Binding polypeptides derived from T cell receptors

Abstract

The present invention relates to a binding polypeptide 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 the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80% identity therewith, preferably consisting of the amino acid sequence of SEQ ID NO: 1 or a sequence having at least 80% identity therewith; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80% identity thereto, preferably consisting of the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80% identity thereto; or wherein (ii) the CDR3 of the first variable TCR domain comprises the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% identity thereto, preferably consisting of the amino acid sequence of SEQ ID NO: 3 or a sequence having at least 80% identity thereto; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO: 4 or a sequence thereof having at least 80% identity, preferably consisting of the amino acid sequence of SEQ ID NO: 4 or a sequence thereof having at least 80% identity; as well as polynucleotides, host cells, methods, uses, kits and devices related thereto.

Description

[0001] The present invention relates to a binding polypeptide 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 the amino acid sequence of SEQ ID NO:1, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:1, or a sequence having at least 80% identity thereto; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:2, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:2, or a sequence having at least 80% identity thereto; or wherein (ii) the CDR3 of the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% identity thereto; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:5. The invention relates to an amino acid sequence of NO:4 or a sequence composed of at least 80% of its identity; and polynucleotides, host cells, methods, uses, kits and devices related thereto.

[0002] Adoptive T cell therapy using genetically engineered T cells expressing chimeric antigen receptors (CARs) against the B cell antigen CD19 has achieved remarkable symptom regression in patients with leukemia and lymphoma. Translating this cell concept to solid tumors has proven difficult, with the main obstacle being the identification of appropriate cell surface antigens. On the other hand, intracellular tumor-associated antigens (TAAs) can also be processed and presented to T cells on the major histocompatibility complex and can therefore be exploited as T cell targets. Despite the successful induction or amplification of T cell responses against TAAs by therapeutic vaccines, the level of cellular effector immune responses required to control tumor growth has not yet 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 an alternative therapeutic option for ex vivo expansion of spontaneous T cell responses. However, nonspecific cytokine-driven ex vivo expansion of TILs inevitably leads to the loss of potential TAA-reactive T cell clones before re-infusion into patients, thereby reducing the efficacy of cell therapy. Here, T cell receptor (TCR) transgenic cell therapy provides a valuable alternative, because the transgenic cells target known and defined antigens, TAA-reactive transgenic cells can be additionally genetically modified to enhance T cell responses, and re-infusion of monoclonal TCR transgenic T cell products allows accurate enumeration of true tumor-reactive T cells compared to TIL therapy.

[0004] Gliomas are typically tumors with a low mutational burden, containing only 30–50 nonsynonymous mutations. Recently, two multinational and multicenter glioblastoma (WHO grade IV glioma) vaccine consortia, the Glioma Active Personalized Vaccine Consortium (GAPVAC) and the NeoVax Consortium, independently reported the success of inducing highly personalized TAA-specific but also tumor-specific antigen (TSA)-specific T cell responses in glioblastoma patients (Hilf et al., supra; Keskin et al., supra). Both phase 1 trials were able to demonstrate vaccine-induced T cell homing to brain tumors. Similarly, a multicenter, single-arm, open-label, first-in-human phase I trial (German Cancer Society Neuro-Oncology Working Group Trial 16 (NOA16), NCT02454634) in 33 patients newly diagnosed with World Health Organization grade 3 and 4 mutant isocitrate dehydrogenase 1 (IDH1R132H)-positive astrocytomas provided proof of concept that target-specific T helper cells can, in principle, home to gliomas (Platten et al., Nature 592:463-468 (2021)). At the same time, evidence from these studies suggests that the magnitude of vaccine-induced specific T cell responses still needs to be improved.

[0005] Therefore, there is still a need for improved cancer treatment. The technical problem of the present invention can be seen as providing means and methods that meet the above needs. The technical problem is solved by the claims and the embodiments described below.

[0006] Accordingly, the present invention relates to binding polypeptides comprising a first variable T cell receptor (TCR) domain and a second variable TCR domain.

[0007] In general, the terms used herein are given their usual and customary meanings for those skilled in the art, and are not limited to special or custom meanings unless otherwise specified. As shown below, the terms "having", "including" or "comprising" or any arbitrary grammatical variant thereof are used in a non-exclusive manner. Therefore, these terms may refer to the situation where there are no further features in the entity described in the context in addition to the features introduced by these terms, or may refer to the situation where one or more further features exist. For example, the expressions "A has B", "A includes B" and "A includes B" may refer to the situation where there are no other elements in A except B (i.e., A is composed solely and exclusively of B), or may refer to the situation where there are one or more other elements (e.g., element C, element C and D, or even further elements) in entity A except B. In addition, as understood by the technician, the expressions "comprising a" and "comprising an" preferably refer to "comprising one or more", i.e., equivalent to "comprising at least one". Accordingly, unless otherwise stated, a statement relating to one of a plurality of items preferably relates to at least one such item, more preferably to a plurality thereof; thus, for example, identifying "a cell" relates to identifying at least one cell, preferably to identifying a plurality of cells.

[0008] Furthermore, as used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "specifically", "more specifically" or similar terms are used in conjunction with optional features without limiting further possibilities. Therefore, the features introduced by these terms are optional features and are not intended to limit the scope of the claims in any way. As the skilled person will recognize, the present invention can be implemented by using optional features. Similarly, features introduced by "in one embodiment" or similar expressions are intended to be optional features, without any limitation as to further embodiments of the invention, without any limitation as to the scope of the invention, nor any limitation as to the possibility of combining the features introduced in the described manner with other optional or non-optional features of the invention.

[0009] The method specified below is preferably an in vitro method. In principle, the method steps can be carried out in any arbitrary order that the skilled person considers suitable, but are preferably carried out in the order indicated; in addition, one or more, preferably all, of the steps can be assisted or carried out by automated devices. Furthermore, the method may contain steps other than those explicitly mentioned above.

[0010] As used herein, the term "standard conditions", if not otherwise specified, refers to IUPAC standard ambient temperature and pressure (SATP) conditions, i.e. preferably a temperature of 25°C and an absolute pressure of 100 kPa; and preferably, standard conditions include a pH of 7. In addition, if not otherwise specified, the term "approximately" refers to an indicated value with a technical accuracy generally accepted in the relevant field, preferably to an indicated value ±20%, more preferably to ±10%, and most preferably to ±5%. In addition, the term "substantially" means that there is no deviation that has an impact on the indicated result or use, i.e., the potential deviation will not cause the indicated result to deviate by more than ±20%, more preferably ±10%, and more preferably ±5%. Therefore, "substantially consisting of..." means including the specified ingredients, but excluding materials present as impurities, materials that are inevitably present due to the process used to provide the ingredients, and ingredients added for purposes other than the technical effects of the present invention, other ingredients are not included. For example, a composition defined using the phrase "substantially consisting of..." includes any known acceptable additives, excipients, diluents, carriers, etc. Preferably, a composition consisting essentially of a group of ingredients 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 one or more non-specified ingredients.

[0011] 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, wherein the sequence fragments in the comparison window may contain additions or deletions (e.g., gaps or overhangs) compared to the comparison sequences used for optimal alignment. The percentage is calculated by determining the number of positions at which the same residues occur in the two sequences (preferably over the full length of the polynucleotide or polypeptide) to produce the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The optimal alignment of the sequence for comparison can be carried out by the local homology algorithm of Smith and Waterman (1981), the homology alignment algorithm of Needleman and Wunsch (1970), the retrieval of the similarity method of Pearson and Lipman (1988), the computerized implementation of these algorithms (TFASTA in GAP, BESTFIT, BLAST, PASTA and Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin) or visual inspection. In view of having determined that two sequences are used for comparison, preferably GAP and BESTFIT are used to determine their optimal alignment, thereby determining the degree of identity. Preferably, the default value of weight gap 5.00 and the default value of gap weight length 0.30 are used. In the context of biological sequences involved herein, the term "substantially identical" refers to a % identity value of at least 80%, preferably at least 90%, more preferably at least 98%, most preferably at least 99%. As will be understood, the term substantially identical includes 100% identity. The above applies mutatis mutandis to the term "substantially complementary".

[0012] The term "fragment" of a biomacromolecule, preferably a "fragment" of a polynucleotide or polypeptide, is used in a broad sense herein and relates to any sub-portion, preferably a subdomain, of the corresponding biomacromolecule comprising the specified sequence, structure and / or function. Thus, the term includes sub-portions resulting from the actual cleavage of the biomacromolecule, as well as sub-portions derived from the corresponding biomacromolecule in an abstract manner (e.g., on a computer). Thus, as used herein, Fc or Fab fragments, as well as, for example, single-chain antibodies, bispecific antibodies, and nanobodies may be referred to as fragments of immunoglobulins.

[0013] Unless otherwise specifically indicated herein, the specified compounds, in particular polynucleotides and polypeptides, may be included in a larger structure, for example, may be covalently or non-covalently linked to further sequences, carrier molecules, retardants and other excipients. In particular, the specified polypeptide may be included in a fusion polypeptide comprising a further peptide, which may be used as a tag, for example, 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 to or introduced into a fusion polypeptide; preferably, the tag is added to the C-terminus or N-terminus of the fusion polypeptide. The amino acid fragment preferably allows the detection of the polypeptide by a specific binding partner (e.g., an antibody that specifically recognizes the tag); or it preferably allows the formation of a functional conformation, such as a chelator; or it preferably allows visualization, such as in the case of a fluorescent tag. Preferred detectable tags are Myc tags, FLAG tags, 6-His tags, HA tags, GST tags or fluorescent protein tags, such as GFP tags. These tags are well known in the art. Other further peptides preferably included in the fusion polypeptide, comprising further amino acids or other modifications, may act as mediators of secretion, as mediators of blood-brain barrier passage, as cell penetrating peptides and / or as immunostimulants. Further polypeptides or peptides that may be fused to the polypeptide are signal sequences and / or transport sequences, such as the IL-2 signal sequence and linker sequences.

[0014] As used herein, the term "polypeptide" refers to a molecule comprising several (usually at least 20) amino acids, which are covalently linked to each other by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are generally considered to be "peptides". Preferably, the polypeptide comprises 50 to 1000 amino acids, more preferably 60 to 1000 amino acids, more preferably 70 to 500 amino acids, and most preferably 80 to 400 amino acids. A polypeptide may be a complex of more than one amino acid chain, i.e., a multimer, such as a dimer, a trimer, etc.; in this case, the complex of more than one amino acid chain may also be referred to as a "polypeptide oligomer" or as a "protein complex". Preferably, the complex of more than one amino acid chain is a heteropolymer, more preferably a heterodimer, preferably the heterodimer comprises at least one first variable TCR domain and at least one second variable TCR domain in a non-covalent complex. In addition, the polypeptide may comprise additional non-peptide structures, such as at least one glycosylation, lipid conjugation, etc. More preferably, the specified polypeptide includes all the specified structural components contained in one continuous covalent peptide chain, therefore, the polypeptide is preferably a fusion polypeptide or is contained in a fusion polypeptide. Unless otherwise explicitly stated, the specific polypeptide mentioned herein preferably includes polypeptide variants.

[0015] As used herein, the term "polypeptide variant" refers to any chemical molecule comprising at least one polypeptide described herein, which has a specified activity but is structurally different from the specific polypeptide. Preferably, the polypeptide variant comprises a polypeptide having a continuous 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 specific polypeptide amino acid sequence. In addition, it should be understood that the polypeptide variant referred to in the present invention should have an amino acid sequence that is different due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, and most preferably at least 99% identity with the amino acid sequence of the specific polypeptide. The degree of identity between the two amino acid sequences can be determined by algorithms well known in the art and as described above. The above-mentioned polypeptide variants can be allelic variants or any other species-specific homologs (homolog), paralogs (paralog) or orthologs (ortholog). In addition, polypeptide variants mentioned herein include fragments of specific polypeptides or polypeptide variants of the above types, as long as these fragments and / or variants have a specified biological activity. These fragments may be or may be derived from, for example, degradation products or splice variants of the polypeptide. Further included are variants that differ due to post-translational modifications, such as phosphorylation, glycosylation, ubiquitinylation, sumoylation or myristylation, by including non-natural amino acids and / or becoming peptidomimetics.

[0016] The term "polynucleotide" as used herein refers to a linear or circular nucleic acid molecule. The polynucleotides of the present invention are preferably provided as isolated polynucleotides (i.e., isolated from their natural environment) or in a genetically modified form (preferably comprising at least one heterologous sequence). The term encompasses single-stranded and double-stranded polynucleotides. In addition, chemically modified polynucleotides are also included, including naturally occurring modified polynucleotides (e.g., glycosylated or methylated polynucleotides) or artificially modified derivatives (e.g., biotinylated polynucleotides, locked nucleic acids, etc.). The polynucleotides of the present invention have the activity of encoding the binding polypeptides described herein. Methods for testing whether a given polynucleotide has the above-mentioned activity are known in the art and are described below. Unless otherwise expressly stated, the specific polynucleotides mentioned herein preferably include polynucleotide variants.

[0017] As used herein, the term "polynucleotide variant" relates to a variant of a polynucleotide as described herein, comprising a nucleic acid sequence characterized in that the sequence can be derived from the above-mentioned specified nucleic acid sequence by at least one nucleotide substitution, addition and / or deletion, wherein the polynucleotide variant should have the activity specified for the specific polynucleotide. Preferably, the polynucleotide variant is an ortholog, a paralog or another homolog of a specific polynucleotide. Also preferably, the polynucleotide variant is or is derived from a non-naturally occurring allele of a specific polynucleotide. Polynucleotide variants also include polynucleotides comprising a nucleic acid sequence that can hybridize with the above-mentioned specific polynucleotide, preferably under stringent hybridization conditions. These stringent conditions are known to the skilled person and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, ny (1989), 6.3.1-6.3.6. The skilled person knows how to determine the required hybridization conditions by reference to textbooks such as the above textbooks or 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 of Oxford University Press, Oxford; Brown (Ed.) 1991, "Essential Molecular Biology: A Practical Approach", IRL Press of Oxford University Press, Oxford. Alternatively, polynucleotide variants can also be obtained by PCR-based techniques, such as DNA amplification based on mixed oligonucleotide primers. In addition, 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%, still more preferably at least 98%, and most preferably at least 99% identical to a specific specified nucleic acid sequence. In addition, also comprise the polynucleotide of the nucleic acid sequence that comprises the amino acid sequence of coding, the identity of described amino acid sequence and specific specified amino acid sequence is at least 70%, more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 98%, most preferably at least 99%.Percent identity value, preferably, is calculated on whole amino acid or nucleic acid sequence region, preferably as described above.Polynucleotide of the present invention can be made up of above-mentioned nucleic acid sequence, be made up of above-mentioned nucleic acid sequence basically or comprise above-mentioned nucleic acid sequence.Therefore, they also can comprise further nucleic acid sequence.Specifically, the polynucleotide of the present invention may encode a fusion protein, wherein one partner of the fusion protein is a polypeptide encoded by the nucleic acid sequence described above. In addition, the polynucleotide may be contained in a vector.

[0018] The term "vector" preferably includes bacteriophages, plasmids, viral vectors or retroviral vectors and artificial chromosomes, such as bacterial artificial chromosomes or yeast artificial chromosomes. In addition, the term also relates to a targeting construct, which allows the targeting construct to be randomly or site-specifically integrated into genomic DNA. Such a targeting construct preferably comprises DNA of sufficient length for homologous or heterologous recombination as described in detail below. The vector comprising the polynucleotide of the present invention preferably further comprises a selectable marker for propagation and / or selection in the host. The vector can be incorporated into a host cell by various techniques well known in the art. For example, a plasmid vector can be introduced in a precipitate (such as a calcium phosphate precipitate or a rubidium chloride precipitate, etc.), or introduced in a complex of a charged lipid, or introduced in a carbon-based cluster (such as fullerene). Alternatively, a plasmid vector can be introduced by heat shock or electroporation techniques. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line before being applied to a host cell. Retroviral vectors can have replication ability or replication defects. In the latter case, viral propagation usually only occurs in a 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 a prokaryotic cell or a eukaryotic cell or a separated part thereof, i.e., preferably, the polynucleotide is contained in an expression vector. The expression of the polynucleotide comprises transcription of the polynucleotide into RNA. Regulatory elements that ensure expression in eukaryotic cells (preferably mammalian cells) are well known in the art. Preferably, it comprises a regulatory factor that ensures transcription initiation and, optionally, a poly-A signal that ensures transcription termination and transcription stability. Other regulatory elements may include transcription enhancers and translation enhancers. Possible regulatory elements that allow expression in prokaryotic host cells include, for example, lac, trp or tac promoters in Escherichia coli (E. coli), and examples of regulatory elements that allow expression in eukaryotic host cells are AOX1 or GAL1 promoters in yeast, or CMV-, SV40-, RSV-promoters (Rouse sarcoma virus), CMV-enhancers, SV40-enhancers or globin introns in mammalian and other animal cells. In addition, inducible expression control sequences can be used in the expression vectors included in the present invention. Such inducible vectors may 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. In addition to the elements responsible for transcription initiation, these regulatory elements may also include transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site downstream of the polynucleotide.In this context, suitable expression vectors are known in the art, such as the 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 and a gene transfer vector or a targeting vector. Expression vectors derived from viruses such as retrovirus, vaccinia virus, adeno-associated virus, herpes virus or bovine papilloma virus can be used to deliver polynucleotides or vectors to a target cell population. Methods well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, 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).

[0019] The term "specific binding" is understood by the skilled person. Preferably, specific binding relates to a binding wherein the affinity of the binding polypeptide to the peptide (preferably a peptide as described elsewhere herein) is at least 10 times, preferably at least 100 times, most preferably at least 1000 times higher than any non-homologous (poly)peptide. Thus, the dissociation constant (K) of any binding polypeptide / non-homologous peptide complex is d ) is preferably at least 10 -6 mol / L, more preferably at least 10 -5 mol / L, most preferably at least 10 -4 mol / L.

[0020] The term "binding polypeptide" as used herein relates to a polypeptide having a structural element as described herein and having the activity of binding to its cognate peptide, preferably specifically binding to said cognate peptide, more preferably specifically binding to said cognate peptide when said peptide is presented on an MHC class I molecule, preferably an HLA-A molecule, more preferably an HLA-A*02 molecule. Preferably, said cognate peptide comprises the amino acid sequence MIWEHNVEV (SEQ ID NO: 14), more preferably consists of the amino acid sequence MIWEHNVEV (SEQ ID NO: 14) or comprises the amino acid sequence NLDTLMTYV (SEQ ID NO: 13), more preferably consists of the amino acid sequence NLDTLMTYV (SEQ ID NO: 13).

[0021] The term "T cell receptor", abbreviated as "TCR" as used herein, refers to a polypeptide complex that mediates recognition of antigenic peptides presented by target cells on the surface of T cells, preferably in the context of MHC molecules or MHC-related molecules, such as MR1 ​​or CD1, more preferably in the context of MHC molecules, also preferably in the context of MHC class I molecules or MHC class II molecules, most preferably in the context of MHC class I molecules. Typically, TCR comprises one TCR-α chain and one TCR-β chain, i.e., an α / β chain heterodimer. However, TCR may also comprise a TCR γ chain and a TCR δ chain instead of a TCR α chain and a β chain. In accordance with common terminology, the complex composed of α and β chains or γ and δ chains is referred to herein as a "T cell receptor" or "TCR", the α chain and / or β chain and the γ chain and / or δ chain are generally or individually referred to as a "TCR polypeptide" or multiple "TCR polypeptides", and the polypeptide complex comprising TCR and auxiliary polypeptides (such as CD3 and CD247) is referred to as a "T cell receptor complex", abbreviated as "TCR complex". Each TCR polypeptide comprises several polypeptide domains, namely a transmembrane region (transmembrane domain), a constant region (constant domain), a connecting region (constant domain) and a variable region (variable domain), and the variable region of each TCR α, β, γ or δ chain comprises three complementary determining regions (CDRs), respectively referred to as CDR1, CDR2 and CDR3. As described herein, the variable region of a TCR polypeptide is referred to as a "variable T cell receptor domain", which can be abbreviated as a "variable TCR domain". In general, the nomenclature of amino acid numbering of TCR domains or immunoglobulin superfamily polypeptides is well known to those skilled in the art, for example, the nomenclature of the international ImMunoGeneTics information system® (IMGT numbering scheme, Lefranc et al., Dev Comparative immuno1 27: 55 (2003)), Kabat numbering, etc. Preferably, the numbering scheme is the IMGT numbering scheme and is used for amino acid numbering herein. As known to the skilled person, in TCR, binding specificity is essentially determined by the variable domain, specifically the CDR3 region of the variable region. Therefore, a TCR having an activity of binding specific homologous peptides can be structurally defined by the amino acid sequence of the variable domain of its CDR3.

[0022] 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 contained in a fusion polypeptide, i.e., preferably in a continuous peptide chain. And preferably, the first variable TCR domain is covalently linked to the first constant TCR domain, and / or the second variable TCR domain is covalently linked to the second constant TCR domain. Preferably, the first variable TCR domain and the second variable TCR domain are covalently linked to a transmembrane domain, preferably a TCR transmembrane domain. Therefore, the binding polypeptide may be a TCR, preferably comprising all structural elements of a TCR as shown in other parts of this article. Preferably, the first variable TCR domain and the second variable TCR domain are covalently linked to a signaling domain.

[0023] Thus, the binding polypeptide may be a chimeric antigen receptor (CAR). CAR and its construction principles are known in the art. 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 may preferably be an immunomodulatory compound. And preferably, the binding polypeptide is a soluble TCR, preferably a soluble TCR tetramer. In addition, the binding polypeptide may comprise a label, such as a coloring and / or fluorescent dye; and / or an effector compound, such as a chemotherapeutic drug, a radioactive compound, etc.

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

[0025] More preferably, the first variable TCR domain comprises 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, 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 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, 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 the first variable TCR domain comprises 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, 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 wherein the second variable TCR domain comprises 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, 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. Even more preferably, the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:5, preferably consists of the amino acid sequence of SEQ ID NO:5 and / or wherein the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:6, preferably consists of the amino acid sequence of SEQ ID NO:6; or the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:7, preferably consists of the amino acid sequence of SEQ ID NO:7; and / or wherein the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:8, 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. And 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 known to those skilled in the art and as described above, due to the degeneracy of the genetic code, a given amino acid sequence can be encoded by multiple nucleic acid sequences; therefore, the skilled person is able to provide an appropriate coding sequence, which can be, for example, an optimized codon for a host cell from a particular species.

[0027] Advantageously, in the work underlying the present invention, it was found that the binding polypeptides are particularly suitable for specific binding to cancer cells, in particular cancer cells expressing PTPRZ1 and / or NLGN4X, and thus present a polypeptide having an amino acid sequence as shown in SEQ ID NO: 13 and / or NO: 14. Therefore, the binding polypeptides provided herein are particularly suitable for cancer therapy, in particular T cell therapy of glioma.

[0028] The above definitions apply mutatis mutandis to the following situations. The other definitions and explanations further provided below also apply mutatis mutandis to all embodiments described in this specification.

[0029] The present invention also relates to a polynucleotide encoding at least one of the first variable TCR domain and the second variable TCR domain of the binding polypeptide of the present invention, and to a vector comprising the polynucleotide.

[0030] The term "polynucleotide" preferably includes polynucleotide variants, as described in detail above, as well as preferred nucleic acid sequences. Preferably, the polynucleotide encodes a first variable TCR domain and a second variable TCR domain of a binding polypeptide, more preferably 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 expression of at least one of the first variable TCR domain and the second variable TCR domain of a binding polypeptide in a suitable host cell.

[0031] The term vector has also been described in detail 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 capable of receiving, and preferably expressing, the binding polypeptide, polynucleotide or vector. Preferably, the host cell is a bacterial cell, more preferably a cell of a common laboratory bacterial strain known in the art, most preferably an Escherichia strain, in particular an Escherichia coli strain. And preferably, the host cell is a eukaryotic cell, preferably a yeast cell, such as a cell of a baker's yeast system, or an animal cell. More preferably, the host cell is an insect cell or a mammalian cell, in particular 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 is known to the skilled person, CD8 TCR is preferably expressed in CD8+ T cells, and CD4 TCR is preferably expressed in CD4+ T cells. And preferably, the host cell is capable of presenting a binding polypeptide as described herein on its surface, preferably a binding polypeptide encoded by a polynucleotide and / or a vector as described herein.

[0034] The present invention also relates to the binding polypeptides of the present invention, the polynucleotides of the present invention, the vectors of the present invention and / or the host cells of the present invention, which are used in medicine, in particular for treating and / or preventing cancer. Therefore, the binding polypeptides, polynucleotides, vectors and / or host cells can be formulated into pharmaceutical compositions.

[0035] The present invention also relates to a method for treating and / or preventing cancer in a subject, the method comprising

[0036] (a) contacting the subject with the binding polypeptide of the present invention, the polynucleotide of the present invention, the vector of the present invention and / or the host cell of the present invention, and

[0037] (b) thereby treating cancer in said subject.

[0038] The term "cancer" as used herein relates to a disease of an animal (including a human) characterized by the uncontrolled growth of a group of body cells ("cancer cells"). The uncontrolled growth may be accompanied by the formation of cell clusters of cancer cells (tumors), invasion and destruction of surrounding tissues (infiltration), and may cause cancer cells to spread to other parts of the body (metastasis). Preferably, the term cancer also includes the recurrence (recurrence) of cancer. Therefore, preferably, the cancer is a solid cancer (including primary tumor), its metastasis and recurrence. However, this cancer may also be a non-solid cancer. Preferably, the cancer is selected from the list consisting of: 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, Intestinal cancer, craniopharyngioma, endometrial cancer, ependymoma, ependymoma, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal stromal cell tumor, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, Kaposi's sarcoma, laryngeal cancer, medulloblastoma, medullary epithelioma, black leukoma, Merkel cell carcinoma, mesothelioma, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, ovarian low malignant potential tumor, pancreatic cancer, papillomatosis, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, chromaffin cells Preferably, the cancer is a solid cancer, which is metastatic or recurrent. More preferably, the cancer is a brain cancer, preferably a glioma, and most preferably a glioblastoma.

[0039] The terms "treating" and "treatment" refer to a significant degree of improvement in a disease or condition described herein or its attendant symptoms; as used herein, the terms include preventing the exacerbation of a disease, condition, or symptom associated therewith. Treatment as described herein also includes complete recovery of health with respect to a disease or condition described herein. It should be understood that treatment, as the term is used herein, may not be effective for all subjects treated. However, the term should require that, preferably, a statistically significant portion of subjects suffering from a disease or condition described herein can be successfully treated. Whether a portion is statistically significant can undoubtedly be determined by a person skilled in the art using various well-known statistical evaluation tools, such as, for example, determination of confidence intervals, determination of p-values, Student's t-tests, rank sum tests, 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 should 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 in a given cohort or population. Preferably, the treatment comprises activating an immune response to a medium that causes or mediates the disease (i.e., to cancer cells). Preferably, treating cancer is to reduce the burden of tumors and / or cancer cells in the subject. As will be appreciated by the skilled person, the effectiveness of treatment of, for example, cancer depends on a variety of factors, including, for example, cancer stage and cancer type. And preferably, cancer treatment further comprises at least one of surgery, chemotherapy, and radiotherapy.

[0040] The terms "preventing" and "prevention" refer to maintaining health relative to the diseases or conditions described herein in a subject for a certain period of time. It should be understood that the period of time may depend on the amount of the drug compound that has been administered and the individual factors of the subject discussed in other parts of this specification. It should be understood that prevention may not be effective in all subjects treated with the compounds according to the present invention. However, the term requires that preferably, a statistically significant portion of subjects in a cohort or population is effectively prevented from suffering from a disease or condition described herein or its associated symptoms. Preferably, in this context, the cohort or population of subjects is envisioned as being generally, that is, in the absence of preventive measures according to the present invention, a disease or condition described herein will be developed. Whether a portion is statistically significant can undoubtedly be determined by a person skilled in the art using various well-known statistical evaluation tools discussed elsewhere in this specification. In the context of cancer treatment, prevention is particularly related to preventing the development of cancer, preventing the formation of metastasis and / or preventing recurrence, preferably to preventing the formation of metastasis and / or preventing recurrence.

[0041] Preferably, the treatment and / or prevention of cancer comprises administering a host cell to a subject to be treated, preferably administering a T cell as described above to a subject to be treated, producing a binding polypeptide as described herein. In principle, methods for conferring the ability of a host cell to produce a binding polypeptide are known in the art, and in particular include introducing at least one expression construct, such as an expression vector, into a host cell. Suitable methods depend on the type of host cell and are known in the art, such as electroporation, infection with a viral vector, etc. In the case of cancer treatment and / or prevention, the host cell may be allogeneic, such as an allogeneic T cell, more preferably autologous, in particular autologous T cell. In addition, the host cell may be expanded before (again) administration to the subject, for example by in vitro culture expansion; and / or may be activated, for example, by co-culturing with cells presenting a homologous peptide and / or cytokine treatment.

[0042] The term "subject" as used herein relates to a vertebrate, preferably a mammal, in particular a livestock, companion or laboratory animal. Most preferably, the subject is a human. Preferably, the subject has been diagnosed with cancer, is suspected of having cancer, or is known to be at risk of cancer, in particular a hereditary cancer. Preferably, the subject's cancer has been identified as being sensitive to treatment with a binding polypeptide, preferably as described below.

[0043] The terms "drug" and "pharmaceutical composition" are used interchangeably herein and are known in principle to the skilled person. As described herein, the terms refer to any composition of matter containing one or more active agents specified as one or more pharmaceutically active compounds and one or more excipients. The one or more pharmaceutically active compounds may be present in liquid or dry (e.g., lyophilized) form. It should be understood that the form and characteristics of pharmaceutically acceptable excipients (e.g., carriers or diluents) are determined by the amount of active ingredient combined therewith, the route of administration, and other well-known variables. One or more excipients must be acceptable in the sense of being compatible with the other ingredients of the formulation and harmless to the recipient. The excipients used may include solids, gels, or liquids. Exemplary solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers are phosphate buffered saline solutions, physiological saline, Ringer's solution, glucose solution, and Hank's solution, syrups, oils, water, emulsions, various types of wetting agents, and the like. Similarly, the carrier or diluent may include time-delay materials well known in the art, such as glyceryl monostearate or glyceryl distearate alone or with wax. Suitable carriers include the above-mentioned carriers and other well-known carriers in the art, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania. One or more excipients are selected so as not to affect the biological activity of the composition. However, excipients may also be selected to improve the entry of the active agent into host cells, particularly the uptake of target cells. Therefore, excipients may also be virions and / or lipid vesicles, preferably virions and / or lipid vesicles known to mediate entry and / or fusion with the target cell of interest.

[0044] The drug is preferably administered by a route as specified above. A therapeutically effective dose refers to the amount of an effector polypeptide or an expression construct encoding the effector polypeptide used in a drug, which prevents, improves or cures the symptoms associated with the disease or condition mentioned in this specification. The therapeutic effect and toxicity of a drug can be determined by standard pharmaceutical procedures in cell culture or experimental animals, such as ED50 (the therapeutically effective dose for 50% of the population) and LD50 (the lethal dose for 50% of the population). The dose ratio between the therapeutic effect and the toxic effect is the therapeutic index, which can be expressed as the ratio of LD50 / ED50. The dosing regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dose for any one patient may depend on many factors, including the patient's size, age, specific formulation of the drug administered, sex, time and route of administration, general health, and other drugs administered simultaneously. The drugs mentioned herein are preferably administered at least once, for example as a bolus. However, the drug may be administered more than once, preferably at least twice, for example, permanently or regularly after a specified time window. Progress can be monitored by regular assessment. Dosage recommendations may be indicated in the prescription or user instructions so that the dosage can be adjusted according to the estimated dosage for the intended recipient.

[0045] The medicine according to the present invention may include other active agents in addition to the one or more active agents mentioned above. Preferably, the pharmaceutically active compound according to the present invention will be administered together with at least one other drug, and therefore can be formulated as a drug together with the at least one other drug. More preferably, in the case of cancer treatment, the at least one other active agent is a chemotherapeutic agent or other immunotherapeutic agent, such as an immune checkpoint regulator. In addition, it should be understood that in order to ensure the quality, drug safety and effectiveness of the drug, the preparation of the pharmaceutical composition is preferably carried out under GMP standardized conditions or similar conditions.

[0046] The present invention also relates to a method for identifying a target cell expressing protein tyrosine phosphatase receptor type Z1 (PTPRZ1) and / or neuroligin-4, X-linked (NLGN4X), comprising:

[0047] (i) contacting the target cell with a binding polypeptide of the present invention;

[0048] (ii) detecting binding of the binding polypeptide to the target cell; and

[0049] (iii) based on the detection in step (ii), identifying target cells expressing PTPRZ1 and / or NLGN4X.

[0050] The method for identifying a target cell expressing PTPRZ1 and / or NLGN4X is preferably an in vitro method. The method can be performed in particular on a cultured cell or sample from a subject, preferably on a sample from a subject, preferably the sample includes or is suspected of containing a cancer cell, preferably a tumor sample. Accordingly, the target cell can be, but is not necessarily, a host cell. Preferably, the term "target cell" as used herein relates 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 also be a cell of an experimental animal, a livestock animal or a companion animal, such as a mouse cell, a rat cell, a guinea pig cell, a cow cell, a sheep cell, a pig cell, a dog cell or a cat cell. Preferably, the target cell is known or suspected to be a cancer cell, preferably a cell contained in a tumor.

[0051] The binding of the binding polypeptide in step (ii) can be detected by any method deemed appropriate by the skilled person, for example by an immunological method. Preferably, in this case, the binding polypeptide comprises a detectable label, such as a dye or an enzyme that catalyzes a detectable chemical reaction. In view of the description herein, the skilled person will understand that the detection of the binding of the binding polypeptide in step (ii) indicates the target cell expressing PTPRZ1 and / or NLGN4X in step (iii). The skilled person will also understand that the detection in step (ii) can be qualitative, preferably semi-quantitative or quantitative.

[0052] The present invention also relates to a method for identifying a cancer that is sensitive to treatment with a binding polypeptide according to the present invention, a polynucleotide according to the present invention, a vector according to the present invention and / or a host cell according to the present invention, comprising:

[0053] (I) identifying target cells expressing PTPRZ1 and / or NLGN4X in a cancer sample; and

[0054] (II) identifying a cancer that is sensitive to treatment based on the identification in step (I).

[0055] The method for identifying a cancer that is sensitive to treatment is preferably an in vitro method, preferably performed on a cancer sample. As used herein, the term "sample" refers to a sample containing cells of a subject; the sample may be a body fluid sample, preferably a sample of blood, saliva, sputum, urine, or a sample containing isolated cells, or a sample from a tissue or organ. The isolated cells can be obtained from body fluids (e.g., lymph, blood, plasma, serum, liquid, etc.), or 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 the term "cancer sample" preferably relates to a sample known or suspected to contain target cells, preferably cancer cells; therefore, preferably, the sample is a sample of a primary tumor, infiltration, metastasis, lymph node or recurrence. However, the cancer sample can also be a single cell sample, for example in the case of a non-solid tumor. The sample can be obtained from a subject by conventional techniques well known to those skilled in the art, for example, venous or arterial puncture or open biopsy, including aspiration of tissue or cell material from a subject. For those areas that are not easily accessible by open biopsy, surgery, preferably minimally invasive surgery, can be performed.

[0056] As will be understood by a person skilled in the art based on the description provided herein, if target cells expressing PTPRZ1 and / or NLGN4X are identified in a cancer sample in step (I), a cancer that is sensitive to treatment is preferably identified in step (II). Preferably, if the proportion of target cells expressing PTPRZ1 and / or NLGN4X in the cancer sample 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%, then it is identified as a cancer that is sensitive to treatment. And preferably, compared to a control, such as a cell known not to express PTPRZ1 and / or NLGN4X, if the binding of the binding polypeptide increases, preferably significantly increases, the cells in the cancer sample are identified as expressing PTPRZ1 and / or NLGN4X.

[0057] The present invention also relates to the use of the binding polypeptide of the present invention, the polynucleotide of the present invention, the vector of the present invention and / or the host cell of the present invention in the preparation of a composition for diagnosing, treating and or preventing a disease, preferably cancer.

[0058] The present invention further relates to the use of the binding polypeptide of the present invention, the polynucleotide of the present invention, the vector of the present invention and / or the host cell of the present invention for detecting target cells expressing neuroligin-4X linked (NLGN4X) and / or protein tyrosine phosphatase receptor type Z1 (PTPRZ1), preferably in vitro.

[0059] Furthermore, the present invention also relates to a kit comprising a binding polypeptide of the present invention, a polynucleotide of the present invention, a vector of the present invention and / or a host cell of the present invention, which is contained in a housing and / or further comprises an administration means.

[0060] The term "kit" as used herein refers to a collection of the above-mentioned compounds, means or reagents, which may or may not be packaged together. The components of the kit may consist of separate vials (i.e., a kit as a separate part) or be provided in a single vial, for example as the above-specified composition. The housing of the kit preferably allows the transfer (translocation) of the compounds of the kit, in particular the common transfer; therefore, the housing may be a removable container containing all the specified components in particular. In addition, it should be understood that the kit of the present invention can be used to implement the above-mentioned method. It is preferably envisaged that all the ingredients are provided in a ready-to-use manner for implementing the above-mentioned method. In addition, the kit preferably includes instructions for carrying out the method. The instructions may be provided by a user manual in paper or electronic form. For example, the manual may include instructions for explaining the results obtained when the above-mentioned method is implemented using the kit. Preferably, the kit contains other compounds, such as reaction buffer, hybridization solution, lysis buffer, etc. Preferably, the kit is suitable for the method of the present invention, and more preferably is suitable for containing all the reagents required for carrying out the one or more methods.

[0061] The application tool may include a delivery unit for applying the compound or composition and a storage unit for storing the compound or composition until administration. However, it is also considered that the application tool may appear as a separate device in this embodiment and is preferably packaged together in the kit. Preferred application tools are those methods that can be applied without the specific knowledge of professional technicians. In a preferred embodiment, the application tool is a syringe containing the compound or composition, more preferably a syringe with a needle. In another preferred embodiment, the application tool is an intravenous infusion (IV) device containing the compound or composition. In another preferred embodiment, the application tool is an inhaler containing the compound of the present invention, wherein, more preferably, the compound is formulated for administration as an aerosol.

[0062] 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.

[0063] As used herein, the term "device" includes any and all distinct devices comprising the components. Preferably, the device is an application tool as described above. Preferably, the device is suitable for performing the method as described herein, in particular the diagnostic method and / or the identification method. Thus, the device may also be a diagnostic device. Thus, the device preferably comprises (i) an analysis unit comprising means for determining the binding of a binding polypeptide to a target cell, and, operably connected thereto, (ii) an evaluation unit comprising tangibly embedded executable instructions for performing the method as described herein. Typical means for determining the binding of a binding polypeptide to a target cell and means for performing said determination are known to those skilled in the art. As will be appreciated by those skilled in the art, means for determining the binding of a binding polypeptide to a target cell include means capable of determining the amount of binding of a binding polypeptide to a target cell, such as an ELISA reader, and means for determining the effect of binding of a binding polypeptide to a target cell, such as an optical unit for detecting the signal of a reporter gene assay. Evaluation means refers to any means capable of providing said analysis; preferably, the evaluation means is a data processing means, such as a microprocessor, a handheld device, such as a mobile phone, or a computer. How the means are operatively connected will depend on the type of means included in the device. In one embodiment, the means are comprised by a single device. The device may thus comprise (i) an analysis unit for determining the binding of a binding polypeptide to a target cell and (ii) a computer unit for processing the data obtained by the evaluation. Preferably, the instructions and interpretations are contained in an executable program code contained in the device, so that, as a result of the determination, the subject is classified as suitable for treatment. Typical devices are those that can be used without the specific knowledge of a professional technician, such as a test strip or an electronic device, which only requires the loading of a sample. The result can be given as an output of raw data, which requires the interpretation of a technician. However, preferably, the output of the device is processed, i.e. evaluated raw data, which does not require the interpretation of a technician. Further typical devices include an analysis unit / device (e.g., a biosensor, an array, a solid support coupled to a ligand that specifically recognizes the binding, a plasma surface resonance device, an NMR spectrometer, a mass spectrometer, etc.) or an evaluation unit / device according to the method of the present invention. Preferably, the device further comprises a memory unit, preferably a database, which comprises at least one reference value for the binding of a binding polypeptide to a target cell.

[0064] In view of the above, the following embodiments are particularly contemplated:

[0065] Embodiment 1: A binding polypeptide comprising a first variable T cell receptor (TCR) domain and a second variable TCR domain.

[0066] Embodiment 2: The binding polypeptide of embodiment 1, (i) wherein the complementarity determining region 3 (CDR3) of the first variable TCR domain comprises the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO: 1, or a sequence having at least 80% identity thereto; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO: 2, or a sequence having at least 80% identity thereto; or (ii) wherein the CDR3 of the first variable TCR domain comprises the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO: 3, or a sequence having at least 80% identity thereto; and / or the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO: 4, or a sequence having at least 80% identity thereto.

[0067] Embodiment 3: The binding polypeptide of embodiment 1 or 2, wherein the first variable TCR domain is a TCR alpha variable domain.

[0068] Embodiment 4: The binding polypeptide of any one of Embodiments 1 to 3, wherein the second variable TCR domain is a TCR beta variable domain.

[0069] Embodiment 5: The binding polypeptide of any one of embodiments 1 to 4, wherein the first variable TCR domain comprises 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, 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 wherein the second variable TCR domain comprises 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, 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.

[0070] Embodiment 6: The binding polypeptide of any one of embodiments 1 to 5, wherein the first variable TCR domain comprises 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, 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 wherein the second variable TCR domain comprises 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, 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.

[0071] Embodiment 7: The binding polypeptide of any one of embodiments 1 to 6, wherein the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:5, preferably consists of the amino acid sequence of SEQ ID NO:5 and / or wherein any one of the second variable TCR domains comprises the amino acid sequence of SEQ ID NO:6, preferably consists of the amino acid sequence of SEQ ID NO:6.

[0072] Embodiment 8: The binding polypeptide of any one of embodiments 1 to 7, wherein the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:7, preferably consists of the amino acid sequence of SEQ ID NO:7 and / or wherein the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:8, preferably consists of the amino acid sequence of SEQ ID NO:8.

[0073] Embodiment 9: The binding polypeptide of any one of embodiments 1 to 8, wherein the amino acid sequence of SEQ ID NO:5 is encoded by the nucleic acid sequence of SEQ ID NO:9.

[0074] Embodiment 10: The binding polypeptide of any one of embodiments 1 to 9, wherein the amino acid sequence of SEQ ID NO:6 is encoded by the nucleic acid sequence of SEQ ID NO:10.

[0075] Embodiment 11: The binding polypeptide of any one of Embodiments 1 to 10, wherein the amino acid sequence of SEQ ID NO:7 is encoded by the nucleic acid sequence of SEQ ID NO:11.

[0076] Embodiment 12: The binding polypeptide of any one of embodiments 1 to 11, wherein the amino acid sequence of SEQ ID NO:8 is encoded by the nucleic acid sequence of SEQ ID NO:12.

[0077] Embodiment 13: The binding polypeptide of 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.

[0078] Embodiment 14: The binding polypeptide of any one of Embodiments 1 to 13, wherein the first variable TCR domain is covalently linked to a first constant TCR domain, and / or wherein the second variable TCR domain is covalently linked to a second constant TCR domain.

[0079] Embodiment 15: The binding polypeptide of any one of Embodiments 1 to 14, wherein the binding polypeptide recognizes its cognate peptide when the peptide is presented on an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A*02 molecule.

[0080] Embodiment 16: The binding polypeptide of any one of embodiments 1 to 15, wherein the binding polypeptide recognizes a peptide having an amino acid sequence of NLDTLMTYV (SEQ ID NO: 13), preferably, when the peptide is presented on an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A*02 molecule, the binding polypeptide recognizes a peptide having an amino acid sequence of NLDTLMTYV (SEQ ID NO: 13).

[0081] Embodiment 17: The binding polypeptide of any one of embodiments 1 to 16, wherein the binding polypeptide recognizes a peptide having the amino acid sequence MIWEHNVEV (SEQ ID NO: 14), preferably, when the peptide is presented on an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A*02 molecule, the binding polypeptide recognizes a peptide having the amino acid sequence MIWEHNVEV (SEQ ID NO: 14).

[0082] Embodiment 18: The binding polypeptide of 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.

[0083] Embodiment 19: The binding polypeptide of any one of Embodiments 1 to 18, wherein the binding polypeptide is a TCR.

[0084] Embodiment 20: The binding polypeptide of any one of Embodiments 1 to 18, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to a signaling domain.

[0085] 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).

[0086] Embodiment 22: The binding polypeptide of any one of Embodiments 1 to 17, wherein the first variable TCR domain and the second variable TCR domain are covalently linked to the Fc domain of an immunoglobulin.

[0087] Embodiment 23: The binding polypeptide of any one of Embodiments 1 to 17 and 22, wherein the binding polypeptide is a soluble TCR, preferably a soluble TCR tetramer.

[0088] Embodiment 24: The binding polypeptide of any one of Embodiments 1 to 23, wherein the binding polypeptide further comprises a label and / or an effector compound.

[0089] Embodiment 25: A polynucleotide encoding at least one of the first variable TCR domain and the second variable TCR domain of the binding polypeptide according to any one of Embodiments 1 to 24.

[0090] Embodiment 26: The polynucleotide of Embodiment 25, wherein the polynucleotide is an expression construct.

[0091] Embodiment 27: The polynucleotide of Embodiment 25 or 26, wherein the polynucleotide is contained in a vector, preferably an expression vector.

[0092] Embodiment 28: A vector comprising the polynucleotide according to any one of Embodiments 25 to 27.

[0093] Embodiment 29: A host cell comprising the binding polypeptide according to any one of embodiments 1 to 24, the polynucleotide according to any one of embodiments 25 to 27 and / or the vector according to embodiment 28.

[0094] Embodiment 30: The host cell of Embodiment 29, wherein the host cell is a T cell, preferably a CD8+ T cell.

[0095] 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.

[0096] Embodiment 32: The binding polypeptide according to any one of embodiments 1 to 24, the polynucleotide according to any one of embodiments 25 to 27, the vector according to embodiment 28 and / or the host cell according to embodiment 29 or 30, for use in treating and / or preventing cancer.

[0097] Embodiment 33: A method of treating and / or preventing cancer in a subject, the method comprising

[0098] (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

[0099] (b) thereby treating cancer in said subject.

[0100] Embodiment 34: A method for identifying a target cell expressing protein tyrosine phosphatase receptor type Z1 (PTPRZ1) and / or neuroligin-4X linked (NLGN4X), comprising

[0101] (i) contacting the target cell with a binding polypeptide according to any one of embodiments 2 to 24, preferably embodiments 22 to 24;

[0102] (ii) detecting binding of the binding polypeptide to the target cell; and

[0103] (iii) based on the detection in step (ii), identifying target cells expressing PTPRZ1 and / or NLGN4X.

[0104] Embodiment 35: A method of identifying a cancer that is sensitive 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

[0105] (i) identifying target cells expressing PTPRZ1 and / or NLGN4X in a cancer sample; and

[0106] (ii) identifying a cancer that is susceptible to treatment based on the identification in step (I).

[0107] Embodiment 36: Use of the binding polypeptide according to any one of embodiments 1 to 24, the polynucleotide according to any one of embodiments 25 to 27, the vector according to embodiment 28 and / or the host cell according to embodiment 29 or 30 in the preparation of a composition for diagnosing, treating, and / or preventing a disease, preferably cancer.

[0108] Embodiment 37: Use of the binding polypeptide according to any one of embodiments 1 to 24, the polynucleotide according to any one of embodiments 25 to 27, the vector according to embodiment 28 and / or the host cell according to embodiment 29 or 30 for detecting target cells expressing neuroligin-4X linked (NLGN4X) and / or protein tyrosine phosphatase receptor type Z1 (PTPRZ1).

[0109] Embodiment 38: The use of Embodiment 37, wherein the use is an in vitro use.

[0110] 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, which is contained in a shell and / or further comprises an application tool.

[0111] 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.

[0112] Embodiment 41: The subject matter of any one of Embodiments 32 to 40, wherein the cancer is brain cancer, preferably glioma, more preferably glioblastoma.

[0113] Embodiment 42: The subject matter of any one of Embodiments 32 to 41, wherein said cancer is identified as being sensitive to treatment according to the method of Embodiment 35.

[0114] All references cited in the present specification are hereby incorporated by reference in their entire disclosure contents as well as the disclosure contents specifically mentioned in the present specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0115] Figure 1 : Binding to PTPRZ1 1814-1822 (A) PTPRZ1 activation-based sorting after peptide vaccination in HLA-A2 positive patients 1814-1822 Single-cell VDJ sequencing of reactive T cells; (B) using PTPRZ1 containing the mouse TCR β chain 1814-1822-TCR transfection of TCR-deficient Jurkat T cells for flow cytometry-based TCR expression analysis and comparison with TCRβ fluorescence minus one (FMO); (C) using T2 cells as antigen-presenting cells and PTPRZ1-loaded 1814-1822 Peptide or containing PTPRZ1 1814-1822 HLA-A2-positive U87 glioma cells expressing tandem minigenes for PTPRZ1 1814-1822 -TCR transgenic Jurkat T cells were used for activation assay based on nuclear factor of activated T cells (NFAT) reporter gene; (D) PTPRZ1 1814-1822 TCR transgenic Jurkat T cells carry PTPRZ1 1814-1822 Pentamer flow cytometry; (E) PTPRZ1 expression in U87 and P3 glioma target cells and T cells from healthy donors 1814-1822 Killing assay (n=3). Experimental conditions are as shown. Flu is a negative control peptide and TCR.

[0116] Figure 2 : Combined with NLGN4X 131-139 (A) Polymer-sorted NLGN4X after peptide vaccination in HLA-A2 positive patients 131-139 Single-cell VDJ sequencing of reactive T cells; (B) NLGN4X 131-139 Intracellular flow cytometry of reactive TCR transgenic healthy donor-derived 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) NLGN4X 131 -139 Primary human T cells transduced with reactive TCR SFG vectors were co-cultured with peptide-loaded U87 glioma cells and U87 cells overexpressing the full-length NLGN4X protein (MOG, myelin oligodendrocyte glycoprotein, negative control peptide); (D) NLGN4X 131-139 Primary human T cells transduced with the reactive TCR pLEX307 vector were co-cultured with peptide-loaded U87 glioma cells 7 days after transduction (MOG, myelin oligodendrocyte glycoprotein, negative control peptide); (E) NLGN4X 131-139 Vital FRassay of TCR-transgenic primary human T cells transduced with reactive TCR SFG vectors targeting expression of NLGN4X at an E:T ratio of 2:1 131-139 Tandem minigene (TMG) U87.

[0117] Figure 3 : NLGN4X-TCR-T has comparable recognition and lysis effects on target cells in vitro as control TCRs used clinically. (A) VDJ analysis of single-cell TCR sequencing of T cells from multimer-sorted patients after vaccination. Frequencies: ft1–90.82%; ft2–1.78%; ft3–0.96%; ft4–0.96%. (B) Exemplary flow cytometric analysis of transfection efficiency of Jurkat T cells transfected with TCRs. (C) Jurkat76 T cells transfected with NLGN4X ft1-4 TCRs were co-cultured with peptide-loaded BOLETHAPCs. Mean values ​​with SEM of three technical replicates. (D) Jurkat76 T cells transfected with NLGN4X TCRs were co-cultured with peptide-loaded HLA-A * 02 + NFAT reporter gene assay of PBMC co-culture. RLU = relative luminescence unit. Mean values ​​with SEM of three technical replicates. (E) Jurkat76 T cells transfected with NLGN4X TCR and peptide-loaded HLA-A*02 + NFAT reporter gene assay of U87 glioma cell co-culture. RLU = relative luminescence unit. Mean values ​​with SEM of three technical replicates.

[0118] Figure 4: Development of manufacturing process for T cell products. (A) Schematic diagram of TCRft1-pLEX307 EF1-α: EF1-α promoter-TCR β chain (including mouse TCR β constant region)-TCR α chain-woodchuck hepatitis virus post-transcriptional regulatory element-puromycin resistance. (B) Exemplary transduction efficiency of human T cells transduced with TCRft1-pLEX307 EF1α compared to Mock-transduced T cells by flow cytometry analysis of mTCRb. (C) Schematic diagram of SFG-IRES-GFP vector: long terminal repeat sequence-Moloney murine leukemia virus-TCR β chain (including mouse TCR β constant region)-TCR α chain-internal ribosome entry site-GFP-long terminal repeat sequence. (D) Exemplary transduction efficiency of human T cells transduced with TCRft1-SFG-IRES-GFP compared to Mock-transduced T cells by flow cytometry analysis of mTCRb. (E) GFP+ human T cells after transduction with TCRft1-SFG-IRES-GFP retroviral vector. Mean values ​​with SEM. (F) mTCRb expression in primary T cells 4 days after transduction with TCRft1-SFG-IRES-GFP retroviral vector. (G) Multicolor flow cytometry evaluation of different phenotypic markers in human T cells after transduction with TCRft1-SFG-IRES-GFP vector. N = 2 biological replicates.

[0119] Figure 5 : The recognition and lysis effects of NLGN4X-TCR-T on target cells in vitro are comparable to those of the control TCR used in clinical practice. (A) 131-139 TCR transduction and interaction with peptide-loaded HLA-A * 02 + Heat map of functional responses (IFN-γ, TNF-α, GrzB) of three different donors in co-culture of K562 leukemia cells. For statistical analysis, comparison Figure 3 BD, n=3 biological replicates. (B) HLA-A*02 loaded with peptide +Preparation of TNF-α by NLGN4X-TCR-T and Mart-1-TCR-T co-cultured with K562 leukemia cells. Target peptide: NLGN4X-TCR-T vs. MART-1 TCR T cells, p = 0.0863. Mean values ​​with SEM of n=3 biological replicates. Two-way ANOVA test. (C) Preparation of IFN-γ by NLGN4X-TCR-T and Mart-1-TCR-T co-cultured with peptide-loaded HLA-A*02+ K562 leukemia cells. Target peptide: NLGN4X-TCR-T vs. MART-1-TCR-T, p = 0.2926. (D) Preparation of IFN-γ by NLGN4X-TCR-T and Mart-1-TCR-T co-cultured with peptide-loaded HLA-A*02 + Granzyme B expression of NLGN4X-TCR-T vs. MART-1-TCR-T cultured with K562 leukemia cells. Target peptide: NLGN4X-TCR-T vs. MART-1-TCR-T, p = 0.3461. Mean values ​​with SEM of n = 3 biological replicates. Two-way ANOVA test. (E) Exemplary overview of the modified Vital-FR assay used in this study: Target cells endogenously expressing the target epitope or exogenously loaded with the corresponding peptide were stained with CellTrace TM FarRed labeled, non-target cells (irrelevant peptide or no target expression) were detected by CellTrace TM Violet labeled and cultured in the same well with target-specific TCR-transduced T cells. (F) After co-culture with NLGN4X-TCR-T or MART-1-TCR-T overnight, the viability of K562 cells loaded or not loaded with target peptide was evaluated by flow cytometry analysis. Mean values ​​with SEM of n=3 biological replicates. Two-way ANOVA test. The target peptide of NLGN4X-TCR-T is NLGN4X 131-139 Peptide, the target peptide of MART-1-TCR-T is 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 MOG (myelin oligodendrocyte glycoprotein) peptide (Sahillioglu AC et al. 2022).

[0120] Figure 6: 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 transduced with NLGN4X TCR and co-cultured with peptide-loaded HLA-A*02+ U87 glioma cells. (B) LDH release was measured by measuring optical density (OD) after 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 overnight. Mean values ​​with SEM of n=3 biological replicates. Two-way ANOVA test. (C) Specific cytotoxicity of NLGN4X-TCR-T co-cultured with peptide-loaded U87 glioma cells or unloaded cells was calculated by using FACS-based tumor cell counting. Mean values ​​with SEM of n=3 biological replicates. Unpaired t-test. (D) Heatmap of functional responses (CD69, 4-1BB, GrzB) of 3 different donors transduced with NLGN4X TCR or Flu TCR (here: negative control TCR) and co-cultured with U87 glioma endogenously expressing NLGN4X protein sequence including the relevant epitope (U87 NLGN4X) or a tandem minigene containing the antigenic sequence of NLGN4X (U87 TMG). (E) LDH release assay of NLGN4X-TCR-T versus Flu-TCR-T (TCR negative control) targeting U87 NLGN4X, U87 TMG or U87 (targeting negative control) glioma cells. Mean values ​​with SEM of n=3 biological replicates. Two-way ANOVA test. The target peptide of NLGN4X-TCR-T is NLGN4X 131-139 Peptide. The control peptide is Flu (influenza) that Flu-TCR-T can react with 58-66 MHC class I peptides (as published by Choo, J Virol, 2014 (VDJdb.cdr3.net).

[0121] Figure 7: Intracerebroventricular delivery of NLGN4X-TCR-T mediates transient tumor regression and improved survival in an intracranial tumor model (A) Schematic overview of the experiment: NSG MHC I / II KO mice were challenged with intracranial gliomas overexpressing the U87 NLGN4X antigen, and after confirmation of tumor growth, NLGN4X-TCR-T or Flu-TCR-T were injected on days 15 and 22 after tumor inoculation, respectively. (B) Preclinical survival of mice treated with NLGN4X-TCR-T or Flu-TCR-T targeting U87 cells overexpressing the NLGN4X antigen intracranially. NTC = no T cell control. n = 9 mice in the NLGN4X-TCR-T group, n = 8 mice in the Flu-TCR-T group, and n = 7 mice in the NTC group. Log-rank test. (C) Assessment of radiological response according to mRANO criteria: between days 11-67, complete response (CR) was defined as -100% change in tumor volume, partial response (PR) <-65%, stable disease (SD) between >-65% and +40%, and progressive disease (PD) >+40%. (D) MRI images of long-term surviving NLGN4X-TCR-T treated animals showed tumor regression at the initial tumor site until day 67 and tumor progression at day 98. (E) Individual growth curves of glioma cells expressing the U87 NLGN4X antigen in animals treated with NLGN4X-TCR-T (I) and Flu-TCR-T (II). Circled mice were analyzed by FACS, as shown in Figure 2. Figure 7 F. Growth on a log10 scale. Therefore, tumor volume at V = 0 μl is not shown in the figure. CR = complete response, PR = partial response, SD = stable disease, PD = progressive disease, D = death. For visualization of tumor growth, the detection limit of tumor volume was set to 0.1 μl. (F) Representative flow cytometric analysis of two animals with late tumor relapse (M1 = mouse 1, M2 = mouse 2) shows the presence of predominantly CD4+ T cells at the tumor site, with a predominantly CCR7-CD45RA effector memory phenotype, and impaired proliferation with high PD-1 expression. Gating 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 animals, or NTC animals at late time points compared to U87 TMG and U87 cells cultured in vitro. Relative to hGAPDH or hβ-actin expression. On a log10 scale. N = 3 biological replicates. (H) Exemplary immunofluorescence staining of HLA-A expression: late time points are shown for one NLGN4X-TCR-T treated animal and one untreated animal.

[0122] Figure 8: NLGN4X-TCR-T exhibits an effector phenotype in the tumor microenvironment after intraventricular delivery. (A) Experimental overview: Gliomas overexpressing the U87 NLGN4X antigen were injected intracranially, and NLGN4X-TCR-T or Flu-TCR-T were injected into the contralateral ventricle. After 6 days, T cells were analyzed by flow cytometry and in vitro activation was evaluated. (B) Exemplary flow cytometry plots showing intratumoral CD3+ T cell, CD4-CD8 distribution, and mTCRb and GFP expression. (C) Normalized counts (for tumor volume) of CD3+CD8+ T cells in the TME. n=8 (NLGN4X-TCR-T), n=7 (Flu-TCR-T). Unpaired t-test. (D) Exemplary FACS plots showing expression of CD45RA and CCR7 as well as Ki67 and PD-1 on intratumoral CD3+CD8+ T cells (TCM = T central memory cells, TN = naïve T cells, TEM = T effector memory cells, TEM-CD45RA+ = TEM re-expressing CD45RA). (E) Heat map 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 test.

[0123] Fig. 9 : In vitro activation and cytotoxicity studies of human T cells engineered with a PTPRZ1-specific TCR. Activation of TCR-T cells. T cells were co-cultured with U87 cells expressing the terminal epitope of the loaded peptide and with a P3 cell line endogenously expressing PTPRZ1. Activation was assessed by flow cytometry gating on CD8+ TCR+ cells.

[0124] Fig.10 : In vivo efficacy of PTPRZ1-specific TCR-engineered human T cells. NSG MHC KO mice were inoculated subcutaneously with U87 cell lines expressing the epitope. Mice received 2 doses of adoptive cell transfer of transduced TCR-T cells. Tumor growth was measured by caliper.

[0125] The following examples are merely illustrative of the present invention and are not to be construed as limiting the scope of the present invention.

[0126] Example 1 Materials and Methods

[0127] 1.1 Single-cell TCR-seq

[0128] Single cell capture and downstream library construction of FACS sorted cells were 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. The constructed scVDJ library was sequenced on the HiSeq2500 fast platform (Illumina).

[0129] 1.2 Modification of cell lines and gene expression

[0130] U87 glioma cells were cultured in DMEM with FBS, penicillin, and streptomycin. U87 NLGN4X was generated by transfection of the NLGN4X full-length protein coding sequence in pMXs-IRES-PuroR using FuGene HD transfection reagent (Promega). U87 cells were seeded at a density of 100,000 cells per well in six-well plates and allowed to rest for 24 h before transfection. Cells were transfected with 2 μg of plasmid DNA, rested for 48 h, and then positively selected in the presence 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 the U87 cell line as above but selected by 2 μg / ml puromycin.

[0131] 1.3 Cloning of human TCR constructs

[0132] The variable chain of the TCR was synthesized by Eurofins and cloned into the S / MAR vector (pSMARTer V8) containing mouse constant α and β chains using NEB Golden Gate assembly mix (New England Biolabs). This construct was used to electroporate TCR-deficient Jurkat T cells. Interventional and control TCRs were cloned into SFG vectors generated using In-Fusion cloning by Takara Bio Europe.

[0133] 1.4 Electroporation of Jurkat T cells

[0134] 2 Mio cells of Jurkat CD8+ TCR-deficient T cells were used per electroporation. 5 μg of TCR encoding vector was used separately, and NFAT reporter gene encoding vector was additionally delivered to Jurkats. (Settings: 1325 V, 3 pulses, 10 ms, Neon transfection system). After 24 h of incubation, TCR expression was determined by flow cytometric analysis of TCRβ-positive cells compared to untransfected T cells.

[0135] 1.5 Isolation of human T cells

[0136] Whole blood samples from healthy donors were obtained from IKTZ in Heidelberg, and PBMCs were isolated by density gradient centrifugation using Lympho-Paque solution (Genaxxon). Subsequently, the obtained PBMCs were washed with PBS supplemented with 1 mM EDTA and finally frozen for subsequent downstream applications. Human T cells were isolated by using MagniSort TM Human T cells were isolated from PBMCs of healthy donors using Human T cell Enrichment Kit (Invitrogen) or Pan T cell Isolation Kit, Human (Miltenyi).

[0137] 1.6 Lentiviral transduction of primary human T cells

[0138] HEK 293 T cells were co-transfected with TCR encoding lentiviral constructs (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 activated directly after isolation from fresh peripheral blood mononuclear cells in Xvivo medium supplemented with human interleukin 7 (0,2 IU / ml), human interleukin 15 (290 IU / ml) and 10 yl / million T cells human T Cell TransAct™ beads (Miltenyi) for 24 hours. After transduction, lentiviral supernatants were spin-loaded onto Retronectin-coated 6-well plates. Subsequently, up to 2 Mio. of activated T cells / well were transduced by spinoculation on virus-loaded plates and incubated overnight. TCR expression was assessed after 48 hours.

[0139] 1.7 Retroviral transduction of primary human T cells

[0140] Briefly, T cells were activated directly after isolation from fresh PBMCs and activated for 48 h in CTL medium (45% RPMI, 45% Click's 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.

[0141] HEK 293T cells were seeded at a density of 3 Mio cells per 10 cm dish and co-transfected with TCR-encoding SFG vectors and RD-114 and PeqPam as helper plasmids using Fugene HD transfection reagent. Viral supernatants were harvested 48 hours later, filtered, and spun onto 24-well plates previously coated with Retronectin. Subsequently, viral supernatants were removed and 0.5 Mio T cells were added to 1 ml CTL medium and incubated for 4 days at 37°C, 5% CO2. TCR transduction efficiency was assessed by flow cytometric analysis of GFP and TCRβ expression in CD3 T cells.

[0142] 1.8 NFAT reporter gene activation assay

[0143] Jurkat TCR-deficient T cells were equipped with TCR and NFAT reporter genes, respectively, by electroporation as described previously. U87 glioma cells or T2 cells were pulsed with a final peptide concentration of 10 µg / ml in 96-well plates at a density of 3 Mio cells / ml for 1 h or overnight. After confirmation of TCR expression, Jurkat T cells were co-cultured with peptide-loaded antigen-presenting cells at a density of 3 Mio cells / ml overnight. Subsequently, Nano-Glo luciferase assay reagent (Promega) was diluted according to the manufacturer's instructions and added to the co-culture. Luminescence was recorded on a PHERAstar FS plate reader and analyzed using MARS data analysis software (BMG Labtech). Activation was evaluated by NFAT reporter assay or flow cytometric assessment of cluster of differentiation (CD) 69 expression in Jurkat cell co-cultures.

[0144] 1.9 Human T cell activation and cytotoxicity assays

[0145] T cells were isolated and activated as described above, and the corresponding TCRs were 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 U87 glioma cells overexpressing NLGN4X as target cells and incubated overnight. Subsequently, cytotoxicity was determined using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, see below), and T cells were extracellularly and intracellularly stained for flow cytometric analysis of TNF-α, IFN-γ, and granzyme B expression.

[0146] Vital-FR assay

[0147] T cell receptor transduced human T cells were used on day 4 after transduction and co-cultured overnight with different target cell lines previously labeled with CellTrace reagents at a dilution of 1:50,000 according to the manufacturer's instructions at a 2:1 effector to target cell ratio. After overnight co-culture, the plates were centrifuged and resuspended in PBS. T cells were removed, 30 μl of trypsin was added to each well, and tumor cells were resuspended after 10 minutes and extracellular FACS staining was performed using eFluor780-conjugated fixable viability dye (1:1000 eBioscience) and PE-conjugated anti-human CD3 (1:1000, BioLegend) for negative selection of tumor cells.

[0148] Vital-FR cytotoxicity assay and flow cytometry-based cytotoxicity assay

[0149] TCR-transduced human T cells were used on day 4 after transduction and co-cultured overnight with different target cell lines at different effector to target cell ratios (E:T) that had been previously expressed by CellTrace at 0.2 µM according to the manufacturer’s instructions. TMFar Red or Violet reagent labeling. If peptide-pulsed target cells were used as antigen presenting cells (APCs), APCs were loaded at the indicated peptide dilutions for 1 hour at 37°C, 5% CO2 before the assay. After overnight co-culture, the plates were centrifuged and resuspended in PBS. T cells were removed, 30µl of trypsin was added to each well, and tumor cells were resuspended 10 minutes later and flow cytometry was performed using eFluor780-conjugated fixable viability dye and PE-conjugated anti-human CD3 for negative selection of tumor cells. 123 counting beads were added to 50µl at a 1:5 dilution. Cytotoxicity was calculated using normalized tumor cell counts. Cytotoxicity (in %) was calculated as the ratio of viable tumor cells after T cell co-culture to tumor cells alone or unloaded tumor cell controls.

[0150] LDH cytotoxicity assay

[0151] After 24 h, the co-cultures were centrifuged and 50 µl of the supernatant was used and incubated with CytoTox 96 Reagent (Promega) for 30 min at room temperature in the dark. After 30 min, stop solution was added and the absorbance at 490 nm was recorded. For subsequent analysis, the minimum LDH release from T cells and target cells and the background signal of the medium were determined.

[0152] 1.10 Flow cytometry

[0153] Intracellular staining Cells were incubated with 5µg / ml Brefeldin A (Sigma-Aldrich) for 5 hours at 37°C, 5% CO2 to allow intracellular enrichment of 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 eBioscience), respectively, and then stained with relevant antibodies for 45 minutes at 4°C. Human T cells were stained in 50µl or 100µl, with an antibody dilution of 1:50.

[0154] 1.11 Mice

[0155] NOD.Cg-Prkdc scid H2-K1 tm1Bpe H2-Ab 1em1Mvw H2-D1 tm1Bpe Iq tm1Wjl / SzJ (NSGMHCI / II KO) mice were kindly provided by L.D. Shultz and maintained at the DKFZ animal facility. All animal procedures were performed in accordance with the institutional guidelines for laboratory animal research and approved by the government agency (Karlsruhe Regional Administration, Germany, file number: G-37 / 18). For experimental groups, mice aged 6 to 28 weeks were matched by age and sex.

[0156] 1.12 Intracranial tumor experiments and intraventricular T cell injection

[0157] For orthotopic tumor cell injection, 1 × 10 5 U87 tandem-minigene (TMG) glioma cells were cultured at 50 × 10 6 T cells were transduced and expanded as described above. After tumor growth was confirmed using magnetic resonance imaging (MRI), up to 5 × 10 6 T cells were injected into the left ventricle of tumor-bearing mice, 0.5 mm lateral to the anterior bregma and 2.2 mm deep, in a total volume of 4 μl PBS. For survival experiments, T cells were injected on days 15 and 22, and once on day 48 to evaluate the intratumoral phenotype. Tumor growth was monitored by repeated MRI, and mice were examined daily for tumor-related symptoms and sacrificed when humane or experimental stop criteria were reached (Karlsruhe Regional Administration, file number: G-37 / 18). The obtained tissues were either directly processed for flow cytometry or frozen fixed and evaluated by immunofluorescence.

[0158] 1.13 Statistical analysis

[0159] All results were analyzed by Prism version 9.4.0. Statistical tests are shown in the respective figure legends. Multiple testing was corrected for ANOVA using Sidak multiple comparison correction. Results were considered significant if the p value was less than 0.05.

[0160] Example 2: Results

[0161] 2.1 PTPRZ1-specific TCR results

[0162] PTPRZ1(1814-1822)-specific TCRs were identified from patients who received a peptide vaccine containing the PTPRZ1(1814-1822) peptide by single-cell sequencing. Single-cell TCR sequencing after enrichment for activated T cells revealed an oligoclonal T cell population ( Figure 1A). The subsequently identified PTPRZ1(1814-1822) TCR containing the mouse TCRβ constant chain was overexpressed in Jurkat T cells, as indicated by cell surface expression of mouse TCRB ( Figure 1 B). HLA-A*02:01 positive and PTPRZ1(1814-1822) peptide loaded T2 as well as glioma cells U87, specifically activated PTPRZ1(1814-1822) TCR transgenic Jurkat T cells in an activation assay based on the nuclear factor of activated T cells (NFAT) reporter gene ( Figure 1 C). In addition, expression of a tandem minigene (TMG) containing PTPRZ1(1814-1822) in U87 cells resulted in strong activation of PTPRZ1(1814-1822) TCR-transgenic Jurkat T cells. In addition, these PTPRZ1(1814-1822) TCR-transgenic Jurkat T cells could be identified by PTPRZ1(1814-1822)-pentamer flow cytometry ( Figure 1 D). Most importantly, healthy donor-derived T cells stably overexpressing the PTPRZ1(1814-1822) TCR robustly killed PTPRZ1(1814-1822)-loaded U87, as well as P3, an HLA-A*02:01-positive patient-derived primary glioblastoma cell line with endogenous PTPRZ1 expression ( Figure 1 E).

[0163] 2.2 NLGN4X-specific TCR results

[0164] NLGN4X(131-139)-specific TCRs were identified from patients who received a peptide vaccine containing the NLGN4X(131-139) peptide by single-cell sequencing. Single-cell TCR sequencing after multimer-based T cell enrichment revealed a monoclonal T cell population ( Figure 2 A). When evaluating target recognition by intracellular flow cytometry, HLA-A2-positive K562 cell line loaded with NLGN4X(131-139) peptide was used to specifically activate NLGN4X(131-139) TCR transgenic primary healthy donor T cells, resulting in the production of multifunctional cytokines ( Figure 2 B). Importantly, loading of U87 glioma cells with NLGN4X(131-139) peptide or expressing full-length NLGN4X resulted in potent multifunctional cytokine production by NLGN4X(131-139) TCR transgenic primary healthy donor T cells ( Figure 2C, D). Most importantly, T cells from healthy donors that stably overexpressed the NLGN4X(131-139) TCR could strongly kill U87 glioma cells expressing a tandem minigene containing NLGN4X(131-139) ( Figure 2 E).

[0165] 2.3 Identification of NLGN4X from vaccinated glioblastoma patients by single-cell TCR-seq 131-139 Reactive TCR

[0166] About NLGN4X 131-139 -HLA-A * 02 + Tetramer-enriched CD45 + CD3 + Single-cell VDJ sequencing (scVDJ-seq) of T cells derived from cells administered with NLGN4X 131-139 After the peptide vaccine, there was an expansion of NLGN4X 131-139 The tetramer-enriched T cell repertoire was highly clonal, with one monoclonal type accounting for 90.82% of the TCR repertoire ( Figure 3 A). Next, free-genotype nanoscaffold matrix attachment region (nano-S / MAR) DNA vectors with the corresponding variable α and β chains of the first four TCR clones (frequotype 1-4 = ft1-4) were generated and transfected with TCR-deficient Jurkat T cells (Jurkat76). TCR surface expression was confirmed by flow cytometric analysis of the mouse TCR β chain (mTCRb), allowing specific detection of the transgenic TCR and excluding mispairing with the endogenous TCR ( Figure 3 B). Jurkat76 transfected with TCR were incubated with BOLETH HLA-A cells loaded with peptides. * 02 + Presenting cells were co-cultured and CD69 expression was evaluated by flow cytometry. 131-139 When the epitope was expressed, the T cell activation marker CD69 was only upregulated in Jurkat76 cells expressing TCRft1 ( Figure 3 C). Therefore, TCRft1 was subsequently used for downstream reactivity assessment. As a mode of action for antigen-dependent T cell stimulation, nuclear translocation of nuclear factor of activated T cells (NFAT) represents a characteristic of TCR signaling. Therefore, Jurkat76 expressing TCRft1 was co-transfected with a luciferase NFAT reporter gene and then incubated with peptide-loaded HLA-A * 02 + Peripheral blood mononuclear cells (PBMCs) and HHLA-A cells that do not endogenously express NLGN4X protein* 02 + U87 glioma cells ( Figure 3 D, E) Co-culture. Using PBMC as antigen presenting cells, NLGN4X 131-139 The induced luminescence signal intensity was similar to that of TCR stimulated by anti-CD3 / CD28 beads ( Figure 3 D).

[0167] 2.4 NLGN4X 131-139 Generation and phenotypic characterization of reactive TCR-transduced human T cells

[0168] Finally, to deliver NLGN4X-reactive TCR, the retroviral SFG-IRES-GFP vector ( Figure 4 C) and used with RD-114 and PeqPam (a different packaging plasmid system), the retroviral SFG-IRES-GFP vector is currently being studied in a clinical trial evaluating anti-CD19 CAR-T cells in patients with relapsed or refractory CD19+ lymphoid disease. Importantly, the use of the retroviral TCRft1-SFG-IRES-GFP vector ( Figure 4 C) The transduction process of primary human T cells resulted in up to 93.2% GFP + CD3 + T cells and resulted in a transduction efficiency as high as 90.4% ( Figure 4 DF). Therefore, 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 re-infusion. To evaluate the phenotype of primary human T cells expressing TCRft1-SFG-IRES-GFP during in vitro expansion, longitudinal flow cytometric analysis of TCR-transduced T cells was performed using phenotypic markers of T cell differentiation. T cells expressing TCRft1-SFG-IRES-GFP acquired a naive T cell-like state with CD45RA + CD45RO - CCR7 + T cell phenotype ( Figure 4 G), indicating that transduction with TCRft1-SFG-IRES-GFP does not lead to T cell differentiation into terminal effector cells. In addition, no relevant increase in the expression of exhaustion markers PD-1 and TIM-3 was observed. Overall, flow cytometric analysis showed that transduced TCRft1-SFG-IRES-GFP primary human T cells displayed a naive T cell phenotype, which has recently been described by Vitanza NA et al. (2021) as conferring superior anti-tumor capacity.

[0169] 2.5 NLGN4X-TCR-T cells lyse glioma cells in vitro

[0170] Then, the aim was to target non-adherent HLA-A*02 with exogenously loaded peptides. + Flow cytometry analysis of NLGN4X expression in K562 leukemia cells after co-culture 131-139 - In vitro function of primary human T cells expressing specific TCRs. As the most relevant effector proteins, the production of granzyme-B (GrzB), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) was evaluated. Independently of healthy T cell donors, expression of NLGN4X was found 131-139 -specific TCR T cells strongly express the highly cytotoxic cytokines TNF-α and GrzB ( Figure 5 AD). Strikingly, when NLGN4X is expressed 131-139 When primary human T cells expressing HER2-specific TCRs were benchmarked against a well-established high-affinity melanoma antigen recognized by a T cell-1 (MART-1)-specific TCR that has been used in Phase I and II clinical trials, similar expression levels of GrzB, IFN-γ, and TNF-α were observed in the co-culture system ( Figure 5 BD). To confirm the expression of NLGN4X 131-139 -specific TCRs strongly expressed cytotoxic proteins leading to target cell killing. A modified version of the Vital-FR assay was used to detect specific lysis of peptide-loaded K562 target cells ( Figure 5 E). Importantly, NLGN4X 131-139 -specific and MART1-specific T cells (MART-1-TCR-T) showed similar cytotoxic activity against peptide-loaded K562 target cells ( Figure 3 F).

[0171] Next, we aimed to evaluate the effect of NLGN4X-TCR-T on HLA-A * 02 + Cytotoxic capacity of adherent glioma cell line U87 loaded with NLGN4X 131-139 -peptide induced specific upregulation of GrzB, CD69, and 4-1BB (CD137) in NLGN4X-TCR-T cells ( Figure 6 A). Consistent with these findings, NLGN4X 131-139 -Specific T cells were able to specifically lyse U87 cells loaded with target peptides in lactate dehydrogenase (LDH)-based and flow cytometry-based killing assays ( Figure 6 B, C). In co-culture assays using synthetic MHC class I-restricted peptides for reactivity testing, MHC molecules are often saturated and external MHC loading may occur.131-139 Previously identified in some glioblastoma patients by HLA-ligandome analysis, the aim was to demonstrate that NLGN4X 131-139 Endogenous expression of NLGN4X in tumor model systems. 131-139 - Antigens can be expressed by using tandem minigenes (TMG) with different MHC class I epitopes, including NLGN4X (U87TMG), or retroviral vectors containing full-length NLGN4X (U87 NLGN4X). Similar to the results of studies using K562 cells as target cells, specific upregulation of GrzB, CD69, and 4-1BB was found when NLGN4X-TCR-T was co-cultured with U87 NLGN4X or U87 TMG target cells ( Figure 6 D). In addition, U87 TMG and U87 NLGN4X were specifically cleaved by NLGN4X-TCR-T ( Figure 6 E). In summary, it was demonstrated that primary human T cells expressing TCRft1-SFG-IRES-GFP specifically recognized and lysed cells expressing NLGN4X in vitro 131-139 epitopes of tumor cells.

[0172] 2.6 NLGN4X-TCR-T promotes tumor regression and increases HLA-A*02 + Survival rate of glioma-bearing mice

[0173] NLGN4X has been proven 131-139 - Human T cells modified with specific TCR can specifically recognize NLGN4X 131-139 - epitope and cleavage of tumor cells of the U87 human glioma cell line expressing NLGN4X-, followed by the aim to evaluate its in vivo therapeutic potential. Therefore, NOD scidγ (NSG) MHC class I and MHC class II knockout (NSG MHCI / II KO) mice, which do not develop graft-versus-host disease after T cell transfer, were challenged with intracranial U87 TMG experimental gliomas. Mice received NLGN4X- ​​by intraventricular transfer 131-139 - Human T cells modified with specific TCR or negative control (Flu) TCR modified human T cells (Flu-TCR-T), or no T cell treatment (NTC = no T cell control) ( Figure 7 A). On days 15 and 22, 5 × 10 cells were transduced to express 86.6-87.4% of mTCRb on the surface. 6 TCR modified primary human HLA-A*02 + T cells were injected twice into the lateral ventricle of the non-tumor hemisphere ( Figure 7A). NLGN4X-TCR-T treatment prolonged the survival of glioma-bearing animals compared with Flu-TCR-T or NTC mice ( Figure 7 B). By using longitudinal MRI, the aim was to investigate whether NLGN4X-TCR-T treatment would result in an objective radiological response, so tumor volumes from day 11 to day 67 were evaluated according to modified RANO criteria. On day 67, the best response time point, 11.1% stable disease, 22.2% partial response (PR), and 22.2% complete response (CR) were observed in NLGN4X-TCR-T treated mice, compared to 0.0% in both Flu-TCR-T and NTC mice, resulting in an objective response rate (ORR: CR + PR) of 44.4% ( Figure 7 C). In addition to NLGN4X 131-139 In addition to evaluating the radiological response to specific TCR-modified T cell therapy, longitudinal MRI can also locally evaluate tumor growth ( Figure 5 Interestingly, two mice with late recurrent U87 TMG tumors ( Figure 7 E) showed radiological response on previous MRI (1 PR, 1 CR) ( Figure 7 E). Therefore, flow cytometric analysis of tumor-infiltrating leukocytes in recurrent advanced tumors was performed ( Figure 7 F). In these tumors, 78 days after the second administration of NLGN4X-TCR-T, CD4+ T cells with low GFP expression were found to be predominant in the experimental tumors. These CD4 + CD45RA - CCR7 - , and had low expression of the proliferation marker Ki67 and high expression of PD-1 ( Figure 7 F).

[0174] Notably, recurrent U87 TMG tumors maintained NLGN4X expression as confirmed by qPCR. 131-139 -antigen expression, in addition, MHC class I expression can still be detected by immunofluorescence staining ( Figure 7 G, H). Collectively, these findings suggest that late recurrence is due to intratumoral cytotoxic NLGN4X 131-139 - Loss of specific CD8+ T cells ( Figure 7 FH).

[0175] 2.7 Phenotypic adaptation of NLGN4X-TCR-T in the tumor microenvironment

[0176] At late relapse, exhausted CD4+ T cells with low TCR transgene expression were found. Therefore, this observation prompted the evaluation of the intratumoral phenotype of NLGN4X-TCR-T at early time points after intraventricular delivery in U87 TMG glioma-bearing animals ( Figure 8 A). Six days after intraventricular transfer, T cells were present in the contralateral experimental glioma and NLGN4X was confirmed on the CD8+ T cell subset. 131-139 -Specific TCR positive ( Figure 8 B). Quantitatively, U87 TMG gliomas treated with NLGN4X-TCR-T and Flu-TCR-T in immunocompromised mice were not differentially infiltrated by CD3+ T cells ( Figure 8 C). CD8+ T cells mainly showed T effector memory (TEM) phenotype, with low expression of exhaustion markers PD-1 and TIM-3, high expression of CD45RA and Ki67 ( Figure 8 D, E). Specific upregulation of various activation and effector cell markers in NLGN4X-TCR-T compared with Flu-TCR-T ( Figure 8 F), showing antigen recognition and specific intratumoral T cell activation. However, the early phenotype of intratumoral NLGN4X-TCR-T cells ( Figure 8 ) and the phenotype found in late relapse ( Figure 7 E) support the hypothesis that loss of cytotoxic CD8+ NLGN4X-TCR-T contributes to late-stage tumor recurrence. In summary, the study demonstrates that targeting NLGN4X using patient-retrieved off-the-shelf 131-139 Human T cells engineered with TCRs specific for antigens were able to lyse tumor cells in vitro and mediate transient tumor control of experimental gliomas.

[0177] References

[0178] Hilf et al., Nature 565:240-245 (2019);

[0179] Keskin et al., Nature 565:234-239 (2019);

[0180] Platten et al., Nature 592:463-468 (2021);

[0181] Ellingson BM et al., Neurotherapeutics, 14(2):307-320 (2017);

[0182] Rohaan MW et al., N engl J Med., 387(23):2113-2125 (2022);

[0183] Morgan RA et al., J Immunother., 36(2):133-51 (2013);

[0184] Chheda ZS et al., J Exp Med., 215(1):141-157 (2018);

[0185] Immisch L et al., J Immunother Cancer, Oct 2022;10(10);

[0186] Kilian M et al., Clin Cancer Res. 28(2):378-389 (2022);

[0187] Bolinger MF et al., Proc NAl Acad Sci USA, 105(17):6421-6 (2008);

[0188] Choe JH et al., Sci Transl Med, 28 2021;13(591) (2021);

[0189] Sahillioglu AC et al., Curr Opin Immunol., 74:190-198 (2022);

[0190] Choo J et al., J Virol., 88(18):10613-23 (2014);

[0191] 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) the complementarity determining region 3 (CDR3) of the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:1, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:1, or a sequence having at least 80% identity thereto; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:2, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:2, or a sequence having at least 80% identity thereto; or wherein (ii) the CDR3 of the first variable TCR domain comprises the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:3, or a sequence having at least 80% identity thereto; and / or wherein the CDR3 of the second variable TCR domain comprises the amino acid sequence of SEQ ID NO:4, or a sequence having at least 80% identity thereto, preferably consists of the amino acid sequence of SEQ ID NO:

5. The amino acid sequence of NO:4 or a sequence having at least 80% identity thereto.

2. The binding polypeptide according to claim 1, wherein (i) the first variable TCR domain comprises 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, 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 wherein the second variable TCR domain comprises 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, 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 wherein (ii) the first variable TCR domain comprises 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, 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 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, 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. NO:8 has an amino acid sequence with at least 70% identity.

3. The binding polypeptide according to claim 1 or 2, wherein the binding polypeptide recognizes a peptide having the amino acid sequence MIWEHNVEV (SEQ ID NO: 14), preferably, when the peptide is presented on an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A*02 molecule, the binding polypeptide recognizes a peptide having the amino acid sequence MIWEHNVEV (SEQ ID NO: 14) or wherein the binding polypeptide recognizes a peptide having the amino acid sequence NLDTLMTYV (SEQ ID NO: 13), preferably, when the peptide is presented on an MHC class I molecule, more preferably an HLA-A molecule, more preferably an HLA-A*02 molecule, the binding polypeptide recognizes a peptide having the amino acid sequence NLDTLMTYV (SEQ ID NO: 13).

4. The 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 wherein the binding polypeptide is a TCR.

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 signaling domain, preferably wherein the binding polypeptide is a chimeric antigen receptor (CAR).

6. The 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 the Fc domain of an immunoglobulin, or wherein the polypeptide is a soluble TCR, preferably a soluble TCR tetramer.

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

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

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

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

11. A method for identifying a cancer that is sensitive 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 (I) identifying target cells expressing PTPRZ1 and / or NLGN4X in cancer samples; and (II) identifying a cancer that is sensitive to treatment based on the identification in step (I).

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

13. A kit comprising a binding polypeptide according to any one of claims 1 to 6, a polypeptide according to claim 7 and / or a host cell according to claim 8, which is contained in a shell and / or further comprises an administration tool.

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 of any one of claims 9 and 11 to 14, wherein the cancer is brain cancer, preferably glioma, more preferably glioblastoma.

Images