Artificial Immune Receptors

Abstract

This disclosure provides a novel polypeptide, termed artificial immune receptor or AlR, which is a novel tool for cell therapy. AIR-expressing regulatory T cells sense these environmental signals and convert them into a TregTCR-like activation program, allowing Treg cells to perform their suppressive and tissue protective functions independent of specific TCR or CAR antigens and independent of endogenous TCR-MHC restriction.

Description

[Background technology]

[0001] Regulatory T cells (Tregs) are a crucial T cell population with various functions in the body. Tregs promote tolerance to self-antigens, allergens, and commensals, thereby limiting immune cell self-reactivity and excessive inflammation. Mutations in the Treg master transcription factor Foxp3 cause lethal multi-organ autoinflammation in both mice and humans (Nat Genet, 2001.27(1):18-20; Nat Genet, 2001.27(1):68-73). In recent years, it has become clear that Tregs exert additional functions in protecting tissue homeostasis and tissue regeneration (Immunology, 2020.161(1):4-17). Based on these facts, Tregs can be considered as a promising live drug for autoimmune disorders, and clinical trials have already proven the safety and efficacy of Treg-based cell therapy (Front Immunol, 2019.10:43; Science, 2018.362(6411):154-155).

[0002] The innovative concept of applying chimeric antigen receptor (CAR) technology to Tregs is now the next step to enhance the efficacy of adoptive Treg cell therapy (Nat Rev Drug Discov, 2019.18(10):749-769.). Preclinical studies have already demonstrated the superiority of engineered Tregs with CAR-guided (self) antigen specificity over Tregs with only the natural polyclonal TCR repertoire in reducing alloimmune responses in graft-versus-host disease (GvHD) and graft rejection after transplantation (Sci Transl Med, 2020.12(557); Am J Transplant, 2020.20(6):1562-1573; J Clin Invest, 2016.126(4):1413-24; Am J Transplant, 2017.17(4):917-930; Am J Transplant, 2017.17(4):p.931-943). CAR-Tregs have also been used effectively to treat asthma, hemophilia A, type 1 diabetes, experimental autoimmune encephalitis (EAE), 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:p.1125; J Autoimmun, 2019.103:p.102289).

[0003] However, in human autoimmune diseases, the involved autoantigens that can serve as potential targets for CAR-Tregs are often unknown, or multiple organs and tissues are affected without uniform antigens. In contrast, mediators of inflammation show high redundancy in many inflammatory diseases and functional importance for the development of such diseases. In particular, cytokines of the tumor necrosis factor (TNF) family are involved in many different inflammatory and autoimmune diseases, and therapeutic intervention of TNF receptor (TNFR) activation is an important treatment option for several inflammatory diseases (Trends Immunol, 2012.33(3):144-52). These considerations led us to develop a new concept for engineered Treg cell therapy, generating artificial immune receptors (AIRs) that target these inflammatory mediators instead of tissue-specific antigens.

[0004] We focused on the target of TNF family and selected the receptors of ligands LIGHT and Lα1β2, TNFα, and TL1A because these cytokines have multifaceted roles in many autoimmune diseases. Disease models and patient data show that LIGHT and Lα1β2 signaling by the corresponding receptors HVEM and LTBR enhances the pathology of IBD, autoimmune hepatitis, asthma, rheumatoid arthritis, multiple sclerosis, and GvHD (reviewed in Immunol Rev, 2008.223:202-20). The role of TL1A and its receptor DR3 has been described in various inflammatory diseases. Studies using blocking antibodies or TL1A and DR3-deficient mice have demonstrated the important involvement of this receptor-ligand system in the induction and maintenance of chronic inflammation in IBD, arthritis, and EAE. Moreover, patient data and genome-wide association studies point to a fundamental impact of TL1A-DR3 in human diseases (reviewed in FEBS Lett, 2017.591(17):2543-2555). TNFα itself, the most prominent member of the TNF family, has been extensively studied since its discovery in 1984. Its pro-inflammatory functions have manifested in multiple diseases such as ankylosing spondylitis, IBD, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and juvenile idiopathic arthritis, among others (reviewed in Crit Rev Immunol, 2019.39(6):439-479).

[0005] AIR, as a new synthetic tool for cell therapy, enables Tregs to sense these environmental signals and convert them into a Treg TCR-like activation program, allowing Treg cells to fulfill their suppressive and tissue-protective functions independent of specific TCR or CAR antigens and independent of endogenous TCR-MHC restriction.

[0006] Certain constructs are known in the art that share certain characteristics with the constructs of the present invention. WO2021 / 051195 for example discloses constructs that lack the CD3 zeta TCR signaling domain. However, the additional CD3 zeta TCR signaling domain offers some advantages over the common functionality of the CD3 T cell signaling domain. Due to the presence of the CD3 zeta domain, the AIRs of the present invention exert their function directly at the main signal of the signaling cascade. As shown herein, AIR constructs that do not contain the CD3 zeta domain are not functional. The constructs of WO2021 / 051195 can only regulate auxiliary signals and cannot regulate TCR-like activation of cells. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2021 / 051195 Brochure [Non-patent literature]

[0008] [Non-Patent Document 1] Nat Genet,2001.27(1):18-20 [Non-Patent Document 2] Nat Genet,2001.27(1):68-73 [Non-Patent Document 3] Immunology,2020.161(1):4-17 [Non-Patent Document 4] Front Immunol, 2019.10:43 [Non-Patent Document 5] Science,2018.362(6411):154-155 [Non-Patent Document 6] Nat Rev Drug Discov,2019.18(10):749-769. [Non-Patent Document 7] Sci Transl Med, 2020.12(557)

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Summary of the Invention

Problems to be Solved by the Invention

[0009] The present disclosure provides novel artificial immune receptors that can activate regulatory T cells into a Treg TCR-like activation program that is independent of the specific TCR antigen or specific CAR antigen, and independent of endogenous TCR-MHC restriction. [Means for solving the problem]

[0010] a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0011] The general concept is illustrated using the extracellular domains of four different tumor necrosis factor receptor superfamily members: TNFR2, DR3, LTBR, ​​and CD40. In certain embodiments, the transmembrane domain is derived from the same protein as the extracellular domain.

[0012] The artificial immune receptor also comprises a cytoplasmic costimulatory signaling domain and a cytoplasmic T cell receptor signaling domain.Any commonly known costimulatory signaling domain and T cell receptor signaling domain can be used in the context of the AIR of the present disclosure.The cytoplasmic costimulatory signaling domain of CD28 and the cytoplasmic T cell receptor signaling domain from CD3 zeta are exemplified.

[0013] A particularly preferred artificial immune receptor comprises the extracellular and transmembrane domains of TNFR2, the cytoplasmic costimulatory signaling domain from CD28, and the cytoplasmic T cell receptor signaling domain from CD3 zeta. Yet another particularly preferred artificial immune receptor comprises the extracellular and transmembrane domains of DR3, the cytoplasmic costimulatory signaling domain from CD28, and the cytoplasmic T cell receptor signaling domain from CD3 zeta. Yet another particularly preferred artificial immune receptor comprises the extracellular and transmembrane domains of LTBR, ​​the cytoplasmic costimulatory signaling domain from CD28, and the cytoplasmic T cell receptor signaling domain from CD3 zeta.

[0014] Also preferred are artificial immune receptors comprising the amino acid sequences of SEQ ID NO:39, SEQ ID NO:35, and SEQ ID NO:37.

[0015] Also preferred are artificial immune receptors comprising the amino acid sequences of SEQ ID NO:41, SEQ ID NO:35, and SEQ ID NO:37.

[0016] Also preferred are artificial immune receptors comprising the amino acid sequences of SEQ ID NO:33, SEQ ID NO:35, and SEQ ID NO:37.

[0017] Also preferred are artificial immune receptors comprising the amino acid sequences of SEQ ID NO:47, SEQ ID NO:35, and SEQ ID NO:37. The present disclosure also provides a nucleic acid encoding the artificial immune receptor described above. The present disclosure also provides a vector comprising the nucleic acid. The present disclosure also provides a host cell comprising the nucleic acid, the vector, and a host cell expressing the artificial immune receptor of the present disclosure.

[0018] The present disclosure also provides an artificial immune receptor for use in medicine. In certain embodiments, the use in medicine is the treatment of cancer or inflammation. In other embodiments, the use in medicine is the treatment of autoimmunity. In yet other embodiments, the use in medicine is the treatment of graft-versus-host disease or in solid organ transplantation. definition

[0019] As used herein, the term "cell" includes a single cell and multiple cells.

[0020] The term "host cell" as used herein refers to a cell that contains a nucleic acid and / or a vector. In the context of the artificial immune receptor of the present disclosure, the term host cell refers to a cell that contains a nucleic acid and / or a vector that encodes AIR. Such host cells express AIR on the cell surface and are suitable for use as medicine. The preferred host cells of the present invention are eukaryotic host cells, such as immune cells.

[0021] 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 called 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 different functions, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, and natural killer T cells. In some embodiments, the T cells are engineered T cells.

[0022] The term "regulatory T cells" or "Tregs" as used herein refers to a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune diseases. These cells generally suppress or downregulate the induction and proliferation of effector T cells. Treg cells are long-lived cells that dominantly and antigen-specifically suppress excessive or uncontrolled immune responses in vivo. Genetic mutations in forkhead box protein 3 (FoxP3), a key transcription factor required for the differentiation of Treg cells, result in severe autoimmunity. Indeed, studies in various animal models have demonstrated that Tregs can be used to treat many autoinflammatory diseases, such as type 1 diabetes, inflammatory bowel disease, systemic lupus erythematosus, multiple sclerosis (MS), rheumatoid arthritis, and autoimmune gastritis. Treg cell therapy can also be used in the control of allogeneic immune responses in the setting of GVHD, as well as in organ and cell transplantation.

[0023] The term "engineered TCR" or "engineered T cell receptor" refers to any TCR that has been modified from its naturally occurring form. An engineered TCR may have modifications to the alpha and / or beta chains, or the gamma and / or delta chains (including substitutions of any of the aforementioned chains) that enable the TCR to recognize a specific antigen (e.g., neoantigen). An engineered TCR may have modifications to any CD3 subunit (e.g., CD3a, as in the case of the TRuC receptor), including the addition of an antigen recognition domain (e.g., antibody, scFv, DARPin). An engineered TCR may have an antigen recognition domain (e.g., antibody, scFv, DARPin) attached to the transmembrane domain of the alpha and / or beta chains, or the gamma and / or delta chains.

[0024] 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 DNA and RNA, can be either double-stranded or single-stranded, and represents the sense or antisense strand. The term also includes modified or substituted sequences that include non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application can be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and can include naturally occurring bases including adenine, guanine, cytosine, thymidine, and uracil. The sequences can also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine, and uracil, as well as xanthine and hypoxanthine. The nucleic acids of the present disclosure can be isolated from biological organisms, formed by recombinant laboratory methods, or obtained by chemical synthesis or other known protocols for making nucleic acids.

[0025] As used herein, the term "isolated polynucleotide" or "isolated nucleic acid sequence" refers to a nucleic acid that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

[0026] The term "recombinant nucleic acid" or "engineered nucleic acid" as used herein refers to a nucleic acid or polynucleotide not found in a biological organism. For example, a recombinant nucleic acid may be formed by laboratory methods of genetic recombination (such as molecular cloning) to create a sequence not otherwise found in nature. A recombinant nucleic acid 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 application described herein to which they are appropriate, as understood by those of skill in the art.

[0027] The term "polypeptide" or "protein" as used herein refers to a chain of amino acids. A polypeptide or protein of the present disclosure may be a peptide, typically characterized by an amino acid chain of 2 to about 30 amino acids. The term protein as used herein also refers to a chain of amino acids having more than 30 amino acids, and may be a protein or a fragment or domain of a full-length protein. Furthermore, as used herein, the term protein may refer to a linear chain of amino acids, or to a chain of amino acids that has been processed and folded into a functional protein. However, it is understood that 30 is an arbitrary number with respect to distinguishing between peptides and proteins, and these terms may be used interchangeably with respect to amino acid chains. Proteins of the present disclosure may be obtained by isolation and purification of proteins from cells in which they are naturally produced, by enzymatic (e.g., proteolytic) cleavage, and / or by recombinant expression of a nucleic acid encoding a protein or fragment of the present disclosure. Proteins and / or fragments of the present disclosure may also be obtained by chemical synthesis or other known protocols for producing proteins and fragments.

[0028] The term "isolated polypeptide" refers to a polypeptide that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or that is substantially free of chemical precursors or other chemicals when chemically synthesized.

[0029] The term "vector" as used herein refers to a polynucleotide that can be used to deliver nucleic acid to the inside of a cell. In one embodiment, the vector is an expression vector that comprises an expression control sequence (e.g., a promoter) operably linked to the nucleic acid that is expressed in the cell. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids, and viruses.

[0030] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired.

[0031] As used herein, the terms "treatment", "treating" and the like refer, in some embodiments, to administering an agent or carrying out a procedure for the purpose of obtaining an effect. The effect may be preventative in that it completely or partially prevents a disease or its symptoms, and / or may be therapeutic in that it affects a partial or complete cure of a disease and / or symptoms of a disease. The term includes the treatment of a disease or disorder (e.g., inflammation) in a mammal, particularly a human, and includes (a) preventing a disease or symptoms of a disease from occurring in a subject who may have a predisposition to the disease, but has not yet been diagnosed as having it (e.g., including diseases that may be related to or caused by a primary disease, (b) inhibiting a disease, i.e., halting its development, and (c) relieving a disease, i.e., causing regression of a disease. The treatment or amelioration of a symptom is based on one or more objective or subjective parameters, including the results of an examination by a physician. Thus, the term "treating" includes the administration of a compound or agent of the present invention to prevent, delay, alleviate, halt, or inhibit the onset of a symptom or condition associated with a disease (e.g., inflammation).

[0032] The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, a symptom of a disease, or a side effect of a disease in a subject.

[0033] As used herein, the term "tumor necrosis factor receptor superfamily," "TNFR superfamily," or "TNFRSF" refers to a protein superfamily of cytokine receptors characterized by the ability to bind to tumor necrosis factor superfamily member (TNFSF) ligands via their extracellular domains. There are numerous members of the TNFR superfamily, including TNFR1, TNFR2, Fas, DR4, DR5, DR3, DR6, EDAR, XEDAR, TROYLTBR, ​​or NGFR. Other members include lymphotoxin beta receptor (CD18), Ox40 (CD134), CD40 (TNFRSF5), decoy receptor 3 (TR6, M68), CD27 (S152, Tp55), CD30 (Ki-1, TNR8), 4-1BB (CD137), decoy receptor 1 (TRAILR3), decoy receptor 2 (TRAILR4), RANK (CD265), osteoprotegerin (OCIF, TR1), TWEAK receptor (Fn14, CD266), TACI (IGAD2, CD267), BAFF receptor (CD268), BAFF, APRIL, herpes virus entry mediator (HVEM, CD270), B cell maturation antigen (TNFRSF13A, CD269), and glucocorticoid-induced TNFR-related protein (AITR, CD357).

[0034] Tumor necrosis factor receptor 1 (NCBI Entrez Gene:7132; also known as TNFR1, tumor necrosis factor receptor superfamily member 1A, TNFRSF1A, or CD120a) is a membrane-bound receptor that binds tumor necrosis factor alpha (TNFα). TNFR1 activates the transcription factor NF-kB, mediates apoptosis, and functions as a regulator of inflammation.

[0035] Tumor necrosis factor receptor 2 (NCBI Entrez Genes:7133; also known as TNFR2, tumor necrosis factor receptor superfamily member 1B, TNFRSF1B, or CD120b9, is a membrane-bound receptor that binds tumor necrosis factor-alpha (TNFα).

[0036] Fas receptor (also known as NCBI Entrez Gene:355, Fas, FasR, apoptosis antigen 1, APO-1, APT, CD95, tumor necrosis factor receptor superfamily member 6, or TNFRSF6) is a protein that is encoded by the FAS gene in humans. Multiple splice variants of Fas have been identified, which translate into seven isoforms of the protein. The apoptosis-inducing Fas receptor is referred to as isoform 1 and is a type 1 transmembrane protein. Many of the other isoforms are rare haplotypes that are usually associated with disease states. Any suitable isoform of Fas is contemplated for use with the embodiments disclosed herein.

[0037] Death receptor 4 (NCBI Entrez Gene:8797, also known as death domain 4, DR4, TRAIL receptor 1, TRAILR1, tumor necrosis factor receptor superfamily member 10A, or TNFRSF10A) is a cell surface receptor of the TNF receptor superfamily that binds TRAIL and mediates apoptosis.

[0038] Death domain 5 (NCBI Entrez Gene:8795, also known as death receptor 5, DR5, TRAIL receptor 2, TRAILR2, tumor necrosis factor receptor superfamily member 10B, or TNFRSF10B) is a cell surface receptor of the TNF receptor superfamily that binds TRAIL and mediates apoptosis.

[0039] Death domain 3 (NCBI Entrez Gene:8718, also known as death receptor 3, DR3, tumor necrosis factor receptor superfamily member 25, or TNFRSF25) is a cell surface receptor of the tumor necrosis factor receptor superfamily that mediates apoptotic signaling and differentiation. Its only known TNFSF ligand is TNF-like protein 1A (TL1A).

[0040] Death domain 6 (NCBI Entrez Gene:27242, also known as death receptor 6, DR6, tumor necrosis factor receptor superfamily member 21, or TNFRSF21) is a cell surface receptor of the tumor necrosis factor receptor superfamily that activates the JNK and NF-κB pathways.

[0041] Ectodiplasin receptor A (NCBI Entrez Gene:10913, also known as ectodermal dysplasia receptor, EDA-A1, or EDAR) is a member of the TNF receptor superfamily. It plays an important role in the process of ectodermal differentiation.

[0042] Ectodiplassin A2 receptor (NCBI Entrez Gene:60401, also known as XEDAR, EDAR2, EDA-A2, or tumor necrosis factor receptor superfamily member 27) is a protein encoded by the EDA2R gene in humans. EDA-A1 and EDA-A2 are two isoforms of ectodiplassin encoded by the anhidrotic ectodermal dysplasia (EDA) gene.

[0043] TROY (NCBI Entrez Gene:55504, also known as tumor necrosis factor receptor superfamily member 19, or TNFRSF19) is a member of the TNF receptor superfamily. This receptor is highly expressed during embryonic development. It has been shown to interact with TNF receptor-associated factor (TRAF) family members and activate the c-Jun N-terminal kinase (JNK) signaling pathway when overexpressed in cells. This receptor can induce apoptosis by a caspase-independent mechanism and is believed to play an essential role in embryonic development.

[0044] NGFR (NCBI Entrez Gene:4804, also known as nerve growth factor receptor, TNFR superfamily member 16, TNFRSF16, LNGFR, or p75 neurotrophin receptor) is a member of the tumor necrosis factor receptor (TNF receptor) superfamily. It is one of two receptor types for neurotrophins, a family of protein growth factors that stimulate neuronal cells to survive and differentiate.

[0045] LTBR (NCBI Entrez Genes:4055, also known as lymphotoxin beta receptor, TNFRSF3, TNFCR, tumor necrosis factor C receptor for TNFR3) plays a role in signal transduction, lipid metabolism, immune response, and programmed cell death during development of the lymphatic system and other organs.

[0046] CD40 (NCBI Entrez Gene:958, also known as Bp50, tumor necrosis factor receptor superfamily member 5, or TNFRSF5) is a costimulatory protein found on antigen-presenting cells and is required for their activation. CD40 is involved in the regulation of T H It binds to CD154 (CD40L) on cells, thereby activating antigen-presenting cells and inducing a variety of downstream effects.

[0047] CD30 (NCBI Entrez Gene:943, D1S166E, also known as tumor necrosis factor receptor superfamily member 8, TNFRSF8, CD30L receptor, or KI-1) is expressed by activated but not resting T and B cells and interacts with TRAF2 and TRAF5.

[0048] BAFF (NCBI Entrez Gene:10673, also known as TALL-1, tumor necrosis factor receptor superfamily member 13B, TNFRSF13B, THANK, or CD257) is a ligand for various receptors. BAFF is expressed in B cell lineage cells and acts as a B cell activator. It has also been shown to play an important role in B cell proliferation and differentiation.

[0049] APRIL (NCBI Entrez Gene:8741, also known as CD256, tumor necrosis factor receptor superfamily member 13, TNFRSF13, TALL-2, TRDL-2, or ZNTF2) is recognized by the cell surface receptor TACI and together with that receptor plays an important role in B cell development.

[0050] The term "costimulatory molecule" or "costimulatory receptor" as used herein refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response in the T cell, such as, but not limited to, activation or proliferation. Costimulatory receptors can be expressed on cells other than T cells, such as NK cells or macrophages. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), Toll-like receptors, and NK cell receptors. Costimulatory molecules include, but are not limited to, 4-IBB (CD137), BAFFR, OX40, CD27, CD28, CD40, ICOS, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1-TLR9 receptors, IL-2, IL-7, and IL-15 receptors.

[0051] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a domain of a costimulatory molecule or costimulatory receptor that is involved in mediating a costimulatory response by T cells. The intracellular signaling domain may include the entire intracellular portion of the molecule from which it is derived, or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0052] The term "T cell receptor signaling domain" or "TCR signaling domain" as used herein refers to a cytoplasmic signaling sequence that acts stimulatorily to induce immune effector functions. In some embodiments, the TCR signaling domain contains a signaling motif known as an 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 (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. Preferred TCR signaling domains are TCR signaling domains selected from CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon. Particularly preferred TCR signaling domains are CD3 zeta, CD3 gamma, CD3 delta, and CD3 epsilon.

[0053] The term "artificial immune receptor" or "AIR" refers to a novel molecule of the present disclosure. AIR contains the extracellular domain, transmembrane domain, cytoplasmic costimulatory signaling domain, and cytoplasmic cell membrane T cell receptor signaling domain of a member of the tumor necrosis factor receptor superfamily.

[0054] The terms "is", "are", "is derived from" and "are derived from" in the context of a polypeptide or polypeptide domain refer to the amino acid sequence of said polypeptide or polypeptide domain and indicate that the amino acid sequence is either identical to a native version of said polypeptide or polypeptide domain, or is a variant of said polypeptide or polypeptide domain that is in a form that is functionally indistinguishable from the native version of said polypeptide or polypeptide domain.

[0055] The term "P2A" as used herein refers to a peptide sequence capable of self-cleavage. P2A belongs to a family of 2A peptides, all capable of self-cleavage. The peptides share a common core sequence motif. Any 2A peptide may be used in place of P2A. The amino acid sequence of P2A is shown in SEQ ID NO: 13. Other commonly used 2A peptides are T2A, E2A, and F2A.

[0056] As used herein, the term "CD90.1" refers to an allelic isoform of mouse CD90. The amino acid sequence of mouse CD90.1 is shown in SEQ ID NO: 15. CD90.1 is a commonly used genetic marker.

[0057] The terms "GVHD", "GvHD" and "graft-versus-host disease" refer to diseases commonly associated with bone marrow and stem cell transplants and characterized by inflammation. White blood cells of the donor's immune system remain within the donor-donated tissue or cells (the graft) and are recognized as non-self by the recipient (the host). [Brief description of the drawings]

[0058] [Figure 1]Schematic diagram of AIR design. The top construct (A) shows an AIR according to the present disclosure. It consists of an extracellular binding domain of a tumor necrosis factor (TNF) superfamily member, a transmembrane domain, and two intracellular domains - one costimulatory domain and one T cell receptor signaling domain. The AIR fusion protein is linked via a P2A peptide cleavage site to a modified CD90.1 that serves as a reporter gene for transduction efficacy. P2A and CD90.1 are not part of AIR. These constructs are cloned into a murine stem cell leukemia virus (MSCV), which serves as a vector and mediates stable integration of the gene of interest into T cell DNA. Constructs designated B, C, D, and G are exemplary AIR according to the present disclosure. Constructs E and F are control constructs. Construct E lacks the extracellular binding domain, and construct F contains an extracellular anti-CD19 single chain antibody (scFv) instead of the binding domain of a tumor necrosis factor (TNF) superfamily member. [Diagram 2] 1 shows the mode of action of AIR according to the present disclosure: Binding of membrane-bound TNF family members via AIR results in translation of cytokine signals into T cell receptor (TCR)-like signals, which initiate T cell activation independently of their endogenous TCR-MHC restriction in an environment / inflammation-dependent manner. [Diagram 3] FIG. 1 demonstrates efficient transduction and expression of AIR according to the present disclosure. Murine regulatory T cells (Tregs) were isolated from spleens and lymph nodes of C57 / BL6 mice, sorted (naive CD4+CD25+CD62L+Tregs or Foxp3+reporter (hCD2+) positive), and expanded by anti-CD3 and anti-CD28 antibody-coupled beads + IL-2 (2000U / ml) stimulation for 6 days. Purity of Treg cultures was determined by flow cytometry. Treg cultures contained approximately 90-99% Foxp3+T cells. Two days after isolation, Tregs were transduced with AIR constructs or anti-CD19 chimeric antigen receptor (α-CD19 CAR) as a control. Three days later, transduction efficacy via CD90.1 staining and surface expression of AIR were confirmed by flow cytometry. [Figure 4]FIG. 10 demonstrates surface expression of AIR according to the present disclosure. Panels A, B, and C show results with AIR carrying the extracellular domains of LTBR, ​​DR3, and TNFR2, respectively. In each panel, AIR is compared to non-transduced cells and cells carrying extracellular anti-CD19 single chain antibody (scFv). [Diagram 5] Figure 6 shows the scientific rationale of the experiment in Example 6. Nr4a1 is a well-known target gene of T cell receptor signaling. We investigated whether AIR of the present disclosure can induce Nr4a1 upregulation in a similar manner to TCR signaling. We used Tregs derived from Nr4a1-eGFP reporter mice. As a control, Tregs were stimulated with anti-CD3 / CD28 antibody-bound beads. [Figure 6] AIR expressing Tregs were stimulated in overnight co-culture with HEK cells expressing murine Light (mLight), mTL1A, or mTNF. As a positive control, Tregs were stimulated with anti-CD3 / CD28 antibody-conjugated beads to mimic TCR signaling. Nr4a1-eGFP expression of AIR positive (CD90.1+) Tregs is shown in the left panel, and Nr4a1-eGFP expression of AIR negative (CD90.1-) Tregs is shown in the right panel. [Figure 7] Figure 1 demonstrates expression of Nr4a1 upon administration of AIR according to the present disclosure (exemplified by AIR carrying the extracellular domain of LTBR). Tregs expressing LTBR AIR according to the present disclosure or an irrelevant CAR carrying an anti-CD19 single chain antibody (scFv) extracellularly were co-cultured with murine EL4 cells known to express Light in the absence or presence of mLight blocking LTBR-Ig fusion protein (50ug / ml) and then stained for intracellular Nr4a1 expression. [Figure 8]1 shows the pre-gating strategy for CD90.1+ cells of LTBR AIR according to the present disclosure, and an irrelevant CAR carrying an anti-CD19 single chain antibody (scFv) extracellularly. Tregs expressing the constructs were left to rest for 24 hours, and then co-cultured with HEK cells or HEK cells expressing mLight for 18 hours. Pre-gating for CD90.1+ cells is shown. [Figure 9] Figure 9. Sorting of Tregs pre-gated as indicated in Figure 8. Cells were sorted as CD4+CD25+CD90.1+ (left and center) or CD4+CD25+CD90.1+Nr4a1+LAP+ (right). [Figure 10] Figure 1 shows RNA expression analysis of differentially expressed transcripts after stimulation with LTBR AIR according to the present disclosure (comparison of LTBR AIRTreg stimulated with HEK-mLight vs. α-CD19 CARTreg stimulated with HEK-mLight) Cells were sorted as outlined in Figures 8 and 9 and Example 7. RNA was then isolated and RNA sequencing was performed. On the left side, a summary of up- and down-regulated genes is shown, comparing α-CD19 CARTreg stimulated with HEK-mLight vs. LTBR AIRTreg stimulated with HEK cells as a control (endogenous mLight signaling) and LTBR AIRTreg stimulated with HEK-mLight vs. α-CD19 CARTreg stimulated with HEK-mLight (specific LTBR AIR signaling). On the right side, a volcano plot showing differentially expressed transcripts is shown. [Figure 11] Similar to FIG. 10 with DR3 AIR according to the present disclosure. DR3 AIR Tregs were stimulated with HEK cells (endogenous mTL1A signaling). DR3 AIR Tregs were stimulated with HEK-mTL1A vs. α-CD19 CAR Tregs stimulated with HEK-mTL1A (specific DR3 AIR signaling). Differentially expressed genes are shown on the left and volcano plots are shown on the right. [Figure 12]Similar to Figure 10 comparing α-CD19 CAR Tregs stimulated with anti-CD3 / CD28 beads with unstimulated Tregs. Differentially expressed genes are shown on the left and volcano plots are shown on the right. [Figure 13] FIG. 13 shows RNA expression data from LTBR AIR or control CAR expressing Tregs for Tnfrsf9, Tigit, Tgfb1, Cd69, Ccr8, and Nr4a1 after 18 hours of co-culture with HEK+ / -mLight (Deseq2, n=3). [Figure 14] Representative flow cytometry analysis from LTBR AIR or control CAR expressing Tregs after 18 hours of co-culture with HEK+ / -mLight. Protein expression of CD137 and Tigit is shown in the upper panel, CD69 and LAP (membrane-bound Tgfβ1) in the lower panel. Data are representative of three independent experiments. [Figure 15] FIG. 13 shows RNA expression data from Treg-expressing DR3 AIR or control CAR for Tnfrsf9, Tigit, Tgfb1 after 18 hours of co-culture with HEK+ / -mLight (Deseq2, n=3). [Figure 16] Representative flow cytometry analysis from DR3 AIR or control CAR expressing Tregs after 18 hours of co-culture with HEK+ / -mTL1A. Protein expression of CD137 and Tigit is shown in the upper panel, CD69 and LAP (Tgfβ1) in the lower panel. Data are representative of three independent experiments. [Figure 17] RNA expression data (top left, Deseq2, n=3) and protein expression (right) from LTBR AIR expressing Tregs against Irf8 or control CARs after 18 hours of co-culture with HEK+ / -mLight. RNA expression data (bottom left, Deseq2, n=3) and protein expression (right) from DR3 AIR expressing Tregs against Irf8 or control CARs after 18 hours of co-culture with HEK+ / -mTL1A. [Figure 18]Figure 1 demonstrates that the disclosed AIR mediates the activation and proliferation of Treg. LTBR AIR or control CAR expressing Treg was left for 24 hours and labeled with CFDA-SE proliferation dye. The engineered and labeled Treg was co-cultured with HEK+ / -mLight in the presence of IL2 for 72 hours and then analyzed for proliferation by flow cytometry. A representative dot plot is shown on the left. Summary data of three experiments performed with three technical replicates is shown on the right (n=3). [Figure 19] Schematic diagram of the (major MHC mismatch) graft-versus-host disease (GvHD) model. Briefly, FACS-sorted Tregs from Foxp3-hCD2 reporter mice were expanded and transduced with LTBR AIR or a truncated construct (lacking the extracellular binding domain shown in FIG. 1a) as a control. 2.5×105 engineered Tregs were transplanted into lethally irradiated Balb / c mice along with 2.5×106 bone marrow cells and 2.5×105 spleen cells from C57 / BL6 mice. As a transplantation control, one group received bone marrow cells (BM) only, and as a disease development control, the other group received BM cells + spleen cells. After transplantation, animals were monitored for 47 days. [Figure 20] Figure 1 shows the results of quality control of engineered Tregs. Prior to transfer into mice, Tregs were stained for CD4, hCD2 (reporter for Foxp3) as well as CD90.1 and LTBR. [Figure 21] Kaplan-Meier curves showing survival of transplanted Balb / c mice treated with LTBR AIR and the respective control constructs. The graph contains data sets from two independent experiments (log-rank test, n=11-12). [Figure 22] FIG. 1 shows the mean GvHD score per group; animals that reached a score of 40 had to be euthanized (dead animals were kept with the highest score across the following time points for statistical analysis, two-way ANOVA). [Figure 23]Representative FACS plots of spleens from surviving animals that received LTBR AIR Tregs are shown on the left, and the percentage of Foxp3+ Tregs in spleens of surviving mice at day 47 is shown on the right. [Figure 24] Analysis of Klrg1+ Tregs in spleen. Left: Representative FACS plots of identified CD45.1+ (BM-derived) and CD45.2+ (transferred AIR Treg) Treg cells. Expression of Klrg1 (marker of tissue-specific phenotype) and hCD2 (Foxp3 reporter) is shown. Center: Klrg1+ expression of engineered CD45.2+ or BM-derived CD45.1+ Tregs in surviving mice. Right: Frequency of Klrg1+hCD2+ Tregs in mice receiving LTBR AIR Tregs (Mann-Whitney U, n=8 survivors vs. n=4 deceased). [Diagram 25] Representative FACS analysis of digested colons of surviving animals that received LTBR AIR Tregs. Differentiation of CD45.1 and CD45.2 cells within the CD4+ population on the left is shown. CD45.2 Tregs are further analyzed for Klrg1 and hCD2 expression on the right. [Figure 26] The percentage of hCD2+ (indicating Foxp3+) cells among the transferred engineered CD45.2+ cells is shown on the left, demonstrating phenotypic stability over 47 days in vivo. On the right, the cell numbers of transferred CD45.2 Tregs in the colon at day 47 are shown. [Figure 27]Figure 1 shows that human LTBR AIR is efficiently transduced and expresses AIR on the cell surface. Human T cells from healthy blood donors were sorted with typical Treg protein markers (TCRb+CD4+CD25+CD127-CD45RA+) and expanded with anti-CD3 / CD28 stimulation (TransACT, Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-2 (500U / ml) for 7 days. Two days after isolation, Tregs were transduced with human LTBR AIR constructs (using a bitropic version of the retroviral expression system MSCV). After 5 days, cultured Tregs were stained for intracellular Foxp3+ to confirm Treg purity and for CD90.1 and hLTBR to analyze transduction efficacy and surface expression of AIR protein. [Figure 28] Figure 1 shows that human LTBR AIR has the expected signaling capacity. Tregs expressing hLTBR AIR or an irrelevant CAR (anti-CEA, carcinoembryonic antigen) were co-cultured with parental HEK cells or HEK cells expressing human Light protein on their surface for 18 hours. Expression of Ccr8, glycoprotein A repeat dominant (GARP), CD137 (4-1BB), and Tigit is shown in CD90.1+Foxp3+Tregs. [Figure 29] At the top is the LTBR "AIR" lacking the CD3 zeta chain, and at the bottom is flow cytometry data confirming surface expression of the construct on Tregs. [Diagram 30] Representative flow cytometry analysis of Tregs expressing full-length LTBR AIR with LTBR "AIR" lacking the CD3 zeta chain after 18 hours of co-culture with HEK+ / -mLight. Protein expression of CD137 and Tigit is shown in the upper panel, CD69 and LAP (membrane-bound Tgfβ1) in the lower panel. [Diagram 31]Figure 1 shows the expression of TIGIT and CD137. Constructs containing CD40 AIR-expressing Treg cells or anti-CD19 CAR-expressing Treg cells were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 hours. Expression of TIGIT and CD137 was measured by flow cytometry. [Diagram 32] Three independent experiments are summarized in Figure 31. Differences are statistically significant as calculated via one-way Anova test. [Diagram 33] Figure 1 shows the expression of LAP (TGFb1) and CD69. Constructs containing CD40 AIR-expressing Treg cells or anti-CD19 CAR-expressing Treg cells were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 hours. Expression of LAP (TGFb1) and CD69 was measured by flow cytometry. [Diagram 34] Figure 34 summarizes three independent experiments shown in Figure 33. Differences are statistically significant as calculated via one-way Anova test. [Diagram 35] FIG. 1 shows that Treg cells derived from Nr4a1-eGFP reporter mice expressing CD40 AIR induce the expression of Nr4a1, an early response gene of TCR signaling. [Diagram 36]

[0036] Figure 36 is a schematic diagram of additional AIRs generated and tested in this disclosure: Figure 36A shows CD40 AIR with an ICOS costimulatory domain, and Figure 36B shows CD40 AIR with a 4-1BB costimulatory domain. [Figure 37] FIG. 1 shows that CD40 AIR with ICOS or 41BB costimulatory domains is efficiently expressed on the surface of regulatory T cells. [Figure 38] FIG. 1 shows that all CD40 AIRs tested, i.e., AIRs carrying the CD28, ICOS, and 41BB costimulatory domains, have the ability to induce Treg cell activation. [Figure 39]Figure 1 shows quality control of Teg cells prior to transplantation: Both constructs, CD40 AIR and control construct, showed high transduction efficiency, but only CD40 AIR-transduced cells exhibited binding to CD40 via the CD40 receptor extracellular domain. [Diagram 40] Kaplan-Meier plot showing survival of transplanted Balb / c mice treated with CD40 AIR and the respective control constructs. The graph contains data sets from two independent experiments (log-rank test, n=10-12). [Diagram 41] Figure 14: Demonstrating the importance of CD3 for the constructs of the invention: Only full-length LTBR AIR, which contains the CD3 zeta domain, but not the other two constructs tested, is able to induce Nr4a1.eGFP upregulation in response to the corresponding ligand mLIGHT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] Artificial Immune Receptors The artificial immune receptors disclosed herein are novel and powerful tools that enable regulatory T cells to perform their suppressive and tissue protective functions independent of specific TCR or CAR antigens and independent of endogenous TCR-pMHC (peptide-loaded MHC) restriction.

[0060] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0061] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) an artificial immune receptor comprising a cytoplasmic T cell receptor signaling domain.

[0062] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the 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.

[0063] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor is one in which the member of the tumor necrosis factor receptor superfamily is selected from TNFR2, DR3, LTBR, ​​and CD40.

[0064] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the 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.

[0065] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor is one in which the member of the tumor necrosis factor receptor superfamily is selected from TNFR2, DR3, LTBR, ​​and CD40.

[0066] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The invention relates to an artificial immune receptor, wherein the transmembrane domain is derived from the same protein as the extracellular domain.

[0067] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The invention relates to an artificial immune receptor, wherein the transmembrane domain and the extracellular domain are derived from the same protein.

[0068] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor is a member of the tumor necrosis factor receptor superfamily and the transmembrane domain is derived from TNFR2.

[0069] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:39.

[0070] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:38.

[0071] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:38.

[0072] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is derived from DR3.

[0073] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:41.

[0074] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO: 40.

[0075] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO: 40.

[0076] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from a LTBR.

[0077] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:33.

[0078] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:31.

[0079] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:31.

[0080] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The invention relates to an artificial immune receptor, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is derived from CD40.

[0081] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:46.

[0082] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:47.

[0083] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:47.

[0084] Costimulatory Signaling Domains In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0085] The cytoplasmic costimulatory signaling domain is selected from 4-IBB (CD137), BAFFR, OX40, CD27, CD28, ICOS, CD40, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY, and TWEAK.

[0086] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0087] The cytoplasmic costimulatory signaling domain is CD28.

[0088] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0089] The cytoplasmic costimulatory signaling domain is derived from CD28.

[0090] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0091] The cytoplasmic costimulatory signaling domain comprises SEQ ID NO:35.

[0092] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain.

[0093] The cytoplasmic costimulatory signaling domain consists of SEQ ID NO:35.

[0094] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain is derived from the same protein as the extracellular domain; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain is selected from CD137, BAFFR, OX40, CD27, CD28, CD40, ICOS, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY, and TWEAK.

[0095] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain is derived from the same protein as the extracellular domain; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain is CD28.

[0096] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain is derived from the same protein as the extracellular domain; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain comprises SEQ ID NO:35.

[0097] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain is derived from the same protein as the extracellular domain; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain consists of SEQ ID NO:35.

[0098] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain and the extracellular domain are derived from TNFR2; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain is CD28.

[0099] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain and the extracellular domain are from DR3, The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain is CD28.

[0100] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain and the extracellular domain are derived from LTBR; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain is CD28.

[0101] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain and the extracellular domain are derived from CD40; The artificial immune receptor, wherein the cytoplasmic costimulatory signaling domain is CD28.

[0102] T Cell Receptor Signaling Domains In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is selected from CD3 zeta, CD3 gamma, and CD3 epsilon.

[0103] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0104] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The present invention relates to an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is derived from CD3 zeta.

[0105] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain comprises SEQ ID NO:37.

[0106] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain consists of SEQ ID NO:37.

[0107] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain is derived from the same protein as the extracellular domain; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0108] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from TNFR2; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0109] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from DR3; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0110] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from LTBR; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0111] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from CD40; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0112] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the transmembrane domain is derived from the same protein as the extracellular domain; the cytoplasmic costimulatory signaling domain is CD28; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0113] Preferred AIR In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from TNFR2; the cytoplasmic costimulatory signaling domain is CD28; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0114] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from DR3; the cytoplasmic costimulatory signaling domain is CD28; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0115] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from LTBR; the cytoplasmic costimulatory signaling domain is CD28; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0116] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from CD40; the cytoplasmic costimulatory signaling domain is CD28; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0117] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from TNFR2 and comprises SEQ ID NO:39; the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0118] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from TNFR2 and consists of SEQ ID NO: 39; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0119] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor comprises SEQ ID NO:38.

[0120] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor is an artificial immune receptor consisting of SEQ ID NO:38.

[0121] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from DR3 and consists of SEQ ID NO: 41; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0122] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor comprises SEQ ID NO: 40.

[0123] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor is of sequence number 40.

[0124] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is derived from LTBR and consists of SEQ ID NO: 33; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0125] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor comprises SEQ ID NO:31.

[0126] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor is of sequence number 31.

[0127] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; said member of the tumor necrosis factor receptor superfamily and said transmembrane domain is derived from CD40 and consists of SEQ ID NO: 46; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0128] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor comprises SEQ ID NO:47.

[0129] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO: 47.

[0130] Nucleic Acids, Vectors, and Host Cells The artificial immune receptors of the present disclosure are encoded by nucleic acids. Thus, in certain embodiments, the present disclosure relates to nucleic acids that encode the artificial immune receptors.

[0131] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a nucleic acid encoding an artificial immune receptor comprising a cytoplasmic T cell receptor signaling domain.

[0132] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from TNFR2 and comprises SEQ ID NO:39; the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35; The nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0133] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from TNFR2 and consists of SEQ ID NO: 39; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The present invention relates to a nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0134] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:38.

[0135] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:38.

[0136] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from DR3 and comprises SEQ ID NO: 41; the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35; The nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0137] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from DR3 and consists of SEQ ID NO: 41; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The present invention relates to a nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0138] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:40.

[0139] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:40.

[0140] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from LTBR and comprise SEQ ID NO: 33; the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35; The nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0141] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is derived from LTBR and consists of SEQ ID NO: 33; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The present invention relates to a nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0142] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, the artificial immune receptor comprising SEQ ID NO:31.

[0143] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:31.

[0144] In certain embodiments, the disclosure relates to a nucleic acid comprising SEQ ID NO:30.

[0145] In certain embodiments, the disclosure relates to a nucleic acid consisting of SEQ ID NO:30.

[0146] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is derived from CD40 and comprises SEQ ID NO: 46; the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35; The nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0147] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; said member of the tumor necrosis factor receptor superfamily and said transmembrane domain is derived from CD40 and consists of SEQ ID NO: 46; the cytoplasmic costimulatory signaling domain is CD28 and consists of SEQ ID NO:35; The present invention relates to a nucleic acid encoding an artificial immune receptor, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta and consists of SEQ ID NO:37.

[0148] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor comprises SEQ ID NO:47.

[0149] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a cytoplasmic T cell receptor signaling domain; The artificial immune receptor relates to a nucleic acid encoding an artificial immune receptor, wherein the artificial immune receptor consists of SEQ ID NO:47.

[0150] In certain embodiments, the disclosure relates to a nucleic acid encoding a protein comprising SEQ ID NO:30.

[0151] In certain embodiments, the disclosure relates to a nucleic acid encoding a protein consisting of SEQ ID NO:30.

[0152] In certain embodiments, the present disclosure relates to a vector comprising a nucleic acid encoding an artificial immune receptor of the present disclosure.

[0153] In certain embodiments, the present disclosure provides a method for producing a pharmaceutical composition comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) a nucleic acid encoding an artificial immune receptor comprising a cytoplasmic T cell receptor signaling domain.

[0154] In certain embodiments, the present disclosure relates to a host cell comprising a nucleic acid or vector encoding an artificial immune receptor according to the present disclosure.

[0155] In certain embodiments, the present disclosure relates to host cells expressing the artificial immune receptors of the present disclosure.

[0156] In certain embodiments, the present disclosure relates to a host cell expressing an artificial immune receptor of the present disclosure on its cell surface.

[0157] In preferred embodiments, the host cell is a eukaryotic host cell. Thus, in certain embodiments, the present disclosure relates to a eukaryotic host cell comprising a nucleic acid or vector encoding an artificial immune receptor according to the present disclosure. In other embodiments, the present disclosure relates to a eukaryotic host cell expressing an artificial immune receptor of the present disclosure. In yet other embodiments, the present disclosure relates to a eukaryotic host cell expressing an artificial immune receptor of the present disclosure on the cell surface.

[0158] therapeutic use The artificial immune receptors of the present disclosure can be used therapeutically for the prevention and treatment of diseases and disorders.

[0159] In certain embodiments, the present disclosure relates to an artificial immune receptor disclosed herein for use in medicine. In certain embodiments, the present disclosure relates to an artificial immune receptor disclosed herein for use in the prevention of treatment of a disease or disorder. In certain embodiments, the disease or disorder is an inflammatory disorder or cancer. In preferred embodiments, the disease or disorder is an autoimmune disorder. In certain embodiments, the disease or disorder is the treatment of graft-versus-host disease or use in solid organ transplantation.

[0160] Certain Specific Embodiments

[0161] 1. An artificial immune receptor comprising: a) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and b) a transmembrane domain; c) a cytoplasmic costimulatory signaling domain; and d) an artificial immune receptor comprising a cytoplasmic T cell receptor signaling domain.

[0162] 2. The artificial immune receptor of claim 1, wherein the 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.

[0163] 3. The artificial immune receptor of claim 1 or 2, wherein the member of the tumor necrosis factor receptor superfamily is selected from TNFR2, DR3, LTBR, ​​and CD40.

[0164] 4. The artificial immune receptor of any one of claims 1 to 3, wherein the transmembrane domain is derived from the same protein as the extracellular domain.

[0165] 5. The artificial immune receptor of any one of claims 1-4, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from TNFR2.

[0166] 6. The artificial immune receptor of claim 5, comprising SEQ ID NO:39.

[0167] 7. The artificial immune receptor of claim 5 or 6, comprising SEQ ID NO:38.

[0168] 8. The artificial immune receptor of any one of claims 1-4, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from DR3.

[0169] 9. The artificial immune receptor of claim 8, comprising SEQ ID NO:41.

[0170] 10. The artificial immune receptor of claim 8 or 9, comprising SEQ ID NO:40.

[0171] 11. The artificial immune receptor of any one of claims 1-4, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from LTBR.

[0172] 12. The artificial immune receptor of claim 11, comprising SEQ ID NO:33.

[0173] 13. The artificial immune receptor of claim 11 or 12, comprising SEQ ID NO:31.

[0174] 14. The artificial immune receptor of any one of claims 1-4, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are derived from CD40.

[0175] 15. The artificial immune receptor of claim 11, comprising SEQ ID NO:46.

[0176] 16. The artificial immune receptor of claim 11 or 12, comprising SEQ ID NO:47.

[0177] 17. The artificial immune receptor of any one of claims 1-16, wherein the cytoplasmic costimulatory signaling domain is selected from 4-IBB (CD137), BAFFR, OX40, CD27, CD28, CD40, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1-TLR9 receptors, IL-2, IL-7, and IL-15 receptors.

[0178] 18. The artificial immune receptor of any one of claims 1-17, wherein the cytoplasmic costimulatory signaling domain is CD28.

[0179] 19. The artificial immune receptor of any one of claims 1-18, wherein the cytoplasmic costimulatory signaling domain comprises SEQ ID NO:35.

[0180] 20. The artificial immune receptor of any one of claims 1-19, wherein the cytoplasmic T cell receptor signaling domain is selected from CD3 zeta, CD3 gamma, and CD3 epsilon.

[0181] 21. The artificial immune receptor of any one of claims 1-20, wherein the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0182] 22. The artificial immune receptor of any one of claims 1-21, wherein the cytoplasmic T cell receptor signaling domain comprises SEQ ID NO:37.

[0183] 23. The artificial immune receptor of any one of claims 1-7 or 17-22, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from TNFR2, the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0184] 24. The artificial immune receptor of any one of claims 1-7 or 17-23, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from TNFR2 and comprises SEQ ID NO:39, the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0185] 25. The artificial immune receptor of any one of claims 1-4, 8-10, or 17-22, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from DR3, the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0186] 26. The artificial immune receptor of any one of claims 1-4, 8-10, 17-22, or 25, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from DR3 and comprises SEQ ID NO:41, the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0187] 27. The artificial immune receptor of any one of claims 1-4, 11-13, or 17-22, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from LTBR, ​​the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0188] 28. The artificial immune receptor of any one of claims 1-4, 11-13, 17-22, or 27, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from LTBR and comprise SEQ ID NO:33, the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0189] 29. The artificial immune receptor of any one of claims 1-4, 14-16, or 17-22, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from CD40, the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta.

[0190] 30. The artificial immune receptor of any one of claims 1-4, 14-16, 17-22, or 29, wherein the member of the tumor necrosis factor receptor superfamily and the transmembrane domain is from CD40 and comprises SEQ ID NO:46, the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3 zeta and comprises SEQ ID NO:37.

[0191] 31. A nucleic acid encoding an artificial immune receptor according to any one of claims 1 to 30.

[0192] 32. A vector comprising the nucleic acid of claim 31.

[0193] 33. A host cell comprising the nucleic acid of claim 31 or the vector of claim 32, or expressing an artificial immune receptor of any one of claims 1 to 30.

[0194] 34. An artificial immune receptor according to any one of claims 1 to 30 or a host cell according to claim 33 for use in medicine.

[0195] 35. The artificial immune receptor of claim 34, wherein the use is in the treatment of an inflammatory disorder.

[0196] 36. The artificial immune receptor of claim 35, wherein the inflammatory disorder is graft-versus-host disease. EXAMPLES

[0197] Example 1

[0198] material Ethics statement Peripheral blood mononuclear cells for T cell enrichment were isolated from leukocyte reduction chambers from healthy platelet donors. The collection of immune cells from these donors was performed in accordance with the Declaration of Helsinki after ethical approval by the local ethical committee and signed informed consent.

[0199] Isolation of peripheral blood mononuclear cells and pre-enrichment of blood lymphocytes

[0200] To isolate T cells from human blood, a leukocyte reduction chamber (provided by a transfusion physician, University Clinics Regensburg) was used. Leukocytes were first diluted with PBS and the resulting mixture of blood and PBS was divided into four fractions and underlaid with an equal volume of Pancoll (PAN Biotech, Aidenbach, Germany). Samples were centrifuged at 1.000 x g for 20 min at room temperature with acceleration set to 4 and braking set to 0. The PBMC layer was isolated and washed twice by centrifugation steps. Cells were pre-enriched with biotinylated anti-human CD25-PE (clone 2A3; BD Biosciences RRID:AB_341011) according to the manufacturer's protocol, followed by column-based magnetic separation with anti-PE ultrapure microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0201] mouse Female BALB / c mice, C57BL / 6 Nr4a1-eGFP mice (JAX stock #016617, C57BL / 6-Tg(Nr4a1-EGFP / cre)820Khog / J) and C57BL / 6 CD45.1+ mice (JAX stock #002014, B6.SJL-PtprcaPepcb / BoyCrl) were obtained from Charles River Breeding Laboratories (Wilmington, MA, USA) or Jackson Laboratory (Bar Harbor, ME, USA). B6N.129(Cg)-Foxp3tm3Ayr mice (Foxp3.IRES-DTR / GFP) were crossed to C57BL / 6CD45.1+ mice and served as bone marrow donors for GvHD experiments. C57BL / 6Foxp3-hCD2 (Foxp3tm1(CD2 / CD52)Shori) mice were a kind gift from S. Hori (P Natl Acad Sci USA, 2009;106(6):1903-8). Mice were kept under specific pathogen-free conditions and the study was approved by the Ethics Committee for Animal Experiments of the Bavarian State Government.

[0202] cell line Phoenix-Eco and Phoenix-Ampho cell lines were purchased from ATCC (catalog number CRL-3214 and number CRL-3213). These are second generation retrovirus producer cell lines for generating ecotropic and amphotropic retroviruses. EL4 lymphoblastoid cell line was purchased from ATCC (catalog number TIB-39).

[0203] Additional resources are listed in Tables 1, 2, 3, and 4.

[0204] [Table 1] JPEG2025517482000003.jpg179168

[0205] [Table 2] JPEG2025517482000005.jpg159169

[0206] [Table 3] JPEG2025517482000007.jpg82169

[0207] [Table 4]

[0208] statistical analysis Data were analyzed using Prism software or algorithms. Statistical details are shown in the figure legends. For survival differences, Kaplan-Meier analysis was performed and the log-rank test was used. P values ​​< 0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). Example 2

[0209] method Digestion of murine tissues for flow cytometric analysis, FACS sorting of cells, and transplantation To isolate cells from colonic tissue, colons were isolated, cleared of feces, and prepared using a lamina propria dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and gentleMACS instrument (Miltenyi Biotec, Bergisch Gladbach, Germany, program 37C_mLPDK_1) according to the manufacturer's instructions. A more detailed protocol for T cell isolation from murine tissue has been published (Eur J Immunol. 2019;49(10):1457-973.).

[0210] To isolate cells from blood, peripheral blood was collected and incubated in heparin buffer. Blood samples were centrifuged and red blood cells were lysed using ACK lysis buffer (ThermoFisher / GIBCO, Waltham, MA, USA, #A1049201).

[0211] To isolate cells from spleen and lymph nodes, tissues were harvested and mechanically disrupted. Samples were centrifuged and red blood cells were lysed using ACK lysis buffer. Samples were pre-enriched with anti-human CD2 (RPA-2.10) microbeads (for Foxp3-hCD2 reporter mice) or anti-CD25 (PC61) antibody staining and anti-PE microbeads.

[0212] To isolate cells from bone marrow for transplantation, femurs were harvested and the femoral head and medial and lateral femoral epicondyles were removed. The bone shaft was flushed with PBS and bone marrow was harvested by centrifugation followed by red blood cell lysis.

[0213] Preparation of samples for flow cytometry Single cell suspensions were prepared and pre-enriched as previously described (Eur J Immunol. 2019;49(10):1457-973). Samples were stained in either 1.5 mL Eppendorf tubes or 96-well plates in FACS buffer (1% FCS in PBS). Surface staining was performed in a chromosomal volume of 50–100 µL for 20 min at 4 °C. Antibodies were used as recommended by the manufacturers unless otherwise indicated. The following antibodies were used for surface staining of murine samples: TCR-β chain (H57-597), CD4 (RM4-5), CD8α (53-6.7), CD19 (6D5), CD25 (PC61), CD45.1 (A20), CD45.2 (104), CD90.1 (OX-7), CD120b (TR79-89), IA / IE / MHCII (M5 / 114.15.2), Klrg1(2F1), Tigit(1G9), DR3(4C12), CD62L(MEL-14), CD137(I7B5), CD69(H1.2F3), LTBR(5G11), LAP(TW7-16B4), H2Kb(AF6-88.5), H2Kd(SF1-1.1), hCD2(RPA-2.10).

[0214] The following antibodies were used for surface staining of human samples: CD4 (OKT4), CD25 (BC96, 2A3), CD127 (A019D5), CD45RO (UCHL1), CD45RA (HI100), CD137 (41BB), TCR-β chain (IP26), CCR8 (433H RUO), and GARP (7B11).

[0215] Intracellular staining was performed using Foxp3 / Transcription Factor Buffer Set (Thermo Fisher / Biosciences, Waltham, MA, USA) according to the manufacturer's protocol with the following adaptations: the intracellular staining step was performed for 60 min at room temperature. Antibodies for intracellular staining included Foxp3 (JFK-16S), Nr4a1 (12.14), Irf8 (V3GYWCH) for mouse samples and Foxp3 (206D) for human samples. Dead cells were excluded using a fixable live / dead dye (eBioscience Fixable Viability Dye eFluor780). All antibodies are listed in the resource table.

[0216] Flow cytometry and FACS sorting of T cells from blood, tissues, and cell cultures Cells were isolated, pre-enriched, and stained as previously described (Eur J Immunol. 2019;49(10):1457-973). Samples were then filtered through a 40 μM filter unit and acquired on a BD FACSymphony™, BD FACSCelesta™, or BD FACSFusion™ flow cytometer (all from Becton Dickinson, Franklin Lakes / NJ, USA). Machine functionality was verified using BD CS&T beads. Fluorescence extravasation compensation was performed with lymphocytes stained with anti-CD4 antibodies of the respective color. Flow cytometry data were analyzed using BD FlowJo™ (version 10.6.2). Sorting was performed using a BD FACSAriaII™ or BD FACSFusion™ cell sorter equipped with a 70 μm nozzle. Post-sorting quality control was performed, if required. For murine Treg cultures, anti-CD25 enriched cells were sorted for naive CD4+CD25+CD62L+ Tregs (Leukemia 2020;34(3):895-908). hCD2 pre-enriched cells were sorted for CD4+CD25+hCD2+. For culture, cells were sorted directly into cell culture medium. All procedures were performed in DNA low binding tubes (Eppendorf, Hamburg, Germany, #0030108051) or 15 ml tubes.

[0217] For bulk RNA sequencing, 4 × 104 to 8 × 10 4 Cells were sorted directly on live CD4+CD25+CD90.1+GFP(Nr4a1)+LAP+ or CD4+CD25+CD90.1 into 500 μL of RLT+ lysis buffer (Qiagen, Hilden, Germany, RNEasy Plus Micro Kit#74034). For human Treg cultures, CD25-enriched cells from human blood were sorted directly on TCR-β chain+CD4+CD25+CD45RA+ into TexMACS medium.

[0218] Murine and human Treg cell cultures For murine Treg cultures, sorted Treg cells were plated at 3 × 10 in 96-well round-bottom plates (Corning, Wiesbaden, Germany) containing anti-CD3 / CD28 beads (Miltenyi Biotec, Bergisch Gladbach, Germany, 4 beads per cell) and 2000 U / ml rhIL-2 (ProleukinS®, Novartis, Basel, Switzerland). 4 ~4×10 4 Cells were seeded at 10 ...

[0219] Retroviral transduction of Treg cells Retroviruses in the pMSCV-Thy1.1 system can be produced in Phoenix-Eco cells, which are pCLEco (packaging plasmid) carrying mutants in HEK293 cells. Thus, Phoenix-Eco cells were cultured in 1.8 × 10 cells in a 6-well plate 6 hours before lipofection. 6Cells were seeded on a gelatin matrix at 1000 cells / well. To produce liposomal particles containing the viral transgene, 3 μg of vector DNA and 1 μg of an additional pCL-Eco packaging plasmid were co-incubated with 12 μL of TransIT-293 transfection reagent (MoBiTec, Göttingen, Germany) for 20 min in OptiMEM medium at room temperature. The liposomes were added to the Phoenix-Eco cells and incubated for an additional 16 h. Afterwards, the medium was changed and the production of viral particles was allowed to proceed for 24 h. The supernatant containing the produced pMSCV retrovirus was then added to the Treg cell culture and mixed gently. Treg cells were transduced for 6.5 h of incubation at 37 °C. Afterwards, the viral supernatant was removed and the cells were incubated with fresh medium supplemented with IL-2 (2000 U / ml) for an additional 72–96 h. Cells were then harvested and anti-CD3 / CD28 antibody-bound beads were removed using a MACSiMAG separation magnet (Miltenyi Biotec, Bergisch Gladbach, Germany). For transduction of human Tregs, the amphoteric Phoenix cell line was used. Phoenix-Ampho cells were seeded at 2 × 10 per well in 6-well plates 6 h prior to lipofection. 6100 cells were seeded on the cells. For the production of liposomal particles containing the viral transgene, 3 μg of vector DNA and 1 μg of an additional pCL-Ampho packaging plasmid were co-incubated with 12 μL of TransIT-293 transfection reagent (MoBiTec, Bergisch Gladbach, Germany) for 20 min in OptiMEM medium at room temperature. The liposomes were added to the Phoenix-Ampho cells and incubated for a further 16 h. Afterwards, the medium was changed and the production of viral particles was allowed to proceed for 24 h. The supernatant containing the produced pMSCV retrovirus was then added to the Treg cell culture and mixed gently. Treg cells were transduced for 6.5 h of incubation at 37 °C. Thereafter, viral supernatant was removed, and cells were incubated for an additional 72–96 h with fresh medium supplemented with IL-2 (500 U / ml), 100 U / ml penicillin-streptomycin, and fresh TransAct (1:100 dilution).

[0220] Transient transfection of HEK293 cells with ligands for TNFSF For production of liposomal particles, 2 μg of plasmid DNA was incubated with 6 μl of TransIT-293 transfection reagent in 100 μl of OptiMEM medium for 20 min at room temperature. The liposomes were then gently added to 5×105 HEK293 cells resuspended in 500 μl of DMEM. After shaking for 1 min, 230 μl of DMEM was added and 100 μl of cell-liposome suspension (approximately 6×10 4 (containing 1000 x 100 cells) were added per well to a 96-well flat-bottom plate. 18 hours after changing the lipofection medium, the transfected HEK293 cells were ready for use in co-culture experiments.

[0221] Treg proliferation assay Anti-CD3 / CD28 beads were removed from transduced Treg cultures via a MACSiMAG separation magnet. Tregs were left in fresh medium supplemented with 100U / ml rhIL-2 for 18 hours. Tregs were then labeled with CellTrace™ CFSE cell proliferation dye (1 μM) and added to HEK293 cells expressing mTNF, mTL1A, or mLIGHT on the cell surface. rhIL-2 (2000U / ml) was added to the cell cultures. After 72 hours of co-incubation, the proliferation of transduced CFSE-labeled Tregs was analyzed by flow cytometry.

[0222] RNA sequencing Total RNA was isolated using Qiagen Rneasy Micro Kit (Qiagen, Hilden, Germany) and RNA was eluted in 14 μL of RNAse-free water. RNA quality was assessed using a Tapestation system 4200 and high-sensitivity RNA screen tape (Agilent). 7 μl of RNA was used to generate RNA-seq libraries using the SMART-seq Stranded Kit from Takara (Mountain View / CA, USA). Indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq550 machine with the NextSeq 500 / 550 High Output Kit v2.5 (75 cycles).

[0223] GvHD Model BALB / c (H2Kd) recipients were irradiated (8 Gy) and administered 5 × 10 5 with or without (BM control) 2.5 × 10 splenocytes 6 Animals in the treatment group were transplanted retrobulbarly with 2.5 × 10 BM cells in vitro. 5 Tregs were expanded and transduced (C57BL / 6, Foxp3-hCD2, congenic CD45.2). Recipients were monitored daily, with weight and GvHD symptoms assessed two or three times weekly by unblinded investigators applying standardized scoring. Example 3

[0224] Recombinant Proteins Murine TNF For production of murine TNF, the expression plasmid BCMGS-L6 was used, encoding the uncleaved mouse TNF protein (JBC 1999, 274(53):38112-8).

[0225] Mouse TL1A To generate a non-cleavable version of TL1A (mTL1A), amino acids 69-93 were deleted from the wild-type sequence as described in J Immunol 2018;200(4):1360-9. The cDNA encoding non-cleavable mTL1A was cloned into pIRES2-DsRed via NheI / SalI. The nucleotide sequence of mTL1A is shown below: (SEQ ID NO:1)

[0226] Human LIGHT The cDNA encoding human LIGHT was cloned into pcDNA3.1neo via BamHI / EcoRV. The nucleotide sequence of hLIGHT is shown below: (SEQ ID NO:2)

[0227] Mouse LIGHT The cDNA encoding mouse LIGHT was cloned into pIRES2-DsRed via NheI / SalI. Amino acids 63-84 were deleted to generate a non-cleavable form of mouse LIGHT. The nucleotide sequence of mLIGHT is shown below: ATGGAGAGTGTGGTACAGCCTTCAGTGTTTGTGGTGGATGGACAGACGGACATCCCATTCAGGCGGCTGGAACAGAACCACCGGAGACGGCGCTGTGGCACTGTCCAGGTCAGCCTGGCCCTGGTGCTGCTGCTAGGTGCTGGGCTGGCCACTCAGGGCTGGTTTCTCCTGAGACTGCATCAACGTCAACGATCTCACCAGGCCAACCCAGCAGCACATCTTACAGGAGCCAACGCCAGCTTGATAGGTATTGGTGGACCTCTGTTATGGGAGACACGACTTGGCCTGGCCTTCTTGAGGGGCTTGACGTATCATGATGGGGCCCTGGTGACCATGGAGCCCGGTTACTACTATGTGTACTCCAAAGTGCAGCTGAGCGGCGTGGGCTGCCCCCAGGGGCTGGCCAATGGCCTCCCCATCACCCATGGACTATACAAGCGCACATCCCGCTACCCGAAGGAGTTAGAACTGCTGGTCAGTCGGCGGTCACCCTGTGGCCGGGCCAACAGCTCCCGAGTCTGGTGGGACAGCAGCTTCCTGGGCGGCGTGGTACATCTGGAGGCTGGGGAAGAGGTGGTGGTCCGCGTGCCTGGAAACCGCCTGGTCAGACCACGTGACGGCACCAGGTCCTATTTCGGAGCTTTCATGGTCTGA(SEQ ID NO: 3)

[0228] Design of Artificial Immune Receptor (AIR) (Example 4)

[0229] Murine and human AIRs were designed based on the published nucleotide sequences of the individual moieties and domains (https: / / www.ensembl.org / ). The extracellular and transmembrane domains of LTbR, DR3, TNFR2, and CD40 were fused to the intracellular signaling domains of CD28 and CD3-zeta chain. Two point mutations were introduced into the murine CD3-zeta chain as shown to increase expression of chimeric antigen receptor (Blood2003;102(13):4320-5). As negative controls, ORFs encoding the transmembrane and signaling domains of CD28 and the signaling domain of CD3-zeta chain were used. Anti-CD19 CAR and anti-CEACAR have been described previously (Blood2010;116(19):3875-86; Mol Ther2019;27(10):1825-35). The cDNAs encoding AIR and anti-CD19 CAR and anti-CEACAR were fused to the congenic marker CD90.1 by linking to the self-cleaving P2A sequence. The cDNAs were synthesized by ThermoFisher / Life Technologies (Waltham, MA, USA) and cloned via NotI / Mlu into pMSCV-Thy1.1 retroviral backbone (Addgene, Cat. No. 17442). An overview of the constructs is shown in FIG. 1. The sequences of the constructs and their parts are shown in Table 5 (N=nucleic acid, P=polypeptide). The CD90.1 marker gene is cleaved via the P2A cleavage site and is not part of AIR.

[0230] [Table 5] JPEG2025517482000010.jpg245166JPEG2025517482000011.jpg247166JPEG20255174820 00012.jpg248166JPEG2025517482000013.jpg239166JPEG2025517482000014.jpg244166 JPEG2025517482000015.jpg248166JPEG2025517482000016.jpg247166JPEG20255174820 00017.jpg236166JPEG2025517482000018.jpg245166JPEG2025517482000019.jpg176166

[0231] The mode of action of AIR is illustrated in Figure 2. Binding of membrane-bound TNF family members via AIR leads to the translation of cytokine signals into T cell receptor (TCR)-like signals, which initiate T cell activation independently of their endogenous TCR-MHC restriction in an environment / inflammation-dependent manner. Example 5

[0232] AIR transduction and surface expression Transduction and expression of AIR according to the present disclosure was investigated in murine regulatory T cells (Tregs). Tregs were isolated from spleens and lymph nodes of C57 / BL6 mice, sorted (naive CD4+CD25+CD62L+Tregs or Foxp3+reporter (hCD2+) positive), and expanded for 6 days by anti-CD3 and anti-CD28 antibody-coupled beads + IL-2 (2000U / ml) stimulation. The purity of Treg cultures was determined by flow cytometry. Treg cultures contained approximately 90-99% Foxp3+T cells. Two days after isolation, Tregs were transduced with AIR constructs or anti-CD19 chimeric antigen receptor (anti-CD19 CAR) as a control. After 3 days, transduction efficacy via CD90.1 staining and surface expression of AIR were confirmed by flow cytometry. The results are shown in Figures 3 and 4. Similar results were obtained with CD40 AIR (93.6% Foxp3+ T cells). It can be demonstrated that all AIRs are efficiently expressed on the surface of regulatory T cells. Example 6

[0233] AIR induces the expression of T cell receptor signaling marker genes To test whether the AIR of the present disclosure induces T cell receptor signaling, we investigated the induction of Nr4a1. Nr4a1 is a well-known target gene of T cell receptor signaling. We used Tregs from Nr4a1-eGFP reporter mice. As a control, Tregs were stimulated with anti-CD3 / CD28 antibody-bound beads. The experimental outline is shown in Figure 5.

[0234] AIR-expressing Tregs were stimulated in overnight co-culture with HEK cells expressing murine Light (mLight), mTL1A, or mTNF. As a positive control, Tregs were stimulated with anti-CD3 / CD28 antibody-conjugated beads to mimic TCR signaling. Results are shown in Figure 6. Only AIR-positive (CD90.1+) Tregs express Nr4a1-eGFP (left), whereas AIR-negative (CD90.1-) Tregs do not (right). In contrast, anti-CD19 CAR-expressing Tregs did not show upregulation of Nr4a1-GFP (lower panel).

[0235] Figure 7 demonstrates the expression of Nr4a1 upon administration of AIR according to the present disclosure, exemplified with AIR bearing the extracellular domain of LTBR. Tregs expressing LTBR AIR according to the present disclosure or an irrelevant CAR bearing an anti-CD19 single chain antibody (scFv) extracellularly were co-cultured with murine EL4 cells known to express LIGHT in the absence or presence of mLight blocking LTBR-Ig fusion protein (50ug / ml) and then stained for intracellular Nr4a1 expression. Example 7

[0236] RNA and protein expression analysis of AIR-stimulated Tregs In this experiment, we tested whether Tregs stimulated with AIR according to the present disclosure exhibited expression patterns consistent with the hypothesized TCR signaling effects, as measured by RNA-seq expression analysis.

[0237] The first cells were sorted accordingly. Tregs expressing AIR or irrelevant CAR carrying extracellular anti-CD19 single chain antibody (scFv) were left to stand for 24 hours, then co-cultured with HEK cells or mLight expressing HEK cells for 18 hours. Figure 8 shows the pre-gating of CD90.1+ cells. The pre-gated cells were then sorted for CD4+CD25+CD90.1+ (left and center) or CD4+CD25+CD90.1+Nr4a1+LAP+ (right). The results are shown in Figure 9.

[0238] After cell sorting, RNA was isolated and RNA sequencing was performed, and the results are shown in Figures 10 (LTBR AIR), 11 (DR3 AIR), and 12 (α-CD19 control CAR).

[0239] On the left side of Figure 10, a summary of up- and down-regulated genes is shown comparing HEK-mLight stimulated α-CD19 CAR Tregs versus LTBR AIR Tregs stimulated with HEK cells as a control (endogenous mLight signaling) and HEK-mLight stimulated LTBR AIR Tregs versus HEK-mLight stimulated α-CD19 CAR Tregs (specific LTBR AIR signaling). On the right side, a volcano plot showing differentially expressed transcripts is shown.

[0240] Similarly, in Figure 11, DR3 AIR Tregs were stimulated with HEK cells (endogenous mTL1A signaling). DR3 AIR Tregs were stimulated with HEK-mTL1A vs. α-CD19 CAR Tregs stimulated with HEK-mTL1A (specific DR3 AIR signaling). Differentially expressed genes are shown on the left and volcano plots are shown on the right.

[0241] Similarly, Figure 12 compares α-CD19 CAR Tregs stimulated with anti-CD3 / CD28 beads versus unstimulated Tregs. Differentially expressed genes are shown on the left and a volcano plot is shown on the right.

[0242] The RNA and / or protein expression levels of various specific marker genes were analyzed in more detail. Figure 13 shows RNA expression data from LTBR AIR or control CAR expressing Tregs for Tnfrsf9, Tigit, Tgfb1, Cd69, Ccr8, and Nr4a1 after 18 hours of co-culture with HEK+ / -mLight (Deseq2, n=3). LTBR AIR (each of the graphs shown on the far right) results in significant upregulation of all six marker genes tested. Figure 14 shows representative flow cytometry analysis from LTBR AIR or control CAR expressing Tregs after 18 hours of co-culture with HEK+ / -mLight. Protein expression of CD137 and Tigit is shown in the upper panel, and CD69 and LAP (membrane-bound Tgfβ1) in the lower panel. Data are representative of three independent experiments. Again, LTBR AIR results in significant upregulation of the marker genes investigated.

[0243] Figure 15 shows RNA expression data from DR3 AIR expressing Tregs or control CARs for Tnfrsf9, Tigit, Tgfb1 after 18 hours of co-culture with HEK+ / -mLight (Deseq2, n=3). DR3 AIR (each of the graphs shown on the far right) results in significant upregulation of all three marker genes tested. Figure 16 shows representative flow cytometry analysis from DR3 AIR expressing Tregs or control CARs after 18 hours of co-culture with HEK+ / -mTL1A. Protein expression of CD137 and Tigit is shown in the top panel, and CD69 and LAP (Tgfβ1) are shown in the bottom panel. Data are representative of three independent experiments. Again, DR3 AIR results in significant upregulation of the marker genes investigated.

[0244] Figure 17 shows RNA expression data (top left, Deseq2, n=3) and protein expression (right) from LTBR AIR or control CAR expressing Treg against Irf8 after 18 hours of co-culture with HEK+ / -mLight. RNA expression data (bottom left, Deseq2, n=3) and protein expression (right) from DR3 AIR or control CAR expressing Treg against Irf8 after 18 hours of co-culture with HEK+ / -mTL1A. Again, all marker genes tested are significantly upregulated.

[0245] Figure 18 demonstrates that the AIR of the present disclosure mediates the activation and proliferation of Treg. LTBR AIR or control CAR expressing Treg was left for 24 hours and labeled with CFDA-SE proliferation dye. The engineered and labeled Treg was co-cultured with HEK+ / -mLight in the presence of IL2 for 72 hours and then analyzed for proliferation by flow cytometry. A representative dot plot is shown on the left. Summary data of three experiments performed with three technical replicates is shown on the right (n=3). Furthermore, all marker genes tested are significantly upregulated.

[0246] In summary, RNA and protein expression analyses clearly demonstrate that the AIR of the present disclosure induces cellular responses consistent with the underlying hypothesis. Example 8

[0247] LTBR AIR ameliorates GvHD pathology in mice The utility of the AIR of the present disclosure was tested in a graft-versus-host disease (GvHD) mouse model. FACS-sorted Tregs from Foxp3-hCD2 reporter mice were expanded and transduced with LTBR AIR or a truncation construct (lacking the extracellular binding domain shown in FIG. 1a) as a control. 2.5×10 5 Engineered Tregs were administered in 2.5 × 10 6 10 bone marrow cells and 2.5 x 10 5The mice were transplanted with 1000 spleen cells into lethally irradiated (8 Gy) Balb / c mice. As a transplantation control, one group received bone marrow cells (BM) only, and as a disease development control, the other group received BM cells + spleen cells. After transplantation, animals were monitored for 47 days. An overview of the experiment is shown in Figure 19. The quality of the engineered Tregs was confirmed before transfer into mice by staining for CD4, hCD2 (reporter for Foxp3), as well as CD90.1 and LTBR. The results are shown in Figure 20. On day 20, blood samples were taken from each animal to verify the recovery of donor-derived (H2kB+)CD19+ B cells.

[0248] Figure 21 shows a Kaplan-Meier plot containing data sets from two independent experiments (log-rank test, n=11-12). As can be seen, LTBR AIR was highly efficient in the GvHD model and significantly increased the survival rate of transplanted Balb / c mice. This can also be seen in Figure 22, which shows the mean GvHD score per treatment group (animals that reached a score of 40 had to be euthanized; dead animals kept the highest score over the following time points for statistical analysis, two-way ANOVA). Figure 23 shows a representative FACS plot (left) of the spleen of a surviving animal that received LTBR AIR Treg. On the right, the percentage of Foxp3+ Treg in the spleen of surviving mice at day 47 is shown. Figure 24 shows the analysis of Klrg1+ Treg in the spleen. On the left, a representative FACS plot of identified CD45.1+ (BM-derived) and CD45.2+ (transferred AIR Treg) Treg cells is shown. Klrg1 is a marker of tissue-specific phenotype; hCD2 is a Foxp3 reporter. In the center, Klrg1 expression of engineered CD45.2+ and BM-derived CD45.1+ Tregs in surviving mice is shown. On the right, the frequency of Klrg1+hCD2+ Tregs in mice that received LTBR AIR Tregs (Mann-Whitney U, n=8 survivors vs. n=4 deceased) is shown. Figure 25 shows a representative FACS analysis of digested colons of surviving animals that received LTBR AIR Tregs. Discrimination between CD45.1 and CD45.2 cells within the CD4+ population on the left is shown. CD45.2 Tregs are further analyzed for Klrg1 and hCD2 expression on the right. Figure 26 shows the percentage of hCD2+ (indicating Foxp3+) cells among the transferred engineered CD45.2+ cells (left), demonstrating phenotypic stability over 47 days in vivo. On the right, the cell numbers of transferred CD45.2 Tregs in the colon at day 47 are shown.

[0249] Taken together, this example demonstrates the clinical utility of the AIR of the present disclosure in an established clinical model for graft-versus-host disease. Example 9

[0250] Human LTBR AIR Human LTBR AIR construct was generated similarly to the murine LTBR construct.The human construct contains the same elements as the murine construct, except that all domains, namely LTBR extracellular domain, LTBR transmembrane domain, cytoplasmic CD28 domain, and cytoplasmic CD3 zeta domain, are of human origin (SEQ ID NO:30 (nucleic acid); SEQ ID NO:31 (polypeptide)).The expression and signal transduction capacity of the human LTBR AIR construct was tested.

[0251] Human T cells from healthy blood donors were sorted with Treg protein markers (TCRb+CD4+CD25+CD127-CD45RA+) and expanded with anti-CD3 / CD28 stimulation (TransACT, Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-2 (500U / ml) for 7 days. Two days after isolation, Tregs were transduced with the human LTBR AIR construct (using a bitropic version of the retroviral expression system MSCV). After 5 days, cultured Tregs were stained for intracellular Foxp3+ to confirm Treg purity and for CD90.1 and hLTBR to analyze transduction efficacy and surface expression of AIR protein. The results are shown in Figure 27. LTBR AIR expression on human Treg cells could be verified.

[0252] In a separate experiment, Tregs expressing human LTBR AIR or an irrelevant CAR (anti-CEA, carcinoembryonic antigen) were co-cultured for 18 h with parental HEK cells or HEK cells expressing human Light protein on their surface. Expression of Ccr8, glycoprotein A repeat dominant (GARP), CD137 (4-1BB), and Tigit is shown in CD90.1+Foxp3+Tregs. Again, expression levels of all marker genes confirm the expected activity of human LTBR AIR. Example 10

[0253] TCR signaling is important for the activity of AIR A control construct was generated that has a functionality comparable to that of the AIR of the present disclosure, but has the important difference that it lacks the CD3 zeta TCR signaling domain. Such a construct that does not contain the CD3 zeta domain is known in the prior art (see WO2021 / 051195). Unlike the construct of WO2021 / 051195, the construct provided by the present invention exerts its function directly in the main signal of the signaling cascade in a CD3 (TCR)-dependent manner.

[0254] The organization of the construct is shown at the top of Figure 29. The amino acid sequence of this construct is shown below: MRLPRASSPCGLAWGPLLLGLSGLLVASQPQLVPPYRIENQTCWDQDKEYYEPMHDVCCSRCPPGEFVFAVCSRSQDTVCKTCPHNSYNEHWNHLSTCQLCRPCDIVLGFEEVAPCTSDRKAECRCQPGMSCVYLDNECVHCEEERLVLCQPGTEAEVTDEIMDTDVNCVPCKPGHFQNTSSPRARCQPHTRCEIQGLVEAAPGTSYSDTICKNPPEPGAMLLLAILLSLVLFLLFTTVLACAWAAANSRRNRGGQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRPID (SEQ ID NO: 48)

[0255] Surface expression of the construct could be demonstrated by flow cytometry. See Figure 29 below. However, unlike the AIR of the present disclosure, the construct lacking CD3 zeta does not induce marker genes. Thus, the AIR of the present disclosure is functionally different from the prior art molecules. Figure 30 shows a representative flow cytometry analysis of Tregs expressing full-length LTBR AIR with LTBR "AIR" lacking CD3 zeta chain after 18 hours of co-culture with HEK+ / -mLight. Protein expression of CD137 and Tigit is shown in the upper panel, and CD69 and LAP (membrane-bound Tgfβ1) are shown in the lower panel.

[0256] The importance of the CD3 zeta domain was further demonstrated using LTBR AIR, LTBR AIR without CD3 zeta chain, and anti-CD19 CAR-expressing Treg cells from Nr4a1.eGFP reporter mice. The cells were rested for 24 hours and then co-cultured with HEK cells or HEK cells expressing mLIGHT for 18 hours. The cells were then analyzed for eGFP and LAP expression by flow cytometry. The results of one of two independent experiments are shown in Figure 41. Only full-length LTBR AIR containing the CD3 zeta domain, but not the other two constructs tested, can induce Nr4a1.eGFP upregulation in response to the corresponding ligand mLIGHT. This finding again demonstrates the importance of the CD3 domain for AIR-mediated TCR-like signaling and Treg cell activation. Example 11

[0257] CD40 AIR leads to expression of functionally related marker genes Similar experiments were performed with murine CD40 AIR of SEQ ID NO: 43. This AIR was tested in comparison with a construct containing an extracellular anti-CD19 single chain antibody (scFv) instead of the binding domain of a tumor necrosis factor (TNF) superfamily member (construct F in FIG. 1). CD40 AIR or anti-CD19 construct expressing Treg cells were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 hours. Cells were then analyzed by flow cytometry for expression of TIGIT and CD137 (FIGS. 31 and 32), as well as LAP (TGFb1) and CD69 (FIGS. 33 and 34).

[0258] Again, the AIR of the present disclosure can be shown to be functionally active and to induce the expected marker genes (Figures 31 and 33). The results are summarized in Figures 32 and 34. The induction of marker genes by CD40 AIR is statistically significant as determined by one-way Anova analysis. Example 12

[0259] CD40 AIR induces expression of Nr4a1, an early response gene in TCR signaling Treg cells from Nr4a1-eGFP reporter mice expressing the construct of Example 11 were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 hours, after which Nr4a1-eGFP expression was measured by flow cytometry.

[0260] Expression of Nr4a1, an early response gene in TCR signaling, was induced by Treg cells expressing CD40 AIR but not by constructs expressing anti-CD19 CAR (Figure 35), again confirming the TCR-like activation mediated by AIR of the present disclosure. Example 13

[0261] Design of AIR with alternative costimulatory domains Further constructs were generated to test whether alternative costimulatory domains are also functional. To do so, the CD28 costimulatory domain of the construct shown in Figure 1G was replaced by ICOS and 4-1BB costimulatory domains. These further constructs are shown in Figure 36A (ICOS) and Figure 36B (4-1BB). The sequences of the constructs and their parts are shown in Table 6 (N=nucleic acid, P=polypeptide). The CD90.1 marker gene is truncated through the P2A cleavage site and is not part of AIR. The constructs were generated as outlined in Example 4.

[0262] [Table 6] JPEG2025517482000021.jpg247166JPEG2025517482000022.jpg178166 Example 14

[0263] Surface expression of CD40 AIR with ICOS and 41BB costimulatory domains Transduction and surface expression of AIR with ICOS and 41BB costimulatory domains was examined as described in Example 5.

[0264] The transduction rates of CD40 AIR with different costimulatory domains (CD28, ICOS, 41BB) were comparable as measured by flow cytometry analysis (data shown). Surface expression of CD40 AIR with ICOS and 41BB costimulatory domains is shown in FIG. 37. It can be demonstrated that, like CD40 AIR with the CD28 costimulatory domain, CD40 AIR with the ICOS or 41BB costimulatory domain is also efficiently expressed on the surface of regulatory T cells. Example 15

[0265] All tested costimulatory domains confer CD40 AIR signaling In this experiment, we compared the signaling capacity of CD40 AIR with three different costimulatory domains. Treg cells expressing CD40 AIR versions with CD28, ICOS, or 41BB costimulatory domains were rested for 24 h and then cocultured with HEK cells or HEK cells expressing mCD40L for 18 h. Afterwards, the expression of CD69 and LAP was analyzed by flow cytometry (one-way ANOVA).

[0266] The results are shown in Figure 38. All constructs tested showed similar ability to induce Treg cell activation, confirming that any co-stimulatory domain can be used. Example 16

[0267] CD40 ameliorates GvHD pathology in mice Similar to LTBR AIR (see Example 8), CD40 AIR was also tested for its therapeutic utility in a graft-versus-host disease (GvHD) mouse model. Prior to transplantation, Treg cells were quality-controlled by flow cytometry analysis for transduction efficiency (CD90.1 expression) and CD40 AIR surface expression in Treg cells 3 days after transduction. As a control, a construct lacking extracellular CD40 binding domain was used). The results are shown in Figure 39. Both constructs, CD40 AIR and the control construct, showed high transduction efficiency, but only CD40 AIR-transfected cells exhibited binding to CD40.

[0268] The results of the experiment are shown in Figure 40. The Kaplan-Meier plot contains data sets from two independent experiments (log-rank test, n=10-12) and shows the survival of transplanted BALB / c mice. As can be seen, CD40 AIR was highly efficient in the GvHD model and significantly increased the survival rate of transplanted Balb / c mice. Taken together, this example validates the clinical utility of the AIR of the present disclosure in an established clinical model of graft-versus-host disease.

Claims

1. b) the extracellular domain of a member of the tumor necrosis factor receptor superfamily; and c) a transmembrane domain; and d) a cytoplasmic costimulatory signaling domain; and e) a cytoplasmic CD3 T cell receptor signaling domain; and An artificial immune receptor.

2. 2. The artificial immune receptor of claim 1, wherein the 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.

3. 3. The artificial immune receptor of claim 1 or 2, wherein the member of the tumor necrosis factor receptor superfamily is selected from TNFR2, DR3, LTBR, ​​and CD40.

4. The artificial immune receptor of any one of claims 1 to 3, wherein the transmembrane domain is derived from the same protein as the extracellular domain.

5. The artificial immune receptor of any one of claims 1 to 4, wherein the artificial immune receptor comprises the amino acid sequence of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:41, or SEQ ID NO:

46.

6. 6. The artificial immune receptor of any one of claims 1-5, wherein the cytoplasmic costimulatory signaling domain is selected from 4-IBB (CD137), BAFFR, OX40, CD27, CD28, ICOS, CD40, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1-TLR9 receptors, IL-2, IL-7, and IL-15 receptors.

7. The artificial immune receptor of any one of claims 1 to 6, wherein the cytoplasmic costimulatory signaling domain is selected from 4-IBB (CD137), CD28, and ICOS.

8. The artificial immune receptor of any one of claims 1 to 7, wherein the cytoplasmic costimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:

35.

9. The artificial immune receptor of any one of claims 1 to 8, wherein the cytoplasmic CD3 T cell receptor signaling domain is selected from CD3 zeta, CD3 gamma, and CD3 epsilon, preferably CD3 zeta.

10. The artificial immune receptor of any one of claims 1 to 9, wherein the cytoplasmic T cell receptor signaling domain comprises the amino acid sequence of SEQ ID NO:

37.

11. a) the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from TNFR2, the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta; b) the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from DR3, the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta; c) the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from LTBR, ​​the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta, or d) the member of the tumor necrosis factor receptor superfamily and the transmembrane domain are from CD40, the cytoplasmic costimulatory signaling domain is CD28, and the cytoplasmic T cell receptor signaling domain is CD3 zeta; The artificial immune receptor of any one of claims 1 to 10.

12. A nucleic acid encoding an artificial immune receptor according to any one of claims 1 to 11.

13. A vector comprising the nucleic acid of claim 12.

14. A host cell comprising the nucleic acid of claim 12 or the vector of claim 13, or expressing an artificial immune receptor of any one of claims 1 to 11.

15. An artificial immune receptor according to any one of claims 1 to 11 or a host cell according to claim 14 for use in medicine.

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