Means and methods for the quantification of target expression

Abstract

The present invention provides a highly sensitive, specific and reproducible assay using new sets of reference genes for determining the expression of a target in a biological sample, such as PRAME expression in a tumor sample.

Description

[0001] MEANS AND METHODS FOR THE QUANTIFICATION OF TARGET EXPRESSION

[0002] TECHNICAL FIELD

[0003] The present invention relates to a highly sensitive, specific and reproducible assay (e.g. RT-qPCR or digital PCR) for determining the expression levels of a target in a biological sample, such as a PRAME antigenic peptide in a tumor sample, which is, inter alia, useful for selecting cancer patients for treatment with therapies that target such antigenic peptides.

[0004] BACKGROUND OF THE INVENTION

[0005] The identification of target peptides that are abundantly expressed and presented on tumor cells, along with the development of binding molecules and cellular therapies that specifically recognize these targets, has significantly expanded the treatment options for various types of cancer. The emerging field of personalized medicine aims to tailor treatments to specific patient groups, enhancing efficacy and minimizing side effects. However, the success of personalized medicine relies on accurate, reproducible, and clinically useful companion diagnostic tests to identify patients who can benefit from targeted therapies.

[0006] Despite the development of various therapies for many types of cancer, numerous patients do not benefit from these treatments. This challenge has led to a deeper understanding of the mechanisms of protective anti-tumor immunity and the development of more effective immunotherapies, which have been shown to increase patient survival and support the longstanding idea that immunity plays a crucial role in cancer pathogenesis and therapy. Immunotherapy is a cancer treatment designed to enhance the body's natural defences to combat cancer.

[0007] Bispecific T cell binding molecules are a novel class of immunotherapy designed to redirect T cells to target and kill cancer cells. These molecules have two binding sites: one attaches to a specific antigen on cancer cells, and the other binds to the CD3 receptor on T cells. This bridging action forms an immunological synapse, activating T cells to release cytotoxic granules and cytokines, ultimately killing the cancer cells. Bispecific binding molecules offer target specificity and do not require genetic modification of T cells, making them less complex and quicker to develop. An example is blinatumomab (Blincyto), which targets CD19 on B-cell leukemia cells and CD3 on T cells, showing significant efficacy in treating relapsed or refractory B-cell acute lymphoblastic leukemia (ALL).

[0008] Adoptive cell therapy (ACT) involves infusing a patient's own or donor immune cells to treat cancer, often involving genetic modification to enhance anti-tumor activity. Types of ACT include Chimeric Antigen Receptor T Cells (CAR T Cells), Tumor-Infiltrating Lymphocytes (TILs), and T cell receptor-engineered T cells (TCR T cells). TCR T and CAR T cells are engineered to express receptors that recognize specific cancer antigens, collected from the patient, modified in the lab, expanded, and reinfused. Other approaches involve the in vivo modification of patient’s T cells with suitable vectors and allogeneic, off-the-shelf approaches. Therapies like tisagenlecleucel (Kymriah) and axicabtagene ciloleucel (Yescarta) have shown success in treating certain blood cancers. TILs, isolated from tumors, are expanded ex vivo and reinfused into the patient after lymphodepletion, showing promise in treating metastatic melanoma. ACT therapies are highly personalized, potentially leading to higher efficacy and fewer side effects, generating a robust and sustained immune response against cancer cells.

[0009] Bispecific T-cell binding molecules and ACT represent significant advancements in cancer immunotherapy. Bispecifics offer a targeted approach to redirect T cells against cancer cells, while ACT harnesses the patient’s own immune cells for a personalized attack on tumors. Both therapies rely on the expression and MHC presentation of cancer-specific target peptides to redirect T cells to malignant cells in the patient’s body.

[0010] Such target peptides can be derived from PRAME (Preferentially Expressed Antigen in Melanoma), a cancer-testis antigen (CTA) that has gained attention as a promising target for T cell-based immunotherapies, including TCR-T cell therapy. PRAME is an attractive target due to its restricted expression pattern and high prevalence in various cancers. It is predominantly expressed in a wide range of cancers, such as melanoma, leukemia, lung cancer, breast cancer, ovarian cancer, and sarcomas, while its expression is largely absent in normal adult tissues except for the testis and some fetal tissues, which do not present antigens to T cells due to the lack of MHC class I expression. This makes PRAME an ideal target for immunotherapy, as targeting it is less likely to cause damage to normal tissues.

[0011] Many peptides, such as PRAME peptides, are recognized by the immune system as a foreign antigen capable of eliciting strong T cell responses. They can be presented by both MHC class I and class II molecules, making them accessible to cytotoxic T lymphocytes (CTLs) and helper T cells, enhancing the overall immune response against tumors expressing said peptides. However, every cancer is not the same. Targeted therapy requires a certain threshold of peptide expression in a patient’s cancer cells. Specifically, patients with tumor indications associated with lower peptide prevalence need to be tested for said peptide expression to enable treatment decisions. For instance, available PRAME assays are either based on PCR (Clin Cancer Res 2016;22(5):1234-42), or IHC, but not developed for treatment decisions and have different cut-offs: The PRAME assay described in Clin Cancer Res 2016;22(5):1234-42 uses RBM23, SAP130, and MRPS21 as reference genes which are expressed at low(er) levels and requires macro-dissection to purify tumor tissue and a pre-amplification step, which adds complexity to the assay. It was developed only for uveal melanoma samples with the purpose to determine metastatic risk, rather than for selecting patients for PRAME-targeted therapy. Moreover, RNA-based assays such as quantitative real-time PCR (RT-qPCR) require RNA in sufficient quantity and quality to produce conclusive results. This is not always possible with RNA from small archived formalin-fixed paraffin-embedded (FFPE) tumor samples, and thus e.g. fresh frozen tissue may be required, which imposes additional burden on patients and clinical practitioners. IHC- based assays detect a target protein, for which the correlation with peptide levels has not been shown. Further, state-of-the art assays typically have been optimized only for a limited number of tumor indications. Thus, a diagnostic assay is required that can reliably analyses target peptide (e.g., PRAME) expression in solid tumor tissues across multiple tumor indications and preferably in formalin-fixed paraffin-embedded (FFPE) samples which are readily available for each patient. Furthermore, the assay should preferably be highly reproducible and require low sample volumes.

[0012] DEFINITIONS

[0013] Where an indefinite or definite article is used when referring to a singular noun, e.g., “a”, “an” or “the”, does not exclude a plurality, unless something else is specifically stated. 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. Accordingly, the articles “a”, “an” or “the” preceding an element or component are intended to be non-restrictive regarding the number of instances (i.e. , occurrences) of the element. Therefore, “a”, “an” or “the” is to be read to include one or at least one, and the singular word form of the element also includes the plural unless the number is obviously meant to be singular.

[0014] The terms “about” or “approximately” mean within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within an acceptable standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to ±20%, ±15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, for instance within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term “about” is implicit and in this context means within an acceptable error range for the particular value. The terms “about” and “approximately” also include the concrete value, e.g., "about 50" includes the value "50".

[0015] The term “adoptive cell therapy” (“ACT”) is a type of immunotherapy in which T cells are administered to a patient to treat a disease, such as cancer. In autologous ACT, T cells that have been extracted from a patient are cultured in vitro and are typically genetically modified and are then returned to the same patient for therapy. Comparatively, allogeneic ACT involves T cells isolated and expanded from a donor or donors different from the patient receiving the T cells for therapy. Alternatively, vectors TCRs (and optionally other proteins of interest) capable of specifically targeting T cells can be administered to the patient (“in vivo ACT”).

[0016] The term “amino acid” refers to one of the 20 naturally occurring amino acids or any non-natural analogues. Preferably, the term “amino acid” refers to one of the 20 naturally occurring amino acids. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation.

[0017] “Amino acid mutations” may be deletions, insertions or substitutions. The term “amino acid substitution” refers to replacing an amino acid residue present in a parent or reference sequence with another amino acid residue. An amino acid can be substituted in a parent or reference sequence, for example, via chemical peptide synthesis or through recombinant methods known in the art. In the context of the present disclosure, substitutions (even when they referred to as amino acid substitution) are typically conducted at the nucleic acid level, i.e. , substituting an amino acid residue with an alternative amino acid residue is conducted by substituting the codon encoding the first amino acid with a codon encoding the second amino acid.

[0018] Accordingly, a reference to a “substitution at position X” refers to the substitution of an amino acid present at position X with an alternative amino acid residue. In some aspects, substitution patterns can be described according to the schema AnY, wherein A is the single letter code corresponding to the amino acid naturally or originally present at position n, and Y is the substituting amino acid residue. In other aspects, substitution patterns can be described according to the schema An(YZ), wherein A is the single letter code corresponding to the amino acid residue substituting the amino acid naturally or originally present at position X, and Y and Z are alternative substituting amino acid residue.

[0019] Amino acid substitutions may be conservative or non-conservative. For instance, and specifically in the context of antigen binding proteins, substitutions may be conservative substitutions, in which one amino acid is substituted for another amino acid with similar structural and / or chemical properties. An amino acid substitution may also be a post- translational modification of the antigen binding protein and is herein also encompassed.

[0020] A conservative amino acid substitution may include the substitution of an amino acid by another amino acid of the same class, for example, (non-polar amino acids substituted by other non-polar amino acids.

[0021] A conservative amino acid substitution may be made in accordance with Table 1. Methods for predicting tolerance to protein modification may be found in, for example, Guo et al., Proc. Natl. Acad. Sci. , USA, 101(25):9205-9210 (2004), the contents of which are incorporated by reference in their entirety.

[0022] Conservative Amino Acid substitutions

[0023]

[0024] An antigen binding protein can comprise synthetic amino acids in place of one or more naturally-occurring amino acids. Such synthetic amino acids are known in the art, and may include, for example, aminocyclohexane carboxylic acid, norleucine, a-amino n- decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4- hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4- carboxyphenylalanine, p-phenylserine p-hydroxyphenylalanine, phenylglycine, a- naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1, 2,3,4- tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a- aminocyclopentane carboxylic acid, a-aminocyclohexane carboxylic acid, a- aminocycloheptane carboxylic acid, a-(2-amino-2-norbornane)-carboxylic acid, a,y- diaminobutyric acid, a,p-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0025] An antigen binding protein or nucleic acid(s) encoding an antigen binding protein can be recombinant, isolated, engineered and / or purified.

[0026] The term "amplification conditions" is generally defined as conditions, which promote hybridizing or annealing of primer sequences to a target sequence and subsequent extension of the primer sequence. It is well known in the art that such annealing is dependent on several parameters, including temperature, ionic strength, sequence length, complementarity and G:C content of the sequences. For example, lowering the temperature in the environment of complementary nucleic acid sequences promotes annealing. For any given set of sequences, melt temperature, or Tm, can be estimated by any of several known methods. Typically, diagnostic applications utilize hybridization temperatures, which are close to (i.e. within 10" C) the melt temperature. Ionic strength or "salt" concentration also impacts the melt temperature, since small cations tend to stabilize the formation of duplexes by negating the negative charge on the phosphodiester backbone. Typical salt concentrations depend on the nature and valency of the cation but are readily understood by those skilled in the art. Similarly, high G:C content and increased sequence length are also known to stabilize duplex formation because G:C pairings involve 3 hydrogen bonds where A:T pairs have just two, and because longer sequences have more hydrogen bonds holding the sequences together. Thus, a high G:C content and longer sequence lengths impact the hybridization conditions by elevating the melt temperature. Once sequences are selected for a given diagnostic application, the G:C content and length will be known and can be accounted for in determining precisely what hybridization conditions will encompass. Since ionic strength is typically optimized for enzymatic activity the only parameter left to vary is the temperature. Generally, the hybridization temperature is selected close to or at the Tm of the primers or probe. Thus, obtaining suitable hybridization conditions for a particular primer, probe, or primer and probe set is well within ordinary skill of one practicing this art. The amplification product produced as above can be detected during or subsequently to the amplification of the target sequence using any suitable method and a probe disclosed in greater detail below.

[0027] The term “analogue” refers to a structurally related polypeptide or nucleic acid molecule having the function of a reference polypeptide or nucleic acid molecule.

[0028] The term “and / or,” as used herein should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. In other words, the term “and / or” is to be interpreted as encompassing that one or more of the cases it connects may occur. Furthermore, “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0029] The term “antibody” as used herein is meant to include native and engineered antibodies. The term “engineered antibody includes functional antibody fragments, single chain antibodies, single domain antibodies, bispecific or multispecific antibodies.

[0030] The term “antigen binding protein” (occasionally abbreviated to “ABP”) herein refers to a polypeptide or a complex of two or more polypeptides comprising an antigen binding site that specifically binds to an antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein, and that polypeptide or the two or more polypeptides comprise(s) the CDRs as herein provided, such as CDRal , CDRa3, and optionally CDRa2, and CDRbl , CDRb3, and optionally CDRb2. The two or more polypeptides of the antigen binding protein may be covalently or non-covalently linked together. As used in the context of the present specification, the term antigen binding protein includes antigen binding proteins that comprise fragment(s) of the herein provided TCRs. The herein provided antigen binding proteins may be used in different formats as also described below, such as membrane bound antigen binding proteins, fusion proteins, monovalent, bivalent and multivalent antigen binding proteins, monospecific, bispecific and multispecific antigen binding proteins.

[0031] The term includes antigen binding proteins having the overall structure of a TCR, an antibody and / or a chimeric antigen receptor (CAR). The antigen binding protein can comprise TCR-derived CDRs, in particular a variable domain Vacomprising TCR-derived CDRal, CDRa3, and optionally CDRa2, and a variable domain Vbcomprising TCR-derived CDRbl, CDRb3, and optionally CDRb2. Antigen binding proteins can comprise a variable domain Vacomprising complementarity determining regions (CDRs) CDRal , CDRa2, and CDRa3, e.g. on a first polypeptide, and a variable domain Vbcomprising CDRbl, CDRb2, and CDRb3, e.g. on a second polypeptide, wherein CDRal , CDRa2, CDRa3, CDRbl, CDRb2 and CDRb3 form an “antigen binding domain A”. “Antigen binding domain A” denotes a binding domain that binds to the antigenic peptide that is in a complex with a major histocompatibility complex (MHC) protein. For instance, the entire Vadomain and / or the entire Vbdomain may be TCR-derived and thus be TCR alpha, beta, gamma or delta variable domains (Va, Vp, Vy or Vb). Preferably, the antigen binding protein may be a TCR or functional fragment(s) thereof, e.g. the variable domains VAand VBof the TCR. In some instances, the antigen binding protein can comprise CDRs and optionally the VAand VBas herein provided, and can comprise further (an) additional domain(s) fused directly or indirectly to VAor VB. The further domains may form (an) additional binding domain(s) or (a) binding site(s). For example and in particular instances, the additional binding domains may form antigen binding domain B. Further binding domains may be comprised that, e.g., form further antigen binding domains, e.g. antigen binding domain C, etc. The additional / further domains comprised in the antigen binding protein may also be a further protein.

[0032] The antigen binding protein thus also includes fusion proteins wherein fragment(s) of the herein provided TCRs further comprise other binding domains. Examples of additional domains comprised in an antigen binding protein of the disclosure that is a fusion protein are listed below.

[0033] The term “antigenic peptide in a complex with an MHC protein”, herein refers to an antigenic peptide that is non-covalently bound to an MHC molecule. In particular, the antigenic peptide is located to a “peptide-binding groove” formed by the MHC molecule. A complex of an MHC molecule and an antigenic peptide is herein also referred to as “peptide- MHC complex” or “pMHC complex”. For instance, in the case of the PRAME antigenic peptide, the complex is also referred to as “PRAME antigenic peptide-MHC complex” or “PRAME:MHC complex”.

[0034] An “antigen-expressing” cancer” (also referred to as an antigen “positive” cancer), is characterized by the over-presentation of the antigenic peptide (e.g., PRAME-004) in cancer cells. Some cancer indications are known to express a certain antigenic peptide. Others reguire testing of the patient. In such cases, a cancer biopsy can be used and the antigenic peptide can be identified using the XPresident® and related methods (according to WO 03 / 100432; WO 2005 / 076009; WO 2011 / 128448; WO 2016 / 107740, US 7,811,828, US 9,791 ,444, and US 2016 / 0187351, the contents of each are hereby incorporated by reference in their entirety.

[0035] An “antigenic peptide presenting cell” or “antigenic peptide:MHC complex presenting cell” refers to a cell that presents on its surface the a certain antigenic peptide (e.g., PRAME- 004) in a complex with an MHC molecule. Specifically, the antigenic peptide:MHC complex presenting cell may be a tumor cell, wherein the tumor is preferably a cancer as defined herein. In the context of the present disclosure, an antigenic peptide:MHC complex is “overpresented” on the cell surface of a an antigenic peptide:MHC complex presenting cell, compared to levels of said complex on the surface of cells in normal (healthy) tissue (also referred to as “healthy cells”) or on the surface of control cells loaded with a different antigen presenting peptide or no peptide. By "over-presented" is meant that the antigenic peptide: MHC complex is present at a level at least 2-fold, preferably between 5-fold to 10- fold of the level present in healthy tissue or control cells.

[0036] The term “approximately,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain instances, the term “approximately” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0037] As used herein with respect to a disease, the term “associated with” means that the symptom, measurement, characteristic, or status in guestion is linked to the diagnosis, development, presence, or progression of that disease. As association can, but need not, be causatively linked to the disease. For example, symptoms, seguelae, or any effects causing a decrease in the guality of life of a patient having cancer are considered associated with the cancer and in some instances of the present disclosure can be treated, ameliorated, or prevented by administering the polynucleotides of the present disclosure to a subject in need thereof.

[0038] The term “at least one” herein refers to one or more of the specified elements such as 1 , 2, 3, 4, 5 or 6 or more of the specified elements. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to 1 , 2, 3, 4, 5 or 6 or more A, with no B present (and optionally including elements other than B); in another embodiment, to 1 , 2, 3, 4, 5 or 6 or more B, with no A present (and optionally including elements other than A); in yet another embodiment, to 1 , 2, 3, 4, 5 or 6 or more A, and 1 , 2, 3, 4, 5 or 6 or more B (and optionally including other elements); etc.

[0039] The term “comprising” is to be interpreted as encompassing all specifically mentioned elements as well optional, additional, unspecified elements. In other words, the term “comprising” does not exclude other elements. For the purposes of the present disclosure, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

[0040] The term “consisting of” refers to the inclusion of exactly one element of a number or list of elements, and preferably excluding more than trace elements of other ingredients other than the element or list of elements.

[0041] The term “consisting essentially of’ when used to define compounds, compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants, e.g., from the isolation and purification method and pharmaceutically acceptable carriers, preservatives, and the like.

[0042] The term “copy number” refers to the number of antigenic peptide / MHC complexes as defined in the context of the present disclosure that are present on the cell surface of a cell, such as a antigenic peptide / MHC presenting cell, for example a cancer cell, or a healthy cell. Copy numbers of a protein can be determined by a variety of art known methods including FACS analysis of diseased cells with fluorescently labelled antigen binding proteins.

[0043] The term "cycle threshold" (Ct) refers to quantification cycle values calculated from the record fluorescence measurements of the PCR.

[0044] The term "disease" or "disorder" refers to any condition that would benefit from treatment with the therapeutic agent(s) of the disclosure. The term may include chronic and acute disorders or diseases including those pathological conditions which predisposes the subject to the disorder in question. A “domain” may be any region of a protein or nucleic acid sequence, generally defined on the basis of sequence homologies and often having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions). The terms “domain” and “region” may be used interchangeably herein.

[0045] The term “engineered” or “genetically engineered” in the context of a nucleic acid, protein, cell or organism, means that the nucleic acid, protein, cell or organism has been modified using biotechnological methods. Such genetically modified nucleic acids, proteins, cells or organisms typically do not occur in nature. For instance, genetically engineered nucleic acids, proteins, cells or organisms can be obtained by introducing a DNA vector or an RNA encoding a heterologous gene.

[0046] As used herein, the term “ex vivo” refers to events that occur outside of an organism (e.g., animal, plant, or microbe or cell or tissue thereof). Ex vivo events may take place in an environment minimally altered from a natural (e.g., in vivo) environment.

[0047] The term "expression" generally refers to the process by which information (e.g., gene-encoded and / or epigenetic information) is converted into the structures present and operating in the cell. Therefore, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and / or polypeptide modifications (e.g., posttranslational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a posttranslational processing of the polypeptide, e.g., by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). A sample or cell that "expresses" a protein of interest is one in which mRNA encoding the protein, or the protein, including fragments thereof, is determined to be present in the sample or cell.

[0048] The terms "expression level" or "expression level" in general are used interchangeably and generally refer to the amount of a polynucleotide or polypeptide of interest in a sample.

[0049] A “format” in relation to an antigen binding protein relates to a defined spatial arrangement of domains, in particular of variable and optionally constant domains. Characteristics of such antigen binding protein formats are the number of polypeptide chains (single chain, double chain or multiple chains), the type and length of linkers connecting different domains, the number of variable domains (and thus the number of valences), the number of different variable domains (and thus the number of specificities for different antigens, e.g. bispecific, or multispecific), and the order and orientation of variable domains (e.g. cross-over, parallel).

[0050] The term “heterologous” refers to an element, such as a nucleic acid sequence, a gene or a protein that is foreign to its genomic location and / or host in that it originates from a different cell, genomic location, organism or species. The term is, inter alia, used in the context of “heterologous” gene expression, which is typically achieved through recombinant DNA technology. Essentially, a gene from one cell or organism is optionally engineered and inserted into a different cell or organism, which his then also referred to as a “recombinant” cell or organism. A “heterologous” proteins is produced in a system (e.g., a cell) different from their native host.

[0051] The term “HLA-A*02” signifies a specific HLA allele, wherein the letter A signifies the allele and “*02” indicates the A2 serotype.

[0052] The terms "hybridized" and "hybridization" refer to the base-pairing interactions between two nucleic acids that result in formation of a duplex. It is not a requirement that two nucleic acids have 100% complementarity over their full length to achieve hybridization.

[0053] The term “in vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[0054] The term “in vivo” refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[0055] The "Limit of Detection" (LoD) is the lowest analyte concentration likely to be reliably distinguished from the blank and at which detection is feasible. LoD is determined by utilizing both the measured LoB and test replicates of a sample known to contain a low concentration of analyte.

[0056] The "Limit of Quantification" (LoQ) refers to the lowest concentration at which the analyte can not only be reliably detected but at which some predefined goals for bias and imprecision are met.

[0057] The “Major Histocompatibility Complex” (MHC) is a set of cell surface proteins essential for the acquired immune system to recognize foreign molecules in vertebrates, which in turn determines histocompatibility. The main function of MHC molecules is to bind to antigens derived from pathogens and display them on the cell surface for recognition by the appropriate T cells. The human MHC is also called the HLA (human leukocyte antigen) complex (or just HLA). Thus, the terms “MHC” and “HLA” can be used interchangeably. The MHC gene family is divided into three subgroups: class I, class II, and class III. Complexes of peptide and MHC class I molecules (MHC I) are usually recognized by CD8-positive T cells (CD8+ T cells) bearing the appropriate T cell receptor (TCR), whereas complexes of peptide and MHC class II molecules (MHC II) are usually recognized by CD4-positive helper- T cells (CD4+ T cells) bearing the appropriate TCR. CD4 and CD8 usually function as coreceptors of a TCR in binding to MHC I and MHC II, respectively. In some exceptional cases, complexes of peptide and MHC I are recognized by CD8-negative (in particular CD8- negative, CD4-positive) T cells (Soto et al., 2013, Cancer Immunol Immunother. 2013 Feb; 62(2): 359-369). Since the responses of CD8-positive and CD4-positive T cells contribute jointly and synergistically to the anti-tumor effect, the identification and characterization of tumor-associated antigens and corresponding T cell receptors is important in the development of cancer immunotherapies such as vaccines and cell therapies. The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger, a-chain, constituent of HLA-A. Variation of HLA-A a-chain is key to HLA function. This variation promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of certain structures, greater variety of HLAs means greater variety of antigens to be 'presented' on the cell surface. The MHC class I HLA protein in the context of the present disclosure may be an HLA-A, HLA-B or HLA-C protein, suitably HLA-A protein, for example HLA-A*02. In the MHC class I dependent immune reaction, peptides not only have to be able to bind to certain MHC class I molecules expressed by tumor cells, they subsequently also have to be recognized by T cells bearing specific T cell receptors (TCR).

[0058] The term "malignant" neoplasm generally refers to a poorly differentiated (anaplasia) neoplasm that has characteristically rapid growth accompanied by progressive infiltration, invasion, and destruction of the surrounding tissue. Furthermore, a malignant neoplasm has the capacity to metastasize to distant sites.

[0059] The term "metastasis" refers to the spread or migration of cancerous cells from a primary (original) tumor to another organ or tissue, and is typically identifiable by the presence of a "secondary tumor" or "secondary cell mass" of the tissue type of the primary (original) tumor and not of that of the organ or tissue in which the secondary (metastatic) tumor is located.

[0060] As used herein, the term "melting temperature" (Tm ) in relation to an oligonucleotide is defined as the temperature at which 50% of the DNA forms a stable double-helix and the other 50% has been separated into single stranded molecules. As known to those of skill in the art, PCR annealing temperature is typically a few degrees less than the Tm , the latter of which is calculated based on oligo and salt concentrations in the reaction.

[0061] The term “messenger RNA” or “mRNA” refers to any polynucleotide (a ribonucleic acid) which encodes a (poly-)peptide of interest and which is capable of being translated to produce the encoded (poly-)peptide of interest in vitro, in vivo, in situ, or ex vivo. Typically, the basic components of an mRNA molecule include a coding region, a 5’UTR, a 3’UTR, a 5’ cap, and a poly-A tail. The term “nucleotide” refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and / or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. Nucleotides are referred to by their commonly accepted single-letter codes. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Nucleobases are referred to herein by their commonly known one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Accordingly, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, U represents uracil.

[0062] As used herein, a "part" or "region" of a polynucleotide or polypeptide is defined as any portion of the polynucleotide or polypeptide that is less than the entire length of the polynucleotide or polypeptide, respectively.

[0063] As used herein, the term "primer" refers to an oligonucleotide which, produced synthetically, is capable of acting as a point of initiation of nucleic acid synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e. , in the presence of nucleotides and an agent for polymerization such as DNA polymerase, reverse transcriptase or the like, and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the agents for polymerization. The exact lengths of the primers will depend on many factors, including temperature and the source of primer. For example, depending on the complexity of the target sequence, a primer typically contains 15 to 25 or more nucleotides, although it can contain fewer nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with a template. In particular, the primers, which are used here, are preferably designed in a way that avoids amplification of genomic DNA or cDNA in order to avoid non-specific amplification of contaminating genomic DNA in the sample.

[0064] The term “sequence identity” refers to a measure of the similarity between two biological sequences, such as polynucleotide or polypeptide sequences. It is expressed as a percentage of the number of identical positions in the two sequences divided by the total number of positions. In other words, sequence identity indicates how much two sequences match each other in terms of the order and composition of their building blocks, such as nucleotides or amino acids. If the two sequences to be compared are not of equal length, they can be aligned to give the best possible fit, allowing the insertion of gaps or alternatively, truncation at the ends of the nucleic acid sequences or amino acid sequences. The skilled person will acknowledge that various means for comparing sequence identity are available (see below). A higher sequence identity may indicate a closer evolutionary relationship between the two sequences, or a higher degree of functional similarity or homology. Sequence identity is commonly used in bioinformatics and molecular biology to compare and analyze the structure and function of biological molecules, such as proteins, and to infer their evolutionary history and relationships.

[0065] For example, in the context of the present disclosure, a sequence that is “at least 85% identical to a reference sequence” may be a sequence having, over its entire length, 85%, or more, in particular 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the entire length of a reference sequence (e.g., a variable domain disclosed herein). Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% Identical” to a reference sequence may comprise mutations such as deletions, insertions and / or substitutions compared to the reference sequence.

[0066] In the context of the present disclosure, the sequence identity can be calculated using a global pairwise alignment (i.e. the two sequences are compared over their entire length). Methods for comparing the identity of two or more sequences are well known in the art. For example, the “needle” program, which uses the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment (including gaps) of two sequences when considering their entire length, may be used. The needle program is for example available on the ebi.ac.uk World Wide Web site and is further described in the following publication (EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp. 276-277). The percentage of identity between two polypeptides or polynucleotides, in accordance with the disclosure, can be calculated using the EMBOSS: needle (global) program with a “Gap Open” parameter equal to 10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

[0067] As used herein, by "subject" or "individual" or "animal" or "patient" or "mammal," is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, prophylaxis or therapy is desired. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; bears, food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. Preferably, the subject is a human subject. The term "substantially" refers to the qualitative condition of exhibiting total or neartotal extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical characteristics rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness in many biological and chemical characteristics.

[0068] The term “TOR” as used herein includes both native and engineered TCRs.

[0069] A “native” TCR refers to a wildtype TCR that can be isolated from nature, whereas an “engineered” TCR may be a protein resembling a native TCR, but comprising further modifications e.g. in the variable and / or constant domains compared to the naturally occurring sequence, e.g. a humanized TCR or a TCR with altered characteristics (e.g. altered binding, heterodimerization or expression level).

[0070] Native TCRs are heterodimeric cell surface proteins of the immunoglobulin super- family, which are associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Native heterodimeric TCRs exist in ap and yb forms, which are structurally similar but have distinct locations and probably functions. The terms “a / p TCR” or a ”y / b TCR” thus refer to a TCR comprising an a-chain and a p-chain as described above, or a y-chain and a b-chain, respectively. Such TCRs may also be described as “full length TCRs” or “conventional TCRs”. An a / p TCR or a y / b TCR may be a native TCR or may be an engineered TCR that retains the structure of a native TCR, i.e. an engineered TCR comprising minor modifications in the variable and / or constant domains as described above, such as a humanized TCR.

[0071] Native, full-length ap heterodimeric TCRs consist of an a-chain and a p-chain. The a- chain comprises a variable region (V region) encoded by a TRAV gene, a joining region (J region) encoded by a TRAJ gene, and a constant region (C region) encoded by a TRAC gene. The p-chain comprises a variable region (V region) encoded by a TRBV gene, a joining region (J region) encoded by a TRBJ gene and a constant region (C region) encoded by a TRBC gene, and usually a short diversity region (D region) encoded by a TRBD gene between the V and J regions, although this D region is often considered as part of the J region (Lefranc, (2001), Curr Protoc Immunol Appendix 1: Appendix 10). The genes encoding different a-chain and p-chain variable, joining and constant regions are referred to in IMGT nomenclature by unique numbers (Folch and Lefranc, (2000), Exp Clin Immunogenet 17(1): 42-54; Scaviner and Lefranc, (2000), Exp Clin Immunogenet 17(2): 83- 96; LeFranc and LeFranc, (2001), "T cell Receptor Factsbook", Academic Press). Further information on TCR genes can be found in the international ImMunoGeneTics information system®, Lefranc M-P et al., (Nucleic Acids Res. 2015 Jan;43(Database issue): D413-22; and http: / / www.imgt.org / ). The alpha chain TRAC constant domain sequence and the beta chain TRBC1 or TRBC2 constant domain are in the following, also referred to as TCR constant domain sequences. The TCR constant domain sequences may be derived from any suitable species, such as any mammal, e.g., human, rat, monkey, rabbit, donkey, or mouse, preferably human. The TCR constant domain sequences may be modified, for example, by the introduction of heterologous sequences, preferably mouse sequences, which may increase TCR expression and stability. Also, further stabilizing mutations as known from the state of the art (e.g. WO 2018 / 104407, PCT / EP2018 / 069151, WO 2011 / 044186, WO 2014 / 018863) may be introduced, such as replacement of unfavorable amino acids in the variable regions and / or the introduction of a disulfide bridge between the TCR C domains and the removal of unpaired cysteine. Thus, the disclosure also relates to proteins that may comprise the antigen binding domains (e.g. the CDRs as provided herein) and further comprise domains / amino acid sequences that are not found in the naturally occurring TCR.

[0072] On the protein level, TCR a-, p-, y- and b-chains comprise two immunoglobulin domains, the variable domain and the constant domain. The variable domain corresponds to the V(D)J region. The constant domain corresponds to the C region. The constant domain is the membrane-proximal domain and in the context of the present disclosure also includes the transmembrane (TM) domain and a short cytoplasmic tail. Each of the constant and variable domains include an intra-chain disulfide bond. The variable domains (Vaand Vpin op TCRs and VYand V5in y<5 TCRs) contain highly polymorphic loops comprising the complementarity determining regions (CDRs).

[0073] Each TCR variable domain comprises three “TCR complementarity determining regions (CDRs)” embedded in a framework sequence, one being the hypervariable region named CDR3. In the context of the present disclosure, CDRal , CDRa2 and CDRa3 denote □-chain CDRs, and CDRbl, CDRb2 and CDRb3 denote p-chain CDRs. The sequences encoding CDRal and CDRa2 are comprised in TRAV, the sequences encoding CDRa3 are comprised in TRAV and TRAJ, the sequences encoding CDRbl and CDRb2 are comprised in TRBV, and the sequences encoding CDRb3 are comprised in TRBV, TRBD and TRBJ. In TCRs, the CDR1 and CDR3 amino acid residues make contact with the antigenic peptide, while the CDR2 amino acid residues mainly contact the HLA molecule (Stadinski et al., J Immunol. 2014 June 15; 192(12): 6071-6082; Cole et al., J Biol Chem. 2014 Jan 10;289(2):628-38). The antigen specificity of a TCR is thus defined by the CDR3 and CDR1 sequences. The CDR2 sequences are not required for the determination of antigen specificity, but may play a role in the overall affinity of a TCR towards a peptide:MHC complex.

[0074] “TCR framework regions” (FRs) refer to amino acid sequences interposed between the CDRs, i.e. to those portions of the variable domains that are to some extent conserved among different TCRs. The a-, p-, y- and b-chain variable domains each have four FRs, herein designated FR1-a, FR2-a, FR3-a, FR4-a (for an a- or y-chain), and FR1-b, FR2-b, FR3-b, FR4-b (for a - or b-chain), respectively. Accordingly, an a-chain or y-chain variable domain may be described as (FR1-a)-(CDRa1)-(FR2-a)-(CDRa2)-(FR3-a)-(CDRa3)-(FR4-a) and a p- or b-chain variable domain may be described as (FR1-b)-(CDRb1)-(FR2-b)- (CDRb2)-(FR3-b)-(CDRb3)-(FR4-b). In the context of the present disclosure, the CDR / FR sequences in an a-, p, y- or b-chain variable domain is determined based on IMGT definition (Lefranc et al., Dev. Comp. Immunol., 2003, 27(1):55-77; www.imgt.org). Accordingly, CDR / FR amino acid positions when related to TCR or TCR-derived domains are indicated according to said IMGT definition. Preferably, the IMGT position of the CDR / FR amino acid positions of the variable domain Va is given in analogy to the IMGT numbering of TRAV24*01 and / or the IMGT position of the CDR / FR amino acid positions of the variable domain Vp is given in analogy to the IMGT numbering of TRBV12-3*01.

[0075] A “fragment” of a TCR” refers to a fragment of a TCR that retains or substantially retains the affinity, functional avidity and / or specificity of the parental TCR from which it is derived for a target antigen. In other words, a “fragment” of a TCR is preferably a “functional” fragment of the TCR. The term “parental TCR” in this context refers to a full length TCR from which a functional fragment may be derived.

[0076] As binding to the target antigenic peptide is defined by the CDR1 and CDR3 sequences, and binding to the target antigenic peptide MHC complex is defined by CDR1 , CDR2 and CDR3, antigen binding proteins comprising the CDR1 and CDR3 and optionally CDR2 sequences of a TCR retain the affinity, functional avidity and / or specificity of the parental TCR for a target antigen. The person skilled in the art is aware that the CDRs have to be interspersed with framework regions (FRs), however the specific amino acid sequences of the framework regions are not directly involved in target antigen specificity. Examples of functional TCR fragments include single variable domains, such as TCR alpha, beta, gamma or delta variable domains, or fragments of the a, p, b or y chain, such as an a, P, b or y chain without transmembrane domain and short cytoplasmic tail. The term “fragment” as used herein refers to naturally occurring fragments (e.g. splice variants or peptide fragments) as well as artificially constructed fragments, in particular to those obtained by gene-technological means.

[0077] A functional fragment of a TCR may have retained or substantially retained the functional avidity for a target antigen, if, for example, the functional avidity for the target antigen is identical to that of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1%. In particular, a functional fragment of a TCR is considered to have retained or substantially retained the functional avidity for a target antigen, if, for example, its cytotoxic activity in response to the target of the parent protein measured in a cytotoxicity assay is identical to the cytotoxic activity of the TCR or is increased or reduced, preferably reduced, no more than 50%, 40%, 30%, 20%, 15%, 10%, 8%, 5%, 3%, 2% or 1 %, preferably 10%, 8%, 5%, 3%, 2% or 1 %.

[0078] A functional fragment of a TCR is considered to have retained or substantially retained the specificity for a target antigen (i.e. the ability to specifically bind to a target antigen), if it does not significantly bind to peptides other than the target antigenic peptide of the TCR.

[0079] “Single chain TCR (scTCR)” as used herein denotes a TCR in which the variable domains of the TCR are located on a single polypeptide. Typically, the variable domains in scTCRs are separated by a linker, wherein said linker typically comprises 10 to 30 amino acids, such as 25 amino acids.

[0080] The term "treating" or "treatment" or "therapy" refers to the application or administration of a composition including one or more therapeutic agents to a subject with the aim of curing, healing, preventing, reducing, delaying, slowing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting a condition, disorder or disease such as cancer and / or its symptoms. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. For instance, cancer treatment may include, without limitation, inhibiting the recurrence of cancer, alleviating its symptoms, diminishing any direct or indirect pathological consequences, preventing or reducing metastases, decreasing the rate of cancer progression, ameliorating or palliation of the disease state, and remission or improved prognosis.

[0081] “Vg” in the context of the present disclosure refers to a variable domain of a TCR a- chain. The Vamay correspond to a naturally occurring Va, or may be modified. Typically, the Vacomprises TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a-chain (Va), p-chain (Vp) , y-chain (VY) or b-chain (V5), preferably from a Va. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, p-chain, y-chain or b-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR a-chain.

[0082] ”Vg” in the context of the present disclosure refers to a variable domain of a TCR p- chain. The ”VP” may correspond to a naturally occurring Va, or may be modified. Typically, the Vp” comprises TCR-derived CDR sequences and TCR-derived framework sequences. The CDR and framework sequences may be derived from a variable domain of a TCR a- chain (Va), p-chain (Vp) , y-chain (VY) or b-chain (V5), preferably from a Vp. The sequences surrounding the CDRs, i.e. the framework sequences, may be derived from a variable domain of a TCR, i.e. a variable domain of a TCR a-chain, p-chain, y-chain or b-chain, or from a variable domain of an antibody, preferably from a variable domain of a TCR - chain. In the examples, various framework and CDR mutations / substitutions are shown.

[0083] The CDR and framework sequences of the variable domains may not necessarily be derived from the same TCR. For example, the CDRs derived from one TCR variable domain (of the donor TCR) are grafted onto another TCR variable domain (of the acceptor TCR). For example, the donor TCR may comprise a VBencoded by TRBV2 and TRBJ2-1, and the acceptor TCR may comprise a VBencoded by TRBV27 and TRBJ1-5.

[0084] CDRs may not only be exchanged / grafted between different alpha variable domains or different beta variable domains, but also may be grafted from a TCR alpha to a TCR beta, gamma or delta variable domain, or from a TCR beta to a TCR alpha, gamma or delta variable domain.

[0085] ”Vk” in the context of the present disclosure refers to a variable domain of an antibody light chain.

[0086] “VH” in the context of the present disclosure refers to a variable domain of an antibody heavy chain.

[0087] The terms "vector" includes "cloning vectors" and "expression vectors" and refers to a vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence. Preferably, and unless specified otherwise, the term “vector” refers to expression vectors.

[0088] The terms “of the disclosure” as used herein are intended to refer to all aspects and embodiments disclosed and / or claimed herein. Any aspects, items or embodiments referred to herein as being “disclosed herein” or “described herein” are to be understood as being aspects, items or embodiments “of the disclosure” or “according to the disclosure”.

[0089] The term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either”, “one of”, “only one of”, or “exactly one of’.

[0090] The terms "polynucleotide" and "oligonucleotide" are used interchangeably. "Oligonucleotide" is a term sometimes used to describe a shorter polynucleotide.

[0091] The term “primer” refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of nucleic acid sequence synthesis when placed under appropriate reaction conditions. One or more of the nucleotides of the primer can be modified, for instance, by addition of a methyl group, a biotin or digoxigenin moiety, a fluorescent tag or by using radioactive nucleotides or universal detectable marker, in which case the primer can act as a probe. The term “probe” refers to an oligonucleotide sequence that is complementary to a specific nucleic acid sequence of interest, and typically comprises a label. A “hydrolysis probe” is a synthetic oligonucleotide construct designed for sequence-specific detection of nucleic acid targets during real-time polymerase chain reaction (qPCR). The probe comprises a nucleotide sequence complementary to a specific region of the nucleic acid of interest, and is typically labeled with a fluorescent reporter moiety at the 5' end and a quencher moiety at the 3' end.

[0092] The term “quantification cycle” or “Cq” "refers to the number of cycles required for the PCR signal to reach the significant threshold. The calculated Cq value is proportional to the log of the number of mRNA copies present in the sample. Because Cq is directly related to the starting concentration of the mRNA of interest, and the difference in Cq values is related to the starting concentration ratio, Cq values are inverse to the amount of mRNA of interest that is in the sample, and correlate to the number of mRNA copies in the sample. Lower Cq values indicate high amounts of the mRNA of interest. Higher Cq values mean lower amounts of mRNA nucleic acid.

[0093] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0094] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Where a range of values is recited, it is to be understood that each intervening integer value, and each fraction thereof, between the recited upper and lower limits of that range is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range can independently be included in or excluded from the range, and each range where either, neither or both limits are included is also encompassed within this disclosure. Where a value is explicitly recited, it is to be understood that values which are about the same quantity or amount as the recited value are also within the scope of this disclosure. Where a combination is disclosed, each subcombination of the elements of that combination is also specifically disclosed and is within the scope of this disclosure. Conversely, where different elements or groups of elements are individually disclosed, combinations thereof are also disclosed. Where any element of an disclosure is disclosed as having a plurality of alternatives, examples of that disclosure in which each alternative is excluded singly or in any combination with the other alternatives are also hereby disclosed; more than one element of an disclosure can have such exclusions, and all combinations of elements having such exclusions are hereby disclosed.

[0095] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of this disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0096] The invention will now be described in more details with reference to the following figures and examples. All literature and patent documents cited herein are hereby incorporated by reference. While the invention has been illustrated and described in detail in the foregoing description, the examples are to be considered illustrative or exemplary and not restrictive.

[0097] *****

[0098] *****

[0099] SUMMARY OF THE INVENTION

[0100] The present invention provides highly sensitive, specific and reproducible means and methods for, directly or indirectly, determining the expression level of a target (such as a PRAME-004 peptide), preferably in a patient’s biological sample (such as a tumor sample), that can, inter alia, be used to predict patients’ responses to targeted therapies, and / or select patients for treatment with targeted therapies.

[0101] The inventive means and method are used to determine (relative) expression levels of targets in a biological sample. To this end, the inventive means and methods determine the expression level of said target, e.g. via detecting target mRNA or amplicons in said sample, and the expression level of at least one reference gene, e.g. via detecting and quantifying the reference gene(s) mRNA or amplicons in said sample. The at least one reference gene is selected from the group of RPL37A, OAZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA. Subsequently, the expression level of said target is typically normalized to the expression level(s) of said reference gene(s) to determine the relative expression level of said target in said sample.

[0102] The invention relates to in vitro methods and are not performed on a human body

[0103] In preferred embodiments, if the target is a target peptide, or a protein, its expression level can be determined indirectly by detecting the target mRNA in said sample (or an amplicons thereof in a PCR amplification mix). Alternatively, target peptide or protein expression levels can be determined directly by detecting the target peptide or protein as such.

[0104] The “target” can be any gene, RNA, protein or peptide of interest. Preferably, the target is a peptide, also referred to as a “target peptide” herein. Target peptides of interest, in particular antigenic peptides, are disclosed throughout the specification and comprise, inter alia, the PRAME-004 peptide.

[0105] The inventive means and methods are particularly useful when applied in the context of polymerase chain reactions (PCR), in particular quantitative real-time PCR (RT-qPCR) and digital PCR. Many benefits of the inventive method are, inter alia, associated with the choice of reference genes.

[0106] The present inventors have evaluated and selected reference genes that are highly and stably expressed across multiple tumor indications (both in fresh frozen and FFPE samples). Stable expression is critical for reference genes, as target (e.g. PRAME-004 peptide) mRNA expression (Ct) is normalized to the average mRNA expression level (Ct) of reference genes; the normalizer should have comparable expression across samples and tissue types. High expression of reference genes is favourable especially for FFPE tissues, where RNA is often degraded. Reference genes with high expression levels enable the testing of FFPE samples without introduction of a pre-amplification step into the RT-qPCR protocol, which would make the assay workflow more complex, costly and potentially error- prone. Reference gene selection was achieved by 3 steps: (1) pre-selecting reference gene candidates based on an extensive analysis of RNASeq databases (Immatics XPRESIDENT®, TCGA, MET500); (2) assessing the selected candidate reference genes by RT-qPCR on FF and FFPE tissues; (3) testing of the selected reference genes for multiplexability using different combinations of reference genes and fluorescent dyes. In step (2) it was surprisingly discovered that 1-2 reference genes are sufficient for assay normalization, as adding more reference genes did not lead to decreased variability of the average reference gene Ct value. Selection of a low number of reference genes also paved the way to establish a multiplex assay (step 3). In a preferred embodiment, a combination of antigenic peptide (e.g., PRAME-004) with two reference genes, OAZ1 and RPL37A is suggested, where each gene is associated with a different fluorescent dye, detected in separate instrument channels. The present inventors have designed and selected new primers and probes, resulting in short amplicon sizes (<100 bp or even <80 bp), to accommodate the increased degradation of RNA in FFPE samples. This optimization resulted in lower Ct values, indicating an increased sensitivity for detection of reference genes. The higher sensitivity leads to higher assay evaluability rates. As noted above, the inventive adaptions now enable using the disclosed assay as a multiplex assay, i.e. PRAME primers and probes are multiplexed with the reference gene primers and probes, such that all genes are analysed in the same well. This leads to (a) less sample consumption, which is favourable for FFPE samples that often are associated with low RNA yield, and is (b) likely the reason for the higher reproducibility of the disclosed assay as compared to previously disclosed PCR-based assays, as deviations in pipetting volumes for the assay reaction mix affect target genes and reference genes equally. The inventive methods thus advantageously not only allows for the use of fresh tissues (e.g., biopsies directly frozen or stored in RNA-preserving agent such as RNAprotect) as sample material, but also of FFPE samples. Fresh tissues are not usually available from cancer patients, so usually a biopsy has to be taken to obtain these sample types which is painful, complex and cost- and time-consuming. To avoid an additional invasive biopsy and, thus, enable broader access of patients to antigenic peptide (e.g., PRAME-004)targeting therapies, the present inventors have developed the method and kit described herein, which is optimized to deliver reliable results in formalin-fixed paraffin- embedded (FFPE) tissues. FFPE is a routine clinical sample type that is typically prepared from tumor samples during medical procedures and is, thus, mostly available from cancer patients. Analysing RNA expression in FFPE tissues is more challenging due to RNA degradation during tissue fixation and storage, which can lead to low RNA yields and lower levels of amplifiable RNA.

[0107] Taken together, the present invention has several benefits that set it apart from protocols known in the art. The choice of suitable reference genes and method design inter alia allows for multiplexing, which increases reproducibility and robustness of the assay and saves time and material. Additionally, it made the method applicable not only to fresh biopsies, but also to (existent) FFPE samples, which is advantageous in that it is not only cost- and time-saving, but also increases patient compliance. This makes the invention clearly superior to prior art practice, which, for instance, rely on B2M (Beta-2 microglobulin) or other household genes that are commonly used in the prior art. The method is also novel in that it reliably enables the selection (or confirmation of selection) of patients for treatment with therapeutic agents that specifically act on the detected target.

[0108] It is thus an object of the present invention to provide an improved method of quantifying a target (e.g., PRAME-004) in a patient’s sample (e.g., from a solid tumor), e.g. via its mRNA expression, to determine the expression level of said target peptide (e.g., PRAME-004) in said sample. It is a further object of the present invention to provide an improved method of selecting a patient for treatment with a therapeutic agent targeting said target, or for confirming their selection for the treatment. These objects is achieved by a method according to the appended claims, specification and drawings.

[0109] Specifically, these objects are achieved by an in vitro method for determining the relative expression level of a target in a biological sample, comprising a) determining the expression level of said target, b) determining the expression levels of at least one reference gene selected from RPL37A, OAZ1, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, and c) normalizing the expression of said target against the expression level(s) of said at least one reference gene(s).

[0110] This method is particularly useful when using polymerase chain technology (PCR) methodologies, such as quantitative real-time PCT (RT-qPCR), in vitro. In RT-qPCR, (relative) expression levels are typically measured and expressed as dCt values.

[0111] In a further aspect, the invention relates to a method for determining the expression level of a target in a biological sample, comprising a) determining the expression level of said target, and b) determining the expression levels of at least one reference gene selected from RPL37A, OAZ1, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA This method is particularly useful when using polymerase chain technology (PCR) methodologies, such as digital PCR, in silico. In such cases, a normalization step is not required, and the reference genes serve as a control. Such methods yield results (expression levels) as copies / uL, i.e. absolute copy numbers of the target in the sample.

[0112] After the (relative) expression level of the target peptide has been determined in the biological sample (e.g., a tissue affected by a disease such as cancer), it can then be compared to the predetermined threshold. In embodiments of the invention, the threshold is determined as described in Fritsche J et al. Translating Immunopeptidomics to Immunotherapy-Decision-Making for Patient and Personalized Target Selection. Proteomics. 2018 Jun;18(12):e1700284. doi: 10.1002 / pmic.201700284. Epub 2018 Apr 10. PMID: 29505699; PMCID: PMC6032917. Alternatively, the threshold can be determined by subjecting a control sample to the inventive method that is known or suspected to comprise no or less of the target and comparing the relative expression levels of said target in the biological sample to the control sample. The control sample can be a healthy tissue. Alternatively, the threshold can be determined by using clinical data. To this end, patients’ responses to targeted therapies are evaluated, and correlated with the patients’ individual target expression levels to determine a relative expression threshold where treatment is effective.

[0113] If said target (relative) expression level exceeds the predetermined threshold, the donor from whom the biological sample was derived can be selected for treatment with a targeted therapy. Alternatively or additionally, it can be confirmed that the donor is eligible for treatment with a targeted therapy. A targeted therapy is a therapeutic treatment or agent targeting the target of interest. For instance, the targeted therapy can be a target-specific adoptive cell therapy, i.e. treatment with T cells targeting a specific target peptide.

[0114] In further aspects, the invention thus relates to methods for treating a patient with a targeted therapy, comprising a) obtaining a sample from said patient, b) determining the relative expression level of said target, c) determining the expression levels of at least one reference gene selected from RPL37A, 0AZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, d) normalizing the expression level of said target against the expression level(s) of said at least one reference gene(s), e) comparing the expression level of said target peptide to a predetermined threshold, and selecting the patient for treatment with a targeted therapy if the expression level of said target peptide exceeds said threshold.

[0115] The inventive methods may also be used to monitor the relative expression level of said target during treatment of the sample donor with a targeted therapy.

[0116] Advantageously, selecting appropriate reference genes enables the use of fewer genes in the reaction.. In the methods of the invention, it is preferably sufficient to determine the expression levels of 3, 2 or 1 reference genes.

[0117] Specifically, the at least one reference gene is selected from RPL37A, 0AZ1 , HNRNPA2B1, RPLO, NONO, HSP90AB1, and ACTB. Preferably, the selected reference genes comprise or consist of RPL37A and either OAZ1 or HNRNPA2B1 , or both. In further preferred embodiments, only one reference gene is detected, which is selected from RPL37A and OAZ1.

[0118] The expression levels of target and reference gene can be detected in the sample using any suitable means. Specifically, expression levels can be detected by measuring a) the mRNA or b) the corresponding peptide or protein of said target peptide and / or reference gene(s) in said sample. Generally, mRNA expression can be measured by using primers and / or probes that specifically bind to the mRNA sequences of said target and reference gene(s), whereas the level of protein expression is measured by using antibodies specific to the corresponding protein of said markers.

[0119] Specifically, the inventive method may comprise a quantitative polymerase chain reaction (PCR), a DNA or RNA array, a nucleotide hybridization technique, western blot, in situ hybridization, ELISA, immunohistochemistry or a protein array. Preferably, the inventive method comprises or consists of a PCR method, in particular real time RT-qPCR, or digital PCR.

[0120] Advantageously, when the method is PCR, and in particular RT-qPCR, the method allows for measuring the expression levels of said target and said reference genes in the same reaction vessel. This is also referred to as multiplexing herein.

[0121] Specifically, and in particular where the inventive method comprises or consists of PCR (in particular RT-qPCR), the method may involve the following steps:

[0122] 1) subjecting the biological sample to reverse transcription using mRNA present in the sample as a template to synthesize a corresponding cDNA sequence,

[0123] 2a) forming a target reaction mixture comprising the sample, nucleic acid amplification reagents, an target primer pair, an target hydrolysis probe, said target primer pair and target hydrolysis probe being capable of hybridizing to a target mRNA sequence,

[0124] 2 b) forming at least a first reference reaction mixture comprising the sample, a first reference primer pair and a first reference hydrolysis probe, said first reference primer pair and first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA sequence,

[0125] 2 c) subjecting the target reaction mixture and the at least one first reference reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, said target sequence corresponding to a target mRNA sequence, and at least one copy of a nucleic acid sequence complementary to a first reference gene mRNA sequence, and

[0126] 2) determining the amount of target mRNA and of the first reference gene mRNA in said sample.

[0127] In embodiments of the inventive method, the method applies to one-step RT-PCR methods such as RT-qPCR or RT-digital PCR (dPCR). In such cases, steps 1) (reverse transcription) and 2) (amplification and quantification) are combined. In other words, the reaction agents needed for reverse transcription and amplification / quantification are all added to the biological sample to form a single-vial target reaction mixture or single-vial reference gene reaction mixture. In a further embodiment, the single-vial target and reference gene(s) reaction mixtures can be combined (multiplexed) to obtain a multiplexed single-vial reaction mixture.

[0128] The inventive methods are advantageously applicable to a variety of samples. The biological sample that is used in the inventive method is preferably a patient’s sample (i.e. , the patient is the donor of said sample), optionally selected from a biopsy sample, including a fresh frozen biopsy sample or a biopsy sample in an RNA-preserving agent, or a formalin- fixed paraffin-embedded (FFPE) sample. The patient may be a cancer patient. The cancer may be a solid tumor. The cancer may be a metastatic cancer and / or advanced cancer and / or unresectable cancer and / or recurrent cancer and / or refractory cancer.

[0129] The target is preferably a target peptide. The target peptide may be an antigenic peptide, which is optionally capable of a) being recognized by the immune system, particularly by antibodies or T-cell receptors, or derivatives thereof; and / or b) eliciting an immune response. Such antigenic peptides may be characterized in that they are a) 8-25 amino acids long; b) capable of being presented on MHC-I molecules; c) capable of being recognized by a component of the immune system, such as a T cell receptor (TCR); and / or d) capable of eliciting a T-cell mediated immune response.

[0130] An antigenic peptide of interest in the context of the present invention is the PRAME- 004 peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 (SLLQHLIGL). Another antigenic peptide of interest is a MAG-003 target peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 2 (KVLEHVVRV).

[0131] The targeted therapy preferably comprises or consists of a therapeutic agent that is capable of binding to the target peptide (optionally in complex with an MHC-I molecule), and may be selected from T cells comprising a T cell receptor (TCR) or chimeric antigen receptor (CAR), an antigen binding protein; or a vector encoding a TCR, CAR or antigen binding protein.

[0132] Specifically, therapeutic agent may be selected a T cell comprising a T cell receptor (TCR) comprising the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 6, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 7, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 8, ): a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 9, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 10, and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 11. The TCR is preferably capable of binding to a PRAME-004 peptide, optionally when presented by a MHC-I molecule, in particular HLA- A*02. The T cell may additionally comprise a heterologous CD8 molecule. In specific embodiments, the T cell comprises a R11P3D3_KE TOR as disclosed in WO2018 / 172533 A1.

[0133] Alternatively, the therapeutic agent may be an antigen binding protein comprising the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 12, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 13, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 14;: a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 15, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 16, and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 17. The antigen binding protein is preferably capable of binding to a PRAME-004 peptide, optionally when presented by a MHC-I molecule, in particular HLA-A*02. The antigen binding protein may additionally comprise a recruiter domain binding to CD3. In specific embodiments, the antigen binding protein is the TTP1295 TCER® as described in WO 2022 / 233956 A1.

[0134] The invention also relates to a kit comprising primers and probes that is useful for putting the method to practice. Specifically, a kit for performing the method of the invention is provided, which comprises a) nucleic acid amplification reagents, b) an target peptide primer pair being capable of hybridizing to a target (such as a PRAME-004 peptide) mRNA, c) an target peptide hydrolysis probe being capable of hybridizing to a target (such as a PRAME-004 peptide) mRNA, d) at least a first reference primer pair capable of hybridizing to a first reference gene mRNA, e) at least a first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA, wherein said first reference gene is selected from the group of RPL37A, OAZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, and f) optionally instructions for performing the in vitro method as disclosed herein.

[0135] The kit may further comprise at least a second reference primer pair capable of hybridizing to a second reference gene mRNA, and at least a second reference hydrolysis probe capable of hybridizing to a second reference gene mRNA, wherein said second reference gene is preferably selected from the group of OAZ1, RPL37A, RPLPO, PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and RPLPO.

[0136] Specifically, in the methods and kits of the invention that are used to detect the PRAME-004 peptide, may employ a forward target primer that comprises or consists of at least one sequence selected from 5'- AGA GGC CGC CTG GAT CAG -3' (SEQ ID NO: 4); a reverse target primer comprises or consists of at least one sequence selected from 5'- COG GCA GTT AGT TAT TGA GAG GG -3' (SEQ ID NO: 5); and / or a target hydrolysis probe that comprises or consists of at least one sequence selected from 5'- TGC TCA GGC ACG TGA TGA ACC CCT TGG -3' (SEQ ID NO: 6). The target and / or reference gene hydrolysis probe(s) may comprise detectable labels, such as fluorophores. The kit can be applied for the same uses as the method of the invention, i.e. determining the relative expression level of a target in a biological sample, and optionally selecting the sample donor for treatment with a targeted therapy; confirming the eligibility of the sample donor for treatment with a targeted therapy; and / or monitoring the expression level of said target during treatment with a therapeutic agents targeting said target peptide.

[0137] DETAILED DESCRIPTION

[0138] In a first aspect, the present invention relates to in vitro methods for determining the relative expression levels of targets, such as antigenic peptides, in a biological sample. As used herein, the term “expression level” refers to the measurable amount of a gene product, preferably an mRNA or alternatively a peptide or protein, produced by a gene in a biological sample. Expression levels may be determined using standard molecular biology techniques such as RT-qPCR and may be expressed as absolute values or relative to a control or reference gene or sample. An expression level that is expressed relative to a reference gene or sample is also referred to as “relative expression level” (or “normalized expression level”) herein.

[0139] The inventive method involves a) determining the expression level of said target, b) determining the expression level(s) of at least one reference gene selected from RPL37A, OAZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA; and c) normalizing the expression level of said target against the expression level(s) of said at least one reference gene(s).

[0140] In this way, the method yields the “relative expression level” of said target. This normalized value reflects the relative abundance of the target transcript in a manner that is independent of sample-to-sample variation, thereby enabling accurate comparison of target peptide expression levels across different samples or experimental conditions.

[0141] As noted previously, the inventive methods may preferably apply RT-qPCR methodologies. In such instances, expression levels are typically expressed as Ct values (i.e., the number of PCR cycles required for the fluorescent signal to cross a defined threshold and become detectable above background). Lower Ct values indicate a higher initial amount of mRNA, while higher Ct values indicate a lower initial amount of mRNA. Alternatively, for instance in digital PCR, expression levels can be expressed in absolute values (e.g., mRNA copy numbers / uL).

[0142] The relative expression level of a target is typically expressed as a delta Ct value (dCt or ACt) and calculated as or

[0143] In preferred embodiments of the invention, the relative expression level of the target is expressed as a dCt value and compared to the predetermined threshold, which is also expressed as a dCt value. Patients are selected for treatment with a targeted therapy - or their eligibility for treatment is confirmed - if their (e.g., tumor-derived) biological sample contain sufficient amounts of target mRNA. In such embodiments, a patient is selected for targeted therapy if the dCt value obtained from the biological sample is lower than the predetermined dCt threshold value (i.e., the relative target mRNA expression level of in the biological sample is higher than a predetermined threshold mRNA expression level). Alternatively, in digital PCR, absolute values (e.g. copy number / uL) in the biological sample, or their ratio to one or multiple reference genes (in copy number / uL), can be determined and compared to a predetermined threshold.

[0144] In further embodiments, the AACt (delta-delta Ct) method may be employed. In this approach, the ACt value of a test sample is compared to the ACt value of a calibrator or control sample. The resulting AACt value reflects the fold change in gene expression between the test and control conditions and may be converted to a relative expression ratio using the formula 2A(-AACt), assuming amplification efficiency is approximately 100%.

[0145] The terms “determining” and “quantifying” are used interchangeably herein. E.g., when an expression level is “determined”, it is also “quantified”. Additionally, “determining / quantifying” of expression levels typically involves “determining / quantifying” the amount of target peptide or target peptide mRNA or target peptide amplicons in a sample or reaction mixture.

[0146] The relative expression level of said target can be compared to a predetermined threshold, as described elsewhere herein.

[0147] Applications

[0148] Advantageously, the inventive method can therefore be used to detect the expression level of a target in biological sample, typically a patient’s sample, and may be employed to assess or confirm whether the patient expresses the target at a level that warrants targeted treatment.

[0149] In preferred embodiments, the inventive methods detect a target mRNA in a sample. The term "detecting" is used herein in the broadest sense to include both qualitative and quantitative measurements of a molecule of interest. Detecting includes determining the mere presence of the molecule in a sample, determining whether the molecule is present in the sample at detectable levels, and determining the expression level of said molecule. Detecting may be direct or indirect. Preferably, the inventive methods involve RT-qPCR or digital PCR. These methods can be used to detect (i.e. determine the presence and quantify) mRNA (e.g., PRAME-004 mRNA) in said sample.

[0150] To confirm a patient’s eligibility for targeted treatment, the patient’s relative target expression level is compared to a predetermined threshold. The threshold will typically differ from target to target. For instance, and in particular in the context of antigenic peptides, it can be determined by analysis of paired mass spectrometry (peptidomics, detection of the target peptide) and mRNA expression data. The approach is thus based on comparing transcriptomic and proteomic data: specifically, it involves identifying the minimum mRNA expression level at which the corresponding peptide could be reliably detected using mass spectrometry (MS). This mRNA-based cutoff serves as a practical proxy for peptide presence, as described in Fritsche J et al. Translating Immunopeptidomics to Immunotherapy-Decision-Making for Patient and Personalized Target Selection. Proteomics. 2018 Jun;18(12):e1700284. doi: 10.1002 / pmic.201700284. Epub 2018 Apr 10. PMID: 29505699; PMCID: PMC6032917. In this way, an RNA-based (e.g. dCt) threshold is established, serving as a quantitative cutoff for assessing whether antigenic peptide levels are adequate to support targeted therapeutic intervention. Alternatively, the threshold can be determined by subjecting a control sample to the inventive method that is known or suspected to comprise no or less of the target and comparing the relative expression levels of said target in the biological sample to the control sample. The control sample is typically a healthy tissue. Alternatively, the threshold can be determined by using clinical data. To this end, patients’ responses to targeted therapies are evaluated, and compared with the patients’ individual target expression levels to determine a relative expression threshold where treatment is effective.

[0151] By comparing the (relative) target expression level to the threshold, the presence of the target (e.g., antigenic peptide) can be established.

[0152] In instances where the target expression level exceeds the predetermined threshold, the target can be considered “present” in the sample (i.e. , the a patient’s tumor sample is found to express a target (antigenic) peptide), and the method may further comprise a step of selecting the sample donor (patient) for targeted therapy. Additionally, the method may further comprise a step of confirming the eligibility of the sample donor (patient) for treatment with a targeted therapy. For instance, for cancer patients with tumors that are known to express a certain antigenic peptide (e.g., PRAME-004), treatment can be initiated and subsequently, samples can obtained to confirm the tumour’s expression of the target peptide while treatment is ongoing. This is particularly beneficial in clinical scenarios requiring the prompt initiation of therapy, wherein the therapeutic intervention is personalized and / or necessitates a lead time for the manufacture or preparation of the therapeutic agent.

[0153] In embodiments of the present invention, the method further comprises a step of monitoring the relative expression level of said target throughout the course of treatment. This is particularly relevant in cases where tumors may reduce the expression of specific target. By tracking these changes, the method enables assessment of whether continued treatment with a targeted therapy remains appropriate, or if an alternative therapeutic strategy should be considered.

[0154] Specifically, the inventive method may be particularly useful for: a) diagnosing and / or prognosing MHC-presented peptide expression (such as PRAME expression) in a subject before administering a MHC-presented peptide-targeting therapy (such as PRAME-targeting therapy); b) predicting efficacy of treatment of malignant neoplastic disease in a subject before adoptive cell therapy; c) selecting patients for MHC-presented peptide-targeting therapy (such as PRAME-targeting therapy) of a malignant neoplastic disease; d) assessing outcome of treatment of malignant neoplastic disease in a subject during and after MHC- presented peptide-targeting therapy (such as such as PRAME-targeting therapy), wherein the subject is a mammal, having or is suspected of having a malignant neoplastic disease; e) assessing the recurrence of malignant neoplastic disease in a subject during and after MHC- presented peptide-targeting therapy (such as PRAME-targeting therapy), wherein the subject is a mammal having, or is suspected of having a malignant neoplastic disease.

[0155] The term “MHC-presented peptide-targeting therapy” (e.g., “PRAME-targeting therapy”) as used herein specifically includes treatment with therapeutic agents, in particular binding molecules, such as bispecific binding molecules that act as T-cell engagers, and adoptive cell therapies, such as TCR-T, that are based on the specific recognition of a MHC- bound peptide (e.g., PRAME peptide) by said T-cell engager or TCR. For instance, the “PRAME-targeting therapy” can be an adoptive cellular therapy with a T cell expressing a T cell receptor capable of specifically binding to PRAME-004 presented on an MHC I protein. Other “PRAME-targeting therapies” may include treatment with antibodies or antibody derivatives, or other TCR derivatives. Specifically, the PRAME targeting therapy may include treatment with a T cell that is engineered to express a T cell receptor as disclosed in WO / 2018 / 172533, or treatment with a bispecific T cell engager as disclosed in WO / 2022 / 233956. The above equally applies to any other MHC-presented peptide-targeting therapies, mutatis mutandis

[0156] Methodology

[0157] The disclosed method may be implemented utilizing any appropriate analytical or detection technology. In various embodiments, the method comprises techniques such as quantitative polymerase chain reaction (qPCR), including digital PCR (dPCR), DNA or RNA microarrays, nucleotide hybridization assays, western blotting, in situ hybridization, enzyme- linked immunosorbent assay (ELISA), immunohistochemistry, or protein microarrays.

[0158] In preferred embodiments, the method employs polymerase chain reaction (PCR), particularly real-time reverse transcription quantitative PCR (RT-qPCR) or digital PCR.

[0159] A standard RT-qPCR method in accordance with the present invention generally incorporates PCR steps commonly known and practiced in the field (see, e.g., Biassoni, R., & Raso, A. (Eds.). (2020). Quantitative Real-Time PCR: Methods and Protocols (2nd ed.). Springer). Typically, RT-qPCR measures mRNA expression levels by using primers and / or probes that specifically bind to the mRNA sequences of said target and reference gene(s). Therefore, in embodiments of the present invention, the method includes the following steps:

[0160] 1) subjecting the biological sample to reverse transcription using mRNA present in said sample as a template to synthesize a corresponding cDNA sequence,

[0161] 2a). forming an target peptide reaction mixture comprising said sample, nucleic acid amplification reagents, an target peptide primer pair, an target peptide hydrolysis probe, said target peptide primer pair and target peptide hydrolysis probe being capable of hybridizing to a target peptide mRNA sequence,

[0162] 2 b) forming at least a first reference reaction mixture comprising said sample, a first reference primer pair and a first reference hydrolysis probe, said first reference primer pair and first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA sequence,

[0163] 2 c) subjecting the target peptide reaction mixture and the at least one first reference reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, said target peptide sequence corresponding to a target peptide mRNA sequence, and at least one copy of a nucleic acid sequence complementary to a first reference gene mRNA sequence, and

[0164] 3) determining the amount (or expression level) of target peptide mRNA and of the first reference gene mRNA in said sample.

[0165] Subsequently, the expression level of target peptide is normalized against the expression level(s) of said reference gene(s) as described elsewhere herein to obtain the relative expression level of the target peptide in question. In the context of RT-qPCR, normalization is typically performed by calculating the difference in quantification cycle (Ct) values between the target peptide and reference gene(s) (ACt), which yields the relative expression level of the target, as described below.

[0166] Multiplexing

[0167] Advantageously, the inventive method allows for multiplexing, particularly if applied in the context of PCR, such as RT-qPCR. In this context, the term “multiplexing” generally refers to the simultaneous amplification and quantitative detection of two or more distinct nucleic acids within a single PCR reaction mixture. In the context of the present invention, it specifically refers to detecting the target and one or more reference genes in the same reaction mixture. This is achieved through the use of multiple sets of specific primers and corresponding fluorescently labelled probes, each possessing a unique spectral signature that enables independent detection and quantification of each target sequence. Specifically, multiplexing allows for increased throughput by reducing the number of reactions required, conservation of sample material, and internal normalization or control through the inclusion of reference genes or internal standards in the same reaction vessel. The term encompasses both duplex (two nucleic acids) and higher-order multiplex (three or more nucleic acids) configurations, and includes systems employing hydrolysis probes, molecular beacons, scorpion probes, or other fluorescence-based detection chemistries compatible with real-time PCR instrumentation.

[0168] Therefore, in embodiments and particularly where the method involves PCR or more specifically RT-qPCR, the expression levels of said target and said reference gene(s) are preferably measured simultaneously in the same reaction vessel.

[0169] Primers / Probes

[0170] The inventive method, particularly when practiced as a PCR method such as RT- qPCR, employs probes, such as target hybridization probes, hydrolysis (TaqMan) probes or molecular Beacon, or SCORPION type probes, for detecting and quantifying of the amplification PCR product (and, thus, the mRNA encoding the target peptide), and / or to ensure specificity. Preferably, the probe is a hydrolysis probe.

[0171] One target hydrolysis probe which is useful in the method and kit of the invention is designed to hybridize to a specific region of the PRAME mRNA sequence. It is typically a 20- 30 bp oligonucleotide with a fluorescent reporter dye. A proximally located quencher dye can reduce the emission intensity of the reporter dye. The hydrolysis probe is added directly to the PCR mix, and conditions are virtually identical to those that are established for a standard PCR. During the extension phase of the PCR cycle, the Taq DNA polymerase cleaves the hydrolysis probe only when it is hybridized to the target, separating the reporter dye from the quencher dye. An increase in fluorescence intensity (due to the release of the quenching effect on the reporter) is the result of hydrolysis probe hydrolysis and is quantitative for the initial amount of the template. The fluorescence intensity specific to the reporter dye increases because of its lack of proximity to the quencher dye. Repeated cycles of denaturation, annealing, and extension result in exponential amplification of the PCR product and of fluorescence intensity.

[0172] The inventive method, particularly in the context PCR or RT-qPCR for determining the expression level of a PRAME-004 peptide, may employ the following primer / probes: a the forward primer that comprises or consists of at least one sequence selected from 5-AGA GGCCGCCTGGATCAG-3' (SEQ ID NO: 4); a reverse primer that comprises or consists of at least one sequence selected from 5-CCGGCAGTTAGTTATTGAGAG GG-3' (SEQ ID NO: 5); and / or a hydrolysis probe that comprises or consists of at least one sequence selected from 5 -TGCTCAGGCACGTGATGAACC CCTTGG-3' (SEQ ID NO: 6).

[0173] In embodiments of the present invention, the target and / or reference gene hydrolysis probes may be conjugated with a label that enables real-time detection and quantification of nucleic acid amplification during RT-qPCR. The label can be either directly detectable (e.g., fluorophores, chemiluminophores, fluorescent particles and the like) or indirectly detectable with specific binding partners and nucleic acids. Preferred labels are directly detectable, and particularly preferred labels are fluorescent dyes, such as FAM, TAMRA, VIC, JOE, ROX, NED, SYBR Green, Cy-5, Cy-3 and Texas Red, and other fluorescent dyes known in the art. The use of distinct fluorophores for the target and reference probes allows for multiplex detection within a single reaction, thereby enhancing assay efficiency and enabling accurate normalization of expression levels.

[0174] Reference Genes

[0175] The selection of appropriate reference genes is a critical determinant in realizing the full advantages of the disclosed methods. A reference gene should ideally be stable and consistently expressed in the cells and tissues of interest, without showing changes under experimental conditions or disease states. These genes are used to normalize the mRNA levels of targets before comparing different samples using RT-qPCR. Reference genes help measure and reduce errors arising from variations among samples, such as differences in extraction and RNA quality, efficiency in cDNA synthesis, internal controls, and the experimental samples, whether they are normal cells or tumor tissue. In the method of the invention, choosing an appropriate reference gene is crucial for accurate quantification of mRNA expression levels, as the quantification cycle (Cq) of the target as compared to the Cq of the reference gene. The present inventors have identified several reference genes that are particularly useful for the purposes of the inventive method. Advantageously, even using only one or two of the disclosed reference genes yields reliable results. However, it is also possible to include more than two reference genes in the inventive method.

[0176] In the inventive methods, the expression level of at least one reference gene is determined, wherein said reference gene is selected from RPL37A, OAZ1, RPLPO, PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA. Preferably, the expression level of no more than three reference genes is measured, more preferably no more than two. In certain embodiments, the expression level of a single reference gene is sufficient for the intended analysis. In embodiments of the present invention, the 1, 2, 3 or more reference genes are selected from RPL37A, OAZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA. In some embodiments, the reference gene is not ACTB. RPL37A is a preferred reference gene for use in the inventive method. OAZ1 is a preferred reference gene for use in the inventive method. Therefore, in embodiments of the invention, the selected 1, 2, 3 or more reference genes comprise RPL37A, and optionally either OAZ1 or HNRNPA2B1 , or both. Alternatively, the selected 1 , 2, 3 or more reference genes comprise or consist of OAZ1 and either RPL37A or HNRNPA2B1, or both. The selected reference genes may therefore comprise or consist of a panel of reference genes including RPL37A and OAZ1, RPL37A and HNRNPA2B1, OAZ1 and HNRNPA2B1 or RPL37A, OAZ1 and HNRNPA2B1. In other embodiments, the selected reference genes comprise or consist of a panel of reference genes including RPL37A, OAZ1 and RPLO. Preferred embodiments of the invention determine the expression levels of the two reference genes RPL37A and OAZ1. In further preferred embodiments, one reference gene is sufficient. This reference gene may be RPL37A. Alternatively, the gene may be OAZ1.

[0177] In embodiments of the present invention, the first reference gene is selected from the group of RPL37A, OAZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, more preferably from the group consisting of RPL37A, OAZ1 , RPLPO and HNRNPA2B1, most preferably from the group consisting of RPL37A, OAZ1 , and HNRNPA2B1. Preferably, the first reference gene may be RPL37A.

[0178] In some embodiments, the method further includes determining the expression level of a second reference gene. In such cases, the method therefore comprises using a second reference primer pair that hybridizes to a reference mRNA sequence of a second reference gene.

[0179] The second reference gene is preferably selected from the group of OAZ1 , RPL37A, RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, more preferably from the group consisting of OAZ1 , RPL37A, RPLPO and HNRNPA2B1, most preferably from the group consisting of OAZ1, RPL37A, and HNRNPA2B1.

[0180] In some instances, the first reference gene is RPL37A and the second reference gene is selected from OAZ1, RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA. In some instances, the first reference gene is OAZ1 and the second reference gene is selected from RPL37A, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA.

[0181] In some instances, the first reference gene is RPLPO and the second reference gene is selected from RPL37A, OAZ1, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA.

[0182] In some instances, the first reference gene is HNRNPA2B1 and the second reference gene is selected from RPL37A, OAZ1, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB and PPIA.

[0183] In some instances, the first reference gene is RPL37A and the second reference gene is selected from OAZ1, RPLPO and HNRNPA2B1.

[0184] In some instances, the first reference gene is OAZ1 and the second reference gene is selected from RPL37A, RPLPO and HNRNPA2B1.

[0185] In some instances, the first reference gene is RPLPO and the second reference gene is selected from RPL37A, OAZ1 and HNRNPA2B1.

[0186] In some instances, the first reference gene is HNRNPA2B1 and the second reference gene is selected from RPL37A, OAZ1 and RPLPO.

[0187] The sample and reference genes used in the inventive assay are listed in Table 1 below:

[0188] Table 1

[0189]

[0190] For instance, the inventive method may include amplifying the mRNA sequences of RPL37A as the first reference gene, and 0AZ1 as the second reference gene by subjecting the respective reference primer pairs and reference hydrolysis probes to appropriate reaction conditions. Notably, the target reaction mixture and reference reaction mixture(s) may be present in different reaction vessels (single-plexing) or the same reaction vessel (multi-plexing). In some instances, multiplexing is preferred.

[0191] Biological samples

[0192] It will be appreciated that the inventive methods described herein are generally applicable to any type of biological sample. The term “biological sample” refers to any material obtained from a biological source that contains nucleic acids, proteins, cells, or other biomolecules suitable for analysis. Biological samples may be derived from human or animal subjects, or from environmental, clinical, or laboratory sources. Biological samples may be derived from donors (which may be humans or animals). Examples of biological samples include blood (such as whole blood, plasma, or serum), saliva, urine, cerebrospinal fluid, tissue biopsies, buccal swabs, sputum, amniotic fluid, cell cultures, microbial cultures, and extracellular vesicles such as exosomes. In embodiments of the present invention, the biological sample is a blood sample or tissue biopsy, collected for diagnostic, prognostic, or research purposes. In such instances, the human donor may be a patient, particularly a cancer patient.

[0193] As noted previously, the inventive method is useful for determining the expression level of a target (such as PRAME-004) in a variety of samples, typically biological samples. Many detection methods require the use of fresh frozen biopsy samples, which is often not available and thus requires scheduling of a doctor’s appointment to obtain the biopsy - a procedure that is time- and cost-consuming and inconvenient for the patient. The present method also allows the use of formalin-fixed paraffin-embedded FFPE samples, which do not require an additional biopsy. Additionally, the method is equally useful for fresh, fresh frozen, circulating tumor cells, ascites and other samples.

[0194] The inventive method is therefore particularly useful for determining relative expression levels of a target in a biological sample, such as a biopsy sample, including a fresh frozen biopsy sample or a biopsy sample in an RNA-preserving agent, or a formalin- fixed paraffin-embedded (FFPE) sample. Specifically, the patient may be a cancer patient, and the biological sample may be a tumor sample. As noted previously, if the (relative) expression level of the target exceeds a predetermined threshold, the cancer patient is eligible for targeted therapy against said target, which is preferably expressed on cancer cells.

[0195] Target peptides

[0196] In preferred embodiments of the invention, the target is a target peptide. The term “target peptide” or “peptide target” refers to a specific amino acid sequence, either naturally occurring or synthetically derived, that is recognized, bound, or otherwise specifically interacts with by a molecule of interest, such as an antibody, receptor, ligand, enzyme, or therapeutic agent. The target peptide may serve as a biomarker, epitope, substrate, or functional domain and is typically selected based on its biological relevance, structural features, or role in a physiological or pathological process. The sequence of the target peptide may be contiguous or discontinuous and may include post-translational modifications, such as phosphorylation, glycosylation, or methylation, where applicable. In preferred embodiments, the target peptide may be an antigenic peptide. The term “antigenic peptide” is used herein to refer to a short sequence of amino acids (typically 8-30 residues) that is capable of being bound or recognized by the immune system, particularly by antibodies or T-cell receptors, or derivatives thereof and preferably of eliciting an immune response. Such peptides may be derived from natural proteins, synthetic sources, or recombinant expression, and are capable of eliciting an immune response. In the context of the present invention, such peptides are typically derived from natural proteins. As previously mentioned, the preferred approach of the inventive method involves measuring the mRNA that encodes the antigenic peptides in the sample (e.g. via RT-qPCR), as this measurement serves as an indirect indicator of the amount of translated antigenic peptide present.

[0197] Preferred “antigenic peptides” are capable of being recognized by T-cell receptors or derivatives thereof. Such antigenic peptides are typically polypeptide fragments comprising a sequence of amino acids, typically ranging from 8 to 25 residues in length, that is capable of being processed and presented by a major histocompatibility complex (MHC) molecule on the surface of an antigen-presenting cell (APC) or other host cell. The antigenic peptide is recognized by a T cell receptor (TCR) on a T lymphocyte, thereby eliciting a T cell-mediated immune response. The peptide may be presented in the context of MHC class I molecules, for recognition by CD8+cytotoxic T lymphocytes, or MHC class II molecules, for recognition by CD4+helper T lymphocytes. In embodiments of the present invention, the antigenic peptide may be selected from a PRAME-004 peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 (SLLQHLIGL), which is capable of being presented by a MHC-I molecule, in particular HLA-A*02. As used herein, PRAME refers to the human PRAME (Melanoma antigen preferentially expressed in tumors) protein (UniProt Acc. No. P78395). The peptide SLLQHLIGL (SEQ ID NO: 1), also referred to herein as PRAME antigenic peptide or PRAME-004, corresponds to amino acids 425-433 of the full length PRAME protein and said peptide is presented on the cell surface in complex with an MHC molecule, in particular HLA-A*02 (Kessler et al., J Exp Med. 2001 Jan 1 ; 193(1):73- 88). PRAME is expressed in tumors but not in normal tissues. Expression of PRAME has been linked to the development and prognosis of various cancers. PRAME-derived peptides, in particular the PRAME-004 peptide ‘SLLQHLIGL’, are presented on the surface of tumor cells by molecules of the major histocompatibility complex (MHC). TCRs or TCR-derived soluble molecules targeting the PRAME-004 peptide ‘SLLQHLIGL’ are promising anti-cancer agents. A “PRAME peptide” is thus antigenic peptide derived from the PRAME protein, which is preferably 8 to 25 amino acid residues long, capable of being processed and presented by a major histocompatibility complex (MHC) molecule on the surface of an antigen-presenting cell (APC) or other host cell, and capable of being recognized by a component of the immune system, in particular a TCR. The term “PRAME peptide” includes “PRAME-004”, the antigenic peptide with the amino acid sequence according to SEQ ID NO: 1, as a preferred embodiment. The presence of PRAME-004 in a biological sample (via detection and quantification of PRAME-004 mRNA), particularly a tumor sample of a cancer patient, may be useful to determine or confirm or monitor the patient’s eligibility for PRAME- 004 targeting treatment for many types of cancer, such as uveal melanoma, ovarian cancer, head and neck cancer, lung cancer, and breast cancer, including triple negative breast cancer. The same applies to other PRAME peptides that are expressed by cancer cells.

[0198] Alternatively, the antigenic peptide may be a MAG-003 antigenic peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 2 (KVLEHVVRV). In embodiments of the present invention, the antigenic peptide may thus be selected from a MAG-003 peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 2 (KVLEHVVRV), which is capable of being presented by a MHC-I molecule, in particular HLA-A*02.

[0199] Alternatively, the antigenic peptide may be a COL6A3 antigenic peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 3 (FLLDGSANV). In embodiments of the present invention, the antigenic peptide may thus be selected from a MAG-003 peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 3 (FLLDGSANV), which is capable of being presented by a MHC-I molecule, in particular HLA-A*02.

[0200] As will be readily understood by a person skilled in the art, the methods described herein are not limited to a specific target peptide, but are broadly applicable to a wide range of target peptides. In particular, the target peptides may include, but are not limited to, antigenic peptides that are known or suspected to be expressed by tumor cells. These antigenic targets may be recognized by components of the immune system, such as T-cell receptors, antibodies, or their derivatives. The selection of such antigenic peptides may be based on their immunogenic properties, tumor-specific expression profiles, or known associations with immune recognition. These antigenic peptides represent valuable targets for therapeutic intervention, particularly in the context of treatments involving agents that are capable of binding to and acting upon such targets, including but not limited to TCR-T cells, CAR-T cells, monoclonal antibodies or T-cell engagers. In the context of the present invention, the patient is therefore typically a cancer patient. The cancer may be a solid tumor, including a metastatic, advanced, unresectable, recurrent and / or refractory solid tumor.

[0201] Uses

[0202] As noted previously, the inventive method is useful for testing or confirming a patient’s eligibility for a treatment with a targeted therapy If the patient’s sample is found to have increased expression of the target, e.g. the PRAME-004 peptide, the treatment T cell expressing a TCR against said PRAME-004 peptide or with an antigen binding protein against said PRAME-004 peptide, or with a vector encoding the TCR or antigen binding protein, can be initiated or commenced. Such treatments can include immune therapy, specifically autologous or allogeneic T-cell Receptor Therapy (TCR-T) or CAR-T. Targeted therapies may comprise or consist of treatment with therapeutic agents including an autologous or allogeneic cell, in particular a T-cell comprising a T cell receptor (TCR) or chimeric antigen receptor (CAR) capable of binding to said target; a antigen binding protein capable of binding to said target; or a vector encoding a TCR, CAR or antigen binding protein.

[0203] Therapeutic agents

[0204] In embodiments of the present invention, the method is used to test or confirm a patient’s eligibility for a treatment with a targeted therapy, e.g. a PRAME-004 targeting therapeutic agent.

[0205] A “targeted therapy” preferably includes administering a therapeutic agent targeting the target, e.g. the antigenic peptide.

[0206] A therapeutic agent “targeting” a target peptide is preferably capable of specifically binding to (or: recognizing) said target peptide, and eliciting an immune response. In the preferred context of antigenic peptides, the therapeutic agent is preferably capable of specifically binding to (recognizing) said antigenic peptide, optionally when it is being presented by (or: in complex with) a MHC class I molecule, such as HLA-A*02. The term “specifically binding” means that the therapeutic agent preferentially binds to its target peptide, and not or to a lesser extent to unrelated molecules.

[0207] In preferred embodiments, the therapeutic agent may be a T cell comprising a T cell receptor (TCR) that specifically binds to the PRAME-004 peptide, optionally in complex with HLA-A*02. The TCR may comprise the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 7, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 8, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 9, a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 10, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 11 , and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 12, wherein one or more of CDR1a, CDR2a, CDR3a, CDR1b, CDR2a and CDR3a may optionally comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0208] The TCR may comprise a variable alpha (Va) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 13. The TCR Va domain preferably comprises CDR1a, CDR2a and CDR3a. The TCR may comprise a variable beta (Vp) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 14. The TCR Vp domain preferably comprises CDR1b, CDR2b and CDR3b.

[0209] The TCR may comprise an alpha (a) chain comprising or consisting of the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 15. The TCR a chain preferably comprises the Va domain with CDR1a, CDR2a and CDR3a, and a constant domain. The TCR may comprise a beta (P) chain comprising or consisting of the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 16. The TCR p chain preferably comprises the Vp domain with CDR1b, CDR2b and CDR3b, and a constant domain.

[0210] In embodiments of the present invention, the therapeutic agent is a T cell comprising the R11 P3D3_KE TCR as disclosed in WQ2018 / 172533 A1 , or a vector encoding the R11 P3D3_KE TCR. In other embodiments, the therapeutic agent is a T cell comprising any other TCR as disclosed in WQ2018 / 172533 A1 , or a vector encoding the same.

[0211] In embodiments of the present invention, the therapeutic agent is a T cell comprising a T cell receptor targeting the target peptide, such as the PRAME-004 TCR disclosed above, and additionally comprises a heterologous CD8 molecule. To this end, the T cell may be genetically modified to include a vector encoding the TCR and a CD8 alpha and beta chain, as described in WO2020 / 243134 A1. The vector may comprise a nucleotide sequence S1 encoding a CD8 alpha chain, a nucleotide sequence S2 encoding a CD8 beta chain, a nucleotide sequence S3 encoding a TCR alpha chain, and a nucleotide sequence S4 encoding a TCR beta chain, wherein the nucleotide sequences are arranged in tandem in a 5’ to 3’ orientation selected from S1-S2-S3-S4, S1-S2-S4-S3, S2-S1-S3-S4, S2-S1-S4-S3, S3-S4-S1-S2, S3-S4-S2-S1 , S4-S3-S1-S2, and S4-S3-S2-S1 , preferably S4-S3-S2-S1. The CD8 alpha and beta chains may have any amino acid sequences known in the art. For instance, the CD8 alpha chain may comprise or consist of the amino acid sequence of SEQ ID NO: 35, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 35. The CD8 beta chain may comprise or consist of the amino acid sequence of SEQ ID NO: 36, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 36..

[0212] In further embodiments of the present invention, the therapeutic agent may be an antigen binding protein, in particular a soluble antigen binding protein. Preferably, the antigen-binding protein is a bispecific antigen-binding protein. The term “bispecific” in connection with the herein described antigen-binding proteins refers to antigen-binding proteins with at least two antigen-binding sites. In this context, it is preferred herein that at least one antigen binding sites is derived from a TCR, more particularly, that at least one antigen-binding site comprises the TCR derived CDRs as described herein. Accordingly, “bispecific” in the context of the present invention may refer to an antigen-binding protein which combines at least one antigen-binding site comprising TCR derived CDRs, and at least one further antigen-binding site, wherein said at least one further antigen-binding site, may be derived from an antibody and thus comprises antibody CDRs, or from a further TCR and thus comprises the CDRs of a further TCR, preferably said further antigen binding site, is derived from an antibody and thus comprises antibody CDRs. As mentioned before, a preferred format is the TCER® format. When it is referred to a bispecific molecule it is, however, possible that e.g. the Fc part of said molecule has an additional binding partner e.g. FcRn.

[0213] As mentioned above, the therapeutic agent may be an antigen binding protein, in particular a bispecific soluble antigen binding protein.. A first antigen binding site may bind to a PRAME antigenic peptide, more preferably an antigenic peptide according to SEQ ID NO: 1. It is preferred that the antigenic peptide is in a complex with a (human) MHC protein, preferably HLA-A*02. Said antigen binding site may be TCR derived. Accordingly, a Va and VP may form a first antigen binding site that specifically binds to a PRAME antigenic peptide according to SEQ ID NO: 1 in a complex with a human MHC protein. Corresponding variable domains and CDRs are described throughout this application.

[0214] A second antigen binding site may bind to a T cell receptor complex, preferably an alpha / beta TCR / CD3 complex. Said antigen binding site may be antibody derived. Accordingly, a VH and VL may form a second antigen binding site that specifically binds to a T cell receptor complex. Binding of the second antigen binding site that specifically binds to a T cell receptor complex may activate the T cell. Corresponding variable domains and CDRs are described throughout this application.

[0215] The antigen-binding protein may further comprise a first polypeptide forming a first antigen binding site that specifically binds to the PRAME-004 peptide, optionally in complex with HLA-A*02.

[0216] Accordingly, the antigen-binding protein may comprise a first polypeptide comprising a CDR1a comprising or consisting of the amino acid sequence according to SEQ ID NO: 37, a CDR2a comprising or consisting of the amino acid sequence according to SEQ ID NO: 38, and a CDR3a comprising or consisting of the amino acid sequence according to SEQ ID NO: 39; a CDR1b comprising or consisting of the amino acid sequence according to SEQ ID NO: 40, a CDR2b comprising or consisting of the amino acid sequence according to SEQ ID NO: 41, and a CDR3b comprising or consisting of the amino acid sequence according to SEQ ID NO: 42, wherein one or more of CDR1a, CDR2a, CDR3a, CDR1b, CDR2a and CDR3a may optionally comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0217] The first polypeptide may comprise a variable alpha (Va) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 43, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 43. The antigen binding protein Va domain preferably comprises CDR1a, CDR2a and CDR3a. The first polypeptide may comprise a beta (VP) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 44, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 44. The antigen binding protein Vp domain preferably comprises CDR1b, CDR2b and CDR3b.

[0218] The antigen-binding protein may further comprise a second polypeptide forming a second antigen binding site that specifically binds to a T cell receptor complex. Accordingly, the antigen binding protein may additionally comprise a second polypeptide comprising a CDRL1 comprising the amino acid sequence according to SEQ ID NO:45, a CDRL2 comprising the amino acid sequence according to SEQ ID NO:46, a CDRL3 comprising the amino acid sequence according to SEQ ID NO:47; a CDRH1 comprising the amino acid sequence according to SEQ ID NO: 48, a CDRH2 comprising the amino acid sequence according to SEQ ID NO: 49, and a CDRH3 comprising the amino acid sequence according to SEQ ID NO: 50; wherein one or more of CDRL1 , CDRL2, CDRL3, CDRH1 , CDRH2, and CDRH3 may comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0219] The second polypeptide may comprise an antibody variable light (VL) chain domain comprising or consisting of the amino acid sequence of SEQ ID NO: 51 , or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 51. The antigen binding protein VL domain preferably comprises CDRL1, CDRL2 and CDRL3. The second polypeptide may comprise an antibody variable heavy (VH) chain domain comprising or consisting of the amino acid sequence of SEQ ID NO: 52, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 52. The antigen binding protein VH domain preferably comprises CDRH1 , CDRH2 and CDRH3.

[0220] The antigen-binding protein may comprise or consists of a first polypeptide comprising a structure represented by the formula: VL-L1-VP [I]; wherein the first polypeptide preferably comprises or consists of the amino acid sequence of SEQ ID NO: 53, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 53; and a second polypeptide comprising a structure represented by the formula:

[0221] Va-L2-VH [II]; wherein the second polypeptide preferably comprises or consists of an amino acid sequence of SEQ ID NO: 54, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 54; and wherein L1 and L2 are linkers.

[0222] The antigen binding protein may also comprise a modification of the N-terminal and / or C-terminal amino acid(s), preferably wherein the N-terminal glutamine is substituted by a pyro-glutamate. Specifically, the therapeutic agent may be the PRAME TCER® TTP1295 as described in WO 2022 / 233956 A1 , or a vector encoding the TCER® TTP1295. In other embodiments, the therapeutic agent is another TCER® disclosed in WO 2022 / 233956 A1, or a vector encoding the same.

[0223] Alternatively, the therapeutic agent may be a T cell comprising a T cell receptor (TCR) that specifically binds to the MAG-003 peptide, optionally in complex with HLA-A*02, or may be a vector encoding said TCR.

[0224] The TCR may comprise the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 17, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 18, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 19, a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 20, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 21 , and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 22, wherein one or more of CDR1a, CDR2a, CDR3a, CDR1b, CDR2a and CDR3a may optionally comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0225] The TCR may comprise a variable alpha (Va) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 23. The TCR Va domain preferably comprises CDR1a, CDR2a and CDR3a. The TCR may comprise a variable beta (VP) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 24. The TCR Vp domain preferably comprises CDR1b, CDR2b and CDR3b.

[0226] Specifically, the therapeutic agent may be a T-cell comprising the R7P1 D5 TCR as described in WQ2017 / 158103, or a vector encoding the R7P1 D5 TCR. In other embodiments, the therapeutic agent is T-cell comprising another TCR disclosed in WQ2017 / 158103, or a vector encoding the same.

[0227] Alternatively, the therapeutic agent may be an antigen-binding molecule that specifically binds to the MAG-003 peptide, optionally in complex with HLA-A*02, or may be a vector encoding said antigen binding molecule. The antigen binding molecule may comprise a first polypeptide comprising the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 55, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 56, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 57, : a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 58, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 59, and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 60, wherein one or more of CDR1a, CDR2a, CDR3a, CDR1b, CDR2a and CDR3a may optionally comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0228] Additionally, the antigen binding molecule may comprise a second polypeptide comprising aa second polypeptide comprising a CDRL1 comprising the amino acid sequence according to SEQ ID NO:45, a CDRL2 comprising the amino acid sequence according to SEQ ID NO:46, a CDRL3 comprising the amino acid sequence according to SEQ ID NO:47; a CDRH1 comprising the amino acid sequence according to SEQ ID NO: 48, a CDRH2 comprising the amino acid sequence according to SEQ ID NO: 49, and a CDRH3 comprising the amino acid sequence according to SEQ ID NO: 50; wherein one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3 may comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0229] The first polypeptide may comprise an alpha (a) chain comprising or consisting of the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 61. The antigen binding protein alpha (a) chain preferably comprises CDR1a, CDR2a and CDR3a. The first polypeptide may comprise a beta (P) chain comprising or consisting of the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 62. The antigen binding protein chain preferably comprises CDR1b, CDR2b and CDR3b.

[0230] The second polypeptide preferably comprises

[0231] Specifically, the therapeutic agent may be the MAG-003 TCER® as described in WO 2021 / 023658 A1, or a vector encoding the MAG-003 TCER®. Alternatively, the therapeutic agent may be a T cell comprising a T cell receptor (TCR) that specifically binds to the COL6A3 peptide, optionally in complex with HLA-A*02, or may be a vector encoding said TCR.

[0232] The TCR may comprise the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 25 or 26, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 27, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 28, a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 29, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 30, and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 31 , wherein one or more of CDR1a, CDR2a, CDR3a, CDR1b, CDR2a and CDR3a may optionally comprise one, two or three amino acid mutations, wherein the mutation may be a deletion, an insertion, or a substitution, preferably a conservative substitution.

[0233] The TCR may comprise a variable alpha (Va) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 32 or 33, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 32 or 33. The TCR Va domain preferably comprises CDR1a, CDR2a and CDR3a. The TCR may comprise a variable beta (VP) domain comprising or consisting of the amino acid sequence of SEQ ID NO: 34, or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 34. The TCR Vp domain preferably comprises CDR1b, CDR2b and CDR3b.

[0234] Specifically, the therapeutic agent may be a T-cell comprising the R4P3F9 TCR (C-5 or C-14 variant) as described in WO 2019 / 086665 A1, or a vector encoding the R4P3F9 TCR (C-5 or C-14 variant). In other embodiments, the therapeutic agent is T-cell comprising another TCR disclosed in WO 2019 / 086665 A1, or a vector encoding the same.

[0235] In further embodiments, the therapeutic agent is a vector or several vectors that encode a target peptide-targeting therapeutic agent, such as a TCR or antigen-binding protein as described above. Suitable vectors are known in the art and include nucleic acids such as RNA, plasmids, and viral vectors, such as lentiviral, adenoviral or adeno-associated viral vectors. Kit

[0236] In further aspects, the invention relates to a kit for performing the method as described herein, comprising a) nucleic acid amplification reagents; b) a target primer pair being capable of hybridizing to a target (preferably PRAME-004) mRNA; c) a target hydrolysis probe being capable of hybridizing to a target (preferably PRAME-004) mRNA; d) at least a first reference primer pair capable of hybridizing to a first reference gene mRNA; e) at least a first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA; and optionally f) optionally instructions for performing the in vitro method as described herein, wherein said first reference gene is selected from the group of RPL37A, 0AZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA.

[0237] In embodiments of the invention, where a second reference gene is detected, the kit further comprises at least a second reference primer pair capable of hybridizing to a second reference gene mRNA, and at least a second reference hydrolysis probe capable of hybridizing to a second reference gene mRNA, wherein said second reference gene is preferably selected from the group of 0AZ1, RPL37A, RPLPO, PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and RPLPO.

[0238] Each one of the embodiments described in the context of the method of the invention is applicable to the inventive kit, mutatis mutandis. Specifically, the kit may comprise primers and / or probes as described in connection with the method above. The kit may be designed to detect and quantify any of the target described in the context of the inventive method. One such targets is the antigenic PRAME-004 peptide. Therefore, the kit may comprise suitable primers and probes, e.g. a forward primer that comprises or consists of at least one sequence selected from 5 -AGA GGC CGC CTG GAT CAG-3' (SEQ ID NO: 4); a reverse primer that comprises or consists of at least one sequence selected from 5-CCG GCA GTT AGT TAT TGA GAG GG-3' (SEQ ID NO: 5); and / or a hydrolysis probe that comprises or consists of at least one sequence selected from 5-TGC TCA GGC ACG TGA TGA ACC CCT TGG-3' (SEQ ID NO: 6).

[0239] The kit additionally comprises suitable primers and probes for detecting at least one reference gene as described in connection with the method above. Specifically, RPL37A is a preferred reference gene for use in the inventive kit. Therefore, in embodiments of the invention, the kit is designed to detect (by including suitable reaction agents, specifically primers and probes) 1, 2, 3 or more reference genes selected from RPL37A, and optionally either 0AZ1 or HNRNPA2B1 , or both. The kit may be designed to detect a panel of reference genes including RPL37A and 0AZ1 , RPL37A and HNRNPA2B1 , or RPL37A, 0AZ1 and HNRNPA2B1. In other embodiments, the reference genes comprise or consist of a panel of reference genes including RPL37A, 0AZ1 and RPLO. Preferred embodiments of the inventive kit are designed to determine the expression levels of the two reference genes RPL37A and 0AZ1.

[0240] In further embodiments of the present invention, the first reference gene is selected from the group of RPL37A, 0AZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, more preferably from the group consisting of RPL37A, OAZ1 , RPLPO and HNRNPA2B1 , most preferably from the group consisting of RPL37A, OAZ1 , and HNRNPA2B1. Preferably, the first reference gene may be RPL37A.

[0241] In some embodiments, the kit is designed to determine the expression level of a second reference gene. In such cases, the kit comprises a second reference primer pair that hybridizes to a reference mRNA sequence of a second reference gene.

[0242] The second reference gene is preferably selected from the group of OAZ1 , RPL37A, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, more preferably from the group consisting of OAZ1 , RPL37A, RPLPO and HNRNPA2B1 , most preferably from the group consisting of OAZ1 , RPL37A, and HNRNPA2B1.

[0243] In some instances, the first reference gene is RPL37A and the second reference gene is selected from OAZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA.

[0244] In some instances, the first reference gene is OAZ1 and the second reference gene is selected from RPL37A, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA.

[0245] In some instances, the first reference gene is RPLPO and the second reference gene is selected from RPL37A, OAZ1 , PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA. In some instances, the first reference gene is HNRNPA2B1 and the second reference gene is selected from RPL37A, OAZ1, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB and PPIA.

[0246] In some instances, the first reference gene is RPL37A and the second reference gene is selected from OAZ1, RPLPO and HNRNPA2B1.

[0247] In some instances, the first reference gene is OAZ1 and the second reference gene is selected from RPL37A, RPLPO and HNRNPA2B1.

[0248] In some instances, the first reference gene is RPLPO and the second reference gene is selected from RPL37A, OAZ1 and HNRNPA2B1.

[0249] In some instances, the first reference gene is HNRNPA2B1 and the second reference gene is selected from RPL37A, OAZ1 and RPLPO.

[0250] Finally, the invention provides the use of the inventive kit for determining the relative expression level of a target in a biological sample, and optionally - as described in connection with the inventive method above - selecting a patient for treatment with a targeted therapy; confirming the eligibility of a patient for treatment with a targeted therapy; and / or monitoring the expression level of said target peptide during treatment with a targeted therapy.

[0251] Specifically, the invention includes a kit for determining the relative expression level of a PRAME-004 peptide comprising or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), wherein said kit comprises a forward primer that comprises or consists of at least one sequence selected from 5-AGA GGC CGC CTG GAT CAG-3' (SEQ ID NO: 4); a reverse primer that comprises or consists of at least one sequence selected from 5-CCG GCA GTT AGT TAT TGA GAG GG-3' (SEQ ID NO: 5); and / or a hydrolysis probe that comprises or consists of at least one sequence selected from 5-TGC TCA GGC ACG TGA TGA ACC CCT TGG-3' (SEQ ID NO: 6), and wherein said kit additionally comprises suitable forward and reverse primers and probes for determining the expression level of RPL37A, and optionally OAZ1 and / or HNRNPA2B1. The kit can be used on a biological sample (fresh biopsy or FFPE) obtained from a cancer patient, where the cancer is known or suspected to express the PRAME peptide. The relative expression level of PRAME-004 in the patient’s cancer can be determined and compared to relative expression levels of PRAME-004 in normal tissues. If the cancer expresses the PRAME-004 peptide, the patient can be selected for treatment with a therapeutic agent targeting said target peptide; in particular a T-cell therapy (TCR-T) with T-cells comprising a T cell receptor (TCR) that specifically binds to the PRAME-004 peptide, optionally in complex with HLA-A*02. The TCR may comprise the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 7, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 8, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 9, a CDR1b comprising or consisting of the amino acid sequence of SEQ ID NO: 10, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 11, and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 12, or or for treatment with an antigen binding protein comprising a first polypeptide comprising the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence according to SEQ ID NO: 37, a CDR2a comprising or consisting of the amino acid sequence according to SEQ ID NO: 38, and a CDR3a comprising or consisting of the amino acid sequence according to SEQ ID NO: 39; a CDR1b comprising or consisting of the amino acid sequence according to SEQ ID NO: 40, a CDR2b comprising or consisting of the amino acid sequence according to SEQ ID NO: 41, and a CDR3b comprising or consisting of the amino acid sequence according to SEQ ID NO: 42, and a second polypeptide comprising a CDRL1 comprising the amino acid sequence according to SEQ ID NO:45, a CDRL2 comprising the amino acid sequence according to SEQ ID NO:46, a CDRL3 comprising the amino acid sequence according to SEQ ID NO:47; a CDRH1 comprising the amino acid sequence according to SEQ ID NO: 48, a CDRH2 comprising the amino acid sequence according to SEQ ID NO: 49, and a CDRH3 comprising the amino acid sequence according to SEQ ID NO: 50;

[0252] The TCR may comprise an alpha chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 13, and / or a beta chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 14. The soluble antigen binding protein may comprise an alpha chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 43, and / or a beta chain variable domain comprising or consisting of the amino acid sequence of SEQ ID NO: 44, and additionally an antibody variable light (VL) chain domain comprising or consisting of the amino acid sequence of SEQ ID NO: 51 , and an antibody variable heavy (VH) chain domain comprising or consisting of the amino acid sequence of SEQ ID NO: 52. Alternatively, treatment may also involve in vivo treatment with suitable vectors encoding the TCR or soluble antigen binding protein disclosed above. The kit may also be used to confirm the eligibility of a patient for treatment with these therapeutic agents, or for monitoring the expression level of the PRAME peptide during treatment with said therapeutic agents.

[0253] In further embodiments, the kit is designed to detect the MAG-003 peptide comprising or consisting of the amino acid sequence KVLEHVVRV (SEQ ID NO: 2) and comprises suitable primers and / or probes for detecting and quantifying the MAG-003 peptide.

[0254] Finally, the invention also relates to a kit for a) amplifying a MHC-presented peptide (such as PRAME) target sequence, b) determining the expression of MHC-presented peptide (such as PRAME) mRNA in a sample, c) diagnosing and / or prognosing malignant neoplastic disease in a subject before MHC-presented peptide (such as PRAME)-targeting therapy, d) predicting efficacy of treatment of malignant neoplastic disease in a subject before MHC-presented peptide (such as PRAME)-targeting therapy, e) selecting a patient for MHC-presented peptide (such as PRAME)-targeting therapy of a malignant neoplastic disease, f) assessing outcome of treatment of malignant neoplastic disease in a subject during and after MHC-presented peptide (such as PRAME)-targeting therapy, and / or g) assessing the recurrence of malignant neoplastic disease in a subject during and after MHC-presented peptide (such as PRAME)-targeting therapy.

[0255] The kit preferably comprises a) nucleic acid amplification reagents, b) a target primer pair being capable of hybridizing to target (such as PRAME) mRNA, c) a target hydrolysis probe being capable of hybridizing to target (such as PRAME) mRNA, d) at least a first reference primer pair capable of hybridizing to a first reference gene mRNA, e) at least a first reference hydrolysis probe being capable of hybridizing to a first reference gene mRNA, wherein said first reference gene is selected from the group of RPL37A, 0AZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, and f) optionally instructions for performing the in vitro method as defined herein.

[0256] The "target primer pair", which is used in the method and kit of the invention, is capable of hybridizing to a sequence of target (such as PRAME) mRNA. In particular the target sequence is the mRNA-transcript of the target (such as PRAME) mRNA sequence. It should be noted that the DNA sequence for PRAME corresponds to the transcribed mRNA sequence of said protein.

[0257] In view of the above, the inventive kit may further comprise: a) a forward target primer for determining the quantity of a target (such as PRAME) in a sample, b) a reverse target primer for determining the quantification of a target (such as PRAME) in a sample, and / or c) a hydrolysis target probe for determining the quantity of a target (such as PRAME) in a sample, wherein said forward target primer, said reverse target primer and said hydrolysis target probe comprise or consist of the sequences disclosed in Table 3.

[0258] Additionally, the inventive kit may further comprise suitable forward and reverse primers and hydrolysis probes to determine the quantity of at least one reference gene in a sample, said at least one reference gene being selected from the group of RPL37A, 0AZ1 , RPLPO, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, more preferably from the group consisting of RPL37A, 0AZ1 , RPLPO and HNRNPA2B1, most preferably from the group consisting of RPL37A, 0AZ1, and RPLPO. In preferred instances, the inventive kit further comprises suitable forward and reverse primers and hydrolysis probes to determine the quantity of RPL37A as a first reference gene and 0AZ1 as a second reference gene in the sample.

[0259] In the context of the present invention "reference primer pair" and "target primer pair" are not the same. In the context of the present invention the term "reference primer pair" is intended to mean a primer pair, which is capable of hybridizing to a sequence of a gene, which is ubiquitous to a given cell. More particularly, the "reference primer pair", which is used in the method, is capable of hybridizing to a sequence of reference gene mRNA sequence. In other words, a "reference primer pair" can be used as an internal control in a method or kit of the invention. In Real-time RT-qPCR a hybridization probe for the quantification of reference gene expression may be used as described previously in relation to the PRAME primer pair. Any reference primers and reference probes can be used as long as they reliably enable the quantification of the respective reference gene in the sample.

[0260] In further aspect the present invention also relates to a method or kit for: a) diagnosing and / or prognosing MHC-presented peptide (such as PRAME) expression in a subject before administering an MHC-presented peptide (such as PRAME) - targeting therapy, e.g. PRAME-specific binding molecules and PRAME-specific adoptive cell therapy, b) predicting efficacy of treatment of malignant neoplastic disease in a subject before adoptive cell therapy, c) selecting patients for MHC-presented peptide (such as PRAME) -targeted therapy of a malignant neoplastic disease, and / or d) assessing outcome of treatment of malignant neoplastic disease in a subject during and after adoptive MHC-presented peptide (such as PRAME) -targeting cell therapy, and e) assessing the recurrence of malignant neoplastic disease in a subject during and after MHC-presented peptide (such as PRAME) -targeting therapy,

[0261] Indications

[0262] As noted above, the inventive methods and kits are particularly useful for predicting treatment efficacy and selecting patients for PRAME-targeting therapy of a malignant neoplastic diseases. The term “cancer” and “malignant neoplastic disease” are used interchangeably herein.

[0263] In embodiments of the invention, the cancer is a metastatic cancer and / or advanced cancer and / or unresectable cancer and / or recurrent cancer and / or refractory cancer. In some embodiments of the invention, the cancer patients have received at least one, two, three, four or more lines of cancer therapy, preferably standard-of-care cancer therapy.

[0264] Generally, the inventive method and kit are particularly useful in cases where a subject suffers from a cancer where fewer than 90%, preferably 85% or fewer, more preferably 80% or fewer of subjects are considered “target-positive”. “Target-positive” (e.g., PRAME-004 positive) means that a patient expresses sufficient numbers of target molecules to justify the administration of a targeted therapy directed against said target. In preferred embodiments of the invention, the cancer is selected from acral melanoma; acute lymphocytic cancer; acute myeloid leukemia (AML); adenocarcinoma nonsmall cell lung carcinoma (adenocarcinoma NSCLC); adenoid cystic carcinoma; adenosquamous NSCLC; adrenocortical carcinoma; alveolar rhabdomyosarcoma; anal canal cancer; anaplastic thyroid carcinoma; and cutaneous melanoma; angiosarcoma; anorectum cancer; anus cancer; ardenocarcinoma; basal cell carcinoma; bladder cancer; bladder urothelial carcinoma; bone cancer; brain cancer; breast cancer; breast carcinoma; cervical adenocarcinoma; cervical adenosquamous cell carcinoma; cervical cancer; cervical carcinoma; cervical squamous cell carcinoma; cervical squamous cell carcinoma and adenocarcinoma; cholangiocarcinoma; chronic lymphocytic leukemia; chronic myeloid cancer; colon cancer; cutaneous melanoma; cutaneous Melanoma (CM); cutaneous squamous cell carcinoma; diffuse large B-cell lymphoma (DLBCL); embryonal rhabdomyosarcoma; endocervical adenocarcinoma; endometrial clear cell carcinoma; endometrial sarcoma; endometrial serous carcinoma; endometrioid endometrial carcinoma; endometrioid epithelial ovarian cancer (EOC); esophageal adenocarcinoma; esophageal adenosquamous carcinoma; esophageal cancer; esophageal carcinoma; esophageal small cell carcinoma; esophageal squamous cell carcinoma; Ewing sarcoma; eye cancer; fibrosarcoma; gallbladder cancer; gastrointestinal carcinoid tumor; glioma; head & neck squamous cell carcinoma (HNSCC); head and neck adenocarcinoma; head and neck cancer; head and neck salivary duct carcinoma; head and neck salivary gland carcinoma; head and neck squamous cell carcinoma (HNSCC); hepatocellular carcinoma (HCC); Her2- enriched breast carcinoma; Hodgkin lymphoma; hypopharynx cancer; intrahepatic bile duct cancer; joint cancer; kidney cancer; kidney carcinoma; kidney renal clear cell carcinoma; kidney renal papillary cell carcinoma; large cell lung carcinoma (LCLC); large cell neuroendocrine lung carcinoma (LCNEC); larynx cancer; liposarcoma; liver cancer; luminal a breast carcinoma; luminal b breast carcinoma; lung cancer; malignant mesothelioma; malignant peripheral nerve sheath tumor (MPNST); malignant rhabdoid tumor; melanoma; Merkel cell carcinoma (MCC); middle ear cancer; mucoepidermoid carcinoma; mucosal melanoma; multiple myeloma; myxoid liposarcoma; nasal cavity; nasopharynx cancer; neck cancer; neuroblastoma; non-Hodgkin lymphoma; non-small cell lung adenocarcinoma; nonsmall cell lung cancer (NSCLC); non-squamous anal carcinoma; non-squamous cell Non- small cell lung cell carcinoma; nose cancer; omentum cancer; oral cavity cancer; oropharynx cancer; osteosarcoma; other squamous cell carcinomas; ovarian cancer; ovarian carcinoma; ovarian clear cell carcinoma; ovarian endometrioid carcinoma; ovarian leiomyosarcoma; ovarian mucinous carcinoma; ovarian serous cystadenocarcinoma; ovarian serous carcinoma; ovarian tube cancer (fallopian tube cancer); pancreatic cancer; pancreatic neuroendocrine adenocarcinoma; penis cancer; peritoneum cancer; pharynx cancerprostate cancer; pleura cancer; primary peritoneal cancer (PPC); prostate neuroendocrine adenocarcinoma; rectal cancer; renal cancer; sarcoma; sarcomatoid carcinoma of the lung; skin cancer; skin melanoma; small cell lung cancer (SCLC); small intestine cancer; soft tissue cancer; spindle cell melanoma; squamous anal carcinoma; squamous cell Non-small cell lung cell carcinoma; squamous cell NSCLC; squamous non-small cell lung cancer (sqNSCLC); stomach adenocarcinoma; stomach cancer; synovial sarcoma; testicular cancer; testicular germ cell tumor (seminoma and non-seminoma); thymoma; thyroid cancer; triple-negative breast cancer (TNBC); undifferentiated pleomorphic sarcoma; ureter cancer; urinary bladder cancer; uterine carcinosarcoma; uterine carcinosarcoma (UCS); uterine cervix cancer; uterine corpus cancer (UCC); uterine endometrial carcinoma; uterus cancer; uveal melanoma (UM); vagina cancer; vulva cancer; well differentiated NET; and Wilms tumor (nephroblastoma).

[0265] In embodiments of the invention, the cancer is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, including triple negative breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, head and neck cancer, malignant mesothelioma, melanoma, including uveal melanoma and skin melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, including uterine endometrial carcinoma and uterine carcinosarcoma, ureter cancer, urinary bladder cancer, cancer of the uterine cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis, preferably a PRAME expressing cancer selected from uveal melanoma, ovarian cancer, head and neck cancer, lung cancer, and breast cancer, in particular triple negative breast cancer.

[0266] In further embodiments of the invention, the cancer is selected from Adrenocortical carcinoma; Ardenocarcinoma; Bladder cancer, including Bladder urothelial carcinoma; Breast cancer, including Breast carcinoma and Triple-negative breast cancer (TNBC); Cervical cancer, including Cervical carcinoma, Cervical squamous cell carcinoma and endocervical adenocarcinoma; Cholangiocarcinoma; Esophageal cancer, including Esophageal carcinoma; Head and neck cancer, including Head and neck adenocarcinoma and Head and neck squamous cell carcinoma (HNSCC); Hepatocellular cancer, including Hepatocellular carcinoma (HCC); Kidney cancer, including Kidney carcinoma, Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma; Lung cancer, including Non-small cell lung cancer (NSCLC), including squamous cell Non-small cell lung cell carcinoma, non- squamous cell Non-small cell lung cell carcinoma and Non-small cell lung adenocarcinoma; Melanoma, including Cutaneous Melanoma (CM), Acral Melanoma, Uveal Melanoma (UM), Mucosal Melanoma, and Melanoma of unknown primary origin; Ovarian cancer, including Ovarian carcinoma, Ovarian serous cystadenocarcinoma, Ovarian tube cancer (Fallopian tube cancer), endometrioid epithelial ovarian cancer (EOC) and Primary peritoneal cancer (PPC); Sarcoma, including Synovial sarcoma; Small cell lung cancer (SLC); and Uterine cancer, including Uterine carcinosarcoma (UCS), Uterine endometrial carcinoma, Uterine corpus cancer (UCC), and Uterine sarcoma.

[0267] In further embodiments of the invention, the cancer is selected from adrenocortical carcinoma, adenoid cystic carcinoma, non-squamous anal carcinoma, squamous anal carcinoma, basal cell carcinoma, bladder urothelial carcinoma, Her2-enriched breast carcinoma, luminal a breast carcinoma, luminal b breast carcinoma, triple negative breast carcinoma (TNBC), cervical adenocarcinoma, cervical adenosquamous cell carcinoma, cervical squamous cell carcinoma, cholangiocarcinoma, diffuse large B-cell lymphoma (DLBCL), esophageal adenocarcinoma, esophageal adenosquamous carcinoma, esophageal small cell carcinoma, esophageal squamous carcinoma, head and neck squamous cell carcinoma (HNSCC), kidney renal clear cell carcinoma, kidney renal papillary cell carcinoma, malignant rhabdoid tumor, Wilms tumor (nephroblastoma), acute myeloid leukemia (AML), hepatocellular carcinoma, large cell lung carcinoma (LCLC), large cell neuroendocrine lung carcinoma (LCNEC), adenocarcinoma non-small cell lung carcinoma (adenocarcinoma NSCLC), squamous cell NSCLC, adenosquamous NSCLC, sarcomatoid carcinoma of the lung, small cell lung cancer (SCLC), acral melanoma, mucosal melanoma, cutaneous melanoma, spindle cell melanoma, uveal melanoma, Merkel cell carcinoma (MCC), mucoepidermoid carcinoma, neuroblastoma, ovarian clear cell carcinoma, ovarian endometrioid carcinoma, ovarian leiomyosarcoma, ovarian mucinous carcinoma, ovarian serous cystadenocarcinoma, pancreatic neuroendocrine adenocarcinoma, prostate neuroendocrine adenocarcinoma, head and neck salivary duct carcinoma, head and neck salivary gland carcinoma, malignant peripheral nerve sheath tumor (MPNST), alveolar rhabdomyosarcoma, angiosarcoma, Ewing sarcoma, fibrosarcoma, liposarcoma, myxoid liposarcoma, osteosarcoma, embryonal rhabdomyosarcoma, synovial sarcoma, undifferentiated pleomorphic sarcoma, cutaneous squamous cell carcinoma, stomach adenocarcinoma, testicular germ cell tumor (seminoma and non-seminoma), anaplastic thyroid carcinoma, thymoma, endometrial clear cell carcinoma, endometrial sarcoma, endometrioid endometrial carcinoma, endometrial serous carcinoma, and uterine carcinosarcoma.

[0268] In embodiments of the invention where, the target peptide is PRAME-004, and the therapeutic agent is a T cell comprising a TCR targeting the PRAME-004 peptide as disclosed herein (e.g., the R11 P3D3_KE TCR as disclosed in WO2018 / 172533 A1), the cancer may be a solid tumor, including a metastatic, advanced, unresectable, recurrent and / or refractory solid tumor. Alternatively, the therapeutic agent can be a vector encoding said TCR

[0269] In embodiments, and in particular when the target peptide is PRAME-004, and the therapeutic agent is a T cell comprising a TCR targeting the PRAME-004 peptide as disclosed herein (e.g., the R11 P3D3_KE TCR as disclosed in WO2018 / 172533 A1) and a heterologous CD8, the cancer may be selected from a solid tumor, including a metastatic, advanced, unresectable, recurrent and / or refractory solid tumor. Alternatively, the therapeutic agent can be a vector encoding said TCR and CD8 molecule.

[0270] In embodiments where the target peptide is PRAME-004, and the therapeutic agent is an antigen-binding molecule targeting the PRAME-004 peptide as disclosed herein (e.g., the TTP1295 TCER® as disclosed in WO 2022 / 233956 A1), the cancer may be a solid tumor, including a metastatic, advanced, unresectable, recurrent and / or refractory solid tumor. . Alternatively, the therapeutic agent can be a vector encoding said TCER.

[0271] In embodiments where the target peptide is MAG-003, and the therapeutic agent is a MAG-003 TCER® as described in WO 2021 / 023658 A1, the cancer may be a solid tumor, such as a metastatic, advanced, unresectable, recurrent and / or refractory solid tumor.

[0272] When selecting patients for a PRAME-004 directed therapy, the method comprises the steps of: a) performing the inventive in vitro method for quantification of the PRAME-004 mRNA in a sample as described herein, b) determining the amount of PRAME-004 mRNA in a said sample, c) normalizing the expression of PRAME-004 with respect to the reference gene expression, d) classifying a patient as “eligible” for a PRAME-004 targeting therapy if the amount of PRAME-004 mRNA exceeds a predetermined threshold, e.g. as disclosed in Fritsche J et al. Translating Immunopeptidomics to Immunotherapy-Decision-Making for Patient and Personalized Target Selection. Proteomics. 2018 Jun;18(12):e1700284. doi: 10.1002 / pmic.201700284. Epub 2018 Apr 10. PMID: 29505699; PMCID: PMC6032917. The same applies to any other target of interest.

[0273] The invention also relates to a method for selecting a patient for treatment with a targeted therapy, or for confirming a patient for eligibility for treatment with a targeted therapy comprising a) obtaining a sample from said patient, b) determining the relative expression level of said target, c) determining the expression levels of at least one reference gene selected from RPL37A, OAZ1, RPLP0, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, d) normalizing the expression level of said target against the expression level(s) of said at least one reference gene(s), e) comparing the expression level of said target to a predetermined threshold, and selecting the patient for treatment if the expression level of said target exceeds said threshold.

[0274] The invention thus also relates to a method of for treating a patient with a targeted therapy, comprising a) obtaining a sample from said patient, b) determining the relative expression level of said target, c) determining the expression levels of at least one reference gene selected from RPL37A, OAZ1, RPLP0, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, d) normalizing the expression level of said target against the expression level(s) of said at least one reference gene(s), e) comparing the expression level of said target to a predetermined threshold, and selecting the patient for treatment if the expression level of said target peptide exceeds said threshold.

[0275] Furthermore, the invention also relates to the kit of for use in treating a patient with targeted therapy, comprising a) obtaining a sample from said patient, b) determining the relative expression level of said peptide, c) determining the expression levels of at least one reference gene selected from RPL37A, OAZ1 , RPLP0, PUM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, d) normalizing the expression level of said target against the expression level(s) of said at least one reference gene(s), e) comparing the expression level of said target to a predetermined threshold, and selecting the patient for treatment if the expression level of said target peptide exceeds said threshold.

[0276] Suitable methodologies and therapeutic agents are disclosed above.

[0277] BRIEF DESCRIPTION OF FIGURES Fig. 1 shows the results of a variance component analysis (VGA) in (A) FFPE and (B) FF samples. The least variable single, two-gene, three-gene, and four-gene combinations are shown. Red bars indicate the variability within groups (indications), cyan bars indicate the variability between groups.

[0278] Fig. 2 shows PRAME-004 expression (dCt) results in 12 FFPE samples using 7 different RNA inputs each.

[0279] Fig. 3 shows PRAME-004 expression (2-DCt) normalized to RPL37A and OAZ1. 114 FFPE samples were analysed (102 tumor samples and 12 normal tissue samples). 18 samples were found to be “inconclusive” due to reference gene Ct values above LOQ. Most of the 84 tumor samples are positive for PRAME-004. All 12 normal tissue samples are negative for PRAME-004.

[0280] SEQUENCES

[0281]

[0282]

[0283]

[0284]

[0285] CDR = complementarity determining region; TCR = T cell receptor

[0286] MATERIALS AND METHODS

[0287] Indications and samples

[0288] A total of 13 tumor indications were analyzed in three different databases (XPRESIDENT®, TCGA and MET500) and compared to normal tissue (adipose, adrenal gland, blood cells, blood vessel, bone, bone marrow, brain, breast, esophagus, eye, head and neck, heart, kidney, large intestine, liver, lung, lymph node, ovary, pancreas, peripheral nerve, peritoneum, pituitary, placenta, pleura, prostate, skeletal muscle, skin, small intestine, spinal cord, spleen, stomach, thymus, thyroid gland, trachea, urinary bladder, uterus).

[0289] Primer and probe designs

[0290] Primers and hydrolysis probes for PRAME and the selected reference genes were de novo in-silico designed. In-silico design was performed by using Beacon Designer v8 and the designs were verified against NCBI Blast. Assays were designed to result in exonspanning amplicons to mitigate the risk of genomic DNA interference with the assay result, low amplicon size, high in silico (BLAST) specificity to the target region, minimal degree of self-dimerization, cross-dimerization and hairpins within and between primer sets, and other design criteria (annealing temperature, melting temperature of maximum + / - 1.5°C between forward and reverse primers, GC content between 30-70%, GO clamp on the 3’ end of each primer, mononucleotide repeat of <= 4, avoiding common SNPs within primer binding sites, primer sequence length between 16-24 nucleotides). Suitable hydrolysis probes were included.

[0291] RT-qPCR assay

[0292] RT-qPCR reaction was performed in the QuantStudio 5 DX instrument (Thermo Fisher Scientific) The cycling protocol was identical for both PRAME and the reference genes: 1) 50°C for 2 min, 2) 95°C for 10 min, 3) 95°C for 15 sec, 4) 6 0°C for 1 min 5) Imaging. Step 3, 4 and 5 are repeated in 40 cycles.

[0293] EXAMPLE 1 Pre-selection of candidate reference genes

[0294] The Immatics XPRESIDENT® database, and the publicly accessible TCGA and MET500 databases were analysed to identify suitable reference gene candidates with low variability of expression and high expression levels. Up to 13 cancer indications (as available per database) as well as samples from 37 normal tissues from the Immatics XPRESIDENT® database were included in the analysis. Only genes with a median transcript-per-million (TPM) >20 in all databases and without missing values were considered. Two thousand one hundred thirty-nine (2139) genes were selected for further analysis.

[0295] Final reference gene candidates were chosen by assessing the CV within each entity (indication) of interest (solid tumor indications as well as normal tissues; ideally CV <100% per entity) and the ratio of the median expression within each entity vs. the global median expression (ideally between 0.5 and 2, i.e., at most a two-fold difference). Final reference gene candidates to be processed in step 2 were: PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1, OAZ1, RPLPO, RPL37A, and PPIA.

[0296] EXAMPLE 2: Design of primers and probes and selection of preferred primers and probes by amplification efficiency testing

[0297] For each of the reference gene candidates and for the target PRAME, up to 5 primer and probe sets (also referred to as assays) were designed. Assays were designed to result in exon-spanning amplicons to mitigate the risk of genomic DNA interference with the assay result, low amplicon size, high in silico (BLAST) specificity to the target region, minimal degree of self-dimerization, cross-dimerization and hairpins within and between primer sets, and other design criteria One or two sets of primers and probes were selected per gene based on testing for suitable amplification efficiency (85-115% efficiency), specificity (in terms of resulting in a single band in the bioanalyzer), potential difference between observed and expected product size (< 15%), and ideally low Ct values at a given cDNA input.

[0298] EXAMPLE 3: Selection of best performing reference genes in RT-qPCR (Reference gene study):

[0299] The selected primer probe sets (also referred to as assays) for PRAME and the reference genes were analysed by RT-qPCR in fresh frozen (FF) and FFPE samples of 10 representative tumor indications. Matched pairs of FF and FFPE (i.e., FF and FFPE derived from the same donor and tumor lesion) were used in order to enable comparison between RT-qPCR results on both sample types. Reference genes candidates with low Ct values (NONO, HSP90AB1, ACTB, RPLPO, RPL37A, OAZ1 , and HNRNPA2B1) were analysed by RT-qPCR. As shown in the tables below, mean expression (Ct) was calculated for each assay and variance component analysis (VCA) was performed for all single genes and gene combinations (up to 4 genes), with gene expression as outcome and “group” (disease indication) as predictor (random factor): Figure 1 shows the stability of expression of reference genes and combinations thereof, in ranked order. Total variability is composed of “between group variability” (between disease indications) and “within group variability” (residual error).

[0300] Table 2: Reference gene assays ranked by mean epression)

[0301] Reference genes were selected based on the following:

[0302] 1) Stability of expression (Figure 1): The best stability of expression was observed for OAZ1_4 in FFPE and HNRNPA2B1_6 in FF, and their combination was the second best performing in both sample types. However, 95% Confidence intervals (Cis) for the best performing sets were overlapping with Cis of other combinations, implying that other reference genes can be considered, e.g. based on their higher expression.

[0303] 2) Expression level (Table 2): RPL37A, OAZ1 , RPLPO, and HNRNPA2B1 resulted in the lowest Ct values (highest expression) in FFPE samples. Expression levels in FF samples were acceptably high for all genes tested. Thus, these 4 genes were defined as preferred reference genes regarding expression level. 3) Number of reference genes: Due to the ranking order (with one or two reference genes being ranked higher than combinations of more reference genes) and the overlapping Cis in the VGA (Figure 1), it can be concluded that surprisingly, there is no advantage of choosing 3 or even 4 reference genes rather than 1 or 2. Choice of less reference genes is favourable as the final assay is to be multiplexed (target and reference genes combined in the same well). Using state of the art equipment, multiplexing of up to 4 dyes I genes is technically feasible, i.e. up to 3 reference genes can be combined with the target gene. However, from experience, choice of less dyes is favourable for successful multiplexing. Altogether, a combination of 2 reference genes was considered to be ideal. Based on the data, even a single reference gene is sufficient for normalization of target expression; a second gene may add a level of safety.

[0304] Thus, finally selected reference gene assays to be processed in step 3 (as combinations of 2) were: RPL37A, OAZ1 , HNRNPA2B1. Other reference genes listed in Table 2 were considered as back-up in case multiplexing was not possible using the selected reference gene assays.

[0305] EXAMPLE 4: Final selection of reference genes by multiplexing efficiency testing:

[0306] The selected primer-probe sets for PRAME and the reference genes were synthesized with different fluorophores in order to combine PRAME with 2 of the reference genes as multiplex assay. 4 sets of primers and probes were tested for amplification efficiency:

[0307] PRAME:Dye_1 combined with

[0308] (1) OAZ1 :Dye_1and HNRNPA2B1:Dye_3,

[0309] (2) OAZ1 :Dye_1and RPL37A:Dye_3,

[0310] (3) HNRNPA2B1:Dye_1and RPL37A:Dye_3, and

[0311] (4) RPL37A:Dye_1 Dye_3.

[0312] For amplification efficiency testing, dilution series of cDNA were prepared from 3 independent samples. cDNA was diluted to obtain a cDNA input per well of 32, 6.4, 1.28, 0.256, 0.0512, and 0.0102 ng. From the slope of the dilution curve (cDNA concentration on a log-scale as independent variable, and Ct values as dependent variable), amplification efficiency was calculated.

[0313] The 4 panels were analysed to select those which showed acceptable amplification efficiency and low difference between Ct values between multiplex and singleplex condition (i.e. all primer probe sets run in separate wells). Based on these criteria, all 4 combinations showed acceptable performance. Panel 4 was selected based on slight superiority over the 3 other panels in terms of amplification efficiency. EXAMPLE 5: Multiplexing verification

[0314] The selected multiplex assay (Panel 4), consisting of primer probe sets for PRAME, 0AZ1 , and RPL37A, was compared to the singleplex condition of each assay based on analysis of 15 matched FF and FFPE samples spanning the dCt range. The difference in dCt values between singleplex and multiplex condition (ddCt) ranged between -0.38 and 0.14, showing very minimal bias associated with the switch to the multiplex condition at the dCt level.

[0315] EXAMPLE 6: RNA I cDNA input range:

[0316] The RNA I cDNA input range was assessed, as use of lower inputs may be needed for some FFPE samples, for which yield from RNA isolation can be low. For that purpose, 12 FFPE tissue resections from multiple tumor indications (skin melanoma, uveal melanoma, lung cancer, triple negative breast cancer, ovarian cancer, HNSCC, uterine cancer) were run at 7 inputs each: RNA input into Reverse Transcription (RT) at 256 ng, 128 ng, 80 ng, 60 ng, 40 ng, 30 ng, and 20 ng. The corresponding cDNA inputs in the qPCR (per well) were: 64 ng, 32 ng, 20 ng, 15 ng, 10 ng, 7.5 ng, and 5 ng.

[0317] Figure 2 shows that dCt values were stable independent of the RNA input used. Thus, all RNA I cDNA inputs tested yielded comparable expression levels. The only sample showing higher variation of dCt values for different inputs had very low levels of PRAME, such that the PRAME Ct value was above its LoQ Ct. As Ct values above LoQ are by definition associated with higher imprecision, this result is expected. Overall, the assay can be run at low cDNA inputs, ranging at least from 5 ng to 256 ng, allowing for analysis of samples with very low RNA yield.

[0318] EXAMPLE 7: PRAME-004 expression was analysed as described above in 114 FFPE samples of various tumor indication and normal tissue. Results are shown in FIG. 3.

[0319] ASPECTS

[0320] In further aspects, the invention is defined as follows:

[0321] 1. An in vitro method for determining the presence of a peptide target in a sample, said method comprising the steps:

[0322] 1. subjecting the sample to reverse transcription using mRNA present in the sample as a template to synthesize a corresponding cDNA sequence, ii. forming a target reaction mixture comprising the sample, nucleic acid amplification reagents, a target primer pair, a target hydrolysis probe, said target primer pair and target hydrolysis probe being capable of hybridizing to a target mRNA sequence, iii a) forming at least a first reference reaction mixture comprising the sample, a first reference primer pair and a first reference hydrolysis probe, said first reference primer pair and first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA sequence, iv. subjecting the target reaction mixture and the at least one first reference reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, said target sequence corresponding to a target mRNA sequence, and at least one copy of a nucleic acid sequence complementary to a first reference gene mRNA sequence, and v. determining the amount of target mRNA and of the first reference gene mRNA in a said sample.

[0323] 2. The method according to aspect 1 , wherein said peptide target is an M Represented peptide target.

[0324] 3. The method according to any one of the preceding aspects, wherein said target is an MHC-presented PRAME peptide, preferably PRAME-004 and has the sequence SLLQHLIGL.

[0325] 4. The method according to any one of the preceding aspects, wherein said first reference gene is selected from the group of RPL37A, 0AZ1 , RPLPO, PLIM1, RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA.

[0326] 5. The method according to any one of the preceding aspects, further comprising a step of iii b) forming a second reference reaction mixture comprising the sample, a second reference primer pair and a second reference hydrolysis probe, said second reference primer pair and second reference hydrolysis probe capable of hybridizing to a second reference gene mRNA sequence, wherein said second reference gene is preferably selected from the group of OAZ1, RPL37A, RPLPO, PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA.

[0327] 6. The method of aspect 5, further comprising a step of iii c) forming a third reference reaction mixture comprising the sample, a third reference primer pair and a third reference hydrolysis probe, said third reference primer pair and third reference hydrolysis probe capable of hybridizing to a third reference gene mRNA sequence, wherein said third reference gene is preferably selected from the group of RPLPO, RPL37A, OAZ1, PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA.

[0328] 7. The method according to any one of the preceding aspects, wherein said first, and optionally said second and third reference gene is selected from the group consisting of NONO, HSP90AB1 , ACTB, RPLPO, RPL37A, OAZ1, and HNRNPA2B1, more preferably from the group consisting of RPL37A, OAZ1, RPLPO and HNRNPA2B1.

[0329] 8. The method according to aspect 7, wherein said first reference gene is RPL37A and said second reference gene is OAZ1 or HNRNPA2B1.

[0330] 9. The method according to any one of the preceding aspects, wherein said target reaction mixture and said first and optionally said second and third reference reaction mixture are in the same reaction vessel.

[0331] 10. The method according to any one of the preceding aspects, wherein the sample is a biological sample, optionally a biopsy sample optionally selected from a fresh frozen biopsy sample or a biopsy sample in an RNA-preserving agent, or a formalin-fixed paraffin-embedded (FFPE) sample.

[0332] 11. The method according to any one of the preceding aspects, wherein the forward target primer and the reverse target primer comprises a sequence as shown in Table 3.

[0333] 12. The method according to any one of the preceding aspects, wherein the target hydrolysis probe and / or any of the reference hydrolysis probes comprises fluorescent particles.

[0334] 13. The method according to any one of the preceding aspects, for: a) diagnosing and / or prognosing PRAME expression in a subject before administering a PRAME-targeting therapy, including PRAME-specific binding molecules and PRAME-specific adoptive cell therapy, b) predicting efficacy of treatment of malignant neoplastic disease in a subject before adoptive cell therapy, c) selecting patients for PRAME-targeting therapy of a malignant neoplastic disease, d) assessing outcome of treatment of malignant neoplastic disease in a subject during and after PRAME-targeting therapy, and / or e) assessing the recurrence of malignant neoplastic disease in a subject during and after PRAME-targeting therapy, wherein the subject is a mammal, having or is suspected of having a malignant neoplastic disease.

[0335] 14. The method according to aspect13, wherein the malignant neoplastic disease is selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, including triple negative breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vagina, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, glioma, Hodgkin lymphoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer, head and neck cancer, malignant mesothelioma, melanoma, including uveal melanoma and skin melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, cancer of the oropharynx, ovarian cancer, cancer of the penis, pancreatic cancer, peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, cancer of the uterus, including uterine endometrial carcinoma and uterine carcinosarcoma, ureter cancer, urinary bladder cancer, cancer of the uterine cervix, oropharynx, anus, anal canal, anorectum, vagina, vulva, or penis, preferably a PRAME expressing cancer selected from uveal melanoma, ovarian cancer, head and neck cancer, lung cancer, and breast cancer, including triple negative breast cancer. 15. A kit for performing the method of any one of the preceding aspects, comprising a) nucleic acid amplification reagents, b) a target primer pair being capable of hybridizing to a peptide, preferably PRAME, mRNA, c) a target hydrolysis probe being capable of hybridizing to peptide, preferably PRAME, mRNA, d) at least a first reference primer pair capable of hybridizing to a first reference gene mRNA, e) at least a first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA, wherein said first reference gene is selected from the group of RPL37A, 0AZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, and f) optionally instructions for performing the in vitro method as defined in aspects 1-14.

[0336] 16. The kit according to aspect 15, further comprising at least a second reference primer pair capable of hybridizing to a second reference gene mRNA, and at least a second reference hydrolysis probe capable of hybridizing to a second reference gene mRNA, wherein said second reference gene is preferably selected from the group of 0AZ1 , RPL37A, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and RPLPO.

[0337] 17. A T cell expressing a T cell receptor capable of specifically binding to PRAME-004 presented on an MHC I protein for use in the treatment of cancer, wherein said cancer comprises cells characterized by the presence of a PRAME target on their surface, wherein said presence is determined by a method according to any one of aspects 1 to 14.

[0338] In additional aspects, the invention is also described by the following aspects:

[0339] 1. An in vitro method for determining the presence of a target mRNA sequence in a sample, said method comprising the steps: i. subjecting the sample to reverse transcription using mRNA present in the sample as a template to synthesize a corresponding cDNA sequence, ii. forming a target reaction mixture comprising the sample, nucleic acid amplification reagents, a target primer pair, a target hydrolysis probe, said target primer pair and target hydrolysis probe being capable of hybridizing to a target mRNA sequence, iii a) forming at least a first reference reaction mixture comprising the sample, a first reference primer pair and a first reference hydrolysis probe, said first reference primer pair and first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA sequence, iv. subjecting the target reaction mixture and the at least one first reference reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, said target sequence corresponding to a target mRNA sequence, and at least one copy of a nucleic acid sequence complementary to a first reference gene mRNA sequence, and v. determining the amount of target mRNA and of the first reference gene mRNA in a said sample.

[0340] 2. The method according to embodiment 1, wherein said target mRNA sequence encodes a MHC-presented peptide target.

[0341] 3. The method according to embodiment 1, wherein said target mRNA sequence encodes an MHC-presented PRAME peptide, preferably PRAME-004 having the sequence SLLQHLIGL.

[0342] 4. The method according to any one of the preceding embodiments, wherein said first reference gene is selected from the group of RPL37A, 0AZ1, RPLPO, PLIM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1, ACTB, HNRNPA2B1 and PPIA.

Claims

CLAIMS1 . An in vitro method for determining the relative expression level of a target in a biological sample, comprising a) determining the expression level of said target, b) determining the expression levels of at least one reference gene selected from RPL37A, 0AZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, and c) normalizing the expression of said target against the expression level(s) of said at least one reference gene(s).

2. The method of claim 1 , further comprising comparing the relative expression level of said target with a predetermined threshold.

3. The method of claim 2, wherein, if the relative expression level of said target exceeds said threshold, said method further comprises one or more steps of selecting the sample donor for treatment with a targeted therapy; and / or confirming the eligibility of the sample donor for treatment with a targeted therapy.

4. The method of any one of the preceding claims, wherein said method further comprises monitoring the relative expression level of said target during treatment with a targeted therapy.

5. The method of any one of the preceding claims, wherein said method includes determining the expression levels of 3, 2 or 1 reference genes.

6. The method of any one of the preceding claims, wherein said at least one reference gene is selected from RPL37A, 0AZ1 , HNRNPA2B1 , RPLO, NONO, HSP90AB1 , and ACTB.

7. The method of any one of the preceding claims, wherein said reference genes comprise or consist of RPL37A and either 0AZ1 or HNRNPA2B1 , or both; or wherein said reference genes comprise or consist of 0AZ1 and either RPL37A or HNRNPA2B1 , or both.

8. The method of any one of the preceding claims, wherein the expression level of only one reference gene is determined, and wherein said reference gene is RPL37A or 0AZ1.

9. The method of any one of the preceding claims, wherein said expression levels are determined by measuring a) the mRNA or b) the corresponding peptide or protein of said target and / or reference gene(s) in said sample.

10. The method of claim 9, wherein the mRNA expression level of said target and reference gene(s) is measured by using primers and / or probes that specifically bind to the sequences of said target and reference gene(s) and / or wherein the level of protein expression is measured by using an antibody specific to the corresponding protein of said markers.11 . The method of any one of the preceding claims, wherein said method is polymerase chain reaction (PCR), a DNA or RNA array, a nucleotide hybridization technique, western blot, in situ hybridization, ELISA, immunohistochemistry or a protein array, preferably PCR, in particular real time RT-qPCR, or digital PCR.

12. The method of claim 11 , wherein said method is RT-qPCR, and wherein the expression levels of said target and said reference gene(s) are preferably measured simultaneously in the same reaction vessel.

13. The method of claim 12, wherein said method determines the expression levels of said target and said at least one reference gene by:1 ) subjecting the biological sample to reverse transcription using mRNA present in the sample as a template to synthesize a corresponding cDNA sequence,2 a), forming a target reaction mixture comprising the sample, nucleic acid amplification reagents, a target primer pair, atarget hydrolysis probe, said target primer pair and target hydrolysis probe being capable of hybridizing to a target mRNA sequence,2 b) forming at least a first reference reaction mixture comprising the sample, a first reference primer pair and a first reference hydrolysis probe, said first reference primer pair and first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA sequence,2 c) subjecting the target reaction mixture and the at least one first reference reaction mixture to amplification conditions optimized to generate at least one copy of a nucleic acid sequence complementary to a target sequence, said target sequence corresponding to a target mRNA sequence, and at least one copy of a nucleic acid sequence complementary to a first reference gene mRNA sequence, and3) determining the amount of target mRNA and of the first reference gene mRNA in said sample.

14. The method of any one of the preceding claims, wherein said biological sample is patient’s sample, optionally selected from a biopsy sample, including a fresh frozen biopsy sample or a biopsy sample in an RNA-preserving agent, or a formalin-fixed paraffin-embedded (FFPE) sample.

15. The method of any one of the preceding claims, wherein said patient is a cancer patient.

16. The method of claim 15, wherein said cancer is a solid tumor.

17. The method of any one of the preceding claims, wherein the target is a target peptide, optionally an antigenic peptide, which is further optionally capable of a) being recognized by the immune system, particularly by antibodies or T-cell receptors, or derivatives thereof; and / or b) eliciting an immune response.

18. The method of claim 17, wherein said antigenic peptide is a) 8-25 amino acids long; b) capable of being presented on an MHC-I molecule; c) capable of being recognized by a component of the immune system, such as a T cell receptor (TCR); and / or d) capable of eliciting a T-cell mediated immune response.

19. The method of any one of the preceding claims, wherein said antigenic peptide is a PRAME-004 peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 1 (SLLQHLIGL) or a MAG-003 peptide, optionally comprising or consisting of an amino acid sequence according to SEQ ID NO: 2 (KVLEHVVRV).

20. The method of any one of claims 3 to 19, wherein said targeted therapy includes administering a therapeutic agent selected from: a T cell comprising a T cell receptor (TCR) or chimeric antigen receptor (CAR) capable of binding to said target peptide; a soluble binding protein capable of binding to said target peptide; or a vector encoding a TCR, CAR or soluble binding protein.21 . The method of claim 20, wherein said therapeutic agent is selected from a T cell comprising a T cell receptor (TCR) comprising the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 7, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 8, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 9, a CDR1 b comprising or consisting of the amino acid sequence of SEQ ID NO: 10, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 11 and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and optionally a heterologous CD8 molecule; or an antigen binding protein comprising the following complementarity determining regions (CDRs): a CDR1a comprising or consisting of the amino acid sequence of SEQ ID NO: 37, a CDR2a comprising or consisting of the amino acid sequence of SEQ ID NO: 38, a CDR3a comprising or consisting of the amino acid sequence of SEQ ID NO: 39, a CDR1 b comprising or consisting of the amino acid sequence of SEQ ID NO: 40, a CDR2b comprising or consisting of the amino acid sequence of SEQ ID NO: 41 , and a CDR3b comprising or consisting of the amino acid sequence of SEQ ID NO: 42.

22. A kit for performing the method of any one of the preceding claims, comprising a) nucleic acid amplification reagents, b) a target primer pair being capable of hybridizing to a target mRNA, c) a target hydrolysis probe being capable of hybridizing to a target mRNA,d) at least a first reference primer pair capable of hybridizing to a first reference gene mRNA, e) at least a first reference hydrolysis probe capable of hybridizing to a first reference gene mRNA, wherein said first reference gene is selected from the group of RPL37A, 0AZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, and f) optionally instructions for performing the ex vivo method as defined in claims 1-19.

23. The kit according to claim 22, further comprising at least a second reference primer pair capable of hybridizing to a second reference gene mRNA, and at least a second reference hydrolysis probe capable of hybridizing to a second reference gene mRNA, wherein said second reference gene is preferably selected from the group of 0AZ1 , RPL37A, RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and RPLPO.

24. The method of any one of claims 10 to 21 , or the kit according to any one of claims 22 or 23, wherein the forward target primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 4; the reverse target primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 5; and / or the target hydrolysis probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 6.

25. The method of any one of claims 10 to 21 or 24, or the kit according to any one of claims 22 to 24, wherein the target hydrolysis probe and / or any of the reference hydrolysis probes comprises a detectable label, such as a fluorophore.

26. Use of a kit according to any one of claims 22 to 25 for quantifying the expression level of a target in a biological sample, and optionally selecting the sample donor for treatment with a targeted therapy; confirming the eligibility of the sample donor for treatment with a targeted therapy; and / or monitoring the expression level of said target during treatment with a targeted therapy.

27. An in vitro method for determining the expression level of a target in a biological sample, comprising a) determining the expression level of said target, b) determining the expression levels of at least one reference gene selected from RPL37A, 0AZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB,HNRNPA2B1 and PPIA, and c) comparing the expression of said target against the expression level(s) of said at least one reference gene(s).

28. A method for treating a patient with a targeted therapy, comprising a) obtaining a sample from said patient, b) determining the relative expression level of said target, c) determining the expression levels of at least one reference gene selected from RPL37A, OAZ1 , RPLPO, PUM1 , RBM23, DHX9, EIF4G2, NONO, HSP90AB1 , ACTB, HNRNPA2B1 and PPIA, d) normalizing the expression level of said target against the expression level(s) of said at least one reference gene(s), e) comparing the expression level of said target to a predetermined threshold, and selecting the patient for treatment if the expression level of said target exceeds said threshold.

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