Terminal deoxynucleotidyl transferase (TdT) binding protein

Abstract

The present invention provides a binding protein capable of specifically binding to human leukocyte antigen (HLA) complex type A2, presenting a peptide having the amino acid sequence ALYDKTKRI (peptide T1) as set forth in SEQ ID NO: 1 or the amino acid sequence ALYDKTKRIFL (peptide T3) as set forth in SEQ ID NO: 15, derived from human terminal deoxynucleotidyl transferase (TdT). The binding protein contains an antigen-binding unit containing an alpha chain variable domain and a beta chain variable domain, each of which contains three complementarity determining regions (CDRs) derived from a TCR that recognizes the peptide. The binding proteins and cells expressing them can be used in cancer therapy.

Description

[Technical field]

[0001] The present disclosure relates to binding proteins based on sequences derived from the T cell receptor (TCR). The binding proteins are directed to human leukocyte antigen A2 (HLA-A2 or HLA-A * 02) in the context of major histocompatibility complex (MHC) class I. The binding proteins disclosed herein and cells expressing them can be used in cancer therapy. [Background technology]

[0002] Cancer therapy based on autologous lymphocytes expressing chimeric antigen receptor (CAR) is well known. Various CARs that target tumor-associated antigens have been disclosed in the past decade. However, the main drawback of traditional CARs is their ability to target only cell surface proteins.

[0003] Acute lymphoblastic leukemia (ALL) is a blood cancer for which new therapies are urgently needed. In B-cell ALL (B-ALL), targeting the B-cell-specific antigen CD19 by CAR T-cell therapy often induces complete remission. However, approximately 40-50% of B ALL patients relapse after anti-CD19 CAR-T cell therapy (Non-Patent Document 1, Non-Patent Document 2), most often due to loss of CD19 from the cancer. (Non-Patent Document 3). Furthermore, because CD19 is not a tumor-specific antigen, normal and malignant B cells are killed by anti-CD19 CAR-T cell therapy alike. B-cell aplasia is relatively well tolerated, but requires lifelong immunoglobulin supplementation, and the long-term effects of persistent CD19-specific CAR-T cells are unknown. Thus, a treatment that maintains normal B-lymphocyte production without the toxicity associated with allogeneic hematopoietic stem cell transplantation is desirable. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2017 / 0290897 [Patent Document 2] WO 91 / 18019 (Squibb & Sons, Dana Farber Cancer Institute) [Patent Document 3] WO 96 / 18105 (Harvard) [Patent Document 4] WO 2000 / 031239 (Yeda R&D) [Patent Document 5] International Publication No. 2019 / 166463 [Patent Document 6] International Publication No. 2004 / 054512 [Patent Document 7] International Publication No. 2016 / 116601 [Patent Document 8] International Publication No. 2018 / 129199 [Patent Document 9] International Publication No. 2014 / 037422 [Non-patent literature]

[0005] [Non-Patent Document 1] Maude et al., New England Journal of Medicine 378:439-448 (2018) [Non-Patent Document 2] SAGrupp et al., Blood 132, 895-895 (2018) [Non-Patent Document 3] Shah and Fry, Nature Reviews Clinical Oncology 16:372-385 (2019) [Non-Patent Document 4] Sellar et al., British Journal of Haematology 181:515-522 (2018) [Non-Patent Document 5] Komori et al., Science 261:1171-1175 (1993) [Non-Patent Document 6] Drexler et al., Acta Haematologia 75:12-17 (1986) [Non-Patent Document 7] Pellin et al., Nature Communications 10:2395 (2019) [Non-Patent Document 8] Li et al., Journal of Experimental Medicine 178:951-960 (1993) [Non-Patent Document 9] Boulter, JM et al. (Protein Eng. Des. Sel. 16(9):707-711 (2003) [Non-Patent Document 10] Cohen et al. (Cancer Research 67(8):3898-903 (2007) [Non-Patent Document 11] Walseng et al., PLoS ONE 10(4):e0119559 (2015) [Non-Patent Document 12] Walseng et al., Scientific Reports 7:Article 10713(2017) [Non-Patent Document 13] Busch and Sassone-Corsi, Trends in Genetics 6:36-40 (1990) [Non-Patent Document 14] Lewis et al., 2015, J Neurosci Methods 256:22-29 [Non-Patent Document 15] Wang et al., 2015 (Scientific Reports 5:Article No.16273) [Non-Patent Document 16] Wigler et al., Cell 11(1):223-232 (1977) [Non-Patent Document 17] Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33-37 (1992) [Non-Patent Document 18] Waelchli et al., PLoS ONE 6(11):e27930 (2011) [Non-Patent Document 19] Saeboee-Larssen et al., J. Immunol. Methods 259:191-203(2002), 191-203(2002) [Non-Patent Document 20] Rice, P. et al., Trends Genet. 16, (6) pp276-277 (2000) [Non-Patent Document 21] Sievers F. et al., Mol. Syst. Biol. 7:539 (2011) [Non-Patent Document 22] Edgar, RC, Nucleic Acids Res.32(5):1792-1797(2004) [Non-Patent Document 23] Becker et al. Cancer Immunology, Immunotherapy 65:477-484 (2016) [Non-Patent Document 24] Spanholtz et al., PLoS ONE 6(6):e20740 (2011) [Non-Patent Document 25] Tosato et al., Current Protocols in Immunology Chapter 7, Unit 7 22 (2007) [Non-Patent Document 26] Ali et al., Nature Protocols 14:1926-1943 (2019) [Non-Patent Document 27] Scheper et al., Nature Medicine 25:89-94 (2019) [Non-Patent Document 28] Stronen et al., Science 352:1337-1341 (2016) [Non-Patent Document 29] Linnemann et al., Nature Medicine 19:1534-1541 (2013) [Non-Patent Document 30] Brochet et al., Nucleic Acids Research 36:W503-508 (2008) [Non-Patent Document 31] Kumari et al., PNAS 111:403-408 (2014) [Non-Patent Document 32] Rapoport et al., Nature Medicine 21:914-921 (2015) [Non-Patent Document 33] Johnson et al., J. Immunol. 177, 6548-6559 (2006) Summary of the Invention [Problem to be solved by the invention]

[0006] Despite the numerous T-cell specific markers, no CAR therapy has been approved for T-cell ALL (T-ALL) that efficiently targets malignant cells across all T-ALL subtypes while sparing mature healthy T cells. Achieving this is crucial, as depletion of healthy T cells may be extremely toxic or incompatible with life. Furthermore, no tumor-specific targets have been identified in T-ALL, and no immunotherapy has proven efficacy in clinical trials. When chemotherapy fails (15-20%), T-ALL has a poor prognosis, with an overall survival rate of less than 25%.

[0007] The present inventors have identified the intracellular enzyme terminal deoxynucleotidyl transferase (TdT) as a target for immunotherapy in B-ALL and T-ALL. The function of TdT is to add N-nucleotides to the VDJ bond during recombination of T cell receptor (TCR) and B cell receptor genes (Non-Patent Document 5), and thus expression of TdT is restricted to the B and T cell lineages. TdT is overexpressed in 80-94% of ALL and lymphoblastic lymphomas derived from B and T cells (Non-Patent Document 6), but is not expressed in hematopoietic stem cells (Non-Patent Document 7), so myelopoiesis, including red blood cell and platelet production, should not be affected by targeting of TdT, and normal mature B and T cells should be spared, since TdT is downregulated during differentiation (Non-Patent Document 8). The present inventors have generated TCRs that recognize TdT-derived peptides that can be used for ALL therapy. Although TdT has been identified as a potential target for cancer immunotherapy, there are no known current therapies that target TdT.

[0008] The TCRs provided herein target cancerous T and B cells in ALL, but only a small subset of healthy T and B cells. Thus, the TCRs provided herein provide a first-line immunotherapy for the treatment of the majority of T-ALL subtypes and an improved immunotherapy for B-ALL that does not cause lifelong immune damage. [Means for solving the problem]

[0009] The present disclosure provides binding proteins capable of specifically binding to human leukocyte antigen complex class I presenting the TdT-peptide as set forth in SEQ ID NO:1 or SEQ ID NO:15. Such binding proteins (or cells expressing them) can be used to target cancer cells expressing TdT, in particular ALL. As demonstrated in the examples, the claimed binding proteins bind to HLA-A antigens presenting either the TdT-peptide as set forth in SEQ ID NO:1 or the TdT-peptide as set forth in SEQ ID NO:15 under physiological conditions. *It has excellent specificity for 02:01. In particular, it has excellent specificity for HLA-A * 02:01 is expressed by the majority of patients of European, Middle Eastern or North African ancestry with ALL, thus providing a novel treatment for T-ALL and B-ALL that may be particularly beneficial for chemotherapy-resistant patients.

[0010] Thus, herein, in a first aspect, there is provided a binding protein capable of specifically binding to human leukocyte antigen (HLA) complex type A2, presenting a peptide having the amino acid sequence ALYDKTKRIFL as set forth in SEQ ID NO: 15; the binding protein comprises an antigen-binding unit comprising an α chain variable domain and a β chain variable domain; The alpha chain variable domain comprises three complementarity determining regions (CDRs: CDR1, CDR2 and CDR3) containing the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively; The beta chain variable domain contains three complementarity determining regions (CDRs: CDR1, CDR2 and CDR3) containing the amino acid sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively; Binding Proteins But provided;

[0011] In the present specification, in a second aspect, a binding protein capable of specifically binding to human leukocyte antigen (HLA) complex type A2, which presents a peptide having the amino acid sequence ALYDKTKRI as set forth in SEQ ID NO: 1, is provided; the binding protein comprises an antigen-binding unit comprising an α chain variable domain and a β chain variable domain; The alpha chain variable domain comprises three complementarity determining regions (CDRs: CDR1, CDR2 and CDR3) containing the amino acid sequences set forth in SEQ ID NOs: 2, 3 and 4, respectively; The beta chain variable domain contains three complementarity determining regions (CDRs: CDR1, CDR2 and CDR3) containing the amino acid sequences set forth in SEQ ID NOs: 5, 6 and 7, respectively; Binding Proteins is provided.

[0012] In a third aspect, there is provided a recombinant nucleic acid molecule encoding a binding protein provided herein.

[0013] In a fourth aspect, there is provided a vector containing a recombinant nucleic acid molecule provided herein.

[0014] A fifth aspect is a kit containing a first recombinant nucleic acid molecule encoding a first chain of a binding protein and a second recombinant nucleic acid molecule encoding a second chain of the binding protein, (i) the first chain contains an alpha chain variable domain of the first aspect of the disclosure and the second chain contains a beta chain variable domain of the first aspect of the disclosure; or (ii) the first chain comprises an alpha chain variable domain of the second aspect of the disclosure and the second chain comprises a beta chain variable domain of the second aspect of the disclosure; kit is provided.

[0015] A sixth aspect provides an immune effector cell that comprises a recombinant nucleic acid molecule provided herein, a vector provided herein, or a pair of recombinant nucleic acid molecules comprised in a kit provided herein, and that expresses a binding protein provided herein in its cell membrane.

[0016] A seventh aspect provides a pharmaceutical composition containing the immune effector cells provided herein.

[0017] An eighth aspect provides an immune effector cell as provided herein, or a pharmaceutical composition as provided herein, for use in therapy.

[0018] A ninth aspect provides an immune effector cell as provided herein, or a pharmaceutical composition as provided herein, for use in treating a cancer that expresses terminal deoxynucleotidyl transferase (TdT).

[0019] Similarly, provided is a method of treating a terminal deoxynucleotidyl transferase (TdT)-expressing cancer in a subject, the method comprising administering to the subject an immune effector cell provided herein, or a pharmaceutical composition provided herein.

[0020] Also provided is the use of an immune effector cell provided herein in the manufacture of a medicament for the treatment of a cancer that expresses terminal deoxynucleotidyl transferase (TdT).

[0021] The cancer to be treated according to the disclosure herein may be, but is not limited to, acute lymphoblastic leukemia (ALL), such as B-cell acute lymphoblastic leukemia (B-ALL) or T-cell acute lymphoblastic leukemia (T-ALL).

[0022] In a tenth aspect, there is provided a method of generating a terminal deoxynucleotidyl transferase (TdT)-specific immune effector cell, the method comprising introducing into said immune effector cell a recombinant nucleic acid molecule provided herein, a vector provided herein, or a pair of recombinant nucleic acid molecules contained in a kit provided herein. [Brief description of the drawings]

[0023] [Figure 1]FIG. 1: Schematic diagram of various TCR constructs. FIG. 1A shows a full-length TCR containing an α-chain and a β-chain. Both chains contain a variable domain with three CDRs (lighter rectangles), an extracellular constant domain, a transmembrane domain and a short cytoplasmic domain. FIG. 1B shows a minimal binding unit containing an α-chain variable domain with three CDRs (lighter rectangles) and a β-chain variable domain with three CDRs (lighter rectangles). FIG. 1C shows a binding unit (herein referred to as TCR-scFv) containing an α-chain variable domain with three CDRs (lighter rectangles) and a β-chain variable domain with three CDRs (lighter rectangles), where the β-chain variable domain is connected to the α-chain variable domain via a peptide linker. FIG. 1D shows a receptor (herein referred to as chimeric TCR) containing a binding unit, an extracellular constant domain, a transmembrane domain and a cytoplasmic signaling domain. Figure IE shows a truncated TCR (herein referred to as a soluble TCR) containing an α-chain and a β-chain, which contains a binding unit, an extracellular constant domain and two cysteine ​​bridges. Figure IF shows a receptor (herein referred to as a TCR-CAR) containing the structure from Figure IE, where one of the chains further contains a transmembrane domain and a cytoplasmic signaling domain. [Diagram 2]Figure 2 - TdT-reactive TCR is restricted to HLA-A2. Figure 2A shows activation of T1 and T3 cells following co-incubation with peptide-pulsed T2 lymphoblastoid cells. EC50 = half the maximal effective concentration. Data are pooled from three independent experiments, where each circle represents the mean of three technical replicates from an individual experiment. Error bars indicate SD. Figure 2B shows CD137 upregulation on CD8+ T1 and T3 cells following co-culture with EBV-LCL derived from one HLA-A2pos and one HLA-A2neg donor. Cell lines were pulsed with the indicated concentrations of peptide-1 (T1) or peptide-3 (T3) or electroporated with mRNA encoding full-length TdT. Data points represent three technical replicates in one experiment representative of two performed. Figure 2C shows activation of T1 and T3 cells after co-culture with various cell lines with the indicated HLA-A2 and TdT expression, loaded or not with TdT peptide (2x10-7M). The suffix +A2 indicates cell lines transduced with HLA-A*02:01. Results are from one experiment representative of 2 or 3 performed with different T cell donors, data points represent technical replicates (2-3) and error bars indicate range. [Diagram 3] Figure 3: TdT-reactive TCRs do not show off-target reactivity. Figure 3A shows heat maps of IFN-γ concentrations in culture supernatants of T1 cells (top) and T3 cells (bottom) co-incubated with EBV-LCLs pulsed with peptides from the mimotope library (peptide concentration: 2 × 10-7 M). Row / column intersections indicate amino acids substituted at a given position, open circles indicate amino acids in the wild-type peptide. IFN-γ concentration range for positive reactions was 500-31254 pg / mL. Repeat once per condition. Figure 3B-C shows IFN-γ production by T1 (B) and T3 (C) cells after co-culture with target cells loaded with the indicated peptides. 9-mer and 11-mer peptides containing amino acids upstream or downstream of wild-type peptide-1 and -3 in the TdT protein sequence were included. Additionally, peptides of 8-12 amino acids in length containing part or all of peptide-1 and -3 were included. Repeat once per condition. [Figure 4] Figure 4: T1 and T3 cells are activated by TdTpos HLA-A2pos leukemic cell lines and effectively kill the leukemic cell lines. Figure 4A shows viable TdTpos HLA-A2pos NALM-6 and BV173 cells after 48 h of co-culture with T1 and T3 cells (1:1 E:T ratio) as a percentage of the corresponding number after mock-transduced T cell treatment, quantified by flow cytometry. Data points represent technical replicates in one experiment representative of three experiments performed. Figure 4B shows flow cytometry plots of BV173 cells co-cultured with mock, T1 and T3 cells for 48 h. The inset numbers indicate event counts within the viable tumor cell gate. [Figure 5-1] Figure 5: T1 and T3 cells efficiently kill leukemia cells in vitro and in vivo in BV173 and NALM-6 animal models. Figure 5A-B and D-E show bioluminescence imaging of leukemia-bearing mice 1 day before, 21 days (BV173, AB) or 14 days after (NALM-6, DE) treatment with 1G4, T1 or T3 (NY-ESO-1 reactive) transduced human T cells. Untreated mice were included as controls. [Figure 5-2] Figures 5C and 5F show survival analysis of leukemia-bearing mice untreated (n=10 (BV173) or n=11 (NALM-6)) or treated with 1G4 (n=7 (BV173) or n=9 (NALM-6)), T1 (n=8 (BV173)) or T3 (n=9 (BV173) or n=11 (NALM-6)) cells. Data shown in Figures 5B and C and Figures 5E and F are pooled from two independent experiments. In the box plots in Figures 5B and E, the interquartile range (25th to 75th percentile) is shown, with the central bar indicating the median and the whiskers indicating the range. Dots represent data from individual mice. P=not significant (ns), **P<0.01, ****P<0.0001, calculated by ordinary one-way ANOVA, corrected for multiple comparisons with Tukey's post-hoc test. Survival analyses (FIGS. 5C and F) were performed by two-tailed log-rank (Mantel-Cox) test, ****P<0.0001. [Figure 5-3] Figure 5G is a flow cytometry plot showing bone marrow tumor burden in T3-treated mice on day 60 (M1-M5) compared to 1G4-treated or untreated mice at the time of sacrifice (day 21). The threshold for leukemia positivity was set as GFP+ cells >0.01% of viable cells. [Figure 6] Figure 6: T1 and T3 cells deplete primary cancer cells while sparing mature B and T lymphocytes and non-lineage committed hematopoietic progenitors. Figure 6A shows representative t-distributed stochastic neighborhood embedding (t-SNE) plots of viable HLA-A2pos, TdTpos B-ALL tumor cells (CD19+CD10+ events, left panel) and T-ALL tumor cells (CD5+CD7+CD99+ and surface CD3-CD4- events, right panel), normal B cells (CD19+CD10-), normal T cells (CD3+ and CD8+ or CD4+) and CD34+lin- progenitors after 72 h of coculture with mock-, T1- or T3-transduced T cells (E:T ratio = 1:1) as quantified by flow cytometry. TCR-transduced cells were excluded from the analysis as CTV-positive events. Figure 6B shows diagnostic samples from 12 patients with HLA-A2pos,TdTposB-ALL or T-ALL assayed as described in 6A. Each dot represents the number of viable tumor cells, normal B cells or T cells after co-culture with T1 (green) or T3 (purple) cells as a percentage of the corresponding number in cultures treated with mock-transduced T cells. Data points represent three or four technical replicates and error bars indicate the range. Data shown are from one experiment representative of at least two experiments performed for each patient sample. [Figure 7]Figure 7: Patient-derived CD8+ T cells transduced with T1 or T3 efficiently kill autologous B-ALL cells. Figure 7A shows t-SNE plots of peripheral blood diagnostic samples from B-ALL patients after 72 h of co-culture with autologous T cells transduced with T1, T3 or mock. Inserted numbers indicate absolute event counts of the indicated cell populations after co-culture with mock, T1 and T3 cells. Figure 7B shows quantification of malignant cells, normal B, T and CD34+lin- progenitor cells after 72 h of flow cytometry-based cytotoxicity assay. Data points represent technical replicates (3-4) from one representative experiment out of two performed, error bars indicate range. [Figure 8-1] Figure 8: T3 cells efficiently eliminate primary B-ALL cells while sparing healthy hematopoiesis in vivo. In Figures 8A-8C, untreated n=5, DMF5-treated n=8, T3-treated n=8. The figures show pooled data from two experiments. [Figure 8-2] Figure 8A shows representative FACS plots of viable mononuclear cells (MNC) from bone marrow (BM) of T3-treated (top)- and DMF5-treated (bottom)-NSG mice engrafted with primary human B-ALL cells. Figure 8B shows the percentage of leukemic cells (hCD45+CD19+CD10+) in BM adjusted for human T cells at baseline and 11 days after T cell infusion. Figure 8C shows the number of leukemic cells present in BM. Figure 8D shows the number of MNC in BM. Data are pooled from two independent experiments and presented as mean ± SEM in terminal analysis 11 days after treatment for untreated, DMF5-treated and T3-treated mice. Populations were identified by flow cytometry according to the gating strategy shown in Figure 8A. Kruskal-Wallis ANOVA with Dunn's multiple comparison test was performed using GraphPad Prism software for statistical analysis (*P<0.05, **P<0.01, ***P<0.001). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The expression profile of TdT is described above. As detailed above, during normal hematopoiesis, both B and T cell lineages transiently express TdT, whereas mature B and T cells and CD34 + Hematopoietic stem cells lack the expression of TdT. This allows the targeting of cancerous TdT-expressing cells while sparing hematopoietic stem cells and mature lymphocytes. Thus, successful TdT-targeting therapy leaves patients with healthy B and T cell compartments and maintains adaptive immune responses. As detailed above, TdT is overexpressed in most ALL. Because TdT is localized intracellularly, it cannot be targeted by conventional CARs that utilize targeting units from antibodies. However, the TCRs and TCR-derived binding proteins provided herein can recognize TdT-derived peptides when presented by MHC class I molecules, and thus target TdT-expressing cells.

[0025] The binding proteins provided herein are derived from TCRs referred to herein as T1 TCR and T3 TCR. Both T1 TCR and T3 TCR are αβ TCRs, i.e., heterodimers containing an α chain and a β chain. The general structure of an αβ TCR is well known in the art and is depicted diagrammatically in FIG. 1A. The binding proteins can be provided in a variety of formats, including classical, or "native", full-length two-chain surface receptor formats (e.g., αβ formats), or formats with single-chain antigen-binding units, or as soluble molecules, etc.

[0026] As is well known, the variable domains of the α and β chains each contain three complementarity determining regions (CDRs), numbered (starting from the N-terminus) as CDR1, CDR2 and CDR3. It is the CDR that directly interacts with the target epitope / MHC complex and thus determines the specificity of the TCR, with CDR3 being the most important in determining TCR specificity. The CDRs are separated by and flanked by framework sequences that form a scaffold for the CDRs so that their spatial arrangement is appropriate for target binding.

[0027] The T3 TCR recognizes a peptide having the amino acid sequence ALYDKTKRIFL as set forth in SEQ ID NO: 15 when presented by class I MHC containing HLA-A2. SEQ ID NO: 15 corresponds to amino acids 475-485 of human TdT (UniProt entry P04053, SEQ ID NO: 27). The α chain of the T3 TCR contains a variable region in which CDR1 (which may be referred to as VαCDR1) has the amino acid sequence TSINN as set forth in SEQ ID NO: 16, VαCDR2 has the amino acid sequence IRSNERE as set forth in SEQ ID NO: 17, and VαCDR3 has the amino acid sequence CADTAGAYSGGGADGLTF as set forth in SEQ ID NO: 18. The β chain of the T3 TCR contains a variable region in which CDR1 (which may be referred to as VβCDR1) has the amino acid sequence MNHEY as set forth in SEQ ID NO: 19, VβCDR2 has the amino acid sequence SMNVEV as set forth in SEQ ID NO: 20, and VβCDR3 has the amino acid sequence CASSLSSSYNEQFF as set forth in SEQ ID NO: 21.

[0028] The T1 TCR recognizes a peptide having the amino acid sequence ALYDKTKRI as set forth in SEQ ID NO: 1 when presented by class I MHC containing HLA-A2. SEQ ID NO: 1 corresponds to amino acids 475-483 of human TdT. The α chain of the T1 TCR contains a variable region in which VαCDR1 has the amino acid sequence VSGLRG as set forth in SEQ ID NO: 2, VαCDR2 has the amino acid sequence LYSAGEE as set forth in SEQ ID NO: 3, and VαCDR3 has the amino acid sequence CAVQASSNSGYALNF as set forth in SEQ ID NO: 4. The β chain of the T1 TCR contains a variable region in which VβCDR1 has the amino acid sequence SQVTM as set forth in SEQ ID NO: 5, VβCDR2 has the amino acid sequence ANQGSEA as set forth in SEQ ID NO: 6, and VβCDR3 has the amino acid sequence CSVEPGYADTQYF as set forth in SEQ ID NO: 7.

[0029] Thus, a first aspect of the present disclosure provides a binding protein as described above that is based on the T3 TCR.

[0030] The alpha and beta chains of the T3 TCR have variable domains that contain the amino acid sequences set forth in SEQ ID NO: 22 and SEQ ID NO: 23, respectively. In a particular embodiment, the T3 TCR based binding protein contains an antigen binding unit that contains an alpha chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90% or 95% sequence identity thereto. In another embodiment, the T3 TCR based binding protein contains an antigen binding unit that contains a beta chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90% or 95% sequence identity thereto. In a particular embodiment, the T3 TCR based binding protein contains an antigen binding unit that contains an alpha chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and a beta chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0031] If the binding protein contains an α chain variable domain containing a variant of SEQ ID NO: 22 (i.e. an amino acid sequence having 90% or more but less than 100% sequence identity to SEQ ID NO: 22), the VαCDR sequences are nevertheless as defined above (i.e. VαCDR1, VαCDR2 and VαCDR3 contain or consist of the amino acid sequences set forth in SEQ ID NO: 16, 17 and 18, respectively). Any sequence modifications are therefore made within the framework regions of the variable domain. If the binding protein contains a β chain variable domain containing a variant of SEQ ID NO: 23 (i.e. an amino acid sequence having 90% or more but less than 100% sequence identity to SEQ ID NO: 23), the VβCDR sequences are nevertheless as defined above (i.e. VβCDR1, VβCDR2 and VβCDR3 contain or consist of the amino acid sequences set forth in SEQ ID NO: 19, 20 and 21, respectively). Any sequence modifications are therefore made within the framework regions of the variable domain.

[0032] A second aspect of the present disclosure provides a binding protein as described above that is based on the T1 TCR.

[0033] The α and β chains of the T1 TCR have variable domains that contain the amino acid sequences set forth in SEQ ID NO:8 and SEQ ID NO:9, respectively. In a particular embodiment, the T1 TCR based binding protein contains an antigen binding unit that contains an α chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least 90% or 95% sequence identity thereto. In another embodiment, the T1 TCR based binding protein contains an antigen binding unit that contains a β chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 90% or 95% sequence identity thereto. In a particular embodiment, the T1 TCR based binding protein contains an antigen binding unit that contains an α chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and a β chain variable domain that contains or consists of the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0034] If the binding protein contains an α chain variable domain containing a variant of SEQ ID NO:8 (i.e. an amino acid sequence having 90% or more but less than 100% sequence identity to SEQ ID NO:8), the VαCDR sequences are nevertheless as defined above (i.e. VαCDR1, VαCDR2 and VαCDR3 contain or consist of the amino acid sequences set forth in SEQ ID NO:2, 3 and 4, respectively). Any sequence modifications are therefore made within the framework regions of the variable domains. If the binding protein contains a β chain variable domain containing a variant of SEQ ID NO:9 (i.e. an amino acid sequence having 90% or more but less than 100% sequence identity to SEQ ID NO:9), the VβCDR sequences are nevertheless as defined above (i.e. VβCDR1, VβCDR2 and VβCDR3 contain or consist of the amino acid sequences set forth in SEQ ID NO:5, 6 and 7, respectively). Any sequence modifications are therefore made within the framework regions of the variable domains.

[0035] A "binding protein" as defined herein is a protein that binds to (or recognizes) a target molecule. The binding proteins provided herein can specifically bind to MHC-I molecules containing peptides derived from human TdT, as described above. "Specific binding" means that the binding proteins specifically bind to their specific molecular partners (the molecular partner of the binding protein derived from T3 TCR is an HLA-A2 molecule presenting the TdT peptide of SEQ ID NO: 15; the molecular partner of the binding protein derived from T1 TCR is an HLA-A2 molecule presenting the TdT peptide of SEQ ID NO: 1). Specific binding to a target can be distinguished from off-target or non-specific binding. For example, binding proteins bind to their molecular partners with higher affinity than they bind to other molecules (or at least most other molecules). Binding of a binding protein to a target can be measured by any suitable method known in the art, such as surface plasmon resonance (SPR).

[0036] In particular, the binding proteins bind to their molecular partners (as defined above) with higher affinity than they bind to MHC-peptide complexes containing peptides derived from proteins other than TdT. The binding proteins show minimal or no binding to non-cognate MHC-peptide complexes, particularly MHC-peptide complexes containing peptides derived from proteins other than TdT. The binding proteins provided herein can specifically bind to their molecular partners under physiological conditions, i.e., conditions found within the body of a host animal, particularly within the body of a human. In particular, the binding proteins can specifically bind to their molecular partners under conditions found within human tumors.

[0037] As detailed above, the T3 TCR specifically binds to an MHC-I complex containing HLA-A2 and the peptide of SEQ ID NO: 15, and the T1 TCR specifically binds to an MHC-I complex containing HLA-A2 and the peptide of SEQ ID NO: 1. Notably, although the peptides of SEQ ID NO: 1 and SEQ ID NO: 15 are very similar (as detailed above, both peptides differ only by a two amino acid extension at the C-terminus of SEQ ID NO: 15), as shown in the examples below, the T3 and T1 TCRs do not show cross-reactivity with their respective binding partners.

[0038] As detailed above, the binding proteins provided herein contain an antigen-binding unit comprising an alpha chain variable domain and a beta chain variable domain. An "alpha chain variable domain" as defined herein is a variable domain derived from, or a polypeptide based on, the alpha chain of a TCR, particularly a human TCR. Similarly, a "beta chain variable domain" as defined herein is a variable domain derived from, or a polypeptide based on, the beta chain of a TCR, particularly a human TCR. Polypeptides based on the variable domains of the alpha or beta chain of a TCR encompass, for example, variants of the variable domains that contain sequence modifications relative to the native variable domains.

[0039] An "antigen-binding unit" as referred to herein is simply the domain of a binding protein that is formed from the α and β chain variable domains and that binds to a target MHC-peptide complex.

[0040] Thus, the minimal binding protein of the present disclosure is an antigen-binding unit containing an α chain variable domain and a β chain variable domain as shown in FIG. 1B.

[0041] The α chain variable domain may be covalently linked to the β chain variable domain to form an antigen-binding unit, for example by a peptide linker, as shown in Figures 1C and 1D. Such antigen-binding units and proteins containing them are disclosed in US Patent No. 5,399,233, US Patent No. 5,399,233 and US Patent No. 5,399,233. Suitable peptide linkers may contain, for example, 1, 2, 3 or 4 amino acids, 5, 10 or 15 amino acids, or other intermediate numbers of amino acids conveniently ranging from 1 to 20. Peptide linkers may be formed from any generally convenient amino acid residues, such as glycine and / or serine. One example of a suitable linker is Gly4Ser. Multimers of such linkers, for example dimers, trimers, tetramers or pentamers, for example (Gly4Ser)2, (Gly4Ser)3, (Gly4Ser)4 or (Gly4Ser)5, may be used. The peptide linker may connect the C-terminus of the α chain variable domain to the N-terminus of the β chain variable domain, or the N-terminus of the α chain variable domain to the C-terminus of the β chain variable domain.

[0042] A binding protein composed of an α chain variable domain linked to a β chain variable domain by a polypeptide linker (as described above) is referred to herein as a TCR-scFv (similar to a standard scFv derived from the variable domains of an antibody). TCR-scFv is the smallest soluble TCR species and is shown diagrammatically in FIG. 1C. In one embodiment, the binding protein is a TCR-scFv. Uses of TCR-scFv are discussed further below.

[0043] In another embodiment, the binding protein provided herein contains a first chain containing an α chain variable domain and a second chain containing a β chain variable domain. That is, the α chain variable domain and the β chain variable domain may be located in separate polypeptide chains (that associate with each other to form the binding protein). Thus, the first and second chains correspond to the α and β chains of a classical full-length αβ TCR, but need not contain all domains naturally occurring in a full-length α or β chain. In one embodiment, the first and second chains may contain all domains of the α or β chain, respectively, i.e., the variable domain, the constant domain, the transmembrane domain, and the cytoplasmic domain (endodomain). In other embodiments, the first and second chains may contain variable and constant domains. In other words, the first chain may contain an α chain variable domain and an α chain constant domain. The second chain may contain a β chain variable domain and a β chain constant domain. In yet other embodiments, the chains may further contain a transmembrane domain, or a transmembrane domain and a cytoplasmic domain. Thus, the first and second chains may correspond to or represent full-length or truncated α and β chains, respectively (or may indeed be referred to as full-length or truncated α and β chains). By "full-length" it is meant that the chain contains all the domains of the α or β chain. In other words, the full-length chain contains the variable domain, the constant domain, the transmembrane domain and the cytoplasmic domain. The transmembrane domain or the cytoplasmic domain may be obtained or derived from an α or β chain, as appropriate, or may be obtained or derived from any other protein. Thus, the full-length α or β chain may be a natural or a synthetic or engineered protein. By "truncated" it is meant that the chain may lack all or part of any one or more of the constant domain, the transmembrane domain or the cytoplasmic domain. Thus, in a full-length or truncated α or β chain according to the present disclosure, the variable and constant domains may be TCR α or β chain domains, while the transmembrane and cytoplasmic domains are not limited to those obtained or derived from a TCR α or β chain.The term "first chain" as used broadly herein means a polypeptide chain that contains at least a variable domain obtained or derived from an α chain, and the term "second chain" as used broadly herein means a polypeptide chain that contains at least a variable domain obtained or derived from a β chain. Thus, the first chain can be considered an "α-based chain" and the second chain can be considered a "β-based chain."

[0044] Nevertheless, in one embodiment, the individual polypeptide chains may be covalently linked (e.g., by one or more disulfide bonds formed between the side chains of cysteine ​​residues). For example, the extracellular constant domains of native αβ TCRs are known to form interchain disulfide bridges that are believed to stabilize the heterodimer. The interchain disulfide bridges are shown in FIG. 1A as solid vertical bars connecting the constant domains (C).

[0045] Thus, as described above, the first chain (i.e., full length or truncated α chain) and / or the second chain (i.e., full length or truncated β chain) may contain an extracellular constant domain. Without being bound by theory, the interaction between the α chain constant domain and the β chain constant domain may stabilize the antigen-binding unit. In one embodiment, the first chain contains an extracellular α chain constant domain (located C-terminal to the variable domain, e.g., directly C-terminal such that the N-terminal amino acid of the constant domain is linked to the C-terminal amino acid of the variable domain by a peptide bond). In another embodiment, the second chain contains an extracellular β chain constant domain (located C-terminal to the variable domain, e.g., directly C-terminal to the variable domain). In one embodiment, both the first chain and the second chain contain an extracellular constant domain.

[0046] Any suitable extracellular constant domain can be used. The constant domain may be a human TCR constant domain or a TCR constant domain from another animal, for example the constant domain may be a mouse constant domain. Alternatively the constant domain may be a synthetic constant domain or a constant domain derived from a synthetic protein. When both the first and second chains contain a constant domain, both constant domains may be from the same species or different species. The extracellular constant domain sequences of the human α chain and β chain types 1 and 2 are set forth in SEQ ID NOs: 28-30, respectively. In one embodiment, the extracellular α chain constant domain comprises an amino acid sequence set forth in SEQ ID NO: 28 or an amino acid sequence having at least 90% or 95% sequence identity thereto, and / or the extracellular β chain constant domain comprises an amino acid sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 30 or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0047] The extracellular constant domain sequences of mouse α chain and β chain types 1 and 2 are respectively set forth in SEQ ID NOs: 31 to 33. In certain embodiments, the extracellular α chain constant domain contains the amino acid sequence set forth in SEQ ID NO: 31, or an amino acid sequence having at least 90% or 95% sequence identity thereto, and / or the extracellular β chain constant domain contains the amino acid sequence set forth in SEQ ID NO: 32 or SEQ ID NO: 33, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0048] It is also known that additional cysteine ​​bridges can be introduced by insertion or by substituting appropriate amino acid residues on each chain with cysteine ​​residues. Figures 1E and 1F show the binding protein with an additional cysteine ​​bridge (shown as a solid vertical bar connecting the constant domains (C)). The additional cysteine ​​residue should be introduced at a position such that the formation of a disulfide bond between the introduced residues results in the formation of a functional complex in which the variable regions are correctly positioned and facing each other, as disclosed, for example, by J. Am. Soc. 1999, 143:1311-1323.

[0049] Known suitable positions in the human TCR chain constant region for the introduction of a cysteine ​​residue are disclosed in Non-Patent Document 10 and Patent Document 4. In the human TCR α chain constant region of SEQ ID NO: 28, an additional cysteine ​​residue can be introduced by substituting a cysteine ​​residue for the threonine residue at position 49. The modified human α chain extracellular constant domain obtained by this Thr49Cys substitution has the amino acid sequence set forth in SEQ ID NO: 34. In the human TCR β chain type 1 constant region of SEQ ID NO: 29 or type 2 constant region of SEQ ID NO: 30, an additional cysteine ​​residue can be introduced by substituting a cysteine ​​residue for the serine residue at position 57. The modified human β chain extracellular constant domains obtained by these Ser57Cys substitutions have the amino acid sequences set forth in SEQ ID NOs: 35 and 36, respectively. Thus, in one embodiment, the first chain contains an extracellular α chain constant domain containing the amino acid sequence set forth in SEQ ID NO: 34, or an amino acid sequence having at least 90% or 95% sequence identity thereto, and the second chain contains an extracellular β chain constant domain containing the amino acid sequence set forth in SEQ ID NO: 35 or 36, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0050] As detailed above, the extracellular constant domains of SEQ ID NOs: 34-36 are modified to form two interchain disulfide bonds. Thus, when the binding proteins provided herein contain a variant of any one of these sequences (i.e., an amino acid sequence having 90% or more but less than 100% sequence identity to any one of SEQ ID NOs: 34-36) as the extracellular constant domain, the cysteine ​​residues involved in disulfide bond formation may be retained (i.e., not substituted or deleted). In the α-chain extracellular domain of SEQ ID NO: 34, the cysteine ​​residues involved in disulfide bond formation are Cys49 and Cys96. In the β-chain extracellular domains of SEQ ID NOs: 35 and 36, the cysteine ​​residues involved in disulfide bond formation are Cys57 and Cys131.

[0051] In the mouse TCR chain extracellular constant domain, the positions equivalent to the above positions in the human TCR constant domain are suitable for the introduction of an additional cysteine ​​residue. Thus, in the mouse TCR α chain constant region of SEQ ID NO: 31, an additional cysteine ​​residue can be introduced by substitution of the threonine residue at position 49 with a cysteine ​​residue. The modified mouse α chain extracellular constant domain obtained by this Thr49Cys substitution has the amino acid sequence set forth in SEQ ID NO: 10. In the mouse TCR β chain, in the type 1 constant region of SEQ ID NO: 32 or the type 2 constant region of SEQ ID NO: 33, an additional cysteine ​​residue can be introduced by substitution of the serine residue at position 57 with a cysteine ​​residue. The modified mouse β chain extracellular constant domain obtained by these Ser57Cys substitutions have the amino acid sequences set forth in SEQ ID NO: 37 and 11, respectively. Thus, in certain embodiments, the first chain contains an extracellular α chain constant domain containing the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 90% or 95% sequence identity thereto, and the second chain contains an extracellular β chain constant domain containing the amino acid sequence set forth in SEQ ID NO: 11 or 37, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0052] With respect to the above modified human sequences, in a variant of any one of SEQ ID NOs: 10, 11 and 37 (i.e., an amino acid sequence having 90% or more but less than 100% sequence identity to any one of SEQ ID NOs: 10, 11 or 37), the cysteine ​​residues involved in disulfide bond formation may be retained (i.e., not substituted or deleted). In the α-chain extracellular domain of SEQ ID NO: 10, the cysteine ​​residues involved in disulfide bond formation are Cys49 and Cys92. In the β-chain extracellular domains of SEQ ID NOs: 35 and 36, the cysteine ​​residues involved in disulfide bond formation are Cys57 and Cys127.

[0053] In the T1 and T3 TCRs, the α chain contains an extracellular α chain constant domain containing the amino acid sequence set forth in SEQ ID NO: 10, and the β chain contains an extracellular β chain constant domain containing the amino acid sequence set forth in SEQ ID NO: 11. Thus, in certain embodiments, the binding protein contains a first chain containing an extracellular α chain constant domain containing, or consisting of, the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence having at least 90% or 95% sequence identity thereto; and a second chain containing an extracellular β chain constant domain containing, or consisting of, the amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0054] The binding protein, containing a first chain and a second chain, each containing a variable domain and an extracellular domain, as provided herein, may be a soluble TCR (i.e., not embedded in a membrane), as shown in FIG. 1E and previously disclosed in J. Immunol. 1999, 103:1311-1323 (2002) and J. Immunol. 1999, 103:1311-1323 (2002). The term "soluble TCR" as used herein means a TCR molecule that contains separate first and second chains, but is not membrane-bound. In other words, a soluble TCR does not contain a TM domain or a cytoplasmic domain. A soluble TCR may in particular contain a first and second chain, each consisting only of an extracellular domain.

[0055] It is an essential aspect of the soluble TCR that the first and second chains are linked. If they were not linked, the chains would diffuse apart in solution and the TCR would function poorly, if at all. The chains can be linked covalently or non-covalently. One way in which the truncated α and β chains can be covalently linked is by one or more disulfide bonds between cysteine ​​residues, as described above. Another way in which the truncated α and β chains of the soluble TCR can be linked is by non-covalent interactions. In one embodiment, a leucine zipper is used to non-covalently link the chains. In this embodiment, both the truncated α and β chains contain a leucine zipper domain at the C-terminus of their extracellular constant domains. Leucine zippers and their sequences are well known in the art and are reviewed, for example, in J. Immunol. 1999, 143:1311-1320. A combination of covalent and non-covalent interactions can also be used to link the truncated α and β chains of the soluble TCR.

[0056] The use of soluble TCRs (and scFv-TCRs, which are also soluble but do not fall within the definition of "soluble TCR" as used herein) is further described below. Soluble TCRs and scFv-TCRs can be encoded with an affinity tag, which can be used in the purification of the soluble TCR or scFv-TCR after its synthesis. In the case of soluble TCRs, in one embodiment, only one chain (i.e., the truncated α or β chain) is produced with an affinity tag. The affinity tag can be located at either end, e.g., the C-terminus, of the truncated α or β chain. The affinity tag can be any suitable tag known to those skilled in the art, such as a FLAG-tag, His-tag, HA-tag, Strep-tag, S-tag, Myc-tag, glutathione S-transferase (GST), maltose binding protein (MBP), etc.

[0057] A linker and / or a protease cleavage site can be located between the binding protein sequence and the affinity tag. Inclusion of a protease cleavage site allows for removal of the affinity tag from the binding protein after purification. Suitable protease cleavage sites are well known to those skilled in the art and include thrombin, factor Xa, enterokinase, human rhinovirus (HRV) 3C and tobacco etch virus (TEV) cleavage sites.

[0058] The first chain and / or the second chain may further comprise a transmembrane domain. In the first or second chain, the transmembrane domain is located C-terminal to the extracellular constant domain.

[0059] Any suitable transmembrane domain can be used. In a particular embodiment, the transmembrane domain is a TCR chain transmembrane domain. That is, if the first chain contains a transmembrane domain, it may be a TCR α chain transmembrane domain; if the second chain contains a transmembrane domain, it may be a TCR β chain transmembrane domain. In one embodiment, the transmembrane domain is a human TCR chain transmembrane domain. In another embodiment, the transmembrane domain is a mouse TCR chain transmembrane domain. In one embodiment, if both the first and second chains contain a TCR chain transmembrane domain, both TCR transmembrane domains are from the same species.

[0060] The human TCR alpha chain transmembrane domain has the amino acid sequence set forth in SEQ ID NO: 38, and thus in one embodiment the first chain contains a transmembrane domain containing the amino acid sequence set forth in SEQ ID NO: 38, or an amino acid sequence having at least 90% or 95% sequence identity thereto. The mouse alpha chain transmembrane domain has the amino acid sequence set forth in SEQ ID NO: 44, and thus in another embodiment the first chain contains a transmembrane domain containing the amino acid sequence set forth in SEQ ID NO: 44, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0061] The transmembrane domains of the human and mouse TCR beta chain types 1 and 2 all have the amino acid sequence set forth in SEQ ID NO: 39. Thus, in certain embodiments, the second chain contains a transmembrane domain that contains the amino acid sequence set forth in SEQ ID NO: 39, or an amino acid sequence having at least 90% sequence identity thereto.

[0062] The T1 and T3 TCRs disclosed in the Examples both have an alpha chain with a transmembrane domain of SEQ ID NO: 44 and a beta chain with a transmembrane domain of SEQ ID NO: 39. Thus, in a particular embodiment, the first and second chains both contain a transmembrane domain, and the transmembrane domain of the first chain of the binding protein contains an amino acid sequence set forth in SEQ ID NO: 44 or an amino acid sequence having at least 90% or 95% sequence identity thereto, and the transmembrane domain of the second chain of the binding protein contains an amino acid sequence set forth in SEQ ID NO: 39 or an amino acid sequence having at least 90% sequence identity thereto.

[0063] Alternatively, and as discussed further below, the transmembrane domain may be based on or derived from the transmembrane domain of any other transmembrane protein. Typically, the transmembrane domain may be or be derived from CD8α, CD28, CD4, CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD134, CD137 or CD154, e.g., from the human version of any one of these proteins. In one embodiment, the transmembrane domain may be or be derived from CD8α, CD28, CD4 or CD3ζ, e.g., from human CD28, CD4 or CD3ζ.

[0064] In one embodiment, the transmembrane domain is the transmembrane domain of human CD28 having the amino acid sequence of SEQ ID NO: 40. That is, the transmembrane domain may contain the amino acid sequence set forth in SEQ ID NO: 40 or an amino acid sequence having at least 95% sequence identity thereto.

[0065] The first chain and / or the second chain may further contain a cytoplasmic domain. As is known in the art, in native TCRs, the α and β chains contain a short cytoplasmic domain located at the C-terminus. Any suitable cytoplasmic domain can be used. In one embodiment, the cytoplasmic domain is the cytoplasmic domain of a TCR chain. That is, if the first chain is an α chain containing a cytoplasmic domain, it may be the cytoplasmic domain of a TCR α chain, and if the second chain is a β chain containing a cytoplasmic domain, it may be the cytoplasmic domain of a TCR β chain.

[0066] In one embodiment, the cytoplasmic domain is a cytoplasmic domain of a human TCR chain. In another embodiment, the cytoplasmic domain is a cytoplasmic domain of a mouse TCR chain. When both the first and second chains contain a TCR chain cytoplasmic domain, the TCR cytoplasmic domains can both be derived from the same species.

[0067] The human and mouse TCR alpha chain cytoplasmic domains have the amino acid sequence set forth in SEQ ID NO: 41, and thus in one embodiment the first chain is an alpha chain containing a cytoplasmic domain containing the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% sequence identity thereto. The human TCR beta chain type 1 cytoplasmic domain has the amino acid sequence set forth in SEQ ID NO: 42, and the human TCR beta chain type 2 cytoplasmic domain has the amino acid sequence set forth in SEQ ID NO: 43. Thus in one embodiment the second chain is a beta chain containing a cytoplasmic domain containing the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0068] The mouse TCR beta chain type 1 cytoplasmic domain has the amino acid sequence set forth in SEQ ID NO: 46 and the mouse TCR beta chain type 2 cytoplasmic domain has the amino acid sequence set forth in SEQ ID NO: 47. Thus, in one embodiment the second chain is a beta chain that contains a cytoplasmic domain that contains the amino acid sequence set forth in SEQ ID NO: 46 or SEQ ID NO: 47, or an amino acid sequence having at least 90% sequence identity thereto.

[0069] The T1 and T3 TCRs both have an alpha chain having a cytoplasmic domain of SEQ ID NO: 41 and a beta chain having a cytoplasmic domain of SEQ ID NO: 47. In certain embodiments, the first and second chains both contain cytoplasmic domains (i.e., they are full-length alpha and beta chains), and the cytoplasmic domain of the alpha chain contains the amino acid sequence set forth in SEQ ID NO: 41 or an amino acid sequence having at least 80% sequence identity thereto, and the cytoplasmic domain of the beta chain contains the amino acid sequence set forth in SEQ ID NO: 47 or an amino acid sequence having at least 90% sequence identity thereto.

[0070] In another embodiment, the first chain and / or the second chain (generally either the first chain or the second chain) contains a cytoplasmic domain that contains an intracellular signaling domain. The term "intracellular signaling domain" as used herein refers to a domain of a binding protein chain that is involved in transducing the message of effective receptor binding to a target antigen-MHC complex inside an immune effector cell expressing that receptor, and inducing effector cell function, such as activation, cytokine production, proliferation and / or cytotoxic activity, including release of cytotoxic factors toward bound target cells, or other cellular responses elicited by binding of the receptor to its target.

[0071] Examples of intracellular signaling domains that can be used include those derived from CD3zeta, FcRy, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the intracellular signaling domain is derived from CD3zeta or FcRgamma, e.g., human CD3zeta or FcRgamma. In one embodiment, the intracellular signaling domain is a human CD3zeta domain containing the amino acid sequence set forth in SEQ ID NO:48 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:48.

[0072] Furthermore, to enable or augment full activation of immune effector cells expressing the receptor, the cytoplasmic domain may further contain a costimulatory domain (in addition to the intracellular signaling domain). Thus, the intracellular signaling domain may initiate antigen-dependent primary activation (i.e., may be a primary cytoplasmic signaling sequence), and the costimulatory domain may act antigen-independently to provide a secondary or costimulatory signal (i.e., may be a secondary cytoplasmic signaling sequence).

[0073] The term "costimulatory signal domain" or "costimulatory domain" refers to the portion of the cytoplasmic domain that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or an Fc receptor that upon binding to an antigen provides a second signal necessary for efficient activation and function of immune effector cells (e.g., T cells). Examples of costimulatory domains that can be used herein include the intracellular domains of CD27, CD28, 4-IBB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS (CD278), LFA-1, CD2, CD7, LIGHT, NKD2C, and B7-H2, including human versions thereof. Costimulatory domains can be used alone or in combination (i.e., one or more costimulatory domains can be included). The inclusion of one or more costimulatory signaling domains can enhance the potency and expansion of immune effector cells expressing the receptors provided herein. In one embodiment, the costimulatory domain may be or may include the intracellular domain of human CD28 having the amino acid sequence of SEQ ID NO:49 or an amino acid sequence having at least 95% sequence identity to SEQ ID NO:49.

[0074] The examples herein demonstrate the functionality of full-length T1 and T3 TCRs, containing the α and β chains (the term "full-length TCR" is interchangeable with the term "TCR" herein). HLA-A * T cells engineered to express these TCRs targeting peptides derived from the lymphoid-specific enzyme terminal deoxynucleotidyl transferase (TdT) in the context of 02:01 have been demonstrated to specifically eliminate primary acute lymphoblastic leukemia (ALL) cells of T and B cell origin both in vitro and in mouse models of disseminated ALL. In contrast, normal, mature T and B cell repertoires and non-lineage-committed hematopoietic progenitors lacking TdT expression are spared. Extensive mapping of TCR reactivity described in the Examples did not identify cross-reactivity with any naturally occurring peptides in the human proteome, demonstrating exceptionally high peptide specificity.

[0075] As described above and in the Examples, the T1 and T3 TCRs contain a human TCR variable domain and a mouse TCR extracellular constant domain, transmembrane domain and cytoplasmic domain. Thus, in certain embodiments, the binding proteins provided herein are TCRs having a structure as illustrated in Figure 1A. In this embodiment, the TCR thus comprises an α chain and a β chain, the α chain containing a TCR α chain variable domain, a TCR α chain extracellular constant domain, a TCR α chain transmembrane domain and a TCR α chain cytoplasmic domain, and the β chain containing a TCR β chain variable domain, a TCR β chain extracellular constant domain, a TCR β chain transmembrane domain and a TCR β chain cytoplasmic domain.

[0076] The various domains may have the sequences described above. In particular, the TCR may contain an α chain and a β chain, each of which contains a human variable domain and a murine extracellular constant domain, a transmembrane domain and a cytoplasmic domain.

[0077] In certain embodiments, the TCR comprises: (i) an alpha chain comprising, from N-terminus to C-terminus, a variable domain comprising an amino acid sequence set forth in SEQ ID NO:22 or an amino acid sequence having at least 90% or 95% sequence identity thereto; an extracellular constant domain comprising an amino acid sequence set forth in SEQ ID NO:10 or an amino acid sequence having at least 90% or 95% sequence identity thereto; a transmembrane domain comprising an amino acid sequence set forth in SEQ ID NO:44 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and a cytoplasmic domain comprising an amino acid sequence set forth in SEQ ID NO:41 or an amino acid sequence having at least 80% sequence identity thereto; (ii) a beta chain comprising, from N-terminus to C-terminus, a variable domain containing an amino acid sequence set forth in SEQ ID NO:23 or an amino acid sequence having at least 90% or 95% sequence identity thereto; an extracellular constant domain containing an amino acid sequence set forth in SEQ ID NO:11 or an amino acid sequence having at least 90% or 95% sequence identity thereto; a transmembrane domain containing an amino acid sequence set forth in SEQ ID NO:39 or an amino acid sequence having at least 90% sequence identity thereto; and a cytoplasmic domain containing an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence having at least 90% sequence identity thereto. It is a T3 TCR that contains

[0078] The full length mature T3 TCR alpha chain has the amino acid sequence set forth in SEQ ID NO: 24. Thus, in one embodiment, the binding protein contains an alpha chain containing the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90% or 95% sequence identity thereto. The full length mature T3 TCR beta chain has the amino acid sequence set forth in SEQ ID NO: 25. Thus, in one embodiment, the binding protein contains a beta chain containing the amino acid sequence set forth in SEQ ID NO: 25, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0079] In certain embodiments, the TCR provided herein comprises: (i) an alpha chain containing the amino acid sequence set forth in SEQ ID NO: 24, or an amino acid sequence having at least 90% or 95% sequence identity thereto; and (ii) a β-chain comprising the amino acid sequence set forth in SEQ ID NO: 25 or an amino acid sequence having at least 90% or 95% sequence identity thereto; It is a T3 TCR that contains

[0080] In another specific embodiment, the TCR is (i) an alpha chain comprising, from N-terminus to C-terminus, a variable domain comprising an alpha chain comprising an amino acid sequence set forth in SEQ ID NO:8 or an amino acid sequence having at least 90% or 95% sequence identity thereto; an extracellular constant domain comprising an amino acid sequence set forth in SEQ ID NO:10 or an amino acid sequence having at least 90% or 95% sequence identity thereto; a transmembrane domain comprising an amino acid sequence set forth in SEQ ID NO:44 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and an alpha chain comprising a cytoplasmic domain comprising an amino acid sequence set forth in SEQ ID NO:41 or an amino acid sequence having at least 80% sequence identity thereto; and (ii) a beta chain comprising, from N-terminus to C-terminus, a variable domain containing an amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 90% or 95% sequence identity thereto; an extracellular constant domain containing an amino acid sequence set forth in SEQ ID NO:11 or an amino acid sequence having at least 90% or 95% sequence identity thereto; a transmembrane domain containing an amino acid sequence set forth in SEQ ID NO:39 or an amino acid sequence having at least 90% sequence identity thereto; and a cytoplasmic domain containing an amino acid sequence set forth in SEQ ID NO:47 or an amino acid sequence having at least 90% sequence identity thereto. is a T1 TCR containing

[0081] The full length mature T1 TCR alpha chain has the amino acid sequence set forth in SEQ ID NO: 12. Thus, in one embodiment, the binding protein contains an alpha chain containing the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 90% or 95% sequence identity thereto. The full length mature T1 TCR beta chain has the amino acid sequence set forth in SEQ ID NO: 13. Thus, in one embodiment, the binding protein contains a beta chain containing the amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0082] In certain embodiments, the TCR provided herein comprises: (i) an alpha chain containing the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence having at least 90% or 95% sequence identity thereto; and (ii) a β-chain comprising the amino acid sequence set forth in SEQ ID NO: 13 or an amino acid sequence having at least 90% or 95% sequence identity thereto; It is a T1 TCR that contains

[0083] As noted above, the cytoplasmic domain of the binding proteins provided herein may contain an intracellular signaling domain (and optionally a costimulatory domain) rather than a TCR cytoplasmic domain. Such alternative cytoplasmic domains can be used in chimeric receptors.

[0084] An exemplary chimeric receptor is a chimeric TCR, the basic structure of which is shown in Figure ID. Chimeric TCRs have been previously described in US Patent No. 5,999,623 and US Patent No. 5,999,623. Chimeric TCRs contain a single polypeptide chain that contains (from N-terminus to C-terminus) a single-chain TCR variable region (scFv-TCR), an extracellular constant domain, a transmembrane domain and an intracellular signaling domain.

[0085] In the chimeric TCRs provided herein, the scFv-TCR is as described above. The extracellular constant domain may be the constant domain of the α chain or the constant domain of the β chain. As described above, any suitable extracellular constant domain of the TCR α chain or β chain may be used, for example, the human or mouse extracellular constant domain of the TCR α chain or β chain. The chimeric TCR contains only a single extracellular constant domain of the TCR chain. The chimeric TCRs provided herein may contain any suitable transmembrane domain, for example, those described above. The transmembrane domain of the chimeric TCR may be the transmembrane domain of the TCR chain as described above, or may be the transmembrane domain of a non-TCR protein as described above. The cytoplasmic domain contains an intracellular signaling domain as described above. The cytoplasmic domain may further contain a costimulatory domain as described above.

[0086] In the context of a chimeric TCR, the transmembrane and cytoplasmic domains together constitute a "signaling tail." In a signaling tail that contains both an intracellular signaling domain and a costimulatory domain, the intracellular signaling domain and the costimulatory domain can be linked in any order in tandem to the C-terminus of the transmembrane domain. In one embodiment, the signaling tail contains, in the following order from N-terminus to C-terminus, a CD28 transmembrane domain, a CD28 intracellular domain, and a CD3ζ intracellular domain.

[0087] Another exemplary chimeric receptor is the TCR-CAR, the basic structure of which is shown in FIG. 1F. TCR-CAR was first described in Non-Patent Document 12. TCR-CAR is based on the same rationale as standard CAR, but the scFv of standard CAR is replaced with a soluble TCR (as described above). This provides a construct with the functional potential of CAR but with the substrate breadth of TCR (i.e., they can be directed against any peptide resulting from cellular proteolysis).

[0088] Thus, a TCR-CAR contains a soluble TCR as described above, but is not a soluble protein, and one chain of the soluble TCR construct contains a signaling tail, as described above in the context of a chimeric TCR. The signaling tail may be located at the C-terminus (C-terminal to the extracellular constant domain) of either the first (i.e., truncated α-) chain or the second (i.e., truncated β-) chain.

[0089] Soluble binding proteins provided herein (i.e., binding proteins lacking a transmembrane domain, e.g., TCR-scFv and soluble TCR, as described above) can be synthesized using a protein expression system, such as a cell expression system using prokaryotic (e.g., bacterial) cells or eukaryotic (e.g., yeast, fungal, insect or mammalian) cells. An alternative protein expression system is a cell-free in vitro expression system, in which a DNA sequence encoding the binding protein is transcribed into mRNA, and the mRNA is translated into a protein in vitro. Cell-free expression system kits are widely available and can be purchased, for example, from Thermo Fisher Scientific (USA). Alternatively, the binding protein can be chemically synthesized in a non-biological system, e.g., by liquid or solid phase synthesis.

[0090] After synthesis, the specific binding molecules are isolated and purified using techniques known in the art. For example, the binding protein can be produced using a host eukaryotic cell. The binding protein can be expressed to contain a leader sequence that directs it to export (as discussed below). In this case, the binding protein is exported from the production cells into the culture medium. The culture medium is then separated from the production cells, for example, by centrifugation. The binding protein can then be purified, for example, by affinity chromatography (if the binding protein contains an affinity tag as described above). If a protease cleavage site is present between the tag and the mature binding protein, the tag can be cleaved using an appropriate protease after purification of the binding protein.

[0091] Soluble binding proteins provided herein (i.e., binding proteins lacking a transmembrane domain, such as TCR-scFv and soluble TCR) can be linked or bound to a therapeutic or diagnostic agent or a carrier that contains or comprises a therapeutic or diagnostic agent. A therapeutic agent is an agent used in therapy. By therapy is meant the treatment or prevention of a disease. A therapeutic agent can be an agent useful in the treatment of neoplastic conditions, particularly cancer.

[0092] The therapeutic agent can be a drug molecule, e.g., a toxin for killing a target cell. A suitable toxin is one that is not capable of entering, killing, or otherwise destroying a human cell by itself, but can exert its toxic effect when taken up into a human cell via a binding molecule. Thus, such a toxin can only be taken up by a cell bound by a soluble binding protein carrying the toxin (such as a soluble TCR carrying the toxin) and exert its target effect on the cell to which the soluble binding protein is taken up.

[0093] Suitable toxins include peptide toxins that lack a targeting domain. For example, it may be a peptide toxin that originally lacks a targeting domain, or it may be a peptide toxin whose original form has been modified to remove its targeting domain. Examples of such toxins include saporin and gelonin, which are ribosome-inactivating proteins (RIPs) of the same family as, for example, ricin, but cannot cross the plasma membrane of a cell. Similarly, enzymatic domains (i.e., catalytic domains) of pathogen cytotoxins may be used, such as enzymatic domains of bacterial cytotoxins, for example, diphtheria toxin, Pseudomonas exotoxin A, or clostridial cytotoxins, for example, TcsL of Clostridium sordellii.

[0094] The soluble binding protein can be encoded as a fusion protein with a toxin located at its C-terminus (in the case of a soluble TCR, the toxin can be located at the C-terminus of either the truncated α or β chain). Alternatively, the toxin can be conjugated to the soluble binding protein using any suitable method known in the art (e.g., using the biotin / streptavidin system).

[0095] The therapeutic agent may optionally be another useful therapeutic agent, such as a chemotherapeutic agent or other anti-cancer agent, or an anti-viral agent, etc.

[0096] A diagnostic agent is an agent useful for diagnostic purposes. Such an agent may be, for example, a tracer or label, i.e., an agent that can be detected to follow its passage through the body. The tracer or label can be detected by a scan, e.g., a PET scan or a CT scan. Many tracers and labels, including radioactive labels, are known in the art. Common radioisotopes 11 C. 13 N, 15 O. 18 F, 99 Tc, 123 I and 125 Any suitable tracer or label can be used, including I. The diagnostic agent can be attached to the soluble binding protein (e.g., soluble TCR) using any suitable label group known in the art, e.g., radiolabeled biotin.

[0097] Soluble binding proteins (e.g., soluble TCRs) linked to a therapeutic agent can be used in therapy. Soluble TCRs linked to a diagnostic agent or a carrier containing a diagnostic agent can be used in in vivo diagnostic methods.

[0098] The insoluble binding proteins provided herein (i.e., binding proteins that contain a transmembrane domain, such as a TCR) are provided in the context of a cell that expresses the binding protein. Such cells may be, in particular, immune effector cells in which functional expression of the binding protein is achieved, as described below.

[0099] Recombinant nucleic acid molecules encoding the binding proteins and / or antigen-binding units provided herein are also provided herein. The nucleic acid molecules provided herein may be isolated nucleic acid molecules and may contain DNA or RNA or chemical derivatives of DNA or RNA. The term "nucleic acid molecule" specifically encompasses single-stranded and double-stranded forms of DNA and RNA. Nucleic acids (e.g., DNA or RNA) may be circular or linear. A "recombinant" nucleic acid molecule is a nucleic acid molecule that has been synthesized using recombinant techniques, e.g., molecular cloning.

[0100] The recombinant nucleic acid molecules provided herein encode the binding proteins provided herein. As detailed above, the binding proteins may contain two polypeptide chains, particularly a first chain (i.e., a full-length or truncated α chain) and a second chain (i.e., a full-length or truncated β chain). A recombinant nucleic acid molecule encoding a binding protein containing two polypeptide chains encodes both of the two chains (i.e., the first chain and the second chain are both encoded by the same recombinant nucleic acid molecule). In this case, the two chains may be encoded as two separate genes, each under the control of its own promoter, such that the two chains are expressed separately. Alternatively, as discussed further below, the two chains may be encoded by a single gene within a single polypeptide.

[0101] In a particular embodiment herein, the recombinant nucleic acid molecule contains a cDNA molecule. In particular, the binding protein or a chain thereof may be encoded by a cDNA. "cDNA" as used herein means DNA in its true and original sense (i.e. DNA synthesized by reverse transcription of mRNA), DNA amplified from the original cDNA, and DNA equivalent to cDNA. DNA equivalent to cDNA is DNA that encodes the binding protein provided herein (or its first or second strand) and lacks introns, and thus resembles the protein coding sequence obtained by reverse transcription of mRNA. In a particular embodiment herein, the nucleic acid molecule encoding the binding protein provided herein is codon-optimized. In particular, the binding protein may be encoded by a codon-optimized cDNA sequence.

[0102] Methods for constructing the nucleotide sequences defined herein are well known in the art and include, for example, conventional polymerase chain reaction (PCR) cloning techniques.

[0103] Methods for the isolation of nucleic acid molecules are also well known in the art. For example, DNA can be isolated using a suitable kit. Plasmid DNA can be isolated from bacteria using Miniprep or Maxiprep kits according to the manufacturer's instructions. Such kits are available, for example, from Qiagen (Germany). Genomic DNA can be extracted from eukaryotic or prokaryotic cells, for example, using the QIAamp DNA Mini Kit (Qiagen) or DNeasy kit (Qiagen), according to the manufacturer's instructions. Alternatively, DNA can be isolated from cells of interest using the traditional method of phenol-chloroform extraction. RNA can be extracted from cells using a kit (for example, RNeasy Mini Kit, Qiagen) or by the traditional method of phenol-chloroform extraction combined with DNase treatment. cDNA can be generated by reverse transcription of RNA, for example, using a kit such as the SuperScript First Strand Synthesis System (Thermo Fisher Scientific, USA).

[0104] The recombinant nucleic acid molecules provided herein can be provided in a recombinant construct that contains the recombinant nucleic acid molecule linked to a heterologous nucleic acid sequence. As used herein, "heterologous" refers to a nucleic acid sequence that is not naturally linked to the nucleic acid molecule described herein, i.e., is not linked to the nucleic acid molecule described herein in nature. The term "linked" as used herein with respect to a construct simply means that the nucleic acid molecule is directly attached to the heterologous nucleic acid sequence. In one embodiment, in the recombinant construct, the nucleic acid molecule provided herein is operably linked to a heterologous expression control sequence.

[0105] The term "expression control sequence" refers to a nucleotide sequence located upstream, within, or downstream of a coding sequence that affects the transcription, RNA processing or stability, or translation of the associated coding sequence (i.e., affects any aspect of the expression of the encoded specific binding molecule). Expression control sequences include promoters, promoter elements such as the TATA box or B recognition element, operators, enhancers, translation leader sequences, terminator sequences, and the like.

[0106] In the construct, the nucleic acid molecule can be operably linked to one or more heterologous expression control sequences. The nucleic acid molecule is typically operably linked to at least a promoter. Suitable promoter sequences include cytomegalovirus (CMV) promoters, such as human CMV (HCMV) promoters, PGK promoters, EF1a promoters, constitutive simian virus 40 (SV40) early promoters, mouse mammary tumor virus (MMTV) promoters, HIV LTR promoters, MoMuLV promoters, avian leukosis virus promoters, EBV immediate early promoters, and Rous sarcoma virus promoters. Human gene promoters can also be used, including, but not limited to, actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. In some embodiments, inducible promoters can be used. These provide a molecular switch that can turn on or off the expression of the nucleic acid molecule. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, or tetracycline promoters.

[0107] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence if it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).

[0108] As detailed above, the recombinant nucleic acid molecules provided herein encode the binding proteins provided herein. When the binding protein contains two polypeptide chains (i.e., a full-length or truncated α chain and a full-length or truncated β chain), for example, when the binding protein is a TCR, both polypeptide chains are encoded by the same nucleic acid molecule. In this case, equimolar expression of the first and second chains can be achieved, for example, by encoding the binding protein as a single polypeptide containing the first chain linked to the second chain by a linker (i.e., the binding protein can be encoded as a single polypeptide chain in which the first and second chains are separated by a linker). The linker can have any suitable amino acid sequence. Suitable linkers are known in the art. The linker can be of any suitable length. For example, it can be 1-30 amino acids long, for example, 1-25 or 1-20 amino acids long. The linker can be cleavable, thereby allowing separation of the first and second polypeptides. If the first and second polypeptides cannot be separated, the two chains may not be able to adopt the correct conformation required for the formation of the antigen-binding site from the variable regions of the first and second chains. Those skilled in the art can select a suitable linker. The linker may contain a protease cleavage site to allow specific post-translational cleavage of the linker and thus separation of the first and second polypeptides. Suitable protease cleavage sites are well known to those skilled in the art and include thrombin, factor Xa, enterokinase, human rhinovirus (HRV) 3C and tobacco etch virus (TEV) cleavage sites.

[0109] In certain embodiments, the linker is self-splicing. A self-splicing linker can catalyze its own cleavage, thus separating the first and second polypeptides without the need for an active cleavage step. No stimulation or induction is required for the self-splicing reaction to occur. The cleavage reaction may completely excise the linker from the single-chain specific binding molecule. Alternatively, the linker or a portion of the linker may remain attached to one or both of the resulting separate polypeptide chains. The self-splicing reaction catalyzed by the linker may occur post-translationally (i.e., it may be an autocatalytic proteolytic reaction) or it may occur co-translationally. Co-translational splicing may occur by preventing the formation of a peptide bond within the linker or between the linker and one of the polypeptide chains on either side of it.

[0110] The self-splicing linker may utilize a ribosome skipping sequence. Such ribosome skipping sequences are well known to those skilled in the art. A suitable self-splicing linker is one derived from the picornavirus self-cleaving 2A peptide. The 2A peptide is approximately 20-25 amino acids long and terminates with the conserved sequence motif Asp-Val / Ile-Glu-X-Asn-Pro-GlyPro (SEQ ID NO:50). The 2A peptide undergoes co-translational self-splicing by preventing the formation of a peptide bond between a conserved glycine residue at the final position in the linker and the C-terminal proline residue, effectively resulting in cleavage of the protein between these two amino acids. After cleavage, the 2A peptide (excluding the C-terminal proline) remains attached to the C-terminus of the upstream protein and the final proline residue remains attached to the N-terminus of the downstream protein. The 2A peptide is described in Non-Patent Document 14.

[0111] The examples herein demonstrate the expression of TCR from a vector encoding an α-chain and a β-chain separated by the porcine tesicovirus-derived 2A sequence of SEQ ID NO: 26. Thus, the nucleic acid molecules provided herein can encode the binding proteins provided herein, where the binding proteins are encoded in the form of a single polypeptide containing a first chain (e.g., an α-chain) linked to a second chain (e.g., a β-chain) by a self-splicing 2A peptide. The 2A peptide linker can have any suitable sequence so as to have self-splicing activity. Several 2A peptide sequences are known (e.g., the 2A sequences disclosed in Non-Patent Document 15 and Patent Document 4), any of which can be used in accordance with the present disclosure. Essentially any sequence can be used as long as the linker contains the 2A peptide motif of SEQ ID NO: 50 at its C-terminus. In one embodiment, the self-splicing linker is a 2A peptide containing the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity thereto.

[0112] The binding proteins provided herein (or, where relevant, the first and second chains of the binding proteins) may be encoded by an N-terminal leader sequence. Such a leader sequence is believed to direct the binding protein (or its chains) to the cell membrane for either export (in the case of soluble binding proteins) or insertion into the membrane (in the case of binding proteins containing a transmembrane domain) and is trimmed to yield the mature protein upon export of the protein or its insertion into the membrane. An exemplary alpha chain N-terminal leader sequence has the amino acid sequence set forth in SEQ ID NO:51, and an exemplary beta chain N-terminal leader sequence has the amino acid sequence set forth in SEQ ID NO:52 and SEQ ID NO:53 (these leader sequences are used in the Examples below). Specifically, SEQ ID NO:52 is used as the leader sequence for the T1 TCR beta chain and SEQ ID NO:53 is used as the leader sequence for the T3 TCR beta chain. While any suitable leader sequence may be used in accordance with the present disclosure, in one embodiment, the first chain (e.g., the α chain) is encoded by a leader sequence containing the amino acid sequence set forth in SEQ ID NO:51, or an amino acid sequence having at least 90% or 95% sequence identity thereto; the second chain (e.g., the β chain) is encoded by a leader sequence containing the amino acid sequence set forth in SEQ ID NO:52 or SEQ ID NO:53, or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0113] In one embodiment, a T1 TCR based binding protein may contain a first chain (e.g., an α chain) having a leader sequence of SEQ ID NO: 51 or an amino acid sequence having 90% or 95% sequence identity thereto; and a second chain (e.g., a β chain) having a leader sequence of SEQ ID NO: 52 or an amino acid sequence having at least 90% sequence identity thereto. In another embodiment, a T3 TCR based binding protein may contain a first chain (e.g., an α chain) having a leader sequence of SEQ ID NO: 51 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and a second (e.g., a β chain) having a leader sequence of SEQ ID NO: 53 or an amino acid sequence having at least 90% or 95% sequence identity thereto. If the binding protein contains only a single polypeptide chain, any leader sequence can be used, for example, a leader sequence from a TCR α chain or a TCR β chain. For example, the leader sequence may contain an amino acid sequence set forth in SEQ ID NO: 51, 52 or 53 or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0114] An exemplary single polypeptide containing a T3 TCR chain has the amino acid sequence set forth in SEQ ID NO: 45. This polypeptide construct contains, from N-terminus to C-terminus, a beta chain of SEQ ID NO: 25 with an N-terminal leader of SEQ ID NO: 53, an alpha chain of SEQ ID NO: 24 with a 2A peptide linker of SEQ ID NO: 26 and an N-terminal leader of SEQ ID NO: 51. An exemplary single polypeptide containing a T1 TCR chain has the amino acid sequence set forth in SEQ ID NO: 14. This polypeptide construct contains, from N-terminus to C-terminus, a beta chain of SEQ ID NO: 13 with an N-terminal leader of SEQ ID NO: 52, an alpha chain of SEQ ID NO: 12 with a 2A peptide linker of SEQ ID NO: 26 and an N-terminal leader of SEQ ID NO: 51. A DNA sequence encoding the T3 single chain TCR polypeptide is set forth in SEQ ID NO: 55 and a DNA sequence encoding the T1 single chain TCR polypeptide is set forth in SEQ ID NO: 54.

[0115] The recombinant nucleic acid molecules or constructs provided herein can be provided in a vector. As used herein, the term "vector" refers to a vehicle into which a nucleic acid molecule or construct provided herein can be introduced (e.g., covalently inserted), from which a specific binding molecule encoded by the nucleic acid molecule can be expressed and / or the nucleic acid molecule / construct can be cloned. Thus, a vector can be a cloning vector or an expression vector.

[0116] Examples of vectors include plasmids, autonomously replicating sequences, and transposable elements. Further exemplary vectors include, but are not limited to, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), bacteriophages such as lambda phage or M13 phage, and animal viruses such as human viruses (i.e., viral vectors). Examples of animal virus categories useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex viruses), pox viruses, baculoviruses, papilloma viruses, and papova viruses (e.g., SV40). Examples of expression vectors are pCl-neo vectors (Promega) for expression in mammalian cells, and pLenti4 / V5-DEST™ and pLenti6 / V5-DEST™ for lentivirus-mediated gene transfer and expression in mammalian cells.

[0117] The vector may be a bacterial or prokaryotic vector (i.e., a vector for use, e.g., for cloning or expression, in bacterial or prokaryotic cells) or a eukaryotic vector (i.e., a vector for use in mammalian cells), e.g., a mammalian vector. The nucleic acid molecules or constructs provided herein can be produced in or introduced into a general-purpose cloning vector, such as a bacterial cloning vector, e.g., an E. coli cloning vector. Typically, the cloning vector is a bacterial plasmid, e.g., an E. coli plasmid. Examples of such cloning vectors include pUC19 (e.g., available from New England Biolabs (NEB), USA), pBluescript vectors (Agilent, USA) and pCR TOPO® vectors from Thermo Fisher Scientific.

[0118] The nucleic acid molecules or constructs provided herein can be subcloned into an expression vector for expression of the specific binding molecules provided herein, such as a mammalian expression vector. The expression vector can contain various expression control sequences. For efficient gene transcription and translation in its respective host cell, the expression vector must have the necessary 5' upstream and 3' downstream regulatory elements, such as a promoter sequence (described above), including a TATA box, a Kozak sequence at the translation initiation site, and a 3'UTR AATAAA polyadenylation signal sequence that signals transcription termination.

[0119] In addition to control sequences governing transcription and translation, vectors may contain additional nucleic acid sequences that perform other functions, including, for example, vector replication, selection markers, etc. Examples of selection markers suitable for selection of bacterial host cells include antibiotic resistance genes, such as ampicillin resistance genes (e.g., β-lactamase), kanamycin resistance genes, or chloramphenicol resistance genes (e.g., chloramphenicol acetyltransferase). Selection markers suitable for use in mammalian host cells include the hygromycin-B phosphotransferase gene (hph), which confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5, which encodes resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene. Such selection markers allow for in vitro selection of cells carrying the vector.

[0120] The vector can contain a marker that renders immune effector cells carrying the vector susceptible to negative selection in vivo. The inclusion of such a marker allows selective destruction of immune effector cells carrying the vector in individuals to whom such cells are administered, such as patients treated with adoptive cell therapy. This can be important, for example, if a patient experiences severe side effects to the treatment. A negatively selectable phenotype can result from the insertion of a gene that confers sensitivity to the administered drug. Negatively selectable genes are known in the art and include, inter alia, the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which confers sensitivity to ganciclovir (Non-Patent Document 16), and bacterial cytosine deaminase, which confers sensitivity to 5-fluorocytosine (Non-Patent Document 17).

[0121] The vector may be a viral vector. The viral vector may be derived from a retrovirus, such as a lentivirus or a spammer virus / foamy virus. As used herein, the term "viral vector" refers to a virus-derived particle that carries a nucleic acid molecule or construct provided herein and can deliver the nucleic acid molecule / construct to a target cell. The viral vector may contain a nucleic acid molecule provided herein in place of or in addition to a non-essential viral gene. The vector may be utilized to transfer DNA, RNA or other nucleic acid into cells, either in vitro or ex vivo.

[0122] Numerous forms of viral vectors are known in the art, and any suitable viral vector can be used in accordance with the present teachings, including both single-stranded and double-stranded RNA viral vectors and DNA viral vectors. Single-stranded viral vectors can be positive sense or negative sense viral vectors. In some embodiments, the viral vector is a retroviral vector (i.e., a viral vector derived from a retrovirus), such as a lentiviral vector (i.e., a viral vector derived from a lentivirus). Because the retroviral genome is integrated into the genome of the infected cell, the retroviral vectors described herein can be used to stably transduce target cells, i.e., to permanently change the genetic makeup of the target cell.

[0123] The viral vector may be a self-inactivating vector or a replication-defective vector. Replication-defective retroviral vectors can be obtained, for example, by modifying (e.g., deleting or replacing) the 3'LTR enhancer-promoter region (known as U3 region) of the viral genome to prevent viral transcription beyond the first round of viral replication. As a result, the vector can only be integrated once after infecting the host genome and cannot pass further.

[0124] Retroviral vectors for use herein may be derived, for example, from any known retrovirus, for example, C-type retroviruses such as Moloney murine sarcoma virus (M-MSV), Harvey murine sarcoma virus (Ha-MuSV), mouse mammary tumor virus (MMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend virus, murine stem cell virus (MSCV) and Rous sarcoma virus (RSV); human T-cell leukemia viruses such as HTLV-1 and HTLV-2; human immunodeficiency viruses HIV-1 and HIV-2, simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine immunodeficiency virus (EIV) and other classes of retroviruses, including the lentivirus family.

[0125] Retroviral packaging cell lines (typically mammalian cell lines) can be used to produce viral vectors, which can then be used for transduction of T cells. Packaging cell lines can be used to produce viral vectors by transfection with one or more vectors (e.g., plasmids) carrying genes required for viral particle assembly. Exemplary viral vectors are described, for example, in Patent Document 5. An exemplary plasmid for the production of retroviral vectors is pMP71, described in Non-Patent Document 18.

[0126] In another embodiment, the vector is an mRNA vector. An mRNA vector is a positive-detecting mRNA strand that contains the above-mentioned nucleic acid molecule or construct. An mRNA vector is a translatable mRNA strand that, when delivered to a target cell, can bind to ribosomes and initiate synthesis of the encoded protein. An mRNA vector is advantageous because it does not require nuclear entry or transcription to initiate production of its encoded protein, but instead can directly bind to cytoplasmic ribosomes and initiate translation. Thus, transfection of target cells with an mRNA vector can be used for rapid production of the encoded specific binding molecule. RNA has a limited half-life due to its inherent instability, and therefore, an mRNA vector can be used for transient transfection of target cells.

[0127] An mRNA vector contains the essential elements for translation, such as a 5' 7-methylguanylic acid head and a polyadenylic acid tail for ribosome recognition. An mRNA vector can be produced from an mRNA expression vector using a cell-based or cell-free system according to methods known in the art. Suitable mRNA expression vectors include pClpA102 (Non-Patent Document 19) and pCIpA120-G (Non-Patent Document 18). Cell-free systems require a DNA template containing the gene to be transcribed and a suitable promoter, and utilize an RNA polymerase, typically a phage RNA polymerase. Kits for performing in vitro transcription (e.g., MEGAscript™ SP6 Transcription Kit) can be obtained, for example, from Thermo Fisher Scientific.

[0128] Also provided herein is a kit containing a first nucleic acid molecule encoding a first strand of a binding protein provided herein and a second nucleic acid molecule encoding a second strand provided herein. Thus, the kit contains a pair of nucleic acid molecules, one encoding the first strand and the other encoding the second strand, as described above.

[0129] The first and second nucleic acid molecules are as described above, e.g., they can be DNA or RNA, double-stranded or single-stranded, linear or circular, etc. The first and second recombinant nucleic acid molecules can be provided in the kit in association with a first and second recombinant construct (a first recombinant construct containing the first nucleic acid molecule and a second recombinant construct containing the second nucleic acid molecule) or in a first and second vector. The recombinant constructs and vectors are described above. The first and second nucleic acid molecules of the kit are provided as separate molecules, i.e., they are not both located in the same construct or vector.

[0130] As mentioned above, the vector may contain a selection marker so that cells that have incorporated the vector can be positively selected. In one embodiment, when the first and second nucleic acid molecules are provided in a first and second vector, the first and second vectors each contain a selection marker. The markers of the first and second vectors are generally different, so that cells that have incorporated both vectors can be selected more than cells that have incorporated only one of the two vectors (or neither vector). Suitable selection markers are discussed above.

[0131] The two nucleic acid molecules of the kit (e.g., the first and second recombinant constructs or the first and second vectors) can be provided in a single container (i.e., a mixture of the two nucleic acid molecules) or in separate containers. The nucleic acid molecules may be provided in an aqueous solution (e.g., water or a suitable buffer such as TE buffer) or in lyophilized form.

[0132] The first and second strands encoded by the nucleic acid molecules in the kit are both derived from either the T1 or T3 TCR (i.e. the kit does not include one nucleic acid molecule encoding a chain derived from the T1 TCR and one nucleic acid molecule encoding a chain derived from the T3 TCR).

[0133] Thus, in a first embodiment, a kit contains a nucleic acid molecule encoding the first and second chains of a T3 TCR-based binding protein, the kit containing: (i) a first nucleic acid molecule encoding a first chain containing a variable domain comprising CDR1, CDR2 and CDR3 containing the amino acid sequences set forth in SEQ ID NOs: 16, 17 and 18, respectively, in particular wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and (ii) a second nucleic acid molecule encoding a second chain containing a variable domain comprising CDR1, CDR2 and CDR3 containing the amino acid sequences set forth in SEQ ID NO: 19, 20 and 21, respectively, in particular wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0134] Thus, the first chain may be any full-length or truncated alpha chain based on the T3 TCR as described above, and the second chain may be any full-length or truncated beta chain based on the T3 TCR as described above.

[0135] Thus, in a second embodiment, a kit contains nucleic acid molecules encoding the first and second chains of a T1 TCR-based binding protein, the kit containing: (i) a first nucleic acid molecule encoding a first chain containing a variable domain containing CDR1, CDR2 and CDR3 containing the amino acid sequences shown in SEQ ID NO:2, 3 and 4, respectively, in particular, the variable domain containing the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least 90% or 95% sequence identity thereto; and (ii) a second nucleic acid molecule encoding a second chain containing a variable domain comprising CDR1, CDR2 and CDR3 containing the amino acid sequences set forth in SEQ ID NO:5, 6 and 7, respectively, in particular wherein the variable domain comprises the amino acid sequence set forth in SEQ ID NO:9 or an amino acid sequence having at least 90% or 95% sequence identity thereto.

[0136] Thus, the first chain may be any full-length or truncated alpha chain based on the T1 TCR as described above, and the second chain may be any full-length or truncated beta chain based on the T1 TCR as described above.

[0137] Sequence identity can be assessed by any convenient method. However, to determine the degree of sequence identity between sequences, computer programs that generate pairwise or multiple alignments of sequences are convenient. For example, EMBOSS Needle or EMBOSS Stretcher (both non-patent literature 20) can be used for pairwise sequence alignment, while Clustal Omega (non-patent literature 21) or MUSCLE (non-patent literature 22) can be used for multiple sequence alignment, although any other suitable program can be used. Another suitable alignment program is BLAST, which uses the blastp algorithm for protein alignment and the blastn algorithm for nucleic acid alignment. The alignment, whether pairwise or multiple, must be performed globally (i.e., across the entire reference sequence) rather than locally.

[0138] Sequence alignments and percent identity calculations can be determined, for example, using standard Clustal Omega parameters: matrix Gonnet, gap opening penalty of 6, gap extension penalty of 1. Alternatively, standard EMBOSS needle parameters can be used: matrix BLOSUM62, gap opening penalty of 10, gap extension penalty of 0.5. Any other suitable parameters can be used instead.

[0139] Where a sequence has less than 100% identity to a reference sequence cited herein, the sequence may be altered by amino acid substitution, deletion or addition compared to the reference sequence (or any combination thereof).

[0140] The nucleic acid molecules, constructs and vectors provided herein can be introduced into cells, such as immune effector cells, by known methods. The first and second nucleic acid molecules of the kits provided herein can be similarly co-introduced into host cells. The nucleic acid molecules, constructs and vectors can be introduced into target eukaryotic (e.g., human) cells by transfection (e.g., electroporation, microinjection, lipofection or biolistic, or any other method known in the art) or transduction using viral vectors. The nucleic acid molecules, constructs and vectors can be introduced into target prokaryotic (e.g., bacterial) cells by any method known in the art, such as transformation, transduction or conjugation.

[0141] Accordingly, further provided herein are cells containing a recombinant nucleic acid molecule or vector provided herein, or a pair of nucleic acid molecules comprised in a kit provided herein.

[0142] The cell may be an effector cell, more specifically an immune effector cell or a precursor or progenitor cell therefor, in particular containing a recombinant nucleic acid molecule or vector provided herein, or a pair of nucleic acid molecules from the kit encoding the above-mentioned binding protein containing a transmembrane domain. The recombinant nucleic acid molecule, vector, or pair of nucleic acid molecules may encode a TCR. Alternatively, a TCR-CAR or a chimeric TCR may be encoded. When cells, such as effector cells, or immune effector cells, express such binding proteins in their cell membrane, they can recognize and target TdT-expressing cells. Thus, cells containing such recombinant nucleic acid molecule, vector, or pair of nucleic acid molecules express the encoded specific binding protein in their cell membrane. That is, the encoded specific binding protein is expressed and localized in the cell membrane and is functional. In certain embodiments, cells, such as effector, or immune effector cells, express a functional TCR in their cell membrane.

[0143] As referred to herein, "immune effector cells" refers to any immune cell that can exert an effector function (e.g., target cell killing by cytotoxicity, cytokine release, etc.) upon activation and can express a functional TCR. More generally, an "effector cell" is any cell that can perform an effector function. This can be any useful effect exerted by the cell or any useful property of the cell. Effector cells include stem cells. Effector cells (which term includes immune effector cells in particular) can be considered or include cells suitable for therapeutic use, such as use in adoptive cell transfer therapy. Thus, effector cells can alternatively be referred to as therapeutic cells or cells for therapy.

[0144] The term "immune effector cells" may specifically encompass mature or fully differentiated immune effector cells, but as noted above, is also within the scope of the disclosure herein and as used herein refers to precursor (or progenitor) cells, including stem cells (e.g., hematopoietic stem cells, HSCs) or cells derived from HSCs or other stem cells, as well as more committed precursors. Thus, precursors of immune effector cells include CD34+ cells derived from hematopoietic tissues, such as bone marrow, umbilical cord blood, or blood, such as mobilised peripheral blood. + The cells may be cells obtained or derived from HSCs contained within the population, which upon administration to a subject may differentiate into mature immune effector cells or may be induced to differentiate into immune effector cells in vivo or in vitro. The stem cells may also be induced pluripotent stem cells (iPSCs) and thus may be precursors of immune effector cells, or may be iPSCs or cells obtained or derived from iPSC cells.

[0145] In certain embodiments, the immune effector cell is a T cell. The T cell can be any type of T cell. For example, it can be a cytotoxic T cell (CD8 + T cells), T helper cells (CD4 + T cells), natural killer T cells (NKT cells), naive T cells, memory T cells or any other type of T cell. The T cells can be helper T cells or cytotoxic T cells. In another specific embodiment, the immune effector cells are natural killer cells (NK cells).

[0146] Thus, the immune effector cells can be selected from cytotoxic T cells, NK cells and T helper cells. In one embodiment, the immune effector cells are NK cells and the expressed binding protein is a TCR. In this case, the additional expression of one or more CD3 chains may be required as disclosed in US Pat. No. 5,993,333 and US Pat. No. 5,993,333.

[0147] T cells or NK cells containing a recombinant nucleic acid molecule, vector or pair of nucleic acid molecules as described herein can be obtained by transfecting or transducing target T cells or NK cells as described above. The T cells or NK cells to be transfected can be derived from an existing cell line or can be primary T cells or NK cells isolated from the subject of interest (which can be the patient to be treated or the donor). The T cells or NK cells can also be activated and stimulated to proliferate in vitro (such activation and stimulation to proliferate can be referred to as proliferation) before and / or after being modified to express the binding molecule. The NK cells can be expanded and activated using the method described in US Pat. No. 5,399,992.

[0148] T cells can be obtained from many sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. T cells can be isolated using any method known in the art. T cells can be obtained from PBMCs, which can be isolated from buffy coats obtained by density gradient centrifugation of whole blood. CD4 + or CD8 + Specific subpopulations of T cells, such as T cells, can be further isolated by positive or negative selection techniques. For example, enrichment of a T cell population by negative selection can be achieved with a combination of antibodies against surface markers unique to the negatively selected cells. Such negative selection can be performed, for example, by fluorescence-activated cell sorting (FACS).

[0149] NK cells can be obtained from the same sources as T cells, using standard methods known in the art, as described above. For example, NK cells from cell lines, such as NK-92 cells, can be used. Primary NK cells can be isolated from blood, such as PBMCs or non-bile umbilical cord blood (UCB), as described, for example, in Non-Patent Document 23 or Non-Patent Document 24.

[0150] The cells, e.g., effector cells or immune effector cells as used herein, can be human. Effector cells, e.g., immune effector cells expressing TCR (or other membrane-bound binding proteins provided herein), can be autologous or allogeneic. That is, when the cells, e.g., effector cells or immune effector cells, are for therapeutic use, they are autologous cells, i.e., derived from the patient to be treated, which ensures histocompatibility and non-immunogenicity, meaning that once genetically modified, they will not induce an immune response from the patient. Alternatively, the cells can be non-autologous cells for therapeutic use (i.e., they are donor cells obtained from an individual other than the patient), in which case they can be allogeneic.

[0151] Therapeutic non-autologous cells may be non-immunogenic, such that when administered to a subject, they do not generate an immune response that affects, interferes with, or prevents the use of the cells in therapy. Non-autologous cells (such as immune effector cells or their precursors) may of course be non-immunogenic if they are HLA-compatible with the patient. Non-autologous cells may be made non-immunogenic by modification to reduce or eliminate expression of MHC molecules, for example, by knocking out or knocking down expression of genes encoding MHC proteins. The cells may be HLA-negative human cells. In one embodiment, disruption of class I MHC expression may be achieved by knocking out the gene encoding β2-microglobulin (β2-m).

[0152] The cells (e.g., effector cells, or more specifically immune effector cells) may alternatively be irradiated prior to administration to the subject. Without wishing to be bound by theory, it is believed that irradiation of the cells causes the cells to be present in the subject only transiently, thus reducing the time available for the subject's immune system to mount an immunological response against the cells. Such cells may express functional MHC molecules on their cell surface, but are also believed to be non-immunogenic. The radiation may be from any source of alpha, beta or gamma radiation, or may be x-ray radiation or ultraviolet light. A radiation dose of 5-10 Gy will be sufficient to inhibit proliferation. Alternatively, the cells may be modified to express a "suicide gene", which allows the cells to be inducibly killed or prevented from replicating in response to an external stimulus.

[0153] The cells provided herein may alternatively be production host cells, where the recombinant nucleic acid molecule, vector or pair of nucleic acid molecules encodes a soluble binding protein (e.g., a soluble TCR) as described above. The production host cell is any cell suitable for use in protein production. Suitable production hosts are known in the art. The production host may be a prokaryote, e.g., an E. coli strain optimized for eukaryotic protein production, such as a Rosetta strain. The production host may be a eukaryotic cell, e.g., a fungal cell, such as a yeast cell, e.g., Pichia pastoris. The production host may be a mammalian cell, such as a human cell or a non-human cell. When a non-human mammalian cell is used, the cell may be optimized for human protein expression. Suitable mammalian cells include Cos cells, e.g., Cos-7 cells, HEK293 cells and CHO cells, although any suitable cell line or type may be used. CHO (Chinese Hamster Ovary) cells are commonly used in the art for protein production and may be used in accordance with the present disclosure. The gene encoding the specific binding molecule can be codon optimized for expression in the selected host.

[0154] The cell provided herein can be one of the pair of nucleic acid molecules provided by the kit or a cloning host used during synthesis of the recombinant nucleic acid or vector provided herein. Prokaryotic cells can be used as cloning hosts for the above-mentioned nucleic acid molecules, constructs or vectors. Prokaryotic cells suitable for use as cloning hosts include eubacteria such as gram-negative or gram-positive organisms, for example Enterobacteriaceae such as Escherichia such as E. coli, and Bacillus subtilis. The cloning host can alternatively be a fungal cell, for example Pichia pastoris, or a yeast cell, or a higher eukaryotic cell such as a mammalian cell.

[0155] Another aspect of the present disclosure provides a method of generating TdT-specific cells, or more specific effector cells, or immune effector cells (i.e., immune effector cells provided herein) or precursors thereof, comprising introducing a recombinant nucleic acid molecule provided herein, a vector provided herein, or a pair of recombinant nucleic acid molecules contained in a kit provided herein into a cell (e.g., an immune effector cell or precursor thereof). The immune effector cell can be any immune effector cell as described above, in particular a T cell (such as a cytotoxic T cell or a T helper cell) or a NK cell, and the precursor can include any stem cell, such as an iPSC or a cell derived therefrom. The nucleic acid molecule or vector can be introduced into the cell by any method known in the art, such as transfection or transduction, as also described above. The cell can be further modified as described above (e.g., to render it non-immunogenic or to express additional proteins of interest, e.g., NK cells can be modified to express both TCR and CD3 chains to express a functional TCR-CD3 complex, as described above). The effector cells can also be expanded as described above. Modification and expansion of the cells can be performed in any order.

[0156] Further provided herein is a pharmaceutical composition containing the cells provided herein, particularly effector cells, more particularly immune effector cells or precursors therefor. Also provided is a pharmaceutical composition containing the soluble binding proteins provided herein (e.g., TCR-scFv or soluble TCR). The pharmaceutical composition containing the cells provided herein can be used in cancer treatment, as further described below. Similarly, the pharmaceutical composition containing the soluble binding proteins provided herein can be used in cancer treatment or diagnosis, as described above and further discussed below.

[0157] The pharmaceutical compositions provided herein contain an active pharmaceutical agent (i.e., a cell or a soluble binding protein) and at least one pharma- ceutically acceptable diluent, carrier or excipient. The compositions can be formulated in any convenient manner, according to techniques and procedures known in the pharmaceutical art. As used herein, the term "pharmaceutical acceptable" refers to a component that is compatible with other components of the composition and physiologically acceptable to the recipient. The properties, dosages, and the like of the composition and carrier or excipient materials can be selected in a routine manner according to the choice and desired route of administration, purpose of treatment, and the like.

[0158] The pharmaceutical composition can be prepared for administration to a subject by any suitable means. Such administration can be, for example, oral, nasal or parenteral. Oral administration as used herein includes buccal and sublingual administration. Parenteral administration as defined herein includes subcutaneous, intramuscular, intravenous, intraperitoneal and intradermal administration.

[0159] The pharmaceutical compositions provided herein include liquid solutions or syrups, solid compositions such as powders, granules, tablets or capsules, creams, ointments and any other style of composition commonly used in the art. Pharmaceutically acceptable diluents, carriers and excipients for use in such compositions are well known in the art.

[0160] For example, suitable excipients include lactose, corn starch or its derivatives, stearic acid or its salts, vegetable oils, waxes, fats and polyols. Suitable carriers or diluents include carboxymethylcellulose (CMC), methylcellulose, hydroxypropylmethylcellulose (HPMC), dextrose, trehalose, liposomes, polyvinyl alcohol, pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose (and other sugars), magnesium carbonate, gelatin, oils, alcohols, surfactants, and emulsifiers such as polysorbates. Stabilizers, wetting agents, emulsifiers, sweeteners, and the like can also be used.

[0161] Liquid pharmaceutical compositions, whether in solution, suspension or other similar form, may contain one or more of the following: water for injection, saline (which may be physiological), Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides that may act as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates, and tonicity adjusting agents such as sodium chloride or dextrose. Parenteral preparations may be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials. Injectable pharmaceutical compositions may be sterile.

[0162] Provided herein are cells as provided herein, particularly effector cells, more particularly immune effector cells or precursors therefor, for use in therapy. Also provided herein are pharmaceutical compositions containing such cells (above) for use in therapy. Also provided herein are soluble binding proteins (e.g., TCR-scFv or soluble TCR) as provided herein for use in therapy or for use in diagnosis. Also provided herein are pharmaceutical compositions containing such soluble binding proteins for use in therapy or for use in diagnosis. In the context of the soluble binding proteins provided herein, soluble binding proteins linked or conjugated to a therapeutic agent (as detailed above) can be used in therapy, and soluble binding proteins linked or conjugated to a diagnostic agent (as detailed above) can be used in diagnosis.

[0163] As used herein, "treatment" refers to the treatment of any medical condition. Such treatment may be prophylactic (i.e., preventative), curative (or treatment intended to cure), or palliative (i.e., treatment designed merely to limit, reduce or ameliorate the symptoms of the condition).

[0164] Therapy using cells or compositions containing cells is adoptive transfer therapy (alternatively known as adoptive transfer). Adoptive transfer therapy can be performed using known techniques. Cells can be formulated for therapy by first harvesting them from their culture medium, then washing and concentrating the cells in a medium and container system (a pharma- ceutically acceptable carrier) suitable for administration in a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically normal saline, e.g., Normosol R (Abbott) or Plasma-Lyte A (Baxter), but 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium can be supplemented with human serum albumin.

[0165] The cells provided for therapeutic use herein that express a membrane-bound specific binding molecule (e.g., TCR) as described above can be administered either alone or as a pharmaceutical composition in combination with a diluent and / or other components such as IL-2 or other cytokines or cell populations. Such pharmaceutical compositions are described above.

[0166] The cells for therapeutic use provided herein and pharmaceutical compositions containing such cells can be administered to a patient by any suitable route. In particular, the cells can be administered intravenously or by intratumoral injection.

[0167] Treatment as described herein can include administration of a pharma- tically effective dose of the cells herein. A pharma- tically effective dose can be, for example, 1×10 cells expressing a binding protein (e.g., TCR). 5 ~1×10 10 A pharma- ceutical effective dose can range from 1×10 cells expressing TCR or more. 7 ~5×10 9 For the uses provided herein, the cells are generally in a volume of 1 liter or less, 500 mL or less, or even 250 mL or 100 mL or less. Thus, the desired cell density is typically 10 6 More than 10 cells / mL, typically 7 More than 10 cells / mL, typically 8 cells / mL or greater. Clinically relevant numbers are cumulative and exceed 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 or 10 12The cell composition may be divided into multiple injections resulting in more than one cell. For example, two, three, four, five, six or more separate injections may be administered to the patient at 24 or 48 hour intervals, or every 3, 4, 5, 6 or 7 days. Infusions may also be given at weekly, biweekly or monthly intervals, or at 6 week or longer intervals. The cell composition may be administered multiple times at doses within the ranges described above. If desired, treatment may also include administration of mitogens (e.g., PHA) or lymphokines, cytokines and / or chemokines (e.g., IFN-γ, IL-2, IL-12, TNF-α, IL-18, and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MGRTα, etc.) to enhance induction of an immune response.

[0168] In the treatment using the soluble binding proteins provided herein, the dosage etc. can be routinely selected according to the choice and desired route of administration, the purpose of treatment etc. The dosage may also depend on the nature of the patient, e.g., age, size and condition, as well as the type and severity of the patient's disease. The appropriate dosage can be determined by clinical trials. The soluble binding proteins provided herein can alternatively be used in in vivo diagnostic methods, in which the soluble binding proteins are linked or conjugated to a diagnostic agent, as described above. Suitable in vivo diagnostic methods are known to the skilled practitioner and include the use of soluble binding proteins conjugated to a tracer or the like, e.g., a radioactive label, in the scanning of the patient.

[0169] The soluble binding proteins can be administered by any route common in the art, such as oral, nasal or parenteral routes as described above.

[0170] The present disclosure broadly provides cells, more specifically effector cells or immune effector cells or precursors therefor, expressing a TCR that specifically binds to an HLA complex presenting a TdT peptide (or a pharmaceutical composition containing said cells), for use in treating cancer of T cell origin. Similarly, the present disclosure provides a method for treating cancer of T cell origin comprising administering a pharmaceutical composition containing a pharma- tically effective dose of cells expressing a TCR that specifically binds to an HLA complex presenting a TdT peptide.

[0171] Further provided herein are cells provided herein, particularly effector cells, more particularly immune effector cells or precursors thereof, soluble binding proteins provided herein, or pharmaceutical compositions provided herein for use in treating cancers expressing TdT. Cancer is broadly defined herein to include any neoplastic condition, whether malignant, premalignant, or non-malignant. However, generally, it is a malignant condition. Both solid and non-solid tumors are included, and the term "cancer cells" can be considered synonymous with "tumor cells." A cancer expressing TdT is a cancer in which the constituent cancer cells express TdT. Whether a cancer expresses TdT can be identified, for example, by analysis of a biopsy sample. A solid or liquid sample is obtained by standard biopsy procedures and analyzed by histology, for example, immunohistochemistry using anti-TdT antibodies, to identify TdT expression. Other immunological methods, for example, Western blot of a biopsy sample, may be used to identify TdT expression. TdT expression can also be identified by mRNA analysis, e.g., qPCR or RNA-Seq. This aspect of the disclosure can be used to treat cancer in patients who carry the HLA-A2 gene, which means that the cancer cells express HLA-A2 (i.e., the cancer is HLA-A2 positive or HLA-A2 positive). * 02 positive, possibly with HLA-A * 02:01 positive). Thus, the cancers treated in accordance with the disclosure are HLA-A2 positive.

[0172] The cancer may be any cancer, but may in particular be a hematological cancer. In a particular embodiment, the hematological cancer is acute lymphoblastic leukemia (ALL). The cancer may be ALL of B-cell origin (B-ALL) or ALL of T-cell origin (T-ALL). In a particular embodiment, the cancer is T-ALL. In another embodiment, the hematological cancer is non-Hodgkin's lymphoma, in particular lymphoblastic lymphoma.

[0173] Similarly, HLA-A expressing TdT * Provided herein are methods for treating immune deficiency virus type 2 (HLA-A) positive cancers, comprising administering to a subject in need thereof a cell, particularly an effector cell, more particularly an immune effector cell or a precursor thereof, a soluble binding protein, or a pharmaceutical composition provided herein. Such a subject may have a disease or condition that is associated with such cancer (i.e., TdT and HLA-A positive cancers). * The subject is a human subject suffering from a cancer that expresses IL-1, IL-2, or IL-3. The cancer may in particular be one of those detailed above.

[0174] In general, we also provide a method for treating TdT-positive cancer in a patient, comprising administering a pharmaceutical composition comprising a cell, in particular an effector cell and more particularly an immune effector cell or a precursor thereof, expressing a receptor specifically bound to an HLA class I molecule presenting a peptide fragment obtainable from TdT. We also provide a method for treating TdT-positive cancer in a patient, comprising administering a pharmaceutical composition comprising a cytotoxic protein specifically bound to an HLA class I molecule presenting a peptide fragment obtainable from TdT. A suitable peptide fragment obtainable from TdT may contain 8 to 12 amino acid residues, preferably unique to TdT.

[0175] Similarly, as used herein, HLA-A expressing TdT in a subject *The present invention provides the use of a cell, particularly an effector cell, more particularly an immune effector cell or precursor therefor, or a soluble binding protein provided herein, in the manufacture of a medicament for the treatment of a 02-positive cancer. The cancer may in particular be a cancer as described above.

[0176] Further, as used herein, HLA-A expressing TdT * The soluble binding protein provided herein is provided for use in diagnosing HLA-A02 positive cancer. The use of the soluble binding protein in diagnosing cancer is described in detail above. The cancer may be any cancer, as described in detail above with respect to the use of the present disclosure in cancer treatment. Similarly, the present invention relates to a soluble binding protein that binds to HLA-A02 positive cancer. * Methods are provided for diagnosing cancer comprising administering a soluble binding protein provided herein to a subject suspected of having a cancer that is 02 positive and expresses TdT.

[0177] The present disclosure may be more fully understood from the following non-limiting examples and with reference to the drawings, in which: EXAMPLES

[0178] Materials and Methods (Primary patient cells and cell lines) Pediatric and young adult patients with relapsed or refractory (r / r) B-ALL were enrolled and treated according to the Chimeric Antigen Receptor (CAR) T-cell trials ClinicalTrials.gov Identifiers NCT02435849 and NCT03123939, and pediatric T-ALL patients according to NOPHO-ALL-2008 (NCT00816049).

[0179] Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation (Axis-Shield) from healthy donor buffy coats obtained from the blood bank of Oslo University Hospital. PBMCs were typed for HLA-A2 expression by flow cytometry. Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCLs) were transfected with HLA-A2 as described in Non-Patent Document 25. pos and HLA A2 neg Thymic tissue was generated from PBMCs. Thymic tissue was cleaned of blood clots, connective tissue, fat tissue, and necrotic tissue, cut into small pieces, which were gently triturated with a 1 mL micropipette attached to a wide-bore pipette tip to release thymocytes into cold RPMI-1640 medium. Thymocytes were washed twice in medium and then cryopreserved.

[0180] The following cell lines were obtained from the American Type Culture Collection (ATCC) or the German Collection of Microvories and Cell Cultures (DSMZ): NALM-6, BV173, REH, HPB-ALL, RS4;11, T2, RD, U-2 OS, FM-6, HeLa, HaCaT, MCF7, K562, COLO 688, EA.hy926, EST149, U-87 MG, Daoy, HCT-116, CHP-212, and Phoenix-AMPHO. Most cell lines were purchased from ATCC and DSMZ for research and were already authenticated and frozen aliquots labeled according to passage. Only low passage cell lines were used to start fresh cultures. The identity of the cell lines obtained prior to these studies was confirmed by short tandem repeat DNA profiling, a service provided by Labcorp DNA Identification Lab (formerly Genetica, NC, USA, https: / / celllineauthentication.com / ). Cell lines were cultured in media as specified by the supplier at 37°C in a humidified cell incubator containing 5% CO2 and were regularly tested for mycoplasma contamination.

[0181] (Induction of antigen-specific T cells) Induction of TdT peptide-reactive T cells and generation of cytotoxic T cell lines and clones were performed as described in Non-Patent Document 26 with some modifications. Briefly, on day 4, monocytes were purified by HLA-A2 / HLA-Asp ... pos PBMCs were isolated from healthy donors and cultured for 3 days in CellGro GMP DC medium (CellGenix) supplemented with 1% (vol / vol) human serum (HS, Trina biotech) and 1% (vol / vol) P / S containing 50 IU / mL interleukin (IL)-4 (PeproTech) and 800 IU / mL GM-CSF (Genzyme). Monocyte-derived dendritic cells (MoDCs) were then matured for 14–16 h by the addition of lipopolysaccharide (LPS; Sigma-Aldrich) and IFN-γ (PeproTech) to final concentrations of 10 ng / mL and 100 IU / mL, respectively. On day 1, naive CD8 + T cells were identified as HLA-A2 neg PBMCs from donors were separated by AutoMACS Pro Separator and mixed with CD45RO- and CD57-reactive beads (Miltenyi Biotecs). + MoDCs were isolated by using a T cell isolation kit. On day 0, moDCs were harvested, electroporated with mRNA encoding full-length TdT, and co-cultured with naïve T cells at a DC:T cell ratio of 1:4 in DC-T cell medium supplemented with 30 ng / mL IL-21 (PeproTech). Parallel control co-cultures were initiated with moDCs transfected with irrelevant mRNA.

[0182] (pMHC multimer + CD8 + T cell selection and cloning Peptide-1 and Peptide-3 specific CD8 +Single cell cloning of T cells was performed as described in Non-Patent Document 26. To assess functionality, T cell clones were stimulated with EBV-LCL pulsed with relevant peptides and the NALM-6 cell line, which naturally expresses TdT, and assessed for upregulation of CD137.

[0183] (TCR sequencing and cloning) The TCRα and TCRβ chain pairs from three clones reactive to peptide-1 and one clone reactive to peptide-3 were amplified using the protocol described in Non-Patent Document 27 and Non-Patent Document 28, modified and adapted for targeted amplification of TCRα and β transcripts. Briefly, RNA was extracted and processed to obtain TCR-specific cDNA. RT PCR was performed for each clone using four pairs of TCRα / β constant domain specific primers, followed by addition of poly-G tail and template switching to obtain double-stranded DNA. Finally, two rounds of nested PCR amplification were performed using additional constant domain primers and an adapter primer annealing to the anchor sequence introduced in the poly-G domain. Libraries were prepared using the kappa Illumina kit and later sequenced on an Illumina MiSeq. Sequencing data was analyzed using MiTCR scripts, and full-length TCR chains were reconstructed using in-house Python scripts TCR primers as described in Non-Patent Document 27 and Non-Patent Document 29. The output was manually verified for each sample in IMGT / V-Quest (30). Variable TCRα and TCRβ fragments of the identified TCRs were codon-optimized, synthesized, and cloned by Genscript.

[0184] (Gene transfer into human PBMCs and cell lines) T1 and T3 TCRs, HLA-A2 pos Donor-derived and patient-derived PBMCs were transduced with 1G4 and DMF5 TCRs, and, as a control for in vivo experiments, with HLA-A2 posDonor-derived PBMCs were transduced. For stimulation of human PBMCs, 6-well or 12-well tissue culture treated plates were coated with anti-CD3 (clone OKT3, eBioscience) and anti-CD28 (clone CD28.6, eBioscience) antibodies. PBMCs (2 × 10 ) in T cell medium supplemented with IL-7 and IL-15 (5 ng / mL each, PeproTech) were transduced. 6 4×10 cells / mL) were added to the antibody-coated plates and incubated at 37° C. with 5% CO for 72 hours. For generation of retroviral supernatant, 4×10 6 Phoenix-AMPHO packaging cells were plated in 10 cm Petri dishes for 24 h, and cells were transfected with γ-retroviral vector DNA and mixed in X-tremeGENE 9 DNA transfection reagent (Roche Diagnostics) and Opti-MEM. The next day, medium was refreshed and cells were incubated at 32°C, 5% CO2 for 24 h. PBMCs were then harvested, resuspended in T cell medium supplemented with IL-7 and IL-15, mixed with retroviral supernatant, placed in non-tissue culture treated 6-well plates pre-coated with Retronectin (20 μg / mL, Takara) and spinoculated at 900 × g for 60 min. The next day, a second spinoculation was performed with fresh retroviral supernatant, and transduction efficiency was determined 3–5 days later by staining with anti-mouse TCR β chain antibodies and / or pMHC multimers followed by flow cytometry. Prior to functional experiments, cells were cultured for 48-72 h in T cell medium containing low concentrations of cytokines (0.5 ng / mL IL-7 and IL-15), and cells were frozen for later experiments.

[0185] Retroviral supernatants containing viral DNA encoding full-length HLA-A2 and TdT were also produced as described above and utilized to transduce REH, RD, HeLa, K562, HaCaT, COLO688, EA.hy926 and HPB-ALL cell lines with HLA-A2, and EBV-LCL with TdT. For in vivo experiments, the BV173 cell line was stably transduced to express firefly luciferase and green fluorescent protein (GFP) and named BV173ffluc-eGFP. All transduced cell lines were then purified by FACS sorting, expanded, and frozen for use in subsequent experiments.

[0186] The cDNAs of TdT and HLA-A2 were cloned into the pClpA102 vector for mRNA production as described in Non-Patent Document 31.

[0187] (Antibodies and Flow Cytometry) Flow cytometry was performed on a BD LSR II flow cytometer (BD Biosciences) and data were analyzed using FlowJo (TreeStar) or FACS DIVA (BD Biosciences) software. For surface antibody staining, antibodies were added to cells for 15-20 min on ice, followed by a washing step. For intracellular staining, cells were suspended in Cytofix / Cytoperm (BD Bioscience) solution for 20 min, washed with Perm / Wash buffer (BD Bioscience), and then stained with antibodies. The following fluorescently conjugated anti-human antibodies were obtained from BD Bioscience or BioLegend unless otherwise stated: Anti-CD14 (HCD14), -HLA-A2 (BB7.2), -CD62L (DREG-56), CD56 (HCD56), -CD57 (HNK-1), -CD45RO (UCHL1), -CD45RA (H I100), -CCR7(150503), -CD137(4B4-1), -CD45(HI30), -TdT(E17-1519), -CD10(HI10a), -CD19(HIB19, SJ25C1) , -CD38 (HIT2), -CD34 (581), -CD1a (HI149), -CD2 (S5.2), -CD3 (UCHT1, OKT3), -CD8a (RPA-T8), -CD4 (RPA-T4), -CD5 (UCHT2, L17F12), -CD7 (M-T701), anti-mouse CD45 (30-F11), -CD99 (DN16, Bio-Rad) and -CD3 (SK7, eBioscience). Anti-mouse TCR β chain PE (H57-597, BD Biosciences) was used to test the transduction efficiency of T1 and T3 TCR in human cells and to monitor the transduced T cells used for in vivo treatment in mice. In all flow cytometry experiments, the Live / Dead Fixable Near-IR Dead Cell Stain kit (Life Technologies) was used to exclude dead cells.

[0188] (T cell activation analysis) The reactivity of T cell clones and TCR-transduced T cells was investigated by measuring CD137 upregulation or IFN-γ release. Briefly, 100,000 cells / well of the indicated target cell lines or primary patient tumor cells were co-incubated with T cell clones or TCR-transduced PBMCs (50,000 cells / well). Where indicated, target cells were pulsed with specific concentrations of peptide for 1-2 h or electroporated with mRNA encoding full-length TdT, washed, and then co-cultured with effector cells. After 14-16 h of co-incubation, plates were centrifuged at 400 × g for 3 min. Culture supernatants were collected for measurement of IFN-γ by ELISA, and the remaining cells were stained for flow cytometry to detect viable CD8 + The upregulation of CD137 on TCR-transduced CD8 + CD137 in T cells + Percentage of CD8+ cells (transduction efficiency >90%) or CD137 + Events are reported as percentages. In some experiments, cells were labeled with 0.75 μM of either CellTrace Violet (CTV, Life Technologies) or carboxyfluorescein succinimidyl ester (CSFE, Life Technologies) fluorescent cell staining dyes to distinguish target from effector cells. The following reagents were obtained from BD Pharmingen or R&D Systems: mouse anti-human IFN-γ capture antibody (NIB42), biotin mouse anti-human IFN-γ detection antibody (4S.B3), streptavidin-HRP, stabilized tetramethylbenzidine and hydrogen peroxide as substrate solution, sulfuric acid as stop solution, and recombinant human IFN-γ protein as standard. Assays were performed according to the manufacturer's instructions.

[0189] Flow cytometry-based cytotoxicity assay using cell lines as targets For cytotoxicity assays against B- and T-ALL cell lines, 50,000 target cells in T cell medium were co-cultured in triplicate with T1 or T3 TCR-transduced PBMCs in round-bottom 96-well plates for 48 h. Effector cells were TCR-transduced CD8 + T cells (typically >90% transduction efficiency, CD8 + 55-65% are T cells, the rest are CD4 + T cells) with an effector to target ratio of 1:1. After co-culture, cells were harvested, washed, and stained with human anti-CD3, -CD8, -CD19 and live / dead fixed near infrared to exclude dead cells. After 15 min, cells were washed and resuspended in 200 μL of FACS buffer containing 10,000 CountBright Absolute Counting Beads (Thermo Fisher). During data acquisition, an equal number of bead events (5000) were recorded from each well. Data were normalized and reported as a percentage of the average live tumor cell count obtained from three parallel wells co-cultured with mock-transduced T cells from the same donor.

[0190] Flow cytometry-based analysis of T cell activation and cytotoxicity using primary human B-ALL and T-ALL samples. Peripheral blood or bone marrow samples from B-ALL and T-ALL patients were thawed and resuspended in T cell medium containing low concentrations of IL-7 and IL-15 (0.5 ng / mL). Cells were transferred to round-bottom 96-well plates for assays measuring CD137 upregulation on TCR-transduced T cells or cytotoxicity on target cells. Individualized antibody panels and gating strategies to identify malignant blast and normal leukocyte populations were designed after reviewing diagnostic phenotyping available in the hospital records. TCR-transduced, allogeneic T cells or, in the case of patient 1N, autologous T cells derived from the patient were used for the experiments. TCR-transduced cells were prelabeled with CTV dye to distinguish them from target cells. Whenever indicated, target cells were loaded with the relevant peptide for 1–2 h, washed, and then co-incubated with TCR-transduced T cells for measurement of CD137 upregulation, as described above.

[0191] For cytotoxicity assays, 50,000 target cells per well were co-incubated with an equal number of effector cells, 2–4 in parallel per condition, for 48–72 h and then stained with individualized antibody panels for flow cytometry. CountBright Absolute Counting Beads were utilized for acquisition normalization, and data were normalized and reported as above. To visually display flow cytometry plots, we utilized unsupervised nonlinear dimensionality reduction algorithms such as t-distributed stochastic neighbor embedding (t-SNE) using FlowJo (TreeStar) software.

[0192] In vivo TdT TCR T cell activity in two xenograft B-ALL cell line models These experiments used male and female NOD-scid IL2Rgnull (NSG) mice, aged 8-10 weeks, bred in-house. On day 11, mice were sublethally irradiated with 2.5 Gy using a MultiRad225 X-ray irradiator (RPS services). On day 10, mice were transfected with 4 × 10 cells of the human B-ALL cell line BV173 expressing GFP and firefly luciferase. 6Cells were injected through the tail vein. After leukemia was established and confirmed by bioluminescence imaging (BLI) on day 1, mice were treated with TCR-transduced PBMCs and transduced with either T1, T3 or control TCR targeting the cancer-testis antigen NY-ESO-1 (1G4) (32). A separate control group of mice did not receive any T cell injection. Mice were injected daily with 2500 IU of IL-2 (R&D Systems) by intraperitoneal injection, and BLI images (IVIS system, PerkinElmer) and blood analysis were performed by flow cytometry at different intervals. For survival analysis, mice were observed for clinical signs of tumor spread and were sacrificed if they developed more than 20% weight loss, hunched posture, wavy fur or quadriplegia. To avoid the risk of graft-versus-host disease, the experiment was terminated 2 months after T cell injection and surviving mice in the treatment group were humanely sacrificed. In one experimental group, bone marrow from surviving T3-treated mice was harvested at the time of sacrifice and processed for flow cytometry to analyze the presence of T cells and tumor cells as well as the expression of TdT and HLA-A2.

[0193] In vivo TdT TCR T cell activity in patient-derived xenograft models To establish primary human B-ALL xenograft mice, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG; Jackson Laboratory stock 005557) mice aged 9-15 weeks were sublethally irradiated by two doses of 1.65 Gy (X-ray source) at 4-h intervals. pos Viable bone marrow cells from B-ALL patient 20O were identified by 7AAD exclusion, and T cell-depleted bone marrow cells were isolated from CD40 mice. 3+ Yield sorting was performed on a BD FACS Aria™ Fusion by cell exclusion. 4×10 5 Viable CD3-negative bone marrow cells were injected via the tail vein of NSG mice 4–6 h after the final radiation dose. Stable engraftment was confirmed by both PB analysis and bone marrow aspirates from all transplanted mice 18–19 and 20–26 days after transplantation, respectively.

[0194] NSG mice were assigned to untreated, DMF5 and T3 T cell groups based on their engraftment levels, such that the mean human leukemia engraftment was comparable between treatment groups. 6 mTCRβ + CD8 + T cells were injected 22-25 days after primary B-ALL cell transplantation and all groups received 2500 IU IL-2 (R&D Systems) intraperitoneally (IP) per mouse daily. Engraftment was monitored in PB 3 and 10 days after T cell injection. At the time of sacrifice of mice 11 days after T cell injection, bone marrow, spleen and PB were subjected to detailed flow cytometric analysis with human anti-CD45, -CD8a, -CD4, -CD3, -CD19, -HLA-A2, -CD10 and mouse anti-CD45, -Ter119 and -TCRβ. For analysis of BM samples, a minimum of 1.8 × 10 5 events were acquired for every mouse, with at least 3 × 10 5 events were acquired for all but two T3 cell-treated mice and MRD levels were determined according to NOPHO guidelines.

[0195] Cell counts in bone marrow (2 tibia, 2 femur, and 2 crest) and spleens of sacrificed mice were measured using a Sysmex blood cell counter. TrueCount beads (BD Biosciences) were added to total PB according to the manufacturer's instructions and stained for mouse CD45.1 and human CD45 to determine absolute MNC counts per μL of blood.

[0196] For each tissue, leukemic burden and TCR-transduced CD8 + The cells were each treated with human CD45 + CD19 + CD10 + and human CD45 + CD3 + CD8 + mTCRβ + Cells were quantified based on frequency and in relation to total cell number.

[0197] In vivo effects of T3 cell treatment on normal human hematopoiesis in humanized NSG mice HLA-A2 pos NSG mice stably engrafted with human umbilical cord blood cells were purchased from Jackson Laboratory. Three mice were sacrificed 21 weeks after transplantation, when human engraftment was confirmed. Single cell suspensions from the spleens of three engrafted NSG mice were transduced with 1G4 or T3 TCR constructs and expanded as described above. The activity of NSG-derived 1G4 and T3 cells was confirmed in vitro by performing a flow cytometry-based cytotoxicity assay on BV173 cells in parallel with the injection into the remaining humanized NSG mice. 10 7 NSG-derived 1G4 or T3 cells were injected into engrafted mice via the tail vein, and the effect of the injected cells on normal hematopoiesis was examined in PB, spleen, thymus, and bone marrow 17 days after T cell injection. Half of the mice did not receive a supportive infusion of IL-2 at a daily IP dose of 500 IU, as the engrafted mice also contained endogenous cord blood-derived T cells capable of producing IL-2 (data from these groups were combined, as no difference was observed with or without IL-2 supportive infusion). Survival of the infused T cells was monitored in PB, and the effect of treatment on mature blood lineages was monitored in PB, spleen, thymus, and bone marrow by flow cytometry using human anti-CD45, -CD8a, -CD19, -CD33, -CD4, -CD3, and mouse anti-CD45, -Ter119, and TCRβ. The effect on human T cell precursors in the mouse thymus was examined by surface and intracellular staining with human anti-TdT, -CD45, -CD8 -CD19, -CD3 (intracellular and surface), -CD4, -HLA-A2, and mouse anti-CD45. Cell counts in bone marrow (two tibias, two femurs, and two crests) of sacrificed mice were performed using a Sysmex blood cell counter.

[0198] In vitro effects on clonogenic potential of normal hematopoietic progenitor cells Four HLA-A2 individuals from Karolinska University Hospital posBone marrow mononuclear cells were obtained from healthy donors with informed consent and medical approval (EPN 2018 / 901-31). + Progenitor cells were identified by DAPI and mature lineage exclusion and sorted on a BD FACS Aria Fusion. + Lineage precursors were isolated from 500 CD4 - CD19 - 1G4, T1 or T3 TCR-transduced T cells (identified by anti-human CD4-PE-Cy5 and anti-human CD19-PE-Cy5 and sorted on a BD FACS Aria Fusion) were incubated with 10% BIT9500 (Stem cell technologies), penicillin / streptavidin (100 U / mL; Hyclone Laboratories), 2-mercaptoethanol (2-ME; 0.1 mM; Sigma-Aldrich), and 0.1% ethanol (0.1 mM; Sigma-Aldrich). Aldrich), stem cell factor (SCF; 10 ng / mL), flt3 ligand (FL; 10 ng / mL), thrombopoietin (TPO; 10 ng / mL), interleukin 3 (IL3; 5 ng / mL), granulocyte colony-stimulating factor (G-CSF; 10 ng / mL), granulocyte-macrophage colony-stimulating factor (GM-CSF; 10 ng / mL), and erythropoietin (EPO; 1 U / mL) were added to StemSpan SFEM (Stem cell technologies) at 37°C and 5% CO2. + Lineage precursors were used as controls.

[0199] After 72 hours, cells from the co-cultures were transferred to cytokine-containing methylcellulose (MethoCult H4434, StemCell Technologies) in Iscove's modified Dulbecco's medium (IMDM; Gibco) supplemented with 20% fetal bovine serum (FBS, Sigma Aldrich), L-glutamine (2 mM; Sigma Aldrich), penicillin / streptavidin (100 U / mL) and 2-ME (0.1 mM) to generate colonies. After 14 days in methylcellulose, colonies were scored under an inverted microscope as myeloid or erythroid. As a positive control, CD34 + Lineage precursors were exogenously challenged with 1 μM peptide-1 or peptide-3 in StemSpan SFEM for 2 h, followed by co-culture with or without transduced T cells in the presence of 100 nM peptide for 48 h, and colonies were scored as described above 10 days after transferring cells to methylcellulose.

[0200] (statistical analysis) Statistical analysis was performed using GraphPad Prism version 6 or 7 (GraphPad Software). For BLI signal analysis, a regular one-way analysis of variance (ANOVA) test with adjustment for multiple comparisons with Tukey's post-test was employed. Survival analysis was performed by Log-rank (Mantel-Cox) test. To clarify the differences between in vivo treatment groups in PDX and humanized NSG mouse models, Kruskal-Wallis ANOVA with Dunn's multiple comparison test and Mann-Whitney test was performed. P<0.05 was considered statistically significant.

[0201] [result] (TCR T1 and TCR T3 specifically recognize the TdT peptide in an HLA-A2 restricted manner) Two TdT-derived peptides identified as HLA-A2 binders (peptide-1 (SEQ ID NO: 1) and peptide-3 (SEQ ID NO: 15)) were used to obtain T cell clones that recognize the TdT protein. The TCR sequences from the three peptide-1 reactive clones analyzed were identical and were designated T1, and the sequence from the peptide-3 reactive clone was designated T3.

[0202] Both TCRs express third-party peripheral blood (PB) CD8 T cells as demonstrated by staining with pMHC multimers or with anti-mouse TCR-β antibodies reactive to mouse constant regions introduced into the TCR. + It was efficiently expressed in T cells (data not shown). All T3-transduced cells and the majority of T1-transduced cells that stained positive with anti-mouse TCR-β were pMHC-multimer positive, indicating preferential pairing of the introduced TCR α and β chains after retroviral transduction (data not shown).

[0203] T1 and T3 recognized their cognate peptides with high sensitivity (T1 EC 50 = 5.8nM and T3 EC 50 = 1.2 nM, Figure 2A).

[0204] T cells transduced with T1 and T3 TCRs (hereafter referred to as T1 and T3 cells) recognized antigens in an HLA-restricted manner. T1 and T3 cells were expressed as TdT pos HLA-A2 neg HLA-A2 presents either an exogenously loaded peptide or an endogenously processed antigen derived from the mRNA encoding full-length TdT, but not by EBV-LCL pos EBV-LCL activated the cells (Fig. 2B). pos But HLA-A2 neg The cell lines REH (B-ALL origin) and HPB-ALL (T-ALL origin) of * 02:01 was introduced into T1 and T3 cells. Furthermore, we transfected T1 and T3 cells with human TdT cells of various tissue origins. negWhen co-cultured with a large panel of cell lines, we did not observe any TCR activation unless they were loaded with the relevant peptide and expressed HLA-A2 either natively or transgenic (Figure 2C). These results indicate that the T1 and T3 TCRs do not react with unintended targets presented on HLA-A2 or on the wide variety of other HLA molecules expressed by the cell lines.

[0205] (Mapping of TCR T1 and TCR T3 reactivity does not reveal cross-recognition peptides) In preclinical testing of candidate TCRs, potential clinically relevant cross-reactivity should be excluded. To this end, we employed a combination of empirical and computational approaches to identify potential off-target toxicity. We first synthesized peptide mimotope libraries encoding peptide-1 and peptide-3 sequences in which the amino acid residue at each position was replaced, one at a time, by all other natural amino acids. T1 and T3 cells were combined with target cells loaded with single mimotopes from the relevant libraries. The resulting T cell activation, as measured by IFN-γ production or CD137 upregulation, was highly correlated (Figure 3A). We then queried the curated human proteome database UniProtKB / Swiss-Prot and Protein Data Bank for all combinations of aa substitutions inducing reactivity in T1 or T3 cells by using the ScanProsite tool (https: / / prosite.expasy.org / scanprosite / ). The search did not identify any naturally occurring 9-mer or 11-mer peptides in the human proteome that matched these combinations.

[0206] Searching the uncurated database UniProtKB / TrEMBL, which contains approximately 261 times more entries than UniProtKB / Swiss-Prot, we found one peptide derived from small integral membrane protein 19 (GLFMYAKRIFG) that matched the combination of reactivity to T3. However, upon functional challenge, this peptide did not induce any response in T3 cells (not shown). Taken together, the data did not provide evidence of off-target reactivity with T1 and T3. Furthermore, peptides shifted upstream and / or downstream of peptide-1 and 3 sequences in the TdT protein sequence were unable to activate T1 and T3 cells unless longer mutants encompassing the entire cognate peptide sequence were used (Figure 3B-C). Furthermore, peptide-1 did not activate T3 cells, whereas peptide-3 activated T1 cells only at high concentrations, possibly due to degradation of peptide-3 to generate peptide-1 in culture.

[0207] T1 and T3 mediate rejection of disseminated leukemia in a mouse model. Next, we investigated whether T1 and T3 cells could kill leukemia cell lines that naturally express TdT. T1 and T3 cells killed the B-cell leukemia cell lines BV173 and NALM6 (which naturally express TdT) as measured by IFN-γ production, proliferation, and killing. pos and HLA-A2 pos ) (96%–99% at low effector-to-target ratios (E:T = 1:1)) (Figure 4A–B).

[0208] To study the in vivo efficacy, BV173ffluc-eGFP cells were engrafted into NOD-scid IL2Rgnull (NSG) mice, and treatment with T1 or T3 cells was initiated after leukemia establishment. In these mice, tumor signals were very low (T1) or absent (T3) on day 21 (BV173, Fig. 5A,B) and day 14 (NALM-6, Fig. 5D,E), and shortly thereafter, untreated and 1G4-treated control mice (control TCR for NY-ESO-1) had to be sacrificed due to high tumor burden (Fig. 5C,F). Notably, in mice treated with T3 cells, none of the mice died of leukemia during the observation period after injection of leukemia cells (Fig. 5C,F). Two T3 cell-treated mice in the NALM-6 model died of unknown causes, unrelated to the spread of leukemia. Bioluminescence imaging (BLI) remained negative in T3 cell-treated mice in the NALM-6 model at day 57, consistent with the absence of tumors in the bone marrow of sacrificed mice (not shown). Only one of five T3-treated BV173 mice had GFP in the bone marrow at the time of sacrifice. + In contrast, 1G4 TCR-transduced T cells initially expanded, presumably due to the injection of IL-2. The survival benefit was also highly significant for BV173 mice treated with T1 cells (Fig. 5C), although some of these mice eventually developed tumors, consistent with the lower peptide sensitivity to T1 compared to T3.

[0209] (T1 and T3 eliminate primary blasts from B-ALL and T-ALL but spare normal lymphocytes and non-lineage-restricted hematopoietic progenitors) We next quantified the ability of T1 and T3 cells to selectively recognize human primary ALL cells in samples containing normal B and T cells and non-lineage-restricted hematopoietic progenitor cells. We then assayed various proportions of leukemic blast cells, normal B and T cells, and normal CD34 + Hematopoietic progenitor cells (CD34 + lin - ) from 9 B-ALL and 3 T-ALL patients. pos and HLA-A2pos Diagnostic samples were co-cultured with T1 or T3 cells. After 48-72 hours of co-culture, T3 cells eliminated an average of 97% and T1 cells 69% of leukemic blasts (T3: average 97%, range 92%-99.9%; T1: average 69%, range 13%-96%, n=12). In contrast, normal B and T cells were unaffected (Figure 6A-B), and non-cancerous CD34 cells were detected in four cases. + lin - cells were also unaffected (Fig. 6A).

[0210] Next, the present inventors investigated HLA-A2 pos TdT pos We showed that introduction of T3 into normal T cells from a B-ALL patient resulted in activation of the T cells and elimination of virtually all autologous tumor cells (Figure 7A). + lin - Progenitor cells were spared (Figure 7A-B). Collectively, these data demonstrate a high degree of target selectivity and therapeutic efficacy of T1 and T3 cells against primary leukemia cells with representative TdT and HLA-A2 expression.

[0211] Further studies show that T3 cells efficiently eliminate primary B-ALL cells while maintaining healthy hematopoiesis in vivo. We next investigated the efficacy of T3 cell treatment in NSG mice stably engrafted with cells from patients with primary B-ALL leukemia. Untreated mice and mice treated with control MART-1 (DMF5) (33) or T3 TCR-transduced T cells had high and comparable leukemic burdens in the bone marrow at baseline (untreated mean 9.7±3.4%, DMF5 mean 15.6±5.7%, T3 mean 15.4±7.2%), providing stringent conditions for validating therapeutic efficacy. We utilized T cells matched for HLA-A2 expression but otherwise not HLA-matched to ALL. Injected T cells were primarily naive and central memory T cells and an average of 34% CD4 T cells, similar to clinical products that drive antitumor reactivity in adoptive T cell therapy. +The mice contained T cells. However, a high proportion of naive T cells as well as a high burden of leukemic cells that are not HLA-matched to the T cells in treated mice also promote alloreactivity. We therefore monitored mice for possible development of a graft-versus-leukemia effect by comparing leukemic engraftment in the blood of DMF5 (control TCR)-treated mice with untreated mice, and performed a detailed end-stage analysis before the results were confounded by significant alloreactivity. This was observed 10-11 days after treatment. At the end point of this experiment, the leukemic burden in the control mice group was very high and comparable in the bone marrow (mean untreated 42.9 ± 3.4%, mean DMF5 40.1 ± 4.5%), but only decreased to 0.009 ± 0.005% after treatment with T3 cells (Figure 8A-D).

[0212] The leukemic burden in peripheral blood and spleen in untreated and DMF5 cell-treated mice was lower than in bone marrow. Nevertheless, a similar and almost complete leukemic cell disappearance was observed in mice treated with T3 cells (PB: mean 0.003±0.001%; ​​spleen: mean 0.002±0.001%) (data not shown). Taken together, the leukemic burden was reduced to a level considered minimal residual disease (MRD) negative (<0.1%) according to the criteria set by the NOPHO-2008 protocol (NCT00816049). TCR-transduced T cells were detected in bone marrow, PB, and spleen of all mice (not shown). Murine hematopoiesis suppressed in the bone marrow of leukemic engrafted mice was significantly higher in T3 mice than in DMF5 mice (not shown).

[0213] Normal peripheral B and T cell repertoires were TdT negative and were not affected by T3 or T1 TCRs during in vitro killing assays or proliferation of transduced cells (see, for example, Figure 6B). 14Studies using C dating support the view that the repertoire of naive T cells is sufficiently diverse to maintain peripheral adaptive T cell immunity throughout life, even in young individuals, and thus potential toxic effects on thymocytes are not of major concern even during prolonged treatment. However, the thymic contribution to the peripheral repertoire is higher in individuals younger than 20 years, raising the possibility that toxicity may be higher in this age group. To answer this question, we studied the expression of TdT and HLA-A2 during human thymocyte differentiation. We found that interestingly, HLA-A2 expression was significantly associated with TdT cell differentiation, including early double negative (DN) and single positive (SP) cells. neg We found that HLA-A2 expression was high only on thymocytes, whereas in late DN cells transitioning into double-positive (DP) cells, HLA-A2 expression was downregulated concomitantly with the upregulation of TdT (data not shown).

[0214] We next performed humanized CD34 T cells injected with autologous T3- or 1G4-transduced human T cells harvested from mouse spleens. + We investigated the effect of T3 cell therapy on thymocytes in NSG mice. T3 cells were treated with TdT in vitro before injection. pos It was demonstrated to effectively kill cell lines (not shown). pos The percentage of thymocytes was similar in T3- and 1G4-treated mice. HLA-A2 levels were significantly higher than surface CD3 in both experimental groups. + TdT neg Compared with thymocytes, TdT pos Correspondingly, only a small proportion of human thymocytes were recruited by umbilical cord blood progenitor cells in the mouse thymus, and TdT was observed in both groups, as was also observed for thymocytes derived from the human thymus. high and HLA-A2 high(not shown). The experiment was terminated 17 days after T cell injection. At this time, TCR-transduced T cells were still detectable in the PB, and the percentage of 1G4-transduced T cells declined faster than that of T3-transduced T cells, possibly suggesting low-level antigen stimulation of T3 T cells (not shown). Analysis of the distribution of human myeloid and lymphoid lineages in the PB, bone marrow, spleen and thymus, and cellularity in the bone marrow, showed no differences between the 1G4 and T3 groups (not shown). Further supporting the lack of toxicity to hematopoietic stem and myeloid progenitor cells, adult CD34 + The formation of myeloid and erythroid colonies from bone marrow progenitor cells was not adversely affected following co-culture with T1 or T3 cells unless the co-culture was performed in the presence of peptides 1 or 3 (not shown).

[0215] [Conclusion] Our studies demonstrate that T cells transduced with a TCR that recognizes peptides derived from the novel cancer target TdT in the context of allogeneic HLA-A2 inhibit TdT of B and T cell origin in vitro. pos HLA-A2 pos The results show that TdT TCR efficiently killed lymphoblastic leukemia cells derived from the patient. pos It was highly efficient in killing ALL cell lines as well as primary ALL cells. Furthermore, the TdT TCR mediated the elimination of engrafted leukemia in vivo in three different mouse models of B-ALL. Our data further indicate that the TCR can be used therapeutically without adverse toxicity.

[0216] The inventors further demonstrated that the specificity for TdT peptides and HLA-A2 restriction is enhanced in mice in which TdT is knocked out or HLA-A2 is absent, pos We have generated data, not presented here, showing that the cognate peptides presented by HLA on primary leukemic cells were directly identified by mass spectrometry, confirmed by loss of reactivity to leukemic cells. neg Various HLA-A2 posNo cell lines were recognized, and mapping of TCR reactivity did not identify naturally occurring 9-mer or 11-mer peptides in the human proteome that matched the substituted peptide combinations to which T1 or T3 were reactive. Taken together, the data demonstrating the high potency and specificity of the TdT TCR combined with the unique expression profile of TdT provide a promising new therapeutic approach to TdT TCR, encompassing a patient population currently with a poor prognosis. pos Opens up new possibilities for the treatment of acute leukemia.

[0217] Description of sequences in the sequence listing [Table 1-1] [Table 1-2]

Claims

1. A TCR-derived binding protein capable of specifically binding to a peptide-presenting human leukocyte antigen (HLA) complex type A2, comprising: (A) the peptide has the amino acid sequence ALYDKTKRIFL set forth in SEQ ID NO: 15; the protein comprises an antigen-binding unit derived from a T3 TCR, the antigen-binding unit comprising an alpha chain variable domain and a beta chain variable domain; wherein the alpha chain variable domain contains three complementarity determining regions (CDRs): CDR1, CDR2, and CDR3, which contain the amino acid sequences set forth in SEQ ID NOs: 16, 17, and 18, respectively; the beta chain variable domain contains three CDRs: CDR1, CDR2 and CDR3, which contain the amino acid sequences set forth in SEQ ID NOs: 19, 20 and 21, respectively; or (B) the peptide has the amino acid sequence ALYDKTKRI set forth in SEQ ID NO: 1; the protein comprises an antigen-binding unit derived from a T1 TCR, the antigen-binding unit comprising an alpha chain variable domain and a beta chain variable domain; wherein the alpha chain variable domain contains three complementarity determining regions (CDRs): CDR1, CDR2, and CDR3, which contain the amino acid sequences set forth in SEQ ID NOs: 2, 3, and 4, respectively; the beta chain variable domain contains three CDRs: CDR1, CDR2 and CDR3, which contain the amino acid sequences set forth in SEQ ID NOs: 5, 6 and 7, respectively; Binding proteins.

2. i) In (A), the α chain variable domain contains the amino acid sequence set forth in SEQ ID NO: 22 or an amino acid sequence having 90% or more sequence identity thereto, and the β chain variable domain contains the amino acid sequence set forth in SEQ ID NO: 23 or an amino acid sequence having 90% or more sequence identity thereto. or ii) In (B), the α chain variable domain contains the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having 90% or more sequence identity thereto, and the β chain variable domain contains the amino acid sequence set forth in SEQ ID NO: 9 or an amino acid sequence having 90% or more sequence identity thereto. The binding protein of claim 1.

3. 2. The binding protein of claim 1, wherein the binding protein comprises a first chain containing the alpha chain variable domain and a second chain containing the beta chain variable domain, and optionally the first chain further contains an extracellular alpha chain constant domain and the second chain further contains an extracellular beta chain constant domain.

4. i) the extracellular alpha chain constant domain contains the amino acid sequence set forth in SEQ ID NO: 10 or an amino acid sequence having 90% or more sequence identity thereto, and The extracellular β chain constant domain contains the amino acid sequence set forth in SEQ ID NO: 11 or an amino acid sequence having 90% or more sequence identity thereto. ii) the first chain and / or the second chain further contain a transmembrane domain; and / or iii) the first chain and / or the second chain further contain a cytoplasmic domain; The binding protein of claim 3.

5. (a) the first chain is an α chain containing the amino acid sequence set forth in SEQ ID NO: 24, and the second chain is a β chain containing the amino acid sequence set forth in SEQ ID NO: 25; or (b) the first chain is an α chain containing the amino acid sequence set forth in SEQ ID NO: 12, and the second chain is a β chain containing the amino acid sequence set forth in SEQ ID NO: 13; The binding protein of claim 3

6. A recombinant nucleic acid molecule encoding a binding protein as defined in any one of claims 1 to 5, optionally wherein said nucleic acid molecule comprises a cDNA molecule.

7. 7. The recombinant nucleic acid molecule of claim 6, wherein the nucleic acid molecule encodes a polypeptide comprising a first chain linked to a second chain, wherein preferably the first chain is linked to the second chain by a self-splicing linker, and optionally the self-splicing linker is a 2A peptide comprising the amino acid sequence set forth in SEQ ID NO: 26 or an amino acid sequence having 50% or greater sequence identity thereto.

8. A vector containing the recombinant nucleic acid molecule of claim 6.

9. 1. A kit containing a first nucleic acid molecule encoding a first chain of a binding protein and a second nucleic acid molecule encoding a second chain of the binding protein, The first chain and the second chain each contain an α chain variable domain and a β chain variable domain as defined in claim 1. kit. (i) a recombinant nucleic acid molecule encoding a binding protein as defined in any one of claims 1 to 5; (ii) a vector comprising the recombinant nucleic acid molecule of (i); or (iii) a first nucleic acid molecule encoding a first chain of a binding protein and a second nucleic acid molecule encoding a second chain of a binding protein, wherein the first chain and the second chain are an α-chain variable domain and a β-chain variable domain, respectively, as defined in any one of claims 1 to 5; and expressing the binding protein in its cell membrane; cell.

11. The cells i) is an immune effector cell or a precursor thereof, or ii) is a T cell or natural killer cell or a precursor thereof, or the cell is a stem cell, and optionally is a T helper cell or a cytotoxic T cell; The cell of claim 10.

12. A pharmaceutical composition comprising the cells defined in claim 10.

13. 11. A pharmaceutical composition comprising cells as defined in claim 10 for the treatment of cancer, wherein the cancer expresses terminal deoxynucleotidyl transferase (TdT).

14. 14. The pharmaceutical composition of claim 13, wherein the cancer is acute lymphoblastic leukemia, preferably acute lymphoblastic leukemia of T-cell or B-cell origin.

15. 1. A method for generating terminal deoxynucleotidyl transferase (TdT)-specific cells, the method comprising: (i) a recombinant nucleic acid molecule as defined in any one of claims 1 to 5; (ii) a vector comprising the recombinant nucleic acid molecule of (i); or (iii) A first nucleic acid molecule encoding a first chain of a binding protein and a second nucleic acid molecule encoding a second chain of a binding protein, wherein the first chain and the second chain contain an α-chain variable domain and a β-chain variable domain, respectively, as defined in any one of claims 1 to 5. The method includes introducing wherein the cell is a T cell or NK cell or a precursor thereof, and preferably the T cell is a T helper cell or a cytotoxic T cell.

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