Engineered T cell receptor constructs for cancer therapy

Abstract

The present invention relates to engineered T cell receptors (TCRs) against cancer-testis antigen Newton esophageal squamous cell carcinoma-1 (NY-ESO-1) useful for the treatment of cancer. In particular, isolated TCR polypeptides, as well as corresponding nucleic acid molecules and cell compositions, are provided which are characterized by improved properties particularly suitable for use in cancer immunotherapy.

Description

Technical Field

[0001] The present invention relates to engineered T cell receptors against NY-ESO-1, nucleic acid constructs encoding them, and their use in cancer therapy. Background Art

[0002] Cancer is the leading cause of death worldwide, causing nearly 10 million deaths, or nearly one in six deaths, in 2020. Traditional cancer therapies such as chemotherapy drugs and radiation therapy often have low specificity and can act on both healthy and diseased tissues, resulting in severe side effects. This low specificity has laid the foundation for the development of targeted treatment approaches, including various immunotherapy modalities. The main approaches used in immunotherapy include monoclonal antibodies, immune system modulators (e.g., cytokines), therapeutic vaccines, immune checkpoint inhibitors, and adoptive cell transfer (ACT).

[0003] One of the major challenges in cancer therapy is the immune evasion ability of tumors. Cancer cells exploit strategies used by the immune system to control the magnitude of immune responses, such as upregulation of checkpoint protein expression, activation of cell death programs, and accumulation of various immunosuppressive cells, to evade immune attack and may employ additional immune tolerance mechanisms.

[0004] It has been recognized that ACT is a promising treatment method for cancer patients. ACT can be used to enhance the function of the immune system or improve the specificity and persistence of immune cells transferred to patients. Cancer ACT usually uses immune cells isolated from the patient's blood (or from a suitable donor), which may have undergone ex vivo cancer-specific selection and / or genetic modification to give these cells anti-tumor function. Subsequently, a large number of expansion steps of the selected or modified cells are usually performed, and the expanded cells are then infused back into the patient to fight the cancer.

[0005] ACT includes a large number of different types of immunotherapy schemes, one of which involves T cells (TCR-T) transduced by T cell receptors (TCRs), wherein the immune cells are genetically modified or manipulated before being administered to a subject to express a TCR with desired tumor antigen specificity. Despite the promise, some setbacks have been encountered in the development of TCR-T compositions for ACT, such as in the expression and / or function of transfected TCRs. For example, it has been reported that the assembly of mismatched endogenous and transfected TCRs produces TCRs with unexpected specificity, including autoreactive TCRs. It has been proposed that the recognition of target antigens on normal tissues by T cells or unrelated antigens caused by TCR cross-reactivity is one of the main challenges in developing TCR-T patterns with sufficient efficacy and safety. Other setbacks involve, for example, insufficient expression, stability and / or function in engineered cells, which may then be accompanied by insufficient functional affinity. In addition, the exhaustion of effectors produced by overstimulation and nervous signaling may further impair the therapeutic outcome.

[0006] Attempts to modify various positions and structural elements in the TCR chain sequence with the aim of improving its functional properties have been reported. For example, in Bialer G. et al. (J. Immunol. 2010, 184: 6232-6241), Cohen CJ et al. (Cancer Res. 2006 September 1; 66 (17): 8878-8886), Johnson LA et al. (J Immunol. 2006 November 1; 177 (9): 6548-6559), Daniel-Meshulam I. et al. (Front. Immunol., 06 July 2012), Haga-Friedman A. et al. (J. Immunol. 2012, 188: 5538-5546), Cohen CJ et al. (Cancer Res. 2007, April 15; 67 (8): 3898-3903), Kuball et al. (Cancer Res. 2007, April 15; 67 (8): 3898-3903), J. et al. (Blood. 2007, 109: 2331-2338) and Ikeda H (International Immunology, Vol. 28, No. 7, pp. 349-353), discussed the development of improved engineered TCR. So far, preclinical and clinical studies have demonstrated the feasibility, safety and efficacy of various levels of using TCR engineered T cells to treat cancer and viral infections, and there is still a need for the development of TCRs with improved expression and function that are compatible with ACT regimens.

[0007] Cancer-associated epitopes specifically expressed on tumor cells for TCR-T have been vigorously sought to reduce cytotoxic effects in healthy tissues of cancer patients. NY-ESO-1 or New York esophageal squamous cell carcinoma 1 is a well-known cancer-testis antigen that is re-expressed in various cancer types. NY-ESO-1 is reportedly expressed in a variety of tumor types, including neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer, and breast cancer. Its ability to elicit spontaneous humoral and cellular immune responses and its restricted expression pattern make it a good candidate target for cancer immunotherapy (Thomas R et al., Front Immunol. 2018, 9: 947). The reactive epitope of human NY-ESO-1 was identified as amino acids 157-167.

[0008] Robbins et al. (J. Immunol. 2008, 180: 6116-6131) disclosed the generation of single and double amino acid substitution variants in the TCR CDRs of three TCRs that recognize tumor-associated antigens. Among the variants generated were variants of the 1G4 TCR that recognized a peptide corresponding to amino acid residues 157-165 of the human cancer-testis Ag NY-ESO-1 in the context of the HLA-A*02 class I allele.

[0009] EP2016102, attributed to some of the present inventors and colleagues, relates to chimeric TCRs and functional variants thereof, the chimeric TCRs comprising the variable regions of a human TCR and the constant region of at least the extracellular domain comprising the constant region of a non-human TCR. The disclosed chimeric TCRs may have antigenic specificity for a cancer antigen selected from MART-I, gp-100, p53 and NY-ESO-1.

[0010] Other TCRs against, inter alia, NY-ESO-1 are disclosed in, for example, US10201597, WO2020188348, US20210163891, US20190100592, US20210317184, WO2008037943, US20200040358, and US8088379.

[0011] Many clinical trials investigating NY-ESO-1-specific engineered TCR-T have been initiated. For example, Robbins et al. (Clin Cancer Res. 2015 March 1; 21(5): 1019-1027), Rapoport A.P. et al. (Nat Med. 2015, August; 21(8): 914-921), Stadtmauer EA et al. (Blood Adv, 2019, 3(13), 2022), Ramachandran I et al. (J Immunother Cancer. 2019, 7: 276) and D'Angelo SP et al. (Cancer Discov. 2018, 8(8): 944-957) reported clinical studies involving NY-ESO-1-directed immunotherapy. Some studies have shown that efficacy and / or safety are impaired. For example, while the treatments have been found to be effective in some patients, they generally result in a lack of response (or lack of a complete response) and / or a short duration of response (e.g., due to poor persistence of adoptively transferred cells) in the majority of patients tested. Reported adverse reactions include immunodeficiency (thrombocytopenia, lymphopenia, leukopenia, neutropenia, anemia), gastrointestinal symptoms (diarrhea, decreased appetite, nausea), fatigue, hypoxia, infection, hypophosphatemia, cytokine release syndrome, autologous graft-versus-host disease (GVHD), immune effector cell-associated neurotoxicity (ICANS), and treatment-related death.

[0012] Therefore, despite reported success in preclinical trials and certain cancer patient groups, many TCR-T models have shown impaired or insufficient therapeutic activity (or other applicability) in clinical practice. To date, no engineered TCR-T therapy products have received regulatory approval for marketing.

[0013] Therefore, there is still a need for other improved means, methods and compositions for NY-ESO-1 TCR-based cancer ACT. In particular, the development of NY-ESO-1 TCR-based ACT treatment modalities that provide enhanced efficacy and / or safety would be very beneficial. Summary of the invention

[0014] The present invention relates to engineered T cell receptors (TCRs) against the cancer-testis antigen New York esophageal squamous cell carcinoma-1 (NY-ESO-1), nucleic acid constructs encoding them, and their use in cancer therapy. In particular, isolated TCR polypeptides and corresponding nucleic acid molecules and cell compositions are provided, characterized by improved properties particularly suitable for cancer immunotherapy.

[0015] The present invention is based, in part, on the development of improved constructs for providing NY-ESO-1 specific lymphocyte compositions suitable for cancer immunotherapy. As disclosed herein, a nucleic acid construct encoding a novel engineered NY-ESO-1 specific TCR was developed. The construct, cloned into a retroviral vector containing a long terminal repeat (LTR) from a murine stem cell virus (MSCV), was characterized in various in vitro and in vivo assays. Notably, transduction of human primary lymphocytes with the vector resulted in a significantly enhanced anti-tumor response compared not only to lymphocytes transduced with an unrelated TCR, but also to lymphocytes transfected with a hitherto known TCR directed against the same epitope of NY-ESO-1. Furthermore, a clinical-grade pharmaceutical composition comprising the construct, manufactured in accordance with GMP requirements, was found to be highly effective and safe in treating tumors in vivo.

[0016] Thus, disclosed herein are the development and preparation of improved therapeutic modalities (including nucleic acid-based pharmaceutical compositions and cell compositions) for cancer immunotherapy. In various embodiments, compositions according to the principles of the present invention can be used to overcome or reduce the persistence defects typically associated with adoptive transfer therapy.

[0017] Thus, the present invention, in its embodiments, relates to an isolated TCR against NY-ESO-1. As disclosed herein, the isolated TCR (also referred to herein as an isolated or engineered TCR complex, molecule or polypeptide) specifically binds to an epitope presented by HLA-A2 comprising the amino acid sequence SLLMWITQC (SEQ ID NO: 21) (which corresponds to residues 157 to 165 of NY-ESO-1). The TCR is characterized by a unique combination of structural elements as described in detail below, providing advantageous functional properties. In some embodiments, the TCR according to the present invention comprises a complementary determining region (CDR) sequence as set forth in Table 1 below. Other structural and functional properties characteristic of the TCR according to embodiments of the present invention are described in further detail below.

[0018] In one aspect of the present invention, there is provided an isolated TCR against NY-ESO-1, comprising:

[0019] (a) a TCR alpha chain comprising:

[0020] i. a variable region (VR) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3;

[0021] ii. a constant region (CR) comprising a cysteine ​​residue at position 47 and an amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0022] (b) a TCR β chain comprising:

[0023] i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and

[0024] ii. CR comprising a cysteine ​​residue at position 57; and

[0025] (c) multiple interchain disulfide bonds between the α and β chains.

[0026] In one embodiment, the TCR comprises a first disulfide bond between a cysteine ​​residue at position 94 of the α chain CR and at position 131 of the β chain CR, and a second disulfide bond between a cysteine ​​residue at position 47 of the α chain CR and at position 57 of the β chain CR. In another embodiment, the transmembrane region of the α chain has the amino acid sequence LLVIVLRILLLKVAGFNLLMT (SEQ ID NO: 23). In another embodiment, the TCR comprises a TCR α chain having an amino acid sequence set forth in SEQ ID NO: 17, optionally without a signal peptide at positions 1-20 thereof. In yet another embodiment, the TCR comprises a TCR β chain having an amino acid sequence set forth in SEQ ID NO: 18, optionally without a signal peptide at positions 1-21 thereof. In another embodiment, each of the α and β chains exhibits at least 95% sequence identity to SEQ ID NOs: 17 and 18, respectively, and the TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21. In yet another embodiment, each of the α and β chains exhibits at least 95% sequence identity to SEQ ID NOs: 17 and 18, respectively, and the TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21 with high functional affinity.

[0027] On the other hand, a nucleic acid construct encoding the isolated TCR is provided. In another embodiment, the nucleic acid construct comprises the nucleic acid sequences encoding the CDR1, CDR2 and CDR3 of the α chain, respectively, as set forth in SEQ ID NO: 7-9, and the nucleic acid sequences encoding the CDR1, CDR2 and CDR3 of the β chain, respectively, as set forth in SEQ ID NO: 10-12. In another embodiment, the nucleic acid construct comprises a first nucleic acid molecule encoding the α chain and having the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the β chain and having the nucleic acid sequence set forth in SEQ ID NO: 20. In another embodiment, the first and second nucleic acid molecules are operably linked to one or more transcriptional control elements. In yet another embodiment, the one or more transcriptional control elements are capable of inducing or enhancing the expression of the TCR chain in human lymphocytes (particularly T cells).

[0028] In another embodiment, the nucleic acid construct comprises a first nucleic acid molecule encoding the alpha chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the beta chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 20, wherein the encoded TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21. In yet another embodiment, the nucleic acid construct comprises a first nucleic acid molecule encoding the alpha chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the beta chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 20, wherein the encoded TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21 with high functional affinity. In yet another embodiment, the encoded TCR is capable of exerting tumor-specific reactivity in a CD8-independent manner. In yet another embodiment, the nucleic acid construct is capable of providing enhanced expression levels and / or duration of the encoded TCR compared to a construct encoding a naturally occurring TCR for an epitope set forth in SEQ ID NO: 21 presented by HLA-A2. In yet another embodiment, the first nucleic acid molecule and the second nucleic acid molecule are linked by a third nucleic acid molecule encoding a linker. In yet another embodiment, the linker is a 2A peptide.

[0029] In another aspect, a nucleic acid construct is provided, comprising at least one nucleic acid molecule selected from SEQ ID NO: 19 and 20. In another aspect, an expression vector is provided, comprising the nucleic acid construct disclosed herein. In another embodiment, the present invention provides a host cell comprising the vector. In another embodiment, a pharmaceutical composition is provided, comprising a therapeutically effective amount of the vector.

[0030] In another aspect, a cellular composition for cancer immunotherapy is provided, comprising a therapeutically effective amount of immune cells (usually and preferably a population of T cells) engineered to express a TCR against NY-ESO-1, wherein the TCR comprises:

[0031] (a) a TCR alpha chain comprising:

[0032] i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3;

[0033] ii. CR comprising a cysteine ​​residue at position 47 and the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0034] (b) a TCR β chain comprising:

[0035] i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and

[0036] ii. CR comprising a cysteine ​​residue at position 57; and

[0037] (c) multiple interchain disulfide bonds between the α and β chains.

[0038] In one embodiment, the cell composition is adapted for adoptive transfer therapy (ACT). In another embodiment, the immune cells are a cell population comprising T cells. In a specific embodiment, the immune cells are a cell population comprising T cells, and the composition is adapted for ACT. In another embodiment, at least 60%, 70%, 80% or 90% of the immune cells are characterized by the engineered TCR (eTCR + In another embodiment, at least 70% of the immune cells are CD3 + eTCR+ cells and contain CD4 + and CD8 + Two kinds of cells. In another embodiment, the immune cell is engineered to express the TCR by administering a nucleic acid construct encoding the TCR, and the cell composition is characterized by an average copy number of 5 or less (e.g., 1, 2, 3, 4, or 5) construct molecules per cell. In another embodiment, the TCR comprises a TCR alpha chain having an amino acid sequence set forth in SEQ ID NO: 17, optionally without a signal peptide at positions 1-20 thereof, and a TCR beta chain having an amino acid sequence set forth in SEQ ID NO: 18, optionally without a signal peptide at positions 1-21 thereof. In another embodiment, the nucleic acid construct comprises a first nucleic acid molecule encoding the alpha chain and having a nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the beta chain and having a nucleic acid sequence set forth in SEQ ID NO: 20.

[0039] In another embodiment, the cell composition is prepared by a method comprising the steps of:

[0040] a. providing a cell population containing T cells,

[0041] b. culturing the cell population in the presence of a CD3-specific antibody and interleukin-2 (IL-2),

[0042] c. engineering the cells obtained from step b. to express the TCR,

[0043] d. expanding the cells obtained from step c. so as to obtain a therapeutically effective amount of expanded engineered cells, and

[0044] e. Harvesting the expanded engineered cells obtained from step d.

[0045] In another embodiment, the cell population containing T cells provided in step a. is a peripheral blood mononuclear cell (PBMC) population. In another embodiment, step b. further comprises culturing the cells with a CD28-specific antibody, and the CD3-specific antibody and the CD28-specific antibody are surface-bound. In another embodiment, step d. is performed by culturing the cells after step c. with IL-2 and optional feeder cells. In a specific embodiment, step d. is performed by culturing the cells after step c. with IL-2 and feeder cells. In another embodiment, step d. is performed without supplementing antibodies. In another embodiment, step d. is performed to obtain at least 10 9 CD3 +eTCR + In another embodiment, step e. is performed within 7-21 days, typically within 14-21 days, of starting step b.

[0046] In another aspect, a method for preparing a cell composition for ACT is provided, the method comprising:

[0047] a. providing a cell population containing T cells,

[0048] b. culturing the cell population in the presence of CD3-specific antibodies and IL-2,

[0049] c. engineering the cells obtained from step b. to express the TCR disclosed herein,

[0050] d. expanding the cells obtained from step c. so as to obtain a therapeutically effective amount of expanded engineered cells, and

[0051] e. Harvesting the expanded engineered cells obtained from step d.

[0052] In one embodiment, the method comprises:

[0053] a. Provide PBMC population,

[0054] b. culturing the cell population in the presence of CD3-specific antibodies and IL-2,

[0055] c. engineering the cell to express the TCR by incubating the cell with a viral vector comprising a nucleic acid construct encoding the TCR,

[0056] d. amplifying the cells after step c. in the presence of IL-2 and feeder cells without supplementing antibodies to obtain at least 10 9 CD3 expressing the TCR + cells, and

[0057] e. Harvest the expanded cells within 14-15 days after starting step b.

[0058] In another embodiment, the resulting cell composition comprises at least 70% CD3 + eTCR + cells, including CD4 + and CD8 + In another embodiment, step b. comprises administering to said cell an expression vector disclosed herein (which encodes a TCR of the invention).

[0059] In another aspect of the present invention, a method of treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a cell composition disclosed herein. In one embodiment, the cells of the composition (obtained from step e.) are autologous cells of the subject.

[0060] In another aspect, provided is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition disclosed herein.

[0061] In another aspect, a method of enhancing anti-tumor immunity in a subject in need thereof is provided, the method comprising administering to the subject a cell composition or a pharmaceutical composition disclosed herein. Each possibility represents a separate embodiment of the present invention.

[0062] In some embodiments of the methods of the invention, the subject has a tumor that expresses NY-ESO-1. In another embodiment, the subject is HLA-A2 positive. In another embodiment, the tumor is selected from melanoma, bladder, ovarian, lung, breast, and prostate tumors. In another embodiment, the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial, and prostate tumors. Each possibility represents a separate embodiment of the invention.

[0063] In another aspect, the invention relates to a cell composition of the invention for use in treating cancer in a subject in need thereof. In another embodiment, the subject has a tumor that expresses NY-ESO-1, and / or the subject is HLA-A2 positive. In another embodiment, the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors. In another embodiment, the cells of the composition are autologous cells of the subject.

[0064] In another aspect, a pharmaceutical composition comprising a therapeutically effective amount of a vector of the invention is provided for treating cancer in a subject in need thereof. In another embodiment, the subject has a tumor expressing NY-ESO-1, and / or the subject is HLA-A2 positive. In another embodiment, the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors.

[0065] In another aspect, a cell composition of the invention is provided for use in enhancing anti-tumor immunity in a subject in need thereof. In another embodiment, the subject has a tumor expressing NY-ESO-1, and / or the subject is HLA-A2 positive. In another embodiment, the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors. In another embodiment, the cells of the composition are autologous cells of the subject.

[0066] In another aspect, a pharmaceutical composition comprising a therapeutically effective amount of a vector of the invention is provided for enhancing anti-tumor immunity in a subject in need thereof. In another embodiment, the subject has a tumor expressing NY-ESO-1, and / or the subject is HLA-A2 positive. In another embodiment, the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors.

[0067] Other objects, features and advantages of the present invention will become apparent from the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figures 1A-1C Depicted are the levels of cytokine secretion after co-culture of cells transduced with eTCR, 1G4, or NGFR with syngeneic target cells (melanoma cell lines A375 and 624.38 expressing NY-ESO-1) or with control melanoma cells (888A2). Levels of tumor necrosis factor (TNFα), interleukin 2 (IL2), and interferon gamma (IFN-γ) in the co-culture supernatant were determined by ELISA. Figure 1A -TNFα concentration. Figure 1B -IL2 concentration. Figure 1C -IFN-γ concentration.

[0069] Figure 2 Depicted are the expression levels of the T cell activation marker OX40 after co-culturing cells transduced with eTCR, 1G4 or NGFR with A375 and 624.38 ("624") target cells and the 888A2 melanoma cell line as a control. The transduced cells were stained with anti-OX40 antibodies and measured by fluorescence activated cell sorting (FACS). The results were normalized to the activity of the NGFR transduced cells against A375 cells.

[0070] Figure 3 Figure 3 shows the expression of CD8 T cells after co-culture of cells transduced with eTCR, 1G4, or NGFR with A375 or 624.38 target cells. +The expression level of T cell activation marker 4-1BB in cells. The transduced cells were stained with differentially labeled anti-4-1BB and anti-CD8 antibodies and measured by FACS.

[0071] Figure 4 The expression levels of eTCR in PG13 cells are depicted, corresponding to the first 12 clones of eTCR. PG13 cells transduced with medium alone were used as negative controls and are shown as PG13. Cells were stained with PE-coupled anti-Vβ13 antibodies against the specific β chain of the eTCR and evaluated by FACS.

[0072] Figure 5 The target-induced IFN-γ secretion levels of PBMCs activated and transduced with eTCR subclones 1.2 and 1.16 of different dilutions (1: 1, 1: 2, 1: 4, 1: 8) are shown. The IFN-γ concentration in the supernatant after co-culture with melanoma A375 cells was determined by ELISA. Undiluted clone 1.3 was used as a positive control, and PG13 cells transduced with culture medium only ("no vector") were used as negative controls.

[0073] Figure 6 .Depicts FACS graphs of caspase-3 levels in A375 cells (expressing NY-ESO-1) after co-culture with PBMC activated and transduced with eTCR subclones. Cells were stained with the cell tracer marker DDAO, and caspase-3 was used as an indicator of apoptosis. The percentage of caspase-3 and DDAO expression in cells was detected by FACS.

[0074] Figure 7A-7B . Depicts the results of PCR for the presence of replication-competent retrovirus (RCR) in 6 random PBMC samples activated and transduced with eTCR vectors. PCR was performed on day 20 after transduction. Fig. 7A , PCR using primers for GALV (viral envelope protein) as an indicator of RCR. Figure 7B , PCR using primers for β-actin (βAct) was used as a positive control.

[0075] Figure 8 Specific subsets and phenotypes of cell compositions prepared under different expansion conditions are depicted. CD3 +T cells were stained with antibodies against CD45RA and CCR7 and analyzed by flow cytometry. Lymphocytes transduced with vector-free supernatant were used as negative controls ("no vector"), and lymphocytes transduced with the cell composition are shown as "eTCR". The expansion protocols tested: anti-CD3 antibody and feeder cells are shown as "aCD3", Transact (Milteny) protocol is shown as "Trans", and DynaBeads (Thermo Scientific) is shown as "Dyna". Effector memory cells lacking expression of both CCR7 and CD45RA are shown as Tem (CD45RA - CCR7 - , gray bars), Temra cells expressing CD45RA but lacking CCR7 expression are shown as T EMRA (CD45RA + CCR7 - , black bars). The results are given in each group (T EM或 T EMRA ) in CD3 + The percentiles among the total number of cells are expressed.

[0076] Fig. 9 Describes how Figure 8 Results of a second analysis of specific subpopulations and phenotypes of cell compositions prepared under different expansion protocols as described in.

[0077] Fig.10 The proportions of different immune cell subsets in each cell composition are depicted. Cells were stained for CD3, CD4, CD8, CD14, CD19, CD83 and CD56 and analyzed by flow cytometry. The results correspond to day 13 after transduction in experiment 2. The different treatment groups were as follows Figure 8 As described in.

[0078] Fig.11 Describes how Fig.10 The ratio of different immune cell subsets in each cell composition described in Fig.10 Same experiment on day 20 after transduction.

[0079] Fig.12 Tumor volumes at different time points after in vivo ACT are depicted. ACT was performed in cohorts of NSG mice inoculated with A375 melanoma cells and transduced with eTCR vectors (black line), negative control PG13 supernatant transduced with cells (no vector, dashed line), or treated with saline (no ACT, dotted line). Tumor volumes were measured on days 7, 10, 15, 18, and 21 after ACT and are expressed as mean ± SD.

[0080] Fig.13 Figure 5. Tumor weights obtained from ACT-treated mice on day 22 after tumor inoculation. Fig.12 Tumors from mice treated with eTCR, no vector, or saline as described were harvested and weighed, and the mean ± SD is presented.

[0081] Figures 14A-14F Figure 3 shows the change in tumor volume over time in individual A375 melanoma-bearing mice according to different ACT test groups. The test groups were treated with increasing doses of eTCR + Cells were treated as ACT. Each line represents a single mouse. Fig.14A , control group mice that did not receive treatment. Fig. 14B , control group mice received non-transduced cells. Fig. 14C , accepts 14x10 6 eTCR per cell / mouse + ACT dosed mice. Fig.14D , eTCR + ACT dose is 21x10 6 cells / mouse. Fig.14E , eTCR + ACT dose is 28x10 6 cells / mouse. Fig.14F , eTCR + ACT dose is 56x10 6 cells / mouse.

[0082] Fig.15 Kaplan-Meier curves of the survival of A375 melanoma-bearing mice after ACT over time are depicted. Survival curves were calculated for each test group (p<0.01): no treatment is shown as tumor only - solid diamonds, negative control (no vector cells) - line with a vertical dash on top, eTCR + Cells: 14x10 6 -Solid square, 21x10 6 - Solid Hexagon, 28x10 6 -Filled circle, 56x10 6 Hollow triangle.

[0083] Fig.16 Depicted are photographic imaging of time-dependent bioluminescence of luciferase-expressing A375 melanoma cells in individual mice following ACT according to the test groups described in Figure 14. Bioluminescence was monitored at days 6, 13, 20, 27, and 34 after tumor inoculation.

[0084] Figures 17A-17B Figure 3. E-TCR in the blood of NSG tumor-bearing mice after ACT. +Flow cytometry plot of cell persistence. 6 eTCR + Blood samples obtained from mice that expressed cells and achieved a complete antitumor response were stained and analyzed by flow cytometry. Fig.17A - Blood samples stained with FITC-conjugated anti-human CD8 antibody. Fig. 17B - from Fig.17A CD8 + Cell populations.

[0085] Fig.18 Depicts administration of eTCRs generated by different regimens + Figure 2. Changes in tumor volume over time in female mice bearing A375 melanoma cells expressing luciferase. The regimens are: no treatment "tumor only" - dotted line with solid circles; no carrier - dashed line with solid triangles; Dynabeads (13x10 6 Positive cells / 17.3x10 6 Total cells) are shown as DYna-black lines with solid squares; Transact (13x10 6 Positive cells / 18.6x10 6 Total cells) are shown as Trans-black and dashed lines; anti-CD3 antibody and feeder cells (13x10 6 Positive cells / 28x10 6 Total cells) are shown as aCD3- black line with solid circles.

[0086] Fig.19 Describes Fig.18 Kaplan-Meier curves of survival of ACT-treated mice as described in. No treatment is shown as "Tumor Only" - dashed line, no vector - dotted line, Dynabeads are shown as DYna - thick line, Transact is shown as Trans - thin line, anti-CD3 antibody and feeder cells are shown as feeder cells - solid squares.

[0087] Fig. 20 Target-specific TNFα secretion of cells expressing eTCR or the hitherto known TCR, 1G4, is depicted. TNFα concentrations were measured after co-culturing eTCR- or 1G4-transduced cells with T2 cells pulsed with different concentrations of NY-ESO-1 peptide (or MART-1 peptide as a control).

[0088] Fig.21Target-induced IFN-γ secretion levels of cell compositions prepared from PBMCs originally isolated from uveal melanoma patients transduced or not with eTCR are shown. IFN-γ secretion in supernatants was measured by ELISA after 24 hours of incubation with T2 cells loaded or not with NY-ESO-1 peptide at a 1:1 effector to target (E:T) ratio. T: T2 - nonspecific T cells (not transduced) incubated with unloaded T2 cells; T: T2-ESO - nonspecific T cells incubated with peptide-loaded T2 cells; eTCR + : T2-eTCR-transduced T cells cultured with unloaded T2 cells; eTCR + :T2-ESO-eTCR incubated with peptide-loaded T2 cells + T cells.

[0089] Figures 22A-22D The persistence of eTCR-transduced cells in the blood of four patients at different time points after ACT is depicted. PBMCs obtained from each tested patient were stained with FITC-conjugated anti-human CD3 antibody. + The cell populations were further stained with PE-conjugated anti-Vβ13 antibody and analyzed by flow cytometry. Fig.22A - Patient 1 (uveal melanoma, 1x10 9 eTCR + ACT dose of cells). Fig. 22B - Patient 2 (synovial sarcoma, 1x10 9 eTCR + ACT dose of cells). Fig. 22C - Patient 3 (triple negative breast cancer, 1x10 9 eTCR + ACT dose of cells). Fig.22D - Patient 4 (uveal melanoma, 5x10 9 eTCR + ACT dose of cells).

[0090] Fig.23 Delineating the eTCR + CD4 + Secretion levels of IFN-γ after co-culture of cells with A375 melanoma target tumor cells expressing NY-ESO-1 antigen (+A375) or in the absence of target cells (-A375). IFN-γ levels in culture supernatants were determined by ELISA.

[0091] Figures 24A-24B Figure 3 shows the expression of eTCR in A375 melanoma cells after co-culture or in the absence of target cells. + CD4 +Expression levels of the cytotoxic activation marker CD107 in cells. Transduced cells were stained with differentially labeled antibodies against CD107 (CD107-APC-) or TCRβ chain (Vb13.3-PE) and measured by FACS. Fig.24A , eTCR cultured in the absence of target cells + cell. Fig. 24B , eTCR cultured with A375 cells + cell. DETAILED DESCRIPTION

[0092] The present invention relates to engineered T cell receptors (TCRs) against the cancer-testis antigen New York esophageal squamous cell carcinoma-1 (NY-ESO-1), nucleic acid constructs encoding them, and their use in cancer therapy. In particular, isolated TCR polypeptides and corresponding nucleic acid molecules and cell compositions are provided, characterized by improved properties particularly suitable for cancer immunotherapy.

[0093] The present invention is based in part on the development of improved constructs for providing NY-ESO-1 specific immunotherapy to cancer patients. As disclosed herein, a nucleic acid construct encoding a novel recombinant (engineered) NY-ESO-1 specific TCR was developed. Surprisingly, the construct not only + T cells, and on CD4 + This provides for stable and long-term expression of the TCR on the surface of T cells, showing exceptionally high transduction levels depending on the number of copies transduced.

[0094] Unexpectedly, the construct provided a significantly enhanced anti-tumor response compared not only to lymphocytes transduced with an unrelated TCR, but also to lymphocytes transfected with a hitherto known TCR directed against the same epitope of NY-ESO-1. Furthermore, clinical-grade cell compositions comprising the construct were found to be highly effective and safe in treating tumors in vivo and exhibited long-term persistence in human patients.

[0095] The present invention, in its embodiments, relates to an isolated TCR against NY-ESO-1. As disclosed herein, the isolated TCR (also referred to herein as an isolated or engineered TCR complex, molecule or polypeptide) specifically binds to an epitope presented by HLA-A2 comprising the amino acid sequence SLLMWITQC (SEQ ID NO: 21) corresponding to residues 157 to 165 of human NY-ESO-1. The TCR is characterized by a unique combination of structural elements as described in detail below, providing advantageous functional properties.

[0096] In one aspect, an isolated TCR against NY-ESO-1 is provided, comprising:

[0097] (a) a TCR alpha chain comprising: i. a variable region (VR) comprising a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; ii. a constant region (CR) comprising a cysteine ​​residue at position 47 and comprising the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0098] (b) a TCR β chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6; and ii. a CR comprising a cysteine ​​residue at position 57; and

[0099] (c) multiple interchain disulfide bonds between the α and β chains.

[0100] In another aspect, a nucleic acid construct encoding the isolated TCR is provided. In another aspect, the present invention provides a nucleic acid construct comprising at least one nucleic acid molecule selected from SEQ ID NO: 19 and 20.

[0101] In another aspect, a pharmaceutical composition is provided, comprising a therapeutically effective amount of an expression vector comprising a nucleic acid construct encoding an isolated TCR against NY-ESO-1, wherein the TCR comprises:

[0102] (a) a TCR alpha chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; ii. a CR comprising a cysteine ​​residue at position 47 and comprising the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0103] (b) a TCR β chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6; and ii. a CR comprising a cysteine ​​residue at position 57; and

[0104] (c) multiple interchain disulfide bonds between the α and β chains.

[0105] In another aspect, the present invention relates to a cellular composition for cancer immunotherapy, comprising a therapeutically effective amount of immune cells engineered to express a TCR against NY-ESO-1, wherein the TCR comprises:

[0106] (a) a TCR alpha chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; ii. a CR comprising a cysteine ​​residue at position 47 and comprising the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0107] (b) a TCR β chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6; and ii. a CR comprising a cysteine ​​residue at position 57; and

[0108] (c) multiple interchain disulfide bonds between the α and β chains.

[0109] In another aspect, a method for preparing a cell composition for adoptive transfer therapy (ACT) is provided, the method comprising:

[0110] a. providing a cell population containing T cells,

[0111] b. culturing the cell population in the presence of CD3-specific antibodies and IL-2,

[0112] c. engineering the cells obtained from step b. to express the TCR defined herein,

[0113] d. expanding the cells obtained from step c. so as to obtain a therapeutically effective amount of expanded engineered cells, and

[0114] e. Harvesting the expanded engineered cells obtained from step d.

[0115] In other aspects, the present invention relates to a method of treating cancer and / or enhancing anti-tumor immunity in a subject in need thereof, the method comprising administering to the subject a cellular composition for cancer immunotherapy comprising a therapeutically effective amount of immune cells engineered to express a TCR against NY-ESO-1, the TCR comprising:

[0116] (a) a TCR alpha chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; ii. a CR comprising a cysteine ​​residue at position 47 and comprising the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0117] (b) a TCR β chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6; and ii. a CR comprising a cysteine ​​residue at position 57; and

[0118] (c) multiple interchain disulfide bonds between the α and β chains.

[0119] In another aspect, the present invention relates to a cell composition as defined herein for use in treating cancer in a subject in need thereof. In another aspect, the present invention relates to a cell composition as defined herein for use in enhancing anti-tumor immunity in a subject in need thereof.

[0120] In other aspects, the present invention relates to a method of treating cancer and / or enhancing anti-tumor immunity in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an expression vector comprising a nucleic acid construct encoding an isolated TCR against NY-ESO-1, the TCR comprising:

[0121] (a) a TCR alpha chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; ii. a CR comprising a cysteine ​​residue at position 47 and comprising the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0122] (b) a TCR β chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6; and ii. a CR comprising a cysteine ​​residue at position 57; and

[0123] (c) multiple interchain disulfide bonds between the α and β chains.

[0124] In another aspect, the present invention relates to a pharmaceutical composition as defined herein for use in treating cancer in a subject in need thereof. In another aspect, the present invention relates to a pharmaceutical composition as defined herein for use in enhancing anti-tumor immunity in a subject in need thereof.

[0125] These and other embodiments are described and exemplified in greater detail below.

[0126] Features

[0127] Without wishing to be limited to a particular theory or mechanism of action, the TCR and constructs of the present invention are characterized by at least one, typically multiple, characteristics as described in detail below. In some embodiments, the characteristics are selected from affinity enhancement, avidity enhancement, functional avidity enhancement, reduced mismatching of α and β chains with non-related (e.g., endogenous) TCR chains, enhanced stability, improved transduction efficiency, increased expression levels, and / or extended surface expression to target epitopes. Therefore, in various embodiments, compositions (including cell compositions) and methods utilizing TCR and constructs of the present invention exhibit or provide at least one, typically multiple, functional characteristics discussed herein.

[0128] In various embodiments of the present invention, the terms "enhanced", "reduced", etc., as used herein, refer to an enhancement or reduction that is considered by a skilled person to be significant, particularly to a statistically significant enhancement or reduction. With respect to the functional properties of the present invention, the change (e.g., enhancement, increase, improvement, reduction or prolongation) is calculated compared to a corresponding control TCR molecule (e.g., a natural TCR directed against NY-ESO-1). According to a specific embodiment, the change in a specific property can be conveniently calculated relative to a value corresponding to the property exhibited by a hitherto disclosed NY-ESO-1 specific TCR (including but not limited to those mentioned in the above-mentioned background section). In another specific embodiment, the naturally occurring or hitherto disclosed TCR is directed against the NY-ESO-1 epitope of SEQ ID NO: 21. For example, but not limited to, the change is conveniently calculated relative to a TCR having the VRα and VRβ chains set forth in SEQ ID NOs: 26 and 27, as disclosed and exemplified in Example 2 below.

[0129] In another embodiment, the TCR is characterized by an enhanced affinity for the target epitope of SEQ ID NO: 21. Affinity is the strength of binding of one molecule to another. Specifically, the term "affinity" as used herein refers to the strength of binding of an antigen molecule to an antigen binding molecule (e.g., an antibody or TCR). In the context of the TCR molecules of the present invention, the term "affinity" refers to the strength of a single interaction between an MHC-bound antigen (e.g., the NY-ESO-1 peptide epitope of SEQ ID NO: 21 presented in an HLA-A2 context) and a TCR. For example, affinity is measured when the antigen of interest is present in a cell-free environment, such as when an HLA-presented peptide is bound to a surface or bead. As a non-limiting example, the binding affinity of a TCR protein to its target can be measured by equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA), staining with peptide / MHC multimers in the presence of a competitor, surface plasmon resonance (SPR), or radioimmunoassay (RIA)) or kinetic methods (e.g., BIACORE TM analysis) to determine.

[0130] In another embodiment, the TCR is characterized by enhanced affinity. The term "avidity" as used herein is a measure of multiple affinity, reflecting the sum of the strengths of two molecules binding to each other at multiple sites, for example, taking into account the valence of the interaction. Specifically, the term "avidity" as used herein refers to the overall strength of the binding between an antigen having an antigenic determinant (e.g., a peptide of interest presented by an antigen presenting cell) and an antigen binding molecule such as a TCR or an antibody (e.g., in the context of a cell expressing a TCR or an antibody). For example, with respect to the TCR molecules of the present invention, the term avidity refers to the overall strength of the binding between the displayed peptide (e.g., the NY-ESO-1 epitope of SEQ ID NO: 21 presented in the context of HLA-A2) and the expressed TCR, or the overall strength of the binding between the cell (e.g., a tumor cell) displaying the peptide and the cell expressing the TCR. Avidity can be measured, for example, by staining cells expressing TCR (usually T cells), using graded concentrations of HLA / peptide multimers and competing or not competing with unlabeled HLA / polypeptide monomers.

[0131] In another embodiment, the TCR is characterized by enhanced functional avidity. As used herein, the term "functional avidity" refers to a cell expressing a TCR (particularly a T cell expressing an engineered TCR described herein, also referred to as an eTCR). + The functional affinity of an eTCR is the ability of an eTCR to respond to a given concentration of a ligand in vitro and is considered to be related to the in vivo effector capacity of a cell expressing the TCR. +The ability of cells to stimulate antigen-induced secretion of cytokines, such as TNFα and / or IFNγ, after co-culture with antigen-negative HLA-A2 expressing target cells (e.g., T2 cells) loaded with different concentrations of the NY-ESO-1 peptide of SEQ ID NO: 21.

[0132] In another embodiment, the TCR is characterized by high functional avidity. By definition, a TCR with high functional avidity + Cells respond to very low antigen doses in in vitro assays, and these cells with low functional avidity produce TCRs that are comparable to those with high functional avidity. + A larger amount of antigen is required before a cell-mediated immune response can occur. Therefore, functional avidity can be considered as TCR + A quantitative determinant of the activation threshold of a cell. It is determined in vitro by exposing such cells to varying amounts of cognate antigen. Thus, a TCR with high functional affinity + cells respond to low antigen doses. For example, if the TCR + The cells were loaded with 10 -6 Up to 10 -12 If a target cell (e.g., T2 cell) expressing the antigen-negative NY-ESO-1 epitope of SEQ ID NO: 21 secretes at least about 200 pg / mL or more (e.g., 1-100, 10-90, 20-80, 10-50, 50-100, 30-70, or 40-60 ng / mL) of IFNγ after co-culture with a low concentration of 1-2 μM, it is generally considered to bind to its antigenic target with "high" functional affinity. + Cells showed less than 10 -8 M, usually 10 -10 -10 -13 M, for example about 10 -12 When an EC50 of about 100 μM (e.g., for IFNγ or TNFα) is less than 100 μM, it can also be considered to bind to its antigenic target with high functional affinity. When used herein, and unless otherwise indicated, the term "about" is intended to encompass variations of ±10%, more usually ±5%.

[0133] In some embodiments, the TCR of the present invention is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence SLLMWITQC (SEQ ID NO: 21) with high functional affinity. In another embodiment, the TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21 with enhanced functional affinity compared to a naturally occurring TCR for NY-ESO-1. For example, but not limited to, the functional affinity may be enhanced by at least 3 times, typically at least 4, 4.5, 5, 5.5, 6, 6.5 or 7 times, such as 3-12 times, 4-15 times, 5-7 times or 10-20 times, compared to a naturally occurring TCR. In various embodiments, functional affinity can be determined using well-known methods based on assays known in the art, including but not limited to target-induced cytokine secretion. Non-limiting examples of such assays and methods and constructs according to the present invention are provided in Example 2 below, which exhibit significantly enhanced high functional affinity compared to TCRs known to date for the same epitope.

[0134] For example, as demonstrated in Example 2, the TCRs of the invention exhibit enhanced functional avidity compared to NY-ESO-1 specific TCRs having the VRα and VRβ sequences set forth in SEQ ID NOs: 26 and 27, respectively, as determined by an antigen-induced cytokine secretion assay using physiologically relevant antigen levels. More specifically, in the presence of 10 -6 Up to 10 -9 At a concentration of 1 M of NY-ESO-1 peptide, it was measured that TNFα secretion was enhanced by about 3-12 times, with an average of about 6 times.

[0135] In another embodiment, the TCR of the present invention is characterized by enhanced stability. Protein stability is a recognized measure of the ability of a protein to maintain one or more physical properties in response to environmental conditions. For example, impaired stability may be structurally reflected as a disturbance of the primary, secondary, tertiary and / or quaternary structures of the protein (or polypeptide complex) in question. As a non-limiting example, as known in the art, protein stability can be determined by assessing thermal stability, unfolding resistance, renaturation yield, sustained surface expression and / or specific binding of ligands (e.g., antigens) under various environmental conditions (e.g., pH, salt concentration, or the presence of detergents or denaturants).

[0136] For the TCR molecules of the present invention, the term "stability" as used herein specifically refers to the stability of the TCR complex under physiological conditions after expression in a suitable cell or expression system. More specifically, the term relates to the ability of the TCR heterodimer of the present invention to maintain structural integrity after expression on the surface of mammalian cells, particularly human T cells. For example, compared with naturally occurring or previously disclosed TCRs, the TCR of the present invention can be characterized as enhanced resistance to unfolding (denaturation), enhanced resistance to one or more TCR chain misfolding, reduced mispairing of TCR heterodimers, and enhanced renaturation ability and / or renaturation yield. In various embodiments, the enhancement of stability can be reflected by the enhancement of the level, duration and / or degree of TCR surface expression in the absence of stabilizing molecules (such as CD8). In other embodiments, the enhanced stability of the TCR complex can be reflected by the enhanced antigen binding ability in the absence of stabilizing molecules (such as CD8).

[0137] In another embodiment, the TCR is characterized by enhanced stability in the expressing cell, whether or not there are other non-related (e.g., endogenous) TCR molecules. In another embodiment, the TCR is characterized by enhanced pairing of its α and β chains (the ratio of the complex of specific pairing in the total production chain is increased). In various embodiments, the pairing of heterodimer chains is enhanced by at least one of the α and β chains and the mismatch reduction (ratio reduction of mismatch complex) of non-related (e.g., endogenous) TCR chains to reflect. In another embodiment, the TCR does not substantially produce mismatched TCR complexes after co-expression in T cells derived from normal donor PBMCs.

[0138] In another embodiment, the TCR is stably expressed on the surface of an immune cell such that the TCR is highly active even in the absence of a CD8 molecule that stabilizes the TCR-MHC complex. In another embodiment, the immune cell is a CD3 + In another embodiment, the immune cell is a T cell. In another embodiment, the TCR is capable of expressing a CD8 - In another embodiment, the encoded TCR is capable of stably expressing on the surface of an immune cell, such as a helper T cell. + and CD8 + Both are stably expressed on T cells (with sufficient stability and functionality to allow antigen-specific responses under physiologically relevant conditions).

[0139] In another embodiment, the TCR is capable of exerting tumor-specific reactivity (e.g., cytotoxicity) in a CD8-independent manner (which does not require co-expression or prior expression of CD8 by the expressing TCR cell to exert the function or activity). Thus, T cells expressing the TCR of the invention are capable of initiating or enhancing TCR-mediated functional activity (e.g., cytotoxicity or cytokine production) specific for tumors expressing NY-ESO-1, compared to their response to non-related cells (which do not express the antigen and are generally and advantageously unaffected). Notably, as disclosed herein, the TCR of the invention is capable of exerting tumor-specific reactivity (e.g., cytotoxicity or cytokine production) not only in CD8 - Immune cells (such as CD4 + cells), and when expressed in these cells, the TCR molecule is able to maintain the antigen-specific activity. Thus, as exemplified herein, the TCR of the present invention, when expressed in CD4 + When expressed on the surface of T cells, it is functionally reactive to tumor cells expressing NY-ESO-1 and exhibits the ability to secrete high levels of IFN-γ in response to target melanoma cells (A375) expressing the antigen. In addition, as demonstrated herein, the CD4 + TCR + The cells were shown to adopt a cytotoxic phenotype characterized by enhanced expression of the cytotoxic effector marker CD107 (CD107 + ), which was further enhanced after recognition with the cognate target A375 tumor cells.

[0140] In some embodiments, the TCR of the invention can be in CD4 + and CD8 + eTCRs are stably expressed on the surface of both T cells, allowing both populations to be clonally (antigen-specifically) expanded in vitro and / or in vivo. Thus, a T cell population derived from a T cell engineered (e.g., transduced) to express the TCR of the invention, in particular a cell composition prepared from healthy human donor PBMCs according to the methods disclosed herein, may advantageously express an eTCR in the presence of its cognate antigen (and in the absence of a subsequent engineering step). +An increase in the relative proportion of cells over time. For example, but not limited to, such an increase can be observed within 20-60 days, 30-50 days, or 35-45 days after transduction (or other engineering), or 15-40 days, 17-35 days, or 19-30 days after adoptive transfer. As a non-limiting example, in vivo stability, retention, and clonal expansion can be conveniently assessed in a suitable animal model accepted in the art, such as in NOD SCID gamma (NSG) mice with tumor xenografts expressing NY-ESO-1, such as described in Example 6 below. According to an exemplary embodiment, the composition according to the present invention can be characterized by CD8 40 days after transduction (or other engineering, such as in the NSG xenograft model of adoptive transfer of cell compositions derived from healthy human donor PBMCs discussed herein). + The average expression of said TCR on the cells is at least 97%, typically at least 98%, more typically at least 99%. In addition, the composition according to the invention may be characterized in that 40 days after transduction (or other engineering, such as in the NSG xenograft model adoptively transferred with a cell composition derived from healthy human donor PBMC as discussed herein), CD4 + The average expression of said TCR on the cells is at least 30%, typically at least 35%, more typically at least 40%.

[0141] In another embodiment, the invention provides an increase in the level and / or duration of expression of the encoded TCR compared to a construct encoding a naturally occurring TCR for an epitope set forth in SEQ ID NO: 21 presented by HLA-A2. In a specific embodiment, the invention provides an increase in the proportion of cells characterized by surface expression of the TCR compared to a corresponding construct encoding a naturally occurring TCR for an epitope set forth in SEQ ID NO: 21 presented by HLA-A2. In another embodiment, the invention provides the preparation of an adoptive cell therapy composition characterized in that the surface expression of the TCR is greater than 50% of the engineered cell population, typically in that the average expression is greater than 53%, 55%, 60%, 70%, 80%, 90% or greater, as disclosed herein.

[0142] In another embodiment, the present invention provides high TCR expression levels per construct copy number. The term "copy number" is used herein in conjunction with a nucleic acid construct (optionally as "CPN", "construct copy number", "vector copy number" or "VCN") to represent the number of copies of a construct such as a plasmid or vector (e.g., a viral vector) in each cell. The copy number of a single cell can be determined, and the copy number of a cell population can also be determined. In the latter case, an average CPN is obtained. Exemplary methods for measuring CPN include PCR procedures (e.g., quantitative polymerase chain reaction (qPCR) and digital droplet polymerase chain reaction (ddPCR)) and flow cytometry. Since the risk of tumorigenesis may be increased if the vector copy number (VCN) per cell is high (especially when using a genomic integration vector), the Administration (FDA) recommends that VCN should be <5 copies per genome.

[0143] According to exemplary embodiments, the present invention can prepare immune cell compositions, wherein at least 60%, 70%, 80% or 90% of immune cells (e.g., derived from healthy donor PBMC) are characterized by surface expression of TCR and the average copy number of the construct encoding the TCR is 5 or less molecules per cell. In contrast, the method reported so far provides an average expression level of 30-50% in the cell composition of the corresponding construct comprising an equivalent average copy number. In another embodiment, the functional properties are described in detail and exemplified herein. Each possibility represents an independent embodiment of the present invention.

[0144] Structural characteristics

[0145] TCR is a membrane-bound heterodimeric protein connected by disulfide bonds expressed on the surface of T cells and is a member of the immunoglobulin superfamily. TCR binds to antigenic peptides complexed with MHC / HLA through its variable region, inducing downstream T cell signaling (further referred to herein as TCR-mediated signaling). TCR generally comprises highly variable α chains and β chains, which are complexed with invariant CD3 chain molecules; a few TCRs comprise variable γ chains and δ chains. Each of the α chain and the β chain comprises two extracellular domains: a variable region (VR) and a constant region (CR). Each variable region (e.g., in the α chain and the β chain) contains three hypervariable regions separated by a framework region (FR), also referred to as "complementarity determining regions" (CDRs). CDR3 is the main CDR responsible for antigen binding. The α chain and the β chain also contain a junction (J) region. The β chain also generally comprises a diversity (D) region between the V region and the J region; however, the D region can be considered as part of the J region.

[0146] In naturally occurring TCRs, the constant region is encoded by the TRAC gene for the TCR α chain and by the TRBC1 or TRBC2 gene for the TCR β chain, while the TCR variable region is encoded by the TRAV or TRAJ gene segment for the TCR α chain and by the TRBV, TRBD or TRBJ gene segment for the TCR β chain. During thymocyte development, the TCR gene segments are assembled through processes including random somatic gene rearrangement (V(D)J recombination) and combined with a selection process for appropriate expression and MHC recognition to produce a diverse T cell library specific to the individual.

[0147] The specific recognition and binding of MHC-presented antigens by clonotype-specific α / β heterodimers leads to the activation of transcriptional events in the expressing T cells and subsequent proliferation and effector functions (e.g., CD8 + Cytotoxic activity and CD4 in T cells + This activation involves other subunits of the receptor complex (e.g., CD3) that couple the extracellular ligand ligation event to downstream signaling pathways (TCR-mediated signaling, such as protein phosphorylation, release of phosphoinositides, and increases in intracellular calcium levels).

[0148] As disclosed herein, the present invention relates to improved TCR molecules, characterized in that the structural characteristics detailed above. In particular, a TCR complex as a heterodimer of a (non-naturally occurring) α and β chain of structural modification (also referred to as "engineered" or "recombinant" α / βTCR) is provided. More specifically, the TCR according to the present invention comprises a complementary determining region (CDR) sequence as described in Table 1 below, wherein CDR1α, CDR2α, and CDR3α correspond to CDR1, CDR2, and CDR3 sequences of α chains, respectively, and CDR1β, CDR2β, and CDR3β correspond to CDR1, CDR2, and CDR3 sequences of β chains, respectively. Specifically, the TCR according to the present invention adopts the use of improved CDR3α as disclosed herein. In addition, Table 1 further lists the favorable nucleic acid sequences encoding the CDRs of nucleic acid constructs particularly suitable for cancer immunotherapy.

[0149] Table 1 - CDR sequences

[0150]

[0151] As further disclosed herein, TCR according to the present invention comprises multiple interchain disulfide bonds. The term "disulfide bond" (also referred to as a disulfide bridge) used herein includes a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​includes a sulfhydryl group, which can form a disulfide bond or bridge with a second sulfhydryl group. Naturally occurring TCR complexes usually contain a single disulfide bond between their TCR chains, which is formed by the cysteine ​​residues present at the specific position of the constant region (CR) of each chain. In contrast, the TCR of the present invention contains multiple disulfide bonds, including engineered (artificial) disulfide bridges disclosed herein in particular. Therefore, the term "interchain disulfide bond" used herein refers to any disulfide bridge formed between the cysteine ​​residues in the CR of TCR alpha chains and TCR beta chains, including both natural and engineered or artificial disulfide bridges.

[0152] In particular, the TCR of the present invention further comprises a second disulfide bond between the α chain and the β chain, which is promoted by replacing specific amino acids on the two chains with cysteine ​​residues. In some embodiments, the second disulfide bond has no equivalent in the natural α / βTCR, but provides the variable domain sequences of the α and β chains with substantially the same mutual orientation as in the natural α / βTCR. Therefore, the engineered TCR of the present invention has improved stability, but retains or even improves their functions, such as their specificity for cognate antigens and their ability to induce TCR-mediated signaling and activate T cells.

[0153] In particular, disclosed herein are TCRs comprising cysteine ​​residues that form disulfide bonds (in the mature TCR complex) at position 182 of the α chain precursor and at position 189 of the β chain precursor. It should be understood that the above positions are proposed for α and β chain precursors including the corresponding signal peptides. Thus, the above cysteine ​​residues correspond to positions 47 and 57 of the CR of the α and β chains, respectively.

[0154] In another embodiment, the TCR further comprises a disulfide bond between the cysteine ​​residue at position 229 of the α chain precursor and the cysteine ​​residue at position 263 of the β chain precursor. In some embodiments, the multiple disulfide bonds include or consist of the first disulfide bond and the second disulfide bond disclosed herein. In another embodiment, the TCR comprises a first disulfide bond between the cysteine ​​residue at position 94 of the α chain CR and the cysteine ​​residue at position 131 of the β chain CR, and a second disulfide bond between the cysteine ​​residue at position 47 of the α chain CR and the cysteine ​​residue at position 57 of the β chain CR.

[0155] For example, the TCR alpha chain precursor of SEQ ID NO: 17 set forth below comprises cysteine ​​residues at positions 182 and 229 thereof (taking into account the signal peptide at positions 1-20), which correspond to positions 47 and 94 of its CR sequence set forth below in SEQ ID NO: 14. Similarly, in another example, the beta chain precursor of SEQ ID NO: 18 set forth below comprises cysteine ​​residues at positions 189 and 263, which correspond to positions 57 and 131 of its CR sequence set forth below in SEQ ID NO: 16. According to an embodiment of the present invention, these cysteine ​​residues participate in the formation of disulfide bonds, in particular the interchain disulfide bonds disclosed herein.

[0156] As also disclosed herein, TCR according to the present invention is characterized in that the incidence of hydrophobic amino acids increases. The term "hydrophobic amino acid" as used herein refers to an amino acid with a side chain that is uncharged, non-polar and generally repelled by an aqueous solution at physiological pH. Exemplary hydrophobic amino acids include isoleucine (Ile or I), phenylalanine (Phe or F), valine (Val or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A) and glycine (Gly or G). Particularly preferred hydrophobic amino acids in the present disclosure are leucine, isoleucine and valine. In another embodiment, the hydrophobic amino acid is selected from alanine, glycine, leucine, isoleucine and valine.

[0157] Specifically, a TCR is provided in which the α chain comprises the LVIVL (SEQ ID NO: 22) sequence. Typically and advantageously, the sequence set forth in SEQ ID NO: 22 is located at the transmembrane region of the α chain. More specifically, the TCR of the present invention comprises the LVIVL sequence located at positions 250-254 of the α chain precursor (including the signal peptide). Therefore, the TCR of the present invention may comprise the LVIVL sequence at positions 115-119 of the α chain CR (CRα). In another embodiment, the transmembrane region of the α chain comprises at least 14 hydrophobic amino acids. The term "transmembrane" used herein in connection with the TCR chain refers to one or more portions of the TCR chain that are embedded in the cytoplasmic membrane. The transmembrane region or domain of the TCR chain is typically about 20-25 amino acids (aa) (e.g., 21) in length and comprises a transmembrane α helix and several conserved polar residues. According to an embodiment of the present invention, the TCR chain according to the present invention comprises a TCR-derived transmembrane domain (e.g., derived from a naturally occurring TCR chain) that is modified to comprise a hydrophobic sequence and / or residue disclosed herein. In another embodiment, the transmembrane region of the alpha chain has the amino acid sequence LLVIVLRILLLKVAGFNLLMT (SEQ ID NO: 23).

[0158] In addition, the TCR chain precursor of the present invention generally contains a modified signal peptide. The term "signal peptide" used herein has its general meaning in the art, and refers to the presence of an N-terminal peptide in a protein precursor form, and directs the polypeptide to enter a propeptide of a cell secretory pathway. The signal peptide is usually cut off from the protein during translation or transport, and is therefore not present in the mature protein. In one embodiment, the signal peptide used herein has a modified sequence (not peculiar to naturally occurring TCR chains), but provides sufficient presentation of the TCR chains on the plasma membrane. Specifically, the α chain precursor according to the present invention can include a phenylalanine residue at position 2 of the signal peptide.

[0159] Thus, the amino acid sequences of advantageous TCRs according to the invention are provided in SEQ ID NOs: 13-18 as shown below:

[0160] Variable region of α chain (VRα, excluding signal peptide):

[0161] KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPY (SEQ ID NO: 13).

[0162] Constant region of α chain (CRα):

[0163] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 14).

[0164] Variable region of the β chain (VRβ, excluding the signal peptide):

[0165] GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVL (SEQ ID NO: 15).

[0166] Constant region of the β chain (CRβ):

[0167] EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 16).

[0168] The amino acid sequences of the full-length α and β chain precursors (including the signal peptide, which is underlined) according to a particularly advantageous embodiment of the present invention are set forth in SEQ ID NOs: 17-18, respectively, as shown below:

[0169] MFETLLGLLILWLQLQWVSS KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPLYGGSYIPTFGRGTSLIVHPYIQNPDPAVYQLRDSKSS DKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS (alpha chain precursor, SEQ ID NO: 17).

[0170] MSIGLLCCAALSLLWAGPVNA GVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYVGNTGELFFGEGSRLTVLEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFF PDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF(beta chain precursor SEQ ID NO: 18).

[0171] Thus, the amino acid sequences of the α and β chains of the mature, processed TCR chains (lacking their corresponding signal peptides) correspond to positions 21-275 of SEQ ID NO: 17 and positions 22-309 of SEQ ID NO: 18, respectively. The term "TCR chain precursor" as used herein refers to a recombinant molecule formed after translation in a suitable host cell or expression system, prior to subsequent processing such as cleavage of the signal peptide, glycosylation, complexing with the corresponding chain (e.g., alignment of the α-β chains), and disulfide bond formation. It should be understood that the term as used herein does not mean to include or require pre-translational processing, such as somatic recombination.

[0172] Nucleic acid sequence

[0173] Furthermore, the TCR according to the invention is advantageously encoded by an improved nucleic acid sequence. Without wishing to be limited to a particular theory or mechanism of action, embodiments of the invention employ nucleic acid sequences that provide improved expression in mammalian cells and expression systems, particularly in human cells such as lymphocytes.

[0174] The nucleic acid sequences encoding the full length α and β chain precursors (including the signal peptide, which is underlined) according to a particularly advantageous embodiment of the present invention are set forth in SEQ ID NOs: 19-20, respectively, as shown below:

[0175] Nucleic acid molecule encoding TCRα precursor -

[0176] ATGTTCGAAACTCTGCTGGGGCTGCTGATTCTGTGGCTGCAGCTGCAGTGGGTGTCATCCAAACAGGAGGTCACTCAGATTCCCGCTGCCCTGAGCGTGCCTGAGGGCGAGAACCTGGTGCTGAATTGCTCCTTCACCGACTCTGCCATCTACAACCTGCAGTGGTTTAGGCAGGATCCAGGCAAGGGCCTGACCAGCCTGCTGCTGATCCAGAGCTCGCAGAGGGAGCAGACATCCGGCCGCCTGAATGCCTCTCTGGACAAGTCTAGCGGCCGGAGCACCCTGTACATCGCAGCAAGCCAGCCAGGCGATTCCGCCACATACCTGTGCGCCGTGCGGCCTCTGTACGGAGGCTCTTATATCCCAACCTTCGGCAGAGGCACAAGCCTGATCGTGCACCCTTACATCCAGAACCCAGACCCCGCCGTGTATCAGCTGCGGGACAGCAAGTCCTCTGATAAGTCCGTGTGCCTGTTCACCGACTTTGATTCCCAGACAAACGTGAGCCAGAGCAAGGACTCTGACGTGTACATCACCGACAAGTGCGTGCTGGATATGAGAAGCATGGACTTTAAGTCCAACTCTGCCGTGGCCTGGAGCAATAAGTCCGATTTCGCCTGCGCCAACGCGTTTAACAATAGCATCATCCCCGAGGATACATTCTTTCCTTCCCCAGAGAGCTCCTGTGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACAGATACAAACCTGAATTTTCAGAACCTGCTGGTCATCGTGCTGCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGTCTAGC(SEQ ID NO:19).

[0177] Therefore, VRα and CRα are encoded by positions 1 - 405 and 406 - 825 of SEQ ID NO: 19, respectively.

[0178] Nucleic acid molecule encoding the TCRβ precursor -

[0179] ATGTCTATCGGCCTGCTGTGCTGTGCCGCCCTGAGCCTGCTGTGGGCAGGACCAGTGAACGCAGGAGTGACCCAGACACCCAAGTTCCAGGTGCTGAAGACCGGCCAGTCTATGACACTGCAGTGCGCCCAGGACATGAATCACGAGTACATGAGCTGGTATCGGCAGGATCCTGGCATGGGCCTGAGACTGATCCACTACTCCGTGGGAGCAGGAATCACCGACCAGGGAGAGGTGCCAAACGGCTATAACGTGAGCAGGAGCACCACAGAGGATTTCCCACTGAGGCTGCTGTCTGCCGCACCTTCTCAGACAAGCGTGTACTTTTGCGCCTCCTCTTATGTGGGCAACACCGGCGAGCTGTTCTTTGGCGAGGGCTCCAGGCTGACAGTGCTGGAGGACCTGAATAAGGTGTTCCCCCCTGAGGTGGCCGTGTTTGAGCCCTCTGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTTTCCTGATCACGTGGAGCTGTCCTGGTGGGTGAACGGCAAGGAGGTGCACTCTGGCGTGTGCACAGACCCACAGCCCCTGAAGGAGCAGCCAGCCCTGAATGACTCgAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA(SEQ ID NO:20).

[0180] Therefore, VRβ and CRβ are encoded by positions 1-396 and 397-930 of SEQ ID NO: 20, respectively.

[0181] In other embodiments, the present invention relates to nucleic acid sequences having a high degree of homology (e.g., at least 95%, 96%, 97%, 98% or 99% sequence identity and / or homology to SEQ ID NO: 19 or 20), wherein the resulting TCR is capable of specifically binding to the epitope of SEQ ID NO: 21 presented by HLA-A2 with high functional affinity. Each possibility represents an independent embodiment of the present invention. Such nucleic acid molecules generally comprise nucleic acid sequences encoding CDR1, CDR2 and CDR3 of the α chain, respectively, as set forth in SEQ ID NO: 7-9, or nucleic acid sequences encoding CDR1, CDR2 and CDR3 of the β chain, respectively, as set forth in SEQ ID NO: 10-12.

[0182] In another embodiment, the nucleic acid molecule of the method and composition of the present invention is a DNA molecule. In another embodiment, the nucleic acid molecule is an RNA molecule. In another embodiment, the molecule is any other type of nucleic acid molecule known in the art. Every possibility represents an independent embodiment of the present invention.

[0183] TCR

[0184] The present invention, in its aspects and embodiments, relates to isolated TCRs against NY-ESO-1. As used herein, the term "isolated" means altered or removed from the natural state. For example, a nucleic acid or polypeptide naturally present in a living animal is not "isolated", but the same nucleic acid or polypeptide partially or completely separated from coexisting materials in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-natural environment, such as a host cell.

[0185] The term "directed against" used herein in conjunction with antigen binding proteins such as TCRs and antibodies refers to the ability to specifically bind to the antigen under physiological conditions. "Specific binding" or "specific for..." as used herein refers to the binding or association of a binding protein (e.g., TCR) with a target molecule (e.g., a peptide epitope presented by MHC) with an affinity or K a (i.e., the equilibrium binding constant for a specific binding interaction, expressed in 1 / M) equal to or greater than 10 5 M -1 , while not significantly binding or associating with any other molecule or component in the sample. Binding proteins or binding domains can be classified as "high affinity" or "low affinity" binding proteins or domains. "High affinity" binding proteins or binding domains are those with a K a For at least 10 7 M -1 , at least 10 8 M -1 , at least 109 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 or at least 10 13 M -1 A "low affinity" binding protein or binding domain is one that has a K a For up to 10 6 M -1 or up to 10 5 M -1 of molecules.

[0186] The term "NY-ESO-1" or "New York Esophageal Squamous Cell Carcinoma 1" refers to the well-known cancer-testis antigen (CTA), also known as cancer / testis antigen 1B (CTAG1B). The human CTAG1B gene maps to the Xq28 region of chromosome X and is silent in normal somatic cells except male testes. However, NY-ESO-1 is aberrantly expressed in many types of cancer cells as a result of epigenetic events involving the tightly controlled recruitment and sequential interactions of histone deacetylases, histone methyltransferases, DNA methyltransferases, and transcription factors. The human NY-ESO-1 sequence is set forth in accession number NP_001318.1.

[0187] More specifically, the TCR according to the invention specifically binds to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21, which corresponds to residues 157 to 165 of human NY-ESO-1. According to a specific embodiment, the TCR of the invention is directed against the peptide epitope of SEQ ID NO: 21 in the context of HLA-A*0201 and / or HLA-A*0206. As used herein, the term "HLA-presented" peptide, epitope or antigen refers to a peptide that is capable of specifically binding to the antigen binding groove of the MHC or a specific allele thereof (e.g., an epitope presented by HLA-A2 is specific for the HLA-A2 allele). Such antigens are generally referred to in the art as being "restricted" by such MHC. Antigens generally have a characteristic size and / or chemical composition, for example, in the case of peptide antigens, a characteristic amino acid length and anchor residue group, respectively, that enable them to specifically bind to the antigen binding groove of a specific MHC haplotype, thereby forming an MHC / antigen complex therewith, the complex having an antigen presenting portion that is capable of specifically binding to the variable region of the cognate TCR. For example, for HLA-A2, the anchor positions are P2 and P9.

[0188] In one aspect, the present invention relates to an isolated TCR against NY-ESO-1, comprising:

[0189] (a) a TCR alpha chain comprising:

[0190] i. CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3;

[0191] ii. CR comprising a cysteine ​​residue at position 47 and a LVIVL sequence at positions 250-254;

[0192] iii. an optional signal peptide comprising a phenylalanine residue at position 2;

[0193] (b) a TCR β chain comprising:

[0194] i. a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and

[0195] ii. CR, which contains a cysteine ​​residue at position 57;

[0196] iii. an optional signal peptide; and

[0197] (c) multiple interchain disulfide bonds between the α and β chains.

[0198] In another aspect, the present invention provides an isolated TCR against NY-ESO-1 comprising:

[0199] (a) a TCR alpha chain comprising:

[0200] i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3;

[0201] ii. CR comprising a cysteine ​​residue at position 47 and the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0202] (b) a TCR β chain comprising:

[0203] i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and

[0204] ii. CR comprising a cysteine ​​residue at position 57; and

[0205] (c) multiple interchain disulfide bonds between the α and β chains.

[0206] In another embodiment, the CDRs are as set forth in SEQ ID NOs: 1-6. In another embodiment, the TCR comprises a TCR alpha chain having an amino acid sequence set forth in SEQ ID NO: 17, optionally excluding a signal peptide at positions 1-20. In another embodiment, the TCR comprises a TCR beta chain having an amino acid sequence set forth in SEQ ID NO: 18, optionally excluding a signal peptide at positions 1-21. In another embodiment, the TCR comprises the alpha and beta chains set forth in SEQ ID NOs: 17 and 18, optionally excluding their corresponding signal peptides.

[0207] In other embodiments, the invention encompasses TCRs comprising certain modifications (e.g., substitutions) to the amino acid sequences set forth in SEQ ID NOs: 17 and 18 that maintain a high degree of homology (e.g., greater than 95%, 96%, 97%, 98% or 99%), as long as the structural elements set forth in clauses (a) to (c) above are maintained. In a specific embodiment, the substitutions are conservative substitutions.

[0208] The terms "homology" and "sequence identity" as used herein refer to the degree of relatedness between two or more amino acid sequences or two or more nucleic acid sequences determined by comparing the sequences. Comparison of sequences and determination of sequence identity or homology can be accomplished using a mathematical algorithm; those skilled in the art will be aware of computer programs that can be used to align two sequences and determine the percent identity between them. The term "homology" specifically refers to the percentage of amino acid residues or nucleotides in the sequence that are identical to the residues of the reference polypeptide or polynucleotide after aligning the sequence with the reference sequence being compared, and introducing spaces where necessary in certain embodiments to achieve the maximum homology percentage, and without considering any conservative substitutions. In the context of two or more nucleic acid or polypeptide sequences, the term "% identity" as used herein can be used interchangeably with the term "sequence identity", and refers to two or more sequences of identical nucleotides or amino acid residues with a specified percentage when compared and aligned to obtain maximum correspondence, the percentage being measured using a suitable sequence comparison algorithm (e.g., BLASTP and BLASTN or other algorithms available to the skilled person) or by visual inspection. Furthermore, with respect to sequence identity as used herein, the total length of the molecules being compared is also taken into account so that the degree of identity is calculated over the entire length of the sequence rather than locally.

[0209] As disclosed herein, TCRs that retain the unique structural elements of the present disclosure (particularly those structural elements set forth in clauses (a) to (c)) and at the same time retain a high degree of homology and / or identity exhibit the advantageous functional properties disclosed in this specification, such as their epitope specificity and functional affinity. For sequence homology or identity, the term "high" refers to at least 90% homology or sequence identity, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or identity, respectively, wherein each possibility represents an independent embodiment of the present invention. In another specific embodiment, each of the α and β chains has at least 95% sequence identity to SEQ ID NOs: 17 and 18, respectively, and the TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence SLLMWITQC (SEQ ID NO: 21) with high functional affinity. In various other specific embodiments, each of the α and β chains has at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and / or homology to SEQ ID NOs: 17 and 18, respectively, such that the resulting TCR is capable of specifically binding to an epitope of SEQ ID NO: 21 presented by HLA-A2 with high functional affinity. Each possibility represents a separate embodiment of the present invention.

[0210] Constructs and vectors

[0211] Polypeptides, peptides and nucleic acid molecules can be easily produced by recombinant technology. Recombinant methods for designing, expressing and purifying proteins, peptides and nucleic acid molecules are known in the art (see, for example, Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York). Nucleic acid molecules may include DNA, RNA or derivatives of DNA or RNA. The isolated nucleic acid sequence encoding a polypeptide or peptide can be obtained from its natural source as a whole (i.e., complete) gene or a part thereof. Nucleic acid molecules can also be produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Nucleic acid sequences include natural nucleic acid sequences and homologues thereof, including but not limited to modified nucleic acid sequences, in which nucleotides are inserted, deleted, replaced and / or inverted in some way, so that such modifications do not substantially interfere with the ability of the nucleic acid molecule to encode a functional product. Polynucleotide or oligonucleotide sequences can be inferred from the genetic code of the protein, but the degeneracy of the code must be considered, and exceptions to the classical base pairing in the third position of the codon are allowed, as given by the so-called "wobble rule". Polynucleotides containing more or fewer nucleotides can produce the same or equivalent proteins. Using recombinant production methods, a selected host cell, such as a cell of a microorganism such as Escherichia coli or yeast, is transformed with a hybrid virus or plasmid DNA vector containing a specific DNA sequence encoding the polypeptide, and the polypeptide is synthesized in the host after transcription and translation of the DNA sequence.

[0212] Such recombinant methods can also be used to prepare nucleic acid constructs, including expression constructs or vectors particularly for delivering and expressing the TCR of the present invention in mammalian hosts such as human T cells, as described herein. The construct comprises nucleic acid molecules of the present invention, and may also comprise regulatory sequences or selective markers as known in the art. The nucleic acid construct (also referred to as vector in some embodiments) may include additional sequences that make the vector suitable for replication and integration in prokaryotes, eukaryotes or optionally both (e.g., shuttle vectors). In addition, typical cloning vectors may also contain transcription and translation initiation sequences, transcription and translation terminators, and polyadenylation signals.

[0213] In another aspect, a nucleic acid construct encoding at least one chain of the isolated TCR is provided. In another embodiment, the nucleic acid construct comprises at least one nucleic acid molecule encoding the TCR alpha chain and TCR beta chain disclosed herein. In another embodiment, the at least one nucleic acid molecule is operably linked to one or more transcription control elements.

[0214] The phrase "operably linked" means that a nucleic acid sequence is linked to a transcription control sequence in a manner that enables the molecule to be expressed when transfected (i.e., transformed, transduced, infected or transfected) into a host cell. Transcription control sequences are sequences that control the initiation, elongation and termination of transcription. Particularly important transcription control sequences are those that control the initiation of transcription, such as promoter, enhancer, operator and repressor sequences.

[0215] In another embodiment, the construct comprises the nucleic acid sequences encoding CDR1, CDR2 and CDR3 of the α chain, respectively, as set forth in SEQ ID NOs: 7-9, and the nucleic acid sequences encoding CDR1, CDR2 and CDR3 of the β chain, respectively, as set forth in SEQ ID NOs: 10-12. In another embodiment, the construct comprises a first nucleic acid molecule encoding the α chain and having the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the β chain and having the nucleotide sequence set forth in SEQ ID NO: 20. In another embodiment, a nucleic acid construct is provided, comprising at least one nucleic acid molecule selected from SEQ ID NOs: 19 and 20.

[0216] In another embodiment, the construct comprises a first nucleic acid molecule encoding a TCR alpha chain and a second nucleic acid molecule encoding a TCR beta chain, the first and second nucleic acid molecules having the nucleic acid sequences set forth in SEQ ID NO: 19-20, respectively, and wherein the first and second nucleic acid molecules are operably linked to one or more transcription control elements. In another embodiment, the first nucleic acid molecule is at the 5' end of the second nucleic acid molecule. In another embodiment, the first nucleic acid molecule is at the 3' end of the second nucleic acid molecule. In another embodiment, the one or more transcription control elements (e.g., promoters) are capable of inducing or enhancing the expression of the TCR chain in human leukocytes. In another embodiment, the one or more transcription control elements (e.g., promoters) are capable of inducing or enhancing the expression of the TCR chain in human lymphocytes (e.g., T cells).

[0217] For example, in an expression vector capable of expressing a nucleic acid molecule in human T cells, an expression control sequence operably linked to a gene product to be expressed includes a promoter and other elements active in human T cells. The promoter may be of genomic origin or may be synthetically produced. Various promoters for T cells are well known in the art. For example, the promoter may be constitutive or inducible, wherein induction is associated with activation or maturation of a specific cell type or a specific level. Alternatively, many well-known viral promoters are also suitable. Promoters of interest include, but are not limited to, beta-actin promoters, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, and Friend spleen lesion formation virus promoters. The promoter may be associated with or not associated with an enhancer, wherein the enhancer may be naturally associated with the specific promoter or may be associated with a different promoter.

[0218] Exemplary strong and well-characterized promoters for inducing expression in human T cells include the human cytomegalovirus (CMV) immediate early promoter (a strong and ubiquitous classically used viral promoter), the murine stem cell virus (MSCV) retroviral LTR promoter (previously described in T cells as mimicking the transcriptional regulation of γ-retroviral vectors), the human phosphoglycerate kinase (PGK) promoter (an endogenous housekeeping promoter, shown to maintain moderate and stable expression levels), the β-2-microglobulin (β2m) promoter (a ubiquitous constitutive promoter, particularly strong in immune cells), the human elongation factor 1α (EF1α) promoter (a strong ubiquitous constitutive promoter, commonly used for stable gene transfer) and other promoters such as hPGK and RPBSA promoters. Such promoters can be conveniently used for ex vivo manipulation of T cells, where constitutive expression of the manipulation is required, such as in the preparation of ACT compositions. In some embodiments, for example, for in vivo gene editing, T cell-specific and / or inducible promoters are preferred. In some embodiments, the promoter is a T cell specific promoter, such as a CD3, CD4 or CD8 promoter. Exemplary inducible promoters that initiate expression of a gene product following lymphocyte or T cell activation include NFAT, AP1 and NR4A promoters.

[0219] In another embodiment, the construct comprises a first nucleic acid molecule encoding the α chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the β chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 20, wherein the encoded TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO: 21 with high functional affinity. In another embodiment, the construct comprises a nucleic acid molecule encoding an engineered TCR of the present invention. According to a specific embodiment, the first and second nucleic acid sequences contained in the construct exhibit a high degree of homology (e.g., at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity and / or homology to SEQ ID NO: 19 or 20, respectively), wherein the resulting TCR retains the structural and functional properties disclosed herein. In another embodiment, the encoded TCR is capable of specifically binding to an epitope of an amino acid sequence of SEQ ID NO: 21 presented by HLA-A2 with high functional affinity. + and CD8 + In another embodiment, the construct is capable of providing enhanced expression levels and / or duration of the encoded TCR compared to a construct encoding a naturally occurring TCR directed against the epitope set forth in SEQ ID NO: 21 presented by HLA-A2.

[0220] In another embodiment, the first nucleic acid molecule and the second nucleic acid molecule are connected by a third nucleic acid molecule encoding a joint. In another embodiment, the joint is a cleavable joint. In another embodiment, the joint makes a self-cleaving joint. The term "cleavable joint" as used herein refers to a joint that can be incorporated into a polycistronic mRNA construct so that multiple genes of equal molar levels can be produced from the same mRNA, typically a peptide joint (e.g., a length of about 5-30 amino acids). The non-limiting examples of cleavable joints include self-cleaving joints that can be used to produce multiple gene products (usually by ribosome jumping) from a single transcript during translation, and enzymatically cleavable joints (e.g., furin cleavable joints).

[0221] In one embodiment, the self-cleaving linker comprises a 2A peptide. In another embodiment, the linker is a 2A peptide (e.g., T2A, P2A, E2A and / or F2A). 2A peptides are typically 18-22 amino acid (aa) long viral oligopeptides that mediate the "cleavage" of polypeptides during eukaryotic cell translation. The name "2A" refers to a specific region of the viral genome, and different viral 2A peptides are usually named after the virus from which they are derived. For example, F2A is a 2A peptide derived from foot-and-mouth disease virus (FMDV), E2A is derived from horse armor rhinitis virus (ERAV), P2A is derived from porcine Teschovirus (PTV-1), and T2A is derived from Thosea asigna virus (TaV). In this specification, the term 2A peptide encompasses viral 2A peptides and 2A-like self-cleaving peptides known in the art. 2A peptide-like sequences are present in various positive-strand and double-stranded RNA virus groups, including Picornaviridae, Flaviviridae, Tetraviridae, Dicistroviridae, Reoviridae and Monocomponent Viridae. Furthermore, the 2A-like peptides used according to the present invention may include viral 2A peptides fused to additional sequences, such as the enzymatic cleavage sites disclosed herein. In a specific embodiment, the 2A peptide is a P2A peptide having the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 28).

[0222] In various embodiments, the 2A linker can be used with or without a furin recognition site. Furin is a ubiquitously expressed protease that is present in the trans-Golgi apparatus and processes protein precursors before secretion. Furin cuts at the COOH-end of its consensus recognition sequence. Various furin consensus recognition sequences (also referred to herein as "furin recognition site" or "furin cleavage site") are known to those skilled in the art. For example, the Arg-X-Lys / Arg-Arg motif can be cut by furin.

[0223] In a particular embodiment, the linker is selected from T2A, P2A, E2A and F2A comprising an added upstream furin recognition site. In a particular embodiment, the linker is a P2A linker (with or without the furin recognition site). In another embodiment, the linker may further include a "GSG" (Gly-Ser-Gly) linker, for example at the N-terminus of the 2A peptide and / or between the 2A peptide and the furin recognition site. According to a preferred embodiment, the linker contains a P2A peptide and a furin recognition sequence Arg-Ala-Lys-Arg, such as a linker encoded by SEQ ID NO: 24. In another embodiment, the encoded linker has the amino acid sequence RAKRSGSGATNFSLLKQAGDVEENPGPR (SEQ ID NO: 29).

[0224] Alternatively, the nucleic acid construct may be included in an internal ribosome entry site (IRES) sequence between the nucleic acid molecules encoding two chains. "Internal ribosome entry site" or "IRES" as used herein refers to an initiation codon (e.g., ATG) that promotes internal ribosomes to enter directly into a protein coding region, thereby causing an element of cap-independent translation of the gene. Various internal ribosome entry sites are known to those skilled in the art, including but not limited to IRES that can be obtained from viral or cell mRNA sources such as immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF), fibroblast growth factor 2, insulin-like growth factor, translation initiation factor eIF4G, yeast transcription factor TFIID and HAP4, and IRES that can be obtained from, for example, cardiovirus, rhinovirus, foot-and-mouth disease virus, HCV, Friend murine leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV). Those skilled in the art will be able to select suitable IRES for the present invention. Every possibility represents an independent embodiment of the present invention.

[0225] In another embodiment, a system is provided, comprising: (i) a first construct comprising a first nucleic acid molecule encoding a TCR alpha chain operably linked to one or more transcriptional control elements, and (ii) a second construct comprising a construct nucleic acid molecule encoding a TCR beta chain operably linked to one or more transcriptional control elements.

[0226] In another embodiment, the present invention provides an expression vector comprising at least one nucleic acid construct as disclosed herein. The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably connected to a nucleotide sequence to be expressed. The expression vector comprises cis-acting elements sufficient for expression; other elements for expression can be provided by a host cell or an in vitro expression system. Expression vectors include suitable vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide. In another embodiment, the vector is capable of expressing the TCR of the present invention in mammalian cells. In another embodiment, the cell is a human cell (e.g., a leukocyte, a lymphocyte, and / or a T cell). In another embodiment, the vector is a viral vector. In a specific embodiment, the vector is a retroviral vector.

[0227] Suitable viral vectors are known in the art and are commercially available. Exemplary retroviral vectors include, but are not limited to, pMSGV1, pMSCV, pBMN, pQXIX, pBullet, pBabe, pMSGV, pRETRO, pMIGR, pMX, and pRET. Exemplary lentiviral vectors include, but are not limited to, pRRL, pCLX, pLenti, pHR, pLVX, and pCAG. Other exemplary vectors include, but are not limited to, pBullet, pLSC, and adeno-associated virus (AAV) vectors, including, but not limited to, pAAV and pX601. In another embodiment, the vector is a retroviral vector comprising LTRs from mouse stem cell viruses (MSCV LTRs), such as pMSGV1 or pMSCV-derived vectors.

[0228] As a non-limiting example, the vector of the present invention may include a lentiviral vector comprising an MSCV LTR operably linked to the nucleic acid sequences encoding the α and β chains set forth in SEQ ID NOs: 19 and 20, respectively, and connected via a P2A linker comprising a 5' furin recognition site.

[0229] In another embodiment, the invention provides a host cell comprising the vector. The term "host cell" means a cell comprising an exogenous nucleic acid of interest, and is intended to include any individual cell or cell culture and its progeny that can be or has become a receptor of a nucleic acid molecule of the present invention or a construct or vector encoding the nucleic acid molecule, as long as the nucleic acid molecule exists. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria, yeast cells and animal cells, such as insect cells, and mammalian cells such as mice, rats or humans. According to a specific embodiment, the host cell is an immune cell, particularly a human immune cell, including but not limited to PBMC or a T cell composition. In another embodiment, the host cell is a colony containing T cells, such as primary lymphocytes of people or a cell colony derived therefrom.

[0230] In another embodiment, a pharmaceutical composition comprising the vector is provided. For example, the pharmaceutical composition may comprise an expression vector or system disclosed herein as an active ingredient, and optionally one or more pharmaceutically acceptable carriers, excipients or diluents. In another example, the pharmaceutical composition may comprise a host cell (e.g., T cell) containing the construct or system, and optionally one or more pharmaceutically acceptable carriers, excipients or diluents.

[0231] According to the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of the present invention comprises a therapeutically effective amount of an active ingredient disclosed herein (e.g., a nucleic acid construct or a vector, a virion, or a cell comprising the nucleic acid construct) and at least one pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition includes a composition for parenteral, transdermal, intraluminal, intravenous, intraarterial, or intrathecal administration or direct injection into a tissue or tumor. It is particularly contemplated that the pharmaceutical composition is administered to a patient by infusion or injection. In other embodiments, the composition is formulated for ex vivo administration to a recipient cell (e.g., PBMC derived from the subject or a suitable donor), which has been engineered ex vivo with the composition and reintroduced into the subject.

[0232] "Effective amount" or "therapeutically effective amount" refers to an amount sufficient to produce beneficial results in the methods of the present invention. More specifically, a therapeutically effective amount means an amount of active ingredient that is effective to prevent, alleviate or improve the symptoms of a disorder (e.g., cancer) or prolong the survival of a treated subject.

[0233] The determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, particularly in light of the detailed disclosure provided herein. For any formulation used in the methods of the present invention, the dosage or therapeutically effective amount can be initially estimated from in vitro and cell culture assays. For example, a formulation can be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine a dosage useful in humans.

[0234] The toxicity and therapeutic efficacy of the active ingredients described herein can be determined in vitro, in cell culture or in experimental animals by standard pharmaceutical procedures. The data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage may vary depending on the dosage form used and the route of administration used. The exact formulation, route of administration and dosage can be selected by an individual physician according to the patient's condition (see, for example, Fingl, E. et al., (1975), The Pharmacological Basis of Therapeutics, Ch. 1, p. 1.).

[0235] For example, but not limited to, the nucleic acid construct (e.g., viral vector) according to the present invention can be used for ex vivo administration at a dose of 1-10 μg / ml, and for adult patients, can be used at a dose of 1-300 or 30-100 μg or 1×10 10 -5x10 10 Doses of infectious units (IU) were used.

[0236] Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (eg, oil / water emulsions), various types of wetting agents, sterile solutions, etc. Compositions containing such carriers can be formulated by well-known conventional methods.

[0237] In another embodiment, the carrier is a targeting carrier, also referred to herein as a delivery carrier or an in vivo delivery system. Examples of delivery carriers particularly suitable for administering nucleic acid agents include, but are not limited to, artificial and natural lipid-containing delivery carriers. Natural lipid-containing delivery carriers include exosomes and cell membranes. Artificial lipid-containing delivery carriers include liposomes and micelles. Other suitable delivery carriers include, for example, in vivo delivery systems based on nanoparticles. The delivery carrier of the present invention can be modified to target a specific site in a subject, thereby targeting the site and utilizing the nucleic acid molecules of the present invention at the site. Suitable modifications include manipulating the chemical formula of the lipid portion of the delivery carrier and / or introducing compounds that can specifically target the delivery carrier to a preferred site (e.g., a preferred cell type) into the carrier. Specific targeting refers to the interaction of a compound in the delivery carrier with a molecule on the surface of a specific cell, so that the carrier is combined with the specific cell. Suitable targeting compounds include ligands that can selectively (i.e., specifically) bind to another molecule at a specific site. Examples of such ligands include antibodies, antigens, receptors, and receptor ligands. For example, compounds that can be used to target T cells include, for example, antibodies (or their antigen binding portions) specific to CD3, CD4, and / or CD8.

[0238] The compositions of the present invention can be administered topically or systemically. Administration is usually parenteral, such as intravenous; DNA can also be administered directly to the target site, such as delivered to an internal or external target site by bioballistics, or delivered to a site within an artery by a catheter. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous carriers include fluid and nutrient supplements, electrolyte supplements (e.g., supplements based on Ringer's dextrose), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, etc., may also be present. It is contemplated that, according to the intended use of the pharmaceutical composition of the present invention, in addition to the active ingredients disclosed herein, the pharmaceutical composition may also include additional active agents. Such drugs may be anticancer drugs, such as immunotherapeutic agents, such as immune checkpoint inhibitors. The term "checkpoint inhibitor" refers to drugs (e.g., antibodies or small molecules) that target and antagonize, neutralize, or otherwise reduce the activity of immunosuppressive checkpoint molecules (e.g., PD-1, PD-L1, and CTLA-4).

[0239] In another embodiment, the pharmaceutical composition is suitable for cancer immunotherapy.In another embodiment, the pharmaceutical composition is suitable for adoptive transfer therapy (ACT).

[0240] Cell compositions and adoptive cell transfer therapy

[0241] In another aspect, the present invention relates to a cell composition expressing a TCR of the present invention. In another embodiment, a cell composition for cancer immunotherapy is provided, characterized in that the surface expression of the TCR of the present invention is provided. In another embodiment, the cell composition is an ACT composition. Therefore, the cell composition expressing the TCR of the present invention (or characterized by the surface expression of the TCR of the present invention) comprises at least one cell population expressing the TCR. In some embodiments, a cell composition for cancer immunotherapy is provided, comprising a therapeutically effective amount of immune cells engineered to express a TCR for NY-ESO-1, the TCR comprising:

[0242] (a) a TCR alpha chain comprising i. VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; ii. CR comprising a cysteine ​​residue at position 47 and comprising the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254;

[0243] (b) a TCR β chain comprising: i. a VR comprising a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6; and ii. a CR comprising a cysteine ​​residue at position 57; and

[0244] (c) multiple interchain disulfide bonds between the α and β chains.

[0245] In another embodiment, the TCR is characterized as disclosed herein.The terms "engineered" and "genetically engineered" are used interchangeably.

[0246] The term "cell composition" as used herein refers to a pharmaceutical composition containing cells or cell materials as active ingredients. Cell compositions generally contain a pharmaceutically acceptable carrier, excipient or diluent, and optional additional components other than cells, such as a culture medium or a preservative solution. The term "cancer immunotherapy" refers to treating a subject with cancer or a risk of cancer recurrence by a method including regulating an immune response in a subject. Specifically, cancer immunotherapy is generally intended to induce and / or stimulate an immune response of the subject to cancer cells. Exemplary agents and regimens used in cancer immunotherapy include immune checkpoint regulators (e.g., checkpoint inhibitors or agonists), tumor-specific antibodies, cytokines, and therapeutic anti-cancer vaccines. Another common form of cancer immunotherapy involves adoptive transfer of effector cells including immune cells (ACT), particularly T cells, NK cells, and / or NK T cells.

[0247] As used herein, and unless otherwise indicated, the term "adoptive transfer" refers to a form of passive immunotherapy in which a previously sensitized immune agent (e.g., cells or serum) is transferred to a recipient. The phrases "adoptive cell transfer," "adoptive transfer immunotherapy," "adoptive transfer therapy," "adoptive cell therapy," and "adoptive cell immunotherapy" are used interchangeably herein to refer to a therapeutic prevention regimen or mode in which effector immune-active cells are administered (adoptively transferred) to a subject in need to mitigate or ameliorate the development or symptoms of cancer or infectious disease.

[0248] The adoptively transferred cells are generally directed against tumors or tumor-associated antigens, for example, the ACT compositions of the present invention are directed against NY-ESO-1. Specifically, the ACT compositions of the present invention contain an effective amount of immune cells (generally including T cells) disclosed herein that are engineered to express the TCR of the present invention. Therefore, the ACT compositions according to the present invention contain an effective amount of engineered cells disclosed herein, which are produced under sterile and suitable (e.g., cGMP grade) conditions and are administered to human subjects with neoplastic diseases as part of an anti-tumor regimen.

[0249] When used herein in conjunction with a cell composition, an effective amount (also referred to herein as a therapeutically effective amount) is an amount sufficient to induce or enhance a beneficial immune response, such as an anti-tumor response, when administered to a subject, e.g., 10 6 Up to 10 12 cells or 10 7 Up to 10 11 Specifically, for the ACT composition of the present invention, the effective amount is an amount sufficient to induce or enhance the immune response after adoptive transfer to a subject in need thereof, and generally comprises at least 10 9 , more usually at least 5x10 9 cells, and up to about 10x10 10 or more cells, e.g. 5x10 9 -10x10 9 A living cell provided by the preparation method disclosed herein.

[0250] T lymphocytes (T cells) are one of the various cell types involved in the immune response. The activity of T cells is regulated by antigens presented to the T cells in the context of major histocompatibility complex (MHC) molecules. The T cell receptor (TCR) then binds to the MHC-antigen complex. Once the antigen is complexed with the MHC, the MHC-antigen complex is bound by a specific TCR on the T cell, thereby changing the activity of the T cell. Proper activation of T lymphocytes by antigen presenting cells requires not only stimulation of the TCR, but also the combination and coordinated engagement of its co-receptors.

[0251] T cells (e.g., α / β T cells) are often characterized by the expression of CD3 and are therefore often considered to be CD3 +Cells. CD3 is a TCR co-receptor composed of four transmembrane subunits (γ, δ, ε and ζ), which plays a role in TCR-mediated activation and participates in it. Specifically, the intracellular portion of the CD3γ, δ, ε and ζ subunits contains a copy of a sequence motif known as ITAM (immunoreceptor tyrosine-based activation motif), which promotes TCR-mediated signal transduction. Therefore, the TCR of the present invention can be optionally engineered to co-express CD3 (in addition to the TCR of the present invention), for example, by a recombinant method disclosed herein, to express in immune cells lacking endogenous CD3 expression.

[0252] Memory (primed) T cells include, for example, central memory cells (T cells) that express CCR7 but not CD45RA. CM ), effector memory lacking expression of both CCR7 and CD45RA ( TEM ) cells and / or T cells expressing CD45RA but lacking CCR7 expression EMRA cell.

[0253] T helper cells (TH cells) assist other white blood cells in immune processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4 + T cells, because they express CD4 glycoprotein on their surface. When MHC class II molecules expressed on the surface of antigen presenting cells (APCs) present peptide antigens to helper T cells, they are activated. Once activated, they rapidly divide and secrete small proteins known as cytokines that regulate or assist active immune responses. CD4 is complexed with the bound TCR and is thought to be involved in the stabilization of the MHC II restricted TCR complex.

[0254] Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells and are also involved in transplant rejection. These cells are also known as CD8 + T cells, because they express the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens bound to MHC class I molecules present on the surface of all nucleated cells. CD8 is complexed with the bound TCR and is thought to be involved in the stabilization of the MHC I-restricted TCR complex.

[0255] However, despite the fact that “CD4 + Cells" and "CD8 + "Cells" are usually used to refer to TH cells and TC cells, respectively, but it is worth noting that additional CD3 + lymphocyte populations. For example, in the absence of CD8 expression, CD4 +CTL is an effector cell with cytotoxic function. According to the embodiments of the present disclosure, the cell composition of the present invention is characterized by a unique subpopulation distribution that is different from other ACT compositions disclosed so far.

[0256] The present invention provides, in its embodiments, the preparation of an ACT composition in which greater than 50% of the cells are characterized by surface expression of the TCR, and are typically characterized by an average expression greater than 53%, 55%, 60%, 70%, 80%, 90% or more, as disclosed herein. Since CPN<5 is recommended for regulatory purposes, it is advantageous to increase the surface expression of the TCR per copy number. The present invention provides, in its embodiments, the preparation of an ACT composition suitable for clinical use, characterized by CPN<5 and enhanced TCR surface expression disclosed herein. In addition, the composition characterized by high eTCR expression disclosed herein can be produced not only when allogeneic immune cells from healthy donors are used, but also when autologous cells from cancer patients are used (due to the presence of a tumor (tumor-derived immunosuppression) and / or previous treatment with other cancer therapies (e.g., chemotherapy or radiation that may induce immune cell depletion or suppression), the autologous cells are typically immunosuppressed or damaged).

[0257] In another embodiment, the composition comprises CD4 + eTCR + Cells and CD8 + eTCR + In another embodiment, the composition comprises CD8 - In another embodiment, the composition comprises a cell population characterized by surface expression of CD4, a TCR of the invention and CD107 (CD4 + eTCR + CD107 + According to an exemplary embodiment, the ACT composition of the present invention may include 5% to 10% of the total CD4 + eTCR + 5-40%, 10-30%, 10-20%, 10-25%, 15-30%, such as about 15% of CD4 + eTCR + CD107 + Cell population. In addition, the ACT composition according to the present invention exhibits the functional properties disclosed herein in terms of the activity of the engineered TCR.

[0258] In another embodiment, at least 60%, 70%, 80% or 90% of the immune cells are characterized by surface expression of the engineered TCR (eTCR+ In another embodiment, at least 70% of the immune cells are CD3 + eTCR + cells, including CD4 + and CD8 + In another embodiment, the composition comprises 20-50%, 15-50%, 15-40%, 40-50%, 20-60% or 20-40% CD4 + cells and 30-70%, 30-85%, 40-50%, 40-85%, 50-70% or 50-85% CD8 + cells, wherein each possibility represents a separate embodiment of the present invention. As disclosed herein, the CD4 + The cells are functionally responsive to tumor cells expressing NY-ESO-1. Additionally or alternatively, the immune cells are engineered to express the TCR by administering a nucleic acid construct encoding the TCR, and the cell composition is characterized by an average copy number of 5 or fewer construct molecules per cell.

[0259] In another embodiment, the composition may further comprise additional active ingredients, such as anticancer drugs or cytokines. In a specific embodiment, the anticancer drug is a cancer immunotherapy, such as an immune checkpoint inhibitor. In another specific embodiment, the cytokine is a cytokine that regulates T cells, such as interleukin-2 (IL-2). In another embodiment, the composition further comprises IL-2 (e.g., 100-500 IU / mL). In another embodiment, the composition comprises at least 10 9 CD3 + eTCR + Cells, for example 10 9 -5x10 10 , 5x10 9 -10 10 or 10 9 -10 10 cells.

[0260] As a non-limiting example, a clinical-grade cell composition for ACT may include at least 10 9 Autologous CD3 + eTCR + cells, the injection medium contained 0.9% sodium chloride, 2% human albumin and IL-2 300 IU / mL, and the total volume was 300 mL.

[0261] In another embodiment, the cell composition is prepared by a method disclosed herein. In another embodiment, a method of preparing a cell composition disclosed herein is provided.

[0262] In another embodiment, the method of the present invention comprises the steps of:

[0263] a. Providing an immune cell population, usually a cell population containing T cells,

[0264] b. culturing the cell population in the presence of CD3-specific antibodies and IL-2,

[0265] c. engineering the cells obtained from step b. to express the TCR of the present invention,

[0266] d. expanding the cells obtained from step c. so as to obtain a therapeutically effective amount of expanded engineered cells, and

[0267] e. Harvesting the expanded engineered cells obtained from step d.

[0268] Step a. is further referred to herein as a sample acquisition step. In another embodiment, the cell colony containing T cells provided in the step a. is a peripheral blood mononuclear cell (PBMC) colony. In another embodiment, the cell colony is obtained by apheresis (e.g., leukocyte apheresis). In another embodiment, the cell may be subjected to an additional step of enrichment or separation of a specific cell colony. In another embodiment, the cell colony may include, for example, isolated T cells, memory T cells, or T cell lines. In another embodiment, the cell colony containing T cells includes or is derived from a tumor infiltrating lymphocyte colony. In another embodiment, the PBMC colony is provided in the form of a non-fractionated PBMC sample. Each possibility represents an independent embodiment of the present invention. In another embodiment, the PBMC colony has been cryopreserved and thawed before step b.

[0269] Peripheral blood mononuclear cells (PBMC) are leukocytes separated from peripheral blood, and include cells with round nuclei, such as lymphocytes, monocytes and dendritic cells. Generally, lymphocytes account for the majority of PBMC colonies. PBMC can be extracted from whole blood using methods known in the art, such as using Ficoll-hypaque density gradient centrifugation. In addition or alternatively, PBMC or its specific leukocyte population can also be separated from peripheral blood using apheresis. Apheresis products may contain lymphocytes (including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes), red blood cells and platelets, or may be apheresis products comprising lymphocytes (including T lymphocytes, monocytes, granulocytes, B cells and other nucleated leukocytes). In one embodiment, the cells collected by apheresis can be cleaned to remove plasma fractions, and the cells are placed in suitable buffer or culture medium for subsequent treatment (such as density gradients or additional purification steps). The cells can be cleaned with PBS or another suitable solution that does not contain calcium, magnesium and most (if not all other) divalent cations. When used herein, and unless otherwise indicated, the term PBMC is further used to denote in particular human PBMCs. Non-limiting examples of PBMC isolation are provided in the Examples below.

[0270] Step b. is also referred to herein as an activation (or stimulation) step, wherein the cells provided in step a. are cultured in the presence of CD3-specific antibodies and IL-2 to induce T cell activation. The term "activation" as used herein refers to a state in which immune cells have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector functions. The term "activated T cells" refers in particular to T cells that are undergoing cell division.

[0271] In another embodiment, step b. further comprises culturing the cells with CD28-specific antibodies, wherein the CD3-specific antibodies and CD28-specific antibodies are surface-bound. For example, but not limited to, the antibodies can be bound to the surface of particles, beads, nanoparticles, culture dishes, etc. In another embodiment, step b. can be performed for 1-3 days, such as 2 days. CD28 refers to the cluster of differentiation 28 protein, which is one of the proteins expressed on T cells and provides the co-stimulatory signals required for T cell activation and survival. CD28 is a receptor for CD80 and CD86 proteins. The sequence of human CD28 is provided in NCBI reference number: NP_006130. In this specification and claims, "anti-CD28 antibody" and "anti-CD3 antibody" refer to anti-CD28 agonist antibodies and anti-CD3 agonist antibodies, respectively, i.e. antibodies that specifically stimulate CD28 receptors and CD3 receptors on cells, respectively.

[0272] The term "antibody" is used in the broadest sense and includes monoclonal antibodies (including full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments long enough to exhibit desired biological activity (e.g., specific binding to CD3 or CD28 (particularly human CD3 or D28) in an exciting manner). Methods for producing monoclonal antibodies and polyclonal antibodies are well known in the art. Antibodies can be produced by any of several known methods, which can be produced by inducing the in vivo production of antibody molecules, screening immunoglobulin libraries, or producing monoclonal antibody molecules by continuous cell lines in culture. These methods include, but are not limited to, hybridoma technology, human B cell hybridoma technology, and Epstein-Barr virus (EBV) hybridoma technology. In addition to conventional methods for producing antibodies in vivo, phage display technology can also be used to produce antibodies in vitro by methods known in the art (e.g., "Modern Methods in Immunology" (Current Protocols inImmunology), edited by Colligan et al., John Wiley & Sons, Inc. (1992-2000), Chapter 17, Section 17.1). In addition, various antibodies specific for human CD3 and CD28 are commercially available, for example, agonistic anti-CD3 antibodies include OKT3 (ATCCCRL-8001 / Invitrogen), UCHT1 (BDPharMingen) and HIT3a (BDPharMingen), and agonistic anti-CD28 antibodies include, for example, CD28.2 (Biologend). As shown in the examples below, anti-CD3 and anti-CD28 antibodies suitable for use in embodiments of the present invention in a surface-bound form can be purchased from Thermo Scientific and Milteny.

[0273] Step c. is also referred to herein as an engineering step, comprising genetically engineering the activated cells obtained from step b. to express the TCR of the present invention. Engineering can be performed by suitable methods known in the art such as transduction, infection, transfection or transformation, so that it can be detected on the surface of at least a portion of the obtained cell population by conventional methods such as flow cytometry. In another embodiment, step c. is performed by cultivating the cells after step b. with a viral vector containing a nucleic acid construct encoding the TCR (so as to transduce the cells with the construct). In some embodiments, fibronectin fragments (e.g., Retronectin) or other agents that promote colocalization of viral vectors and intended target cells can be used to improve transduction efficiency.

[0274] Step d. Also referred to herein as the expansion step, which includes further culturing the engineered cells under specific conditions to enhance the eTCR of the desired phenotype+ The total number of cells. For example, expansion can use a regulatory T cell cytokine such as IL-2 that induces T cell proliferation, which is generally used in combination with other T cell stimulators disclosed herein. In another embodiment, step d. is performed by culturing the cells after step c. with IL-2 and optional feeder cells (e.g., attenuated allogeneic cells, such as irradiated PBMCs). In another embodiment, step d. is performed without supplementing antibodies. In another embodiment, step d. is performed to obtain at least 10 9 CD3 + eTCR + Cells. In another embodiment, step e. is performed within 7-21 days, typically 14-21 days, after starting step b. In some embodiments, step e. is performed within 20, 19, 18, 17, 16, 15 or 14 days after starting step b., wherein each possibility represents an independent embodiment of the present invention. In a specific embodiment, step e. is performed within 14 to 15 days after starting step b. In other specific embodiments, step e is performed within 13, 12, 11, 10, 9, 8 or 7 days after starting step b., wherein each possibility represents an independent embodiment of the present invention.

[0275] The term "feeder cells" generally refers to a type of cell that is co-cultured with a second type of cell to provide an environment in which the second type of cell can be maintained and proliferated. For the purposes of the present invention, the term specifically refers to an auxiliary cell carrying an Fc receptor, which is generally alloreactive with a population containing T cells to be propagated. In other words, the feeder cells do not need to be tissue compatible with the population containing T cells to be propagated, and in certain advantageous embodiments, the two populations are generally HLA mismatched. A typical example of a feeder cell used in an embodiment of the present invention is an allogeneic normal donor peripheral blood mononuclear cell (PBMC). For example, but not limited to, an ACT composition can be prepared using irradiated PBMCs (which cannot proliferate) as feeder cells, for example, by attenuating the cells by exposing them to 6000RAD for irradiation.

[0276] Step e. is also referred to herein as the harvesting step. This step includes separating and collecting cells from the culture medium of the cells and preparing the cells for therapeutic applications. Harvesting generally includes washing the cells with a suitable buffer (e.g., a saline solution containing BSA), and optionally resuspending the cells in a buffer suitable for the intended application (e.g., for infusion) (e.g., suspended in 100-400 ml of a saline solution containing 1-5% human albumin and 100-500 IU / ml IL-2, e.g., in 200-400 ml of a 0.9% saline solution containing 2% human albumin and 300 IU / ml IL-2).

[0277] It is well within the knowledge of the skilled artisan to determine the therapeutically effective amount of the expanded engineered cells obtained and harvested by the methods of the present invention. For example, the total number of viable cells can be calculated by staining a cell sample with a viability dye (e.g., trypan blue) and counting the cells using a hemocytometer. + eTCR + The number of cells can be assessed by flow cytometry (e.g. after staining with appropriate antibodies and / or NY-ESO-1-specific tetramers). + eTCR + The percentile of cells was multiplied by the total number of viable cells to calculate the viable CD3 + eTCR + An effective amount of cells.

[0278] Thus, advantageous methods according to the invention provide cell compositions having a desired phenotype that exhibit elevated levels of activation markers and reduced levels of exhaustion markers compared to compositions reported to date. In a particular embodiment, the activation markers include cytotoxic effector markers (e.g., CD107). According to some embodiments, the ACT compositions of the invention are characterized by elevated levels of activation markers including CD134, CD137, and / or CD107 compared to compositions known to date.

[0279] As described herein, the present specification provides and exemplifies suitable conditions for effecting the methods of the present invention. The present specification demonstrates the implementation of these methods and conditions to provide a therapeutically effective amount of live CD3+ cells characterized by the described favorable phenotype. + eTCR + Cells suitable for the production of autologous and allogeneic ACT compositions.

[0280] In another embodiment, the method comprises the steps of:

[0281] a. Provide PBMC population,

[0282] b. culturing the cell population in the presence of CD3-specific antibodies and IL-2,

[0283] c. engineering the cell (obtained from step b.) to express the TCR by incubating the cell with a viral vector comprising a nucleic acid construct encoding the TCR,

[0284] d. amplifying the cells after step c. in the presence of IL-2 and feeder cells without supplementing antibodies to obtain at least 10 9 CD3 expressing the TCR+ cells, and

[0285] e. Harvesting the expanded cells within 7-21 or 14-21 (eg 15) days after starting step b.

[0286] In another embodiment, the steps as described above include culturing / expanding the cells in the presence of only the antibodies, cells and / or cytokines described in steps b. and d. above without adding other antibodies, cells, cytokines. For example, but not limited to, step b. advantageously includes culturing the cell population in the presence of 10-100, such as 50 ng / mL of CD3-specific antibodies (e.g., OKT3) and 100-500, such as 300 IU / mL of IL-2 as the only activator, and step d. advantageously includes culturing the obtained cells with irradiated allogeneic PBMCs at a ratio of 25:1 to 100:1 (e.g., 25:1 to 75:1, 40:1 to 75:1 or about 50:1) of PBMC to engineered cells, and culturing in the presence of 1500-4500, such as 3000 IU / mL IL-2 without supplementing antibodies or other cytokines. However, in other embodiments, other methods for activating and / or expanding the cells can be used, which methods substantially retain the parameters of the obtained cell composition disclosed herein. For example, but not limited to, as described in detail herein, in the activation and expansion steps, a colloidal polymer nanomatrix (e.g., TRANSACT) covalently attached to a humanized recombinant agonist for human CD3 and CD28 can be used in combination with IL-2. In another example, as described in detail herein, in the activation and expansion steps, a uniform inert superparamagnetic bead (e.g., DYNABEADS) having a diameter of 4.5 μm, which is close to that of antigen presenting cells and covalently coupled to anti-CD3 and anti-CD28 antibodies, can be used in combination with IL-2. As a non-limiting example, surface-bound anti-CD3 and anti-CD28 antibodies can be used in a concentration range of 0.2 μg / ml to 5 μg / ml.

[0287] For example, the following examples demonstrate the use of the following method: sample acquisition by providing an autologous or allogeneic PBMC population; activation by culturing the cell population for 2-3 days in the presence of CD3-specific antibodies and IL-2 as the only activating agents under the conditions described above; engineering by transduction with a viral vector encoding a TCR of the present invention (e.g., a retroviral vector disclosed herein) in the presence of a surface-bound fibronectin fragment; expansion by culturing the obtained cells with IL-2 and irradiated allogeneic PBMC as feeder cells for 11-13 days under the conditions described above, and further suspending the harvested cells in a saline solution for injection containing BSA and IL-2. It is demonstrated herein that the method can be used to obtain at least 10 9 A CD3 phenotype characterized as disclosed herein + eTCR + Cells, providing improved ACT. Other examples of suitable reaction conditions (eg when using surface-bound CD3-specific antibodies and CD28-specific antibodies) are further provided in the Examples section below.

[0288] In addition, as disclosed herein, the ACT compositions of the present invention provide significant persistence in the blood circulation of tumor-bearing individuals using animal models and human cancer patients. According to some embodiments, the ACT compositions of the present invention are detectable in the blood of a recipient subject in need for more than 30 days, more typically at least 45, 60, 75, or 90 days or at least 4, 5, 6, 7, or 8 months, up to about 9 months or longer, for example, in human subjects for 45 days to 9 months, 2-9 months, or 3-9 months.

[0289] Therapeutic Uses

[0290] On the other hand, a method for treating cancer in a subject in need is provided, the method comprising administering an ACT composition of the present invention to the subject. In various embodiments, the cells are autologous, allogeneic tissue compatible, allogeneic partially tissue compatible (e.g., substantially tissue compatible) or allogeneic non-tissue compatible for the subject. Typically and advantageously, it is contemplated to use autologous and allogeneic tissue compatible ACT compositions. In a specific embodiment, the cells are autologous cells of the subject.

[0291] The term "autologous" as used herein refers to any material derived from a subject that will be reintroduced into the same subject later. According to certain preferred embodiments, the cell composition is compatible with the subject tissue to be treated (e.g., autologous cells or allogeneic cells of MHC II matching). The term "histocompatibility" refers to the similarity of tissues between different individuals. The level of tissue compatibility describes the degree of matching between a patient and a donor. The main determinant of tissue compatibility is human leukocyte antigen (HLA). HLA typing is performed between a potential donor and a potential recipient to determine the degree of HLA matching between the two. The term "histocompatible" as used herein refers to an embodiment in which all six HLA antigens (2 A antigens, 2 B antigens, and 2 DR antigens) are identical between a donor and a recipient. However, in other embodiments, although the donor and the recipient are not fully matched, "mismatched" on two or more antigens, such as 5 of 6 antigens or in other embodiments 4 of 6 antigens or 3 of 6 antigens matching donors and recipients, can be covered by certain embodiments of the present invention. As used herein, the term "substantially histocompatible" refers to embodiments in which 5 of the 6 HLA antigens are identical between the donor and recipient.

[0292] In another aspect, a method for treating cancer in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition comprising a construct of the present invention. For example, but not limited to, the construct may be delivered to the subject in the form of an expression vector exhibiting cell type or tissue specificity (e.g., for immune cells disclosed herein) or in the form of an RNA molecule delivered by nanoparticles or other suitable in vivo delivery systems.

[0293] For example, but not limited to, the method of the present invention can be achieved by administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising a therapeutically effective amount of an expression vector, the expression vector comprising a nucleic acid construct encoding the TCR of the present invention, for example, comprising a first nucleic acid sequence encoding a TCR α chain (wherein the CDR is encoded by a nucleic acid sequence set forth in SEQ ID NO: 7-9) and a second nucleic acid sequence encoding a TCR β chain (wherein the CDR is encoded by a nucleic acid sequence set forth in SEQ ID NO: 10-12). In various exemplary embodiments, the vector exhibits T cell specificity, for example, using a T cell-specific promoter disclosed herein.

[0294] In another aspect, the present invention provides a method of enhancing anti-tumor immunity in a subject in need thereof, the method comprising administering to the subject a cell composition of the present invention, or in other embodiments administering a pharmaceutical composition comprising a construct of the present invention. Each possibility represents an independent embodiment of the present invention. When used herein, enhancing anti-tumor immunity includes inducing or enhancing a tumor-specific immune response (also referred to herein as tumor-specific immunoreactivity) in the subject. In another embodiment, the response includes a T cell-mediated response that triggers or enhances TCR-mediated functional activity (e.g., cytotoxicity or cytokine production), particularly against tumors expressing NY-ESO-1 disclosed herein. In other embodiments, the enhancement of anti-tumor immunity includes enhancement of tumor-specific CD8-independent cytotoxicity. In another embodiment, the enhancement of anti-tumor immunity is manifested as a significant tumor-specific immune response for more than 30 days, more typically at least 45, 60, 75 or 90 days, or at least 4, 5, 6, 7 or 8 months, up to about 9 months or longer, such as 45 days to 9 months, 2-9 months, or 3-9 months. In another embodiment, the enhancement of anti-tumor immunity comprises reducing the defects associated with the previous or previously administered adoptive transfer therapy. In another embodiment, the method of the present invention provides for inducing or enhancing a tumor-specific immune response characterized by the advantageous functional properties disclosed herein. Each possibility represents an independent embodiment of the present invention.

[0295] In some embodiments, the methods of the invention using ACT compositions may generally include a step of lymphocyte clearance prior to adoptive transfer of cells to a recipient subject. Lymphocyte clearance protocols are well known, easily performed by the treating physician, and adjusted according to the patient's clinical status and medical history. For example, but not limited to, lymphocyte clearance may be performed by, for example, a non-myeloablative lymphocyte clearance protocol (e.g., using cyclophosphamide, with or without fludarabine) within 5-6 days before ACT administration. Non-limiting examples of such protocols are provided in Example 9.

[0296] Typically, in the methods of the invention, the subject has a tumor that expresses NY-ESO-1. In addition, the subject to be treated by the methods of the invention is typically HLA-A2 positive. The terms "expressed" or "positive" are well known in the art and mean that the gene product is expressed in an amount that can be detected by methods known in the art. Proteins can be detected using immunological assays (e.g., ELISA, immunostaining) or flow cytometry (e.g., using labeled antibodies). Transcripts can be detected using methods for amplifying and / or detecting nucleic acids (e.g., RT-PCR, microarrays, biochips, or RNAseq).

[0297] As used herein, the terms "NY-ESO-1 expressing tumor", "NY-ESO-1 cancer" and "NY-ESO-1 expressing tumor cells" refer to tumors comprising cells expressing NY-ESO-1. These cells are typically characterized by surface expression of at least one epitope of a naturally occurring NY-ESO-1 polypeptide in the context of an HLA molecule, particularly an HLA class I molecule. More typically, the HLA molecule is an HLA-A2 molecule.

[0298] HLA-A2 is a specific class I major histocompatibility complex (MHC) allele at the HLA-A locus; the alpha chain is encoded by the HLA-A*02 allele and the beta chain is encoded by the beta2-microglobulin or B2M locus. The following A2 alleles are known: A*02:01, A*02:02, A*02:03, A*02:04, A*02:05, A*02:06(A2.4A), A*02:07, A*02:08, A*02:09, A*02:10, A*02:11(A2.5), A*02: 12. A*02:13(A*02SLU), A*02:16, A*02:17, A*02:18(A2K), A*02:19, A*0 2:20, A*02:21, A*02:31, A*02:34 (A*AAT), A*02:35, A*02:36 and A*02:37. The most common allele is the A*02:01 allele, and thus most subjects identified as having HLA-A2 carry the A*02:01 allele signal (also referred to as HLA-A*201 or HLA-A2*01).

[0299] In some embodiments, the tumor is selected from: melanoma, bladder, ovarian, lung, breast and prostate tumors (typically tumors expressing NY-ESO-1). In other embodiments, the subject to be treated by the method of the present invention may suffer from, for example, neuroblastoma, myeloma, metastatic melanoma, synovial sarcoma, bladder cancer, esophageal cancer, hepatocellular carcinoma, head and neck cancer, non-small cell lung cancer, ovarian cancer, prostate cancer or breast cancer. In other embodiments, the tumor can be, for example, a melanoma (e.g., uveal melanoma), an ovarian tumor (e.g., ovarian cancer), a synovial tumor (e.g., synovial sarcoma) or a breast tumor (e.g., triple negative breast cancer, TNBC). According to an exemplary embodiment, the tumor can be, for example, a tumor expressing NY-ESO-1, which is selected from uveal melanoma, synovial sarcoma, TNBC and ovarian cancer. In other specific embodiments, the subject suffers from a tumor selected from melanoma (e.g., uveal melanoma), a sarcoma (e.g., synovial sarcoma) and a cancer (e.g., bladder cancer, ovarian cancer, lung cancer, breast cancer and prostate cancer). In yet another embodiment, the tumor may be a melanoma, bladder, ovarian, lung, breast, prostate or synovial tumor expressing NY-ESO-1. In another embodiment, the tumor is selected from the group consisting of: melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors. Each possibility represents a separate embodiment of the present invention.

[0300] In another embodiment, the method of the invention further comprises the step of determining the expression of HLA-2 and / or NY-ESO-1 in a sample of the subject prior to treatment (e.g., determining whether the subject has a tumor expressing an NY-ESO1 epitope presented by HLA-2), for example, by an immunoassay or amplification method disclosed herein. Thus, according to some embodiments, a subject determined to have an HLA-2 and NY-ESO-1 positive tumor is treated with a pharmaceutical composition or cellular composition of the invention. According to a specific embodiment, the tumor to be treated by the method of the invention expresses the peptide epitope of SEQ ID NO: 21 in the context of HLA-A*0201 and / or HLA-A*0206.

[0301] In another embodiment, the tumor is a solid tumor. In another embodiment, the tumor is metastatic. In another embodiment, the tumor is a locally advanced tumor. In another embodiment, the tumor is a refractory (treatment resistant) tumor. In another embodiment, the tumor is a recurrent tumor or a malignant tumor. In yet another embodiment, the tumor is a metastatic cancer or a locally advanced refractory or recurrent malignant tumor that is not originally suitable for curative treatment.

[0302] In another embodiment, the patient has received prior treatment with cancer therapy, such as chemotherapy and / or immunotherapy. In another embodiment, the patient has failed to respond to at least one course of chemotherapy and / or immunotherapy. In another embodiment, the patient has a tumor that is resistant to the treatment. In another embodiment, the treatment is immunotherapy using an immune checkpoint inhibitor. In another embodiment, the checkpoint inhibitor is directed to PD-1 and / or CTLA-4. In another embodiment, the patient has received prior treatment with an immune checkpoint inhibitor directed to PD-1 and / or CTLA-4 and has failed to respond. Each possibility represents an independent embodiment of the present invention.

[0303] The term "resistance" refers to the characteristic that a cancer (or tumor) does not respond to a given treatment. A tumor may already be resistant to a particular therapy at the beginning of treatment (primary resistance). Alternatively, a tumor may develop resistance during treatment (acquired resistance, also known as secondary resistance). Resistance to therapy is generally understood as unsatisfactory effectiveness of treatment, usually leading to disease progression. In the case of acquired resistance, resistance can also be manifested as a reduction in the amount of tumor regression at the same dose or an increase in the dose required for the same amount of tumor regression. For example, in the case of resistance to immune checkpoint inhibitors, the term "resistance" refers to a tumor that fails or has failed to respond to treatment with immune checkpoint inhibitors for more than 3 months or more, responding adequately in a manner that is favorable in terms of tumor shrinkage or stabilization or duration of shrinkage.

[0304] The term "suitable for treatment" means that the tumor is predicted or determined to respond favorably to the treatment. Suitability for treatment can be determined by a treating physician or oncologist by evaluating the clinical response to the treatment (or to a treatment of similar effects of the same category), or by a professional and technical personnel by evaluating cell viability or apoptosis induction activity after contacting a suitable anticancer agent with a tumor or tumor sample derived from a subject. Professional and technical personnel know that there are standard assays for determining treatment suitability or screening for resistant cancers, such as MTT assays, ATP measurements and / or apoptosis assays, such as TUNEL, cytochrome C release or cleavage of caspase-3 assays. In another embodiment, the method of the present invention further comprises administering cancer immunotherapy to the subject. In another embodiment, the immunotherapy is administered before, simultaneously with, or after the adoptive transfer of the ACT composition of the present invention, wherein each possibility represents an independent embodiment of the present invention. In a specific embodiment, the cancer immunotherapy is a T cell-mediated immunotherapy (intended to induce, enhance, or otherwise regulate the activity of T cells in the subject). Exemplary T cell-mediated immunotherapies include, but are not limited to, T cell regulatory cytokines, immune checkpoint inhibitors, and cellular composition immunotherapies disclosed herein (e.g., adoptively transferred cell compositions).

[0305] For example, but not limited to, the subject further receives treatment with one or more cytokines (e.g., IL-2, IL-7, IL-12, IL-15, and IL-21). In other exemplary embodiments, it is contemplated that combined treatment with immune checkpoint inhibitors (e.g., for PD-1, CTLA4, TIGIT, TIM3, PD-L1, 41BB, FAS, GITR, BTLA, ICOS, OX40, LAG-3, GLECTIN 9, CD96, VISTA, H-VEM, P-selectin, or PCGL) is used. In another embodiment, the tumor is resistant to treatment using one or more of the above-mentioned cytokines or immune checkpoint inhibitors, wherein each possibility represents an independent embodiment of the present invention. In another embodiment, the tumor is an immunotherapy-resistant tumor, which is not suitable for immunotherapy treatments such as cytokines and immune checkpoint inhibitors.

[0306] In order to more fully illustrate some embodiments of the present invention, the following examples are given. However, they should in no way be construed as limiting the broad scope of the present invention.

[0307] Example

[0308] Example 1. Generation of a novel engineered TCR targeting NY-ESO-1

[0309] A novel engineered TCR that recognizes NY-ESO-1 in the context of an HLA-A*0201 class I restriction element was constructed. The TCR construct encodes human α and β chains having the amino acid sequences set forth in SEQ ID NOs: 17 and 18, respectively. The construct contains nucleic acid sequences encoding the α and β chains set forth in SEQ ID NOs: 19 and 20, respectively, connected by a P2A linker comprising a 5' end furin recognition site, the linker having the amino acids set forth in SEQ ID NO: 29 and the nucleic acid sequence set forth in SEQ ID NO: 24, as shown below:

[0310] AGGGCAAAGCGCAGCGGATCCGGAGCAACAAACTTTAGCCTGCTGAAGCAGGCAGGCGACGTGGAGGAGAATCCAGGACCGCGG (SEQ ID NO: 24).

[0311] Thus, the sequence encoding the linked α and β chains construct has the nucleic acid sequence set forth in SEQ ID NO: 25, as shown below (wherein the signal peptide is underlined and the linker is in bold):

[0312]

[0313]

[0314]

[0315] The resulting engineered TCR encoded by the construct is referred to as eTCR hereinafter. The DNA sequence (SEQ ID NO: 25) of the construct encoding the eTCR chain was cloned into the MSGV-1 retroviral expression vector. MSGV-1 is a derivative of the spliced ​​gag vector (pMSGV) based on the mouse stem cell virus (MSCV), which uses the MSCV long terminal repeat (LTR). Stable retroviral production clones were generated by transfection into Eco-Phoenix cells, and the vector supernatant was subsequently used to transduce PG13 packaging cells. In brief, DH5α Escherichia coli bacteria were used to produce eTCR plasmid stocks for transfection. Transfection into Eco-Phenix cells was performed by Jetprime reagent and 2 μg of purified eTCR construct plasmid. Viruses were collected from Eco-Phoenix cells two days after transfection. Then, transduction of PG13 cells was performed using a spinoculation method using a retronectin reagent. Subsequently, viral particles collected from selected PG13 cells were inoculated by centrifugation with retronectin and used to transduce healthy donor PBMCs.

[0316] As described in Example 2 below, the function of the eTCR was evaluated and determined to be highly effective against tumor cells expressing NY-ESO-1. Example 3 describes the selection of eTCR subclones for the preparation of clinical-grade cell compositions, and the generation of parent cell banks that stably express the subclones. Examples 4-5 describe the development and characterization of clinical-grade cell compositions and their preparation protocols, and Examples 6-9 describe in vivo preclinical and clinical evaluations of these cell compositions.

[0317] Example 2 . Engineered TCRs show improved functional activity

[0318] In order to evaluate the function of the eTCR, its ability to induce antigen-specific stimulation and activity in transduced lymphocytes was evaluated and compared with a control TCR. To this end, primary peripheral blood mononuclear cells (PBMCs) from human donors were activated with anti-CD3 antibodies (OKT3) and IL-2 and subsequently transduced with a retroviral vector encoding the TCR tested as described in more detail in Example 3 below. In addition to cells transduced with a vector encoding the eTCR, control cells were transduced with a retroviral vector encoding a truncated version of the nerve growth factor receptor (NGFR) as a negative control, or with a natural NY ESO-1TCR (referred to as 1G4) known to date for the same epitope as the eTCR as a positive control. The nucleic acid sequences encoding the α and β chain variable regions (VRα and VRβ) of the 1G4 control TCR are as follows:

[0319] ATGGAGACCCTCTTGGGCCTGCTTATCCTTTGGCTGCAGCTGCAATGGGTGAGCAGCAAACAGGAGGTGACGCAGATTCCTGCAGCTCTGAGTGTCCCAGAAGGAGAAAACTTGGTTCTCAACTGCAGTTTCACTGATAGCGCTATTTACAACCTCCAGTGGTTTAGGCAGGACCCTGGGAAAGGTCTCACATCTCTGTTGCTTATTCAGTCAAGTCAGAGAGAGCAAACAAGTGGAAGACTTAATGCCTCGCTGGATAAATCATCAGGACGTAGTACTTTATACATTGCAGCTTCTCAGCCTGGTGACTCAGCCACCTACCTCTGTGCTGTGAGGCCCACATCAGGAGGAAGCTACATACCTACATTTGGAAGAGGAACCAGCCTTATTGTTCATCCG (1G4 VRα, SEQ ID NO: 26); and

[0320] ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACGTCGGGAACACCGGGGAGCTGTTTTTTGGAGAAGGCTCTAGGCTGACCGTACTG (1G4 VRβ, SEQ ID NO: 27).

[0321] Next, the transduced cells were co-cultured with target tumor cells that either expressed or did not express the NY-ESO-1 target antigen. The target cells included two types of melanoma tumor lines, A375 and 624.38, which are NY-ESO-1 / HLA-A-0201 positive lines with different levels of NY-ESO-1 expression. As a negative control, the 888A2 melanoma cell line that does not express NY-ESO-1 was used. The cells were incubated for 24 hours at a 1:1 effector cell:tumor cell ratio (100,000 cells per colony). After incubation, the levels of cytokines secreted into the culture medium and the levels of activation markers on the transduced lymphocytes were assessed.

[0322] Cytokine secretion

[0323] The secretion levels of tumor necrosis factor (TNFα), interleukin 2 (IL2), and interferon gamma (IFNγ) in the co-culture supernatant were measured by ELISA. The results are presented in Figures 1A-1C In which 1G4 TCR is shown as 1G4. Figure 1A In the Figures, TNFα concentrations are presented as mean ± SEM, n = 3, using three different donors; normalized to the activity of the 1G4 construct against A375 melanoma cells (mean secretion of 567 pg / ml). Figure 1B IL2 concentrations are presented as mean ± SEM, n = 2, using two different donors; normalized to the activity of the negative control construct against A375 cells (mean secretion of 119 pg / ml). Figure 1C In the table, the IFNγ concentrations of the above co-cultures or the negative control without target cells are presented in pg / ml.

[0324] As can be seen in Figures 1A-1C As seen in Figure 3, transduction of eTCR or 1G4TCR in the presence of cells expressing NY-ESO-1 resulted in enhanced cytokine secretion compared to the amounts secreted in the presence of irrelevant tumor cells. In contrast, transduction with the negative control NGFR construct did not result in enhanced cytokine secretion, and these cells maintained low basal levels in the presence of all target cell types.

[0325] Furthermore, as can be seen in these figures, cells transduced with the eTCR secreted significantly higher levels of cytokines in the presence of their cognate target cells compared to cells transduced with the 1G4 TCR. Figure 1A As seen in Figure 3, a statistically significant difference was observed between the levels of TNFα secreted by eTCR-transduced cells and 1G4 TCR-transduced cells (p=0.0026) using paired Student's t-test calculations. Figure 1B and 1C Significant enhancement in the magnitude of antigen-induced IL2 and IFNγ secretion between the eTCR- and 1G4-transduced groups was shown.

[0326] Activation markers

[0327] OX40 (CD134) and 4-1BB (CD137) are cell surface proteins expressed on activated T cells, so they are considered to be T cell activation markers, where their expression levels serve as indicators of T cell activation. Therefore, after co-culturing T cells transduced with eTCR, 1G4TCR or NGFR with target cells as described above, the expression levels of OX40 and 4-1BB were determined. To this end, OX40 and 41BB were stained with suitable antibodies and measured by fluorescence activated cell sorting (FACS). The results of OX40 are shown in Figure 2 Presented as mean + SEM, n = 3, using three different donors; normalized to the activity of the NGFR constructs against A375, where cell line 624.38 is shown as 624. Figure 3 The results show that CD8 + FACS plot of 4-1BB expression in cells.

[0328] As can be seen in Figure 2 As can be seen in Figure 3, it is clear that the eTCR-transduced cells exhibit high OX40 expression levels, which are enhanced in the presence of tumor cells expressing NY-ESO-1. Surprisingly, OX40 expression in eTCR-transduced cells is significantly higher than in 1G4-transduced cells, which are only slightly elevated compared to NGFR-transduced cells.

[0329] As can be seen in Figure 3 As seen in Figure 3, similar differences were detected for 4-1BB expression levels; notably, eTCR-transduced cells exhibited higher 4-1BB expression levels compared to 1G4-transduced cells co-cultured with either of the specific target cell lines A375 and 624.38.

[0330] CD8-independent activity

[0331] Isolation of CD4 from PBMCs by magnetic beads + The purified population was transduced as described above and found to express the eTCR at high levels. Functionally, these cells showed the ability to secrete IFN-γ (2174 pg / mL) in response to target melanoma cells A375 expressing the antigen. These results suggest that CD4 expressing the NY-ESO-1 TCR +T cells can be functionally reactive to melanoma tumor cells, CD8 + The same was true for cells (which secreted 2779 pg / mL IFN-γ under the same experimental conditions).

[0332] Functional affinity

[0333] like Fig. 20 As shown in , T cells expressing 1G4 TCR or the eTCR were co-cultured with T2 cells pulsed with different concentrations of NY-ESO-1 peptide (SEQ ID NO: 21) or with MART-1 peptide (1 μM) as a negative control. After overnight co-culture, the secretion of TNFα in the culture supernatant was measured. The results are shown in Fig. 20 , where TNFα levels (TNFa%) were plotted as a function of log[peptide concentration] and normalized to the levels measured in 1G4 expressing cells at 1 μM concentration. Results are plotted as mean+SEM (n=4).

[0334] As can be seen in Fig. 20 As seen in Figure 2, both cells expressing eTCR and cells expressing 1G4 showed a dose-dependent enhancement of TNFα secretion in the presence of cognate antigen. -6 M to about 1x10 -10 In the case of T2 cells pulsed with NY-ESO-1 peptide concentrations over a range of 1 μM, expression of the eTCR was associated with significantly higher target-induced TNFα secretion compared to cells expressing 1G4. Notably, in the presence of all tested NY-ESO-1 peptide concentrations, an enhancement of TNFα secretion of approximately 3-12 fold, with an average of approximately 6 fold, could be measured.

[0335] Thus, as demonstrated herein, the constructs of the invention were found to be characterized by high functional avidity. Furthermore, the constructs exhibited significant enhancement in functional avidity compared to hitherto known TCRs for the same antigen, with an average of about 6-fold improvement at physiologically relevant antigen levels.

[0336] In summary, transduction of the eTCR into lymphocytes derived from human PBMCs significantly enhanced tumor-specific responses compared not only to an irrelevant TCR (NGFR) but also to a NY-ESO-1 TCR (1G4) directed against the same epitope as the eTCR. Transduction with the novel engineered construct resulted in enhanced secretion of cytokines, major players in anti-tumor immunity, improved functional avidity, and enhanced expression of activation markers, indicating improved function of the eTCR. The results also demonstrated that the eTCR is remarkably capable of exerting tumor-specific reactivity in a CD8-independent manner.

[0337] Example 3 .Subcloning and production of viral vectors for clinical manufacturing

[0338] Essentially as described in Example 1, a stock of the eTCR expression vector for transfection was generated using DH5α E. coli bacteria (ThermoFischer, catalog number 18265-017) and transfected into Phoenix-ECO cells. The ability of the vector to transduce PG13 cells was confirmed and 12 single cell clones were generated. The expression and functional potential of the generated clones and subsequent subclones were studied as described in detail below.

[0339] In order to select clones with high expression of eTCR, the transduction efficiency was evaluated by measuring the expression of the β chain of the eTCR on PBMCs transduced with supernatants derived from each tested clone. To this end, cryopreserved PBMCs from healthy donors were thawed, suspended in culture medium (433 ml RPMI culture medium, 50 ml human serum, 5 ml GlutaMAX-ICTS (100X), 0.5 ml β-mercaptoethanol, 12 ml Hepes and 300 IU / ml IL-2) and washed by centrifugation. The cells were then plated at 2x10 6 The cells were resuspended in culture medium at a concentration of 1 cell / mL and activated for two days in the presence of anti-CD3 antibody (OKT3, 50ng / mL) and IL2. The transduction of the activated cells was performed in a 24-well plate pre-coated with Retronectin and blocked with 2% BSA in PBS. Next, 2mL of PG13-derived viral supernatant was added to the coated wells, centrifuged at 2000G for 2 hours, the supernatant was collected and treated with sodium hypochlorite (to inactivate the remaining retroviral particles). Cells activated from PBMC were added (500,000 cells per well), the plate was centrifuged and incubated at 37°C. The next day, the cells were washed, resuspended in culture medium plated in 6-well plates, and incubated at 37°C for 72 hours. The eTCR expression of the obtained cells was analyzed on the 4th day after transduction.

[0340] Figure 4 Shown are exemplary results of a flow cytometric assay measuring the expression levels of eTCRs corresponding to the initial 12 clones using a PE-conjugated anti-Vβ13 antibody directed against the specific β chain of the eTCR. Figure 4 PG13 cells transduced with medium alone were used as a negative control, and the percentile of Vβ13-PE staining is shown in each graph.

[0341] The selected clones were further subcloned in PG13 cells, and these subclones (all determined to show sufficiently high expression levels as their original clones) were used to transduce lymphocytes to examine their functional efficacy. For IFN-γ secretion assays, the vectors were also titrated and transduced with vectors from selected clones diluted 1:1, 1:2, 1:4 or 1:8. Next, the transduced lymphocytes prepared from thawed healthy donor PBMCs as described in detail above were co-cultured with melanoma A375 cells expressing NY-ESO-1 at a 1:1 effector cell to target cell (E:T) ratio for 24 hours. After cultivation, the secretion of IFN-γ in the supernatant was detected by ELISA. In addition, the killing effect of transduced lymphocytes (using undiluted vectors) on melanoma A375 cells was determined by culturing the transduced lymphocytes with A375 cells at a 1:1 E:T ratio for 1.5 hours. Melanoma cells were stained with the cell tracking marker DDAO, and caspase-3 was used as an indicator of apoptosis. The percentage of caspase-3 and DDAO in the cells was detected by FACS. Representative results of the IFN-γ secretion assay are shown in Figure 5 In the present invention, clones 1.2 and 1.16 are shown at different dilutions (1:1, 1:2, 1:4 or 1:8), undiluted clone 1.3 is used as a positive control, and PG13 cells transduced with medium only without vector (hereinafter referred to as vector-free) are used as a positive control. Figure 5 ) or PG13( Figure 6 )) was used as a negative control. Representative results of caspase-3 measurement are shown in Figure 6 , where “Poly” refers to the original polyclonal eTCR supernatant from the initial transduction with PEG13 before subcloning.

[0342] The results showed that the novel constructs of the present invention promoted the expression of eTCR in an efficient manner, which enabled the transduced cells to exert specific anti-cancer activity. Specifically, clone 1.16 was determined to provide high expression levels, high IFN-γ secretion levels, and high apoptosis induction levels in melanoma A375 cells, as measured by caspase-3 levels, and was therefore selected for further manufacturing of clinical-grade virus master cell banks (MCBs).

[0343] The selected PG13 clones were expanded to produce MCBs (each stock contained an average of at least 300 x 10 6 cells), tested for the absence of mycoplasma and sterility, and the retroviral supernatant was collected and used in the following examples.

[0344] Example 4 Transduction conditions for preparing clinical-grade cell compositions

[0345] Clone 1.16 generated from MCB served as the source of eTCR vectors for the experiments described below as part of the development and production of a GMP clinical grade composition. To determine appropriate transduction conditions, the following experiments were performed.

[0346] Several batches of eTCR supernatants harvested from PG13 cells grown at different temperatures (32°C or 37°C) at different times (12 hours or 24 hours) were diluted differently (1:2, 1:5 or 1:10). PBMCs obtained from apheresis were activated in the presence of anti-CD3 antibody and IL-2, and two days later, the vectors collected in different batches were transduced at different dilutions using a centrifugal seeding method using Retronectin, essentially as described in Example 3. On days 6, 13, and 20 after transduction, eTCR expression and function were assessed as described below.

[0347] Different dilutions of eTCR vector harvests under different transduction conditions were assessed for transduction efficacy using Vβ13-PE antibody and analyzed by FACS essentially as described in Example 3. Table 2 shows exemplary results of eTCR expression levels after transduction, presented as a percentage of Vβ13 expression.

[0348] Table 2 - Changes in eTCR expression levels after transduction with different transduction conditions

[0349]

[0350]

[0351] In Table 2, "No Dilution" indicates undiluted PG13 supernatant obtained from MCB. "No Vector" indicates PG13 supernatant obtained from cells without TCR expression vector. "HVB1" indicates undiluted stock of 1.16 vector derived from MCB used in Example 5. ND - not determined, NA - not available.

[0352] The results showed that MCB-derived eTCRs displayed unexpectedly high transduction efficiencies at 1:2 and 1:5 dilutions under all conditions. The highest transduction efficiencies were detected for eTCR vectors harvested at 24-hour intervals.

[0353] Function and activity were assessed by measuring IFN-γ secretion and caspase-3 levels, essentially as described in Example 3. In brief, transduced cells were co-cultured with A375 cells (expressing NY-ESO-1) at an E:T ratio of 1:1 for 24 hours. After co-culture, supernatants were collected from the cells at days 6, 13, and 20 after transduction, and IFN-γ secretion levels were measured by ELISA. To assess tumor killing ability, transduced PBMCs were co-cultured with A375 cells expressing NY-ESO-1 at an E:T ratio of 1:1 for 1.5 hours. After co-culture, caspase-3 in A375 melanoma cells was stained for FACS detection at days 6, 13, and 20 after transduction as an indicator of apoptosis.

[0354] The results confirmed that the IFN-γ secretion level was significantly higher than that of the negative control, indicating the activity of specific target induction. Different conditions had only a small effect on the secretion level, and the secretion level of the harvest at 24h intervals in the group transduced at 32°C increased slightly. Similar results were obtained by caspase-3 staining assay. Compared with the negative control, the level was higher, indicating the activity of specific target induction, and a decrease in caspase-3 levels was detected on the 20th day after transduction.

[0355] The copy number (CPN) of the eTCR vector in cells transduced with supernatants from 16 different harvests (different harvesting times and culture temperatures) was further determined. PBMCs from two healthy donors were transduced with eTCR vectors and co-cultured with melanoma A375 cells. The copy number was detected using RT-PCR assay. The results are presented in Table 3 as CPN / transduced cells, n=2.

[0356] Table 3 - Average copy number (CPN)

[0357] Harvest / dilution 1:02 1:05 1:10 1 5.51±0.93 5.79±0.55 1.80±2.55 2 4.89±1.56 2.39±0.04 2.23±1.11 3 2.89±0.15 2.65±0.59 2.28±0.81 4 3.75±0.87 2.42±0.37 1.66±1.075 5 2.66±0.59 2.69±0.67 2.40±0.05 6 3.12±0.71 2.75±0.06 2.92±0.79

[0358] As can be seen in Table 3, most of the conditions tested produced CPN values ​​of about 5 CPN or less. Since CPN ≤ 5 is the regulatory standard in clinical trials, it is clear that the eTCR construct derived from the MCB provides high surface expression and sufficient function and activity as described above while meeting regulatory standards.

[0359] The multiplicity of infection (MOI) was calculated using the following formula:

[0360] P(k)=e -m m k / k!

[0361] where "P(k)" is the fraction of cells infected with k viral particles and "m" is the MOI.

[0362] MOI was measured on day 6 after transduction, n = 2. Mean results for different batches ranged from 1.14 ± 0.02 to 1.47 ± 0.06 at 1:2 dilution, from 1.21 ± 0.06 to 1.03 ± 0.12 at 1:5 dilution, and from 0.74 ± 0.02 to 0.94 ± 0.07 at 1:10 dilution.

[0363] Finally, on day 20 after transduction, the presence of replication-competent retrovirus (RCR) was tested in 6 random samples by PCR using primers for GALV (viral envelope protein) as an indicator of RCR and primers for β-actin (βAct) as a positive control. PCR products were run on a 1% agarose gel. The results are presented in Figure 7A-7B middle. Fig. 7A , PCR using primers for GALV (viral envelope protein) as an indicator of RCR. Figure 7B , PCR using primers for β-actin (βAct) as a positive control. It is clear from the results that none of the samples expressed GALV, indicating that RCR was not present in any of the samples tested.

[0364] Based on the experimental results discussed herein, the following transduction conditions were selected for the experiments described in the Examples below: transduction temperature of 32°C, viral particle harvest time of 24 hours, vector dilution of 1:5, and MOI=1.

[0365] Example 5 . Expansion conditions for preparing clinical-grade cell compositions

[0366] Next, various protocols for preparing clinical-grade adoptive cell transfer (ACT) compositions were investigated. To this end, fresh or thawed PBMC samples obtained from healthy donors by apheresis were subjected to various activation and expansion protocols as described in further detail below and summarized in Table 4. Under the conditions described in Example 4, transduction was performed using the eTCR vector after the activation step and before the expansion step.

[0367] The first protocol involves activation with anti-CD3 antibody (OTK3) and IL-2 at the concentrations shown in Table 4 for two days (in complete medium containing RPMI, 10% human serum, glutamine, Hepes and b-mercaptoethanol), and expansion in the presence of increased IL-2 levels and irradiated allogeneic PBMCs as feeder cells. In this protocol, antibodies are not supplemented during the expansion phase, and the only cell stimulants supplemented are IL-2 and feeder cells. During the expansion step, the cell culture medium is gradually changed from complete medium to AIMV (Gibco) containing 5% human serum. This protocol is referred to herein as "anti-CD3+feeder cells". Lymphocytes transduced with vector-free supernatant ("vector-free") are used as negative controls.

[0368] The second and third protocols involved activation and expansion using surface-bound anti-CD3 and anti-CD28 antibodies and IL-2. Specifically, the second protocol used the commercially available product TCell TransAct TM (Milteny), which comprises anti-CD3 and anti-CD28 antibodies coupled to nanoparticles (collectively referred to herein as "TransAct"), and the third scheme uses DynaBeads (Thermo Scientific), using anti-CD3 and anti-CD28 antibodies coupled to superparamagnetic beads. In these schemes, surface-bound antibodies are introduced only in the activation phase, and the expansion step only includes supplementing IL-2 and gradually replacing the culture medium, as described in the first scheme. These schemes are also referred to as "Transact" and "DynaBeads" schemes, respectively.

[0369] Table 4 - Activation and amplification protocols

[0370]

[0371] On days 6, 13 and 20 after transduction, surface expression of eTCR after different activation and expansion protocols was assessed by measuring Vβ13 using flow cytometry essentially as described in Example 3. Exemplary results of two independent experiments (Experiment 1 and Experiment 2) are presented in Table 5 below.

[0372] Table 5 - eTCR expression after different amplification steps

[0373]

[0374] As can be seen in Table 5, all protocols resulted in very high transduction levels with surprisingly persistent retention of surface expression. Throughout the experiment and up to day 20 post-transduction, and in all protocols, eTCR expression levels were high, with the Dynabeads protocol providing the highest levels and only a modest decline by the end of the other protocols.

[0375] Next, the functional capacity of the cells generated by the different protocols was determined by evaluating antigen-induced IFN-γ secretion. To this end, the obtained cells were co-cultured with T2 cells loaded with 1 μg of NY-ESO-1 peptide at an E:T ratio of 1:1 for 24 hours. After co-culture, supernatants were collected from the cells on days 6, 13 and 20 after transduction and the level of IFN-γ secretion was tested by ELISA. Exemplary results are presented in Table 6.

[0376] Table 6 - Target-specific IFN-γ secretion after different activation and expansion protocols. IFN-γ concentrations are shown in fg / transduced cell.

[0377]

[0378] In Table 6, "Exp" indicates the experiment number (corresponding to the experiment described in Table 5 above), "Avg" indicates the mean value, and "SD" indicates the standard deviation, and the production protocol is as detailed in Table 4 above.

[0379] As can be seen in Table 6, target-induced IFN-γ secretion was detected in each of the eTCR-transduced groups, particularly in the cell compositions generated by the Transact protocol and the anti-CD3+ feeder cell protocol.

[0380] To further examine the effects of the activation and expansion protocols on the function of the GMP eTCR cell compositions, the cytotoxic activity of the cells was assessed by detecting apoptosis in homologous target cells. To this end, the cells produced as described above were co-cultured with T2 cells loaded with 1 μg of NY-ESO-1 peptide at an E:T ratio of 1:1 for 1.5 hours. Subsequently, caspase-3 in co-cultured T2 cells was stained at days 6, 13, and 20 after transduction and detected by FACS. Exemplary results are presented in Table 7.

[0381] Table 7 - Expression of caspase-3 in target cells loaded with NY-ESO-1 (%)

[0382]

[0383]

[0384] As can be seen in Table 7, antigen-specific target cell apoptosis was demonstrated in eTCR-transduced cells made according to each of the protocols tested, with particularly strong activity observed in the Dynabeads group.

[0385] In summary, the results presented herein demonstrate that all manufacturing protocols support the production of cell compositions characterized by high and durable eTCR expression and potent anti-tumor activity.

[0386] Next, the presence of specific subsets and phenotypes in the compositions were further characterized. To characterize the different T cell subsets in the cell compositions prepared by the above protocol, the CD3 + Cells were stained with different antibodies against CD45RA and CCR7 and analyzed by flow cytometry. The analysis used a mixture of the following fluorophore-conjugated antibodies: CCR7-rPE, CD45RA-ECD, CD28-PC5.5, CD3-PC7, CD8-APC, CD62L-AF700, CD4-APC-Cy7, and CD27-BV421.

[0387] The staining provides characterization of four groups of T cells as follows: naive T cells (Tn), which express both CCR7 and CD45RA, and three groups of primed (memory) cells, central memory cells (Tn), which express CCR7 but not CD45RA. CM ), effector memory cells lacking expression of both CCR7 and CD45RA (T EM , gray bars) and T cells expressing CD45RA but lacking CCR7 expression EMRA cells (black bars). Figure 8 and Fig. 9 The exemplary results presented in FIG. 2 correspond to experiments 1 and 2, respectively, on day 20 after transduction. Results are presented for each group (T EM or T EMRA ) in CD3 + Percentage of total cells.

[0388] As can be seen in Figure 8-9 As can be seen in Figure 2, the resulting T cells adopted a differentiated effector phenotype, with negligible amounts of naive cells detected on day 20. As can be further seen, T cells generated by anti-CD3+ feeder cells and the Transact protocol mainly adopted a T EM (CD45RA - CCR7 - ) phenotype, T EMRA (CD45RA + CCR7 - ) cell population was smaller, while T cells generated by the Dynabeads protocol showed different phenotypes, T EMRA (CD45RA + CCR7 - ) cells increased.

[0389] In addition, the proportions of different immune cell subsets in various cell compositions were analyzed at days 13 and 20 after transduction. Cells were stained for CD3, CD4, CD8, CD14, CD19, CD83, and CD56 and analyzed by flow cytometry. Figure 10-11 2 presents exemplary results corresponding to day 13 and day 20 of Experiment 2, respectively (similar results were obtained in Experiment 1).

[0390] As can be seen in these figures, the resulting cell composition was dominated by CD3 + Cells with very little CD14 + 、CD83 + or CD19 + Impurities (representing other cells such as macrophages and B cells). It can also be seen that all eTCR-transduced groups surprisingly include a significant proportion of CD4 + Cells and CD8 + It can also be seen that compared with the other groups, CD8 + Cells account for the entire T cell population (CD3 + ) has decreased significantly.

[0391] Finally, qPCR was used to evaluate the copy number analysis of the eTCR vector in the cell compositions obtained from the different protocols of Experiment 1. The copy number was 1.82 for the anti-CD3+ feeder cell protocol, 0.93 for the Transact protocol, and 1.89 for the Dynabeads protocol.

[0392] In summary, the present invention describes the production of a clinical-grade ACT composition expressing a newly designed NY-ESO-1 specific TCR. The composition is characterized by exhibiting improved properties, including but not limited to: exceptionally high and long-term expression of the transgenic gene at low vector copy number, + and CD8 + The cells expressed the desired phenotype and exhibited a high proportion of effector cells, as well as strong antigen-specific activity.

[0393] Based on these results and considering all of the above factors, a CD3+ feeder cell protocol was selected to prepare clinical-grade ACT compositions for examination in the in vivo experiments described in Examples 6-8 below (Compositions 1-3, respectively). Example 8 further describes the in vivo evaluation of ACT compositions produced using other preparation methods as detailed above.

[0394] The characteristics of the ACT compositions produced for in vivo testing are presented in Table 8 below.

[0395] Table 8 - Characteristics of ACT compositions

[0396]

[0397]

[0398] Example 6. In vivo efficacy

[0399] The first in vivo study was conducted in 21 female and 4 male NOD SCID gamma (NSG) mice, each inoculated with 1x10 6 On day 7 after inoculation, the tumor-bearing mice were divided into three experimental groups and infused (adoptively transferred) with the test cell composition or control as follows:

[0400] Group 1 (n=8) received 2x10 7 eTCR-expressing cells (composition 1 in Table 8 above). Group 2 (n=7) was infused with 2x10 7 Group 3 (n=6) of mice were transduced with saline. Blood and spleen samples were analyzed for the presence of transduced cells on day 15 after adoptive transfer (ACT) as described in detail below, followed by assessment of tumor volume and weight.

[0401] In order to evaluate the presence of cells expressing eTCR in blood and spleen samples, CD3 and Vβ13 staining was performed on the leukocytes obtained from the samples, and analyzed by flow cytometry, essentially as described above. In short, spleen lymphocyte samples were prepared by crushing the spleen in 20 mL PBS, centrifuging at 1000 rpm for 6 minutes, and treating the resulting sediment with 5 mL potassium ammonium chloride (ACK) for 5 minutes. In order to prepare blood samples, 100 μl of whole blood was treated with ACK as described above. The obtained cells were washed, and 300,000 cells from each sample (spleen and blood) were used for flow cytometry analysis. To this end, cells were seeded in 96-well plates, centrifuged, stained with an antibody mixture containing Vβ13-specific marker antibodies and CD3-specific marker antibodies, and resuspended in FACS buffer. The results are presented in Table 9 as the percentage of CD3 and Vβ13 positive cells in the total cells treated.

[0402] Table 9 - eTCR expression in mouse blood and spleen 15 days after ACT

[0403]

[0404] As can be seen in Table 9, eTCR +The cells showed remarkable persistence in the blood and spleen of mice, and eTCR expression remained high, with an average of 43% in the blood and 48% in the spleen measured 15 days after the start of ACT.

[0405] To evaluate the effect of treatment on tumor development, mice were sacrificed on day 22 after tumor inoculation and tumors were harvested and examined. Tumor volume measurements are presented in Fig.12 In the figure, A375 tumor volumes determined in cohorts of mice treated with cells transduced with eTCR vectors (black line, n=8), cells transduced with negative control PG13 supernatant (no vector, dashed line, n=7), or saline (no ACT, dotted line, n=6) are presented. Tumor weights are presented in Fig.13 In the middle, means ± SD are indicated.

[0406] As can be seen in Fig.12 and 13 As seen in the figure, compared with the two control groups, the eTCR + Significant reductions in tumor volume and weight were observed in cell-treated mice. Fig.12 A highly significant difference in tumor volume between the eTCR group and the saline-treated group (**p<0.001), as well as a significant difference between the eTCR group and the group treated with no vector cells (*p<0.01) are shown. Fig.13 Significantly reduced tumor weights between the two control groups and the eTCR group are shown (**p<0.01) (all calculated using paired Student's t-test).

[0407] In summary, the results demonstrate that the ACT compositions of the present invention induce a strong tumor-specific response characterized by prolonged persistence of adoptively transferred cells and significant inhibition of tumor progression.

[0408] Example 7 Characterization of in vivo responses

[0409] A second in vivo experiment was performed to evaluate the composition prepared using clinical-grade viral constructs, in which different doses of eTCR + After addition of luciferase or vector-free cells, A375 melanoma cells carrying a luciferase reporter gene were monitored as detailed in Table 10 below.

[0410] For this reason, A375 cells are genetically modified to express luciferase and puromycin resistance genes as reporter proteins. In order to select cells expressing luciferase, before tumor cell inoculation, transfected cells are cultured for two weeks in the presence of 5 μg / mL puromycin. The cells obtained are luminous after being exposed to luciferase substrate luciferin, so after treatment, mice are injected with luciferin to promote the imaging of tumors in vivo. Luciferin (VivoGlo) is used IV in the first 5 minutes of imaging.

[0411] Male NSG mice were administered increasing doses of cells formulated in saline + 2% human albumin intraperitoneally (IP).Because tumor development varies among individual mice, treatment groups were constructed so that mean tumor volumes were similar in all groups.

[0412] Table 10 - Treatment and dosing regimen

[0413]

[0414]

[0415] *Group 6 - The dose is divided into two injections, administered one week apart.

[0416] Three treated mice from each group were sacrificed on day 6 after ACT. Tumors were weighed and fixed in buffered formalin and analyzed by immunohistochemistry (IHC) for the human marker CD3. Organs (heart, lung, kidney, spleen, brain, and liver) were preserved in formalin or snap-frozen and stored at -80°C to examine the presence of adoptively transferred cells by IHC and qRT-PCR, respectively. Various parameters, including antitumor efficacy, eTCR + Persistence of cells in the blood of treated mice, eTCR close to the time of administration (day 6 after ACT) + The tumor infiltration of the cells and their tumorigenic potential during the 4-month follow-up period.

[0417] A. Anti-tumor effect

[0418] Figures 14A-14F Graphs of tumor volumes (calculated based on caliper measurements) in individual mice from test groups 1-6 as detailed in Table 10 are presented separately. The results demonstrate that eTCRs produced in a controlled manner using GMP-grade viral supernatants + The ACT combination was an effective treatment to reduce tumor volume in this NSG melanoma xenograft model. + The combination successfully reduced tumor burden even at the lowest dose examined, which correlated with the absolute number of tumor cells administered, at 56x10 per mouse. 6 eTCR+ Complete inhibition of tumor development was measured at doses of 1.5 wt %.

[0419] Calculate the mean survival rate over time for each test group. Fig.15 Kaplan-Meier survival curves for each group are presented (p<0.01): untreated is shown as tumor only - line with solid diamonds, negative control (no vector cells) - line with a vertical dash on top, eTCR + Cells: 14x10 6 -Line with solid squares, 21x10 6 -Line with solid hexagons, 28x10 6 - Line with solid circles, 56x10 6 - Line with hollow triangle.

[0420] The results revealed a clear dose-dependent antitumor activity, as compared with the vector-free or untreated mice groups, all eTCR + Delayed tumor growth and overall survival were observed in all mice treated with ACT. Notably, the use of 28x10 6 eTCR cells and 56x10 6 The eTCR cell-treated group showed an outstanding 100% survival rate.

[0421] The bioluminescence of luciferase-expressing A375 melanoma cells was monitored until day 34 after inoculation and the results are presented in Fig.16 In the 6th, 13th, 20th, 27th and 34th day, it can be seen that compared with the control, the eTCR + The cell-treated groups showed a reduction or delay in tumor growth, which appeared to be dose-dependent. Of note, tumor growth in most mice in the untreated group reached a volume that necessitated the sacrifice of the mice for ethical reasons before day 34.

[0422] B. Biodistribution, persistence, and characterization of adoptively transferred cells

[0423] Evaluation of eTCR in the blood of NSG tumor-bearing mice at different time points after ACT + To this end, blood samples obtained from mice on days 6, 19, and 33 were stained with FITC-conjugated anti-human CD8 antibodies. + The cell populations were further stained with PE-conjugated anti-Vβ13 antibody and analyzed by flow cytometry. Figures 17A-17B The results show that the 56x10 6 eTCR +The results of staining CD8 and Vβ13 in mice.

[0424] As can be seen, on day 33, after infusion of 56x10 6 eTCR + All CD8 cells circulating in the blood of mice that achieved a complete anti-tumor response + The cells were all positive for eTCR, indicating that the adoptively transferred cells had a striking and significant persistence. In addition, these findings indicate that eTCR + The cells were cloned in vivo, since the initial transduction percentage was 45% under the experimental conditions used. Surprisingly, after receiving the maximum dose of eTCR + In mice with PD-1+ cells, the expression of the activation marker 4-1BB was further detected in human lymphocytes circulating in the peripheral blood of mice.

[0425] Finally, T cell populations in the peripheral blood of mice after ACT were characterized. Blood from different treatment groups was sampled on days 6, 19, and 33 after ACT. Human CD4 + (hCD4 + ) or human CD8 + (hCD8 + ) cells and analyzed by FACS. The results are presented in Table 11, where "2ACT" means that the cells were divided into two 28x10 6 doses, and administered one week apart.

[0426] Table 11 - Characterization of T cells in peripheral blood of mice after ACT

[0427]

[0428]

[0429] The results showed that on days 19 and 33, eTCR + The proportion of human T cells in the peripheral blood of ACT-treated mice increased significantly and continuously over time, demonstrating active expansion of the infused cells.

[0430] In summary, the results of this experiment confirmed that clinical-grade eTCR + The ACT combination demonstrated a dramatic dose-dependent efficacy with prolonged persistence and in vivo clonal expansion over time. The results further demonstrated adequate safety and significantly prolonged survival of treated animals compared to controls.

[0431] Example 8 In vivo evaluation of cell compositions produced by different preparation protocols

[0432] Clinical-grade cell compositions generated by different protocols as described in Example 5 were examined in a xenograft in vivo model essentially as described in Examples 6 and 7. Briefly, luciferase-expressing A375 melanoma cells were implanted into female NSG mice and infused with various ACT compositions summarized in Table 12 below.

[0433] Table 12 - Treatment regimens using cells generated using different expansion protocols

[0434]

[0435] More specifically, the preparation method is described in more detail in Table 4 above, and the specific cell composition administered to Group 3 corresponds to Composition 3 of Table 8.

[0436] Fig.18 and 19 Results for tumor volume and survival rate in different groups are shown separately. Fig.18 In the figure, untreated is shown as tumor only - dotted line with solid circles, no vector - dashed line with solid triangles, Dynabeads is shown as DYna - black line with solid squares, Transact is shown as Trans - black line, anti-CD3-aCD3, black line with solid circles. Fig.19 Kaplan-Meier curves are presented in , where no treatment is shown as tumor only - dashed line, no vector - dotted line, Dynabeads are shown as DYna - thick line, Trans - thin line, and anti-CD3 are shown as feeder cells - squares.

[0437] As can be seen, all production protocols produced ACT compositions that were effective in eradicating tumor growth. + ACT-treated mice survived the entire monitoring period, whereas all mice in the control group died by day 40.

[0438] Finally, the GMP compatibility protocol used in the clinical trial was further evaluated in a GMP-grade facility using healthy donor PBMCs transduced and expanded according to the "anti-CD3+ feeder cell" protocol. The results presented in Table 13 below show a remarkable transduction efficiency in the preparation of the allogeneic ACT composition, averaging about 63% after 13 days of culture, and even higher and more consistent at day 15, reaching a transduction rate of about 70% (average greater than 69%). Therefore, a 15-day expansion period was used in the clinical trial using the autologous ACT composition described below.

[0439] Table 13 - Transduction Rates in Clinical Trial Facilities

[0440]

[0441] *- Average of two samples

[0442] **- Prepare one bag containing the dose of product.

[0443] Example 9 Clinical trials

[0444] A clinical trial was conducted in human patients with metastatic cancer expressing NY-ESO-1, in which autologous PBMCs were obtained from the patients, modified ex vivo to obtain the ACT composition disclosed herein, and then infused back into the patients. The ACT composition and viral source materials were prepared and formulated according to a GMP-compatible protocol essentially as described in the above examples and described in more detail below.

[0445] Briefly, the preparation procedure of the ACT composition comprises the following main steps, wherein the total length of the production procedure (after collecting the source materials) is approximately 14-21 days. These steps are described in more detail below.

[0446] 1. Collect source material (leukocyte apheresis);

[0447] 2. Purify PBMCs by density gradient;

[0448] 3. In vitro stimulation;

[0449] 4. Transduce stimulated cells with retroviral vector;

[0450] 5. amplifying the transduced cells;

[0451] 6. harvesting the cells and preparing a cell composition; and

[0452] 7. Prepare the final formulation (dosage form) for infusion.

[0453] Patients (aged 18 to 70 years) were screened for NY-ESO-1 expression by immunohistochemistry of resected tissue, and those expressing tumor-associated NY-ESO-1 epitopes were identified as eligible for treatment. Screened patients had solid tumors, and additional inclusion criteria were, inter alia, metastatic cancer or locally advanced refractory / recurrent malignancies not amenable to curative therapy, and HLA-A*0201 or A*0206 expression.

[0454] Eligible patients undergo leukapheresis to process approximately twice the patient's total blood volume and collect at least 2.5 x 10 9 After leukocyte apheresis, the collected material was analyzed for cell number and viability, CD3 +The number and percentage of cells, sterility and mycoplasma testing. Then, 50 ng / mL soluble anti-CD3 antibody (OKT3) was used to activate (also referred to as stimulation in this article) the PBMCs collected from the apheresis by density gradient for two days in the presence of 300 IU / mL recombinant IL-2. Optionally, the collected PBMCs were cryopreserved and thawed before stimulation.

[0455] Next, cells activated from PBMCs were transduced with clinical grade PG13 supernatant containing retrovirus encoding eTCR using retronectin, essentially as described in Example 1. Briefly, wells of a 6-well plate were coated with 25 μg / mL Retronectin in 1.5 mL PBS and blocked with 2.5% human albumin in PBS. The Retronectin-coated wells were then coated with PG13 supernatant diluted in medium without IL-2 by centrifugal seeding (including centrifugation at 2000G for 2 hours at 23°C). The cells obtained from the 2-day activation were washed in medium and incubated at 0.5x10 6 The cells were plated at a density of 10 cells / mL in CM containing 300 IU / mL IL-2. The plates were centrifuged at 1000 G for 10 minutes at room temperature. The plates were incubated overnight at 37°C in a 5% CO2 humidified incubator.

[0456] The transduced cells were expanded by culturing with irradiated allogeneic PBMCs used as feeder cells. For this purpose, allogeneic PBMCs were collected under the informed consent procedure, kept frozen before use in the preparation process, and irradiated at 7000 RAD before use. Next, the transduced cells and feeder cells were cultivated in AIMV medium containing IL-2 (3000 IU / mL) at a ratio of 1:50 (transduced cells to feeder cells). After the rapid expansion step, 1×10 9 Up to 1×10 10 Clinical dose of transduced expanded cells.

[0457] To prepare the final GMP cell composition for infusion, the expanded cells were harvested and suspended in 300 ml of 0.9% saline containing 2% human albumin and 300 IU / mL IL-2 to obtain a formulation suitable for intravenous (IV) administration to patients. The resulting ACT composition was then packaged in infusion bags (Macopharma, 600 ml VSE 4001XA) and labeled with patient identifiers, relevant product information, expiration dates, warnings and precautionary statements, including "for autologous use only," according to local regulatory requirements.

[0458] The processes are performed by specially trained personnel according to a series of standard operating procedures (SOPs). A quality management system (QMS) has been established, which monitors all aspects of manufacturing, including raw materials, intermediate and final products (receiving, inspection, testing, handling, labeling, traceability storage and inventory); facilities and equipment (maintenance and calibration); personnel training and responsibilities; batch production SOPs; batch records; packaging, transportation, stability and shelf life.

[0459] At various stages of the manufacturing process, a battery of quality control (QC) tests were performed to assess viability, cell number, identity, purity, percent transduction, potency, replication-competent virus (RCR), mycoplasma, and sterility. Preliminary results for sterility, potency, and percent transduced cells were available on day 6 of manufacturing, prior to initiating the lymphodepletion protocol (day 10).

[0460] Prior to administering the cell composition to the patient, non-myeloablative lymphocyte depletion was initiated 5 or 6 days prior to ACT administration. Depending on the patient's clinical status and bone marrow recovery history, the patient was treated on days 5, 4, and 3 prior to administration according to one of the following two lymphocyte depletion regimens:

[0461] 1. Cyclophosphamide (CYTOXAN) 250 mg / m 2 and fludarabine 25 mg / m 2 .

[0462] 2. Cyclophosphamide 250 mg / m 2 .

[0463] Then 1x10 9 cells to 1x10 10 The cell composition is infused at a dose of 10 cells, and each selected predetermined dose is administered to a single patient. This dose escalation scheme is used to determine the maximum tolerated dose. IL-2 can be infused continuously (e.g., 18x10 cells are administered 24 hours after the ACT infusion). 6 IU / 24h for 5 days or until dose-limiting toxicity is observed).

[0464] Safety and tumor progression are monitored (e.g., by tumor detection imaging and scanning methods and blood profiling). In addition, the presence and persistence of transduced cells in the patient's blood is assessed.

[0465] The details of the five treated patients are summarized in Table 14 below.

[0466] Table 14 - Patient details

[0467]

[0468]

[0469] Evaluation of the resulting ACT composition indicated the presence of CD4 + and CD8 + eTCR + Both cells, CD8 + Cells and CD4 + The proportion of cells with eTCR + In addition, as detailed above, even when using PBMCs from immunocompromised or otherwise compromised patients, transduction efficiency can be maintained at an average of about 53% (a rate significantly higher than the regulatory requirement of >10%) and can be increased to as high as about 82% by increasing the construct copy number (CPN) per transduced cell.

[0470] Prior to treatment, the functionality and efficacy of the ACT composition was assessed by measuring the secretion of IFN-γ following target recognition, essentially as described in detail in Example 3. Briefly, activated, transduced or untransduced PBMCs were co-cultured with T2 cells loaded with NY-ESO-1 peptide or unloaded at a 1:1 effector cell to target cell (E:T) ratio for 24 hours. After culture, IFN-γ secretion in the supernatant was detected by ELISA (on day 6 after activation). Exemplary results for an ACT composition prepared for Patient 1 (uveal melanoma, previously treated with a combination of PD-1 and CTLA4 checkpoint inhibitors) are presented in Fig.21 In the figure, nonspecific T2 cells cultured with unloaded T2 cells are represented as "T:T2", nonspecific T cells cultured with T2 cells loaded with peptides are represented as "T:T2-ESO", and eTCR cells cultured with T2 cells are represented as "T:T2-ESO". + T cells are denoted as "eTCR + :T2", eTCR cultured with T2 cells loaded with peptide + T cells are called "eTCR + :T2-ESO".

[0471] As can be seen in Fig.21 As seen in the eTCR +The ACT compositions secreted high levels of IFN-γ upon specific recognition of the NY-ESO-1 peptide, with no detectable secretion observed in any of the control groups. In addition, the measured IFN-γ levels of ACT compositions prepared from autologous PBMCs of cancer patients previously treated with chemotherapy or immunomodulatory therapy were found to be comparable to those of ACT compositions prepared from PBMCs of healthy donors using the constructs of the present invention, indicating potent anti-tumor activity as described in Example 2 of the autologous ACT model in a clinical setting (and an improvement over compositions reported to date).

[0472] Furthermore, the ACT composition was well tolerated and exhibited a remarkable safety profile with no serious adverse events associated with the transferred cells.

[0473] Monitoring of eTCR in the blood of treated patients at different time points after ACT + To this end, PBMCs obtained from each test patient were stained with FITC-conjugated anti-human CD3 antibody. + The cell populations were further stained with PE-conjugated anti-Vβ13 antibody and analyzed by flow cytometry. Figures 22A-22D The accepted 1x10 9 or 5x10 9 eTCR + Cells expressing eTCR in PBMCs of patients 1 to 4 at the ACT dose + CD3 + as shown in Table 14.

[0474] As can be seen in Figures 22A-22D As seen in Figure 2, high levels of eTCR expression were detected in all patients after ACT. + CD3 + cells, which gradually stabilized to remain in the range of 3-10% of PBMCs. Notably, eTCR expression can be detected in peripheral blood for a long time. + CD3 + cells, even at the latest time point monitored in each patient, which was up to 9 months after the start of ACT ( Fig.22A ). These results indicate that the transduced cells adopted a highly durable phenotype.

[0475] In summary, the results show that ACT compositions can be prepared not only from the blood of healthy donors, but also from PBMCs of patients with refractory solid tumors who have failed to respond to treatment with chemotherapy or immunotherapy. These patients are usually immunocompromised due to the presence of tumors and / or previous treatments, but are still suitable for the preparation of ACT compositions according to the present invention. The results further demonstrate that the obtained ACT compositions have significant in vivo safety and persistence in peripheral blood; in contrast to many compositions reported to date with persistence of up to 30 days, the compositions according to the present invention can even be detected in the peripheral blood of patients for up to 1.5-9 months. Finally, in vitro evaluation of the functional capacity of the composition showed that it was able to exert improved anti-tumor activity.

[0476] Example 10 .eTCR + CD4 + Characterization of cells

[0477] As described in detail below, eTCR + CD4 + The function of the cells (CD8-independent activity) was further evaluated and characterized. CD4 + Cells. Two days before transduction with eTCR viral vectors, the resulting cells were activated with anti-CD3 and anti-CD28 antibodies (OKT3 and CD28.2 clones, from Invitrogen and Biologend, respectively, at a concentration of 1 μg / ml). After transduction, the cells were maintained in a medium containing 300 IU / ml IL-2 for 9 days until target-induced cytokine secretion was determined.

[0478] IFN-γ secretion was assessed as described in Example 3. Briefly, transduced cells were cultured with or without A375 melanoma target tumor cells (expressing the NY-ESO-1 target antigen). The cells were cultured for 24 hours at a 1:1 effector cell:tumor cell ratio (100,000 cells per population). After incubation, IFN-γ levels secreted into the culture supernatant were measured by ELISA. The results are presented in Fig.23 In the figure, cells incubated with A375 melanoma target tumor cells are denoted as "+A375" and control cells incubated in the absence of target cells are denoted as "-A375".

[0479] The results showed that in the presence of target tumor cells expressing NY-ESO-1, eTCR + CD4 +The cells were able to secrete high levels of IFN-γ, whereas in the absence of tumor cells, no detectable levels of IFN-γ were measured. These results indicate that eTCR not only plays a role in the CD4 + T cells (eTCR + CD4 + ) are detectable on the surface, and the constructs and protocols employed enable the eTCR to be expressed in a functional manner.

[0480] Next, the CD4 + The cells were further evaluated for phenotype. CD107 (lysosomal associated membrane protein-1, LAMP-1) is a cell surface protein involved in the activation of CD8 + Degranulation process of T cells and NK cells. As detailed above, cells were incubated for 6 hours in the presence or absence of A375 target cells. After incubation, CD107, CD4 and eTCRβ chain (Vb13.3) were stained with differentially labeled antibodies and measured by FACS. The results after incubation in the presence or absence of A375 melanoma cells are presented in Fig.24A and 24B middle.

[0481] As can be seen in Fig.24A As seen in Figure 2, even in the absence of target cells, eTCR + CD4 + The cells also surprisingly showed significant levels of CD107 expression (5.48%), while the gated eTCR - Less than 1% of cells showed surface expression of CD107. Fig. 24B As seen in Figure 2, in the presence of target cells expressing NY-ESO-1, eTCR + CD4 + The surface expression of CD107 in the cells was further enhanced by about three times to 17.1%, indicating an enhanced target specificity of the cytotoxic effector marker CD107. + Similar results were obtained when cells were transduced and co-cultured with tumor cells, where the cells were specifically gated for CD4 + CD107 levels on cells were assessed.

[0482] CD107 is considered a T cell activation and cytotoxicity marker, where its expression level serves as an indicator of T cell activation and effector function (e.g., degranulation) in cytotoxic lymphocytes.+ cells (such as Th1 and Th2 cells) and only after activation adopt CD4 + Thus, without being limited to a particular theory or mechanism of action, the results suggest that eTCR + CD4 + Cells adopt a cytotoxic effector phenotype, which is not usually CD8 - Cells (such as helper (CD4 + )T cells). It was further found that among the total CD4 + eTCR + In, CD8 - Effector cell population (CD4 + eTCR + CD107 + cells, and CD4 + The incidence of CTL phenotype consistency is about 15%.

[0483] In summary, the results show that the constructs and protocols of the present invention provide a + and CD4 + eTCR + Production of cell composition of cell populations, and the CD4 + eTCR + cells were able to exert CD8-independent NY-ESO-1-specific activities (which can also be considered as MHC II-independent). In addition, the results showed that the transduced CD4 + eTCR + The cells adopted a unique phenotype characterized by surface expression of CD107, demonstrating the potential to enhance the overall cytotoxic activity of the ACT composition.

[0484] The description of the above specific embodiments will fully reveal the general nature of the present invention, so that others can easily modify and / or adapt these specific embodiments to adapt to various applications by applying current knowledge without excessive experimentation and without departing from the general concept, and therefore, such adaptations and modifications should and are intended to be understood within the equivalent meaning and scope of the disclosed embodiments. It should be understood that the phrases or terms used herein are for the purpose of description rather than limitation. Without departing from the present invention, the means, materials and steps for performing the various disclosed functions can take various alternative forms.

Claims

1. An isolated T cell receptor (TCR) against NY-ESO-1, comprising: (a) a TCR alpha chain comprising: i. a variable region (VR) comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3; ii. a constant region (CR) comprising a cysteine ​​residue at position 47 and an amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254; (b) a TCR β chain comprising: i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and ii. CR comprising a cysteine ​​residue at position 57; and (c) multiple interchain disulfide bonds between the α and β chains.

2. The isolated TCR according to claim 1, comprising a first disulfide bond between the cysteine ​​residues at position 94 of the α chain CR and at position 131 of the β chain CR, and a second disulfide bond between the cysteine ​​residues at position 47 of the α chain CR and at position 57 of the β chain CR.

3. The isolated TCR of claim 1, wherein the transmembrane region of the α chain has the amino acid sequence LLVIVLRILLLKVAGFNLLMT (SEQ ID NO: 23).

4. The isolated TCR according to claim 1, comprising a TCR alpha chain having the amino acid sequence set forth in SEQ ID NO: 17, optionally excluding the signal peptide at positions 1-20 thereof.

5. The isolated TCR according to claim 1 or 4, comprising a TCR beta chain having the amino acid sequence set forth in SEQ ID NO: 18, optionally excluding the signal peptide at positions 1-21 thereof.

6. An isolated TCR according to claim 1, wherein each of the α and β chains exhibits at least 95% sequence identity to SEQ ID NOs: 17 and 18, respectively, and wherein the TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence SLLMWITQC (SEQ ID NO: 21).

7. A nucleic acid construct encoding the isolated TCR according to any one of claims 1-6.

8. The nucleic acid construct according to claim 7, comprising the nucleic acid sequences set forth in SEQ ID NOs: 7-9 encoding CDR1, CDR2 and CDR3 of the α chain, respectively, and the nucleic acid sequences set forth in SEQ ID NOs: 10-12 encoding CDR1, CDR2 and CDR3 of the β chain, respectively.

9. The nucleic acid construct according to claim 8, comprising a first nucleic acid molecule encoding the α chain and having the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the β chain and having the nucleic acid sequence set forth in SEQ ID NO:

20.

10. The nucleic acid construct according to any one of claims 7 to 9, wherein the first and second nucleic acid molecules are operably linked to one or more transcription control elements.

11. The nucleic acid construct of claim 10, wherein the one or more transcriptional control elements are capable of inducing or enhancing the expression of the TCR chain in human lymphocytes.

12. The nucleic acid construct of claim 7, comprising a first nucleic acid molecule encoding the α chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the β chain and having at least 95% identity to the nucleic acid sequence set forth in SEQ ID NO: 20, wherein the encoded TCR is capable of specifically binding to an epitope presented by HLA-A2 comprising the amino acid sequence of SEQ ID NO:

21.

13. The nucleic acid construct of claim 7, wherein the encoded TCR is capable of exerting tumor-specific reactivity in a CD8-independent manner.

14. The nucleic acid construct of claim 7, which is capable of providing enhanced expression levels and / or duration of the encoded TCR compared to a construct encoding a naturally occurring TCR directed against the epitope set forth in SEQ ID NO: 21 presented by HLA-A2.

15. according to the nucleic acid construct described in any one in claim 7-14, wherein said first nucleic acid molecule and said second nucleic acid molecule are connected through the third nucleic acid molecule encoding a joint. The nucleic acid construct according to claim 15 , wherein the linker is a 2A peptide.

17. A nucleic acid construct comprising at least one nucleic acid molecule selected from the group consisting of SEQ ID NOs: 19 and 20.

18. An expression vector comprising the nucleic acid construct according to any one of claims 7 to 17.

19. A host cell comprising the vector according to claim 18.

20. A pharmaceutical composition comprising a therapeutically effective amount of the vector according to claim 18.

21. A cellular composition for cancer immunotherapy, comprising a therapeutically effective amount of immune cells engineered to express a TCR against NY-ESO-1, wherein the TCR comprises: (a) a TCR alpha chain comprising: i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3; ii. CR comprising a cysteine ​​residue at position 47 and the amino acid sequence set forth in SEQ ID NO: 22 at positions 250-254; (b) a TCR β chain comprising: i. VR comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6; and ii. CR comprising a cysteine ​​residue at position 57; and (c) multiple interchain disulfide bonds between the α and β chains.

22. The cell composition of claim 21, wherein the immune cells are a cell population containing T cells, and the composition is adapted for adoptive transfer therapy (ACT).

23. A cell composition according to any one of claims 21-22, wherein the TCR comprises a TCR alpha chain having an amino acid sequence set forth in SEQ ID NO: 17, optionally excluding a signal peptide at positions 1-20 thereof, and a TCR beta chain having an amino acid sequence set forth in SEQ ID NO: 18, optionally excluding a signal peptide at positions 1-21 thereof.

24. The cell composition of claim 23, wherein the nucleic acid construct comprises a first nucleic acid molecule encoding the α chain and having the nucleic acid sequence set forth in SEQ ID NO: 19, and a second nucleic acid molecule encoding the β chain and having the nucleic acid sequence set forth in SEQ ID NO:

20.

25. The cell composition according to any one of claims 21-24, prepared by a method comprising the steps of: a. providing a cell population containing T cells, b. culturing the cell population in the presence of a CD3-specific antibody and interleukin-2 (IL-2), c. engineering the cells obtained from step b. to express the TCR, d. expanding the cells obtained from step c. so as to obtain a therapeutically effective amount of expanded engineered cells, and e. Harvesting the expanded engineered cells obtained from step d.

26. The cell composition according to claim 25, wherein the T cell-containing cell population provided in step a. is a peripheral blood mononuclear cell (PBMC) population.

27. The cell composition of claim 25, wherein step b. further comprises culturing the cells with a CD28-specific antibody, and the CD3-specific antibody and the CD28-specific antibody are surface-bound.

28. The cell composition according to claim 25, wherein step d. is performed by incubating the cells after step c. with IL-2 and optionally feeder cells.

29. The cell composition of claim 28, wherein step d. is performed without supplementation with antibodies.

30. The cell composition according to claim 25, wherein step d. is performed to obtain at least 10 9 CD3 + eTCR + cell.

31. The cell composition of claim 25, wherein step e. is performed within 7-21 days after starting step b.

32. A method for preparing a cell composition for ACT, the method comprising: a. providing a cell population containing T cells, b. culturing the cell population in the presence of CD3-specific antibodies and IL-2, c. engineering the cells obtained from step b. to express a TCR according to any one of claims 1 to 6, d. expanding the cells obtained from step c. to obtain a therapeutically effective amount of expanded engineered cells, and e. Harvesting the expanded engineered cells obtained from step d.

33. The method of claim 32, comprising: a. Provide PBMC population, b. culturing the cell population in the presence of CD3-specific antibodies and IL-2, c. engineering the cell to express the TCR by incubating the cell with a viral vector comprising a nucleic acid construct encoding the TCR, d. Expand the cells after step c. in the presence of IL-2 and feeder cells without supplementing antibodies to obtain at least 10 9 CD3 expressing the TCR + cells, and e. Harvest the expanded cells within 14-15 days after starting step b.

34. The method according to any one of claims 32-33, wherein step b. comprises administering to the cell an expression vector according to claim 18.

35. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a cellular composition according to any one of claims 21-31.

36. The method of claim 35, wherein the cells of the composition are autologous cells of the subject.

37. The method of claim 35, wherein the subject has a tumor that expresses NY-ESO-1.

38. The method of claim 37, wherein the subject is HLA-A2 positive.

39. The method of claim 35 or 38, wherein the tumor is selected from the group consisting of melanoma, myeloma, bladder, ovarian, lung, breast, synovial, and prostate tumors.

40. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 20.

41. The method of claim 40, wherein the subject has a tumor expressing NY-ESO-1, and / or wherein the subject is HLA-A2 positive.

42. The method of claim 41, wherein the tumor is selected from the group consisting of melanoma, myeloma, bladder, ovarian, lung, breast, synovial, and prostate tumors.

43. A method of enhancing anti-tumor immunity in a subject in need thereof, the method comprising administering to the subject the cell composition of claim 21 or the pharmaceutical composition of claim 20.

44. The method of claim 43, wherein the subject has a tumor that expresses NY-ESO-1, and / or wherein the subject is HLA-A2 positive.

45. The method of claim 44, wherein the tumor is selected from the group consisting of melanoma, myeloma, bladder, ovarian, lung, breast, synovial, and prostate tumors.

46. ​​A cellular composition according to any one of claims 21-31 for use in treating cancer in a subject in need thereof.

47. The cell composition for use according to claim 46, wherein the subject suffers from a tumor expressing NY-ESO-1, and / or wherein the subject is HLA-A2 positive.

48. A cell composition for use according to claim 46 or 47, wherein the tumor is selected from the group consisting of melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors.

49. A cell composition for use according to any one of claims 46-48, wherein the cells of the composition are autologous cells of the experimenter.

50. The pharmaceutical composition of claim 20, for use in treating cancer in a subject in need thereof.

51. The composition for use according to claim 50, wherein the subject suffers from a tumor expressing NY-ESO-1, and / or wherein the subject is HLA-A2 positive.

52. A composition for use according to claim 50 or 51, wherein the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors.

53. A cellular composition according to any one of claims 21-31 for use in enhancing anti-tumor immunity in a subject in need thereof.

54. The cell composition for use according to claim 53, wherein the subject suffers from a tumor expressing NY-ESO-1, and / or wherein the subject is HLA-A2 positive.

55. A cell composition for use according to claim 53 or 54, wherein the tumor is selected from the group consisting of melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors.

56. A cell composition for use according to any one of claims 53-55, wherein the cells of the composition are autologous cells of the subject.

57. The pharmaceutical composition of claim 20, for use in enhancing anti-tumor immunity in a subject in need thereof.

58. The composition for use according to claim 57, wherein the subject suffers from a tumor expressing NY-ESO-1, and / or wherein the subject is HLA-A2 positive.

59. A composition for use according to claim 57 or 58, wherein the tumor is selected from melanoma, myeloma, bladder, ovarian, lung, breast, synovial and prostate tumors.

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