Regulatory T cells expressing chimeric antigen receptors

Abstract

The present invention provides regulatory T (Treg) cells expressing a chimeric antigen receptor (CAR), which comprises: a) at least one antigen-binding domain; b) a transmembrane domain; and c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least the costimulatory signaling domain of CD137, wherein the antigen-binding domain specifically binds to an antigen expressed on the surface of a target cell, or a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen. Also disclosed are compositions comprising the Treg cells and methods for enriching and analyzing activated Tregs expressing the CAR.

Description

[Technical Field]

[0001] The present invention relates to the field of chimeric antigen receptors expressed on immune cells, in particular on regulatory T cells. [Background technology]

[0002] Conventional T cells (Tcon) can be engineered to improve pathogen or tumor recognition and destruction. Regulatory T cells (Treg) comprise a subset of T cells with immunosuppressive functions. Treg can be engineered to prevent or treat autoimmunity, transplant rejection, allergies, and chronic inflammatory diseases.

[0003] Chimeric antigen receptors (CARs) have emerged as a promising option for generating antigen-specific regulatory T cells (Tregs). In addition to TCRs, CARs are artificial receptors that contain antibody-type specificity, allowing them to bind to surface antigens independently of MHC. Specific recognition of a particular antigen by CAR-T cells is mediated by an antibody-derived single-chain variable fragment (scFv) with an extracellular spacer domain linked to an intracellular TCR-derived signaling domain via a transmembrane region (Gross et al., 1989; Kuwana et al., 1987). In mouse models, redirected CAR-Tregs reactive against myelin basic protein (PB) can ameliorate EAE (Mekala and Geiger 2005), and CAR-Tregs specific for 2,4,6-trinitrophenol (TNP) or carcinoembryonic antigen (CEA) have been successfully redirected to the colon, where they are highly potent in suppressing colitis and the associated development of colorectal cancer (Blat et al. 2014; Elinav et al. 2009; Elinav et al. 2008). Recently, human CAR-Tregs redirected against HLA-A2, a commonly mismatched antigen in transplantation, have been shown to suppress xenogeneic GvHD (MacDonald et al. 2016; Noyan et al. 2017; Boardman et al. 2017). Furthermore, it has been demonstrated that CAR-Tregs have the potential to ameliorate allergic bronchitis (Skuljec et al. 2017) and prevent neutralizing immune responses to factor VIII in mice (Yoon et al. 2017). McGovern et al. (2017, Frontiers in Immunology, Vol. 8, Paper 1517, pp. 1-6) review the current state of the art regarding CAR-expressing Tregs.

[0004] To the best of our knowledge, the CAR constructs used so far for Tregs have a CD28 costimulatory domain.

[0005] Identifying disease-related target antigens as essential for in vitro generation of antigen-specific Tregs remains a major challenge. Furthermore, functional assays for analyzing CAR-Treg efficacy are limited by the lack of markers that allow specific identification of Tregs and testing their specific activation requirements. This is important for creating optimal Treg CAR constructs and optimizing Treg grafts, i.e., maximizing CAR-associated Treg functional activity and minimizing the contamination of effector T cells. Therefore, the requirements for activation and proliferation of CAR-Tregs, which may differ significantly from those of Tregs, remain poorly understood.

[0006] There is a need in the art for regulatory T cells that express a CAR that can be used to treat subjects to protect against or treat various immune-related disorders, such as autoimmunity, transplant rejection, allergy, or chronic inflammatory disease.

[0007] There is also a need in the art for the generation of highly purified and functionally optimized regulatory T cells that express a CAR that can be used to treat subjects to protect against or treat the immune-related disorders. Summary of the Invention

[0008] For therapeutic application, it is extremely important to have a pure population of Treg cells and be able to evaluate the effect of CAR constructs on Treg function, which may differ from the requirements of Tcon.However, in vitro Treg populations are generally contaminated with Tcon cells, and both populations are not easily separated from each other.Identifying and isolating true activated Treg from samples containing activated regulatory T cells and activated normal T cells is a requirement for the effect of specific CAR constructs on Treg in vitro and for the subsequent analysis of their functionality in vivo.Therefore, the problem of defining the conditions for optimal CAR-mediated activation of Treg functional activity and creating optimal CAR Treg has not yet been solved.

[0009] Such identification and separation can be carried out by methods such as those disclosed in EP2306191B1.

[0010] In this patent, the inventors compare various signaling domains known to activate conventional T cells (Tcon) and surprisingly show that use of the CD137 (4-1BB) costimulatory domain has a selective advantage in stimulating and expanding human regulatory T cells, which is important for optimizing Treg function for adoptive Treg therapy.

[0011] Figure 4 shows that, surprisingly, when a CAR contains a CD137 (4-1BB) costimulatory signaling domain (also referred to herein as a "CD137 CAR"), Tregs in vitro are better activated via the CAR (as indicated by CAR-ligand (antigen)-induced expression and proliferation of CD137) compared with a CAR with a CD28 costimulatory signaling domain (also referred to herein as a "CD28 CAR"), while the opposite is true for Tregs. Thus, the use of a CD137 CAR allows optimal stimulation of Tregs and can be utilized for in vitro selection of activated Tregs after CAR-ligand (antigen) stimulation to generate highly purified CAR-ligand (antigen)-reactive Tregs. It is also expected that CD137 CARs in vivo are better activated and expanded by CAR-ligand (antigen) than Tregs with a CD28 CAR, because in vivo Treg function strictly depends on the activation of a functional antigen receptor, which is mimicked by the CAR construct. Transduction or transfection of Tregs with a CAR as disclosed herein (a CAR with a CD137 costimulatory domain) allows for efficient in vitro activation of the engineered Treg cells via the CAR and provides a means for subsequent sorting of activated Tregs expressing the CAR. This allows for the generation of pure and functional Treg CAR populations that are beneficial for therapeutic use in subjects in need thereof, as they will offer better safety due to less contamination in vivo and improved therapeutic activity due to better CAR-ligand (antigen)-induced activation and proliferation.Furthermore, in certain embodiments of the present invention, CD137 CARs provide improved in vivo activation of Tregs in response to soluble antigens, such as dextran, applied as an external stimulus to a subject, compared to CD28 CARs or CARs with other signaling domains that do not respond to such external stimuli, and also enhance the in vivo activity of transferred Tregs, because they result in increased Treg proliferation and Treg activity, which in turn will result in improved responsiveness to endogenous TCRs or Tregs. Thus, CAR activation can be used to boost the natural regulatory or suppressive activity of Tregs against endogenous Treg antigens, without the need to know the specific Treg antigen target.

[0012] Tregs as disclosed herein are well suited for the prevention or treatment of subjects suffering from immune-mediated diseases such as autoimmunity, transplant rejection, allergy, or chronic inflammatory diseases.

[0013] In one embodiment of the present invention, the CD137 CAR expressed on Tregs is specific for the exogenous soluble antigen dextran. Administration of dextran with the dextran-specific CAR to a subject in need of treatment can enable a regulated and sustained immune response of Treg cells expressing the CAR for the prevention or treatment of autoimmunity, transplant rejection, allergy, or chronic inflammatory disease. [Brief explanation of the drawings]

[0014] [Figure 1] Generation of dextran-specific CAR-Tregs with different intracellular signaling domains. (A) Schematic of CAR constructs with different signaling domains. (B) LNGFR expression in CD25-enriched Tregs after lentiviral transduction is shown (n = 10-19 from 3-6 independent experiments). (C) Binding of soluble FITC-dextran is shown (n = 7-21 from 2-7 independent experiments). [Figure 2]Activation of dextran-specific CAR-Tregs with different intracellular signaling domains. (A) CD137 expression was analyzed after 6 hours of restimulation with bead-bound dextran, and CD137 expression in unstimulated samples was subtracted (n = 7-26, 2-8 separate experiments performed). (B) ZAP70 phosphorylation in LNGFR+ and LNGFR- Tregs was analyzed after 5 minutes of incubation with soluble dextran (n = 7, 2 independent experiments performed). [Figure 3] Expansion of dextran-specific CAR-Tregs with different intracellular signaling domains. Tregs were expanded in the presence of (A,C) anti-CD3 / CD28 or (B,D) bead-bound dextran. (A,B) The enrichment of LNGFR+ cells at day 17 was calculated as the ratio of LNGFR- / LNGFR+ Tregs at day 0 × the ratio of LNGFR+ / LNGFR- Tregs at day 17 (n = 13–18 from 4–6 independent experiments). (C,D) Tregs with different signaling domains were pooled, and the relative expression of the different signaling domains was quantified by qPCR (n = 7, 3 independent experiments). [Figure 4] Comparison of different intracellular signaling domains in Tregs and Tcon. (A) Dextran binding of CAR-Tregs and CAR-Tcon is shown. (B) Analysis of CAR-Treg (CD137 expression) and CAR-Tcon (CD154 expression) activation after 6 hours of stimulation with bead-bound dextran; expression in LNGFR- Tregs was subtracted for each sample as background, and CD137 and CD154 expression was normalized to the percentage of dextran+ cells in each culture. [Figure 5]Isolation of CAR-Tregs by activation-induced CD137 expression. Unstimulated LNGFR+ Tregs or CD137+ LNGFR+ Tregs after 6 hours of stimulation with bead-coupled dextran were sorted and expanded with anti-CD3 / 28 (sorted LNGFR+) or without further stimulation (sorted CD137+) for 14 days, followed by restimulation with bead-coupled dextran and staining for (A) LNGFR, (B) dextran, and (C) CD137 expression (n=12, 4 independent experiments for sorted LNGFR; n=15, 5 different experiments for sorted CD137). DETAILED DESCRIPTION OF THE INVENTION

[0015] In a first aspect, the present invention provides a regulatory T (Treg) cell expressing a chimeric antigen receptor (CAR), comprising: a) at least one antigen-binding domain; b) a transmembrane domain, c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least a costimulatory signaling domain of CD137 wherein the antigen-binding domain specifically binds to an antigen expressed on the surface of a target cell, a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen.

[0016] The CAR, wherein the at least one primary cytoplasmic signaling domain can be CD3ζ.

[0017] The CAR, wherein the antigen-binding domain can directly bind to an antigen expressed on the cell surface of a target cell. The target cell can be a cell in a disease state that expresses a disease-related autologous or allogeneic antigen. The disease can be an autoimmune disease, a chronic inflammatory disease, an allergy, a transplant rejection, GvHD, or a viral, bacterial, or parasitic infection in the subject.

[0018] Alternatively, the antigen-binding domain of the CAR can bind to the tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell. As a result, the antigen-binding domain of the CAR indirectly binds to the antigen expressed on the surface of a target cell. Such an adapter CAR approach is disclosed, for example, in US9,233,125B2. The tag can be a hapten such as biotin or FITC. The tagged polypeptide that binds to an antigen expressed on the surface of a cell can be an antibody or its antigen-binding fragment.

[0019] Alternatively, and preferably, the antigen binding domain of the CAR may be specific for a soluble antigen, such that binding of the soluble antigen to the antigen binding domain of the CAR allows activation of the Treg cell, preferably without the Treg cell binding to another cell.

[0020] The soluble antigen can be an exogenous antigen that is not naturally present in the blood or tissues of a subject, preferably a human, to whom the Treg cells expressing the CAR are to be applied. More preferably, the exogenous antigen that is not naturally present in the blood or tissues of a subject to whom the Treg cells expressing the CAR are to be applied does not preferentially bind to a target in the subject other than the antigen-binding domain of the CAR.

[0021] The exogenous soluble antigen may be a non-pathogenic antigen that does not induce harm to a subject when applied to the subject.

[0022] The (exogenous) soluble antigen may be dextran.

[0023] The antigen-binding domain of the CAR can comprise the sequences of SEQ ID NO: 1 and SEQ ID NO: 2. SEQ ID NO: 1 represents the variable domain of an immunoglobulin heavy chain (VH) and SEQ ID NO: 2 represents the variable domain of an immunoglobulin light chain (VL).

[0024] Alternatively, antigens (monovalent or multivalent) can be attached to biological surfaces, such as the surface of cells, or to tissue matrix compounds using specific adhesion molecules. In this way, antigens can be directed to specific surfaces, cell types, or organs in vivo, and attachment to the surface also improves CAR cross-linking. This results in localized and improved Treg activation.

[0025] In one embodiment, the present invention provides a composition comprising: i) Regulatory T (Treg) cells expressing a chimeric antigen receptor (CAR), a) at least one antigen-binding domain; b) a transmembrane domain, c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least a costimulatory signaling domain of CD137 wherein the antigen-binding domain specifically binds to a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell or a soluble antigen; ii) the tagged polypeptide to provide.

[0026] In a further aspect, the present invention provides Treg cells expressing a CAR as disclosed herein for use in treating or preventing autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, chronic inflammatory diseases such as inflammatory bowel disease, or chronic viral, bacterial, or parasitic infections in a subject.

[0027] In another aspect, the present invention provides a composition comprising a population of Treg cells that express a CAR as disclosed herein.

[0028] The composition of a population of Treg cells expressing a CAR as disclosed herein can comprise at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% Treg cells. The Treg cells can be CD25 + CD127 - FoxP3+ They may be characterized by a phenotype and / or a >80% demethylated TSDR (Treg Specific Demethylated Region) and / or lack of CD137 and CD154 expression after 5-7 hours of polyclonal stimulation with, for example, anti-CD3 / anti-CD28 molecules or pharmacological T cell activators such as PMA / ionomycin.

[0029] The composition, wherein the population of Treg cells expressing a CAR as disclosed herein can be a population of activated Tregs obtainable by a method for enriching activated Treg cells expressing a CAR as disclosed herein.

[0030] The composition may be a pharmaceutical composition, optionally including a pharmaceutically acceptable carrier.

[0031] In a further aspect, the present invention provides a pharmaceutical composition combination comprising: a) a population of Treg cells expressing a CAR as disclosed herein together with a pharmaceutically acceptable carrier; and b) a soluble antigen as disclosed herein The present invention provides a pharmaceutical composition comprising:

[0032] The population of Treg cells expressing a CAR as disclosed herein can comprise at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% Treg cells. + CD127 - FoxP3 + They can be characterized by phenotype and / or >80% demethylated TSDR and / or lack of CD137 and CD154 expression after 5-7 hours of polyclonal stimulation with, for example, anti-CD3 / anti-CD28 molecules or pharmacological T cell activators such as PMA / ionomycin.

[0033] Said pharmaceutical composition combination for the treatment or prevention of autoimmune diseases, allergies, transplant rejection, graft versus host disease, chronic inflammatory diseases such as inflammatory bowel disease, or chronic viral, bacterial or parasitic infections in a subject.

[0034] The soluble antigen may be dextran.

[0035] The combination of said pharmaceutical compositions, wherein said population of Treg cells expressing a CAR as disclosed herein can be a population of activated Tregs obtainable by a method for enriching activated Treg cells expressing a CAR as disclosed herein.

[0036] In another aspect, the present invention provides a method for enrichment of activated Treg cells expressing a CAR, wherein the CAR is i) at least one antigen-binding domain ii) transmembrane domain iii) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least the costimulatory signaling domain of CD137. wherein the antigen-binding domain specifically binds to an antigen expressed on the surface of a target cell, a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen; The method comprises: a) Providing a sample containing regulatory T cells b) genetically modifying the regulatory T cells of the sample to express the CAR c) activating the genetically modified regulatory T cells by contacting them with an antigen bound by the antigen-binding domain of the CAR for 6 to 16 hours. d) α) The cells of step c) I) contacting a molecule that binds to CD154 with CD154 + depleting T cells; or II) contacting the cells with a molecule that binds to a marker for regulatory T cells or activated regulatory T cells, and positively selecting cells that bind to the binding molecule; and β) I) contacting the cells of step α) I) with a molecule that binds to a marker for regulatory T cells or activated regulatory T cells, and positively selecting cells that bind to the binding molecule, thereby obtaining a population of activated regulatory T cells that express the CAR; or II) contacting the cells of step α)II) with a molecule that binds to CD154, + depleting T cells, thereby obtaining a population of activated regulatory T cells that express the CAR. and isolating the activated Treg cells in step c) by The present invention provides a method comprising:

[0037] Optionally, the method may comprise the step of expanding the genetically modified regulatory T cells after step b) and before step c).

[0038] The CAR and the antigen of the method may have the characteristics and properties of the CAR and antigen as already described above and disclosed herein. All of the above-disclosed variations and embodiments of the CAR and antigen as disclosed herein may also be applied to the method.

[0039] The method, wherein the marker for regulatory T cells is selected from the group of markers CD25 and GITR, and the marker for activated regulatory T cells is selected from the group of markers CD137, latent TGF-β (LAP), GARP (LRRC32), and CD121a / b.

[0040] The aforementioned method, wherein the molecule that binds to CD154, or a marker of regulatory T cells or activated regulatory T cells, can be an antibody or an antigen-binding fragment thereof.

[0041] The method, wherein the genetically modified regulatory T cells (step c) are expanded in the presence of anti-CD3 / CD28 and / or an antigen that binds to the antigen-binding domain of a CAR as disclosed herein and the addition of an appropriate growth factor, such as IL-2.

[0042] The method, wherein the isolation (separation) is performed using flow cytometry or magnetic cell sorting.

[0043] The above method, wherein the molecule that binds to CD154 and / or the molecule that binds to a marker for regulatory T cells or activated regulatory T cells is linked to a fluorescent dye, a hapten, and / or a magnetic particle.

[0044] The method, wherein the provided sample (step a) is derived from whole blood, PBMC, umbilical cord blood, lymph node tissue, bone marrow, or leukapheresis.

[0045] The method, wherein genetic modification of the regulatory T cells of the sample to express the CAR (step b) can be performed by methods well known in the art (e.g., viral-based systems, physical methods, biological methods, chemical methods).

[0046] The genetic modification of the Treg cells can be carried out by transduction, transfection, or electroporation.Preferably, the transduction is carried out by using lentivirus, gamma-, alpha-retrovirus, or adenovirus, or by electroporation or transfection with nucleic acid (DNA, mRNA, miRNA, antagomir, ODN), protein, site-specific nuclease (zinc finger nuclease, TALEN, CRISP / R), self-replicating RNA virus (e.g., equine encephalopathy virus), or integration-deficient lentiviral vector.More preferably, the genetic modification of the Treg cells can be carried out by transducing the cells with lentiviral vector.

[0047] The method, wherein the enriched population of activated Treg cells expressing the CAR comprises at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% Treg cells. + CD127 - FoxP3 + They are characterized by a phenotype and / or a lack of CD137 and CD154 expression after 5-7 hours of polyclonal stimulation with pharmacological T cell activators, e.g., CD3 / CD28 or PMA / ionomycin.

[0048] The method may be carried out in a closed system.

[0049] The method is an automated method in a closed system.

[0050] The present invention also provides the use of CD137 CARs expressed on Treg cells to select from various CARs the CAR that best enables Treg activation, by using at least two CARs that contain a CD137 signaling domain (CD137 CAR) but differ in at least one other component of the CAR, and compare their ability to activate Tregs expressing said CAR, for example, by contacting the CAR-expressing Tregs with a CAR ligand (antigen) and measuring and comparing the degree of induction of CD137 surface expression or another Treg activation marker known to be expressed after activation of Treg cells.

[0051] Therefore, in a further aspect, the present invention provides the use of a CAR expressed in regulatory T (Treg) cells for analyzing the activation efficiency of Treg cells, wherein said CAR a) at least one antigen-binding domain; b) a transmembrane domain, c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least a costimulatory signaling domain of CD137 wherein the antigen-binding domain specifically binds to an antigen expressed on the surface of a target cell, or to a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or to a soluble antigen.

[0052] In a further aspect, the present invention provides a method for analyzing (comparing) the activation efficiency of at least two Treg cells, wherein at least a first Treg cell is: a) at least one antigen-binding domain; b) a transmembrane domain, c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least a costimulatory signaling domain of CD137 expressing a first chimeric antigen receptor (CAR) comprising: at least a second Treg cell expresses a second CAR that differs from the first CAR in at least one domain of the first CAR, but expresses the costimulatory signaling domain of CD137; and the antigen-binding domain of the at least first CAR and the antigen-binding domain of the at least second CAR specifically bind to an antigen expressed on the surface of a target cell, or a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen; The method comprises: a) Providing a sample containing regulatory T cells b) genetically modifying said at least a first Treg cell to express said at least a first CAR, and genetically modifying said at least a second Treg cell to express said at least a second CAR. c) activating the genetically modified at least first Treg cells and at least second Treg cells by contacting them with an antigen bound by the antigen-binding domain of the at least first CAR and the at least second CAR for 6 to 16 hours. d) measuring the expression levels of Treg activation markers in the at least first Treg cells and the at least second Treg cells, wherein different expression levels indicate different activation efficiencies of the at least first CAR and the at least second CAR in Treg cells. The present invention provides a method comprising:

[0053] Comparing the strength of activation induced by contacting CARs with their ligands (antigens) allows for the optimization of CARs for use with Tregs by selecting CARs with the strongest activation capacity.

[0054] As used herein in the context of comparing the activation efficiency of at least two CARs in Treg cells, the term "domain of a CAR" refers to any domain that can be used in a functional CAR, and such domains can be, for example, the extracellular domain, transmembrane domain, and intracellular domain of a CAR. The extracellular domain can be at least one antigen-binding domain, a linker such as (G4 / S)3, and / or a spacer / hinge such as a CD8 hinge. The intracellular domain can be at least one stimulatory signaling domain and / or at least one costimulatory signaling domain. Differences that may exist between at least two CARs compared with each other can include modifications of domains that may be functionally related to the CAR, i.e., affecting stability, expression level, antigen binding, or ligand (antigen)-induced CAR-mediated signaling cascade, which ultimately affect CAR-mediated Treg activation. The term "modification" includes, for example, complete or partial replacement or deletion of such domain, adjusting the position of the domain within the CAR, or simply modifying the amino acid sequence of the domain, i.e., replacing, inserting, removing, etc., at least one amino acid in such domain.

[0055] The terms "Treg activation" or "activation efficiency in Treg cells" refer to changes in Treg gene expression patterns or functional changes induced by antigen receptor triggering. Treg activation is required in vivo to enable Tregs to exert their physiological functions, such as suppressing inappropriate or pathological immune responses, such as allergies, autoimmunity, graft-versus-host disease and graft rejection, IBD, and other chronic inflammatory diseases. Various parameters and methods are known in the art for measuring Treg activation, such as the expression of activation markers. The Treg activation markers can be selected from the group consisting of CD137, latent TGF-β (LAP), GARP (LRRC32), CD121a / b, or IL-10. Alternatively, functional assays, such as the suppression of responder T cell proliferation by co-culture with activated Tregs, are used. One specific parameter of Treg activation is the induction of CD137 and the simultaneous absence of CD154 after 4-7 hours of stimulation, which is a highly specific Treg activation signature. This can be measured by standard techniques known to those skilled in the art, such as fluorescent antibody staining and flow cytometry. Quantitative differences in Treg activation can be either the amount of CD137 expressed on a single cell, or the number or percentage of cells that are induced to express that marker.

[0056] The first CAR and the antigen of the method may have the characteristics and properties of the CAR and antigen as already described above and disclosed herein. All the variants and embodiments disclosed above for the CAR and antigen as disclosed herein may also be applied to the method.

[0057] The second CAR may be a variant of the first CAR, having at least one modification compared to the first CAR that may affect antigen binding characteristics and / or signaling capabilities, e.g., resulting in altered Treg cell activation properties of the second CAR compared to the first CAR.

[0058] The modification of the second CAR compared to the first CAR can be, for example, a different spacer, a different antigen-binding domain specific for the same antigen, a different transmembrane domain, and / or a different signaling domain.

[0059] The method, wherein the provided sample (step a) can be derived from whole blood, PBMC, umbilical cord blood, lymph node tissue, bone marrow, or leukapheresis.

[0060] The method, wherein genetic modification of the regulatory T cells of the sample to express the CAR (step b) can be performed by methods well known in the art (e.g., viral-based systems, physical methods, biological methods, chemical methods).

[0061] The genetic modification of the Treg cells can be carried out by transduction, transfection, or electroporation.Preferably, the transduction is carried out by using lentivirus, gamma-, alpha-retrovirus, or adenovirus, or by electroporation or transfection with nucleic acid (DNA, mRNA, miRNA, antagomir, ODN), protein, site-specific nuclease (zinc finger nuclease, TALEN, CRISP / R), self-replicating RNA virus (e.g., equine encephalopathy virus), or integration-deficient lentiviral vector.More preferably, the genetic modification of the Treg cells can be carried out by transducing the cells with lentiviral vector.

[0062] The present invention also provides a method for testing various antigen preparations for inducing Treg activation by contacting the antigen with a CD137 CAR, for example, by contacting the antigen sample with Tregs expressing a CAR having an antigen-binding domain, and comparing the Treg activation induced by the various antigen samples with at least a second antigen sample different from the first antigen sample, to determine the ability of the first antigen sample to activate CD137 CAR-expressing Tregs.

[0063] Therefore, in a further aspect, the present invention provides a method for analyzing (comparing) the activation efficiency of a first antigen sample to activate a CAR expressed in a Treg cell, together with a second antigen sample different from said first antigen sample, wherein said CAR is a) at least one antigen-binding domain; b) a transmembrane domain, c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least a costimulatory signaling domain of CD137 wherein the antigen-binding domain specifically binds to an antigen of the first antigen sample expressed on the surface of a target cell, or a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen; The method comprises: a) Providing a sample containing regulatory T cells b) genetically modifying the Treg cells to express the CAR c) activating the genetically modified Treg cells by contacting the antigen-binding domain of the CAR with the first antigen sample for 6 to 16 hours, and activating the genetically modified Treg cells by contacting the antigen-binding domain of the CAR with the second antigen sample for 6 to 16 hours. d) measuring Treg activation of the Treg cells contacted with the first antigen sample and the Treg cells contacted with a second antigen sample, wherein different activation levels indicate different efficiencies of the first antigen sample and the second antigen sample to activate the functional activity of the CAR expressed in Treg cells.

[0064] The first antigen sample and the second antigen sample can differ in the formulation of the antigen, e.g., soluble, monomeric versus multimerized antigen, antigen attached to various supports, e.g., culture dishes, microbeads of varying sizes, e.g., but not limited to, 50 nm to 50 μm, or biocompatible polymer matrices, e.g., dextran or other polysaccharides, or different antigens that can be bound by the same antigen-binding domain of a CAR, but with, e.g., different affinities or different conformational changes induced in the CAR.

[0065] A DNA or RNA construct (nucleic acid molecule) encoding a CAR as disclosed herein can be transfected or transduced into a host cell by methods well known in the art (e.g., viral-based systems including retroviruses and lentiviruses, physical methods including electroporation, biological methods, chemical methods). Regardless of the method used to integrate, preferably stably integrate, the resulting DNA encoding a CAR as disclosed herein in the host cell, the host cell will express the CAR as disclosed herein.

[0066] Alternatively, the nucleic acid sequence can be produced synthetically.

[0067] Engineered cells expressing an antigen-binding receptor as disclosed herein can be isolated (enriched or separated) from non-transfected / transduced cells following the transfection / transduction process to generate such engineered cells by methods well known in the art, for example, fluorescence-based separation technologies such as FACS®, or magnetic cell separation methods such as MACS® (Miltenyi Biotec GmbH).

[0068] Generally, cells such as immune cells, preferably T cells, can be obtained from a subject to generate engineered cells expressing antigen-binding receptors as disclosed herein. Cells such as immune cells, preferably T cells, can be obtained from various sources, such as whole blood, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, or other tissues containing T cells. These cells can be enriched using methods well known in the art, such as centrifugation through Ficoll™ or PERCOLL™ gradients, or positive / negative selection techniques, such as fluorescent sorting (e.g., FACSsort) or magnetic sorting (e.g., MACS™).

[0069] Illustratively, Tregs from a subject's blood or tissue sample are magnetically labeled, e.g., with magnetic beads linked to antibodies specific for CD25, washed, magnetically enriched, and collected. These Tregs can then be engineered to express antigen-binding receptors on their cell surface as disclosed herein.

[0070] In one embodiment of the present invention, isolated / enriched engineered cells, e.g., immune cells, preferably Treg cells, expressing an antigen-binding receptor as disclosed herein can be activated before or after genetic modification and expanded to expand the number of engineered cells using methods well known in the art, e.g., polyclonal stimulation of Tregs with a Treg expansion kit (Miltenyi Biotec) consisting of micron-sized particles conjugated to CD3- and CD28-binding antibodies in the presence of appropriate growth factors, such as IL-2. Preferably, the number of engineered immune cells, e.g., T cells, can be expanded to a therapeutically effective amount.

[0071] Genetically modified Treg cells expressing a CAR as disclosed herein can be produced in a closed, automated process. In one embodiment of the present invention, the process for producing genetically modified Treg cells expressing a CAR as disclosed herein comprises, for example, the following steps: a) Providing a sample containing regulatory T cells b) genetically modifying the regulatory T cells of the sample to express the CAR c) optionally expanding the genetically modified regulatory T cells d) activating the expanded genetically modified regulatory T cells by contacting them with a CAR ligand for 6 to 16 hours. e) α) The cells of step d) I) contacting a molecule that binds to CD154 with CD154 + depleting T cells; or II) contacting the cells with a molecule that binds to a marker for regulatory T cells or activated regulatory T cells, such as CD137, and positively selecting cells that bind to the binding molecule; and β) I) contacting the cells of step α) I) with a molecule that binds to a marker for regulatory T cells or activated regulatory T cells, such as CD137, to positively select cells that bind to the binding molecule, thereby obtaining a population of activated regulatory T cells that express the CAR; or II) contacting the cells of step α)II) with a molecule that binds to CD154, + depleting T cells, thereby obtaining a population of activated regulatory T cells that express the CAR. Step d) isolating activated Treg cells by may include:

[0072] All or some of these steps may be carried out automatically in a closed system, preferably a closed sterile system.

[0073] The process is particularly suitable for preparing genetically modified Treg cells, where enriched Treg cells are genetically modified by using viral and / or non-viral vectors.

[0074] Any of these steps may be added, omitted, or performed in a different order.

[0075] A fully automated cell processing device, CliniMACS Prodigy®, and associated tubing sets (Miltenyi Biotec GmbH, Germany) can be used as a closed system for cell modification (WO2009 / 072003). This closed system can meet the requirements for GMP-grade processing of almost any type of cell product, reduce cleanroom requirements, improve technology transfer, and enable harmonization of cell manufacturing processes.

[0076] In one embodiment of the present invention, engineered Tregs expressing a CAR as disclosed herein may be used to treat subjects suffering from disorders such as autoimmune, transplant rejection, allergy, or chronic inflammatory disease.

[0077] Tregs can be isolated from a subject, preferably a human, or an established immune cell line can be used. The subject can be suffering from the disorder or can be a healthy subject. These Treg cells are genetically modified in vitro to express a CAR as disclosed herein. These engineered Treg cells can be activated and expanded in vitro into a therapeutically effective population of cells expressing a CAR as disclosed herein, which can be further enriched to a higher purity by methods as disclosed herein before or after their modification. In cell therapy, these engineered Treg cells can be infused into a recipient in need thereof as a pharmaceutical composition (or a formulation of a therapeutically effective cell population expressing a CAR as disclosed herein) in addition to a second pharmaceutical composition, a soluble antigen that functions as an external stimulus for the Treg cells. The infused Treg cells in the recipient can suppress the subject's inflammatory immune response or at least reduce the effects and / or symptoms of the disorder during treatment. The recipient can be the same subject from whom the cells were obtained (autologous cell therapy), Or its cells, or from another subject of the same species (allogeneic cell therapy).

[0078] A population of Treg cells expressing a CAR as disclosed herein can be formulated for administration to a subject using techniques known to those of skill in the art.

[0079] The formulation comprising a therapeutically effective Treg cell population expressing a CAR as disclosed herein can contain a pharmaceutically acceptable excipient (carrier or diluent). The excipients included in the formulation have different purposes, for example, depending on the nature of the antigen-binding domain of the CAR as disclosed herein. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricants.

[0080] A formulation of a therapeutically effective Treg cell population expressing a CAR as disclosed herein can include one Treg cell population expressing a CAR as disclosed herein, or more than one cell population expressing a CAR as disclosed herein. Different Treg cell populations expressing a CAR as disclosed herein can differ, for example, based on the identity of the antigen-binding domain and / or the identity of the activation domain of the CAR used.

[0081] The preparation comprising the therapeutically effective Treg cell population that expresses CAR as disclosed herein can be administered to the subject by using methods and techniques known to those skilled in the art.Exemplary methods include but are not limited to intravenous injection.Other methods include but are not limited to intratumoral, intradermal, subcutaneous (sc, sq, sub-Q, Hypo), intramuscular (im), intraperitoneal (ip), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal and intrathecal (spinal fluid).

[0082] A formulation comprising a therapeutically effective Treg cell population expressing a CAR as disclosed herein that is administered to a subject comprises a number of Treg cells that express a CAR as disclosed that are effective in treating a particular indication or disorder.

[0083] Generally, about 1 x 10 cells expressing a CAR as disclosed herein 4 From about 1 x 10 10 A formulation containing between about 1 x 10 Treg cells expressing a CAR as disclosed herein can be administered. 5 From about 1 x 10 9 Between about 5 x 10 Treg cells expressing a CAR as disclosed herein 5 From about 5 x 10 8 up to about 1 x 10 Treg cells, or up to about 1 x 10 expressing a CAR as disclosed herein 6 From about 1 x 10 7 The number of Treg cells that can be administered to a subject and express a CAR as disclosed herein can vary within a wide range, depending on the location, source, identity, degree, and severity of the injury, the age and condition of the individual to be treated, etc. A physician can ultimately determine the appropriate dosage to be used.

[0084] Soluble antigens such as dextran can be formulated for administration to a subject using techniques known to those skilled in the art. Formulations of soluble antigens such as dextran can include pharmaceutically acceptable excipients (carriers or diluents). The excipients included in the formulation have different purposes, depending on, for example, the properties of the soluble antigen and the mode of administration. Examples of commonly used excipients include, but are not limited to, saline, buffered saline, dextrose, water for injection, glycerol, ethanol, and combinations thereof, stabilizers, solubilizers and surfactants, buffers and preservatives, tonicity agents, bulking agents, and lubricants.

[0085] The formulation of soluble antigen may contain one type of soluble antigen, or more than one type of soluble antigen.

[0086] Soluble antigens such as dextran can be administered to subjects by methods and techniques known to those skilled in the art.Exemplary methods include, but are not limited to, intravenous, intraperitoneal and intratumoral injection.Other methods include, but are not limited to, intradermal, subcutaneous (sc, sq, sub-Q, hypo), intramuscular (im), intraarterial, intramedullary, intracardiac, intraarticular (joint), intrasynovial (joint fluid area), intracranial, intraspinal and intrathecal (spinal fluid).

[0087] A formulation containing a soluble antigen, such as dextran, is administered to a subject in an amount effective to treat a particular indication or disorder. Generally, a formulation containing at least about 1 μg / kg to about 100 mg / kg of a soluble antigen, such as dextran, can be administered to a subject in need of treatment. In most cases, the dosage can be from about 100 μg / kg to about 10 mg / kg of a soluble antigen, such as dextran, daily, weekly, or monthly, taking into account the route of administration, symptoms, etc. However, the amount of a soluble antigen, such as dextran, in a formulation administered to a subject can vary within a wide range, depending on the location, source, identity, extent, and severity of the disorder, the age and condition of the individual being treated, etc. A physician can ultimately determine the appropriate dosage to be used.

[0088] All definitions, characteristics and embodiments defined herein in relation to one aspect of the invention, for example the first aspect of the invention, also apply mutatis mutandis in relation to other aspects of the invention as disclosed herein.

[0089] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0090] Generally, a CAR may comprise an extracellular domain (extracellular portion) containing an antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (intracellular signaling domain). The extracellular domain may be linked to the transmembrane domain by a linker. The extracellular domain may also comprise a signal peptide. In some embodiments of the present invention, the antigen-binding domain of a CAR binds to a hapten linked to a polypeptide (a "haptenized" or "tagged" polypeptide), which may bind to a disease-associated antigen such as an autoantigen, or an antigen derived from an innocuous exogenous substance, e.g., an airborne particle such as a microbiota, plant pollen, or fungal spore. Such a CAR may also be termed an "anti-tag" CAR, as disclosed, for example, in US9233125B2. In other embodiments of the present invention, the extracellular portion of a CAR may comprise a linker / tag epitope (LLE)-binding domain as an antigen-binding domain that binds to a linker / tag epitope (LLE) that is part of a TCBM. Such CARs may be termed anti-LLE CARs, as disclosed in European Patent Application No. EP16196487.9. Both types of CARs are versatile and / or adaptable CARs. Both haptens and LLEs are "tags" that are directly or indirectly linked to a polypeptide (tagged polypeptide), which can bind to disease-associated antigens, such as autoantigens, expressed on the surface of target cells, or to harmless exogenous substances, such as airborne particles, such as microbiota, plant pollen, or fungal spores. In another embodiment of the present invention, the antigen-binding domain of the CAR binds to a soluble antigen, as disclosed herein.

[0091] A "signal peptide" refers to a peptide sequence that directs the transport and localization of a protein within a cell, for example, to a particular cellular organelle (eg, the endoplasmic reticulum) and / or to the cell surface.

[0092] Generally, "antigen-binding domain" refers to the region of a CAR that specifically binds to an antigen, e.g., a soluble antigen. The CAR of the present invention may comprise one or more antigen-binding domains. Generally, the targeting region on a CAR is extracellular. The antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof. The antigen-binding domain may comprise, for example, a full-length heavy chain, a Fab fragment, a single-chain Fv (scFv) fragment, a bivalent single-chain antibody, or a diabody. Any molecule that specifically binds to a predetermined antigen, such as an affibody, or a ligand-binding domain derived from a naturally occurring receptor, may be used as the antigen-binding domain. Often, the antigen-binding domain is an scFv. Typically, in an scFv, the variable regions of an immunoglobulin heavy chain and a light chain are fused by a flexible linker to form the scFv. Such a linker may be, for example, a "(G4 / S)3 linker."

[0093] In some cases, it is beneficial that the antigen binding domain is derived from the same species as the species that CAR is used in.For example, when it is planned to be used in human therapeutically, it can be beneficial that the antigen binding domain of CAR comprises human or humanized antibody or its antigen binding fragment.Human or humanized antibody or its antigen binding fragment can be produced by various methods well known in the art.

[0094] As used herein, "spacer" or "hinge" refers to a hydrophilic region located between the antigen-binding domain and the transmembrane domain. The CAR of the present invention may include an extracellular spacer domain, but may also exclude such a spacer. The spacer may include, for example, an Fc fragment or fragment thereof of an antibody, a hinge region or fragment thereof of an antibody, a CH2 or CH3 region of an antibody, an accessory protein, an artificial spacer sequence, or a combination thereof. A notable example of a spacer is the CD8α hinge. The transmembrane domain of the CAR may be derived from any desired natural or synthetic source for such a domain. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The transmembrane domain may be derived from, for example, CD8α or CD28. The important signaling module (domain) and antigen recognition module (domain) are located on two (or even more) polypeptides, and as a result, the CAR may have two (or more) transmembrane domains. Its separate key signaling and antigen recognition modules allow small molecule-dependent, titratable, and reversible regulation of CAR cell expression by small molecule-dependent heterodimerization domains in each polypeptide of the CAR (Wu et al., 2015, Science 350:293-303).

[0095] The cytoplasmic signaling domain (or intracellular signaling domain) of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. "Effector function" refers to the specialized function of a cell; for example, in Tregs, effector function can be suppressive or regulatory activity, including secretion of immunosuppressive cytokines such as IL-10, IL-35, TGF-β, or expression of inhibitory molecules such as TIGIT, CTLA4, and competitive cytokine receptors such as IL-2 receptor. The intracellular signaling domain refers to the portion of a protein that transmits an effector function signal and instructs the cell expressing the CAR to perform a specialized function. The intracellular signaling domain can include any complete, mutated, or truncated portion of the intracellular signaling domain of a given protein sufficient to transmit a signal that initiates or blocks immune cell effector function.

[0096] Prominent examples of intracellular signaling domains used in CARs include the cytoplasmic signaling sequences of the T cell receptor (TCR) and co-receptors, which initiate signal transduction following antigen receptor binding.

[0097] Generally, T cell activation can be mediated by two different classes of cytoplasmic signaling sequences: first, those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences, primary cytoplasmic signaling domains), and second, those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences, costimulatory signaling domains). Thus, the intracellular signaling domain of a CAR can comprise one or more primary cytoplasmic signaling domains and / or one or more secondary cytoplasmic signaling domains.

[0098] The primary cytoplasmic signaling domain, which acts in a stimulatory manner, may contain an ITAM (immunoreceptor tyrosine-based activation motif).

[0099] Examples of ITAM-containing primary cytoplasmic signaling domains often used in CARs are those derived from TCRζ (CD3ζ), FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, with the CD3ζ-derived sequence being the most prominent.

[0100] The cytoplasmic domain of the CAR can be designed to include a CD3ζ signaling domain alone or in combination with any other desired cytoplasmic domain. The cytoplasmic domain of the CAR can include a CD3ζ chain portion and a costimulatory signaling region (domain). The costimulatory signaling region refers to the portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for the efficient response of lymphocytes to antigens. Examples of costimulatory molecules are CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0101] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in a random or specified order, with or without a linker. A short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the linkage. A prominent linker is a glycine-serine doublet.

[0102] For example, the cytoplasmic domain may comprise the signaling domain of CD3ζ and the signaling domain of CD28. In another example, the cytoplasmic domain may comprise the signaling domain of CD3ζ and the signaling domain of CD137. In a further example, the cytoplasmic domain may comprise the signaling domain of CD3ζ, the signaling domain of CD28, and the signaling domain of CD137.

[0103] As used herein, the term "CD137 CAR" includes at least a CD137 costimulatory signaling domain in addition to at least a primary cytoplasmic signaling domain. As used herein, the term "CD28 CAR" includes at least a CD28 costimulatory signaling domain in addition to at least a primary cytoplasmic signaling domain. As mentioned above, either the extracellular portion of the transmembrane domain or the cytoplasmic domain of the CAR may also include a heterodimerization domain for the purpose of separating the important signaling and antigen recognition modules of the CAR.

[0104] CARs can be further modified to include one or more operating elements at the level of the nucleic acid encoding the CAR to eliminate CAR-T cells or Treg cells by a suicide switch. Suicide switches can include, for example, an apoptosis-inducing signaling cascade or a drug that induces cell death. In one embodiment, the nucleic acid that expresses and encodes the CAR can be further modified to express an enzyme such as thymidine kinase (TK) or cytosine deaminase (CD).

[0105] The CARs of the present invention can be designed to include any part or portions of the above-referenced domains as described herein, in any order and / or combination that results in a functional CAR, i.e., a CAR that mediates an immune effector response of an immune effector cell expressing the CAR, but that includes at least a CD137 costimulatory domain.

[0106] As used herein, the term "antibody" is used in the broadest sense to encompass various forms of antibody structures, including, but not limited to, monoclonal and polyclonal antibodies (including full-length antibodies), multispecific antibodies (e.g., bispecific antibodies), antibody fragments, i.e., antigen-binding fragments of antibodies, immunoadhesins that specifically recognize (i.e., bind) a target antigen, and antibody-immunoadhesin chimeras. An "antibody fragment" comprises a portion of a full-length antibody, preferably the variable domain thereof or at least the antigen-binding site thereof ("antigen-binding fragment of an antibody"). Examples of antibody fragments include Fab (fragment antigen binding), scFv (single-chain fragment variable), single-domain antibodies, diabodies, dsFv, Fab', diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0107] As used herein, the term "antigen" is intended to include a substance that binds to or elicits the production of one or more antibodies, and may include, but is not limited to, proteins, peptides, polypeptides, oligopeptides, lipids, carbohydrates such as dextrans, haptens, and combinations thereof, e.g., glycosylated proteins or glycolipids.

[0108] The term antigen may refer to an antigen expressed on the cell surface of a target cell. However, the term may also refer to an antigen that is not expressed or present on the surface of a cell in a subject that can be treated with, for example, an engineered Treg expressing a CAR as disclosed herein. As a result, the antigen is referred to herein as a "soluble antigen." As used herein, the terms "soluble antigen" and "free antigen" can be used interchangeably and mean that a soluble antigen that can be bound by the antigen-binding domain of a CAR as disclosed herein is not naturally expressed or present on the surface of cells in the subject, preferably a human, when the Treg expressing the CAR is administered to the subject. Preferably, the soluble antigen is not present in the blood or tissues of the subject to which the Treg cells expressing the CAR are administered. More preferably, it also does not have a specific affinity or preference for binding to another molecule in the subject rather than to the antigen-binding domain of the CAR. Preferably, the soluble antigen may be an exogenous antigen. The exogenous antigen can be applied to a subject that also receives or has received the Treg expressing the CAR for the treatment of a disorder as disclosed herein, and is an external stimulator (external stimulus) that can bind to the antigen-binding domain of the CAR as disclosed herein and then activate the Treg cells expressing the CAR. The exogenous soluble antigen can be a non-pathogenic antigen that does not cause harm to the subject when applied to the subject. The exogenous soluble antigen can be, for example, a macromolecule such as a polypeptide or polysaccharide that is preferably not naturally occurring in the subject that can be treated as disclosed herein.

[0109] The soluble antigen may preferably be selected from the group consisting of macromolecules such as proteins, polysaccharides, oligo- or polynucleotides, polyethylene glycol, or any other biocompatible polymeric compounds that can be applied to humans, or derivatives of such molecules, such as small molecule "haptens" linked to larger macromolecules. The soluble antigen may be applied in a manner that allows activation of the CAR, which is typically achieved by cross-linking; that is, the macromolecule used may contain more than one copy, ideally several copies, of the actual domain bound by the antigen-binding domain of the CAR. Such multivalent molecules can induce cross-linking and CAR activation. Preferably, the only requirement for the (exogenous) soluble antigen is that it can circulate in the circulatory system, e.g., the blood system or lymphatic system, and / or tissues of the subject to be treated as disclosed herein, and is not part of the surface of the subject's cells. As a result, the (exogenous) soluble antigen can only be bound by the antigen-binding domain of the CAR as disclosed herein expressed by the Treg cells when the Treg cells are applied to the subject. Thus, (exogenous) soluble antigens can also be immobilized on structures such as beads (nanobeads, microbeads), allowing the antigens immobilized on such structures to circulate in the circulatory system, e.g., blood system, of said subject, which can also be (exogenous) soluble antigens within the meaning of the present invention.

[0110] A preferred soluble antigen is dextran (dextran molecule), which can be administered to a subject in need of treatment with the Tregs expressing the CAR as a free dextran molecule or as a dextran molecule immobilized on particles such as microbeads or nanobeads.

[0111] Dextran is a complex branched glucan (a polysaccharide made up of many glucose molecules) composed of chains of various lengths (from 3 kilodaltons to 2000 kilodaltons). Dextran of any length, for example, from 3 kDa to 2000 kDa, can be used in the applications disclosed herein. Preferably, the dextran used may be greater than 5 kDa, greater than 10 kDa, greater than 20 kDa, greater than 100 kDa, or greater than 200 kDa. In some embodiments of the present invention, the dextran used may be a dextran from 60 kDa to 200 kDa. Soluble dextran as used herein can present multiple antigens to CAR that bind to the dextran as disclosed herein. Dextran can be a polyantigen instead of a monoantigen to anti-dextran CAR.

[0112] The dextran can be unconjugated dextran, i.e., free dextran, soluble dextran, or dextran conjugated to colloidal nano- or microparticles, and can be administered to a patient in need thereof who has Treg cells as disclosed herein to activate the Treg cells under regulatable conditions.

[0113] The dextran can also be administered to a subject as part of a pharmaceutical composition (e.g., Deltadex; 10% dextran in 40 NaCl; e.g., injection of 1.5 g or less of dextran per kg of body weight). Alternatively, the dextran can be conjugated to an antibody or another attachment structure, such as an extracellular matrix attachment peptide, that allows specific targeting of the dextran to the surface of a cell or tissue matrix in vivo.

[0114] Tregs (also termed "regulatory T cells" or "Treg cells" herein) typically express Foxp3, which also expresses CD25 and lacks expression of CD127. + CD4 +Tregs are defined herein as T cells. Tregs are further characterized by selective expression of CD137 upon activation, but lack of CD154 expression within a 4-8 hour time frame of activation, as well as lack of effector cytokine expression, e.g., IL-2-IFNγ, IL-17, IL-4, etc. Tregs are also characterized by selective demethylation of specific DNA regions, e.g., within the foxp3 gene region (Treg-specific demethylation region, TSDR; see also Huehn, J. et al., 2009, Nat Rev Immunol 9, 83-9), as well as other specific methylation patterns in other regions, such as the CD25, CTLA4, FANK1, CD137, and CD154 gene regions. They represent a distinct T cell lineage with high immunosuppressive function required to maintain tolerance to self-antigens and harmless foreign antigens. Tcon cells, as defined herein, are all CD4 T cells that are not Tregs. + Contains T cells.

[0115] As used herein, the term "target cell" refers to a cell that can express an antigen on its cell surface that is to be recognized (bound) directly or indirectly (e.g., via a tagged polypeptide) by a CAR as disclosed herein.

[0116] The target cells can be cells in disease states that cause autoimmunity, transplant rejection, allergy, and chronic inflammatory diseases in a subject.

[0117] Autoimmunity refers to a condition in which immune cells are directed against the self, resulting in an immune response against endogenous structures, which can lead to autoimmune disease. Graft rejection refers to the development of an immune response against transplanted tissue that is recognized as foreign by the host's immune system, resulting in rejection of the transplanted tissue.

[0118] Allergy refers to the development of an inappropriate immune response to a harmless foreign antigen, which may be derived, for example, from the environment or food, when the subject comes into contact with it, for example, by inhalation, ingestion, or skin contact.

[0119] Chronic inflammatory disease refers to the development of an immune response against antigens that remain in the system. Examples include, for example, an immune response against bacteria during inflammatory bowel disease, an immune response against viruses during chronic infection, or an immune response against endogenous structures during autoimmune reactions. Examples also include chronic inflammation resulting from an allergic response to foreign antigens. Autoimmune diseases are conditions resulting from autoimmunity that can affect multiple different organ systems. Examples include rheumatoid arthritis, multiple sclerosis, neuromyelitis optica, systemic lupus erythematosus, and type 1 diabetes. Chronic infection of a subject with a virus, bacteria, or parasite refers to the invasion of a subject by a disease-causing agent, such as a virus, bacteria, or parasite, followed by their replication.

[0120] The CAR (polypeptide), nucleic acid molecule encoding a CAR, recombinant expression vector, cell expressing a CAR, and cell population expressing a CAR as disclosed herein can be isolated and / or purified. The term "isolated" means changed or removed from a natural state. For example, an isolated cell population refers to the enrichment of such cells and their separation from other cells that normally accompany the isolated cells in their naturally occurring state. An isolated cell population refers to a substantially purified population of cells that is a more homogeneous cell population than that found in nature. Preferably, an enriched cell population contains at least about 90% of a selected cell type. In certain embodiments, a cell population contains at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% of a selected cell type.

[0121] For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can also exist in a non-native environment, such as, for example, a host cell.

[0122] As used herein, the term "subject" refers to a mammal, such as a mouse, rat, cow, pig, goat, chicken, dog, monkey, or human. Preferably, the subject is a human. The subject may be a subject (patient) suffering from a disorder such as an autoimmune disease, allergy, transplant rejection, or chronic inflammation, but the subject may also be a healthy subject.

[0123] As used herein, the term "autologous" refers to any material that originated from the same subject into which it is later reintroduced.

[0124] As used herein, the term "allogeneic" refers to any material that originated from a different subject of the same species as the subject into which the material is being reintroduced.

[0125] The term "therapeutically effective amount" or "therapeutically effective population" refers to the amount of a cell population that provides a therapeutic benefit in a subject.

[0126] For example, when used in a CAR as disclosed herein, the term "specifically binds" or "specific for" with respect to an antigen-binding domain of an antibody or antigen-binding fragment thereof refers to an antigen-binding domain that recognizes and binds to a specific antigen in a sample but does not substantially recognize or bind to other molecules. An antigen-binding domain that specifically binds to an antigen from one species may also bind to that antigen from another species. This interspecies cross-reactivity is typical for many antibodies and therefore does not violate the definition of the antigen-binding domain as specific. An antigen-binding domain that specifically binds to an antigen may also bind to different allelic forms of the antigen (such as allelic variants, splice variants, isoforms, etc.) or homologous variants of this antigen from the same gene family. This cross-reactivity is typical for many antibodies and therefore does not violate the definition of the antigen-binding domain as specific.

[0127] As used herein, the terms "engineered cells" and "genetically modified cells" can be used interchangeably. The terms refer to cells that contain and / or express a foreign gene or nucleic acid sequence that subsequently modifies the genotype and / or phenotype of the cell or its progeny. In particular, the terms refer to the fact that cells, preferably immune cells, can be engineered by recombinant methods well known in the art to stably or transiently express peptides or proteins that are not naturally expressed in these cells. For example, immune cells can be engineered to express artificial constructs, such as chimeric antigen receptors, on their cell surface.

[0128] The term "disorder" means a functional abnormality or disturbance in a subject, such as cancer, an autoimmune disorder, or a viral, bacterial, parasitic, or other infection.

[0129] As used herein, the term "treating" a disorder means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0130] Immunotherapy is a medical term defined as "the treatment of disease by inducing, enhancing, or suppressing an immune response." Immunotherapies designed to induce or amplify an immune response are classified as activating immunotherapies, while immunotherapies that reduce or suppress the immune response are classified as suppressing immunotherapies. Cancer immunotherapy, as an activating immunotherapy, attempts to stimulate the immune system to reject and destroy tumors. Adoptive cell transfer uses a cell-based cytotoxic response to attack cancer cells. Immune cells, such as T cells, with natural or genetically engineered reactivity against a patient's cancer are generated in vitro and then transferred back into the cancer patient.

[0131] As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter in a cell.

[0132] The amino acid sequences of SEQ ID NO:1 and SEQ ID NO:2 as shown in the sequence listing protocol are partial sequences of CAR as disclosed herein. The sequences of SEQ ID NO:1 and SEQ ID NO:2 may also include variants of these sequences that have some amino acid deletions, additions, or substitutions while still retaining the intended function as described herein. Thus, variants of the amino acid sequences in SEQ ID NO:1 and SEQ ID NO:2 are included in this definition, such as amino acid sequences that are essentially similar to SEQ ID NO:1 and SEQ ID NO:2, respectively, and have at least 70%, or at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity at the amino acid sequence level. Generally, all amino acid variations that do not result in an intended change in the intended function of the sequences of SEQ ID NO:1 and SEQ ID NO:2 are included in this definition. In the context of the present invention, "sequence identity" can be determined by pairwise alignment using amino acid sequence alignment programs well known in the art.

[0133] The "circulatory system" is the organ system of interest that enables blood to circulate and transport nutrients (e.g., amino acids and electrolytes), oxygen, carbon dioxide, hormones, and blood cells to and from cells in the body to provide nutrients and help fight disease, stabilize temperature and pH, and maintain homeostasis. The circulatory system includes two separate systems: the cardiovascular system, which distributes blood, and the lymphatic system, which circulates lymphatic fluid.

[0134] As used herein, the term "automated method" or "automated process" refers to any process that would otherwise be or can be performed manually by an operator and that is automated through the use of a device and / or a computer and computer software. An automated method (process) does not require significant human intervention or human time for delivery. In some cases, a method is automated if at least one step of the method is performed without any human assistance or intervention. Preferably, a method is automated if all steps of the method are performed without human assistance or intervention.

[0135] As used herein, the term "particle" refers to a solid phase such as a colloidal particle, a microsphere, a nanoparticle, or a bead. Methods for producing such particles are well known in the art. The particles may be magnetic particles. The particles may be in a solution or suspension, or they may be lyophilized before use in the present invention. The lyophilized particles are then reconstituted in a convenient buffer before contacting with the sample to be treated in accordance with the present invention.

[0136] Particularly powerful sorting technology is magnetic cell sorting.Methods for magnetically separating cells are commercially available from several suppliers.In a preferred embodiment, for example, for enriching, sorting and / or detecting cells in cell-containing biological samples for Treg cells and other cells (immune cells), monoclonal antibodies or their antigen-binding fragments are used together with colloidal superparamagnetic microparticles, for example, with polysaccharide organic coating (Magnetic Activated Cell Sorting (MACS®) technology (Miltenyi Biotec, Bergisch Gladbach, Germany)).

[0137] Another sorting technology uses flow cytometry. Flow cytometry is a laser- or impedance-based biophysical technology used for cell sorting and biomarker detection, for example, by suspending cells in a fluid stream and passing them through an electronic detection device. Flow cytometry allows for simultaneous multiparameter analysis of the physical and chemical properties of up to thousands of particles per second. Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence properties of each cell.

[0138] As used herein, the terms "depletion," "depleting," and the like in the context of isolating, purifying, or enriching cells have their ordinary meaning in the art and refer to the removal of specialized cells (e.g., CD154) from a sample containing Tregs and other (immune) cells. + Depletion refers to the removal of specialized cells (e.g., CD154 cells) in a population. Methods for depletion are well known in the art and described herein, and include, for example, the removal of specialized cells (e.g., CD154 cells) in a population. +Examples of such techniques include FACS or MACS sorting, in which specialized cells (e.g., IgG, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG1, IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG1, IgG1, IgG2, IgG4 ...4, IgG5, IgG6, IgG1, IgG1, IgG2, IgG4, IgG4, IgG4, IgG4, IgG5, IgG6, IgG1, IgG1, IgG2, IgG4, IgG4, IgG4, IgG4, IgG4, IgG5, IgG6, IgG1, IgG1, IgG2, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4, IgG5, IgG6, IgG6, IgG1, IgG1, IgG1, IgG2, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4, IgG4,

[0139] As used herein, the term "positive selection" in the context of cell isolation, purification, or enrichment has its usual meaning in the art and refers to the isolation, purification, or enrichment of specialized cells (e.g., CD137) from a sample containing Tregs and optionally other (immune) cells. + Positive selection refers to the enrichment of specialized cells (e.g., CD137 cells) in a population. Methods for positive selection are well known in the art and described herein, and include, for example, enrichment of specialized cells (e.g., CD137 cells) in a population. + Examples include FACS or MACS sorting, in which specialized cells are labeled and isolated from a cell population, and the isolated cells give rise to a new population in which the specialized cells are present in a higher proportion than in the starting population.

[0140] Embodiment In one embodiment of the present invention, Tregs are generated that express CARs, which comprise CD3ζ and CD137 signaling domains and antigen binding domains specific to exogenous antigens, such as dextran.The DNA construct encoding CARs can be transfected or transduced into Treg cells by methods well known in the art (for example, virus-based systems, physical methods, biological methods, chemical methods).Regardless of the method used to integrate, preferably stably integrate, the nucleic acid encoding CARs into Treg cells, the resulting Treg cells express CARs.These Treg cells can be activated in vitro and in vivo by adding dextran to cells in cell culture or cells circulating in the blood of the subject to which they are applied.

[0141] In another embodiment of the invention, Tregs expressing a CAR of the invention are isolated by isolation of cells expressing CD137 in combination with or without CD154 by either magnetic or fluorescent sorting after antigen-specific activation with the respective antigen, e.g., dextran.

[0142] In embodiments of the present invention, Tregs can be obtained from various sources, such as peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, or thymus tissue. Enrichment of these cells can be performed using methods well known in the art, such as centrifugation through Ficoll™ or PERCOLL™ gradients, or positive / negative selection techniques such as fluorescent sorting (e.g., FACSsort) or magnetic sorting (e.g., MACS®).

[0143] In one embodiment, a given source of Tregs from a subject are magnetically labeled, washed, magnetically enriched, and collected, e.g., with magnetic beads linked to antibodies specific for CD4 and / or CD25 and / or CD127 and / or CD154 and / or CD137. These Treg cells can then be engineered to express CD137-CD3ζ-CAR on their cell surface.

[0144] In another embodiment of the invention, engineered Tregs expressing a CAR of the invention are isolated after the transfection / transduction process by methods well known in the art, for example, fluorescence-based separation technologies such as FACS®, or magnetic cell separation methods such as MACS®.

[0145] In one embodiment of the present invention, engineered Tregs expressing CD137-CD3ζ-CAR are expanded in the presence of exogenous antigen (e.g., dextran) or polyclonal stimulation with anti-CD3 / anti-CD28 to increase the number of engineered Tregs and to increase the purity of Tregs expressing CD137-CD3ζ-CAR. Preferably, the amount of engineered Tregs is increased to a therapeutically effective amount.

[0146] In one embodiment of the present invention, highly pure Tregs (e.g., >80% FoxP3 expression) are genetically engineered to express CD137-CD3ζ-CAR.

[0147] In one embodiment of the present invention, highly pure Tregs (eg, >80% FoxP3 expression) are isolated by expression of CD137 in combination with other markers, eg, CD25, CD127, CD154.

[0148] In one embodiment of the present invention, CD137-CD3ζ-CAR is used to treat subjects with an inflammatory or autoimmune disease, such as inflammatory bowel disease, rheumatoid arthritis, multiple sclerosis, or transplant rejection or graft-versus-host disease (GvHD).

[0149] In one embodiment of the present invention, CD137-CD3ζ-CAR is activated by application of exogenous antigen (e.g., dextran) in soluble or bead-immobilized form, either at a local site or systemically, preferably in patients with inflammatory or autoimmune diseases, such as IBD, rheumatoid arthritis, MS, graft rejection, GvHD. [Example]

[0150] Generation of dextran-specific CAR-Tregs with different intracellular signaling domains The CAR construct contains a specific binding fragment derived from an antibody specific for an exogenous antigen (e.g., dextran). The hinge region can be derived from an IgG domain, CD8a, or CD28 and can include an epitope / tag that allows for detection of the CAR. For example, as shown in Figure 1A, the transmembrane domain can be derived from, for example, CD8a or CD28, followed by one to three signaling domains containing CD3ζ and CD137. Tregs can be engineered to express CD137-CD3ζ-CAR, which can be determined by expression of LNGFR (Figure 1B). Antigen binding of CD137-CD3ζ-CAR can be determined by incubation with the respective antigen, which can be labeled (e.g., fluorescently), as shown for dextran in Figure 1C. [Example]

[0151] Activation of CAR-Tregs with different intracellular signaling domains CAR-Tregs specific for exogenous antigens (e.g., dextran) can be activated by their respective antigens, and activation can be analyzed by CD137 expression. CAR-Treg activation after stimulation with bead-bound dextran is shown in Figure 2A. CD137-CD3ζ-CAR was more potent in inducing CD137 expression in CAR-Tregs (Figure 2A). The functionality of other tested CAR constructs with the same specificity was analyzed by ZAP70 phosphorylation. Phosphorylated ZAP70 was detected in CAR-Tregs with CD28-CD3ζ signaling, for example (Figure 2B), but only CD137-CD3ζ-CAR induced Treg activation (Figure 2A). [Example]

[0152] Expansion of dextran-specific CAR-Tregs with different intracellular signaling domains CAR-Tregs specific for exogenous antigens (e.g., dextran) can proliferate in the presence of anti-CD3 / CD28 (Figure 3A, C) or their respective antigens, e.g., bead-bound dextran (Figure 3B, D). Only CAR-Tregs with CD137-CD3ζ-CAR proliferated, indicating superior functionality of CD137-CD3ζ-CAR. [Example]

[0153] Comparison of different intracellular signaling domains in Tregs and Tcons CAR-Treg and CAR-Tcon constructs expressing anti-dextran CARs with different costimulatory domains in combination with CD3ζ were generated. Dextran binding was similar between constructs (Figure 4A), but the different signaling domains resulted in different effects on Treg and Tcon activation. Treg activation was analyzed by CD137 expression, and Tcon activation was analyzed by CD154 expression. CAR-Tregs were most efficiently activated with CD137-CD3ζ, and CAR-Tcon with CD28-CD3ζ (Figure 4B). [Example]

[0154] Isolation of antigen-specific CAR-Tregs CAR-Tregs bearing the CD137-CD3ζ CAR were isolated by LNGFR or CD137 expression after 6 hours of dextran stimulation. Although transgene (Figure 5A) and receptor expression (Figure 5B) were similar between both sorting strategies, antigen-specific restimulation was highly efficient when CAR-Tregs were sorted by CD137 expression (Figure 5C).

[0155] method CAR constructs All CAR constructs contained the AC146-derived scFv, CD8 transmembrane domain, XS IgG4 hinge, and P2A-linked ΔLNGFR for transfection and detection of transduced cells. Lentiviral supernatants were generated by co-transfection of HEK293T cells with the expression vector and packaging plasmids. One day before transfection, 3 × 10 6HEK293T cells were seeded in 10 cm cell culture dishes in complete DMEM (cDMEM) consisting of DMEM (Gibco®) + 10% FCS + 100 U / ml penicillin, 100 μg / ml streptomycin + 50 μM 2-mercaptoethanol (all from Thermo Fisher Scientific, Schwerte, Germany). Cells were transiently transfected with 0.84 μg pMDG-2.VSV-G, 5.16 μg pCMVΔR8.74, and 3.35 μg dextran-CAR plasmid diluted in ddH2O supplemented with 2.5 M CaCl2. While aerating, 2 ml of 2x HBS buffer (136.89 mM NaCl, 4.96 mM KCl, 1.76 mM NaHPO, 20.98 mM HEPES, pH 6.75-6.76 in ddH2O) was slowly added to the solution, and 2 ml of the transfection solution was added dropwise to the cells. The medium containing the transfection solution was removed after 4 hours, and the cells were washed twice with prewarmed PBS, followed by the addition of fresh cDMEM. After 48 hours, the lentiviral supernatant was harvested, filtered (0.45 μm), and used immediately or stored at -80°C for up to 6 months.

[0156] Treg isolation and transduction Leukapheresis products from healthy donors were obtained from Charite University Hospital, Berlin, Germany, in accordance with ethical guidelines and with informed consent. PBMCs were obtained by Ficoll-Paque (GE Healthcare Life Sciences, Freiburg, Germany) gradient centrifugation. CD25 +Tregs were isolated from PBMCs using CD25 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's recommendations. Tregs were cultured in "Treg expansion medium" consisting of TexMACS medium (Miltenyi Biotec, Bergisch Gladbach, Germany) + 5% (v / v) human AB serum (Sigma-Aldrich, Schnelldorf, Germany) + 100 U / ml IL-2 + 100 nmol rapamycin (both Miltenyi Biotec, Bergisch Gladbach, Germany) and 100 U / ml penicillin / 100 μg / ml streptomycin (Gibco®, Thermo Fisher Scientific, Schwerte, Germany) in the presence of Treg expansion beads (Miltenyi Biotec, Bergisch Gladbach, Germany) at a bead-to-cell ratio of 4:1. CD4 +Tcon cells were activated in TexMACS medium (Miltenyi Biotec, Bergisch Gladbach, Germany) + 5% (v / v) human AB serum (Sigma-Aldrich, Schnelldorf, Germany) + 200 U / ml IL-2 in the presence of 30 ng / ml anti-CD3 and 1 μg / ml anti-CD28. On day 3, the medium was replaced with the respective lentiviral supernatant supplemented with 4 μg / ml protamine sulfate, and cells were spun in retronectin-coated 96-well plates at 800 x g for 90 minutes at 32°C. After centrifugation, the viral supernatant was removed, and fresh medium was added to the cells. Transduction efficiency was assessed by staining for LNGFR on the cell surface on days 2 or 3 posttransduction. Tregs and Tcon cells were expanded for 10–12 days, with medium changes every 2–3 days. Cells were cultured in RPMI-1640 (Gibco®, Thermo Fisher Scientific) for 6 h before restimulation with Treg expansion beads (4:1 bead-to-cell ratio, Miltenyi Biotec, Bergisch Gladbach, Germany), soluble FITC dextran (MW: 2,000,000, 2 μg / ml, Sigma-Aldrich, Schnelldorf, Germany), bead-bound dextran (1:100; dextran-coated microbeads in PBS, Miltenyi Biotec, Bergisch Gladbach, Germany), or 10 ng / ml PMA and 500 ng / ml ionomycin (Sigma-Aldrich, Schnelldorf, Germany). The cells were then allowed to rest for 2 days without stimulation in 5% (v / v) human AB serum (Sigma-Aldrich, Schnelldorf, Germany) + 100 U / ml penicillin / 100 μg / ml streptomycin (Gibco®, Thermo Fisher Scientific, Schwerte, Germany).

[0157] Flow cytometry Cells were stained with different combinations of the following antibodies according to the manufacturer's recommendations: CD4-PE-Vio770, CD4-APC-Vio-770, CD4-FITC, CD4-VioBlue (VIT4), CD25-VioBright FITC (4E3), CD127-FITC, CD127-PE-Vio770 (MB15-18C9), CD271(LNGFR)-PE, CD271(LNGFR)-PE-Vio770 (ME20.4-1.H4), CD137-PE (4B4-1), CD154-APC, and CD154-VioBlue (5C8) (all from Miltenyi Biotech, Bergisch Gladbach, Germany). Viobility 405 / 520 Fixable Dye (Miltenyi Biotech, Bergisch Gladbach, Germany) or propidium iodide (Sigma-Aldrich, Schnelldorf, Germany) was used to exclude dead cells. For staining of CAR surface expression, Tregs were incubated with 2 μg / ml FITC-labeled dextran (MW: 2,000,000, Sigma-Aldrich, Schnelldorf, Germany) and other surface molecule labels for 10 min at 4°C. All data were acquired on a FACS Canto / LSRII (BD, Heidelberg, Germany) or MACS Quant Analyzer (Miltenyi Biotec, Bergisch Gladbach, Germany), and FACS sorting was performed on an Aria I, Aria II, or Influx Cell Sorter (BD, Heidelberg, Germany). FlowJo (TreeStar, Inc., Ashland, OR, USA) was used for data analysis.

[0158] Quantification of gene expression The competitive growth of Dex-CAR constructs with different signal transduction domains was analyzed by quantitative real-time PCR.DNA was isolated by Zymo Research Quick-DNA™ Miniprep Kit (Zymo Research, Freiburg, Germany) according to the manufacturer's instructions, and gene expression was analyzed using 1×SYBR® Green PCR Master Mix (Thermo Fisher Scientific, Schwerte, Germany) and 500nMol forward and reverse primers (TIB MOLBIOL, Berlin), respectively.Gene expression was analyzed using StepOne® Real-Time PCR System (Thermo Fisher Scientific, Schwerte), and normalized to the expression of GAPDH.

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Claims

1. A human regulatory T (Treg) cell expressing a chimeric antigen receptor (CAR), a) at least one antigen-binding domain; b) a transmembrane domain; c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least a costimulatory signaling domain of CD137. Including, the antigen-binding domain specifically binds to an antigen expressed on the surface of a target cell, or a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen; A Treg cell, wherein said at least one primary cytoplasmic signaling domain is CD3zeta.

2. 2. The Treg cell of claim 1, wherein the antigen-binding domain of the CAR is specific for a soluble antigen, thereby allowing activation of the Treg cell by binding of the soluble antigen to the antigen-binding domain of the CAR.

3. The Treg cell of claim 2 , wherein the soluble antigen is an exogenous antigen that does not naturally occur in the blood or tissues of a subject to which the Treg cells are applied.

4. The Treg cell of claim 2 or 3, wherein the soluble antigen is dextran.

5. The Treg cell of claim 4, wherein the antigen-binding domain of the CAR comprises the sequences of SEQ ID NO: 1 and SEQ ID NO:

2.

6. A pharmaceutical composition comprising a population of Treg cells expressing the CAR of any one of claims 1 to 5, for use in the treatment or prevention of autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, chronic inflammatory diseases including inflammatory bowel disease, or chronic infection by viruses, bacteria, or parasites in a subject.

7. A composition comprising a population of Treg cells expressing the CAR according to any one of claims 1 to 5.

8. The composition according to claim 7 or the pharmaceutical composition according to claim 6, wherein the population of CAR-expressing Treg cells is a population of activated Treg cells obtainable by a method comprising the steps of: a) providing a sample containing human Treg cells b) genetically modifying the Treg cells of the sample to express the CAR c) activating the genetically modified Treg cells by contacting them with an antigen bound by the antigen-binding domain of the CAR for 6 to 16 hours. d) α) The cells of step c) I) contacting a CD154-binding molecule with CD154 ＋ depleting T cells; or II) contacting the cells with a molecule that binds to a marker for Treg cells or activated Treg cells, and positively selecting cells that bind to the binding molecule; and β) I) contacting the cells of step α) I) with a molecule that binds to a marker of Treg cells or activated Treg cells, and positively selecting cells that bind to the binding molecule, thereby obtaining a population of activated Treg cells that express the CAR; or II) contacting the cells of step α) II) with a molecule that binds to CD154, ＋ depleting T cells, thereby obtaining a population of activated Treg cells that express the CAR. and isolating the activated Treg cells of step c).

9. A pharmaceutical composition combination comprising: a) a population of Treg cells expressing the CAR of any one of claims 1 to 5 together with a pharmaceutically acceptable carrier; and b) Soluble antigen wherein the antigen-binding domain of the CAR is specific for the soluble antigen, thereby enabling activation of the Treg cell upon binding of the soluble antigen to the antigen-binding domain of the CAR, and the soluble antigen is dextran.

10. 10. The pharmaceutical composition combination of claim 9 for the treatment or prevention of autoimmune diseases, allergies, transplant rejection, graft-versus-host disease, chronic inflammatory diseases such as inflammatory bowel disease, or chronic viral, bacterial or parasitic infections in a subject.

11. A method for enriching human activated regulatory T (Treg) cells expressing a CAR, the method comprising: i) at least one antigen-binding domain ii) transmembrane domain iii) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least the costimulatory signaling domain of CD137. Including, the antigen-binding domain specifically binds to an antigen expressed on the surface of a target cell, or a tag on a tagged polypeptide that binds to an antigen expressed on the surface of a target cell, or a soluble antigen; the at least one primary cytoplasmic signaling domain is CD3zeta; and the method comprises: a) providing a sample containing human Treg cells b) genetically modifying the Treg cells of the sample to express the CAR c) activating the genetically modified Treg cells by contacting them with an antigen bound by the antigen-binding domain of the CAR for 6 to 16 hours. d) α) The cells of step c) I) contacting a CD154-binding molecule with CD154 ＋ depleting T cells; or II) contacting the cells with a molecule that binds to a marker for Treg cells or activated Treg cells, and positively selecting cells that bind to the binding molecule; and β) I) contacting the cells of step α) I) with a molecule that binds to a marker of Treg cells or activated Treg cells, and positively selecting cells that bind to the binding molecule, thereby obtaining a population of activated Treg cells that express the CAR; or II) contacting the cells of step α) II) with a molecule that binds to CD154, ＋ depleting T cells, thereby obtaining a population of activated Treg cells that express the CAR. and isolating the activated Treg cells of step c) by A method comprising:

12. 12. The method of claim 11, wherein the antigen-binding domain of the CAR is specific for a soluble antigen, and the soluble antigen is dextran.

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