T-cell receptor-derived binding polypeptides

Abstract

The present invention relates to binding polypeptides comprising a first variable T-cell receptor (TCR) domain and a second variable TCR domain, wherein (i) the complementarity determining region 3 (CDR3) of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 or a sequence at least 80% identical thereto; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2 or a sequence at least 80% identical thereto; or (ii) the CDR3 of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3 or a sequence at least 80% identical thereto; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4 or a sequence at least 80% identical thereto; and polynucleotides, host cells, methods, uses, kits and devices related thereto.

Description

[Technical field]

[0001] The present invention relates to binding polypeptides comprising a first variable T-cell receptor (TCR) domain and a second variable TCR domain, wherein (i) the complementarity determining region 3 (CDR3) of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 or a sequence at least 80% identical thereto; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2 or a sequence at least 80% identical thereto; or (ii) the CDR3 of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3 or a sequence at least 80% identical thereto; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4 or a sequence at least 80% identical thereto; and polynucleotides, host cells, methods, uses, kits and devices related thereto. [Background technology]

[0002] Adoptive T cell therapy using engineered T cells expressing chimeric antigen receptors (CARs) against the B cell antigen CD19 has achieved significant regressions in leukemia and lymphoma patients. Applying this cellular concept to solid tumors has proven difficult, with a major obstacle being the identification of appropriate cell surface antigens. On the other hand, intracellular tumor-associated antigens (TAAs) can also be processed and presented on the major histocompatibility complex to T cells and thus exploited as T cell targets. Despite successful induction or amplification of T cell responses against TAAs by therapeutic vaccination, the level of cellular effector immune responses required to control tumor growth has not been achieved [e.g., Hilf et al., Nature 565:240-245 (2019); Keskin et al., Nature 565:234-239 (2019)].

[0003] Tumor infiltrating leukocyte (TIL) therapy is another therapeutic option to amplify spontaneous T cell responses ex vivo. However, the non-specific cytokine-driven in vitro expansion of TILs inevitably causes the loss of potential TAA-reactive T cell clones before reinfusion into patients, attenuating the efficacy of cell therapy. Here, T cell receptor (TCR) transgenic cell therapy offers a beneficial alternative since transgenic cells target known defined antigens, TAA-reactive transgenic cells can be further genetically modified to enhance T cell responses, and in contrast to TIL therapy, reinfusion of monoclonal TCR transgenic T cell products allows for accurate enumeration of truly tumor-reactive T cells.

[0004] Gliomas, tumors with a generally low mutational burden, contain only 30–50 nonsynonymous mutations. Recently, two multinational, multicenter glioblastoma (WHO classification IV glioma) vaccine consortia, The Glioma Actively Personalized Vaccine Consortium (GAPVAC) and The NeoVax Consortium, each independently reported successful induction of T cell responses specific to highly personalized TAAs, but also to tumor-specific antigens (TSAs), in glioblastoma patients (Hilf et al., loc. cit.; Keskin et al., loc. cit.). Both phase 1 trials were able to demonstrate homing of vaccine-induced T cells to brain tumors. Similarly, a multicenter, single-arm, open-label, first-in-human phase 1 study conducted in 33 patients with newly diagnosed World Health Organization grade 3 and 4 mutant isocitrate dehydrogenase 1 (IDH1R132H)-positive astrocytomas [Clinical Trial 16 (NOA16) of the Neurooncology Working Group of the German Cancer Society, NCT02454634] provided proof of concept that it is in principle possible to home target-specific helper T cells to gliomas [Platten et al., Nature 592:463-468 (2021)]. At the same time, evidence from these studies suggests that the strength of vaccine-induced specific T cell responses can still be improved. Summary of the Invention

[0005] Therefore, there is still a need for improving cancer treatment. The technical problem underlying the present invention can be seen as the provision of means and methods for meeting the above-mentioned needs. This technical problem is solved by the embodiments characterized in the claims and hereinafter.

[0006] Thus, the present invention relates to a binding polypeptide comprising a first variable T-cell receptor (TCR) domain and a second variable TCR domain. [Brief description of the drawings]

[0007] [Figure 1-1] FIG. 1 shows HLA-A2-restricted TCRs that bind PTPRZ11814-1822. (A) Single cell VDJ sequencing of activation-based sorted PTPRZ11814-1822-reactive T cells from HLA-A2 positive patients after peptide vaccination; (B) Exemplary flow cytometry analysis following transfection of TCR-deficient Jurkat T cells with PTPRZ11814-1822-TCR containing mouse TCR beta chain compared to TCRβ fluorescence minus one (FMO) for flow cytometry-based analysis of TCR expression; (C) Nuclear factor of activated T cells (NFAT) reporter-based activation assay of PTPRZ11814-1822-TCR transgenic Jurkat T cells using T2 cells as antigen presenting cells and HLA-A2 positive U87 glioma cells loaded with PTPRZ11814-1822 peptide or PTPRZ11814-1822-containing tandem minigenes, respectively; (D) (E) PTPRZ11814-1822-pentamer flow cytometry for PTPRZ11814-1822 TCR transgenic Jurkat T cells; (F) PTPRZ11814-1822 killing assay using U87 and P3 as glioma target cells and T cells from healthy donors (n=3). Experimental conditions are indicated in the figure. Flu is negative control peptide and TCR. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 2-1]HLA-A2-restricted TCR-binding NLGN4X131-139. (A) Single cell VDJ sequencing of multimer-based sorted NLGN4X131-139-reactive T cells from HLA-A2 positive patients after peptide vaccination; (B) Intracellular flow cytometry of NLGN4X131-139-reactive TCR transgenic healthy donor T cells (n=3) after overnight co-culture with peptide-loaded K562 antigen presenting cells (MOG, myelin oligodendrocyte glycoprotein, negative control peptide; CD3 / CD28, positive control); (C) NLGN4X131-139-reactive TCR SFG vector transduced human primary T cells were co-cultured with peptide-loaded U87 glioma cells and U87 cells overexpressing full-length NLGN4X protein (MOG, myelin oligodendrocyte glycoprotein, negative control peptide); (D) 7 days after transduction, NLGN4X131-139-reactive TCR (E) Vital FR assay using TCR transgenic primary human T cells transduced with NLGN4X131-139-reactive TCR SFG vector targeting U87 expressing NLGN4X131-139-containing tandem minigene (TMG) at an E:T ratio of 2:1: pLEX307 vector-transduced primary human T cells co-cultured with peptide-loaded U87 glioma cells (MOG, myelin oligodendrocyte glycoprotein, negative control peptide); (F) Vital FR assay using TCR transgenic primary human T cells transduced with NLGN4X131-139-reactive TCR SFG vector targeting U87 expressing NLGN4X131-139-containing tandem minigene (TMG) at an E:T ratio of 2:1: [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 3-1]Figure 1: NLGN4X-TCR-T recognized and lysed target cells in vitro as effectively as clinically used control TCR. (A) VDJ analysis of single cell TCR sequencing of multimer-sorted patient T cells after vaccination. Frequency: ft1-90.82%; ft2-1.78%; ft3-0.96%; ft4-0.96%. (B) Exemplary flow cytometry analysis of transfection efficiency of TCR-transfected Jurkat T cells. (C) Jurkat76 T cells transfected with NLGN4X ft1-4 TCR co-cultured with peptide-loaded BOLETH APC. Mean and SEM of three technical replicates. (D) NFAT-reporter assay of NLGN4X TCR-transfected Jurkat76 T cells co-cultured with peptide-loaded HLA-A*02+ PBMC. RLU = relative luminescence units. Mean and SEM of three technical replicates. (E) NFAT-reporter assay of NLGN4X TCR transfected Jurkat76 T cells co-cultured with peptide-loaded HLA-A*02+ U87 glioma cells. RLU = relative luminescence units. Mean and SEM of three technical replicates. [Figure 3-2] Continued from Figure 3-1. [Figure 4-1]Development of a manufacturing process for T cell products. (A) Schematic overview of TCRft1-pLEX307 EF1-alpha: EF1-alpha promoter-TCR beta chain (including mouse TCR beta constant region)-TCR alpha chain-Woodchuck Hepatitis Virus post-transcriptional regulatory element-Puromycin resistance. (B) Exemplary transduction efficiency of human T cells transduced with TCRft1- pLEX307 EF1-alpha by flow cytometric analysis of mTCRb compared to mock transduced T cells. (C) Schematic overview of SFG-IRES-GFP vector: long terminal repeat-Moloney murine leukemia virus-TCR beta chain (including mouse TCR beta constant region)-TCR alpha chain-internal ribosome entry site-GFP-long terminal repeat. (D) Exemplary transduction efficiency of human T cells transduced with TCRft1-SFG-IRES-GFP compared to mock-transduced T cells by flow cytometric analysis of mTCRb. (E) GFP+ human T cells after transduction with TCRft1-SFG-IRES-GFP retroviral vector. Mean and SEM. (F) mTCRb expression in primary T cells 4 days after transduction with TCRft1-SFG-IRES- GFP retroviral vector. (G) Multicolor flow cytometric assessment of different phenotypic markers in human T cells after transduction with TCRft1-SFG-IRES-GFP vector. n=2 biological replicates. [Figure 4-2] Continued from Figure 4-1. [Figure 4-3] Continued from Figure 4-2. [Figure 5-1]NLGN4X-TCR-T recognized and lysed target cells in vitro as effectively as a clinically used control TCR. (A) Heatmap of functional responses (IFNγ, TNFα, GrzB) of three different donors transduced with NLGN4X131-139 TCR and co-cultured with peptide-loaded HLA-A*02+ K562 leukemia cells. For statistical analysis, compare Figures 3B-D. n=3 biological replicates. (B) TNFα production of NLGN4X-TCR-T and Mart-1-TCR-T cultured with peptide-loaded HLA-A*02+ K562 leukemia cells. Target peptide: NLGN4X-TCR-T vs. MART-1 TCR T cells, p = 0.0863. Mean and SEM of n=3 biological replicates. Two-way ANOVA. (C) IFNγ production of NLGN4X-TCR-T and Mart-1-TCR-T cultured with peptide-loaded HLA-A*02+ K562 leukemia cells. Target peptide: NLGN4X-TCR-T vs. MART-1-TCR-T p = 0.2926. (D) Granzyme B expression of NLGN4X-TCR-T vs. MART-1-TCR-T cultured with peptide-loaded HLA-A*02+ K562 leukemia cells. Target peptide: NLGN4X-TCR-T vs. MART-1-TCR-T p = 0.3461. Mean and SEM of n=3 biological replicates. Two-way ANOVA. (E) Exemplary outline of the modified Vital FR assay used in this study: target cells endogenously expressing the target epitope or exogenously loaded with the respective peptide were labeled with CellTrace™ FarRed, non-target cells (no expression of irrelevant peptide or target) were labeled with CellTrace™ Violet and cultured with target-specific TCR-transduced T cells in the same well. (F) Viability of K562 cells, either loaded or not with target peptide, was assessed by flow cytometry analysis after overnight co-culture with NLGN4X-TCR-T or MART-1-TCR-T. Mean and SEM of n=3 biological replicates. Two-way ANOVA.The target peptide for NLGN4X-TCR-T is the NLGN4X131-139 peptide, and for MART-1-TCR-T it is the MART-1 peptide (Ellingson BM et al., 2017; Rohaan MW et al., 2022; Morgan RA et al., 2013; Chheda ZS et al., 2018; Immisch L et al., 2022; Kilian M et al., 2022; Bolliger MF et al., 2008; Choe JH et al., 2021; Sahillioglu AC et al., 2022). The control peptide is the MOG (myelin oligodendrocyte glycoprotein) peptide (Sahillioglu AC et al., 2022). [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 6-1]NLGN4X TCR specifically recognizes and lyses glioma cells expressing the NLGN4X target epitope. (A) Heatmap of functional responses (CD69, 4.1BB, GrzB) of three different donors co-cultured with NLGN4X TCR-transduced and peptide-loaded HLA-A*02+ U87 glioma cells. (B) Detection of LDH release measured by optical density (OD) after overnight co-culture of NLGN4X-TCR-T and Flu (MHCI epitope) TCR transgenic T cells (Flu-TCR-T) with NLGN4X or Flu peptide-loaded U87 glioma cells. Mean and SEM of n=3 biological replicates. Two-way ANOVA. (C) NLGN4X-TCR-T were co-cultured with peptide-loaded or unloaded U87 glioma cells and specific cytotoxicity was calculated using FACS-based counting of tumor cells. Mean and SEM of n=3 biological replicates. Unpaired t-test. (D) Heatmap of functional responses (CD69, 4-1BB, GrzB) of three different donors transduced with NLGN4X TCR or Flu TCR (here: negative control TCR) and co-cultured with U87 glioma endogenously expressing NLGN4X protein sequence containing the relevant epitope (U87 NLGN4X) or tandem minigene containing antigenic sequence of NLGN4X (U87 TMG). (E) LDH release assay of NLGN4X-TCR-T versus Flu-TCR-T (TCR negative control) targeting either U87 NLGN4X, U87 TMG or U87 (targeting negative control) glioma cells. Mean and SEM of n=3 biological replicates. Two-way-RM-ANOVA. The target peptide for NLGN4X-TCR-T is the NLGN4X131-139 peptide. The control peptide is the Flu (influenza) 58-66 MHC class I peptide (disclosed in Choo, J Virol, 2014), against which Flu-TCR-T is reactive (VDJdb.cdr3.net). [Figure 6-2] Continued from Figure 6-1. [Figure 6-3] Continued from Figure 6-2. [Figure 7-1]Intraventricular delivery of NLGN4X-TCR-T mediates transient tumor regression and enhanced survival in an intracranial tumor model. (A) Schematic experimental outline: NSG MHC I / II KO mice were challenged with intracranially U87 NLGN4X antigen-overexpressing gliomas and tumor growth was confirmed before injection of NLGN4X-TCR-T or Flu-TCR-T at days 15 and 22 post-tumor inoculation. (B) Preclinical survival of mice treated with either NLGN4X-TCR-T or Flu-TCR-T targeting intracranial NLGN4X antigen-overexpressing U87 cells. NTC = no T cell control. n = 9 mice for NLGN4X-TCR-T, n = 8 mice for Flu-TCR-T, n = 7 for NTC. Log-rank test. (C) Radiation response assessment by mRANO criteria: Complete response (CR) was defined as -100%, partial response (PR) as <-65%, stable disease (SD) as >-65% to +40%, and progressive disease (PD) as >+40% change in tumor volume from day 11 to day 67. (D) MRI image of one long-term survivor NLGN4X-TCR-T treated animal showing tumor regression at the initial tumor site until day 67 and tumor progression at day 98. (E) Individual growth curves of U87 NLGN4X antigen expressing glioma cells from NLGN4X-TCR-T (I) and Flu-TCR-T (II) treated animals. Encircled mice were analyzed by FACS as shown in Figure 7F. Growth in Log10 scale. Tumor volume at V=0 μL is therefore not shown in the graph. CR=complete response, PR=partial response, SD=stable disease, PD=progressive disease, D=death. To visualize tumor growth, the detection limit of tumor volume was set at 0.1 μL. (F) Representative flow cytometry analysis of two animals with late tumor recurrence (M1=mouse 1, M2=mouse 2) shows persistence of primary CD4+ T cells with a predominant CCR7-CD45RA- effector memory phenotype at the tumor site, impaired proliferation with high PD-1 expression. Gating was done on live hCD3+ T cells.(G) Real-time quantitative PCR of U87 TMG plasmid sequences in tumors of NLGN4X-TCR-T, Flu-TCR-T treated or NTC animals at late time points compared to in vitro cultured U87 TMG and U87 cells. Relative expression to hGAPDH or hβ-actin. Log10 scale. n=3 biological replicates. (H) Exemplary immunofluorescence staining of HLA-A expression: one NLGN4X-TCR-T treated and one untreated animal at terminal time points are shown. [Figure 7-2] Continued from Figure 7-1. [Figure 7-3] Continued from Figure 7-2. [Figure 8-1] NLGN4X-TCR-T has an effector phenotype in the tumor microenvironment after intraventricular delivery. (A) Experimental overview: U87 gliomas overexpressing NLGN4X antigen were injected intracranially and NLGN4X-TCR-T or Flu-TCR-T were injected into the contralateral ventricle. Six days later, T cells were analyzed by flow cytometry to assess ex vivo activation. (B) Exemplary flow cytometry plots showing intratumoral CD3+ T cells, CD4-CD8 distribution, and mTCRb and GFP expression. (C) Number of CD3+CD8+ T cells in the TME, normalized (to tumor volume). n=8 (NLGN4X-TCR-T), n=7 (Flu-TCR-T). Unpaired t-test. (D) Exemplary FACS plots showing CD45RA and CCR7 expression and Ki67 and PD-1 expression in intratumoral CD3+CD8+ T cells (TCM=central memory T cells, TN=naive T cells, TEM=effector memory T cells, TEM-CD45RA+=CD45RA re-expressing TEM). (E) Heatmap of phenotypic markers of intratumoral CD3+CD8+ T cells. n=8 (NLGN4X-TCR-T), n=7 (Flu-TCR-T). (F) Evaluation of activation and effector cell markers of intratumoral CD8+ T cells. n=8 (NLGN4X-TCR-T), n=7 (Flu-TCR-T). Two-way ANOVA. [Figure 8-2] Continued from Figure 8-1. [Figure 8-3] Continued from Figure 8-2. [Figure 8-4] Continued from Figure 8-3. [Figure 9] Figure 1 shows in vitro activation and cytotoxicity of PTPRZ1-specific TCR-engineered human T cells. Activation of TCR-T cells. T cells were co-cultured with peptide-loaded terminal epitope expressing U87 cells and endogenous PTPRZ1 expressing P3 cell line. Activation was assessed by flow cytometry gated on CD8+ TCR+ cells. [Figure 10] Figure 1 shows the efficacy of PTPRZ1-specific TCR-engineered human T cells in vivo. NSG MHC KO mice are subcutaneously inoculated with U87 cell lines expressing the epitope. Mice received two doses of adoptive cell transfer of transduced TCR-T cells. Tumor growth is measured with calipers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] In general, the terms used herein shall be given their usual and customary meanings to those skilled in the art, and shall not be limited to special or customized meanings unless otherwise indicated. When used below, the terms "have", "comprise" or "include" or any grammatical variants thereof shall be used non-exclusively. That is, these terms may refer to both the situation where no further features are present in the entity described in this context, other than the features introduced by these terms, and the situation where one or more further features are present. As an example, the expressions "A has B", "A contains B" and "A includes B" may refer to both the situation where no other elements are present in A, other than B (i.e., the situation where A is solely and exclusively composed of B), and the situation where one or more further elements are present in entity A, other than B (such as element C, elements C and D, or further elements). Also, as will be appreciated by those skilled in the art, the phrases "comprising a" and "comprising an" preferably mean "comprising one or more," i.e., equivalent to "comprising at least one." Thus, a phrase referring to one of a plurality of items, unless otherwise indicated, preferably relates to at least one such item, more preferably a plurality thereof; thus, for example, identifying a "cell" relates to identifying at least one cell, preferably identifying a multiplicity of cells.

[0009] Furthermore, when used below, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "particularly", "more particularly" or similar terms are used with optional features without further limiting the possibilities. That is, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. The invention can be implemented by using alternative features, as the skilled person will recognize. Similarly, features introduced by "in one embodiment" or similar expressions are intended to be optional features, without any limitations on further embodiments of the invention, without any limitations on the scope of the invention, and without any limitations on the possibility of combining the features introduced in such a way with other optional or non-optional features of the invention.

[0010] The method specified herein below is preferably an in vitro method.The steps of this method can be carried out in any order that is considered suitable by those skilled in the art, but are preferably carried out in the order shown; and one or more of said steps, preferably all of them, can be assisted or carried out by an automated device.Furthermore, the method can include steps in addition to those explicitly mentioned above.

[0011] As used herein, the term "standard conditions" refers to IUPAC standard ambient temperature and pressure (SATP) conditions, unless otherwise specified, i.e., preferably at a temperature of 25° C. and an absolute pressure of 100 kPa; also preferably, the standard conditions include a pH of 7. Furthermore, unless otherwise indicated, the term "about" refers to the indicated value with the technical precision generally accepted in the relevant field, preferably to the indicated value ±20%, more preferably ±10%, most preferably ±5%. Furthermore, the term "essentially" indicates that there is no deviation having an effect on the indicated result or use, i.e., possible deviations do not cause deviations of more than ±20%, more preferably ±10%, most preferably ±5% of the indicated result. In other words, "consisting essentially of" means including the specified components, but excluding other components, except for materials present as impurities, unavoidable materials present as a result of the process used to provide the components, and components added for purposes other than achieving the technical effect of the present invention. For example, a composition defined using the phrase "consisting essentially of" includes any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a set of components will contain less than 5% by weight, more preferably less than 3% by weight, even more preferably less than 1% by weight, and most preferably less than 0.1% by weight of an unspecified component.

[0012] The degree of identity (e.g., expressed as "% identity") between two biological sequences, preferably DNA, RNA, or amino acid sequences, can be determined by algorithms well known in the art. Preferably, the degree of identity is determined by comparing two optimally aligned sequences over a comparison window, where the fragments of the sequences in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the sequences compared for optimal alignment. The percentage is calculated by determining the number of positions at which identical residues exist in both sequences, preferably over the entire length of the polynucleotide or polypeptide, to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970), by the similarity search method of Pearson and Lipman (1988), by computer implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), or by visual inspection. Given that two sequences are specified for comparison, GAP and BESTFIT are preferably used to determine their optimal alignment, i.e., degree of identity. Preferably, default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used. In the context of biological sequences referred to herein, the term "essentially identical" denotes a percent identity value of at least 80%, preferably at least 90%, more preferably at least 98%, and most preferably at least 99%.As will be understood, the term "essentially identical" includes 100% identity. The above applies mutatis mutandis to the term "essentially complementary."

[0013] The term "fragment" of a biopolymer, preferably a polynucleotide or polypeptide, is used herein in a broad sense to refer to any subpart, preferably a subdomain, of the respective biopolymer, including the sequence, structure and / or function shown. Thus, the term includes subparts generated by actual fragmentation of the biopolymer, but also subparts derived from the respective biopolymer in a conceptual manner, e.g., in silico. Thus, as used herein, Fc or Fab fragments, but also, for example, single chain antibodies, bispecific antibodies and nanobodies, can be referred to as fragments of immunoglobulins.

[0014] Unless otherwise indicated herein, the compounds specified, particularly polynucleotides and polypeptides, may be included in a larger structure, for example, covalently or non-covalently linked to additional sequences, carrier molecules, retarders, and other excipients.In particular, the polypeptides as specified may be included in a fusion polypeptide that includes additional peptides, which may serve, for example, as a tag for purification and / or detection, as a linker, or to extend the in vivo half-life of the compound.The term "detectable tag" refers to a stretch of amino acids that is added or introduced into the fusion polypeptide; preferably, the tag is added to the C-terminus or N-terminus of the fusion polypeptide.The stretch of amino acids preferably allows the detection of the polypeptide by a specific binding partner, for example, an antibody that specifically recognizes the tag; or preferably allows the formation of a functional conformation, for example, a chelator; or preferably allows visualization, for example, in the case of a fluorescent tag.Preferred detectable tags are Myc tag, FLAG tag, 6-His tag, HA tag, GST tag, or fluorescent protein tag, for example, GFP tag. These tags are all well known in the art.Other additional peptides that are preferably included in fusion polypeptide include additional amino acids or other modifications that can serve as secretion mediators, blood-brain barrier crossing mediators, cell-penetrating peptides, and / or immune stimulants.Additional polypeptides or peptides that can be fused to polypeptides are signal and / or transport sequences, such as IL-2 signal sequences, and linker sequences.

[0015] The term "polypeptide" as used herein means a molecule comprising several, typically at least 20, amino acids covalently linked to each other by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are usually considered to be "peptides". Preferably, a polypeptide is composed of 50-1000, more preferably 60-1000, even more preferably 70-500, and most preferably 80-400 amino acids. A polypeptide may be a complex of two or more amino acid chains, i.e. a multimer, e.g. a dimer, a trimer, etc.; in such cases, a complex of two or more amino acid chains may also be referred to as a "polypeptide oligomer" or a "protein complex". Preferably, a complex of two or more amino acid chains is a heteromultimer, more preferably a heterodimer, preferably comprising at least one first variable TCR domain and at least one second variable TCR domain in a non-covalent complex. A polypeptide may also comprise additional, non-peptidic structures, e.g. at least one glycosylation, lipid conjugation, etc. More preferably, a polypeptide as specified comprises all components as indicated comprised in one continuous covalent peptide chain, and thus preferably the polypeptide is or is comprised in a fusion polypeptide. Unless specifically stated otherwise, reference herein to a particular polypeptide preferably includes polypeptide variants.

[0016] As used herein, the term "polypeptide variant" refers to any chemical molecule comprising at least one polypeptide as specified herein, which has the indicated activity but differs in structure from said specific polypeptide.Preferably, the polypeptide variant comprises a polypeptide having a contiguous amino acid sequence corresponding to at least 50%, preferably at least 75%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% of the amino acid sequence of the specifically indicated polypeptide.Furthermore, it should be understood that the polypeptide variant as referred to in accordance with the present invention will have an amino acid sequence that differs due to at least one amino acid substitution, deletion and / or addition, and the amino acid sequence of said variant is still preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98%, and most preferably at least 99% identical to the amino acid sequence of the specific polypeptide.The degree of identity between two amino acid sequences can be determined by algorithms well known in the art and described herein above. The polypeptide variants referred to above may be allelic variants or any other species-specific homologs, paralogs or orthologs. Furthermore, the polypeptide variants referred to herein include fragments of a particular polypeptide or polypeptide variants of the above-mentioned types, so long as these fragments and / or variants have the specified biological activity. Such fragments may be, or may be derived from, for example, degradation products or splicing variants of the polypeptide. Furthermore, variants that differ by post-translational modifications such as phosphorylation, glycosylation, ubiquitination, sumoylation, or myristylation, by including unnatural amino acids, and / or by being peptidomimetics are included.

[0017] As used herein, the term "polynucleotide" refers to a linear or circular nucleic acid molecule. The polynucleotide of the present invention will preferably be provided as an isolated polynucleotide (i.e., isolated from its natural context) or in a genetically modified form, preferably including at least one heterologous sequence. The term encompasses single-stranded polynucleotides as well as double-stranded polynucleotides. In addition, naturally occurring modified polynucleotides, such as glycosylated or methylated polynucleotides, or chemically modified polynucleotides, including artificially modified derivatives, such as biotinylated polynucleotides, locked nucleic acids, etc., are also included. The polynucleotide of the present invention has the activity of encoding a binding polypeptide as specified herein. Methods for testing whether a given polynucleotide has the above-mentioned activity are known to those skilled in the art and are described herein below. Unless otherwise specified, preferably, a reference to a particular polynucleotide herein includes polynucleotide variants.

[0018] The term "polynucleotide variant" as used herein refers to a variant of the polynucleotides referred to herein, including a nucleic acid sequence characterized in that the sequence can be derived from the specific nucleic acid sequence described above by at least one nucleotide substitution, addition and / or deletion, and the polynucleotide variant will have an activity as specified for the specific polynucleotide. Preferably, the polynucleotide variant is an ortholog, paralog or other homolog of the specific polynucleotide. Also preferably, the polynucleotide variant is or is derived from a non-naturally occurring allele of the specific polynucleotide. Polynucleotide variants also encompass polynucleotides that comprise a nucleic acid sequence capable of hybridizing to the specific polynucleotide described above, preferably under stringent hybridization conditions. These stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Those skilled in the art know how to determine the required hybridization conditions by referring to textbooks such as those mentioned above, or to the following textbooks: Sambrook et al., "Molecular Cloning", Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, "Nucleic Acids Hybridization: A Practical Approach", IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press at Oxford University Press, Oxford. Alternatively, polynucleotide variants can be obtained by PCR-based techniques such as mixed oligonucleotide primer-based DNA amplification.Moreover, variants include polynucleotides comprising a nucleic acid sequence that is at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% or most preferably at least 99% identical to the specifically indicated nucleic acid sequence. Furthermore, polynucleotides comprising a nucleic acid sequence that encodes an amino acid sequence that is at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% or most preferably at least 99% identical to the specifically indicated amino acid sequence are also encompassed. The percent identity values ​​are preferably calculated over the entire amino acid or nucleic acid sequence region, preferably as specified above. The polynucleotides of the present invention either consist of, consist essentially of or comprise the above-mentioned nucleic acid sequences. That is, the polynucleotides of the present invention can further comprise additional nucleic acid sequences. In particular, the polynucleotides of the present invention can encode a fusion protein, where one partner of the fusion protein is a polypeptide encoded by the above-mentioned nucleic acid sequence. The polynucleotides may also be included in a vector.

[0019] The term "vector" preferably includes phage, plasmid, viral or retroviral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes. Furthermore, the term also relates to targeting constructs that allow random or site-specific integration of the targeting construct into genomic DNA. Such targeting constructs preferably contain DNA of sufficient length for either homologous or non-homologous recombination, as described in detail below. A vector comprising a polynucleotide of the present invention preferably further comprises a selectable marker for propagation and / or selection in a host. A vector can be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced into a precipitate, such as a calcium phosphate precipitate or a rubidium chloride precipitate, or into a complex containing a charged lipid, or into a carbon-based cluster, such as fullerenes. Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line before application to a host cell. A retroviral vector may be replication-competent or replication-deficient. In the latter case, the proliferation of the virus generally occurs only in the complementary host / cell. More preferably, in the vector of the present invention, the polynucleotide is operably linked to an expression control sequence that allows expression in prokaryotic or eukaryotic cells or their isolated fractions, i.e., preferably, the polynucleotide is contained in an expression vector. The expression of the polynucleotide comprises the transcription of the polynucleotide into RNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known in the art. They preferably comprise a regulatory sequence that ensures the initiation of transcription, and optionally a polyA signal that ensures the termination of transcription and the stabilization of the transcript. Additional regulatory elements include transcriptional as well as translational enhancers.Possible regulatory elements allowing expression in prokaryotic host cells include, for example, the lac, trp, or tac promoters in E. coli, and examples of regulatory elements allowing expression in eukaryotic host cells are the AOX1 or GAL1 promoters in yeast or the CMV, SV40, RSV promoters (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin introns in mammalian and other animal cells. Furthermore, inducible expression control sequences can be used in the expression vectors encompassed by the present invention. Such inducible vectors can include tet or lac operator sequences, or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. Besides the elements responsible for initiation of transcription, such regulatory elements can also include transcription termination signals downstream of the polynucleotide, such as the SV40-polyA site or the tk-polyA site. In this context, suitable expression vectors are known in the art, such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (InVitrogene) or pSPORT1 (GIBCO BRL).Preferably, the vector is an expression vector as well as a gene transfer or targeting vector.Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papilloma viruses can be used to deliver polynucleotides or vectors to target cell populations.Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0020] The term "specific binding" is understood by those skilled in the art. Preferably, specific binding relates to binding in which the affinity of a binding polypeptide to a peptide, preferably a peptide as specified anywhere herein, is at least 10-fold, preferably at least 100-fold, most preferably at least 1000-fold higher than the affinity to any non-cognate (poly)peptide. Thus, the dissociation constant (K d ) is preferably at least 10 -6 mol / L, more preferably at least 10 -5 mol / L, most preferably at least 10 -4 It is in moles / L.

[0021] As used herein, the term "binding polypeptide" refers to a polypeptide having structural elements as specified herein, having the activity of binding to a cognate peptide, preferably specifically binding to said cognate peptide, and more preferably binding to an MHC class I molecule, preferably an HLA-A molecule, more preferably an HLA-A * The present invention relates to a polypeptide that specifically binds to its cognate peptide when presented on a .O2 molecule. Preferably, the cognate peptide comprises, more preferably consists of, the amino acid sequence MIWEHNVEV (SEQ ID NO: 14), or comprises, more preferably consists of, the amino acid sequence NLDTLMTYV (SEQ ID NO: 13).

[0022] As used herein, the term "T cell receptor", abbreviated as "TCR", refers to a polypeptide complex on the surface of a T cell that mediates the recognition of antigenic peptides presented by target cells, preferably in the context of an MHC molecule or an MHC-related molecule, such as MR1 ​​or CD1, more preferably in the context of an MHC molecule, even more preferably in the context of an MHC class I or MHC class II molecule, and most preferably in the context of an MHC class I molecule. Typically, a TCR comprises one TCR alpha chain and one TCR beta chain, i.e., an alpha / beta chain heterodimer. However, a TCR may comprise a TCR gamma and a TCR delta chain instead of the TCR alpha and beta chains. According to conventional nomenclature, the complex consisting of alpha and beta chains or gamma and delta chains is referred to herein as "T cell receptor" or "TCR", the alpha and / or beta chains and the gamma and / or delta chains are generally or singly referred to as "TCR polypeptide" or "TCR polypeptides", and the polypeptide complex comprising the TCR and accessory polypeptides, such as CD3 and CD247, is referred to as "T cell receptor complex" and abbreviated as "TCR complex". Each TCR polypeptide comprises several polypeptide domains, namely a transmembrane region (transmembrane domain), a constant region (constant domain), a joining region (constant domain) and a variable region (variable domain), and the variable region of each TCR alpha, beta, gamma or delta chain comprises three complementarity determining regions (CDRs), called CDR1, CDR2 and CDR3, respectively. As referred to herein, the variable region of the TCR polypeptide is referred to as "variable T cell receptor domain" and may be abbreviated as "variable TCR domain". Nomenclature for numbering amino acids within a TCR domain or immunoglobulin superfamily polypeptide is generally known to those of skill in the art, such as the international ImMunoGeneTics information system® (IMGT numbering scheme, Lefranc et al., Dev Comparative Immunol 27:55 (2003)), Kabat numbering, etc.Preferably, the numbering scheme is the IMGT numbering scheme, and is used in the amino acid numbering herein.As known to those skilled in the art, in TCR, binding specificity is determined primarily by variable domain, and in particular by the CDR3 region of variable domain.Therefore, the TCR that has the activity of binding to a specific cognate peptide can be structurally defined by the amino acid sequence of the CDR3 of its variable domain.

[0023] The binding polypeptide comprises a first variable TCR domain and a second variable TCR domain. Preferably, the first variable TCR domain and the second variable TCR domain are comprised in a fusion polypeptide, i.e. preferably in a continuous peptide chain. Also, preferably, the first variable TCR domain is covalently connected to the first constant TCR domain and / or the second variable TCR domain is covalently connected to the second constant TCR domain. Preferably, the first variable TCR domain and the second variable TCR domain are covalently connected to a transmembrane domain, preferably a TCR transmembrane domain. Thus, the binding polypeptide may be a TCR, preferably comprising all the structural elements of a TCR as shown elsewhere herein. Also, preferably, the first variable TCR domain and the second variable TCR domain are covalently connected to a signaling domain. Thus, the binding polypeptide may be a chimeric antigen receptor (CAR). CARs and their construction principles are known to those skilled in the art. Also preferably, the first variable TCR domain and the second variable TCR domain are covalently linked to the Fc domain of an immunoglobulin; thus, the binding polypeptide can preferably be an immunomodulatory compound.Also preferably, the binding polypeptide is a soluble TCR, preferably a soluble TCR tetramer.Furthermore, the binding polypeptide may comprise a label, such as a colored dye and / or a fluorescent dye; and / or an effector compound, such as a chemotherapeutic agent, a radioactive compound, etc.

[0024] Preferably, the variable TCR domain of the binding polypeptide comprises a CDR3 sequence that mediates or contributes to the activity as specified herein above.Thus, preferably, (i) the complementarity determining region 3 (CDR3) of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 or a sequence at least 80% identical thereto; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2 or a sequence at least 80% identical thereto; or (ii) the CDR3 of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3 or a sequence at least 80% identical thereto; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4 or a sequence at least 80% identical thereto. More preferably, (i) the CDR3 of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 or a sequence at least 80% identical thereto; the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2 or a sequence at least 80% identical thereto; or (ii) the CDR3 of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3 or a sequence at least 80% identical thereto; the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4 or a sequence at least 80% identical thereto. Even more preferably, (i) the complementarity determining region 3 (CDR3) of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2; or (ii) the CDR3 of the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3; and / or the CDR3 of the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4.

[0025] More preferably, the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:5 or an amino acid sequence at least 70% identical to SEQ ID NO:5, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:6 or an amino acid sequence at least 70% identical to SEQ ID NO:6, or the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:7 or an amino acid sequence at least 70% identical to SEQ ID NO:7, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:8 or an amino acid sequence at least 70% identical to SEQ ID NO:8, even more preferably, the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:5, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:6, or the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:7, and / or said second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO:8.

[0026] Preferably, the amino acid sequence of SEQ ID NO:5 is encoded by the nucleic acid sequence of SEQ ID NO:9, and the amino acid sequence of SEQ ID NO:6 is encoded by the nucleic acid sequence of SEQ ID NO:10. Also, preferably, the amino acid sequence of SEQ ID NO:7 is encoded by the nucleic acid sequence of SEQ ID NO:11, and the amino acid sequence of SEQ ID NO:8 is encoded by the nucleic acid sequence of SEQ ID NO:12. As those skilled in the art will recognize and as specified herein above, a given amino acid sequence may be encoded by multiple nucleic acid sequences due to the degeneracy of the genetic code; therefore, those skilled in the art will be able to generate an appropriate coding sequence, for example, the coding sequence may be codon optimized for a host cell from a particular species.

[0027] Advantageously, the binding polypeptides as identified in the studies underlying the present invention have been found to be particularly well suited for specific binding to cancer cells, in particular cancer cells expressing PTPRZ1 and / or NLGN4X and thus presenting peptides having the amino acid sequences shown in SEQ ID NO: 13 and / or SEQ ID NO: 14. The binding polypeptides provided herein are therefore particularly suited for cancer therapy, in particular T-cell therapy of gliomas.

[0028] The definitions given above apply mutatis mutandis below. The additional definitions and explanations further given below also apply mutatis mutandis to all embodiments described herein.

[0029] The present invention also relates to polynucleotides encoding at least one of the first and second variable TCR domains of a binding polypeptide of the invention, and vectors comprising said polynucleotides.

[0030] The term "polynucleotide", which preferably includes polynucleotide variants, has been specified herein above as having preferred nucleic acid sequences.Preferably, the polynucleotide encodes the first variable TCR domain and the second variable TCR domain of the binding polypeptide, and more preferably encodes a fusion polypeptide comprising said first variable TCR domain and said second variable TCR domain.Preferably, the polynucleotide is an expression construct, i.e., preferably causes the expression of at least one of the first variable TCR domain and the second variable TCR domain of the binding polypeptide in a suitable host cell.

[0031] The term vector is likewise specified herein above. Preferably, the vector is an expression vector.

[0032] The present invention also relates to a host cell comprising a binding polypeptide of the invention, a polynucleotide of the invention and / or a vector of the invention.

[0033] As used herein, the term "host cell" refers to any cell that can receive and preferably express a binding polypeptide, polynucleotide or vector as specified. Preferably, the host cell is a bacterial cell, more preferably a cell of a common laboratory bacterial lineage known to those skilled in the art, most preferably an Escherichia lineage, especially an E. coli lineage. Also preferably, the host cell is a eukaryotic cell, preferably a yeast cell, such as a cell of a strain of Baker's yeast, or an animal cell. More preferably, the host cell is an insect cell or a mammalian cell, especially a mouse or rat cell. Most preferably, the host cell is a human cell. Preferably, the host cell is a T cell, more preferably a CD8+ T cell or a CD4+ T cell, more preferably a CD8+ T cell. As the skilled artisan will understand, the CD8 TCR is preferably expressed in CD8+ T cells, and the CD4 TCR is preferably expressed in CD4+ T cells. Also, preferably, the host cell is capable of displaying on its surface a binding polypeptide as specified herein, preferably encoded by a polynucleotide and / or a vector as specified herein.

[0034] The present invention also relates to a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention and / or a host cell of the invention for use in medicine, in particular for use in the treatment and / or prevention of cancer. Thus, the binding polypeptide, polynucleotide, vector and / or host cell may be formulated as a pharmaceutical composition.

[0035] The present invention provides a method of treating and / or preventing cancer in a subject, comprising: (a) contacting the subject with a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, and / or a host cell of the invention; and (b) thereby treating cancer in said subject. The present invention also relates to a method, comprising:

[0036] The term "cancer" as used herein relates to a disease of animals, including humans, characterized by the uncontrolled growth of a group of somatic cells ("cancer cells"). This uncontrolled growth may involve the formation of a mass of cancer cells (tumor), the invasion and destruction of surrounding tissues (infiltration) and possibly the spread of cancer cells to other locations in the body (metastasis). Preferably, the term cancer also includes the recurrence of cancer (recurrence). Thus, preferably, the cancer is a solid cancer, including the primary tumor, metastasis and its recurrence. However, the cancer may also be a non-solid cancer. Preferably, the cancer is acute lymphocytic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytoma, atypical teratoma, basal cell carcinoma, bile duct cancer, bladder cancer, brain stem glioma, breast cancer, Burkitt's lymphoma, carcinoid tumor, cerebellar astrocytoma, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colon cancer, colorectal cancer, craniopharyngioma, intrauterine cancer, Membrane cancer, ependymoblastoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, hypothalamic and optic nerve glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, medulloblastoma, ependymoma, melanoma, Merkel cell carcinoma, mesothelioma, oral Cavity cancer, Combined endocrine neoplastic syndrome, Multiple myeloma, Mycosis fungoides, Nasal and paranasal sinus cancer, Nasopharyngeal cancer, Neuroblastoma, Non-Hodgkin's lymphoma, Non-small cell lung cancer, Oral cavity cancer, Oropharyngeal cancer, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Low malignant potential ovarian tumor, Pancreatic cancer, Papillomatosis, Paranasal sinus and nasal cancer, Parathyroid cancer, Penile cancer, Pheochromocytoma, Pituitary tumor, Pleuropulmonary blastoma, Primary The cancer is selected from the list consisting of central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell cervical cancer, testicular cancer, throat cancer, thymus cancer, thymoma, thyroid cancer, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. More preferably, the cancer is a solid cancer, metastasis or recurrence thereof. More preferably, the cancer is a brain cancer, preferably a glioma, more preferably a glioblastoma.

[0037] The terms "treating" and "treatment" refer to the improvement of the disease or disorder referred to herein or symptoms associated therewith to a significant degree; as used herein, this term includes the prevention of the worsening of the disease, disorder, or symptoms associated therewith. As used herein, the treating also includes the complete recovery of the health status related to the disease or disorder referred to herein. As used herein, it should be understood that treating may not be effective in all subjects to be treated. However, this term will preferably require that a statistically significant portion of subjects suffering from the disease or disorder referred to herein can be successfully treated. Whether the portion is statistically significant or not can be determined by those skilled in the art without further ado using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98% or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the treatment will be effective for at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the subjects of a given cohort or population. Preferably, treating comprises activating an immune response against disease-causing or mediating factors, i.e., against cells. Preferably, treating cancer comprises reducing tumor and / or cancer cell load in the subject. As will be understood by those skilled in the art, the effectiveness of a treatment, e.g., of cancer, depends on various factors, including, e.g., the stage and type of cancer. Also preferably, the cancer treatment further comprises at least one of surgery, chemotherapy, and radiation therapy.

[0038] The terms "prevent" and "prevention" refer to maintaining the health of a subject with respect to a disease or disorder as referred to herein for a certain period of time. It will be understood that the period of time may depend on the amount of drug compound administered and the subject's personal factors as discussed elsewhere herein. It should be understood that prevention may not be effective in all subjects treated with a compound according to the present invention. However, the term preferably requires that a statistically significant portion of the subjects of a cohort or population is effectively prevented from suffering from a disease or disorder as referred to herein or its associated symptoms. Preferably, in this context, a cohort or population of subjects is contemplated that would normally, i.e., develop a disease or disorder as referred to herein without the use of a preventive measure according to the present invention. Whether or not a portion is statistically significant can be determined without further ado by those skilled in the art using various well-known statistical evaluation tools as discussed elsewhere herein. In the context of cancer treatment, in particular, preventing relates to preventing the onset of cancer, preventing the formation of metastases and / or preventing recurrence, preferably to preventing the formation of metastases and / or preventing recurrence.

[0039] Preferably, treating and / or preventing cancer comprises administering to the subject to be treated host cells, preferably T cells as specified herein above, which produce a binding polypeptide as specified herein. Methods of endowing a host cell with the ability to produce a binding polypeptide are in principle known to the skilled artisan and in particular comprise introducing at least one expression construct, such as an expression vector, into said host cell. Suitable methods depend on the type of host cell and are known to the skilled artisan, such as electroporation, infection with viral vectors, etc. In the case of treating and / or preventing cancer, the host cell may be allogeneic, such as allogeneic T cells, or more preferably autologous, in particular autologous T cells. Also, the host cell may be expanded, for example by in vitro culture, before being (re)administered to the subject; and / or may be activated, for example by co-culture with cells presenting the cognate peptide and / or by cytokine treatment.

[0040] The term "subject" as referred to herein relates to a vertebrate, preferably a mammal, in particular a livestock, pet or laboratory animal. Most preferably, the subject is a human. Preferably, the subject is diagnosed with cancer, suspected of having cancer, or known to be at risk of having cancer, in particular a genetic form of cancer. Preferably, the subject's cancer is identified as being susceptible to treatment with a binding polypeptide, preferably as specified herein below.

[0041] The terms "medicament" and "pharmaceutical composition" are used herein essentially interchangeably and are known in principle to those skilled in the art. When referred to herein, these terms relate to any composition that includes an active agent identified as a pharmacoactive compound and one or more excipients. The pharmacoactive compound may be present in liquid or dry form, for example in lyophilized form. It will be understood that the form and characteristics of an acceptable pharmaceutical excipient, such as a carrier or diluent, will be determined by the amount of active ingredient with which it will be combined, the route of administration, and other well-known variables. An excipient must be acceptable in the sense of being compatible with the other ingredients of the formulation and not harmful to the recipient thereof. The excipients used may include solids, gels, or liquids. Exemplary solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers are phosphate buffered saline solution, physiological saline solution, Ringer's solution, dextrose solution and Hank's solution, syrup, oil, water, emulsion, various kinds of wetting agents, etc. Similarly, carrier or diluent can comprise time-delay material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with wax. The suitable carriers include those mentioned above and others known in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. The excipient is selected so as not to affect the biological activity of the combination. However, the excipient can be selected to improve the uptake of the active agent into host cells, particularly target cells. Thus, the excipient can also be viral particles and / or lipid vesicles, preferably viral particles and / or lipid vesicles known to mediate entry and / or fusion into the target cells of interest.

[0042] Medicaments are preferably administered by the routes as specified herein above. Therapeutically effective dose refers to the amount of effector polypeptide or expression construct encoding it used in the medicament that prevents, ameliorates or cures symptoms associated with the disease or condition referred to herein. The therapeutic efficacy and toxicity of a drug can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, such as ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio, LD50 / ED50. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical art, the dosage for any one patient depends on many factors, including the patient's size, age, the specific formulation of the medicament administered, sex, time and route of administration, overall health and other drugs administered at the same time. Medicaments referred to herein are preferably administered at least once, for example as a bolus. However, the medicament may be administered multiple times, preferably at least twice, for example, permanently or periodically after a specified time frame.Progress may be monitored by regular evaluation.Recommended dosage may be indicated in instructions or instructions, to predict dosage adjustment according to the intended recipient.

[0043] The medicament according to the present invention may comprise an additional active agent in addition to the above-mentioned active agent.Preferably, the pharmaceutically active compound as specified herein is applied together with at least one additional drug, and therefore may be formulated as a medicament together with this at least one additional drug.More preferably, in the case of cancer treatment, said at least one additional active agent is a chemotherapeutic drug or an additional immunotherapeutic drug, such as an immune checkpoint modulator.It should also be understood that the formulation of pharmaceutical composition is preferably carried out under GMP standardized conditions, etc., in order to ensure the quality, pharmaceutical safety and efficacy of the medicament.

[0044] The present invention provides a method for identifying a target cell expressing protein tyrosine phosphatase receptor type Z1 (PTPRZ1) and / or neuroligin-4, X-linked (NLGN4X), comprising: (i) contacting said target cell with a binding polypeptide of the invention; (ii) detecting binding of the binding polypeptide to the target cell; and (iii) identifying a target cell expressing PTPRZ1 and / or NLGN4X based on the detection in step (ii). The present invention also relates to a method, comprising:

[0045] The method of identifying target cells expressing PTPRZ1 and / or NLGN4X is preferably an in vitro method. The method may be performed in particular in cultured cells or samples from a subject, preferably in samples from a subject, preferably in samples that contain or are suspected to contain cancer cells, preferably in tumor samples. Thus, the target cell may be, but need not necessarily be, a host cell. Preferably, the term "target cell" as used herein refers to a vertebrate cell, more preferably a mammalian cell, even more preferably a primate cell, most preferably a human cell. However, the target cell may be a cell of a laboratory animal, livestock or pet, such as a mouse cell, a rat cell, a guinea pig cell, a bovine cell, a sheep cell, a pig cell, a dog cell or a cat cell. Preferably, the target cell is a cell that is known or suspected to be a cancer cell, preferably a cell contained in a tumor.

[0046] The binding of the binding polypeptide in step (ii) can be detected by any method considered appropriate by the skilled artisan, for example, immunological method.Preferably, in such a case, the binding polypeptide comprises a detectable label, for example, a dye or an enzyme that catalyzes a detectable chemical reaction.As the skilled artisan will understand in view of the description herein, the detection of the binding of the binding polypeptide in step (ii) is indicative of the target cell expressing PTPRZ1 and / or NLGN4X in step (iii).As the skilled artisan will also understand, the detection in step (ii) can be qualitative, but is preferably semi-quantitative or quantitative.

[0047] The present invention provides a method for identifying a cancer susceptible to treatment with a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, and / or a host cell of the invention, comprising the steps of: (I) identifying target cells expressing PTPRZ1 and / or NLGN4X in a cancer sample; and (II) identifying a cancer that is susceptible to treatment based on the identification in step (I). The present invention also relates to a method, comprising:

[0048] The method of identifying cancers susceptible to treatment is preferably an in vitro method, preferably performed on a cancer sample. The term "sample" herein means a sample containing cells of a subject; the sample may be a body fluid sample, preferably a blood, saliva, sputum, urine sample, or a sample containing separated cells or a sample from a tissue or organ. Separated cells may be obtained from body fluids, such as lymph, blood, plasma, serum, liquor, etc., or from tissues or organs by separation techniques such as centrifugation or cell sorting. More preferably, the sample is a sample known or suspected to contain cancer cells. Preferably, the sample is a cancer sample, and preferably the term "cancer sample" relates to a sample known or suspected to contain target cells, preferably cancer cells; thus, preferably, the sample is a sample of a primary tumor, an invasion, a metastasis, a lymph node or a recurrence. However, the cancer sample may also be a sample of a single cell, for example in the case of a non-solid tumor. Samples can be obtained from a subject by routine techniques well known to those of skill in the art, such as venous or arterial puncture or open biopsy, e.g., aspiration of tissue or cellular material from a subject. For areas not easily accessible by open biopsy, surgery, preferably minimally invasive surgery, can be performed.

[0049] As will be understood by those skilled in the art in view of the description provided herein, if target cells expressing PTPRZ1 and / or NLGN4X are identified in cancer samples in step (I), cancers that are sensitive to treatment are preferably identified in step (II).Preferably, if the fraction of target cells expressing PTPRZ1 and / or NLGN4X in cancer samples is at least 10%, preferably at least 20%, more preferably at least 30%, even more preferably at least 40%, and most preferably at least 50%, cancers that are sensitive to treatment are identified.Also preferably, if the binding of binding polypeptide is increased, preferably significantly increased, compared to control, for example, cells known not to express PTPRZ1 and / or NLGN4X, cells in cancer samples are identified as expressing PTPRZ1 and / or NLGN4X.

[0050] The present invention also relates to the use of a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention and / or a host cell of the invention in the manufacture of a composition for diagnosing, treating and / or preventing a disease, preferably cancer.

[0051] The present invention further relates to the use, preferably in vitro, of a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention and / or a host cell of the invention for detecting target cells expressing neuroligin-4, X-linked (NLGN4X) and / or protein tyrosine phosphatase receptor type Z1 (PTPRZ1).

[0052] Furthermore, the present invention relates to kits comprising a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention, and / or a host cell of the invention contained in a housing and / or further comprising a means of administration.

[0053] The term "kit" as used herein means a collection of the above-mentioned compounds, means or reagents, which may or may not be packaged together. The components of the kit may be included by separate vials (i.e. as a kit of separate parts) or may be provided in a single vial, for example as the composition specified herein above. In one embodiment, the housing of the kit allows the transfer, particularly the general transfer, of the components of the kit; thus, the housing may be a transferable container that includes all the components specified in particular. Furthermore, it should be understood that the kit of the present invention may be used to carry out the methods mentioned herein above. It is preferably contemplated that all components are provided in a ready-to-use format to carry out the methods mentioned above. Furthermore, the kit preferably contains instructions for carrying out the methods. The instructions can be provided by a user manual in paper or electronic form. For example, the manual can include instructions for interpreting the results obtained when carrying out the above-mentioned methods using the kit. Preferably, the kit includes additional compounds, such as reaction buffers, hybridization solutions, lysis buffers, etc. Preferably, the kits are adapted for use in the methods of the invention, and more preferably are configured to contain all the reagents necessary to carry out the method or methods.

[0054] The means of administration can include a delivery unit for administration of the compound or composition, and a storage unit for storing the compound or composition until administration. However, it is also intended that the means of administration can appear as separate devices in such embodiments, and are preferably packaged together in the kit. The preferred means of administration is one that can be applied without the specific knowledge of a specialized technician. In a preferred embodiment, the means of administration is a syringe, more preferably a syringe with a needle, containing the compound or composition as specified. In another preferred embodiment, the means of administration is an intravenous injection (IV) device containing the compound or composition. In yet another preferred embodiment, the means of administration is an inhaler containing the compound of the present invention, and more preferably, the compound is formulated for administration as an aerosol.

[0055] The present invention also relates to a device comprising a binding polypeptide of the invention, a polynucleotide of the invention, a vector of the invention and / or a host cell of the invention.

[0056] As used herein, the term "device" includes any device that includes the components specified. Preferably, the device is an administration means as specified herein above. Preferably, the device is adapted to perform the method as specified herein, in particular the diagnostic method and / or the identification method. Thus, the device may be a diagnostic device. Thus, the device preferably includes (i) an analysis unit that includes a means for determining the binding of the binding polypeptide to the target cell, and (ii) an evaluation unit that is operatively connected thereto and includes executable instructions specifically embedded therein for performing the method as specified herein. Exemplary means for determining the binding of the binding polypeptide to the target cell and means for making the determination are known to those skilled in the art. As will be understood by those skilled in the art, the means for determining the binding of the binding polypeptide to the target cell include means that can determine the amount of the binding polypeptide that is bound to the target cell, such as an ELISA reader, and means for determining the effect of the binding of the binding polypeptide to the target cell, such as an optical unit that detects the signal of a reporter gene assay. The evaluation means is any means capable of obtaining the analysis as specified; preferably, the evaluation means is a data processing means, such as a microprocessor, a portable device, such as a mobile phone, or a computer. The way in which the means are linked in the operating mode will depend on the type of means contained in the device. In one embodiment, the means are contained in a single device. Thus, the device can include (i) an analysis unit for measuring the binding of the binding polypeptide to the target cell and (ii) a computer unit for processing and evaluating the obtained data. Preferably, instructions and interpretations are contained in the executable program code contained in the device, with the purpose of receiving a decision and classifying the subject as suitable for therapy. Typical devices are those that can be applied without the special knowledge of a specialist technician, such as a test strip or an electronic device, which only requires loading the sample. The result may be obtained as a raw data output that requires interpretation by a technician.Preferably, the output of the device is, however, processed, i.e., evaluated raw data, and does not require a technician for its interpretation. Further exemplary devices include the above-described analysis units / devices (e.g., biosensors, arrays, solid supports coupled to ligands that specifically recognize binding, surface plasmon resonance devices, NMR spectrometers, mass spectrometers, etc.) or evaluation units / devices according to the methods of the present invention. Preferably, the device further includes a memory device, preferably a database that includes at least one reference value for the binding of the binding polypeptide to the target cell.

[0057] In view of the above, the following embodiments are specifically contemplated: Embodiment 1: A binding polypeptide comprising a first variable T-cell receptor (TCR) domain and a second variable TCR domain.

[0058] Embodiment 2: A binding polypeptide according to embodiment 1, wherein (i) the complementarity determining region 3 (CDR3) of said first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 or a sequence at least 80% identical thereto; and / or the CDR3 of said second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2 or a sequence at least 80% identical thereto; or (ii) the CDR3 of said first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3 or a sequence at least 80% identical thereto; and / or the CDR3 of said second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4 or a sequence at least 80% identical thereto.

[0059] Embodiment 3: A binding polypeptide according to embodiment 1 or 2, wherein said first variable TCR domain is a TCR alpha variable domain.

[0060] Embodiment 4: A binding polypeptide according to any one of embodiments 1 to 3, wherein the second variable TCR domain is a TCR beta variable domain.

[0061] Embodiment 5: A binding polypeptide according to any one of embodiments 1 to 4, wherein the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 5, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 6.

[0062] Embodiment 6: A binding polypeptide according to any one of embodiments 1 to 5, wherein the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 7, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 8.

[0063] Embodiment 7: A binding polypeptide according to any one of embodiments 1 to 6, wherein the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 5 and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 6.

[0064] Embodiment 8: A binding polypeptide according to any one of embodiments 1 to 7, wherein the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 7 and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 8.

[0065] Embodiment 9: A binding polypeptide according to any one of embodiments 1 to 8, wherein the amino acid sequence of SEQ ID NO:5 is encoded by a nucleic acid sequence of SEQ ID NO:9.

[0066] Embodiment 10: A binding polypeptide according to any one of embodiments 1 to 9, wherein the amino acid sequence of SEQ ID NO: 6 is encoded by a nucleic acid sequence of SEQ ID NO: 10.

[0067] Embodiment 11: A binding polypeptide according to any one of embodiments 1 to 10, wherein the amino acid sequence of SEQ ID NO: 7 is encoded by a nucleic acid sequence of SEQ ID NO: 11.

[0068] Embodiment 12: A binding polypeptide according to any one of embodiments 1 to 11, wherein the amino acid sequence of SEQ ID NO: 8 is encoded by a nucleic acid sequence of SEQ ID NO: 12.

[0069] Embodiment 13: A binding polypeptide according to any one of embodiments 1 to 12, wherein the first variable TCR domain and the second variable TCR domain are comprised in a fusion polypeptide.

[0070] Embodiment 14: A binding polypeptide according to any one of embodiments 1 to 13, wherein the first variable TCR domain is covalently linked to a first constant TCR domain and / or the second variable TCR domain is covalently linked to a second constant TCR domain.

[0071] Embodiment 15: The binding polypeptide is an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A * 15. A binding polypeptide according to any one of embodiments 1 to 14, which recognizes its cognate peptide when displayed on a .O2 molecule.

[0072] Embodiment 16: The binding polypeptide is preferably an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A * 16. A binding polypeptide according to any one of embodiments 1 to 15, which recognizes a peptide having the amino acid sequence NLDTLMTYV (SEQ ID NO: 13) when presented on a .O2 molecule.

[0073] Embodiment 17: The binding polypeptide is preferably an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A *17. The binding polypeptide of any one of embodiments 1 to 16, which recognizes a peptide having the amino acid sequence MIWEHNVEV (SEQ ID NO: 14) when presented on a .O2 molecule.

[0074] Embodiment 18: A binding polypeptide according to any one of embodiments 1 to 17, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to a transmembrane domain, preferably a TCR transmembrane domain.

[0075] Embodiment 19: A binding polypeptide according to any one of embodiments 1 to 18, wherein the binding polypeptide is a TCR.

[0076] Embodiment 20: A binding polypeptide according to any one of embodiments 1 to 18, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to a signalling domain.

[0077] Embodiment 21: The binding polypeptide of any one of embodiments 1 to 18 and 20, wherein the binding polypeptide is a chimeric antigen receptor (CAR).

[0078] Embodiment 22: A binding polypeptide according to any one of embodiments 1 to 17, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to an immunoglobulin Fc domain.

[0079] Embodiment 23: A binding polypeptide according to any one of embodiments 1 to 17 and 22, wherein said binding polypeptide is a soluble TCR, preferably a soluble TCR tetramer.

[0080] Embodiment 24: A binding polypeptide according to any one of embodiments 1 to 23, wherein said binding polypeptide further comprises a label and / or an effector compound.

[0081] Embodiment 25: A polynucleotide encoding at least one of the first and second variable TCR domains of a binding polypeptide according to any one of embodiments 1 to 24.

[0082] Embodiment 26: The polynucleotide of embodiment 25, wherein the polynucleotide is an expression construct.

[0083] Embodiment 27: The polynucleotide of embodiment 25 or 26, wherein said polynucleotide is comprised in a vector, preferably an expression vector.

[0084] Embodiment 28: A vector comprising a polynucleotide according to any one of embodiments 25 to 27.

[0085] Embodiment 29: A host cell comprising a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, and / or a vector according to embodiment 28.

[0086] Embodiment 30: The host cell of embodiment 29, wherein the host cell is a T cell, preferably a CD8+ T cell.

[0087] Embodiment 31: A binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28 and / or a host cell according to embodiment 29 or 30 for use in medicine.

[0088] Embodiment 32: A binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28 and / or a host cell according to embodiment 29 or 30 for use in the treatment and / or prevention of cancer.

[0089] Embodiment 33: A method of treating and / or preventing cancer in a subject, comprising: (a) contacting the subject with a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28, and / or a host cell according to embodiment 29 or 30; and (b) thereby treating cancer in said subject. A method comprising:

[0090] Embodiment 34: A method for identifying a target cell expressing Protein Tyrosine Phosphatase Receptor Type Z1 (PTPRZ1) and / or Neuroligin-4, X-linked (NLGN4X), comprising: (i) contacting said target cells with a binding polypeptide according to any one of embodiments 2 to 24, preferably embodiments 22 to 24; (ii) detecting binding of the binding polypeptide to the target cell; and (iii) identifying a target cell expressing PTPRZ1 and / or NLGN4X based on the detection in step (ii). A method comprising:

[0091] Embodiment 35: A method for identifying a cancer susceptible to treatment with a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28, and / or a host cell according to embodiment 29 or 30, comprising: (I) identifying target cells expressing PTPRZ1 and / or NLGN4X in a cancer sample; and (II) identifying a cancer that is susceptible to treatment based on the identification in step (I). A method comprising:

[0092] Embodiment 36: Use of a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28 and / or a host cell according to embodiment 29 or 30 in the manufacture of a composition for diagnosing, treating and / or preventing a disease, preferably cancer.

[0093] Embodiment 37: Use of a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28, and / or a host cell according to embodiment 29 or 30 for detecting target cells expressing neuroligin-4, X-linked (NLGN4X) and / or protein tyrosine phosphatase receptor type Z1 (PTPRZ1).

[0094] Embodiment 38: The use of embodiment 37, wherein said use is an in vitro use.

[0095] Embodiment 39: A kit comprising a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28, and / or a host cell according to embodiment 29 or 30, contained in a housing and / or further comprising a means of administration.

[0096] Embodiment 40: A device comprising a binding polypeptide according to any one of embodiments 1 to 24, a polynucleotide according to any one of embodiments 25 to 27, a vector according to embodiment 28, and / or a host cell according to embodiment 29 or 30.

[0097] Embodiment 41: The subject matter of any one of embodiments 32-40, wherein the cancer is a brain cancer, preferably a glioma, more preferably a glioblastoma.

[0098] Embodiment 42: The subject matter of any one of embodiments 32-41, wherein the cancer has been identified as susceptible to treatment according to the method of embodiment 35.

[0099] All references cited herein are incorporated herein by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein. EXAMPLES

[0100] The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention in any way.

[0101] [Example 1] material and method 1.1 Single-cell TCR-seq Single-cell capture and downstream library construction of FACS-sorted cells was performed using Chromium Single Cell V(D)J Reagent kit v1 chemistry (10x Genomics; PN-1000006, PN-1000020, PN-1000005, PN-120262) according to the manufacturer's protocol. Constructed scVDJ libraries were sequenced on a HiSeq2500 rapid platform (Illumina).

[0102] 1.2 Cell Lines and Modification of Gene Expression U87 glioma cells were cultured in DMEM containing FBS, penicillin and streptomycin. U87 NLGN4X was generated by transfection with the full-length NLGN4X protein coding sequence in pMXs-IRES-PuroR using FuGene HD transfection reagent (Promega). Prior to transfection, U87 cells were seeded at a density of 100.000 cells per well in 6-well plates and rested for 24 h. Cells were transfected with 2 μg of plasmid DNA each, rested for 48 h, and then positively selected under application of 5 μg / mL puromycin. TMG containing epitopes of PTPRZ1 (1814–1822) and NLGN4X (131–139) were cloned into pMXs-IRES-PuroR and transfected into U87 cell lines as above, but selected with 2 μg / mL puromycin.

[0103] 1.3 Cloning of human TCR constructs The variable chains of the TCRs were synthesized by Eurofins and cloned into an S / MAR vector (pSMARTer V8) containing mouse constant alpha and beta chains using NEB Golden Gate assembly mix (NEW England Biolabs). This construct was used for electroporation of TCR-deficient Jurkat T cells. Intervention and control TCRs were cloned into SFG vectors generated using In-Fusion cloning from Takara Bio Europe.

[0104] 1.4 Electroporation of Jurkat T cells 2 million Jurkat CD8 per electroporation +TCR-deficient T cells were used. 5 μg of vector encoding the TCR and a vector encoding the NFAT reporter were additionally delivered to Jurkats (Settings: 1325V, 3 pulses, 10 ms, Neon transfection system). After 24 h of incubation, TCR expression was determined by flow cytometric analysis of TCR beta positive cells compared to non-transfected T cells.

[0105] 1.5 Isolation of human T cells Healthy donor whole blood samples were obtained from IKTZ Heidelberg and PBMCs were isolated by density gradient-based centrifugation using Lympho-Paque solution (Genaxxon). The resulting PBMCs were then washed with PBS supplemented with 1 mM EDTA and finally frozen for later downstream applications. Human T cells were isolated from healthy donor PBMCs using MagniSort™ Human T cell Enrichment Kit (Invitrogen) or Pan T Cell Isolation Kit, human (Miltenyi).

[0106] 1.6 Lentiviral transduction of primary human T cells HEK 293T cells were co-transfected with a lentiviral construct encoding the TCR (pLEX307) and Ready-to-Use Lentiviral Packaging Plasmid Mix (Cellecta) using Fugene transfection reagent. Virus-containing supernatants were harvested after 24, 48 and 72 hours. T cells were directly activated after isolation from fresh peripheral blood mononuclear cells and activated for 24 hours in Xvivo medium supplemented with human interleukin 7 (0.2 IU / mL), human interleukin 15 (290 IU / mL), and 10 μL(yl) human T Cell TransAct™ beads (Miltenyi) per million T cells. After transduction, lentiviral supernatants were spin-loaded onto retronectin-coated 6-well plates. Up to 2 million activated T cells / well were then transduced by spinoculation on the virus-loaded plates and incubated overnight. TCR expression was assessed 48 hours later.

[0107] 1.7 Retroviral transduction of primary human T cells Briefly, T cells were activated directly after isolation from fresh PBMCs and activated for 48 h in CTL medium (45% RPMI, 45% Click medium, 10% FBS supplemented with 10 ng / mL hIL-7 and 5 ng / mL hIL-15) and Transact Beads (Miltenyi) according to the manufacturer's instructions. HEK 293T cells were seeded at a density of 3 million cells per 10 cm dish and co-transfected with SFG vector encoding the TCR and RD-114 and PeqPam as a helper plasmid using Fugene HD transfection reagent. After 48 hours, viral supernatants were harvested, filtered, and spin-loaded onto pre-retronectin-coated 24-well plates. The viral supernatant was then discarded and 0.5 million T cells were added in 1 mL CTL medium and incubated at 37°C, 5% CO 2The cells were incubated at 4°C for 4 days. The efficiency of TCR transduction was assessed by flow cytometric analysis of GFP and TCR beta expression in CD3 T cells.

[0108] 1.8 NFAT reporter-based activation assays Jurkat TCR-deficient T cells were equipped with the respective TCR and NFAT reporters by electroporation as previously shown. U87 glioma cells or T2 cells were pulsed with peptides for 1 h or overnight at a density of 3 million cells / mL and a final peptide concentration of 10 μg / mL in 96-well plates. After confirming the expression of TCR, Jurkat T cells at a density of 3 million cells / mL were co-cultured overnight with peptide-loaded antigen-presenting cells. Nano-Glo Luciferase assay reagents (Promega) were then diluted according to the manufacturer's instructions and added to the co-cultures. Luminescence was recorded on a PHERAstar FS plate reader and analyzed using MARS Data analysis software (BMG Labtech). Activation was assessed by NFAT reporter assay or flow cytometric assessment of cluster of differentiation (CD)69 expression on Jurkat cell co-cultures.

[0109] 1.9 Human T cell activation and cytotoxicity assays T cells were isolated and activated as described above, and the respective TCR was delivered by retroviral transduction. Four days after transduction, TCR-T cells were used for in vitro T cell activation and cytotoxicity assays using peptide-loaded U87 glioma cells or K562 cells, U87 TMG-expressing glioma cells or NLGN4X-overexpressing U87 glioma cells as target cells and incubated overnight. Cytotoxicity was then measured using CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, see below), and T cells were stained extracellularly and intracellularly for flow cytometry analysis of TNF alpha, IFNy and granzyme B expression.

[0110] Vital FR Assay Four days after transduction, T cell receptor-transduced human T cells were used to co-culture overnight with various target cell lines pre-labeled with CellTrace reagent diluted 1:5000 according to the manufacturer's instructions at an effector to target cell ratio of 2:1. After overnight co-culture, plates were centrifuged and resuspended in PBS. T cells were discarded, 30 μL trypsin was added per well, and after 10 min, tumor cells were resuspended and subjected to extracellular FACS staining using eFluor780-conjugated fixable viability dye (1:1000, eBioscience) and PE-conjugated anti-human CD3 (1:100, BioLegend) to negatively select tumor cells.

[0111] Vital FR Cytotoxicity Assay and Flow Cytometry-Based Cytotoxicity Assay Four days after transduction, TCR-transduced human T cells were used to co-culture overnight with different target cell lines pre-labeled with 0.2 μM CellTrace™ Far Red or Violet reagents according to the manufacturer's instructions at different effector to target (E:T) cell ratios. When peptide-pulsed target cells were used as antigen-presenting cells (APCs), the APCs were incubated with the indicated peptide dilutions at 37° C., 5% CO prior to the assay. 2 After overnight co-culture, plates were centrifuged and resuspended in PBS. T cells were removed, 30 μL trypsin was added per well, and tumor cells were resuspended after 10 min and subjected to flow cytometry using eFluor780-conjugated fixable viability dye and PE-conjugated anti-human CD3 to negatively select tumor cells. 123 Counting beads were added in 50 μL at a 1:5 dilution. Cytotoxicity was calculated using the normalized number of tumor cells. Cytotoxicity (%) was calculated as the quotient of viable tumor cells after T cell co-culture against either tumor cells alone or unloaded tumor cell control.

[0112] LDH cytotoxicity assay After 24 hours, the co-cultures were centrifuged and 50 μL of the supernatant was used to incubate with CytoTox 96 Reagent (Promega) for 30 minutes at room temperature in the dark. After 30 minutes, stop solution was added and absorbance at 490 nm was recorded. The minimal LDH release of T cells and target cells as well as the medium background signal were measured for subsequent analysis.

[0113] 1.10 Flow cytometry For intracellular staining, cells were incubated with 5 μg / mL brefeldin A (Sigma-Aldrich) at 37 °C and 5% CO 2 The cells were incubated at 4°C for 5 hours to allow intracellular enrichment of the target proteins. For human T cells, PBS supplemented with 10% human serum was used for blocking and cells were subsequently stained. For intracellular staining of cytokines, cells were fixed and permeabilized using IC fixation buffer and IC permeabilization buffer (both from eBioscience), respectively, and then stained with the relevant antibodies for 45 min at 4°C. Human T cells were stained in 50 μL or 100 μL containing a 1:50 dilution of antibody.

[0114] 1.11 Mouse NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab 1em1Mvw H2-D1 tm1Bpe Il2rg tm1Wjl / SzJ (NSG MHCI / II KO) mice were kindly provided by LD Shultz and bred in the DKFZ animal facility. All animal procedures were performed in accordance with the institutional guidelines for laboratory animal research and approved by the governmental agency (Regional Administrative Authority Karlsruhe, Germany, file number G-37 / 18). For the experimental groups, 6- to 28-week-old mice were age- and sex-matched.

[0115] 1.12 Intracranial tumor experiments and intraventricular injection of T cells For orthotopic tumor cell injections, 1 × 10 5U87 tandem minigene (TMG) glioma cells were implanted in the right hemisphere of NSG MHCI / II KO mice, 1 mm anterior to the coronal suture, 2 mm lateral to the bregma and 3 mm deep, in 1 mL of PBS. 6 T cells were transduced and expanded as described above. After confirming tumor growth using magnetic resonance imaging (MRI), tumors were expanded up to 5 × 10 6 T cells were injected into the left lateral ventricle of tumor-bearing mice, 0.5 mm lateral to the anterior apex and 2.2 mm deep, in a total volume of 4 μL PBS. In survival experiments, T cells were injected on days 15 and 22, and for evaluation of intratumoral phenotype, a single injection was performed on day 48. Tumor growth was repeatedly monitored by MRI, mice were checked daily for tumor-associated pathology, and sacrificed when humane or experimental termination criteria (Regional commission Karlsruhe, file number G-37 / 18) were met. Resulting tissues were either directly processed for flow cytometry or cryofixed and evaluated by immunofluorescence.

[0116] 1.13 Statistical analysis All results were analyzed with Prism version 9.4.0. Statistical tests are indicated in the legends of each figure. ANOVA tests were corrected for multiple testing using the Sidac® multiple comparison correction. Results were considered significant when p-values ​​were less than 0.05.

[0117] [Example 2] result 2.1 Results regarding PTPRZ1-specific TCR PTPRZ1(1814-1822)-specific TCRs were identified by single-cell sequencing of patients who received a multi-peptide vaccine containing the PTPRZ1(1814-1822) peptide. Single-cell TCR sequencing after T cell activation-based enrichment revealed oligoclonal T cell populations (Figure 1A). The subsequently identified PTPRZ1(1814-1822) TCR, carrying the mouse TCR beta constant chain, was overexpressed in Jurkat T cells as demonstrated by mouse TCRB cell surface expression (Figure 1B). HLA-A * Both 02:01-positive and PTPRZ1(1814-1822) peptide-loaded T2 and glioma cells U87 specifically activated PTPRZ1(1814-1822) TCR transgenic Jurkat T cells in a nuclear factor of activated T cells (NFAT) reporter-based activation assay (Figure 1C). In addition, expression of PTPRZ1(1814-1822)-containing tandem minigene (TMG) in U87 caused strong activation of PTPRZ1(1814-1822) TCR transgenic Jurkat T cells. Furthermore, these PTPRZ1(1814-1822) TCR transgenic Jurkat T cells could be identified by PTPRZ1(1814-1822) pentamer flow cytometry (Figure 1D). Most importantly, T cells from healthy donors stably overexpressing the PTPRZ1(1814-1822) TCR were able to express PTPRZ1(1814-1822)-loaded U87 and P3, HLA-A TCRs with endogenous PTPRZ1 expression. * 02:01 potently killed a positive patient-derived primary glioblastoma cell line ( Fig. 1E ).

[0118] 2.2 Results regarding NLGN4X-specific TCR NLGN4X(131-139)-specific TCRs were identified by single-cell sequencing of patients who received a multipeptide vaccine containing the NLGN4X(131-139) peptide. Single-cell TCR sequencing after multimer-based enrichment of T cells revealed a monoclonal T cell population (Figure 2A). Using the NLGN4X(131-139) peptide-loaded HLA-A2-positive K562 cell line, specifically activated NLGN4X(131-139) TCR transgenic healthy donor primary T cells that result in multifunctional cytokine production upon target recognition were assessed by intracellular flow cytometry (Figure 2B). Importantly, U87 glioma cells loaded with the NLGN4X(131-139) peptide or expressing full-length NLGN4X result in robust multifunctional cytokine production by NLGN4X(131-139) TCR transgenic healthy donor primary T cells (Figure 2C, Figure 2D). Most importantly, healthy donor-derived T cells stably overexpressing the NLGN4X(131-139) TCR potently killed U87 glioma cells expressing an NLGN4X(131-139)-containing tandem minigene (Figure 2E).

[0119] 2.3 NLGN4X identified by TCR sequencing of single cells from vaccinated glioblastoma patients 131-139 Reactive TCR NLGN4X 131-139 Following administration of a multi-peptide vaccine containing NLGN4X 131-139 NLGN4X from a patient with expanded reactive T cells 131-139 -HLA-A * 02 + Tetrameric enriched CD45 + CD3 +T cells were subjected to single cell VDJ sequencing (scVDJseq). The tetramer-enriched T cell pool was highly clonal, with one single clonotype constituting 90.82% of the TCR repertoire (Figure 3A). We then generated episomal nano-scaffold matrix-binding region (nano-S / MAR) DNA vectors containing the alpha and beta variable chains of each of the top four [frequotypes 1–4 = ft1–4] TCR clones and transfected TCR-deficient Jurkat T cells (Jurkat76). Surface expression of the TCR was confirmed by flow cytometry analysis of the murine TCR beta chain (mTCRb), which allows specific detection of the transgenic TCR and excludes mispairing with endogenous TCR (Figure 3B). TCR-transfected Jurkat76 were then transfected with peptide-loaded BOLETH HLA-A * 02 + CD69 expression was assessed by flow cytometry after co-culture with presenter cells. 131-139 When exposed to the epitope, TCRft1 was only upregulated in TCRft1-expressing Jurkat76 (Figure 3C). Therefore, TCRft1 was consequently used to evaluate downstream reactivity. As a mechanism of action for antigen-dependent T cell stimulation, nuclear translocation of nuclear factor of activated T cells (NFAT) represents a hallmark of TCR signaling. Therefore, TCRft1-expressing Jurkat76 was co-transfected with a luciferase NFAT reporter and subsequently peptide-loaded HLA-A * 02 + Peripheral blood mononuclear cells (PBMCs) and HLA-A do not endogenously express the NLGN4X protein * 02 + When PBMCs were used as antigen-presenting cells, NLGN4X was co-cultured with U87 glioma cells (Figure 3D, Figure 3E). 131-139 The intensity of the induced luminescence signal was similar to that of TCR stimulation with anti-CD3 / CD28 beads (FIG. 3D).

[0120] 2.4 NLGN4X 131-139Generation and phenotypic characterization of reactive TCR-transduced human T cells Finally, to deliver NLGN4X-reactive TCRs, we have developed a method to target relapsed or refractory CD19 + The retroviral SFG-IRES-GFP vector (Figure 4C), currently being investigated in clinical trials evaluating anti-CD19 CAR T cells in patients with lymphoid diseases, was chosen and used with different packaging plasmid systems, RD-114 and PeqPam. Importantly, the transduction process of primary human T cells using the retroviral TCRft1-SFG-IRES-GFP vector (Figure 4C) resulted in up to 93.2% GFP expression. + CD3 + T cells, resulting in a transduction efficiency of up to 90.4% (Figure 4D-F). Consequently, the SFG-IRES-GFP vector was used for all subsequent in vitro and in vivo experiments. Engineered autologous T cells were routinely expanded after transduction before reinfusion. To assess the phenotype of TCRft1-SFG-IRES-GFP-expressing primary human T cells during in vitro expansion, TCR-transduced T cells were subjected to longitudinal flow cytometric analysis by using a phenotypic marker for T cell differentiation. TCRft1-SFG-IRES-GFP-expressing T cells resembled the naive T cell state and were CD45RA suggesting that transduction with TCRft1-SFG-IRES-GFP does not result in T cell differentiation into terminal effector cells. + CD45RO - CCR7 + T cell phenotype (Figure 4G). Moreover, no associated increase in the expression of exhaustion markers PD-1 and TIM-3 was observed. Overall, flow cytometry profiling suggests that transduced TCRft1-SFG-IRES-GFP primary human T cells display a naive T cell phenotype that was recently described in Vitanza NA et al. (2021) to provide superior antitumor capabilities.

[0121] 2.5 NLGN4X-TCR-T lyses glioma cells in vitro Exogenously peptide-loaded non-adherent HLA-A * 02 + NLGN4X in vitro using flow cytometric profiling after co-culture with K562 leukemia cells 131-139 We then aimed to evaluate the functionality of specific TCR-expressing primary human T cells. As the most relevant effector proteins, granzyme B (GrzB), interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) production were evaluated. Independently of healthy T cell donors, a strong expression of the highly cytotoxic cytokines TNFα and GrzB was observed in NLGN4X T cells. 131-139 This was seen by specific TCR-expressing T cells (Figures 5A-5D). 131-139 Similar expression levels of GrzB, IFNγ and TNFα (Figure 5B-D) were observed in the co-culture system when NLGN4X-specific TCR-expressing primary human T cells were compared with the well-established high affinity melanoma antigen 1 (MART-1)-specific TCR recognized by T cells used in phase I and II clinical trials. 131-139 To confirm that potent expression of cytotoxic proteins by specific TCR-expressing primary human T cells results in target cell killing, we utilized a modified version of the Vital FR assay to detect specific lysis of peptide-loaded K562 target cells (Figure 5E). Importantly, NLGN4X 131-139 Specific T cells and MART1-specific T cells (MART-1-TCR-T) exhibited cytotoxic activity comparable to that of peptide-loaded K562 target cells (Figure 3F).

[0122] Next, HLA-A * 02 + We aimed to evaluate the cytotoxic potential of NLGN4X-TCR-T against the adherent glioma cell line U87. 131-139Peptide-loaded U87 wild-type cells induced specific upregulation of GrzB, CD69, and 4-1BB (CD137) in NLGN4X-TCR-T cells (Figure 6A). 131-139 Specific T cells were able to specifically lyse U87 cells loaded with the target peptide in lactate dehydrogenase (LDH) and flow cytometry-based killing assays (Figure 6B, Figure 6C). In co-culture assays where synthetic MHC class I-restricted peptides are applied for reactivity testing, MHC molecules are usually saturated and loading of exogenous MHC may occur. To date, NLGN4X 131-139 have been identified by HLA-ligandome analysis in some glioblastoma patients, 1 , NLGN4X in tumor model systems 131-139 Therefore, we aimed to demonstrate endogenous expression of NLGN4X. 131-139 Antigens were expressed by using either tandem minigenes (TMG) containing different MHC class I epitopes including NLGN4X (U87 TMG) ​​or a retroviral vector containing full-length NLGN4X (U87 NLGN4X). Similar to the findings using K562 cells as target cells, specific upregulation of GrzB, CD69 and 4-1BB was observed when NLGN4X-TCR-T was co-cultured with U87 NLGN4X or U87 TMG target cells (Figure 6D). Furthermore, U87 TMG and U87 NLGN4X were specifically lysed by NLGN4X-TCR-T (Figure 6E). In summary, primary human T cells expressing TCRft1-SFG-IRES-GFP in vitro were able to upregulate NLGN4X. 131-139 It was demonstrated that it specifically recognizes and lyses tumor cells expressing the epitope.

[0123] 2.6 NLGN4X-TCR-T promotes tumor regression and inhibits HLA-A * 02 + Improves survival in mice with glioma NLGN4X 131-139 NLGN4X by specific TCR engineered human T cells 131-139Having demonstrated specific recognition of the epitope and tumor cell lysis of the NLGN4X-expressing U87 human glioma cell line, we next intended to evaluate their therapeutic potential in vivo. Therefore, NOD scid gamma (NSG) MHC class I and MHC class II knockout (NSG MHCI / II KO) mice, which do not develop graft-versus-host rejection after T cell transplantation, were challenged with intracranial U87 TMG experimental gliomas. Mice were challenged with NLGN4X by intraventricular transplantation. 131-139 received either specific TCR-engineered human T cells or negative control [influenza (Flu)] TCR-engineered human T cells (Flu-TCR-T), or did not receive any T cell treatment [(NTC) = no T cell control] (Figure 7A). * 02 + T cells 5 x 10 6 The NLGN4X-TCR-T was injected twice into the lateral ventricle of the non-tumor-bearing hemisphere on days 15 and 22 (Figure 7A). Treatment with NLGN4X-TCR-T resulted in a prolonged survival of glioma-bearing animals compared to Flu-TCR-T or NTC mice (Figure 7B). We intended to investigate whether treatment with NLGN4X-TCR-T resulted in an objective radiological response by using longitudinal MRI, and therefore assessed tumor volume from days 11 to 67 according to modified RANO criteria. At day 67, the time point of the best response, we observed an objective response rate of 44.4% (ORR: CR+PR) with stable disease in 11.1% of NLGN4X-TCR-T-treated mice, partial response (PR) in 22.2%, and complete response (CR) in 22.2%, compared to 0.0% in both Flu-TCR-T and NTC mice (Figure 7C). 131-139In addition to assessing radiation response with specific TCR-engineered T cell therapy, longitudinal MRI also allowed for the local assessment of tumor growth (Figure 5D, Figure 5E). Interestingly, two mice with late recurrence of U87 TMG tumors (Figure 7E) had previously demonstrated radiation response (1 PR, 1 CR) in MRI (Figure 7E). Therefore, flow cytometric analysis of tumor-infiltrating leukocytes in recurrent late tumors was performed (Figure 7F). In these tumors, 78 days after the second administration of NLGN4X-TCR-T, CD4+ with low GFP expression was observed in the experimental tumors. + T cells were predominantly found. These CD4 + T cells are CD45RA - CCR7 - , expression of the proliferation marker Ki67 was low, and expression of PD-1 was high ( Fig. 7F ). Notably, recurrent U87 TMG tumors were NLGN4X, as demonstrated by qPCR. 131-139 In addition, MHC class I expression was still detectable by immunofluorescence staining (Figure 7G, H). Overall, these findings suggest that late recurrence is due to intratumoral cytotoxicity of NLGN4X. 131-139 specific CD8 + This suggests that this is due to a lack of T cells (Figures 7F to 7H).

[0124] 2.7 NLGN4X-TCR-T is phenotypically compatible with the tumor microenvironment Exhausted CD4 with low TCR transgene expression in late relapse + Therefore, this observation prompted the evaluation of the intratumoral phenotype of NLGN4X-TCR-T at early time points after intraventricular delivery in U87 TMG-bearing glioma animals (Figure 8A). Six days after intraventricular implantation, T cells were present within the contralateral experimental glioma and expressed NLGN4X 131-139 Specific TCR positive CD8 + Quantitatively, NLGN4X-TCR-T and Flu-TCR-T treated U87 TMG gliomas upregulated CD3 + CD8 T cells were not differentially infiltrated (Figure 8C). +T cells predominantly displayed a T effector memory (TEM) phenotype with expression of CD45RA and high expression of Ki67 compared to low expression of exhaustion markers PD-1 and TIM-3 (Figure 8D, Figure 8E). The specific upregulation of various activation and effector cell markers in NLGN4X-TCR-T compared to Flu-TCR-T (Figure 8F) suggests antigen recognition and specific intratumoral T cell activation. However, a fundamental difference between the early phenotype of intratumoral NLGN4X-TCR-T (Figure 8) and that seen in late relapse (Figure 7E) is the expression of cytotoxic CD8 + These results support the hypothesis that loss of NLGN4X-TCR-T leads to tumor recurrence. 131-139 It has been demonstrated that antigen-targeting, readily available, patient-derived TCR-engineered human T cells can lyse tumor cells in vitro and mediate transient tumor control in experimental gliomas.

[0125] Literature: Hilf et al., Nature 565:240-245 (2019); Keskin et al., Nature 565:234-239 (2019); Platten et al., Nature 592:463-468 (2021); Ellingson BM et al., Neurotherapeutics, 14(2):307-320 (2017); Rohaan MW et al., N engl J Med., 387(23):2113-2125 (2022); Morgan RA et al., J Immunother., 36(2):133-51 (2013); Chheda ZS et al., J Exp Med., 215(1):141-157 (2018); Immisch L et al., J Immunother Cancer, Oct 2022;10(10); Kilian M et al., Clin Cancer Res. 28(2):378-389 (2022); Bolinger MF et al., Proc NAl Acad Sci USA, 105(17):6421-6 (2008); Choe JH et al., Sci Transl Med, 28 2021;13(591) (2021); Sahillioglu AC et al., Curr Opin Immunol., 74:190-198 (2022); Choo J et al., J Virol., 88(18):10613-23 (2014); Vitanza NA et al., Nat Med, 27(9):1544-1552 (2021).

Claims

1. A binding polypeptide comprising a first variable T-cell receptor (TCR) domain and a second variable TCR domain, wherein (i) complementarity determining region 3 (CDR3) of said first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 1 or a sequence at least 80% identical thereto; and / or CDR3 of said second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 2 or a sequence at least 80% identical thereto; or (ii) CDR3 of said first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 3 or a sequence at least 80% identical thereto; and / or CDR3 of said second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 4 or a sequence at least 80% identical thereto.

2. 2. A binding polypeptide according to claim 1, wherein (i) the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 5, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 6, or (ii) the first variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 70% identical to SEQ ID NO: 7, and / or the second variable TCR domain comprises, preferably consists of, the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is at least 70% identical to SEQ ID NO:

8.

3. Preferably, the binding polypeptide is an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A * or wherein the binding polypeptide recognizes a peptide having the amino acid sequence MIWEHNVEV (SEQ ID NO: 14) when presented on an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A molecule. * 3. The binding polypeptide of claim 1 or 2, which recognizes a peptide having the amino acid sequence NLDTLMTYV (SEQ ID NO: 13) when presented on a .O2 molecule.

4. 4. A binding polypeptide according to any one of claims 1 to 3, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to a transmembrane domain, preferably a TCR transmembrane domain, preferably said binding polypeptide is a TCR.

5. 5. The binding polypeptide of any one of claims 1 to 4, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to a signalling domain, preferably wherein the binding polypeptide is a chimeric antigen receptor (CAR).

6. 6. A binding polypeptide according to any one of claims 1 to 5, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to an Fc domain of an immunoglobulin; or wherein the binding polypeptide is a soluble TCR, preferably a soluble TCR tetramer.

7. A polynucleotide encoding at least one of the first and second variable TCR domains of a binding polypeptide according to any one of claims 1 to 6.

8. A host cell comprising a binding polypeptide according to any one of claims 1 to 6 and / or a polynucleotide according to claim 7.

9. A binding polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7 and / or a host cell according to claim 8 for use in medicine, preferably for use in the treatment and / or prevention of cancer.

10. 1. A method for identifying a target cell expressing protein tyrosine phosphatase receptor type Z1 (PTPRZ1) and / or neuroligin-4, X-linked (NLGN4X), comprising: (i) contacting said target cell with a binding polypeptide according to any one of claims 1 to 6; (ii) detecting binding of the binding polypeptide to the target cell; and (iii) identifying a target cell expressing PTPRZ1 and / or NLGN4X based on the detection in step (ii). A method comprising:

11. A method for identifying a cancer susceptible to treatment with a binding polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, and / or a host cell according to claim 8, comprising the steps of: (i) identifying target cells expressing PTPRZ1 and / or NLGN4X in a cancer sample; and (ii) identifying a cancer susceptible to treatment based on the identification in step (i). A method comprising:

12. 10. The in vitro use of a binding polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, and / or a host cell according to claim 8 for detecting target cells expressing neuroligin-4, X-linked (NLGN4X) and / or protein tyrosine phosphatase receptor type Z1 (PTPRZ1).

13. A kit comprising a binding polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, and / or a host cell according to claim 8, contained in a housing and / or further comprising an administration means.

14. A device comprising a binding polypeptide according to any one of claims 1 to 6, a polynucleotide according to claim 7, and / or a host cell according to claim 8.

15. The subject matter according to any one of claims 9 and 11 to 14, wherein the cancer is a brain cancer, preferably a glioma, more preferably a glioblastoma.

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