, the subject's health, weight, and / or response to drugs, and / or the judgment of the treating physician.

[0236] The compositions of the present disclosure may also include one or more adjuvants. Adjuvants are substances that non-specifically enhance or potentiate immune responses, such as immune responses to antigens mediated by CD8 positive T cells and helper T (TH) cells, and would therefore be considered useful in the medicaments of the present invention. Suitable adjuvants include 1018ISS, aluminum salts, AMPLIVAX®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or flagellin-derived TLR5 ligands, 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 pegylated derivatives thereof, IS patch, ISS, ISCOMATRIX, ISCOM, JuvImmune®, LipoVac, MALP2, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide Adjuvants include, but are not limited to, ISA-51, water-in-oil and oil-in-water emulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system, poly(lactide)-co-glycolide) [PLG]-based and dextran microparticles, talactoferrin SRL172, virosomes and other virus-like particles, YF-17D, VEGF traps, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon derived from saponin, mycobacterial extracts and synthetic bacterial cell wall mimics, and other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred. Several immunological adjuvants specific for dendritic cells and their preparations (eg, MF59) have been previously described (Allison and Krummel, 1995). Cytokines may also be used.Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-), promoting the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1, IL-4) (U.S. Patent No. 5,849,589, incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12, IL-15, IL-23, IL-27, IFN-alpha, IFN-beta).

[0237] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine settings. Without wishing to be bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced activation of TH1 cells and potent cytotoxic T lymphocyte (CTL) generation even in the absence of CD4 T cell help. The TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which normally promote a TH2 bias. CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations, which are particularly necessary to induce strong responses when the antigen is relatively weak. They accelerate immune responses and also allow the antigen dose to be reduced by approximately two orders of magnitude, with some experiments showing antibody responses equivalent to full-dose vaccines without CpG (Krieg, 2006). US Patent No. 6,406,705 B1 describes the combination of CpG oligonucleotides, non-nucleic acid adjuvants and antigens to induce antigen-specific immune responses. The CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR binding molecules, such as RNA-binding TLR7, TLR8 and / or TLR9, can also be used.

[0238] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), dsRNA analogs such as poly(I:C) and derivatives thereof (e.g., AmpliGen®, Hiltonol®, poly(ICLC), poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA, mimetics of the bacterial lipopeptide Pam3Cys-Ser-Ser such as Pam3Cys-GDPKHPKSF(XS15). See (Gouttefangeas and Rammensee, 2018; Rammensee et al., 2019), the contents of which are incorporated herein by reference for the purposes of enabling disclosure.

[0239] Other examples of useful adjuvants include immune checkpoint inhibitors, including cyclophosphamide, sunitinib, ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab, which may act therapeutically and / or as adjuvants, bevacizumab®, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib, VEGF trap, ZD2171, AZD2171, immunoactive small molecules and antibodies such as anti-CTLA4, other antibodies targeting key structures of the immune system (e.g. anti-CD40, anti-TGF-beta, anti-TNF-alpha receptor) and SC58175. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation.

[0240] Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, and particle formulations including poly(lactide-co-glycolide) (PLG), virosomes, and / or interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.

[0241] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony stimulating factors such as granulocyte-macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, resiquimod, and interferon-alpha.

[0242] In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is selected from the group consisting of colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod, and resiquimod. In a preferred embodiment of the pharmaceutical composition according to the invention, the adjuvant is cyclophosphamide, imiquimod or resiquimod. Further preferred adjuvants are Montanide IMS1312, Montanide ISA206, Montanide ISA50V, Montanide ISA-51, poly ICLC (Hiltonol®) and anti-CD40 mAb, or a combination thereof.

[0243] The engineered αβ and / or γδ T cells of the present disclosure may be used to treat a subject in need of treatment for a condition, e.g., a cancer as described herein.

[0244] Methods of treating a condition (e.g., a disease) in a subject with engineered αβ T cells and / or γδ T cells may include administering a therapeutically effective amount of engineered αβ T cells and / or γδ T cells to a subject. The engineered αβ T cells and / or γδ T cells of the present disclosure may be administered in a variety of dosing regimens (e.g., timing, concentration, dosage, interval between treatments, and / or formulations). The subject may also be preconditioned, e.g., with chemotherapy, radiation, or a combination of both, prior to receiving the engineered αβ T cells and / or γδ T cells of the present disclosure. The engineered αβ T cell and / or γδ T cell population may be frozen or cryopreserved prior to administration to the subject. The engineered αβ T cell and / or γδ T cell population may include two or more cells expressing the same tumor recognition moiety, different tumor recognition moieties, or a combination of the same tumor recognition moiety and different tumor recognition moieties. For example, an engineered αβ T cell and / or γδ T cell population may comprise several different engineered αβ T cells and / or γδ T cells designed to recognize different antigens or different epitopes of the same antigen.

[0245] In one embodiment, the engineered αβ T cells and / or γδ T cells of the present disclosure may be used to treat infectious diseases. In another embodiment, the engineered αβ T cells and / or γδ T cells of the present disclosure may be used to treat infectious diseases, which may be caused by a virus. In yet another embodiment, the engineered αβ T cells and / or γδ T cells of the present disclosure may be used to treat immune diseases, such as autoimmune diseases.

[0246] Treatment with the αβ T cells and / or γδ T cells of the present disclosure may be provided to a subject before, during, and after clinical onset of a condition. Treatment may be provided to a subject 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of a disease. Treatment may be provided to a subject more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more after clinical onset of a disease. Treatment may be provided to a subject less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of a disease. Treatment may also include treatment of humans in clinical trials. Treatment may include administering to a subject a pharmaceutical composition comprising the engineered αβ T cells and / or γδ T cells of the present disclosure.

[0247] In another embodiment, administration of the engineered αβ T cells and / or γδ T cells of the present disclosure to a subject can modulate the activity of endogenous lymphocytes in the subject's body. In another embodiment, administration of the engineered αβ T cells and / or γδ T cells to a subject can provide antigen to endogenous T cells and enhance an immune response. In another embodiment, the memory T cells can be CD4+ T cells. In another embodiment, the memory T cells can be CD8+ T cells. In another embodiment, administration of the engineered αβ T cells and / or γδ T cells of the present disclosure to a subject can activate the cytotoxicity of another immune cell. In another embodiment, the other immune cell can be a CD8+ T cell. In another embodiment, the other immune cell can be a natural killer T cell. In another embodiment, administration of the engineered αβ T cells and / or γδ T cells of the present disclosure to a subject can suppress a regulatory T cell. In another embodiment, the regulatory T cell can be a FOX3+ Treg cell. In another embodiment, the regulatory T cell can be a FOX3- Treg cell. Non-limiting examples of cells whose activity may be modulated by the engineered αβ T cells and / or γδ T cells of the present disclosure may include hematopoietic stem cells; B cells; CD4; CD8; red blood cells; white blood cells; dendritic cells, including dendritic antigen presenting cells; white blood cells; macrophages; memory B cells; memory T cells; monocytes; natural killer cells; neutrophil granulocytes; helper T cells; and T killer cells.

[0248] During most bone marrow transplants, a combination of cyclophosphamide and total body irradiation may be conventionally employed to prevent rejection of hematopoietic stem cells (HSCs) in the transplant by the subject's immune system. In one embodiment, ex vivo incubation of donor bone marrow with interleukin-2 (IL-2) may be performed to enhance production of killer lymphocytes in the donor bone marrow. Interleukin-2 (IL-2) is a cytokine that may be required for the growth, proliferation, and differentiation of wild-type lymphocytes. Current studies on adoptive transfer of αβ and / or γδ T cells into humans may require co-administration of αβ and / or γδ T cells with interleukin-2. However, both low and high doses of IL-2 may have highly toxic side effects. IL-2 toxicity may be manifested in multiple organs / systems, most notably the heart, lungs, kidneys, and central nervous system. In another aspect, the disclosure provides a method for administering engineered αβ T cells and / or γδ T cells to a subject without co-administration of natural cytokines or modified forms thereof, such as IL-2, IL-15, IL-12, IL-21, etc. In another aspect, the engineered αβ T cells and / or γδ T cells may be administered to a subject without co-administration with IL-2. In another aspect, the engineered αβ T cells and / or γδ T cells may be administered to a subject during a treatment such as a bone marrow transplant without co-administration of IL-2.

[0249] Method of administration In general, therapies, including vaccines, antibodies, TCRs, bispecific or multispecific molecules and T cells, may be administered via any feasible mode of administration.

[0250] In one embodiment, the treatment is administered by im (intramuscular), iv (intravenous) or sc (subcutaneous) injection or infusion. In one embodiment, the treatment is not administered into the lymphatics. In one embodiment, the treatment is administered by im (intramuscular), iv (intravenous) or sc (subcutaneous) injection or infusion, but is not administered into the lymphatics.

[0251] One or more engineered αβ T cells and / or γδ T cell populations can be administered to a subject in any order or simultaneously. If simultaneously, multiple engineered αβ T cells and / or γδ T cells can be provided in a single unified form, such as an intravenous injection, or in multiple forms, such as multiple intravenous infusions, sc injections, or tablets. The engineered γδ T cells can be packaged together or separately, in a single package or multiple packages. One or all of the engineered αβ T cells and / or γδ T cells can be given in multiple doses. If not simultaneously, the timing between multiple doses can vary by one week, one month, two months, three months, four months, five months, six months, or up to about a year.

[0252] In another aspect, the engineered αβ T cells and / or γδ T cells may be expanded in vivo within the subject after administration to the subject. The engineered αβ T cells and / or γδ T cells may be frozen to provide cells for multiple treatments with the same cell preparation. The engineered αβ T cells and / or γδ T cells of the present disclosure, and pharmaceutical compositions comprising same, may be packaged as kits. The kits may include instructions (e.g., instructions) for use of the engineered αβ T cells and / or γδ T cells and compositions comprising same. In another aspect, a method of treating cancer includes administering a therapeutically effective amount of engineered αβ T cells and / or γδ T cells to a subject, where the administration treats the cancer. In another embodiment, a therapeutically effective amount of engineered αβ T cells and / or γδ T cells may be administered for at least about 10 seconds, 30 seconds, 1 minute, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or 1 year. In another aspect, a therapeutically effective amount of engineered αβ T cells and / or γδ T cells may be administered for at least 1 week. In another aspect, a therapeutically effective amount of engineered αβ T cells and / or γδ T cells may be administered for at least 2 weeks.

[0253] The engineered αβ T cells and / or γδ T cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administering the pharmaceutical composition comprising the engineered αβ T cells and / or γδ T cells can vary. For example, the engineered αβ T cells and / or γδ T cells can be used as a prophylactic and can be administered continuously to a subject prone to a condition or disease to reduce the likelihood of the onset of the disease or condition. The engineered αβ T cells and / or γδ T cells can be administered to a subject during or as soon as possible after the onset of symptoms. Administration of the engineered αβ T cells and / or γδ T cells can be initiated soon after the onset of symptoms, within the first 3 hours from the onset of symptoms, within the first 6 hours from the onset of symptoms, within the first 24 hours from the onset of symptoms, within 48 hours from the onset of symptoms, or within any time period from the onset of symptoms. Initial administration can be via any practical route, such as by any route described herein, using any formulation described herein. In another embodiment, administration of the engineered αβ T cells and / or γδ T cells of the present disclosure may be intravenous. One or more doses of engineered αβ T cells and / or γδ T cells may be administered as soon as practicable after onset of cancer, infectious disease, immune disease, sepsis, or in conjunction with a bone marrow transplant for the length of time required to treat the immune disease, e.g., from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months, etc. In the treatment of cancer, one or more doses of engineered αβ T cells and / or γδ T cells may be administered many years after onset of the cancer, before or after other treatments.In another embodiment, the engineered αβ T cells and / or γδ T cells may be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The duration of treatment may vary from subject to subject.

[0254] keep In one embodiment, αβ T cells and / or γδ T cells may be formulated in freezing medium and placed in a cryogenic storage unit, such as a liquid nitrogen freezer (−196° C.) or a deep freezer (−65° C., −80° C., −120° C., or −150° C.), for long-term storage of at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, or at least 5 years. The freezing medium may include dimethyl sulfoxide (DMSO), and / or sodium chloride (NaCl), and / or dextrose, and / or dextran sulfate, and / or hydroxyethyl starch (HES), along with a physiological pH buffer to maintain a pH between about 6.0 to about 6.5, about 6.5 to about 7.0, about 7.0 to about 7.5, about 7.5 to about 8.0, or about 6.5 to about 7.5. Cryopreserved αβT cells and / or γδT cells can be thawed and further treated by stimulation with antibodies, proteins, peptides, and / or cytokines as described herein. Cryopreserved αβT cells and / or γδT cells can be thawed and genetically modified by viral vectors (including retroviruses, adeno-associated viruses (AAV), and lentiviral vectors) or non-viral means (including RNA, DNA, e.g., transposons, and proteins) as described herein. Modified αβT cells and / or γδT cells can be further cryopreserved to generate cell banks in quantities of at least about 1, 5, 10, 100, 100, 150, 200, 500 vials of at least about 101, 102, 103, 104, 105, 106, 107, 108, 109, or at least about 1010 cells / mL in freezing medium. Cryopreserved cell banks can retain their functionality and can be thawed to further stimulate and expand.

[0255] In another embodiment, the thawed cells can be stimulated and expanded in a suitable closed vessel, such as a cell culture bag and / or a bioreactor, to generate large quantities of cells as an allogeneic cell product. The cryopreserved αβ and / or γδ T cells can maintain their biological function for at least about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 15 months, 18 months, 20 months, 24 months, 30 months, 36 months, 40 months, 50 months, or at least about 60 months under cryogenic storage conditions. In another embodiment, no preservatives may be used in the formulation. The cryopreserved αβ and / or γδ T cells can be thawed and infused into multiple patients as an allogeneic, pre-made cell product. In one embodiment, the engineered αβ and / or γδ T cells described herein can be administered in a volume of at least 1×10 3 Cells / ml, at least 2 × 10 3 Cells / ml, at least 3 × 10 3 Cells / ml, at least 4 × 10 3 Cells / ml, at least 5 × 10 3 Cells / ml, at least 6 × 10 3 Cells / ml, at least 7 × 10 3 Cells / ml, at least 8 × 10 3 Cells / ml, at least 9 × 10 3 Cells / ml, at least 1 × 10 4 Cells / ml, at least 2 × 10 4 Cells / ml, at least 3 × 10 4 Cells / ml, at least 4 × 10 4 Cells / ml, at least 5 × 10 4 Cells / ml, at least 6 × 10 4 Cells / ml, at least 7 × 10 4 Cells / ml, at least 8 × 10 4 Cells / ml, at least 9 × 10 4 Cells / ml, at least 1 × 10 5 Cells / ml, at least 2 × 10 5 Cells / ml, at least 3 × 10 5 Cells / ml, at least 4 × 10 5 Cells / ml, at least 5 × 10 5Cells / ml, at least 6 × 10 5 Cells / ml, at least 7 × 10 5 Cells / ml, at least 8 × 10 5 Cells / ml, at least 9 × 10 5 Cells / ml, at least 1 × 10 6 Cells / ml, at least 2 × 10 6 Cells / ml, at least 3 × 10 6 Cells / ml, at least 4 × 10 6 Cells / ml, at least 5 × 10 6 Cells / ml, at least 6 × 10 6 Cells / ml, at least 7 × 10 6 Cells / ml, at least 8 × 10 6 Cells / ml, at least 9 × 10 6 Cells / ml, at least 1 × 10 7 Cells / ml, at least 2 × 10 7 Cells / ml, at least 3 × 10 7 Cells / ml, at least 4 × 10 7 Cells / ml, at least 5 × 10 7 Cells / ml, at least 6 × 10 7 Cells / ml, at least 7 × 10 7 Cells / ml, at least 8 × 10 7 Cells / ml, at least 9 × 10 7 Cells / ml, at least 1 × 10 8 Cells / ml, at least 2 × 10 8 Cells / ml, at least 3 × 10 8 Cells / ml, at least 4 × 10 8 Cells / ml, at least 5 × 10 8 Cells / ml, at least 6 × 10 8 Cells / ml, at least 7 × 10 8 Cells / ml, at least 8 × 10 8 Cells / ml, at least 9 × 10 8 Cells / ml, at least 1 × 10 9 cells / ml, at least 1 x 10 3 Cells / ml ~ at least about 1 x 10 8 cells / ml, approximately 1×10 5 Cells / ml ~ at least about 1 x 108 cells / ml, or approximately 1 x 10 6 Cells / ml ~ at least about 1 x 10 8 It may be present in the composition in the amount of cells / ml.

[0256] In one aspect, the methods described herein can be used to produce autologous or allogeneic products according to one aspect of the present disclosure.

[0257] According to one embodiment of the invention, the antibody according to the above or the T cell receptor according to the above a) a toxin, or b) Immune Modulators The compound further comprises an effector moiety selected from the group consisting of:

[0258] Immune modulators are known. These are molecules that induce or stimulate an immune response through direct or indirect activation of the humoral or cellular arms of the immune system, such as by activation of T cells. Examples include IL-1, IL-1α, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-11, IL-12, IL-13, IL-15, IL-21, IL-23, TGF-β, IFN-γ, TNFα, anti-CD2 antibodies, anti-CD3 antibodies, anti-CD4 antibodies, anti-CD8 antibodies, anti-CD44 antibodies, anti-CD45RA antibodies, anti-CD45RB antibodies, anti-CD45RO antibodies, anti-CD49a antibodies, anti-CD49b antibodies, anti-CD49c antibodies, anti-CD49d antibodies, anti-CD49e antibodies, anti-CD49f antibodies, anti-CD16 antibodies, anti-CD28 antibodies, anti-IL-2R antibodies, viral proteins and peptides, and bacterial proteins or peptides. Where the immune modulator polypeptide is an antibody, it may specifically bind to an antigen presented by a T cell and may be an scFv antibody.

[0259] In one embodiment, the immune modulator is an anti-CD3 antibody. In one embodiment, the immune modulator binds to CD3γ, CD3δ, or CDε. In one embodiment, the immune modulator is the anti-CD3 antibody OKT3. In one embodiment, the immune modulator is the anti-CD3 antibody UCHT-1, or its humanized variant hUCHT-1. In one embodiment, the immune modulator is the anti-CD3 antibody BMA031. In one embodiment, the immune modulator is the anti-CD3 antibody 12F6. In some embodiments, fragments of these antibodies, such as V H and V L A person skilled in the art can easily identify the V domain from a published antibody. H and V L I know how to get a domain.

[0260] The humanized antibody hUCHTI is disclosed in (Zhu and Carter, 1995), the contents of which are incorporated herein by reference. In particular, the VHIT1 variants UCHT1-V17, UCHT1-V17opt, UCHT1-V21, or UCHT1-V23, preferably UCHT1-V17, are preferred. H and V L Further preferred embodiments and variants of this antibody are disclosed elsewhere herein.

[0261] Antibody BMA031, which targets the TCRα / β CD3 complex, and humanized versions thereof, are disclosed in (Shearman et al., 1991). In particular, the BMA031 variant BMA031(V36), or a V variant derived from BMA031(V10), preferably derived from BMA031(V36), is disclosed. H and V L Further preferred embodiments and variants of this antibody are disclosed elsewhere herein.

[0262] In a further embodiment, the immune modulator binds to a cell surface antigen selected from the group consisting of CD4, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, and / or CD42a.

[0263] Toxins that can be used to bind targeting domains are also known, see, for example, (Storz, 2015), the contents of which are incorporated herein by reference.

[0264] In one embodiment, the toxin is an auristatin (MMAE, MMAF). In one embodiment, the toxin is a maytansinoid, and in one embodiment, the toxin is an anthracycline or derivative thereof. In one embodiment, the toxin is a calicheamicin. In one embodiment, the toxin is a duocarmycin. In one embodiment, the toxin is a taxane. In one embodiment, the toxin is a pyrrolobenzodiazepine. In one embodiment, the toxin is α-amanitin. In one embodiment, the toxin is a ribotoxin or RNase. In one embodiment, the toxin is a tubulysin. In one embodiment, the toxin is a benzodiazepine derivative.

[0265] According to one embodiment of the invention, a T cell receptor as above is provided for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with, suffering from, or at risk of developing, (i), a metastasis or metastatic lesion.

[0266] The T cell receptor comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain is selected from the group consisting of SEQ ID NOs: 184, 187, 189, 190, 195, 206, 208, 210, 212, 216, 218, 219, 220, 221, 222, 229, 230, 232, 234, 236, 238, 240, 241, 242, 243, 244, 246, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 298, 299, 300, 302, or 304, and comprising the complementarity determining regions (CDRs) of said sequences; and the second polypeptide chain comprises a second hinge domain and / or a second Fc domain. and the second polypeptide comprises SEQ ID NOs: 179, 180, 181, 182, 183, 185, 186, 188, 191, 194, 203, 205, 213, 214, 215, 217, 223, 224, 225, 226, 227, 228, 231, 233, 235, 237, 239, 245, 247, 248, 249, 264, 266, 267, 268 , 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 301, or 303 and comprises the CDRs of said sequences.

[0267] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0268] The method includes administering to the patient a T cell receptor comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain is selected from the group consisting of SEQ ID NOs: 184, 187, 189, 190, 195, 206, 208, 210, 212, 216, 218, 219, 220, 221, 222, 229, 230, 232, 234, 236, 238, 240, 241, 242, and the like. , 243, 244, 246, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 265, 298, 299, 300, 302, or 304, and comprising the complementarity determining regions (CDRs) of said sequences; and the second polypeptide chain comprises a second hinge domain and / or a second F domain. c domain, and the second polypeptide is selected from SEQ ID NOs: 179, 180, 181, 182, 183, 185, 186, 188, 191, 194, 203, 205, 213, 214, 215, 217, 223, 224, 225, 226, 227, 228, 231, 233, 235, 237, 239, 245, 247, 248, 249, 264, 266, 267, 268, 95% identity to any one of 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 301, or 303 and including the CDRs of said sequences.

[0269] The sequence is a T cell receptor variable domain. The CDRs of the T cell receptor variable domain can be determined based on (Lefranc et al., 2003), the contents of which are incorporated herein by reference. Further disclosure can be found at http: / / www.imgt.org / IMGTScientificChart / Numbering / IMGTIGVLsuperfamily.html

[0270] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or metastatic lesions comprising such a T cell receptor as an active ingredient.

[0271] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0272] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0273] In one embodiment, the first polypeptide chain is fused to the second polypeptide chain by a covalent and / or non-covalent bond between the first and second hinge domains and / or between the first and second Fc domains.

[0274] In one embodiment, the first polypeptide chain is fused to the second polypeptide chain by a covalent and / or non-covalent bond between the first and second hinge domains and / or between the first and second Fc domains.

[0275] In one embodiment, the first and second Fc domains each comprise at least one Fc effector function-silencing mutation.

[0276] For example, the Fc domain of one or both, preferably both, polypeptide chains may contain one or more modifications that inhibit Fc gamma receptor (FcyR) binding. Such modifications may include L234A, L235A.

[0277] In a further embodiment, the Fc domain of one or both, preferably both, polypeptide chains may contain an N297Q mutation to remove an N-glycosylation site within the Fc portion, such a mutation abolishes Fc-gamma receptor interaction.

[0278] In one embodiment, the first and second Fc domains each comprise a CH3 domain that comprises at least one mutation that promotes heterodimer formation.

[0279] Thus, in some embodiments, the Fc domain of one of the polypeptides, e.g., Fc1, comprises the amino acid substitutions S354C and T366W (knob) in its CH3 domain, and the Fc domain of the other polypeptide, e.g., Fc2, comprises the amino acid substitutions Y349C, T366S, L368A and Y407V (hole) in its CH3 domain, or vice versa. This set of amino acid substitutions can be further expanded by including the amino acid substitution K409A in one polypeptide and F405K in the other polypeptide, as described in (Wei et al., 2017). Thus, in some embodiments, the Fc domain of one of the polypeptides, e.g., Fc1, comprises or further comprises the amino acid substitution K409A in its CH3 domain, and the Fc domain of the other polypeptide, e.g., Fe2, comprises or further comprises the amino acid substitution F405K in its CH3 domain, or vice versa.

[0280] Thus, in one embodiment, the Fc domain of one of the polypeptides, e.g., Fc1, comprises or further comprises charge pair substitutions E356K, E356R, D356R, or D356K and D399K or D399R, and the Fc domain of the other polypeptide, e.g., Fc2, comprises or further comprises charge pair substitutions R409D, R409E, K409E, or K409D and N392D, N392E, K392E, or K392D, or vice versa.

[0281] In one embodiment, said first and second Fc domains each comprise a CH2 and CH3 domain comprising at least two additional cysteine ​​residues.

[0282] Such cysteine ​​residues can lead to the formation of disulfide bridges, which can improve the stability of antigen-binding proteins without optimally interfering with the binding properties of antigen-binding proteins. Such cysteine ​​bridges can further improve heterodimerization. Further amino acid substitutions, such as charge pair substitutions, to improve the heterodimerization of the resulting proteins are described in the art, for example in EP 2970484.

[0283] Some embodiments of the present disclosure include a method of treating a metastatic lesion presenting a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), comprising, for example, identifying a metastatic lesion and administering a T lymphocyte of the present disclosure or an activated T lymphocyte produced by a method described herein to the metastatic lesion, wherein the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, gastric adenocarcinoma, gastric cancer, gastrointestinal ... The method may include a method of treating a cancer selected from the group consisting of carcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0284] Some embodiments of the present disclosure include a method of treating a metastatic lesion presenting a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), comprising, for example, identifying a metastatic lesion and treating the metastatic lesion with a T lymphocyte population that binds to and / or is specific for SLLQHLIGL (SEQ ID NO: 310), wherein the metastasis or metastatic lesion is selected from the group consisting of: adrenocortical sarcoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, , gastric adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0285] Other embodiments of the present disclosure include a method of treating a metastatic lesion presenting a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), e.g., treating the metastatic lesion with a T lymphocyte population that binds and / or is specific for SLLQHLIGL (SEQ ID NO: 310), wherein the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, gastric adenocarcinoma, The method may include a method of treating a cancer selected from the group consisting of renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0286] Other embodiments of the present disclosure include a method of treating a metastatic lesion that presents on its cell surface a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), comprising, for example, selecting a patient having a metastatic lesion and administering to the patient a composition comprising the T lymphocytes of the present disclosure or activated T lymphocytes produced by the methods described herein, wherein the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, or the like. In some embodiments, the method may include a method of treating a cancer selected from the group consisting of gastrointestinal cancer, gastric adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0287] Some embodiments of the present disclosure include a method of generating an immune response against a metastatic lesion presenting a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), comprising, for example, identifying a metastatic lesion and administering a T lymphocyte of the present disclosure or an activated T lymphocyte produced by a method described herein into the metastatic lesion, wherein the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, or the like. The method may include a method derived from a cancer selected from the group consisting of cancer, gastric adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0288] Some embodiments of the present disclosure include a method of generating an immune response against a metastatic lesion presenting a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), comprising, for example, identifying a metastatic lesion and treating the metastatic lesion with a T lymphocyte population that binds and / or is specific for SLLQHLIGL (SEQ ID NO: 310), wherein the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, or the like. In some embodiments, the method may include a method of treating a cancer selected from the group consisting of gastrointestinal cancer, gastric adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0289] Another embodiment of the present disclosure is a method of generating an immune response against a metastatic lesion presenting on its cell surface a peptide comprising, consisting essentially of, or consisting of SLLQHLIGL (SEQ ID NO: 310), comprising, for example, selecting a patient having a metastatic lesion and administering to the patient a composition comprising the T lymphocytes of the present disclosure or activated T lymphocytes produced by the methods described herein, wherein the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, ovarian serous cystadenocarcinoma, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon The method may include a method derived from a cancer selected from the group consisting of cancer, gastrointestinal cancer, gastric adenocarcinoma, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, uterine carcinosarcoma, endometrial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0290] Some embodiments of the present disclosure may include administering to the patient at least one adjuvant selected from the group consisting of anti-CD40 antibodies, imiquimod, resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, particle formulations comprising poly(lactide-co-glycolide) (PLG), virosomes, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-21 (IL-21), and interleukin-23 (IL-23).

[0291] Some embodiments of the present disclosure may include a method of preparing a T cell population comprising obtaining a T cell population from PBMCs, activating the obtained T cell population, transducing the activated T cell population with a nucleic acid of the present disclosure, and expanding the transduced T cell population, wherein the activating, transducing, and expanding steps are performed with or without a histone deacetylase inhibitor (HDACi) in the presence of IL-21.

[0292] In one embodiment, the present disclosure provides a method for the production of antigen-specific effector T cells (T EFF cells) into central memory T cells (T CM The present invention provides a method for reprogramming a T cell from a subject, the method comprising: EFF obtaining a starting population of lymphocytes comprising T cells; EFF From a starting population of lymphocytes containing T cells EFF Preparing a cell-enriched sample; EFF The starting population of lymphocytes containing cells or T EFF The cell-enriched samples were EFF T cells CMand culturing the subject in the presence of a HDACi and IL-21 in amounts sufficient to reprogram the T cells obtained from the subject, wherein reprogramming results in T cells obtained from the subject. EFF The starting population of lymphocytes containing T cells CM T compared to the number of cells CM A cell-enriched lymphocyte population is produced.

[0293] In some embodiments, T EFF Obtaining a starting population of lymphocytes comprising cells may include obtaining a sample of tumor infiltrating lymphocytes (TILs) or a sample comprising peripheral blood mononuclear cells (PBMCs) from the subject. In some embodiments, the method comprises obtaining a starting population of lymphocytes comprising TILs or a sample comprising peripheral blood mononuclear cells (PBMCs) from the subject. EFF From a starting population of lymphocytes containing T cells EFF The method may further include preparing a cell-enriched sample. EFF From a starting population of lymphocytes containing T cells EFF The step of preparing the cell-enriched sample includes the steps of: EFF From the starting population of lymphocytes containing CD8 + T EFF It may include a step of isolating the cells.

[0294] In some embodiments, IL-21, HDACi, or a combination thereof may be utilized in the field of cancer treatment with the methods described herein and / or with the ACT process described herein. In one embodiment, the present disclosure provides a method for reprogramming effector T cells to a central memory phenotype, comprising culturing the effector T cells with at least one HDACi together with IL-21. Representative HDACi include, for example, trichostatin A, trapoxin B, phenylbutyric acid, valproic acid, vorinostat (suberanilohydroxamic acid or SAHA), belinostat, panobinostat, dacinostat, entinostat, tacedinaline, and mocetinostat. In certain aspects, the HDACi may be SAHA. In other aspects, the HDACi may be panobinostat.

[0295] Bispecific molecules against PRAME-004 The molecules of the present disclosure generally comprise a first polypeptide chain and a second polypeptide chain, which together provide a variable domain of an antibody specific for an epitope of an immunomodulator cell surface antigen, and a variable domain of a TCR specific for an MHC-associated peptide epitope, such as SLLQHLIGL (PRAME-004) (SEQ ID NO: 310). The variable domains from the antibody and the TCR are stabilized by covalent and non-covalent bonds formed between the Fc moieties or parts thereof located on both polypeptide chains. The bispecific polypeptide molecule is thus capable of binding simultaneously to a cell receptor and an MHC-associated peptide epitope.

[0296] As discussed, the variable domains of antibodies include CD3γ, CD3δ, CD3ε, CDζ, CD4, CD7, CD8, CD10, CD11b, CD11c, CD14, CD16, CD18, CD22, CD25, CD28, CD32a, CD32b, CD33, CD41, CD41b, CD42a, CD42b, CD44, CD45RA, CD49, CD55, CD56, CD61, CD64, CD68, CD94 , CD90, CD117, CD123, CD125, CD134, CD137, CD152, CD163, CD193, CD203c, CD235a, CD278, CD279, CD287, Nkp46, NKG2D, GITR, FcεRI, TCRα / β, TCRγ / δ, and HLA-DR.

[0297] In the context of the present invention, the variable domain is derived from an antibody capable of recruiting human immune modulator cells by specifically binding to a surface antigen of said effector cells. In one particular embodiment, said antibody specifically binds to an epitope of the TCR-CD3 complex of human T cells comprising the peptide chains TCRα, TCRβ, CD3γ, CD3δ, CD3δ, and CD3δ.

[0298] In the context of the present invention, a dual affinity polypeptide molecule according to the invention is exemplified by a construct which binds to the SLLQHLIGL peptide (SEQ ID NO: 310) when presented as a peptide-MHC complex.

[0299] For example, dual affinity polypeptide molecules of the present disclosure can include those disclosed in U.S. Patent Application Publication Nos. 20190016801, 20190016802, 20190016803, and 20190016804, the contents of which are incorporated by reference in their entireties.

[0300] The bispecific polypeptide molecules according to the present invention preferably bind both the immunomodulator cellular antigen and the specific antigen epitope presented as a peptide-MHC complex with high specificity, e.g. with a binding affinity (KD) of about 100 nM or less, about 30 nM or less, about 10 nM or less, about 3 nM or less, about 1 nM or less, as measured, e.g., by biolayer interferometry or determined by flow cytometry.

[0301] Preferred are bispecific polypeptide molecules according to the invention, where the knob-into-hole mutations are selected from T366W as knob of the CH3 domain, T366'S, L368'A and Y407'V as holes (see for example WO 98 / 50431). This set of mutations can be further expanded by including the mutations K409A and F405'K as described in (Wei et al., 2017). Another knob can be T366Y and the hole is Y407'T.

[0302] Engineering was performed according to the methods described in (Reiter et al., 1994) to incorporate knob-into-hole mutations in the CH3 domain with or without additional interchain disulfide bond stabilization, to remove N-glycosylation sites in CH2 (e.g., N297Q mutation), to introduce Fc silencing mutations, and to introduce additional disulfide bond stabilization in VL and VH, respectively. An overview of the bispecific TCR / mAb diabodies produced, the mutants, and the corresponding sequences are listed in Table 1.

[0303] Preferred is a bispecific polypeptide molecule according to the invention, wherein said first and second polypeptide chains further comprise at least one hinge domain and / or Fc domain or a part thereof. In an antibody, "hinge" or "hinge region" or "hinge domain" refers to the flexible part of the heavy chain located between the CH1 and CH2 domains. It is approximately 25 amino acids long and is divided into the "upper hinge", "middle hinge" or "core hinge" and the "lower hinge". "Hinge subdomain" refers to the upper hinge, the middle (or core) hinge or the lower hinge. The amino acid sequence of the hinge of an IgG1 molecule is IgG1:EPKSCDKTHTCPPCPAPELLG (SEQ ID NO: 129), where E is E216 according to EU (http: / / www.imgt.org / IMGTScientificChart / Numbering / Hu IGHGnber.html) numbering.

[0304] Preferred are bispecific polypeptide molecules according to the invention comprising at least one IgG fragment crystallizable (Fc) domain, i.e. a fragment crystallizable region (Fc region) which is the tail region of an antibody that interacts with the Fc receptor and some proteins of the complement system. The Fc region comprises two or three heavy chain constant domains (CH domains 2, 3, and 4) in each polypeptide chain. The Fc region of IgG also has highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is essential for activity via the Fc receptor. The small size of bispecific molecule formats such as BiTE® and DART (approximately 50 kD) may result in fast clearance and short half-life. Therefore, for improved pharmacokinetic properties, TCR variable-only region (scTv)-cell receptor (e.g. CD3) bispecific polypeptide molecules may be fused to (human IgG1) Fc domains, thereby increasing the molecular weight. Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q / M428L and M252Y / S254T / T256E+H433K / N434F, have been shown to increase the binding affinity to the neonatal Fc receptor (FcRn) and the half-life of IgG1 in vivo, which may further extend the serum half-life of Fc-containing molecules.

[0305] In the bispecific polypeptide molecule of the invention, the Fc domain may comprise a CH2 domain comprising at least one Fc effector function silencing mutation. These mutations are preferably introduced in the ELLGGP (SEQ ID NO: 130) sequence of human IgG1 (residues 233-238) or the corresponding residues of other isotypes) known to be associated with effector function. As a rule, one or more mutations corresponding to residues from IgG2 and / or IgG4 are introduced in the IgG1 Fc. Preference is given to E233P, L234V, L235A and the absence of the residue or G at position 236. Another mutation is P331S. EP 1075496 discloses a recombinant antibody comprising a chimeric domain derived from two or more human immunoglobulin heavy CH2 domains, these human immunoglobulins being selected from IgG1, IgG2 and IgG4, the chimeric domain being a human immunoglobulin heavy chain CH2 domain having the following blocks of amino acids at defined positions: 233P, 234V, 235A, and a residue or G deletion at position 236, as well as 327G, 330S and 331S according to the EU numbering system, which is at least 98% identical to the CH2 sequence (residues 231-340) from human IgG1, IgG2 or IgG4 with said modified amino acids.

[0306] A bispecific polypeptide molecule of the invention according to the invention is exemplified herein by a bispecific polypeptide molecule comprising a first polypeptide chain comprising SEQ ID NO: 131 and a second polypeptide chain comprising SEQ ID NO: 132, or a bispecific polypeptide molecule comprising a first polypeptide chain comprising SEQ ID NO: 133 and a second polypeptide chain comprising SEQ ID NO: 134.

[0307] In one aspect, the disclosure provides a polypeptide having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:131, 132, 133, or 134.

[0308] In another embodiment, the polypeptide or bispecific polypeptide molecule disclosed herein may be modified by substitution of one or more residues at different, possibly selective, sites in the peptide chain. Such substitutions may be of a conservative nature, where an amino acid is replaced by an amino acid of similar structure and characteristics, for example, where a hydrophobic amino acid is replaced by another hydrophobic amino acid. More conservative would be the replacement of an amino acid of the same or similar size and chemical properties, such as where leucine is replaced by isoleucine. In the study of sequence variants in a family of naturally occurring homologous proteins, some amino acid substitutions are often tolerated better than others, and these are often correlated with the similarity of size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, which is the basis for defining "conservative substitution".

[0309] In another aspect of the present invention, the above object is solved by providing a nucleic acid(s) encoding the first and / or second polypeptide chain as disclosed herein, or an expression vector(s) comprising such a nucleic acid.

[0310] In another aspect of the present invention, the above object is solved by providing a host cell comprising the vector(s) as defined herein.

[0311] In another aspect of the present invention, the above object is solved by providing a method for producing a bispecific polypeptide molecule according to the invention, comprising a suitable expression of said expression vector(s) comprising the disclosed nucleic acid(s) in a suitable host cell and a suitable purification of the molecule(s) from the cell and / or its medium.

[0312] In another aspect of the present invention, the above object is solved by providing a pharmaceutical composition comprising a bispecific polypeptide molecule according to the present invention, a nucleic acid or expression vector(s) according to the present invention, or a cell according to the present invention, together with one or more pharma- ceutically acceptable carriers or excipients.

[0313] In another aspect of the invention, the invention relates to a bispecific polypeptide molecule according to the invention, a nucleic acid(s) or expression vector(s) according to the invention, a cell according to the invention or a pharmaceutical composition according to the invention for use in medicine.

[0314] In another aspect of the invention, the invention relates to a bispecific polypeptide molecule according to the invention, a nucleic acid or expression vector(s) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention for use in the treatment of a disease or disorder as disclosed herein, in particular selected from cancer and infectious diseases.

[0315] In another aspect of the invention, the invention relates to a method for the treatment of a disease or disorder comprising the step of administering a therapeutically effective amount of a bispecific polypeptide molecule according to the invention, a nucleic acid or expression vector(s) according to the invention, a cell according to the invention, or a pharmaceutical composition according to the invention.

[0316] In another aspect of the invention, the invention relates to a method for raising an immune response in a patient or subject comprising the step of administering a therapeutically effective amount of a bispecific polypeptide molecule according to the invention or a pharmaceutical composition according to the invention.

[0317] In another aspect, the present invention relates to a method for killing target cells in a patient or subject comprising the step of administering to the patient an effective amount of a bispecific polypeptide molecule according to the invention.

[0318] Examples of such bispecific molecules are shown in Table 2b.

[0319] [Table 2b]

[0320] In one embodiment, the first and second variable domains as defined herein may comprise an amino acid substitution at position 44 according to the IMGT numbering. In a preferred embodiment, said amino acid at position 44 is substituted with another suitable amino acid to improve pairing. In a particular embodiment, said antigen binding protein is a TCR, said mutation improves, for example, chain pairing (i.e. α and β chain pairing or γ and δ pairing). In a preferred embodiment, the amino acid present at position 44 of the variable domain is substituted with one amino acid selected from the group consisting of Q, R, D, E, K, L, W, and V.

[0321] In one embodiment the first variable domain of the antigen binding protein of the invention comprises: - a CDRa1 comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences DRGSQS (SEQ ID NO: 135) and DRGSQL (SEQ ID NO: 136), and / or - a CDRa2 comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences IYSNGD (SEQ ID NO: 137) and IYQEGD (SEQ ID NO: 138), and / or - Amino acid sequence CAAVINNPSGGMLTF (SEQ ID NO: 139), CAAVIDNSNGGILTF (SEQ ID NO: 140), CAAVIDNPSGGILTF (SEQ ID NO: 141), CAAVIDNDQGGILTF (SEQ ID NO: 142), CAAVIPNPPGGKLTF (SEQ ID NO: 143), CAAVIPNPGGGALTF (SEQ ID NO: 144), CAAVIPNSAGGRLTF (SEQ ID NO: 145), CAAVIPNLEGGSLTF (SEQ ID NO: 146), CAAVIPNRLGGYLTF (SEQ ID NO: 147), CAAVIPNTDGGRLTF (SEQ ID NO: 148), CAAVIPNQRGGALTF (SEQ ID NO: 149), CAAVIPNVVGGILTF (SEQ ID NO: 150), CAAVITNIAGGSLTF (SEQ ID NO: 151), CAAVIPNNDGGYLTF (SEQ ID NO: 152)), CA CDRa3 comprising or consisting of an amino acid sequence selected from the group consisting of AVIPNGRGGLLTF (SEQ ID NO: 153), CAAVIPNTHGGPLTF (SEQ ID NO: 154), CAAVIPNDVGGSLTF (SEQ ID NO: 155), CAAVIENKPGGPLTF (SEQ ID NO: 156), CAAVIDNPVGGPLTF (SEQ ID NO: 157), CAAVIPNNNGGALTF (SEQ ID NO: 158), CAAVIPNDQGGILTF (SEQ ID NO: 159), CAAVIPNVVGGQLTF (SEQ ID NO: 160), CAAVIPNSYGGLLTF (SEQ ID NO: 161), CAAVIPNDDGGLLTF (SEQ ID NO: 162), CAAVIPNAAGGLLTF (SEQ ID NO: 163), CAAVIPNTIGGLLTF (SEQ ID NO: 164) and CAAVIPNTRGGLLTF (SEQ ID NO: 165). and the second variable domain comprises - a CDRb1 comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences SGHRS (SEQ ID NO: 166) and PGHRA (SEQ ID NO: 167), and / or - a CDRb2 comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences YFSETQ (SEQ ID NO: 169), YVHGEE (SEQ ID NO: 170) and YVHGAE (SEQ ID NO: 171), and / or - CDRb3 comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences CASSPWDSPNEQYF (SEQ ID NO: 172) and CASSPWDSPNVQYF (SEQ ID NO: 173). Includes.

[0322] The inventors of the present invention have identified in the examples disclosed herein TCR variants "HiAff1" and "LoAff3", whose CDR amino acid sequences notably enhance the binding affinity, stability and specificity of antigen binding proteins comprising these CDRs compared to reference proteins when used in antigen binding proteins of the present invention, in particular in bispecific antigen binding proteins, more particularly in Fc-containing bispecific TCR / mAb (anti-CD3) diabody formats.

[0323] Such reference proteins include, for example, antigen binding proteins comprising the CDRs of the parent / wild type TCR R16P1C10 as disclosed in WO 2018 / 172533, such as F-TCRs described herein comprising the CDRs of said TCR R16P1C10. c The reference protein may be a bispecific TCR / mAb (anti-CD3) diabody comprising said TCR R16P1C10 CDRs, or may be an antigen binding protein comprising the CDRs of said TCR R16P1C10, and of the same format as the antigen binding protein to be compared. Such a reference protein may be, for example, an antigen binding protein comprising the CDRs of "CDR6", e.g., a F-binding protein as described herein comprising the CDRs of "CDR6". c The reference protein may be a bispecific TCR / mAb (anti-CD3) diabody comprising the CDRs of "CDR6", and is of the same format as the antigen binding protein to which it is being compared, wherein the CDRs of "CDR6" are disclosed herein above.

[0324] The inventors have further demonstrated that the antigen binding proteins of the invention comprising the above CDRs have improved stability compared to an antigen binding protein comprising the CDRs of a reference antigen binding protein, designated "CDR6", which comprises the following alpha and beta CDRs: CDRa1 comprising or consisting of the amino acid sequence DRGSQS (SEQ ID NO: 135), CDRa2 comprising or consisting of the amino acid sequence IYSNGD (SEQ ID NO: 137), CDRa3 consisting of the amino acid sequence CAAVIDNDQGGILTF (SEQ ID NO: 142), CDRb1 comprising or consisting of the amino acid sequence PGHRA (SEQ ID NO: 167), CDRb2 comprising or consisting of the amino acid sequence YVHGEE (SEQ ID NO: 170), and CDRb3 comprising or consisting of the amino acid sequence CASSPWDSPNVQYF (SEQ ID NO: 173).

[0325] In one particular embodiment, the present invention refers to an antigen binding protein comprising the CDRs of the so-called "HiAff1" and "LoAff3" variants and variants thereof. Thus, in one preferred embodiment, the antigen binding protein of the present invention comprises: a) a first polypeptide chain comprising a first variable domain comprising three complementarity determining regions (CDRs) CDRa1, CDRa2, and CDRa3, CDRa1 comprises or consists of the amino acid sequence DRGSQS (SEQ ID NO: 135) or an amino acid sequence at least 85% identical to SEQ ID NO: 135), CDRa2 comprises or consists of the amino acid sequence IYQEGD (SEQ ID NO: 138), CDRa3 comprises or consists of the amino acid sequence CAAVIDNDQGGILTF (SEQ ID NO: 142), A first polypeptide chain; b) a second polypeptide chain comprising a second variable domain comprising three complementarity determining regions (CDRs) CDRb1, CDRb2 and CDRb3, CDRb1 comprises or consists of the amino acid sequence PGHRA (SEQ ID NO: 167) or PGHRS (SEQ ID NO: 168), preferably PGHRA (SEQ ID NO: 167) or an amino acid sequence at least 85% identical to SEQ ID NO: 167) or SEQ ID NO: 168), preferably SEQ ID NO: 167), CDRb2 comprises or consists of the amino acid sequence YVHGEE (SEQ ID NO: 170) or an amino acid sequence at least 85% identical to SEQ ID NO: 170, CDRb3 comprises or consists of the amino acid sequence CASSPWDSPNEQYF (SEQ ID NO: 172) or CASSPWDSPNVQYF (SEQ ID NO: 173), preferably CASSPWDSPNVQYF (SEQ ID NO: 173) or an amino acid sequence at least 85% identical to SEQ ID NO: 172) or SEQ ID NO: 173), preferably CASSPWDSPNVQYF (SEQ ID NO: 173); A second polypeptide chain; Includes.

[0326] [Table 3]

[0327] All positions and CDR definitions are according to the Kabat numbering system. TCRs consisting of Valpha and Vbeta domains were designed, produced and tested in single chain (scTCR) format coupled to the Fab fragment of the humanized UCHT1-antibody (Table 4). Vectors for expression of recombinant proteins were designed as pUC19 derivatives, controlled by a monocistronic, HCMV-derived promoter element. Plasmid DNA was amplified in E. coli according to standard culture methods and then purified using a commercial kit (Macherey & Nagel). Purified plasmid DNA was used for transient transfection of CHO cells. Transfected CHO cells were cultured at 32°C to 37°C for 10-11 days.

[0328] [Table 4] In this table, the term "α chain" refers to V, except for TPP-70, TPP-71, TPP-72, TPP-73 and TPP74. α The term "β chain" refers to a polypeptide chain that contains a variable domain derived from the TCR α chain. β , i.e., a polypeptide chain that contains a variable domain derived from the TCR beta chain. In TPP-70, TPP-71, TPP-72, TPP-73 and TPP74, the "alpha chain" does not contain a TCR-derived variable domain, while the "beta chain" contains two TCR-derived variable domains, one derived from the TCR alpha chain and one derived from the TCR beta chain.

[0329] The present disclosure provides an antigen binding protein for use in (the manufacture of a medicament for) the treatment of metastasis or metastatic lesions, the antigen binding protein being selected from the group consisting of TPP-1295, TPP-1298, TPP-230, TPP-669, or TPP-1333.

[0330] Alternatively or additionally, there is provided a method of treating a patient with metastasis or a metastatic lesion having been diagnosed with (i), suffering from (ii), or at risk of developing (iii), the method comprising administration of one or more therapeutically effective doses of an antigen binding protein selected from the group consisting of TPP-1298, TPP-1295, TPP-230, TPP-669, or TPP-1333.

[0331] According to one embodiment, the antigen binding protein is TPP-1295 and comprises the following set of sequences:

[0332] JPEG2024537844000007.jpg96167

[0333] According to one embodiment, the antigen binding protein comprises a first and a second polypeptide chain linked together forming a first and a second antigen binding domain, The first antigen-binding domain is A complementarity determining region (CDR) a1 comprising the amino acid sequence of SEQ ID NO: 320; Optionally, a CDRa2 comprising the amino acid sequence of SEQ ID NO: 321, and CDRa3 comprising the amino acid sequence of SEQ ID NO: 322 a T cell receptor (TCR) alpha variable domain comprising CDRb1 comprising the amino acid sequence of SEQ ID NO: 325, Optionally, CDRb2 comprises the amino acid sequence of SEQ ID NO: 326, and CDRb3 comprising the amino acid sequence of SEQ ID NO: 327 and a TCRβ variable domain comprising Includes.

[0334] The first antigen-binding domain of the antigen-binding protein binds to an MHC molecule, preferably HLA-A * It binds to a peptide comprising or consisting of the amino acid sequence SLLQHLIGL in a complex with 02.

[0335] The antigen binding protein may have a TCR alpha variable domain comprising SEQ ID NO: 323, and a TCR beta variable domain comprising SEQ ID NO: 328. The antigen binding protein may have a first polypeptide chain comprising SEQ ID NO: 324, and a second polypeptide chain comprising SEQ ID NO: 329.

[0336] According to one embodiment, the antigen binding protein is TPP-1298 and comprises the following set of sequences:

[0337] JPEG2024537844000008.jpg92165

[0338] According to one embodiment, the antigen binding protein comprises a first and a second polypeptide chain linked together forming a first and a second antigen binding domain, The first antigen-binding domain is CDRa1 comprising the amino acid sequence of SEQ ID NO: 330, Optionally, a CDRa2 comprising the amino acid sequence of SEQ ID NO: 331, and CDRa3 comprising the amino acid sequence of SEQ ID NO: 332 a TCR alpha variable domain comprising CDRb1 comprising the amino acid sequence of SEQ ID NO: 335, Optionally, CDRb2 comprises the amino acid sequence of SEQ ID NO: 336, and CDRb3 comprising the amino acid sequence of SEQ ID NO: 337 and a TCRβ variable domain comprising Includes.

[0339] The first antigen-binding domain of the antigen-binding protein binds to an MHC molecule, preferably HLA-A * It binds to a peptide comprising or consisting of the amino acid sequence SLLQHLIGL in a complex with 02.

[0340] The antigen binding protein may have a TCR alpha variable domain comprising SEQ ID NO: 333, and a TCR beta variable domain comprising SEQ ID NO: 338. The antigen binding protein may have a first polypeptide chain comprising SEQ ID NO: 334, and a second polypeptide chain comprising SEQ ID NO: 339.

[0341] According to one embodiment, the antigen binding protein is TPP-230 and comprises the following set of sequences:

[0342] JPEG2024537844000009.jpg92166

[0343] According to one embodiment, the antigen binding protein comprises a first and a second polypeptide chain linked together forming a first and a second antigen binding domain, The first antigen-binding domain is CDRa1 comprising the amino acid sequence of SEQ ID NO: 340, Optionally, a CDRa2 comprising the amino acid sequence of SEQ ID NO: 341, and CDRa3 comprising the amino acid sequence of SEQ ID NO: 342 a TCR alpha variable domain comprising CDRb1 comprising the amino acid sequence of SEQ ID NO: 345, Optionally, CDRb2 comprises the amino acid sequence of SEQ ID NO: 346, and CDRb3 comprising the amino acid sequence of SEQ ID NO: 347 and a TCRβ variable domain comprising Includes.

[0344] The first antigen-binding domain of the antigen-binding protein binds to an MHC molecule, preferably HLA-A * It binds to a peptide comprising or consisting of the amino acid sequence SLLQHLIGL in a complex with 02.

[0345] The antigen binding protein may have a TCR alpha variable domain comprising SEQ ID NO: 343, and a TCR beta variable domain comprising SEQ ID NO: 348. The antigen binding protein may have a first polypeptide chain comprising SEQ ID NO: 344, and a second polypeptide chain comprising SEQ ID NO: 349.

[0346] According to one embodiment, the antigen binding protein is TPP-669 and comprises the following set of sequences:

[0347] JPEG2024537844000010.jpg92166

[0348] According to one embodiment, the antigen binding protein comprises a first and a second polypeptide chain linked together forming a first and a second antigen binding domain, The first antigen-binding domain is CDRa1 comprising the amino acid sequence of SEQ ID NO: 350, Optionally, a CDRa2 comprising the amino acid sequence of SEQ ID NO: 351, and CDRa3 comprising the amino acid sequence of SEQ ID NO: 352 a TCR alpha variable domain comprising CDRb1 comprising the amino acid sequence of SEQ ID NO: 355, Optionally, CDRb2 comprises the amino acid sequence of SEQ ID NO: 356, and CDRb3 comprising the amino acid sequence of SEQ ID NO: 357 and a TCRβ variable domain comprising Includes.

[0349] The first antigen-binding domain of the antigen-binding protein binds to an MHC molecule, preferably HLA-A * It binds to a peptide comprising or consisting of the amino acid sequence SLLQHLIGL in a complex with 02.

[0350] The antigen binding protein may have a TCR alpha variable domain comprising SEQ ID NO: 353, and a TCR beta variable domain comprising SEQ ID NO: 358. The antigen binding protein may have a first polypeptide chain comprising SEQ ID NO: 354, and a second polypeptide chain comprising SEQ ID NO: 359.

[0351] According to one embodiment, the antigen binding protein is TPP-1333 and comprises the following set of sequences:

[0352] JPEG2024537844000011.jpg92163

[0353] According to one embodiment, the antigen binding protein comprises a first and a second polypeptide chain linked together forming a first and a second antigen binding domain, The first antigen-binding domain is CDRa1 comprising the amino acid sequence of SEQ ID NO: 360, Optionally, a CDRa2 comprising the amino acid sequence of SEQ ID NO: 361, and CDRa3 comprising the amino acid sequence of SEQ ID NO: 362 a TCR alpha variable domain comprising CDRb1 comprising the amino acid sequence of SEQ ID NO: 365, Optionally, CDRb2 comprises the amino acid sequence of SEQ ID NO: 366, and CDRb3 comprising the amino acid sequence of SEQ ID NO: 367 and a TCRβ variable domain comprising Includes.

[0354] The first antigen-binding domain of the antigen-binding protein binds to an MHC molecule, preferably HLA-A *It binds to a peptide comprising or consisting of the amino acid sequence SLLQHLIGL in a complex with 02.

[0355] The antigen binding protein may have a TCR alpha variable domain comprising SEQ ID NO: 363, and a TCR beta variable domain comprising SEQ ID NO: 368. The antigen binding protein may have a first polypeptide chain comprising SEQ ID NO: 364, and a second polypeptide chain comprising SEQ ID NO: 369. Purification and quality control of the antigen binding proteins provided herein may be performed as exemplified below.

[0356] According to some embodiments, the metastasis or metastatic lesion is: ACC metastasis BLCA metastasis BRCA Translocation TNBC metastasis CRC metastasis HNSCC metastasis HNAC metastasis MEL metastasis SKCM metastasis UVM Transition LC metastasis NSCLC metastasis ·NSCLC glandular metastasis NSCLC squamous metastasis NSCLC and other metastases ·SCLC metastasis CHOL metastasis ESCA Metastasis CESC metastasis OC transfer OV metastasis LIHC transfer RCC metastasis KIRC metastasis KIRP Metastasis SARC metastasis ·FS Transition LPS transfer MPNST metastasis SS Transition STAD metastasis TGCT metastasis THYM Metastasis UCS metastasis UCEC metastasis, and / or UEC metastasis At least one selected from the group consisting of:

[0357] According to some embodiments, the metastasis or metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma ... The cancer is derived from a cancer selected from the group consisting of: lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0358] Conditioned cell supernatants were clarified by filtration (0.22 μm) using a Sartoclear Dynamics® Lab Filter Aid (Sartorius). Bispecific molecules were purified using an Akta Pure 25 L FPLC system (GE Lifesciences) equipped to perform affinity and size exclusion chromatography in series. Affinity chromatography was performed on a Protein L column (GE Lifesciences) according to standard affinity chromatography protocols. Elution from the affinity column (pH 2.8) was immediately followed by size exclusion chromatography on a Superdex 200 pg 16 / 600 column (GE Lifesciences) according to standard protocols to obtain highly pure monomeric protein. Protein concentrations were determined on a NanoDrop system (Thermo Scientific) using the extinction coefficient calculated according to the predicted protein sequence. If necessary, the concentration was adjusted using a Vivaspin device (Sartorius). Finally, the purified molecule was stored at a temperature of 2-8 °C at a concentration of approximately 1 mg / mL in phosphate buffered saline. The final product yield was calculated after completion of purification and formulation.

[0359] The quality of the purified bispecific molecules was determined by HPLC-SEC on a MabPac SEC-1 column (5 μm, 4×300 mm) run in 50 mM sodium phosphate pH 6.8 containing 300 mM NaCl in a Vanquish uHPLC system.

[0360] Stress stability testing was performed by incubation of the molecules formulated in PBS for up to 2 weeks at 40° C. Integrity, aggregate content, and monomer recovery were analyzed by HPLC-SEC analysis.

[0361] The inventors demonstrate by LDH release assay that antigen-binding proteins, in particular TCER® molecules, cause cell lysis in T2 cells loaded with the target peptide PRAME-004 (Table 5). The inventors further demonstrate by LDH release assay that antigen-binding proteins, in particular TCER® molecules, cause cell lysis in PRAME-positive tumor cell lines, while PRAME-negative tumor cell lines were not affected by co-incubation with TCER® molecules (Figures 35-37). These in vitro experiments further prove the safety of the antigen-binding proteins of the invention and demonstrate that the cytotoxic effect is highly selective for PRAME-positive tumor tissues. Thus, the molecules of the invention show a beneficial safety profile.

[0362] TCER® slot III mutants TPP-214, -222, -230, -666, -669, -871, -872, -876, -879, -891, -894 were further characterized for their ability to kill T2 cells loaded with various levels of target peptides. T2 cells were loaded with the respective concentrations of PRAME-004 for 2 h, after which peptide-loaded T2 cells were co-cultured with human PBMCs at an E:T ratio of 5:1 for 48 h in the presence of increasing concentrations of TCER® mutants. Levels of LDH released in the supernatant were quantified using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega). All TCER® mutants showed sub-picomolar EC 50 The EC 50 However, even at a very low loading concentration of PRAME-004 of 10 pM, killing was induced by all TCER® mutants except TPP-214.

[0363] [Table 5]

[0364] According to yet another aspect of the present invention, at least one active agent comprising: Peptides from the above -Antibodies or fragments thereof according to the above -T cell receptor or its fragment - a nucleic acid or expression vector according to the above a host cell according to the above, recombinant T-lymphocytes according to the above, and / or - Activated T lymphocytes due to the above and a pharmaceutical acceptable carrier. The composition is for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with, suffering from, or at risk of developing (iii) metastasis or a metastatic lesion.

[0365] Alternatively or additionally, methods of treating a patient diagnosed with metastasis or a metastatic lesion (i), suffering from (ii), or at risk of developing (iii) are provided.

[0366] This method is Peptides from the above -Antibodies or fragments thereof according to the above -T cell receptor or its fragment - a nucleic acid or expression vector according to the above a host cell according to the above, recombinant T-lymphocytes according to the above, and / or - Activated T lymphocytes due to the above The method includes administering to a patient one or more therapeutically effective doses of at least one active ingredient selected from the group consisting of at least one of the following:

[0367] Alternatively or additionally, there is provided a pharmaceutical composition for treating metastasis or metastatic lesions comprising such an active ingredient as an active ingredient.

[0368] In one embodiment, the metastasis or metastatic lesion is PRAME positive. In one embodiment, the metastasis or metastatic lesion displays on the surface of at least one of its cells an amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL), or a peptide comprising said amino acid sequence bound to the major histocompatibility complex.

[0369] In one embodiment, the patient is * 02 positive. This is due in particular to the haplotype HLA-A * 02:01, HLA‐A * 02:02, HLA‐A * 02:03, HLA‐A * 02:05, HLA‐A * 02:06, HLA‐A * 02:07, and HLA‐A * In one embodiment, the patient is * 02:01Positive.

[0370] In various embodiments of the invention, the metastasis or metastatic lesion is · ACC (adrenal cortical carcinoma) metastasis ·BLCA (bladder urothelial carcinoma) metastasis ·BRCA (breast cancer) metastasis -TNBC (triple negative breast cancer) metastasis CRC (colorectal cancer) metastasis ·HNSCC (head and neck squamous cell carcinoma) metastasis Head and neck adenocarcinoma (HNAC) metastasis ·MEL (melanoma) metastasis ·SKCM (cutaneous melanoma) metastasis UVM (Uveal Melanoma) Metastasis LC (lung cancer) metastasis NSCLC (non-small cell lung cancer) metastasis ·NSCLC squamous (non-small cell lung squamous cell carcinoma) metastasis ·NSCLC gland (non-small cell lung adenocarcinoma) metastasis Other NSCLC (metastases in NSCLC samples that were not clearly assigned to NSCLC glandular or NSCLC squamous) ·SCLC (small cell lung cancer) metastasis ·CHOL (bile duct carcinoma) metastasis ESCA (Esophageal Carcinoma) Metastasis ·CESC (cervical squamous cell carcinoma and cervical adenocarcinoma) metastasis ·OC (ovarian carcinoma) metastasis Metastasis to ovarian serous cystadenocarcinoma LIHC (Liver Hepatocellular Carcinoma) Metastasis RCC (renal cell carcinoma) metastasis KIRC (clear cell renal cell carcinoma) metastasis ·KIRP (papillary renal cell carcinoma) metastasis SARC (sarcoma) metastasis FS (fibrosarcoma) metastasis LPS (liposarcoma) metastasis MPNST (malignant peripheral nerve sheath tumor) metastasis · SS (synovial sarcoma) metastasis -STAD (gastric adenocarcinoma) metastasis ·TGCT (testicular germ cell tumor) metastasis Thymoma metastasis UCS (uterine carcinosarcoma) metastasis and / or ·UEC (endometrial carcinoma) metastasis At least one selected from the group consisting of at least one of the following:

[0371] According to further embodiments, the following is provided:

[0372] 1. An in vitro method for producing specific activated T lymphocytes for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with, suffering from, or at risk of developing (i) metastasis or a metastatic lesion, the method comprising the steps of providing a synthetic or recombinant peptide consisting of the amino acid sequence of SEQ ID NO: 310, and contacting in vitro T cells with an antigen-loaded human class I major histocompatibility complex (MHC) molecule expressed on the surface of a suitable antigen-presenting cell or an artificial construct that mimics an antigen-presenting cell for a period of time sufficient to activate said T cells in an antigen-specific manner, wherein said antigen is a peptide consisting of the amino acid sequence of SEQ ID NO: 310.

[0373] 2. A cell line of activated T lymphocytes produced by the method described in item 1, characterized in that the cell line is capable of selectively recognizing metastatic cells presenting a peptide consisting of the amino acid sequence of SEQ ID NO: 310.

[0374] 3. An in vitro method for producing a soluble T cell receptor comprising: (i) selecting specific T cell clones expressing a T cell receptor that binds to an HLA ligand consisting of a synthetic or recombinant peptide comprising the amino acid sequence of SEQ ID NO: 310, optionally wherein said peptide is bound to MHC, and optionally wherein said T cell clones have been generated by immunizing a non-human mammal transgenic with the entire human TCR locus with a peptide comprising the amino acid sequence of SEQ ID NO: 310, or with a peptide-MHC complex comprising such a peptide, optionally selecting from a library of TCR or CDR variants, e.g. by yeast, phage or T cell display, specific T cell receptors that bind to a synthetic or recombinant peptide comprising the amino acid sequence of SEQ ID NO: 310, optionally when bound to MHC, or (ii) selecting specific T cell receptors that bind to an HLA ligand consisting of a synthetic or recombinant peptide consisting of the amino acid sequence of SEQ ID NO: 310, optionally wherein said peptide binds to MHC from a phage display system; Including, The T cell receptor is capable of reacting with an HLA ligand consisting of the peptide of SEQ ID NO: 310 presented by metastatic cells by binding to a peptide-MHC complex that contains a peptide comprising SEQ ID NO: 310 bound to an MHC molecule. A method comprising:

[0375] 4. An in vitro method for producing a recombinant antibody that specifically binds to human major histocompatibility complex (MHC) class I complexed with a peptide of the amino acid sequence of SEQ ID NO: 310, comprising: (i) immunizing a non-human mammal transgenic with an entire human immunoglobulin locus with a peptide comprising the amino acid sequence of SEQ ID NO: 310, or with a peptide-MHC complex comprising such a peptide; (ii) isolating mRNA molecules from said non-human mammalian antibody-producing cells; (iii) producing a phage display library that displays the protein molecules encoded by the mRNA molecules; (iv) isolating at least one phage from the phage display library, wherein at least one phage comprises the antibody that specifically binds to a peptide comprising SEQ ID NO: 310 bound to an MHC class I molecule; Including, said antibody is capable of specifically recognizing said peptide of SEQ ID NO: 310 when complexed with an MHC molecule by binding to a peptide-MHC complex comprising a peptide comprising SEQ ID NO: 310 bound to an MHC class I molecule; The peptide of SEQ ID NO: 310 is expressed on the surface of metastatic cells. A method comprising:

[0376] 5. A pharma- ceutically acceptable salt of a peptide consisting of the amino acid sequence of SEQ ID NO: 310, characterized in that the salt is an acetate salt, a trifluoroacetate salt, or a chloride salt.

[0377] 6. A pharmaceutical composition comprising a cell line produced according to the method of item 2, a TCR produced according to the in vitro method of item 3, or an antibody produced according to the in vitro method of item 4, and a pharma- ceutically acceptable carrier.

[0378] According to a further aspect of the invention there is provided a nucleic acid comprising at least one coding sequence encoding at least one antigenic peptide consisting of SLLQHLIGL (SEQ ID NO:310).

[0379] In one embodiment, the nucleic acid comprises two or more repeats ("concatamers") of the coding sequence separated by short stretches of nucleotides ("spacers").

[0380] The nucleic acid may be or include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA including LNA with b-D-ribo configuration, a-LNA with a-L-ribo configuration (diastereomer of LNA), 2'-amino-LNA with 2'-amino functionalization, and 2'-amino-a-LNA with 2'-amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0381] According to one embodiment, the nucleic acid is mRNA.

[0382] According to one embodiment, the mRNA comprises a 5' untranslated region (UTR) and / or a 3'UTR.

[0383] In some embodiments, the 3'-UTR comprises or consists of a nucleic acid sequence derived from the 3'-UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1, and RPS9, or from a homolog, fragment, or variant of any one of these genes.

[0384] In some embodiments, the 5'-UTR comprises or consists of a nucleic acid sequence derived from the 5'-UTR of a gene selected from HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2, or from a homolog, fragment, or variant of any one of these genes.

[0385] In some embodiments, the 5'-UTR and the heterologous 3'UTR are selected from UTR designs a-1 (HSD17B4 / PSMB3), a-3 (SLC7A3 / PSMB3), e-2 (RPL31 / RPS9), and i-3 (- / muag), and are selected from UTR designs a-1 (HSD17B4 / PSMB3) and i-3 (- / muag).

[0386] According to one embodiment, the mRNA includes modified nucleosides instead of uridine.

[0387] According to one embodiment, the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U).

[0388] According to one embodiment, the nucleic acid comprises a coding sequence that has been codon-optimized and / or has an increased G / C content and a decreased uridine content compared to a wild-type coding sequence, and preferably the codon-optimization and / or increased G / C content does not change the sequence of the encoded amino acid sequence.

[0389] The generation of G / C content optimized nucleic acid sequences (RNA or DNA) may be carried out using the methods described in WO 2002 / 098443, the disclosure of which is incorporated in its entirety by the present invention in this regard.

[0390] In a preferred embodiment, the nucleic acid may be modified so that the codons of at least one coding sequence are adapted to human codon usage (herein referred to as a "human codon usage adapted coding sequence").

[0391] Codons encoding the same amino acid occur at different frequencies in humans. Therefore, it is preferred that the coding sequence of the nucleic acid is modified so that the frequency of the codons encoding the same amino acid corresponds to the natural frequency of the codon according to human codon usage. For example, for the amino acid alanine, the wild type or reference coding sequence is preferably adapted so that the codon "GCC" is used at a frequency of 0.40, the codon "GCT" is used at a frequency of 0.28, the codon "GCA" is used at a frequency of 0.22, the codon "GCG" is used at a frequency of 0.10, etc. Thus, such a procedure (as exemplified by alanine) is applied to each amino acid encoded by the coding sequence of the nucleic acid to obtain a sequence adapted to human codon usage.

[0392] According to some embodiments, the nucleic acid is SEQ ID NO: 314 (PRAME mRNA) 315 (PRAME mRNA GC enriched) 316 (PRAME cDNA) 317 (PRAME 004 mRNA) ·318(PRAME 004 mRNA GC enrichment) 319(PRAME 004 cDNA) At least one selected from the group consisting of:

[0393] According to another aspect of the invention there is provided a composition or pharmaceutical preparation comprising a nucleic acid according to above.

[0394] In one embodiment, the composition comprises a peptide that is a fragment of prostate specific membrane antigen (PSMA), in particular PSMA. 288‐297 (GLPSIPVHPI, SEQ ID NO: 376) or PSMA 288‐297 It does not include a nucleic acid encoding 1297V (GLPSIPVHPV, SEQ ID NO: 377).

[0395] According to one embodiment, the composition comprises mRNA with RNA integrity of 70% or greater.

[0396] The term "RNA integrity" generally refers to whether or not a complete RNA sequence is present in a liquid composition.Low RNA integrity may be due to, among others, RNA degradation, RNA cleavage, incorrect or incomplete chemical synthesis of RNA, incorrect base pairing, incorporation of modified nucleotides or modification of already incorporated nucleotides, lack of capping or incomplete capping, lack of polyadenylation or incomplete polyadenylation, or incomplete in vitro transcription of RNA.RNA is a fragile molecule that can be easily degraded by, for example, temperature, ribonuclease, pH, and other factors (e.g., nucleophilic attack, hydrolysis, etc.), which can reduce the integrity of RNA and thus the functionality of RNA.

[0397] According to one embodiment, the composition comprises mRNA with a degree of capping of 70% or more, preferably at least 70%, 80% or 90% of the mRNA species contain Cap1 structures.

[0398] 5'-capping of polynucleotides may be completed simultaneously during in vitro transcription reactions using the following chemical RNA cap analogs: 3'-O-Me-m7G(5')ppp(5')G [ARCA cap]; G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA) to generate a 5'-guanosine cap structure according to the manufacturer's protocol. 5'-capping of modified RNA may be completed post-transcriptionally using Vaccinia Vims capping enzyme to generate the "cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Both Vaccinia Vims capping enzyme and 2'-O methyltransferase may be used to generate m7G(5')ppp(5')G-2'-O-methyl to generate the Cap1 structure. From the Cap1 structure, the Cap2 structure may be generated by subsequent 2'-O-methylation of the third nucleotide from the 5' end with 2'-O-methyltransferase. From the Cap2 structure, the Cap3 structure may be generated by subsequent 2'-O-methylation of the fourth nucleotide from the 5' end with 2'-O-methyltransferase. The enzymes may be from recombinant sources.

[0399] According to some embodiments, at least one nucleic acid is complexed or associated with one or more lipids or lipid-based carriers, thereby forming liposomes, lipid nanoparticles (LNPs), lipoplexes, and / or nanoliposomes that preferably encapsulate at least one nucleic acid.

[0400] According to one embodiment, the LNP comprises: (i) at least one cationic lipid; (ii) at least one neutral lipid; (iii) at least one steroid or steroid analog; and (iv) at least one polymer-conjugated lipid, preferably a PEG-lipid; Includes.

[0401] According to one embodiment, (i)-(iv) have a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG-lipid.

[0402] According to some embodiments, the cationic lipid is at least one selected from the group consisting of:

[0403] JPEG2024537844000013.jpg83158

[0404] According to some embodiments, the polymer-conjugated lipid comprises:

[0405] JPEG2024537844000014.jpg105159

[0406] According to one embodiment, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0407] According to one embodiment, the steroid or steroid analog is cholesterol.

[0408] According to one embodiment, the composition or pharmaceutical preparation is a vaccine.

[0409] According to another aspect of the present invention, there is provided a method of eliciting an immune response against a tumor or metastatic lesion presenting a peptide comprising SLLQHLIGL (SEQ ID NO: 310) on its cell surface, the method comprising administering to a patient a composition as described above.

[0410] According to another aspect of the invention there is provided a composition as above for use in (the manufacture of a medicament for) the treatment of a patient diagnosed with, suffering from, or at risk of developing (iii) a tumour or metastatic lesion which presents a peptide comprising SLLQHLIGL (SEQ ID NO: 310) on its cell surface.

[0411] According to some embodiments, the tumor is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma ... The cancer is selected from the group consisting of lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma.

[0412] According to some embodiments thereof, the metastatic lesion is: ACC metastasis BLCA metastasis BRCA Translocation TNBC metastasis CRC metastasis HNSCC metastasis HNAC metastasis MEL metastasis SKCM metastasis UVM Transition LC metastasis NSCLC metastasis ·NSCLC glandular metastasis NSCLC squamous metastasis NSCLC and other metastases ·SCLC metastasis CHOL metastasis ESCA Metastasis CESC metastasis OC transfer OV metastasis LIHC transfer RCC metastasis KIRC metastasis KIRP Metastasis SARC metastasis ·FS Transition LPS transfer MPNST metastasis SS Transition STAD metastasis TGCT metastasis THYM Metastasis UCS metastasis UCEC metastasis, and / or UEC metastasis At least one selected from the group consisting of:

[0413] According to some embodiments thereof, the metastatic lesion is selected from the group consisting of adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mesothelioma, breast cancer, breast carcinoma, triple negative breast cancer, primary brain cancer, ovarian cancer, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma ... The cancer is derived from a cancer selected from the group consisting of: epithelial cell tumor, bladder cancer, bladder urothelial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. pylori-induced MALT non-Hodgkin's lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma. [Brief description of the drawings]

[0414] [Figure 1] Figure 1 shows the expansion of γδ T cells with zoledronate (Zometa) in defined medium containing IL-2, IL-15, and amphotericin B. The fold increase in absolute numbers of γδ T cells is 3,350-fold, 11,060-fold, and 31,666-fold from days 0 to 17, 0 to 22, and 0 to 29, respectively, for donor 20. Similarly, the fold increase in absolute numbers of γδ T cells is 4,633-fold, 12,320-fold, and 32,833-fold from days 0 to 17, 0 to 22, and 0 to 29, respectively, for donor 21.

[0415] In contrast, as described above, classical Vγ9δ2 T cell expansion protocols can only result in a 100-fold increase in total Vγ9δ2 T cells within 14 days, after which increased cell death can cause a decline in the expansion rate. In one embodiment, using the above-mentioned method, the increase in the absolute number of γδ T cells after expansion on day 29 compared to day 0 is about 1000-fold to about 40,000-fold, about 3000-fold to about 35,000-fold, about 5000-fold to about 35,000-fold, about 6000-fold to about 35,000-fold, about 7000-fold to about 35,000-fold, about 8000-fold to about 30,000-fold, about 10,000-fold to about 35,000-fold, about 15,000-fold to about 35,000-fold, about 20,000-fold to about 25,000-fold, about 30,000-fold to about 35,000-fold, about 40,000-fold to about 40,000-fold, about 50,000-fold to about 35,000-fold, about 60,000-fold to about 35,000-fold, about 70,000-fold to about 35,000-fold, about 80,000-fold to about 30,000-fold, about 10,000-fold to about 35,000-fold, about 15,000-fold to about 35 ...5,000-fold to It may be 5,000-fold, about 15,000-fold to about 35,000-fold, about 20,000-fold to about 35,000-fold, about 25,000-fold to about 35,000-fold, about 30,000-fold to about 35,000-fold, more than about 10,000-fold, more than about 15,000-fold, more than about 20,000-fold, more than about 25,000-fold, more than about 30,000-fold, more than about 40,000-fold, or more than about 40,000-fold.

[0416] [Figure 2A] We showed that 34.9% of Vγ9δ2 T cells transduced with αβTCR retrovirus and CD8αβ retrovirus (αβTCR+CD8) stained positively by peptide-MHC-dextramer (TAA / MHC-dex) and anti-CD8 antibody (CD8) compared with Vγ9δ2 T cells without viral transduction (mock), suggesting the generation of Vγ9δ2 T cells expressing both αβTCR and CD8αβ on the cell surface (αβTCR+CD8αβ engineered Vg9d2 T cells).

[0417] The principle of the CD107a degranulation assay is based on the killing of target cells via a granule-dependent pathway that utilizes preformed lytic granules within the cytoplasm of cytotoxic cells. The lipid bilayer surrounding these granules contains lysosome-associated membrane glycoproteins (LAMPs), including CD107a (LAMP-1). Upon recognition of a target cell via the T-cell receptor complex, apoptosis-inducing proteins such as granzymes and perforin are released into the immune synapse, a process called degranulation. This exposes the transmembrane protein CD107a to the cell surface, where it can be stained by specific monoclonal antibodies.

[0418] [Figure 2B] Compared with Vγ9δ2 T cells without viral transduction (mock), 23.1% of Vγ9δ2 T cells transduced with αβTCR retrovirus and CD8αβ retrovirus (αβTCR+CD8) incubated with target cells, e.g., A375 cells, were positively stained by anti-CD107a antibody, suggesting that αβTCR+CD8αβ engineered Vg9d2 T cells are cytolytic by degranulating when exposed to A375 cells. IFN-γ release assay measures cell-mediated responses against antigen-presenting cells, e.g., A375 cells, through the level of IFN-γ released when the TCR of T cells specifically binds to the peptide-MHC complex of antigen-presenting cells on the cell surface.

[0419] [Figure 2C]We show that 19.7% of Vγ9δ2 T cells transduced with αβTCR retrovirus and CD8αβ retrovirus (αβTCR+CD8) were positively stained by anti-IFN-γ antibody compared to Vγ9δ2 T cells without viral transduction (mock), suggesting that αβTCR+CD8αβ engineered Vγ9δ2 T cells are cytolytic by releasing IFN-γ when exposed to A375 cells. Cytolytic activity was assessed by gating apoptosis of non-CD3 T cells, i.e., A375 cells, 24 hours after exposure to A375 cells. Apoptosis was assessed by staining harvested cultures with live / dead dye.

[0420] [Figure 2D] We show that αβTCR+CD8αβ-engineered Vγ9δ2 T cells (αβTCR+CD8) induced apoptosis in 70% of A375 cells compared to Vγ9δ2 T cells without viral transduction (mock), suggesting that αβTCR+CD8αβ-engineered Vγ9δ2 T cells are cytolytic by killing A375 cells. Cytolytic activity was also assessed in real time during the 84-h co-culture assay. Non-transduced and αβTCR+CD8αβ-transduced γδ T cells were co-cultured with target-positive A375-RFP tumor cells at an effector-to-target ratio of 3:1. Lysis of target-positive A375-RFP tumor cells was assessed in real time by an IncuCyte® live cell analysis system (Essen BioScience). Tumor cells alone and non-transduced and αβTCR-transduced αβ T cells were used as negative and positive controls, respectively.

[0421] [Figure 2E]While non-transduced γδ T cells exhibited cytotoxicity due to the inherent anti-tumor properties of γδ T cells, αβTCR+CD8αβ-transduced γδ T cells exhibited similar cytotoxicity compared to αβTCR-transduced αβ T cells, suggesting that αβTCR+CD8αβ-transduced γδ T cells can be engineered to target and kill tumor cells. These data suggest that engineered Vγ9δ2 T cells produced by the disclosed methods are functional and can be used to kill target cells, e.g., cancer cells, in a peptide-specific manner.

[0422] [Diagram 3] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R11P3D3 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-040 and IFN-041 were analyzed.

[0423] [Figure 4]IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R16P1C10 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-046 and IFN-041 were analyzed.

[0424] [Diagram 5] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R16P1E8 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-040 and IFN-041 were analyzed.

[0425] [Figure 6]IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P1A9 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-040 and IFN-041 were analyzed.

[0426] [Figure 7] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P1D7 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-040 and IFN-041 were analyzed.

[0427] [Figure 8]IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P1G3 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-046 and IFN-041 were analyzed.

[0428] [Figure 9] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P2B6 after co-incubation with T2 target cells loaded with PRAME 004 peptide (SEQ ID NO: 310) or similar but unrelated peptides TMED9-001, CAT-001, DDX60L-001, LRRC70-001, PTPLB-001, HDAC5-001, VPS13B-002, ZNF318-001, CCDC51-001, IFIT1-001, or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells from two different healthy donors. CD8+ T cells electroporated with RNA alone or in co-incubation with unloaded target cells were used as controls. Different donors for IFN-040 and IFN-041 were analyzed.

[0429] [Figure 10]IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R11P3D3 following co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 310) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data was obtained on CD8+ T cells derived from two different healthy donors. Different donors, TCRA-0003 and TCRA-0017, were analyzed.

[0430] [Figure 11] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R16P1C10 following co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 310) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data was obtained on CD8+ T cells derived from two different healthy donors. Different donors, TCRA-0003 and TCRA-0017, were analyzed.

[0431] [Figure 12] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R16P1E8 following co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 310) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data was obtained on CD8+ T cells derived from two different healthy donors. Different donors, TCRA-0003 and TCRA-0017, were analyzed.

[0432] [Figure 13] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P1D7 following co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 310) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data was obtained on CD8+ T cells derived from two different healthy donors. Different donors were analyzed: TCRA-0003 and TCRA-0017.

[0433] [Figure 14] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P1G3 following co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 310) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data was obtained on CD8+ T cells derived from two different healthy donors. Different donors were analyzed: TCRA-0003 and TCRA-0017.

[0434] [Figure 15] IFNγ release from CD8+ T cells electroporated with alpha and beta chain RNA of TCR R17P2B6 following co-incubation with T2 target cells loaded with PRAME-004 peptide (SEQ ID NO: 310) at various peptide loading concentrations from 10 μM to 10 pM. IFNγ release data was obtained on CD8+ T cells derived from two different healthy donors. Different donors were analyzed: TCRA-0003 and TCRA-0017.

[0435] [Figure 16] HLA-A*02 / PRAME-004 (sequence number 310) tetramer or HLA-A*02 / NYESO1-001 (sequence number 311) tetramer staining of CD8+ T cells electroporated with alpha and beta chain RNA of TCR R16P1C10, CD8+ T cells electroporated with RNA of 1G4 TCR (sequence numbers 85-96) which specifically binds to the HLA-A*02 / NYESO1-001 (sequence number 311) complex, and mock-electroporated CD8+ T cells used as controls.

[0436] [Figure 17]TCR following co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO: 310) or the similar (identical to PRAME-004 at positions 3, 5, 6, and 7) but unrelated peptides ACPL-001, HSPB3-001, UNC7-001, SCYL2-001, RPS2P8-001, PCNXL3-003, AQP6-001, PCNX-001, AQP6-002, TRGV10-001, NECAP1-001, FBXW2-001, or the control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release from CD8+ T cells lentivirally transduced with R11P3D3 (D103805 and D191451) or non-transduced cells (D103805NT and D191451NT). IFNγ release data was obtained in CD8+ T cells derived from two different healthy donors, D103805 and D191451.

[0437] [Figure 18] IFNγ release from CD8+ T cells lentivirally transduced with TCR R11P3D3 following co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO: 310) or a similar (identical to PRAME-004 at positions 3, 5, 6, and 7) but unrelated peptide or the control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells derived from two different healthy donors, TCRA-0087 and TCRA-0088.

[0438] [Figure 19]IFNγ release from CD8+ T cells lentivirally transduced with TCR R11P3D3 (D103805 and D191451) or non-transduced cells (D103805NT and D191451NT) following co-incubation with different primary cells (HCASMC (coronary artery smooth muscle cells), HTSMC (tracheal smooth muscle cells), HRCEpC (renal cortical epithelial cells), HCM (cardiomyocytes), HCMEC (cardiac microvascular endothelial cells), HSAEpC (small airway epithelial cells), HCF (cardiac fibroblasts)) and iPSC-derived cell types (HN (neurons), iHCM (cardiomyocytes), HH (hepatocytes), HA (astrocytes)). The tumor cell lines UACC-257 (derived from a primary melanoma, high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (derived from peripheral blood of a myeloma patient, very low PRAME-004), and MCF-7 (no PRAME-004) present different amounts of PRAME-004 per cell. T cells alone were used as control. IFNγ release data were obtained with CD8+ T cells derived from two different healthy donors, D103805 and D191451.

[0439] [Figure 20]TCR after co-incubation with different primary cells (NHEK (epidermal keratinocytes), HBEpC (bronchial epithelial cells), HDMEC (dermal microvascular endothelial cells), HCAEC (coronary artery endothelial cells), HAoEC (aortic endothelial cells), HPASMC (pulmonary artery smooth muscle cells), HAoSMC (aortic smooth muscle cells), HPF (pulmonary fibroblasts), SkMC (skeletal muscle cells), HOB (osteoblasts), HCH (chondrocytes), HWP (white preadipocytes), hMSC-BM (mesenchymal stem cells), NHDF (dermal fibroblasts) IFNγ release from CD8+ T cells lentivirally transduced with R11P3D3. The tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (no PRAME-004) present different copy numbers of PRAME-004 per cell. T cells alone were used as control. IFNγ release data were obtained in CD8+ T cells derived from two different healthy donors, TCRA-0084 and TCRA-0085.

[0440] [Figure 21] Enhanced TCR following co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO: 310) or the similar (identical to PRAME-004 at positions 3, 5, 6, and 7) but unrelated peptides ACPL-001, HSPB3-001, UNC7-001, SCYL2-001, RPS2P8-001, PCNXL3-003, AQP6-001, PCNX-001, AQP6-002, TRGV10-001, NECAP1-001, FBXW2-001, or the control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release from CD8+ T cells lentivirally transduced with R11P3D3_KE (D103805 and D191451) or non-transduced cells (D103805NT and D191451NT). IFNγ release data was obtained in CD8+ T cells derived from two different healthy donors, D103805 and D191451.

[0441] [Figure 22]IFNγ release from CD8+ T cells lentivirally transduced with enhanced TCR R11P3D3_KE following co-incubation with T2 target cells loaded with 100 nM PRAME-004 peptide (SEQ ID NO: 310) or a similar (identical to PRAME-004 at positions 3, 5, 6, and 7) but unrelated peptide or control peptide NYESO1-001 (SEQ ID NO: 311). IFNγ release data was obtained with CD8+ T cells derived from two different healthy donors, TCRA-0087 and TCRA-0088.

[0442] [Diagram 23] Enhanced IFNγ release from CD8+ T cells lentivirally transduced with TCR R11P3D3_KE (D103805 and D191451) or non-transduced cells (D103805NT and D191451NT) after co-incubation with different primary cells (HCASMC (coronary artery smooth muscle cells), HTSMC (tracheal smooth muscle cells), HRCEpC (renal cortical epithelial cells), HCM (cardiomyocytes), HCMEC (cardiac microvascular endothelial cells), HSAEpC (small airway epithelial cells), HCF (cardiac fibroblasts)) and iPSC-derived cell types (HN (neurons), iHCM (cardiomyocytes), HH (hepatocytes), HA (astrocytes)). Tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (no PRAME-004) present different copy numbers of PRAME-004 per cell. T cells alone were used as control. IFNγ release data were obtained in CD8+ T cells derived from two different healthy donors, D103805 and D191451.

[0443] [Figure 24]Enhanced TCR after co-incubation with different primary cells (NHEK (epidermal keratinocytes), HBEpC (bronchial epithelial cells), HDMEC (dermal microvascular endothelial cells), HCAEC (coronary artery endothelial cells), HAoEC (aortic endothelial cells), HPASMC (pulmonary artery smooth muscle cells), HAoSMC (aortic smooth muscle cells), HPF (pulmonary fibroblasts), SkMC (skeletal muscle cells), HOB (osteoblasts), HCH (chondrocytes), HWP (white preadipocytes), hMSC-BM (mesenchymal stem cells), NHDF (dermal fibroblasts) IFNγ release from CD8+ T cells lentivirally transduced with R11P3D3_KE. The tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (no PRAME-004) present different copy numbers of PRAME-004 per cell. T cells alone were used as control. IFNγ release data were obtained in CD8+ T cells derived from two different healthy donors, TCRA-0084 and TCRA-0085.

[0444] [Diagram 25] IFNγ release from lentivirally transduced CD8+ T cells with TCR R11P3D3 or enhanced TCR R11P3D3_KE or non-transduced cells after co-incubation with tumor cell lines UACC-257 (high PRAME-004), Hs695T (moderate PRAME-004), U266B1 (very low PRAME-004), and MCF-7 (no PRAME-004), presenting different amounts of PRAME-004 per cell. T cells alone were used as control. IFNγ release of both TCRs correlates with the presentation of PRAME-004, with R11P3D3_KE inducing a higher response compared to R11P3D3.

[0445] [Figure 26]Potency assay evaluating the cytolytic activity of lentiviral-transduced T cells expressing TCR R11P3D3 or enhanced TCR R11P3D3_KE against PRAME-004 positive tumor cells. Cytotoxic response of R11P3D3 and R11P3D3_KE transduced and non-transduced (NT) T cells measured against A-375 (primary skin cancer cell line, low PRAME-004) or U2OS (primary osteosarcoma, moderate PRAME-004) tumor cells. Assays were performed in a 72-hour fluorescence microscopy-based cytotoxicity assay. Results are shown as fold tumor growth over time.

[0446] [Figure 27] Potency assay evaluating the cytolytic activity of lentiviral-transduced T cells expressing TCR R11P3D3 or enhanced TCR R11P3D3_KE against PRAME-004 positive tumor cells. Cytotoxic responses of R11P3D3 and R11P3D3_KE transduced and non-transduced (NT) T cells measured against A-375 (low PRAME-004) or U2OS (moderate PRAME-004) tumor cells. Assays were performed in a 72-hour fluorescence microscopy-based cytotoxicity assay. Results are shown as fold tumor growth over time.

[0447] [Figure 28] Figure 1 shows the results of an LDH release assay with bispecific TCR / mAb diabody construct IA_5 targeting tumor-associated peptide PRAME-004 (SEQ ID NO: 310) presented on HLA-A*02. CD8 positive T cells isolated from healthy donors were co-incubated with cancer cell lines UACC-257, SW982 (primary synovial sarcoma cell line) and U2OS presenting different amounts of PRAME-004:HLA-A*02-1 complexes on their cell surface (approximately 1100, approximately 780 and approximately 240 copies per cell, respectively, as determined by targeted MS analysis) at an effector:target ratio of 5:1 in the presence of increasing concentrations of TCR / mAb diabody molecules. After 48 hours of co-culture, target cell lysis was quantified using an LDH release assay according to the manufacturer's instructions (Promega).

[0448] [Figure 29] Figure 1 shows the results of an LDH release assay with bispecific TCR / mAb diabody constructs IA_5 and IA_6 utilizing a stability / affinity matured TCR and its enhanced version, respectively, against the tumor-associated peptide PRAME-004 (SEQ ID NO: 310) presented on HLA-A*02. CD8 positive T cells isolated from healthy donors were co-incubated with the cancer cell line U2OS, which presents approximately 240 copies of the PRAME-004:HLA-A*02:1 complex per cell, or with unloaded PRAME-004 negative T2 cells (effector:target ratio 5:1) in the presence of increasing concentrations of TCR / mAb diabody molecules. After 48 hours of co-culture, target cell lysis was quantified using an LDH release assay according to the manufacturer's instructions (Promega).

[0449] [Diagram 30] The results of a heat stress stability study with TCR / mAb diabody constructs IA_5 and IA_6 utilizing a stability / affinity matured TCR and its enhanced version, respectively, against the tumor associated peptide PRAME-004 (SEQ ID NO: 310) presented on HLA-A*02 are shown. For this, the protein was formulated in PBS at a concentration of 1 mg / mL and then stored at 40° C. for 2 weeks. Protein integrity and recovery were assessed using HPLC-SEC, which determined the amount of high molecular weight species according to the percentage of peak area eluted before the main peak. The recovery of monomeric protein was calculated by comparing the main peak area of ​​unstressed and stressed samples.

[0450] [Diagram 31]Binding kinetics of bispecific molecules containing different R16P1C10 variants. FAB2G sensor was used for scTCR-Fab format (20 μg / ml 120 sec loading) and AHC sensor for diabody-Fc format (improved variants 10 μg / ml 120 sec loading; stabilized variants LoAff3, CDR6, HiAff1 5 μg / ml 120 sec loading). Analyzed HLA-A*02 / PRAME-004 concentrations are expressed in nM. Graph shows curve of measured data and calculated fitting values.

[0451] [Diagram 32] Lysis of PRAME-positive tumor cell lines induced by bispecific molecules containing CDR6, HiAff1 or LoAff3 TCR variants, respectively, in the presence of CD8+ T cells derived from two healthy donors (HBC-887 and HBC-889). Lysis was determined by quantification of released LDH after 48 h of co-incubation. CDR6 is shown as filled circles, HiAff1 as light grey squares, LoAff3 as dark grey triangles and the control group without bsTCR as open inverted triangles.

[0452] [Diagram 33] Lysis of PRAME-negative tumor cell lines induced by bispecific molecules containing CDR6, HiAff1 or LoAff3 TCR variants, respectively, in the presence of CD8+ T cells derived from two healthy donors (HBC-887 and HBC-889). Lysis was determined by quantification of released LDH after 48 h of co-incubation. CDR6 is shown as filled circles, HiAff1 as light grey squares, LoAff3 as dark grey triangles and the control group without bsTCR as open inverted triangles.

[0453] [Diagram 34]In vivo efficacy. NOG mice bearing Hs695T tumors of approximately 50 mm3 were implanted with human PBMCs and treated iv twice weekly with PBS (group 1), 0.5 mg / kg body weight HiAff1 / anti-CD3 diabody-Fc (group 2) or 0.5 mg / kg anti-HIV / anti-CD3 diabody-Fc (group 3). Tumor volumes were measured with calipers and calculated as length × width 2 / 2.

[0454] [Diagram 35] In vitro cytotoxicity of TCER® molecules against target-positive and target-negative tumor cell lines. PBMCs from healthy HLA-A*02 positive donors were incubated with the target-positive tumor cell line Hs695T (●) or the target-negative but HLA-A*02 positive tumor cell line T98G (glioblastoma cell line (negative control) (◯) at a ratio of 1:10, respectively, in the presence of increasing concentrations of TCER®. After 48 h of co-culture, TCER®-induced cytotoxicity was quantified by measurement of released LDH. Results of experiments evaluating TPP-93 and TPP-79 are shown in the upper and lower panels, respectively.

[0455] [Diagram 36] In vitro cytotoxicity of the TCER® molecule TPP-105 against target-positive and target-negative tumor cell lines. PBMCs from healthy HLA-A*02 positive donors were incubated with the target-positive tumor cell line Hs695T (●) or the target-negative but HLA-A*02 positive tumor cell line T98G (◯) at a ratio of 1:10, respectively, in the presence of increasing concentrations of TPP-105. After 48 h of co-culture, TCER®-induced cytotoxicity was quantified by measurement of released LDH.

[0456] [Figure 37]Summary of cytotoxicity data for TCER® slot III molecules. EC50 values ​​of dose-response curves obtained in LDH release assays were calculated using nonlinear 4-point curve fitting. For each TCER® molecule evaluated, calculated EC50 values ​​are shown against the target-positive tumor cell lines Hs695T (●), U2OS (◯), and the target-negative but HLA-A*02-positive tumor cell line T98G (*). Thus, each symbol represents one assay using PBMCs derived from different HLA-A*02-positive donors. For TPP-871 / T98G, EC50 is estimated since T98G was not recognized by TPP-871.

[0457] [Figure 38] In vitro cytotoxicity of TCER® slot III mutants against T2 cells loaded with different concentrations of target peptide. Cytotoxicity was determined by quantifying LDH released in the supernatant. Human PBMCs were used as effector cells at an E:T ratio of 5:1. Readout was performed after 48 hours.

[0458] [Figure 39] Normal tissue cell safety analysis of selected TCER® slot III variants. TCER®-mediated cytotoxicity against five different normal tissue cell types expressing HLA-A*02 was evaluated in comparison to cytotoxicity directed against PRAME-004 positive Hs695T tumor cells. PBMCs from healthy HLA-A*02+ donors were co-cultured with normal tissue cells or Hs695T tumor cells at a 10:1 ratio (in triplicates) in a 1:1 mixture of respective normal tissue cell medium (4, 10a or 13a) and T cell medium (LDH-AM) or in T cell medium alone. After 48 hours, lysis of normal tissue cells and Hs695T cells was assessed by measuring LDH release (LDH-Glo™ Kit, Promega).

[0459] [Diagram 40] Over-presentation of SEQ ID NO: 310 in various tumor metastases

[0460] This figure shows the over-representation of SEQ ID NO: 310 in various tumor metastases compared to normal tissues. Top: HLA-A * Median MS signal intensities from technical replicate measurements of a single normal sample (grey dots, figure left) and metastatic sample (black dots, figure right) in which sequence ID number 310 on 02 was identified are plotted as points. Boxes represent the median, 25th and 75th percentiles of normalized signal intensity, while whiskers extend to the lowest data point that falls within 1.5 interquartile range (IQR) of the lower quartile and the highest data point that falls within 1.5 IQR of the upper quartile. Bottom: Relative peptide detection frequency in each organ is shown as a spine plot. Numbers below the panels indicate the frequency of peptide detection in each organ (N=762) or metastatic indication (HLA-A * 02 Positive metastasis samples indicate the number of samples in which the peptide was detected out of the total number of samples analyzed (N=102).

[0461] If a peptide was detected in a sample but could not be quantified for technical reasons, the sample is included in this display of detection frequency but does not have a dot at the top of the figure.

[0462] Tissues (from left to right): Normal sample: adipose; adrenal gl; bile duct; bladder; blood cells; bloodvess; bone marrow; brain; breast; esophagus; eye; gall bl; head and neck; heart; intest.la; intest.sm; kidney; liver; lung; lymph node; nerve cent; nerve periph; ovary; pancreas; parathyroid; perit; pituit; placenta; pleura; prostate; skel.mus; skin; spinal cord; spleen; stomach; testis; thymus; thyroid; trachea; ureter; uterus.

[0463] Metastasis samples: BRCA (breast cancer metastasis); CCC (cholangiocarcinoma metastasis); CRC (colorectal cancer metastasis); GC (gastric cancer metastasis); HCC (hepatocellular carcinoma metastasis); HNSCC (head and neck squamous cell carcinoma metastasis); MEL (melanoma metastasis); NHL (non-Hodgkin's lymphoma metastasis); NSCLC adenocarcinoma (non-small cell lung cancer adenocarcinoma metastasis); NSCLC squamous (squamous non-small cell lung cancer metastasis); OC (ovarian cancer metastasis); OSCAR (esophageal cancer metastasis); PACA (pancreatic cancer metastasis); PRCA (prostate cancer metastasis); RCC (renal cell carcinoma metastasis); SARC (sarcoma metastasis); SCLC (small cell lung cancer metastasis); UBC (bladder carcinoma metastasis); UEC (endometrial cancer metastasis).

[0464] [Diagram 41] PRAME expression profile

[0465] Tumor (black dots) and normal (grey dots) samples are grouped according to the organ of origin. Box plots represent the median, 25th and 75th percentiles (boxes) with whiskers extending to the lowest data point that falls within 1.5 IQR of the lower quartile and the highest data point that falls within 1.5 IQR of the upper quartile.

[0466] Tissues (from left to right): Normal sample: adipose; adrenal gl; bile duct; bladder; blood cells; bloodvess; bone marrow; brain; breast; esoph; eye; gall bl; head and neck; heart; intest.la (large intestine); intest.sm (small intestine); kidney; liver; lung; lymph node; nerve periph (peripheral nerve); ovary; pancreas; parathyroid; perit (peritoneum); pituit (pituitary gland); placenta; pleura; prostate; skel.mus (skeletal muscle); skin; spinal cord; spleen; stomach; testis; thymus; thyroid; trachea; ureter; uterus.

[0467] Metastatic samples: AML (acute myeloid leukemia metastases); BRCA (breast cancer metastases); CCC (cholangiocarcinoma metastases); CRC (colorectal cancer metastases); GBC (gallbladder cancer metastases); GC (gastric cancer metastases); HCC (hepatocellular carcinoma metastases); HNSCC (head and neck squamous cell carcinoma metastases); MEL (melanoma metastases); NHL (non-Hodgkin's lymphoma metastases); NSCLC adenocarcinoma (non-small cell lung cancer adenocarcinoma metastases); NSCLC other (NSCLC samples that could not be clearly assigned to NSCLC adenocarcinoma or NSCLC squamous); NSCLC squamous (squamous non-small cell lung cancer metastases); OC (ovarian cancer metastases); OSCAR (esophageal cancer metastases); PACA (pancreatic cancer metastases); PRCA (prostate cancer metastases); RCC (renal cell carcinoma metastases); SCLC (small cell lung cancer metastases); UBC (bladder carcinoma metastases); UEC (endometrial cancer metastases).

[0468] [Diagram 42] Presentation of KRT5-004 (SEQ ID NO: 312) on primary tumors and metastases.

[0469] Comparing HNSCC primary tumors to HNSCC metastases reveals a complete loss of presentation of SEQ ID NO: 312. SEQ ID NO: 312 is detectable in nearly 50% of primary HNSCC tumor samples, but is completely absent from metastatic HNSCC tumor samples analyzed.

[0470] [Diagram 43] Presentation of PRAME-004 (SLLQHLIGL) (SEQ ID NO: 310) on normal tissues, primary tumors, and metastatic cancer tissues.

[0471] Metastasis samples: BRCA (breast cancer metastasis); CCC (cholangiocarcinoma metastasis); CRC (colorectal cancer metastasis); GC (gastric cancer metastasis); HCC (hepatocellular carcinoma metastasis); HNSCC (head and neck squamous cell carcinoma metastasis); MEL (melanoma metastasis); NHL (non-Hodgkin's lymphoma metastasis); NSCLC adenocarcinoma (non-small cell lung cancer adenocarcinoma metastasis); NSCLC squamous (squamous non-small cell lung cancer metastasis); OC (ovarian cancer metastasis); OSCAR (esophageal cancer metastasis); PACA (pancreatic cancer metastasis); PRCA (prostate cancer metastasis); RCC (renal cell carcinoma metastasis); SARC (sarcoma metastasis); SCLC (small cell lung cancer metastasis); UBC (bladder carcinoma metastasis); UEC (endometrial cancer metastasis).

[0472] [Diagram 44] Presentation of PRAME-004 (SLLQHLIGL) (SEQ ID NO: 310) on normal tissues and on cancer tissues combining primary and metastatic cancer tissues.

[0473] [Diagram 45] Presentation of PRAME-004 (SLLQHLIGL) (SEQ ID NO: 310) on normal tissues, primary triple-negative breast cancer (TNBC), and metastases considered to be TNBC.

[0474] [Figure 46] PRAME-004 (SLLQHLIGL) (SEQ ID NO: 310) presentation on normal tissues and TNBC combined with primary TNBC and metastases considered TNBC.

[0475] [Figure 47A] Baseline PRAME expression in tumor biopsies from PRAME-positive patients

[0476] The patients were enrolled in a clinical trial and treated with engineered T cells expressing PRAME-004 specific TCR. Arrows indicate PRAME expression in Patient 1 and Patient 2 with head and neck adenocarcinoma, who had the best overall response in the trial (see Figure 47B).

[0477] [Figure 47B] Preliminary results from clinical trials

[0478] Patients 1 and 2 with head and neck adenocarcinoma treated with engineered T cells expressing PRAME-004-specific TCR in the study experienced 9.7% and 13.1% tumor reduction, respectively, compared to baseline.

[0479] [Figure 48] In vivo efficacy in metastatic pancreatic cancer patient-derived xenograft (PDX) models.

[0480] Approximately 80mm 3 Female NOG mice bearing 1000 mg PAXF1657 (lung metastasis of pancreatic cancer) tumors were implanted with human PBMCs and treated with 5 mL / kg body weight of PBS (groups 1, 2) or 0.25 mg / kg body weight of TCER® TPP-1295 (groups 3, 4) on days 1, 8, and 15. Tumor volumes were measured with calipers and expressed as (length × width). 2 ) / 2, calculated as length > width.

[0481] [Figure 49A] In vivo efficacy in metastatic non-small cell lung carcinoma patient-derived xenograft (PDX) models.

[0482] Approximately 80mm 3 Female NOG mice bearing LXFL1176 (lymph node metastasis of non-small cell large cell lung carcinoma) tumors were implanted with human PBMCs and treated with 5 mL / kg body weight of PBS (groups 1, 2) or 0.25 mg / kg body weight of TCER® TPP-1295 (groups 3, 4) on days 1, 8, 15, and 22. Tumor volumes were measured with calipers and expressed as (length × width). 2 ) / 2, calculated as length > width.

[0483] [Figure 49B] In vivo efficacy in metastatic non-small cell lung adenocarcinoma patient-derived xenograft (PDX) models.

[0484] Approximately 80mm 3Female NOG mice bearing LXFA1125 (ovarian metastasis of non-small cell lung adenocarcinoma) tumors were implanted with human PBMCs and treated with 5 mL / kg body weight of PBS (groups 1, 2) or 0.25 mg / kg body weight of TCER® TPP-1295 (groups 3, 4) on days 1, 8, and 15. Tumor volumes were measured with calipers and expressed as (length × width). 2 ) / 2, calculated as length > width.

[0485] [Figure 50] Prevalence of PRAME-004 in metastatic cancer patients with various tumor indications.

[0486] Tumor positivity will be determined from tumor biopsy samples of metastatic cancer patients using a dedicated targeted PRAME-004 qPCR assay (IMADetect®). Paired PRAME-004 immunopeptidomic mass spectrometry and exon expression data will be used to determine the threshold for PRAME-004 positivity (Fritsche et al., 2018).

[0487] The table in Figure 50 lists the PRAME positivity results in patient-derived metastatic tumor samples. ≧1~<25%=+ ≧25=++ ≧50=+++ ≧75=++++

[0488] The number of patient-derived metastatic tumor samples evaluated is shown.

[0489] PRAME-004 positivity could also be established for the following tumor indications. The number of samples with PRAME positivity is shown: squamous cell anal carcinoma (5), gastric cancer (2), tonsillar carcinoma (1), bronchial carcinoma (2), mucosal melanoma (1), esophageal melanoma (1), anal melanoma (1), rectal carcinoma (1), pancreatic neuroendocrine tumor (1), tongue carcinoma (1), malignant peripheral nerve sheath tumor (1).

[0490] [Figure 51] Prevalence of PRAME-004 in cancer patients with various tumor indications.

[0491] Tumor positivity was determined from tumor biopsy samples of cancer patients analyzed by immunohistochemical staining for PRAME. Tumor samples with a P score ≥ 1 (%) were considered PRAME positive.

[0492] The table in FIG. 51 lists the results of PRAME positivity in patient-derived metastatic tumor samples assessed by immunohistochemistry. ≧1~<25%=+ ≧25=++ ≧50=+++ ≧75=++++

[0493] The number of patient-derived tumor samples evaluated is indicated.

[0494] [Figure 52] Immunohistochemical staining of PRAME-positive cancer

[0495] Exemplary PRAME-positive tissue sections of anal carcinoma (left image), small cell lung cancer (middle image), and uterine carcinosarcoma (right image). EXAMPLES

[0496] While the invention has been illustrated and described in detail in the drawings and the foregoing description, such illustration and description must be considered as illustrative or explanatory and not restrictive, and the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art of practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. In the claims, the word "comprising" does not exclude other elements or steps, and the indefinite articles "a" or "an" do not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0497] All amino acid sequences disclosed herein are presented from N-terminus to C-terminus, and all nucleic acid sequences disclosed herein are presented from 5' to 3'.

[0498] Example 1: T cell receptor R11P3D3

[0499] TCR R11P3D3 (SEQ ID NOs: 12-23 and 120) is a marker for HLA-A * 02 is bound to presentation PRAME-004 (SEQ ID NO: 310) (see FIG. 3).

[0500] R11P3D3 is a novel TCR that is expressed by human primary CD8+ T cells expressing this TCR, which express the PRAME-004 peptide or a different peptide that shows a high degree of sequence similarity to PRAME-004, and expresses the HLA-A * It specifically recognizes PRAME-004, releasing IFNγ upon co-incubation with HLA-A 02+ target cells (Figure 3). The NYESO1-001 (SEQ ID NO: 311) peptide is used as a negative control. TCR R11P3D3 specifically recognizes PRAME-004, releasing IFNγ upon co-incubation with HLA-A 02+ target cells (Figure 3). * EC of 0.74 nM for 02 presentation PRAME-004 (SEQ ID NO: 310) 50 (Figure 10) and binding affinities (K D ).

[0501] Re-expression of R11P3D3 in human primary CD8+ T cells is associated with HLA-A * TCR R11P3D3 did not react with any of the 25 healthy, primary, or iPSC-derived cell types tested (Figures 19 and 20), and furthermore, 67 similar peptides (57 of which were identical to PRAME-004 at positions 3, 5, 6, and 7) but not HLA-A * Cross-reactivity to unrelated peptides was tested in view of 02 (Figs. 3, 17, and 18).

[0502] Example 2: T cell receptor R16P1C10

[0503] TCR R16P1C10 (SEQ ID NOs: 24-35 and 121) is a marker for HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 310) (see FIG. 4).

[0504] R16P1C10 was identified by a novel TCR-dependent transcription factor (TCR) that was expressed by human primary CD8+ T cells loaded with either the PRAME-004 peptide or a different peptide showing high sequence similarity to PRAME-004, respectively, in the HLA-A * Upon coincubation with HLA-A 02+ target cells, it released IFNγ and * TCR R16P1C10 specifically recognizes PRAME-004 (Figure 4) because it binds to the 02 tetramer (Figure 16). The NYESO1-001 (SEQ ID NO: 311) peptide is used as a negative control. TCR R16P1C10 has an EC 50 (Figure 11).

[0505] Example 3: T cell receptor R16P1E8

[0506] TCR R16P1E8 (SEQ ID NOs: 36-47 and 122) is a marker for HLA-A * 02 is bound to presentation PRAME-004 (SEQ ID NO: 310) (see FIG. 5).

[0507] R16P1E8 was expressed by human primary CD8+ T cells re-expressing this TCR, and was expressed by HLA-A loaded with either the PRAME-004 peptide or alanine or a different peptide showing high sequence similarity to PRAME-004. * It specifically recognizes PRAME-004 and releases IFNγ upon coincubation with 02+ target cells (Figure 5). NYESO1-001 (SEQ ID NO: 311) peptide (SLLMWITQV, SEQ ID NO: 311) is used as a negative control. TCR R16P1E8 has an EC 50 (Figure 12).

[0508] Example 4: T cell receptor R17P1A9

[0509] TCR R17P1A9 (SEQ ID NOs: 48-59 and 123) is a marker for HLA-A * R17P1A9 is restricted to HLA-A 02-presenting PRAME-004 (SEQ ID NO: 310) (see FIG. 6). Human primary CD8+ T cells re-expressing this TCR were challenged with either the PRAME-004 peptide or a different peptide showing high sequence similarity to PRAME-004. * It specifically recognizes PRAME-004, thus releasing IFNγ upon co-incubation with 02+ target cells ( FIG. 6 ). The NYESO1-001 (SEQ ID NO: 311) peptide is used as a negative control.

[0510] Example 5: T cell receptor R17P1D7

[0511] TCR R17P1D7 (SEQ ID NOs: 60-71 and 124) is a nucleotide analog of HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 310) (see FIG. 7).

[0512] R17P1D7 was identified in human primary CD8+ T cells re-expressing this TCR by HLA-A loading with either the PRAME-004 peptide or alanine or a different peptide showing high sequence similarity to PRAME-004. * It specifically recognizes PRAME-004, releasing IFNγ upon co-incubation with 02+ target cells (Figure 7). NYESO1-001 (SEQ ID NO: 311) peptide is used as a negative control. TCR R17P1D7 has an EC of approximately 1.83 nM. 50 (Figure 13).

[0513] Example 6: T cell receptor R17P1G3

[0514] TCR R17P1G3 (SEQ ID NOs: 72-83 and 125) is a marker for HLA-A * 02 is constrained against presentation PRAME-004 (SEQ ID NO: 310) (see FIG. 8).

[0515] R17P1G3 was identified as a TCR expressed by human primary CD8+ T cells loaded with either the PRAME-004 peptide or a different peptide showing high sequence similarity to PRAME-004, and was expressed by HLA-A * Specifically recognizes PRAME-004, releasing IFNγ upon co-incubation with 02+ target cells (Figure 8). NYESO1-001 (SEQ ID NO: 311) peptide is used as a negative control. TCR R17P1G3 has an EC of approximately 8.63 nM. 50 (Figure 14).

[0516] Example 7: T cell receptor R17P2B6

[0517] TCR R17P2B6 (SEQ ID NOs: 84-95 and 126) is a marker for HLA-A * 02 is bound to presentation PRAME-004 (SEQ ID NO: 310) (see FIG. 9).

[0518] R17P2B6 is a novel TCR expressed by human primary CD8+ T cells expressing this TCR, which express either the PRAME-004 peptide or alanine or a different peptide showing high sequence similarity to PRAME-004, in response to HLA-A stimulation. * Specifically recognizes PRAME-004, releasing IFNγ upon co-incubation with HLA-A 02+ target cells (Figure 9). NYESO1-001 (SEQ ID NO: 311) peptide is used as a negative control. TCR R17P2B6 specifically recognizes PRAME-004, releasing IFNγ upon co-incubation with HLA-A 02+ target cells (Figure 9). * EC2 of 2.11 nM for PRAME-004 50 (Figure 15) and a binding affinity (K D ).

[0519] Example 8: Enhanced T cell receptor R11P3D3_KE

[0520] A mutant "pairing-enhanced" TCR R11P3D3_KE, in which the naturally occurring α and β variable domains of αW44 / βQ44 are mutated to αK44 / βE44, is introduced as a variant of R11P3D3. The double mutation is selected from the list present in European Patent Application No. 2017 / 081745, which is expressly incorporated herein by reference. It is specifically designed to restore optimal interaction and shape complementarity to the TCR scaffold.

[0521] Compared to the parental TCR R11P3D3, the enhanced TCR R11P3D3_KE shows superior sensitivity in recognition of PRAME-004. Compared to the parental TCR R11P3D3, the enhanced TCR R11P3D3_KE shows stronger responses against PRAME-004-presenting tumor cell lines (Figure 25). Furthermore, the cytolytic activity of R11P3D3_KE is stronger compared to R11P3D3 (Figure 27). The observed improvement in functional response of the enhanced TCR R11P3D3_KE is consistent with that of Example 1 (R11P3D3, K D = 18 to 26 μM) and Example 8 (R11P3D3_KE, K D = 5.3 μM) (Fig. 2A).

[0522] Example 9: Generation of cancer-targeting bispecific TCR / mAb diabody molecules

[0523] To further validate the platform capacity of the bispecific TCR / mAb diabody construct, the TCR-derived variable domains were exchanged with those of a TCR that had been stability / affinity matured by yeast display according to a method previously described (Smith, Harris, and Kranz, 2015). The TCR variable domains were expressed as HLA-A *It specifically binds to the tumor-associated peptide PRAME-004 (SEQ ID NO: 310) bound to UCHT1.02. Additionally, the variable domains of hUCHT1 (Var17), a humanized version of the UCHT1 antibody, were used to generate the PRAME-004-targeting TCR / mAb diabody molecule IA_5 (comprising SEQ ID NO: 131 and SEQ ID NO: 132). This molecule was expressed, purified, and characterized. The purity and integrity of the final preparation was greater than 96% by HPLC-SEC analysis.

[0524] PRAME‐004:HLA‐A * The binding affinity of the bispecific TCR / mAb diabody construct to HLA-A 02 was determined by biolayer interferometry. Measurements were performed on an Octet RED384 system using the settings recommended by the manufacturer. Briefly, purified bispecific TCR / mAb diabody molecules were loaded onto a biosensor (AHC) and then bound to HLA-A 02. * Serial dilutions of 02 / PRAME-004 were analyzed.

[0525] HLA-A on the tumor cell surface as determined by LDH release assay * The activity of this PRAME-004-targeted TCR / mAb diabody construct in inducing tumor cell lysis was evaluated by assessing human CD8+ T cell-mediated lysis of the human cancer cell lines UACC-257, SW982, and U2OS, which present different copy numbers of the PRAME-004 peptide in the context of 02 (UACC-257-approximately 1100, SW982-approximately 780, and U2OS-approximately 240 copies of PRAME-004 per cell as determined by quantitative MS analysis).

[0526] As shown in Figure 28, the PRAME-004-targeted TCR / mAb diabody construct IA_5 induced concentration-dependent lysis of PRAME-004-positive tumor cell lines. Even tumor cells U2OS, which express only 240 copies of PRAME-004 per tumor cell, were efficiently lysed by this TCR / mAb diabody molecule. These results further demonstrate that the TCR / mAb diabody format is applicable as a molecular platform that allows the introduction of variable domains of various TCRs as well as variable domains of various T cell-engaging antibodies.

[0527] Example 10: Engineering of TCR / mAb diabody constructs

[0528] The variable TCR domains utilized in construct IA_5 were further enhanced for affinity to PRAME-004 and TCR stability and used for incorporation into a TCR / mAb diabody scaffold to generate construct IA_6 (comprising SEQ ID NO: 133 and SEQ ID NO: 134). TCR / mAb diabody molecules IA_5 and IA_6 were expressed, purified, and characterized. The purity and integrity of the final preparation was greater than 97% by HPLC-SEC analysis.

[0529] The efficacy of the stability- and affinity-enhanced TCR / mAb diabody mutant IA6 against PRAME-004 was evaluated by comparing the efficacy of the mutant with a low concentration of PRAME-004:HLA-A as target cells. * The 02-presenting tumor cells were evaluated in cytotoxicity experiments with the tumor cell line U2OS or unloaded T2 cells and human CD8 positive T cells as effector cells.

[0530] As shown in Figure 29, we observed increased cytotoxic potency of the TCR / Ab diabody molecule IA_6, which contains the variable domains of the stability / affinity enhanced TCR mutants, compared to the precursor construct IA_5. No cytolysis of target negative T2 cells was detected with both constructs IA_5 and IA_6, confirming PRAME-004-dependent lysis.

[0531] The stability of PRAME-004-specific TCR / mAb diabody mutants IA_5 and IA_6 was analyzed by subjecting the protein constructs to further heat stress at 40°C for up to 2 weeks. HPLC-SEC analysis after heat stress revealed a significant improvement in the stability of mutant IA_6 when compared to the precursor construct IA_5 (see Figure 30). The temperature-induced increase in the high molecular weight species of the construct (i.e. eluting before the main peak) was less pronounced for IA_6 than for IA_5. Consistent with this result, the recovery of intact monomeric protein after heat stress was 87% and 92% for IA_5 and IA_6, respectively.

[0532] These exemplary engineering data demonstrate that highly potent, stable TCR / mAB diabody constructs can be further improved by incorporating stability / affinity enhancing TCR variable domains, resulting in therapeutic proteins with superior characteristics.

[0533] Example 11: Binding Affinity of Mature TCR Mutants

[0534] Mature R16P1C10 TCR mutants expressed as soluble bispecific molecules (Stabilized, improved: scTCR / anti-CD3 Fab format; Stabilized, improved, CDR6, HiAff1 and LoAff3: TCR / anti-CD3 diabody-F c format) to HLA-A * The binding affinity to the HLA-A 02 / PRAME-004 monomer was analyzed by biolayer interferometry. Measurements were performed on an Octet RED384 system using the settings recommended by the manufacturer. Briefly, binding kinetics were measured at 30°C and a shaking speed of 1000 rpm using PBS, 0.05% Tween-20, 0.1% BSA as buffer. The bispecific molecules were loaded onto the biosensor (FAB2G or AHC) and then assayed for binding affinity to the HLA-A 02 / PRAME-004 monomer. * A serial dilution of 02 / PRAME-004 was analyzed. The stabilized R16P1C10 showed an affinity of approximately 1 μM (1.2 μM for scTCR-Fab and 1.2 μM for diabody-F). c930 nM as a ribozyme), and all mutants containing mature CDRs had significantly lower K D To further verify that the affinity of the TCR mutants is only slightly affected by the format, the K values ​​of the affinity matured TCR mutants were determined (Table 5, Figure 31). D Values ​​were calculated using scTCR-Fab or diabody-F c The formats were measured: scTCR-Fab format and diabody-F c The formats are 10 nM and 8.7 nM K D values, further highlighting the good comparability between the different formats (Table 5, Figure 31).

[0535] Example 12: Killing of target-positive and target-negative tumor cell lines

[0536] TCR / anti-CD3 diabody-F c The mature R16P1C10 TCR mutant was expressed as a soluble bispecific molecule using the ELISA format. The cytotoxic activity of the bispecific molecule against PRAME-positive and PRAME-negative tumor cell lines was analyzed by LDH release assay, respectively. * Tumor cell lines presenting PRAME-004 on the cell surface were co-incubated with CD8+ T cells isolated from two healthy donors in the presence of increasing concentrations of the bispecific molecule. After 48 hours, lysis of the target cell lines was measured using CytoTox 96 Non-Radioactive Cytotoxicity Assay Kits (Promega). As shown in Figure 32, highly efficient induction of lysis was detected in all PRAME-positive cell lines tested, clearly dependent on the concentration of the bispecific molecule. HLA-A * In a similar experiment utilizing a cell line expressing PRAME-002 but not presenting detectable levels of the peptide PRAME-004, FIG. 33 shows that there was no or only limited target lysis induced by the bispecific molecule, suggesting TCR domain specificity.

[0537] Example 13: In vivo efficacy

[0538] TCR / anti-CD3 diabody-F c The mature R16P1C10 TCR mutant HiAff1 and the HIV-specific high affinity control TCR were expressed as soluble bispecific molecules using the ELISA format. A pharmacodynamic study designed to test the ability of the bispecific TCR molecule to recruit and induce activity of human cytotoxic CD3+ T cells against the PRAME-positive tumor cell line Hs695T in the hyperimmunodeficient NOG mouse strain was performed. The NOG mouse strain was host to subcutaneously injected human tumor cell line Hs695T and intravenously injected human peripheral blood mononuclear cell xenografts. Human peripheral blood mononuclear cells (5 × 10 6 cells / mouse, intravenous injection) in individual tumors with a volume of 50 mm 3 Immunotherapy was performed within 24 hours when the mice reached a 100% naïve state. Treatment was initiated within 1 hour after transplantation of human blood cells. Four to five female mice per group received an intravenous bolus injection (5 mL / kg body weight, twice weekly dosing up to a maximum of 7 doses, starting 1 day after randomization) into the tail vein. The injection dose of PRAME-targeted bispecific TCR molecule was 0.5 mg / kg body weight per injection (group 2), PBS was used in the vehicle control group (group 1), and HIV-targeted control TCR bispecific molecule (0.5 mg / kg body weight per injection) was used in the negative control substance group (group 3). At the indicated time points, the tumor tissue was measured with a vernier caliper and measured length × width. 2 The mean tumor volume for each group was calculated based on the individual tumor volumes calculated as 1 / 2. Treatment with PRAME-targeted bispecific TCR molecules increased tumor volumes from basal levels (start of randomization) to 65-409 mm at day 23. 3 The increase in tumor volume from baseline levels observed in the vehicle control group was 69 to 1266 mm 3 and an increase of 66 to 1686 mm from the basal level observed in the negative control group. 3 The results showed that IFN-γ inhibited tumor growth as suggested by the decrease compared to the increase in IFN-γ (Figure 34).

[0539] Example 14: Production and characterization of soluble scTCR-Fab molecules

[0540] PRAME-004: The variable domain of a TCR that binds to an MHC complex may be selected from: V A comprises or consists of the amino acid sequence of SEQ ID NO: 305; B comprises or consists of the amino acid sequence of SEQ ID NO: 306; V A comprises or consists of the amino acid sequence of SEQ ID NO: 305; B comprises or consists of the amino acid sequence of SEQ ID NO: 307; V A comprises or consists of the amino acid sequence of SEQ ID NO: 305; B comprises or consists of the amino acid sequence of SEQ ID NO: 308; V A comprises or consists of the amino acid sequence of SEQ ID NO: 309; B comprises or consists of the amino acid sequence of SEQ ID NO: 306; V A comprises or consists of the amino acid sequence of SEQ ID NO: 309; B comprises or consists of the amino acid sequence of SEQ ID NO: 307; or V A comprises or consists of the amino acid sequence of SEQ ID NO: 309; B comprises or consists of the amino acid sequence of SEQ ID NO:306.

[0541] V A comprises or consists of the amino acid sequence of SEQ ID NO: 305; B Most preferably, V comprises or consists of the amino acid sequence of SEQ ID NO: 306. For targeting the TCR-CD3 complex, V is derived from the CD3-specific humanized antibody hUCHT1 (Zhu and Carter, 1995). H and V L domain, in particular the V domain derived from the UCHT1 variants UCHT1-V17, UCHT1-V17opt, UCHT1-V21, or UCHT1-V23, preferably derived from UCHT1-V17. H and V Ldomain, more preferably comprising or consisting of SEQ ID NO: 193 H and V comprising or consisting of SEQ ID NO: 192 L Alternatively, V from antibody BMA031, which targets the TCRα / β CD3 complex, may be used. H and V L domain, as well as humanized versions thereof (Shearman et al., 1991), in particular the V domain derived from the BMA031 variants BMA031(V36) or BMA031(V10), preferably derived from BMA031(V36). H and V L More preferably, the V domain comprises or consists of SEQ ID NO: 196, or SEQ ID NO: 198, (A02) or SEQ ID NO: 199, (D01), or SEQ ID NO: 200, (A02_H90Y) or SEQ ID NO: 201, (D01_H90Y). H and V comprising or consisting of SEQ ID NO: 197 L Alternatively, the V-antibody derived from the CD3ε-specific antibody H2C (described in EP 2155783) may be used. H and V L V comprising or consisting of the domain, in particular SEQ ID NO: 202, or SEQ ID NO: 207, (N100D) or SEQ ID NO: 209, (N100E) or SEQ ID NO: 211, (S101A) H and V comprising or consisting of SEQ ID NO: 204 L You may also use.

[0542] Example 15: Identification and quantification of tumor-associated peptides displayed on the cell surface

[0543] Tissue samples Patient tissues were obtained from BiolVT (Detroit, MI, USA and Royston, Herts, UK); BioOptions 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); University of Bonn (Bonn, Germany); Asklepios Clinic St. Georg (Hamburg, Germany); Val d'Hebron University Hospital (Barcelona, ​​Spain); Center for cancer immune therapy (CCIT), Herlev Hospital (Herlev, Denmark); Leiden University Medical Center (LUMC) (Leiden, The Netherlands); Istituto Nazionale Tumori “Pascale”, Molecular Biology and Viral Oncology Unit (Naples, Italy); Stanford Cancer Center (Palo Alto, CA, USA); Geneva University Hospitals (Geneva, Switzerland); Heidelberg University Hospital (Heidelberg, Germany); University Hospital Munich (Munich, Germany); University Hospital Tübingen (Tübingen, Germany).

[0544] All patients gave written informed consent before surgery or dissection. Tissues were shock frozen immediately after resection and stored at or below -70°C until isolation of TUMAPs.

[0545] Isolation of HLA peptides from tissue samples HLA-A was assayed according to a slightly modified protocol (Falk et al., 1991; Seeger et al., 1999). * HLA peptide pools from shock-frozen tissue samples were obtained by immunoprecipitation from solid tissues using 02-specific antibody BB7.2, HLA-A, -B, -C-specific antibody w6 / 32, HLA-DR-specific antibody L243, and HLA-DP-specific antibody B7 / 21, CNBr-activated Sepharose, acid treatment, and ultrafiltration.

[0546] mass spectrometry The resulting HLA peptide pools were separated according to their hydrophobicity by reversed-phase chromatography (nanoAcquity UPLC system, Waters) and the eluted peptides were analyzed on an LTQ Velos and Fusion hybrid mass spectrometer (Thermo) equipped with an ESI source. The peptide pool was directly loaded onto an analytical fused silica microcapillary column (75 μm i.d. × 250 mm) packed with 1.7 μm C18 reversed-phase material (Waters) applying a flow rate of 400 nL / min. Peptides were subsequently separated using a two-step 180 min binary gradient from 10% to 33% B at a flow rate of 300 nL / min. The gradient consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). A gold-coated glass capillary (PicoTip, New Objective) was used for introduction into the nanoESI source. The LTQ-Orbitrap mass spectrometer was operated in data-dependent mode using the TOP5 strategy. Briefly, a scan cycle started with a full scan at high mass accuracy in the orbitrap (R = 30000), followed by an MS / MS scan of the five most abundant precursor ions in the orbitrap (R = 7500) with dynamic exclusion of previously selected ions. Tandem mass spectra were interpreted by SEQUEST with a fixed false discovery rate (q ≤ 0.05) and additional manual controls. In cases where the identified peptide sequence was uncertain, it was further verified by comparison of the generated native peptide fragmentation patterns with those of synthetic sequence-identical reference peptides.

[0547] Label-free relative LC-MS quantification was performed by ion counting, i.e., by extraction and analysis of LC-MS features (Mueller et al., 2007). This method assumes that the LC-MS signal area of ​​a peptide correlates with its abundance in the sample. The extracted features were further processed by charge state deconvolution and retention time alignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MS features were cross-referenced with sequence identification results to combine quantitative data of the different samples with tissue peptide presentation profiles. Quantitative data were normalized in a two-step manner according to central tendency to account for variability within technical and biological replicates. Thus, each identified peptide can be associated with quantitative data, allowing relative quantification across samples and tissues. In addition, all quantitative data obtained for peptide candidates were manually inspected to ensure data integrity and to verify the accuracy of the automated analysis. Presentation profiles showing the average sample presentation and replicate variability were calculated. The profiles juxtapose BRCA (breast cancer metastases); CCC (cholangiocarcinoma metastases); CRC (colorectal cancer metastases); GC (gastric cancer metastases); HCC (hepatocellular carcinoma metastases); HNSCC (head and neck squamous cell carcinoma metastases); MEL (melanoma metastases); NHL (non-Hodgkin's lymphoma metastases); NSCLC adenocarcinoma (non-small cell lung cancer adenocarcinoma metastases); NSCLC squamous (squamous non-small cell lung cancer metastases); OC (ovarian cancer metastases); OSCAR (esophageal cancer metastases); PACA (pancreatic cancer metastases); PRCA (prostate cancer metastases); RCC (renal cell carcinoma metastases); SCLC (small cell lung cancer metastases); UBC (bladder carcinoma metastases); and UEC (endometrial cancer metastases) samples to a baseline of normal tissue samples. The presentation profile of SEQ ID NO: 310 is shown in Figure 40. The plot shows only the identification of the peptide as dots generated on the respective HLA allotype positive tissue samples treated with HLA specific antibodies.

[0548] The peptide presentation on various indications for SEQ ID NO: 310 is shown in Table 6. This table lists all indications in which each peptide was identified at least once, independent of the HLA typing of the sample or the antibody used to treat said sample.

[0549] Example 16: Identification and absolute quantification of tumor-associated peptides displayed on the cell surface

[0550] The generation of conjugates such as antibodies and / or TCRs is a laborious process that can only be performed for a few selected targets. In the case of tumor-associated and specific peptides, the selection criteria include, but are not limited to, the exclusivity of presentation and the density of peptides presented on the cell surface. In addition to the isolation and relative quantification of peptides described in the examples, the inventors analyzed the absolute peptide copy number per cell as described in WO2016 / 107740. Quantification of TUMAP copies per cell in solid tumor samples requires absolute quantification of isolated TUMAPs, the efficiency of the TUMAP isolation process, and the cell number of the tissue sample to be analyzed.

[0551] Peptide quantification by nanoLC-MS / MS For accurate quantification of peptides by mass spectrometry, two different isotopically labeled peptide variants (including one or two isotopically labeled amino acids during TUMAP synthesis) were used to generate a calibration curve for SEQ ID NO:310 / PRAME-004. These isotopically labeled variants differ from the tumor-associated peptide only in their mass but no other physicochemical properties (Anderson et al., 2012). For peptide calibration curves, a series of nanoLC-MS / MS measurements were performed to determine the MS / MS signal ratios of titrated (single-isotopically labeled peptide) versus stable (dual-isotopically labeled peptide) isotopically labeled peptides.

[0552] A dual-isotopically labeled peptide, also called an internal standard, was further spiked into each MS sample, and all MS signals were normalized to the MS signal of the internal standard to level out potential technical errors between MS experiments.

[0553] Calibration curves were prepared in at least three different matrices, i.e., HLA peptide eluates from natural samples as well as routine MS samples, and each preparation was measured in duplicate MS runs. For evaluation, MS signals were normalized to the signal of the internal standard and the calibration curves were calculated by logistic regression.

[0554] For quantification of tumor-associated peptides from tissue samples, the respective samples were also spiked with an internal standard, and the MS signal was normalized to the internal standard and quantified using a peptide calibration curve.

[0555] Efficiency of peptide-MHC isolation As with any protein purification process, the isolation of proteins from tissue samples involves some loss of the protein of interest. To determine the efficiency of TUMAP isolation, peptide-MHC complexes were generated for all TUMAPs selected for absolute quantification. To be able to distinguish the spiked ones from the native peptide-MHC complexes, monoisotopically labeled versions of TUMAPs were used, i.e. one isotope-labeled amino acid was included in the TUMAP synthesis. These complexes were spiked into freshly prepared tissue lysates, i.e. at the earliest possible time point in the TUMAP isolation procedure, and then captured like native peptide-MHC complexes in the subsequent affinity purification. Thus, by measuring the recovery of the monolabeled TUMAPs, conclusions can be made regarding the isolation efficiency of the individual native TUMAPs.

[0556] The efficiency of isolation was analyzed in a small sample set and was comparable between these tissue samples. In contrast, the isolation efficiency differs between individual peptides. This suggests that the isolation efficiency, although determined only in a limited number of tissue samples, can be extrapolated to any other tissue preparation. However, it is necessary to analyze each TUMAP individually, since the isolation efficiency cannot be extrapolated from one peptide to another.

[0557] Determination of cell number in solid frozen tissue

[0558] To determine the cell number of tissue samples subjected to absolute peptide quantification, we applied DNA content analysis. This method is applicable to a wide range of samples of various origins, and most importantly, to frozen samples (Alcoser et al., 2011; Forsey and Chaudhuri, 2009; Silva et al., 2013). During the peptide isolation protocol, tissue samples are processed into homogenous lysates from which small lysate aliquots are taken. The aliquots are divided into three parts from which DNA is isolated (QiaAmp DNA Mini Kit, Qiagen, Hilden, Germany). Total DNA content from each DNA isolate is quantified in at least two replicates using a fluorescence-based DNA quantification assay (Qubit dsDNA HS Assay Kit, Life Technologies, Darmstadt, Germany).

[0559] To calculate cell number, a DNA standard curve from aliquots of isolated healthy blood cells from multiple donors at a series of defined cell numbers is generated. The standard curve is used to calculate the total cell content from the total DNA content from each DNA isolation. The average total cell number of tissue samples used for peptide isolation is then extrapolated given the known volumes of the lysate aliquots and the total lysate volume.

[0560] Peptide copy number per cell

[0561] Using the data from the previous experiment, we calculated the TUMAP copy number per cell by dividing the total peptide amount by the total cell number of the sample, then dividing by the isolation efficiency. The copy cell number of SEQ ID NO: 310 is shown in Table 7. [Table 7]

[0562] Absolute copy count:

[0563] This table lists the results of absolute peptide quantification in metastatic samples. ≧1~<25=+ ≧25=++ ≧50=+++ ≧75=++++

[0564] The number of samples for which evaluable high-quality MS data were available is indicated.

[0565] More elaborate disclosures of methods for absolute quantification of peptides are disclosed in International Patent Application Publication No. 2016107740A1 and U.S. Patent Application No. 14 / 969,423, the contents of both of which are incorporated herein by reference.

[0566] Example 17: Expression profiling of genes encoding peptides of the invention

[0567] Over- or specific presentation of a peptide on tumor cells compared to normal cells is sufficient for its utility in immunotherapy, and some peptides are tumor-specific even though their source proteins also occur in normal tissues. Nevertheless, mRNA expression profiling provides an additional layer of safety in the selection of peptide targets for immunotherapy. In particular, for therapeutic options with high safety risks, such as affinity matured TCRs, ideal target peptides are derived from proteins that are unique to tumors and not found on normal tissues.

[0568] RNA sources and preparations After obtaining written informed consent from each patient, surgically removed tissue specimens were provided as described above (see Example 1). Tumor tissue specimens were flash frozen immediately after surgery and then homogenized with a mortar and pestle under liquid nitrogen. Total RNA was prepared from these samples using TRI Reagent (Ambion, Darmstadt, Germany) followed by cleanup with RNeasy (QIAGEN, Hilden, Germany), both methods being performed according to the manufacturer's protocols.

[0569] Total RNA from healthy human tissues for RNASeq experiments was obtained from Asterand (Detroit, MI, USA and Royston, Herts, UK); BioOptions Inc. (Brea, CA, USA); Geneticist Inc. (Glendale, CA, USA); ProteoGenex Inc. (Culver City, CA, USA); and Tissue Solutions Ltd (Glasgow, UK).

[0570] Total RNA from tumor tissues for RNASeq experiments was obtained from Asterand (Detroit, MI, USA and 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); and University Hospital Heidelberg (Heidelberg, Germany).

[0571] The quality and quantity of all RNA samples were assessed using the RNA6000Pico LabChip kit (Agilent) on an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany).

[0572] RNAseq experiments

[0573] Gene expression analysis of tumor and normal tissue RNA samples was performed by next generation sequencing (RNAseq) by GENEWIZ Germany GmbH (Leipzig, Germany). Briefly, sequencing libraries were prepared from total RNA using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina, which includes mRNA selection, RNA fragmentation, cDNA conversion and addition of sequencing adapters, according to the manufacturer's instructions (New England BioLabs, Ipswich, Massachusetts, USA). For sequencing, libraries were multiplexed and loaded onto an Illumina NovaSeq 6000 sequencer (Illumina Inc., San Diego, CA, USA) according to the manufacturer's instructions to generate a minimum of 80 million 150 bp paired-end raw reads per sample. After quality control, adapter trimming and mapping to the reference genome, RNA reads supporting the peptides were counted and sequenced for the following subtypes: AML (acute myeloid leukemia metastases); BRCA (breast cancer metastases); CCC (cholangiocarcinoma metastases); CRC (colorectal cancer metastases); GBC (gallbladder cancer metastases); GC (gastric cancer metastases); HCC (hepatocellular carcinoma metastases); HNSCC (head and neck squamous cell carcinoma metastases); MEL (melanoma metastases); NHL (non-Hodgkin's lymphoma metastases); NSCLC Adenocarcinoma (non-small cell lung cancer adenocarcinoma metastases); NSCLC Other ( Exemplary expression profiles of peptides of the present invention that are highly overexpressed or exclusively expressed in NSCLC samples that could not be clearly assigned to NSCLC gland or NSCLC squamous metastasis; NSCLC squamous (squamous non-small cell lung cancer metastasis); OC (ovarian cancer metastasis); OSCAR (esophageal cancer metastasis); PACA (pancreatic cancer metastasis); PRCA (prostate cancer metastasis); RCC (renal cell carcinoma metastasis); SCLC (small cell lung cancer metastasis); UBC (bladder carcinoma metastasis); and UEC (endometrial cancer metastasis) are shown (Figure 41).

[0574] Example 18: In vivo efficacy in metastatic patient-derived xenograft models

[0575] With TCER® TPP-1295, we performed a pharmacodynamic study designed to test the ability of bispecific TCR molecules to recruit and induce activity of human cytotoxic CD3+ T cells against PRAME-positive tumors. Most importantly, these metastatic / metastatic tumors are patient-derived xenografts (PDX), providing the opportunity for efficacy testing in a preclinical model of tumor biology that is as close as possible to the in vivo situation in patients. The main genetic and histological characteristics of the patient's tumor remain unchanged over a period of time (murine passage), making PDX models superior, for example, in terms of predictive value of patient response, compared to cell line-derived xenografts (CDX) (Hidalgo et al., 2014; Johnson et al., 2001; Gillet et al., 2011).

[0576] Pharmacodynamic evaluation of TCER® TPP-1295 was performed on three different metastatic PDX models in the highly immunodeficient NOG mouse strain: PAXF1657 (lung metastasis of pancreatic cancer), LXFL1176 (lymph node metastasis of non-small cell large cell lung carcinoma), and LXFA1125 (ovarian metastasis of non-small cell lung adenocarcinoma). Human tumor pieces were implanted subcutaneously (and unilaterally) in the right dorsal flank and tumor volumes were measured with calipers and (length × width). 2 ) / 2. Individual tumor volumes were approximately 80 mm 3 Once the mice reached 100 mg / kg, they were randomized and administered human peripheral blood mononuclear cells (PBMCs) (1 × 10 7PDX models were humanized with 100% T cells / mouse, intravenously. To address donor variability, PBMCs from two different healthy randomized donors were used (PBMC donor 1: groups 1 and 3; PBMC donor 2: groups 2 and 4). Treatment was initiated within 24 hours of randomization, and three female mice per group (1-4 for each PDX model) were dosed weekly with an intravenous bolus injection (5 mL / kg body weight) into the tail vein (PAXF1657: days 1, 8, and 15; LXFL1176: days 1, 8, 15, and 22; LXFA1125: days 1, 8, and 15). The injection dose of PRAME-targeted bispecific TCER® molecule TPP-1295 molecule was 0.25 mg / kg body weight per injection (groups 3 and 4), and PBS was used as a control vehicle (groups 1 and 2). Individual tumor volumes were measured twice weekly (at the indicated time points, see Figures 48, 49A, and 49B). Based on individual tumor volumes, mean tumor volumes were calculated for all groups as well as treatment groups (control vehicle [PBS]: Groups 1 and 2; TCER® TPP-1295 0.25 mg / kg body weight: Groups 3 and 4). Treatment with PRAME-targeted bispecific TCER® molecules inhibited tumor growth, as suggested by the reduced increase in tumor volume from basal levels (start of randomization). In metastatic pancreatic cancer PDX model PAXF1657 treated with 0.25 mg / kg TCER® TPP-1295 (Groups 3 and 4), the mean basal tumor volume was 80 mm 2 , which was less than that observed in the vehicle control (PBS; Groups 1 and 2). 3 (Base level on day 0) to 1705mm 3 (day 20) compared with an increase of 81 mm 3 (Day 0) to 873mm 3 In metastatic non-small cell large cell lung carcinoma PDX model LXFL1176 treated with 0.25 mg / kg TCER® TPP-1295 (Groups 3 and 4), the mean basal tumor volume was 86 mm 2 compared to the 86 mm 2 observed in the vehicle control (PBS; Groups 1 and 2). 3 (Day 0) to 1065mm 3 (Day 30) growth rate was 83 mm. 3 (Day 0) to 122mm 3In metastatic non-small cell lung adenocarcinoma PDX model LXFA1125 treated with 0.25 mg / kg TCER® TPP-1295 (Groups 3 and 4), the mean basal tumor volume decreased from 144 mm observed in the vehicle control (PBS; Groups 1 and 2). 3 (Day 0) to 707mm 3 (Day 34) growth rate of 145 mm 3 (Day 0) to 261mm 3 (Day 34) (Figure 49B).

[0577] These data plausibly suggest that treatment of PRAME-positive metastases or metastatic lesions with the pharmaceutical agents disclosed herein is a promising option.

[0578] Example 19: Immunohistochemical (IHC) staining of PRAME

[0579] Staining was performed with an automated IHC staining system (Leica Bond Max) according to the manufacturer's instructions. FFPE tissue samples were stained using the following protocol. - Bake at 60℃ Wax removal, 3 times at 60°C Alcohol rinse, 3 times Bond cleaning, 3 times for 5 minutes each Epitope retrieval, 100°C for 20 min Bond wash, 35°C, 3 min, 4 times Peroxide block, 5 minutes at a time Bond cleaning, 3 times for 5 minutes each PRAME staining (PRAME clone EPR20330, Abcam), 15 min Bond cleaning, 3 times Secondary (poly-HRP anti-mouse), 8 min Bond cleaning, 3 times for 2 minutes Polymer (poly-HRP anti-rabbit IgG), 8 min Bond cleaning, 2 times for 2 minutes Deionized water, 1 time ·DAB staining, 10 minutes Deionized water, 3 times Hemotoxylin, 8 minutes Deionized water, 1 time Bond cleaning, 1 time Deionized water, 1 time Dehydration of slides and coverslipping with cytoseal

[0580] The results are shown in Figures 51 and 52.

[0581] Example 20 - TCER® Mutant (Sl...

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL) is bound to the peptide, For use in the treatment of patients diagnosed with metastasis or metastatic lesions (i), suffering from (ii), or at risk of developing (iii), T cell receptor or a functional fragment thereof.

2. The T cell receptor according to claim 1, provided as a soluble molecule.

3. It binds to a peptide consisting of the amino acid sequence (SLLQHLIGL) of SEQ ID NO: 310, Nucleic acids encoding T cell receptors or functional fragments thereof for use in the treatment of patients diagnosed with metastatic or metastatic lesions (i), suffering from (ii), or at risk of developing (iii).

4. A peptide consisting of the amino acid sequence of SEQ ID NO: 310 (SLLQHLIGL) is bound to the peptide, Recombinant T lymphocytes expressing T cell receptors for use in the treatment of patients diagnosed with metastatic or metastatic lesions (i), suffering from (ii), or at risk of developing (iii).

5. The aforementioned T cell receptor is (1) CDR1α containing the amino acid sequence of SEQ ID NO: 12, CDR2α containing the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 120, CDR3α containing the amino acid sequence of SEQ ID NO: 14, CDR1β containing the amino acid sequence of SEQ ID NO: 18, CDR2β containing the amino acid sequence of SEQ ID NO: 19, and CDR3β containing the amino acid sequence of SEQ ID NO: 20, or (2) CDR1α containing the amino acid sequence of SEQ ID NO: 24, CDR2α containing the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 121, CDR3α containing the amino acid sequence of SEQ ID NO: 26, CDR1β containing the amino acid sequence of SEQ ID NO: 30, CDR2β containing the amino acid sequence of SEQ ID NO: 31, and CDR3β containing the amino acid sequence of SEQ ID NO: 32, or (3) CDR1α containing the amino acid sequence of SEQ ID NO: 36, CDR2α containing the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 122, CDR3α containing the amino acid sequence of SEQ ID NO: 38, CDR1β containing the amino acid sequence of SEQ ID NO: 42, CDR2β containing the amino acid sequence of SEQ ID NO: 43, and CDR3β containing the amino acid sequence of SEQ ID NO: 44, or (4) CDR1α containing the amino acid sequence of SEQ ID NO: 48, CDR2α containing the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 123, CDR3α containing the amino acid sequence of SEQ ID NO: 50, CDR1β containing the amino acid sequence of SEQ ID NO: 54, CDR2β containing the amino acid sequence of SEQ ID NO: 55, and CDR3β containing the amino acid sequence of SEQ ID NO: 56 (5) CDR1α containing the amino acid sequence of SEQ ID NO: 60, CDR2α containing the amino acid sequence of SEQ ID NO: 61 or SEQ ID NO: 124, CDR3α containing the amino acid sequence of SEQ ID NO: 62, CDR1β containing the amino acid sequence of SEQ ID NO: 66, CDR2β containing the amino acid sequence of SEQ ID NO: 67, and CDR3β containing the amino acid sequence of SEQ ID NO:

68. (6) CDR1α containing the amino acid sequence of SEQ ID NO: 72, CDR2α containing the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 125, CDR3α containing the amino acid sequence of SEQ ID NO: 74, CDR1β containing the amino acid sequence of SEQ ID NO: 78, CDR2β containing the amino acid sequence of SEQ ID NO: 79, and CDR3β containing the amino acid sequence of SEQ ID NO: 80 (7) CDR1α containing the amino acid sequence of SEQ ID NO: 84, CDR2α containing the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 126, CDR3α containing the amino acid sequence of SEQ ID NO: 86, CDR1β containing the amino acid sequence of SEQ ID NO: 90, CDR2β containing the amino acid sequence of SEQ ID NO: 91, and CDR3β containing the amino acid sequence of SEQ ID NO: 92 The T cell receptor is HLA-A ＊ It can bind to a peptide consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO: 310) in a complex with 02. Recombinant T lymphocytes according to claim 4.

6. The aforementioned T cell receptor is (1) an α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 15, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 21, or (2) An α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 27, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 33, or (3) an α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 39, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45, or (4) An α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 51, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 57, or (5) An α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 63, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 69, or (6) An α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 75, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 81, or (7) An α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 87, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 93, or (8) An α-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 111, and a β-chain variable domain containing an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 117 The T cell receptor is HLA-A ＊ It can bind to a peptide consisting of the amino acid sequence of SLLQHLIGL (SEQ ID NO: 310) in a complex with 02. Recombinant T lymphocytes according to claim 4.

7. Selectively recognize cells that present a peptide consisting of the amino acid sequence (SLLQHLIGL) of SEQ ID NO: 310, For use in the treatment of patients diagnosed with metastasis or metastatic lesions (i), suffering from (ii), or at risk of developing (iii), These are activated T lymphocytes, The activated T lymphocytes are produced by an in vitro method comprising the step of contacting T cells with an antigen-loaded human class I MHC molecule expressed on the surface of an appropriate antigen-presenting cell or an artificial construct mimicking an antigen-presenting cell for a period of time sufficient to activate the T lymphocytes in an antigen-specific manner, wherein the antigen is a peptide consisting of the amino acid sequence (SLLQHLIGL) of SEQ ID NO:

310. Activated T lymphocytes.

8. For use in the treatment of patients who have been diagnosed with metastasis or metastatic lesion (i), who have (ii), or who are at risk of developing (iii), - The T cell receptor or fragment thereof according to claim 1 or 2, - The nucleic acid described in claim 3, • Recombinant T lymphocytes according to any one of claims 4 to 6, and / or - Activated T lymphocytes according to claim 7 A pharmaceutically acceptable carrier comprising at least one active agent selected from the group consisting of at least one of the following, Pharmaceutical composition.

9. The metastasis or metastatic lesion is at least one selected from the group consisting of at least one of ACC metastasis, BLCA metastasis, BRCA metastasis, TNBC metastasis, CRC metastasis, HNSCC metastasis, NHAC metastasis, MEL metastasis, SKCM metastasis, UVM metastasis, LC metastasis, NSCLC metastasis, NSCLC glandular metastasis, NSCLC squamous metastasis, NSCLC other metastasis, SCLC metastasis, CHOL metastasis, ESCA metastasis, CESC metastasis, OC metastasis, OV metastasis, LIHC metastasis, RCC metastasis, KIRC metastasis, KIRP metastasis, SARC metastasis, FS metastasis, LPS metastasis, MPNST metastasis, SS metastasis, STAD metastasis, TGCT metastasis, THYM metastasis, UCS metastasis, and / or UEC metastasis, the T cell receptor or fragment thereof according to claim 1 or 2, the nucleic acid or expression vector according to claim 3, the recombinant T lymphocyte according to any one of claims 4 to 6, or the activated T lymphocyte according to claim 7.

10. The metastatic or metastatic lesion is adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mucosal melanoma, esophageal melanoma, anal melanoma, mesothelioma, breast cancer, breast carcinoma, triple-negative breast cancer, primary brain cancer, ovarian cancer, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. Cancers selected from the group consisting of pylori-induced MALT non-Hodgkin lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma, A T cell receptor or fragment thereof according to claim 1 or 2, a nucleic acid or expression vector according to claim 3, a recombinant T lymphocyte according to any one of claims 4 to 6, or an activated T lymphocyte according to claim 7.

11. The patient is further administered at least one adjuvant selected from the group consisting of anti-CD40 antibody, imiquimod, reciquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, poly(lactidocoglycolide) (PLG), virosoms, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-21 (IL-21), and interleukin-23 (IL-23). A T cell receptor or fragment thereof according to claim 1 or 2, a nucleic acid or expression vector according to claim 3, a recombinant T lymphocyte according to any one of claims 4 to 6, or an activated T lymphocyte according to claim 7.

12. Recombinant T lymphocytes according to any one of claims 4 to 6, or activated T lymphocytes according to claim 7, which are autologous or allogeneic to the patient.

13. The pharmaceutical composition according to claim 8, which corresponds to one or more of the following (A) to (D). (A) The metastasis or metastatic lesion is at least one selected from the group consisting of at least one of the following: ACC metastasis, BLCA metastasis, BRCA metastasis, TNBC metastasis, CRC metastasis, HNSCC metastasis, NHAC metastasis, MEL metastasis, SKCM metastasis, UVM metastasis, LC metastasis, NSCLC metastasis, NSCLC glandular metastasis, NSCLC squamous metastasis, NSCLC other metastasis, SCLC metastasis, CHOL metastasis, ESCA metastasis, CESC metastasis, OC metastasis, OV metastasis, LIHC metastasis, RCC metastasis, KIRC metastasis, KIRP metastasis, SARC metastasis, FS metastasis, LPS metastasis, MPNST metastasis, SS metastasis, STAD metastasis, TGCT metastasis, THYM metastasis, UCS metastasis, and / or UEC metastasis. (B) The metastatic or metastatic lesions include adrenocortical carcinoma, lung cancer, non-small cell lung cancer, non-small cell lung adenocarcinoma, non-small cell lung squamous cell carcinoma, small cell lung cancer, melanoma, cutaneous melanoma, uveal melanoma, mucosal melanoma, esophageal melanoma, anal melanoma, mesothelioma, breast cancer, breast carcinoma, triple-negative breast cancer, primary brain cancer, ovarian cancer, uterine carcinoma, uterine carcinosarcoma, head and neck squamous cell carcinoma, head and neck adenocarcinoma, colon cancer, gastrointestinal cancer, renal cell carcinoma, clear cell renal cell carcinoma, papillary renal cell carcinoma, sarcoma, fibrosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma, germ cell tumor, lymphoma, testicular cancer, testicular germ cell tumor, bladder cancer, bladder urothelial carcinoma, prostate cancer, oral carcinoma, oral squamous cell carcinoma, acute myeloid leukemia, H. Cancers selected from the group consisting of pylori-induced MALT non-Hodgkin lymphoma, glioblastoma, cervical carcinoma, cervical squamous cell carcinoma and cervical adenocarcinoma, hepatocellular carcinoma, hepatocellular carcinoma of the liver, Ewing's sarcoma, endometrial cancer, laryngeal epithelial carcinoma, esophageal carcinoma, oral carcinoma, atypical meningioma, papillary thyroid carcinoma, thymoma, brain tumor, salivary duct carcinoma, and extranodal T / NK cell lymphoma, (C) The patient is further administered at least one adjuvant selected from the group consisting of anti-CD40 antibody, imiquimod, reciquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab, atezolizumab, interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives, poly(I:C) and derivatives, RNA, sildenafil, poly(lactidocoglycolide) (PLG), virosoms, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-21 (IL-21), and interleukin-23 (IL-23). (D) The pharmaceutical composition contains autologous or allogeneic recombinant T lymphocytes or activated T lymphocytes for the patient.