A chimeric human CD95 switch receptor, T cells expressing the receptor together with a modified T cell receptor, their respective vectors, kits, pharmaceutical compositions, and a method for treating a patient with a disease.

Abstract

The present invention relates, in particular, to T cells expressing a polypeptide-containing chimeric CD95 receptor, wherein the polypeptide comprises at least one CD95-derived polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 wild-type receptor, the human CD95 polypeptide region comprises a CD95 extracellular ligand-binding domain, and further comprises at least one non-CD95-derived costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the T cells further express the modified T cell receptor.

Description

Technical Field

[0001] (Cross - reference to related applications) This application claims priority based on European Patent Application No. 23193416.7 filed on August 25, 2023, the content of which is hereby incorporated herein by reference in its entirety.

[0002] (Technical Field) The present invention relates to isolated T cells that express a chimeric CD95 receptor comprising a polypeptide, the polypeptide comprising at least one CD95 polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the wild - type human CD95 receptor, the human CD95 polypeptide region comprising the CD95 extracellular ligand - binding domain. The present invention also relates to vectors comprising nucleic acids encoding the chimeric CD95 receptor and isolated T cells into which the vectors have been introduced. Further, the present invention relates to kits for preparing the (isolated) T cells of the present invention and pharmaceutical compositions comprising the T cells. The present invention relates to methods of modulating T cells for immunotherapy, methods of treating a patient having a disease comprising administering a pharmaceutical composition, and / or methods of increasing the cytotoxicity of T cells in adoptive cell therapy comprising introducing a vector into the T cells. Further, the present invention relates to a chimeric CD95 receptor comprising a polypeptide, the polypeptide comprising at least one CD95 polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the wild - type human CD95 receptor, the human CD95 polypeptide region comprising the CD95 extracellular ligand - binding domain, and further, the polypeptide comprising at least one domain, region or motif of a non - CD95 co - stimulatory cytoplasmic polypeptide.

Background Art

[0003] T cells are known to be an important vehicle for adaptive cell-mediated immune responses. Adoptive T cell therapy (ACT) using T cells expressing native or transgenic αβ T cell receptors (TCRs) is a promising treatment for cancer because TCRs cover a wide range of potential target antigens [Chandran and Klebanoff, 2019]. Native TCR specificity has been successfully utilized for ACT using tumor-infiltrating lymphocytes (TILs) for melanoma [Dafni et al., 2019] and other tumors [Chandran and Klebanoff, 2019], or virus-specific T cells (VSTs) for virus-associated malignancies [Leung and Heslop, 2019]. Transgenic TCR-based ACTs can genetically redirect T cell specificity in a highly specific and reproducible manner, and have shown promising results in melanoma and several solid tumors [Robbins et al., 2015], multiple myeloma (MM) [Rapoport et al., 2015], as well as virus-associated malignancies [Doran et al., 2019] and acute myeloid leukemia (AML) [Chapuis et al., 2019]. Another promising option in ACT is chimeric antigen receptor (CAR)-T cell therapy, which has produced significant clinical responses in certain subsets of B-cell leukemia or lymphoma [Sterner and Sterner, 2019]. Promising results have also been reported in multiple myeloma.

[0004] T cell antigen recognition and subsequent T cell activation are known to depend on the interaction between T cell receptors (TCRs) and peptides (major histocompatibility complex (pMHC) molecules) [Davis and Bjorkman, 1988]. In particular, the CD8 coreceptor plays a major role in CD8 T cell activation, while the CD4 coreceptor stabilizes the interaction between the TCR in CD4 T cells and MHC class II molecules in antigen-presenting cells (APCs). Recently, in adoptive therapy experiments, the effect of highly binding active CD4 T cells in providing protective tumor immunity was reported to be similar to the therapeutic effect observed with CD8 T cells. Specifically, co-transfer of class ITCR and CD8 coding genes has been described as generating highly binding active CD4 T cells [Xue et al., 2013].

[0005] Furthermore, in addition to antigens, T cells need to receive positive signals to trigger an effective immune response. Co-signaling molecules are known to play important roles in regulating T cell activation, subset differentiation, effector function, and survival. For example, CD28 is constitutively expressed in native CD4 and CD8 T cells and has been shown to act as a positive co-stimulatory molecule. The involvement of CD28 in immunological synapses reduces the amount of antigen required to trigger T cell activation [Kamphorst et al., 2015].

[0006] In addition to CD28, many other co-stimulatory molecules have been identified in recent years. Many co-signaling molecules are members of the immunoglobulin superfamily (IgSF) and the tumor necrosis factor receptor superfamily (TNFRSF). For example, TNFRSF co-signaling receptors with co-stimulatory function include HVEM (herpesvirus entry medium), death receptor 3 (DR3; also known as TNFRSF25), CD40 (also known as TNFRSF5), and lymphotoxin β receptor (LTBR; also known as TNFRSF3) [Chen and Flies, 2013]. Furthermore, V-type receptors, i.e., all branched receptor families (including 4-1BB (also known as CD137 or TNFRSF9), OX40 (also known as TNFSF4), CD27 (also known as TNFRSF7), glucocorticoid-induced TNFR-related protein (GITR; also known as TNFRSF18), and CD30 (also known as TNFRSF8)), also primarily function as costimulatory molecules [Croft et al., 2012]. For example, in addition to CD28, IgSF co-signaling receptors with costimulatory function include, for example, costimulatory receptor-induced T cell costimulatory molecule (ICOS), CD226, CRTAM, TIM1, CD2, SLAM, CD84, Ly9, and CRACC [Chen and Flies, 2013].

[0007] Furthermore, other receptor families may also play a role in T cell costimulation. For example, although Toll-like receptors (TLRs) are highly expressed by innate immune cells, particularly antigen-presenting cells, the earliest reports on human TLRs also described their expression and function within T cells. By acting directly on T cells, TLR agonists can enhance cytokine production by activated T cells, increase T cell sensitivity to T cell receptor stimulation, promote long-term T cell memory, and reduce the inhibitory activity of regulatory T cells.

[0008] While progress has been made in the development of specific ACTs targeting tumor cell-specific antigen genes, several challenges remain for ACTs, including tumor heterogeneity, antigen evasion, T cell migration, and the immunosuppressive tumor microenvironment. For example, solid tumors can cleverly evade expected immune responses, including T cell therapy, by expressing various inhibitory molecules that can inhibit T cell function. For instance, while it is mentioned above that receptors in the immunoglobulin superfamily (IgSF) and tumor necrosis factor receptor superfamily (TNFRSF) are known to have co-stimulatory functions, some family members are known to be bound by inhibitory molecules (for example, in the immunosuppressive tumor microenvironment) and transmit inhibitory effects to T cells. Within IgSG, for example, PD1, TIGIT, and TIM-3 are described as transmitting inhibitory signals from solid tumors that may inhibit activation and / or promote T cell exhaustion. Within TNFRSF, for example, CD95 (Fas receptor), when bound by its ligand CD95L (also known as FASL), is known to transmit inhibitory signals that inhibit T cell activation, promote exhaustion, and / or induce apoptosis.

[0009] Recently, it has been reported that fusing specific costimulatory domains, particularly the 4-1BB costimulatory domain, to the CD95 receptor can enhance proliferative signaling, proliferation, antitumor function, and altered metabolism in vivo [Oda SK et al., 2020]. However, considering the diversity of the immunosuppressive tumor microenvironment and the large number of related agents that can positively or negatively suppress the suppressive activity of T cells in adoptive cell therapy, the difficult challenge of providing effective T cells that exhibit sufficient cytotoxicity, especially in the immunosuppressive tumor microenvironment, remains.

[0010] Therefore, the object of the present invention is to improve the above-mentioned inconvenience. [Overview of the project]

[0011] This objective is achieved, in particular, by (isolated) T cells, vectors, pharmaceutical compositions, kits, methods, and chimeric CD95 receptors, each possessing the features of an independent claim.

[0012] In a first embodiment, the present invention provides isolated T cells expressing a polypeptide-containing chimeric CD95 receptor, wherein the polypeptide comprises at least one CD95(derived) polypeptide region having at least 60% sequence identity with the polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in SEQ ID NO: 1, the CD95(derived) polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprising at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the T cells further express a modified T cell receptor.

[0013] In a second embodiment, the present invention provides isolated T cells expressing a polypeptide-containing chimeric CD95 receptor, wherein the polypeptide comprises one CD95(derived) polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprises at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the T cells further express the modified T cell receptor.

[0014] The inventors have found that T cells containing both a chimeric CD95 receptor and a modified T cell receptor, which are thus modified for use as a switch receptor, can, for example, convert negative signals present in the tumor microenvironment into positive signals for T cell activation. This is advantageous because the T cells provided herein express both the modified T cell receptor and the specifically modified recombinant chimeric CD95 receptor, thereby combining the costimulatory domain described herein with at least one CD95-derived polypeptide region that remains capable of binding to its native ligand. The T cells provided herein exhibit relatively low TCR-T exhaustion and apoptotic loss, and show stimulated TCR-T proliferation and functional activity. Therefore, the expression "region of tumor necrosis factor receptor superfamily proteins" as used herein may refer to a protein portion that retains the ability to propagate co-stimulatory signaling of functional co-stimulatory tumor necrosis factor receptor superfamily proteins similar to wild-type tumor necrosis factor receptor superfamily proteins. The expression "motif of tumor necrosis factor receptor superfamily proteins" as used herein may refer to a protein portion that is a recognizable region of the protein structure, which may be defined by a specific chemical or biological function and also retains the ability to propagate co-stimulatory signaling of functional co-stimulatory tumor necrosis factor receptor superfamily proteins similar to wild-type tumor necrosis factor receptor superfamily proteins.

[0015] The T cells provided herein contain a chimeric CD95 switch receptor lacking the cytoplasmic repressive motif / domain / region of the wild-type CD95 receptor. Therefore, for example, in the tumor microenvironment, the binding of CD95L to the CD95 switch receptor does not lead to, for example, inhibition of activation, promotion of exhaustion, and / or induction of apoptosis of T cells expressing the chimeric CD95 switch receptor, but instead co-stimulates the T cells, thereby enhancing their cytotoxic effects.

[0016] In a third embodiment, the present invention provides a vector comprising a nucleic acid comprising a nucleic acid sequence encoding a chimeric CD95 receptor comprising a polypeptide, wherein the polypeptide comprises at least one CD95(derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in SEQ ID NO: 1, wherein the human CD95 polypeptide region comprises a CD95 extracellular ligand-binding domain, further comprising at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the vector further comprises a nucleic acid comprising a nucleic acid sequence encoding a modified T cell receptor.

[0017] In a fourth embodiment, the present invention provides a vector comprising a nucleic acid comprising a nucleic acid sequence encoding a chimeric CD95 receptor comprising a polypeptide, wherein the polypeptide comprises a CD95 (derived) polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprises at least one non-CD95 (derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the vector further comprises a nucleic acid comprising a nucleic acid sequence encoding a modified T cell receptor.

[0018] In some embodiments, in addition to comprising the nucleic acid sequence encoding the chimeric CD95 receptor and the nucleic acid sequence encoding the modified T cell receptor, the vector may further comprise a nucleic acid encoding a CD8 coreceptor, such as a wild-type CD8 coreceptor or a chimeric CD8 coreceptor.

[0019] For example, overexpression of the chimeric human CD95 receptor described herein in T cells provided herein, in parallel with a modified transgenic αβ T cell receptor TCR, and in some embodiments, in parallel with, for example, a CD8 coreceptor, may provide several advantages of this method and expand therapeutic possibilities.

[0020] The present invention optionally provides a CD8 coreceptor along with the chimeric CD95 switch receptor and modified T cell receptor in T cells, which may enable the effective introduction of, for example, CD4 cells into TCR-T cell therapy in a way that enhances the anti-tumor immune response by utilizing their unique properties.

[0021] For example, CD4 cells have the ability to control the functions of other immune cells such as CD8 cytotoxic T cells, dendritic cells, macrophages, and B cells by providing biological signals through cytokine secretion and direct cell-to-cell interactions. This known helper function may be important for enhancing the persistence and efficacy of TCR-T cells in the tumor microenvironment.

[0022] In a fifth aspect, the invention provides an isolated T cell, wherein the T cell comprises the vector of the invention.

[0023] In a sixth aspect, the invention provides an isolated T cell, into which the vector of the invention has been introduced (e.g., transfected, transduced, or transformed).

[0024] In a seventh aspect, the invention provides an isolated T cell, which has been subjected to treatments such as transfection, transduction, or transformation to express the chimeric CD95 receptor described herein together with the modified T cell receptor.

[0025] In an eighth aspect, the invention provides a kit comprising means for preparing the isolated and / or modified T cells of the invention.

[0026] In a ninth aspect, the invention provides a pharmaceutical composition comprising the isolated T cells of the invention.

[0027] In a tenth aspect, the invention provides a method for preparing T cells for immunotherapy, the method comprising: - isolating T cells from a human subject, - introducing the vector provided herein into the T cells, - and expanding the T cells into which the vector has been introduced.

[0028] In an eleventh aspect, the invention provides a pharmaceutical composition comprising T cells expressing the chimeric CD95 receptor and the modified T cell receptor described herein.

[0029] In a twelfth aspect, the present invention provides a method for treating a patient having a disease, the method comprising administering a pharmaceutical composition of the present invention to the patient.

[0030] In a thirteenth aspect, the present invention provides a method for treating a patient having a disease, the method comprising introducing the vector provided herein into the T cells of the patient in vivo.

[0031] In a fourteenth aspect, the present invention provides a method for increasing the cytotoxicity of T cells in adoptive cell therapy, the method comprising introducing a vector into the T cells, the vector comprising a nucleic acid encoding a chimeric CD95 receptor as described herein and further encoding a modified T cell receptor.

[0032] In a 15th aspect, the present invention provides a chimeric CD95 receptor comprising a polypeptide, wherein the polypeptide comprises at least one CD95(derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in SEQ ID NO: 1, wherein the human CD95 polypeptide region comprises a CD95 extracellular ligand-binding domain, and further comprises at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif, wherein the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is selected from the group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

[0033] In a 17th aspect, the present invention provides a chimeric CD95 receptor comprising a polypeptide, wherein the polypeptide comprises at least one CD95(derived) polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprises at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif, wherein the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is selected from the group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

[0034] These chimeric CD95 receptors have been shown to, for example, convert negative signals present in the tumor microenvironment into positive signals for T cell activation. The chimeric CD95 receptors provided herein are advantageous in conferring resistance to the immunosuppressive tumor microenvironment to T cells, preventing TCR-T exhaustion and apoptosis loss, and stimulating TCR-T proliferation and functional activity by binding a specific costimulatory cytoplasmic polypeptide domain / region / motif of CD40L, CD2, TLR2, TLR4, or IL6R subunit beta to at least one CD95-derived polypeptide region that is still capable of binding to a native ligand.

[0035] In further embodiments, the present invention provides vectors and T cells comprising a chimeric CD95 receptor according to the thirteenth embodiment. Furthermore, as described herein, the chimeric CD95 receptor according to the thirteenth embodiment may be used in the compositions, kits, and methods of the present invention.

[0036] All aspects of the present invention provide the above advantages and improvements in relation to providing T cells comprising both a modified T cell receptor and a chimeric CD95 receptor, the chimeric CD95 receptor comprising the fusion of a specific costimulatory cytoplasmic domain of a specific non-CD95 (derived) polypeptide described herein to a human CD95 receptor polypeptide region comprising a functional CD95 extracellular ligand-binding domain, thereby constituting a switch receptor capable of converting a negative signal into a positive signal (e.g., with respect to the activation state and / or cytotoxic performance of a T cell). [Brief explanation of the drawing]

[0037] The present invention will be better understood by referring to the detailed description in conjunction with the non-limiting embodiments and drawings. [Figure 1] Figure 1 shows a graph of the results of flow cytometry analysis of T cells transduced with a chimeric CD95 receptor polypeptide. [Figure 2] Figure 2 shows a graph of the results of the in vitro T cell killing assay of the present invention, in which HeLa cells were transduced with modified T cell receptors and chimeric CD95 receptor polypeptides. [Figure 3] Figure 3 shows a graph of the results of the in vitro T cell killing assay of the present invention, in which modified T cell receptors and chimeric CD95 receptor polypeptides were transduced into NCIH2030 cells. [Figure 4] Figure 4 shows a graph of the results of the in vitro T cell killing assay of the present invention, in which HeLa cells were transduced with modified T cell receptors, chimeric CD8 coreceptors, and chimeric CD95 receptor polypeptides. [Modes for carrying out the invention]

[0038] As described above, in a first aspect of the present invention, the present invention relates to an isolated T cell, the T cell expressing a polypeptide-containing chimeric CD95 receptor, the polypeptide comprising at least one CD95(derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the wild-type human CD95 receptor (e.g., SEQ ID NO: 1), the CD95(derived) polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprising at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the T cell further expressing a modified T cell receptor.

[0039] For example, a T cell may express a chimeric CD95 receptor comprising a polypeptide, the polypeptide comprising at least one CD95(derived) polypeptide region having one, two, or three high-cysteine ​​domains (CRDs) of the human CD95 receptor, the one, two, or three CRDs being selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, the human CD95 polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprising at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the T cell further expresses the modified T cell receptor. A special example of a CRD that would be included in the chimeric CD95 receptor may be the CRD defined in SEQ ID NO: 3.

[0040] Expression of the human wild-type CD95 receptor is understood to be related to a protein having the amino acid sequence according to UniProtKB database entry number P25445·TNR6_HUMAN, as defined by Sequence ID No. 1.

[0041] As used in this invention, the terms "sequence identity" or "identity" refer to the ratio of pairs of identical residues to the total number of residues in the longer of the two sequences, after homology alignment of the polypeptide and / or nucleic acid sequences of the invention with the sequence in question.

[0042] The degree of sequence homology, or sequence identity, is determined, for example, in this application, using the BLASTP program, version blastp2.2.5 (November 16, 2002, see Altschul, SF et al. (1997) Nucl. Acids Res. 25, 3389-3402). The degree of homology is determined using the entire polypeptide sequence containing each sequence (matrix: BLOSUM62, gap cost: 11.1, 10 -3 It is based on the alignment of the cutoff value set. This is calculated as the ratio of the number of "positive" (homologous amino acids) shown as a result of the BLASTP program output divided by the total number of amino acids for the alignment selected by the program.

[0043] In this context, it is first discovered that a chimeric CD95 receptor, comprising both a non-CD95-derived costimulatory cytoplasmic polypeptide domain, region, or motif of at least one tumor necrosis factor receptor superfamily protein (including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptor, and / or IL6 receptor family proteins) and a functional extracellular CD95 receptor ligand-binding domain, can convert a negative signal (e.g., present in the tumor microenvironment) into a positive signal for T cell activation when co-expressed in T cells with a modified T cell receptor. Advantageously, by substituting, for example, at least the cytoplasmic "death domain" (amino acids 230-314 of SEQ ID NO: 1) of wild-type CD95, characterized by the ability to initiate the implementation of pro-apoptotic signals, for example, by recruiting Fas-associated death domain-containing proteins (FADDs) via isomorphic interactions, or by substituting, for example, the entire cytoplasmic domain of CD95 with at least one non-CD95-derived costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, T cells containing chimeric CD95 receptors and modified T cell receptors can evade the suppressive effects of FASL expressed by the tumor microenvironment and thus develop resistance to tumor-mediated immunosuppression. Secondly, the T cells provided herein thus contain a chimeric CD95 receptor, which may act as a molecular switch that redirects the signaling pathway induced by FAS linkage with FASL. Instead of inducing apoptosis and T cell death, the fusion of the CD95 receptor ligand-binding domain to the costimulatory domain alters the intercellular signaling event, promoting T cell activation, persistence, and an enhanced antitumor response.By introducing T cells provided herein, containing a modified chimeric CD95 receptor, together with a modified T cell receptor, into T cell therapy, FASL expression by solid tumors is neutralized in terms of inhibiting T cell function. This innovative method enables the T cells provided herein to resist the immune evasion mechanisms deployed by solid tumors, allowing them to better recognize and eliminate tumor cells.

[0044] According to one embodiment, the extracellular ligand-binding domain of the T cell chimeric CD95 receptor provided herein may have the ability to bind to FAS-ligand (CD95L) or any other protein / polypeptide that has the ability to bind to the wild-type CD95 receptor ligand-binding domain. For example, scFv and Fab have been described as having the ability to bind to the ligand-binding domain of the wild-type CD95 receptor.

[0045] According to one embodiment, the at least one CD95 (derived) polypeptide region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD95 receptor (e.g., SEQ ID NO: 1). According to one embodiment, the at least one CD95 (derived) polypeptide region may have one or more conserved amino acid substitutions with respect to the amino acid sequence of the wild-type CD95 receptor.

[0046] For example, the at least one CD95 (derived) polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD95 receptor (e.g., SEQ ID NO: 1).

[0047] A human CD95 polypeptide region containing a CD95 extracellular ligand-binding domain may generally have a sufficient portion of the human wild-type CD95 extracellular ligand-binding domain in order to have the function of binding to FASL. For example, at least one CD95-derived polypeptide region having at least 60% sequence identity with the CD95-derived FASL-binding domain of the human wild-type CD95 receptor may contain the whole or a substantial portion of the human wild-type CD95 FASL-binding domain and / or all amino acids at each amino acid position of the wild-type CD95 receptor that are necessary and sufficient for FASL to bind to the CD95 receptor.

[0048] As used herein, the terms “domain region,” “binding site region,” and “motif region” are understood to refer to regions of the chimeric CD95 receptor polypeptide necessary and / or sufficient for the biological function of the chimeric receptor, regions of the chimeric CD95 receptor defined by their localization to the cell, or structurally defined units of the chimeric CD95 receptor polypeptide. Furthermore, as used herein, the terms “cytoplasmic polypeptide domain” and “cytoplasmic polypeptide motif” may be understood to refer to regions of the chimeric CD95 receptor defined by their position in the cytoplasm of a cell and necessary and / or sufficient for the biological function of the chimeric receptor.

[0049] The chimeric CD95 receptor polypeptide may also be a single-chain polypeptide.

[0050] According to one embodiment, the CD95(derived) polypeptide region may further comprise at least one, at least two, or at least three CD95(derived) high-cysteine ​​domains (CRDs). For example, at least one CD95(derived) CRD may comprise CRD1, CRD2, and / or CRD3 of the wild-type CD95 receptor. CRD1 of the CD95 wild-type receptor as used herein may relate to a polypeptide having the amino acid sequence defined in SEQ ID NO: 2. CRD2 of the CD95 wild-type receptor as used herein may relate to a polypeptide having the amino acid sequence defined in SEQ ID NO: 3. CRD3 of the CD95 wild-type receptor as used herein may relate to a polypeptide having the amino acid sequence defined in SEQ ID NO: 4. For example, the CD95(derived) polypeptide region may comprise CRD1, CRD2, and CRD3 of the wild-type CD95 receptor. In this specification, the CRD1, 2, and 3 polypeptide regions contained in the T cell chimeric CD95 receptor provided herein may have one or more conserved amino acid substitutions to the CRD1, 2, and 3 polypeptide regions of the wild-type CD95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wild-type CRD domain are conceivable. For example, Gary C. Starling et al., 1998, which is incorporated in whole by reference, discloses mutagenesis studies to identify amino acid residues that contribute to the Fas-FasL interaction. Therefore, those skilled in the art are aware of potential amino acid substitutions that maintain functional activity and the protein portions of the CRD1, CRD2, and / or CRD3 of the wild-type CD95 receptor. For example, the CD95 polypeptide region may include the CRD2 of the CD95 wild-type receptor as defined herein, and the CRD2 may relate to a polypeptide having the amino acid sequence defined in Sequence ID No. 3 of this application (optionally having one or more conserved amino acid substitutions to the CRD2 polypeptide region of the wild-type CD95 receptor).

[0051] According to one embodiment, at least one CD95 (derived) polypeptide region further comprises an extracellular N-terminal preligand assembly domain (PLAD) region. The N-terminal PLAD region of the CD95 wild-type receptor as used herein may relate to a polypeptide comprising amino acid sequences 17-82 of UniProtKB database entry number P25445·TNR6_HUMAN, as defined by Sequence ID No. 1. For example, the N-terminal PLAD region as used herein may relate to a polypeptide having the amino acid sequence defined by Sequence ID No. 5. In this specification, the PLAD region contained in the T cell chimeric CD95 receptor provided herein may have one or more conserved amino acid substitutions to the PLAD region of the wild-type CD95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wild-type PLAD region are considered.

[0052] According to another embodiment, the at least one CD95-derived polypeptide region may further comprise a CD95 wild-type isotypic interaction domain. The CD95 isotypic interaction domain as defined herein may, for example, relate to a polypeptide comprising amino acid sequences 59-82 of UniProtKB database entry number P25445·TNR6_HUMAN, as defined in Sequence ID No. 1. For example, the isotypic interaction domain as defined herein may relate to a polypeptide having the amino acid sequence defined in Sequence ID No. 6. It is considered herein that the isotypic interaction domains contained in the T cell chimeric CD95 receptor provided herein may have one or more conserved amino acid substitutions relative to the wild-type CD95 receptor isotypic interaction domain. In particular, all amino acid substitutions that maintain the functional activity of the wild-type isotypic interaction domain are considered.

[0053] According to another embodiment, the at least one CD95 (derived) polypeptide region may include a CD95-derived transmembrane region. The transmembrane region of the CD95 wild-type receptor as used herein may relate to a polypeptide comprising amino acid sequences 174-190 of UniProtKB database entry number P25445·TNR6_HUMAN, as defined in SEQ ID NO: 1. For example, the transmembrane region of wild-type CD95 as used herein may relate to a polypeptide having the amino acid sequence defined in SEQ ID NO: 7. In this specification, the transmembrane domains included in the T cell chimeric CD95 receptor provided herein may have one or more conserved amino acid substitutions to the transmembrane domain of the wild-type CD95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wild-type transmembrane domain are considered.

[0054] Therefore, the T cell chimeric CD95 receptor polypeptides provided herein, in addition to comprising a CD95 (derived) ligand-binding domain and a costimulatory domain of CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, may further include domain regions / motif regions / binding site regions from the wild-type human CD95 receptor in all conceivable combinations to establish a functional chimeric CD95 receptor polypeptide. In this context, “functional” chimeric CD95 receptor refers to a chimeric CD95 receptor that redirects the signaling pathway induced by FAS association with FASL so that FASL binding promotes T cell activation, the sustained and enhanced antitumor response of T cells provided herein (instead of inducing apoptosis and T cell death). For example, any test of the functionality of a chimeric CD95 receptor may be an in vitro T cell killing assay using FASL-expressing cells, for example, as described by Kalbasi, A., Siurala, M., Su, LL et al., “Enhancement of adoptive cell therapy using synthetic IL-9 receptors,” Nature 607, 360-365 (2022). Therefore, the expression “all possible combinations” of the CD95 receptor region above means excluding combinations with wild-type human CD95 receptor domains / regions / motifs that are repressive and / or promote apoptosis and T cell death. For example, the T cell chimeric CD95 receptor polypeptides provided herein may lack the “death domain” of wild-type CD95 (amino acids 230-314 of SEQ ID NO: 1), or may contain only a modified “death domain” that no longer performs the function of promoting T cell death and / or apoptosis due to mutations that invalidate any repressive and / or apoptosis-promoting functionality of the “death domain.”

[0055] According to one embodiment, the CD95 (derived) polypeptide region may include the complete wild-type CD95 receptor extracellular domain. For example, the "wild-type CD95 receptor extracellular domain" as used herein may relate to a polypeptide comprising amino acid sequences 26-173 of UniProtKB database entry number P25445·TNR6_HUMAN, as defined in Sequence ID No. 1. For example, the wild-type CD95 receptor extracellular domain as used herein may relate to a polypeptide having the amino acid sequence defined in Sequence ID No. 8. In this specification, the CD95 receptor extracellular domain included in the T cell chimeric CD95 receptor provided herein may have one or more conserved amino acid substitutions to the transmembrane domain of the wild-type CD95 receptor. In particular, all amino acid substitutions that maintain the functional activity of the wild-type CD95 receptor extracellular domain are considered.

[0056] According to one embodiment, the CD95-derived polypeptide region may include the complete wild-type CD95 receptor extracellular domain and the entire CD95-derived transmembrane domain.

[0057] According to some embodiments, the transmembrane domain of the T cell chimeric CD95 receptor provided herein may be derived from other proteins containing transmembrane domains. For example, the transmembrane domain may be derived from a co-stimulatory molecule. In principle, any transmembrane domain that is functional and enables surface-detectable expression of the chimeric CD95 receptor is considered herein.

[0058] The T cell chimeric CD95 receptors provided herein may further comprise at least one linker region, which may be, for example, a polypeptide linker region. One or more such linkers may be located, for example, between functional domains / regions / motifs of the chimeric CD95 receptor. It may be a linker region that arises spontaneously in, for example, the wild-type CD95 receptor, or in a co-stimulatory protein, or in a co-stimulatory protein derived from the receptor's co-stimulatory domain. For example, a polypeptide linker region may be located between the transmembrane domain and the ligand-binding domain, and / or between the transmembrane domain and the CRD domain of the chimeric CD95 receptor, and / or between individual CRDs (including two or more CRDs in embodiments), and / or between the transmembrane domain and the intercellular co-stimulatory domain, and / or between individual co-stimulatory domains (including two or more co-stimulatory domains in embodiments).

[0059] Such linker regions may contain 1 to 100 amino acids, or for example, 1 to 80 amino acids, 1 to 50 amino acids, or 5 to 100 amino acids.

[0060] According to one embodiment, the linker region of the T cell chimeric CD95 receptor provided herein is SEQ ID NO: 9(GGGS) n Or sequence number 10 (GGGGS) n It may contain a specified amino acid sequence, where n is 0-20, or n is 0-10, 0-5, or 3-5.

[0061] However, as a general rule, each (polypeptide) linker known in the said technology is considered to be potentially included in the chimeric CD95 switch receptor of the present invention.

[0062] Next, returning to the co-stimulatory domain, according to one embodiment, the T cell chimeric CD95 receptor polypeptide provided herein may comprise a cytoplasmic polypeptide motif or at least one cytoplasmic polypeptide domain of CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins. The authors have for the first time discovered that the cytoplasmic costimulatory domain of a family member of immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or Il6 receptor family proteins, or alternatively, any one of the TNF-family members CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​when fused with at least one CD95-derived polypeptide region containing a functional CD95 receptor extracellular ligand-binding domain, generates a functional chimeric CD95 switch receptor that can redirect the signaling pathway induced by FAS linkage with FASL to promote T cell activation, the persistence and enhanced antitumor response of T cells provided herein, when T cells simultaneously contain / express the modified T cell receptor (instead of inducing apoptosis and T cell death). Thus, for example, the expression “T cell costimulatory domain or motif” as used herein may refer to a protein portion necessary and / or sufficient to demonstrate the ability to propagate costimulatory signals to the costimulatory molecules / proteins described herein. For example, Hong Ye et al., 1999, incorporated herein by reference, discloses TRAF-2 binding sites of various TNF family members that may function as T cell costimulatory domains or motifs in the sense of this application (as an example). Those skilled in the art will further recognize several “T cell costimulatory domains or motifs” described in the literature. As merely an example, an amino acid sequence necessary and / or sufficient to retain the ability to propagate costimulatory signals to a costimulatory molecule / protein in the sense of this invention may include, for example, amino acids 170-177 of SEQ ID NO: 28 disclosed herein, or consist solely of these.

[0063] According to one embodiment, the T cell chimeric CD95 receptor provided herein may comprise, for example, at least one complete cytoplasmic domain of an immunoglobulin superfamily (IgSF) protein, a Toll-like receptor, and / or an IL6 receptor family protein, a tumor necrosis factor receptor superfamily member CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR.

[0064] According to some embodiments, the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of CD40, CD40L, CD27, ICOS, HVEM, GITR, CD30, CD2, OX40, LTBR, ​​CD28, TLR2, TLR4, and IL6R subunit beta is at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or less than each functional polypeptide domain or functional polypeptide motif of wild-type human CD40, CD40L, CD27, ICOS, HVEM, GITR, CD30, CD2, OX40, LTBR, ​​CD28, TLR2, TLR4, or IL6R subunit beta. It may have an amino acid sequence having at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity.

[0065] According to one embodiment, the chimeric CD95 receptor polypeptide and the modified T cell receptor may enhance the cytotoxicity of the T cells provided herein.

[0066] According to one embodiment, the chimeric CD95 receptor polypeptide and the modified T cell receptor may enhance the activation, proliferation, and / or production of activating cytokines in T cells / in the

[0067] According to one embodiment, the chimeric CD95 receptor and the modified T cell receptor may increase the resistance of the T cells provided herein to CD95L-expressing cancer cells.

[0068] The T cell chimeric CD95 receptors provided herein may include any functional combination of one or more domains, one or more motifs and / or one or more regions of a cytoplasmic polypeptide that is co-stimulated by a domain, region or motif of a cytoplasmic polypeptide selected from the group consisting of CD40, CD40L, CD27, ICOS, HVEM, 4-1BB, GITR, CD30, CD2, OX40, LTBR, ​​CD28, TLR2, TLR4, and IL6R subunit beta.

[0069] According to one embodiment, the T cell chimeric CD95 receptor provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of wild-type CD40. For example, the “wild-type human CD40 cytoplasmic domain” as used herein may relate to a polypeptide comprising amino acid sequences 216-277 of UniProtKB database entry number P25942·TNR5_HUMAN, as defined in SEQ ID NO: 11. It is considered herein that the wild-type CD40 cytoplasmic polypeptide domain, region, or motif contained in the T cell chimeric CD95 receptor provided herein may have one or more conserved amino acid substitutions relative to the amino acid sequence defined in SEQ ID NO: 11. In particular, all amino acid substitutions that maintain the functional activity of the wild-type CD40 cytoplasmic polypeptide domain, region, or motif are considered.

[0070] In some embodiments in which the co-stimulatory region, motif, or domain of CD40 is included in the T cell chimeric CD95 receptor provided herein, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 12.

[0071] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of CD28. For example, the cytoplasmic polypeptide region of CD28 may include the entire cytoplasmic domain of wild-type human CD28. In other embodiments, the cytoplasmic polypeptide region of CD28 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human CD28 cytoplasmic domain. For example, the “wild-type human CD28 cytoplasmic domain” as used herein may relate to a polypeptide containing amino acid sequences 180-220 of UniProtKB database entry number P10747·CD28_HUMAN, as defined in Sequence ID No. 13. It is also considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type CD28 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in Sequence ID No. 13. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type CD28 are considered.

[0072] According to one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of 4-1BB. For example, the cytoplasmic polypeptide region of 4-1BB may include the entire cytoplasmic domain of wild-type human 4-1BB. In other embodiments, the cytoplasmic polypeptide region of 4-1BB included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human 4-1BB cytoplasmic domain. For example, the “wild-type human 4-1BB cytoplasmic domain” as used herein may relate to a polypeptide containing amino acid sequences 214-255 of UniProtKB database entry number Q07011·TNR9_HUMAN, as defined in SEQ ID NO: 14. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type 4-1BB included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 14. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type 4-1BB are considered.

[0073] In some embodiments in which the 4-1BB co-stimulatory region, motif, or domain is included in the chimeric CD95 receptor provided herein, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 15.

[0074] In one embodiment, the chimeric CD95 receptor of T cells provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of ICOS. For example, the cytoplasmic polypeptide region of ICOS may include the entire cytoplasmic domain of wild-type human ICOS. In other embodiments, the cytoplasmic polypeptide region of ICOS included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human ICOS cytoplasmic domain. For example, "wild-type human ICOS cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 162-199 of UniProtKB database entry number Q9Y6W8·ICOS_HUMAN, as defined in SEQ ID NO: 16. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type ICOS included in the chimeric CD95 receptor of T cells provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 16. In particular, all amino acid substitutions that maintain the functional activity of the wild-type ICOS cytoplasmic polypeptide domain, region, or motif are considered.

[0075] In some embodiments in which the ICOS co-stimulatory region, motif, or domain is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 17.

[0076] According to one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of HVEM. For example, the cytoplasmic polypeptide region of HVEM may include the entire cytoplasmic domain of wild-type human HVEM. In other embodiments, the cytoplasmic polypeptide region of HVEM included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human HVEM cytoplasmic domain. For example, "wild-type human HVEM cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 224-283 of UniProtKB database entry number Q92956·TNR14_HUMAN, as defined in SEQ ID NO: 18. Hereinafter, the cytoplasmic polypeptide domain, region, or motif of wild-type HVEM included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 18. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type HVEM are considered.

[0077] In some embodiments in which a co-stimulatory region, motif, or domain of HVEM is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 19.

[0078] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of OX40. For example, the cytoplasmic polypeptide region of OX40 may include the entire cytoplasmic domain of wild-type human OX40. In other embodiments, the cytoplasmic polypeptide region of OX40 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human OX40 cytoplasmic domain. For example, "wild-type human OX40 cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 236-277 of UniProtKB database entry number P43489·TNR4_HUMAN, as defined in SEQ ID NO: 20. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type OX40 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 20. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type OX40 are considered.

[0079] In some embodiments in which the co-stimulatory region, motif, or domain of OX40 is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 21.

[0080] According to one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of CD27. For example, the cytoplasmic polypeptide region of CD27 may include the entire cytoplasmic domain of wild-type human CD27. In other embodiments, the cytoplasmic polypeptide region of CD27 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human CD27 cytoplasmic domain. For example, the “wild-type human CD27 cytoplasmic domain” as used herein may relate to a polypeptide comprising amino acid sequences 213-260 of UniProtKB database entry number P26842·CD27_HUMAN, as defined in SEQ ID NO: 22. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type CD27 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 22. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type CD27 are considered.

[0081] In some embodiments in which a CD27 co-stimulatory region, motif, or domain is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 23.

[0082] According to one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of CD40L. For example, the cytoplasmic polypeptide region of CD40L may include the entire cytoplasmic domain of wild-type human CD40L. In other embodiments, the cytoplasmic polypeptide region of CD40L included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human CD40L cytoplasmic domain. For example, the “wild-type human CD40L cytoplasmic domain” as used herein may relate to a polypeptide comprising amino acid sequences 1-22 of UniProtKB database entry number P29965·CD40L_HUMAN, as defined in SEQ ID NO: 24. Hereinafter, the cytoplasmic polypeptide domain, region, or motif of wild-type CD40L included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 24. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type CD40L are considered.

[0083] In some embodiments in which the co-stimulatory region, motif, or domain of CD40L is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 25.

[0084] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of GITR. For example, the cytoplasmic polypeptide region of GITR may include the entire cytoplasmic domain of wild-type human GITR. In other embodiments, the cytoplasmic polypeptide region of GITR included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human GITR cytoplasmic domain. For example, "wild-type human GITR cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 184-241 of UniProtKB database entry number Q9Y5U5·TNR18_HUMAN, as defined in SEQ ID NO: 26. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type GITR included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 26. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type GITR are considered.

[0085] In several embodiments in which a co-stimulatory region, motif, or domain of GITR is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 27.

[0086] According to one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of CD30. For example, the cytoplasmic polypeptide region of CD30 may include the entire cytoplasmic domain of wild-type human CD30. In other embodiments, the cytoplasmic polypeptide region of CD30 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human CD30 cytoplasmic domain. For example, "wild-type human CD30 cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 407-595 of UniProtKB database entry number P28908·TNR8_HUMAN, as defined in SEQ ID NO: 28. Hereinafter, the cytoplasmic polypeptide domain, region, or motif of wild-type CD30 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 28. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type CD30 are considered.

[0087] In some embodiments in which a co-stimulatory region, motif, or domain of CD30 is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 29.

[0088] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of CD2. For example, the cytoplasmic polypeptide region of CD2 may include the entire cytoplasmic domain of wild-type human CD2. In other embodiments, the cytoplasmic polypeptide region of CD2 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human CD2 cytoplasmic domain. For example, "wild-type human CD2 cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 236-351 of UniProtKB database entry number P06729·CD2_HUMAN, as defined in SEQ ID NO: 30. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type CD2 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 30. In particular, all amino acid substitutions that maintain the functional activity of the wild-type CD2 cytoplasmic polypeptide domain, region, or motif are considered.

[0089] In some embodiments in which a CD2 co-stimulatory region, motif, or domain is included in the chimeric CD95 receptor, for example, the polypeptide of the chimeric CD95 receptor may have an amino acid sequence having at least 85% identity with the amino acids defined by SEQ ID NO: 31.

[0090] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of LTBR. For example, the cytoplasmic polypeptide region of LTBR may include the entire cytoplasmic domain of wild-type human LTBR. In other embodiments, the cytoplasmic polypeptide region of LTBR included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human LTBR cytoplasmic domain. For example, "wild-type human LTBR cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 249-435 of UniProtKB database entry number P36941·TNR3_HUMAN, as defined in SEQ ID NO: 32. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type LTBR included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 32. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type LTBR are considered.

[0091] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of TLR2. For example, the cytoplasmic polypeptide region of TLR2 may include the entire cytoplasmic domain of wild-type human TLR2. In other embodiments, the cytoplasmic polypeptide region of TLR2 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human TLR2 cytoplasmic domain. For example, "wild-type human TLR2 cytoplasmic domain" as used herein may relate to a polypeptide comprising amino acid sequences 610-784 of UniProtKB database entry number O60603·TLR2_HUMAN, as defined in SEQ ID NO: 33. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type TLR2 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 33. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type TLR2 are considered.

[0092] According to one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of TLR4. For example, the cytoplasmic polypeptide region of TLR4 may include the entire cytoplasmic domain of wild-type human TLR4. In other embodiments, the cytoplasmic polypeptide region of TLR4 included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human TLR4 cytoplasmic domain. For example, "wild-type human TLR4 cytoplasmic domain" as used herein may relate to a polypeptide containing amino acid sequences 653-839 of UniProtKB database entry number O00206·TLR4_HUMAN, as defined in SEQ ID NO: 34. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type TLR4 included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 34. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type TLR4 are considered.

[0093] In one embodiment, the chimeric CD95 receptor of a T cell provided herein may include at least one cytoplasmic polypeptide domain, region, or motif of IL6R subunit beta. For example, the cytoplasmic polypeptide region of IL6R subunit beta may include the entire cytoplasmic domain of wild-type human IL6R subunit beta. In other embodiments, the cytoplasmic polypeptide region of IL6R subunit beta included in the chimeric CD95 receptor may include at least one motif / domain / region having a co-stimulatory function of the entire wild-type human IL6R subunit beta cytoplasmic domain. For example, "wild-type human IL6R subunit beta cytoplasmic domain" as used herein may relate to a polypeptide comprising amino acid sequences 642-918 of UniProtKB database entry number P40189·IL6RB_HUMAN, as defined in SEQ ID NO: 35. It is considered herein that the cytoplasmic polypeptide domain, region, or motif of wild-type IL6R included in the chimeric CD95 receptor of a T cell provided herein may have one or more conserved amino acid substitutions to the amino acid sequence defined in SEQ ID NO: 35. In particular, all amino acid substitutions that maintain the functional activity of the cytoplasmic polypeptide domain, region, or motif of wild-type IL6R are considered.

[0094] Therefore, the expression “CD95-derived polypeptide region” as used in this application may be used synonymously with the expression “CD95 polypeptide region” which refers to the (functional) portion of the wild-type CD95 protein (or its variants). With respect to wild-type CD95 variants, such variants may have at least 60% sequence identity with respect to the corresponding portion of the amino acid sequence of SEQ ID NO: 1 (e.g., at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more). For example, the wild-type CD95 variants described herein may include an extracellular domain, which retains the ability to bind FASL. Appropriate methods for determining the functional ability (e.g., surface plasmon resonance assays) are known to those skilled in the art.

[0095] According to some embodiments, the T cell chimeric CD95 receptors provided herein may comprise a polypeptide having an amino acid sequence having at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity with the amino acids defined by, for example, one of the SEQ ID NOs: 12, 15, 17, 19, 21, 23, 25, 27, 29, and 31. According to some embodiments, the T cell chimeric CD95 receptors provided herein may have one or more conserved amino acid substitutions with respect to the amino acid sequence defined by one of the SEQ ID NOs: 12, 15, 17, 19, 21, 23, 25, 27, 29, and 31.

[0096] In a further embodiment, the present invention provides a vector comprising a nucleic acid comprising a nucleic acid sequence encoding a polypeptide-containing chimeric CD95 receptor, wherein the polypeptide comprises at least one CD95(derived) polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in SEQ ID NO: 1, wherein the human CD95 polypeptide region comprises a CD95 extracellular ligand-binding domain, and further comprises at least one non-CD95(derived) costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and the vector further comprises a nucleic acid comprising a nucleic acid sequence encoding a modified T cell receptor.

[0097] As used herein, the terms “polynucleotide” or “nucleic acid” include polyribonucleotides and polydeoxyribonucleotide sequences, for example, modified or unmodified RNA or DNA, each in single-stranded and / or double-stranded form, linear or cyclic, or hybrid molecules, and mixtures thereof. Accordingly, the nucleic acids according to the present invention include DNA (dsDNA, ssDNA, cDNA, etc.), RNA (dsRNA, ssRNA, mRNA, ivtRNA, etc.), combinations thereof, and their derivatives (RNA, etc.).

[0098] The polynucleotide may contain conventional phosphodiester bonds or non-conventional bonds (e.g., amide bonds, as found in peptide nucleic acids (RNA)). The polynucleotide of the present invention may also contain one or more modified bases, such as abnormal bases, such as tritylated bases and inosine. Other modifications, including chemical, enzymatic, or metabolic modifications, are also possible, insofar as the binding molecule of the present invention can be expressed from the polynucleotide. The polynucleotide may be provided in an isolated form as defined elsewhere in this application. The polynucleotide may contain regulatory sequences such as transcriptional regulatory elements (including promoters, enhancers, operators, repressors, and transcription termination signals), ribosome binding sites, or introns.

[0099] For example, the present invention provides a polynucleotide comprising or consisting of a nucleic acid, wherein the nucleic acid is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to a reference polynucleotide sequence selected from the group consisting of sequences described in SEQ ID NOs. 36 to 45.

[0100] The polynucleotide described above may or may not include additional or modified nucleotide sequences, for example, encoding modified amino acid residues. The polynucleotide may further encode fusion polypeptides, fragments, variants, and other derivatives of the chimeric CD95 receptor described herein.

[0101] The nucleic acid sequences of the vectors of the present invention may be codon-optimized for optimal expression in desired host T cells, such as human lymphocytes, or for expression in bacterial, yeast, or insect T cells, which are particularly considered for expression of the soluble TCR of the present invention. Codon optimization means replacing codons that are generally rare in high-expression genes of any species in the sequence of interest with codons that are generally frequent in high-expression genes of such species (such codons encoding the same amino acids as the codons being replaced). Therefore, the selection of optimized codons depends on the codon usage of the host genome and the presence of multiple desirable and undesirable sequence motifs.

[0102] As understood in this specification, “vector” refers to a nucleic acid molecule used as a vehicle for transcribing (foreign) genetic material into host T cells, which may, for example, be replicated and / or expressed.

[0103] The vector may be a viral vector or a non-viral vector.

[0104] Viral vectors may be selected from adenoviruses, smallpox viruses, alphaviruses, arenaviruses, flaviviruses, rhabdoviruses, retroviruses, lentiviruses, herpesviruses, paramyxoviruses, picornaviruses, and combinations thereof. Viruses used for T cell transfer may include naturally occurring viruses as well as artificial viruses. Viruses may be enveloped or non-enveloped viruses. Parvoviruses (such as AAV) are an example of non-enveloped viruses. Viruses may also be enveloped viruses. Viruses used for T cell transfer may be retroviruses, particularly lentiviruses. Viral envelope proteins capable of promoting viral infection of eukaryotic cells may include HIV-1-derived lentiviral vectors (LV) pseudotyped with envelope glycoproteins from vesicular stomatitis virus (VSV-G), modified feline endogenous retrovirus (RD114TR), and modified gibbon leukemia virus (GALVTR). These envelope proteins can efficiently facilitate the entry of adeno-associated viruses (AAVs) and other viruses such as parvoviruses, thereby demonstrating the broad efficiency of envelope proteins.For example, other viral envelope proteins include Moloney mouse leukemia virus (MLV) 4070env (e.g., Merten et al., J. Virol. 79:834~840, 2005, which is incorporated into this application by reference), RD114env, chimeric envelope protein RD114pro or RDpro (this is an RD114-HIV chimera constructed by substituting the R peptide cleavage sequence of RD114 with the HIV-1 matrix / capsid (MA / CA) cleavage sequence, e.g., Bell et al., Experimental Biology and You may also use bacrovirus GP64env (for example, Wang et al., J. Virol. 81:10869-10878, 2007, which is incorporated into this application by reference), or GALVenv (for example, Merten et al., J. Virol. 79:834-840, 2005, which is incorporated into this application by reference), or derivatives thereof.

[0105] In particular, the term “vector” as used herein includes, but is not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia virus vectors, polyoma genome vectors, and adenovirus-associated vectors (AAVs)), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is generally a nucleotide sequence, typically a DNA sequence including an insertion (transgene), and a larger sequence that functions as the “backbone” of the vector. Modified vectors typically include a starting point for autonomous proliferation in hosT cells (where stable expression of polynucleotides is desired), a selection marker, and restriction enzyme cleavage sites (e.g., multiple clonal sites, MCSs). The vector may further include a promoter, a genetic marker, a reporter gene, a target sequence, other regulatory elements, and / or a protein purification tag. As is known to those skilled in the art, a large number of suitable vectors are known to those skilled in the art, and many are commercially available.

[0106] In one embodiment, the vector may further contain nucleic acids encoding a T cell receptor including TCRα and TCRβ chains. For example, the modified T cell receptor may be a recombinant T cell receptor.

[0107] In a further embodiment, the vector may further contain a nucleic acid encoding a CD8 coreceptor. For example, the CD8 coreceptor may be a wild-type CD8 coreceptor. The nucleic acid may, for example, encode CD8α and CD8β coreceptors. The advantage of incorporating a CD8 coreceptor into the vector is that it provides an option to achieve coordinated CD4+ and CD8+ TCR-T cell responses in adoptive cell therapy, thereby broadening and deepening clinical responses. Alternatively, the CD8 coreceptor may be a chimeric CD8 coreceptor. For example, the chimeric CD8 coreceptor may include a polypeptide, wherein the polypeptide includes at least one CD8α (derived) polypeptide region having at least 60% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD8α coreceptor (e.g., SEQ ID NO: 46), wherein the at least one CD8α-derived polypeptide region includes a CD8α-derived IG-like domain region, and further, the polypeptide includes at least one CD8β (derived) polypeptide region having at least 60% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD8β coreceptor (e.g., SEQ ID NO: 47), wherein the at least one CD8β-derived polypeptide region includes a CD8β-derived IG-like domain region.

[0108] Expression of the human wild-type CD8α coreceptor is understood to refer to a protein having the amino acid sequence specified by UniProtKB database entry number P01732·CD8A_HUMAN, as defined by SEQ ID NO: 46. Expression of the human wild-type CD8β coreceptor is understood to refer to a protein having the amino acid sequence specified by UniProtKB database entry number P10966·CD8B_HUMAN, as defined by SEQ ID NO: 47.

[0109] Chimeric human CD8 coreceptor polypeptides containing both CD8β(derived)IG-like domain regions and CD8α(derived)IG-like domain regions are thought to be able to maintain the function of the CD8α(derived)IG-like domain regions and CD8β(derived)IG-like domain regions present in the individual, isolated polypeptides of the wild-type CD8αβ coreceptor. Therefore, as used herein, the expression "Ig-like domain" may (in principle) refer to polypeptide regions homologous to the V and / or C domains in immunoglobulin proteins.

[0110] According to one embodiment, the at least one CD8α (derived) polypeptide region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with the functional polypeptide domain or functional polypeptide motif of a wild-type human CD8α coreceptor (e.g., SEQ ID NO: 46).

[0111] For example, the at least one CD8α (derived) polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD8α coreceptor (e.g., SEQ ID NO: 46).

[0112] According to one embodiment, the at least one CD8β (derived) polypeptide region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD8β coreceptor (SEQ ID NO: 47).

[0113] For example, the at least one CD8β (derived) polypeptide region may have at least 70%, or at least 71%, or at least 72%, or at least 73%, or at least 74%, or at least 75%, or at least 76%, or at least 77%, or at least 78%, or at least 79%, or at least 80%, or at least 81%, or at least 82%, or at least 83%, or at least 84%, or at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity with the functional polypeptide domain or functional polypeptide motif of the wild-type human CD8β coreceptor (e.g., SEQ ID NO: 47).

[0114] According to one embodiment, the chimeric human CD8 coreceptor polypeptide is a single-chain polypeptide.

[0115] According to one embodiment, the chimeric CD8 coreceptor comprises a CD8α (derived) IG-like domain region, a CD8β (derived) IG-like domain region, a stalk region (typically between the transmembrane domain and the IG-like domain region of the CD8 receptor), a transmembrane domain region, and an intracellular / cytoplasmic domain region, wherein the intracellular domain region comprises a palmitoylation motif region and an LCK binding site region.

[0116] In this specification, the stalk region, transmembrane region, and intracellular domain of the chimeric CD8 coreceptor may be derived from other proteins or from CD8α or CD8β. Therefore, in addition to containing the CD8α(derived)IG-like domain region and the CD8β(derived)IG-like domain region, the chimeric CD8 coreceptor polypeptide may further contain domain regions / motif regions / binding site regions from one or both of the wild-type human CD8α coreceptor and / or the wild-type human CD8β coreceptor, and / or from other proteins, in all possible combinations to establish a functional chimeric CD8 coreceptor polypeptide.

[0117] For example, according to one embodiment, the chimeric CD8 coreceptor may include a CD8α-derived transmembrane region.

[0118] According to one embodiment, the chimeric CD8 receptor may include the cytoplasmic region of the CD4 coreceptor (derived from). For example, the chimeric CD8 coreceptor may include the entire cytoplasmic region of the wild-type CD4 coreceptor. For example, the "wild-type human CD4 coreceptor cytoplasmic region" as used herein may relate to a polypeptide containing amino acid sequences 419-458 of UniProtKB database entry number P01730·CD4_HUMAN, as defined in Sequence ID No. 48.

[0119] According to one embodiment, the chimeric CD8 coreceptor may further comprise at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of tumor necrosis factor receptor superfamily proteins, immunoglobulin superfamily (IgSF) proteins, and / or ITAM-related receptors.

[0120] The enhanced chimeric CD8 coreceptor, in some embodiments, is encoded by the vector of the present invention and binds an additional costimulatory domain to the human CD8 receptor. Co-stimulation associated with such costimulatory domains, provided by a fusion cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of tumor necrosis factor receptor superfamily proteins and / or immunoglobulin superfamily (IgSF) proteins and / or ITAM-associated receptors, complements TCR signaling and leads to more potential TCR-T cell production.

[0121] Therefore, the provision of T cells provided herein, including chimeric CD95 receptors, modified T cell receptors, and furthermore, chimeric CD8 coreceptors, enhances T cell activation, proliferation, cytokine production, and cytotoxicity, ultimately improving the therapeutic effect of TCR-T cell therapy.

[0122] In one embodiment, the chimeric CD8 coreceptor includes a CD4(derived) cytoplasmic domain progenitor. In this embodiment, at least one CD8α(derived) polypeptide region may further include a CD8α(derived) transmembrane domain region, and at least one CD8β(derived) polypeptide region may include a CD8β(derived) stalk domain region. In this embodiment, the CD8α(derived) IG-like domain region may be located closer to the N-terminus of the enhanced chimeric CD8 coreceptor polypeptide than the CD8β(derived) IG-like domain region. For example, the chimeric CD8 coreceptor may further include a co-stimulatory CD30 motif provided herein, for example, as defined in SEQ ID NO: 49. Furthermore, the chimeric CD8 coreceptor according to this embodiment includes a CD40 cytoplasmic domain. For example, according to this embodiment, the polypeptide linker sequence GGGS is inserted between the CD30 co-stimulatory motif and the CD40 cytoplasmic domain. The CD30 motif may be included in the C-terminus of the chimeric CD8 coreceptor. The enhanced chimeric CD8 coreceptor according to this embodiment may include a polypeptide having an amino acid sequence that has at least 85%, or at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity with the amino acids defined in, for example, SEQ ID NO: 50 (pTK-0638).

[0123] In some embodiments, the transgene may further comprise one or more polycistronic factors, which may be positioned between any two nucleic acid sequences encoding a chimeric CD95 receptor, TCRα, TCRβ, and any CD8 coreceptor. In some embodiments, the one or more polycistronic factors may comprise a ribosome skipping factor selected from T2A, P2A, E2A, or F2A, or a sequence encoding an internal ribosome translocation site (IRES).

[0124] As used herein, the term “self-cleaving 2A peptide” refers to a relatively short peptide (approximately 20 amino acids in length, depending on the origin virus) that acts cotranslatically, preventing the formation of a normal peptide bond between glycine and the final proline, causing the ribosome to skip to the next codon, and the newly synthesized peptide to cleave between Gly and Pro. After cleavage, the short 2A peptide remains fused to the C-terminus of the “upstream” protein, while the proline is added to the N-terminus of the “downstream” protein. The self-cleaving 2A peptide may be selected from porcine tesiovirus-1 (P2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), foot-and-mouth disease virus (F2A), or any combination thereof. Adding a linker sequence (GSG or SGSG [SEQ ID NO: 51]) before the self-cleaving 2A sequence may enable the efficient synthesis of bioactive proteins such as TCRs and the chimeric CD95 receptor described herein.

[0125] In a further embodiment, isolated T cells are also provided, comprising a nucleic acid encoding the chimeric human CD95 receptor of the present invention, wherein the nucleic acid further encodes the modified T cell receptor.

[0126] In the context of this invention, the term "modified T cell receptor" is distinguished from "CAR" T cell receptors. For example, unlike chimeric antigen receptors (CARs), modified TCRs recognize HLA-presenting peptides derived from proteins in all intracellular compartments. Furthermore, the term "modified T cell receptor" is understood to include TCRs that are not naturally expressed by the listed T cells (e.g., TCRs that are exogenous to T cells and introduced into the T cell genome by the genetic engineering techniques described herein).

[0127] In another embodiment, T cells may express the chimeric CD95 receptor and modified T cell receptor described herein.

[0128] In some embodiments, T cells may further express CD8 coreceptors, such as wild-type CD8 coreceptors or chimeric CD8 coreceptors. The chimeric CD8 coreceptor may be a chimeric receptor having the functionality of the wild-type CD8 coreceptor. For example, the chimeric CD8 coreceptor may have the same MHC complex binding function as the wild-type CD8 coreceptor.

[0129] For example, the T cells may be CD4 T cells, and furthermore, the CD4 T cells may additionally express recombinant human CD8 coreceptors such as CD8α and CD8β receptors, or chimeric CD8 coreceptors.

[0130] According to some embodiments, the vector may be introduced into T cells as described herein.

[0131] The (isolated) T cells may be generated by various methods, including those recognized in the literature. For example, polynucleotides encoding an expression cassette containing tumor recognition or another type of recognition site, and also encoding the chimeric CD95 receptor and modified T cell receptor and optionally a CD8 coreceptor as described herein, may be stably introduced into T cells by virus-based gene transfer systems such as transposon / transposase systems, lentiviral or retroviral systems, or by other suitable methods such as transfusion, electroporation, transduction, lipofection, nanofabricated materials such as calcium phosphate (CaPCU) and organically modified silica (Ormosil), mRNA-based therapies, and viral delivery methods including adenoviruses, retroviruses, lentiviruses, and adeno-associated viruses. T cells may also be generated by introducing nucleic acids into T cells in vivo, for example, using DNA or mRNA and nanoparticles such as lipid nanoparticles.

[0132] T cells may be transfused by means known in the art, including lipofection (transfusion by liposomes), electroporation, calcium phosphate transfusion, biolithographic particle delivery (e.g., gene guns), microinjection, or a combination thereof. Various methods for transfusing cells are known in the art. See, for example, Sambrook & Russell (Eds.) Molecular Cloning: A Laboratory Manual (3rd edition) Vols. 1-3 (2001) Cold Spring Harbor Laboratory Press, and Ramamoorth & Narvekar "Non-viral vectors in gene therapy (summary)" J Clin Diagn Res. (2015) 9(1):GE01-GE06.

[0133] According to one embodiment, the cells may be αβT cells, γδT cells, and / or natural killer T cells.

[0134] For example, αβT cells may be CD4T cells, or αβT cells may be CD8T cells, or δT cells may contain, for example, a Vγ1 chain or a Vγ2 chain, or for example, a Vγ9Vδ2+ T cell.

[0135] T cells may express the chimeric CD95 receptor described herein, as well as the modified T cell receptor. In embodiments, the T cells may also be CD4 T cells, which further express CD8 coreceptors such as both CD8α and CD8β coreceptors, or any modified protein that expresses coreceptor functionality.

[0136] According to the present invention, T cells further express a modified T cell receptor. The modified T cells of this disclosure can be used to treat subjects who need to be treated for a condition such as cancer as described herein. The T cells may be αβT cells or γδT cells expressing the chimeric CD95 receptor polypeptide described herein, or they may be modified TCRs. Optionally, the T cells may express a CD8 coreceptor, such as a wild-type or chimeric CD8 coreceptor. The T cells described herein may be used to treat cancers, including solid tumors and malignant hematological disorders. The T cells provided herein may be used to treat, for example, "hot" tumors or "cold" tumors.

[0137] For example, the modified T cell receptor described herein may specifically bind to a member of the MAGE antigen family, such as MAGE-A1 or Mage-A4, or in this case, the modified T cell receptor may specifically bind to an antigen selected from the group consisting of PRAME antigen, NY-ESO-1 antigen, GP100 antigen, AFP antigen, Col6A3 antigen, HPV-16 antigen, WT1 antigen, HA1 antigen, HA2 antigen, mutant KRAS antigen, mutant NRAS antigen, mutant HRAS antigen, mutant TP53 antigen, and EGFR antigen.

[0138] In this context, the term "mutation" used in the present invention to refer to a known mutation within the epitope region of each protein, polypeptide, or peptide that correlates with expression in human cancer.

[0139] According to one embodiment, the T cells described herein may be used to treat an infectious disease, which may be caused by a virus. The T cells described herein may be used to treat an immunological disease, such as an autoimmune disease. The T cells may be αβT cells or γδT cells expressing the chimeric CD95 receptor and modified TCR described herein, and optionally a CD8 coreceptor such as a wild-type or chimeric CD8 coreceptor.

[0140] In some embodiments, T cells may be derived from induced pluripotent stem cells (iPSCs).

[0141] In another embodiment, the present invention provides a kit comprising means for preparing the above-mentioned T cells.

[0142] In yet another aspect, the present invention relates to a pharmaceutical composition comprising T cells provided by the present invention.

[0143] In this specification, pharmaceutical compositions may further include adjuvants, excipients, buffers, diluents, carriers, stabilizers, or combinations thereof.

[0144] In a further embodiment, a pharmaceutical composition comprising T cells expressing the chimeric CD95 receptor and modified T cell receptor described herein is provided.

[0145] According to one embodiment, the pharmaceutical composition may further contain CD4 T cells that express the chimeric CD95 receptor, the modified T cell receptor, and also express recombinant CD8 coreceptors such as the CD8α receptor and the CD8β receptor, or the chimeric CD8 receptor.

[0146] The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers. Any pharmaceutically acceptable carrier may be used, provided that it does not affect the viability of the T cells to which it will be administered and is suitable for the chosen route of administration of the pharmaceutical composition. The pharmaceutically acceptable carrier is physiological saline and may optionally comprise components such as human serum albumin, which can improve the viability of T cells expressing the chimeric CD95 receptor. The chimeric CD95 receptors expressing T cells may also be stored in a frozen form after their manufacture, for example, at a temperature of -20°C to -80°C. In this case, the pharmaceutical composition may comprise cryoprotective substances added to protect the cells from damage caused by the freezing process. Examples of antifreeze agents that may be used herein for freezing pharmaceutical compositions containing transduced T cells include glycerol and DMSO. These antifreeze agents may be used with commercially available crystalloid solutions such as HypoThermosol or PlasmaLyte-A, which are approved for intravenous infusion and available in pharmaceutical grade. Other possible media that can be used as carriers in pharmaceutical compositions include media from the "CryoStor family," commercially available, animal protein-free, clearly defined cryopreservation media from Biolife Solutions, such as CyroStor2 (CS2, an optimized cryopreservation medium pre-prepared with 2% DMSO), CyroStor5 (CS5, an optimized cryopreservation medium pre-prepared with 5% DMSO), or CyroStor10 (CS10, an optimized cryopreservation medium pre-prepared with 10% DMSO).

[0147] In a further embodiment, a method for preparing T cells for immunotherapy is provided, and this method is -T cells were isolated from human subjects, -The method comprises introducing a vector containing a nucleic acid encoding a chimeric CD95 receptor, wherein the polypeptide comprises at least one CD95 polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of a human CD95-derived receptor as defined in SEQ ID NO: 1, the human CD95 polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprising at least one non-CD95-derived costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, and further comprising a nucleic acid encoding a modified T cell receptor in T cells, and the method further comprises - This includes increasing the transduced T cells.

[0148] For example, the method may include transmuting, transtransferring, or transducing the vector into the isolated T cells.

[0149] In a further embodiment, a method for treating a patient having a disease is also provided, the method comprising administering the pharmaceutical composition of the present invention to the patient.

[0150] In a further embodiment, a method is provided for treating a patient having a disease, the method comprising introducing the vector disclosed herein into the T cells of the patient in vivo.

[0151] According to one embodiment, the nucleic acid may be DNA or mRNA.

[0152] For in vivo administration, the vector may be, for example, a non-replicating viral vector.

[0153] According to one embodiment, the nucleic acid may be mRNA, and the mRNA may be introduced in vivo into the patient's T cells using nanoparticles such as lipid nanoparticles.

[0154] In the methods for treating a patient provided herein, the disease may be, for example, an autoimmune disease or cancer.

[0155] In the methods for treating patients provided herein, for example, the cancers treated by these methods may be selected from the group consisting of non-small cell lung cancer, small cell lung cancer, pancreatic cancer, ovarian cancer, melanoma, breast cancer, liver cancer, kidney cancer, esophageal cancer, brain tumor, gastric cancer, Merkel cell carcinoma, leukemia, bladder cancer, uterine cancer, colorectal cancer, gallbladder cancer, bile duct cancer, and prostate cancer.

[0156] For example, the cancer being treated may be a solid tumor. In the exemplary embodiments of the types of solid tumors described above, the lung cancer may be, but is not limited to, squamous cell carcinoma of the lung, adenocarcinoma of the lung, large cell carcinoma of the lung, and other histological types of NSCLC or small cell lung cancer, including but not limited to these. In other exemplary examples, the breast cancer may be, but is not limited to, ductal carcinoma, ductal invasive breast cancer, invasive breast cancer, tubular breast cancer, medullary breast cancer, or a combination thereof. In yet another exemplary example, the gastric cancer may be gastric adenocarcinoma or squamous cell carcinoma. For sarcoma, the sarcoma may be, but is not limited to, chondrosarcoma, osteosarcoma, or a combination thereof. The adenoma may be, but is not limited to, gastric adenocarcinoma, pancreatic adenocarcinoma, or a combination thereof.

[0157] In a further embodiment, as described above, a method for increasing the cytotoxicity of T cells in adoptive cell therapy is also provided, and this method is - The process involves introducing a vector into T cells, the vector comprising a nucleic acid encoding a modified T cell receptor and further encoding a chimeric CD95 receptor comprising a polypeptide, the polypeptide comprising at least one CD95 polypeptide region having at least 60% sequence identity with a polypeptide domain, polypeptide region, or polypeptide motif of a human CD95-derived receptor as defined in SEQ ID NO: 1, the human CD95 polypeptide region comprising a CD95 extracellular ligand-binding domain, and further comprising at least one non-CD95-derived costimulatory cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins.

[0158] For example, the nucleic acid may further encode a recombinant CD8 coreceptor, such as a wild-type CD8 coreceptor or a chimeric CD8 coreceptor.

[0159] According to one embodiment, the T cell receptor may be a recombinant T cell receptor that specifically binds to a tumor-specific antigen. In some embodiments, this may be a MAGE antigen, such as the MAGE-A1 antigen, as an example.

[0160] The present invention will be further illustrated by the following non-limiting experimental examples.

[0161] The sequences used in this specification are shown in Table 1 below.

[0162] [Table 1] JPEG2026529164000002.jpg228170JPEG2026529164000003.jpg227170JPEG2026529164000004.j pg227170JPEG2026529164000005.jpg227170JPEG2026529164000006.jpg227170JPEG20265291640 00007.jpg227170JPEG2026529164000008.jpg227170JPEG2026529164000009.jpg227170JPEG202 6529164000010.jpg226170JPEG2026529164000011.jpg230170JPEG2026529164000012.jpg182170 [Examples]

[0163] Example 1: In vitro T cell killing analysis of T cells of the present invention transduced with chimeric CD95 receptor polypeptide and modified T cell receptor. To assess the suitability of T cells expressing a chimeric CD95 receptor construct along with the modified T cell receptor described herein for adoptive T cell therapy (ACT), and / or the increased cytotoxicity of generated T cells expressing a chimeric CD95 switch receptor and a modified T cell receptor, CD8 T cells were transduced with an HLA-I restriction TCR grown against MAGE-A1 using a chimeric CD95 receptor construct. The purified transduced T cells were used in in vitro T cell killing assays using NCI-H2030FASL-expressing cells and HeLaFASL-expressing cells, respectively, to evaluate the cytotoxicity of the transduced T cells.

[0164] 1.1 Materials and Methods Cloning of chimeric human CD95 receptor and chimeric CD8 coreceptor creations Chimeric human CD95 receptor and chimeric CD8 coreceptor compounds were generated using standard cloning techniques. Table 2 below summarizes the cloned base plasmids for the chimeric CD95 compounds.

[0165] Table 2 below provides a general overview of the chimeric CD95 receptor creations produced by the inventors.

[0166] [Table 2] JPEG2026529164000014.jpg102151

[0167] In use in Table 2, "EC" refers to the origin of the extracellular domain of the chimeric receptor, "TM" refers to the origin of the transmembrane domain of the chimeric receptor, and "CYP" refers to the origin of the cytoplasmic domain of the generated product.

[0168] Furthermore, Table 3 below summarizes the types of chimeric CD95 receptors used in the T cell killing assay shown in Figure 4, and the types of chimeric CD8 coreceptors used in the T cell killing assay shown in Figure 4.

[0169] [Table 3]

[0170] In use in Table 3, the term "EC" refers to the origin of the extracellular domain of the chimeric receptor. "TM" refers to the origin of the transmembrane domain of the chimeric receptor, and "CYP" refers to the origin of the cytoplasmic domain of the generated artifact. Furthermore, the type of coreceptor relates to the type of CD8 coreceptor that is co-transduced into T cells, which applies to all artifacts on page 638 of Table 3. Therefore, the co-stimulatory domains of CD30 (CD30 motif) and CD40 both encode chimeric CD8 coreceptors fused to the cytoplasmic domain of CD4.

[0171] CD8 cell generation PBMCs from healthy donor buffy coats were isolated by density gradient centrifugation using Lymphoprep. Purified polyclonal CD8 T cells were obtained by negative selection using anti-CD4 microbeads to deplete the C4 population. CD3 T cells were activated using TransAct in the presence of IL-7 / IL-15. Two days after activation, CD8 T cells were transduced individually, either alone or with different versions of the switch receptor, with HLA-I restriction TCRs grown against MAGE-A1 (MAGE-A1_TCR). The HLA-I restriction TCRs grown against MAGE-A1 (MAGE-A1_TCR) used herein are described in WO2014 / 118236, which is incorporated herein by reference in its entirety. In particular, the HLA-restriction TCRs grown against MAGE-A1 used herein relate to "TCR1367" described in WO2014 / 118236. The CDR sequences of each α and β strand of "TCR1367" used herein are further described, for example, in WO2023 / 083864, which are incorporated herein by reference in their entirety.

[0172] Transduced CD8 T cells were further proliferated, and the transduced fraction was positively selected on day 9 using CD34 microbeads. Purified transduced T cells were cultured for further proliferation, harvested on day 10, and cryopreserved. T cell characterization was based on transgene expression levels using FACS and killing assays.

[0173] Cell killing assay in NCIH2030 cells expressing FASL In vitro T cell killing assays were performed based on the method described in, for example, "Enhancement of adoptive cell therapy with synthetic IL-9 receptor" by Kalbasi, A., Siurala, M., Su, LL et al., Nature 607, 360-365 (2022). In particular, the human TCRT cell repeated killing assay was performed using IncuCyte live cell analysis. 1 × 10⁶ cells per well. 4NCIH2030 tumor cells were seeded into 96-well plates. Human T cells, either untransduced (simulated) or transduced (MAGE_TCR transduced alone, or MAGE_TCR transduced with a chimeric CD95 receptor), were added in a cubed E:T ratio of 1 to 1 or 1:2.

[0174] Cell killing assay in FASL-expressing HeLa cells In vitro T cell killing assays were performed based on the method described in, for example, "Enhancement of adoptive cell therapy with synthetic IL-9 receptor" by Kalbasi, A., Siurala, M., Su, LL et al., Nature 607, 360-365 (2022). In particular, the human TCRT cell repeated killing assay was performed using IncuCyte live cell analysis. 1 × 10⁶ cells per well. 4 HeLa tumor cells were seeded in 96-well plates. Human T cells that were not transduced (simulated) or transduced (MAGE_TCR transduced alone, or MAGE_TCR transduced with a chimeric CD95 receptor) were added in a cube ratio of 1 to 2 E:T ratios. For the second and third stimuli, tumor cells (1 × 10⁶) were added, respectively. 4 Individual HeLa cells were added to each well, typically 50 to 100 hours after the start of the experiment, after the cancer cells had been killed since the previous stimulation.

[0175] Long-term cell killing assay in NCIH2030 cells In vitro T cell killing assays were performed based on the method described in, for example, Kalbasi, A., Siurala, M., Su, LL et al., "Enhancement of adoptive cell therapy with synthetic IL-9 receptors," Nature 607, 360-365 (2022). PBMCs from healthy donor buffy coats were isolated by density gradient centrifugation using Lymphoprep. Purified polyclonal CD8+ T cells were obtained by positive selection with anti-CD8+ microbeads. CD3+ T cells were activated using TransAct in the presence of IL-7 / IL-15. Two days after activation, CD8 T cells were transduced individually with HLA-I restriction TCRs (MAGE-A1_TCRs) grown against MAGE-A1, either alone or with different versions of the switch receptor. Where indicated (results in Figure 4), chimeric CD8 coreceptors were cotransduced (CoR). The chimeric CD8 coreceptor used in the experiment is the chimeric CD8 coreceptor polypeptide (pTK-0638) having the amino acid sequence defined by SEQ ID NO: 50.

[0176] Flow cytometry Extracellular surface staining was performed in flow cytometry FACS buffer (BDBioscience) at 4°C for 30 minutes. The following antibodies were used: BioLegend's CD8a (clone HIT8a), Invitrogen's CD34 (clone QBEND10), CD34 (clone 4H11), Miltenyi Biotec's CD8a (clone REA734), CD95 (clone DX2), and BeckmanCoulter's TCRBV3S1Vβ3. PE-labeled HLA-A*02:01 specific MAGE-A1MHC tetramer (KVLEYVIKV) (SEQ ID NO: 52) (TB-M070-1) was added along with the cell surface staining antibody. Live and dead cells were distinguished using the ZombieYellow® Fixed Viability Kit. Chimeric CD95 receptor expression was determined in CD8 cells transduced with different chimeric CD95 receptors provided herein.

[0177] 1.2. T-cell killing assay Relative cell growth was observed for each transduced T cell fraction. The results are shown in Figures 2, 3, and 4. Figure 2 shows the results of the chimeric CD95 receptor cell killing assay in HeLa cells, and Figure 3 shows the results of the chimeric CD95 receptor cell killing assay in NCIH2030 cells. In this specification, "simulated" refers to the simulated-transduced T cell fraction, "MAGE_TCR" refers to the CD8 T cell fraction transduced with an HLA-I restriction TCR grown against MAGE-A1 (MAGE-A1_TCR), and "MAGE-A1_TCR-SwR_CD40" refers to the transduced HLA-I restriction TCR grown against MAGE-A1 (MAGE-A1_TCR) together with a chimeric CD95 receptor containing the CD40 cytoplasmic domain. Regarding the CD8 T cell fractions, "MAGE-A1_TCR-SwR_CD40" is a CD8 T cell fraction in which an HLA-I restriction TCR was transduced to MAGE-A1 (MAGE-A1_TCR) along with a chimeric CD95 receptor containing the CD40L cytoplasmic domain. Regarding the CD8 T cell fractions in which an HLA-I restriction TCR was transduced, "MAGE-A1_TCR-SwR_CD27" is a CD8 T cell fraction in which an HLA-I restriction TCR was transduced to MAGE-A1 (MAGE-A1_TCR) along with a chimeric CD95 receptor containing the CD27 cytoplasmic domain. Regarding the CD8 T cell fraction into which an HLA-I restriction TCR developed against MAGE-A1 has been transduced, "MAGE-A1_TCR-SwR_ICOS" is a chimeric CD95 receptor containing the ICOS cytoplasmic domain, and regarding the CD8 T cell fraction into which an HLA-I restriction TCR developed against MAGE-A1 (MAGE-A1_TCR) has been transduced, "MAGE-A1_TCR-SwR_HVEM" is a chimeric CD95 receptor containing the HVEM cytoplasmic domain. Regarding the CD8 T cell fraction into which an HLA-I restriction TCR developed against MAGE-A1 (MAGE-A1_TCR) has been transduced, "MAGE-A1_TCR-SwR_4-1BB" is, and regarding the CD8 T cell fraction into which an HLA-I restriction TCR developed against MAGE-A1 (MAGE-A1_TCR) has been transduced together with a chimeric CD95 receptor containing the 4-1BB cytoplasmic domain, "MAGE-A1_TCR-SwR_GITR" is,Regarding the CD8 T cell fraction in which an HLA-I restriction TCR was transduced into MAGE-A1 (MAGE-A1_TCR) along with a chimeric CD95 receptor containing the GITR cytoplasmic domain, "MAGE-A1_TCR-SwR_CD30" is a variant of "MAGE-A1_TCR" in which an HLA-I restriction TCR was transduced into MAGE-A1 (MAGE-A1_TCR) along with a chimeric CD95 receptor containing the CD30 cytoplasmic domain. "-SwR_CD2" refers to a CD8 T cell fraction transduced with an HLA-I restriction TCR developed against MAGE-A1 (MAGE-A1_TCR) along with a chimeric CD95 receptor containing a CD2 cytoplasmic domain, while "MAGE-A1_TCR-SwR_OX40" refers to a CD8 T cell fraction transduced with an HLA-I restriction TCR developed against MAGE-A1 (MAGE-A1_TCR) along with a chimeric CD95 receptor containing an OX40 cytoplasmic domain.

[0178] Figure 4 shows the results of the long-term killing assay, where T cells were either simulated, or MAGE-A1TCR (MAGEA1TCR), or MAGE-A1TCR with wild-type CD8 coreceptor (MageA1TCRWT_CoR), or MAGE-A1TCR with chimeric CD8 coreceptor (MageA1TCRESC_CoR), or MAGE-A1T cells transduced with one of the following: chimeric CD8 coreceptor and chimeric CD95 receptor constructs described herein. These are CRs (MageA1TCRESC_CoRFASCD40, MageA1TCRESC_CoRFASCD30, MageA1TCRESC_CoRFASOX40, MageA1TCRESC_CoRFASCD2, MageA1TCRESC_CoRFASCD27, MageA1TCRESC_CoRFASCD40L, MageA1TCRESC_CoRFASHVEM, and MageA1TCRESC_CoRFASICOS, respectively).

[0179] 1.3 Flow Cytometry Analysis CD8 cells transduced with vectors containing nucleic acids encoding different chimeric CD95 receptors provided herein have been shown to express the chimeric CD95 receptors. As can be seen in Figure 1, only the chimeric CD95 receptors containing the cytoplasmic domains of 41BB, GITR, and CD40L respectively showed a relatively low proportion of Fas-high expression in the population, while all other chimeric CD95 receptors tested showed a high proportion of Fas-high expression in the population.

[0180] 1.4 Results As can be seen in Figures 2 and 3, co-transduction of a modified HLA-I restriction TCR grown against MAGE-A1 into CD8 cells, along with a chimeric CD95 receptor containing a domain or motif of a non-CD95-derived costimulatory cytoplasmic polypeptide provided herein, results in increased toxicity of the modified T cells compared to T cells transduced only with an HLA-I restriction TCR grown against MAGE-A1, as observed in both HeLa and NCIH2030 cells.

[0181] As can be seen in Figure 4, T cells transduced with a modified HLA-I restriction TCR grown against MAGE-A1, along with the chimeric CD95 receptor and chimeric CD8 coreceptor provided herein, exhibit increased toxic activity compared to simulant transduced T cells and / or T cells simply transduced with an HLA-I restriction TCR grown against MAGE-A1, as observed in NCIH2030 cells.

[0182] Summary and Conclusion The results described above demonstrate, in principle, the suitability of adoptive cell therapy (ACT) for T cells involving both the chimeric CD95 receptor polypeptide and the modified T cell receptor provided herein. Specifically, the chimeric CD95 receptor polypeptide of the present invention, in combination with the modified T cell receptor, may have the function of providing enhanced resistance to T cells in an immunosuppressive tumor microenvironment, prevention of T cell exhaustion and / or loss by apoptosis, and stimulation of T cell proliferation and functional activity, such as increased cytotoxicity.

[0183] Therefore, the fusion of at least one cytoplasmic costimulatory motif, costimulatory domain, or costimulatory polypeptide region, such as tumor necrosis factor receptor superfamily proteins including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins, with a chimeric CD95 receptor containing a CD95 ligand-binding domain is thought to act like a “switch” receptor, by converting a negative signal to a positive signal if the T cell further contains the modified T cell receptor, thereby enhancing the cytotoxicity of T cells in the presence of tumor cells expressing ligand FASL. Modified T cells expressing the chimeric CD95 receptor polypeptide together with the modified T cell receptor provided herein exhibit improved killing activity compared to control samples in the presence of FASL.

[0184] It will be readily apparent to those skilled in the art that substitutions and modifications may be made to the invention as described herein without departing from the scope and spirit of the invention.

[0185] All patents and publications referenced herein indicate the level of skill of those skilled in the art to which this invention belongs. All patents and publications are incorporated by reference to the same extent as each individual publication is specifically and individually incorporated by reference.

[0186] The present invention, as illustrated herein, may be adequately implemented with any one or more elements not specifically disclosed herein, or without one or more limitations. Therefore, terms such as “includes,” “equip,” and “possess” should be interpreted broadly and not limitably. Furthermore, the terms and expressions used herein are for illustrative purposes only, not limitation. In using such terms and expressions, it is not intended to exclude any equivalent of any illustrated or described feature or part thereof, and it is recognized that various modifications are possible within the scope of the claimed invention. Therefore, although the invention has been described more concretely by preferred embodiments and optional features, modifications and changes to the embodiments of the invention disclosed herein may be made by those skilled in the art, and such modifications and changes should be interpreted as being within the scope of the invention. The invention has been described broadly and generally herein. Each of the narrower groups of species and subgenerics within the general disclosure is also part of the invention. This includes the comprehensive description of the invention, with any proviso or negative requirement excluding any subject matter from a genus, whether or not the excluded subject matter is specifically described herein. Furthermore, if any feature or aspect of the present invention is described in terms of the Markush Group, a person skilled in the art will recognize that the present invention also describes any individual member or subgroup member of the Markush Group. Further embodiments of the present invention will become apparent from the following claims.

Claims

1. Isolated T cells, wherein the T cells express a chimeric CD95 receptor containing a polypeptide, The polypeptide comprises at least one CD95 polypeptide region having at least 60% sequence identity with the polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in Sequence ID No.

1. The human CD95 polypeptide region includes a CD95 extracellular ligand binding domain. Furthermore, the polypeptide comprises at least one non-CD95 costimulated cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins. T cells that further express a modified T cell receptor.

2. Isolated T cells, wherein the T cells express a chimeric CD95 receptor containing a polypeptide, The polypeptide comprises at least one CD95 polypeptide region including a CD95 extracellular ligand-binding domain, Furthermore, the polypeptide comprises at least one non-CD95 costimulated cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins. T cells that further express a modified T cell receptor.

3. A T cell according to claim 1 or 2, The extracellular ligand-binding domain of the chimeric CD95 receptor has the function of binding to FAS-ligand (CD95L) or any other protein / polypeptide capable of binding to the wild-type CD95 receptor ligand-binding domain, in T cells.

4. A T cell according to any one of claims 1 to 3, The polypeptide is a single-chain polypeptide, a T cell.

5. A T cell according to any one of claims 1 to 4, The CD95 polypeptide region further comprises at least one, at least two, or at least three CD95 high-cysteine ​​domains (CRDs) in the T cell.

6. A T cell according to any one of claims 1 to 5, The T cell wherein the at least one CD95 polypeptide region further comprises an extracellular N-terminal PLAD region.

7. A T cell according to any one of claims 1 to 6, The T cell wherein the at least one CD95 polypeptide region further comprises a CD95 isomorphic interaction domain.

8. A T cell according to any one of claims 1 to 7, The aforementioned at least one CD95 polypeptide region includes a CD95 transmembrane region in a T cell.

9. A T cell according to any one of claims 1 to 8, The CD95 polypeptide region includes the CD95 extracellular domain in T cells.

10. A T cell according to claim 9, The CD95 polypeptide region includes a CD95 extracellular domain and a CD95 transmembrane domain in T cells.

11. A T cell according to any one of claims 1 to 10, The CD95 polypeptide region comprises at least one linker region in the T cell.

12. A T cell according to any one of claims 1 to 11, The polypeptide comprises the complete cytoplasmic domain of the tumor necrosis factor receptor superfamily protein, immunoglobulin superfamily (IgSF) protein, Toll-like receptor, and / or IL-6 receptor family protein in T cells.

13. A T cell according to any one of claims 1 to 12, T cells, wherein the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif of an immunoglobulin superfamily (IgSF) protein, a Toll-like receptor, and / or an IL6 receptor family protein is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of ICOS, CD28, TLR2, TLR4, and IL6R subunit beta.

14. A T cell according to any one of claims 1 to 13, T cells, wherein the polypeptide has an amino acid sequence having at least 85%, optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the amino acids defined by any one of the sequences selected from the group consisting of SEQ ID NOs. 12, 15, 17, 19, 21, 23, 25, 27, 29, and 31.

15. A T cell according to any one of claims 1 to 14, A T cell wherein the at least one CD95 polypeptide region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 97% sequence identity with the functional polypeptide domain or functional polypeptide motif of the human CD95 receptor (SEQ ID NO: 1).

16. The T cell according to any one of claims 1 to 15, T cells with enhanced cytotoxicity.

17. A T cell according to any one of claims 1 to 16, T cells exhibiting enhanced resistance to cancer cells expressing CD95L.

18. A T cell according to any one of claims 1 to 17, T cells that further express the CD8 coreceptor.

19. A T cell according to claim 18, CD4 T cells are T cells.

20. A T cell according to claim 18 or 19, The CD8 coreceptors are wild-type CD8 coreceptors such as CD8α and CD8β coreceptors, which are found on T cells.

21. A T cell according to claim 18 or 19, The aforementioned CD8 coreceptor is a chimeric CD8 coreceptor, specifically a T cell.

22. A T cell according to claim 21, The chimeric CD8 coreceptor is a T cell having an amino acid sequence that is at least 85% identical to the amino acids defined in SEQ ID NO:

50.

23. A T cell according to any one of claims 1 to 22, The modified T cell receptor may specifically bind to a MAGE antigen family member such as MAGE-A1 or Mage-A4, or in this case, the modified T cell receptor specifically binds to an antigen selected from the group consisting of PRAME antigen, NY-ESO-1 antigen, GP100 antigen, AFP antigen, Col6A3 antigen, HPV-16 antigen, WT1 antigen, HA1 antigen, HA2 antigen, mutant KRAS antigen, mutant NRAS antigen, mutant HRAS antigen, mutant TP53 antigen, and EGFR antigen, thereby enabling T cells.

24. A vector comprising nucleic acid encoding a chimeric CD95 receptor containing polypeptides, The polypeptide comprises at least one CD95 polypeptide region having at least 60% sequence identity with the polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in Sequence ID No.

1. The human CD95 polypeptide region includes a CD95 extracellular ligand binding domain. Furthermore, the polypeptide comprises at least one non-CD95 costimulated cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins. A vector further containing nucleic acids encoding a modified T cell receptor.

25. A vector comprising nucleic acid encoding a chimeric CD95 receptor containing polypeptides, The polypeptide comprises at least one CD95 polypeptide region including a CD95 extracellular ligand-binding domain, Furthermore, the polypeptide comprises at least one non-CD95 costimulated cytoplasmic polypeptide domain, region, or motif of tumor necrosis factor receptor superfamily proteins, including CD40, CD40L, CD27, HVEM, GITR, CD30, OX40, and / or LTBR, ​​immunoglobulin superfamily (IgSF) proteins, Toll-like receptors, and / or IL6 receptor family proteins. A vector further containing nucleic acids encoding a modified T cell receptor.

26. A vector according to claim 24 or 25, A vector, which can be a viral or nonviral vector.

27. A vector according to claim 26, The viral vector is selected from adenovirus, smallpox virus, alphavirus, arenavirus, flavivirus, rhabdovirus, retrovirus, lentivirus, herpesvirus, paramyxovirus, picornavirus, and combinations thereof.

28. A vector according to any one of claims 24 to 27, The modified T cell receptor is a vector comprising a TCRα chain and a TCRβ chain.

29. The vector according to claim 28, The aforementioned T cell receptor is a recombinant T cell receptor vector.

30. A vector according to any one of claims 24 to 29, A vector further comprising nucleic acids encoding a CD8 coreceptor, such as wild-type or modified CD8 coreceptors.

31. A vector according to claim 30, The nucleic acid is a vector that encodes a wild-type CD8 coreceptor, such as CD8α and CD8β coreceptors, or the nucleic acid is a vector that encodes a chimeric CD8 coreceptor.

32. The vector according to claim 31, The nucleic acid described above encodes a chimeric CD8 coreceptor, Furthermore, the receptor is a vector having at least 85%, optionally at least 86%, or at least 87%, or at least 88%, or at least 89%, or at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or 100% identity with the amino acid defined in Sequence ID No.

50.

33. Isolated T cells, A T cell into which the vector according to any one of claims 24 to 32 has been introduced.

34. A T cell according to claim 1 to 23 or 33, T cells, which are αβ T cells, γδ T cells, and / or natural killer T cells.

35. A T cell according to claim 34, The αβ T cell is a CD4 T cell, or the αβ T cell is a CD8 T cell, or the γδ T cell is a Vγ9Vδ2+ T cell, or the γδ T cell includes a Vδ1 T cell.

36. A T cell according to any one of claims 1 to 23 or 33 to 35, The aforementioned T cells are T cells derived from induced pluripotent stem cells (iPSCs).

37. A kit comprising means for preparing the T cells according to any one of claims 1 to 23 or 33 to 36.

38. A pharmaceutical composition comprising T cells according to any one of claims 1 to 23 or 33 to 36.

39. A pharmaceutical composition according to claim 38, A pharmaceutical composition further comprising an adjuvant, excipient, buffer, diluent, carrier, stabilizer, or combination thereof.

40. A method for preparing T cells for immunotherapy, -T cells were isolated from human subjects, - The vector according to any one of claims 24 to 32 is introduced into the T cells, - A method comprising increasing the T cells.

41. In the method according to claim 40, A method comprising transmuting, translocating, or introducing the isolated T cells with the vector.

42. A pharmaceutical composition according to claim 38 or 39, A pharmaceutical composition comprising T cells expressing the chimeric CD95 receptor and the modified T cell receptor.

43. A pharmaceutical composition according to claim 42, A pharmaceutical composition further comprising CD4 cells expressing the chimeric CD95 receptor and further expressing a recombinant CD8 coreceptor.

44. A pharmaceutical composition according to claim 43, The aforementioned CD8 coreceptor is a chimeric CD8 coreceptor in this pharmaceutical composition.

45. A method for treating patients with a disease, A method comprising administering the composition according to claim 38, 39, 42, 43, or 44 to the patient.

46. A method for treating patients with a disease, A method comprising introducing the vector described in any one of claims 24 to 32 into the T cells of the patient in vivo.

47. The method according to claim 46, The method wherein the vector is DNA or mRNA.

48. The method according to claim 46, The vector is a non-replicating viral vector, in this method.

49. The method according to claim 46 or 47, The vector is mRNA, and the mRNA is introduced into the patient's T cells using nanoparticles.

50. A method according to any one of claims 45 to 49, The disease is cancer or an autoimmune disease, in this manner.

51. The method according to claim 50, The cancer is selected from the group consisting of non-small cell lung cancer, small cell lung cancer, pancreatic cancer, ovarian cancer, melanoma, breast cancer, liver cancer, kidney cancer, esophageal cancer, brain tumor, gastric cancer, Merkel cell carcinoma, leukemia, bladder cancer, uterine cancer, colorectal cancer, gallbladder cancer, bile duct cancer, and prostate cancer.

52. A method according to claim 50 or 51, A method for cancer cells to express FasL.

53. A method for increasing the cytotoxicity of T cells in adoptive cell therapy, - A method comprising introducing the vector according to any one of claims 24 to 32 into the T cells.

54. The method according to claim 53, The vector encodes a CD8 coreceptor, such as a human wild-type CD8 coreceptor or a chimeric CD8 coreceptor.

55. The method according to claim 53 or 54, The method wherein the T cell receptor is a recombinant T cell receptor that specifically binds to a tumor-specific antigen.

56. It is a chimeric human CD95 receptor, Contains polypeptides, The polypeptide comprises at least one CD95 polypeptide region having at least 60% sequence identity with the polypeptide domain, polypeptide region, or polypeptide motif of the human CD95 receptor as defined in Sequence ID No.

1. The human CD95 polypeptide region includes a CD95 extracellular ligand binding domain. Furthermore, the polypeptide comprises at least one domain, region, or motif of a non-CD95 costimulated cytoplasmic polypeptide. A chimeric human CD95 receptor, wherein the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

57. It is a chimeric human CD95 receptor, Contains polypeptides, The polypeptide comprises at least one CD95 polypeptide region including a CD95 extracellular ligand-binding domain, Furthermore, the polypeptide comprises at least one domain, region, or motif of a non-CD95 costimulated cytoplasmic polypeptide. A chimeric human CD95 receptor, wherein the at least one cytoplasmic polypeptide domain or cytoplasmic polypeptide motif is a cytoplasmic polypeptide domain or cytoplasmic polypeptide motif selected from the group consisting of CD40L, CD2, TLR2, TLR4, and IL6R subunit beta.

58. A chimeric CD95 receptor according to claim 56 or 57, The extracellular ligand-binding domain is a chimeric CD95 receptor that has the functionality to bind FAS-ligand.

59. A chimeric CD95 receptor according to claim 56, 57, or 58, The polypeptide is a single-chain polypeptide, a chimeric CD95 receptor.

60. A chimeric CD95 receptor according to any one of claims 56 to 59, The CD95 polypeptide region further comprises at least one, at least two, or at least three CD95 high-cysteine ​​domains (CRDs), thereby a chimeric CD95 receptor.

61. A chimeric CD95 receptor according to any one of claims 56 to 60, The chimeric CD95 receptor comprising at least one CD95 polypeptide region further comprising an extracellular N-terminal PLAD region.

62. A chimeric CD95 receptor according to any one of claims 56 to 61, The CD95 receptor wherein the at least one CD95 polypeptide region further comprises a CD95 isomorphic interaction domain.

63. A chimeric CD95 receptor according to any one of claims 56 to 62, The CD95 receptor comprises at least one CD95 polypeptide region including a CD95 transmembrane region.

64. A chimeric CD95 receptor according to any one of claims 56 to 63, The CD95 polypeptide region is a CD95 receptor containing the CD95 extracellular domain.

65. A chimeric CD95 receptor according to claim 64, The CD95 polypeptide region is a chimeric CD95 receptor comprising a CD95 extracellular domain and a CD95 transmembrane domain.

66. A chimeric CD95 receptor according to any one of claims 56 to 65, The CD95 polypeptide region comprises at least one linker region, thus forming a chimeric CD95 receptor.

Images