cell

Abstract

To provide a manipulated cell that expresses a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) and a Fas ligand (FasL)-bound receptor.SOLUTION: Provided is a cell containing: (a) CAR or TCR; and (b) (i) a Fas ectodomain and a TNFR endodomain where the TNFR endodomain includes a signaling portion of a decoy receptor 2(DcR2), GITR, CD30, XEDAR, CD40, CD27, BCMA or a Fn14 endodomain, or (ii) a FasL-bound receptor (FLBR) including a membrane-binding decoy receptor 3(DcR3).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to engineered cells that express a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) and a Fas ligand (FasL) binding receptor. [Background technology]

[0002] The tumor microenvironment (TME) provides tumor cells with signals essential for survival, proliferation, and immune resistance. The TME is immunosuppressive and can inhibit the persistence and survival of immune cells in cancer immunotherapy, such as CAR T cell therapy. Immunosuppressive mechanisms used by the TME include, for example, upregulation of immune checkpoint signals, such as PDL-1 and / or CTLA-4, and secretion of cytokines, such as IL-6 and / or TGF-β.

[0003] Recent evidence suggests that the immunosuppressive TME also upregulates death ligands, such as Fas ligand (FasL), which induces apoptosis of immune cells that express the Fas death receptor, such as tumor-infiltrating lymphocytes (TILs).

[0004] In addition to the TME upregulating death ligands, it has also been shown that CAR-T cells themselves, upon activation and transduction of CAR constructs, upregulate death receptors and their ligands, triggering activation-induced cell death (AICD), further exacerbating the problem of CAR-T cell persistence in the TME. Furthermore, FasL is not only expressed by activated T cells, but is also upregulated by exposure to IFNγ produced by activated T cells.

[0005] In particular, third-generation CARs with two costimulatory endodomains appear to be particularly susceptible to AICD as a result of increased FasL expression (Non-Patent Document 1, Non-Patent Document 2).

[0006] Scientists have attempted to block the Fas / FasL interaction using antibody technology, but in vivo data have shown that significant amounts of antibody are required every 2-3 days and that substantial side effects occur (Non-Patent Document 3).

[0007] Furthermore, because FasL is an important weapon for T cells to exert cell killing, blocking FasL with an antibody can interfere with the cell-killing effect of CAR T cells (Non-Patent Document 4).

[0008] Therefore, alternative approaches are needed to suppress death receptor stimulation on CAR T cells to alleviate this immune checkpoint and improve the efficacy of engineered cells in persisting and surviving in the TME. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Xu et al., 2017, Hum Vaccin Immunother. 13(7):1548-1555 [Non-patent document 2] Benmebarek et al., 2019, In ​​J Mol Sci.20(6): 1283 [Non-patent document 3] Ma et al., 2016, PLoS Pathog.12(5): e1005642 [Non-patent document 4] Gargett et al., 2016, Mol Ther.24:1135-49 Summary of the Invention

[0010] The present inventors have determined that cells expressing receptors that competitively bind to FasL and neutralize the Fas-FasL pathway can reduce or prevent Fas-induced apoptosis. Co-expression of these receptors with CAR or transgenic TCR improves the infiltration of CAR / TCR-expressing cells into the tumor vasculature and their persistence within the TME.

[0011] We propose two alternative approaches to achieve neutralization of the Fas-FasL pathway, both of which competitively bind to FasL, blocking its ability to induce trimerization and recruit a protein termed Fas-associated death domain (FADD) through homotypic interactions of their respective death domains.

[0012] The first approach involves fusing the extracellular domain of the Fas receptor to the tumor necrosis factor receptor (TNFR) intracellular signaling domain, and the second approach involves expressing membrane-bound decoy receptor 3 (DcR3), which binds to FasL.

[0013] Thus, in a first aspect, the present invention provides a cell comprising (a) a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) and (b) a FasL-binding receptor (FLBR) comprising (i) a Fas ectodomain and a TNFR endodomain, wherein the TNFR endodomain comprises the signaling portion of decoy receptor 2 (DcR2), GITR, CD30, XEDAR, CD40, CD27, BCMA, or Fn14 endodomain, or (ii) membrane-bound decoy receptor 3 (DcR3).

[0014] Unlike the endodomain of the Fas receptor, the TNFR endodomain of the present invention cannot bind to the death domain on the Fas-associated death domain (FADD) through homotypic interaction, thus inhibiting the assembly of the death-inducing signaling complex (DISC) and ultimately the Fas-FasL pathway leading to apoptosis. Similarly, membrane-bound DcR3 outcompetes the binding of endogenous Fas receptor to FasL, neutralizing the Fas-FasL pathway.

[0015] In the first approach described above, the FLBR of the invention has the general structure: Fas Exo-TM-TNFR End (In the formula, Fas exo is the Fas ectodomain, TM is the transmembrane domain, The TNFR endodomain may comprise a TNFR endodomain.

[0016] In particular, the FLBR may comprise a Fas ectodomain and a CD40 endodomain. The CD40 endodomain may comprise the sequence shown in SEQ ID NO:4.

[0017] The FLBR of the present invention may comprise a Fas transmembrane domain. Alternatively, the transmembrane domain of the FLBR may comprise a CD28 transmembrane domain, a CD8a transmembrane domain, a DcR2 transmembrane domain, a TYRP-1 transmembrane domain, or an EGFR transmembrane domain.

[0018] In the second approach described above, the FLBR has the general structure: DcR3-spacer-TM-end (In the formula, DcR3 contains the FasL-binding domain of DcR3, the spacer is a spacer sequence that links DcR3 to the transmembrane domain; TM is the transmembrane domain, The end may have an optional intracellular sequence).

[0019] Membrane-bound DcR3 can be tethered to the membrane through the CD8 transmembrane stalk. Alternatively, membrane-bound DcR3 can be membrane-tethered through any sequence capable of anchoring the otherwise soluble decoy receptor to the plasma membrane.

[0020] Membrane-bound DcR3 can contain mutations in the C-terminal heparin-binding domain (HBD), because the HBD binds to heparan sulfate proteoglycans (HSPGs), which can induce apoptosis in dendritic cells through cross-linking. More specifically, the HBD C-terminal portion of membrane-bound DcR3 can contain mutations at one or more of positions K256, R258, and R259, with respect to the sequence shown below as SEQ ID NO: 9. Mutation of these otherwise basic amino acids to alanine residues abolishes binding to HSPGs.

[0021] FLBR may comprise a sequence selected from Fas-DcR2 (SEQ ID NO: 35), Fas-GITR (SEQ ID NO: 36), Fas-CD30 (SEQ ID NO: 37), Fas-XEDAR (SEQ ID NO: 38), Fas-CD27 (SEQ ID NO: 39), Fas-BCMA (SEQ ID NO: 40), Fas-CD40 (SEQ ID NO: 41), Fas-Fn14 (SEQ ID NO: 42), DcR3-CD8STK (SEQ ID NO: 43), and DcR3-mutant-CD8STK (SEQ ID NO: 44), or a variant having at least 80% sequence identity to any of SEQ ID NOs: 35-44. FLBR may also comprise two or more TNFR endodomains. For example, the sequence of FLBR may be Fas-GITR-GITR, Fas-GITR-CD30, Fas-GITR-XEDAR, or Fas-GITR-DcR2. Alternatively, the FLBR can be, for example, DcR3-41BB, DcR3-OX40, DcR3-XEDAR, DcR3-GITR, DcR3-CD40, DcR3-CD27, DcR3-BCMA, or DcR3-Fn14.

[0022] In a second aspect, the present invention provides a FasL-binding receptor (FLBR) comprising a Fas ectodomain and a TNFR endodomain, wherein the TNFR endodomain comprises the signaling portion of a decoy receptor 2 (DcR2), GITR, CD30, XEDAR, CD40, CD27, BCMA, or Fn14 endodomain. In particular, the TNFR endodomain can be a CD40 endodomain.

[0023] In a third aspect, the present invention provides a nucleic acid sequence encoding a FLBR of the present invention.

[0024] In a fourth aspect, the present invention provides a nucleic acid construct comprising (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), and (b) a second nucleic acid sequence encoding a FasL-binding receptor (FLBR) as defined above.

[0025] The first and second nucleic acid sequences can be separated by a co-expression moiety, for example a sequence encoding a self-cleaving peptide.

[0026] In a fifth aspect, the present invention provides a kit of nucleic acid sequences comprising (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR) and (b) a second nucleic acid sequence encoding a FasL-binding receptor (FLBR) as defined above.

[0027] In a sixth aspect, the present invention provides a vector comprising a nucleic acid sequence according to the third aspect of the invention or a nucleic acid construct according to the fourth aspect of the invention.

[0028] In a seventh aspect, the present invention provides a method for producing a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), comprising: (a) a first vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); and (b) a first vector comprising the above. and a second vector comprising a nucleic acid sequence encoding a FasL-binding receptor (FLBR) as defined above.

[0029] In an eighth aspect, the present invention provides a pharmaceutical composition comprising a plurality of cells according to the first aspect of the invention.

[0030] In a ninth aspect, the present invention provides a pharmaceutical composition according to the eighth aspect of the invention for use in treating and / or preventing disease.

[0031] In a tenth aspect, the present invention provides a method for treating and / or preventing a disease, the method comprising administering to a subject in need thereof a pharmaceutical composition according to the eighth aspect of the invention.

[0032] The method may comprise the steps of (i) isolating a cell-containing sample, (ii) transducing or transfecting the cells with a nucleic acid sequence according to the third aspect of the invention, a nucleic acid construct according to the fourth aspect of the invention, a kit of nucleic acid sequences according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention or a kit of vectors according to the seventh aspect of the invention, and (iii) administering the cells from (ii) to a subject.

[0033] The cells can be autologous or allogeneic.

[0034] In an eleventh aspect, the present invention provides the use of a pharmaceutical composition according to the eighth aspect of the invention in the manufacture of a medicament for the treatment and / or prevention of a disease.

[0035] The disease may be cancer.

[0036] In a twelfth aspect, the present invention provides a method for producing a cell according to the first aspect of the invention, the method comprising the step of introducing into a cell in vitro a nucleic acid sequence according to the third aspect of the invention, a nucleic acid construct according to the fourth aspect of the invention, a kit of nucleic acid sequences according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention or a kit of vectors according to the seventh aspect of the invention.

[0037] The cells may be derived from a sample isolated from a subject.

[0038] Surprisingly, the inventors have found that expression of the FasL-binding receptor (FLBR) of the present invention by a cell confers greater resistance to FasL-mediated apoptosis than cells expressing a Fas receptor that simply contains a truncated Fas intracellular death domain. The inventors propose to exploit this finding to generate CAR- and TCR-expressing cells that are more effectively resistant to FasL induced within the TME, thus providing superior immunotherapy. [Brief explanation of the drawings]

[0039] [Figure 1] (a) Schematic diagram illustrating classical CARs. (b)-(d): Different generations and permutations of CAR endodomains. (b) The first designs transduced only ITAM signals through FcεR1-γ or CD3ζ endodomains, while later designs transduced additional (c) one or (d) two costimulatory signals within the same composite endodomain. [Figure 2] FIG. 1 is a schematic diagram illustrating wild-type Fas-FasL-induced apoptosis (a), and the Fas-L-binding receptors (FLBRs), Fas-DcR2 (b), Fas-TNFR (c), membrane-bound DcR3 (d), and DcR3 structure (e). [Figure 3] Figure 1 shows an overview of the TNF superfamily: Fas and TRAIL receptor molecules both use FADD as an adaptor molecule, while TNFR1 and D3 use TRADD as an adaptor molecule. [Figure 4] Schematic diagram of Fas-FasL interactions in the tumor microenvironment (TME). FasL is expressed by T cells / CAR-T cells and overactivated T cells leading to activation-induced cell death (AICD) (a), MDSCs (b), Tregs (c), tumor endothelial cells (d), cancer cells (e), and cancer-secreted exosomes (f). [Figure 5] FIG. 1 is a schematic diagram of a T cell co-culture assay. [Figure 6]Flow cytometry data of total cell viability comparing CAR co-expressed with FasL-binding constructs to cell counts of Fmc63 (anti-CD19 CAR)-transduced PBMCs. The FasL-binding receptor (FLBR) constructs Fas-DcR2, Fas-GITR, and Fas-CD30, and DcR3-CD8stk, show superior cell death rescue compared to other constructs. [Figure 7] 1 shows flow cytometry data comparing total cell viability of CAR co-expressed with the FasL-binding constructs DcR3 and DcR3-CD8stk. Membrane-bound DcR3-CD8stk showed superior cell viability to soluble DcR3. [Figure 8] Flow cytometry data comparing the survival of transduced RQR8-positive cells. The FasL-binding receptor (FLBR) constructs Fas-DcR2, Fas-GITR, and Fas-CD30 demonstrate superior cell death rescue compared to other constructs. The dotted line indicates the mean survival of PBMCs transduced to co-express Fmc63, FasΔDD, and FasL. [Figure 9] Flow cytometry data of total cell viability using immobilized recombinant soluble Fas ligand to induce cell death comparing CAR co-expressed with FasL-binding constructs to Fmc63. The FasL-binding receptor (FLBR) constructs Fas-DcR2 and Fas-GITR show superior cell death rescue compared to other constructs. [Figure 10] Flow cytometry data of transduced RQR8-positive cell survival using immobilized recombinant soluble Fas ligand to induce cell death, comparing CAR co-expressed with a FasL-binding construct to Fmc63. [Figure 11] Flow cytometry data comparing the viability of absolute RQR8-positive cells using immobilized soluble recombinant Fas ligand to induce cell death. The FasL-binding receptor construct, Fas-XEDAR, constitutively expands over a 5-day time course and rescues FasL-induced cell death. [Figure 12]Flow cytometry data using immobilized soluble Fas ligand to induce cell death comparing the proliferation fold difference in absolute RQR8-positive cell numbers between day 5 and day 0. The FasL-binding receptor construct, Fas-XEDAR, shows a much higher proliferation fold difference than the truncated Fas intracellular death domain construct. [Figure 13] Flow cytometry data comparing the percentage of transduced RQR8-positive cells using immobilized soluble Fas ligand to induce cell death. The FasL-binding receptor (FLBR) construct, Fas-XEDAR, shows a higher enrichment of RQR8-positive cells than the excised Fas intracellular death domain at days 0 and 5. [Figure 14] Flow cytometry data comparing survival of transduced RQR8-positive cells using immobilized soluble Fas ligand to induce cell death. The FasL-binding receptor (FLBR) construct, Fas-XEDAR, shows protection from FasL-mediated cell death as evident under "FasL + anti-Fmc63" conditions. [Figure 15] Flow cytometry data using immobilized soluble Fas ligand to induce cell death show that the FasL-binding receptor (FLBR) construct, Fas-XEDAR, secretes significantly higher basal interferon-γ than the truncated Fas intracellular death domain. [Figure 16] 10 is flow cytometry data showing that the FasL-binding receptor (FLBR) construct, Fas-XEDAR, secretes significantly more inducible IL-2 than the truncated Fas intracellular death domain. [Figure 17] Flow cytometry data using immobilized soluble Fas ligand to induce cell death comparing viability and absolute cell numbers of transduced RQR8-positive cells. Surprisingly, PBMCs transduced with the FLBR constructs Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 have higher absolute RQR8-positive cell numbers compared to FasΔDD and Fas-41BB. [Figure 18]Flow cytometry data using immobilized soluble Fas ligand to induce cell death comparing absolute cell numbers of transduced RQR8-positive cells to transduced PBMCs incubated on PBS-treated wells. PBMCs transduced with the FLBR constructs Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 have superior proliferation when incubated with immobilized FasL compared to FasΔDD. [Figure 19] Flow cytometry data using immobilized soluble Fas ligand to induce cell death comparing the proliferation fold difference of transduced RQR8-positive cells (absolute cell counts on day 5 versus absolute cell counts on day 0). PBMCs transduced with FLBR constructs Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 have superior proliferation when incubated with immobilized FasL compared to FasΔDD and Fas-41BB. FLBR constructs Fas-CD40, Fas-BCMA, and Fas-Fn14 also induce homeostatic proliferation (compared to PBS conditions). [Figure 20] Flow cytometry data using immobilized soluble Fas ligand to induce cell death showing that the FLBR constructs Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 secrete significantly more interferon-γ than FasΔDD when incubated with Fas ligand. The FLBR constructs Fas-CD40, Fas-BCMA, and Fas-Fn14 also induce basal interferon-γ secretion (compared to PBS conditions). [Figure 21]Figure 1 shows that Fas-XEDAR, Fas-CD40, Fas-BCMA, and Fas-Fn14 coexpressed with GD2-targeting CAR T cells increase cytotoxicity after repeated antigen encounters. GD2-targeting CAR T cells were cocultured with SupT1 GD2 targets at a 1:1 effector-to-target ratio. Every 3 or 4 days, CAR T cells were restimulated with 0.5 x 105 SupT1 GD2 cells / well. Target cell killing was quantified by FACS before each new restimulation. Remaining viable target cells were defined by Sytox Blue exclusion and the absence of CD2 and CD3 expression, while T cells were defined by CD2 and CD3 expression. Lines represent median values ​​from three separate PBMC donors. DETAILED DESCRIPTION OF THE INVENTION

[0040] Fas-FasL pathway The Fas receptor (CD95, tumor necrosis factor receptor superfamily member 6, Uniprot number P25445) is a type 1 transmembrane glycoprotein receptor with a relative molecular weight of approximately 45,000 that is located on the surface of a variety of cells, including lymphocytes and hepatocytes. The Fas receptor triggers signaling pathways that lead to apoptosis, and Fas expression can be increased by lymphocyte activation and by cytokines such as IFNγ and TNF. The interaction of Fas with its ligand FasL (FasL / CD95L, Uniprot number P48023) regulates many physiological and pathological processes mediated through programmed cell death.

[0041] Both Fas and FasL are members of the TNF-R superfamily and contain one to five extracellular cysteine-rich domains (CRDs) and a death domain (DD) in their cytoplasmic tails, consisting of a motif 80 to 100 residues in length.

[0042] Binding of Fas to FasL involves receptor trimerization and the homodimerization of its death domain (DD). This induces the recruitment of a protein called Fas-associated death domain (FADD) through a type-II interaction. FADD then recruits procaspase-8 to the activated receptor, and the resulting death-inducing signaling complex (DISC) proteolytically activates caspase-8, which initiates the subsequent cascade of caspases (aspartate-specific cysteine ​​proteases) that mediate apoptosis (Figures 2a and 3).

[0043] FasL is an important immune checkpoint because it is overexpressed by many cells within the TME (Figure 4). FasL has been reported to be expressed by many cancers themselves, including melanoma, lung cancer, hepatocellular carcinoma, esophageal cancer, and colon cancer.

[0044] Furthermore, it has been shown that tumor endothelial cells, which line blood vessels and regulate blood and nutrient flow and leukocyte trafficking, express FasL, whereas normal vasculature does not.

[0045] Furthermore, FasL is expressed by myeloid-derived suppressor cells (MDSCs), a heterogeneous population of cells that expand during cancer, chronic inflammation, autoimmune, and infectious diseases, dampening the immune response and thereby promoting tumor growth. Finally, FasL has also been reported to be expressed by cancer-associated fibroblasts (CAFs) and CD4+CD25+ regulatory T cells.

[0046] FasL is also expressed by T cells and has been shown to be further upregulated in CAR-T cells, meaning that CAR T cells are sensitive to fratricide. Finally, FasL is not only expressed by activated T cells, but is also upregulated by exposure to IFNγ produced by activated T cells.

[0047] As a mechanism of T cell homeostasis, constantly activated T cells die through a mechanism called activation-induced cell death (AICD), and the Fas-FasL pathway has been characterized as the cause of this. Despite improved cytolytic activity and cytokine production, third-generation CAR-T cells are more susceptible to AICD as a result of increased FasL expression.

[0048] Thus, avoiding apoptosis induced through the Fas-FasL pathway provides adoptively transferred cells with a significant advantage, both in terms of invasion (through the tumor vasculature) and persistence within the TME.

[0049] FasL-binding receptor (FLBR) The present invention relates to a FasL-binding receptor (FLBR) comprising a Fas ectodomain and a tumor necrosis factor receptor (TNFR) endodomain. FLBR has the general structure: Fas Exo-TM-TNFR End (In the formula, Fas exo is the extracellular domain of Fas, TM is the transmembrane domain, TNFR endodomain is the endodomain of the TNF receptor.

[0050] Fas ectodomain The sequence of human Fas is available from Uniprot (accession number P25445) and is shown below as SEQ ID NO: 49. In this sequence, residues 26-173 form the extracellular domain (SEQ ID NO: 50), residues 174-190 form the transmembrane domain (SEQ ID NO: 20), and residues 191-335 form the cytoplasmic domain (SEQ ID NO: 51). In SEQ ID NO: 51, the portion of the sequence deleted in the truncated Fas (FasΔDD) described in the Examples is underlined.

[0051] SEQ ID NO: 49 (human Fas) MLGIWTLLPLVLTSVARLSSKSVNAQVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKE EGSRSNLGWLCLLLLPIPLIVWVKRKEVQKTCRKHRKENQGSHESPTLNPETVAINLSDVDLSKYITTIAGVMTLSQVKGFVRKNGVNEAKIDEIKNDNVQDTAEQKVQLLRNWHQLHGKKEAYDTLIKDLKKALCTLAEKIQTIILKDITSDSENSNFRNEIQSLV

[0052] SEQ ID NO: 50 (Fas extracellular domain) QVTDINSKGLELRKTVTTVETQNLEGLHHDGQFCHKPCPPGERKARDCTVNGDEPDCVPCQEGKEYTDKAHFSSKCRRCRLCDEGHGLEVEINCTRTQNTKCRCKPNFFCNSTVCEHCDPCTKCEHGIIKECTLTSNTKCKEEGSRSN

[0053] SEQ ID NO: 51 (Fas cytoplasmic domain) TIFF2025170345000001.tif19170

[0054] The FLBR of the present invention may comprise the Fas extracellular domain shown as SEQ ID NO: 50, or a variant thereof that is at least 80%, 90%, 95%, or 99% identical to SEQ ID NO: 50, provided that the resulting FLBR molecule competes with endogenous Fas for binding to FasL and has no or a reduced ability to bind FADD.

[0055] The percent identity between two polypeptide sequences can be calculated at http: / / blast.ncbi.nlm.nih.gov This can be readily determined by programs such as BLAST, which is freely available. Suitably, percent identity is determined across the entire reference and / or query sequence.

[0056] FLBR transmembrane domain FLBR contains a transmembrane domain that spans the membrane. The transmembrane domain can be any protein structure that is thermodynamically stable in the membrane, e.g., does not dimerize. It is typically an alpha helix composed of several hydrophobic residues. The transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion. The presence and span of a transmembrane domain of a protein can be determined by one skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Furthermore, the transmembrane domain of a protein can be determined by one skilled in the art using the TMHMM algorithm (http: / / www.cbs.dtu.dk / services / TMHMM-2.0 / ). Artificially designed TM domains can also be used, provided the domain is a relatively simple structure, i.e., a polypeptide sequence predicted to form a hydrophobic alpha helix of sufficient length to span the membrane (U.S. Pat. No. 7,052,906 describes transmembrane components).

[0057] The transmembrane domain may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from Fas. The transmembrane domain may comprise the sequence set forth as SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity.

[0058] SEQ ID NO: 20 (Fas transmembrane domain) LGWLCLLLLPIPLIVWV

[0059] Variants may have at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 20, provided that the variant sequence retains the ability to cross membranes.

[0060] The transmembrane domain may be based on a transmembrane domain from a TNFR, such as a TNFR as described herein. Suitably, the transmembrane domain is an endoderm present in FLBR. May be based on the same TNFR as the main.

[0061] Suitably, the transmembrane domain may comprise any one of SEQ ID NOs: 20 to 34, or a variant thereof having at least 80% sequence identity. Variants may have at least 90%, 95%, 98% or 99% sequence identity with SEQ ID NOs: 21 to 34, provided that the variant sequence retains the ability to cross the membrane.

[0062] SEQ ID NO: 21 (DcR2 transmembrane domain) YLIIIVVLVIILAVVVVGFSC

[0063] SEQ ID NO: 22 (GITR transmembrane domain) LGWLTVVLLAVAACVLLLTSA

[0064] SEQ ID NO: 23 (CD30 transmembrane domain) PVLFWVILVLVVVVGSSAFLL

[0065] SEQ ID NO: 24 (CD8 transmembrane domain) APTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0066] SEQ ID NO: 25 (CD28 transmembrane domain) FLFVLLGVGSMGVAAIVWGAW

[0067] SEQ ID NO: 26 (4-1BB transmembrane domain) IISFFLALTSTALLFLLFFLTLRFSVV

[0068] SEQ ID NO: 27 (DR3 transmembrane domain) MFWVQVLLAGLVVPLLLGATL

[0069] SEQ ID NO: 28 (OX40 transmembrane domain) VAAILGLGLVLGLLGPLAILL

[0070] SEQ ID NO: 29 (CD70 transmembrane domain) VLRAALVPLVAGLVICLVVCI

[0071] SEQ ID NO: 30 (CD40 transmembrane domain) ALVVIPIIFGILFAILLVLVFI

[0072] SEQ ID NO: 31 (XEDAR transmembrane domain) LVALVSSLLVVF TLAFLGLFF

[0073] SEQ ID NO: 32 (Fn14 transmembrane domain) ILGGALSLTFVLGLLSGFLVW

[0074] SEQ ID NO: 33 (BCMA transmembrane domain) ILWTCLGLSLIISLAVFVLMFLL

[0075] SEQ ID NO: 34 (CD27 transmembrane domain) ILVIFSGMFLVFTLAGALFLH

[0076] TNFR endodomain Structural motifs in the cytoplasmic domains of the TNF superfamily are classified into two groups based on their signaling properties: those containing a death domain (DD) and those that associate with TNFR-associated factors (TRAFs). There is a third group that lacks a membrane anchor domain and is either proteolytically cleaved from the surface or anchored through glycolipid linkages, termed "decoy receptors."

[0077] A list of TNFRs is provided in Table 1.

[0078] [Table 1] TIFF2025170345000003.tif74170

[0079] The FLBR of the present invention may comprise the endodomain of a TNFR or a signaling portion thereof. The TNFR may be selected from the group consisting of GITR, DcR2, CD30, XEDAR, CD40, CD27, BCMA, or Fn14.

[0080] Glucocorticoid-inducible TNF receptor (GITR) FLBR may contain the endodomain of GITR (Uniprot number Q9Y5U5). GITR is a cell surface receptor constitutively expressed on T cells, and its surface expression is increased upon CD3 / 28 stimulation. GITR is a costimulatory receptor, and its activation, through TRAF2 / 5 recruitment, leads to NFκB and MAP kinase signaling, resulting in upregulation of CD25 and secretion of IL-2 and IFNγ.

[0081] The GITR endodomain is shown in SEQ ID NO: 1. FLBR can comprise SEQ ID NO: 1 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1 and retaining the ability to induce GITR-mediated signaling.

[0082] SEQ ID NO: 1 (GITR endodomain) QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

[0083] CD30 endodomain FLBR may contain the endodomain of CD30 (Uniprot number P28908). CD30, also known as TNFRSF8, is a cell membrane protein of the TNFR family and a tumor marker. This receptor is expressed by activated T cells and B cells. TRAF2 and TRAF5 can interact with this receptor and mediate signaling that leads to activation of NFκB. This receptor is a positive regulator of apoptosis and has also been shown to limit the proliferative potential of autoreactive CD8 effector T cells, protecting the body against autoimmunity. Two alternatively spliced ​​transcript variants of this gene, encoding distinct isoforms, have been reported.

[0084] The CD30 endodomain is shown in SEQ ID NO: 2. FLBR can comprise SEQ ID NO: 2 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 2 and retaining the ability to induce CD30-mediated signaling.

[0085] SEQ ID NO: 2 (CD30 endodomain) HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK

[0086] XEDAR End Domain FLBR may contain the endodomain of XEDAR (Uniprot number QPHAV5). XEDAR, also known as TNFRSF27 or EDA2R (ectodysplasin A2 receptor), is a type III transmembrane protein of the TNFR (tumor necrosis factor receptor) superfamily that contains three cysteine-rich repeats and a single transmembrane domain but lacks an N-terminal signal peptide. This protein mediates activation of the NFκB and JNK pathways. Activation is mediated by binding to TRAF3 and TRAF6.

[0087] The XEDAR endodomain is shown in SEQ ID NO: 3. FLBR can comprise SEQ ID NO: 3 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 3 and retaining the ability to induce XEDAR-mediated signaling.

[0088] SEQ ID NO: 3 (XEDAR endodomain) TSNTKCKEEGSRSNLGWLCLLLLPIPLIVWVLYCKQFFNRHCQRGGLLQFEADKTAKEESLFPVPPSKETSAESQVSENIFQTQPLNPILEDDCSSTSGFPTQESFTMASCTSESHSHWVHSPIECTELDLQKFSSSASYTGAETLGGNTVESTGDRLELNVPFEVPSP

[0089] CD40 endodomain FLBR may contain the endodomain of CD40 (Uniprot number P25942). CD40 (cluster of differentiation 40) or TNFRSF5 (tumor necrosis factor superfamily member 5) is a costimulatory protein found on antigen-presenting cells and is required for their activation. This receptor has been found to be essential in mediating a wide variety of immune and inflammatory responses, including T cell-dependent immunoglobulin class switching, memory B cell development, and germinal center formation. CD40 transduces TRAF6- and MAP3K8-mediated signals that activate ERK in macrophages and B cells, leading to the induction of immunoglobulin secretion.

[0090] The CD40 endodomain is shown in SEQ ID NO: 4. FLBR can comprise SEQ ID NO: 4 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 4 and retaining the ability to induce CD40-mediated signaling.

[0091] SEQ ID NO: 4 (CD40 endodomain) KKVAKKPTNKAPHPKQEPQEINFPDDLPGSNTAAPVQETLHGCQPVTQEDGKESRISVQERQ

[0092] CD27 endodomain FLBR may contain the endodomain of CD27 (Uniprot number QPHAV5). CD27, also known as TNFRSF7 (tumor necrosis factor receptor superfamily 7) or T-cell activation antigen CD27, is a transmembrane protein required for the generation and long-term maintenance of T-cell immunity. CD27 binds to the ligand CD70 and plays a key role in regulating B-cell activation and immunoglobulin synthesis. This receptor transduces signals that lead to the activation of NFκB and MAPK8 / JNK. The adaptor proteins TRAF2 and TRAF5 have been shown to mediate the signaling process of this receptor. The pro-apoptotic protein CD27-associated protein (SIVA) binds to this receptor. and is thought to play an important role in apoptosis induced by this receptor.

[0093] The CD27 endodomain is shown in SEQ ID NO: 5. FLBR may comprise SEQ ID NO: 5 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 5 and retaining the ability to induce GITR-mediated signaling.

[0094] SEQ ID NO: 5 (CD27 endodomain) QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

[0095] BCMA endodomain FLBR may contain an endodomain derived from BCMA (Uniprot number QO2223). BCMA (B-cell maturation antigen) is also known as TNFRSF17 (tumor necrosis factor receptor superfamily member 17), a protein encoded by the TNFRSF17 gene in humans. TNFRSF17 is a cell surface receptor of the TNF receptor superfamily that recognizes B-cell activating factor (BAFF / TNFSF13B) and A-growth-inducing ligand (APRIL / TNFSF13). This receptor promotes B-cell survival and plays a role in regulating humoral immunity. This receptor also activates NFκB and JNK.

[0096] The BCMA endodomain is shown in SEQ ID NO: 6. FLBR may comprise SEQ ID NO: 6 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 6 and which retains the ability to induce GITR-mediated signaling.

[0097] SEQ ID NO: 6 (BCMA endodomain) RKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLEYTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKSISAR

[0098] Fn14 endodomain FLBR may contain the endodomain of Fn14 (Uniprot number Q9NP84). Fn14, also known as TNFRSF12A, is a receptor for TNFSF12 / TWEAK and is a weak inducer of apoptosis in certain cell types. Fn14 may also promote angiogenesis and endothelial cell proliferation and regulate cell adhesion to matrix proteins.

[0099] The Fn14 endodomain is shown in SEQ ID NO: 7. FLBR may comprise SEQ ID NO: 7 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 7 and retaining the ability to induce Fn14-mediated signaling.

[0100] SEQ ID NO: 7 (Fn14 endodomain) RRCRRREKFTTPIEETGGEGCPAVALIQ

[0101] DcR2 (decoy receptor 2) FLBR may comprise a portion of the endodomain of DcR2 (Uniprot number Q9UBN6), also known as TRAIL4 or TNFRSF10D (tumor necrosis factor receptor superfamily 10D), which is the cell surface receptor for the cytotoxic ligand TRAIL.

[0102] The FLBR of the present invention can comprise the intracellular domain of DcR2, which lacks a functional death domain. For example, the DcR2 endodomain contains a truncated death domain that is unable to bind FADD. Fusing the extracellular binding domain of Fas to the intracellular domain of DcR2, which has a non-functional death domain, results in FLBR, which competes with Fas for binding to FasL but is unable to bind FADD and inhibits the FasL-induced apoptotic pathway.

[0103] The DcR2 intracellular domain induces NF-κB signaling and provides pro-survival and anti-apoptotic functions, features exploited in cancer. Therefore, the combination of the Fas ectodomain and the DcR2 endodomain in the FLBR of the present invention is beneficial in counteracting the pro-apoptotic signal induced by FasL and converting the pro-apoptotic signal to a pro-survival outcome through NFκB.

[0104] The DcR2 endodomain, including the truncated death domain, is shown in SEQ ID NO: 8. FLBR can comprise SEQ ID NO: 8 or a variant thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 8 and retaining the ability to induce DcR2-mediated signaling.

[0105] SEQ ID NO: 8 (DcR2 endodomain) RKKFISYLKGICSGGGGGPERVHRVLFRRRSCPSRVPGAEDNARNETLSNRYLQPTQVSEQEIQGQELAELTGVTVESPEEPQRLLEQAEAEGCQRRRLLVPVNDADSADISTLLDASATLEEGHAKETIQDQLVGSEKLFYEEDEAGSATSCL

[0106] The FLBR of the present invention can comprise two or more TNFR endodomains. For example, the Fas ectodomain and TNFR endodomain of FLBR can be fused to the signaling portion of an additional TNFR endodomain selected from any one of the TNFR endodomains listed in Table 1. The FLBR construct can, for example, comprise a Fas receptor ectodomain and two TNFR endodomains (e.g., Fas-CD30-41BB or Fas-CD30-OX40).

[0107] Below is a list of the complete FLBR sequence, including the signal peptide (normal text), Fas ectodomain (bold), transmembrane domain (italic), and TNFR endodomain (underlined). FLBRs can contain one or part of these entire sequences. For example, a FLBR can contain a combination of the Fas ectodomain and TNFR, but with a different signal peptide and / or transmembrane domain.

[0108] SEQ ID NO: 35 (human Fas-Dcr2) TIFF2025170345000004.tif40170

[0109] SEQ ID NO: 36 (human Fas-GITR) TIFF2025170345000005.tif32170

[0110] SEQ ID NO: 37 (human Fas-CD30) TIFF2025170345000006.tif46170

[0111] SEQ ID NO: 38 (human Fas-XEDAR) TIFF2025170345000007.tif39170

[0112] SEQ ID NO: 39 (human Fas-CD27) TIFF2025170345000008.tif26170

[0113] SEQ ID NO: 40 (human Fas-BCMA) TIFF2025170345000009.tif33170

[0114] SEQ ID NO: 41 (human Fas-CD40) TIFF2025170345000010.tif32170

[0115] SEQ ID NO: 42 (human Fas-Fn14) TIFF2025170345000011.tif25170

[0116] FLBR of the present invention can comprise any one of SEQ ID NOs: 35-42 or a variant thereof having at least 80% sequence identity. The variant can have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NOs: 35-42, provided that the variant sequence retains the ability to inhibit FasL-induced apoptosis when expressed by a cell.

[0117] Membrane-bound DcR3 DcR3 (decoy receptor 3, TNFRSF6B, Uniprot number O95407) is a type I transmembrane glycoprotein that undergoes alternative splicing to generate a soluble member of the TNFR superfamily. This soluble receptor binds to FasL and neutralizes its biological function, thus inhibiting FasL-induced apoptosis. DcR3 also binds to the ligands TL1A and LIGHT, which are also involved in apoptosis induction through the receptors DR3 and HVEM, respectively.

[0118] To overcome FasL-induced CAR-T cell apoptosis in the TME, the FLBR of the present invention may contain the FasL-binding portion of DcR3, which would compete with Fas by binding to FasL. However, because DcR3 is a soluble protein, secretion of DcR3 by CAR-T may have confounding effects on host T cells (neutralizing FasL-expressing host T cell responses).

[0119] The DcR3 of the present invention is a membrane-bound DcR3 in order to reduce cell-cell contact between DcR3 and FasL present on host T cells. Membrane-bound DcR3 has the general structure: DcR3-spacer-TM-end (In the formula, DcR3 contains the FasL-binding domain of DcR3, the spacer is a spacer sequence that links DcR3 to the transmembrane domain; TM is the transmembrane domain, The end may have an optional intracellular sequence).

[0120] FLBR may comprise a Dcr3 domain having SEQ ID NO:9 or a variant thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:9 that retains the ability to bind FasL.

[0121] SEQ ID NO: 9 (DcR3) VAETPTYPWRDAETGERLVCAQCPPGTFVQRPCRRDSPTTCGPCPPRHYTQFWNYLERCRYCNVLCGEREEEARACHATHNRACRCRTGFFAHAGFCLEHASCPPGAGVIAPGTPSQNTQCQPCPPGTFSASSSS SEQCQPHRNCTALGLALNVPGSSSHDTLCTSCTGFPLSTRVPGAEECERAVIDFVAFQDISIKRLQRLLQALEAPEGWGPTPRAGRAALQLKLRRRLTELLGAQDGALLVRLLQALRVARMPGLERSVRERFLPVH

[0122] The structure of DcR3 contains four N-terminal cysteine-rich domains (CRDs) that bind to FasL, and a C-terminal heparan-binding domain (HBD) that binds to heparan sulfate proteoglycans (HSPGs) (Fig. 2e). The HBDs of DcR3 mediate the cross-linking of HSPGs to dendritic cells. It has been shown that the FasL-binding CRD induces apoptosis in cells, but not in FasL-binding CRD (You et al., 2008, Blood 111, 1480-1488).

[0123] The HBD of DcR3 consists of three basic amino acids (K 256 , R 258 and R 259 ) and binds to HSPGs through a binding motif containing residue K. 256 , R 258 and R 259 The mutation(s) may reduce or abolish binding to HSPGs. The or each mutation may be a substitution mutation. The or each mutation may include a substitution of an amino acid with alanine. FLBR may be a mutant form of DcR3 containing a mutation in one or more of the following: K, K(A), K(B), K(C), K(D ... 256 rank, R 258 Rank and R 259 The sequence may comprise the sequence shown as SEQ ID NO: 10, which contains an alanine mutation at each of the positions.

[0124] SEQ ID NO: 10 (mutant DcR3) TIFF2025170345000012.tif32170

[0125] Spacer The spacer sequence can be any sequence that physically distances the DcR3 domain from the cell membrane and / or provides a degree of flexibility. Spacer sequences commonly used in CARs can be used in the membrane-bound DcR3 FLBR of the present invention. For illustrative purposes only, a list of suitable sequences is provided in Table 2.

[0126] [Table 2]

[0127] The membrane-bound DcR3 may comprise any one of the spacers shown as SEQ ID NOs: 11-17, or variants thereof having at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NOs: 11-17.

[0128] The FLBR may comprise a short stretch of amino acids that connects the DcR3 to a spacer. The linker may comprise, for example, between 2 and 10 amino acids or between 3 and 5 amino acids. The linker may be a "standard" SDP linker sequence.

[0129] The FLBR described herein can comprise a modified DcR3 that is bound to the plasma membrane through a CD8 transmembrane stalk (CD8stk), as shown in Figures 2d and 5b.

[0130] Membrane-bound DcR3 TM domain The membrane-bound DcR3 also contains a transmembrane domain. As explained above, the transmembrane domain can be any protein structure that is thermodynamically stable in the membrane, and is typically an alpha helix containing several hydrophobic residues. The transmembrane domain can be derived from any transmembrane protein. When the spacer is derived from a transmembrane protein, such as CD8 or CD28, the TM domain can preferably be derived from the same protein.

[0131] The sequences of CD28 and CD8α are shown below as SEQ ID NOs: 52 and 53, respectively.

[0132] SEQ ID NO: 52 (CD28 TM domain) FWVLVVVGGVLACYSLLVTVAFIIFWV

[0133] SEQ ID NO: 53 (CD8 TM domain) IYIWAPLAGTCGVLLLSLVIT

[0134] The transmembrane domain may comprise the sequence shown as SEQ ID NO: 52 or 53, or a variant thereof with at least 90%, 95% or 99% sequence identity which retains the ability to span the membrane.

[0135] Membrane-bound DcR3 endodomain FLBR, including membrane-bound DcR3 mutants, may contain an intracellular polarity anchor that provides a polar region in close proximity to the plasma membrane. The polarity anchor sequence is shown below as SEQ ID NO: 18.

[0136] Alternatively, or in addition, FLBR may comprise an intracellular rigid linker. This sequence is less flexible than the commonly used glycine-serine linker and is preferred at the C-terminus of a recombinant protein sequence. The rigid linker sequence is shown below as SEQ ID NO: 19.

[0137] SEQ ID NO: 18 (polar anchor) RKKR

[0138] SEQ ID NO: 19 (rigid linker containing excision) LEAEAAAKEAAAKEAAAAKEAAAKALEAEAAAAKEAAAKEAAAKEAAAKALE

[0139] The membrane-bound DcR3 characteristic of the FLBR of the present invention may also include a TNFR endodomain in addition to or instead of the polar anchor / rigid linker. The TNFR endodomain may be selected from any one of the TNFRs listed in Table 1. The TNFR endodomain may include one of the sequences set forth as SEQ ID NOs: 1-8.

[0140] Below are two complete FLBR sequences based on membrane-bound DcR3. The sequences include the signal peptide (normal text), DcR3 (bold), regular linker (bold and underlined), spacer (italic), transmembrane domain (underlined), polar anchor (double underlined), and rigid linker (bold and italic). FLBRs can contain one of these entire sequences or portions thereof. For example, a FLBR can contain DcR3 and the polar anchor / rigid anchor, but with a different signal peptide, spacer, and / or transmembrane domain.

[0141] SEQ ID NO: 43 (Human DcR3-CD8 stalk / TM / rigid linker) TIFF2025170345000014.tif46170

[0142] SEQ ID NO: 44 (Human mutant DcR3-CD8 stalk / TM / rigid linker) TIFF2025170345000015.tif45170

[0143] The FLBR of the present invention may comprise SEQ ID NO: 43 or 44, or a variant thereof having at least 80% sequence identity. The variant may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with SEQ ID NO: 43 or 44, provided that the variant sequence retains the ability to inhibit FasL-induced apoptosis when expressed by a cell.

[0144] Nucleic acid sequence The present invention provides (i) a Fas ectodomain and a TNFR endodomain, wherein the TNFR endodomain comprises a signaling portion of a decoy receptor 2 (DcR2), GITR, CD30, XEDAR, CD40, CD27, BCMA, or Fn14 endodomain; or (ii) membrane-bound decoy receptor 3 (DcR3); The present invention provides a nucleic acid sequence encoding a FasL-binding receptor (FLBR) comprising:

[0145] The nucleic acid sequence is an "exogenous polynucleotide" in the sense that the polynucleotide that expresses the TNFR is not part of the endogenous genome of the cell. For example, the exogenous polynucleotide can be part of an engineered nucleic acid construct or vector.

[0146] As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are intended to be synonymous with each other.

[0147] It will be understood by those skilled in the art that many different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. Furthermore, one skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed.

[0148] Nucleic acids according to the present invention can include DNA or RNA. They can be single-stranded or double-stranded. They can also be polynucleotides that include synthetic or modified nucleotides therein. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. It is understood that for the purposes of the uses described herein, polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of the polynucleotide of interest.

[0149] The terms "variant," "homologue," or "derivative" in reference to a nucleotide sequence include any variant of the sequence. This includes any substitution, mutation, modification, substitution, deletion or addition of one or more nucleic acids to these or to the sequence.

[0150] nucleic acid construct In one aspect, the present invention provides a nucleic acid construct comprising: (i) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); and (ii) a second nucleic acid sequence as defined above.

[0151] Nucleic acid constructs have the general structure: CAR / TCR-coexpressing-FLBR, or FLBR-coexpressing CAR / TCR (In the formula, CAR / TCR is a nucleic acid sequence encoding a CAR or TCR, The co-expression is a nucleic acid sequence that allows for the co-expression of the CAR / TCR and the FLBR as separate polypeptides, FLBR is a nucleic acid sequence encoding a FasL-binding receptor as described herein).

[0152] In the above structure, "co-expressing" refers to a nucleic acid sequence that allows the two polypeptides to be co-expressed as separate entities. This may be a sequence that encodes a cleavage site(s), such that the nucleic acid construct produces both polypeptides linked by the cleavage site(s). The cleavage site may be self-cleaving, so that when the polypeptides are produced, they are immediately cleaved into individual peptides without the need for any external cleavage activity.

[0153] The cleavage site can be any sequence that allows the two polypeptides to be separated.

[0154] The term "cleavage" is used herein for convenience, however, a cleavage site may cause separation of a peptide into individual entities by mechanisms other than classical cleavage. For example, with regard to the foot-and-mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed to account for the "cleavage" activity: proteolysis by host cell proteinases, autoproteolysis, or translational effects (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041). When a cleavage site is placed between nucleic acid sequences encoding a protein, The precise mechanism of such "cleavage" is not important for the purposes of the present invention, so long as it results in the proteins being expressed as separate entities.

[0155] The cleavage site may be, for example, a furin cleavage site, a tobacco etch virus (TEV) cleavage site, or may encode a self-cleaving peptide.

[0156] A "self-cleaving peptide" refers to a peptide that functions such that once the protein and polypeptide comprising the self-cleaving peptide are produced, they are immediately "cleaved" or separated into distinct and separate first and second polypeptides without the need for any external cleavage activity.

[0157] The self-cleaving peptide can be a 2A self-cleaving peptide from an aphthovirus or a cardiovirus. The primary 2A / 2B cleavage of aphthoviruses and cardioviruses is mediated by 2A "cleavage" at its own C-terminus. In aphthoviruses, such as foot-and-mouth disease virus (FMDV) and equine rhinitis A virus, the 2A region is a short section of approximately 18 amino acids that, together with the N-terminal residue of protein 2B (a conserved proline residue), represents an autonomous element that can mediate "cleavage" at its own C-terminus (Donelly et al., supra). (2001)).

[0158] "2A-like" sequences have been found in picornaviruses other than aphthoviruses or cardioviruses, "picornavirus-like" insect viruses, type C rotaviruses, and repetitive sequences within Trypanosoma species and bacterial sequences (Donnelly et al (2001) as cited above).

[0159] The cleavage site may comprise the 2A-like sequence (RAEGRGSLLTCGDVEENPGP) shown in SEQ ID NO:54.

[0160] The present invention further provides a kit comprising (i) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR), and (ii) a second nucleic acid sequence that is an exogenous polynucleotide capable of expressing a FasL-binding receptor (FLBR) as defined herein.

[0161] Chimeric antigen receptor (CAR) Classical CARs, shown schematically in Figure 1, are chimeric type I transmembrane proteins linking an extracellular antigen-recognition domain (binder) to an intracellular signaling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but can be other formats containing antibody-like antigen-binding sites or based on ligands of the target antigen. A spacer domain may be required to separate the binder from the membrane and allow for its proper orientation. A common spacer domain used is the Fc of IgG1. Depending on the antigen, more compact spacers, such as the stalk from CD8α and even the IgG1 hinge alone, may be sufficient. The transmembrane domain anchors the protein in the cell membrane and links the spacer to the endodomain.

[0162] Early CAR designs had endodomains derived from either the γ chain of FcεR1 or the intracellular portion of CD3ζ. Consequently, these first-generation receptors were sufficient to transmit immunological signals1 and induce T cell killing of cognate target cells. They failed to fully activate T cells to proliferate and survive. To overcome this limitation, composite endodomains have been constructed. Fusing the intracellular portion of a T cell costimulatory molecule to that of CD3ζ results in second-generation receptors that can simultaneously transmit activation and costimulatory signals after antigen recognition. The most commonly used costimulatory domain is that of CD28, which provides the most potent costimulatory signal, i.e., immunological signal 2, which induces T cell proliferation. Several receptors have also been described, including TNF receptor family endodomains, such as the closely related OX40 and 4-1BB, which transmit survival signals. Even more potent third-generation CARs have now been described, which have endodomains capable of transmitting activation, proliferation, and survival signals.

[0163] CAR-encoding nucleic acid can be transferred to T cells, for example, using a retroviral vector. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. When CAR binds to a target antigen, it causes the transmission of an activation signal to the T cells on which the target antigen is expressed. Thus, CAR directs the specificity and cytotoxicity of T cells to cells that express the targeted antigen.

[0164] antigen-binding domain The antigen-binding domain is the part of a classical CAR that recognizes the antigen.

[0165] Many antigens, including those based on the antigen-binding site of antibodies, antibody mimetics, and T-cell receptors Binding sites are known in the art. For example, antigen-binding domains can be derived from single-chain variable fragments (scFv) derived from monoclonal antibodies, natural ligands of the target antigen, peptides with sufficient affinity for the target, single domain binders such as those derived from camelids, etc. , artificial single binders such as Darpins, or single chains derived from T cell receptors.

[0166] A variety of tumor-associated antigens (TAA) are known, some of which are shown in Table 3 below. The antigen-binding domain used in the present invention may be a domain capable of binding to a TAA such as those shown in this table.

[0167] [Table 3]

[0168] Transmembrane domain The transmembrane domain is the classical CAR sequence that spans the membrane. This domain may contain a hydrophobic alpha helix. The transmembrane domain may be derived from any transmembrane protein, as described above, or may be synthetic. The TM domain of the CAR may be derived from CD28, which confers superior receptor stability. Alternatively, the transmembrane domain may be derived from the melanosomal protein Tryp-1.

[0169] signal peptide A CAR can include a signal peptide that, when expressed in a cell, e.g., a T cell, directs the nascent protein to the endoplasmic reticulum and then to the cell surface where it is expressed.

[0170] The core of a signal peptide may contain a long stretch of hydrophobic amino acids that have a tendency to form a single alpha helix. The signal peptide may begin with a short, positively charged stretch of amino acids, which helps enforce the proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by a signal peptidase, which may cleave during or after translocation is complete, to yield a free signal peptide and a mature protein. The free signal peptide is then digested by a specific protease.

[0171] spacer domain CARs may contain a spacer sequence connecting the antigen-binding domain and the transmembrane domain. The flexible spacer allows the antigen-binding domain to be oriented in different directions to facilitate binding.

[0172] The spacer sequence may comprise, for example, an IgG1 Fc region, an IgG1 hinge, or a human or mouse CD8 stalk. Alternatively, the spacer may comprise another linker sequence with similar length and / or domain spacing characteristics as the IgG1 Fc region, IgG1 hinge, or CD8 stalk. The human IgG1 spacer may be modified to remove the Fc binding motif. The CAR may comprise one of the spacers listed in Table 2.

[0173] Intracellular signaling domains The intracellular signaling domain is the signaling portion of a classical CAR. The intracellular signaling domain can be or can include a T cell signaling domain.

[0174] The intracellular signaling domain may contain one or more immunoreceptor tyrosine-based activation motifs (ITAMs). ITAMs are conserved sequences of four amino acids that are repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system. This motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, resulting in the signature YxxL / I. Two of these signatures are typically separated in the molecular tail by between six and eight amino acids (YxxL / Ix). (6~8) YxxL / I).

[0175] ITAMs are important for signal transduction in immune cells. Thus, they are found in important cell signaling molecules, such as the CD3 and ζ chains of the T cell receptor complex, the CD79 α and β chains of the B cell receptor complex, and the tails of certain Fc receptors. Tyrosine residues within these motifs are phosphorylated after interaction of the receptor molecule with its ligand, forming docking sites for other proteins involved in cell signaling pathways.

[0176] The most commonly used signaling domain component is the CD3-ζ endodomain, which contains three ITAMs. This transduces activation signals to T cells after antigen binding. CD3-ζ may not provide a fully competent activation signal, and additional costimulatory signaling may be required. There are two main types of costimulatory signals: those belonging to the Ig family (CD28, ICOS) and those belonging to the TNF family (OX40, 41BB, CD27, GITR, etc.; see Table 1). For example, chimeric CD28 and OX40 may be used together with CD3-ζ to transduce proliferation / survival signals, or all three may be used together (illustrated in Figure 1B).

[0177] The endodomain may comprise the sequences set forth as SEQ ID NOs: 45-48, or variants thereof having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity, provided that the variant sequence retains the ability to transmit an activation signal to a cell.

[0178] SEQ ID NO: 45 (CD3-zeta endodomain) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0179] SEQ ID NO: 46 (4-1BB and CD3-zeta endodomain) MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHS PQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0180] SEQ ID NO: 47 (CD28 and CD3-zeta endodomain) SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0181] SEQ ID NO: 48 (CD28, OX40 and CD3-zeta endodomain) SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0182] Transgenic T cell receptor (TCR) T cell receptors (TCRs) are molecules found on the surface of T cells that are involved in the recognition of fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules.

[0183] The TCR is a heterodimer composed of two different protein chains: in humans, on 95% of T cells the TCR consists of an α chain and a β chain (encoded by TRA and TRB, respectively), while on 5% of T cells the TCR consists of a γ chain and a δ chain (γ / δ, encoded by TRG and TRD, respectively).

[0184] When the TCR engages with an antigenic peptide and MHC (peptide / MHC), the T lymphocyte is activated through signal transduction.

[0185] In contrast to the target antigens against which conventional antibodies are directed, antigens recognized by TCRs can include all potential intracellular proteins that are processed and delivered to the cell surface as peptide / MHC complexes.

[0186] Cells can be engineered to express heterologous (i.e., non-natural) TCR molecules by artificially introducing the TRA and TRB genes or the TRG and TRD genes into the cells using vectors. For example, genes for engineering TCRs can be reintroduced into autologous T cells and then transferred back into the patient for adoptive T cell therapy. Such "heterologous" TCRs may also be referred to herein as "transgenic TCRs."

[0187] The present invention relates to cells that co-express a transgenic TCR and a FLBR as defined herein.

[0188] vector The present invention also provides vectors, or kits of vectors, comprising one or more nucleic acid sequences encoding the FLBRs described in the present invention. Such vectors may be used to introduce the nucleic acid sequences into host cells such that they express the CAR / TCR and FLBR as defined herein.

[0189] The vector may be, for example, a plasmid or a viral vector, such as a retroviral or lentiviral vector, or a transposon-based vector or synthetic mRNA.

[0190] The vector may be capable of transfecting or transducing a cell, for example a T cell or an NK cell.

[0191] cell The present invention provides cells that co-express a CAR / TCR and a FLBR as defined herein.

[0192] The cell may contain a nucleic acid or vector of the invention.

[0193] The cell can be a cytolytic immune cell, such as a T cell or an NK cell.

[0194] T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are various types of T cells, as summarized below.

[0195] T helper cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells are activated when presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or THF, which secrete different cytokines to promote different types of immune responses.

[0196] Cytolytic T cells (TC cells, or CTLs) destroy virus-infected cells and tumor cells and also play a role in transplant rejection. CTLs express CD8 on their surface. These cells recognize their targets by binding to antigens associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine, and other molecules secreted by regulatory T cells, CD8+ cells can be inactivated to an anergic state, which prevents autoimmune diseases such as experimental autoimmune encephalomyelitis.

[0197] Memory T cells are an antigen-specific T cell subset that persists for a long period after an infection has cleared. They rapidly expand into large numbers of effector T cells upon re-exposure to cognate antigen, thus providing the immune system with a "memory" of past infection. Memory T cells include three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells can be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

[0198] Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity toward the end of an immune response and to suppress autoreactive T cells that have escaped the negative selection process in the thymus.

[0199] Two major classes of CD4+ Treg cells have been described: naturally occurring Treg cells and adaptive Treg cells.

[0200] Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and differentiate into myeloid (CD11c+) and thymocytes that are activated with TSLP. It has been implicated in interactions between both stromal cell-like (CD123+) dendritic cells and developing T cells. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations in the FOXP3 gene can prevent regulatory T cell development and cause the fatal autoimmune disease IPEX.

[0201] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) can arise during a normal immune response.

[0202] The cells may be natural killer cells (or NK cells). NK cells form part of the innate immune system. NK cells provide a rapid response to innate signals from virus-infected cells in an MHC-independent manner.

[0203] NK cells (belonging to the innate lymphoid group) are defined as large granular lymphocytes (LGLs) and constitute the third type of cell that differentiates from a common lymphoid precursor that generates B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus, and then enter the circulation.

[0204] The cells of the present invention can be any of the cell types described above.

[0205] The cells described in the present invention can be generated ex vivo either from the patient's own peripheral blood (first party), or in the setting of a hematopoietic stem cell transplant from donor peripheral blood (second party), or from peripheral blood from an unrelated donor (third party).

[0206] Alternatively, cells can be obtained from inducible or embryonic precursor cells, for example, by ex vivo differentiation into T cells or NK cells. Alternatively, immortalized T cell lines that retain lytic function and can act as therapeutic agents may be used.

[0207] In all of these embodiments, chimeric polypeptide-expressing cells are generated by introducing DNA or RNA encoding the chimeric polypeptide by one of a number of means, including transduction with a viral vector or transfection with DNA or RNA.

[0208] The cells of the present invention may be ex vivo cells derived from a subject. The cells may be derived from a peripheral blood mononuclear cell (PBMC) sample. The cells may be activated and / or expanded, for example, by treatment with an anti-CD3 monoclonal antibody, before being transduced with a nucleic acid encoding a molecule providing a chimeric polypeptide according to the first aspect of the present invention.

[0209] The cells of the present invention are (i) isolation of a cell-containing sample from a subject or other source listed above; (ii) transduction or transfection of cells with one or more nucleic acid sequences encoding a CAR / TCR and a FLBR as defined herein; It can be made by

[0210] The cells can then be purified and selected, for example, based on expression of the antigen binding domain of the CAR / TCR or expression of the Fas ectodomain.

[0211] Pharmaceutical Composition The present invention also relates to a pharmaceutical composition comprising one or more cells of the invention. In particular, the present invention relates to a pharmaceutical composition containing cells according to the invention.

[0212] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more additional pharmaceutically active polypeptides and / or compounds. Such a formulation may be, for example, in a form suitable for intravenous infusion.

[0213] treatment The invention provides methods for treating and / or preventing disease, comprising administering to a subject a cell of the invention (eg, in a pharmaceutical composition as described above).

[0214] Suitably, the methods of the invention for treating and / or preventing disease may comprise administering to a subject a cell of the invention (eg in a pharmaceutical composition as described above).

[0215] Methods of treating disease relate to the therapeutic use of the cells of the invention, in which the cells may be administered to a subject with an existing disease or condition to attenuate, reduce or ameliorate at least one symptom associated with the disease and / or to slow, reduce or block the progression of the disease.

[0216] Methods for preventing disease involve the prophylactic use of the cells of the present invention. In this regard, the cells may be administered to a subject who is not yet afflicted with the disease and / or who does not exhibit any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent the development of at least one symptom associated with the disease. The subject may be predisposed to the disease or considered to be at risk of developing the disease.

[0217] The method is: (i) isolating a cell-containing sample; (ii) transducing or transfecting such cells with a nucleic acid sequence or vector provided by the present invention; (iii) administering cells from (ii) to a subject; may include:

[0218] The invention provides the cells of the invention for use in treating and / or preventing disease.

[0219] The invention also relates to the use of the cells in the manufacture of a medicament for the treatment and / or prevention of disease.

[0220] The disease to be treated and / or prevented by the methods of the present invention may be cancer.

[0221] The cancer can be, for example, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[0222] The disease can be multiple myeloma (MM), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL), neuroblastoma, T-cell acute lymphoblastic leukemia (T-ALL), or diffuse large B-cell lymphoma (DLBCL).

[0223] The cells of the present invention, particularly CAR cells, may be capable of killing target cells, such as cancer cells. The target cells may be recognizable by expression of a TAA, such as the TAA provided in Table 3 above. The cancer may be a cancer listed in Table 3.

[0224] How to make cells The CAR or transgenic TCR-expressing cells of the present invention are prepared by expressing DNA or RNA encoding the CAR or TCR and the FasL-binding receptor (FLBR) in the form of a viral vector. They may be produced by introduction by one of a number of means, including transduction, transfection with DNA or RNA.

[0225] The cells of the present invention are (i) isolation of a cell-containing sample from a subject or one of the other sources listed above; (ii) transduction or transfection of cells in vitro or ex vivo with one or more nucleic acid sequences or nucleic acid constructs as defined above; It can be made by

[0226] The cells can then be purified, for example, selected based on expression of the antigen-binding domain of the antigen-binding polypeptide.

[0227] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges include the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation, and any amino acid sequence is written left to right in amino to carboxy orientation, respectively.

[0228] Where a range of values ​​is given, unless the context clearly dictates otherwise, it should be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range is also specifically disclosed. Every smaller range between any specified or intervening value in a stated range and any other specified or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range where either limit, neither limit, or both limits are included within the smaller range is also included within the disclosure, subject to any specifically excluded limits within the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0229] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Please note that

[0230] As used herein, "comprising", "comprises", and The term "comprised of" is synonymous with "including," "includes," "containing," or "contains." "Comprising," "comprises," and "comprises" are non-exclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. and the terms "comprised of" include the term "consisting only of."

[0231] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0232] The present invention will now be further illustrated by examples, which are intended to be used to assist those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. [Example]

[0233] Example 1 - In vitro testing of constructs binding to FasL A cell panel expressing the constructs listed in Table 4 was generated. All cells expressed a second-generation anti-CD19 CAR (Fmc63). Two versions of the cell panel were generated: one in which the construct contained a gene encoding Fas ligand and therefore the cells co-expressed FasL (+FasL in Figures 6-8), and one in which the construct did not contain a gene encoding Fas ligand (-FasL in Figures 6-8). The cells were cultured and tested for their ability to resist FasL-induced cell death.

[0234] [Table 4]

[0235] More specifically, freshly isolated primary human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 and anti-CD28 antibodies (0.5 μg / ml) for 24 hours. IL-2 (100 IU / ml) was then added to the PBMCs for an additional 24 hours. 300,000 of the activated PBMCs were transduced with crude retroviral supernatant containing the gene(s) of interest in a 24-well plate.

[0236] The PBMCs were spun at 1000 g for 40 minutes, at which point the cells were transferred to an incubator and cultured for 3 days. The cells were then resuspended by pipetting, and 100 μl was transferred to a 96-well plate. Cell viability was analyzed on a flow cytometer by counting the number of viable PBMCs.

[0237] Absolute cell numbers were normalized to PBMCs engineered to express Fmc63 alone.

[0238] The results for constructs 1 to 8 are shown in Figure 6. Cells expressing CAR together with Fas-DcR2, Fas-GITR, Fas-CD30, or membrane-bound DcR3 expressed Fas-41BB, F It can clearly be seen that they are far more effective at resisting cell death than cells co-expressing CAR with asOX40 or FasΔDD.

[0239] Similarly, Figure 7 shows increased cell survival for cells expressing the membrane-bound DcR3 of the present invention over comparable cells expressing soluble DcR3 (compare constructs 8 and 9).

[0240] The above methodology was repeated for constructs 1-7, except that in this case the constructs also expressed the short suicide gene RQR8 described in WO 2013 / 153391. RQR8 was used as a marker for transduced cells (RQR8-positive cells). Cells expressing one of the FLBRs, Fas-DcR2, Fas-CD30, or Fas-GITR, were found to have higher numbers of transduced cells than cells expressing Fas-ΔDD, Fas-OX40, or Fas-41BB.

[0241] Example 2 - In vitro testing of FasL-binding receptor (FLBR) with immobilized Fas ligand The cell panel described in Example 1, but not transduced to express FasL, was tested for its ability to resist FasL-induced cell death using immobilized soluble Fas ligand. PBMCs were transduced with retroviral vectors expressing constructs 2, 3, 5, and 6 in Table 4 above, i.e., CAR+FasΔDD, Fas-DcR2, Fas-GITR, and Fas-OX40.

[0242] Soluble Fas ligand (2.5 μg) was immobilized onto a 96-well plate overnight at 4°C. The Fas ligand-immobilized plate was washed four times with PBS. 50,000 transduced PBMCs were added to the wells containing immobilized Fas ligand and cultured in an incubator for 5 days. The cells were spun down by centrifugation, stained for CD3 and CD34, and analyzed by flow cytometry. CountBright™ absolute counting beads (Thermo The absolute number of viable PBMCs was determined using a 100-well platelet count (PBC) assay. Absolute cell numbers were normalized to PBMCs transduced with Fmc63 alone.

[0243] The results are shown in Figures 9 and 10. An increase in cell number was observed for cells expressing Fas-DcR2 and Fas-GITR cultured with immobilized FasL than was seen for cells expressing Fas-OX40 or dnFas.

[0244] Example 3 - In vitro testing of FasL-binding receptor (FLBR) with immobilized Fas ligand A panel of cells expressing the constructs listed in Table 5 was generated. All cells expressed an anti-CD19 CAR (Fmc63). Cells were tested for their ability to resist FasL-induced cell death using immobilized soluble Fas ligand. Cells were then tested in the presence or absence of an anti-Fmc63 anti-idiotypic antibody.

[0245] [Table 5]

[0246] PBMCs were transduced with retroviral vectors expressing constructs as described in Example 1.

[0247] Soluble Fas ligand (2.5 μg) was immobilized on a 96-well plate overnight at 4°C, either alone or in combination with 1 μg anti-Fmc63 Ab. PBS was added to a separate well as a control. The Fas ligand / anti-Fmc63 Ab-immobilized plate was washed four times with PBS. Transduced PBMCs (50,000 cells) were added to the wells and cultured in an incubator for 5 days.

[0248] At the time of seeding of cells into 96-well plates (day 0), cells were spun down by centrifugation, stained for CD3 and CD34 (to detect RQR8 and thus transduced cells), and analyzed by flow cytometry to determine the percentage of cells expressing RQR8. Absolute numbers of PBMCs were determined using CountBright™ absolute counting beads.

[0249] On day 5 of incubation, the cells were spun down by centrifugation, and 100 μl of supernatant was removed for cytokine analysis. The cells were stained for CD3 and CD34, and then analyzed by flow cytometry to determine the percentage of cells expressing RQR8. Absolute numbers of viable PBMCs were determined using CountBright™ absolute counting beads. Absolute cell numbers on day 5 of incubation were compared to the day 0 analysis to measure the fold difference in proliferation. Absolute cell numbers were normalized to PBMCs cultured on PBS-treated wells.

[0250] The results are shown in Figures 11 to 16.

[0251] Expression of Fas-XEDAR can induce homeostatic proliferation of PBMCs in the absence of CAR stimulation and prevent FasL-induced cell death (FIGS. 11 and 12).

[0252] Fmc63-CD3z-transduced cells are susceptible to FasL-mediated cell death. This is particularly evident when transduced cells are co-incubated with FasL and anti-fmc63 idiotypic Ab (Figure 13, blue circles). As shown in Figures 13 and 14, cells expressing Fas-XEDAR are more resistant to FasL-induced death than cells expressing dominant-negative Fas (i.e., Fas containing a truncated death domain (FasΔDD)). The cell population transduced with the vector expressing Fas-XEDAR was enriched for transduced cells over time (Figure 13).

[0253] After 5 days of incubation of transduced PBMCs on plates containing either immobilized Fas ligand alone or Fas ligand / anti-Fmc63, the cells were spun and 100 μL of Supernatants were removed for cytokine analysis. ELISA MAX™ Deluxe Set Human IFN-γ (BioLegend) and ELISA M were used according to the manufacturer's instructions. AX™ Deluxe Set Human IL-2 (BioLegend) was used to sample the supernatant. The inflammatory cytokine concentration was measured.

[0254] Cells expressing Fas-XEDAR showed significantly higher release of IL2 and interferon-γ than cells expressing truncated Fas (FasΔDD) (Figures 15 and 16). Increased IFN-γ was observed even in the absence of immobilized Fas ligand incubation (Figure 15).

[0255] Example 4 - Further in vitro screening of TNFR FasL-binding receptor (FLBR) using immobilized Fas ligand A panel of cells was generated expressing the constructs listed in Table 6. All cells expressed the anti-CD19 CAR (Fmc63-CD3z). Cells were tested for their ability to resist FasL-induced cell death using immobilized soluble Fas ligand.

[0256] [Table 6]

[0257] Immobilized soluble Fas ligand was used to induce cell death, repeating the methodology described in Example 2. The results are shown in Figures 17-20.

[0258] Expression of Fas-TNFR chimeras was shown to prevent FasL-induced cell death and induce proliferation of PBMCs upon FasL engagement. In particular, cells expressing Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 had the highest mean IL-1 expression. The cell counts are shown in Figure 17. Figure 18 shows the absolute cell counts of transduced PBMCs cultured in the presence of immobilized FasL relative to transduced PBMC cultures in the absence of FasL, confirming that expression of each of the screened Fas-TNFRs induced proliferation. The fold difference in proliferation compared to day 0 analysis (Figure 19) showed that expression of Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 induced greater proliferation of transduced PBMCs than expression of Fas-41BB and FasΔDD.

[0259] After 5 days of incubation of transduced PBMCs with Fas ligand, cells were spun and 100 μl of supernatant was removed for cytokine analysis using ELISA MAX™ Deluxe Set human IFN-γ (BioLegend) according to the manufacturer's instructions. The supernatants were then assayed for cytokine concentrations. As shown in Figure 20, cells expressing Fas-CD40 exhibited significantly higher release of interferon-γ than cells expressing FasΔDD. Increased IFN-γ release was observed for cells expressing Fas-CD40 FLBR, even in the absence of exposure to immobilized FasL. Cells expressing the FLBRs Fas-CD27, Fas-CD40, Fas-BCMA, and Fas-Fn14 also exhibited higher interferon-γ release (pg / ml) when incubated with immobilized Fas ligand compared to that of the FasΔDD construct.

[0260] Example 5 - Testing the cytotoxic potential of cells co-expressing anti-GD2 CAR and TNFR FasL-binding receptor (FLBR) in a restimulation assay To evaluate the cytotoxic benefit of the Fas-TNFR chimera in the context of a CAR, transduced PBMCs were subjected to successive rounds of restimulation (Figure 21). PBMCs were transduced to express the GD2-targeting CAR either by itself or coexpressed with either truncated Fas (FasΔDD), Fas-41BB, Fas-XEDAR, Fas-CD40, Fas-CD27, Fas-BCMA, or Fas-Fn14 via the 2A self-cleaving peptide. Non-transduced (NT) and transduced PBMCs were cocultured with GD2-expressing SupT1 target cells at a 1:1 effector-to-target ratio for 4 days, at which point the percentage of remaining viable target cells was quantified. Every 3 or 4 days, 0.5 × 10 CAR-T cells were transduced into PBMCs. 5 GD2 CAR-T cells co-expressing Fas-XEDAR, Fas-CD40, Fas-BCMA, and Fas-Fn14 (Figures 21E, 21F, 21H, and 21I) performed significantly better than control GD2 CAR-T cells and GD2 CAR-T cells co-expressing FasΔDD, Fas-41BB, and Fas-CD27 (Figures 21B, 21C, 21D, and 21G) when measured for their ability to maintain cytotoxicity and expansion after repeated encounters with GD2-positive SupT1 tumor cells in an in vitro continuous co-culture cell killing assay. Both control GD2 CAR-T cells and GD2 CAR-T cells co-expressing FasΔDD, Fas-41BB, or Fas-CD27 began to fail to clear targets and lost their ability to expand and eliminate GD2-positive tumor cells by the fifth restimulation. In contrast, GD2 CAR-T cells co-expressing Fas-XEDAR, Fas-CD40, Fas-BCMA, and Fas-Fn14 remained responsive to antigen stimulation for much longer and were therefore superior at clearing target cells.

[0261] Cell culture and reagents All cell lines and primary T cells used in the experiments were cultured in RPMI 1640 medium (Lonza) supplemented with 10% fetal bovine serum (FBS, Biosera) and 1% L-glutamine (GlutaMAX, Gibco). SupT1 cells were purchased from ATCC. Cells were obtained from the National Health Service Blood and Transplant (NHSBT, Colindale, UK). Transduced T cells were generated from PBMCs obtained from the 1990s. Transduced T cells were cultured in the same medium as above, supplemented with 100 U / mL interleukin-2 (IL-2). Bright™ absolute counting beads (Thermo Fisher) were used to count the absolute number of viable PBMCs. The logarithm was determined. ELISA MAX™ Deluxe Set Human IFN-γ (BioLegend, 430104) and ELISA MAX™ were used according to the manufacturer's instructions. Deluxe Set human IL-2 (BioLegend, 431804) was used to The concentration of HCl was measured.

[0262] Transduction The plasmid encoding gag-pol (pEQ-Pam3-E36), the plasmid encoding RD114 envelope (RDF37), and the desired retroviral transfer vector plasmid were transfected into the 29-well plate using GeneJuice (Millipore). Retroviruses were produced by transient transfection of 3T cells. Transduction was performed using Retronectin (Takara) as previously described. Transduction efficiency for the different constructs was assessed by flow cytometry based on the expression of RQR8 staining, performed using QBEND / 10 mAb. Flow cytometry analysis was performed using a MACSQuant Analyzer 10 (Miltenyi). Flow sorting was performed using a BD FACS.

[0263] All publications cited in the above specification are incorporated herein by reference. It will be apparent to those skilled in the art that various modifications and variations can be made in the described methods and systems of the invention without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. (a) a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); (b) a FasL-binding receptor (FLBR) comprising the human Fas ectodomain and the human CD40 endodomain; Cells containing

2. A FasL-binding receptor (FLBR) comprising a human Fas ectodomain and a human CD40 endodomain.

3. A nucleic acid having a sequence encoding the FLBR of claim 2.

4. (a) a first nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); (b) a second nucleic acid sequence encoding a FasL binding receptor (FLBR) as defined in claim 2; and A nucleic acid construct comprising:

5. (a) a first nucleic acid having a sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); (b) a second nucleic acid having a sequence encoding a FasL binding receptor (FLBR) as defined in claim 2; A nucleic acid kit comprising:

6. A vector comprising the nucleic acid of claim 3, the nucleic acid construct of claim 4, or the nucleic acid of claim 5.

7. (a) a first vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) or a transgenic T cell receptor (TCR); (b) a second vector comprising a nucleic acid sequence encoding a FasL-binding receptor (FLBR) as defined in claim 2; and A vector kit comprising:

8. A pharmaceutical composition comprising a plurality of cells according to claim 1, wherein the plurality of cells is a T cell and / or a NK cell.

9. (i) isolating a cell-containing sample; (ii) transducing or transfecting the cells with a nucleic acid according to claim 3, a nucleic acid construct according to claim 4, a kit of nucleic acids according to claim 5, a vector according to claim 6, or a kit of vectors according to claim 7; (iii) administering the cells from (ii) to a subject; The pharmaceutical composition according to claim 8, which is used for a method for treating and / or preventing a disease, comprising:

10. 10. Use of the pharmaceutical composition according to claim 8 in the manufacture of a medicament for the treatment and / or prevention of a disease.

11. A method for producing the cell described in claim 1, comprising the step of introducing the nucleic acid described in claim 3, the nucleic acid construct described in claim 4, the nucleic acid kit described in claim 5, the vector described in claim 6, or the vector kit described in claim 7 into the cell ex vivo.

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