HLA-independent t-cell receptors against the urokinase receptor upar

Abstract

The present invention relates to tumor- and / or senescence-associated antigens, which elicit a T-cell response independently from a presentation by MHC. Human uPAR was found to be a target of naturally occurring CD4- and CD8-positive T-cell clones that could detect their target antigen on the surface of HLA I or II-negative tumor cells. Thus, the invention provides proteins, protein fragments and peptides of the novel antigens for use in medicine, for example for the treatment, diagnosis and prevention of tumor diseases and disease-causing accumulation of senescent cells in tumor and other diseases. Also provided are nucleic acids expressing the antigens of the invention, binding agents specific for the antigens of the invention, such as T-cell receptor chains and isolated T cells which are reactive against the antigens of the invention, or that express the T-cell receptors of the invention. The invention further pertains to pharmaceutical compositions, especially vaccine compositions, comprising the antigens, nucleic acids, binding agents or T cells in accordance with the invention, and methods for the generation of T cells specifically reactive to the antigens of the invention in an MHC-independent manner.

Description

[0001] HLA-INDEPENDENT T-CELL RECEPTORS AGAINST THE UROKINASE RECEPTOR UPAR

[0002] The present invention relates to tumor- and / or senescence-associated antigens, which elicit a T-cell response independently from a presentation by MHC. Human uPAR was found to be a target of naturally occurring CD4- and CD8-positive T-cell clones that could detect their target antigen on the surface of HLA I or Il-negative tumor cells. Thus, the invention provides proteins, protein fragments and peptides of the novel antigens for use in medicine, for example for the treatment, diagnosis and prevention of tumor diseases and disease-causing accumulation of senescent cells in tumor and other diseases. Also provided are nucleic acids expressing the antigens of the invention, binding agents specific for the antigens of the invention, such as T-cell receptor chains and isolated T cells which are reactive against the antigens of the invention, or that express the T-cell receptors of the invention. The invention further pertains to pharmaceutical compositions, especially vaccine compositions, comprising the antigens, nucleic acids, binding agents or T cells in accordance with the invention, and methods for the generation of T cells specifically reactive to the antigens of the invention in an MHC-independent manner.

[0003] Background of the invention

[0004] One of the major mechanisms by which cancer cells evade the immune system is via down regulation and loss of their major histocompatibility complex class I (MHC -I) or II (MHC-II) molecules (e.g. (26)) (aka human leukocyte antigens (HLAs)). Normally, MHC-positive tumor cells would be targeted by T-cells with T-cell receptors (TCRs) recognizing tumor-specific peptides displayed by the MHC molecules. The recognition and binding of tumor-cell surface peptide-loaded MHCs (pMHCs) by TCRs results in the formation of a cytolytic synapse between the T-cell and cancer cell, leading to the directed massive release of cytotoxic proteins such as perforin and granzymes, as well as clonal T-cell activation and proliferation. Optimal activation of the T cells in this and other synaptic interactions requires two signals, the TCR-MHC interaction, known as “Signal 1” and a costimulatory signal (“Signal 2”) through one of several costimulatory receptorson T cells (e.g., CD28, CD137, 0X40, CD27, ICOS, GITR) and their cognate ligands (e.g., CD80 / 86, CD137L, OX40L, CD70, ICOS-L, GITR-L) on the targeted cells or professional antigen-presenting cell (APC). A third signal, production of immunostimulatory cytokines, helps to drive T-cell differentiation and expansion.

[0005] Loss or downregulation of the MHC molecules allow those cells to escape recognition and killing by tumor-infiltrating T cells which are key components of anti-tumor immunological responses. Additionally, loss of costimulatory molecules (e.g., CD86, CD54), overproduction of checkpoint inhibitory molecules (e.g., PD-1, CTLA4) and tumor production of the tryptophan degrading-enzyme indoleamine 2,3 -dioxygenase (IDO), which eliminates tryptophan, a key amino acid required for T-cell proliferation, are other examples of mechanisms utilized by tumors to evade cytotoxic T cells.

[0006] Today, various therapeutic strategies seek to harness the killing power of T cells in a TCR functionality-independent manner, bypassing the limitation of HLA-restricted antigen recognition. Two of the most important TCR function-independent T cell-based therapeutic strategies employed today are T-cell redirecting bispecific antibodies (TRBAs) and chimeric antigen receptor (CAR)-T cells.

[0007] Chimeric antigen receptors (CARs) engage antigen independently of HLA and enable sustained T-cell proliferation when they are endowed with both activating and costimulatory functions. Nevertheless, while remission rates have been noticeably elevated in numerous clinical trials targeting CD 19, CD22 or BCMA, relapses are still common.

[0008] With TRBAs the epsilon (e) domain of cluster of differentiation 3 (CD3), a component of the TCR complex, is targeted with one combining (i.e., binding) domain, while a second binding domain (hence, “bispecific” antibody) binds a tumor-cell surface antigen. These TRBAs function to bring the T cells and targeted cells into close proximity to form a cytolytic synapse resulting in tumor-cell death. In the case of CAR-T cells, a cancer¬ cell surface antigen-targeting antibody fragment, fused to T-cell activating intracellular domains, is expressed as a neoreceptor on the surface of the T cells. These tumor antigen¬ recognizing “armed” T-cells will then identify, bind and kill the targeted cancer cells.Both of these strategies rely on antibodies to replace the function of the TCR, making them independent of the TCR and its cognate MHC / peptide recognition and both can be employed to recognize and target tumor antigens outside the realm of MHC-displayed peptides (Strohl WR, Naso M. Bispecific T-Cell Redirection versus Chimeric Antigen Receptor (CAR)-T Cells as Approaches to Kill Cancer Cells. Antibodies (Basel). 2019 Jul 3;8(3):41. doi: 10.3390 / antib8030041. PMID: 31544847; PMCID: PMC6784091).

[0009] Hazini et al. (27) consider the mechanisms by which HLA function may be lost in clinical disease. They assess the implications for current immunotherapy approaches using checkpoint inhibitors and examine the prognostic impact of HLA loss demonstrated in clinical trials so far. Finally, they propose strategies that might be explored for possible patient stratification.

[0010] uPAR is a GPI-anchored cell surface receptor for the extracellular serine protease uPA (abbreviation for "urokinase-type plasminogen activator"). The receptor essentially consists of three domains (D1D2D3, see Figure 1). The D3 domain is C-terminal and is connected to the cell membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. The N-terminal domain DI binds the natural ligand uPA. In addition, vitronectin, among others, binds to the uPAR domains DI and D2 (1), although it requires the presence of intact uPAR. uPA and other proteases can cleave off DI, so that in addition to intact uPAR D1D2D3, the variant uPAR D2D3 is also present on the cell surface. Therefore, for therapeutic targeting of membrane-bound uPAR it might be important that the targeting agent does not depend on the presence of domain DI. After cleavage of the GPI anchor, D1D2D3 and D2D3 also exist as soluble variants (suPAR) (2).

[0011] In combination with its ligand uPA, uPAR accelerates plasmin-dependent proteolysis and leads to efficient degradation of the extracellular matrix. Thus, uPAR plays an important role in physiological tissue homeostasis. In cancer, uPAR is involved in various cellular processes, such as invasion, intravasation, migration, metastasis and also in the development of multidrug resistance to cancer drugs of various types (3-6). uPAR is expressed not only by tumor cells, but also by non-malignant cells.The prognostically unfavorable impact of the uPAR / uPA system on malignancies has been reported for a number of tumor entities (7). uPAR is considered as a relevant target for both treatment and imaging of cancer (6, 8).

[0012] Metrangolo et al. (28) describe that the urokinase-type plasminogen activator receptor (uPAR) has now firmly established itself as a versatile molecular target holding promise for the treatment of aggressive malignancies. The copious abundance of uPAR in virtually all human cancerous tissues versus their healthy counterparts has fostered a gradual shift in the therapeutic landscape targeting this receptor from function inhibition to cytotoxic approaches to selectively eradicate the uPAR-expressing cells by delivering a targeted cytotoxic insult. Multiple avenues are being explored in a preclinical setting, including the more innovative immune- or stroma-targeting therapies. The review discusses the current state of these strategies, their potentialities, and challenges, along with future directions in the field of uPAR targeting.

[0013] In recent years, it has been found that uPAR is induced during senescence (9, 10). The accumulation of senescent cells during tumor therapy or in the context of aging processes is considered as detrimental and contributes to therapy resistance or age-related diseases, respectively. It has been shown that CAR-T cells directed against uPAR can reduce or reverse such negative effects through their senolytic effect (9, 10).

[0014] Enhancing or generating T-cell responses against HLA-independently recognizable antigens would circumvent immune escape due to HLA loss and thus represent a substantial expansion of the immunotherapeutic repertoire. Thus, there is a need to provide targets leading to HLA-independent anti -turn or alpha / betaT-cell responses in patients with HLA-negative disease.

[0015] It is therefore an object of the present invention to provide novel HLA-independent antigens recognized by cytotoxic T-lymphocytes (CTL) that are stably expressed in a high proportion of senescent cells and / or tumor cells and is therefore suitable for a broad application. It is a further object of this invention to provide HLA-independent T-cell receptors (TCRs) against these antigens, as well as compositions comprising the TCRs,as well as uses thereof. Other objects of the present invention will become apparent to the person of skill when studying the specification of the present invention.

[0016] In a first aspect thereof, the object of the present invention is solved by providing a pharmaceutical composition comprising human uPAR of SEQ ID NO: 10 or a functional homolog thereof having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof, or an immunogenically active peptide fragment comprising a consecutive sequence of at least 8 amino acids of human uPAR or said functional homolog thereof, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof, or a nucleic acid encoding said human uPAR, said functional homolog thereof or said peptide fragment; and b) at least one pharmaceutically acceptable carrier for use in the prevention or treatment of a cancer and / or senescent cell irrespective of the HL A type and preferably in the presence of down-regulation or loss of HLA expression. Preferred is the pharmaceutical composition for use according to the present invention, which is an anticancer and / or anti-senescence vaccine, and / or wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response.

[0017] In a second aspect thereof, the object of the present invention is solved by providing a binding agent comprising the amino acid sequence of a binding region of a T-cell receptor binding to a cancer cell expressing human uPAR (SEQ ID No. 10), preferably either an intact surface polypeptide or expressing a fragment comprising domains D1 / D2 / D3, or comprising domains D2 / D3, preferably after cleavage of domain DI, or an amino acid sequence having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR or the fragment thereof, wherein said binding is independent of HLA. Preferred is the binding agent according to the present invention, wherein said T-cell receptor is selected from a TCR-construct comprising a TCR alpha or beta chain comprising a sequence according to any one of SEQ ID NOs: 1 to 9. In a preferred embodiment, the binding agent accordingto the present invention is selected from a TCR, a modified TCR, such as a chimeric T cell receptor comprising a murinized constant region and a human variable region, a single-chain TCR molecule, and a bispecific molecule, such as a bispecific antibody or a CAR. In another preferred embodiment, the binding agent according to the present invention is selected from a fusion protein comprising the binding agent according to the present invention fused to an antibody effector function, such as an scFv, also called ImmTAC®. Preferred are fusion proteins comprising the anti-uP AR-TCR-derived binder fused to an anti-CD3 antibody fragment.

[0018] The present invention further provides a nucleic acid molecule encoding for a binding agent according to the present invention, or a vector comprising the nucleic acid molecule, such as, for example, an expression vector comprising and expressing the nucleic acid molecule. The present invention further provides a recombinant or genetically modified cell comprising a nucleic acid molecule or vector according to the present invention. Preferably, the recombinant or genetically modified T cell expressing a TCR that binds to human uPAR or an amino acid sequence having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR, preferably of the domains DI, D2 / D3 or D1 / D2 / D3 thereof, wherein said binding is independently from HLA, such as, for example, a TCR-construct comprising a sequence according to any one of SEQ ID NOs: 1 to 9, or a fragment thereof binding human uPAR.

[0019] In a third aspect thereof, the object of the present invention is solved by providing an in-vitro method for generating HLA-independent T cells, a) providing a first cell that expresses human uPAR (SEQ ID No. 10) or an amino acid sequence having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to uPAR or an immunogenic protein fragment thereof, preferably wherein the polypeptide is full length human uPAR or preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof, b) bringing a population of peripheral blood mononuclear cells (PBMCs) into contact with said first cell, and thereby stimulating said PBMCs, and c) selecting from the population of stimulated PBMCs Tcells that have the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell, wherein preferably in step c) the ability of a T cell to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell is determined by testing the reactivity of said T-cell against a cell expressing the human uPAR or an immunogenic protein fragment thereof, wherein i) the cell expressing the human uPAR or an immunogenic protein fragment thereof lacks MHC class I or MHC class I and II expression, and / or ii) the T-cell is tested for its reactivity against said cell expressing the human uPAR or an immunogenic protein fragment thereof in the presence of antibodies against MHC class I or II; and / or iii) the T-cell is tested for its reactivity against xenogenic cells transfected with DNA or RNA encoding the human uPAR or an immunogenic protein fragment thereof, wherein in i), ii) and / or iii) a T-cell that shows reactivity is a T-cell having the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independent of the expression of HLA in said cell.

[0020] In a fourth aspect thereof, the object of the present invention is solved by providing an HLA-independent T cell, generated according to a method according to the present invention.

[0021] In a fifth aspect thereof, the object of the present invention is solved by providing a pharmaceutical composition, comprising a binding agent according to the present invention, a nucleic acid or vector according to the present invention, a cell according to the present invention, or a T-cell according to the present invention, and at least one pharmaceutically acceptable carrier. Preferred is the pharmaceutical composition according to the present invention for use as a medicament, preferably for use in the prevention or treatment of a uPAR-positive cancer cell and / or the accumulation of senescent cells irrespective of the HLA type and / or in case of a down-regulation or loss of HLA expression. Further preferred is the pharmaceutical composition for use according to the present invention, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response. Also preferred is the pharmaceutical composition for use according to the present invention, wherein the cancer and / or senescent cell expresses human uPAR (SEQ ID No. 10) or anamino acid sequence having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof.

[0022] In a sixth aspect thereof, the object of the present invention is solved by providing a method for eliciting a CD4- or CD8-positive T-cell response in a subject independently from a presentation of an antigen by MHC, comprising administering to said subject the pharmaceutical composition for use according to the present invention or the pharmaceutical composition according to the present invention.

[0023] In a seventh aspect thereof, the object of the present invention is solved by providing a kit comprising: a) the pharmaceutical composition for use according to the present invention or the pharmaceutical composition according to the present invention, and b) a composition comprising at least one second active ingredient.

[0024] In an eighth aspect thereof, the object of the present invention is solved by providing a method of preventing or treating a cancer and / or a senescent cell irrespective of its HL A type and even when exhibiting down-regulation or loss of HLA expression, wherein the cancer and / or senescent cell furthermore expresses human uPAR (SEQ ID No. 10) or an amino acid sequence having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domains thereof, the method comprising administering to subject suffering or prospectively suffering from said cancer an effective amount of the pharmaceutical composition for use according to the present invention, or of the pharmaceutical composition according to the present invention. Preferred is the method according to the present invention, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC) class I- and II-independent T-cell response. Further preferred is the method according to the present invention, wherein the cancer and / or senescent cell is from a tumor carrying or expressing human uPAR.All known anti-uP AR effector molecules are derived from monoclonal antibodies. This is associated with an inherent limitation of antigen sensitivity compared to T-cell / TCR recognition. However, there is currently no certain answer to the question as to whether TCRs have a clinically relevant therapeutic advantage over CARs or comparable constructs. The different nature of their established ligands (HLA / peptide complexes versus intact surface molecules) makes a direct comparison very difficult. In a first approach to this difficult-to-answer question, Mansilla-Soto et al. compared CARs and so-called HIT receptors, i.e. artificial HLA-independent TCRs. They reported that HIT receptors offer advantages over CARs in the therapeutic targeting of surface antigens with low expression levels (23). These observations suggest that the higher antigen sensitivity of TCRs compared to antibodies can also be transferred to the comparison with CARs.

[0025] Established adoptive T-cell therapies address tumor-associated or tumor-specific antigens and are therefore limited to effects directly on tumor cells. However, cells of the tumor microenvironment and also senescent malignant and non-malignant cells play a major role in tumorigenesis, tumor growth and also therapy resistance (19, 24). But targeting of the tumor stroma or senescent cells has not been established, yet.

[0026] Searching for target antigens of HLA-independent tumor-reactive T cells that can be used for cancer immunotherapy, even when tumors do not (or no longer) express HLA class I and HLA class II molecules, the present inventors found that the urokinase receptor uPAR (abbreviation for "urokinase-type plasminogen activator receptor") is recognized by naturally occurring HLA-independent T cells. Synonyms are PLAUR (abbreviation for "plasminogen activator, urokinase receptor", also the preferred name of the gene coding for uPAR) and CD87 (CD is the abbreviation for "cluster of differentiation"). uPAR is considered as an important target structure for tumor therapy and diagnostics as well as for senolytic approaches (see below). Accordingly, HLA-independent T-cell receptors (TCRs) against uPAR can be used for the adoptive transfer of genetically modified T cells. It is also possible to enhance such natural T-cell responses by vaccination.

[0027] In the context of the present invention, the term “human uPAR” or “hu uPAR” shall mean the human uPAR polypeptide, including fragments thereof comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof, or an immunogenically active peptide fragment comprisinga consecutive sequence of at least 8 amino acids of human uPAR or a functional homolog thereof, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof. The term shall also include glycosylated and non-glycosylated proteins and fragments, and respective amino acid sequences having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to the human or murine uPAR amino acid sequence.

[0028] Human uPAR comprises three domains, here designated as DI, D2 and D3, that are found between amino acids 23-114 (Ly6-1), 115-213 (Ly6-2), and 214-305 (Ly6-3), respectively (see also Figure 1).

[0029] The amino acid sequence of human uPAR can be found in the Uniprot database (status 11 / 24) at Q03405.

[0030] MGHPPLLPLLLLLHTCVPASWGLRCMQCKTNGDCRVEECALGQDLCRTTIVRL WEEGEELELVEKSCTHSEKTNRTLSYRTGLKITSLTEVVCGLDLCNQGNSGRAV TYSRSRYLECISCGSSDMSCERGRHQSLQCRSPEEQCLDVVTHWIQEGEEGRPK DDRHLRGCGYLPGCPGSNGFHNNDTFHFLKCCNTTKCNEGPILELENLPQNGR QCYSCKGNSTHGCSSEETFLIDCRGPMNQCLVATGTHEPKNQSYMVRGCATAS MCQHAHLGDAFSMNHIDVSCCTKSGCNHPDLDVQYRSGAAPQPGPAHLSLTIT LLMTARLWGGTLLWT (SEQ ID NO: 10).

[0031] In addition, the uPAR binding agents as described herein may be generated using the murine uPAR protein or fragments thereof, as described below.

[0032] An identification of uPAR as a target antigen of HLA-independent T cells was performed in healthy individuals. These uPAR-reactive T cells and their TCRs act independently of the HLA type of their target cells. Furthermore, domain DI -independent uPAR-targeting - as achieved with TCRs against domains D2 / D3 - is advantageous, because DI may be physiologically cleaved off by uPA and other proteases.

[0033] In the context of the present invention, nine TCRs that recognize uPAR in an HLA-independent manner were cloned from four umbilical cord blood donations. These TCRsare directed against different domains of uPAR. The HLA-independent recognition of uPAR makes it a broadly applicable target antigen for active and passive immunotherapy in various types of tumors and possibly also in senescence-associated diseases.

[0034] TCRs directed against uPAR are attractive candidates for TCR-transfer experiments and offer several substantial advantages. They are not restricted to the presence of a particular HLA allele and allow bypassing HLA loss. In addition, they also recognize senescent malignant and non-malignant cells and can contribute to overcoming therapy resistance mechanisms (5, 25).

[0035] An immediate therapeutic application is a transfer of HLA-independent TCRs. Naturally occurring HLA-independent anti-uPAR T-cell responses can also be enhanced by therapeutic vaccination. For this, appropriate vaccine techniques are available.

[0036] The use of uPAR-reactive TCR or T cells is important not only for the treatment of tumor diseases but also for the prophylaxis or treatment of age-related metabolic dysfunctions (10).

[0037] The inventors observed that all four TCRs against domain DI (TCR1, TCR2, TCR4 and TCR5, see Table I), originating from two distinct donors, carry the gene TRBV5-1*01 in the ß-chain. The dominance of individual chains in the recognition of HLA-restricted peptide antigens has been described (26). Therefore, uPAR DI recognition seems mainly be mediated by the ß chains carrying the segment encoded by TRBV5-1*01. This also suggests that uPAR DI -reactive HLA-independent TCRs are predominantly found among such TCRs, which in turn would allow for a selective enrichment of uPAR-reactive T cells by expansion of TRBV5-1*01-positive T cells, both ex vivo and in vivo. Equivalent observations have not been made for D2D3-reactive TCRs originating from three distinct donors (TCR3, TCR6, TCR7, TCR8 and TCR9, see Table I).

[0038] As described above, the present invention relates to a pharmaceutical composition comprising human uPAR (SEQ ID No. 10) or a functional homolog thereof having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequenceidentity or at least 98 or 99% sequence identity thereto or an immunogenically active peptide fragment comprising a consecutive sequence of at least 8 amino acids of human uPAR or said functional homolog thereof, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof, or a nucleic acid encoding said human uPAR, said functional homolog thereof or said peptide fragment; and b) at least one pharmaceutically acceptable carrier for use in the prevention or treatment of cancer cells and / or senescent cells regardless / irrespective of the HLA type and even when exhibiting down-regulation or loss of HLA expression.

[0039] Preferred is the pharmaceutical composition for use according to the present invention, that is an anti-cancer vaccine. For the composition and uses, see, for example, Fan et al. (29).

[0040] Preferred is the pharmaceutical composition for use according to the present invention, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC) class I- and II-independent T-cell response.

[0041] Further preferred is the pharmaceutical composition for use according to the present invention, wherein the cancer or senescent cell expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity thereto. Further preferred is the pharmaceutical composition for use according to the present invention, wherein the cancer and / or senescent cell is from a tumor carrying or expressing uPAR. Further preferred is the pharmaceutical composition for use according to the present invention, wherein the vaccine composition further comprises an immunogenic protein and / or fragment thereof that is not human uPAR or the immunogenically active peptide fragment thereof as described herein.

[0042] The pharmaceutical composition for use according to the present invention may comprise a suitable adjuvant. The adjuvant may be selected from the group consisting of bacterial DNA-based adjuvants, oil / surf actant based adjuvants, viral dsRNA-based adjuvants and imidazochinilines, and / or a Montanide ISA adjuvant. Further preferred is thepharmaceutical composition for use according to the present invention, wherein the vaccine composition comprises antigen-presenting cells comprising the immunogenically active peptide fragment or a nucleic acid encoding said immunogenically active peptide fragment, as described herein, wherein preferably the antigen-presenting cell is a dendritic cell.

[0043] As mentioned above, the present invention relates to a binding agent comprising the amino acid sequence of a binding region of a T-cell receptor binding to a cancer cell expressing human uPAR (SEQ ID No. 10), preferably expressing a fragment comprising domains D1 / D2 / D3, and more preferably comprising domains DI or D2 / D3 thereof, or an amino acid sequences having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to the human or murine uPAR amino acid sequence or the fragment thereof, wherein said binding is independent of HL A.

[0044] Preferred is the binding agent according to the present invention, wherein said T-cell receptor is selected from a TCR-construct comprising a TCR alpha or beta chain comprising a sequence according to any one of SEQ ID NOs: 1 to 9 or a mixture or combination thereof.

[0045] The binding agent according to the present invention may be any suitable binding agent, preferably said binding agent is selected from a TCR, a modified TCR, such as a chimeric T-cell receptor comprising murine constant regions and human variable regions, a singlechain TCR molecule, and a bispecific molecule, such as a bispecific antibody or a CAR.

[0046] Another aspect of the present invention relates to a nucleic acid molecule encoding for a binding agent according to the present invention, a vector comprising the nucleic acid molecule, such as, for example, an expression vector comprising and expressing the nucleic acid molecule. The nucleic acid may be DNA, RNA, PNA or combinations thereof.Another aspect of the present invention relates to a recombinant or genetically modified cell comprising a nucleic acid molecule or vector according to the present invention. The cell may be a bacterial or eukaryotic cell, such as a yeast, human, mouse or insect cell.

[0047] Preferred is the recombinant or genetically modified cell according to the present invention, which is a T cell expressing a TCR that binds to human uPAR or a protein having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to uPAR, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof, wherein said binding is independently from HLA, such as, for example, a TCR according to any one of SEQ ID NOs: 1 to 9. Preferred are antigen-presenting cells comprising the immunogenically active peptide fragment as described herein (DI, D2 / D3 or D1 / D2 / D3) or a nucleic acid encoding said immunogenically active peptide fragment, wherein preferably the antigen-presenting cell is a dendritic cell.

[0048] Another aspect of the invention relates to an in-vitro method for generating HLA-independent T cells, comprising a) providing a first cell that expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR or an immunogenic protein fragment thereof, preferably wherein the polypeptide is full length human uPAR or preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof. The cell can be a cell as described herein. Then, b) a population of peripheral blood mononuclear cells (PBMCs) is brought into contact with said first cell, and thereby said PBMCs are stimulated. Then, c) from the population of stimulated PBMCs T cells are selected that have the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a), independently of the expression of HLA in said cell.

[0049] Preferably in step c) the ability of a T cell to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell is determined by testing the reactivity of said T-cell against a cellexpressing the human uPAR or an immunogenic protein fragment thereof. Also, i) the cell expressing the human uPAR or an immunogenic protein fragment thereof lacks MHC class I or MHC class I and II expression, and / or ii) the T-cell is tested for its reactivity against said cell expressing the human uPAR or an immunogenic protein fragment thereof in the presence of antibodies against MHC class I or II; and / or iii) the T-cell is tested for its reactivity against xenogenic cells transfected with DNA or RNA encoding the human uPAR or an immunogenic protein fragment thereof. Also, in i), ii) and / or iii) a T-cell that shows reactivity is a T-cell having the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independent of the expression of HL A in said cell.

[0050] Thus, preferred is the method according to the present invention, wherein in step c) the ability of a T cell to recognize a cell expressing uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell is determined by testing the reactivity of said T-cell against a cell expressing uPAR or an immunogenic protein fragment thereof, wherein i) the cell expressing uPAR or an immunogenic protein fragment thereof lacks MHC class I or MHC class I and II expression, and / or ii) the T-cell is tested for its reactivity against said cell expressing uPAR or an immunogenic protein fragment thereof in the presence of antibodies against MHC class I or II; and / or iii) the T-cell is tested for its reactivity against xenogenic cells transfected with DNA or RNA encoding uPAR or an immunogenic protein fragment thereof, wherein in i), ii) and / or iii) a T-cell that shows reactivity is a T-cell having the ability to recognize a cell expressing uPAR or an immunogenic protein fragment thereof used in a) independent of the expression of HLA in said cell.

[0051] Another aspect of the present invention relates to an HLA independent T cell, generated according to a method according to the present invention as above.

[0052] Another aspect of the present invention relates to a pharmaceutical composition, comprising a binding agent according to the present invention, a nucleic acid or vector according to the present invention, a cell according to the present invention, or a T-cell according to the present invention, and at least one pharmaceutically acceptable carrier.The term “pharmaceutically or therapeutically acceptable excipient or carrier” refers to a solid or liquid filler, diluent or encapsulating substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not substantially toxic to the host, which may be either humans or animals, to which it is administered. Depending upon the particular route of administration, a variety of pharmaceutically acceptable carriers such as those well known in the art may be used. Non-limiting examples include sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water. Pharmaceutically acceptable carriers or excipients also include diluents (fillers, bulking agents, e.g. lactose, microcrystalline cellulose), disintegrants (e.g. sodium starch glycolate, croscarmellose sodium), binders (e.g. PVP, HPMC), lubricants (e.g. magnesium stearate), glidants (e.g. colloidal SiO2), solvents / co-solvents (e.g. aqueous vehicle, Propylene glycol, glycerol), buffering agents (e.g. citrate, gluconates, lactates), preservatives (e.g. Na benzoate, parabens (Me, Pr and Bu), BKC), anti -oxidants (e.g. BHT, BHA, Ascorbic acid), wetting agents (e.g. polysorbates, sorbitan esters), thickening agents (e.g. methylcellulose or hydroxyethylcellulose), sweetening agents (e.g. sorbitol, saccharin, aspartame, acesulfame), flavoring agents (e.g. peppermint, lemon oils, butterscotch, etc.), humectants (e.g. propylene, glycol, glycerol, sorbitol). Other suitable pharmaceutically acceptable excipients are inter alia described in Remington's Pharmaceutical Sciences, 15thEd., Mack Publishing Co., New Jersey (1991) and Bauer et al., Pharmazeutische Technologic, 5thEd., Govi-Verlag Frankfurt (1997). The person skilled in the art knows suitable formulations for the compounds according to the present invention and will readily be able to choose suitable pharmaceutically acceptable carriers or excipients, depending, e.g., on the formulation and administration route of the pharmaceutical composition.

[0053] All suitable modes of administration are contemplated according to the invention. Administration of the composition as a medicament may be via oral, subcutaneous, direct intravenous, slow intravenous infusion, continuous intravenous infusion, intravenous or epidural patient controlled analgesia (PCA and PCEA), intramuscular, intrathecal, epidural, intracistemal, intraperitoneal, transdermal, topical, buccal, sublingual, transmucosal, inhalation, intra-atricular, intranasal, rectal or ocular routes, abuse deterrentand abuse resistant formulations, sterile solutions suspensions and depots for parenteral use, and the like, administered as immediate release, sustained release, delayed release, controlled release, extended release and the like. In the context of the present invention, preferred is an anti-cancer or anti-senescent vaccine as mentioned above.

[0054] In addition to the aforementioned compounds of the invention, the pharmaceutical composition can contain two or more compounds according to the present invention and also other therapeutically active substances.

[0055] The dosage of the pharmaceutical composition according to the present invention can be appropriately selected according to the route of administration, the subject to be administered, the target disease and its severity, age, sex weight, individual differences and disease state. Dosage may be repeated several times a day.

[0056] Another aspect relates to the pharmaceutical composition according to the present invention for use as a medicament, preferably for use in the prevention or treatment of a cancer and / or senescent cell irrespective of its HLA type and even when exhibiting downregulation or loss of HLA expression. Preferred is the pharmaceutical composition for use according to the present invention, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response.

[0057] Further preferred is the pharmaceutical composition for use according to the present invention, wherein the cancer or senescent cell expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domain thereof.

[0058] Preferred is the pharmaceutical composition for use according to the present invention, wherein the cancer or senescent cell is from a tumor carrying or expressing human uPAR.

[0059] Elevated uPAR levels, both in resected lesions or in body fluids (blood, plasma, urine, and ascites) as shed soluble suPAR have been detected in almost all human malignanciesinvestigated to date and are frequently associated with highly invasive phenotypes, poor prognosis, and adverse clinical outcomes. The broad gene expression profile of uPAR in human cancer was reported in the Gene Expression Profiling Interactive Analysis (GEPIA) database.

[0060] Pancreatic cancer (PAAD) is the one exhibiting the highest mean levels of uPAR mRNA, as well as the largest expression separation between neoplastic and normal tissues, compared to all other cancer types. Therefore, the present invention is of interest for the treatment of pancreatic cancer. Also, other cancer entities with overexpression of uPAR in tumor cells or cells of the tumor-supporting microenvironment can be targeted. Since primary malignant cells are readily available in leukemia, we have tested primary blasts from 19 acute myeloid leukemia (AML) patients and found strong recognition by T cells transduced with a TCR against uPAR in a high proportion (see Figure 8). Therefore, we also consider AML -when refractory to standard treatment - as a disease candidate.

[0061] Another aspect of the present invention relates to a therapeutic or non-therapeutic method for eliciting a CD4- or CD8-positive T-cell response in a subject independently from a presentation of an antigen by MHC, comprising administering to said subject the pharmaceutical composition for use according to the present invention or the pharmaceutical composition according to the present invention.

[0062] Another aspect of the present invention relates to a kit comprising; a) the pharmaceutical composition for use according to the present invention or the pharmaceutical composition according to the present invention, and b) a composition comprising at least one second active ingredient. Preferred is the kit according to the present invention, wherein the second active ingredient is an anti-cancer agent, such as an anti-cancer agent selected from a chemotherapeutic agent, for example selected from: Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid,Temozolomide, Teniposide, Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine.

[0063] Preferred is the kit according to the present invention, where the provided compositions are to be administered simultaneously or sequentially.

[0064] Another aspect of the present invention relates to a method of preventing or treating a cancer and / or senescent cell exhibiting down-regulation or loss of HLA expression, wherein the cancer cell furthermore expresses uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity, more preferably at least 80% sequence identity, even more preferably at least 90% sequence identity, and more preferably at least 95% sequence identity or at least 98 or 99% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domain thereof, the method comprising administering to subject suffering or prospectively suffering from said malignant and / or senescence-associated disorder an effective amount of the pharmaceutical composition for use according to the present invention, or of the pharmaceutical composition according to the present invention.

[0065] Preferred is the method according to the present invention, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response. Further preferred is the method according to the present invention, wherein the cancer and / or senescent cell is from a tumor carrying or expressing uPAR, e.g. pancreatic cancer.

[0066] Preferred is the method according to the present invention, wherein the method is combined with a further cancer treatment, wherein preferably the further treatment is selected from the group consisting of chemotherapy, radiotherapy, treatment with immunostimulating substances, gene therapy, treatment with antibodies and treatment using dendritic cells.

[0067] The present invention relates to the following items:

[0068] Item 1. A binding agent comprising the amino acid sequence of a binding region of a T-cell receptor binding to a cancer cell expressing human uPAR (SEQ ID No. 10), preferably expressing a fragment comprising domains D1 / D2 / D3, and more preferablycomprising domains DI or D2 / D3 thereof, or an amino acid sequence having at least 75% sequence identity to human uPAR or the fragment thereof, wherein said binding is independent of HL A.

[0069] Item 2. The binding agent according to Item 1, wherein said T-cell receptor is selected from a TCR-construct comprising a TCR alpha or beta chain comprising a sequence according to any one of SEQ ID NOs: 1 to 9.

[0070] Item 3. A nucleic acid molecule encoding a binding agent according to Items 1 or 2, a vector comprising the nucleic acid molecule, such as, for example, an expression vector comprising and expressing the nucleic acid molecule, or a recombinant or genetically modified cell comprising the nucleic acid molecule or vector.

[0071] Item 4. A recombinant or genetically modified T cell expressing a TCR that binds to human uPAR or a protein having at least 75% sequence identity to human uPAR, preferably of the domains DI, D2 / D3 or D1 / D2 / D3 thereof, wherein said binding is independently from HLA, such as, for example, a TCR-construct comprising a sequence according to any one of SEQ ID NOs: 1 to 9, or a fragment thereof binding human uPAR.

[0072] Item 5. An in-vitro method for generating HLA independent T cells, comprising a) providing a first cell that expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to uPAR or an immunogenic protein fragment thereof, preferably wherein the polypeptide is full length human uPAR or preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof,

[0073] b) bringing a population of peripheral blood mononuclear cells (PBMCs) into contact with said first cell, and thereby stimulating said PBMCs, and

[0074] c) selecting from the population of stimulated PBMCs T cells that have the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell, wherein preferably in step c) the ability of a T cell to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell is determined by testing the reactivity of said T cell against a cell expressing the human uPAR or an immunogenic protein fragment thereof, whereini) the cell expressing the human uPAR or an immunogenic protein fragment thereof lacks MHC class I or MHC class I and II expression, and / or

[0075] ii) the T-cell is tested for its reactivity against said cell expressing the human uPAR or an immunogenic protein fragment thereof in the presence of antibodies against MHC class I or II; and / or

[0076] iii) the T-cell is tested for its reactivity against xenogenic cells transfected with DNA or RNA encoding the human uPAR or an immunogenic protein fragment thereof, wherein in i), ii) and / or iii) a T cell that shows reactivity is a T cell having the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independent of the expression of HL A in said cell.

[0077] Item 6. An HLA-independent T cell, generated according to a method according to Item 5.

[0078] Item 7. A pharmaceutical composition, comprising a binding agent according to any one of Items 1 or 2, a nucleic acid or vector according to Item 3, or a T cell according to Item 4, and at least one pharmaceutically acceptable carrier, preferably the pharmaceutical composition for use as a medicament, preferably for use in the prevention or treatment of a cancer cell even when exhibiting down-regulation or loss of HLA expression and / or the accumulation of senescent cells, wherein preferably said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response.

[0079] Item 8. The pharmaceutical composition for use according to Item 7, wherein the cancer and / or senescent cell expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to human uPAR, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof.

[0080] Item 9. A pharmaceutical composition comprising human uPAR of SEQ ID NO: 10 or a functional homologue thereof at least 75% identical thereto or an immunogenically active peptide fragment comprising a consecutive sequence of at least 8 amino acids of human uPAR or said functional homolog thereof, preferably of the DI, D2 / D3 or D1 / D2 / D3 domains thereof, or a nucleic acid encoding said uPAR, said functional homolog thereofor said peptide fragment; and b) at least one pharmaceutically acceptable carrier for use in the prevention or treatment of a cancer cell even when exhibiting down-regulation or loss of HLA expression and / or the accumulation of senescent cells, which preferably is an anti-cancer vaccine.

[0081] Item 10. The pharmaceutical composition for use according to Item 9, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response, wherein preferably the cancer cell and / or the senescent cell expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domains thereof.

[0082] Item 11. The pharmaceutical composition for use according to Item 9 or 10, wherein the vaccine composition comprises antigen-presenting cells comprising the immunogenically active peptide fragment or a nucleic acid encoding said immunogenically active peptide fragment, wherein preferably the antigen-presenting cell is a dendritic cell.

[0083] Item 12. Method for eliciting a CD4- or CD8-positive T-cell response in a subject independently from a presentation of an antigen by MHC, comprising administering to said subject the pharmaceutical composition for use according to any one of Items 7 to 11.

[0084] Item 13. A kit comprising; a) the pharmaceutical composition for use according to any one of Items 7 to 11, and b) a composition comprising at least one second pharmaceutically active ingredient.

[0085] Item 14. The kit according to Item 13, wherein the second active ingredient is an anticancer agent, such as an anti-cancer agent selected from a chemotherapeutic agent, for example selected from: Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide, Thioguanine, Valrubicin,Vinblastine, Vincristine, Vindesine, and Vinorelbine, wherein preferably the provided compositions are to be administered simultaneously or sequentially.

[0086] Item 15. A method of preventing or treating a cancer and / or a senescent cell ebven when exhibiting down-regulation or loss of HLA expression, wherein the cancer and / or senescent cell furthermore expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domains thereof, the method comprising administering to subject suffering or prospectively suffering from said cancer and / or senescent cells an effective amount of the pharmaceutical composition for use according to any one of Items 7 to 11, wherein preferably the method is combined with a further cancer and / or anti-senescence cancer treatment, wherein preferably the further treatment is selected from the group consisting of chemotherapy, radiotherapy, treatment with immunostimulating substances, gene therapy, treatment with antibodies and treatment using dendritic cells.

[0087] The invention will now be described further in the following examples with reference to the accompanying figures and the attached sequence listing, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited are incorporated by reference in their entireties.

[0088] Figure 1 shows the schematic diagram of uPAR-expression constructs in pcDNA3.1 encoding GPI-anchored full-length human uPAR D1D2D3 or GPI-anchored domains uPAR DI and uPAR D2D3, respectively. N-glycosylation sites are indicated (Nx).

[0089] Figure 2 shows the blocking assay with three uPAR DI -specific antibodies. Antibodies VIM5, REA892 and MM0597 are known to bind to uPAR-domain DI. Effectors were buffy coat-derived T cells transduced with the indicated anti-uP AR TCRs. Target was Ma-Mel-86b_KOr / / / l_huPAR. Data of a 20h IFNg ELISPOT assay. For assignments of TCRs 1-5 see Table I.

[0090] Figure 3 shows A) the domain specificity of anti -uPAR TCRs 1-5. HLA-negative 293T_KO / y2' / ' / / / '1cells were transiently transfected with uPAR-expression constructs in pcDNA3.1 encoding GPI-anchored full-length human huPAR D1D2D3 or GPI-anchoreddomains huPAR DI and huPAR D2D3 (Figure 1). Effectors were buffy coat-derived T cells transduced with the indicated anti-uP AR TCRs. Data of a 20h IFNg ELISPOT assay. For assignments of TCRs see Table I. B) Domain specificity of anti-uP AR TCRs 6-9. HLA I / II-negative Ma-Mel-86b_KOr / / / cells were retrovirally transduced with uPAR-expression constructs in retroviral vector pMX encoding GPI-anchored huPAR D2D3 and muPAR D2D3 (Figure 1). Effectors were buffy coat-derived T cells transduced with the indicated anti-uP AR TCRs. TCR3 served as reference control (see Figure 3a). Data of a 20h IFNg ELISPOT assay. For assignments of TCRs see Table I.

[0091] Figure 4 shows the role of uPARN-glycosylation for recognition by anti-uP AR TCRs 1-5. HLA-negative Ma-Mel-86b_KOr / / / l_huPAR cells were treated with tunicamycin (5pg / ml, overnight). Untreated target cells were negative controls. Effectors were buffy coat-derived T cells transduced with the indicated anti-uP AR TCRs. Data of a 20h IFNg ELISPOT assay. For assignments of TCRs 1-5 see Table I.

[0092] Figure 5 shows the cytolysis by CD3-positive T cells transduced with anti-uP AR TCRs 1-3. Data of a 20-hour Fluc / D-luciferin bioluminescence lysis assay. Targets are indicated. All of them were HLA Land HLA Il-negative and stably expressed Firefly luciferase (Flue). Ma-Mel-86f_KOr / / / lFlue served as negative control (A). Ma-Mel-86b_KOCIITA_huPAR DlD2D3_Fluc stably expressed intact human uPAR (B), whereas Ma-Mel-86b_KOCIITA_huPAR D2D3_Fluc expressed D2D3 without DI (C). Effectors were CD3-positive buffy coat-derived T cells that were transduced with three of the TCRs listed in Table I: TCR2 (orange), TCR3 (grey) or TCR1 (yellow). Negative control effectors were untransduced CD3-positive T cells.

[0093] Figure 6 shows the functional analysis of CD4+ / CD8+ T cells engineered to express the anti-uPAR TCR1 (see Table I) compared with the parental CD4+ T-cell clone UCB55#74, from which TCR1 has been derived. Target cells were 293T_KOB2M / CIITAcells transfected with titrated amounts of uPAR-encoding cDNA. Effector cells were buffy coat-derived CD4+ or CD8+ T cells transduced with TCR1. Data of a 20h IFNg ELISPOT assay. Values are means of duplicates.Figure 7 shows the fine specificity of TCR1 (see Table I). Amino acid residues involved in the binding of uPA to uPAR (asterisks) and known N-glycosylati on sites (filled circles) [see left scheme and Refs. 1 and 2, below] were modified by site-directed mutagenesis as indicated. Recognition of these uPAR mutants by a-uPAR TCR1-T cells was analyzed in ELISPOT assays. Surface expression of mutated uPAR mutant proteins was confirmed by flow cytometry. Effectors were buffy coat-derived T cells transduced with TCR1 (see Table I). Targets were HLA-negative Ma-Mel-86b_KOr / / / cells stably transfected with unmodified or mutated uPAR cDNA. Refs.: [1] Gardsvoll and Ploug, J Biol Chem 2007;

[0094] [2] Ploug et al. Biochem Soc Trans 2002).

[0095] Figure 8 shows the recognition of primary AML blast populations by anti -uPAR TCR1. (A) Buffy coat-derived CD3-positive T lymphocytes were transduced with TCR1 (see Table I) and tested for recognition of 19 primary AML blast populations obtained from patients with AML FAB M4 / M5. To exclude allo-HLA I reactivity, anti-HLA I antibody W6 / 32 was applied for inhibition. Negative control target was Ma-Mel-86b_KOr / / / '', positive control target was Ma-Mel-86b_KOr / / / l_huPAR stably expressing huPAR D1D2D3 after gene transfer. (B) Blocking with anti -uPAR DI antibody REA892 was performed on three AML blast populations. Data of 20h IFNg ELSPOT assays. Values are means of duplicates.

[0096] SEQ ID NOs: 1 to 9 show the full amino acid sequences, respectively, of chimerized uPAR-reactive T-cell receptor (TCR) chain constructs 1 to 9 according to the invention.

[0097] SEQ ID NO: 10 shows the full-length amino acid sequence of human uPAR.

[0098] Examples

[0099] Blood lymphocytes from healthy umbilical cord blood (UCB) donors were stimulated with HL A I- and HL A Il-negative human tumor cells that stably expressed uPAR after retroviral transduction. HLA-independent alpha / beta T-cell responses against uPAR were found among the responder lymphocytes of these mixed lymphocyte / tumor-cell cultures (MLTCs). uPAR-reactive TCRs were cloned from MLTC responders using different methods as outlined below.Stimulator and target cells for the expansion and detection of HLA-independent T cells against uPAR

[0100] The melanoma cell line Ma-Mel-86b, established from a lymph node metastasis, does not carry HLA I molecules on the cell surface due to biallelic β2-microglobulin gene (B2M) mutations (11). In this cell line both MHC-class II transactivator gene (CIITA) alleles were knocked out by genome editing using the transcription activator-like (TAL) effector technology. A resulting clonal HLA I- and HLA Il-negative cell line was designated Ma-Mel-86b_KOr / / / .

[0101] HLA I- and HLA Il-negative variant clones were also produced from cell lines HEK293T and K562 by biallelic TALEN knockout of the genes B2M and CIITA (designated HEK293T_KOB2MC / ™ and K562_KOB2MC / ™).

[0102] The HLA I / II-deficient cell line Ma-Mel-86b_KOr / / / was stably transduced with CD80-and CD83 -encoding cDNA in retroviral expression constructs to improve its stimulation capacity (designation: Ma-Mel-86b_KOr / / / l_CD80 / 83).

[0103] Ma-Mel-86b_KOr / / / l_CD80 / 83 was further stably transduced with retroviral expression constructs containing cDNA for human uPAR (huPAR), either huPAR D1D2D3 or huPAR D2D3, or murine uPAR D1D2D3 (muPAR D1D2D3) to generate:

[0104] Ma-Mel-86b_KOr / / / l_CD80 / 83_huPAR D1D2D3,

[0105] Ma-Mel-86b_KOr / / / l_CD80 / 83_huPAR D2D3, and

[0106] Ma-Mel-86b_KOr / / / l_CD80 / 83_muP AR D 1D2D3.

[0107] For bioluminescence assays, some cell lines were additionally stably equipped with the gene for firefly luciferase (Flue).

[0108] Enrichment of uPAR-reactive T cells with HLA-negative stimulator cells in blood lymphocytes of healthy donors

[0109] CD3-positive lymphocytes from umbilical cord blood (UCB) donations were stimulated once a week with Ma-Mel-86b_KOCIITA_CD80 / 83_huPAR D1D2D3 or Ma-Mel-86b_KOCIITA_CD80 / 83_huPAR D2D3 in MLTCs. At the beginning of the stimulations, 3-5xl0e4 lymphocytes per MLTC unit were used. The antigen specificity of the MLTC-responder lymphocytes of each unit was regularly tested in IFNg ELISPOT assays, for the first time approx. 5 days after the 2nd stimulation (day 12) with the HLA I / II-negative target cells Ma-Mel-86b_KOCIITA_CD80 / 83 and Ma-Mel-86b_KOCIITA_CD80 / 83_huPAR D1D2D3 or Ma-Mel-86b_KOCIITA_CD80 / 83_huPAR D2D3.

[0110] MLTC-responder populations with HLA-independent anti-uP AR reactivity were detected in all samples tested - also in buffy coat (BC) lymphocytes from healthy donors (not shown).

[0111] Cloning and functional testing of HLA-independent alpha / beta TCRs

[0112] After stimulation of lymphocytes from umbilical cord blood donor UCB55 with HLA I / II-negative cells expressing huPAR D1D2D3 the MLTC responder population #74 with HLA-independent anti-uP AR reactivity was cloned via limiting dilution. CD4-positive anti-uP AR T-cell clones were found. From one of them, the TCR UCB55#74, referred to as TCR1, was cloned according to (12) into a bicistronic retroviral construct (see below).

[0113] The stimulation of lymphocytes from another umbilical cord blood donor, UCB54, with HLA I / II-negative cells expressing huPAR D1D2D3 resulted in several MLTC responder populations with HLA-independent anti-uP AR reactivity. From four of them, a total of four aPTCRs targeting uPAR were identified via single-cell RNA sequencing (scRNA-seq, 10x Genomics). They were designated as UCB54#15 (TCR2), UCB54#19 (TCR3), UCB54#11 (TCR4), and UCB54#13 (TCR5).

[0114] The stimulation of lymphocytes from two further umbilical cord blood donors, UCB80 and UCB81, with HLA I / II-negative cells expressing huPAR D2D3 (without the domain DI) resulted in the cloning of four other aPTCRs again via single-cell RNA sequencing (scRNA-seq, 10x Genomics). They were designated as UCB80#l 17 (TCR6), UCB81#24 (TCR7), UCB81#37 (TCR8), and UCB81#151 (TCR9).

[0115] All TCRs were cloned into a vector framework with integrated optimizations to increase TCR functionality (13). These include replacing the constant regions of the humanaPTCRs with codon-optimized mouse homologs for chimerization, adding a second disulfide bond between the chains and arranging the TCR chains in the order P-P2A-a.

[0116] Table I summarizes the origin and properties of the nine anti-uPAR TCRs.

[0117] Table I: Origin, designations und properties of anti-uPAR TCRs 1-9.

[0118] Origin TCR TCR Vbeta genes Valpha genes Domain Cross Depen designation code specificity reactivit dency y with on murine N- uPAR glycos ylation UCB5 UCB55#74 TCR1 TRBV5-1*01 TRAV10*01 DI yes yes 5 (SEQ ID TRBJ2-3*01 TRAJ35*01

[0119] NO: 1) TRBD1*01

[0120] UCB5 UCB54#15 TCR2 TRBV5-1*01 TRAV8- DI yes yes 4 (SEQ ID TRBJ1-2*01 2*01

[0121] NO: 2) TRBD1*01 TRAJ15*01

[0122] UCB5 UCB54#19 TCR3 TRBV5-5*02 TRAV3*01 D2D3 no no 4 (SEQ ID TRBJ2-l*01 TRAJ21*01

[0123] NO: 3) TRBD2*01

[0124] UCB5 UCB54#11 TCR4 TRBV5-1*01 TRAV17*01 DI yes yes 4 (SEQ ID TRBJ1-1*01 TRAJ29*01

[0125] NO: 4) TRBD1*01

[0126] UCB5 UCB54#13 TCR5 TRBV5-1*01 TRAV26- DI yes yes 4 (SEQ ID TRBJ2-2*01 1*01

[0127] NO: 5) TRBD1*01 TRAJ40*01

[0128] UCB8 - TCR6 TRBV12-5*01 TRAV8*01 D2D3 no n.t.

[0129] 0 (SEQ ID TRBD2*01 TRAJ20*01

[0130] NO: 6) TRBJ2-3*01

[0131] UCB8 - TCR7 TRBV27*01 TRAV3*01 D2D3 no n.t.

[0132] 1 (SEQ ID TRBJ2-2*01 TRAJ23*01

[0133] NO: 7)

[0134] UCB8 - TCR8 TRBV5-l*01 TRAV3*01 D2D3 no n.t.

[0135] 1 (SEQ ID TRBJ1-3*01 TRAJ11*01

[0136] NO: 8)

[0137] UCB8 - TCR9 TRBV19*01 TRAV 14 / DV D2D3 no n.t.

[0138] 1 (SEQ ID TRBJ1-2*01 4TRAJ21*01

[0139]

[0140] NO: 9)

[0141] UCBXY: umbilical cord blood donor; n.t.: not tested

[0142] In a first attempt to determine the fine specificity of the TCRs against uPAR, blockade experiments were performed on TCRs 1-5 with three different commercially available monoclonal antibodies: VIM5 (14), REA892 (Miltenyi Biotec), MM0597 (Abeam). Allthree antibodies are known to target the domain DI of uPAR. Recognition of 4 of the 5 tested anti -uP AR TCRs was blocked by each of the antibodies. In contrast, the antibodies had no effect on recognition by TCR3 (UCB54#19) suggesting that TCR3 is not directed against domain DI (Figure 2).

[0143] In a second attempt TCRs 1-9 were tested for their recognition of target cells transfected with plasmids encoding single uPAR domains or domain groups (Figures 3a and 3b). To summarize both analyses TCR1, TCR2, TCR4 and TCR5 recognize domain DI, whereas TCR3, TCR6, TCR7, TCR8 and TCR9 recognize uPAR D2D3 in the absence of domain DI. All Dl-reactive TCRs cross-react with murine uPAR D1D2D3, whereas none of the TCRs against human D2D3 cross-reacts with murine D2D3. Notably, TCRs with both domain specificities have been identified in a single donor, namely UCB54.

[0144] Functionally active uPAR is strongly glycosylated (15). Five N-glycosylation sites of uPAR D1D2D3 are indicated in Figure 1. Pretreatment of uPAR-positive target cells with tunicamycin inhibits N-glycosylation. Recognition of all TCRs against uPAR DI was suppressed by tunicamycin pretreatment. In contrast, tunicamycin had no effect on the recognition by anti-D2D3 TCR3 (Figure 4). The effect of tunicamycin on the recognition by the other anti-D2D3 TCRs has not been tested, yet.

[0145] The ability of anti-uP AR TCRs to trigger a cytolytic reaction was tested and confirmed in a bioluminescence assay. This applied to TCRs 1-2 against domain DI and to TCR3 against domains D2D3 (Figure 5).

[0146] Further investigations were carried out with TCR1 (UCB55#74), the results of which are described below.

[0147] Both the avidity of TCR1 -transduced T cells and the dependence of TCR1 on CD4 and CD8 were investigated. For this purpose, titrated amounts of a plasmid coding for huPAR D1D2D3 were transiently transfected into HLA-negative HEK293T_KO / y2l / / / / '1cells. TCR-transduced T cells were found to recognize even target cells transfected with only 1.5 ng of plasmid DNA. This is consistent with our observations with HLA-restricted TCR. Recognition by TCR-transduced CD4+ or CD8+ T cells was equivalent. TCR-transduced T cells showed higher avidity than the parental T-cell clone UCB55#74 (Figure 6).

[0148] To investigate the role of ligand uPA bound to uPAR D1D2D3 and the importance of N-glycosylation for the recognition of uPAR DI by HLA-independent TCRs, point mutations were inserted at the residues that are known to be relevant for the binding of uPA to DI and for the N-glycosylation of uPAR D1D2D3, respectively (Figure 7, left). None of the mutations interfered with the surface expression of uPAR D1D2D3. Mutation of several uPA binding sites abolished recognition by TCR1. The same was true for mutation of the N-glycosylation site located in domain DI at amino acid position asparagine (N) 52. Reduction or abolition of N-glycosylation of domains D2 and D3 resulted in impaired recognition of TCR1. This was most pronounced when N-glycosylation was abolished at four positions (Figure 7, right). This led to the conclusion that recognition by TCR1 required not only domain DI, but also the presence of ligand uPA and an at least partially intact N-glycosylation.

[0149] The recognition of primary malignant cells by naturally occurring anti-uP AR TCR was exemplarily tested on AML blasts from 19 patients with acute myeloid leukemia (AML). Effectors were buffy coat (BC) lymphocytes transduced with TCR1. Sixteen of 19 AML blast populations were recognized. Allo-HLA I reactivity was ruled out by the fact that the antibody W6 / 32 directed against HLA class I did not block recognition by TCR1-transduced T cells in any case. Specificity for uPAR was confirmed by almost complete blockade with an antibody directed against the DI domain in all three AML blast populations tested (Figure 8, right).

[0150] Amino acid sequences of preferred TCR-constructs according to the present invention:

[0151] UCB55#74 (TCR1) (SEQ ID NO: 1):

[0152] Variable regions of the beta chain [TRBV5-l*01 - TRBJ2-3*01 - TRBD1*01, including CDR3 (CASSLWTGGTDTQYFGPGTRLTVL)] and variable regions of the alpha chain [TRAV10*01 - TRAJ35*01, including CDR3 (CVVSPIGFGNVLHCGSGTQVIVLPD)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGOOVTLSCSPISGHRSVSW YQOTPGOGLOFLFEYFSETQRNKGNFPGRFSGROFSNSRSEMNVSTLELGDSAL YLCASSLWTGGTDTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKOKAT LVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSA TFWHNPRNHFRCQVQFHGPERRGQVARGQPQARDPEHLRRGWGRADCGITSA SYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLKQ AGDVEENPGPMKKHLTTFLVILXLYFYRGNGKNOVEQSPOSLIILEGKNCTLOC NYT VSPF SNLRW YKQDTGRGP VSLTIMTF SENTKSNGRYT ATLD ADTKQ S SLHI TASOLSDSASYICVVSPIGFGNVLHCGSGTQVIVLPDIQNPEPAVYQLKDPRSQD STLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTC QDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFN LLMTLRLWSS UCB54#15 (TCR2) (SEQ ID NO: 2):

[0153] Variable regions of the beta chain [TRBV5-l*01 - TRBJ1-2*01 - TRBD1*01, including CDR3 (CASSYLDRVGYGYTFGSGTRLTVV)] and variable regions of the alpha chain [TRAV8-2*01 - TRAJ15*01, including CDR3 (CVVRLHQAGTALIFGKGTTLSVSSN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0154] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGOOVTLSCSPISGHRSVSW YOOTPGOGLOFLFEYFSETQRNKGNFPGRFSGROFSNSRSEMNVSTLELGDSAL YLCASSYLDRVGYGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKOKAT LVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSA TFWHNPRNHFRCQVQFHGPERRGQVARGQPQARDPEHLRRGWGRADCGITSA SYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLKQ AGDVEENPGPMLLLLVPVLEVIFTLGGTRAOSVTOLDSHVSVSEGTPVLLRCNY SSSYSPSLFWYVOHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPS AHMSDAAEYFCVVRLHQAGTALIFGKGTTLSVSSNIQNPEPAVYQLKDPRSQDS TLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQ DIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNL LMTLRLWSS UCB54#19 (TCR3) (SEQ ID NO: 3):

[0155] Variable regions of the beta chain [TRBV5-5*02 - TRBJ2-l*01 - TRBD2*01, including CDR3 (CASSLGGPTRSSGPYNEQFFGPGTRLTVL)] and variable regions of the alpha chain [TRAV3*01 - TRAJ21*01, including CDR3 (CAVRSIYNFNKFYFGSGTKLNVKPN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0156] MPTLYKKAGFATMGPGLLCWVLLCLLGAGPVDAGVTQSPTHLIKTRGOHVTL RCSPISGHKSVSWYQOVLGOGPQFIFOYYEKEERGRGNFPDRFSAROFPNYSSE LNVNALLLGDSALYLCASSLGGPTRSSGPYNEQFFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAY KESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNI SAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMyKK KNSGSGATNFSLLKOAGDVEENPGPMASAPISMLAMLFTLSGLRAOSVAQPED OVNVAEGNPLTVKCTYSVSGNPYLFWYVOYPNRGLQFLLKYITGDNLVKGSY GFEAEFNKSOTSFHLKKPSALVSDSALYFCAVRSIYNFNKFYFGSGTKLNVKPNI QNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAM DSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQ NLS VMGLRILLLKVAGFNLLMTLRLWS S UCB54#11 (TCR4) (SEQ ID NO: 4):

[0157] Variable regions of the beta chain [TRBV5-l*01 - TRBJ1-1*O1 - TRBD1*01, including CDR3 (CASTRAQGAVEAFFGQGTRLTVV)] and variable regions of the alpha chain [TRAV17*01 - TRAJ29*01, including CDR3 (CAFRNSGNTPLVFGKGTRLSVIAN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0158] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGOOVTLSCSPISGHRSVSW YOOTPGOGLOFLFEYFSETQRNKGNFPGRFSGROFSNSRSEMNVSTLELGDSAL YLCASTRAQGAVEAFFGOGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATF WHNPRNHFRCQVQFHGPERRGQVARGQPQARDPEHLRRGWGRADCGITSASY HQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLKQAG DVEENPGPMETLLGVSLVILWLOLARVNSOOGEEDPOALSIOEGENATMNCSY KTSINNLOWYRQNSGRGLVHLILIRSNEREKHSGRLRVTLDTSKKSSSLLITASR AADTASYFCAFRNSGNTPLVFGKGTRLSVIANIONPEPAVYQLKDPRSODSTLC LFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIF KETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLM TLRLWSS UCB54#13 (TCR5) (SEQ ID NO: 5):

[0159] Variable regions of the beta chain [TRBV5-l*01 - TRBJ2-2*01 - TRBD1*01, including CDR3 (CASSSRNTGELFFGEGSRLTVL)] and variable regions of the alpha chain [TRAV26-l*01 - TRAJ40*01, including CDR3 (CIVSPTTSGTYKYIFGTGTRLKVLAN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0160] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGOOVTLSCSPISGHRSVSW YOOTPGOGLOFLFEYFSETQRNKGNFPGRFSGROFSNSRSEMNVSTLELGDSAL YLCASSSRNTGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVC LARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFW HNPRNHFRCQVQFHGPERRGQVARGQPQARDPEHLRRGWGRADCGITSASYH QGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLKQAGDVEENPGPMRLVARVTVFLTFGTIIDAKTTOPPSMDCAEGRAANLPCNHSTISGN EYVYWYRQIHSOGPOYIIHGLKNNETNEMASLIITEDRKSSTLILPHATLRDTAV YYCIVSPTTSGTYKYIFGTGTRLKVLANIONPEPAVYQLKDPRSODSTLCLFTDF DSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNA TYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLW SS UCB80#117 (TCR6) (SEQ ID NO: 6):

[0161] Variable regions of the beta chain [TRBV12-5*01- TRBD2*01- TRBJ2-3*01, including CDR3 (CASGLGTSGASDTQYFGPGTRLTVL)] and variable regions of the alpha chain [TRAV8*01-TRAJ20*01, including CDR3 (CAVESWENDYKLSFGAGTTVTVRAN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0162] MATRLLCCVVLCLLGEELIDARVTOTPRHKVTEMGOEVTMRCOPILGHNTVFW YROTMMOGLELLAYFRNRAPLDDSGMPKDRFSAEMPDATLATLKIQPSEPRDS AVYFCASGLGTSGASDTQYFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKO KATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLR VSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGIT SASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLL KQ AGP VEENPGPGMLLELIPLLGIHFVLRT ARAO SVTQPDIHITVSEGASLELRC NYSYGATPYLFWYVOSPGOGLQLLLKYFSGDTLVOGIKGFEAEFKRSOSSFNLR KPSVHWSDAAEYFCAVESWENDYKLSFGAGTTVTVRANIONPEPAVYQLKDP RSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQ TSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKV AGFNLLMTLRLWS S UCB81#24 (TCR7) (SEQ ID NO: 7):

[0163] Variable regions of the beta chain [TRBV27*01- TRBJ2-2*01, including CDR3 (CASSLSVAGFARQFFGPGTWLTVL)] and variable regions of the alpha chain [TRAV3*01-TRAJ23*01, including CDR3 (CAVRGIYNQGGKLIFGQGTELSVKPN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0164] MGPOLLGYVVLCLLGAGPLEAOVTQNPRYLITVTGKKLTVTCSONMNHEYMS WYRODPGLGLROIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNOT SLYFCASSLSVAGFARQFFGPGTWLTVLEDLRNVTPPKVSLFEPSKAEIANKOK ATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRV SATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITS ASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLK OAGDVEENPGPGMASAPISMLAMLFTLSGLRAOSVAQPEDOVNVAEGNPLTV KCTYSVSGNPYLFWYVOYPNRGLQFLLKYITGDNLVKGSYGFEAEFNKSOTSF HLKKPSALVSDSALYFCAVRGIYNOGGKLIFGOGTELSVKPNIONPEPAVYOLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSN QTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLK VAGFNLLMTLRLWS S UCB81#37 (TCR8) (SEQ ID NO: 8):

[0165] Variable regions of the beta chain [TRBV5-1*01- TRBJ1-3*01, including CDR3 (CASSPGTGTGNTIYFGEGSWLTVV)] and variable regions of the alpha chain [TRAV3*01-TRAJ11*01, including CDR3 (CAPGWDSGYSTLTFGKGTMLLVSPD)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0166] MPTLYKKAGFATMGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGOOVTL SCSPISGHRSVSWYOOTPGOGLOFLFEYFSETQRNKGNFPGRFSGROFSNSRSE MNVSTLELGDSALYLCASSPGTGTGNTIYFGEGSWLTWEDLRNVTPPKVSLFE PSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESN YSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEA WGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSG SGATNFSLLKQAGDVEENPGPGMASAPISMLAMLFTLSGLRAOSVAQPEDOVN VAEGNPLTVKCTYSVSGNPYLFWYVOYPNRGLQFLLKYITGDNLVKGSYGFEA EFNKSOTSFHLKKPSALVSDSALYFCAPGWDSGYSTLTFGKGTMLLVSPDIONP EPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKS NGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSV MGLRILLLK VAGFNLLMTLRLWS S UCB81#151 (TCR9) (SEQ ID NO: 9):

[0167] Variable regions of the beta chain [TRBV19*01- TRBJ1-2*01, including CDR3 (CASSIQGNYGYTFGSGTRLTVV)] and variable regions of the alpha chain [TRAV14 / DV4-TRAJ21*01, including CDR3 (CAMREFYNFNKFYFGSGTKLNVKPN)] are underlined. Not underlined are the murine beta and alpha constant regions and the P2A element.

[0168] MSNOVLCCVVLCLLGANTVDGGITOSPKYLFRKEGONVTLSCEQNLNHDAMY WYRODPGOGLRLIYYSOIVNDFOKGDIAEGYSVSREKKESFPLTVTSAQKNPTA FYLCASSIQGNYGYTFGSGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKOKATL VCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATF WHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYH QGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSGSGATNFSLLKQAGD VEENPGPGMSLSSLLKVVTASLWLGPGIAOKITOTQPGMFVOEKEAVTLDCTY DTSDPSYGLFWYKQPSSGEMIFLIYOGSYDOONATEGRYSLNFOKARKSANLVI SASOLGDSAMYFCAMREFYNFNKFYFGSGTKLNVKPNIONPEPAVYOLKDPRS QDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSF TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAG FNLLMTLRLWSSLiterature as cited

[0169] (1) Madsen CD, Ferraris GM, Andolfo A, Cunningham O, Sidenius N. uPAR-induced cell adhesion and migration: vitronectin provides the key. J Cell Biol. 2007;177(5):927-39.

[0170] (2) Manfredi M, Van Hoovels L, Benucci M, De Luca R, Coccia C, Bernardini P, et al. Soluble Urokinase Plasminogen Activator Receptor (suPAR) in Autoimmune Rheumatic and Non Rheumatic Diseases. J Pers Med. 2023;13(4).

[0171] (3) Blasi F, Carmeliet P. uPAR: a versatile signalling orchestrator. Nat Rev Mol Cell Biol.

[0172] 2002;3(12):932-43.

[0173] (4) Mondino A, Blasi F. uPA and uPAR in fibrinolysis, immunity and pathology. Trends Immunol. 2004;25(8):450-5.

[0174] (5) Gonias SL, Hu J. Urokinase receptor and resistance to targeted anticancer agents. Front Pharmacol. 2015;6:154.

[0175] (6) Zhai BT, Tian H, Sun J, Zou JB, Zhang XF, Cheng JX, et al. Urokinase-type plasminogen activator receptor (uPAR) as a therapeutic target in cancer. J Transl Med.

[0176] 2022;20(1):135.

[0177] (7) Lv T, Zhao Y, Jiang X, Yuan H, Wang H, Cui X, et al. uPAR: An Essential Factor for Tumor Development. Journal of Cancer. 2021;12(23):7026-40.

[0178] (8) Lawaetz M, Christensen A, Juhl K, Lelkaitis G, Karnov K, Carlsen EA, et al. Diagnostic Value of Preoperative uPAR-PET / CT in Regional Lymph Node Staging of Oral and Oropharyngeal Squamous Cell Carcinoma: A Prospective Phase II Trial. Diagnostics (Basel). 2023; 13(21).

[0179] (9) Amor C, Feucht J, Leibold J, Ho YJ, Zhu C, Alonso-Curbelo D, et al. Senolytic CAR T cells reverse senescence-associated pathologies. Nature. 2020.

[0180] (10) Amor C, Fernandez-Maestre I, Chowdhury S, Ho YJ, Nadella S, Graham C, et al. Prophylactic and long-lasting efficacy of senolytic CAR T cells against age-related metabolic dysfunction. Res Sq. 2023.

[0181] (11) Zhao F, Sucker A, Horn S, Heeke C, Bielefeld N, Schrors B, et al. Melanoma Lesions Independently Acquire T-cell Resistance during Metastatic Latency. Cancer Res.

[0182] 2016;76(15):4347-58.

[0183] (12) Birkholz K, Hofmann C, Hoyer S, Schulz B, Harrer T, Kampgen E, et al. A fast and robust method to clone and functionally validate T-cell receptors. J Immunol Methods.

[0184] 2009;346(1-2):45-54.

[0185] (13) Kropp KN, Schaufele TJ, Fatho M, Volkmar M, Conradi R, Theobald M, et al. A bicistronic vector backbone for rapid seamless cloning and chimerization of aPT-cell receptor sequences. PLoS One. 2020;15(9):e0238875.

[0186] (14) Sillaber C, Baghestanian M, Hofbauer R, Virgolini I, Bankl HC, Fureder W, et al. Molecular and functional characterization of the urokinase receptor on human mast cells. J Biol Chem. 1997;272(12):7824-32.

[0187] (15) Behrendt N, Ronne E, Ploug M, Petri T, Lober D, Nielsen LS, et al. The human receptor for urokinase plasminogen activator. NH2 -terminal amino acid sequence and glycosylation variants. J Biol Chem. 1990;265(11):6453-60.

[0188] (16) Tran E, Robbins PF, Lu YC, Prickett TD, Gartner JJ, Jia L, et al. T-Cell Transfer Therapy Targeting Mutant KRAS in Cancer. N Engl J Med. 2016;375(23):2255-62. (17) Sari G, Rock KL. Tumor immune evasion through loss of MHC class-I antigen presentation. Curr Opin Immunol. 2023;83: 102329.(18) Stupia S, Heeke C, Bruggemann A, Zaremba A, Thier B, Kretz J, et al. HLA class II loss and JAK1 / 2 deficiency coevolve in melanoma leading to CD4 T cell and IFNgamma cross-resistance. Clin Cancer Res. 2023.

[0189] (19) Guha P, Heatherton KR, O'Connell KP, Alexander IS, Katz SC. Assessing the Future of Solid Tumor Immunotherapy. Biomedicines. 2022; 10(3).

[0190] (20) Qin L, Wang L, Zhang J, Zhou H, Yang Z, Wang Y, et al. Therapeutic strategies targeting uPAR potentiate anti-PD-1 efficacy in diffuse-type gastric cancer. Sci Adv.

[0191] 2022;8(21):eabn3774.

[0192] (21) Zhou Y, Yu S, Chen D, Li H, Xu P, Yuan C, et al. Nafamostat Mesylate in Combination with the Mouse Amino-Terminal Fragment of Urokinase-Human Serum Albumin Improves the Treatment Outcome of Triple-Negative Breast Cancer Therapy. Mol Pharm. 2023;20(2):905-17.

[0193] (22) Han Y, Tu L, Zhang Y, Xu L, Sun Z. A novel urokinase plasminogen activator receptor-targeted peptide-based probe for in-vivo molecular imaging of glioblastoma. Nucl Med Commun. 2023;44(2): 142-9.

[0194] (23) Mansilla-Soto J, Eyquem J, Haubner S, Hamieh M, Feucht J, Paillon N, et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med.

[0195] 2022.

[0196] (24) He S, Sharpless NE. Senescence in Health and Disease. Cell. 2017; 169(6): 1000-11. Carpenter VJ, Patel BB, Autorino R, Smith SC, Gewirtz DA, Saleh T. Senescence and castration resistance in prostate cancer: A review of experimental evidence and clinical implications. Biochim Biophys Acta Rev Cancer. 2020; 1874(2): 188424.

[0197] (25) Nakatsugawa M, Yamashita Y, Ochi T, Tanaka S, Chamoto K, Guo T, et al. Specific roles of each TCR hemichain in generating functional chain-centric TCR. J Immunol.

[0198] 2015;194(7):3487-500.

[0199] (26) Stupia et al., Clin Cancer Res 2023, PMID: 37199727, DOI: 10.1158 / 1078-0432. CCR-23-0099.

[0200] (27) Hazini et al. Deregulation of HLA-I in cancer and its central importance for immunotherapy. J Immunother Cancer. 2021 Aug;9(8):e002899. doi: 10.1136 / jitc-2021-002899. PMID: 34353849; PMCID: PMC8344275.

[0201] (28) Metrangolo et al. The Urokinase Receptor (uPAR) as a " Trojan Horse" in Targeted Cancer Therapy: Challenges and Opportunities. Cancers (Basel). 2021 Oct 27;13(21):5376. doi: 10.3390 / cancersl3215376. PMID: 34771541; PMCID: PMC8582577.

[0202] (29) Fan et al. Therapeutic cancer vaccines: advancements, challenges and prospects. Sig Transduct Target Ther 8, 450 (2023). https: / / doi.org / 10.1038 / s41392-023-01674-3.

Claims

Claims1. A binding agent comprising the amino acid sequence of a binding region of a T-cell receptor binding to a cancer cell expressing human uPAR (SEQ ID No. 10), preferably expressing a fragment comprising domains D1 / D2 / D3, and more preferably comprising domains DI or D2 / D3 thereof, or an amino acid sequence having at least 75% sequence identity to human uPAR or the fragment thereof, wherein said binding is independent of HLA.

2. The binding agent according to claim 1, wherein said T-cell receptor is selected from a TCR-construct comprising a TCR alpha or beta chain comprising a sequence according to any one of SEQ ID NOs: 1 to 9.

3. A nucleic acid molecule encoding a binding agent according to claims 1 or 2, a vector comprising the nucleic acid molecule, such as, for example, an expression vector comprising and expressing the nucleic acid molecule, or a recombinant or genetically modified cell comprising the nucleic acid molecule or vector.

4. A recombinant or genetically modified T cell expressing a TCR that binds to human uPAR or a protein having at least 75% sequence identity to human uPAR, preferably of the domains DI, D2 / D3 or D1 / D2 / D3 thereof, wherein said binding is independently from HLA, such as, for example, a TCR-construct comprising a sequence according to any one of SEQ ID NOs: 1 to 9, or a fragment thereof binding human uPAR.

5. An in-vitro method for generating HLA independent T cells, comprisinga) providing a first cell that expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to uPAR or an immunogenic protein fragment thereof, preferably wherein the polypeptide is full length human uPAR or preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof,b) bringing a population of peripheral blood mononuclear cells (PBMCs) into contact with said first cell, and thereby stimulating said PBMCs, andc) selecting from the population of stimulated PBMCs T cells that have the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell, wherein preferably in step c) the ability of a T cell to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independently of the expression of HLA in said cell is determined by testing the reactivity of said T-cell against a cell expressing the human uPAR or an immunogenic protein fragment thereof, whereini) the cell expressing the human uPAR or an immunogenic protein fragment thereof lacks MHC class I or MHC class I and II expression, and / orii) the T-cell is tested for its reactivity against said cell expressing the human uPAR or an immunogenic protein fragment thereof in the presence of antibodies against MHC class I or II; and / oriii) the T-cell is tested for its reactivity against xenogenic cells transfected with DNA or RNA encoding the human uPAR or an immunogenic protein fragment thereof, wherein in i), ii) and / or iii) a T-cell that shows reactivity is a T-cell having the ability to recognize a cell expressing the human uPAR or an immunogenic protein fragment thereof used in a) independent of the expression of HLA in said cell.

6. An HLA-independent T cell, generated according to a method according to claim 5.

7. A pharmaceutical composition, comprising a binding agent according to any one of claims 1 or 2, a nucleic acid or vector according to claim 3, or a T-cell according to claim 4, and at least one pharmaceutically acceptable carrier, preferably the pharmaceutical composition for use as a medicament, preferably for use in the prevention or treatment of the accumulation of senescent cells and / or a cancer cell exhibiting down-regulation or loss of HLA expression, wherein preferably said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response, preferably an MHC class Lindependent T-cell response, or an MHC class I- and II-independent T-cell response.

8. The pharmaceutical composition for use according to claim 7, wherein the senescent and / or cancer cell expresses human uPAR (SEQ ID No. 10) or a protein having at least75% sequence identity to human uPAR, preferably a fragment comprising the DI, D2 / D3 or D1 / D2 / D3 domains thereof.

9. A pharmaceutical composition comprising human uPAR of SEQ ID NO: 10 or a functional homologue thereof at least 75% identical thereto or an immunogenically active peptide fragment comprising a consecutive sequence of at least 8 amino acids of human uPAR or said functional homolog thereof, preferably of the DI, D2 / D3 or D1 / D2 / D3 domains thereof, or a nucleic acid encoding said PD-L1, said functional homolog thereof or said peptide fragment; and b) at least one pharmaceutically acceptable carrier for use in the prevention or treatment of the accumulation of senescent cells and / or a cancer cell exhibiting down-regulation or loss of HLA expression, which preferably is an anti -cancer vaccine.

10. The pharmaceutical composition for use according to claim 9, wherein said prevention or treatment comprises inducing a major histocompatibility complex (MHC)-independent T-cell response, preferably an MHC class I-independent T-cell response, or an MHC class I- and Il-independent T-cell response, wherein preferably the senescent cell and / or cancer cell expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domains thereof.

11. The pharmaceutical composition for use according to claim 9 or 10, wherein the vaccine composition comprises antigen-presenting cells comprising the immunogenically active peptide fragment or a nucleic acid encoding said immunogenically active peptide fragment, wherein preferably the antigen-presenting cell is a dendritic cell.

12. Method for eliciting a CD4- or CD8-positive T-cell response in a subject independently from a presentation of an antigen by MHC, comprising administering to said subject the pharmaceutical composition for use according to any one of claims 7 to 11.

13. A kit comprising; a) the pharmaceutical composition for use according to any one of claims 7 to 11, and b) a composition comprising at least one second pharmaceutically active ingredient.

14. The kit according to claim 13, wherein the second active ingredient is an anti -cancer agent, such as an anti-cancer agent selected from a chemotherapeutic agent, for example selected from: Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Revlimid, Temozolomide, Teniposide, Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine, and Vinorelbine, wherein preferably the provided compositions are to be administered simultaneously or sequentially.

15. A method of preventing or treating a senescent and / or a cancer cell exhibiting downregulation or loss of HL A expression, wherein the senescent and / or cancer cell furthermore expresses human uPAR (SEQ ID No. 10) or a protein having at least 75% sequence identity to human uPAR, preferably the DI, D2 / D3 or D1 / D2 / D3 domains thereof, the method comprising administering to subject suffering or prospectively suffering from said senescent cells and / or cancer an effective amount of the pharmaceutical composition for use according to any one of claims 7 to 11, wherein preferably the method is combined with a further anti-senescence and / or cancer treatment, wherein preferably the further treatment is selected from the group consisting of chemotherapy, radiotherapy, treatment with immunostimulating substances, gene therapy, treatment with antibodies and treatment using dendritic cells.

Images