Engineered TNFR ligands

Abstract

The present invention provides a new therapeutic approach for the treatment of diseases, such as auto- inflammatory and tumor diseases. It makes use of recombinant T cells expressing engineered chimeric antigen receptors (CARs), artificial immune receptors (AIRs) or recombinant TCRs and an engineered synthetic, transmembrane protein comprising the binding domain of a TNFR ligand. The transmembrane protein comprising the binding domain of a TNFR ligand is engineered in a manner which allows the TNFR ligand to bind to its receptor on the same T cell, thereby stimulating its own survival via Nfkb activation and other pro-survival signaling pathways.

Description

[0001] Engineered TNFR ligands

[0002] Field of the invention

[0003] The present invention provides a new therapeutic approach for the treatment of diseases, such as auto- inflammatory and tumor diseases. It makes use of recombinant T cells expressing engineered chimeric antigen receptors (CARs), artificial immune receptors (AIRs) or recombinant TCRs and an engineered synthetic, transmembrane protein comprising the binding domain of a TNFR ligand. The transmembrane protein comprising the binding domain of a TNFR ligand is engineered in a manner which allows the TNFR ligand to bind to its receptor on the same T cell, thereby stimulating its own survival via Nfkb activation and other pro-survival signaling pathways.

[0004] Background

[0005] CD4+ regulatory T (Treg) cells suppress self-reactive immune responses and excessive inflammation in humans and mice (Annu Rev Immunol (2020) 38: 541-66). In addition to these immunoregulatory functions, subpopulations of tissue-resident Treg cells have tissue repair and homeostatic roles (Immunity (2021) 54(4): 702-20, Immunity (2020) 52(2): 295-312). These functions make the Treg cell an interesting candidate for cell therapies against unwanted immune responses. Treg cells are characterized by the expression of the key transcription factor FOXP3 (Annu Rev Immunol (2020) 38: 541-66.). Fatal autoimmunity in patients with immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome or in scurfy mice (both lacking functional Treg cells due to mutations in the FOXP3 gene) shows the importance of FOXP3- positive Treg cells for immune control and homeostasis (Nat Rev Immunol (2014) 14(5): 343-9). Several scientific and technological advances led to the translation of these preclinical findings into adoptive Treg cell therapy trials. In particular, the engineering of T cells for adoptive T cell therapy has been important, allowing the transfer of large numbers of clinical-grade antigen-specific T cells into recipients. This development was spearheaded by chimeric antigen receptors (CARs), which are specific for tumour antigens and are expressed on effector CD8+ and CD4+ T cells to attack tumors (Cancer Discov (2022) 12(7): 1625-33). Innovative concepts applying the CAR technology to Treg cells are now the next step to improve the efficacy of adoptive Treg cell therapy. But also other Treg engineering strategies such as using artificial immune receptors (AIRs) or recombinant TCRs are promising tools to generate novel cellular immunotherapies for treatment of autoimmune and chronic inflammatory diseases. Preclinical studies have already demonstrated the superiority of engineered Tregs with AIR-, TCR- or CAR-mediated specificity over Tregs with only a natural polyclonal TCR repertoire in reducing alloimmune responses in graft-versus- host disease (GvHD) and graft rejection after transplantation (Proc Natl Acad Sci U S A (2022) 119(40): e2208436119, Sci Transl Med (2020) 12(557): eaaz3866, Am J Transplant (2020) 20(6): 1562-73, J Clin Invest (2016) 126(4): 1413-24; Am J Transplant (2017) 17(4): 917-30, Am J Transplant (2017) 17(4) :931-43). Engineered Treg cells have also been effectively used as treatment for asthma, haemophilia A, type 1 diabetes, experimental autoimmune encephalitis (EAE, model for multiple sclerosis), systemic lupus erythematosus (SLE) and inflammatory bowel disease (IBD) in preclinical models (J Neuroinflammation (2012) 9:112, J Immunol Methods (2021) 488: 112931., Mol Ther (2014) 22(5): 1018-28, Front Immunol (2017) 8: 1125, J Autoimmun (2019) 103: 102289, Nat Commun (2024) 15(1): 2542). This engineered Treg cell therapy approach is potentially also effective in chronic and acute inflammatory or inflammation- associated diseases such as myocardial infarction, stroke or cardiovascular diseases, lung inflammation, kidney and liver inflammation, arthritis, rheumatoid diseases, neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis, Parkinson’ disease.

[0006] One of the main challenges in adoptive T cell therapy is the long-term persistence of cells after transfer. In order to create space and a survival benefit for transferred cells preconditioning of patients with drugs such as cyclophosphamide and fludarabine is used in CAR-T cell protocols. This holds true for both, patients suffering from autoimmunity but also malignant or cancer diseases (Nat Med (2022) 28(10): 2124- 32, In: he EBMT / EHA CAR-T Cell Handbook [Internet], Cham (CH): Springer; 2022, Chapter 25, 2022 Feb 7 „ N Engl J Med (2024) 390(8): 687-700). But especially in the setting of Treg cell therapy, where long-lasting, maybe live-long lasting, persistence of transferred cells is mandatory for therapeutic efficacy, the survival of engineered cell is a main difficulty.

[0007] To solve this problem, the present invention provides novel synthetic proteins, derived from TNFR ligands, that can be expressed together with or without a recombinant TCR, CAR or AIR in conventional CD4+ and CD8+ T cells, but also in Treg cells, and that improve the survival capacity of the engineered immune cells. TNFR ligands are typically not expressed on the same cell as their receptors.

[0008] Furthermore, after incorporating linker sequences into the natural TNFR ligand sequence, it was found that such linker-modified TNFR ligand (GITRL in our example) show a higher expression level than the wildtype version of the ligand, and have an increased capacity to bind to their corresponding receptors, which is endogenously expressed on the same cell, and stimulates cell survival via Nfkb activation and other pro-survival signaling pathways.

[0009] WO 2022 / 136681 discloses T cells comprising a CAR and a co-expressed protein comprised of a TNFR ligand, a transmembrane domain and a heterologous cytoplasmic signaling domain. Thus WO 2022 / 136681 provides bi-directional signaling induced by the TNFR ligand and the heterologous cytoplasmic signaling domain. In contrast the present invention uses cytoplasmic domains which do not induce intracellular Nfkb signaling and thus stimulates the T-cell only via the TNFR ligand.

[0010] Expression of such TNFR ligands results in better survival of the engineered cells and leads to higher and more stable expression of the transgenic receptor compared to cells without TNFR ligand expression or compared to cells expressing only wildtype TNFR ligands. This enhanced transgenic receptor expression potentially correlates with higher activity (and better functionality) of the transgenic receptor, respectively results in better functionality of the engineered cell.

[0011] Summary of the invention

[0012] The present disclosure relates to a transmembrane protein comprising the binding domain of a TNFR ligand.

[0013] The present disclosure further relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0014] In certain embodiments, said transmembrane protein comprises the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain. In certain embodiments, said transmembrane protein comprises the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain, wherein the cytoplasmatic domain does not induce NF-kB activation. In certain embodiments, said transmembrane protein comprises the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain, wherein the cytoplasmatic domain is a silent cytoplasmatic domain.

[0015] In certain embodiments, said extracellular domain is selected from GITRL, TL1A, OX40L TNFa, LIGHT, LTa3, LTa1 b2, CD40L, FasL, CD30L, 4-1 BBL, CD27L, TWEAK, APRIL, BAFF, RANKL, TRAIL, EDA1 and EDA2, preferably GITRL, TL1A and GX40L. In certain embodiments, said linker enables the binding of the TNFR ligand to its receptor on the same cell. In certain embodiments, said linker facilitates the binding of the TNFR ligand to its receptor on the same cell. In certain embodiments, said linker is a glycine-serine linker. In certain embodiments, said glycine-serine linker is a (G4S)3(SEQ ID No. 23) - (G4S)I2(SEQ ID No. 24) linker or a (G4S)6linker (SEQ ID No. 25). In certain embodiments, said (G4S)3has the amino acid sequence of SEQ ID No.23, said (G4S)I2linker has the amino acid sequence of SEQ-ID No.24 and said (G4S)6linker has the amino acid sequence of SEQ-ID No.25.

[0016] In certain embodiments said transmembrane protein comprising the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain further comprises one or more additional linkers. In certain embodiments the transmembrane protein comprises the extracellular binding domain of a TNFR ligand, one or more linkers, a transmembrane domain and a cytoplasmatic domain. In certain embodiments the transmembrane protein comprises the extracellular binding domain of a TNFR ligand, two linkers, a transmembrane domain and a cytoplasmatic domain. In certain embodiments, said one or more additional linkers enable the binding of the TNFR ligand to its receptor on the same cell. In certain embodiments, said one or more additional linkers facilitate the binding of the TNFR ligand to its receptor on the same cell. In certain embodiments, said one or more additional linkers are a glycine-serine linker. In certain embodiments, said glycine-serine linker is a (G4S)3-(G4S)I2linker or (G4S)6linker.

[0017] In certain embodiments said transmembrane protein comprising the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain further comprises a multimerization domain.

[0018] In certain embodiments the transmembrane protein comprises the extracellular binding domain of a TNFR ligand, one or more linkers, a multimerization domain, a transmembrane domain and a cytoplasmatic domain. In certain embodiments the transmembrane protein comprises the extracellular binding domain of a TNFR ligand, two linkers, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0019] In certain embodiments said multimerization domain is a human multimerization domain.

[0020] The present disclosure further relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein as disclosed herein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0021] In certain embodiments, said chimeric antigen receptor, artificial immune receptor orT cell receptor is an artificial immune receptor. In certain embodiments, said artificial immune receptor comprises the extracellular domain of member of the tumor necrosis factor receptor superfamily selected from TNFR1 , TNFR2, Fas, DR4, DR5, DR3, DR6, EDAR, XEDAR, TROY, LTBR, NGFR, CD18, CD134, CD40, CD27, CD30, CD137, TRAILR3, TRAILR4, CD265, osteoprotegerin, CD266, TACI, BAFF, BAFF receptor, APRIL, CD270, CD269 and CD357, preferably CD40. In certain embodiments, said artificial immune receptor comprises a CD28 costimulatory domain and / or a CD3 zeta domain.

[0022] In certain embodiments, said chimeric antigen receptor, artificial immune receptor or T cell receptor is a chimeric antigen receptor. In certain embodiments, said chimeric antigen receptor comprises a single chain antibody (scFv) that binds to an antigen selected from the group including, but not limited to, the CD19, CD20, HER2, NY-ESO-1 , MUC1 , CD123, FLT3, B7-H3, CD33, IL1 RAP, CLL1 (CLEC12A)PSA, CEA, VEGF, VEGF-R2, CD22, ROR1 , mesothelin, c-Met, Glycolipid F77, FAP, EGFRvlll, MAGE A3, 5T4, WT1 , KG2D ligand, folate receptor (FRa), Wnt1 antigens, HLA-A2 (and other HLA variants), IL-23R, and citrullinated antigens.

[0023] In certain embodiments, said chimeric antigen receptor, artificial immune receptor or T cell receptor is a T cell receptor.

[0024] In certain embodiments, said transmembrane protein comprising the binding domain of a TNFR ligand comprises the amino acid sequence of SEQ ID No’s 12, 16 or 18. In certain embodiments, said T cell is a cytotoxic T cell. In certain embodiments said cytotoxic T cell is a NK cell. In certain embodiments said cytotoxic T cell is a NKT cell. In certain embodiments said cytotoxic T cell is an iNKT cell. In certain embodiments, said T cell is a regulatoryT cell.

[0025] In certain embodiments, the present disclosure provides aforementioned T cells for use in medicine. In certain embodiments, said use in medicine is the treatment of an auto-inflammatory disease, such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis or auto-immune gastritis, inflammation-associated diseases such as myocardial infarction, stroke or cardiovascular diseases, lung inflammation, kidney and liver inflammation, neurodegenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis or Parkinson’ disease .

[0026] In certain embodiments, said use in medicine is the treatment of cancer e.g. leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma, glioblastoma, colorectal cancer and gastrointestinal cancers, melanoma, pancreatic cancer, kidney / renal cell cancer, lung cancer, breast cancer, prostate cancer and others. In certain embodiments, said use in medicine is the treatment of graft versus host disease or the use in solid organ transplantation.

[0027] Definitions

[0028] The term "cell" as used herein includes a single cell as well as a plurality of cells.

[0029] The term "host cell" as used herein refers to a cell comprising a nucleic acid and / or a vector. In the context of the artificial immune receptors of the present disclosure, the term host cell refers to a cell comprising a nucleic acid and / or a vector encoding for an AIR. Such host cell will express the AIR on the cell surface and is suitable to be used as medicine. Preferred host cells of the present invention are eukaryotic host cells, such as immune cells.

[0030] The term "T cell" as used herein refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells, also referred to as T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor (TCR) on the cell surface. There are several subsets of T cells with distinct functions, including but not limited to, T helper cells, cytotoxic T cells, memoryT cells, regulatoryT cellsand natural killer T cells. In some embodiments, theT cell is an engineered T cell.

[0031] The terms "regulatoryT cell" or "Treg" as used herein refer to a subpopulation of T cells which modulate the immune system, maintain tolerance to self-antigens, and abrogate autoimmune diseases. These cells generally suppress or downregulate induction and proliferation of effector T cells. Treg cells are long-lived cells that suppress excessive or uncontrolled immune responses in vivo in a dominant and antigen-specific manner. Genetic mutations in the forkhead box protein 3 (FoxP3), a key transcription factor required for differentiation of Treg cells, lead to severe autoimmunity. Indeed, research in a variety of animal models has demonstrated that Tregs can be used to treat many auto-inflammatory diseases such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis, and auto-immune gastritis. Treg cell therapy can also be used in controlling alloimmune responses in the context of GVHD, as well as organ and cell transplantation.

[0032] The terms "polynucleotide" and / or "nucleic acid sequence" and / or "nucleic acid" as used herein refer to a sequence of nucleoside or nucleotide monomers consisting of bases, sugars and intersugar (backbone) linkages. The term includes DNAand RNAand can be either double stranded or single stranded, and represents the sense or antisense strand. The term also includes modified or substituted sequences comprising non- naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed by laboratory methods of genetic recombination or obtained by chemical synthesis or other known protocols for creating nucleic acids.

[0033] The terms "isolated polynucleotide" or "isolated nucleic acid sequence" as used herein refer to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0034] The terms "recombinant nucleic acid" or "engineered nucleic acid" as used herein refer to a nucleic acid or polynucleotide that is not found in a biological organism. For example, recombinant nucleic acids may be formed by laboratory methods of genetic recombination (such as molecular cloning) to create sequences that would not otherwise be found in nature. Recombinant nucleic acids may also be created by chemical synthesis or other known protocols for creating nucleic acids. Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0035] The term "polypeptide" or "protein" as used herein describes a chain of amino acids. A polypeptide or protein of this disclosure can be a peptide, which usually describes a chain of amino acids of from two to about 30 amino acids. The term protein as used herein also describes a chain of amino acids having more than 30 amino acids and can be a fragment or domain of a protein or a full length protein. Furthermore, as used herein, the term protein can refer to a linear chain of amino acids or it can refer to a chain of amino acids that has been processed and folded into a functional protein. It is understood, however, that 30 is an arbitrary number with regard to distinguishing peptides and proteins and the terms can be used interchangeably for a chain of amino acids. The proteins of the present disclosure can be obtained by isolation and purification of the proteins from cells where they are produced naturally, by enzymatic (e.g., proteolytic) cleavage, and / or recombinantly by expression of nucleic acid encoding the proteins or fragments of this disclosure. The proteins and / or fragments of this disclosure can also be obtained by chemical synthesis or other known protocols for producing proteins and fragments.

[0036] The term "isolated polypeptide" refers to a polypeptide substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

[0037] The term “multimerization domain” refers to a protein, polypeptide, peptide or amino-acid sequence capable of associating with a second multimerization domain having the same or similar structure to facilitate formation of a multimer (i.e. , the formation of a dimer, trimer, or multimeric complex).

[0038] The term "vector" as used herein refers to a polynucleotide that can be used to deliver a nucleic acid to the inside of a cell. In one embodiment, a vector is an expression vector comprising expression control sequences (for example, a promoter) operatively linked to a nucleic acid to be expressed in a cell. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids and viruses.

[0039] The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0040] As used herein, the terms “treatment,” “treating,” and the like, in some embodiments, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of affecting a partial or complete cure for a disease and / or symptoms of the disease. The terms include treatment of a disease or disorder (e.g. inflammation) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g, including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g. inflammation).

[0041] The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0042] The terms “tumor necrosis factor receptor ligand”, “TNFR ligand” or “TNFRL” as used herein refer to a ligand that binds to the respective receptor of the tumor necrosis factor super family. Exemplary TNFR ligands include TL1A, OX40L, GITRL, TNFa, LIGHT, LTa3, LTa1 |32, CD40L, FasL, CD30L, 4-1 BBL, CD27L, TWEAK, APRIL, BAFF, RANKL, TRAIL, EDA1 and EDA2. GITRL (UniProt: Q9UNG2; also known as Glucocorticoid-Induced TNF-Related Ligand, TNFSF18 or TNF Superfamily Member 18) binds to TNFRSF18 / GITR and is involved in the regulation of T cell responses. It can functions as a costimulator and lower the threshold for T-cell activation and T-cell proliferation. It has also been reported to be important for interactions between activated T-lymphocytes and endothelial cells, the mediation of activation of NF-kappa-B, triggering increased phosphorylation of STAT1 and the upregulation of expression of VCAM1 and ICAM1.

[0043] Human GITRL has the following amino acid sequence:

[0044] MCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLP SKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSK IQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS ( SEQ ID No . 1 )

[0045] Murine GITRL has the following amino acid sequence:

[0046] MEEMPLRESS PQRAERCKKSWLLCIVALLLMLLCSLGTLIYGSTSLKPTAIESCMVKFELSSSKWH MTSPKPHCVNTTSDGKLKILQSGTYLIYGQVIPVDKKYIKDNAPFWQIYKKNDVLQTLMNDFQIL PIGGVYELHAGDNIYLKFNSKDHIQKTNTYWGIILMPDLPFIS ( SEQ ID No . 2 )

[0047] TL1A (UniProt: 095150; also known as TNF Ligand-Related Molecule 1 , TNFSF15 or TNF Superfamily Member 15) binds to TNFRSF25 and TNFRSF6B and is involved in the mediation of activation of NF-kappa- B, the inhibition of vascular endothelial growth and angiogenesis and the activation of caspases and apoptosis.

[0048] Human TL1A has the following amino acid sequence:

[0049] MAEDLGLSFGETASVEMLPEHGSCRPKARSSSARWALTCCLVLLPFLAGLTTYLLVSQLRAQGEAC VQFQALKGQEFAPSHQQVYAPLRADGDKPRAHLTVVRQTPTQHFKNQFPALHWEHELGLAFTKNRM NYTNKFLLIPESGDYFIYSQVTFRGMTSECSEIRQAGRPNKPDS ITWITKVTDSYPEPTQLLMGT KSVCEVGSNWFQPIYLGAMFSLQEGDKLMVNVSDISLVDYTKEDKTFFGAFLL ( SEQ ID No .

[0050] 3 )

[0051] Murine TL1A has the following amino acid sequence:

[0052] MAE ELGLGFGEGVPVEVLPEGC RHR PE ARAGL AARS KAC LALTCCLLSFPI L AGL S T L LMAGQL RV PGKDCMLRAITEERSEPSPQQVYSPPRGKPRAHLTIKKQTPAPHLKNQLSALHWEHDLGMAFTKNG MKYINKSLVIPESGDYFIYSQITFRGTTSVCGDISRGRRPNKPDSITMVITKVADSYPEPARLLTG SKSVCEISNNWFQSLYLGATFSLEEGDRLMVNVSDISLVDYTKEDKTFFGAFLL ( SEQ ID No .

[0053] 4 )

[0054] GX40L (UniProt: P23510; also known as CD252, TNFSF4 orTNFSuperfamily Member 4) binds to TNFRSF4 and is involved in the co-stimulation of T-cell proliferation and cytokine production.

[0055] Human GX40L has the following amino acid sequence: MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQF TEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKV RSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL ( SEQ ID No . 5 )

[0056] Murine OX40L has the following amino acid sequence:

[0057] MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGIKGAGMLLCFIYVCLQLSSS PAKDPPIQRLRGA VTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDLHFREDHNPIS IPML NDGRRIVFTWASLAFKDKVYLTVNAPDTLCEHLQINDGELIWQLTPGYCAPEGSYHSTVNQVPL

[0058] ( SEQ ID No . 6 )

[0059] The terms “tumor necrosis factor receptor” or “TNFR” as used herein refer to the receptors to which the engineered TNFR ligand of the present disclosure bind.

[0060] The terms “costimulatory molecule" or “costimulatory receptor” as used herein refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response in the T cell, such as, but not limited to, activation or proliferation. A co-stimulatory receptor may be expressed on cells other than T cells, such as NK cells or macrophages. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), Tolllike receptors and NK cell receptors. Costimulatory molecules include but are not limited to 4-IBB (CD137), BAFFR, 0X40, CD27, CD28, CD40, ICOS, 2B4, GITR, HVEM, 0X40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1 to TLR9 receptors, IL-2, IL-7 and IL-15 receptors.

[0061] The terms “costimulatory signaling domain” or “costimulatory domain” as used herein refer to the domain of a costimulatory molecule or costimulatory receptor responsible for mediating a costimulatory response by the T cell. The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.

[0062] The terms “T cell receptor signaling domain” or “TCR signaling domain” as used herein refers to cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector functions. In some embodiments, the TCR signaling domain contains a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM. In some embodiments, the primary intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1 G) , FcR beta (Fc Epsilon Rib) , CD79a, CD79b, Fcgamma Rlla, DAP10, and DAP12. A preferred TCR signaling domain is a TCR signaling domain selected from CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon. A particularly preferred TCR signaling domain CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon.

[0063] The terms “artificial immune receptor” or “AIR” refer to artificial immune receptors comprising an extracellular domain of a member of the tumor necrosis factor receptor superfamily, a transmembrane domain, a cytoplasmic costimulatory signaling domain, and a cytoplasmic T cell receptor signaling domain.

[0064] The terms “chimeric antigen receptor” and “CAR” are art recognized and refers to an engineered receptor that has binding specificity for an antigen or other ligand on an immune effector cell (e.g., a T cell, NK cell, a NKT cell, an iNKT cell). A chimeric antigen receptor typically comprises at least an extracellular ligand binding domain or moiety capable of specifically binding an antigen and an intracellular domain that comprises one or more signaling domains and / or co- stimulatory domains. The extracellular ligand-binding domain of a CAR is typically in the form of a binding protein, such as a single chain antibody (scFv), but may also be a small molecule, a peptide, or another a targeting agent.

[0065] CARs typically comprise an intracellular signaling domain that transmit an activation signal to the cell following binding of the extracellular domain. An intracellular signaling domain can be any intracellular signaling domain of interest that is known in the art. Such cytoplasmic signaling domains can include, without limitation, CD3. Intracellular co stimulatory domains can include, without limitation, CD27, CD28, CD8, 4-1 BB (CD137), 0X40, CD30, CD40, CD127, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, Nl, N6, or any combination thereof. A commonly used co-stimulatory domain in CARs is 4-1 BB (CD137).

[0066] The terms "T cell receptor" and "TCR" are art recognized and refer to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. A TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. A TCR comprises a heterodimer of an alpha (a) and beta ( ) chain, although in some cells the TCR comprises gamma and delta (y / 8) chains. TCRs may exist in alpha / beta and gamma / delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain comprises two extracellular domains, a variable and constant domain. In some embodiments, a TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

[0067] The terms “binding domain” as used herein refers to the a portion of a polypeptide that is responsible for the specific binding of the polypeptide to a target molecule (e.g. an antigen), but which does not substantially recognize or bind other molecules .

[0068] The term “linker” is art recognized and refers to peptide linker linking two different domains or modules of a protein and providing a certain degree of flexibility. In the context of the present invention the linker is introduced between the transmembrane domain and the extracellular domain of the TNFR ligand. Such modified TNFR ligands have a higher capacity to bind to their receptor(s) on the same cell.

[0069] Preferably, the flexible linker is hydrophilic and does not interact with other surfaces. Commonly used flexible linkers are glycine-serine linkers (Biochemistry 56(50):6565-6574 (2017)). Glycine and serine are flexible and the adjacent protein domains are free to move relative to one another. Such flexible linkers are referred to herein as “glycine-serine linkers”. Other amino acids commonly used in respective linkers are proline, asparagine and threonine. Often the linker contains several repeats of a sequence of amino acids. A flexible linker used in the present disclosure is a (Gly4Ser)x-linker comprising one or more Gly4Ser units according to amino acids GGGGS (SEQ ID No. 26), i.e. a linker containing four repeats of the sequence glycine- glycine- glycine- glycine- serine. Preferred linkers are (G4S)3according to amino acids GGGGSGGGGSGGGGS (SEQ ID No. 23) - (G4S)12according to amino acids GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No. 24) linkers. Most preferred is a (G4S)6linker according to amino acids GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No. 25). Other linkers that could be used in accordance with the present disclosure include but are not limited to PAS linkers, i.e. linkers containing repeats of the sequence proline- alanine- serine (Protein Eng Des Sei (2013) 26, 489-501 and charged linkers. The terms “Gly4Ser”, “Gly(4)Ser“, “G4S” are used interchangeably herein and refer to amino acids GGGGS (SEQ ID No. 26). The Gly(4)Ser linker (SEQ ID No. 26) was incorporated 2 times (2x Gly(4)Ser) (SEQ ID No. 27), 3 times (3x Gly(4)Ser) (SEQ ID No. 23), 6 times (6xGly(4)Ser) (SEQ ID No. 25) or 12 times (12xGly(4)Ser) (SEQ ID No. 24). The terms “(G4S)3”, “3x Gly(4)Ser“, “(G4S)3” are used interchangeably herein and refer to a 3-fold Gly4Ser linker according to amino acids GGGGSGGGGSGGGGS (SEQ ID No. 23).

[0070] The term “(G4S)3-(G4S)i2“linker as used herein refers to a group of G4S linkers comprising 3 to 12 G4S units. Such “(G4S)3-(G4S)12“ linker is either a (G4S)3(G4S)4(G4S)s (G4S)3, (G4S)2(G4S)g (G4S)gt(G4S)I o, (G4S)ntor (G4S)I2linker.

[0071] The terms “(G4S)6”, “6x Gly(4)Ser“, “(G4S)6” are used interchangeably herein and refer to a 6-fold Gly4Ser linker according to amino acids GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No. 25).

[0072] The terms “(G4S)I2”, “12x Gly(4)Ser“, “(G4S)12” are used interchangeably herein and refer to a 12-fold Gly4Ser linker according to amino acids

[0073] GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID No. 24).

[0074] In embodiments any of the disclosed glycine-serine linkers could be extended and comprise an additional glycine and serine at the N-terminus of the glycine-serine linker and / or an additional glycine and serine at the C-terminus the glycine-serine linker. Typically, such additional glycine and serine are introduced because of the use of certain restriction enzymes (i.e BamHI).

[0075] For example, such extended glycine-serine linkers are:

[0076] Gly(4)Ser-E according to amino acids GSGGGGSGS (SEQ ID No. 28).

[0077] 3xGly(4)Ser-E according to amino acids GSGGGGSGGGGSGGGGSGS (SEQ ID No. 29).

[0078] 6xGly(4)Ser-E according to amino acids GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGS (SEQ ID No. 30)

[0079] 12xGly(4)Ser-E according to amino acids GSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGS (SEQ ID No. 31).

[0080] The terms “is”, “are”, “is derived from” and “are derived from” in the context of a polypeptide or domain of a polypeptide refers to the amino acid sequence of said polypeptide or domain of a polypeptide and indicates that the amino acid sequence is either identical to the native version of said polypeptide or domain of a polypeptide, or a variant of said polypeptide or domain of a polypeptide which is functionally indistinguishable form from the native version of said polypeptide or domain of a polypeptide.

[0081] The term “silent cytoplasmatic domain” as used herein refers to a cytoplasmatic domain that does not directly participate in signaling (e.g., does not bind a signaling pathway component or undergo a chemical or structural change as part of a signaling pathway).

[0082] Figure legends

[0083] Figure 1 depicts the underlying concept of the present invention.

[0084] Figure 2 shows that cell counts for AIR only transduced Treg cells did not change during the resting period, whereas additional expression of modified GITRL-6xGly(4)Ser-linker and GX40L-6xGly(4)Ser-linker significantly increased cell yield (about 2-fold). TL1A-6xGly(4)Ser-linker expression only tendentially increased cell count (RM-one-way ANOVAwith Dunnett's multiple comparisons test; GITRL: n=3, p=0.0056; GX40L n=3, p=0.001 ; TL1A n=3, p=0.1498).

[0085] Figure 3 shows the differences in absolute cell counts between day 0 to day 5 (“resting period”) from the experiment shown in Figure 2 (RM-one-way ANOVA with Dunnett's multiple comparisons test; GITRL: n=3, p= 0.0010; GX40L n=3, p= 0.0002; TL1A n=3, p= 0.1445).

[0086] Figure 4 shows the differences in absolute cell counts for Treg cells transduced with various constructs between day 0 to day 5 (“resting period”) from the experiment shown in Figure 2. Panel A: AIR only transduced Treg cells (paired t-test, p=0.4785); panel B: AIR plus GITRL-6xGly(4)Ser-linker transduced Treg cells (paired t-test, p=0.0195); panel C: AIR plus GX40L-6xGly(4)Ser-linker transduced Treg cells (paired t- test, p=0.0027); panel D: AIR plus TL1A -6xGly(4)Ser-linker transduced Treg cells (paired t-test, p= 0.1467). Clearly, all Treg cells transduced with a modified ligand of the Tumor-Necrosis-Factor (TNF) superfamily in addition to the AIR had a significantly increase cell count.

[0087] Figure 5 shows that additional expression of the modified GITRL significantly increased cell yield, whereas expression of wildtype GITRL did not increase cell yield significantly, indicating improved functionality of the GITRL with 6xGly(4)Ser-linker. (paired t-test, modified GITRL p=0.0156; wildtype GITRL p=0.0817).

[0088] Figure 6 shows the frequency of hCD2 / FOXP3 positive cells among CD4+ T cells. No differences in hCD2 / FOXP3 expression between wildtype ( non transduced) Treg cells or Treg cells transduced with an AIR only or an AIR plus a wildtype version of GITRL or an AIR plus Gly(4)Ser-linker modified version of GITRL were detectable (one-way ANOVA).

[0089] Figure 7 shows that GITRL protein expression on the Treg cell surface was only detectable in Treg cells transduced with an AIR plus Gly(4)Ser-linker and not in Treg cells transduced with AIR plus wildtype version of GITRL.

[0090] Figure 8 shows a summary plot of the data shown in Figure 7.

[0091] Figure 9 shows a summary plot demonstrating that CD40-AIR expression was enhanced in Treg cells with Gly(4)Ser-linker-modified GITRL.

[0092] Figure 10 shows that AIR protein expression on the cell surface was increased in Treg cells transduced with an AIR plus Gly(4)Ser-linker modified ligand (GITRL, GX40L, TL1A).

[0093] Figure 11 shows that CD40-AIR signaling was comparable in all variants transduced cells with a full length version of the CD40-AIR irrespective of the presence of a Gly(4)Ser-modified ligand (GITRL, GX40L, TL1A). No signaling (Nr4a1 .eGFP expression) in Treg cells expressing a truncated version of the AIR plus Gly(4)Ser- modified ligand (GITRL, GX40L, TL1 A) was detectable.

[0094] Figure 12 shows that cell counts for AIR only transduced Treg cells did not change during the resting period, whereas additional expression of modified GITRL-6xGly(4)Ser-linker and GX40L-6xGly(4)Ser-linker significantly increased cell yield (about 2-fold). TL1A-6xGly(4)Ser-linker expression only tendentially increased cell count.

[0095] Figure 13 shows the difference between living cell numbers on day 0 and harvested cells after 4 days resting without TCR stimulation (same data set as Figure 12).

[0096] Figure 14 shows the difference between living cell numbers on day 0 and harvested cells after 4 days resting as fold increase (same data set as Figure 12).

[0097] Figure 15 validates the results of Figure 6 also for TL1A- and GX40L-Gly(4)Ser AIRs.

[0098] Figure 16 shows the statistical analysis of a repeat of the experiment shown in Figure 10.

[0099] Figure 17 shows the impact of modified GITRL expression with various linkers on the expression of the AIR.

[0100] Figure 18 shows the killing efficiency of anti CEA CAR T cells transduced with various constructs.

[0101] Figure 19 illustrates the structure of different engineered TNFR ligands. TNFR ligand (A), TNFR ligand with linker (B), and TNFR-ligand with linkers and human tenascin (hTNC) multimerization domain (C)

[0102] Figure 20 shows CD40 expression in murine Treg cells transduced with various constructs after a 24 hours resting period. Figure 21 : CD69 and 41 BB expression in murine Treg cells transduced with various constructs after a 24 hours resting period followed by 18 hours of activation with CD40 ligand expressing HEK cells to stimulate the CD40-AIR biosensor.

[0103] Figure 22: Cell counts of human Treg cells transduced with various constructs after a 5 day resting period.

[0104] Figure 23 shows hCD40 expression in human Treg cells transduced with various constructs after a 5 day resting period.

[0105] Embodiments of the invention

[0106] The present disclosure relates to a transmembrane protein comprising the binding domain of a TNFR ligand.

[0107] The present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0108] Engineered TNFR ligand

[0109] The present disclosure makes use of natural and engineered TNFR ligands. While natural TNFR ligands principally are capable to bind to their natural receptors, this typically does not occur on the same cell since expression of the ligands and the receptors is separated. In the present disclosure expression of the TNFR ligands and their receptors occurs simultaneously. Binding of the TNFR ligands to their receptors can further be increased by incorporating appropriate linkers between the transmembrane domain and the extracellular domain of the TNFR ligand. Such engineered TNFR ligands are modified in a way, that they have an increased capacity to bind to receptors on the same cell. The present disclosure provides transmembrane proteins comprising such engineered TNFR ligands. The present disclosure provides transmembrane proteins comprising the binding domain of a TNFR ligand. The present disclosure provides transmembrane proteins comprising the extracellular binding domain of a TNFR ligand.

[0110] In embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain. In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of a TNFR ligand, one or more linkers, a transmembrane domain and a cytoplasmatic domain. In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of a TNFR ligand, one or more linkers, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0111] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of a TNFR ligand, a first linker, a multimerization domain, a second linker, a transmembrane domain and a cytoplasmatic domain.

[0112] In further embodiments the transmembrane protein as disclosed herein comprises

[0113] (i) the extracellular binding domain of a TNFR ligand,

[0114] (ii) a linker,

[0115] (iii) a transmembrane domain and

[0116] (iv) a cytoplasmatic domain, wherein (i) to (iv) are arranged in sequential order.

[0117] In further embodiments the transmembrane protein as disclosed herein comprises

[0118] (i) the extracellular binding domain of a TNFR ligand,

[0119] (ii) a linker,

[0120] (iii) a multimerization domain,

[0121] (iv) a transmembrane domain and

[0122] (v) a cytoplasmatic domain, wherein (i) to (v) are arranged in sequential order.

[0123] In further embodiments the transmembrane protein as disclosed herein comprises

[0124] (i) the extracellular binding domain of a TNFR ligand,

[0125] (ii) a first linker,

[0126] (iii) a multimerization domain,

[0127] (iv) a second linker,

[0128] (v) a transmembrane domain and

[0129] (vi) a cytoplasmatic domain, wherein (i) to (vi) are arranged in sequential order.

[0130] As used herein, “in sequential order” means that the recited domains or linkers occur sequentially, in a continuous series, along the polypeptide chain, while permitting additional sequences, in between, before or after, to be present. In further embodiments the transmembrane protein as disclosed herein consists essentially of the extracellular binding domain of a TNFR ligand, a first linker, a multimerization domain, a second linker, a transmembrane domain and a cytoplasmatic domain.

[0131] In further embodiments the transmembrane protein as disclosed herein consists essentially of

[0132] (i) the extracellular binding domain of a TNFR ligand,

[0133] (ii) a linker,

[0134] (iii) a transmembrane domain and

[0135] (iv) a cytoplasmatic domain, wherein (i) to (iv) are arranged in sequential order.

[0136] In further embodiments the transmembrane protein as disclosed herein consists essentially of

[0137] (i) the extracellular binding domain of a TNFR ligand,

[0138] (ii) a linker,

[0139] (iii) a multimerization domain,

[0140] (iv) a transmembrane domain and

[0141] (v) a cytoplasmatic domain, wherein (i) to (v) are arranged in sequential order.

[0142] In further embodiments the transmembrane protein as disclosed herein consists essentially of

[0143] (i) the extracellular binding domain of a TNFR ligand,

[0144] (ii) a first linker,

[0145] (iii) a multimerization domain,

[0146] (iv) a second linker,

[0147] (v) a transmembrane domain and

[0148] (vi) a cytoplasmatic domain, wherein (i) to (vi) are arranged in sequential order.

[0149] The term “consist essentially of’ when used herein in connection with a construct, composition, use or method, denotes that additional features, elements and / or method steps can be present, but that these additions do not materially affect the basic and novel characteristic(s)” of the claimed invention.

[0150] Cytoplasmatic domain

[0151] In embodiments the cytoplasmatic domain of the transmembrane protein lacks a signaling domain. In some embodiments, the cytoplasmatic domain does not induce NF-kB activation. In some embodiments, the cytoplasmatic domain does not induce IkB phosphorylation. In embodiments the cytoplasmatic domain of the transmembrane protein is a silent cytoplasmatic domain.

[0152] In certain embodiments the cytoplasmatic domain of the transmembrane protein is derived from a TNFR ligand as the extracellular binding domain. In certain embodiments the cytoplasmatic domain of the transmembrane protein is an endogenous cytoplasmatic domain derived from a TNFR ligand. In certain embodiments the cytoplasmatic domain of the transmembrane protein is an endogenous cytoplasmatic domain or a variant thereof derived from a TNFR ligand.

[0153] In certain embodiments the cytoplasmatic domain of the transmembrane protein is a cytoplasmatic domain derived from the same TNFR ligand as the extracellular binding domain.

[0154] In another embodiment, the cytoplasmatic domain of the transmembrane protein is derived from the same TNFR ligand as the extracellular binding domain and is a variant of the endogenous cytoplasmatic domain. In certain embodiments the variant of the cytoplasmatic domain is a silent cytoplasmatic domain.

[0155] Multimerization domain

[0156] In embodiments the transmembrane protein further comprises a multimerization domain. In embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain, a cytoplasmatic domain and a multimerization domain.

[0157] In certain embodiments said multimerization domain is a human multimerization domain. In embodiments, the multimerization domain is derived from an extracellular matrix protein. In certain embodiments, the multimerization domain comprises a tenascin multimerization domain, such as an N- terminal coiled-coil domain of a tenascin family member (e.g., tenascin-C, tenascin-X, or tenascin-W). In certain embodiments, the multimerization domain comprises human tenascin. In an embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDIKDLLSRLEELEGLVSSLREQ (SEQ ID NO: 32) or the amino acid sequence ACGCAAAPDVKELLSRLEELENLVSSLREQ (SEQ ID NO: 33) or a sequence having an identity of at least 70%, preferably of at least 80%, more preferably of at least 90%, and even more preferably of at least 93% to SEQ ID NO:32 or SEQ ID NO:33. In other embodiments, the multimerization domain is derived from other structural proteins that naturally form oligomers, including but not limited to, a collagen triple helix domain, a leucine zipper or other coiled-coil motif, a SAM (sterile alpha motif) domain, a trimerization domain from a viral glycoprotein, a death domain, CARD, or DED oligomerization motif, a p-propeller scaffold (e.g., WD40 repeat domain). In some embodiments, the multimerization domain is synthetic or engineered, and may be designed de novo to confer a desired oligomerization state. In certain embodiments, the multimerization domain is covalently stabilized (e.g., by disulfide bonds) or non-covalently stabilized (e.g., by hydrophobic or electrostatic interactions). In further embodiments, the multimerization domain may be modified, truncated, or otherwise engineered to alter its stability, valency, or orientation of the fused functional domains. In still further embodiments, the polypeptide comprises two or more multimerization domains of the same or different type, thereby allowing the assembly of higher-order structures.

[0158] In some embodiments, the multimerization domain is positioned at the N-terminus of the polypeptide, while in other embodiments it is positioned at the C-terminus, or internally within the polypeptide sequence.

[0159] Additional linkers

[0160] In embodiments, transmembrane protein further comprises one or more additional linkers. In certain embodiments the transmembrane protein comprises the extracellular binding domain of a TNFR ligand, one or more linkers, a transmembrane domain and a cytoplasmatic domain. In certain embodiments the transmembrane protein comprises the extracellular binding domain of a TNFR ligand, two linkers, a transmembrane domain and a cytoplasmatic domain. In certain embodiments, said one or more additional linkers enable the binding of the TNFR ligand to its receptor on the same cell. In certain embodiments, said one or more additional linkers facilitate the binding of the TNFR ligand to its receptor on the same cell. In certain embodiments the linker has a length of 3-60 amino acids, particularly a length of 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, ,56, 58 or 60 amino acids. The linker is preferably a glycine / serine linker, i.e. , a peptide linker substantially consisting of the amino acids glycine and serine. In certain embodiments, said one or more additional linkers are a glycine-serine linker. In certain embodiments, said glycine-serine linker is a (G4S)3-(G4S)I2linker or (G4S)6linker .

[0161] In certain embodiments said transmembrane protein comprising the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain further comprises a multimerization domain and one or more additional linkers.

[0162] Transmembrane domain

[0163] As used herein, a ‘transmembrane domain’ refers to a region of a protein that spans or is embedded within the phospholipid bilayer of a cellular membrane. In an embodiment a transmembrane domain of a transmembrane protein as provided herein is embedded in the phospholipid bilayer of a cell expressing the transmembrane protein.

[0164] In some embodiments, the transmembrane domain of a transmembrane protein is derived from a TNFR ligand. In some embodiments, the transmembrane domain of a transmembrane protein is the endogenous transmembrane domain of a TNFR ligand. In some embodiments, the transmembrane domain and the extracellular binding domain of a transmembrane protein are derived from the same protein. In some embodiments, having a transmembrane domain and an extracellular binding domain derived from the same protein provides rigidity to the transmembrane protein. In some embodiments, the transmembrane domain comprises human sequence. In some embodiments, the transmembrane domain is a portion of a human membrane-bound protein.

[0165] In some embodiments, a transmembrane domain is at least 5 amino acids long (e.g., at least 5, at least 10, at least 12, at least 15, at least 20, at least 25, or at least 30 amino acids long). In some embodiments, a transmembrane domain is at most 100 amino acids long (e.g., at most 100, at most 80, or at most 50 amino acids long). In some embodiments, a transmembrane domain is 10-100 amino acids long (e.g., 10- 100, 10-80, 10-70, 10-60, 10-50, 10-40, 20-40, 20-30, 25-35, 20-40, 25-50, 30-50, 30-35, 30-40, 40-50, 40- 60, 40-80, or 40- 100 amino acids long). In some embodiments, a transmembrane domain is 2-20 nm (e.g., 2- 20, 2-15, 2-10, 4-10, 4-15, 4-20, 5-15, 5-10, 7-10, 7-15 or 7.5-12.5 nm) long. In some embodiments, a transmembrane domain is 5-10 nm long, for example, 6 nm, 7 nm, 8 nm, or 9 nm.

[0166] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the binding domain of human GITRL. In other embodiments the transmembrane protein comprising a binding domain of a TNFR ligand, comprises the binding domain of a polypeptide with the amino acid sequence of SEQ ID No. 1 .

[0167] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the binding domain of human GITRL, wherein said transmembrane protein has a linker between the transmembrane domain and the extracellular domain of human GITRL, wherein said linker enables said transmembrane protein to bind to a receptor of human GITRL on the same cell in which said transmembrane protein is expressed. Preferably said linker is a glycine-serine linker. More preferably said linker is a (G^S)^(SEO ID No. 23) -(G^S)io (SEO ID No. 24) linker. Most preferably said linker is a (G^S)B(SEO ID No. 25) linker.

[0168] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the amino acid sequence of SEQ ID No. 12. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence of SEQ ID No.12.

[0169] In certain embodiments, the transmembrane protein comprises the extracellular domain of human GITRL, a linker, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0170] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of human GITRL, one or more linkers, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0171] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of human GITRL, a first linker, a multimerization domain, a second linker, a transmembrane domain and a cytoplasmatic domain.

[0172] In further embodiments the transmembrane protein as disclosed herein comprises (i) the extracellular binding domain of human GITRL,

[0173] (ii) a linker,

[0174] (iii) a transmembrane domain and

[0175] (iv) a cytoplasmatic domain, wherein (i) to (iv) are arranged in sequential order.

[0176] In further embodiments the transmembrane protein as disclosed herein comprises

[0177] (i) the extracellular binding domain of human GITRL,

[0178] (ii) a linker,

[0179] (iii) a multimerization domain,

[0180] (iv) a transmembrane domain and

[0181] (v) a cytoplasmatic domain, wherein (i) to (v) are arranged in sequential order.

[0182] In further embodiments the transmembrane protein as disclosed herein comprises

[0183] (i) the extracellular binding domain of human GITRL,

[0184] (ii) a first linker,

[0185] (iii) a multimerization domain,

[0186] (iv) a second linker,

[0187] (v) a transmembrane domain and

[0188] (vi) a cytoplasmatic domain, wherein (i) to (vi) are arranged in sequential order.

[0189] In certain embodiments said linker, or first or second linker are a glycine-serine linker or glycine-serine linkers. Preferably said linker, or first or second linker is a (G^Sh (SEO ID No. 23) -(G4S)I7(SEO ID No. 24) linker or an extended glycine-serine linker. Most preferably said first linker is a is a (G4S)6(SEO ID No. 25) linker and the second linker is a (G4S)Q (SE0 ID No. 27) .

[0190] In certain embodiments said multimerization domain is a human multimerization domain. In embodiments, the multimerization domain is derived from an extracellular matrix protein. In certain embodiments, the multimerization domain comprises a tenascin multimerization domain, such as an N- terminal coiled-coil domain of a tenascin family member (e.g., tenascin-C, tenascin-X, or tenascin-W). In certain embodiments, the multimerization domain comprises human tenascin. In a preferred embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDIKDLLSRLEELEGLVSSLREQ (SEQ ID NO:32) or the amino acid sequence ACGCAAAPDVKELLSRLEELENLVSSLREQ (SEQ ID NO:33) or a sequence having an identity of at least 70%, preferably of at least 80%, more preferably of at least 90%, and even more preferably of at least 93% to SEQ ID NO:32 or SEQ ID NO:33. In a more preferred embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDVKELLSRLEELENLVS SLREQ (SEQ ID NO:33).

[0191] In certain embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID No. 35. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence of SEQ ID No.35.

[0192] In certain embodiments, the transmembrane protein comprises the amino acid sequence encoded by the nucleic acid according to SEQ ID No. 34. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence encoded by the nucleic acid according to SEQ ID No. 34.

[0193] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising any of the aforementioned and the extracellular binding domain of human GITRL, wherein said binding domain of human GITRL is capable of binding to a receptor on the same T cell.

[0194] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising any of aforementioned binding domain of human GITRL, wherein said binding domain of human GITRL is capable of binding to a receptor on the same T cell.

[0195] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the binding domain of human TL1A. In other embodiments the transmembrane protein comprising a binding domain of a TNFR ligand, comprises the binding domain of a polypeptide with the amino acid sequence of SEQ ID No. 3.

[0196] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the binding domain of human TL1A, wherein said transmembrane protein has a linker between the transmembrane domain and the extracellular domain of human TL1A, wherein said linker enables said transmembrane protein to bind to a receptor of human TLIA on the same cell in which said transmembrane protein is expressed. Preferably said linker is a glycine-serine linker. More preferably said linker is a (G4S)3(SEQ ID No. 23) -(G4S)i? (SEQ ID No. 24) linker. Most preferably said linker is a (G4S)6(SEQ ID No. 25) linker.

[0197] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the amino acid sequence of SEQ ID No. 16. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence of SEQ ID No. 16. In certain embodiments, the transmembrane protein comprises the extracellular domain of human TL1 A, a linker, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0198] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of human TL1A, one or more linkers, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0199] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of human TL1A, a first linker, a multimerization domain, a second linker, a transmembrane domain and a cytoplasmatic domain.

[0200] In further embodiments the transmembrane protein as disclosed herein comprises

[0201] (i) the extracellular binding domain of human TL1 A,

[0202] (ii) a linker,

[0203] (iii) a transmembrane domain and

[0204] (iv) a cytoplasmatic domain, wherein (i) to (iv) are arranged in sequential order.

[0205] In further embodiments the transmembrane protein as disclosed herein comprises

[0206] (i) the extracellular binding domain of human TL1A,

[0207] (ii) a linker,

[0208] (iii) a multimerization domain,

[0209] (iv) a transmembrane domain and

[0210] (v) a cytoplasmatic domain, wherein (i) to (v) are arranged in sequential order.

[0211] In further embodiments the transmembrane protein as disclosed herein comprises

[0212] (i) the extracellular binding domain human TL1 A,

[0213] (ii) a first linker,

[0214] (iii) a multimerization domain,

[0215] (iv) a second linker,

[0216] (v) a transmembrane domain and

[0217] (vi) a cytoplasmatic domain, wherein (i) to (vi) are arranged in sequential order.

[0218] In certain embodiments said linker, or first or second linker are a glycine-serine linker or glycine-serine linkers. Preferably said linker, or first or second linker is a (G^Sh (SEO ID No. 23) -(G^S)I7(SEO ID No. 24) linker or an extended glycine-serine linker. Most preferably said first linker is a is a (G4S)6(SEO ID No. 25) linker and the second linker is a (G4S)? (SE0 ID No. 27) .

[0219] In certain embodiments said multimerization domain is a human multimerization domain. In embodiments, the multimerization domain is derived from an extracellular matrix protein. In certain embodiments, the multimerization domain comprises a tenascin multimerization domain, such as an N- terminal coiled-coil domain of a tenascin family member (e.g., tenascin-C, tenascin-X, or tenascin-W). In certain embodiments, the multimerization domain comprises human tenascin. In a preferred embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDIKDLLSRLEELEGLVSSLREQ (SEQ ID NO:32) or the amino acid sequence ACGCAAAPDVKELLSRLEELENLVSSLREQ (SEQ ID NO:33) or a sequence having an identity of at least 70%, preferably of at least 80%, more preferably of at least 90%, and even more preferably of at least 93% to SEQ ID NO:32 or SEQ ID NO:33. In a more preferred embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDVKELLSRLEELENLVS SLREQ (SEQ ID NO:33)

[0220] In certain embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID No. 39. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence of SEQ ID No.39.

[0221] In certain embodiments, the transmembrane protein comprises the amino acid sequence encoded by the nucleic acid according to SEQ ID No. 38. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence encoded by the nucleic acid according to SEQ ID No. 38.

[0222] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising any of the aforementioned and the extracellular binding domain of human TL1A, wherein said binding domain of human TL1A is capable of binding to a receptor on the same T cell.

[0223] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising any of aforementioned binding domain of human TL1 A, wherein said binding domain of human TL1A is capable of binding to a receptor on the same T cell.

[0224] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the binding domain of human QX40L. In other embodiments the transmembrane protein comprising a binding domain of a TNFR ligand, comprises the binding domain of a polypeptide with the amino acid sequence of SEQ ID No. 5. In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the binding domain of human OX40L, wherein said transmembrane protein has a linker between the transmembrane domain and the extracellular domain of human OX40L, wherein said linker enables said transmembrane protein to bind to a receptor of human OX40L on the same cell in which said transmembrane protein is expressed. Preferably said linker is a glycine-serine linker. More preferably said linker is a (G^S)JSEO ID No. 23) -(G^S)io (SEO ID No. 24) linker. Most preferably said linker is a (G^S)B(SEO ID No. 25) linker.

[0225] In certain embodiments, the transmembrane protein comprising a binding domain of a TNFR ligand comprises the amino acid sequence of SEQ ID No. 18. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence of SEQ ID No. 18.

[0226] In certain embodiments, the transmembrane protein comprises the extracellular domain of human OX40L, a linker, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0227] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of human GX40L, one or more linkers, a multimerization domain, a transmembrane domain and a cytoplasmatic domain.

[0228] In further embodiments the transmembrane protein as disclosed herein comprises the extracellular binding domain of human GX40L, a first linker, a multimerization domain, a second linker, a transmembrane domain and a cytoplasmatic domain.

[0229] In further embodiments the transmembrane protein as disclosed herein comprises

[0230] (i) the extracellular binding domain of human GX40L,

[0231] (ii) a linker,

[0232] (iii) a transmembrane domain and

[0233] (iv) a cytoplasmatic domain, wherein (i) to (iv) are arranged in sequential order.

[0234] In further embodiments the transmembrane protein as disclosed herein comprises

[0235] (i) the extracellular binding domain of human GX40L,

[0236] (ii) a linker,

[0237] (iii) a multimerization domain,

[0238] (iv) a transmembrane domain and

[0239] (v) a cytoplasmatic domain, wherein (i) to (v) are arranged in sequential order.

[0240] In further embodiments the transmembrane protein as disclosed herein comprises

[0241] (i) the extracellular binding domain human GX40L, (ii) a first linker,

[0242] (iii) a multimerization domain,

[0243] (iv) a second linker,

[0244] (v) a transmembrane domain and

[0245] (vi) a cytoplasmatic domain, wherein (i) to (vi) are arranged in sequential order.

[0246] In certain embodiments said linker, or first or second linker are a glycine-serine linker or glycine-serine linkers. Preferably said linker, or first or second linker is a (SEO ID No. 24) linker or an extended glycine-serine linker. Most preferably said first linker is a is a (G4S)6(SEO ID No. 25) linker and the second linker is a (G4S)Q (SE0 ID No. 27) .

[0247] In certain embodiments said multimerization domain is a human multimerization domain. In embodiments, the multimerization domain is derived from an extracellular matrix protein. In certain embodiments, the multimerization domain comprises a tenascin multimerization domain, such as an N- terminal coiled-coil domain of a tenascin family member (e.g., tenascin-C, tenascin-X, or tenascin-W). In certain embodiments, the multimerization domain comprises human tenascin. In a preferred embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDIKDLLSRLEELEGLVSSLREQ (SEQ ID NO:32) or the amino acid sequence ACGCAAAPDVKELLSRLEELENLVSSLREQ (SEQ ID NO:33) or a sequence having an identity of at least 70%, preferably of at least 80%, more preferably of at least 90%, and even more preferably of at least 93% to SEQ ID NO:32 or SEQ ID NO:33. In a more preferred embodiment, the tenascin multimerization domain comprises the amino acid sequence ACGCAAAPDVKELLSRLEELENLVS SLREQ (SEQ ID NO:33)

[0248] In certain embodiments, the transmembrane protein comprises the amino acid sequence of SEQ ID No. 37. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence of SEQ ID No.37.

[0249] In certain embodiments, the transmembrane protein comprises the amino acid sequence encoded by the nucleic acid according to SEQ ID No. 36. In certain embodiments the transmembrane protein comprising a binding domain of a TNFR ligand consists of the amino acid sequence encoded by the nucleic acid according to SEQ ID No. 36.

[0250] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising any of the aforementioned and the extracellular binding domain of human QX40L, wherein said binding domain of human QX40L is capable of binding to a receptor on the same T cell.

[0251] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising any of aforementioned binding domain of human OX40L, wherein said binding domain of human OX40L is capable of binding to a receptor on the same T cell.

[0252] TheTNFR ligands may also be further derivatized or mutated. For example, mutations can be incorporated into the extracellular domain of the TNFR ligand which increase the binding of the ligand to its receptor. Alternative, mutations or deletions can be incorporated into the transmembrane protein comprising the binding domain of the TNFR ligand which prevent cleavage of the extracellular part of the transmembrane protein, and thereby the formation of soluble forms of the protein. Such approaches are known in the art. See for example J Immunol (2018) 200 (4): 1360-1369. Exemplary non-cleavable, murine constructs are shown in the following table.

[0253] Table 1 :

[0254] Chimeric antigen receptors, artificial immune receptors and T cell receptors

[0255] The TNFR ligand engineered according to the present disclosure allows the TNFR ligand to bind to its receptor on the same T cell, thereby stimulating its own survival via Nfkb and other pro-survival signaling pathways. It was found that a linker-modified TNFR ligand (such as GITRL) itself shows a higher expression level than the wildtype version of the ligand. The modified ligand is capable of binding to its corresponding receptor, which is endogenously expressed on the same cell, and stimulates cell survival via Nfkb and other pro-survival signaling pathways. Expression of the linker-modified TNFR ligands results in better survival of the engineered cells and leads to higher and more stable expression of the transgenic receptor (such as CD40-AIR) compared to cells without TNFR ligand expression or compared to cells with only wildtype TNFR ligand sequence. This enhanced transgenic receptor expression potentially correlates with higher activity (and better functionality) of the transgenic receptor, respectively results in better functionality of the engineered cell.

[0256] Principally any chimeric antigen receptor, artificial immune receptor orT cell receptor can be used within the spirit of the present invention.

[0257] In certain embodiments, said chimeric antigen receptor comprises an extracellular ligand binding domain that specifically binds to an antigen, a transmembrane domain, and an intracellular domain that comprises one or more signaling domains and / or costimulatory domains.

[0258] In certain embodiments said extracellular ligand binding domain is a single chain antibody (scFv). In certain embodiments said extracellular ligand binding domain binds to an antigen selected from the group including, but not limited to, the CD19, CD20, HER2, NY-ESO-1 , MUC1 , CD123, FLT3, B7-H3, CD33, IL1 RAP, CLL1 (CLEC12A)PSA, CEA, VEGF, VEGF-R2, CD22, ROR1 , mesothelin, c-Met, Glycolipid F77, FAP, EGFRvlll, MAGE A3, 5T4, WT1 , KG2D ligand, folate receptor (FRa), Wnt1 antigens, HLA-A2 (and other HLA variants), IL-23R, and citrullinated antigens.

[0259] In certain embodiments, said signaling domain acts in a stimulatory manner to induce immune effector functions. In certain embodiments, said signaling domain contains a signaling motif known as Immunoreceptor Tyrosine-based Activation Motif, or ITAM. In certain embodiments, the intracellular signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1 G) , FcR beta (Fc Epsilon Rib) , CD79a, CD79b, Fcgamma Rlla, DAP10, and DAP12. A preferred TCR signaling domain is a TCR signaling domain selected from CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon. A particularly preferred TCR signaling domain CD3 zeta, CD3 gamma, CD3 delta and CD3 epsilon.

[0260] In certain embodiments, said costimulatory domain refer to the domain of a costimulatory molecule or costimulatory receptor responsible for mediating a costimulatory response by the T cell. Generally, a costimulatory domain can be derived from a transmembrane co- stimulatory receptor, particularly from an intracellular portion of a co- stimulatory receptor. Non-limiting examples of co-stimulatory polypeptides include, but are not limited CD27, CD28, CD8, 4-1 BB (CD137), 0X40, CD30, CD40, CD127, PD-1 , IGOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, Nl, N6, or any combination thereof. A commonly used co-stimulatory domain in CARs is 4-1 BB (CD137).

[0261] In certain embodiments, the present disclosure relates to a) a chimeric antigen receptor as characterized herein above, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0262] In certain embodiments, the present disclosure relates to a) an artificial immune receptor as characterized herein above, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0263] In certain embodiments, said artificial immune receptor comprises an extracellular domain of a member of the tumor necrosis factor receptor superfamily, a transmembrane domain, a cytoplasmic costimulatory signaling domain, and a cytoplasmic T cell receptor signaling domain.

[0264] In certain embodiments, said member of the tumor necrosis factor receptor superfamily is selected from TNFR1 , TNFR2, Fas, DR4, DR5, DR3, DR6, EDAR, XEDAR, TROY, LTBR, NGFR, CD18, CD134, CD40, CD27, CD30, CD137, TRAILR3, TRAILR4, CD265, osteoprotegerin, CD266, TACI, BAFF, BAFF receptor, APRIL, CD270, CD269 and CD357.

[0265] In certain embodiments, said cytoplasmic costimulatory signaling domain is selected from 4-IBB (CD137), BAFFR, 0X40, CD27, CD28, IGOS, CD40, 2B4, GITR, HVEM, 0X40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1 to TLR9 receptors, IL-2, IL-7 and IL-15 receptors.

[0266] In certain embodiments, said T cell receptor signaling domain is selected from CD3 zeta, CD3 gamma and CD3 epsilon, preferably CD3 zeta.

[0267] In certain embodiments, the present disclosure relates to a) an artificial immune receptor as characterized herein above, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0268] In certain embodiments, the present disclosure relates to a) a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0269] A mouse CD40-AIR, containing extracellular and transmembrane domain of murine CD40, a CD28 costimulatory domain and a CD3-zeta-chain was used to exemplify the present invention. This mouse CD40-AIR contains the following sequences.

[0270] Table 2: T cells

[0271] In certain embodiments, the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0272] In certain embodiments, said T cell is selected from a T helper cell, a cytotoxic T cell, a memory T cell, a regulatory T cells and a natural killer T cells. In some embodiments, the T cell is an engineered T cell. Preferably said T cell is a regulatory T cell.

[0273] Therefore, in certain embodiments the present disclosure relates to a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell, and wherein said T cell is selected from a T helper cell, a cytotoxic T cell, a memory T cell, a regulatory T cells and a natural killer T cells. In some embodiments, the T cell is an engineered T cell. Preferably said T cell is a regulatory T cell. in certain embodiments the present disclosure relates to a regulatory T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.

[0274] In certain embodiments, said T cell is a cytotoxic T cell. In certain embodiments said cytotoxic T cell is a NK cell. In certain embodiments said cytotoxic T cell is a NKT cell. In certain embodiments said cytotoxic T cell is an iNKT cell. In certain embodiments, said T cell is a regulatory T cell.

[0275] Nucleic acids, vectors and host cells

[0276] The chimeric antigen receptors, the artificial immune receptors, the T cell receptors and the transmembrane protein comprising the binding domain of a TNFR ligand of the present disclosure are encoded by nucleic acids. Therefore, the in certain embodiments the present disclosure relates to nucleic acids encoding aforementioned components.

[0277] In certain embodiments the present disclosure relates a vector comprising aforementioned nucleic acids.

[0278] In certain embodiments the present disclosure relates to a host cell comprising aforementioned nucleic acids or vectors.

[0279] In certain embodiments the present disclosure relates to a host cell expressing aforementioned components.

[0280] In preferred embodiments, said host cell is a eukaryotic host cell. Therefore, in certain embodiments the present disclosure relates to a eukaryotic host cell comprising a nucleic acid or a vector encoding the components of the present disclosure.

[0281] Therapeutic use

[0282] Transmembrane proteins as disclosed herein can be used therapeutically for the prevention and treatment of diseases and disorders. In embodiments transmembrane proteins as disclosed herein are for use in medicine. In certain embodiments, said use in medicine is the treatment of an auto-inflammatory disease, such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis or auto-immune gastritis. In certain embodiments, said use in medicine is the treatment of an inflammatory disorder or cancer e.g. leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma, glioblastoma, colorectal cancer and gastrointestinal cancers, melanoma, pancreatic cancer, kidney / renal cell cancer, lung cancer, breast cancer, prostate cancer and others. In certain embodiments, said use in medicine is the treatment of graft versus host disease or the use in solid organ transplantation.

[0283] T cells equipped with the components of the present invention can be used therapeutically for the prevention and treatment of diseases and disorders.

[0284] In certain embodiments , the present disclosure provides a T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein comprising the binding domain of a TNFR ligand, wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell, for use in medicine. In certain embodiments, said use in medicine is the treatment of an auto-inflammatory disease, such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis or auto-immune gastritis. In certain embodiments, said use in medicine is the treatment of an inflammatory disorder or cancer e.g. leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma, glioblastoma, colorectal cancer and gastrointestinal cancers, melanoma, pancreatic cancer, kidney / renal cell cancer, lung cancer, breast cancer, prostate cancer and others. In certain embodiments, said use in medicine is the treatment of graft versus host disease or the use in solid organ transplantation.

[0285] Examples

[0286] Example 1 : General methods, constructs and schematic overview

[0287] The present invention is based on the hypothesis that the expression of membrane-anchored, synthetically modified ligands of the Tumor-Necrosis-Factor (TNF) superfamily in T cells could improve the survival of the cells after adoptive cell transfer. In order to facilitate binding of the ligand to the receptor on the same cell, a linker is inserted between the transmembrane domain and the extracellular domain of the ligand of the TNF family (such as GITRL, 41 BBL, OX40L or TL1A). This linker may be any conventionally used linker, such as a glycine-serine linker. The modified ligand is genetically inserted into the DNA of a T cell by viral or non-viral genetic engineering techniques. When the modified ligand is expressed, it will bind to corresponding endogenous TNF receptor of the same T cell and stimulate its own survival via Nfkb and other pro-survival signaling pathways. The concept is depicted in Figure 1 .

[0288] The engineered TNFR ligands in the following Table were generated and utilized in the present disclosure. As controls served the natural TNFR ligands.

[0289] Table 3:

[0290] Example 2: Survival of regulatory T cells significantly increases when a synthetic receptor is co-expressed with a modified ligand

[0291] Regulatory T cells (Treg cells) were isolated, expanded via anti-CD3 / CD28 antibody-coupled beads and IL2 and transduced with a retrovirus encoding genetic information for an artificial immune receptor (AIR) only, or for an AIR additionally comprising a modified ligand (GITRL, OX40L, TL1A). The AIR could be theoretically be replaced by a chimeric antigen receptor (CAR) or T cell receptor (TCR) or any kind of synthetic receptor.

[0292] FACS-sorted Treg cells were seeded at 3 x 104- 4 x 104cells per well in a 96 well round-bottom plate with a-CD3 / CD28 beads (Miltenyi Biotec, 4 beads per cell) and 2000 U / ml rhlL-2 (Proleukin S, Novartis). DMEM (Gibco / lnvitrogen) was used as culture medium supplemented with 10% FCS, 2 mM l-glutamine, 5 x 10-5M 2-ME, MEM-Vitamin Sol., 10 mM HEPES, 100mM Natrium-Pyruvat, 1 % NEAA (PAN Biotech), 100 U / ml penicillin-streptomycin (Gibco). Murine Treg cells were transduced 48h after isolation. Retroviral transduction with Murine Stem CellViurs (MSCV) of Treg cells was done according to the following protocol:

[0293] Phoenix-Eco cells were seeded on a gelatin matrix at 1 .3 x 106cells per well in a six well plate 6 hours before lipofection. To produce liposomal particles containing the viral transgene, 3 pg of vector DNA and 1 pg of additional pCL-Eco packaging plasmid were coincubated with 12 pL of TranslT-293 transfection reagent (MoBiTec) for 20 minutes in OptiMEM medium at RT. Liposomes were added to Phoenix-Eco cells and incubated for an additional 16 hours. Afterwards medium was exchanged and production of viral particles could proceed for 24 h. Then supernatant with produced pMSCV retrovirus was added to Treg cell cultures (virus supernatant : remaining cell culture medium 1 :1) together with Polybrene (final concentration 3pg / ml) and mixed gently. Treg cells were transduced for 6.5 hours incubation at 37°C. Afterwards, viral supernatant was removed and cells were incubated with fresh medium supplemented with IL-2 (2000 U / ml) for another 72 to 96 hours.

[0294] Treg cells were deprived of anti-CD3 / CD28 antibody-coupled beads 3 days after transduction and seeded in full-medium with low dose of IL-2, with a defined cell number, for 5 days of “resting” in a 96-well U-bottom plate. After 5 days of resting, living Treg cells were counted with an automated cell counter. Results are shown in Figure 2.

[0295] Cell counts for AIR only transduced Treg cells did not change during the resting period, whereas additional expression of modified GITRL-6xGly(4)Ser-linker and GX40L-6xGly(4)Ser-linker significantly increased cell yield (about 2-fold). TL1 A-6xGly(4)Ser-linker expression only tendentially increased cell count (RM-one-way ANOVA with Dunnett's multiple comparisons test; GITRL: n=3, p=0.0056; OX40L n=3, p=0.001 ; TL1A n=3, p=0.1498).

[0296] Figure 3 shows the differences in absolute cell counts between day 0 to day 5 (“resting period”) from the experiment shown in Figure 2 (RM-one-way ANOVA with Dunnett's multiple comparisons test; GITRL: n=3, p= 0.0010; GX40L n=3, p= 0.0002; TL1A n=3, p= 0.1445).

[0297] Figure 4 shows the differences in absolute cell counts for Treg cells transduced with various constructs between day 0 to day 5 (“resting period”) from the experiment shown in Figure 2. Panel A: AIR only transduced Treg cells (paired t-test, p=0.4785); panel B: AIR plus GITRL-6xGly(4)Ser-linker transduced Treg cells (paired t-test, p=0.0195); panel C: AIR plus GX40L-6xGly(4)Ser-linker transduced Treg cells (paired t- test, p=0.0027); panel D: AIR plus TL1A -6xGly(4)Ser-linker transduced Treg cells (paired t-test, p= 0.1467). Clearly, all Treg cells transduced with a modified ligand of the Tumor-Necrosis-Factor (TNF) superfamily in addition to the AIR had a significantly increase cell count.

[0298] The experiment was repeated, with the difference that 50,000 Treg cells were seeded and rested for 4 days after transduction. Cell counts (Figure 12) for AIR only transduced Treg cells did not change during the resting period, whereas additional expression of modified GITRL-6xGly(4)Ser-linker and GX40L-6xGly(4)Ser -linker significantly increased cell yield (about 2-fold). TL1 A-6xGly(4)Ser-linker expression only tendentially increased cell count (RM-one-way ANOVA with Dunnett's multiple comparisons test: AIR vs. AIR+GITRL- Gly(4)Ser, p=0,0002; AIR vs. AIR+GX40L-Gly(4)Ser, p<0.0001 ; AIR vs. AIR+TL1A-Gly(4)Ser, p=0.8999). Figure 13 shows the difference between living cell numbers on day 0 and harvested cells after 4 days resting (AIR vs. AIR+GITRL-Gly(4)Ser, p=0.0002; AIR vs. AIR+GX40L-Gly(4)Ser, p<0.0001 ; AIR vs. AIR+TL1A-Gly(4)Ser, p=0.8999). Figure 14 shows the difference between living cell numbers on day 0 and harvested cells after 4 days resting as fold increase (AIR vs. AIR+GITRL-Gly(4)Ser, p=0.0002; AIR vs. AIR+GX40L-Gly(4)Ser, p<0.0001; AIR vs. AIR+TL1A-Gly(4)Ser, p=0.8976).

[0299] Figure 22 provides data on cell counts of human Treg cells transduced with various constructs after a 5 day resting period. Here in Panel A cell numbers of CD40 AIR only transduced Treg cells are compared with CD40 AIR plus GITRL-6xGly(4)Ser-linker transduced Treg cells and CD40 AIR plus GITRL-6xGly(4)Ser-linker with hTNC multimerization domain transduced Treg cells. Panel B compares cell numbers of CD40 AIR only transduced human Treg cells with CD40 AIR plus GX40L-6xGly(4)Ser-linker transduced Treg cells and CD40 AIR plus GX40L-6xGly(4)Ser-linker with hTNC multimerization domain transduced Treg cells. Panel C compares cell numbers of CD40 AIR only transduced human Treg cells with CD40 AIR plus TL1A- 6xGly(4)Ser-linker transduced Treg cells and CD40 AIR plus TL1A-6xGly(4)Ser-linker with hTNC multimerization domain transduced Treg cells. The GX40L and TL1A constructs show higher human Treg cells counts after 5 days of resting compared to the CD40 AIR only transduced Treg cells. Example 3: A modified linker is required to generate regulatory T cells with an increased survival

[0300] In this experiment the effect of the linker was investigated. Regulatory T cells transduced with an AIR and a modified GITRL ligand containing a 6x(Gly(4)Ser) linker (SEO ID No. 25) were compared to regulatory T cells transduced with an AIR and a natural GITRL ligand (i.e. GITRL ligand without an additional linker). The AIR could be theoretically be replaced by a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0301] Regulatory T cells (Treg cells) were isolated, expanded via anti-CD3 / CD28 antibody-coupled bead stimulation and IL-2 and transduced with a retrovirus encoding the constructs mentioned above. Treg cells were deprived of anti-CD3 / CD28 antibody-coupled beads 3 days after transduction and seeded in fullmedium without IL-2 for 4 days of “resting” in a 96-well U-bottom plate (starting with 50.000 cells per well). After 4 days of resting, living Treg cells were counted with an automated cell counter.

[0302] Results are shown in Figure 5. Additional expression of the modified GITRL significantly increased cell yield, whereas expression of wildtype GITRL did not increase cell yield significantly, indicating improved functionality of the GITRL with 6xGly(4)Ser-linker (SEO ID No. 25). (paired t-test, modified GITRL p=0.0156; wildtype GITRL p=0.0817).

[0303] Example 4: Characterization of Treg cells engineered with linker-modified TNFR ligands

[0304] Treg cells from FOXP3-hCD2 reporter mice were isolated and expanded via anti-CD3 / CD28 antibody- coupled bead stimulation and IL-2 (2,000 U / ml). 3 days after transduction, the purity of Treg cell cultures were assessed by flow cytometry.

[0305] Figure 6 shows the frequency of hCD2 / FOXP3 positive cells among expanded CD4+T cells. No differences in hCD2 / FOXP3 expression between wildtype (untransduced) Treg cells orTreg cells transduced with an AIR only or an AIR plus a wildtype version of GITRL or an AIR plus Gly(4)Ser-linker modified version of GITRL were detectable (one-way ANOVA). This shows that purity and FOXP3 expression levels of the Treg cell cultures are not altered by expression of Gly(4)Ser-linker modified version of GITRL. Therefore, Treg cell suppressive activity and functionality should be stable when Gly(4)Ser-linker modified version of GITRL is expressed in a Treg cell. The experiment was repeated also including TL1A- and GX40L-Gly(4)Ser Al Rs, confirming these findings (Figure 15; one-way ANOVA: AIR vs. AIR+GITRL-Gly(4)Ser, p=0.8607; AIR vs. AIR+GX40L-Gly(4)Ser, p=0.3356; AIR vs. AIR+TL1A-Gly(4)Ser, p=0.2127).

[0306] Untransduced and transduced Treg cells were also stained with antibodies for flow cytometric analysis to measure AIR (CD40) expression and GITRL expression. Results are shown in Figure 7. GITRL protein expression on the Treg cell surface was only detectable in Treg cells transduced with an AIR plus Gly(4)Ser- linker and not in Treg cells transduced with AIR plus wildtype version of GITRL. A summary plot is shown in Figure 8. Statistical significance of the data is shown in the following table (Mean fluorescence intensity (MFI) for GITRL staining (stained in PE); one-way ANOVA with Tukey's multiple comparisons test; measured from 4 biological replicates).

[0307] Table 4:

[0308] Comparison Summary Adjusted P Value wildtype vs. AIR ns 0,9581 wildtype vs. AIR+GITRL-Gly(4)Ser wildtype vs. AIR+GITRL-wt ns 0,3761

[0309] AIR vs. AIR+GITRL-Gly(4)Ser

[0310] AIR vs. AIR+GITRL-wt ns 0,6500

[0311] AIR+GITRL-Gly(4)Ser vs. AIR+GITRL-wt

[0312] Interestingly, also CD40-AIR expression was enhanced in Treg cells with Gly(4)Ser-linker-modified GITRL. A summary plot is shown in Figure 9. Statistical significance of the data is shown in the following table (Mean fluorescence intensity (MFI) for CD40 staining (stained in PE / Cy7); one-way ANOVA with Tukey's multiple comparisons test; measured from 4 biological replicates).

[0313] Table 5:

[0314] Comparison Summary Adjusted P Value wildtype vs. AIR *** 0,0009

[0315] AIR vs. AIR+GITRL-wt ns 0,0638

[0316] Al R+GITRL-Gly(4)Ser vs. AIR+GITRL-wt *** 0,0002

[0317] Figure 20 provides further data on CD40 expression in murine Treg cells transduced with various constructs after a 24 hours resting period. Panel A in Figure 20 shows representative histograms depicting CD40 expression in murine Treg cells transduced with CD40 AIR only, with CD40 AIR plus GITRL- 6xGly(4)Ser-linker, and with CD40 AIR plus GITRL-6xGly(4)Ser-linker with human tenascin (hTNC) multimerization domain (above) and quantification thereof (below). Panel B shows representative histograms depicting CD40 expression in murine Treg cells transduced with CD40 AIR only, with CD40 AIR plus OX40L-6xGly(4)Ser-linker, and with CD40 AIR plus OX40L-6xGly(4)Ser-linker with hTNC multimerization domain (above) and quantification thereof (below). Panel C shows representative histograms depicting CD40 expression in murine Treg cells transduced with CD40 AIR only, with CD40 AIR plus TL1A-6xGly(4)Ser-linker, and with CD40 AIR plus TL1A-6xGly(4)Ser-linker with hTNC multimerization domain (above) and quantification thereof (below). The human tenascin (hTNC) multimerization domain containing constructs for GITRL and GX40L are superior over the CD40 AIR plus GITRL-6xGly(4)Ser-linker or the CD40 AIR plus GX40L-6xGly(4)Ser-linker construct, respectively. All human tenascin (hTNC) multimerization domain containing constructs for GITRL, GX40L and TLIA are superior to the CD40 AIR only construct.

[0318] Figure 23 shows data on hCD40 expression in human Treg cells transduced with various constructs after a 5 day resting period. Here, Panel A shows representative flow cytometry plots depicting hCD40 expression in human Treg cells transduced with CD40 AIR plus GITRL-6xGly(4)Ser-linker (left) or CD40 AIR plus GITRL- 6xGly(4)Ser-linker with hTNC multimerization domain (right). Panel B shows the quantification of hCD40 expression in human Treg cells transduced with CD40 AIR plus GITRL-6xGly(4)Ser-linker or CD40 AIR plus GITRL-6xGly(4)Ser-linker with hTNC multimerization domain (left), CD40 AIR plus CX40L-6xGly(4)Ser-linker or CD40 AIR plus CX40L-6xGly(4)Ser-linker with hTNC multimerization domain (left), and CD40 AIR plus TL1A-6xGly(4)Ser-linker or CD40 AIR plus TL1A-6xGly(4)Ser-linker with hTNC multimerization domain (right). The human tenascin (hTNC) multimerization domain containing constructs for GITRL, CX40L and TL1A are superior in all three donors over the CD40 AIR plus GITRL-6xGly(4)Ser-linker or the CD40 AIR plus CX40L-6xGly(4)Ser-linker or the CD40 AIR plus TL1A-6xGly(4)Ser-linker constructs, respectively.

[0319] Example 5: Engineered TNFR ligands least to an increased expression of AIRs

[0320] Regulatory T cells (Treg cells) were isolated, expanded via anti-CD3 / CD28 antibody-coupled beads and IL-2, and transduced with a retrovirus encoding genetic information for an artificial immune receptor (AIR) only or an AIR with a Gly(4)Ser-modified ligand (GITRL, CX40L, TL1A). Treg cells were stained with antibodies for flow cytometric analysis to measure AIR (CD40) expression. Results are shown in Figure 10. AIR protein expression on the cell surface was increased in Treg cells transduced with an AIR plus Gly(4)Ser-linker modified ligand (GITRL, CX40L, TL1A).

[0321] The experiment was repeated with essentially the same result. See Figure 16. AIR protein expression on the cell surface was increased in Treg cells transduced with an AIR plus Gly(4)Ser-linker modified ligand (GITRL, CX40L, TL1A). One-way ANOVA with Tukey's multiple comparisons test (AIR vs. AIR+GITRL- Gly(4)Ser, p=0.0019; AIR vs. AIR+CX40L-Gly(4)Ser, p<0.0001 ; AIR vs. AIR+TL1A-Gly(4)Ser, p=0.0350; AIR+GITRL-Gly(4)Ser vs. AIR+GX40L-Gly(4)Ser, p=0.0039; AIR+GITRL-Gly(4)Ser vs. AIR+TL1A-Gly(4)Ser, p>0.9999; AIR+GX40L-Gly(4)Ser vs. AIR+TL1A-Gly(4)Ser, p=0.0149).

[0322] Example 6: Specificity of the response and absence of uncontrolled signaling

[0323] Nr4a1 is a downstream target gene of the TCR signaling pathway. Therefore, Nr4a1.eGFP expression visualizes ongoing TCR-signaling, but can also report AIR or CAR signaling that is mediated via the CD3- zeta-chain.

[0324] Treg cells were isolated from transgenic Nr4a1.eGFP reporter mice, expanded via anti-CD3 / CD28 antibody-coupled beads and IL-2 and transduced with a retrovirus encoding genetic information for a CD40L-specific CD40 AIR only or a CD40-AIR with a Gly(4)Ser-modified ligand (GITRL, GX40L, TL1A), or a truncated version of the CD40-AIR (lacking the extracellular domain of CD40 receptor) with a Gly(4)Ser- modified ligand (GITRL, GX40L, TL1A). After 24 h resting without via anti-CD3 / CD28 antibody-coupled beads, Treg cells were co-cultured with HEK cells or HEK cells expressing CD40L on their surface. Nr4a1 .eGFP expression in Treg cells was measured after 18 hours via flow cytometry.

[0325] Results are shown in Figure 11 . CD40-AIR signaling was comparable in all variants transduced cells with a full length version of the CD40-AIR irrespective of the presence of a Gly(4)Ser-modified ligand (GITRL, GX40L, TL1A). No signaling (Nr4a1.eGFP expression) in Treg cells expressing a truncated version of the AIR plus Gly(4)Ser-modified ligand (GITRL, GX40L, TL1 A) was detectable.

[0326] These results illustrate that the specificity of the CD40 AIR for CD40Lwas maintained and that no negative impact or uncontrolled (tonic) signaling was induced through additional expression of synthetically modified ligands of the Tumor-Necrosis-Factor (TNF) superfamily (Gly(4)Ser-modified ligand GITRL, GX40L, TL1A).

[0327] Example 7: Evaluation of various linkers

[0328] In this experiment, GITRL-AIRs with various linkers were compared. Regulatory T cells (Treg cells) were isolated, expanded via anti-CD3 / CD28 antibody-coupled beads and IL-2, and transduced with a retrovirus encoding genetic information for an artificial immune receptor (AIR) only or an AIR plus wildtype GITRL or an AIR with a Gly(4)Ser-linker modified GITRL. The Gly(4)Ser linker (SEO ID No. 26) was incorporated 3 times (3x Gly(4)Ser) (SEO ID No. 23). 6 times (6xGlv(4)Ser) (SEO ID No. 25) or 12 times (12xGly(4)Ser) (SEO ID No. 24) to test different length versions of the linker. Treg cells were stained 3 days after transduction with antibodies for flow cytometric analysis to measure AIR (CD40) expression. Results are shown in Figure 17. AIR protein expression on the cell surface was significantly increased in Treg cells when different versions of GITRL were co-expressed. The 6xGly(4)Ser linker incorporation into GITRL sequence resulted in the highest expression of the AIR receptor, indicating highest functionality of the engineered T cell (one-way ANOVA with Tukey's multiple comparisons test: AIR vs. AIR+GITRL-6xGly(4)Ser, p<0.0001 ; AIR vs. AIR+GITRLwt, p<0.0001 ; AIR vs. AIR+GITRL-3xGly(4)Ser, p<0.0001 ; AIR vs. AIR+GITRL-12xGly(4)Ser, p=0.0012; AIR+GITRL-6xGly(4)Ser vs. AIR+GITRLwt, p=0.0003; AIR+GITRL-6xGly(4)Ser vs. AIR+GITRL- 3xGly(4)Ser, p=0.0297; AIR+GITRL-6xGly(4)Ser vs. AIR+GITRL-12xGly(4)Ser, p<0.0001 ; AIR+GITRLwt vs. AIR+GITRL-3xGly(4)Ser, p=0.1871 ; AIR+GITRLwt vs. AIR+GITRL-12xGly(4)Ser, p=0.3466; AIR+GITRL- 3xGly(4)Ser vs. AIR+GITRL-12xGly(4)Ser, p=0.0052).

[0329] Example 8: Kill efficiency of anti-CEA CAR T cells transduced with various constructs.

[0330] Murine CD4 and CD8 T cells were transduced with (i) a CEA CAR construct, (ii) a CEA CAR construct plus GITRL-6xGly(4)Ser-linker, or (iii) a CEA CAR construct plus GX40L-6xGly(4)Ser-linker. Cells were rested for 24hours before adding them to the CEA-expressing coloncarcinoma cell line MC38-CEA. Caspase 3 / 7 green substrate was added to quantify apoptotic cells. As cells dye, they release active caspase 3 / 7. The fluorescent dye is activated upon cleavage by these activated enzymes. Once cleaved, the dye intercalates with the double-stranded DNA and emits fluorescence, which allows the identification and quantification of cells undergoing apoptosis. Cells were imaged every hour in the Incucyte® Live-Cell Analysis System to detect this green fluorescence.

[0331] Both the CAR T cells containing the GX40L and GITRL constructs were superior in killing capacity compared to CAR T cells alone (left). Quantification of kill activity as area under the curve (AUG) documents significant differences (right), p values calculated using one-way ANOVA. p <0,0001 (Figure 18). T cells expressing the CEA CAR construct plus GITRL-6xGly(4)Ser-linker or the CEA CAR construct plus GX40L- 6xGly(4)Ser-linker, both, are superior in killing tumor cells as compared to the CEA CAR construct alone, proving that ligands, when expressed on CAR T cells enhance the function of the CAR T cells

[0332] Example 9: Activation of Treg cells transduced with various constructs

[0333] CD69 and 41 BB expression was detected as activation markers in murine Treg cells transduced with various constructs after a 24 hours resting period followed by 18 hours of activation with CD40 ligand expressing HEK cells to stimulate the CD40-AIR biosensor.

[0334] In Figure 21 , Panel A in shows representative FACS plots depicting 41 BB and CD69 expression, as activation marker after CD40-AIR stimulation, in murine Treg cells transduced with CD40 AIR only, with CD40 AIR plus GITRL-6xGly(4)Ser-linker, and with CD40 AIR plus GITRL-6xGly(4)Ser-linker with hTNC multimerization domain (left) and quantification thereof (right). Panel B shows representative FACS plots depicting 41 BB and CD69 expression, as activation marker after CD40-AIR stimulation, in murine Treg cells transduced with CD40 AIR only, with CD40 AIR plus OX40L-6xGly(4)Ser-linker, and with CD40 AIR plus OX40L-6xGly(4)Ser-linker with hTNC multimerization domain (left) and quantification thereof (right). Panel C shows representative FACS plots depicting 41 BB and CD69 expression, as activation marker after CD40- AIR stimulation, in murine Treg cells transduced with CD40 AIR only, with CD40 AIR plus TL1A-6xGly(4)Ser- linker, and with CD40 AIR plus TL1A-6xGly(4)Ser-linker with hTNC multimerization domain (left) and quantification thereof (right). No ligand line in the quantification bar graphs depicts background activation levels of murine Treg cells transduced with the CD40 AIR construct and co-cultured on CD40L negative HEK cells.

[0335] The human tenascin (hTNC) multimerization domain containing constructs for GITRL and CX40L are superior over the CD40 AIR plus GITRL-6xGly(4)Ser-linker or the CD40 AIR plus CX40L-6xGly(4)Ser-linker construct, respectively, in supporting activation of the Treg cells after CD40-AIR stimulation. All human tenascin (hTNC) multimerization domain containing constructs for GITRL, CX40L and TL1Aare superior over the CD40 AIR only construct.

Claims

Claims1. A transmembrane protein comprising the extracellular binding domain of a TNFR ligand, a linker, a transmembrane domain and a cytoplasmatic domain.

2. The transmembrane protein according to claim 1 , wherein the cytoplasmatic domain does not induce NF-kB activation.

3. The transmembrane protein according to any one of the preceding claims, wherein said extracellular domain is selected from GITRL, TL1A, GX40LTNFa, LIGHT, LTa3, LTa1 b2, CD40L, FasL, CD30L, 4-1 BBL, CD27L, TWEAK, APRIL, BAFF, RANKL, TRAIL, EDA1 and EDA2, preferably GITRL, TL1A and GX40L.

4. The transmembrane protein according to any one of the preceding claims, wherein said linker is a glycine-serine linker.

5. The transmembrane protein according to any one of the preceding claims, wherein said transmembrane protein comprises one or more additional glycine-serine linker.

6. The transmembrane protein, according to any one of the preceding claims, wherein said transmembrane protein further comprises a_multimerization domain.

7. The transmembrane protein according to claim 6, wherein said multimerization domain comprises human tenascin, preferably wherein said multimerization domain comprisesthe amino acid sequence of SEQ ID No. 32 or 33.

8. The transmembrane protein according to any one of the claims 1 to 4, wherein the transmembrane protein comprises(i) the extracellular binding domain of a TNFR ligand,(ii) a linker,(iii) a transmembrane domain and(iv) a cytoplasmatic domain, and wherein (i) to (iv) are arranged in sequential order, preferably wherein said transmembrane protein comprises the amino acid sequence of SEQ ID No’s 12, 16 or 18.

9. The transmembrane protein according to any one of the claims 7 to 8, wherein the transmembrane protein comprises(i) the extracellular binding domain of a TNFR ligand,(ii) a first linker,(iii) a multimerization domain,(iv) a second linker,(v) a transmembrane domain and(vi) a cytoplasmatic domain, and wherein (i) to (vi) are arranged in sequential order, preferably wherein said transmembrane protein comprises the amino acid sequence of SEQ ID No’s 35, 37 or 39.

10. T cell comprising a) a chimeric antigen receptor, an artificial immune receptor or a T cell receptor, and b) a transmembrane protein according to any one of the claims 1 to 9 wherein said binding domain of the TNFR ligand is capable of binding to a receptor on the same T cell.11 . The T cell according to claim 13, wherein said chimeric antigen receptor, artificial immune receptor or T cell receptor is an artificial immune receptor, wherein said artificial immune receptor comprises the extracellular domain of member of the tumor necrosis factor receptor superfamily selected from TNFR1 , TNFR2, Fas, DR4, DR5, DR3, DR6, EDAR, XEDAR, TROY, LTBR, NGFR, CD18, CD134, CD40, CD27, CD30, CD137, TRAILR3, TRAILR4, CD265, osteoprotegerin, CD266, TACI, BAFF, BAFF receptor, APRIL, CD270, CD269 and CD357, more preferably CD40.

12. The T cell according to claim 14 or 15, where said artificial immune receptor comprises a CD28 costimulatory domain and / or a CD3 zeta domain.

13. TheT cell according to any one of the claims 13 to 16, wherein said chimeric antigen receptor, artificial immune receptor or T cell receptor is a chimeric antigen receptor, preferably wherein said chimeric antigen receptor comprises a single chain antibody (scFv) that binds to an antigen selected from the group including, but not limited to, the CD19, CD20, HER2, NY-ESO-1 , MUC1 , CD123, FLT3, B7-H3, CD33, IL1 RAP, CLL1 (CLEC12A)PSA, CEA, VEGF, VEGF-R2, CD22, ROR1 , mesothelin, c-Met, Glycolipid F77, FAP, EGFRvlll, MAGE A3, 5T4, WT1 , KG2D ligand, folate receptor (FRa), Wnt1 antigens, HLA-A2 (and other HLA variants), IL-23R, and citrullinated antigens.

14. The T cell according to any one of the claims 10 to 13, wherein said T cell is a regulatory T cell.

15. The transmembrane protein according to any of the claims 1 to 9 or the T cell according to claim I Q- 14 for use in medicine, preferably wherein said use in medicine is the treatment of an auto- inflammatory disease, such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis or auto-immune gastritis, inflammation- associated diseases such as myocardial infarction, stroke or cardiovascular diseases, lung inflammation, kidney and liver inflammation, neurodegenerative diseases such as Alzheimer’s disease, amyotrophic lateral sclerosis or Parkinson’ disease, or in cancer, such as leukemia, Hodgkin lymphoma and non-Hodgkin lymphoma, glioblastoma, colorectal cancer and gastrointestinal cancers, melanoma, pancreatic cancer, kidney / renal cell cancer, lung cancer, breast cancer, prostate cancer and others.

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