Artificial immune receptors

Abstract

The present disclosure provides a novel polypeptide, a so-called artificial immune receptor or AIR, which is a novel tool for cell therapy. Regulatory T cells expressing AIR sense these environmental signals and translate them into a Treg TCR-like activation procedure, enabling Treg cells to achieve their inhibition and tissue protection functions, independent of the specific TCR-or CAR-antigens and independent of the endogenous TCR-MHC restriction.

Description

Background Art

[0001] Regulatory T cells (Treg) are key T cell populations with various functions in vivo. Treg promotes tolerance to self-antigens, allergens, and commensals, thereby limiting the autoreactivity and excessive inflammation of immune cells. Mutations in the Treg master transcription factor Foxp3 lead to lethal multi-organ autoinflammation in mice and humans ((Nat Genet, 2001.27 (1): 18-20; Nat Genet, 2001.27 (1): 68-73). In the past few years, it has become apparent that Treg plays another role in protecting tissue homeostasis and tissue regeneration (Immunology, 2020.161 (1): 4-17). Based on these facts, Treg can be considered as a promising existing drug for autoimmune disorders, and clinical trials have demonstrated the safety and efficacy of Treg-based cell therapy (Front Immunol, 2019.10: 43; Science, 2018.362 (6411): 154-155).

[0002] The new concept of applying chimeric antigen receptor (CAR) technology to Treg is the next step to enhance the efficacy of adoptive Treg cell therapy (Nat Rev Drug Discov, 2019.18(10):749-769). Preclinical studies have demonstrated the superiority of engineered Tregs with CAR-directed (self-)antigen specificity over Tregs with only natural polyclonal TCR lineage in eliminating allogeneic immune 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-Treg has also been effectively used 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, in many cases, the involved autoantigens that can be used as potential targets for CAR-Treg are unknown, or affect multiple organs and tissues in the absence of consistent antigens. In contrast, inflammatory mediators show high redundancy in many inflammatory diseases and are functionally important 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 interventions activated by TNF receptors (TNFRs) are important treatment options for many inflammatory diseases (Trends Immunol, 2012.33 (3): 144-52). These considerations guide us to develop new concepts for engineering Treg cell therapy and generate artificial immune receptors (AIR) that target these inflammatory mediators rather than tissue-specific antigens.

[0004] We are committed to the target of TNF family and select the receptors of ligands LIGHT and Lα1β2, TNFα and TL1A, because these cytokines have pleiotropic effects in many autoimmune diseases. Preclinical disease models and patient data show that LIGHT and Lα1β2 signaling through the corresponding receptors HVEM and LTBR enhance 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 key involvement of this receptor-ligand system in the induction and maintenance of chronic inflammation in IBD, arthritis and EAE. In addition, patient data and genome-wide association studies point to the fundamental influence 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 function is manifested in a variety of diseases, including ankylosing spondylitis, IBD, rheumatoid arthritis, psoriasis, systemic lupus erythematosus, and juvenile idiopathic arthritis (reviewed in Crit Rev Immunol, 2019.39(6):439-479).

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

[0006] Certain constructs are known in the art, which share certain features with the constructs of the present invention. For example, WO2021 / 051195 discloses a construct lacking a CD3ζTCR signaling domain. However, additional CD3ζTCR signaling domains provide multiple advantages beyond the common functions of the CD3T cell signaling domains. Due to the presence of the CD3ζ domain, the AIR of the present invention directly exerts their functions on the main signals of the signal cascade. As shown herein, AIR constructs without the CD3ζ domain are non-functional. The constructs of WO 2021 / 051195 are only able to regulate auxiliary signals, but not TCR-like activation of cells. Summary of the invention

[0007] The present disclosure provides a new artificial immune receptor capable of activating regulatory T cells into a Treg TCR-like activation program. This program is independent of specific TCR- or CAR-antigens and also independent of endogenous TCR-MHC restriction.

[0008] Disclosed herein are artificial immune receptors comprising

[0009] a) the extracellular domain of members of the tumor necrosis factor receptor superfamily,

[0010] b) transmembrane domain,

[0011] c) a cytoplasmic co-stimulatory signaling domain, and

[0012] d) Cytoplasmic T cell receptor signaling domain.

[0013] The general concept is illustrated using the extracellular domains of four different tumor necrosis factor receptor superfamily members, namely TNFR2, DR3, LTBR and CD40. In certain embodiments, the transmembrane domain is derived from the same protein as the extracellular domain.

[0014] The artificial immune receptor also comprises a cytoplasmic co-stimulatory signaling domain and a cytoplasmic T cell receptor signaling domain. Any known co-stimulatory signaling domain and T cell receptor signaling domain can be used in the context of the AIR of the present disclosure. Examples are the cytoplasmic co-stimulatory signaling domain of CD28 and the cytoplasmic T cell receptor signaling domain from CD3ζ.

[0015] Particularly preferred artificial immunoreceptors comprise the extracellular domain and transmembrane domain of TNFR2, the cytoplasmic co-stimulatory signaling domain from CD28, and the cytoplasmic T cell receptor signaling domain from CD3ζ. Still other particularly preferred artificial immunoreceptors comprise the extracellular domain and transmembrane domain of DR3, the cytoplasmic co-stimulatory signaling domain from CD28, and the cytoplasmic T cell receptor signaling domain from CD3ζ. Still other particularly preferred artificial immunoreceptors comprise the extracellular domain and transmembrane domain of LTBR, ​​the cytoplasmic co-stimulatory signaling domain from CD28, and the cytoplasmic T cell receptor signaling domain from CD3ζ.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] Also preferred are artificial immune receptors comprising the amino acid sequences SEQ ID NO:47, SEQ ID NO:35 and SEQ ID NO:37. The present disclosure also provides nucleic acids encoding the above artificial immune receptors. The present disclosure also provides vectors comprising the nucleic acids. The present disclosure also provides host cells comprising the nucleic acids, the vectors and host cells expressing the artificial immune receptors of the present disclosure.

[0020] The present disclosure also provides artificial immune receptors for use in medicine. In certain embodiments, the use in medicine is to treat cancer or inflammation. In other embodiments, the use in medicine is to treat autoimmunity. In other embodiments, the use in medicine is to treat graft-versus-host disease or solid organ transplantation.

[0021] definition

[0022] The term "cell" as used herein includes a single cell as well as a plurality of cells.

[0023] The term "host cell" as used herein refers to a cell comprising a nucleic acid and / or a vector. In the context of the artificial immune receptors disclosed herein, the term host cell refers to a cell comprising a nucleic acid and / or a vector encoding AIR. Such host cells will express AIR on the cell surface and are suitable for use as a drug. Preferred host cells of the present invention are eukaryotic host cells, such as immune cells.

[0024] 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 known as T lymphocytes, can be distinguished from other lymphocytes such as B cells and natural killer cells by the presence of a T cell receptor (TCR) on the cell surface. There are multiple 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 cell is an engineered T cell.

[0025] The term "regulatory T cells" or "Treg" as used herein refers to a T cell subset that regulates the immune system, maintains tolerance to self-antigens, and eliminates autoimmune diseases. These cells typically inhibit or downregulate the induction and proliferation of effector T cells. Treg cells are long-lived cells that suppress excessive or uncontrolled immune responses in vivo in a dominant and antigen-specific manner. Genetic mutations in Forkhead box protein 3 (FoxP3), a key transcription factor required for Treg cell differentiation, lead to severe autoimmunity. In fact, studies in a variety of animal models have shown that Treg 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 to control allogeneic immune responses in the context of GVHD, together with organ and cell transplantation.

[0026] The term "engineered TCR" or "engineered T cell receptor" means any TCR modified from its naturally occurring form. Engineered TCRs can modify (including replacing any of the above chains) alpha chains and / or beta chains or gamma chains and / or delta chains, which enables TCRs to recognize specific antigens (e.g., new antigens). Engineered TCRs can modify any CD3 subunit (e.g., CD3a, as in the case of TRuC receptors), including adding antigen recognition domains (e.g., antibodies, scFv, DARPin). Engineered TCRs can have antigen recognition domains (e.g., antibodies, scFv, DARPin) connected to transmembrane domains of alpha chains and / or beta chains, or gamma chains and / or delta chains.

[0027] As used herein, the terms "polynucleotide" and / or "nucleic acid sequence" and / or "nucleic acid" refer to a sequence of nucleoside or nucleotide monomers consisting of a base, a sugar, and an intermediate sugar (backbone) bond. The term includes DNA and RNA, and may be double-stranded or single-stranded, and represents a sense strand or an antisense strand. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequence of the present application may be a deoxyribonucleic acid sequence (DNA) or a ribonucleic acid sequence (RNA), and may include naturally occurring bases, including adenine, guanine, cytosine, thymine, and uracil. These sequences may also include modified bases. Examples of such modified bases include aza and deazaadenine, guanine, cytosine, thymine, and uracil; as well as xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed by laboratory methods of genetic recombination, or obtained by chemical synthesis or other known protocols for producing nucleic acids.

[0028] 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 substantially free of chemical precursors or other chemicals when chemically synthesized.

[0029] As used herein, the term "recombinant nucleic acid" or "engineered nucleic acid" refers to a nucleic acid or polynucleotide not found in a biological organism. For example, a recombinant nucleic acid can be formed by laboratory methods of genetic recombination (such as molecular cloning) to produce a sequence that cannot be found in nature by other means. Recombinant nucleic acids can also be produced by chemical synthesis or other known protocols for producing nucleic acids. As will be understood by those skilled in the art, unless otherwise indicated, the definitions and embodiments described in this section and other sections are intended to apply to all embodiments and aspects of the present application described herein to which they apply.

[0030] The term "polypeptide" or "protein" as used herein describes an amino acid chain. Polypeptides or proteins of the present disclosure may be peptides, which generally describe an amino acid chain of 2 to about 30 amino acids. The term protein as used herein also describes an amino acid chain with more than 30 amino acids, and may be a fragment or domain or full-length protein of a protein. In addition, as used herein, the term protein may refer to a linear chain of amino acids, or it may refer to an amino acid chain that has been processed and folded into a functional protein. However, it should be understood that 30 is an arbitrary number for distinguishing between peptides and proteins, and these terms may be used interchangeably for amino acid chains. Proteins of the present disclosure may be obtained recombinantly by isolating and purifying proteins from cells, by enzymatic (e.g., proteolysis) cutting and / or by expressing nucleic acids encoding proteins of the present disclosure or fragments. Proteins of the present disclosure and / or fragments may also be obtained by chemical synthesis or other known schemes for producing proteins and fragments.

[0031] 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 substantially free of chemical precursors or other chemicals when chemically synthesized.

[0032] The term "vector" as used herein refers to a polynucleotide that can be used to deliver nucleic acid to the interior of a cell. In one embodiment, a vector is an expression vector comprising an expression control sequence (e.g., a promoter) operably linked to a nucleic acid to be expressed in a cell. Vectors known in the art include, but are not limited to, plasmids, bacteriophages, cosmids, and viruses.

[0033] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

[0034] As used herein, in some embodiments, the terms "treat", "treatment", and the like refer to the administration of an agent or the performance of a procedure for the purpose of obtaining an effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of affecting a partial or complete cure of a disease and / or symptoms of a disease. The term includes the treatment of a disease or condition (e.g., inflammation) in a mammal, particularly a human, and includes: (a) preventing the disease or disease symptoms from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease (e.g., including a disease that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e., preventing its development; and (c) alleviating the disease, i.e., causing the disease to regress. The treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a physician's examination. Thus, the term "treatment" includes the administration of a compound or agent of the invention to prevent, delay, alleviate, prevent or inhibit the development of symptoms or conditions associated with the disease (e.g., inflammation).

[0035] 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.

[0036] 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 ligands of tumor necrosis factor superfamily members (TNFSF) via the extracellular domain. There are many members of the TNFR superfamily, including TNFR1, TNFR2, Fas, DR4, DR5, DR3, DR6, EDAR, XEDAR, TROY LTBR, ​​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).

[0037] 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-α (TNFα). TNFR1 activates the transcription factor NF-kB, mediates apoptosis, and functions as a regulator of inflammation.

[0038] Tumor necrosis factor receptor 2 (NCBI Entrez gene: 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 (TNFa).

[0039] The Fas receptor (NCBI Entrez Gene: 355, also known as Fas, FasR, apoptosis antigen 1, APO-1, APT, CD95, tumor necrosis factor receptor superfamily member 6 or TNFRSF6) is a protein encoded by the FAS gene in humans. Multiple splice variants of Fas have been identified, which are translated into seven isoforms of the protein. The Fas receptor that induces apoptosis is called isoform 1 and is a type 1 transmembrane protein. Many other isoforms are rare haplotypes that are often associated with disease states. Any suitable isoform of Fas is contemplated for use in the embodiments disclosed herein.

[0040] 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 TNFRSF 10A) is a cell surface receptor of the TNF-receptor superfamily that binds TRAIL and mediates apoptosis.

[0041] 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.

[0042] 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).

[0043] 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-κΒ pathways.

[0044] Ectodermal dysplasia 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 a key role in the process of ectodermal differentiation.

[0045] Ectodermal dysplasia receptor 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 ectodermal dysplasia encoded by the anhidrotic ectodermal dysplasia (EDA) gene.

[0046] 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 members of the TNF receptor-associated factor (TRAF) family and activate the c-Jun N-terminal kinase (JNK) signaling pathway when overexpressed in cells. This receptor is able to induce apoptosis through a caspase-independent mechanism and is thought to play an essential role in embryonic development.

[0047] 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 types of receptors for neurotrophins, a family of protein growth factors that stimulate neuronal cell survival and differentiation.

[0048] LTBR (NCBI Entrez Gene: 4055, also known as lymphotoxin beta receptor, TNFRSF3, TNFCR, tumor necrosis factor C receptor of TNFR3) plays a role in signaling during development of lymphoid and other organs, lipid metabolism, immune responses, and programmed cell death.

[0049] CD40 (NCBI Entrez Gene: 958, also known as Bp50, tumor necrosis factor receptor superfamily member 5 or TNFRSF5) is a co-stimulatory protein found on antigen presenting cells and is essential for their activation. H CD154 (CD40L) on cells, thereby activating antigen-presenting cells and inducing multiple downstream effects.

[0050] CD30 (NCBI Entrez Gene: 943, also known as D1S166E, tumor necrosis factor receptor superfamily member 8, TNFRSF8, CD30L receptor, or KI-1) is expressed by activated but not by resting T and B cells and interacts with TRAF2 and TRAF5.

[0051] 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 multiple receptors. BAFF is expressed in cells of the B cell lineage and acts as a B cell activator. It has also been shown to play an important role in the proliferation and differentiation of B cells.

[0052] 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 its receptor, plays an important role in B cell development.

[0053] As used in this article, the term "costimulatory molecule" or "costimulatory receptor" refers to a cognate binding partner on a T cell, which specifically binds to a costimulatory ligand, thereby mediating a costimulatory response in a 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 in 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 to TLR9 receptors, IL-2, IL-7 and IL-15 receptors.

[0054] As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to a domain of a costimulatory molecule or costimulatory receptor that is responsible for mediating a costimulatory response by a T cell. An intracellular signaling domain may comprise the entire intracellular portion of a molecule or functional fragment or derivative thereof from which it is derived or the entire native intracellular signaling domain.

[0055] As used herein, the term "T cell receptor signaling domain" or "TCR signaling domain" refers to a cytoplasmic signaling sequence that acts in a stimulatory manner to induce immune effector function. In some embodiments, the TCR signaling domain comprises 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 CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. A preferred TCR signaling domain is a TCR signaling domain selected from CD3ζ, CD3γ, CD3δ, and CD3ε. Particularly preferred TCR signaling domains are CD3ζ, CD3γ, CD3δ, and CD3ε.

[0056] The term "artificial immune receptor" or "AIR" refers to the novel molecules of the present disclosure. AIR comprises the extracellular domain of a tumor necrosis factor receptor superfamily member, a transmembrane domain, a cytoplasmic co-stimulatory signaling domain, and a cytoplasmic T cell receptor signaling domain.

[0057] In the context of a polypeptide or polypeptide domain, the terms "is", "is", "derived from" and "derived from" refer to the amino acid sequence of the polypeptide or polypeptide domain and indicate that the amino acid sequence is identical to the native form of the polypeptide or polypeptide domain, or to a variant of the polypeptide or polypeptide domain that is in a functionally indistinguishable form from the native form of the polypeptide or polypeptide domain.

[0058] As used herein, the term "P2A" refers to a peptide sequence capable of self-cleavage. P2A belongs to a family of 2A peptides, all of which are capable of self-cleavage. "A peptides share a common core sequence motif. Any 2A peptide can be used instead 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.

[0059] 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.

[0060] The terms "GVHD," "GvHD," and "graft versus host disease" refer to a disease commonly associated with bone marrow and stem cell transplants that is characterized by inflammation. White blood cells from the donor's immune system remain within the donated tissue or cells (the graft) and are recognized as non-self by the recipient (the host). BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 It is a schematic diagram of AIR design. The structure of the top (A) shows AIR according to the present disclosure. It consists of an extracellular binding domain, a transmembrane domain and two intracellular domains (a co-stimulatory domain and a T cell receptor signaling domain) of a member of the tumor necrosis factor (TNF) superfamily. The AIR fusion protein is connected to a modified CD90.1 via a P2A peptide cleavage site, and the modified CD90.1 serves as a reporter gene for transduction efficacy. P2A and CD90.1 are not part of AIR. These constructs are cloned into a mouse stem cell leukemia virus (MSCV), which serves as a vector and mediates the stable integration of the gene of interest into T cell DNA. The constructs labeled B, C, D and G are exemplary AIR according to the present disclosure. Constructs E and F are control constructs. Construct E lacks an extracellular binding domain, and construct F contains an extracellular anti-CD19 single-chain antibody (scFv) instead of a binding domain of a member of the tumor necrosis factor (TNF) superfamily.

[0062] Figure 2 The mode of action of AIR according to the present disclosure is shown. The TNF family members bound to the membrane via AIR cause the cytokine signal to be translated into a T cell receptor (TCR)-like signal. This initiates T cell activation in an environment / inflammation-dependent manner independent of its endogenous TCR-MHC restriction.

[0063] Figure 3 Efficient transduction and expression of AIR according to the present disclosure are shown. Murine regulatory T cells (Treg) were isolated from the spleen and lymph nodes of C57 / BL6 mice, sorted (natural CD4+CD25+CD62L+Treg or Foxp3+ reporter gene (hCD2+) positive) and stimulated to expand for 6 days via beads coupled to anti-CD3 and anti-CD28 antibodies plus IL-2 (2000U / ml). The purity of Treg cultures was determined by flow cytometry. Treg cultures contain about 90%-99% Foxp3+T cells. On the second day after separation, Treg was transduced with AIR constructs or anti-CD19 chimeric antigen receptors (α-CD19 CAR) as controls. Three days later, the transduction efficacy and surface expression of AIR stained by CD90.1 were examined by flow cytometry.

[0064] Figure 4 Surface expression of AIR according to the present disclosure is shown. Sub-figures A, B and C show the results of AIR carrying the extracellular domains of LTBR, ​​DR3 and TNFR2, respectively. In each sub-figure, AIR is compared with untransduced cells and with cells carrying extracellular anti-CD19 single-chain antibodies (scFv).

[0065] Figure 5 The scientific rationale for the experiments in Example 6 is depicted. Nr4a1 is a well-known target gene for T cell receptor signaling. It was investigated whether AIR of the present disclosure can induce Nr4a1 upregulation in a manner similar to TCR signaling. Tregs from Nr4a1-eGFP reporter mice were used. As a control, Tregs were stimulated with beads coupled to anti-CD3 / CD28 antibodies.

[0066] Figure 6 : Tregs expressing AIR were stimulated overnight and co-cultured with HEK cells expressing mouse Light (mLight), mTL1A or mTNF. As a positive control, Tregs were stimulated with beads coupled to anti-CD3 / CD28 antibodies to simulate TCR signaling. The left side shows the Nr4a1-eGFP expression of AIR positive (CD90.1+) Tregs, and the right sub-figure shows the Nr4a1-eGFP expression of AIR negative (CD90.1-) Tregs.

[0067] Figure 7 The expression of Nr4a1 is shown when an AIR according to the present disclosure (exemplified by an AIR carrying the extracellular domain of LTBR) is administered. Tregs expressing an LTBR AIR according to the present disclosure or an unrelated CAR carrying an anti-CD19 single-chain antibody (scFv) extracellularly were co-cultured with mouse EL4 cells known to express Light in the absence or presence of LTBR-Ig fusion protein (50ug / ml) blocking mLight, and then stained for intracellular Nr4a1 expression.

[0068] Figure 8 The pre-gating strategy for CD90.1+ cells of LTBR AIR and extracellular unrelated CAR carrying anti-CD19 single-chain antibody (scFv) according to the present disclosure is shown. Tregs expressing the construct were left to rest for 24h and then co-cultured with HEK cells or HEK cells expressing mLight for 18h. Pre-gating of CD90.1+ cells is shown.

[0069] Fig. 9 Shows Figure 8Sorting of pre-gated Tregs as indicated. Cells were sorted on CD4+CD25+CD90.1+ (left and middle) or CD4+CD25+CD90.1+Nr4a1+LAP+ (right).

[0070] Fig.10 Depicted is RNA expression analysis of transcripts differentially expressed after stimulation with LTPR AIR according to the present disclosure (LTPR AIR Tregs stimulated with HEK-mLight versus α-CD19 CAR Tregs stimulated with HEK-mLight). Figure 8 and Fig. 9 And the sorted cells as outlined in Example 7. Then, RNA was isolated and RNA sequencing was performed. Shown on the left is a summary of up-regulated and down-regulated genes comparing α-CD19 CAR Treg stimulated with HEK-mLight relative to LTBR AIR Treg stimulated with HEK cells (as a control) (endogenous mLight signaling) and LTBR AIR Treg stimulated with HEK-mLight relative to α-CD19 CAR Treg stimulated with HEK-mLight (specific LTBR AIR signaling). Shown on the right is a volcano plot showing differentially expressed transcripts.

[0071] Fig.11 Similar to Fig.10 , with DR3 AIR according to the present disclosure. DR3 AIR Tregs stimulated with HEK cells (endogenous mTL1A signaling). DR3 AIR Tregs stimulated with HEK-mTL1A versus 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.

[0072] Fig.12 Also similar to Fig.10 , comparing α-CD19 CAR Tregs stimulated with anti-CD3 / CD28 beads versus unstimulated Tregs. Differentially expressed genes are shown on the left and the volcano plot is shown on the right.

[0073] Fig.13 Shown are RNA expression data for Tnfrsf9, Tigit, Tgfb1, Cd69, Ccr8, and Nr4a1 from Tregs expressing LTBR AIR or control CAR after 18 h of co-culture with HEK+ / -mLight (Deseq2, n=3).

[0074] Fig.14Representative flow cytometry analysis of Tregs expressing LTBR AIR or control CAR after 18 hours of co-culture with HEK+ / -mLight is shown. 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. Data are representative of three independent experiments.

[0075] Fig.15 Shown are RNA expression data for Tnfrsf9, Tigit, Tgfb1 from Tregs expressing DR3 AIR or control CAR after 18 h of co-culture with HEK+ / -mLight (Deseq2, n=3).

[0076] Fig.16 Representative flow cytometry analysis of Tregs expressing DR3AIR or control CAR after 18 hours of co-culture with HEK+ / -mTL1A is shown. Protein expression of CD137 and Tigit is shown in the upper panel, and CD69 and LAP (Tgfβ1) are shown in the lower panel. Data are representative of three independent experiments.

[0077] Fig.17 RNA expression data (upper left, Deseq2, n=3) and protein expression (right) for Irf8 from Tregs expressing LTBR AIR or control CAR after 18 h of co-culture with HEK+ / -mLight are shown. RNA expression data (lower left, Deseq2, n=3) and protein expression (right) for Irf8 from Tregs expressing DR3 AIR or control CAR after 18 hours of co-culture with HEK+ / -mTL1A.

[0078] Fig.18 AIR of the present disclosure mediates Treg activation and proliferation. Tregs expressing LTBR AIR or control CAR were left to rest for 24 hours and labeled with CFDA-SE proliferation dye. Engineered and labeled Tregs were co-cultured with HEK+ / -mLight in the presence of IL2 for 72 hours and then analyzed for proliferation by flow cytometry. Representative dot plots are shown on the left. Summary data (n=3) from three experiments performed in three technical replicates are shown.

[0079] Fig.19 A schematic diagram of the (major MHC mismatch) graft-versus-host disease (GvHD) model is shown. Briefly, FACS-sorted Tregs from Foxp3-hCD2 reporter mice were expanded and co-transfected with LTBR AIR or a truncated construct (lacking the extracellular binding domain, e.g. Figure 1 a) was used as a control.5 Engineered Tregs and 2.5x10 from C57 / BL6 mice 6 Bone marrow cells and 2.5x10 5 The spleen cells were transplanted together into lethally irradiated Balb / c mice. As a transplantation control, one group received bone marrow cells (BM) alone, and as a disease development control, one group received bone marrow cells plus spleen cells. After transplantation, the animals were monitored for 47 days.

[0080] Fig. 20 Results of quality control of engineered Tregs are shown. Tregs were stained for CD4, hCD2 (reporter gene for Foxp3), as well as CD90.1 and LTBR before transfer into mice.

[0081] Fig.21 Kaplan-Meier curves showing the survival of transplanted Balb / c mice treated with LTBR AIR and corresponding control constructs are shown. The figure contains data sets from two independent experiments (log-rank test, n=11-12).

[0082] Fig. 22 The mean GvHD score for each group is shown, animals reaching a score of 40 had to be euthanized (animals that died kept the highest score at subsequent time points for statistical analysis, two-way ANOVA).

[0083] Fig.23 Representative FACS images of spleens from surviving animals that received LTBR AIR Tregs are shown on the left. Shown on the right is the percentage of Foxp3+ Tregs in spleens of surviving mice at day 47.

[0084] Fig.24 Analysis of Klrg1+Tregs in the spleen is shown. Left: Representative FACS plots of differentiated CD45.1+ (BM-derived) and CD45.2+ (transferred AIR Tregs) Treg cells. Expression of Klrg1 (a marker for tissue-specific phenotype) and hCD2 (Foxp3 reporter gene) is shown. Middle: 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, survivors n=8 vs. dead n=4).

[0085] Fig.25Representative FACS analysis of digested colon from surviving animals receiving LTBR AIR Tregs is shown. The distinction between CD45.1 and CD45.2 cells within the CD4+ population is shown on the left. CD45.2 Tregs were further analyzed for Klrg1 and hCD2 expression on the right.

[0086] Fig.26 On the left are shown the percentage of hCD2+ (indicating Foxp3+) cells among the transferred engineered CD45.2+ cells, demonstrating in vivo phenotypic stability over 47 days. On the right are cell counts of transferred CD45.2 Tregs in the colon at day 47.

[0087] Fig. 27 Show that people LTBR AIR is effectively transduced and expresses AIR on the cell surface. Human T cells from healthy blood donors are sorted on typical Treg protein markers (TCRb+CD4+CD25+CD127-CD45RA+), and stimulated with anti-CD3 / CD28 (TransACT, Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-2 (500U / ml) for 7 days. On the 2nd day after separation, Tregs were transduced with people LTBR AIR constructs (using the double tropism form of the retroviral expression system MSCV). After 5 days, for intracellular Foxp3+, cultured Tregs were stained to check Treg purity and CD90.1 and hLTBR, ​​to analyze the transduction efficacy and surface expression of AIR protein.

[0088] Fig.28 It is shown that human LTBR AIR has the expected signal transduction ability. Tregs expressing hLTBR AIR or irrelevant CAR (anti-CEA, carcinoembryonic antigen) were co-cultured with parental HEK cells or HEK cells expressing human Light protein on the surface for 18h. The expression of Ccr8, glycoprotein A repeat dominant protein (GARP), CD137 (4-1BB) and Tigit was shown in CD90.1+Foxp3+Treg.

[0089] Fig.29 At the top, the LTBR "AIR" lacking the CD3ζ chain is shown. At the bottom, flow cytometry data are shown, which confirm that the construct is surface expressed in Tregs.

[0090] Fig.30Representative flow cytometry analysis of Tregs expressing full-length LTBR AIR and LTBR "AIR" lacking the CD3ζ chain after 18 h of co-culture with HEK + / -mLight is shown. Protein expression of CD137 and Tigit is shown in the upper panel, CD69 and LAP (membrane-bound ) is shown in the figure below.

[0091] Fig.31 The expression of TIGIT and CD137 is shown. Treg cells expressing CD40 AIR or containing constructs expressing anti-CD19 CAR were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 h. The expression of TIGIT and CD137 was measured by flow cytometry.

[0092] Cells

[0093] Fig.32 Summarizes Fig.31 Three independent experiments shown. Differences were statistically significant as calculated via one-way Anova test.

[0094] Fig.33 The expression of LAP (TGFb1) and CD69 is shown. Treg cells expressing CD40 AIR or containing constructs expressing anti-CD19 CAR were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 h. The expression of LAP (TGFb1) and CD69 was measured by flow cytometry.

[0095] Fig.34 Summarizes Fig.33 Three independent experiments shown. Differences were statistically significant as calculated via one-way Anova test.

[0096] Fig.35 Treg cells from Nr4a1-eGFP reporter mice expressing CD40AIR were shown to trigger the expression of Nr4a1, an early response gene for TCR signaling.

[0097] Fig.36 Shown are schematics of additional AIRs generated and tested in this disclosure. Fig.36 A shows CD40 AIR with ICOS costimulatory domain, Fig.36 B shows CD40 AIR with a 4-1BB co-stimulatory domain.

[0098] Fig.37 It was shown that CD40 AIR with ICOS or 41BB co-stimulatory domains is efficiently expressed on the surface of regulatory T cells.

[0099] Fig.38 It was shown that all tested CD40 AIR, ie AIR with CD28, ICOS and 41BB co-stimulatory domains, had the ability to induce Treg cell activation.

[0100] Fig.39 Quality control of Teg cells before transplantation is shown. Both constructs, CD40AIR and control construct, showed high transduction efficiency, but only cells transduced with CD40AIR showed binding to CD40 via its CD40 receptor extracellular domain.

[0101] Fig.40 Shown are Kaplan-Meier curves showing the survival of transplanted Balb / c mice treated with CD40 AIR and corresponding control constructs. The figure contains data sets from two independent experiments (log-rank test, n=10-12).

[0102] Fig.41 The importance of CD3 for the constructs of the invention is shown. Only the full-length LTBR AIR containing the CD3 zeta domain, but not the other two constructs tested, was able to induce Nr4a1.eGFP upregulation in response to the corresponding ligand mLIGHT. DETAILED DESCRIPTION

[0103] Artificial immune receptors

[0104] The artificial immunoreceptors disclosed herein are new and superior tools that enable regulatory T cells to achieve their suppressive and tissue protective functions independently of specific TCR- or CAR-antigens and independently of endogenous TCR-pMHC (peptide-loaded MHC) restriction.

[0105] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0106] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0107] b) transmembrane domain,

[0108] c) a cytoplasmic co-stimulatory signaling domain, and

[0109] d) Cytoplasmic T cell receptor signaling domain.

[0110] In certain embodiments, the present disclosure relates to an artificial immune receptor consisting of:

[0111] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0112] b) transmembrane domain,

[0113] c) a cytoplasmic co-stimulatory signaling domain, and

[0114] d) Cytoplasmic T cell receptor signaling domain.

[0115] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0116] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0117] b) transmembrane domain,

[0118] c) a cytoplasmic co-stimulatory signaling domain, and

[0119] d) a cytoplasmic T cell receptor signaling domain,

[0120] The tumor necrosis factor receptor superfamily member 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.

[0121] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0122] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0123] b) transmembrane domain,

[0124] c) a cytoplasmic co-stimulatory signaling domain, and

[0125] d) a cytoplasmic T cell receptor signaling domain,

[0126] The tumor necrosis factor receptor superfamily member is selected from TNFR2, DR3, LTBR and CD40.

[0127] In certain embodiments, the present disclosure relates to an artificial immune receptor consisting of:

[0128] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0129] b) transmembrane domain,

[0130] c) a cytoplasmic co-stimulatory signaling domain, and

[0131] d) a cytoplasmic T cell receptor signaling domain,

[0132] The tumor necrosis factor receptor superfamily member 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.

[0133] In certain embodiments, the present disclosure relates to an artificial immune receptor consisting of:

[0134] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0135] b) transmembrane domain,

[0136] c) a cytoplasmic co-stimulatory signaling domain, and

[0137] d) a cytoplasmic T cell receptor signaling domain,

[0138] The tumor necrosis factor receptor superfamily member is selected from TNFR2, DR3, LTBR and CD40.

[0139] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0140] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0141] b) transmembrane domain,

[0142] c) a cytoplasmic co-stimulatory signaling domain, and

[0143] d) a cytoplasmic T cell receptor signaling domain,

[0144] The transmembrane domain is derived from the same protein as the extracellular domain.

[0145] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0146] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0147] b) transmembrane domain,

[0148] c) a cytoplasmic co-stimulatory signaling domain, and

[0149] d) a cytoplasmic T cell receptor signaling domain,

[0150] wherein the transmembrane domain and the extracellular domain are derived from the same protein.

[0151] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0152] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0153] b) transmembrane domain,

[0154] c) a cytoplasmic co-stimulatory signaling domain, and

[0155] d) a cytoplasmic T cell receptor signaling domain,

[0156] The tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from TNFR2.

[0157] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0158] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0159] b) transmembrane domain,

[0160] c) a cytoplasmic co-stimulatory signaling domain, and

[0161] d) a cytoplasmic T cell receptor signaling domain,

[0162] The artificial immune receptor comprises SEQ ID NO:39.

[0163] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0164] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0165] b) transmembrane domain,

[0166] c) a cytoplasmic co-stimulatory signaling domain, and

[0167] d) a cytoplasmic T cell receptor signaling domain,

[0168] The artificial immune receptor comprises SEQ ID NO:38.

[0169] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0170] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0171] b) transmembrane domain,

[0172] c) a cytoplasmic co-stimulatory signaling domain, and

[0173] d) a cytoplasmic T cell receptor signaling domain,

[0174] The artificial immune receptor consists of SEQ ID NO:38.

[0175] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0176] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0177] b) transmembrane domain,

[0178] c) a cytoplasmic co-stimulatory signaling domain, and

[0179] d) a cytoplasmic T cell receptor signaling domain,

[0180] The tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from DR3.

[0181] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0182] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0183] b) transmembrane domain,

[0184] c) a cytoplasmic co-stimulatory signaling domain, and

[0185] d) a cytoplasmic T cell receptor signaling domain,

[0186] The artificial immune receptor comprises SEQ ID NO:41.

[0187] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0188] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0189] b) transmembrane domain,

[0190] c) a cytoplasmic co-stimulatory signaling domain, and

[0191] d) a cytoplasmic T cell receptor signaling domain,

[0192] The artificial immune receptor comprises SEQ ID NO:40.

[0193] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0194] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0195] b) transmembrane domain,

[0196] c) a cytoplasmic co-stimulatory signaling domain, and

[0197] d) a cytoplasmic T cell receptor signaling domain,

[0198] The artificial immune receptor consists of SEQ ID NO:40.

[0199] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0200] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0201] b) transmembrane domain,

[0202] c) a cytoplasmic co-stimulatory signaling domain, and

[0203] d) a cytoplasmic T cell receptor signaling domain,

[0204] Wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from LTBR.

[0205] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0206] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0207] b) transmembrane domain,

[0208] c) a cytoplasmic co-stimulatory signaling domain, and

[0209] d) a cytoplasmic T cell receptor signaling domain,

[0210] The artificial immune receptor comprises SEQ ID NO:33.

[0211] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0212] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0213] b) transmembrane domain,

[0214] c) a cytoplasmic co-stimulatory signaling domain, and

[0215] d) a cytoplasmic T cell receptor signaling domain,

[0216] The artificial immune receptor comprises SEQ ID NO:31.

[0217] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0218] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0219] b) transmembrane domain,

[0220] c) a cytoplasmic co-stimulatory signaling domain, and

[0221] d) a cytoplasmic T cell receptor signaling domain,

[0222] The artificial immune receptor consists of SEQ ID NO:31.

[0223] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0224] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0225] b) transmembrane domain,

[0226] c) a cytoplasmic co-stimulatory signaling domain, and

[0227] d) a cytoplasmic T cell receptor signaling domain,

[0228] The tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from CD40.

[0229] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0230] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0231] b) transmembrane domain,

[0232] c) a cytoplasmic co-stimulatory signaling domain, and

[0233] d) a cytoplasmic T cell receptor signaling domain,

[0234] The artificial immune receptor comprises SEQ ID NO:46.

[0235] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0236] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0237] b) transmembrane domain,

[0238] c) a cytoplasmic co-stimulatory signaling domain, and

[0239] d) a cytoplasmic T cell receptor signaling domain,

[0240] The artificial immune receptor comprises SEQ ID NO:47.

[0241] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0242] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0243] b) transmembrane domain,

[0244] c) a cytoplasmic co-stimulatory signaling domain, and

[0245] d) a cytoplasmic T cell receptor signaling domain,

[0246] The artificial immune receptor consists of SEQ ID NO:47.

[0247] co-stimulatory signaling domain

[0248] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0249] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0250] b) transmembrane domain,

[0251] c) a cytoplasmic co-stimulatory signaling domain, and

[0252] d) Cytoplasmic T cell receptor signaling domain.

[0253] wherein the cytoplasmic co-stimulatory signaling domain is selected from 4-IBB (CD137), BAFFR, OX40, CD27, CD28, ICOS, CD40, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY and TWEAK.

[0254] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0255] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0256] b) transmembrane domain,

[0257] c) a cytoplasmic co-stimulatory signaling domain, and

[0258] d) Cytoplasmic T cell receptor signaling domain.

[0259] Wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0260] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0261] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0262] b) transmembrane domain,

[0263] c) a cytoplasmic co-stimulatory signaling domain, and

[0264] d) Cytoplasmic T cell receptor signaling domain.

[0265] Wherein the cytoplasmic co-stimulatory signaling domain is derived from CD28.

[0266] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0267] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0268] b) transmembrane domain,

[0269] c) a cytoplasmic co-stimulatory signaling domain, and

[0270] d) Cytoplasmic T cell receptor signaling domain.

[0271] wherein the cytoplasmic co-stimulatory signaling domain comprises SEQ ID NO:35.

[0272] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0273] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0274] b) transmembrane domain,

[0275] c) a cytoplasmic co-stimulatory signaling domain, and

[0276] d) Cytoplasmic T cell receptor signaling domain.

[0277] wherein the cytoplasmic co-stimulatory signaling domain consists of SEQ ID NO:35.

[0278] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0279] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0280] b) transmembrane domain,

[0281] c) a cytoplasmic co-stimulatory signaling domain, and

[0282] d) a cytoplasmic T cell receptor signaling domain,

[0283] wherein the transmembrane domain is derived from the same protein as the extracellular domain, and

[0284] wherein the cytoplasmic co-stimulatory signaling domain is selected from the group consisting of CD137, BAFFR, OX40, CD27, CD28, CD40, ICOS, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY and TWEAK.

[0285] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0286] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0287] b) transmembrane domain,

[0288] c) a cytoplasmic co-stimulatory signaling domain, and

[0289] d) a cytoplasmic T cell receptor signaling domain,

[0290] wherein the transmembrane domain is derived from the same protein as the extracellular domain, and

[0291] Wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0292] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0293] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0294] b) transmembrane domain,

[0295] c) a cytoplasmic co-stimulatory signaling domain, and

[0296] d) a cytoplasmic T cell receptor signaling domain,

[0297] wherein the transmembrane domain is from the same protein as the extracellular domain, and wherein the cytoplasmic co-stimulatory signaling domain comprises SEQ ID NO: 35. In certain embodiments, the present disclosure relates to an artificial immune receptor comprising

[0298] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0299] b) transmembrane domain,

[0300] c) a cytoplasmic co-stimulatory signaling domain, and

[0301] d) a cytoplasmic T cell receptor signaling domain,

[0302] wherein the transmembrane domain is from the same protein as the extracellular domain, and wherein the cytoplasmic co-stimulatory signaling domain consists of SEQ ID NO: 35. In certain embodiments, the present disclosure relates to an artificial immune receptor comprising

[0303] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0304] b) transmembrane domain,

[0305] c) a cytoplasmic co-stimulatory signaling domain, and

[0306] d) a cytoplasmic T cell receptor signaling domain,

[0307] wherein the transmembrane domain and the extracellular domain are from TNFR2, and wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0308] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0309] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0310] b) transmembrane domain,

[0311] c) a cytoplasmic co-stimulatory signaling domain, and

[0312] d) a cytoplasmic T cell receptor signaling domain,

[0313] wherein the transmembrane domain and the extracellular domain are from DR3, and wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0314] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0315] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0316] b) transmembrane domain,

[0317] c) a cytoplasmic co-stimulatory signaling domain, and

[0318] d) a cytoplasmic T cell receptor signaling domain,

[0319] wherein the transmembrane domain and the extracellular domain are from LTBR, ​​and wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0320] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0321] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0322] b) transmembrane domain,

[0323] c) a cytoplasmic co-stimulatory signaling domain, and

[0324] d) a cytoplasmic T cell receptor signaling domain,

[0325] wherein the transmembrane domain and the extracellular domain are from CD40, and wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0326] T cell receptor signaling domain

[0327] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0328] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0329] b) transmembrane domain,

[0330] c) a cytoplasmic co-stimulatory signaling domain, and

[0331] d) a cytoplasmic T cell receptor signaling domain, and

[0332] wherein the cytoplasmic T cell receptor signaling domain is selected from CD3ζ, CD3γ and CD3ε.

[0333] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0334] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0335] b) transmembrane domain,

[0336] c) a cytoplasmic co-stimulatory signaling domain, and

[0337] d) a cytoplasmic T cell receptor signaling domain, and

[0338] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0339] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0340] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0341] b) transmembrane domain,

[0342] c) a cytoplasmic co-stimulatory signaling domain, and

[0343] d) a cytoplasmic T cell receptor signaling domain, and

[0344] The cytoplasmic T cell receptor signaling domain is derived from CD3ζ.

[0345] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0346] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0347] b) transmembrane domain,

[0348] c) a cytoplasmic co-stimulatory signaling domain, and

[0349] d) a cytoplasmic T cell receptor signaling domain, and

[0350] wherein the cytoplasmic T cell receptor signaling domain comprises SEQ ID NO:37.

[0351] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0352] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0353] b) transmembrane domain,

[0354] c) a cytoplasmic co-stimulatory signaling domain, and

[0355] d) a cytoplasmic T cell receptor signaling domain, and

[0356] wherein the cytoplasmic T cell receptor signaling domain consists of SEQ ID NO:37.

[0357] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0358] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0359] b) transmembrane domain,

[0360] c) a cytoplasmic co-stimulatory signaling domain, and

[0361] d) a cytoplasmic T cell receptor signaling domain, and

[0362] wherein the transmembrane domain is derived from the same protein as the extracellular domain, and

[0363] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0364] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0365] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0366] b) transmembrane domain,

[0367] c) a cytoplasmic co-stimulatory signaling domain, and

[0368] d) a cytoplasmic T cell receptor signaling domain, and

[0369] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from TNFR2, and

[0370] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0371] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0372] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0373] b) transmembrane domain,

[0374] c) a cytoplasmic co-stimulatory signaling domain, and

[0375] d) a cytoplasmic T cell receptor signaling domain, and

[0376] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from DR3, and

[0377] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0378] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0379] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0380] b) transmembrane domain,

[0381] c) a cytoplasmic co-stimulatory signaling domain, and

[0382] d) a cytoplasmic T cell receptor signaling domain, and

[0383] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from LTBR, ​​and

[0384] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0385] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0386] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0387] b) transmembrane domain,

[0388] c) a cytoplasmic co-stimulatory signaling domain, and

[0389] d) a cytoplasmic T cell receptor signaling domain, and

[0390] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from CD40, and

[0391] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0392] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0393] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0394] b) transmembrane domain,

[0395] c) a cytoplasmic co-stimulatory signaling domain, and

[0396] d) a cytoplasmic T cell receptor signaling domain, and

[0397] wherein the transmembrane domain is derived from the same protein as the extracellular domain,

[0398] wherein the cytoplasmic co-stimulatory signaling domain is CD28, and

[0399] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0400] Preferred AIR

[0401] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0402] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0403] b) transmembrane domain,

[0404] c) a cytoplasmic co-stimulatory signaling domain, and

[0405] d) a cytoplasmic T cell receptor signaling domain, and

[0406] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from TNFR2,

[0407] wherein the cytoplasmic co-stimulatory signaling domain is CD28, and

[0408] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0409] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0410] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0411] b) transmembrane domain,

[0412] c) a cytoplasmic co-stimulatory signaling domain, and

[0413] d) a cytoplasmic T cell receptor signaling domain, and

[0414] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from DR3,

[0415] wherein the cytoplasmic co-stimulatory signaling domain is CD28, and

[0416] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0417] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0418] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0419] b) transmembrane domain,

[0420] c) a cytoplasmic co-stimulatory signaling domain, and

[0421] d) a cytoplasmic T cell receptor signaling domain, and

[0422] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from LTBR,

[0423] wherein the cytoplasmic co-stimulatory signaling domain is CD28, and

[0424] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0425] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0426] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0427] b) transmembrane domain,

[0428] c) a cytoplasmic co-stimulatory signaling domain, and

[0429] d) a cytoplasmic T cell receptor signaling domain, and

[0430] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from CD40,

[0431] wherein the cytoplasmic co-stimulatory signaling domain is CD28, and

[0432] Wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0433] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0434] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0435] b) transmembrane domain,

[0436] c) a cytoplasmic co-stimulatory signaling domain, and

[0437] d) a cytoplasmic T cell receptor signaling domain, and

[0438] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2 and include SEQ ID NO: 39,

[0439] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO: 35, and

[0440] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0441] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0442] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0443] b) transmembrane domain,

[0444] c) a cytoplasmic co-stimulatory signaling domain, and

[0445] d) a cytoplasmic T cell receptor signaling domain, and

[0446] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2 and consist of SEQ ID NO: 39,

[0447] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0448] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0449] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0450] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0451] b) transmembrane domain,

[0452] c) a cytoplasmic co-stimulatory signaling domain, and

[0453] d) a cytoplasmic T cell receptor signaling domain, and

[0454] Wherein the artificial immune receptor comprises SEQ ID NO:38.

[0455] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0456] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0457] b) transmembrane domain,

[0458] c) a cytoplasmic co-stimulatory signaling domain, and

[0459] d) a cytoplasmic T cell receptor signaling domain, and

[0460] Wherein the artificial immune receptor consists of SEQ ID NO:38.

[0461] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0462] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0463] b) transmembrane domain,

[0464] c) a cytoplasmic co-stimulatory signaling domain, and

[0465] d) a cytoplasmic T cell receptor signaling domain, and

[0466] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from DR3 and consist of SEQ ID NO: 41,

[0467] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0468] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0469] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0470] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0471] b) transmembrane domain,

[0472] c) a cytoplasmic co-stimulatory signaling domain, and

[0473] d) a cytoplasmic T cell receptor signaling domain, and

[0474] Wherein the artificial immune receptor comprises SEQ ID NO:40.

[0475] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0476] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0477] b) transmembrane domain,

[0478] c) a cytoplasmic co-stimulatory signaling domain, and

[0479] d) a cytoplasmic T cell receptor signaling domain, and

[0480] Wherein the artificial immune receptor consists of SEQ ID NO:40.

[0481] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0482] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0483] b) transmembrane domain,

[0484] c) a cytoplasmic co-stimulatory signaling domain, and

[0485] d) a cytoplasmic T cell receptor signaling domain, and

[0486] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from LTBR and consist of SEQ ID NO: 33,

[0487] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0488] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0489] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0490] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0491] b) transmembrane domain,

[0492] c) a cytoplasmic co-stimulatory signaling domain, and

[0493] d) a cytoplasmic T cell receptor signaling domain, and

[0494] Wherein the artificial immune receptor comprises SEQ ID NO:31.

[0495] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0496] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0497] b) transmembrane domain,

[0498] c) a cytoplasmic co-stimulatory signaling domain, and

[0499] d) a cytoplasmic T cell receptor signaling domain, and

[0500] Wherein the artificial immune receptor consists of SEQ ID NO:31.

[0501] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0502] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0503] b) transmembrane domain,

[0504] c) a cytoplasmic co-stimulatory signaling domain, and

[0505] d) a cytoplasmic T cell receptor signaling domain, and

[0506] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from CD40 and consist of SEQ ID NO: 46,

[0507] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0508] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0509] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0510] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0511] b) transmembrane domain,

[0512] c) a cytoplasmic co-stimulatory signaling domain, and

[0513] d) a cytoplasmic T cell receptor signaling domain, and

[0514] Wherein the artificial immune receptor comprises SEQ ID NO:47.

[0515] In certain embodiments, the present disclosure relates to artificial immune receptors comprising

[0516] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0517] b) transmembrane domain,

[0518] c) a cytoplasmic co-stimulatory signaling domain, and

[0519] d) a cytoplasmic T cell receptor signaling domain, and

[0520] Wherein the artificial immune receptor consists of SEQ ID NO:47.

[0521] Nucleic acids, vectors and host cells

[0522] The artificial immune receptors of the present disclosure are encoded by nucleic acids. Thus, in certain embodiments, the present disclosure relates to nucleic acids encoding artificial immune receptors.

[0523] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0524] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0525] b) transmembrane domain,

[0526] c) a cytoplasmic co-stimulatory signaling domain, and

[0527] d) Cytoplasmic T cell receptor signaling domain.

[0528] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0529] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0530] b) transmembrane domain,

[0531] c) a cytoplasmic co-stimulatory signaling domain, and

[0532] d) a cytoplasmic T cell receptor signaling domain, and

[0533] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2 and include SEQ ID NO: 39,

[0534] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO: 35, and

[0535] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0536] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0537] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0538] b) transmembrane domain,

[0539] c) a cytoplasmic co-stimulatory signaling domain, and

[0540] d) a cytoplasmic T cell receptor signaling domain, and

[0541] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2 and consist of SEQ ID NO: 39,

[0542] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0543] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0544] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0545] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0546] b) transmembrane domain,

[0547] c) a cytoplasmic co-stimulatory signaling domain, and

[0548] d) a cytoplasmic T cell receptor signaling domain, and

[0549] Wherein the artificial immune receptor comprises SEQ ID NO:38.

[0550] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0551] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0552] b) transmembrane domain,

[0553] c) a cytoplasmic co-stimulatory signaling domain, and

[0554] d) a cytoplasmic T cell receptor signaling domain, and

[0555] Wherein the artificial immune receptor consists of SEQ ID NO:38.

[0556] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0557] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0558] b) transmembrane domain,

[0559] c) a cytoplasmic co-stimulatory signaling domain, and

[0560] d) a cytoplasmic T cell receptor signaling domain, and

[0561] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from DR3 and include SEQ ID NO: 41,

[0562] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO: 35, and

[0563] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0564] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0565] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0566] b) transmembrane domain,

[0567] c) a cytoplasmic co-stimulatory signaling domain, and

[0568] d) a cytoplasmic T cell receptor signaling domain, and

[0569] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from DR3 and consist of SEQ ID NO: 41,

[0570] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0571] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0572] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0573] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0574] b) transmembrane domain,

[0575] c) a cytoplasmic co-stimulatory signaling domain, and

[0576] d) a cytoplasmic T cell receptor signaling domain, and

[0577] Wherein the artificial immune receptor comprises SEQ ID NO:40.

[0578] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0579] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0580] b) transmembrane domain,

[0581] c) a cytoplasmic co-stimulatory signaling domain, and

[0582] d) a cytoplasmic T cell receptor signaling domain, and

[0583] Wherein the artificial immune receptor consists of SEQ ID NO:40.

[0584] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0585] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0586] b) transmembrane domain,

[0587] c) a cytoplasmic co-stimulatory signaling domain, and

[0588] d) a cytoplasmic T cell receptor signaling domain, and

[0589] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from LTBR and comprise SEQ ID NO: 33,

[0590] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO: 35, and

[0591] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0592] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0593] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0594] b) transmembrane domain,

[0595] c) a cytoplasmic co-stimulatory signaling domain, and

[0596] d) a cytoplasmic T cell receptor signaling domain, and

[0597] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from LTBR and consist of SEQ ID NO: 33,

[0598] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0599] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0600] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0601] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0602] b) transmembrane domain,

[0603] c) a cytoplasmic co-stimulatory signaling domain, and

[0604] d) a cytoplasmic T cell receptor signaling domain, and

[0605] Wherein the artificial immune receptor comprises SEQ ID NO:31.

[0606] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0607] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0608] b) transmembrane domain,

[0609] c) a cytoplasmic co-stimulatory signaling domain, and

[0610] d) a cytoplasmic T cell receptor signaling domain, and

[0611] Wherein the artificial immune receptor consists of SEQ ID NO:31.

[0612] In certain embodiments, the disclosure relates to a nucleic acid comprising SEQ ID NO:30.

[0613] In certain embodiments, the present disclosure relates to a nucleic acid consisting of SEQ ID NO:30.

[0614] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0615] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0616] b) transmembrane domain,

[0617] c) a cytoplasmic co-stimulatory signaling domain,

[0618] d) a cytoplasmic T cell receptor signaling domain, and

[0619] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from CD40 and comprise SEQ ID NO: 46,

[0620] wherein the cytoplasmic costimulatory signaling domain is CD28 and comprises SEQ ID NO: 35, and

[0621] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0622] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0623] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0624] b) transmembrane domain,

[0625] c) a cytoplasmic co-stimulatory signaling domain, and

[0626] d) a cytoplasmic T cell receptor signaling domain, and

[0627] wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from CD40 and consist of SEQ ID NO: 46,

[0628] wherein the cytoplasmic co-stimulatory signaling domain is CD28 and consists of SEQ ID NO: 35, and

[0629] wherein the cytoplasmic T cell receptor signaling domain is CD3ζ and consists of SEQ ID NO:37.

[0630] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0631] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0632] b) transmembrane domain,

[0633] c) a cytoplasmic co-stimulatory signaling domain, and

[0634] d) a cytoplasmic T cell receptor signaling domain, and

[0635] Wherein the artificial immune receptor comprises SEQ ID NO:47.

[0636] In certain embodiments, the present disclosure relates to a nucleic acid encoding an artificial immune receptor comprising

[0637] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0638] b) transmembrane domain,

[0639] c) a cytoplasmic co-stimulatory signaling domain, and

[0640] d) a cytoplasmic T cell receptor signaling domain, and

[0641] Wherein the artificial immune receptor consists of SEQ ID NO:47.

[0642] In certain embodiments, the present disclosure relates to a nucleic acid encoding a protein comprising SEQ ID NO:30.

[0643] In certain embodiments, the present disclosure relates to a nucleic acid encoding a protein consisting of SEQ ID NO:30.

[0644] In certain embodiments, the present disclosure relates to vectors comprising a nucleic acid encoding an artificial immune receptor of the present disclosure.

[0645] In certain embodiments, the present disclosure relates to a vector comprising a nucleic acid encoding an artificial immune receptor, the artificial immune receptor comprising

[0646] a) Extracellular domain of members of the tumor necrosis factor receptor superfamily;

[0647] b) transmembrane domain,

[0648] c) a cytoplasmic co-stimulatory signaling domain, and

[0649] d) Cytoplasmic T cell receptor signaling domain.

[0650] In certain embodiments, the present disclosure relates to a host cell comprising a nucleic acid or a vector encoding an artificial immune receptor according to the present disclosure.

[0651] In certain embodiments, the present disclosure relates to host cells expressing the artificial immune receptors of the present disclosure.

[0652] In certain embodiments, the present disclosure relates to host cells that express the disclosed artificial immune receptors on the cell surface.

[0653] In preferred embodiments, the host cell is a eukaryotic host cell. Thus, in certain embodiments, the disclosure relates to a eukaryotic host cell comprising a nucleic acid or vector encoding an artificial immune receptor according to the disclosure. In other embodiments, the disclosure relates to a eukaryotic host cell expressing an artificial immune receptor of the disclosure. In other embodiments, the disclosure relates to a eukaryotic host cell expressing an artificial immune receptor of the disclosure on the cell surface.

[0654] Therapeutic Uses

[0655] The artificial immunoreceptors of the present disclosure can be used to therapeutically prevent and treat diseases and disorders.

[0656] In certain embodiments, the present disclosure relates to artificial immunoreceptors as disclosed herein for use in medicine. In certain embodiments, the present disclosure relates to the use of artificial immunoreceptors as disclosed herein for preventing or treating a disease or condition. In certain embodiments, the disease or condition is an inflammatory condition or cancer. In preferred embodiments, the disease or disorder is an autoimmune condition. In certain embodiments, the disease or condition is the treatment of graft-versus-host disease or use in solid organ transplantation.

[0657] Certain specific embodiments

[0658] 1. An artificial immune receptor comprising

[0659] a) the extracellular domain of members of the tumor necrosis factor receptor superfamily,

[0660] b) transmembrane domain,

[0661] c) a cytoplasmic co-stimulatory signaling domain, and

[0662] d) Cytoplasmic T cell receptor signaling domain.

[0663] 2. An artificial immune receptor according to item 1, wherein the tumor necrosis factor receptor superfamily member 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.

[0664] 3. The artificial immunoreceptor according to item 1 or 2, wherein the tumor necrosis factor receptor superfamily member is selected from TNFR2, DR3, LTBR and CD40.

[0665] 4. The artificial immunoreceptor according to any one of items 1 to 3, wherein the transmembrane domain is from the same protein as the extracellular domain.

[0666] 5. The artificial immunoreceptor according to any one of items 1 to 4, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are derived from TNFR2.

[0667] 6. The artificial immune receptor according to item 5, wherein said artificial immune receptor comprises SEQ ID NO: 39.

[0668] 7. The artificial immune receptor according to item 5 or 6, wherein the artificial immune receptor comprises SEQ ID NO: 38.

[0669] 8. The artificial immunoreceptor according to any one of items 1 to 4, wherein said tumor necrosis factor receptor superfamily member and said transmembrane domain are derived from DR3.

[0670] 9. The artificial immune receptor according to item 8, wherein the artificial immune receptor comprises SEQ ID NO: 41.

[0671] 10. The artificial immunoreceptor according to item 8 or 9, wherein the artificial immunoreceptor comprises SEQ ID NO: 40.

[0672] 11. The artificial immunoreceptor according to any one of items 1 to 4, wherein said tumor necrosis factor receptor superfamily member and said transmembrane domain are derived from LTBR.

[0673] 12. The artificial immune receptor according to item 11, wherein the artificial immune receptor comprises SEQ ID NO: 33.

[0674] 13. The artificial immune receptor according to item 11 or 12, wherein the artificial immune receptor comprises SEQ ID NO: 31.

[0675] 14. The artificial immunoreceptor according to any one of items 1 to 4, wherein said tumor necrosis factor receptor superfamily member and said transmembrane domain are derived from CD40.

[0676] 15. The artificial immune receptor according to item 11, wherein the artificial immune receptor comprises SEQ ID NO:46.

[0677] 16. The artificial immune receptor according to item 11 or 12, wherein the artificial immune receptor comprises SEQ ID NO: 47.

[0678] 17. The artificial immunoreceptor according to any one of items 1 to 16, wherein the cytoplasmic co-stimulatory signaling domain is selected from 4-IBB (CD137), BAFFR, OX40, CD27, CD28, CD40, 2B4, GITR, HVEM, OX40, RELT, TACI, TROY, TWEAK, KIR receptors, TLR1 to TLR9 receptors, IL-2, IL-7 and IL-15 receptor.

[0679] 18. The artificial immunoreceptor according to any one of items 1 to 17, wherein the cytoplasmic co-stimulatory signaling domain is CD28.

[0680] 19. The artificial immunoreceptor according to any one of items 1 to 18, wherein the cytoplasmic co-stimulatory signaling domain comprises SEQ ID NO: 35.

[0681] 20. The artificial immunoreceptor according to any one of items 1 to 19, wherein the cytoplasmic T cell receptor signaling domain is selected from the group consisting of CD3ζ, CD3γ and CD3ε.

[0682] 21. The artificial immunoreceptor according to any one of items 1 to 20, wherein the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0683] 22. The artificial immunoreceptor according to any one of items 1 to 21, wherein the cytoplasmic T cell receptor signaling domain comprises SEQ ID NO: 37.

[0684] 23. The artificial immunoreceptor according to any one of items 1-7 or 17-22, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2, the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0685] 24. An artificial immunoreceptor according to any one of items 1-7 or 17-23, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2 and comprise SEQ ID NO:39, the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0686] 25. The artificial immunoreceptor according to any one of items 1-4, 8-10 or 17-22, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from DR3, the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0687] 26. An artificial immunoreceptor according to any one of items 1-4, 8-10, 17-22 or 25, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from DR3 and include SEQ ID NO:41, the cytoplasmic co-stimulatory signaling domain is CD28 and includes SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3ζ and includes SEQ ID NO:37.

[0688] 27. An artificial immunoreceptor according to any one of items 1-4, 11-13 or 17-22, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from LTBR, ​​the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0689] 28. An artificial immunoreceptor according to any one of items 1-4, 11-13, 17-22 or 27, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from LTBR and comprise SEQ ID NO:33, the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3ζ and comprises SEQ ID NO:37.

[0690] 29. The artificial immunoreceptor according to any one of items 1-4, 14-16 or 17-22, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from CD40, the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ.

[0691] 30. An artificial immunoreceptor according to any one of items 1-4, 14-16, 17-22 or 29, wherein the tumor necrosis factor receptor superfamily member and the transmembrane domain are from CD40 and comprise SEQ ID NO:46, the cytoplasmic co-stimulatory signaling domain is CD28 and comprises SEQ ID NO:35, and the cytoplasmic T cell receptor signaling domain is CD3 zeta and includes SEQ ID NO:37.

[0692] 31. A nucleic acid encoding an artificial immune receptor according to any one of items 1-30.

[0693] 32. A vector comprising the nucleic acid according to item 31.

[0694] 33. A host cell comprising the nucleic acid according to item 31 or the vector according to item 32, or expressing the artificial immune receptor according to any one of items 1 to 30.

[0695] 34. An artificial immunoreceptor according to any one of items 1 to 30 or a host cell according to item 33, for use in medicine.

[0696] 35. The artificial immunoreceptor according to item 34, wherein the use is for the treatment of inflammatory disorders.

[0697] 36. The artificial immunoreceptor according to item 35, wherein the inflammatory disorder is graft-versus-host disease.

[0698] Example

[0699] Example 1: Materials

[0700] Ethical Statement

[0701] Peripheral blood mononuclear cells for T cell enrichment were isolated from healthy donors providing platelets in a leukocyte reduction chamber. Collection of immune cells from those donors was performed in compliance with the Helsinki Declaration after ethical approval by the local ethics committee and written informed consent.

[0702] Isolation of peripheral blood mononuclear cells and pre-enrichment of blood lymphocytes

[0703] To isolate T cells from human blood, a leukocyte removal chamber (provided by Transfusion Medicine, University Clinics Regensburg) was used. Leukocytes were first diluted with PBS, and the resulting blood and PBS mixture was divided into four parts and covered with an equal amount of Pancoll (PAN Biotech, Aidenbach, Germany) at the bottom. The samples were centrifuged at 1.000xg for 20 minutes at RT, with the acceleration set to 4 and the brake set to zero. The PBMC layer was separated and washed twice by the centrifugation step. The cells were pre-enriched with biotinylated anti-human CD25-PE (clone 2A3; BD Biosciences RRID: AB_341011), followed by column-based magnetic separation with anti-PE ultrapure microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol.

[0704] Mouse

[0705] Female BALB / c mice, C57BL / 6Nr4a1-eGFP mice (JAX stock#016617, C57BL / 6-Tg(Nr4a1-EGFP / cre)820Khog / J) and C57BL / 6CD45.1+ 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 bred to C57BL / 6CD45.1+ mice and served as bone marrow donors for GvHD experiments.

[0706] C57BL / 6Foxp3-hCD2 (Foxp3tm1 (CD2 / CD52) Shori) mice were a gift from S. Hori (P Natl Acad Sci USA. 2009; 106(6): 1903-8). The mice were maintained under specific pathogen-free conditions and the study was approved by the Committee on Ethics of Animal Experiments of the Bavarian Government.

[0707] Cell lines

[0708] Phoenix-Eco and Phoenix-Ampho cell lines were purchased from ATCC (Cat#CRL-3214 and #CRL-3213). These are second generation retroviral producer cell lines used to produce ecotropic and amphotropic retroviruses. EL4 lymphoblastoid cell line was purchased from ATCC (Cat#TIB-39).

[0709] Additional resources are listed in Tables 1, 2, 3 and 4.

[0710] Table 1: Antibodies

[0711]

[0712]

[0713] Table 2: Chemicals, plasmids and recombinant proteins

[0714]

[0715] Table 3: Commercial assays

[0716]

[0717]

[0718] Table 4: Software and Algorithms

[0719]

[0720] Statistical analysis

[0721] Data were analyzed using Prism software or algorithms. Statistical details are shown in the legend. For survival differences, Kaplan-Meier analysis was performed and the log-rank test was used. P value 0.05 was considered significant (*p<0.05; **p<0.01; ***p<0.001).

[0722] Example 2: Method

[0723] Digestion of mouse tissue for flow cytometry analysis, FACS sorting of cells, and transplantation

[0724] To isolate cells from colon tissue, the colon was isolated using a lamina propria dissociation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and a gentleMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany, program 37C_mLPDK_1) according to the manufacturer's instructions, feces were removed and prepared. A more detailed protocol for isolating T cells from murine tissue is disclosed ((Eur J Immunol. 2019; 49(10): 1457-973).

[0725] To isolate cells from blood, peripheral blood was collected and incubated in heparin buffer. The blood samples were centrifuged and erythrocytes were lysed using ACK lysis buffer (Thermo Fisher / GIBCO, Waltham, MA, USA, #A1049201).

[0726] In order to separate cells from spleen and lymph nodes, tissues were harvested and mechanically decomposed. Samples were centrifuged and erythrocytes 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.

[0727] To isolate cells from the bone marrow for transplantation, the femurs were harvested and the femoral heads and medial and lateral femoral epicondyles removed. The bone shafts were rinsed with PBS and the bone marrow was harvested by centrifugation followed by lysis of the erythrocytes.

[0728] Preparing samples for flow cytometry

[0729] Single cell suspensions were prepared and pre-enriched as previously described (Eur J Immunol. 2019; 49(10): 1457-973). Samples were stained in FACS buffer (1% FCS in PBS) in 1.5 mL Eppendorf tubes or 96-well plates. Surface staining was performed in 50-100 μL chromosome volumes at 4°C for 20 minutes. Antibodies were used as recommended by the manufacturer if not otherwise indicated. The following antibodies were used for surface staining of mouse 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).

[0730] 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).

[0731] Intracellular staining was performed with Foxp3 / transcription factor buffer set (Thermo Fisher / Biosciences, Waltham, MA, USA) according to the manufacturer's protocol with the following modifications: The intracellular staining step was performed at room temperature for 60 minutes. Antibodies used for intracellular staining included Foxp3 (JFK-16S), Nr4a1 (12.14), Irf8 (V3GYWCH) for mouse, and Foxp3 (206D) for human samples. Dead cells were excluded with a fixable live / dead dye (eBioscience Fixable ViabilityDye eFluor780). All antibodies are listed in the Resources table.

[0732] Flow cytometry and FACS sorting of T cells from blood, tissue, and cell culture

[0733] Cells were isolated, pre-enriched and stained as previously described (Eur J Immunol. 2019; 49(10): 1457-973). Samples were then filtered with a 40 μM filter unit and analyzed on a BD FACSymphony TM BD FACSCelesta TM or BDFACSFusion TM The data were acquired on a flow cytometer (all from Becton Dickinson, Franklin Lakes / NJ, USA). BD CS&T beads were used to verify the machine function. Fluorescence spillover compensation was performed using lymphocytes stained with anti-CD4 antibodies in the corresponding colors. BD FlowJo TM Flow cytometry data were analyzed using the BD FACSAria II with a 70 μm nozzle (version 10.6.2). TM or BD FACSFusion TM Cell sorting was performed using a cell sorter. Post-sorting quality control was performed, if appropriate. For mouse Treg cultures, cells enriched in anti-CD25 were sorted for natural 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.

[0734] For bulk RNA sequencing, 4 x 10 4 -8x 104 Cells were sorted directly into 500 μL RLT+ lysis buffer (Qiagen, Hilden, Germany, RNEasy Plus Micro Kit #74034). For human Treg culture, CD25 pre-enriched cells from human blood were sorted directly on TCR-β-chain+CD4+CD25+CD45RA+ into TexMACS medium.

[0735] Murine and human Treg cell cultures

[0736] For mouse Treg culture, sorted Treg cells were cultured at 3 x 10 4 -4x 10 4 10 cells / well were seeded in a 3% culture medium with anti-CD3 / CD28 beads (Miltenyi Biotec, Bergisch Gladbach, Germany, 4 beads / well) and 2000 U / ml rhIL-2 (Proleukin Novartis, Basel, Switzerland) in 96-well round-bottom plates (Corning, Wiesbaden, Germany). The culture medium is DMEM (Gibco / Invitrogen) supplemented with 10% FCS, 2mM l-glutamine, 5x10-5M 2-ME, 10mM HEPES, 1% NEAA (PANBiotech, Aidenbach, Germany), 100U / ml penicillin-streptomycin (Gibco / Sigma-Aldrich, St.Louis / MO, USA). Mouse Treg cells were transduced 48h after separation. Human sorted Treg cells were cultured in TexMACS culture medium supplemented with 500U / ml rhIL-2 and TransAct (1:100 dilution). Human Treg cells were transduced 48h after separation.

[0737] Retroviral transduction of Treg cells

[0738] Retroviruses in the pMSCV-Thyl.1 system can be produced in Phoenix-Eco cells, a variant of HEK293 cells carrying pCLEco (packaging plasmid). Therefore, Phoenix-Eco cells were plated at 1.8 x 10 6 To generate liposome particles containing viral transgenes, 3 μg of vector DNA and 1 μg of additional pCL-Eco packaging plasmid were mixed with 12 μL of TransIT-293 transfection reagent (MoBiTec, Germany) in OptiMEM medium at room temperature for 20 minutes. Liposomes are added to Phoenix-Eco cells and incubated for another 16 hours. Then, the culture medium is replaced, and the production of viral particles can be carried out for 24 hours. The supernatant with the generated pMSCV retrovirus is then added to the Treg cell culture and mixed gently. Treg cells are transduced for 6.5 hours at 37°C incubation. Then, the viral supernatant is removed and the cells are incubated with fresh culture medium supplemented with IL-2 (2000U / ml) for another 72 to 96 hours. Then, the cells are harvested and the coupled beads of anti-CD3 / CD28 antibodies are removed by using a MACSiMAG separator magnet (Miltenyi Biotec, Bergisch Gladbach, Germany). For the transduction of human Treg, a dual-tropic Phoenix cell line is used. 6 hours before lipid transfection, Phoenix-Ampho cells are lysed at 2x10 6 To produce liposome particles containing viral transgenes, 3 μg of vector DNA and 1 μg of additional pCL-Ampho packaging plasmid were mixed with 12 μL of TransIT-293 transfection reagent (MoBiTec, Germany) in OptiMEM culture medium at room temperature for 20 minutes. Liposomes are added to Phoenix-Ampho cells and incubated for another 16 hours. Then, the culture medium is replaced and the production of viral particles can be carried out for 24 hours. Then the supernatant with the generated pMSCV retrovirus is added to the Treg cell culture and mixed gently. Treg cells are transduced for 6.5 hours and incubated at 37°C. Then, the viral supernatant is removed and the cells are incubated for another 72 to 96 hours with fresh culture medium supplemented with IL-2 (500U / ml), 100U / ml penicillin-streptomycin and fresh TransAct (1:100 dilution).

[0739] HEK293 cells were transiently transfected with TNFSF ligand

[0740] To generate liposome particles, 2 μg of plasmid DNA was incubated with 6 μl of TransIT-293 transfection reagent in 100 μl of OptiMEM medium at room temperature for 20 minutes. The liposomes were then gently added to 5 x 10 5 After shaking for 1 minute, 230 μl of DMEM was added, and 100 μl of cell-liposome suspension (containing approximately 6 x 10 418 hours after changing the lipid transfection medium, the transfected HEK293 cells were ready for co-culture experiments.

[0741] Treg proliferation assay

[0742] Anti-CD3 / CD28 beads were removed from the transduced Treg cultures by MACSiMAG separation magnet. Tregs were rested for 18 hours in fresh medium supplemented with 100 U / ml rhIL-2. TM CFSE cell proliferation dye (1 μM) marks Tregs and is added to HEK293 cells expressing mTNF, mTL1A or mLIGHT on the cell surface. rhIL-2 (2000 U / ml) is added to the cell culture. After 72 hours of co-incubation, the proliferation of transduced CFSE-labeled Tregs is analyzed by flow cytometry.

[0743] RNA sequencing

[0744] Total RNA was isolated using the Qiagen RNeasy Micro kit (Qiagen, Hilden, Germany), and RNA was eluted in 14 μL of RNase-free water. RNA quality was assessed using the Tapestation system 4200 and a high-sensitivity RNA screening band (Agilent). RNA-seq libraries were generated using 7 μl of RNA using the SMART-seqStranded kit from Takara (Mountain View / CA, USA). Index libraries were pooled in equimolar ratios and sequenced (75 cycles) on an Illumina NextSeq 550 machine using the NextSeq 500 / 550 High Output Kit v2.5.

[0745] GvHD Model

[0746] BALB / c (H2Kd) recipients were irradiated (8 Gy) and treated with 5 × 10 5 In the case of spleen cells (H2Kb, CD45.1), 2.5×10 6 After BM cell transplantation, animals in the treatment group received 2.5×10 5 In vitro expanded and transduced Tregs (C57BL / 6, Foxp3-hCD2, congenic CD45.2). Recipients were monitored daily, and body weight and GvHD symptoms were assessed two or three times per week by an unblinded investigator using standardized scores.

[0747] Example 3: Recombinant protein

[0748] Mouse TNF

[0749] To produce murine TNF, the expression plasmid BCMGS-L6 encoding the uncleavable mouse TNF protein was used (JBC 1999, 274(53):38112-8).

[0750] Mouse TL1A

[0751] To generate an uncleavable form 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 the uncleavable mTL1A was cloned into pIRES2-DsRed via NheI / SalI. The nucleotide sequence of mTL1A is described as follows:

[0752]

[0753] PEOPLE LIGHT

[0754] The cDNA encoding human LIGHT was cloned into pcDNA3.1 neo via BamHI / EcoRV. The nucleotide sequence of hLIGHT is described as follows:

[0755]

[0756] MouseLIGHT

[0757] The cDNA encoding mouse LIGHT was cloned into pIRES2-DsRed via NheI / SalI. Amino acids 63-84 were deleted to produce an uncut form of mouse LIGHT. The nucleotide sequence of mLIGHT is described as follows:

[0758]

[0759] Example 4: Design of Artificial Immune Receptor (AIR)

[0760] Mouse and human AIR were designed based on the nucleotide sequences of the individual parts and domains disclosed (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-ζ chains. Two point mutations were introduced into the mouse CD3-ζ chain because they were shown to increase the expression of chimeric antigen receptors (Blood 2003; 102(13): 4320-5). As a negative control, ORFs encoding the transmembrane and signaling domains of CD28 and the signaling domain of the CD3-ζ chain were used. Anti-CD19 CAR and anti-CEA CAR have been previously described (Blood 2010; 116(19): 3875-86; Mol Ther 2019; 27(10): 1825-35). The cDNAs encoding AIR, anti-CD19 CAR and anti-CEA CAR were fused to the cognate marker CD90.1 by linking to the self-cleaving P2A sequence. cDNA was synthesized by ThermoFisher / Life Technologies (Waltham, MA, USA) and cloned into the pMSCV-Thyl.1 retroviral backbone (Addgene, cat# 17442) via NotI / Mlu. An overview of these constructs is shown in Figure 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.

[0761] Table 5:

[0762]

[0763]

[0764]

[0765]

[0766]

[0767]

[0768]

[0769]

[0770]

[0771]

[0772]

[0773]

[0774]

[0775]

[0776] exist Figure 2 The mode of action of AIR is shown in . Binding of membrane-bound TNF family members via AIR results in the translation of cytokine signals into T cell receptor (TCR)-like signals. This initiates T cell activation in a context / inflammation-dependent manner independent of its endogenous TCR-MHC restriction.

[0777] Example 5: Transduction and surface expression of AIR

[0778] The transduction and expression of AIR according to the present disclosure were studied in murine regulatory T cells (Treg). Tregs were isolated from the spleen and lymph nodes of C57 / BL6 mice, sorted (natural CD4+CD25+CD62L+Treg or Foxp3+ reporter gene (hCD2+) positive) and stimulated to expand for 6 days via beads coupled to anti-CD3 and anti-CD28 antibodies plus IL-2 (2000U / ml). The purity of Treg cultures was determined by flow cytometry. Treg cultures contain approximately 90%-99% Foxp3+T cells. On the second day after separation, Tregs were transduced with AIR constructs or anti-CD19 chimeric antigen receptors (anti-CD19 CAR) as controls. Three days later, the transduction efficacy and surface expression of AIR by CD90.1 staining were examined by flow cytometry. The results are shown in Figure 3 and Figure 4 Similar results were obtained with CD40 AIR (93.6% Foxp3+ T cells). It could be demonstrated that all AIR were efficiently expressed on the surface of regulatory T cells.

[0779] Example 6: AIR induces the expression of T cell receptor signaling marker genes

[0780] To test whether the disclosed AIR induces T cell receptor signaling, the induction of Nr4a1 was investigated. Nr4a1 is a well-known target gene for T cell receptor signaling. Tregs from Nr4a1-eGFP reporter mice were used. As a control, Tregs were stimulated with anti-CD3 / CD28 antibody-coupled beads. An overview of the experiment is in Figure 5 Shown in.

[0781] Tregs expressing AIR were stimulated overnight and co-cultured with HEK cells expressing mouse Light (mLight), mTL1A, or mTNF. As a positive control, Tregs were stimulated with beads coupled to anti-CD3 / CD28 antibodies to mimic TCR signaling. The results are shown in Figure 6 Middle. Only AIR-positive (CD90.1+) Tregs expressed Nr4a1-eGFP (left), but not AIR-negative (CD90.1-) Tregs (right). In contrast, Tregs expressing anti-CD19 CAR showed no upregulation of Nr4a1-GFP (bottom).

[0782] Figure 7 The expression of Nr4a1 when AIR according to the present disclosure is applied is shown, taking AIR carrying the extracellular domain of LTBR as an example. In the absence or presence of LTBR-Ig fusion protein (50ug / ml) blocking mLight, Tregs expressing LTBR AIR according to the present disclosure or unrelated CAR carrying anti-CD19 single-chain antibody (scFv) extracellularly were co-cultured with mouse EL4 cells known to express LIGHT, and then stained for intracellular Nr4a1 expression.

[0783] Example 7: RNA and protein expression analysis of Treg stimulated with AIR

[0784] In this experiment, it was tested whether Tregs stimulated with AIR according to the present disclosure exhibited expression patterns consistent with putative TCR signaling effects as measured by RNA-seq expression analysis.

[0785] The first cells were sorted accordingly. Tregs expressing AIR or an irrelevant CAR carrying an anti-CD19 single-chain antibody (scFv) extracellularly were left to rest for 24 h and then co-cultured with HEK cells or HEK cells expressing mLight for 18 h. Figure 8 Pre-gating for CD90.1+ cells is shown. Pre-gated cells were then sorted on CD4+CD25+CD90.1+ (left and center) or CD4+CD25+CD90.1+Nr4a1+LAP+ (right). Results are shown in Fig. 9 middle.

[0786] After cell sorting, RNA was isolated and subjected to RNA sequencing. The results are shown in Fig.10 (LTPR AIR), Fig.11 (DR3AIR) and Fig.12 (α-CD19 control CAR).

[0787] Fig.10Shown on the left is a summary of up-regulated and down-regulated genes comparing α-CD19 CAR Tregs stimulated with HEK-mLight relative to LTBR AIR Tregs stimulated with HEK cells (as a control) (endogenous mLight signaling) and LTBRAIR Tregs stimulated with HEK-mLight relative to α-CD19 CAR Tregs stimulated with HEK-mLight (specific LTBR AIR signaling). Shown on the right is a volcano plot showing differentially expressed transcripts.

[0788] Similarly, in Fig.11 In the figure, DR3 AIR Tregs were stimulated with HEK cells (endogenous mTL1A signaling). DR3 AIR Tregs were stimulated with HEK-mTL1A versus α-CD19 CARTregs stimulated with HEK-mTL1A (specific DR3 AIR signaling). Differentially expressed genes are shown on the left and volcano plots are shown on the right.

[0789] Similarly, Fig.12 α-CD19 CAR Tregs stimulated with anti-CD3 / CD28 beads were compared to unstimulated Tregs. Differentially expressed genes are shown on the left and the volcano plot is shown on the right.

[0790] Analyze the RNA and / or protein expression levels of various specific marker genes in more detail. Fig.13 RNA expression data from Tregs expressing LTBRAIR or control CAR for Tnfrsf9, Tigit, Tgfb1, Cd69, Ccr8, and Nr4a1 after 18 h of co-culture with HEK+ / -mLight are shown (Deseq2, n=3). LTBRAIR (in each figure shown on the far right) resulted in significant upregulation of all six marker genes tested. Fig.14 Representative flow cytometry analysis of Tregs expressing LTBR AIR or control CAR after 18 hours of co-culture with HEK+ / -mLight is shown. Protein expression of CD137 and Tigit is shown in the upper figure, and CD69 and LAP (membrane-bound Tgfβ1) are shown in the lower figure. The data are representative of three independent experiments. Again LTBR AIR resulted in significant upregulation of the marker genes studied.

[0791] Fig.15RNA expression data from Tregs expressing DR3 AIR or control CAR for Tnfrsf9, Tigit, Tgfb1 after 18 h of co-culture with HEK+ / -mLight are shown (Deseq2, n=3). DR3 AIR (in each figure shown on the far right) resulted in significant upregulation of all three marker genes tested. Fig.16 Representative flow cytometry analysis of Tregs expressing DR3 AIR or control CAR after 18 hours of co-culture with HEK+ / -mTL1A is shown. Protein expression of CD137 and Tigit is shown in the upper figure, and CD69 and LAP (Tgfβ1) are shown in the lower figure. The data are representative of three independent experiments. In addition, DR3 AIR leads to significant upregulation of the marker genes studied.

[0792] Fig.17 RNA expression data (upper left, Deseq2, n=3) and protein expression (right) for Irf8 from Tregs expressing LTBR AIR or control CAR after 18h co-culture with HEK+ / -mLight are shown. RNA expression data (lower left, Deseq2, n=3) and protein expression (right) for Irf8 from Tregs expressing DR3 AIR or control CAR after 18 hours of co-culture with HEK+ / -mTL1A. Again, all marker genes tested were significantly upregulated.

[0793] Fig.18 AIR of the present disclosure mediates Treg activation and proliferation. Tregs expressing LTBR AIR or control CAR were left to rest for 24 hours and labeled with CFDA-SE proliferation dye. Engineered and labeled Tregs were co-cultured with HEK+ / -mLight for 72h in the presence of IL2, and then analyzed for proliferation by flow cytometry. Representative dot plots are shown on the left. Summary data (n=3) from three experiments performed in three technical replicates are shown on the right. Again, all marker genes tested were significantly upregulated.

[0794] In summary, RNA and protein expression analysis clearly demonstrated that the AIR of the present disclosure elicited cellular responses consistent with the underlying hypothesis.

[0795] Example 8: LTBRAIR ameliorates GvHD pathology in mice

[0796] The effectiveness of the disclosed AIR was tested in a graft-versus-host disease (GvHD) mouse model. FACS-sorted Tregs from Foxp3-hCD2 reporter mice were expanded and treated with LTBR AIR or a truncated construct (lacking the extracellular binding domain, shown in Figure 1 a) Transduction was used as a control. 2.5x105 Engineered Tregs and 2.5x10 from C57 / BL6 mice 6 Bone marrow cells and 2.5x10 5 The spleen cells were transplanted into lethally irradiated (8 Gy) Balb / c mice. As a transplantation control, one group received bone marrow cells (BM) alone, and as a disease progression control, one group received BM cells plus spleen cells. After transplantation, the animals were monitored for 47 days. An overview of the experiment is depicted in Fig.19 Before transfer into mice, the quality of engineered Tregs was checked by staining for CD4, hCD2 (reporter gene for Foxp3) and for CD90.1 and LTBR before transfer into mice. The results are shown in Fig. 20 On day 20, blood samples were collected from each animal to verify the recovery of donor-derived (H2kB+)CD19+ B-cells.

[0797] Fig.21 Kaplan-Meier curves containing data sets from two independent experiments (log-rank test, n=11-12) are shown. As can be seen, LTBR AIR is highly effective in the GvHD model, resulting in a significant increase in the survival of transplanted Balb / c mice. This can also be seen in Fig. 22 Seen in Fig. 22 Mean GvHD scores for each treatment group are shown (animals reaching a score of 40 had to be euthanized; animals that died retained the highest score at subsequent time points for statistical analysis, two-way ANOVA). Fig.23 Representative FACS images of spleens from surviving animals that received LTBR AIR Tregs are shown (left). Shown on the right is the percentage of Foxp3+ Tregs in spleens of surviving mice at day 47. Fig.24 Analysis of Klrg1+Tregs in the spleen is shown. Shown on the left are representative FACS images of differentiated CD45.1+ (BM-derived) and CD45.2+ (transferred AIR Tregs) Treg cells. Klrg1 is a marker for tissue-specific phenotypes; hCD2 is a Foxp3 reporter gene. Shown in the middle is Klrg1+ expression of engineered CD45.2+ and BM-derived CD45.1+Tregs in surviving mice. Shown on the right are the frequencies of Klrg1+hC2+Tregs in mice receiving LTBR AIR Tregs (Mann-Whitney-U, survivors n=8 versus dead n=4). Fig.25Representative FACS analysis of digested colon from surviving animals receiving LTBR AIR Tregs is shown. The distinction between CD45.1 and CD45.2 cells within the CD4+ population is shown on the left. CD45.2 Tregs were further analyzed for Klrg1 and hCD2 expression on the right. Fig.26 The percentage of hCD2+ (indicating Foxp3+) cells among the transferred engineered CD45.2+ cells is shown (left), demonstrating phenotypic stability in vivo over 47 days. Shown on the right are cell counts of transferred CD45.2 Tregs in the colon at day 47.

[0798] In summary, this example demonstrates the clinical usefulness of the disclosed AIR in an established clinical model for graft-versus-host disease.

[0799] Example 9: Human LTBRAIR

[0800] Human LTBR AIR constructs were generated similar to the mouse LTBR constructs. The human constructs contained the same elements as the mouse constructs, except that all domains, i.e., the LTBR extracellular domain, the LTBR transmembrane domain, the cytoplasmic CD28 domain, and the cytoplasmic CD3ζ domain were of human origin (SEQ ID No. 30 (nucleic acid); SEQ ID No. 31 (polypeptide)). The human LTBR AIR constructs were tested for expression and signaling capabilities.

[0801] Human T cells from healthy blood donors were sorted on Treg protein markers (TCRb+CD4+CD25+CD127-CD45RA+) and expanded for 7 days with anti-CD3 / CD28 stimulation (TransACT, Miltenyi Biotec, Bergisch Gladbach, Germany) and IL-2 (500U / ml). On the second day after isolation, Tregs were transduced with human LTBR AIR constructs (double-addicted form using the retroviral expression system MSCV). After 5 days, cultured Tregs were stained for intracellular Foxp3+ to check Treg purity and CD90.1 as well as hLTBR to analyze the transduction efficacy and surface expression of AIR protein. The results are shown in Fig. 27 In. LTBR AIR expression on human Treg cells can be verified.

[0802] In another experiment, Tregs expressing human LTBR AIR or irrelevant CAR (anti-CEA, carcinoembryonic antigen) were co-cultured with parental HEK cells or HEK cells expressing human Light protein on the surface for 18 hours. The expression of Ccr8, glycoprotein A repeat dominant protein (GARP), CD137 (4-1BB) and Tigit was shown in CD90.1+Foxp3+Treg. Again, the expression levels of all marker genes confirmed the putative activity of human LTBR AIR.

[0803] Example 10: TCR signaling is important for the activity of AIR

[0804] Control constructs comparable to the AIR functions disclosed herein were generated, with the important difference being that they lacked the CD3ζTCR signaling domain. Such constructs without the CD3ζ domain are known in the art (see WO 2021 / 051195). Unlike the constructs of WO2021 / 051195, the constructs provided by the present invention function directly on the primary signal of the signaling cascade and in a CD3 (TCR) dependent manner.

[0805] The architecture of this construct is Fig.29 The amino acid sequence of the construct is described as follows:

[0806]

[0807] Surface expression of the constructs can be confirmed by flow cytometry. Fig.29 , bottom. However, unlike the AIR of the present disclosure, the construct lacking CD3ζ does not induce marker genes. Therefore, the AIR of the present disclosure is functionally different from the molecules of the prior art. Fig.30 Representative flow cytometry analysis of Tregs expressing full-length LTBR AIR and LTBR "AIR" lacking the CD3ζ chain is shown after 18h 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.

[0808] The importance of the CD3ζ domain was further demonstrated using Treg cells expressing LTBR-AIR, LTBR-AIR without the CD3ζ chain, and anti-CD19CAR from Nr4a1.eGFP reporter mice. The cells were left to rest for 24 hours and then co-cultured with HEK cells or HEK cells expressing mLIGHT for 18 hours. Afterwards, the cells were analyzed for eGFP and LAP expression by flow cytometry. The results of one of two independent experiments are shown in Fig.41Full-length LTBR AIR containing only the CD3ζ domain, but not the other two constructs tested, was able to 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.

[0809] Example 11: CD40 AIR leads to expression of function-related marker genes

[0810] Similar experiments were performed with the murine CD40 AIR of SEQ ID NO: 43. Figure 1 This AIR was tested compared to construct F). Treg cells expressing CD40 AIR or anti-CD19 constructs were co-cultured with HEK cells or HEK cells expressing CD40L (CD154) for 18 h. The cells were then analyzed by flow cytometry for TIGIT and CD137 ( Fig.31 and 32 ) and LAP (TGFb1) and CD69 ( Fig.33 and 34 ) expression.

[0811] Furthermore, it can be shown that the AIR of the present disclosure is functionally active and induces the expected marker genes ( Fig.31 and Fig.33 The results are summarized in Fig.32 and 34 Induction of marker genes by CD40 AIR was statistically significant as determined by one-way Anova analysis.

[0812] Example 12: CD40AIR triggers the expression of Nr4a1, an early response gene for TCR signaling

[0813] 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 h. Then, the expression of Nr4a1-eGFP was measured by flow cytometry.

[0814] Expression of Nr4a1, an early response gene for TCR signaling, was induced by Treg cells expressing CD40 AIR but not by constructs expressing anti-CD19 CAR ( Fig.35 ), again confirming the TCR-like activation mediated by AIR of the present disclosure.

[0815] Example 13: Design of AIR with alternative co-stimulatory domains

[0816] To test whether alternative costimulatory domains were also functional, additional constructs were generated. Figure 1 The CD28 co-stimulatory domain of the construct shown in G was replaced with an ICOS co-stimulatory domain and a 4-1BB co-stimulatory domain. These additional constructs are shown in Fig.36 A (ICOS) and 36B (4-1BB). The sequences of these constructs and their parts are shown in Table 6 (N = nucleic acid, P = polypeptide). The CD90.1 marker gene is cut off via the P2A cleavage site and is not part of AIR. The constructs were generated as outlined in Example 4.

[0817] Table 6:

[0818]

[0819]

[0820]

[0821]

[0822] Example 14: Surface expression of CD40AIR with ICOS and 41BB co-stimulatory domains

[0823] Transduction and surface expression of AIR with ICOS and 41BB co-stimulatory domains were tested as described in Example 5.

[0824] The transduction efficiency of CD40 AIR with different co-stimulatory domains (CD28, ICOS, 41BB) was comparable as measured by flow cytometry analysis (data shown). Surface expression of CD40 AIR with ICOS and 41BB co-stimulatory domains is shown in Fig.37 It could be demonstrated that, like CD40 AIR with a CD28 costimulatory domain, CD40 AIR with an ICOS costimulatory domain or a 41BB costimulatory domain is also efficiently expressed on the surface of regulatory T cells.

[0825] Example 15: All costimulatory domains tested resulted in CD40AIR signaling

[0826] In this experiment, the signal transduction ability of CD40 AIR with three different costimulatory domains was compared. Treg cells expressing CD40-AIR forms with CD28, ICOS or 41BB costimulatory domains were left to stand for 24 hours and then co-cultured with HEK cells or HEK cells expressing mCD40L for 18 hours. Then, the expression of CD69 and LAP was analyzed by flow cytometry (one-way ANOVA).

[0827] The results are shown in Fig.38All constructs tested showed similar ability to induce Treg cell activation, confirming that any co-stimulatory domain can be used.

[0828] Example 16: CD40 ameliorates GvHD pathology in mice

[0829] As with LTBR AIR (see Example 8), the therapeutic utility of CD40 AIR was also tested in a mouse model of graft-versus-host disease (GvHD). Prior to transplantation, quality control of Treg cells was performed by flow cytometric analysis for transduction efficiency (CD90.1 expression) and CD40 AIR surface expression on Treg cells three days after transduction. As a control, a construct lacking the extracellular CD40 binding domain was used. The results are shown in Fig.39 Both constructs (CD40 AIR and control construct) showed high transduction efficiency, but only cells transfected with CD40 AIR showed binding to CD40.

[0830] The results of the experiment are shown in Fig.40 The Kaplan-Meier curve contains data sets from two independent experiments (log-rank test, n=10-12) and shows the survival of transplanted BALB / c mice. It can be seen that CD40 AIR is highly effective in the GvHD model, resulting in a significant increase in the survival of transplanted Balb / c mice. In summary, this example validates the clinical usefulness of the AIR disclosed herein in an established clinical model for graft-versus-host disease.

Claims

1. An artificial immune receptor comprising: b) the extracellular domain of members of the tumor necrosis factor receptor superfamily, c) transmembrane domain, d) a cytoplasmic co-stimulatory signaling domain, and e) Cytoplasmic CD3 T cell receptor signaling domain.

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

4. The artificial immunoreceptor of any one of claims 1-3, wherein the transmembrane domain is from the same protein as the extracellular domain.

5. The artificial immune receptor of any one of claims 1-4, 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. The artificial immunoreceptor of any one of claims 1-5, wherein the cytoplasmic co-stimulatory 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 to TLR9 receptors, IL-2, IL-7, and IL-15 receptors.

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

8. The artificial immunoreceptor of any one of claims 1-7, wherein the cytoplasmic co-stimulatory signaling domain comprises the amino acid sequence of SEQ ID NO:

35.

9. The artificial immunoreceptor of any one of claims 1-8, wherein the cytoplasmic CD3 T cell receptor signaling domain is selected from CD3ζ, CD3γ and CD3ε, preferably CD3ζ.

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

37.

11. The artificial immune receptor according to any one of claims 1 to 10, wherein a) the tumor necrosis factor receptor superfamily member and the transmembrane domain are from TNFR2, the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ, b) the tumor necrosis factor receptor superfamily member and the transmembrane domain are from DR3, the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ, c) the tumor necrosis factor receptor superfamily member and the transmembrane domain are from LTBR, ​​the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ, or d) the tumor necrosis factor receptor superfamily member and the transmembrane domain are from CD40, the cytoplasmic co-stimulatory signaling domain is CD28 and the cytoplasmic T cell receptor signaling domain is CD3ζ.

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

13. A vector comprising the nucleic acid according to claim 12.

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

15. The artificial immunoreceptor according to any one of claims 1 to 11 or the host cell according to claim 14, for use in medicine.

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