PRAME TCR receptor and its use

Abstract

The present invention relates to a T cell receptor (TCR) capable of binding to the PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), or a portion thereof, or an HLA-A2-binding form thereof. Also included in the invention are nucleic acids encoding the TCR, vectors containing the nucleic acids, and host cells containing the TCR, the nucleic acid sequence, or the vector. Also included are methods, pharmaceutical or diagnostic compositions for obtaining the TCRs described herein, and in vitro methods for detecting the presence of cancer in a subject. Furthermore, the present invention relates to the use of the TCRs, nucleic acids, and / or vectors to obtain modified lymphocytes.

Description

[Technical Field]

[0001] The present invention relates to a T cell receptor (TCR) having the ability to bind to the PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), or a portion thereof, or its HLA-A2 binding form. The present invention also includes nucleic acids encoding the TCR, vectors containing the nucleic acid, and host cells containing the TCR, the nucleic acid sequence, or the vector. Further included are methods for obtaining the TCR described herein, pharmaceutical or diagnostic compositions containing the TCR, nucleic acid, vector, and / or host cells, and methods for detecting the presence of cancer in a subject in vitro, comprising using the TCR, host cells, and / or pharmaceutical composition. Furthermore, the present invention also relates to using the TCR, nucleic acid, and / or vector to generate modified lymphocytes. [Background technology]

[0002] T lymphocytes (or T cells), which form part of the cell-mediated immune system, play a major role in eliminating pathogens. T cells develop in the thymus and express T cell receptor molecules on their surface that enable them to recognize peptides (antigen presentation) presented on major histocompatibility complex (MHC) molecules expressed on nucleated cells. Pathogen antigens, i.e., foreign antigens presented by MHC molecules, will trigger a potent T cell response. On the other hand, self-antigen-specific T cells undergo negative selection in the thymus during their development, so self-antigens usually do not elicit a T cell response. Therefore, the immune system can distinguish between nucleated cells presenting foreign antigens and nucleated cells presenting self-antigens, and specifically target infected cells, eliminating them through the potent cytokine release and cytotoxic mechanisms of T cells.

[0003] The capabilities of the immune system are recognized as a promising tool for future cancer treatment. Over the past decade, research has begun to leverage the unique properties of T cells through adoptive cell transfer (ACT). In ACT, donor-derived lymphocytes are proliferated ex vivo and administered. ACT is an attractive concept for cancer treatment because it does not require the patient's immune capacity, has low immunogenicity due to the absence of mutations, and allows for the specificity of the transferred lymphocytes to target tumor antigens that typically fail to effectively induce an autologous T-cell response. Although ACT has shown promise as a treatment for various types of cancer, the need to specifically isolate and characterize tumor-specific T cells from each patient (a process that can be difficult and time-consuming, and often fails to yield highly avidity T cells) is an obstacle to its widespread clinical application (Non-Patent Literature 1, Non-Patent Literature 2).

[0004] Genetically introducing tumor antigen-specific T cell receptors (TCRs) into primary T cells allows for the rapid generation of tumor-reactive T lymphocytes with defined antigen specificity, even in immunocompromised patients, thereby overcoming some of the current limitations of ACT. However, identifying appropriate TCR-possessing T cell clones that specifically recognize tumor antigens and exhibit desired antitumor effects in vivo remains an ongoing research challenge. Given that there were approximately 14.1 million new cancer cases worldwide in 2012, and cancer currently accounts for about 14.6% of all human deaths globally, there is an urgent need for novel and effective treatment options. Meeting these needs is the objective of this invention.

[0005] PRAME is a tumor-associated antigen expressed in various tumors, preferably melanoma. Furthermore, PRAME has been described as an independent biomarker of metastasis in uveal melanoma and other tumors (Non-Patent Literature 3), and as a prognostic marker for DLBCL (Non-Patent Literature 4). PRAME is not expressed in normal tissue except in the testes. This expression pattern is similar to that of other cancer-testis (CT) antigens (MAGE, BAGE, and GAGE, etc.). However, unlike these other CT antigens, this gene is also expressed in acute leukemia. The encoded protein is thought to act as a repressor of the retinoic acid receptor, thereby conferring a proliferative advantage to cancer cells. Alternative splicing results in multiple transcript variants. Overexpression of PRAME in triple-negative breast cancer has also been shown to promote cancer cell motility through the induction of epithelial-mesenchymal transition (Non-Patent Literature 5). While PRAME deletion has been reported in chronic lymphocytic leukemia, this is not functionally related, as this gene is not expressed in B cells. This deletion is a result of physiological rearrangement of the immunoglobulin light chain. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Xue et al.,Clin Exp Immunol.2005 February;139(2):167-172 [Non-Patent Document 2] Schmitt et al.,Hum Gene Ther.2009 November;20(11):1240-1248 [Non-Patent Document 3] Fiedl et al., Clin Cancer Res 2016 March;22(5):1234-1242 [Non-Patent Document 4] Mitsuhashi et al.,Hematology 2014,1 / 2014 [Non-Patent Document 5] Al-Khadairi et al., Journal of Translational Medicine 2019;17:9 [Overview of the project]

[0007] This invention relates to a novel T cell receptor (TCR) having the ability to specifically recognize the tumor-associated antigen PRAME. Specifically, the identified TCR is the PRAME amino acid sequence SLLQHLIGL (PRAME as specified herein). SLL It specifically recognizes the PRAME peptide. This invention is at least in part based on the surprising discovery that this isolated T cell receptor, which binds to this specific PRAME peptide, has superior properties compared to PRAME TCRs known in the prior art. Specifically, the T cell receptor of the present invention, which has the ability to bind to the PRAME peptide SLLQHLIGL, also provides high functional avidity as well as advantageous tumor cell recognition and tumor cell killing properties. In contrast to tumor cells, normal cells and unrelated peptides are not recognized by the TCR. Furthermore, this T cell receptor recognizes the peptide presented on HLA molecules (human leukocyte antigens (HLA)), particularly the HLA subalele HLA-A * 02:01, HLA-A * 02:02, and HLA-A * PRAME recognizes the peptide presented on the HLA molecule encoded by 02:04. SLL Low-frequency HLA-A * This could even make it possible to treat cancer patients who express the 02 allele.

[0008] In a first aspect, the present invention relates to a T cell receptor (TCR) having the ability to bind to a PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), or a part thereof, or its HLA-A2 binding form, wherein the TCR comprises a CDR3 of the TCR alpha chain variable region comprising or consisting of the amino acid sequence of (SEQ ID NO: 6), or an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 6, and / or a CDR3 of the TCR beta chain variable region comprising or consisting of the amino acid sequence of (SEQ ID NO: 7), or an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 7.

[0009] In another aspect, the present invention relates to a T cell receptor (TCR) having the ability to bind to a PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), or a part thereof, or its HLA-A2 binding form, wherein this TCR is a) CDR3 of the TCR alpha chain variable region comprising or consisting of the amino acid sequence of (SEQ ID NO: 6), or an amino acid sequence that is at least 80%, preferably at least 85%, more preferably 90% or 95% identical to SEQ ID NO: 6; CDR1 of the TCR alpha chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and CDR2 of the TCR alpha chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 4. b) CDR3 of the TCR beta chain variable region comprising or consisting of the amino acid sequence of (SEQ ID NO: 7), or an amino acid sequence that is at least 80%, preferably at least 85%, more preferably 90% or 95% identical to SEQ ID NO: 7, CDR1 of the TCR beta chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and CDR2 of the TCR beta chain region comprising or consisting of the amino acid sequence of SEQ ID NO: 5, Includes.

[0010] HLA-A2 is HLA-A * 02:01, HLA-A *02:02, or HLA-A * It is assumed to be a molecule encoded by 02:04.

[0011] Specifically, when the TCR binds to the sequence SLLQHLIGL (SEQ ID NO: ^{-1}) or preferably a functional portion thereof, or its HLA-A2 binding form, it is assumed that the secretion of IFN-γ is induced by the cells into which the TCR has been transduced or transfected.

[0012] When measured by an IFN-γ immunoassay, the concentration (EC 50 value) that gives 50% of the maximum relative IFN-γ secretion amount is preferably less than 10 -7 M.

[0013] The TCR of the present invention a) a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 2, a CDR2 having the amino acid sequence of SEQ ID NO: 4, and a CDR3 having the amino acid sequence of SEQ ID NO: 6, and / or b) a TCR beta chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 3, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 7 is also assumed to be included.

[0014] Based on the present invention, the TCR referred to herein may include a TCR alpha chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8, and / or a TCR beta chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 9.

[0015] It is also assumed that the TCR of the present invention includes a TCR alpha chain constant region and / or a TCR beta chain constant region.

[0016] Preferably, the TCR of the present invention a) A TCR alpha chain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 10, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 10, and / or b) A TCR beta chain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 11, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 11. Includes.

[0017] The TCR of the present invention may also include at least one TCR alpha chain and at least one TCR beta chain, which are covalently linked to each other to form a TCR heterodimer or TCR polymer.

[0018] In light of the present invention, it is assumed that the TCRs referred to herein may be selected from natural TCRs, TCR variants, TCR fragments, or TCR constructs.

[0019] In some embodiments, the TCR of the present invention comprises one or more fusion elements optionally selected from Fc receptors, Fc domains (including IgA, IgD, IgG, IgE, and IgM), cytokines (including IL-2 or IL-15), toxins, antibodies or their antigen-binding fragments (including anti-CD3 antibodies, anti-CD28 antibodies, anti-CDS antibodies, anti-CD16 antibodies, or anti-CD56 antibodies, or their antigen-binding fragments), CD247 (CD3-zeta) domains, CD28 domains, CD137 domains, or CD134 domains, or combinations thereof, and optionally further comprises at least one linker.

[0020] The TCRs referred to herein preferably comprise at least one TCR alpha chain (as defined herein) and / or at least one TCR beta chain (as defined herein), and / or an antibody or single-chain antibody fragment (scFv) against an antigen or epitope on the surface of a lymphocyte, wherein the TCR alpha chain(s) and TCR beta chain(s) are linked together and fused to the antibody or scFv via an optional linker. The antigen may be selected from CD3, CD28, CD5, CD16, or CD56.

[0021] The TCR of the present invention preferably comprises at least one molecular marker. Preferably, the TCR of the present invention is soluble.

[0022] In another aspect, the present invention relates to nucleic acids encoding TCRs as referred to herein.

[0023] The nucleic acid may contain the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0024] In another aspect, the present invention relates to a vector comprising nucleic acids as defined herein.

[0025] In another aspect, the present invention relates to a host cell comprising a TCR as defined herein, a nucleic acid sequence as defined herein, or a vector as defined herein. The host cell can be selected from lymphocytes, and such lymphocytes include, but are not limited to, cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, and gamma / delta T cells.

[0026] In another aspect, the present invention relates to a method for obtaining a TCR as defined herein, the method comprising incubating a host cell as defined herein under conditions that express the TCR, and purifying the TCR.

[0027] In another embodiment, the present invention relates to a pharmaceutical composition or diagnostic composition comprising one or more of the following: TCRs as defined herein, nucleic acids as defined herein, vectors as defined herein, and / or host cells as defined herein, and an optional number of pharmaceutical excipients. The pharmaceutical composition may further comprise a checkpoint inhibitor. The checkpoint inhibitor may be selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors, LAG3 inhibitors, ICOS inhibitors, TIM3 inhibitors, VISTA inhibitors, and CEACAM1 inhibitors.

[0028] In another aspect, the present invention relates to a TCR as defined herein, a nucleic acid as defined herein, a vector as defined herein, and / or a host cell as defined herein, for use as a pharmaceutical. Preferably, this use is in the detection, diagnosis, prognosis, prevention, and / or treatment of cancer. In this regard, cancer is expected to be selected from the group consisting of melanoma, bladder cancer, colon cancer, and mammary gland adenocarcinoma, sarcoma, prostate cancer, uterine cancer, uveal cancer, uveal melanoma, head and neck squamous cell carcinoma, synovial cancer, Ewing's sarcoma, triple-negative breast cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia. Preferably, cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, or colorectal cancer, sarcoma, or osteosarcoma.

[0029] Furthermore, the use of TCRs, nucleic acids, vectors, and / or host cells as defined herein in the prevention and / or treatment of cancer is permitted. (a) supplying one or more of (i) TCRs as described elsewhere in this Spec., (ii) nucleic acids as described elsewhere in this Spec., (iii) vectors as described elsewhere in this Spec., (iv) host cells as described elsewhere in this Spec., and (v) pharmaceutical compositions as described elsewhere in this Spec. (b) Administer at least one of (i) to (v) to the person in need. It is assumed that this will be included.

[0030] Preferably, the TCRs, nucleic acids, vectors, and / or host cells as defined herein, for use in the prevention and / or treatment of cancer, (a) supplying the target sample, the sample containing lymphocytes, (b) supplying one or more of the following: (i) TCRs as described elsewhere in this Spec., (ii) nucleic acids as described elsewhere in this Spec., (iii) vectors as described elsewhere in this Spec., (iv) host cells as described elsewhere in this Spec., and (v) pharmaceutical compositions as described elsewhere in this Spec. (c) Introduce one or more of (i) to (v) of step (b) into the lymphocytes of step (b) to obtain modified lymphocytes. (d) Administer the modified lymphocytes of step (c) to the subject or patient who needs them. Includes.

[0031] In another aspect, the present invention relates to a method for detecting the presence of cancer in a subject in vitro, the method being: (a) supplying a sample, the sample containing one or more cells, (b) Contacting the sample with (i) a TCR as described elsewhere in this Spec., (ii) a host cell as described elsewhere in this Spec., and / or (iii) a pharmaceutical composition as described elsewhere in this Spec., thereby forming a complex, (c) detecting a complex, wherein the detection of the complex indicates the presence of cancer in the subject, Includes.

[0032] In yet another aspect, the present invention relates to using a TCR, a nucleic acid, and / or a vector as defined herein to generate modified lymphocytes. [Brief explanation of the drawing]

[0033] [Figure 1] Peptide specificity was demonstrated. T2 cells were loaded with either a specific SLL peptide (SLLQHLIGL) or an unrelated peptide (GLSNTHVL). These cells were then co-cultured with TCR-transduced T cells. After 20 hours, IFN-gamma levels in the cell culture supernatant were measured using IFN-gamma ELISA. The specific SLL peptide, upon loading onto T2 cells, was recognized by all TCR-transduced effector cells except for negative control TCRs, while the unrelated peptide was not recognized. [Figure 2] Functional avidity is observed. In co-cultures containing T2 cells loaded with gradually increasing doses of SLL peptide (10⁻⁵ M to 10⁻¹² M), the functional avidity of the TCR transgenic T cell population was measured as the concentration that yields 50% of the maximum relative IFN-gamma release (EC50 value). Cells transduced with TCR 027-004 showed higher functional avidity compared to T cells transduced with TCR 3825. [Figure 3A] This shows the TCR recognition motif (serine scan). TCR-transduced T cells and T2 cells loaded with a 10⁻⁵M peptide were co-cultured in vitro with an effector:target (E:T) ratio of 1:1 (10,000 effector cells / 96 wells). In this experiment, the only difference in the peptide loaded onto the T2 cells was that one amino acid position was sequentially substituted with the amino acid serine. T cells transduced with TCR (027-004 TCR) (A) show a different recognition motif with fewer fixed positions compared to TCR clone 3825 transduced T cells (B). [Figure 3B]This shows the TCR recognition motif (serine scan). TCR-transduced T cells and T2 cells loaded with a 10⁻⁵M peptide were co-cultured in vitro with an effector:target (E:T) ratio of 1:1 (10,000 effector cells / 96 wells). In this experiment, the only difference in the peptide loaded onto the T2 cells was that one amino acid position was sequentially substituted with the amino acid serine. T cells transduced with TCR (027-004 TCR) (A) show a different recognition motif with fewer fixed positions compared to TCR clone 3825 transduced T cells (B). [Figure 4] This demonstrates the recognition of tumor cells. Both TCR-transduced T cell populations (transduced from either TCR 027-004 or TCR 3825) recognize PRAMESLL-positive tumor cell lines. Recognition of PRAMESLL-positive cells is stronger with 027-004 TCR-transduced T cells compared with 3825 TCR-transduced T cells. PRAMESLL-negative tumor cells are not recognized by either of the TCRs tested. [Figure 5-1] This demonstrates tumor cell death. All TCR-transduced effector cells lyse PRAMESLL-positive (PRAME-positive) tumor cells and do not affect the proliferation of PRAMESLL-negative tumor cells (PRAME-negative). Effector cells transduced with TCR 027-004 kill PRAMESLL-positive cell lines more effectively than T cells transduced with TCR 3825. [Figure 5-2] Continuation of Figure 5-1. [Figure 5-3] Continuation of Figure 5-2. [Figure 5-4] Continuation of Figure 5-3. [Figure 5-5] Continuation of Figure 5-4. [Figure 5-6] Continuation of Figure 5-5. [Figure 6-1]This demonstrates the recognition of normal cells. The TCR-transduced T cell population does not recognize unloaded normal cells, resulting in high levels of IFN-gamma secretion. On the other hand, when cells are loaded with the specific PRAMESLL peptide, these cells are recognized by TCR 027-004. Only when co-cultured with the RPTEC cell line (an endogenously PRAME-positive cell line) does both effector preparations produce minimal IFN-gamma, even without loading. [Figure 6-2] Continuation of Figure 6-1. [Figure 7] This shows the allele frequency of HLA-A*02. Allele frequencies for American / European Caucasian racial groups (http: / / www.allelefrequencies.net). [Figure 8] This shows the precise HLA-A*02 typing of TCR 027-004. T cells transduced to TCR 027-004 were co-cultured in vitro with selected HLA-A2 subalele-positive lymphoblastoid cell lines (LCL; EBV-transformed B cells) at an effector:target (E:T) ratio of 1:2 (10,000 effector cells / 96 wells). Specific TCR subalele recognition was determined by loading each individual LCL with SLL peptide (10⁻⁵M). Unloaded target cells were used as negative controls. After 20 hours of co-culture, IFN-gamma secretion levels were determined using standard ELISA. TCR 027-004 efficiently recognizes PRAME peptides presented by 3 of the 10 HLA-A2 subaleles (A*02:xx) tested, with HLA-A*02 subaleles A*02:02 and A*02:04 being recognized at comparable levels to A*02:01. [Figure 9]This study demonstrates the precise HLA-A*02 typing of TCR 3825. T cells transduced to TCR 3825 were co-cultured in vitro with selected HLA-A*02 subalele-positive lymphoblastoid cell lines (LCL; EBV-transformed B cells) at an E:T ratio of 1:2 (10,000 effector cells / 96 wells). Specific TCR subalele recognition was determined by loading each individual LCL with SLL peptide (10⁻⁵M). Unloaded target cells served as negative controls. IFN-gamma secretion levels were determined using standard ELISA after 20 hours of co-culture. Control TCR 3825 efficiently recognized the PRAME peptide presented by one of the 10 HLA-A2 subaleles (A*02:xx) tested, specifically recognizing only HLA-A2 subalele A*02:01. [Figure 10] Figures 8 and 9 summarize the results. [Figure 11-1] This graph shows the proliferation of effector cells after transduction with TCR. The cell count of effector cells was determined using a hemocytometer on the day of transduction and after 13 days of proliferation. Each graph represents one donor. Since no effector cell proliferation was observed with TCR ImCorePrefCombi1, it was not possible to include TCR ImCorePrefCombi1 in further experiments. [Figure 11-2] Continuation of Figure 11-1. [Figure 12-1]This study demonstrates that TCR T23.8-2.1027-004 has a different recognition motif from the most recent TCRs in the relevant field. T2 cells loaded with PRAME-SLL-peptide (far right), or T2 cells loaded with an SLL-peptide variant in which a single amino acid was replaced with threonine, were co-cultured with TCR transduction effectors (effector cells). For readout, the supernatant was collected after 20 hours and analyzed by IFN-gamma ELISA. Each graph represents one TCR. The letters on the X-axis indicate the position of the amino acid substitution. The "fixed" position of the recognition motif is highlighted by a box on the X-axis. In TCR 46SLL, recognition of T2 cells loaded with the original PRAME-SLL peptide was not observed, while in TCR ImCore_Scaffold, a decrease in the recognition of T2 cells loaded with PRAME-SLL was observed. To avoid misunderstanding, please note that while the TCR of this invention is abbreviated as TCR 027-004 in this description, its full name is TCR T23.8-2.1-027-004. [Figure 12-2] Continuation of Figure 12-1. [Figure 12-3] Continuation of Figure 12-2. [Figure 13-1] This study demonstrates that the TCR T23.8-2.1-027-004 transduction effector exhibits higher functional avidity compared to effectors transduced from the most recent TCRs in the relevant field. The TCR T23.8-2.1-027-004 transduction effector (black) and other TCR transduction effectors (gray) were co-cultured with T2 cells loaded with gradually increasing amounts of PRAME-SLL-peptide. For readout, the supernatant was collected after 20 hours and analyzed by IFN-gamma ELISA. The dashed line indicates the peptide concentration required to yield 50% of the maximum relative IFN-gamma secretion. The graph shows a nonlinear regression curve representing optical density values. The EC50 values ​​of different TCR transduction effectors were calculated using nonlinear regression analysis. Each graph represents a comparison of TCR T23.8-2.1-027-004 with one of the most recent TCRs in the relevant field. [Figure 13-2] Continuation of Figure 13-1. [Figure 13-3]Continuation of Figure 13-2. [Figure 13-4] Continuation of Figure 13-3. [Figure 14] This study demonstrates that the T23.8-2.1-027-004 transduced effector strongly recognizes HLA-A*02:01 / PRAME double-positive tumor cell lines compared to effectors transduced with the latest TCRs in the relevant field. Six different TCR transduced effectors, as well as a non-transduced control effector, were co-cultured with the HLA-A*02:01 / PRAME double-positive tumor cell lines MelA375, NCI-H1650, and NCI-H1703. The supernatant of the co-culture was collected after 20 hours and analyzed by ELISA to determine the amount of IFN-gamma secreted. [Figure 15-1] This study demonstrates that the T23.8-2.1-027-004 transduction effector efficiently mediates the lysis of PRAME-positive tumor cells compared to effectors transduced with the latest TCRs in the relevant field. Tumor cell lines MelA375 and NCI-H1650 (HLA-A*02:01 positive / PRAME positive) transduced with NuclightRed were seeded in 96-well flat-bottom plates and co-cultured for 144 hours with non-transduced effectors (shown in gray), T23.8-2.1-027-004 transduction effectors (black circles), or effectors transduced with other TCRs. Apoptosis of tumor cells was visualized by the disappearance of red fluorescence. Non-transduced effectors were used as negative controls. The time course of the number of red cells (1 / mm2) obtained in three series is shown. [Figure 15-2] Continuation of Figure 15-1. [Modes for carrying out the invention]

[0034] The inventors of this invention have identified cells expressing the tumor-associated antigen PRAME (the full-length PRAME shown in SEQ ID NO: 33) and, in particular, the amino acid sequence SLLQHLIGL (SEQ ID NO: 1) (PRAME as shown herein). SLLWe have identified a T cell receptor (TCR) clone that has the ability to recognize cells expressing (also known as PRAME). This sequence is recognized in a specific manner, while unrelated peptides cannot be recognized (Figure 1). This specificity arises from the recognition of only PRAME-positive cancer cell lines (Figures 4 and 5), and normal cells that are PRAME-negative and not loaded with PRAME are not recognized by this TCR receptor. However, even in normal cells, PRAME SLL The peptide is recognized upon loading (Figure 6). This selective recognition is achieved by the recognition motif of the T cell receptor, which has only a very small number of fixed positions (Figure 3). The amino acids LLQ and especially HLI of the sequence SLLQHLIGL (Sequence ID 1) are part of this recognition motif. These amino acids exhibit advantageous effector functions (such as the high accumulation of multiple affinities to the PRAME peptide, resulting in increased strength (e.g., functional avidity, Figure 2)).

[0035] In short, the T cell receptor identified by the inventors of this invention is tumor-associated antigen (TAA) PRAME and especially PRAME SLL It has the ability to specifically recognize cells expressing HLA-A. The TCR exhibits advantageous effector functions (such as cytokine production and target cell lysis). Therefore, the T cell receptor is a promising tool for highly specific and effective cancer treatment. Thus, the identified PRAME-specific TCR is suitable for adoptive T cell therapy for cancer. As mentioned above, it is possible to arm T cells ex vivo and reintroduce them into donors, thereby enabling such T cells to efficiently recognize and specifically eliminate PRAME-expressing cancer cells (Figures 4 and 5). In this regard, the recognition of different HLA subaleles may be advantageous in that it is possible to include patients with different subalele types in the study cohort and cover frequency differences in the global population (Figure 7). The TCR receptor described herein is HLA-A * 02:01 and HLA-A * 02:02 and HLA-A* 02:04 recognizes the molecule encoded by (Figures 8-10). Therefore, cancer treatment may be possible for a large population of cancer patients with tumors expressing PRAME. Furthermore, the antigen-binding region of the novel TCR provided herein can be used to design soluble constructs that include additional functional portions (such as drugs, labels, or other binding domains that attract other immune cells) that are readily available for direct administration.

[0036] To further demonstrate the effectiveness of the TCR of the present invention in light of the latest developments in the relevant field, the inventors compared the recognition motif, tumor cell recognition, functional avidity, and killing ability of the TCR with those of TCRs known in the relevant art. Two TCRs (46SLL and 54SLL) were selected from WO2016142783, two TCRs (ImCore_Scaffold and ImCorePrefCombi1) from WO2018234319, and two additional TCRs (R11P3D3 and R11P3D3_KE) described in WO2018172533. Table 2 shows an overview of these selected clones and their respective publications.

[0037] Figure 11 clearly shows that effector cell proliferation was not observed with TCR ImCorePrefCombi1, and therefore it was impossible to include TCR ImCorePrefCombi1 in subsequent experiments. The selected TCRs were analyzed by threonine scanning (Figure 12). This threonine scanning made it possible to demonstrate that the amino acid compositions forming each recognition motif were different. Importantly, all of the selected TCRs showed a relative IFN-gamma emission amount at lower peptide concentrations compared to the TCR of the present invention (T23.8-2.1-027-004) under the same experimental setup. 50 The value was never reached. Figure 13 shows the EC of the prior art TCR with the highest capability. 50 The values ​​are plotted against the TCR of the present invention. EC 50 The peptide concentration that reached the target value was 3.57 × 10⁻⁶ in TCR 54SLL. -8M, TCR ImCore_Scaffold uses 1.31×10 -7 In contrast to M, TCR T23.8-2.1-027-004 described herein is 1.16 × 10 -8 It was M. Other TCRs also did not exhibit the same good performance as the TCR of the present invention, and EC was performed with those TCRs. 50 The peptide concentration that reached the value was 8.27 × 10⁻⁶. -8 M(R11P3D3) and 5.73×10 -8 The result was M(R11P3D3_KE). Therefore, the TCR of the present invention induces higher functional avidity compared to the TCR of the prior art.

[0038] To further investigate tumor cell recognition, three PRAME-expressing tumor cell lines were co-cultured with effector cells transduced with different TCRs, and their IFN-gamma release was measured 20 hours after co-culture (Figure 14 and Table 3). Effector cells transduced with the TCR of the present invention showed the highest IFN-gamma release after co-culture with the tumor cell lines MelA375, NCI-H1650, and NCI-H1703.

[0039] Finally, tumor cell death was analyzed using tumor cell lines expressing PRAME (MelA375_NuclightRed and NCI-H1650_NuclightRed). As shown in Figure 15 and Table 4, effector cells transduced with the TCR of the present invention efficiently lysed tumor cells compared to effector cells transduced with other state-of-the-art TCRs.

[0040] Based on the data described above, it can be concluded that the TCR of the present invention has higher functional avidity compared to TCRs disclosed in the prior art, best recognizes the tumor cell lines tested, and more efficiently lyses PRAME-positive tumor cells.

[0041] Variable region CDR3 domain In a first aspect, the present invention relates to a T cell receptor (TCR) having the ability to bind to a PRAME peptide having the amino acid sequence SLLQHLIGL (SEQ ID NO: 1), or a portion thereof, or its HLA-A2 binding form, wherein the TCR comprises a CDR3 in the TCR alpha chain variable region comprising or consisting of the amino acid sequence of (SEQ ID NO: 6), and / or a CDR3 in the TCR beta chain variable region comprising or consisting of the amino acid sequence of (SEQ ID NO: 7). Further TCR sequence variants are also conceivable, comprising a CDR3 alpha comprising or consisting of an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 6, provided that the TCR retains the advantageous capabilities of the TCR evaluated in the appended examples, i.e., the TCR has the ability to bind to the antigenic targets specified herein.

[0042] As used herein, the terms “T cell receptor” or “TCR” include, in all grammatical forms, the native TCR, as well as TCR variants, TCR fragments, and TCR constructs. Therefore, the term includes heterodimers comprising TCR alpha and TCR beta chains, as well as multimers and single-stranded constructs, which may optionally include additional domains and / or portions.

[0043] In its natural form, the TCR exists on the surface of T cells as a complex of several proteins. The T cell receptor consists of two (separate) protein chains, which are produced from independent T cell receptor alpha (TCRα) and T cell receptor beta (TCRβ) genes and are called the alpha (α-) chain and the beta (β-) chain. Each chain of the TCR has one N-terminal immunoglobulin-like (Ig) variable (V) domain / region, one Ig stationary-like (C) domain / region, one transmembrane / transmembrane region that anchors the chain to the cell membrane, and one short cytoplasmic tail located at the C-terminus.

[0044] Antigen specificity is conferred by the variable regions of the alpha and beta chains. Both the TCR alpha and beta chains contain three hypervariable regions or complementarity-determining regions (CDR1 alpha / beta, CDR2 alpha / beta, and CDR3 alpha / beta) surrounded by a framework (FR) region. CDR3 is the primary determinant of antigen recognition and specificity (i.e., the ability to recognize and interact with a specific antigen), while CDR1 and CDR2 primarily interact with MHC molecules presenting antigenic peptides.

[0045] The TCRs provided herein have the ability to recognize and specifically recognize PRAME, specifically PRAME in its MHC-bound form, as will be discussed in detail elsewhere herein. Antigenic peptides are said to exist in their “MHC-bound form” when they form a complex with an MHC molecule (this may be present on the surface of antigen-presenting cells (such as dendritic cells) or tumor cells, or they may be immobilized (for example, by coating beads or plates)).

[0046] Natural TCRs recognize antigenic peptides bound to major histocompatibility complex (MHC) molecules (displayed on them) on the surface of antigen-presenting cells. Antigenic peptides displayed on MHC molecules are also referred to herein as “peptide:MHC complexes.” Two distinct classes of MHC molecules exist (MHCI and MHCII), which present peptides derived from different cellular compartments. MHC class I molecules are expressed on the surface of all nucleated cells throughout the human body and display peptides or protein fragments derived from intracellular compartments to cytotoxic T cells. In humans, MHC is also called human leukocyte antigen (HLA). There are three major types of MHC class I (HLA-A, HLA-B, and HLA-C). When TCRs bind to their specific peptide:MHC complexes, T cells are activated and exert biological effector functions.

[0047] The terms "binding" and "recognizing" are used interchangeably herein in all grammatical forms. The antigenic target is an MHC class I molecule, specifically an HLA-A molecule, preferably an HLA-A molecule. * It is particularly assumed that when bound by the O2 molecule, it will be recognized by the TCR of the present invention. Specifically, the antigenic target is HLA-A * 02:01 Aller, HLA-A * 02:02 Allele, or HLA-A * 02:04 When presented by the HLA- molecule encoded by the allele, it is recognized by the TCR of the present invention. The MHC molecule, i.e., HLA-A * 02:01 Aller, HLA-A * 02:02 Alleles and HLA-A * 02:04 The MHC molecule encoded by the allele can be presented on the surface of a cell (e.g., on the surface of a tumor cell) or on a (solid-phase) carrier. In relation to the present invention, PRAME SLL Peptides are HLA-A * 02:01, HLA-A * 02:02, or HLA-A *It is particularly assumed that when the molecule encoded by 02:04 is bound by HLA-A2, it is recognized by the TCR of the present invention. In a preferred embodiment of the present invention, the TCR is HLA-A * 02:01, HLA-A * 02:02, and HLA-A * When PRAME is bonded by the molecule coded by 02:04 SLL It can specifically recognize peptides, that is, these HLA-A * It has the ability to bind to all three molecules encoded by the 02 allele. This means that the TCR of the present invention is an HLA allele, HLA-A * 02:01, HLA-A * 02:02, and HLA-A * This means that each of the molecules encoded by 02:04 has the ability to bind. However, it is not assumed that all HLA-A2 molecules will be recognized simultaneously in a single patient, and these HLA-A2 molecules will be understood as substitutes.

[0048] CDR1 domain and CDR2 domain As mentioned above, CDR1 and CDR2 of the TCR alpha and TCR beta chains are thought to be mainly involved in MHC recognition. HLA-A * The "pool" of CDR1 and CDR2 sequences known to be involved in restrictive antigen recognition is limited, and it is assumed that the CDR3 domain of the present invention can, in principle, be combined with any of the CDR1 and CDR2 domains shown in Sequence IDs 2-5, however, the TCR must be its antigenic target, preferably the HLA-A of its antigenic target. * 02(HLA-A * 02:01, HLA-A * 02:02, and HLA-A *02:04) The condition is that the binding form retains the ability to recognize the antigenic target to the same degree, the same degree, or a stronger degree than TCR 3825 evaluated in the examples. Useful examples of CDR1 and CDR2 domains include CDR1 alpha containing or consisting of the sequence shown in SEQ ID NO: 2, CDR2 alpha containing or consisting of the sequence shown in SEQ ID NO: 4, CDR1 beta containing or consisting of the sequence shown in SEQ ID NO: 3, and CDR2 beta containing or consisting of the sequence shown in SEQ ID NO: 5. These CDR sequences are also shown in Table 1.

[0049] As described above, the present invention provides a TCR comprising two polypeptide chains, each of which comprises a human variable region comprising at least one complementarity-determining region of the TCR (i.e., specifically CDR3, and preferably CDR1 and / or CDR2). A TCR having certain advantageous properties (as shown in the appended examples) comprises a first polypeptide chain comprising CDR1 (CDR1 alpha) comprising or consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 (CDR2 alpha) comprising or consisting of the amino acid sequence of SEQ ID NO: 4, and CDR3 (CDR3 alpha) comprising or consisting of the amino acid sequence of SEQ ID NO: 6, and / or a second polypeptide chain comprising CDR1 (CDR1 beta) comprising or consisting of the amino acid sequence of SEQ ID NO: 3, CDR2 (CDR2 beta) comprising or consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 (CDR3 beta) comprising or consisting of the amino acid sequence of SEQ ID NO: 7.

[0050] Further TCR sequence variants of the TCR of the present invention are also conceivable, comprising CDR1 alpha, which contains or comprises an amino acid sequence having at least about 60% identity with SEQ ID NO: 2, preferably at least 80%, and / or CDR1 beta, which contains or comprises an amino acid sequence having at least about 60% identity with SEQ ID NO: 3, preferably at least 80%, provided that the TCR retains the advantageous capabilities of the TCR evaluated in the appended examples, i.e., the TCR has the ability to bind to the antigenic targets specified herein. Further TCR sequence variants of the TCR of the present invention are also conceivable, comprising CDR2 alpha, which comprises or consists of an amino acid sequence having at least about 70% identity with SEQ ID NO: 4, preferably at least 85% identity with SEQ ID NO: 4, and / or CDR2 beta, which comprises or consists of an amino acid sequence having at least about 65% identity with SEQ ID NO: 5, preferably at least 80% identity with SEQ ID NO: 5, provided that the TCR retains the advantageous capabilities of the TCR evaluated in the appended examples, i.e., the TCR has the ability to bind to the antigenic targets specified herein.

[0051] Complete variable region The present invention further provides a TCR comprising a TCR alpha chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 8, and / or a TCR beta chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 9. The alpha chain sequence and beta chain sequence are also shown in Table 1.

[0052] TCR sequence variants are also envisioned herein that include an alpha chain variable region containing an amino acid sequence having at least 80%, more preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 8, and / or a TCR beta chain variable region containing or consisting of an amino acid sequence having at least 80%, more preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 9, provided that the TCR retains the advantageous capabilities of the TCR evaluated in the attached examples, i.e., the TCR has the ability to bind to the antigenic targets identified herein.

[0053] Steady-state region The TCR of the present invention comprises an alpha chain constant region and / or a TCR beta chain constant region. The constant region may be a human constant region or derived from another species, and if the constant region is derived from another species, a “chimeric” TCR is obtained. For example, the human alpha chain and / or human beta chain can be replaced with their mouse counterparts (“mouserization”), and “mouserization” has been shown to enhance the surface expression of the human TCR by supporting selective pairing between the TCR alpha chain and the TCR beta chain, as well as improved stability of binding to the CD3 coreceptor. Suitable constant regions for the alpha chain can be selected from, for example, SEQ ID NO: 26 (human), SEQ ID NO: 29 (minimally moused), and SEQ ID NO: 31 (mouse). Suitable constant regions for the beta chain can be selected from SEQ ID NO: 27 (human), SEQ ID NO: 28 (human), SEQ ID NO: 30 (minimally moused), and SEQ ID NO: 32 (mouse). The two TCR beta constant regions shown in SEQ ID NO: 27 and SEQ ID NO: 28 are human sequences that differ by a few amino acids. They will be understood as substitutes. Instead of replacing the entire human constant region with its mouse counterpart, it is also possible to replace only certain amino acids in the human constant region with corresponding amino acids in the mouse constant region ("minimizing mouse regions"). This is further described in the "TCR Sequence Variants" section of this specification. The present invention further envisions that the constant region and the variable region can be combined in a manner suitable for these purposes. In this scenario, the constant region and the variable region can be derived from humans or mice, or achieved by the minimizing mouse region process described above.

[0054] Alpha chain and beta chain Useful examples of the TCR of the present invention include those comprising an alpha chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 10, and / or a beta chain containing or consisting of the amino acid sequence shown in SEQ ID NO: 11.

[0055] TCR sequence variants are also envisioned herein, including an alpha chain comprising an amino acid sequence having at least 80%, more preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 10, and / or a TCR beta chain comprising or comprising an amino acid sequence having at least 80%, more preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 11, provided that the TCR retains the advantageous capabilities of the TCR evaluated in the attached examples, i.e., the TCR has the ability to bind to the antigenic targets identified herein.

[0056] antigenic target The TCR provided herein is (human) PRAME (SEQ ID NO: 1) (PRAME SLL It has the ability to favorably bind to the PRAME peptide shown in SEQ ID NO: 1. SLL It is specific to (also known as ). In the context of this invention, the term "specific to" means that the TCR specifically binds to the target. PRAME (antigen selectively expressed in melanoma, Uniprot acceptance number P78395), also known as MAPE (melanoma antigen selectively expressed in tumors) and OIP4 (OPA-interacting protein 4), has been reported as a cancer-testis antigen (CTA) of unknown function. PRAME is a gene that encodes a protein associated with melanoma and leukemia, as well as chronic myeloid leukemia. Gene ontology (GO) annotation for this gene includes retinoic acid receptor binding. The PRAME protein functions as a transcriptional repressor and inhibits retinoic acid signaling via the retinoic acid receptors RARA, RARB, and RARG. This prevents cell proliferation arrest, differentiation, and apoptosis from being induced by retinoic acid.

[0057] Preferably, the TCR of the present invention specifically binds to its antigenic target. Specifically, the present invention provides a TCR having the ability to bind to a peptide contained in the PRAME amino acid sequence shown in SEQ ID NO: 1 (see Table 1). The term "has the ability to bind" means that the TCR specifically binds to the peptide. The term "specific binding" generally indicates that the TCR readily binds to its intended antigenic target via its antigen-binding site compared to a random, unrelated non-target antigen. Specifically, the term "specifically binds" indicates that the binding specificity of the TCR to its antigenic target is at least about 5 times, preferably 10 times, more preferably 25 times, even more preferably 50 times, and most preferably 100 times or more higher than the binding specificity of the TCR to a non-target antigen. The PRAME peptide, consisting of the amino acid sequence shown in SEQ ID NO: 1, is also referred to herein as the "antigenic target" or "SLL peptide." Therefore, the PRAME peptide, consisting of the amino acid sequence shown in SEQ ID NO: 1, is either a target epitope of the TCR of the present invention or constitutes a target epitope of the TCR of the present invention.

[0058] The term "epitope" generally refers to a part of an antigen (typically a (poly)peptide) recognized by a binding domain. The term "binding domain," in its broader sense, refers to an "antigen-binding site," that is, a domain in a molecule that binds to / interacts with a specific epitope on an antigenic target. An antigenic target may contain a single epitope, but typically contains at least two, and can contain any number of epitopes depending on the size, three-dimensional structure, and type of the antigen. The term "epitope" generally encompasses linear epitopes and structural epitopes. Linear epitopes are continuous epitopes contained in the primary amino acid sequence, and typically contain at least two or more amino acids. Structural epitopes are formed by discontinuous amino acids that come together through the folding of the target antigen, specifically the target (poly)peptide.

[0059] The inventors of the present invention have shown that the smallest amino acid sequence recognized by the TCR of the present invention corresponds to the amino acid sequence of PRAME (SEQ ID NO: 1). Specifically, the TCR of the present invention has been shown to (specifically) recognize an amino acid sequence containing or consisting of the amino acid sequence SLLQHLIGL (SEQ ID NO: 1) or its HLA-A2 binding form (as shown in the attached examples). This selective recognition is achievable through the recognition motif of the T cell receptor, which has a very small number of fixed positions (Figure 3). The amino acids LLQ and especially HLI of the sequence SLLQHLIGL (SEQ ID NO: 1) are part of this recognition motif. Specifically, the TCR described herein is expected to recognize at least one epitope contained in the aforementioned amino acid sequence. Furthermore, the TCR of the present invention has a recognition motif that is significantly different from the recognition patterns of other TCRs known in the art (Figure 12).

[0060] The natural TCRs described herein exhibit high functional avidity and their antigenic targets (i.e., preferably HLA-A) * 02:01, HLA-A * 02:02, or HLA-A *02:04 is assumed to bind to PRAME) presented by antigen-presenting cells on the molecule encoded by 02:04. The term “functional avidity” refers to the ability of TCR-expressing cells (specifically, T cells expressing the innate TCR described herein) to respond in vitro to a given concentration of ligand and is thought to correlate with the in vivo effector capacity of TCR-expressing cells. By definition, TCR-expressing cells with high functional avidity respond to very low antigen doses in in vitro tests, while such cells with low functional avidity require more antigen to initiate an immune response similar to that of TCR-expressing cells with high avidity. Thus, functional avidity can be considered a quantitative determinant of the activation threshold of TCR-expressing cells. Functional avidity is determined by exposing such cells in vitro to different amounts of corresponding antigens. TCR-expressing cells with high functional avidity respond to low doses of antigen.

[0061] For example, TCR-expressing cells take approximately 10 units of PRAME peptide, which has a molecular weight of 956 g / mol. -5 ~about 10 -11 If antigen-negative HLA-A2-expressing target cells are co-cultured with low PRAME peptide concentrations in the range of M (i.e., approximately 0.05 ng / mL to approximately 5 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, or 5 ng / mL), and interferon-gamma (IFN-gamma) is secreted at least approximately 200 pg / mL or higher (e.g., ≥200 pg / mL, ≥300 pg / mL, ≥400 pg / mL, ≥500 pg / mL, ≥600 pg / mL, ≥700 pg / mL, ≥1000 pg / mL, ≥5,000 pg / mL, ≥7,000 pg / mL, ≥10,000 pg / mL, or ≥20,000 pg / mL), then it is typically assumed that the cells will bind to their antigenic target with "high" functional avidity. Therefore, the TCR of the present invention provides a concentration (EC) that gives 50% of the maximum relative IFN-gamma secretion. 50 The value) is 10 when measured by IFN-gamma-immunoassay.-7 It is a high avidity TCR with a value of less than M. Preferably, it is a concentration (EC) that gives 50% of the maximum relative IFN-gamma secretion. 50 The value) is 10 when measured by IFN-gamma-immunoassay. -8 It is less than M (Figure 2). The high avidity of the TCR of the present invention is compared to other TCRs disclosed in the art with the EC of each TCR. 50 This is further proven by determining the value (Figure 13).

[0062] The present invention includes the fact that the secretion of IFN-gamma by cells transfected or transfected with a TCR is induced by binding to the sequence SLLQHLIGL (SEQ ID NO: 1) or a part thereof, or to its HLA-A2 binding form. The TCR of the present invention expressed on effector cells is induced by binding to the amino acid sequence (HLA-A2) of SEQ ID NO: 1. * 02:01, HLA-A * 02:02, or HLA-A * The amount of IFN-gamma secreted, induced by binding to the molecule encoded by 02:04, is the amino acid sequence of SEQ ID NO: 1 (HLA-A * 02:01, HLA-A * 02:02, or HLA-A * In the case of binding to the molecule encoded by 02:04, an unrelated peptide (amino acid sequence GLSNTHVL shown in SEQ ID NO: 25) (HLA-A * 02:01, HLA-A * 02:02, or HLA-A * Compared to binding to the molecule encoded by 02:04, the amount can be more than 100 times greater, preferably more than 500 times greater, and more preferably more than 2000 times greater. The amount of IFN-gamma secreted can be, for example, more than 100 pg / ml (more than 500 pg / ml or more than 2000 pg / ml).

[0063] Cytokine release (such as IFN-gamma secretion) can be measured using an in vitro assay, in which K562 cells (Greiner et al., 2006, Blood. 2006 Dec 15;108(13):4109-17) are transfected or transduced with ivtRNA to express either the amino acid sequence of SEQ ID NO: 1 or an unrelated peptide, respectively, and the CD8 cells expressing the TCR to be examined are analyzed. + Concentrated PBMC and / or non-CD8 + These cells are incubated with enriched PBMCs. Alternatively, T2 cells are externally loaded with either SEQ ID NO: 1 or an unrelated peptide, and then the CD8 cells expressing the TCR to be examined are incubated. + Concentrated PBMC and / or non-CD8 + Cytokine release (such as IFN-gamma secretion) can also be measured in an in vitro assay in which the T2 cells are co-incubated with enriched PBMCs.

[0064] Some embodiments refer to the isolated TCR described herein, the polypeptide described herein, or the polyvalent TCR complex described herein, wherein the TCR of the present invention expressed on effector cells has the amino acid sequence of SEQ ID NO: 1, or specifically, HLA-A * 02:01, HLA-A * 02:02, or HLA-A * The amount of IFN-gamma secreted by binding to the amino acid sequence of Sequence ID No. 1 presented by the molecule encoded by 02:04 is below a predetermined threshold. The threshold can be determined by making the ratio of a particular effector to the target at least 2:1. “Effector cells” may be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). Typically, effector cells are immune effector cells (such as T cells). Specific suitable effector cells include cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, and gamma / delta T cells, as described elsewhere herein.

[0065] HLA-A * 02:01, HLA-A * 02:02, or HLA-A * When the TCR of the present invention expressed on effector cells binds to the amino acid sequence of SEQ ID NO: 1 presented by the molecule encoded by 02:04, the secretion of IFN-γ is at least 10 -7 M, preferably at least 10 -8 M, more preferably 10 -9 M of PRAME SLL can be induced by the peptide concentration. In certain embodiments, for example, when the ratio of TCR transgenic T cells to T2 cells is 2:1, HLA-A * When the TCR of the present invention expressed on effector cells binds to the amino acid sequence of SEQ ID NO: 1 presented by the molecule encoded by 02:01, the secretion of IFN-γ that occurs is at least 10 -7 M, preferably at least 10 -8 M, more preferably 10 -9 M of PRAME SLL can be induced by the peptide concentration. Other methods for determining specific binding of the TCR of the present invention include the <51>Cr release assay described by Gertner-Dardenne et al., J Immunol 188(9):4701-4708, CD107a / b surface expression described by Leisegang et al., Clin. Cancer Res 2010.16:2333-2343, and peptide:MHC multimer binding analysis described by Wilde et al., J Immunol 2012;189:598-605.

[0066] variant As stated above, the term "TCR" encompasses TCR variants, including TCR sequence variants, TCR fragments, and TCR constructs. All TCR variants are assumed to be functional variants of the TCR of the present invention. As used herein, the term "functional variant" refers to a TCR, polypeptide, or protein that exhibits substantial or significant sequence identity or similarity with a parent TCR, its variable region, or its antigen-binding region, and that shares its biological activity, i.e., its ability to specifically bind to antigenic targets to which the parent TCR of the present invention has antigen specificity, to the same degree, or even more strongly, than the TCR disclosed herein and evaluated in the appended examples. The present invention also encompasses TCR sequence variants.

[0067] The term “TCR variant” includes a “sequence variant” of the TCR disclosed herein, which is a variant that substantially comprises the amino acid sequence of the TCR of the present invention (also referred to as the “parent” TCR) but includes at least one amino acid modification (i.e., substitution, deletion, or insertion) compared to the “parent” TCR amino acid sequence, provided that such a variant preferably retains the antigen specificity of the “parent” TCR of the present invention. TCR sequence variants of the present invention are typically prepared by introducing appropriate nucleotide modifications into the nucleic acid encoding the “parent” TCR, or by peptide synthesis. Generally, the aforementioned amino acid modifications are introduced into, or may be present in, the variable or constant region of the TCR and may help modulate properties such as binding strength and specificity, post-translational processing (e.g., glycosylation), thermodynamic stability, solubility, surface expression, or TCR construction.

[0068] As described above, amino acid modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the parent TCR. An example of an insertion variant of the TCR of the present invention is a fusion product of the TCR with an enzyme or another functional polypeptide. An example of a substitution variant of the TCR of the present invention includes amino acid substitutions in the variable regions or CDRs, framework regions, or constant regions of the alpha and / or beta chains. Specifically, conserved amino acid substitutions are assumed herein. Conserved amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is replaced with another amino acid having the same chemical or physical properties. For example, conservative amino acid substitutions may include substitution of an acidic amino acid with another acidic amino acid (e.g., Asp or Glu), substitution of an amino acid with a nonpolar side chain with another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), substitution of a basic amino acid with another basic amino acid (Lys, Arg, etc.), or substitution of an amino acid with a polar side chain with another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.). These substitutions may be made, for example, based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved.

[0069] Generally, a TCR sequence variant is envisioned to include at least one of the following: CDR1, CDR2, CDR3, alpha chain variable region, beta chain variable region, alpha chain, and / or beta chain, or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with the amino acid sequences disclosed herein, provided that the binding properties exhibited by the variant are equivalent to, the same as, or improved compared to the TCRs evaluated in the accompanying examples.

[0070] As used herein, the term “sequence identity” refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in alignment, and is often expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies of exactly the same sequence have 100% identity, while sequences with low degree of conservation and containing deletions, additions, or substitutions may have a lower degree of identity. Those skilled in the art will recognize that several algorithms are available for determining sequence identity using standard parameters. Examples of such algorithms include Blast (Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al., (1990) J.Mol.Biol. 215:403-410), Smith-Waterman (Smith et al., (1981) J.Mol.Biol. 147:195-197), and ClustalW.

[0071] Therefore, the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 11 could be, for example, a "target sequence" or "reference sequence," while a different amino acid sequence of CDR3 could be a "query sequence."

[0072] The term “position,” as used in accordance with this disclosure, means the position of an amino acid in an amino acid sequence or a nucleotide in a nucleic acid sequence as shown herein. The term “corresponding,” as used herein, includes not only the determination of position by the number of nucleotides / amino acids that precede them in sequence, but also the determination of position in relation to the surrounding portion of the sequence. Thus, the position of a given amino acid or nucleotide in this disclosure may change due to the deletion of an amino acid or nucleotide at another position in the sequence or the addition of an amino acid or nucleotide at another position in the sequence. Therefore, where a position is referred to as a “corresponding position,” it will be understood that, according to this disclosure, nucleotides / amino acids may differ with respect to certain numbers, but still have similar adjacent nucleotides / amino acids. To determine whether an amino acid residue (or nucleotide) in a given sequence corresponds to a particular position in the amino acid sequence of a “parent” amino acid / nucleotide sequence, a person skilled in the art may use means and methods well known in the art (e.g., sequence alignment), either manually or by using a computer program (such as those exemplified herein).

[0073] The TCR variant used herein as a control is TCR receptor 3825. This variant is based on PRAME-immunized mice (mouse ID 3825) expressing the TCR receptor for PRAME and has the CDR3 alpha sequence (CAVEPGGSYIPTF) shown in SEQ ID NO: 23 and the CDR3 beta sequence (CASSPGLSYEQYF) shown in SEQ ID NO: 24. Recombinant expression and analysis of this TCR were possible via a TCR library containing codon-optimized oligonucleotides (Weis, Manon (2015): Characterization of Antigen-specified T-cells after Induction in TCR-humanized Mousens. Dissertation, LMU Munchen Tierarztliche Fakultat: Veterinary Faculty Ludwigs University of Munich).

[0074] Cysteine ​​modification It has been reported that adding a disulfide bond to the constant region promotes the correct pairing of the TCR alpha chain and the TCR beta chain (Kuball J et al., Blood. 2007 Mar. 15; 109(6): 2331-8.). Therefore, it is also conceivable in this specification to add one or more cysteine ​​bonds to the constant region.

[0075] Mouse transformation As mentioned above, mouse-like TCRs (i.e., exchanging the human constant regions in the alpha and beta chains with their mouse counterparts) are a commonly applied technique to improve the cell surface expression of TCRs in host cells. While we do not wish to be constrained by any particular theory, it is thought that mouse-like TCRs express TCRs with desired antigen specificity while still retaining and expressing their "original" TCRs, as they bind more efficiently to CD3 coreceptors and / or selectively pair with each other, and have a lower tendency to form mixed TCRs on ex vivo engineered human T cells.

[0076] Recently, nine amino acids involved in improving the expression of the mouse-like TCR have been identified (Sommermeyer and Uckert, J Immunol. 2010 Jun. 1; 184(11): 6223-31), and it is hypothesized that one or all of these amino acid residues in the constant region of the TCR alpha chain and / or the constant region of the TCR beta chain should be replaced with their mouse-compatible residues. This method, also known as "minimal mouseization," offers the advantage of enhancing cell surface expression while simultaneously reducing the number of "foreign" amino acid residues in the amino acid sequence, thereby reducing the risk of immunogenicity.

[0077] Constructs and Fragments As used herein, the term “TCR” further includes TCR constructs. The term “construct” includes proteins or polypeptides comprising at least one antigen-binding domain of the TCR of the present invention, but such proteins or polypeptides do not necessarily share the basic structure of the natural TCR (i.e., TCR alpha and TCR beta chains with variable domains incorporated, and these chains forming a heterodimer). TCR constructs and TCR fragments are typically obtained by a given genetic engineering method and are often artificially constructed to include additional functional protein domains or functional polypeptide domains. As described above, it is assumed that the TCR constructs and TCR fragments of the present invention comprise at least one CDR3 alpha (as disclosed elsewhere herein) and / or at least one CDR3 beta (as disclosed elsewhere herein). This specification further envisions constructs and fragments comprising, optionally, at least one CDR1 alpha, at least one CDR2 alpha, at least one CDR1 beta, at least one CDR2 beta, at least one alpha chain variable region, at least one beta chain variable region, at least one alpha chain, and / or at least one beta chain, or a combination thereof, in combination with additional protein domains or portions exemplified herein. The TCR constructs and TCR fragments provided herein are envisioned to have the ability to specifically bind to the same antigenic targets as the TCRs of the present invention described above and evaluated in the appended examples.

[0078] polymer The TCR constructs of the present invention encompass heterodimers and polymers in which at least one TCR alpha-chain variable region or a TCR alpha chain and at least one TCR beta-chain variable region are covalently linked to each other to form a TCR heterodimer or TCR polymer. As used in the present invention, “polymer” describes a molecule of various subunits or functional entities, while a heterodimer contains only two functional entities. In its simplest form, the polyvalent TCR constructs according to the present invention include polymers in which two, three, four or more TCRs are linked to each other (preferably via a linker molecule) (e.g., covalently or in other ways). In this context, “covalently linked” means that a chemical bond exists between the two molecules (sharing electron pairs) and that a stable balance is maintained between the atomic bonds.

[0079] A suitable linker for spheres, preferably homogeneous beads, more preferably polystyrene beads, most preferably biocompatible polystyrene beads. Such a TCR construct may also consist of the TCR of the present invention and beads incorporating a predetermined fluorescent dye. Suitable linker molecules include, but are not limited to, polyvalent binding molecules (such as avidin, streptavidin, neutravidin, and extravidin (each having four binding sites to biotin)). Thus, biotinylated TCRs may form multimers having multiple TCR binding sites. The number of TCRs in the multimer will depend on the amount of TCR relative to the amount of linker molecule used in the preparation of the multimer, as well as the presence or absence of any other biotinylated molecules. Examples of multimers include dimeric TCR constructs, trimer TCR constructs, tetrameric TCR constructs, or pentameric TCR constructs, or higher-order multimeric TCR constructs. The polymer of the present invention may also include additional functional entities (such as labels, drugs, or (solid-phase) carriers).

[0080] Fusion protein A TCR heterodimer or TCR multimer also relates to a fusion protein or fusion polypeptide comprising at least one TCR alpha chain, at least one TCR alpha chain variable region, or at least one CDR3 alpha and / or at least one TCR beta chain, at least one TCR beta chain variable region, or at least one CDR3 beta, and one or more additional fusion elements. A TCR heterodimer or TCR multimer may be at least one TCR alpha chain (as defined herein) and / or at least one TCR beta chain (as defined herein), and / or an antibody or single-chain antibody fragment (scFv) against an antigen or epitope on the surface of a lymphocyte, wherein the TCR alpha chain(s) and TCR beta chain(s) are linked to each other and fused to the antibody or scFv via a linker of choice. Useful elements include Fc receptors, Fc domains (derived from IgA, IgD, IgG, IgE, and IgM), cytokines (such as IL-2 or IL-15), toxins, antibodies or their antigen-binding fragments (such as anti-CD3 antibodies, anti-CD28 antibodies, anti-CD5 antibodies, anti-CD16 antibodies, or anti-CD56 antibodies, or their antigen-binding fragments), CD247 (CD3-zeta), CD28 domains, CD137 domains, CD134 domains, or any combination thereof.

[0081] Examples of antibody fragments that can be used as fusion elements include full-length antibody fragments ((s)dAb, Fv, Fd, Fab, Fab', F(ab')2, or "r IgG" ("half-antibody")), modified antibody fragments (scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabody, single-stranded diabody, tandem diabody (Tandab), tandem di-scFv, tandem tri-scFv, minibody, multibody (such as triabody or tetrabody), and single-domain antibodies (such as nanobody, or single variable-domain antibodies containing only one variable domain (which may be VHH, VH, or VL))).

[0082] By fusing the TCR construct of the present invention with one or more antibodies or antibody fragments, monovalent constructs, bivalent constructs, and polyvalent / multivalent constructs can be obtained, and thus monospecific constructs (which specifically bind to only one target antigen), bispecific constructs, and polyspecific / multispecific constructs (which specifically bind to multiple types of target antigens (e.g., two, three, or more types) via different antigen-binding sites) can be obtained.

[0083] Linkers can be optionally introduced between one or more domains or regions of the TCR construct of the present invention, i.e., between the TCR alpha chain CDR3, the TCR alpha chain variable region, and / or the TCR alpha chain, the TCR beta chain CDR3, the TCR beta chain variable region, and / or the TCR beta chain, and / or one or more fusion elements (as described herein). Linkers are known in the art and are outlined, in particular, by Chen et al., Adv Drug Deliv Rev. 2013 Oct. 15;65(10):1357-1369. Generally, linkers include mobile linkers, cleavable linkers, and rigid linkers, and such linkers will be selected depending on the type of construct and the intended use / application. For example, for therapeutic applications, non-immunogenic mobile linkers are often preferred to ensure some degree of mobility or interaction between domains while reducing the risk of adverse immunogenic reactions. Such linkers are generally composed of small nonpolar amino acids (e.g., Gly) or polar amino acids (e.g., Ser or Thr), and such linkers include "GS" linkers consisting of a Gly residue and a Ser residue interval.

[0084] A particularly useful TCR construct envisioned in accordance with the present invention comprises at least one TCR alpha chain, at least one TCR alpha chain variable region, or at least one CDR3 alpha (as defined herein), at least one TCR beta chain, at least one TCR beta chain variable region, or at least one CDR3 beta (as defined herein), which are optionally linked together and optionally fused with at least one antibody or antibody fragment (such as a single-chain antibody fragment (scFv)) against an antigen or epitope on the surface of a lymphocyte via a linker. Useful antigenic targets recognized by the antibody or antibody fragment (e.g., scFv) include CD3, CD28, CD5, CD16, and CD56. The construct can generally have any structure, provided that the “TCR moiety” (i.e., the TCR alpha and TCR beta chains or their variable regions or CDR3) retains the ability to recognize the antigenic target as defined herein, and that the “antibody moiety” binds to the desired surface antigen or epitope, thereby recruiting the respective lymphocytes and making the target cells the targets of those lymphocytes. Such a construct can be advantageous as an “adapter” that links antigen-presenting cells displaying antigenic targets (such as tumor cells) with lymphocytes (such as cytotoxic T cells or NK cells). An example of such a fusion protein is a construct manipulated according to the principle of a bispecific T cell inducer (BiTE®), consisting of two single-stranded variable fragments (scFv) derived from different antibodies present in a single peptide chain of approximately 55 kilodaltons (kDa). Accordingly, the TCR construct of the present invention may include at least one TCR antigen-binding domain (as described herein) linked to an scFv (or other binding domain) having a desired binding specificity (e.g., CD3 or CD56) (e.g., a fusion of a TCR variable alpha chain and a TCR variable beta chain). The scFv (or other binding domain) binds to T cells, for example, via the CD3 receptor, or to CD56 for NK cell activation, while the other binds to tumor cells via an antigenic target specifically expressed on tumor cells.A tribody comprising at least one TCR antigen-binding domain (as described herein), an scFv (or other binding domain), and additional domains (e.g., for targeting a site of action in the body for the construct) (e.g., an Fc domain) is also envisioned herein.

[0085] Isolated form The TCRs of the present invention may be provided in an “isolated” or “substantially pure” form. “Isolated” or “substantially pure,” as used herein, means that the TCR has been identified, separated, and / or recovered from components of its production environment, and as a result, the “isolated” TCR does not contain, or substantially contains, other contaminants from its production environment that could interfere with its therapeutic or diagnostic use. Contaminants may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Therefore, the “isolated” TCR will be prepared by a method for obtaining a TCR, which involves incubating host cells under conditions in which the TCR is expressed and purifying the TCR, and thus includes at least one purification step to remove or substantially remove such contaminants. The foregoing definitions are equally applicable to “isolated” polynucleotides / nucleic acids.

[0086] soluble form The TCR of the present invention may be provided in a soluble form. Soluble TCRs are useful as diagnostic tools and, furthermore, as carriers or "adapters" that specifically target therapeutic agents or effector cells (e.g., cancer cells expressing antigenic targets recognized by the soluble TCR). A soluble TCR (sTCR) is typically a fragment or construct comprising the TCR alpha chain and / or TCR beta chain or its variable region or CDR, which is optionally stabilized via disulfide bonds or covalently linked via a suitable linker molecule (e.g., those described above in relation to the TCR construct of the present invention). sTCRs typically do not include, for example, a transmembrane region. Depending on the circumstances, (if desired) amino acid modifications may be made to the polypeptide sequence to enhance the solubility of the molecule and / or to ensure proper folding and pairing of the alpha and beta chains, specifically when the polypeptide sequence is produced in a recombinant host where the aforementioned features are not obtained. When E. coli is used as the production host cell, for example, the folding and pairing of the TCR alpha and TCR beta chains are typically achieved in vitro. Therefore, the TCR according to the present invention may include additional cysteine ​​residues, as described elsewhere in this specification.

[0087] Other useful modifications besides additional cysteine ​​crosslinking include, for example, adding leucine zipper and / or ribosome skipping sequences (e.g., the picornavirus-derived 2A sequence described in Walseng et al., (2015), PLoS ONE 10(4):e0119559) to increase the folding, expression, and / or pairing of the TCR alpha and / or TCR beta chains.

[0088] Modification The TCR of the present invention may further include one or more of the modifications described below. The modifications described below are typically covalent modifications and can be achieved using standard methods known in the art. In some circumstances, amino acid modifications in the TCR may be required to facilitate the introduction of such modifications.

[0089] Molecular markers The TCR of the present invention, specifically (soluble) TCR, can be labeled with at least one molecular marker. Useful molecular markers are known in the art, and such molecular markers can be coupled to TCR or TCR varian via optionally selected linkers of various lengths using predetermined methods.

[0090] Generally, different markers are classified into various classes depending on the assay in which they are detected, and examples include, but are not limited to, isotope markers (radioisotopes or heavy isotopes (radioisotopes or radionuclides (e.g., <3> H, <14> , <15> N, <35> S, <89> Zr, <90> Y, <99> Tc, <111> In, <125> I, <131> Examples include magnetic markers (e.g., magnetic particles), redox active moieties, optical dyes (including, but not limited to, chromophores, phosphors, and fluorophores) (e.g., fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores (which may be either “small molecule” fluorophores or proteinaceous fluorophores)), enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), biotinylating groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites to secondary antibodies, metal-binding domains, epitope tags, etc.). Labeling with molecular markers is particularly conceivable when the TCR, TCR variant, or especially a soluble TCR construct (e.g., one containing at least one TCR alpha chain and / or TCR beta chain (as described herein)) is intended for diagnostic use.

[0091] Functional part The TCR of the present invention, specifically soluble TCRs, may be modified by adding additional functional moieties. The purpose of these modifications is, for example, to reduce immunogenicity, improve hydrodynamic size (size in solution), solubility, and / or stability (e.g., by enhancing protection against proteolysis), and / or extend the serum half-life.

[0092] Examples of functional portions for use in accordance with the present invention include peptide or protein domains that bind to other proteins in the human body (such as serum albumin, immunoglobulin Fc region, or fetal Fc receptor (FcRn)), polypeptide chains of varying lengths (e.g., XTEN technology or PASylation®), non-proteinoid polymers (including, but not limited to, various polyols (such as polyethylene glycol (PEGylated), polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol), or carbohydrate polymers (such as hydroxyethyl starch (e.g., HESylation®)) or polysialic acid (e.g., PolyXen® technology)).

[0093] Other useful functional components include “suicide switches” or “safety switches” that can be used to eliminate effector host cells possessing the TCR of the present invention in a patient’s body. One example is the inducible caspase 9 (iCasp9) “safety switch” described by Gargett and Brown Front Pharmacol. 2014;5:235. Briefly, effector host cells are modified by a well-known method to express a caspase 9 domain (whose dimerization depends on a small molecule dimerizing agent such as AP1903 / ClP), resulting in rapid induction of apoptosis in the modified effector cells. This system is described, for example, in EP2173869(A2). Other examples of "suicide switches" and "safety switches" are known in the art, such as those involving the expression of herpes simplex virus thymidine kinase (HSV-TK) or CD20 followed by depletion using an anti-CD20 antibody, or the myc tag (Kieback et al., Proc Natl Acad Sci USA. 2008 Jan. 15; 105(2): 623-8). The TCR of the present invention may also be modified by introducing an inducible so-called "on switch" (e.g., as described in WO2019175209A1), in which case the modified alpha and beta chains of the TCR of the present invention will dimerize only upon interaction with a small dimerizing agent, resulting in a functional TCR that is expressed on the cell surface only in the presence of the dimerizing agent.

[0094] Glycosylation In this specification, TCRs with altered glycosylation patterns are also considered. As is known in the art, the glycosylation pattern may depend on the amino acid sequence (e.g., the presence or absence of certain glycosylated amino acid residues discussed below) and / or the host cell or organism in which the protein is produced. Polypeptide glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the addition of a carbohydrate moiety to the side chain of an asparagine residue. The addition of an N-linked glycosylation site to a binding molecule is conveniently achieved by altering the amino acid sequence to include one or more tripeptide sequences selected from asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid other than proline). O-linked glycosylation sites can be introduced by adding one or more serine or threonine residues to the starting sequence, or by substituting one or more serine or threonine residues into the starting sequence.

[0095] Another means of glycosylation of TCRs is by chemically or enzymatically coupling the glycoside with the protein. Depending on the coupling method used, the sugar(s) may be added to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group (such as that of cysteine), (d) a free hydroxyl group (such as that of serine, threonine, or hydroxyproline), (e) an aromatic residue (such as that of phenylalanine, tyrosine, or tryptophan), or (f) the amide group of glutamine.

[0096] Similarly, deglycosylation (i.e., removal of the carbohydrate portion present on the binding molecule) can be achieved chemically, for example, by exposing the TCR to trifluoromethanesulfonic acid, or enzymatically by using endoglycosidases and exoglycosidases.

[0097] Drug complex It is also conceivable to add a drug (such as a small molecule compound) to a TCR, specifically the soluble TCR of the present invention. Binding can be achieved via covalent or non-covalent interactions (such as electrostatic interactions). Various linkers are known in the art, and such linkers can be used to form drug complexes.

[0098] tag The TCRs of this disclosure, specifically soluble TCRs, may be modified to introduce additional domains (tags) that are useful for the identification, tracking, purification, and / or isolation of each molecule. Examples of such tags include, but are not limited to, peptide motifs known as Myc tags, HAT tags, HA tags, TAP tags, GST tags, chitin-binding domains (CBD tags), maltose-binding proteins (MBP tags), Flag tags, Strep tags and their variants (e.g., StrepII tags), His tags, CD20, Her2 / neu tags, myc tags, FLAG tags, T7 tags, HA (hemagglutinin) tags, or GFP tags.

[0099] Epitope tags are a useful example of tags that can be incorporated into the TCRs of this disclosure. Epitope tags are short amino acid segments that enable the binding of specific antibodies and therefore allow the identification and tracking of the binding and movement of soluble TCRs or host cells or cultured (host) cells in a patient's body. Detection of epitope tags, and thus the detection of tagged TCRs, can be achieved using several different techniques. Examples of such techniques include immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting ("Western"), and affinity chromatography. Epitope tags may have an amino acid length of, for example, 6 to 15, specifically 9 to 11. It is also possible to include multiple epitope tags in the TCRs of the present invention.

[0100] Furthermore, the tag can also be used to stimulate and proliferate host cells possessing the TCR of the present invention by culturing the cells in the presence of a binding molecule (antibody) specific to the tag.

[0101] nucleic acid The present invention further provides nucleic acids encoding the TCR described herein, or polynucleotides encoding the TCR described herein. Such nucleic acids are codon-optimized, meaning that a single protein can be encoded by many alternative nucleic acid sequences that can be selected. Codon selection (bias in codon usage frequency) differs from organism to organism and can make recombinant protein expression in heterologous expression systems difficult, resulting in reduced expression levels and uncertainty. This can also apply to autoexpression, because wild-type sequences are not necessarily optimized not only for expression yield but also for degradation, regulation, and other properties. Therefore, codon optimization is used herein to obtain efficient protein expression. Table 1 below shows the nucleotide sequences encoding each amino acid sequence. [Table 1-1] [Table 1-2] [Table 1-3]

[0102] Specifically, polynucleotides encoding TCR alpha chains or TCR beta chains, TCR alpha chain variable regions or TCR beta chain variable regions, as well as TCR CDR3 alpha and TCR CDR3 beta, as well as TCR variants, TCR constructs, and TCR fragments, are provided herein, the sequences of which are shown in SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.

[0103] As used herein, the terms “polynucleotide” or “nucleic acid” include sequences of polyribonucleotides and polydeoxyribonucleotides (e.g., modified or unmodified RNA or DNA) that exist in linear, cyclic, or mixed single-stranded and / or double-stranded forms, including hybrid molecules. Accordingly, the nucleic acids according to the present invention include DNA (dsDNA, ssDNA, cDNA, etc.), RNA (dsRNA, ssRNA, mRNA, ivtRNA, etc.), combinations thereof, or derivatives thereof (PNA, etc.).

[0104] Polynucleotides may contain conventional phosphodiester bonds or unconventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The polynucleotides of the present invention may also contain one or more modified bases (e.g., tritylated bases and unusual bases, such as inosine). Other modifications are also possible, including chemical, enzymatic, or metabolic modifications, provided that the binding molecule of the present invention can be expressed from the polynucleotide. Polynucleotides may be provided in isolated forms as defined elsewhere in this specification. Polynucleotides may contain regulatory sequences (such as transcriptional regulatory elements (including promoters, enhancers, operators, repressors, and transcription termination signals)), ribosome binding sites, introns, or similar.

[0105] Specifically, the present invention provides a polynucleotide comprising or comprising nucleic acids having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identity with a reference polynucleotide sequence selected from the group consisting of sequences shown in SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.

[0106] The polynucleotides described herein may or may not include additional or modified nucleotide sequences, such as those encoding altered amino acid residues, signal peptides leading to the secretion of the encoded TCR, constant regions, or other heterologous polypeptides (as described herein). Thus, such polynucleotides may encode fusion polypeptides, fragments, variants, and other derivatives of the binding molecules described herein.

[0107] The nucleic acid sequences of the present invention can be subjected to codon optimization to optimize expression in desired host cells (e.g., human lymphocytes) or to induce expression in bacterial, yeast, or insect cells where expression of the soluble TCR of the present invention is particularly anticipated. Codon optimization refers to replacing a sequence containing a target codon that is generally infrequently found in high-expression genes of a given species with a codon that is generally frequently found in such high-expression genes, where the replacement codon encodes the same amino acid as the codon being replaced. Therefore, the selection of the optimal codon depends on the codon usage frequency of the host genome and the number of desirable and undesirable sequence motifs present.

[0108] vector This specification further provides vectors comprising one or more of the nucleic acids described herein. A “vector” is a nucleic acid molecule used as a medium for introducing (foreign) genetic material into a host cell (for example, one in which replication and / or expression of such (foreign) genetic material occurs).

[0109] The term "vector" is not limited to plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenovirus vectors, vaccinia virus vectors, polyomavirus vectors, and adeno-associated virus vectors (AAVs)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). A vector is generally a nucleotide sequence itself, which is typically a DNA sequence containing an insertion fragment (transgene) and a longer sequence that acts as the vector's "backbone." An engineered vector typically includes a starting point for self-replication in host cells (if stable expression of polynucleotides is desired), a selection marker, and restriction enzyme cleavage sites (e.g., multicloning sites (MCS)). A vector may additionally include a promoter, a genetic marker, a reporter gene, a target-directed sequence, and / or a protein purification tag. As is known to those skilled in the art, many suitable vectors are known to those skilled in the art and many are commercially available.

[0110] Target-directed vector Target-directed vectors can be used to integrate polynucleotides into the chromosomes of host cells by methods known in the art (such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012)). Briefly, suitable means include homologous recombination or its use by hybrid recombinases that specifically target the integration site in the sequence. Target-directed vectors are typically circular or linear before use for homologous recombination. Alternatively, the exogenous polynucleotide may be DNA fragments linked by fusion PCR or synthetically constructed DNA fragments, which are then introduced into host cells by recombination. It is also possible to use non-homologous recombination, which results in random or non-target-directed integration.

[0111] Expression vector The vector of the present invention may also be an expression vector. An "expression vector" or "expression construct" can be used to transcribe a heterologous polynucleotide sequence (e.g., one encoding the TCR of the present invention) and translate its mRNA in a suitable host cell. This process is also referred to herein as "expression" of the TCR of the present invention.

[0112] In addition to the origin of replication, selection markers, and restriction enzyme cleavage sites, expression vectors typically include one or more regulatory sequences functionally linked to the heterologous polynucleotide to be expressed.

[0113] The term "regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a functionally linked (heterogeneous) polynucleotide coding sequence in a particular host organism or host cell, and therefore includes transcriptional and translational regulatory sequences. Typically, regulatory sequences required for the expression of a heterogeneous polynucleotide sequence in prokaryotes include a promoter(s), optionally operator(s), and optionally ribosome-binding sites(s). In eukaryotes, a promoter, polyadenylation signal, enhancer, and optionally splice signal are typically required. Furthermore, specific initiation and secretion signals may also be introduced into the vector to enable the secretion of the target polypeptide into the culture medium.

[0114] Nucleic acids are "functionally linked" when they are given a functional association with another nucleic acid sequence, specifically another nucleic acid sequence on the same polynucleotide molecule. For example, a promoter is functionally linked to a coding sequence if it can influence the expression of that coding sequence of a heterologous gene. Promoters are typically located upstream of the gene encoding the target polypeptide and regulate the expression of that gene.

[0115] Examples of regulatory sequences for mammalian host cells include viral elements that express proteins at high levels in mammalian cells. These viral elements include promoters and / or enhancers derived from cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), promoters and / or enhancers derived from simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), promoters and / or enhancers derived from adenoviruses (e.g., adenovirus major late promoter (AdMLP)), and promoters and / or enhancers derived from polyomas. As already mentioned, expression vectors may also include origins of replication and selectable markers.

[0116] As described above, the vector of the present invention may further include one or more selection markers. Selection markers suitable for use in eukaryotic hosts include, but are not limited to, the herpes simplex virus thymidine kinase (TK) gene, the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) gene, and the adenine phosphoribosyltransferase (APRT) gene. Other genes include DHFR (methotrexate resistance), GPT (mycophenolate resistance), NEO (G-418 resistance), and HYGRO (hygromycin resistance). The vector may be amplified to increase the expression level. Generally, the selection marker gene can be directly ligated to the polynucleotide sequence to be expressed or introduced into the same host cell by co-transformation.

[0117] Therefore, in light of the above, the present invention further provides a vector in which one or more of the nucleotide sequences described herein are inserted (i.e., included in the vector). Specifically, the present invention provides a (replicable) vector comprising a nucleotide sequence encoding the TCR of the present invention, or its alpha or beta chain, or an alpha or beta variable domain, or CDR3 alpha or CDR3 beta, ligated to a promoter in a functional manner.

[0118] Those skilled in the art can easily select a suitable expression vector, which is made, for example, based on the host cell in which TCR expression is intended. Examples of suitable expression vectors are viral vectors, such as retroviral vectors (e.g., MP71 vector or retroviral SIN vector) and lentiviral vectors or lentiviral SIN vectors. Viral vectors containing polynucleotides encoding the TCR of the present invention have the ability to infect lymphocytes, for example, which are expected to express heterologous TCRs after infection. Another example of a suitable expression vector is the Sleeping Beauty (SB) transposon transposase DNA plasmid system (SB DNA plasmid). The nucleic acids and / or specifically the expression constructs of the present invention can also be introduced into cells by transiently transfecting RNA.

[0119] Viral vectors currently used for the expression of the natural TCR typically link the TCR-alpha and TCR-beta chain genes in a single vector using either an internal ribosome entry site (IRES) sequence or a 2A peptide sequence derived from porcine tesiovirus. As a result, expression occurs from a single messenger RNA (mRNA) molecule under the control of a viral promoter within the transduced cell.

[0120] host cell The present invention further provides host cells comprising the TCR, nucleic acid, or vector described herein.

[0121] Various host cells may be used in accordance with the present invention. As used herein, the term “host cell” includes cells that may be recipients of the polynucleotides or vectors described herein, or that are recipients of the polynucleotides or vectors described herein, and / or that express and (optionally secrete) the TCRs of the present invention. The terms “cell” and “cell culture” are used interchangeably to indicate the source of the TCRs unless otherwise expressly specified. The term “host cell” also includes host cell lines. Generally, the term “host cell” includes prokaryotic or eukaryotic cells, and also includes, but is not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells (such as insect cells and mammalian cells (e.g., mouse cells, rat cells, macaque cells, or human cells)).

[0122] Accordingly, in light of the foregoing, the present invention provides, in particular, a host cell comprising a polynucleotide or vector (e.g., an expression vector) comprising a nucleotide sequence encoding a TCR or TCR construct as described herein. The polynucleotide and / or vector of the present invention can be introduced into a host cell using a predetermined method known in the art (e.g., by transfect, transform, or similar).

[0123] "Transfect" is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. One example is RNA transfect, which is the process of introducing RNA (such as in vitro transcribed RNA (ivtRNA)) into host cells. This term is most often used for non-viral methods in eukaryotic cells. The term "transduction" is often used to describe the viral introduction of nucleic acid molecules or polynucleotides. In animal cell transfect, typically, a transient opening of pores or "holes" in the cell membrane is performed to allow uptake of the substance. Transfect can be performed using calcium phosphate, by electroporation, by cell squeezing, or by mixing cationic lipids with the substance to produce liposomes (which fuse with the cell membrane and release their cargo inward). Examples of methods for transfecting eukaryotic hosts include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate / DNA coprecipitation method), microinjection, and electroporation.

[0124] The term "transformation" is used to describe the nonviral introduction of nucleic acid molecules or polynucleotides (including vectors) into bacteria, and even non-animal eukaryotic cells (including plant cells). Thus, transformation is a genetic modification of a bacterial or non-animal eukaryotic cell, which occurs when exogenous genetic material (nucleic acid molecules) is directly taken up from the environment of such a bacterial or non-animal eukaryotic cell via the cell membrane and subsequently incorporated. Transformation can be induced by artificial means. For transformation to occur, the cell or bacterium must be in a state of transformation-receptiveness, which can occur as a time-delayed response to environmental conditions (such as starvation or cell density). For prokaryotic cell transformation, techniques may include heat shock-mediated incorporation, bacterial protoplast fusion with intact cells, microinjection, and electroporation. Techniques for transforming plant cells include the introduction of Agrobacterium-mediated agents (such as those using A. tumefaciens), high-speed injection of tungsten or gold nanoparticles, electroporation, microinjection, and polyethylene glycol-mediated incorporation.

[0125] Therefore, in light of the above, the present invention further provides a host cell comprising at least one polynucleotide sequence and / or a vector (as described herein).

[0126] For the expression of the TCR of the present invention, host cells may be selected that regulate the expression of the inserted polynucleotide sequence as desired and / or modify and process the gene product (i.e., RNA and / or protein) as desired. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the gene product may be important for the function of the TCR. The mechanisms for post-translational processing and modification of the gene product are characteristic and specific to the host cell. Appropriate cell lines or host systems may be selected to ensure that the modification and processing of the product occurs correctly and reliably. For this purpose, eukaryotic host cells having cellular mechanisms for appropriately processing the primary transcript, glycosylation and phosphorylation of the gene product may be used.

[0127] (a) a host cell for expressing and obtaining the TCR of the present invention, specifically the soluble form of the TCR of the present invention ("production host cell"), and (b) a host cell that expresses the TCR of the present invention and has effector function ("effector host cell"). Such "effector host cells" are particularly useful for therapeutic purposes and are intended to be administered to subjects in need. Preferred "effector host cells" include lymphocytes (such as cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma / delta T cells, etc.).

[0128] "Producing host cells" cell The "producing host cells" used for the expression of the soluble TCR in the present invention preferably have the ability to express a large amount of recombinant protein.

[0129] Based on the above, possible expression systems (i.e., host cells containing the expression vectors mentioned above) include microorganisms (bacteria (e.g., E. coli, B. subtilis)) transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA expression vectors, or cosmid DNA expression vectors, yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors, insect cell lines infected with recombinant virus expression vectors (e.g., baculovirus), plant cell lines infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV), tobacco mosaic virus (TMV)), or plant cell lines transformed with recombinant plasmid expression vectors (e.g., Ti plasmid). Mammalian expression systems possessing recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, cytomegalovirus (CMV) major early promoter (MIEP) promoter) are often preferred. Suitable mammalian host cells can be selected from known cell lines (e.g., COS cells, CHO cells, BLK cells, 293 cells, 3T3 cells), but lymphocytes (cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, gamma / delta T cells, etc.) may also be used.

[0130] Examples of mammalian host cells that can be used as "productive host cells" include Chinese hamster ovary cells (CHO cells) (including DHFR-negative CHO cells (such as DG44 cells and DUXBl1 cells)), NSO cells, COS cells (derived cells of CVI with the SV40T antigen), HEK293 (human kidney) cells, and SP2 (mouse myeloma) cells. Examples of other host cell lines, but not limited to, include HELA (human cervical cancer), CVI (monkey kidney cell line), VERY, BHK (baby hamster kidney), MDCK, 293, WI38, R1610 (Chinese hamster fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney cell line), P3x63-Ag3.653 (mouse myeloma), BFA-lcIBPT (bovine endothelial cells), and RAJI (human lymphocytes). Host cell lines are typically available from commercial services, the American Tissue Culture Collection (ATCC), or published literature.

[0131] Non-mammalian cells (such as bacterial cells, yeast cells, insect cells, or plant cells) are also readily available and can be used as the "productive host cells" described above. Examples of bacterial host cells include Enterobacteriaceae (Escherichia coli, Salmonella, etc.), Bacillaceae (Bacillus subtilis, etc.), Pneumococcus, Streptococcus, and Haemophilus influenzae. Other host cells include yeast cells (Saccharomyces cerevisiae and Pichia pastoris, etc.). Insect cells include, but are not limited to, Spodoptera frugiperda cells.

[0132] As described above, the present invention also provides a method for producing and obtaining the TCR described herein, the method comprising (a) incubating host cells (i.e., producing host cells) under conditions in which the TCR is expressed, and (b) purifying the TCR.

[0133] culture Host cells containing the expression vector are grown under conditions suitable for the production of the TCR provided herein, specifically the alpha and / or beta chains as described elsewhere herein, and assayed for the synthesis of the alpha and / or beta chain proteins. For the expression of double-stranded TCRs, vectors encoding both the alpha and beta chains may be co-expressed in host cells to express the entire molecule.

[0134] purification Once the TCR of the present invention is expressed, it can be purified by any purification method known in the art, such as chromatography (e.g., ion exchange chromatography (e.g., hydroxyapatite chromatography), affinity chromatography, specifically protein A affinity chromatography, protein G affinity chromatography, or lectin affinity chromatography, size exclusion column chromatography), centrifugation, methods utilizing solubility differences, hydrophobic interaction chromatography, or any other standard protein purification method. Those skilled in the art will be able to easily select an appropriate purification method based on the individual characteristics of the TCR to be recovered.

[0135] "Effector host cell" As described above, the present invention also provides “effector host cells” containing the nucleotide sequence, vector, or TCR of the present invention. These effector host cells are intended to be modified using a predetermined method to contain the nucleic acid sequence encoding the TCR of the present invention, and to specifically express the TCR described herein on the cell surface. For the purposes of the present invention, “modified host cells expressing the TCR of the present invention” generally refers to host cells that have been treated or modified (effector or producing) to express the TCR according to the present invention, and this treatment or modification is carried out, for example, by RNA transfection as described in the appended examples. Other modification or transfection or transduction methods (such as those described elsewhere in this specification) are also intended. Therefore, the term “modified host cells” includes “transfected” host cells, “transduced” host cells, and “genetically modified” host cells, such host cells preferably express the TCR of the present invention.

[0136] Preferably, such “(modified) effector host cells” (specifically, “(modified) effector lymphocytes”) have the ability to mediate effector functions via intracellular signaling when the TCR binds to its specific antigenic target. Such effector functions include, for example, the release of perforin (which creates holes in the target cell membrane), the release of granzymes (proteases that act intracellularly to induce apoptosis), the expression of Fas ligands (which activate apoptosis in target cells having FAS), and the release of cytokines, preferably Th1 / Tc1 cytokines (such as IFN-gamma, IL-2, and TNF-α). Therefore, effector host cells engineered to express the TCR of the present invention, which has the ability to recognize and bind to antigenic targets in the target to be treated, are expected to exert the above-described effector functions, thereby killing target (e.g., cancer) cells. The lysis of target cells can be evaluated, and this evaluation is performed, for example, using a CTL fluorescence death assay (CTL, USA) that detects the disappearance of fluorescently labeled target cells in co-culture with recipient T cells transfected with a TCR.

[0137] Based on the above, effector host cells preferably express a functional TCR, which typically includes the TCR alpha and TCR beta chains described herein, as well as signaling subunits (CD3 gamma, CD3 delta, CD3 epsilon, and CD3 zeta) (CD3 complex). Furthermore, expression of co-receptor CD4 or co-receptor CD8 may also be desirable. In general, lymphocytes, i.e., T cells, that possess necessary genes involved in antigen binding, receptor activation, and downstream signaling (e.g., Lck, FYN, CD45, and / or Zap70) are particularly suitable as effector host cells. On the other hand, effector host cells that express the TCR of the present invention as a "binding domain" without having CD3 signaling subunits and / or the aforementioned downstream signaling molecules (i.e., those that have the ability to recognize the antigenic targets described herein, but do not exhibit the functions mediated by CD3 and / or the aforementioned downstream signaling molecules) are also envisioned herein. Such effector cells are expected to have the ability to recognize the antigenic targets described herein and, optionally, the ability to perform other functions unrelated to CD3 signaling and / or signaling of the aforementioned downstream signaling molecules. Examples include NK cells or NKT cells expressing the TCR of the present invention, which, for example, have the ability to release cytotoxic granules upon recognition of their antigenic target.

[0138] Therefore, cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, and gamma / delta T cells are considered to be useful lymphocyte effector host cells. Such lymphocytes expressing the recombinant TCR of the present invention are also referred to herein as "modified effector lymphocytes." On the other hand, it will be readily apparent to those skilled in the art that, in general, any component of the TCR signaling pathway that elicits a desired effector function can be introduced into suitable host cells by recombinant gene manipulation methods known in the art.

[0139] The effector host cells, specifically lymphocytes (such as T cells), may be autohost cells obtained from a target to be treated and transformed or transduced to express the TCR of the present invention. Typically, recombinant expression of the TCR will be achieved using the viral vector described in the accompanying examples. Methods for obtaining and isolating cells from patients are known in the art.

[0140] As stated herein, the effector host cells provided herein are particularly intended for therapeutic use. Further genetic modification of the host cells may be desirable to enhance therapeutic efficacy. For example, when using autologous CD8+ T cells as “effector host cells,” appropriate additional modifications include downregulation of the expression of endogenous TCRs, CTLA-4, and / or PD-1, and / or amplification of costimulatory molecules (such as CD28, CD134, and CD137). Means and methods for achieving the aforementioned genetic modifications have been reported in the art.

[0141] In this field, methods for targeted genomic manipulation of host cells are known, and these methods include gene knockdown with siRNA, as well as the use of so-called "programmable nucleases," which include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) derived from bacterial clustered short repeat palindromic sequence (CRISPR)-Cas (CRISPR-related) systems (particularly those outlined in Kim & Kim Nature Reviews Genetics 15, 321-334 (2014)). For example, programmable nucleases (such as TALENs) can be used to cleave DNA regions encoding "undesirable" proteins (such as PD-1, CTLA-4, or endogenous TCRs), thereby reducing their expression. When T cells are used as (effector) host cells, downregulation of endogenous TCRs has the advantage of reducing undesirable "false pairing" between endogenous TCR alpha / beta chains and exogenous TCR alpha / beta chains.

[0142] Pharmaceutical composition The present invention further provides pharmaceutical compositions comprising one or more TCRs, nucleic acids, vectors, and / or host cells as described herein as active agents, and one or more optionally selected pharmaceutical additives. Accordingly, it is also envisioned herein that such TCRs, nucleic acids, vectors, and host cells may be used in the manufacture of pharmaceutical compositions or drugs.

[0143] The term "pharmaceutical composition" specifically refers to compositions suitable for administration to humans. However, compositions suitable for administration to non-human animals are also generally included in this term.

[0144] The pharmaceutical compositions envisioned by the present invention may further comprise one or more checkpoint inhibitors, which are preferably selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. All of the aforementioned inhibitors are immune checkpoint inhibitors that have the ability to downregulate the immune response. Cytotoxic lymphocyte-associated protein 4 (CTLA-4) inhibitors are protein receptors that are constitutively expressed in regulatory T cells but are upregulated only after activation in normal T cells. PD-1 inhibitors and PD-L1 inhibitors work by inhibiting programmed cell death ligand 1 (PD-L1) from binding to its receptor (programmed cell death protein 1 (PD-1)). These interactions of cell surface proteins are involved in the suppression of the immune system and, occurring after infection, limit the death of bystander host cells and prevent autoimmune diseases. Therefore, it is preferable to combine these checkpoint inhibitors with the pharmaceutical compositions according to the present invention.

[0145] Additional checkpoint inhibitors included in the present invention are LAG3, ICOS, TIM3, VISTA, and CEACAM1. LAG3 is an inhibitory receptor on antigen-activated T cells. The ICOS protein belongs to the CD28 and CTLA-4 cell surface receptor family. The ICOS protein forms homodimers and plays an important role in intercellular signaling, immune responses, and cell proliferation regulation. TIM3, i.e., the hepatitis A virus cell receptor, encodes a protein belonging to the immunoglobulin superfamily and the TIM protein family. CD4-positive T helper lymphocytes can be classified into type 1 (Th1) and type 2 (Th2) based on their cytokine secretion patterns. VISTA, i.e., the V-Set immunomodulatory receptor, encodes an immunomodulatory receptor that inhibits the T cell response. The CEACAM1 gene encodes a member of the carcinoembryonic antigen (CEA) gene family (belonging to the immunoglobulin superfamily). These checkpoint inhibitors can also be combined with pharmaceutical compositions.

[0146] The pharmaceutical composition and its components (i.e., the active agent and optional pharmaceutical excipients) are preferably pharmaceutically acceptable, that is, they have the ability to induce a desired therapeutic effect without causing any undesirable local or systemic effects in the recipient. The pharmaceutically acceptable compositions of the present invention can, for example, be sterilized. Specifically, the term “pharmaceutically acceptable” may mean that its use in animals, and more specifically in humans, is approved by a regulatory body or other generally recognized pharmacopoeia.

[0147] The aforementioned active agent (e.g., host cells or TCR) is preferably present in the pharmaceutical composition in a therapeutically effective amount. “Therapeutally effective amount” means the amount of active agent that induces the desired therapeutic effect. Therapeutic efficacy and toxicity can be determined by standard procedures (e.g., in cell culture or test animals) (e.g., ED). 50 (A therapeutically effective dose for 50% of the population) and LD 50 (A lethal dose in 50% of the population). The dose-to-therapeutic ratio is the therapeutic index, ED 50 / LD 50 It can be expressed as a ratio. Pharmaceutical compositions with a large therapeutic index are preferred.

[0148] dose The precise dose of TCR polynucleotides, vectors, or host cells will be determined by those skilled in the art using known methods. The appropriate dose will deliver a sufficient amount of the active agent of the present invention, preferably therapeutically effective, i.e., inducing the desired therapeutic effect.

[0149] As is known in the art, it may be necessary to adjust the treatment objective (e.g., maintenance of remission or acute disease relapse), route of administration, time of administration, and frequency of administration, time and frequency of formulation administration, age, weight, overall health, sex, diet, severity of the disease, concomitant medications (multiple), sensitivity to response, and tolerance / response to treatment. An appropriate dose range (e.g., for soluble TCRs as described herein) can be determined using data obtained from cell culture assays and animal studies, and ED 50 It may include. Typically, doses may vary between 0.1 and 100,000 micrograms depending on the route of administration, with a total dose of up to approximately 2 g. Examples of doses of the active agent of the present invention are in the range of approximately 0.01 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 5 mg / kg, approximately 0.01 mg / kg to approximately 1 mg / kg, or approximately 0.1 mg / kg to approximately 1 mg / kg. Guidance on specific delivery doses and delivery methods is provided in the literature. It will be recognized that in treatment, it may be necessary to administer the active agent of the present invention as a single dose or multiple doses in its therapeutically effective dose. For example, depending on the pharmaceutical composition, depending on the formulation, half-life, and clearance rate of the particular composition, it may be administered once every 3-4 days, once every week, once every 2 weeks, or once every month. As described above, the pharmaceutical composition may optionally contain one or more pharmaceutical excipients and / or additional active agents.

[0150] Pharmaceutical additives The term "pharmaceutical additives" includes bulking agents, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, flow enhancers, preservatives, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, viscosity modifiers, surfactants, diluents, humectants, carriers, diluents, preservatives, emulsifiers, stabilizers, and osmotic pressure modifiers. Selecting pharmaceutical additives suitable for the preparation of the desired pharmaceutical composition of the present invention is within the scope of the skills of those skilled in the art. Examples of carriers for use in the pharmaceutical compositions of the present invention include physiological saline, buffered physiological saline, dextrose, and water. Typically, the selection of appropriate pharmaceutical additives will depend, among other things, on the active agent used, the disease to be treated, and the desired formulation of the pharmaceutical composition.

[0151] Additional active agents The present invention further provides pharmaceutical compositions comprising one or more of the above-described active agents of the present invention (e.g., host cells or TCR constructs) and one or more additional active agents suitable for the treatment and / or prevention of the disease to be treated. Examples of preferred active ingredients suitable for combination include known anticancer agents (cisplatin, mytansine derivatives, rachelmycin, calichemycin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate). Examples include glucuronates, auristatin E, vincristine, and doxorubicin, as well as peptide cytotoxins (lysine, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNAase, and RNAase, etc.), radionuclides (iodine-131, rhenium-186, indium-111, yttrium-90, bismuth-210 and bismuth-213, actinium-225, and astatine-213, etc.), prodrugs (antibody-directing enzyme prodrugs, etc.), immunostimulants (IL-2, etc.), chemokines (IL-8, platelet factor 4, melanoma growth-stimulating protein, etc.), antibodies or their fragments (anti-CD3 antibodies or their fragments, etc.), complement activators, heterologous protein domains, allogeneic protein domains, viral / bacterial protein domains, and viral / bacterial peptides.

[0152] Administration Various routes are applicable to the administration of the pharmaceutical composition according to the present invention. Typically, administration will be achieved parenterally. Methods of parenteral delivery include local administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intrathecal administration, subarachnoid administration, intraventricular administration, intravenous administration, intraperitoneal administration, intrauterine administration, intravaginal administration, sublingual administration, or intranasal administration.

[0153] formulation The pharmaceutical compositions of the present invention can be formulated in various forms, particularly depending on the active agent used (e.g., soluble TCR), for example, in solid, liquid, gaseous, or lyophilized form, and in particular, in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for a desired method of administration. The processes for producing the drugs themselves are described in the 22nd edition of Remington's Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa., 2012), and such processes may include, for example, conventional mixing processes, dissolution processes, granulation processes, sugar-coated tablet preparation processes, micronization processes under wet conditions, emulsification processes, encapsulation processes, or lyophilization processes. The pharmaceutical composition (for example, one comprising host cells or soluble TCRs as described herein) is typically provided in liquid form and preferably contains a pharmaceutically acceptable buffer.

[0154] The pharmaceutical composition of the present invention may, after its preparation, be placed in a suitable container and labeled for therapeutic use under indicated conditions. Such a label would include, for example, dosage, frequency of administration, and method of administration.

[0155] treatment Therefore, in light of the foregoing, the present invention provides TCRs, nucleic acids, vectors, and / or host cells described herein for use as agents in the detection, diagnosis, prognosis prediction, prevention, and / or treatment of cancer.

[0156] TCRs, nucleic acids, vectors, and / or host cells may generally be used for the therapeutic detection, diagnosis, prognosis prediction, prevention, and / or treatment of disease or disorder. The term “treatment” in all its grammatical forms includes therapeutic or prophylactic treatment of the subject in need. “Therapeutic or prophylactic treatment” includes prophylactic treatment aimed at complete prevention of clinical and / or pathological signs, or therapeutic treatment aimed at improvement or remission of clinical and / or pathological signs. Thus, the term “treatment” also includes improvement or prevention of disease.

[0157] Such diseases that are expected to be treated using the pharmaceutical compositions of the present invention are preferably cancers selected from the group consisting of melanoma, bladder cancer, colon cancer, and mammary gland adenocarcinoma, sarcoma, prostate cancer, uterine cancer, uveal cancer, uveal melanoma, head and neck squamous cell carcinoma, synovial cancer, Ewing's sarcoma, triple-negative breast cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia, and preferably the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, or colorectal cancer, sarcoma, or osteosarcoma.

[0158] The terms “subject,” “individual,” “animal,” or “patient” are used interchangeably herein to refer to any subject to which treatment is desired, specifically mammalian subjects. Mammalian subjects generally include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and similar species. On the other hand, it will be readily apparent that the TCRs, nucleic acids, vectors, host cells, and pharmaceutical compositions provided herein are particularly intended for the treatment of human subjects, specifically HLA-A2 positive human subjects.

[0159] Direct administration For therapeutic purposes, the TCR (specifically, the soluble TCR of the present invention), nucleic acids, vectors (such as viral vectors), or host cells of the present invention can be administered directly to a subject in need. Accordingly, the present invention provides TCRs, nucleic acids, vectors, or host cells for use in methods for detecting, diagnosing, predicting prognosis, preventing, and / or treating cancer. Such methods may include (a) supplying one or more of the (i) TCR, (ii) nucleic acids, (iii) vectors, (iv) host cells, and / or (v) pharmaceutical compositions of the present invention, and (b) administering one or more of (i) to (v) to a subject in need. Optionally, the method may further include a step of cancer treatment (e.g., radiation therapy or administration of one or more anticancer agents).

[0160] Treatment with ExVivo The treatment according to the present invention may also include (a) a step of supplying a sample of a subject, wherein the sample comprises lymphocytes; (b) a step of supplying one or more of the (i) TCR, (ii) nucleic acids, (ii) vectors, (iv) host cells, and / or (v) pharmaceutical compositions of the present invention; (c) a step of introducing one or more of (i) to (v) of step (b) into the lymphocytes of step (a) to obtain modified lymphocytes; and (d) a step of administering the modified lymphocytes of step (c) to a subject or patient in need.

[0161] The lymphocytes supplied in step (a) are specifically assumed to be the aforementioned "effector host cells," including T cells, NK cells, and / or NKT cells, particularly CD8 cells. +T cells, are preferably selected and can be obtained in a prior step from a sample of a subject, specifically a blood sample of the subject, by a predetermined method known in the art. On the other hand, it is also conceivable to use other lymphocytes (preferably those having the ability to express the TCR of the present invention and exhibiting the desired biological effector functions described herein). Furthermore, such lymphocytes will typically be selected such that compatibility with the subject's immune system is obtained, i.e., such lymphocytes will preferably not induce an immunogenic response. For example, it is conceivable to use "universal recipient cells", i.e., lymphocytes that are universally compatible, exhibit the desired biological effector functions, and can grow and proliferate in vitro. Thus, the use of such cells would eliminate the need to obtain and supply the subject's own lymphocytes in step (a).

[0162] The ex vivo introduction in step (c) can be carried out by introducing the nucleic acid or vector described herein into the lymphocytes by electroporation or by infecting the lymphocytes with the viral vectors (such as lentiviral or retroviral vectors) described above in the context of effector host cells. Other conceivable methods include those using transfection reagents (such as liposomes) or those transiently transfecting RNA. Introducing an antigen-specific TCR gene (e.g., by a (retro)viral vector) into (primary) T cells or transiently transfecting RNA has been a promising tool in generating tumor-associated antigen-specific T cells, which can then be reintroduced into the donor, where they specifically target and destroy tumor cells expressing the antigen. In the present invention, the tumor-associated antigen is PRAME as defined herein, specifically the HLA-A * 02-bound form of PRAME.

[0163] The treatment according to the present invention comprises (a) a step of supplying a sample of a subject, the supply step of which the sample comprises lymphocytes, the treatment comprising (b) a step of supplying one or more of (i) TCRs, (ii) nucleic acids, (iii) vectors, (iv) host cells, and (v) pharmaceutical compositions, (c) a step of introducing one or more of (i) to (v) of step (b) into the lymphocytes of step (b) to obtain modified lymphocytes, and (d) a step of administering the modified lymphocytes of step (c) to a subject or patient in need.

[0164] Therefore, in light of the foregoing, another aspect of the present invention is to use TCRs, nucleic acid sequences, vectors, and / or host cells described elsewhere in this specification to generate modified lymphocytes. Means and methods for introducing (e.g., nucleic acids and vectors) into lymphocytes are described elsewhere in this specification.

[0165] Diagnostic compositions The present invention also provides a diagnostic composition comprising one or more diagnostic agents and a TCR, nucleic acid, vector, and / or host cell as described herein. Typically, the diagnostic agent will include means for detecting that it has bound to its antigenic target (e.g., a label as described in relation to the TCR construct of the present invention). With respect to host cells, for example, modified host cells containing a dye or contrast agent released (instead of cytotoxic granules) upon antigen recognition may be used.

[0166] use The present invention envisions the use of the aforementioned diagnostic agents to achieve in vivo or in vitro detection, diagnosis, and / or prognosis prediction of cancer in a subject.

[0167] Accordingly, the present invention provides a diagnostic composition for use in the in vivo detection and diagnosis of cancer in a subject, the composition comprising the TCR, nucleic acid, vector, and / or host cell of the present invention as a diagnostic agent. The method typically comprises (a) administering the diagnostic agent to a subject, and (b) detecting that the diagnostic agent has bound to its antigenic target.

[0168] Furthermore, the present invention provides a method for detecting, diagnosing, and / or predicting the prognosis of cancer in a subject in vitro. Accordingly, the present invention also provides a method for detecting the presence of cancer in a subject, the method comprising: (a) supplying a sample of the subject, wherein the sample contains one or more cells; (b) contacting the sample with the TCR, host cells, and / or pharmaceutical composition of the present invention to form a complex; and (c) detecting the complex. The complex is intended to indicate that the diagnostic agent has bound to its antigenic target, and that there are (cancer) cells expressing the antigenic target.

[0169] In both methods, the binding of the diagnostic agent to its antigenic target can be detected by using predetermined methods known in the art, and in particular, will depend on the specific diagnostic agent used. Suitable labels that can be coupled with the diagnostic agents of the present invention are exemplified in the section relating to labeled TCR constructs.

[0170] Furthermore, the present invention also envisions the use of TCRs, nucleic acids, or vectors described herein to generate modified lymphocytes. As described elsewhere herein, preferred lymphocytes include, but are not limited to, cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, and gamma / delta-T cells.

[0171] It should be noted that the singular forms “a,” “an,” and “the” as used herein include multiple references unless otherwise clearly indicated in the context. Therefore, for example, a reference to “reagent” includes one or more such different reagents, and a reference to “method” includes a reference to equivalent steps and methods known to those skilled in the art that may be modified for or substitute for the methods described herein.

[0172] Unless otherwise specified, the term “at least” preceding a set of elements will be understood to refer to each of those elements. Those skilled in the art will recognize, or can verify, by performing the prescribed experiments, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0173] The term "and / or" includes, wherever it is used herein, the meanings of "and," "or," and "all or any other combination of the elements connected by that term."

[0174] As used herein, the terms “about” or “approximately” mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, this term also includes nominal numbers; for example, “about 20” includes 20.

[0175] The terms "less than" or "greater than" include nominal numbers. For example, less than 20 means less than or equal to. Similarly, "more than" or "greater than" means more than or equal to or greater than or equal to, respectively.

[0176] Throughout this specification and the claims described herein, unless the context requires a different interpretation, the word “comprise” and its variations (such as “comprises” and “comprising”) will be understood to imply that they include the integer or step or group of integers or steps described, but do not exclude any other integer or step or group of integers or steps. Where used herein, the term “comprising” may be replaced by the terms “containing” or “including,” and where used herein, the term “having” may be replaced in some cases.

[0177] As used herein, “consisting of” excludes any element, step, or component not specified in the claim. As used herein, “essentially consisting of” does not exclude any substance or step that does not substantially affect the basic and novel features of the claim.

[0178] In their respective uses herein, the terms “including,” “essentially consisting of,” and “consisting of” are all interchangeable with any of the other two terms.

[0179] It should be understood that the present invention is not limited to the specific methodologies, protocols, materials, reagents, and substances described herein, and is therefore subject to change. The terminology used herein is intended solely to describe specific embodiments and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0180] All publications and patents cited throughout the text of this Specified (including all patents, patent applications, scientific publications, manufacturer specifications, instructions, etc.) are incorporated herein by reference in their entirety, regardless of where they are cited. This Specified shall not be construed as acknowledging, on the grounds of prior art, that no prior rights are granted to the present invention by such disclosures. To the extent that any material incorporated by reference is inconsistent with or contradicts this Specified, this Specified shall prevail over any such material.

[0181] The following examples will provide a deeper understanding of the present invention and its advantages; however, these examples are provided for illustrative purposes only. These examples are not intended to limit the scope of the present invention in any way. [Examples]

[0182] The following embodiments are illustrative of the present invention and should not be construed as limiting the scope of the invention. [Table 2]

[0183] Example 1: Peptide specificity Either a specific SLL peptide (SLLQHLIGL) or an unrelated peptide (GLSNTHVL) is added at a concentration of 10. -5 As M, T2 cells were loaded at 37°C for 1.5 hours. Next, TCR-transduced T cells and the above cells were co-cultured with an effector:target ratio of 1:1 (10,000 effector cells / 96 wells). After 20 hours, the level of IFN-gamma in the cell culture supernatant was measured using a standard IFN-gamma ELISA. The specific SLL peptide was recognized by all TCR-transduced effector cells except for the negative control TCR upon loading onto T2 cells, while unrelated peptides were not recognized (Figure 1).

[0184] Example 2: Functional Avidity This experiment aimed to measure the functional avidity of SLL peptide-specific TCRs. Functional avidity refers to the combined strength of multiple affinity interactions (such as the interaction between transgenic TCRs and pMHC complexes). The functional avidity of the TCR transgenic T cell population was measured by stepwise (escalating) loading of SLL peptides (10 -5 M~10 -12 In a co-culture of T2 cells (effector:target = 1:1, 10,000 effector cells / 96 wells) subjected to loading at 37°C for 1.5 hours, the concentration (EC) that yielded 50% of the maximum relative IFN-gamma release was determined. 50 It is measured as a value. Standard IFN-gamma ELISA was performed 20 hours after co-culture (values ​​exceeding 4000 pg were extrapolated using a cubic polynomial). result: Compared to 3825 TCR transduced T cells, 027-004 TCR transduced T cells showed higher functional avidity, indicating greater sensitivity to very low amounts of target peptides (Figure 2). Compared to T cells transduced with TCRs disclosed in the art, 027-004 TCR-transduced T cells exhibited higher functional avidity, indicating greater sensitivity to very low amounts of target peptides (Figure 13).

[0185] Example 3: TCR recognition motif (serine scan and threonine scan) This experiment identifies critical residues in the SLL epitope sequence that are essential for direct recognition by the TCR, or HLA-A *The objective is to evaluate the critical residues essential for the binding of the peptide to the molecule encoded by 02:01. An amino acid substitution scan was used to define the critical amino acid in the epitope sequence. If an amino acid is critical, its replacement with a serine or threonine amino acid will result in the loss of TCR recognition. These "fixed" amino acids allow for the definition of a specific TCR recognition motif. By using serine or threonine residues, individual amino acids in the PRAME peptide are systematically replaced (serine scan and threonine scan). TCR-transduced T cells and T2 cells were co-cultured in vitro with an E:T ratio of 1:1 (10,000 effector cells / 96 wells), and 10 -5 Different peptides of M were loaded separately (at 37°C for 1.5 hours). Readout: Standard IFN-gamma ELISA was performed 20 hours after co-culture (values ​​above 4000 pg were extrapolated using a cubic polynomial). result: In serine scanning, 027-004 TCR transduced T cells exhibit different recognition motifs with fewer fixed sites compared to 3825 TCR transduced T cells (Figure 3). In threonine scanning, transduced T cells of the 027-004 TCR exhibit different recognition motifs compared to other TCRs disclosed in the art (Figure 12). [Table 3] [Table 4]

[0186] Example 4: Recognition of tumor cells and elimination of tumor cells Regarding the recognition of tumor cells, effector cells transduced with either TCR 027-004 or TCR 3825 and PRAME SLL Positive tumor cells or PRAME SLLNegative tumor cells and effector cells were co-cultured at 37°C for 20 hours under a 6% CO2 atmosphere with an E:T ratio of 1:1 (10,000 effector cells / 96 wells). The amount of IFN-gamma secreted by effector cells was determined using a standard IFN-gamma ELISA (values ​​exceeding 4000 pg were extrapolated using a cubic polynomial). result: TCR 027-004 transduction effector cells recognize tumor cells (e.g., MelA375) better than 3825 TCR transduction T cells (Figure 4). TCR 027-004 transduction effector cells recognize tumor cells (MelA375, NCI-H1650, and NCI-H1703) better than T cells transduced with TCRs known in the art (Figure 14). To eliminate tumor cells, IncuCyte® NucLight Red lentivirus-transduced tumor cells were seeded in flat-bottomed wells and co-cultured after one day. After adding 20,000 effector cells per well, tumor cells (2,500 cells for 647V, and 5,000 cells for all other tumor cell lines) were added. The culture plates were then transferred to an IncuCyteZOOM® device, and the proliferation of red fluorescent cells was monitored for 100 hours at 37°C under a 6% CO2 atmosphere, with photographs taken every 4 hours during that time. All TCR transduction effector cells are PRAME SLL It lyses positive tumor cells (PRAME positive), but PRAME SLL It does not affect the proliferation of negative tumor cells (PRAME-negative). result: TCR 027-004 transduction effector cells kill tumor cells (e.g., MelA375) more effectively than 3825 TCR transduction T cells (Figure 5). TCR 027-004 transduction effector cells effectively kill tumor cells (MelA375 and NCI-H1650) compared to T cells transduced with TCRs known in the art (Figure 15).

[0187] Example 5: Recognition of normal cells This series of experiments aims to evaluate the potential on-target / off-tumor toxicity and off-target toxicity that can be caused by PRAME-specific TCR transduction effector cells. For this purpose, HLA-A * We tested whether primary cells expressing 02:01, as well as induced pluripotent stem cell (iPS)-derived cell lines representing essential tissues or organs, could be recognized by TCR-transduced T cells. In vitro co-culture experiments (40,000 effector cells / 96 wells) were performed with appropriate E:T ratios according to each target cell type. Cells were seeded at the manufacturer's specified cell density 1-7 days before the start of co-culture, in accordance with the characteristics of each target, and then cultured in a monolayer in flat-bottomed wells. HLA-A2 expression on neurons was induced by adding a low dose of IFN-gamma to the culture medium. To distinguish between on-target / off-tumor toxicity and potential off-target toxicity, PRAME mRNA expression was analyzed in all normal cells tested by quantitative real-time polymerase chain reaction (qPCR). 10 -5 Target cells loaded with M peptide were used as an internal positive control (SLL peptide). Readout: Standard IFN-gamma / IL-2 ELISA was performed 20 hours after co-culture. Results: The TCR-transduced T cell population does not recognize unloaded normal cells in a manner that produces high levels of IFN-gamma. On the other hand, when the cells are loaded with a specific SLL peptide, they are recognized. In co-culture with RPTEC, minimal IFN-gamma production occurs in both samples even without loading. This production is due to the known endogenous PRAME expression in this cell type (Figure 6).

[0188] Example 6: HLA-A * 02 Precision Typing The purpose of this experiment is HLA-A * PRAME outside of 02:01 SLLA common HLA-A2 subalele (HLA-A) that can present an epitope and be recognized by individual SLL-specific TCRs. * We decided to determine the allele naming (HLA-restricted precision typing) using 02:xx; www.hla.alleles.org. Therefore, if a patient expresses such a recognized HLA-A2 subalele, it is possible to add such patients to the study cohort (Figure 7). TCR-transduced T cells and selected HLA-A2 subalele-positive lymphoblastoid cell lines (LCL; EBV-transformed B cells) were co-cultured in vitro at 37°C for 20 hours under a 6% CO2 atmosphere with an E:T ratio of 1:2 (10,000 effector cells / 96 wells). TCR-transduced PBL cells from a single donor were used as effector cells. For each individual LCL, 10 -5 The specific TCR subalele recognition of transduced T cells was determined by loading M-type SLL peptides at 37°C for 1.5 hours, co-culturing these LCLs with their respective effector cells, and testing IFN-gamma secretion. Unloaded target cells were used as negative controls. Readout: Standard IFN-gamma ELISA was performed 20 hours after co-culture. Results: 027-004 was the 10 HLA-A2 subaleles tested (A * It efficiently recognizes the PRAME peptide presented by three of the 02:xx molecules, and HLA-A2 subalele A * 02:02 and A * 02:04 is A * Recognized at the same level compared to 02:01. Control 3825 tested 10 HLA-A2 subaleles (A * It efficiently recognizes the PRAME peptide presented by one of the 02:xx) molecules, i.e., HLA-A2 subalele A * 02:01 is recognized (Figures 8-10).

[0189] References: Altschul,et al.,(1997)Nucleic Acids Res.25:3389-3402, Altschul, et al., (1990) J. Mol. Biol. 215:403-410, Chen et al.,Adv Drug Deliv Rev.2013 Oct.15;65(10):1357-1369 Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012) EP2173869(A2) Gargett and Brown Front Pharmacol. 2014;5:235 Kieback et al, Proc Natl Acad Sci USA.2008 Jan.15;105(2):623-8 Maack Publishing Co., Easton, Pa., 2012 Sambrook et al., Molecular Cloning: A Laboratory Manual (4th edition), Schmitt et al., Hum Gene Ther. 2009 November;20(11):1240-1248 Smith, et al., (1981) J. Mol. Biol. 147:195-197 Sommermeyer and Uckert,J Immunol.2010 Jun.1;184(11):6223-31 Walseng et al., (2015), PLoS ONE 10(4):e0119559 Weis, Manon(2015): Characterization of antigen-specific T cells after induction in TCR-humanized mice. Dissertation, LMU Munchen Tierarztliche Facultat: Veterinary Faculty Ludwigs University of Munich. Xue et al.,Clin Exp Immunol.2005 Feb;139(2):167-172; Fiedl et al.,Clin Cancer Res 2016 Mar;22(5):1234-1242 for DLBCL Mitsuhashi et al.,Hematology 2014,1 / 2014 Al-Khadairi et al.,Journal of Translational Medicine 2019;17:9 WO2019 / 175209A1

Claims

1. It has the amino acid sequence SLLQHLIGL (SEQ ID NO: 1) and is HLA-A * 02:01 HLA-A * 02:02, or HLA-A * A T cell receptor (TCR) having the ability to bind to a PRAME peptide present on a molecule encoded by HLA-A2 which is 02:04, wherein the TCR is a) A TCR alpha chain variable region comprising CDR1 containing or consisting of the amino acid sequence of SEQ ID NO: 2, CDR2 containing or consisting of the amino acid sequence of SEQ ID NO: 4, and CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 6, b) A TCR beta chain variable region comprising CDR1 containing or consisting of the amino acid sequence of SEQ ID NO: 3, CDR2 containing or consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 7, The TCR, including the aforementioned TCR.

2. The TCR according to claim 1, wherein binding to the sequence SLLQHLIGL (Sequence ID 1) induces IFN-gamma secretion by cells transfected or transfected with the TCR.

3. When measured by IFN-gamma immunoassay, the semi-maximal effective concentration of the IFN-gamma secretion is 10 －７ The TCR according to claim 2, wherein the TCR is less than M.

4. The aforementioned TCR, a) A TCR alpha chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 2, CDR2 having the amino acid sequence of SEQ ID NO: 4, and CDR3 having the amino acid sequence of SEQ ID NO: 6, b) A TCR beta chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 3, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 7, A TCR according to any one of claims 1 to 3, including the TCR.

5. a) A TCR alpha chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8, b) A TCR beta chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 9, A TCR according to any one of claims 1 to 4, including the TCR.

6. a) A TCR alpha chain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 10, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 10, b) A TCR beta chain comprising or consisting of an amino acid sequence selected from SEQ ID NO: 11, or an amino acid sequence having at least 80%, preferably at least 85%, more preferably 90% or 95% identity with SEQ ID NO: 11, A TCR according to any one of claims 1 to 5, including the TCR.

7. A TCR according to any one of claims 1 to 6, comprising at least one TCR alpha chain and at least one TCR beta chain, which are covalently linked to each other to form a TCR heterodimer or a TCR polymer.

8. The TCR according to any one of claims 1 to 7, wherein the TCR is selected from a natural TCR, a TCR variant, a TCR fragment, or a TCR construct.

9. A TCR according to any one of claims 1 to 8, further comprising one or more fusion elements optionally selected from an Fc receptor, an Fc domain (including IgA, IgD, IgG, IgE, and IgM), a cytokine (including IL-2 or IL-15), a toxin, an antibody or its antigen-binding fragment (including an anti-CD3 antibody, anti-CD28 antibody, anti-CDS antibody, anti-CD16 antibody, or anti-CD56 antibody, or its antigen-binding fragment), a CD247 (CD3-zeta) domain, a CD28 domain, a CD137 domain, a CD134 domain, or a combination thereof, and optionally further comprising at least one linker.

10. The aforementioned TCR, a) At least one TCR alpha chain according to any one of claims 1 to 9, and b) At least one TCR beta chain according to any one of claims 1 to 9, and c) Antibodies or single-chain antibody fragments (scFv) directed against antigens or epitopes on the surface of lymphocytes. Includes, The TCR according to any one of claims 1 to 9, wherein the TCR alpha chain(s) and the TCR beta chain(s) are linked to each other and fused with the antibody or the scFv via a linker of any choice.

11. The TCR according to claim 10, wherein the antigen is selected from CD3, CD28, CD5, CD16, or CD56.

12. The TCR according to any one of claims 1 to 11, further comprising at least one molecular marker.

13. A soluble TCR according to any one of claims 1 to 12.

14. A nucleic acid encoding a TCR according to any one of claims 1 to 13.

15. The nucleic acid according to claim 14, comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO:

22.

16. A vector comprising the nucleic acid according to claim 14 or claim 15.

17. A host cell comprising a TCR according to any one of claims 1 to 13, a nucleic acid sequence according to claim 14 or claim 15, or a vector according to claim 16.

18. The host cell according to claim 17, wherein the host cell is selected from lymphocytes.

19. The host cell according to claim 18, wherein the lymphocytes include cytotoxic T lymphocytes (CTLs), CD8+ T cells, CD4+ T cells, natural killer (NK) cells, natural killer T (NKT) cells, or gamma / delta T cells.

20. A method for obtaining the TCR described in any one of claims 1 to 19, a) Incubating the host cells according to claim 17 or claim 18 under conditions in which the TCR is expressed, b) Purify the TCR, The method, including the method described above.

21. a) The TCR according to any one of claims 1 to 13, b) The nucleic acid according to claim 14 or claim 15, c) The vector according to claim 16, or d) The host cell according to claim 17 or claim 18 One or more of the following, Optionally, you can select (multiple) pharmaceutical additives and A pharmaceutical composition or diagnostic composition containing the following:

22. The pharmaceutical composition according to claim 21, further comprising a checkpoint inhibitor.

23. The pharmaceutical composition according to claim 22, wherein the checkpoint inhibitor is selected from the group consisting of CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors.

24. A TCR according to any one of claims 1 to 13, a nucleic acid according to claim 14 or claim 15, a vector according to claim 16, or a host cell according to claim 17 or claim 18, for use as a pharmaceutical agent.

25. A TCR according to any one of claims 1 to 13, a nucleic acid according to claim 14 or 15, a vector according to claim 16, or a host cell according to claim 17 or 18, for use in the detection, diagnosis, prognosis, prevention, and / or treatment of cancer.

26. The TCR, nucleic acid, vector, or host cell according to claim 25, wherein the cancer is preferably selected from the group consisting of melanoma, bladder cancer, colon cancer, and breast cancer, sarcoma, prostate cancer, uterine cancer, uveal malignancy, uveal melanoma, head and neck squamous cell carcinoma, synovial cancer, Ewing's sarcoma, triple-negative breast cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, bile duct cancer, breast cancer, bladder cancer, myeloid leukemia, and acute lymphoblastic leukemia, and preferably the cancer is selected from the group consisting of NSCLC, SCLC, breast cancer, ovarian cancer, or colorectal cancer, sarcoma, or osteosarcoma.

27. Cancer prevention and / or treatment, a) (i) The TCR according to any one of claims 1 to 13, (ii) The nucleic acid according to claim 14 or claim 15, (iii) The vector according to claim 16, (iv) The host cell according to claim 17 or claim 18, and (v) The pharmaceutical composition according to any one of claims 21 to 23 To supply one or more of the following, b) Administering at least one of (i) to (v) to a subject in need of it, The TCR, nucleic acid, vector, or host cell according to claim 25, comprising:

28. Cancer prevention and / or treatment, a) Supplying the target sample, wherein the sample contains lymphocytes, b) (i) The TCR according to any one of claims 1 to 13, (ii) The nucleic acid according to claim 14 or claim 15, (iii) The vector according to claim 16, (iv) The host cell according to claim 17 or claim 18, and (v) The pharmaceutical composition according to any one of claims 21 to 23 To supply one or more of the following, c) Introducing one or more of (i) to (v) of step (b) into the lymphocytes of step (a) thereby obtaining modified lymphocytes, d) Administering the modified lymphocytes of step (c) to a subject or patient who needs them, A TCR, nucleic acid, vector, or host cell according to any one of claims 25 to 27, comprising:

29. A method for detecting the presence of cancer in a subject in vitro, (a) A sample containing one or more cells (i) The TCR according to any one of claims 1 to 13, (ii) The host cell according to claim 17 or claim 18, or (iii) Pharmaceutical composition according to claim 21 To bring it into contact with and thereby form a complex, and (b) To detect the composite, Includes, The method for detecting the complex, which indicates the presence of the cancer in the subject.

30. Use of a TCR according to any one of claims 1 to 13, a nucleic acid according to claim 14 or 15, or a vector according to claim 16, for the manufacture of a pharmaceutical product comprising lymphocytes expressing the TCR.

Images