Chimeric antigen receptors

Abstract

Disclosed herein are chimeric antigen receptors (CARs), cells (particularly immune cells, such as regulatory T cells) expressing the CARs, nucleic acids or vectors encoding the CARs, and various uses of the CARs, cells, nucleic acids or vectors. In particular, chimeric antigen receptors (CARs) comprising an antigen recognition domain that specifically binds to GLP1R are provided.

Description

Technical Field

[0001] This disclosure relates generally to the field of chimeric antigen receptors (CARs) and related therapies, such as the treatment of autoimmune or inflammatory diseases (particularly type 1 diabetes). More specifically, this disclosure provides CARs comprising an antigen recognition domain that binds to GLP1R and is expressed in immune cells (particularly Tregs). Such immune cells have therapeutic use in diseases and conditions associated with cells that express GLP1R on their surface. This disclosure also provides nucleic acid molecules encoding such CARs and vectors containing these nucleic acid molecules, which can be used to modify host cells (e.g., immune cells) to express the CAR. Background Technology

[0002] Immunotherapy is becoming a valuable tool for treating a wide range of conditions, from cancer, autoimmune and inflammatory diseases to preventing solid organ transplant rejection. Specifically, in the field of adoptive cell immunotherapy (ACT), particularly in regulatory T-cell (Treg) cell therapy within the areas of autoimmunity and inflammation, clinical activity has increased.

[0003] CD4+FOXP3+ regulatory T cells (Tregs) are a subset of lymphocytes essential for maintaining dominant immune tolerance by suppressing the function of various effector immune cell subsets, such as T effector cells. Furthermore, Tregs are known to promote tissue repair and regeneration. Tregs can confer immune tolerance through a variety of contact-dependent and contact-independent mechanisms. These mechanisms include the production of anti-inflammatory soluble mediators such as IL-10, TGF-β, and IL-35, the depletion of IL-2, the expression of negatively regulated cell surface receptors such as CTLA-4, and direct or indirect targeting of T cells via APCs. Importantly, once activated, Tregs can suppress the immune response in a non-antigen-specific manner (bystander suppression), meaning that once activated, Tregs possess the ability to modulate the local immune microenvironment and suppress inflammation. In addition, Tregs can confer suppressive phenotypes on other cells of the immune system, a process known as "infection tolerance."

[0004] Type 1 diabetes is a chronic autoimmune disease in which the pancreatic beta cells responsible for producing insulin are destroyed by the immune system. This is triggered by both genetic and environmental factors. The destruction of beta cells reduces or eliminates the body's production of insulin and leads to inflammation of the pancreas. Insulin is a hormone required to regulate glucose levels in the bloodstream, and before treatment, individuals with type 1 diabetes will have excessively high blood sugar levels (hyperglycemia). Type 1 diabetes is a serious and lifelong condition. People with type 1 diabetes currently require close monitoring of their blood sugar levels and to take appropriate doses of insulin (e.g., by injection or pump). This treatment is not a cure and must be administered continuously. Over time, irregular blood sugar levels, including large fluctuations, can lead to long-term complications such as damage to the heart, eyes, feet, and kidneys, as well as a shortened life expectancy. Therefore, type 1 diabetes places a considerable burden on healthcare systems worldwide. Furthermore, epidemiological data show a steady increase in the prevalence of autoimmune diseases such as type 1 diabetes in Western societies over the past few decades. The prevalence of type 1 diabetes in Western Europe and North America is approximately 0.5%, or about 2 million people, with a steady upward trend.

[0005] In mouse models of type 1 diabetes and other autoimmune disorders, mounting evidence suggests that dysregulation of Treg-mediated effector T cell (Teff) suppression contributes to disease development (see Visperas and Vignali, J Immunol. 2016; 197(10): 3762-3770). For example, defects in Treg phenotypes and suppressive capacity have been observed in samples obtained from patients with type 1 diabetes. Furthermore, in mouse models of type 1 diabetes, Treg depletion has been shown to accelerate the development of autoimmune diabetes.

[0006] The prospect of improving immunopathology and re-establishing immune tolerance in inflammatory diseases has spurred increasing attention to the clinical development of Treg-based immunotherapies. However, for Treg immunotherapy to be successful, it is beneficial to develop strategies that promote Treg transport to sites of tissue damage and induce their activation in situ.

[0007] Autologous polyclonal Tregs have been administered to patients with type 1 diabetes with promising results, demonstrating the safety and potential of adoptive Treg therapy in this disease setting (see Marek-Trzonkowski et al., ClinImmunol. 2014; 153: 23-30 and Bluestone et al., Sci Transl Med. 2015; 7:315ra189). However, polyclonal Tregs may be associated with unwanted effects, such as systemic immunosuppression, due to their lack of specificity. Furthermore, obtaining sufficient quantities of polyclonal Tregs for delivery to the disease site may be difficult. A recent large clinical trial using polyclonal Tregs conducted by Caladrius Biosciences (Sanford Project) failed to demonstrate efficacy in type 1 diabetes.

[0008] Tregs expressing islet antigen-specific TCRs from transgenic mice have been used to prevent or reverse diabetes in NOD mice (see Tang et al., J Exp Med. 2004; 199: 1455-65). However, this requires Treg activation via TCR, and due to MHC restriction, this transgenic model cannot be transferred to patients, as different patients will require different TCRs.

[0009] Chimeric antigen receptors (CARs) have been used to confer antigen specificity to cells. CARs typically consist of an extracellular antigen-binding domain (e.g., scFv specific to the target antigen), a transmembrane domain, and an intracellular signaling domain that sends a signal to the cell and activates it upon antigen binding to the extracellular antigen-binding domain. CAR-T cell therapy has been approved for the treatment of certain blood cancers. However, CARs are engineered artificial molecules introduced into cells, and their sensitivity is lower than that of TCRs. This difference stems in part from the number of molecules involved in the TCR mechanism: CD4 / CD8 co-receptors, the immunoreceptor tyrosinase-rich activation motif (ITAM), and subunits within the receptor complex. Therefore, selecting suitable targets for CAR-T cell therapy, which can deliver and subsequently activate CAR-T cells to produce a therapeutic effect, is challenging. For example, although insulin-specific CAR-Tregs can produce a proliferative response to insulin and are inhibitory in vitro, they cannot prevent spontaneous diabetes in NOD mice (Tenspolde et al., J Autoimmunity. 2019; 103:102289). Summary of the Invention

[0010] The inventors have determined that therapies for treating autoimmune or inflammatory diseases can be developed by providing immune cell subsets with chimeric antigen receptors (CARs) containing antigen recognition domains specific for GLP1R. Expression of such CARs on the surface of Tregs can provide a universal therapy for treating autoimmune or inflammatory diseases where GLP1R is locally expressed at the disease site, based on the well-known bystander effect of Tregs and their ability to reduce the immune response and modulate the activation state of other immune cell subsets once activated. Specifically, the inventors have developed Tregs expressing anti-GLP1R CARs for the treatment of type 1 diabetes.

[0011] Therefore, the inventors have identified GLP1R as a surprisingly effective target for CAR-Treg therapy, particularly for type 1 diabetes and other pancreatic autoimmune or inflammatory disorders. Specifically, the inventors have found that cells expressing GLP1R-specific CARs are activated in the presence of antigens. Surprisingly, the inventors have identified both ligand-based and scFv-based CARs that are activated in the presence of antigens. Furthermore, the inventors have found that T effector cells expressing GLP1R-specific CARs can induce the onset of type 1 diabetes, indicating that these cells can be transported to the pancreas.

[0012] While GLP1R expression in pancreatic tissue has been reported, its expression in other tissues (brain, heart, abdominal organs) has also been reported in the art, making the selection of GLP1R as a target for specific transport (thus enabling Tregs to be activated to levels sufficient to treat type 1 diabetes) not immediately obvious. Specifically, RNA expression of GLP1R in many other cell types and tissues has been reported in the art. Therefore, the inventors' discovery that the GLP1R protein is an effective pancreatic target is particularly surprising.

[0013] Therefore, in one aspect, the present invention provides a regulatory T cell (Treg) comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain that specifically binds to GLP1R (e.g., specifically binds to human GLP1R and / or mouse GLP1R).

[0014] In some implementations, the CAR may include: a. An outer domain containing an antigen recognition domain; b. Transmembrane domains; and c. Intracellular domain, which contains intracellular signal transduction domains.

[0015] CARs may also include hinge domains and / or one or more co-stimulatory domains.

[0016] The term "hinge domain" generally refers to the outer domain portion that connects the antigen recognition domain to the transmembrane domain. The hinge domain may be selected from CD28, CD8α, CD4, CD7, CH2CH3, the hinge region of an immunoglobulin, or portions or variants thereof. Preferably, the CAR may contain a CD8α or CH2CH3 hinge domain.

[0017] The co-stimulatory domain may be selected from intracellular domains of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or portions thereof or variants thereof. Preferably, the CAR may contain a CD28 co-stimulatory domain.

[0018] The CAR may contain one or more transmembrane domains, which may be selected from transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or portions thereof or variations thereof. Preferably, the CAR contains a CD4, CD28, or CD8α transmembrane domain.

[0019] A CAR (particularly the intracellular domain of a CAR) may contain one or more intracellular signaling domains selected from any one of the CD3ζ signaling domains or its homologs, CD3 peptides, syk family tyrosine kinases, src family tyrosine kinases, CD2, CD5, and CD28, or portions or variants thereof. Preferably, the CAR may contain a CD3ζ signaling domain.

[0020] In one embodiment, the CAR may include a CD8α or CH2CH3 hinge domain (i.e., a hinge domain derived from CD8α or CH2CH3); a CD8α or CD28 transmembrane domain (i.e., a transmembrane domain derived from CD8α or CD28); a CD28 costimulatory domain (i.e., a costimulatory domain derived from CD28); and a CD3ζ signaling domain (i.e., a signaling domain derived from CD3ζ), wherein when the hinge domain is a CD8α hinge domain, the transmembrane domain is a CD8α transmembrane domain, and when the hinge domain is a CH2CH3 hinge domain, the transmembrane domain is a CD28 transmembrane domain. Alternatively, in one embodiment, the CAR may include a CD8α hinge domain, a CD8α transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain. In a separate embodiment, the CAR may include a CH2CH3 hinge domain, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3ζ signaling domain. In addition, CARs can contain CD28 transmembrane domains (i.e., transmembrane domains derived from CD28), especially in combination with CD28 costimulatory domains.

[0021] The CAR may contain a signal peptide and / or a reporter peptide. In one embodiment, the polynucleotide sequence encoding the CAR may contain an additional polynucleotide sequence that encodes a reporter peptide linked by a self-cleaving or cleavage domain.

[0022] The antigen recognition domain of a CAR can be an antibody, an antibody fragment, or an antibody-derived domain. Preferably, the antigen recognition domain is a single-chain antibody (scFv).

[0023] The antigen recognition domain of a CAR can be a ligand of GLP1R. For example, the ligand of GLP1R can be a GLP1R agonist.

[0024] CARs may, for example, include an antigen recognition domain, which contains: (i) (a) the VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 152, 2 and 3 respectively, or (b) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 153, 2 and 3 respectively, or (c) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 154, 2 and 3 respectively, and (a) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 156, 161 and 162 respectively, or (b) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 156, 161 and 163 respectively; (ii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 7, 8 and 9 respectively, and (a) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 156, 161 and 12 respectively, or (b) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 157, 161 and 12 respectively, or (c) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 158, 161 and 12 respectively, or (d) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 160, 161 and 12 respectively; (iii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 13, 14 and 155 respectively, and (a) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 157, 161 and 162 respectively, or (b) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 158, 161 and 162 respectively, or (c) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 159, 161 and 162 respectively; (iv) (a) as shown in SEQ ID NO: 151, 20 and 21 respectively, or (b) as shown in SEQ ID NO: 152, 20 and 21 respectively, or (c) the VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 153, 20 and 21 respectively, and the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 22, 23 and 24 respectively; or (v) (a) the VH CDR1, 2 and 3 sequences shown as SEQ ID NO: 151, 26 and 155 respectively or (b) the VL CDR1, 2 and 3 sequences shown as SEQ ID NO: 154, 26 and 155 respectively; and (a) the VL CDR1, 2 and 3 sequences shown as SEQ ID NO: 159, 161 and 163 respectively or (b) the VL CDR1, 2 and 3 sequences shown as SEQ ID NO: 160, 161 and 163 respectively. One or more of the CDR sequences in (i) to (v) may optionally include one to three amino acid modifications relative to the CDR sequence, specifically, one or more of the CDR sequences may optionally be modified by substitution, addition or deletion of one to three amino acids.

[0025] CARs may, for example, include an antigen recognition domain, which contains: (i) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 1, 2 and 3 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 4, 5 and 6 respectively; (ii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 7, 8 and 9 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 10, 11 and 12 respectively; (iii) The VH CDR1, 2 and 3 sequences shown in SEQ ID NO: 13, 14 and 15 respectively, and the VL CDR1, 2 and 3 sequences shown in SEQ ID NO: 16, 17 and 18 respectively; (iv) VH CDR1, 2, and 3 sequences as shown in SEQ ID NO: 19, 20, and 21, respectively, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 22, 23, and 24, respectively; or (v) The VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 25, 26 and 27, respectively, and the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 28, 29 and 30, respectively; One or more of the CDR sequences in (i) to (v) may optionally include one to three amino acid modifications relative to the CDR sequence, specifically, one or more of the CDR sequences may optionally be modified by substitution, addition or deletion of one to three amino acids.

[0026] Similarly, in this respect, the antigen recognition domain of CAR can include: (i) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 31 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 32 or a sequence having at least 70% sequence identity with it; (ii) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 33 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 34 or a sequence having at least 70% sequence identity with it; (iii) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 35 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 36 or a sequence having at least 70% sequence identity with it; (iv) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 37 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 38 or a sequence having at least 70% sequence identity with it; or (v) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 39 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 40 or a sequence having at least 70% sequence identity with it.

[0027] Antigen recognition domains may include, for example: (i) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 41 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 41, and the VL domain contains a sequence encoded by SEQ ID NO: 42 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 42; (ii) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 43 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 43, and the VL domain contains a sequence encoded by SEQ ID NO: 44 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 44; (iii) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 45 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 45, and the VL domain contains a sequence encoded by SEQ ID NO: 46 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 46; (iv) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 47 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 47, and the VL domain contains a sequence encoded by SEQ ID NO: 48 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 48; or (v) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 49 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 49, and the VL domain contains a sequence encoded by SEQ ID NO: 50 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 50.

[0028] In addition, the antigen recognition domain of a CAR may contain or consist of the following items: (i) A sequence as shown in SEQ ID NO: 51 or a sequence having at least 80% sequence identity with it; (ii) A sequence as shown in SEQ ID NO: 52 or a sequence having at least 80% sequence identity with it; (iii) A sequence as shown in SEQ ID NO: 53 or a sequence having at least 80% sequence identity with it; (iv) A sequence as shown in SEQ ID NO: 54 or a sequence having at least 80% sequence identity with it; or (v) A sequence as shown in SEQ ID NO: 55 or a sequence having at least 80% sequence identity with it.

[0029] An antigen recognition domain may contain or consist of, for example, the following: (i) A sequence encoded by the sequence shown in SEQ ID NO: 56 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 56; (ii) A sequence encoded by the sequence shown in SEQ ID NO: 57 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 57; (iii) A sequence encoded by the sequence shown in SEQ ID NO: 58 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 58; (iv) A sequence encoded by the sequence shown in SEQ ID NO: 59, or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 59; or (v) A sequence encoded by the sequence shown in SEQ ID NO: 60 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 60.

[0030] The antigen recognition domain may, for example, contain or consist of the following: the sequence shown in SEQ ID NO: 61 or a sequence having at least 70% sequence identity with it.

[0031] The antigen recognition domain may, for example, include or consist of the following: a sequence encoded by the sequence shown in SEQ ID NO: 62 or SEQ ID NO: 63, or a sequence having at least about 70% identity with the sequence encoded by the sequence shown in SEQ ID NO: 62 or SEQ ID NO: 63.

[0032] In a further aspect, the present invention provides a CAR comprising an antigen recognition domain that specifically binds to GLP1R. The CAR may conform to any of the embodiments described herein with respect to a first aspect of the invention.

[0033] In one particular aspect, the present invention provides a CAR comprising an antigen recognition domain that specifically binds to GLP1R, wherein the antigen recognition domain is a ligand of GLP1R. In some embodiments, the ligand is a GLP1R agonist. In some embodiments, the antigen recognition domain of the CAR comprises or consists of the following: an amino acid sequence of SEQ ID NO: 61 or a sequence differing therefrom by up to 10 amino acids, for example, a sequence differing by 1 to 5 amino acids.

[0034] In another particular aspect, the present invention provides a CAR comprising an antigen recognition domain that specifically binds to GLP1R, wherein the antigen recognition domain comprises: (i) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 1, 2 and 3 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 4, 5 and 6 respectively; (ii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 7, 8 and 9 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 10, 11 and 12 respectively; (iii) The VH CDR1, 2 and 3 sequences shown in SEQ ID NO: 13, 14 and 15 respectively, and the VL CDR1, 2 and 3 sequences shown in SEQ ID NO: 16, 17 and 18 respectively; (iv) VH CDR1, 2, and 3 sequences as shown in SEQ ID NO: 19, 20, and 21, respectively, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 22, 23, and 24, respectively; or (v) The VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 25, 26 and 27, respectively, and the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 28, 29 and 30, respectively; One or more of the CDR sequences in (i) to (v) may optionally include one to three amino acid modifications relative to the CDR sequence described above. Specifically, one or more of the CDR sequences may optionally be modified by substitution, addition, or deletion of one to three amino acids. Alternatively or otherwise, the antigen recognition domain may be encoded by one of other VH and / or VL sequences or by one of the nucleotide sequences defined herein.

[0035] In a further aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a CAR according to the present invention.

[0036] In a further aspect, the present invention provides a carrier comprising a nucleic acid molecule according to the present invention. The carrier may also comprise a nucleic acid molecule containing a nucleotide sequence encoding a FOXP3 polypeptide or a derivative or variant thereof.

[0037] In a further aspect, the present invention provides a cell comprising a CAR, nucleic acid molecule, or vector according to the present invention. The cell may also comprise an exogenous FOXP3 polypeptide or an exogenous nucleic acid encoding FOXP3. The cell may be an immune cell or its progenitor cell or precursor. Preferably, the cell may be a T cell or its precursor or stem cell. Specifically, the cell may be a Treg or its precursor or iPSC cell. The cell may be a production host cell. In another specific embodiment, the present invention provides a T effector cell (Teff) comprising a CAR, wherein the antigen recognition domain of the CAR specifically binds to GLP1R.

[0038] The cells can be provided in the form of a cell population, which forms another aspect of the invention. Specifically, the cell population can contain multiple cells according to the invention, particularly multiple T cells according to the invention (e.g., multiple Tregs or multiple Teffs). Specifically, the multiple T cells (particularly Tregs) according to the invention can have polyclonal TCRs. Specifically, the multiple T cells (particularly Tregs) according to the invention can have polyclonal endogenous TCRs. Specifically, the clonality of the TCRs of the multiple T cells (particularly Tregs) according to the invention may not be modified in vitro.

[0039] The present invention also provides a pharmaceutical composition comprising cells, cell populations or carriers according to the present invention.

[0040] In another aspect, the present invention provides cells, cell populations, or pharmaceutical compositions according to the invention for use in therapies (e.g., for treating and / or preventing autoimmune or inflammatory diseases, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration). The therapy may be adoptive cell transfer therapy.

[0041] Alternatively, the present invention provides a method for treating and / or preventing autoimmune or inflammatory diseases, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration, wherein the method comprises administering cells (particularly Treg cells), cell populations, or pharmaceutical compositions according to the present invention, particularly pharmaceutical compositions comprising Treg cells.

[0042] In this regard, the method may include the following steps: (i) Isolate or provide Treg-rich cell samples from the subject; (ii) Introducing the nucleic acid molecule or vector of the present invention into Treg cells; and (iii) Administer Treg cells from (ii) to the subject.

[0043] The present invention also provides the use of cells, cell populations or pharmaceutical compositions according to the invention in the preparation of medicaments for treating and / or preventing autoimmune or inflammatory diseases in subjects, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration, specifically wherein the cells are Treg cells.

[0044] Autoimmune or inflammatory diseases can be particularly associated with type 1 diabetes (T1D), such as recently-onset type 1 diabetes.

[0045] In another aspect, the present invention provides a method for preparing cells according to the invention, the method comprising the step of introducing a nucleic acid molecule or vector according to the invention into cells (e.g., transducing or transfecting cells with it). The cells may be Treg cells, and the method may include, before or after the step of introducing the nucleic acid molecule or vector into the cells, isolating or providing a cell-containing sample containing Tregs, and / or enriching or generating Tregs from the cell-containing sample. The present invention also provides cells that can be obtained by this method, which forms another aspect of the invention.

[0046] In a further aspect, the present invention provides the use of CAR-Treg in reducing pancreatic β-cell death (e.g., reducing mortality) or preventing pancreatic β-cell death in a subject. In a further aspect, the present invention provides the use of CAR-Treg in maintaining or increasing fasting blood insulin levels in a subject. In a further aspect, the present invention provides the use of CAR-Treg in reducing or preventing hyperglycemia in a subject. In a further aspect, the present invention provides the use of CAR-Treg in maintaining or reducing fasting blood glucose levels in a subject. In a further aspect, the present invention provides the use of CAR-Treg in maintaining or reducing HbA1c levels in a subject. The CAR is the CAR of the present invention, i.e., the CAR contains an antigen recognition domain that specifically binds to GLP1R (e.g., human GLP1R and / or mouse GLP1R), and the CAR may have any of the characteristics of a CAR as disclosed herein. The subject may, for example, have type 1 diabetes or be at risk of developing type 1 diabetes; specifically, the subject may have recently developed type 1 diabetes. The subject may, for example, not be receiving exogenous insulin. Alternatively, a reduced dose of insulin may be administered to the subject compared to the insulin dose required prior to CAR-Treg administration. Attached Figure Description

[0047] Figure 1 The binding of various scFvs to HEK293T cells expressing human GLP1R (first row) or mouse GLP1R (second row) is shown.

[0048] Figure 2 Immunohistochemical staining of mouse pancreatic sections using various GLP1R-specific scFvs is shown.

[0049] Figure 3 The activation of mouse hybridoma cells expressing GLP1R-specific CARs by human GLP1R protein, HEK cells expressing human GLP1R antigen, and HEK cells expressing mouse GLP1R antigen is demonstrated.

[0050] Figure 4 The activation of mouse hybridoma cells expressing GLP1R-specific CARs by human GLP1R protein, HEK cells expressing human GLP1R antigen, and HEK cells expressing mouse GLP1R antigen is demonstrated.

[0051] Figure 5 An overview of the experimental protocol for observing diabetes induced by GLP1R-specific CARs is shown.

[0052] Figure 6Diabetes induction in C57Bl / 6.RAG mice was demonstrated using T effector cells expressing a GLP1R-specific CAR.

[0053] Figure 7 The configurations of the scFv CAR used in the examples are shown—(A) shows a schematic structure of a CAR with a mutated Fc-IgG hinge and a CD8 hinge, and (B) shows a schematic structure of the construct used in a γ retroviral vector for transducing cells.

[0054] Figure 8 The configurations of the CAR ligands used in the examples are shown—(A) shows a schematic structure of a CAR with a CD8 hinge, (B) shows the sequence of the GLP1R ligand used in the antigen recognition domain, and (C) shows a schematic structure of the construct used in a γ retroviral vector for transducing cells.

[0055] Figure 9 An overview of experimental protocols for observing the prevention of diabetes onset in NOD.SCID mice administered Teff from BDC2.5 NOD mice and NOD Treg expressing a ligand-based GLP1R-specific CAR is provided.

[0056] Figure 10 shows the proportion of NOD.SCID mice without diabetes after administration of Teff from BDC2.5.NOD mice and NOD Treg expressing a ligand-based GLP1R-specific CAR. Figure 10A ) and survival rate ( Figure 10B ).

[0057] Figure 11 The activation of CAR-Tregs exposed to the target (GLP1R) was demonstrated compared to the control antigen and anti-CD3 / CD28 beads.

[0058] Figure 12 The study showed the proliferation of CAR-Tregs exposed to the target (GLP1R) compared to the control antigen and anti-CD3 / CD28 beads. Detailed Implementation

[0059] This invention provides GLP1R-specific CAR-Tregs that are activated in the presence of a GLP1R antigen, which is specifically expressed on pancreatic β-cells. Therefore, these CAR-Tregs have therapeutic potential in treating autoimmune and inflammatory disorders where GLP1R is locally expressed at the disease site. Specifically, these CAR-Tregs have therapeutic potential for type 1 diabetes. Specifically, due to the bystander effect of Treg cells, the antigen (GLP1R) can simply be present and / or expressed at the site of inflammation or disease.

[0060] "Chimeric antigen receptor," "CAR," or "CAR construct" refers to an engineered receptor that can specifically confer antigen-specificity to cells (e.g., immune cells, such as Tregs). Specifically, a CAR enables cells to specifically bind to a particular antigen (e.g., a target molecule, such as a target protein), thereby generating a signal in the CAR's intracellular domain (containing an intracellular signaling domain), such as a signal that triggers cell activation. CARs are also known as artificial T-cell receptors, chimeric T-cell receptors, or chimeric immune receptors.

[0061] The structure of CARs is well known in the art, and several generations of CARs have been produced. For example, at a minimum, a CAR may contain an extracellular antigen-specific targeting region, an antigen-binding domain, a target-binding domain or a ligand-binding domain (which is part of or forms part of the CAR's outer domain), a transmembrane domain, and an intracellular signaling domain (which is an intracellular domain or is contained within an intracellular domain). However, CARs may contain other domains to improve their function, such as one or more co-stimulatory domains to improve T cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence.

[0062] Therefore, chimeric receptors or CAR constructs typically include a binding domain (which can be considered an antigen (i.e., target) or ligand-binding domain, and the terms binding domain, antigen recognition domain, antigen-binding domain, and ligand-binding domain are used interchangeably herein), an optional hinge domain (which acts as a spacer to extend the binding domain to the plasma membrane of the cell (e.g., an immune cell) from which it is expressed), a transmembrane domain, an intracellular signaling domain (e.g., a signaling domain or equivalent of the ζ chain (CD3ζ) of the CD3 molecule from the TCR complex), and optional one or more co-stimulatory domains that may contribute to signaling or functionality in the cell expressing the CAR. The CAR may also include a signaling or leader sequence or domain that functions to target the protein to the membrane and may form part of the outer domain of the CAR. Different domains may be directly linked or linked via adapters, and / or may be present within different polypeptides, for example, within two polypeptides associated with each other.

[0063] When a CAR binds to its target antigen (i.e., GLP1R), this results in the transmission of activation signals to cells that express it. Thus, the CAR specifically directs engineered cells toward GLP1R, particularly cells that express GLP1R.

[0064] The terms "targeted" or "directed" are synonymous with "specific to" or "anti-". In other words, CARs recognize GLP1R target molecules. Therefore, this means that CARs are capable of specifically binding to GLP1R. Specifically, the antigen-binding domain of a CAR is capable of specifically binding to GLP1R (more specifically, when the CAR is expressed on the surface of cells, especially immune effector cells). Specific binding can be distinguished from non-specific binding to non-target molecules or antigens. Thus, cells expressing CARs are directed or redirected to specifically bind to target cells expressing GLP1R, particularly target cells expressing GLP1R on their cell surface. Specifically, "specific" binding means binding only or primarily to GLP1R, without binding to other proteins or peptides (i.e., binding to other proteins or peptides is insignificant or weak). Some cross-reactivity with other proteins may occur, but this level of binding can be considered background. As described above, CARs are capable of binding to GLP1R and transducing signals to cells expressing it. The cells can then be activated and can exert an inhibitory effect within the local environment. Activation of CAR-expressing cells upon antigen binding can be determined by an increase in CD69 levels compared to the same CAR-expressing cells in the absence of the antigen. For example, CD69 is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. CD69 expression levels can be determined using standard techniques, such as FACS, or using commercially available antibodies (e.g., FITC anti-human CD69 antibody, Biolegend). Therefore, intracellular CAR function can be determined by the activation state of CAR-expressing cells, for example, by determining CD69 expression.

[0065] GLP1R (glucagon-like peptide-1 receptor) is a member of the glucagon receptor family of G protein-coupled receptors. GLP1R is expressed on pancreatic β cells and participates in the control of blood glucose levels by enhancing insulin secretion. The amino acid sequence of human GLP1R is shown in SEQ ID NO: 64, and the amino acid sequence of mouse GLP1R is shown in SEQ ID NO: 65.

[0066] The antigen-binding domain of a CAR can be derived from or acquired from any protein or polypeptide that self-binds to (i.e., has an affinity for) GLP1R (e.g., can bind any region or portion of GLP1R, or alternatively bind any epitope within GLP1R, in isolated protein form or when expressed on a cell). Specifically, the antigen-binding domain of a CAR can be derived from or acquired from any protein or polypeptide that self-binds to (i.e., has an affinity for) the extracellular domain of GLP1R. This can be, for example, a ligand of GLP1R, or a physiologically binding protein of GLP1R or a portion thereof, or a synthetic or derived protein. The target molecule (i.e., GLP1R) can typically be expressed on the cell surface, such as a target cell (e.g., a pancreatic β cell) or a cell near the target cell (for the bystander effect), but is not required.

[0067] The antigen-binding domain of a CAR most commonly originates from the antibody variable chain (e.g., usually in the form of scFv), but can also be generated by other molecules (such as ligands or other binding molecules).

[0068] CARs are typically expressed as peptides that also include a signal sequence (also known as a leader sequence), and specifically a signal sequence that targets the CAR to the cell's plasma membrane. This is usually located next to or near the antigen-binding domain, typically upstream of it. The extracellular domain of the CAR can therefore contain both the signal sequence and the antigen-binding domain, essentially consisting of or composed of them.

[0069] As described above, the antigen-binding domain can be any protein or peptide capable of specifically recognizing and binding to GLP1R. Antigen-binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinant binding partner targeting GLP1R. Exemplary antigen-specific targeting domains include antibodies or antibody fragments or derivatives, or soluble or membrane-bound ligands of GLP1R.

[0070] In one embodiment, the antigen recognition domain comprises or is composed of a ligand of GLP1R. The ligand can be a natural ligand of GLP1R (i.e., a naturally occurring ligand, which may be referred to as a physiological ligand) or an artificial ligand of GLP1R (i.e., a non-naturally occurring synthetic ligand, which is not a physiological ligand). The artificial ligand of GLP1R can be derived from the natural ligand of GLP1R. The artificial ligand of GLP1R can have at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, or 96%) identity with the natural ligand of GLP1R. The artificial ligand of GLP1R can differ from the natural ligand of GLP1R by up to 10 amino acids, for example, up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid. For example, the artificial ligand of GLP1R can differ from the natural ligand by at least 1, 2, or 3 amino acids.

[0071] The antigen recognition domain of a CAR can be derived from a ligand of GLP1R, which can be either an agonist or an antagonist. However, activation of GLP1R leads to increased insulin synthesis and release. Therefore, the ligand of GLP1R can be, in particular, a GLP1R agonist.

[0072] Glucagon-like peptide-1 (GLP1) and glucagon are natural ligands of GLP1R. The antigen recognition domain may contain or consist of GLP1 or glucagon or a peptide derived from GLP1 or glucagon. A peptide derived from GLP1 or glucagon may have at least about 70% (e.g., at least about 75%, 80%, 85%, 90%, 95%, or 96%) identity with GLP1 or glucagon, respectively. A peptide derived from GLP1 or glucagon may differ from GLP1 or glucagon by up to 10 amino acids, for example, up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid. For example, a peptide derived from GLP1 or glucagon may differ from GLP1 or glucagon by at least 1, 2, or 3 amino acids. As described below, any antigen recognition domain containing a peptide derived from GLP1 or glucagon, or composed of such peptides, retains its ability to bind GLP1R.

[0073] Various GLP1 analogs are discussed in Gupta, Indian J Endocrinol Metab., 2013; 17(3): 413-421, which is incorporated herein by reference. Any of the GLP1 analogs or their derivatives discussed herein can be used for antigen recognition domains of CARs.

[0074] Both GLP1 and glucagon are derived from proglucagon, which is encoded by the GCG gene. The amino acid sequence of human proglucagon is shown in SEQ ID NO: 66. Proglucagon undergoes tissue-specific post-translational processing in pancreatic α cells and intestinal L cells. In pancreatic α cells, glucagon is produced by cleavage of PCSK2 / PC2. The amino acid sequence of human glucagon is shown in SEQ ID NO: 67. Therefore, the antigen recognition domain may contain or consist of the sequence of SEQ ID NO: 67 or a sequence differing from it by 1, 2, 3 or more amino acids, for example, differing by up to 10 amino acids, for example, differing by 1 to 5 amino acids. In intestinal L cells, PCSK1 / PC1 produces GLP1. GLP1 undergoes further N-terminal truncation in intestinal L cells via post-translational processing to produce the biologically active forms GLP1(7-37) and GLP1(7-36)amide. The amino acid sequence of the starting product GLP1 (1-37) is shown in SEQ ID NO: 68. The amino acid sequence of GLP1 (7-37) is shown in SEQ ID NO: 69. Therefore, the antigen recognition domain may contain or consist of the following: the sequence of SEQ ID NO: 68 or SEQ ID NO: 69, or a sequence that differs from SEQ ID NO: 68 or SEQ ID NO: 69 by 1, 2, 3 or more amino acids, for example, by up to 10 amino acids, or by 1 to 5 amino acids.

[0075] The peptide bond between the 8th Ala residue and the 9th Glu residue of GLP1 is cleaved in vivo by dipeptidyl peptidase-4 (DPP-4), which significantly reduces the amount of GLP1 in circulation. Therefore, modifying one or more of the 8th Ala and 9th Glu residues of SEQ ID NO: 68 (corresponding to the 2nd and 3rd residues of SEQ ID NO: 69) may be beneficial in preventing cleavage of the antigen recognition domain of the CAR. Therefore, the antigen recognition domain may comprise or consist of a sequence derived from SEQ ID NO: 68 or SEQ ID NO: 69, wherein one or both of the 8th Ala residue of SEQ ID NO: 68 (the 2nd residue of SEQ ID NO: 69) and the 9th Glu residue of SEQ ID NO: 68 (the 3rd residue of SEQ ID NO: 69) are modified (e.g., by substitution, deletion, or insertion). For example, the 8th Ala residue of SEQ ID NO: 68 (the 2nd residue of SEQ ID NO: 69) may be modified (e.g., by substitution). For example, position 8 of SEQ ID NO: 68 (position 2 of SEQ ID NO: 69) Ala can be substituted with Gly. Other residues that can be modified (e.g., by substitution, insertion, or deletion) to help prevent cleavage include His at position 7, Gly at position 10, Phe at position 12, Thr at position 13, and Asp at position 15 of SEQ ID NO: 68. Any one or more of these residues can be modified (e.g., by substitution, insertion, or deletion), for example, except for modification of one or both of Ala at position 8 and Glu at position 9 of SEQ ID NO: 68.

[0076] The ligand of GLP1R may constitute part or all of the antigen recognition domain of the CAR described herein, and the ligand of GLP1R may contain one or more modifications (e.g., substitution, insertion, or deletion) to alter its affinity for GLP1R. For example, the ligand of GLP1R may contain one or more modifications (e.g., substitution, insertion, or deletion) to increase its affinity for GLP1R. For example, the ligand of GLP1R may contain one or more modifications (e.g., substitution, insertion, or deletion) to increase its affinity by 10%, 20%, 30%, 40%, 50%, or more. The affinity of the ligand for its receptor (e.g., the balance binding affinity (K)) is also considered. dThe antigen recognition domain can be determined by methods known in the art, such as flow cytometry. For example, the antigen recognition domain may contain or consist of a sequence derived from SEQ ID NO: 68 or SEQ ID NO: 69, wherein one or both of position 22 of SEQ ID NO: 68 (position 16 of SEQ ID NO: 69) Gly and position 36 of SEQ ID NO: 68 (position 30 of SEQ ID NO: 69) Arg are modified. For example, position 36 of SEQ ID NO: 68 (position 30 of SEQ ID NO: 69) Arg may be replaced by, for example, Gly. For example, position 22 of SEQ ID NO: 68 (position 16 of SEQ ID NO: 69) Gly may be replaced by, for example, Glu.

[0077] In one embodiment, the antigen recognition domain of the CAR comprises or consists of the following: the amino acid sequence of SEQ ID NO: 61, or a sequence differing from it by 1, 2, 3 or more amino acids, for example, a sequence differing by up to 10, 5 or 3 amino acids. For example, the sequence may differ by 1 to 5 amino acids. When the amino acid sequence differs from SEQ ID NO: 61 by one or more amino acids, in some embodiments, modifications at positions 2 and / or 16 and / or 30 relative to SEQ ID NO: 69 may be retained. For example, when the amino acid sequence differs from SEQ ID NO: 61 by one or more amino acids, in some embodiments, at least position 2 (Gly) and / or position 16 (Glu) and / or position 30 (Gly) of SEQ ID NO: 61 may be retained. Specifically, at least position 2 (Gly) may be retained.

[0078] In one embodiment, the antigen recognition domain of the CAR comprises or consists of the amino acid sequence of SEQ ID NO: 61, or a sequence having at least about 70% identity with it. For example, the antigen recognition domain of the CAR may have at least about 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 61. When the amino acid sequence differs from SEQ ID NO: 61, in some embodiments, modifications at positions 2 and / or 16 and / or 30 of SEQ ID NO: 69 may be retained. For example, when the amino acid sequence differs from SEQ ID NO: 61, in some embodiments, at least position 2 (Gly) and / or position 16 (Glu) and / or position 30 (Gly) of SEQ ID NO: 61 may be retained. Specifically, at least position 2 (Gly) may be retained.

[0079] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence encoded by the sequence shown in SEQ ID NO: 62 or an amino acid sequence having at least 70% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 62. For example, the antigen-recognition domain of the CAR may have at least about 75%, 80%, 85%, 90%, or 95% identity with the amino acid sequence encoded by SEQ ID NO: 62.

[0080] In one embodiment, the antigen-binding domain of the CAR comprises or consists of the following: an amino acid sequence encoded by the sequence shown in SEQ ID NO: 63 or an amino acid sequence having at least 70% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 63. For example, the antigen-recognition domain of the CAR may have at least about 75%, 80%, 85%, 90%, or 95% identity with the amino acid sequence encoded by SEQ ID NO: 63. For example, the antigen-binding domain of the CAR may comprise or consist of the following: an amino acid sequence or a variant thereof encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., the myc tag and / or the his tag) from SEQ ID NO: 63.

[0081] In a further embodiment, the CAR construct may contain or consist of the following: the sequence shown in any one of SEQ ID NO:126 or 150 or a variant thereof (e.g., a sequence having at least 70% identity with it).

[0082] In one embodiment, the antigen-binding domain is or is derived from an antibody. As used herein, the term "antibody" refers broadly to any immunobinding agent or molecule containing an antigen-binding domain, including polyclonal and monoclonal antibodies. Intact antibodies are classified into one of five main categories based on the type of constant domain in the heavy chain: IgA, IgD, IgE, IgG, and IgM, and antibodies as described herein can belong to any of these categories. Several of these antibodies are further subdivided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, etc. Typically, IgG or IgM antibodies are the most common antibodies used in physiological settings. As those skilled in the art will understand, the term "antibody" encompasses all antibodies, including intact antibodies, dimer, trimer, and multimer antibodies; bispecific antibodies; chimeric antibodies; recombinant and engineered antibodies and fragments thereof.

[0083] The binding domain derived from the antibody can be a fragment of the antibody or a genetically engineered product of one or more fragments of the antibody that participates in binding to the antigen. Examples include variable regions (Fv), complementarity-determining regions (CDR), Fab or F(ab')2, or light and heavy chain variable regions can be linked together to form a single chain (e.g., scFv), and in any orientation (e.g., V). L -V H or V H -V L V L and / or V H The sequence can be modified. Specifically, the frame region can be modified (e.g., substituted, for example, to humanize the antigen-binding domain). Other examples include heavy chain variable regions (VH), light chain variable regions (VL), and single-domain antibodies (sdAbs) that can be called nanobodies. An example of a single-domain antibody is a camelid heavy chain antibody (HCAb), which has an antigen recognition site formed by a single domain (called VHH).

[0084] In a preferred embodiment, the antigen-binding domain is a single-chain antibody (scFv). The scFv can be mouse, human, or humanized scFv.

[0085] In an alternative preferred embodiment, the antigen-binding domain is derived from a camelid heavy chain antibody (HCAb), for example, the antigen-binding domain may be the VHH domain of an HCAb. The VHH has a structure similar to the VH domain of conventional IgG and contains three variable CDRs, although CDRs 1 and 3 typically have more amino acids than the VH domain. In some embodiments, the VHH domain may contain one or more CDRs comprising or consisting of the following: sequences shown in SEQ ID NOs: 1 to 30 as specified herein, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with them. For example, the VHH domain may contain one, two, or three VH CDRs comprising or consisting of the following: sequences shown in SEQ ID NOs: 1 to 30 as specified herein, or sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with them. When the VHH domain contains two or three VH CDRs of SEQ ID NO: 1 to 30 or 151 to 163, these CDRs may be derived from the same binder described herein (e.g., all derived from binders A2, A4, A9, etc.).

[0086] The "complementarity-determining region" or "CDR" of an antibody or its antigen-binding fragment refers to a highly variable loop within the variable region of the antibody's heavy or light chain. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). Both the heavy chain and light chain variable regions contain three CDRs. The "heavy chain variable region" or "VH" refers to a segment of the antibody heavy chain containing three CDRs inserted between flanking extensions (called framework regions), which are more conserved than the CDRs and form a scaffold supporting them. The "light chain variable region" or "VL" refers to a segment of the antibody light chain containing three CDRs inserted between framework regions.

[0087] "Fv" refers to the smallest fragment of an antibody that carries a complete antigen-binding site. An Fv fragment consists of a single light chain variable region that binds to a single heavy chain variable region. A "single-chain Fv antibody" or "scFv" refers to an engineered antibody composed of a light chain variable region and a heavy chain variable region, which are linked to each other directly or via peptide linker sequences in any orientation.

[0088] Antibodies that specifically bind to a predetermined antigen (i.e., GLP1R) can be prepared using methods known in the art. Such methods include phage display, methods for generating human or humanized antibodies, or methods using transgenic animals or plants engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available, and antibodies or fragments thereof that can bind to the target molecule (i.e., GLP1R) can be screened. Phage display libraries of human antibodies are also available. Once identified, the amino acid or polynucleotide sequence encoding the antibody can be isolated and / or determined.

[0089] An antigen recognition domain can bind, appropriately and specifically, to one or more regions or epitopes within a GLP1R. An epitope, also known as an antigenic determinant, is a portion of an antigen that is recognized by an antigen recognition domain (e.g., an antibody). In other words, an epitope is a specific fragment of an antigen that an antibody binds to. Appropriately, the antigen recognition domain binds, appropriately and specifically, to a region or epitope within a GLP1R.

[0090] The antigen recognition domain may contain at least one CDR (e.g., CDR3), which can be predicted by an antibody binding to the antigen (i.e., GLP1R) (or a variant of such a predicted CDR (e.g., a variant with one, two, or three amino acid substitutions)). It should be understood that molecules containing three or fewer CDR regions (e.g., a single CDR or even a portion thereof) may be able to retain the antigen-binding activity of the antibody from which the CDR is derived. Molecules containing two CDR regions are described in the art as being able to bind target antigens, for example, in the form of minibody (Vaughan and Sollazzo, 2001, Combinational Chemistry & High Throughput Screening, 4, 417-430). Molecules containing a single CDR have been described that can exhibit strong binding activity to a target (Nicaise et al., 2004, Protein Science, 13: 1882-91).

[0091] In this respect, the antigen-binding domain may contain one or more variable heavy chain CDRs, for example, one, two, or three variable heavy chain CDRs. Alternatively or otherwise, the antigen-binding domain may contain one or more variable light chain CDRs, for example, one, two, or three variable light chain CDRs. The antigen-binding domain may contain three heavy chain CDRs and / or three light chain CDRs (and more specifically, a heavy chain variable region containing three CDRs and / or a light chain variable region containing three CDRs), wherein at least one CDR (preferably all CDRs) may be derived from an antibody binding to GLP1R.

[0092] The antigen-binding domain may comprise any combination of variable heavy chain and variable light chain CDRs, such as one variable heavy chain CDR in conjunction with one variable light chain CDR, two variable heavy chain CDRs in conjunction with one variable light chain CDR, two variable heavy chain CDRs in conjunction with two or three variable light chain CDRs, three variable heavy chain CDRs in conjunction with one or two variable light chain CDRs, one variable heavy chain CDR in conjunction with two or three variable light chain CDRs, or three variable heavy chain CDRs in conjunction with three variable light chain CDRs. Preferably, the antigen-binding domain comprises three variable heavy chain CDRs (CDR1, CDR2, and CDR3) and / or three variable light chain CDRs (CDR1, CDR2, and CDR3).

[0093] One or more CDRs present within an antigen-binding domain may not all originate from the same antibody, as long as the domain possesses the desired binding activity. Therefore, one CDR can be predicted by the heavy or light chain of an antibody binding GLP1R, while another present CDR can be predicted by different antibodies binding GLP1R. Combinations of CDRs from different antibodies (especially those binding the same desired region or epitope) can be used.

[0094] In a particularly preferred embodiment, the antigen-binding domain comprises three CDRs predicted by the variable heavy chain sequence of the antibody binding GLP1R and / or three CDRs predicted by the variable light chain sequence of the antibody binding GLP1R (preferably the same antibody).

[0095] In one embodiment, the antigen-binding domain comprises (a) the VH CDR1, 2, and 3 sequences as shown in SEQ ID NO: 152, 2, and 3, respectively, or (b) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 153, 2, and 3, respectively, or (c) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 154, 2, and 3, respectively, or (a) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 156, 161, and 162, respectively, or (b) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 156, 161, and 163, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0096] In one embodiment, the antigen-binding domain comprises VHCDR1, 2, and 3 sequences as shown in SEQ ID NO. 1, 2, and 3, respectively, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO. 4, 5, and 6, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0097] More specifically, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence as shown in SEQ ID NO: 31 or a sequence having at least 70% sequence identity therewith, and the VL domain comprises a sequence as shown in SEQ ID NO: 32 or a sequence having at least 70% sequence identity therewith.

[0098] For example, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence encoded by SEQ ID NO: 41 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 41, and the VL domain comprises a sequence encoded by SEQ ID NO: 42 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 42.

[0099] In one embodiment, the antigen-binding domain comprises the VHCDR1, 2, and 3 sequences as shown in SEQ ID NO: 7, 8, and 9, respectively, and (a) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 156, 161, and 12, respectively; or (b) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 157, 161, and 12, respectively; or (c) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 158, 161, and 12, respectively; or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0100] In one embodiment, the antigen-binding domain comprises VHCDR1, 2, and 3 sequences as shown in SEQ ID NO. 7, 8, and 9, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO. 10, 11, and 12, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0101] More specifically, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence as shown in SEQ ID NO: 33 or a sequence having at least 70% sequence identity therewith, and the VL domain comprises a sequence as shown in SEQ ID NO: 34 or a sequence having at least 70% sequence identity therewith.

[0102] For example, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence encoded by SEQ ID NO: 43 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 43, and the VL domain comprises a sequence encoded by SEQ ID NO: 44 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 44.

[0103] In one embodiment, the antigen-binding domain comprises the VH CDR1, 2, and 3 sequences as shown in SEQ ID NO: 13, 14, and 155, respectively, and (a) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 157, 161, and 162, respectively; or (b) the VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 158, 161, and 162, respectively; or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0104] In one embodiment, the antigen-binding domain comprises VHCDR1, 2, and 3 sequences as shown in SEQ ID NO. 13, 14, and 15, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO. 16, 17, and 18, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0105] More specifically, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence as shown in SEQ ID NO: 35 or a sequence having at least 70% sequence identity therewith, and the VL domain comprises a sequence as shown in SEQ ID NO: 36 or a sequence having at least 70% sequence identity therewith.

[0106] For example, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence encoded by SEQ ID NO: 45 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 45, and the VL domain comprises a sequence encoded by SEQ ID NO: 46 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 46.

[0107] In one embodiment, the antigen-binding domain comprises (a) the VHCDR1, 2, and 3 sequences shown in SEQ ID NO: 151, 20, and 21, respectively; (b) the VHCDR1, 2, and 3 sequences shown in SEQ ID NO: 152, 20, and 21, respectively; or (c) the VHCDR1, 2, and 3 sequences shown in SEQ ID NO: 153, 20, and 21, respectively; and the VLCDR1, 2, and 3 sequences shown in SEQ ID NO: 22, 23, and 24, respectively. Alternatively, the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0108] In one embodiment, the antigen-binding domain comprises VHCDR1, 2, and 3 sequences as shown in SEQ ID NO. 19, 20, and 21, respectively, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO. 22, 23, and 24, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0109] More specifically, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence as shown in SEQ ID NO: 37 or a sequence having at least 70% sequence identity therewith, and the VL domain comprises a sequence as shown in SEQ ID NO: 38 or a sequence having at least 70% sequence identity therewith.

[0110] For example, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence encoded by SEQ ID NO: 47 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 47, and the VL domain comprises a sequence encoded by SEQ ID NO: 48 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 48.

[0111] In one embodiment, the antigen-binding domain comprises (a) the VH CDR1, 2, and 3 sequences shown as in SEQ ID NO: 151, 26, and 155, respectively, or (b) the VL CDR1, 2, and 3 sequences shown as in SEQ ID NO: 154, 26, and 155, respectively, and (a) the VL CDR1, 2, and 3 sequences shown as in SEQ ID NO: 159, 161, and 163, respectively, or (b) the VL CDR1, 2, and 3 sequences shown as in SEQ ID NO: 160, 161, and 163, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0112] In one embodiment, the antigen-binding domain comprises VHCDR1, 2, and 3 sequences as shown in SEQ ID NO. 25, 26, and 27, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO. 28, 29, and 30, respectively, or the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0113] More specifically, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence as shown in SEQ ID NO: 39 or a sequence having at least 70% sequence identity therewith, and the VL domain comprises a sequence as shown in SEQ ID NO: 40 or a sequence having at least 70% sequence identity therewith.

[0114] For example, in such embodiments, the antigen-binding domain of the CAR comprises a VH domain and a VL domain, wherein the VH domain comprises a sequence encoded by SEQ ID NO: 49 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 49, and the VL domain comprises a sequence encoded by SEQ ID NO: 50 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 50.

[0115] If the CDR does contain amino acid sequence modifications, this can be the deletion, addition, or substitution of amino acid residues in the CDR sequence listed in SEQ ID NO above. More specifically, the modification can be amino acid substitution, for example, conserved amino acid substitution, such as those listed above. Longer CDRs can tolerate more amino acid residue modifications. In the case of CDRs of 5 or more, or 7 or more amino acid residues in length, the modification can be 0, 1, 2, or 3 residues, for example, 2 residues. Typically, any given CDR sequence can have 0, 1, 2, or 3 modifications. Furthermore, in one embodiment, CDR1 and 2 can be modified, and CDR3 can be unmodified. In another embodiment, all three CDRs can be modified. In yet another embodiment, the CDRs are unmodified.

[0116] The antigen-binding domain can be in the form of an scFv containing VH and VL domain sequences as listed above, in any order, for example, VH-VL. The VH and VL sequences can be linked by a linker sequence.

[0117] Suitable linkers can be easily selected and can have any suitable length, such as 1 amino acid (e.g., Gly) to 30 amino acids, such as any of 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids to any of 12, 15, 18, 20, 21, 25, 30 amino acids, such as 5-30, 5-25, 6-25, 10-15, 12-25, 15-25, etc.

[0118] Exemplary connectors include glycine polymers (G), glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible connectors known in the art, as described above. The connector may contain one or more “GS” domains as described above.

[0119] Linker sequences can be flexible linker sequences. Flexible linkers are a class of linker sequences well-known and described in the art. Linker sequences are generally referred to as sequences that can be used to link or join proteins or protein domains together to produce, for example, fusion proteins or chimeric proteins, or multifunctional proteins or peptides. They can have different properties and can be, for example, flexible, rigid, or cleavable. Protein linkers have been reviewed, for example, in Chen et al., 2013, Advanced Drug Delivery Reviews 65, 1357-1369, which compares the class of flexible linkers with the class of rigid and cleavable linkers. Flexible linkers are also described in Klein et al., 2014, Protein Engineering Design and Selection, 27(10), 325-330; van Rosmalen et al., 2017, Biochemistry, 56, 6565-6574; and Chichili et al., 2013, Protein Science, 22, 153-167.

[0120] Flexible linkers are linkers that allow a degree of movement between the connected domains or components. They are typically composed of small nonpolar (e.g., Gly) or polar (e.g., Ser or Thr) amino acid residues. The small size of the amino acids provides flexibility and allows the connected parts (domains or components) to be mobile. The incorporation of polar amino acids can maintain the stability of the linker in an aqueous environment by forming hydrogen bonds with water molecules.

[0121] The most commonly used flexible linkers have sequences consisting primarily of Ser and Gly residues (the so-called "GS linkers"). However, many other flexible linkers have also been described (e.g., see Chen et al., 2013, above), which may contain additional amino acids (such as Thr and / or Ala, and / or Lys and / or Glu, which can improve solubility). Any flexible linker known in the art and reported therein can be used.

[0122] Although the length of the linker is not critical, in some embodiments, a shorter linker sequence may be desirable. For example, the length of the linker sequence may not exceed 25 amino acids, preferably not exceeding 24, 23, 22, or 21 amino acids.

[0123] In other implementations, longer connector sequences may be desired, for example, consisting of or containing multiple repetitions of the GS domain.

[0124] In some embodiments, the linker length can be any from 2, 3, 4, 5, or 6 amino acids to any from 24, 23, 22, or 21 amino acids. In other embodiments, the linker length can be any from 2, 3, 4, 5, or 6 amino acids to any from 21, 20, 19, 18, 17, 16, or 15 amino acids. In other embodiments, the linker length can be between these ranges, for example, 6 to 21, 6 to 20, 7 to 20, 8-20, 9-20, 10-20, 8-18, 9-18, 10-18, 9-17, 10-17, 9-16, 10-16, etc. Therefore, the linker length can be within a range consisting of any integers listed above.

[0125] Using GS-linkers, or more specifically GS (“Gly-Ser”) domains, in linkers allows for easy variation of linker length by changing the number of GS domain repeats, and thus such linkers represent a preferred class of linkers. However, flexible linkers are not limited to those based on “GS” repeats, and other linkers including Ser and Gly residues dispersed throughout the linker sequence have been reported (including in the literature of Chen et al., see above).

[0126] Therefore, in one embodiment, the linker sequence may contain at least 40% Gly or Gly and Ser residues.

[0127] In another embodiment, the linker sequence may contain Ser and / or Gly residues, and no more than 15 other amino acid residues, preferably no more than 14, 13, 12, 11, 10, 9, 8, 6, 7, 5, or 4 other amino acid residues. It should be understood that "other" amino acid residues may be any amino acid that is not Ser or Gly.

[0128] Pro residues in the linker tend to impart rigidity, so in one embodiment, the linker sequence does not contain any Pro residues. However, this is not absolute, as flexible linker sequences may contain one or more Pro residues depending on the sequence context.

[0129] In a preferred embodiment, the linker sequence includes at least one Gly-Ser domain consisting only of Ser and Gly residues. In such embodiments, the linker may contain no more than 15 other amino acid residues, preferably no more than 14, 13, 12, 11, 10, 9, 8, 6, 7, 5, or 4 other amino acid residues.

[0130] The Gly-Ser domain can have the following formula: (S)q-[(G)m-(S)m]n-(G)p Where q is 0 or 1; m is an integer from 1 to 8; n is an integer at least 1 (e.g., 1 to 8, or more specifically 1 to 6); and p is an integer from 0 to 1 to 3.

[0131] More specifically, the Gly-Ser domain can have the following formula: (i) S-[(G)mS]n; (ii) [(G)mS]n; or (iii) [(G)mS]n-(G)p Where m is an integer from 2 to 8 (e.g., 3 to 4); n is an integer at least 1 (e.g., 1 to 8, or more specifically 1 to 6); and p is an integer from 0 to 1 to 3.

[0132] In a representative example, the Gly-Ser domain can have the following formula: S-[GGGGS]n Where n is an integer of at least 1 (preferably 1 to 8 or 1-6, 1-5, 1-4 or 1-3). In the above formula, the sequence GGGGS is SEQ ID NO: 73.

[0133] The representative sequence GGGS is shown in SEQ ID NO: 70.

[0134] The linker sequence may consist solely of one or more Gly-Ser domains as described or defined above, or it may consist solely of one or more Gly-Ser domains as described or defined above. However, as described above, in another embodiment, the linker sequence may include one or more Gly-Ser domains and additional amino acids. The additional amino acids may be located at one or both ends of a Gly-Ser domain, or at one or both ends of a repeating Gly-Ser domain. Thus, additional amino acids, which may be other amino acids, may be located at one or both ends of the linker sequence; for example, they may be located flanking a Gly-Ser domain. In other embodiments, the additional amino acids may be located between Gly-Ser domains. For example, two Gly-Ser domains may be located flanking a segment of other amino acids in the linker sequence. Furthermore, also as described above, in other linkers, the GS domain does not need to be repeated, and short domains such as G and / or S residues or GS may simply be distributed along the length or sequence.

[0135] Representative exemplary connector sequences are listed below: ETSGGGGSRL (SEQ ID NO. 71) SGGGGSGGGGSGGGGS (SEQ ID NO. 72) S(GGGGS) 1-5 (Where GGGGS is SEQ ID NO. 73) (GGGGS) 1-5 (Where GGGGS is SEQ ID NO. 73) S(GGGS) 1-5 (GGGS is SEQ ID NO. 70) (GGGS) 1-5 (GGGS is SEQ ID NO. 70) S(GGGGGS) 1-5 (Where GGGGGS is SEQ ID NO. 74) (GGGGGS) 1-5 (Where GGGGGS is SEQ ID NO. 74) S(GGGGGGS) 1-5 (Where GGGGGGS is SEQ ID NO. 75) (GGGGGGS) 1-5 (Where GGGGGGS is SEQ ID NO. 75) GGGGSGGGGSGGGGS (SEQ ID NO. 76) GGGGG (SEQ ID NO. 77) GGGGSGGGGS (SEQ ID NO. 78) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 79) GGGGGG (SEQ ID NO. 80) G6 (SEQ ID NO. 81) G8 (SEQ ID NO. 82) KESGSSVSSEQLAQFRSLD (SEQ ID NO. 83) EGKSSGSGSESKST (SEQ ID NO. 84) GSAGSAAGSGEF (SEQ ID NO. 85) SGGGGSAGSAAGSGEF (SEQ ID NO. 86) SGGGLLLLLLLLGGGS (SEQ ID NO. 87) SGGGAAAAAAAAGGGS (SEQ ID NO. 88) SGGGAAAAAAAAAAAAAAAAGGGS (SEQ ID NO. 89) SGALGGLALAGLLLAGLGLGAAGS (SEQ ID NO. 90) SLSLSPGGGGGPAR (SEQ ID NO. 91) SLSLSPGGGGGPARSLLSLSPGGGGG (SEQ ID NO. 92) GSSGSS (SEQ ID NO. 93) GSSSSSS (SEQ ID NO. 94) GGSSSS (SEQ ID NO. 95) GSSSSS (SEQ ID NO. 96) SGGGGS (SEQ ID NO. 97).

[0136] In some implementations, the connector has sequence (GGGGS)3 (SEQ ID NO: 76).

[0137] Although the linker sequences of the present invention peptides as defined above are flexible sequences, this disclosure also includes other peptides, including those that contain non-flexible linkers and / or do not meet the definitions and requirements listed above.

[0138] Another example of a linker that can be used to connect the VH and VL domains is KLEEGEFSEARV (SEQ ID NO: 98) or a sequence having at least about 60% identity with it, for example, a sequence having at least about 65% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or at least about 95% identity with it. Alternatively, the linker may have the sequence of SEQ ID NO: 98 or a sequence that differs from it by no more than 6 amino acids, for example, a sequence that differs from it by no more than 5 or no more than 4 or no more than 3 or no more than 2 or no more than 1 amino acid. Specifically, the linker may be truncated or lengthened at the N-terminus and / or C-terminus (e.g., see Schmiedl A. et al., Protein Eng. 2000 Oct; 13(10): 725-34, where the linker is shortened by one amino acid at each end compared to SEQ ID NO: 98). To improve the stability of the linker and eliminate the two putative trypsin cleavage sites (lysine and arginine), the linker can be modified by exchanging lysine (K) for isoleucine (I), arginine (R) for glycine (G), and valine (V) for cysteine ​​(C). The resulting linker has the amino acid sequence ILEEGEFSEAXC (SEQ ID NO: 99). Any peptide linker carrying the shared amino acid sequence X1LEEGEFSEAX2X3 (SEQ ID NO: 100) can also be used, where X1 is K or I, X2 is R or G, and X3 is V or C.

[0139] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of a VH sequence as shown in SEQ ID NO: 31, which is linked to the VL sequence of SEQ ID NO: 32 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0140] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of a VH sequence as shown in SEQ ID NO: 33, which is linked to the VL sequence of SEQ ID NO: 34 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0141] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of a VH sequence as shown in SEQ ID NO: 35, which is linked to the VL sequence of SEQ ID NO: 36 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0142] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of the VH sequence as shown in SEQ ID NO: 37, which is linked to the VL sequence of SEQ ID NO: 38 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0143] Therefore, in one embodiment, the antigen-binding domain may comprise or consist of a VH sequence as shown in SEQ ID NO: 39, which is linked to the VL sequence of SEQ ID NO: 40 via a linker of sequence (X)n, wherein X is any amino acid and n is an integer between 15 and 25.

[0144] In this regard, in one embodiment, the antigen-binding domain of the CAR comprises or consists of the sequence shown in SEQ ID NO: 51 or a sequence having at least 80% identity with it.

[0145] In another embodiment, the antigen-binding domain of the CAR comprises or consists of the sequence shown in SEQ ID NO: 52 or a sequence having at least 80% identity with it.

[0146] In a further embodiment, the antigen-binding domain of the CAR includes or consists of the following: the sequence shown in SEQ ID NO: 53 or a sequence having at least 80% identity with it.

[0147] In a further embodiment, the antigen-binding domain of the CAR includes or consists of the following: the sequence shown in SEQ ID NO: 54 or a sequence having at least 80% identity with it.

[0148] In a further embodiment, the antigen-binding domain of the CAR comprises or consists of the following: the sequence shown in SEQ ID NO: 55 or a sequence having at least 80% identity with it.

[0149] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence encoded by the sequence shown in SEQ ID NO: 56 or an amino acid sequence having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 56. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence or a variant thereof encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., the myc tag and / or his tag) from SEQ ID NO: 56.

[0150] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence encoded by the sequence shown in SEQ ID NO: 57 or an amino acid sequence having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 57. For example, the antigen-binding domain of the CAR may comprise or consists of an amino acid sequence or a variant thereof encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., the myc tag and / or his tag) from SEQ ID NO: 57.

[0151] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence encoded by the sequence shown in SEQ ID NO: 58 or an amino acid sequence having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 58. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence or a variant thereof encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., the myc tag and / or his tag) from SEQ ID NO: 58.

[0152] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence encoded by the sequence shown in SEQ ID NO: 59 or an amino acid sequence having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 59. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence or a variant thereof encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., the myc tag and / or his tag) from SEQ ID NO: 59.

[0153] In one embodiment, the antigen-binding domain of the CAR comprises or consists of an amino acid sequence encoded by the sequence shown in SEQ ID NO: 60, or an amino acid sequence having at least 80% identity with the amino acid sequence encoded by the sequence shown in SEQ ID NO: 60. For example, the antigen-binding domain of the CAR may comprise or consist of an amino acid sequence or a variant thereof encoded by the sequence remaining after removing the sequence encoding the signal sequence and / or any tag sequence (e.g., the myc tag and / or his tag) from SEQ ID NO: 60.

[0154] In a further embodiment, the CAR construct may contain or consist of the following: a sequence or a variant thereof shown in any one of SEQ ID NO: 101 to 105 or 145 to 149 (e.g., a sequence having at least 70% identity with it).

[0155] The variant sequences disclosed and described herein, including variant CAR, VH, VL, and antigen-binding domain sequences, may have at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% sequence identity with the specified SEQ ID NO.

[0156] The CAR preferably also includes a hinge domain to decouple the extracellular domain (particularly the antigen-binding domain) from the cell surface, and also includes a transmembrane domain. The hinge and transmembrane domains can comprise hinge and transmembrane sequences from any protein having a hinge domain and / or a transmembrane domain, including any type I, II, or III transmembrane protein. The hinge domain can be selected from CD28, CD8α, CD4, CD7, CH2CH3, the hinge region of an immunoglobulin, or a portion thereof or a variant thereof. Typically, the hinge can be derived from CD8 (particularly CD8α) or from CH2CH3. In one embodiment, the hinge can contain one or more cysteine ​​residues, for example, to allow disulfide bond bonding. For example, the CD8 hinge can contain one or more cysteine ​​residues, for example, one cysteine ​​residue, two cysteine ​​residues, or three cysteine ​​residues.

[0157] The transmembrane domain of the CAR may also contain artificial hydrophobic sequences. The transmembrane domain of the CAR can be selected to prevent dimerization. The addition of transmembrane domains will be apparent to those skilled in the art. Examples of transmembrane (TM) regions used in CAR constructs are: 1) CD28 TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41; Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35; Casucci et al., Blood, 2013, Nov 14;122(20):3461-72.); 2) OX40 TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41); 3) 4-1BB TM region (Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35); 4) CD3ζ TM region (Pule et al., Mol Ther, 2005, Nov;12(5):933-41;Savoldo B, Blood, 2009, Jun 18;113(25):6392-402.;5) CD8α™ region (Maher et al., Nat Biotechnol, 2002, Jan;20(1):70-5;Imai C, Leukemia, 2004, Apr;18(4):676-84;Brentjens et al., CCR, 2007, Sep 15;13(18 Pt 1):5426-35;Milone et al., Mol Ther, 2009, Aug;17(8):1453-64.). Other transmembrane domains that may be used include those from ICOS, CD4, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86 or CD154. Preferably, the transmembrane domain can be derived from CD4, CD28, or CD8α.

[0158] In one embodiment, the CAR may not contain a dimerizing domain that binds a regulatory molecule. A regulatory molecule is any molecule capable of binding at least one dimerizing domain in the CAR and capable of preventing or inducing an interaction between a pair of dimerizing domains. Examples of regulatory molecules include soluble proteins (e.g., cytokines, TGF-β, VEGF) or small molecules. In a further embodiment, dimerization may be uncontrolled when it occurs with other CAR molecules. In another embodiment, monovalent binding of the CAR to an antigen may allow activation of cells expressing the CAR.

[0159] The hinge domain can be readily derived from the same protein as the transmembrane domain. In one implementation, when the transmembrane domain originates from the CD8α transmembrane domain, the hinge domain originates from the CD8α hinge domain.

[0160] Alternatively, the hinge domain can be derived from a protein different from the transmembrane domain. For example, the hinge domain can be derived from the CH2CH3 hinge domain, and the transmembrane domain can be derived from the CD28 transmembrane domain.

[0161] For example, the hinge domain may be derived from the CD8α hinge domain and may contain the amino acid sequence shown in SEQ ID NO: 106 or a variant thereof having at least 80% identity with SEQ ID NO: 106. Suitablely, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 106. An example of a modified CD8α hinge domain is shown in SEQ ID NO: 107.

[0162] For example, the hinge domain may comprise the amino acid sequence shown in SEQ ID NO: 108 or the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 109, or a variant thereof, which has at least 80% identity with the sequence encoded by SEQ ID NO: 108 or SEQ ID NO: 109. Suitably, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the sequence encoded by SEQ ID NO: 108 or SEQ ID NO: 109.

[0163] For example, the transmembrane domain may be derived from the CD8α transmembrane domain and may contain the amino acid sequence shown in SEQ ID NO: 110, representing amino acids 183 to 203 of human CD8α, or a variant having at least 80% identity with SEQ ID NO: 110. Suitablely, the variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 110.

[0164] The CD8α transmembrane domain can be combined with the CD8α hinge domain. In one embodiment, the CAR comprises a CD8α hinge and transmembrane domain sequence as shown in SEQ ID NO. 111 or SEQ ID NO: 112, or a variant thereof, having at least 80% sequence identity. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 111 or SEQ ID NO: 112, respectively. SEQ ID NO. 112 comprises a modified hinge domain, which, relative to the wild-type CD8α hinge sequence, contains an amino acid modification of two cysteine ​​residues. The modified CD8α hinge domain sequence is shown in SEQ ID NO. 107. The wild-type CD8α hinge and transmembrane domain sequence is shown in SEQ ID NO. 111. The six amino acids at the ends of SEQ ID NO. 111 and 112 (when present) are not located in the membrane and form part of the intracellular domain of the CAR.

[0165] For example, the hinge domain may be derived from the CH2CH3 hinge domain and may contain the sequence shown in SEQ ID NO. 113 or SEQ ID NO. 114 or a variant thereof, the variant having at least 80% identity with SEQ ID NO. 113 or 114, respectively. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 113 or 114, respectively.

[0166] Alternatively, examples of CD28 hinge and transmembrane sequences that may be used are SEQ ID NO: 115 or variants thereof, wherein the variants have at least 80% identity with SEQ ID NO: 115. Variants may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 115.

[0167] As another example, a CAR may contain a natural or modified CD8α hinge domain and a CD28 transmembrane domain, or a CD28 hinge domain and a CD8α transmembrane domain, for example based on the sequence given above.

[0168] In one embodiment, a CH2CH3 hinge sequence containing one or more cysteine ​​residues may be used. For example, a CH2CH3 hinge sequence containing one, two, three, four, or more cysteine ​​residues. Other hinge domains that may be used include hinge domains derived from CD4, CD7, or immunoglobulins, or portions thereof or variants thereof. These hinge domains may contain one or more cysteine ​​residues, for example, one, two, three, four, or more cysteine ​​residues.

[0169] In one embodiment, the transmembrane domain may be derived from the CD4 transmembrane domain and may contain the sequence shown in SEQ ID NO: 116 or a variant thereof, the variant having at least 80% identity with SEQ ID NO: 116. The variant may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 116.

[0170] The CAR may also contain a signal sequence (or alternatively a leader sequence) that targets the endoplasmic reticulum pathway for expression on the cell surface. An illustrative signal / leader sequence is MALPVTALLLPLALLLHAAAP as shown in SEQ ID NO. 117. This sequence contains a single amino acid substitution compared to the wild-type CD8α sequence MALPVTALLLPLALLLHAARP (as shown in SEQ ID NO. 118). Any sequence or a variant sequence having at least 70% sequence identity with it may be used. For example, the variant sequence may have at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with it.

[0171] As described herein, the intracellular domain of a CAR contains motifs necessary for transducing effector functional signals and directing CAR-expressing cells to perform their specific functions after antigen binding. Specifically, the intracellular domain may contain one or more (e.g., two or three) immune receptor tyrosine-based activation motifs (ITAMs), typically containing the amino acid sequence YXXL / I, where X can be any amino acid. Examples of intracellular signaling domains include, but are not limited to, the ζ-chain intracellular domain of the T cell receptor or any of its homologs (e.g., the η-chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), the CD3 polypeptide domain (… Intracellular signaling domains may include the human CD3ζ chain intracellular domain, FcyRIII, FcsRI, the cytoplasmic tail of the Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) with a cytoplasmic receptor, or a combination thereof.

[0172] Typically, intracellular signal transduction domains include those of the human CD3ζ chain. The sequence of the human CD3ζ chain intracellular signal transduction domain is shown in SEQ ID NO. 119. A CAR may contain a CD3ζ signal transduction domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO. 119. In one embodiment, the signal transduction domain comprises or consists of SEQ ID NO. 119.

[0173] Other signal transduction domains that may be used include CD28 or CD27 signal transduction domains or variations thereof. Additional intracellular signal transduction domains will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the invention. In one embodiment, the CAR of the present invention may not contain a co-stimulatory domain derived from 4-1BB within its intracellular domain.

[0174] The CAR of the present invention may comprise a complex intracellular domain comprising a fusion of the intracellular portion of a T cell costimulatory molecule and, for example, the intracellular portion of CD3ζ. Such a complex intracellular domain may be referred to as a second-generation CAR, which can simultaneously deliver activation and costimulatory signals upon antigen recognition. The most commonly used costimulatory domain is the costimulatory domain of CD28. This provides the most effective costimulatory signal, namely immune signal 2, which triggers T cell proliferation. The CAR intracellular domain may also comprise one or more TNF receptor family signaling domains (such as the signaling domains of ICOS, (CD134)OX40, 4-1BB, CD27, or TNFRSF25, or portions thereof or variants thereof), although preferably the CAR may not comprise an intracellular domain comprising the signaling domains of both CD28 and 4-1BB.

[0175] Intracellular signaling domains of CD28 that can be used as co-stimulatory domains are shown in SEQ ID NO. 121. Exemplary sequences of the OX40, 4-1BB, ICOS, and TNFRSF25 signaling domains are shown in SEQ ID NOs: 122 to 125. A CAR may comprise one or more co-stimulatory domains comprising or consisting of the following: sequences of any one of SEQ ID NOs: 121, 122, 123, 124, and 125, or variants thereof, wherein the variants have at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity with the CAR.

[0176] In some embodiments, the transmembrane domain and the intracellular signaling domain derived from T-cell costimulatory molecules may be derived from the same protein. For example, in some embodiments, the transmembrane domain and the intracellular signaling domain derived from T-cell costimulatory molecules may be derived from CD28. For example, the CAR may comprise the CD28 transmembrane and CD28 intracellular signaling domains, or variants thereof, as shown in SEQ ID NO: 120, having at least 80% sequence identity with it. The variants may have at least 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NO: 120.

[0177] In one embodiment, the CAR comprises a human CD8 hinge domain or a variant thereof and a human CD8 transmembrane domain. Alternatively or otherwise, the CAR comprises an intracellular domain comprising, or substantially comprises, a human CD28 co-stimulatory domain and a human CD3ζ signaling domain.

[0178] In a preferred embodiment, the CAR comprises a hinge, a transmembrane domain, and an intracellular (or intracellular) domain: (i) A CH2CH3 hinge sequence comprising or consisting of the following: the sequence shown in SEQ ID NO. 113 or SEQ ID NO. 114 or a sequence having at least 80% sequence identity with it; (ii) CD28 transmembrane domain and costimulatory domain, the transmembrane domain and costimulatory domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 120 or a sequence having at least 80% sequence identity with it; (iii) CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0179] In an alternative preferred embodiment, the CAR comprises a hinge, a transmembrane domain, and an intracellular (or intracellular) domain: (i) CD8α hinge and transmembrane domain sequence, which comprises or consists of the following: the sequence shown in SEQ ID NO. 111 or a sequence having at least 80% sequence identity with it; (ii) CD28 costimulatory domain, which comprises or consists of the following: the sequence shown in SEQ ID NO. 121 or a sequence having at least 80% sequence identity with it; (iii) CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0180] In another preferred embodiment, the CAR comprises a hinge, a transmembrane domain, and an intracellular (or intracellular) domain: (i) CD8α hinge and transmembrane domain sequence, which comprises or consists of the following: the sequence shown in SEQ ID NO. 112 or a sequence having at least 80% sequence identity with it; (ii) CD28 costimulatory domain, which comprises or consists of the following: the sequence shown in SEQ ID NO. 121 or a sequence having at least 80% sequence identity with it; (iii) CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0181] The encoded and expressed CAR may also contain a leader sequence that includes or consists of the following: a sequence as shown in SEQ ID NO. 117 or SEQ ID NO: 118 or a sequence having at least 80% sequence identity with it.

[0182] The antigen-binding domain of a CAR may contain or consist of the following: sequences as shown in SEQ ID NO. 51, 52, 53, 54, 55, 61 or sequences having at least 80% sequence identity with them, which are capable of binding GLP1R.

[0183] Therefore, in general, a preferred representative CAR may include: i. A leader sequence comprising or consisting of the following: the sequence shown in SEQ ID NO. 117 or 118 or a sequence having at least 80% sequence identity with it; ii. An antigen-binding domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 51, 52, 53, 54, 55, 61 or a sequence having at least 80% sequence identity with it; iii. A CH2CH3 hinge sequence comprising or consisting of the following: the sequence shown in SEQ ID NO. 113 or 114 or a sequence having at least 80% sequence identity with it; iv. The CD28 transmembrane domain and costimulatory domain, which comprises or consists of the following: the sequence shown in SEQ ID NO. 120 or a sequence having at least 80% sequence identity with it; v. CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0184] Alternative preferred representative CARs may include: i. A leader sequence comprising or consisting of the following: the sequence shown in SEQ ID NO. 117 or 118 or a sequence having at least 80% sequence identity with it; ii. An antigen-binding domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 51, 52, 53, 54, 55, 61 or a sequence having at least 80% sequence identity with it; iii. CD8 hinge and transmembrane domain sequence, which comprises or consists of the following: the sequence shown in SEQ ID NO. 111 or 112 or a sequence having at least 80% sequence identity with it; iv. A CD28 costimulatory domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 121 or a sequence having at least 80% sequence identity with it; v. CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0185] The CAR of the present invention may comprise or consist of any one or more sequences of the sequences shown in SEQ ID NOs 101 to 105, 126, and 145 to 150, or variants thereof, wherein the variants have at least about 80% identity with the CAR. For example, any variant may have at least about 85%, 90%, 95%, 97%, 98%, or 99% identity with SEQ ID NOs 101 to 105, 126, or 145 to 150. The CAR of SEQ ID NOs 101 to 105, 126, 145 to 150 or variants thereof may also comprise a signal sequence, for example, a signal sequence having the sequence shown in SEQ ID NO: 117 or 118.

[0186] CARs can bind to GLP1R and transduce signals to cells that express it.

[0187] Cells can express only one type of CAR, meaning that when a cell expresses more than one CAR molecule, the amino acid sequences of each of these expressed molecules are identical to each other.

[0188] The intracellular domain of the CAR described herein may include other domains. For example, the CAR may include a domain that confers the ability to deliver productive IL signaling to cells expressing the CAR in an antigen-specific manner without the need for exogenous IL administration. For example, the CAR may include a domain that contains a STAT5-associating motif, a JAK1-binding motif, and / or a JAK2-binding motif, and optionally a JAK3-binding motif. In such embodiments, the intracellular domain may contain one or more sequences from the intracellular domain of a cytokine receptor (e.g., an interleukin receptor (IL) receptor). Such CARs are described in WO2020 / 044055 (also incorporated herein by reference). Exemplary amino acid sequences derived from the IL-2 receptor β, containing a STAT5-associating motif and a JAK-binding motif, are provided in SEQ ID NO: 127-129. Containing such domains confers the ability of the CAR to deliver productive IL signaling to cells expressing the CAR in an antigen-specific manner without the need for exogenous IL administration. For example, IL-2 is important for the survival, proliferation, and persistence of Treg cells, but IL-2 levels may often be low or impaired in patients requiring treatment. Therefore, a CAR may contain all or part of a sequence corresponding to the β-chain intracellular domain of an IL receptor or its variants, such as the IL2 receptor, optionally combined with the γ-chain intracellular domain of an IL receptor or its variants, such as the IL2 receptor.

[0189] Alternatively or otherwise, additional nucleic acid sequences or peptides may be introduced into cells or cell populations to improve cell persistence or survival in vivo, for example, by providing cells with productive IL signaling without the need for exogenous IL administration. This IL signaling can be constitutive or inducible. Exemplary techniques may involve, for example, the use of engineered or chimeric receptors that can deliver IL signaling without the need for exogenous IL administration. For example, inducible engineered receptors, such as those described in WO 2018 / 111834, WO 2019 / 169290 and WO 2020 / 264039; or constitutive engineered receptors, such as those described in WO 2018 / 038954, WO 2019 / 102207, WO 2019 / 053420, WO 2020 / 180694 and WO 2017 / 218850; chimeric cytokine receptors, such as WO 2020 / 183131, WO 2017 / 029512, WO 2012 / 138858, WO 2014 / 172584, WO 2017 / 068360, WO 2021 / 023987, WO 2020 / 180664 and WO Those described in 2020 / 044239; or engineered receptors with chain-activating molecules, such as those described in WO 2017 / 201432 and WO 2019 / 183389.

[0190] As described above, the cells or cell populations of the present invention may also contain additional polypeptides, particularly exogenous polypeptides, such as FOXP3 and / or safety switch polypeptides. The polypeptides of the present invention (e.g., CAR, FOXP3, and safety switch) may be encoded by a single nucleic acid molecule. The nucleic acid molecule may contain nucleotide sequences encoding self-cleaving sequences between the encoded polypeptides, thereby allowing the polypeptides to be expressed and / or generated as individual or discrete components. This means that although the polypeptides are encoded by a single nucleic acid molecule, they can be expressed or generated as individual polypeptides during or after translation by “cleavage” at the encoded cleavage site, and thus, at the end of protein production in the cell, they can exist in the cell as individual entities or individual polypeptide chains. Alternatively, additional exogenous polypeptides may be encoded by individual nucleic acid molecules or vectors.

[0191] "Discrete" or "separate" polypeptides mean that the polypeptides are not connected to each other and are physically distinct. In fact, after expression, they are located in different or separate cellular locations. Therefore, CAR, FOXP3, and the safety switch polypeptide are ultimately expressed as single and separate components. CAR is expressed as a cell surface molecule. The safety switch polypeptide can be expressed intracellularly or on the cell surface. In one specific embodiment, the safety switch polypeptide and CAR are expressed on the surface of cells intended for use in ACT. FOXP3 is expressed intracellularly, whereby FOXP3 can perform its role as a transcription factor to regulate cell development and / or activity, as further described below.

[0192] The safety switch peptide provides a cell, or a cell on which a suicide component is expressed. This is useful as a safety mechanism, allowing the deletion of cells that have been administered to a subject when needed, or more generally, as desired or required, for example, once the cell has performed or completed its therapeutic effect.

[0193] Suicide moieties possess the inducible ability to cause cell death, or more generally, to lead to the elimination or deletion of cells. An example of a suicide moiety is a suicide protein encoded by a suicide gene, which can be expressed in or on a cell along with a desired gene (in this case, a CAR), and when expressed, allows for cell deletion to shut down CAR expression. In this context, a suicide moiety is a suicide peptide, which is a peptide capable of causing cell deletion under permissive conditions (i.e., conditions that are induced or activated).

[0194] The suicide moiety can be a polypeptide or amino acid sequence that can be activated by an activator administered to a subject to perform cell deletion activity, or it can have cell deletion activity in the presence of a substrate that can be administered to a subject. In a particular embodiment, the suicide moiety can represent a target of a separate cell deletion agent administered to a subject. By binding to the suicide moiety, the cell deletion agent can target the cell to be deleted. Specifically, the suicide moiety can be recognized by an antibody, and the binding of the antibody to a safety switch polypeptide (when expressed on the cell surface) results in the elimination or deletion of the cell.

[0195] The suicide moiety can be HSV-TK or iCasp9. However, it is preferred that the suicide moiety is or contains an epitope recognized by a cell deletion antibody or other binding molecule capable of inducing cell deletion. In such embodiments, the safety switch peptide is expressed on the cell surface.

[0196] As used in the context of cell deletion in this article, the term “deletion” is synonymous with “removal,” “ablation,” or “elimination.” This term is used to encompass the inhibition of cell killing or cell proliferation, resulting in a reduction in the number of cells in a subject. 100% complete removal may be desirable but may not necessarily be achieved. Reducing the number of cells in a subject or inhibiting their proliferation may be sufficient to have a beneficial effect.

[0197] Specifically, the suicide portion can be a CD20 epitope recognized by the antibody rituximab. Therefore, in a safety switch peptide, the suicide portion can include a minimal epitope based on an epitope derived from a CD20 epitope recognized by the antibody rituximab. Biosimilars of rituximab are available and usable. Those skilled in the art can readily prepare antibodies with rituximab binding specificity using conventional methods and their available amino acid sequences. CAR cells specific to GLP1R (which also express a safety switch peptide containing this sequence) can be selectively killed using the antibody rituximab or an antibody with rituximab binding specificity. The safety switch peptide is expressed on the cell surface, and when the expressed peptide is exposed to or in contact with rituximab or an antibody with the same binding specificity, the cell subsequently dies.

[0198] Therefore, rituximab or antibodies with their binding specificity can be combined with the cells of the present invention for ACT. Cells or nucleic acids or vectors or constructs used to generate cells and rituximab or equivalent antibodies can be provided in a kit or as a combination product.

[0199] For example, the suicide constructs of WO2013 / 153391 or WO2021 / 239812 (both incorporated herein by reference) can be used in cells or cell populations (e.g., Tregs or Treg populations) as described herein.

[0200] The nucleic acid molecule of the present invention can be designed to increase FOXP3 expression in cells by introducing a nucleotide sequence encoding FOXP3 into cells (e.g., Treg cells), wherein the term "FOXP3" is synonymous with the term "FOXP3 polypeptide". Therefore, the nucleic acid molecule, as well as constructs and vectors containing the nucleic acid molecule, provides a means for increasing FOXP3 in cells (e.g., Treg or CD4+ cells). As described above, a single nucleic acid molecule can encode the CAR and FOXP3 polypeptide of the present invention, or the CAR and FOXP3 can be encoded by individual or discrete nucleic acid molecules. Therefore, the present invention provides cells comprising a nucleic acid molecule containing a nucleotide sequence encoding a CAR and a nucleic acid molecule containing a nucleotide sequence encoding FOXP3, particularly pluripotent cells (e.g., iPSCs), HPC cells (e.g., expressing CD34), CD4+ T cells, or Treg cells.

[0201] "FOXP3" is an abbreviation for Forkhead Box P3 protein. FOXP3 is a member of the FOX protein family of transcription factors and plays a major regulator of regulatory pathways in the development and function of regulatory T cells. As used in this article, "FOXP3" encompasses variants, isotypes, and functional fragments of FOXP3.

[0202] "Increased FOXP3 expression" refers to increasing the level of FOXP3 mRNA and / or protein in cells (or cell populations) compared to corresponding cells (or cell populations) that have not been modified by introducing nucleic acid molecules, constructs, or vectors. For example, the level of FOXP3 mRNA and / or protein in cells (or populations of such cells) modified according to the present invention can be increased to at least 1.5 times, at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, or at least 150 times higher than the level in corresponding cells (or populations of such cells) not modified according to the present invention. Preferably, the cells are Treg cells, or the cell population is a Treg population.

[0203] Suitable, the levels of FOXP3 mRNA and / or protein in the modified cells (or populations of such cells) may be increased to at least 1.5, 2, or 5 times higher than the levels in the corresponding unmodified cells (or populations of such cells). Preferably, the cells are Treg cells, or the cell population is a Treg population.

[0204] Techniques for measuring the levels of specific mRNAs and proteins are well known in the art. mRNA levels in cell populations (such as Treg cells) can be measured using techniques such as Affymetrix eBioscience Prime Flow RNA assay, Northern blotting, Sequential Gene Expression Analysis (SAGE), or Quantitative Polymerase Chain Reaction (qPCR). Protein levels in cell populations can be measured using techniques such as flow cytometry, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), Western blotting, or enzyme-linked immunosorbent assay (ELISA).

[0205] "FOXP3 polypeptide" is a polypeptide with FOXP3 activity, that is, a polypeptide capable of binding to FOXP3 target DNA and acting as a transcription factor regulating the development and function of Tregs. Specifically, the FOXP3 polypeptide may have the same or similar activity as wild-type FOXP3 (SEQ ID NO. 130), for example, it may have at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of wild-type FOXP3 polypeptide. Therefore, the FOXP3 polypeptide encoded by the nucleotide sequence in the nucleic acid, construct, or vector described herein may have increased or decreased activity compared to wild-type FOXP3. Techniques for measuring transcription factor activity are well known in the art. For example, transcription factor DNA-binding activity can be measured by ChIP. The transcriptional regulatory activity of a transcription factor can be measured by quantifying the expression level of the gene it regulates. Gene expression can be quantified by measuring the levels of mRNA and / or protein produced by a gene using techniques such as Northern blotting, SAGE, qPCR, HPLC, LC / MS, Western blotting, or ELISA. Genes regulated by FOXP3 include cytokines such as IL-2, IL-4, and IFN-γ (Siegler et al., Annu. Rev. Immunol. 2006, 24: 209-26, which is incorporated herein by reference). As discussed in detail below, FOXP3 or FOXP3 peptides include their functional fragments, variants, and isoforms, such as the functional fragments, variants, and isoforms of SEQ ID NO. 130.

[0206] A “functional fragment of FOXP3” can refer to a portion or region of a FOXP3 polypeptide or a polynucleotide (i.e., nucleotide sequence) encoding a FOXP3 polypeptide, which has the same or similar activity as the full-length FOXP3 polypeptide or polynucleotide. The functional fragment may have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the activity of the full-length FOXP3 polypeptide or polynucleotide. Those skilled in the art will be able to generate functional fragments based on known structural and functional characteristics of FOXP3. Such descriptions are found, for example, in Song, X. et al., 2012. Cell reports, 1(6), pp. 665-675; Lopes, JE et al., 2006. The Journal of Immunology, 177(5), pp. 3133-3142; and Lozano, T. et al., 2013. Frontiers in oncology, 3, p. 294. Furthermore, N-terminal and C-terminal truncated FOXP3 fragments, for example having the sequence SEQ ID NO. 131, are described in WO2019 / 241549 (incorporated herein by reference), as discussed below.

[0207] "FOXP3 variants" may include an amino acid or nucleotide sequence having at least 50%, at least 55%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% identity (preferably at least 95%, at least 97%, or at least 99%) with the same as, or similar to, the wild-type FOXP3 polypeptide or polynucleotide; for example, having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150% of the activity of the wild-type FOXP3 polypeptide or polynucleotide. Those skilled in the art will be able to generate FOXP3 variants based on the known structural and functional characteristics of FOXP3 and / or using conserved substitutions. Compared to wild-type FOXP3, FOXP3 variants may have similar or identical turnaround times (or degradation rates) in Treg cells, for example, at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the turnaround time (or degradation rate) of wild-type FOXP3 in Tregs. Some FOXP3 variants may have reduced turnaround times (or degradation rates) compared to wild-type FOXP3, for example, FOXP3 variants with amino acid substitutions (e.g., S418E and / or S422A) at amino acid positions 418 and / or 422 of SEQ ID NO. 130, as described in WO2019 / 241549 (incorporated herein by reference) and listed in SEQ ID NO. 132 to 134, which represent aa418, aa422, and aa418 and aa422 mutants, respectively.

[0208] Appropriately, FOXP3 polypeptides encoded by nucleic acid molecules, constructs or vectors as described herein may include polypeptide sequences of human FOXP3 (such as UniProtKB accession number Q9BZS1 (SEQ ID NO: 130)) or functional fragments or variants thereof, or constitute thereof.

[0209] In some embodiments of the invention, the FOXP3 polypeptide comprises or is composed of an amino acid sequence having at least 70% identity with or consisting of a functional fragment of SEQ ID NO: 130 or thereof. Suitably, the FOXP3 polypeptide comprises or is composed of an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with or consisting of a functional fragment of SEQ ID NO: 130 or thereof. In some embodiments, the FOXP3 polypeptide comprises or is composed of a functional fragment of SEQ ID NO: 130 or thereof.

[0210] In some embodiments, as described above, the FOXP3 peptide may contain a mutation at residues 418 and / or 422 of SEQ ID NO. 130, as shown in SEQ ID NO. 132, SEQ ID NO. 133 or SEQ ID NO. 134.

[0211] In some embodiments of the present invention, the FOXP3 polypeptide may be truncated at the N-terminus and / or C-terminus, resulting in the generation of a functional fragment. Specifically, the N-terminal and C-terminal truncated functional fragment of FOXP3 may comprise or consist of the following: the amino acid sequence of SEQ ID NO. 131 or its functional variant having at least 80%, 85%, 90%, 95%, or 99% identity with it.

[0212] Suitablely, the FOXP3 peptide may be a variant of SEQ ID NO: 130, for example, a native variant. Suitablely, the FOXP3 peptide is an isotype of SEQ ID NO: 130. For example, the FOXP3 peptide may include a deletion of amino acids 72-106 relative to SEQ ID NO: 130. Alternatively, the FOXP3 peptide may include a deletion of amino acids 246-272 relative to SEQ ID NO: 130.

[0213] Appropriately, the FOXP3 polypeptide includes SEQ ID NO: 135 or a functional fragment thereof. SEQ ID NO: 135 represents an exemplary FOXP3 polypeptide.

[0214] Suitably, the FOXP3 polypeptide comprises or is composed of an amino acid sequence having at least 70% identity with or consisting of a functional fragment of SEQ ID NO: 135 or thereof. Suitably, the FOXP3 polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with or consisting of a functional fragment of SEQ ID NO: 135 or thereof. In some embodiments, the FOXP3 polypeptide comprises or is composed of a functional fragment of SEQ ID NO: 135 or thereof.

[0215] Suitablely, the FOXP3 peptide may be a variant of SEQ ID NO: 135, for example, a native variant. Suitablely, the FOXP3 peptide is an isotype of SEQ ID NO: 135 or a functional fragment thereof. For example, the FOXP3 peptide may include a deletion of amino acids 72-106 relative to SEQ ID NO: 135. Alternatively, the FOXP3 peptide may include a deletion of amino acids 246-272 relative to SEQ ID NO: 135.

[0216] Suitable, the polynucleotide encoding the FOXP3 polypeptide comprises or consists of the nucleotide sequence shown in SEQ ID NO: 136, which represents an exemplary FOXP3 nucleotide sequence.

[0217] In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a nucleotide sequence having at least 70% identity with SEQ ID NO: 136 or a fragment thereof encoding a functional FOXP3 polypeptide. Suitably, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 136 or a fragment thereof encoding a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises or is composed of SEQ ID NO: 136 or a fragment thereof encoding a functional FOXP3 polypeptide.

[0218] Suitable, the polynucleotide encoding the FOXP3 polypeptide includes or consists of the polynucleotide sequence shown in SEQ ID NO: 137, which represents another exemplary FOXP3 nucleotide.

[0219] In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a nucleotide sequence having at least 70% identity with SEQ ID NO: 137 or a fragment thereof encoding a functional FOXP3 polypeptide. Suitably, the polynucleotide encoding a FOXP3 polypeptide or variant comprises a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 137 or a fragment thereof encoding a functional FOXP3 polypeptide. In some embodiments of the invention, the polynucleotide encoding a FOXP3 polypeptide or variant comprises or is composed of SEQ ID NO: 137 or a fragment thereof encoding a functional FOXP3 polypeptide.

[0220] Those skilled in the art will understand that FOXP3 expression within a Treg can be indirectly increased by introducing a polynucleotide into the cell that encodes a protein that increases FOXP3 transcription and / or translation, or increases FOXP3 half-life (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%), or function (e.g., determined by the repressive capacity of the transduced Treg, as measured previously). For example, a polynucleotide can be introduced into the Treg that increases the transcription of endogenous FOXP3 by interacting with the endogenous FOXP3 promoter or with a non-coding sequence (CNS, e.g., CNS1, 2, or 3) found upstream of the coding region.

[0221] Appropriately, the polynucleotide encoding the FOXP3 polypeptide or a functional fragment or variant thereof may be codon-optimized for expression in human cells.

[0222] As described above, nucleic acid molecules can contain nucleotide sequences encoding self-cleaving sequences. Specifically, self-cleaving sequences are self-cleaving peptides. Such sequences are automatically cleaved during protein production. Self-cleaving peptides that can be used are 2A peptides or 2A-like peptides known and described in the art, for example, in Donnelly et al., Journal of General Virology, 2001, 82, 1027-1041, which is incorporated herein by reference. 2A and 2A-like peptides are thought to induce ribosome skipping and result in a form of cleavage in which the ribosome skips the formation of a peptide bond between the end of the 2A peptide and the downstream amino acid sequence. The “cleavage” occurs between glycine and proline residues at the C-terminus of the 2A peptide, meaning that the upstream cistron will add some additional residues to the end, while the downstream cistron will begin with proline. Therefore, the term “cleavage” as used herein includes skipping peptide bond formation.

[0223] Suitable self-cleaving domains include the P2A, T2A, E2A, and F2A sequences as shown in SEQ ID NO: 138 to 141, respectively. The sequences can be modified to include the amino acid GSG at the N-terminus of the 2A peptide. Therefore, as a possible option, sequences corresponding to SEQ ID NO: 138-141 are also included, but with a GSG at their N-terminus. Such modified alternative 2A sequences are known and reported in the art. Alternative 2A-like sequences that can be used are shown in Donnelly et al. (see above), for example, the TaV sequence.

[0224] The self-cleavage sequences included in the nucleic acid molecule may be the same or different. In one embodiment, they are all 2A sequences, particularly P2A and / or T2A sequences.

[0225] The self-cleaving sequence may include additional cleavage sites that can be cleaved by common enzymes present in the cell. This can help achieve complete removal of the 2A sequence post-translation. Such additional cleavage sites may include, for example, furin protease cleavage sites RXXR (SEQ ID NO: 142), such as RRKR (SEQ ID NO: 143).

[0226] In one representative embodiment, the nucleic acid molecule may contain a nucleotide sequence encoding a CAR targeting GLP1R, the CAR having a sequence of any one of SEQ ID NO. 101 to 105, 126 or 145 to 150 or a variant thereof as described herein, a nucleotide sequence encoding a safety switch, and a nucleotide sequence encoding FOXP3.

[0227] In such implementations, the CAR may include: (a) A leader sequence comprising or consisting of the following: the sequence shown in SEQ ID NO. 117 or 188 or a sequence having at least 80% sequence identity with it; (b) An antigen-binding domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 51, 52, 53, 54, 55, 61 or a sequence having at least 80% sequence identity with it; (c) CD8α hinge and transmembrane domain sequence, which comprises or consists of the following: the sequence shown in SEQ ID NO. 111 or 112 or a sequence having at least 80% sequence identity with it; (d) CD28 costimulatory domain, which comprises or consists of the following: the sequence shown in SEQ ID NO. 121 or a sequence having at least 80% sequence identity with it; (e) CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0228] In an alternative implementation, the CAR may include: (a) A leader sequence comprising or consisting of the following: the sequence shown in SEQ ID NO. 117 or 118 or a sequence having at least 80% sequence identity with it; (b) An antigen-binding domain comprising or consisting of the following: the sequence shown in SEQ ID NO. 51, 52, 53, 54, 55, 61 or a sequence having at least 80% sequence identity with it; (c) A CH2CH3 hinge domain sequence, which comprises or consists of the following: the sequence shown in SEQ ID NO. 113 or 114 or a sequence having at least 80% sequence identity with it; (d) CD28 transmembrane and costimulatory domain, which comprises or consists of the following: the sequence shown in SEQ ID NO. 120 or a sequence having at least 80% sequence identity with it; (e) CD3ζ signal transduction domain, which contains or consists of the following: the sequence shown in SEQ ID NO. 119 or a sequence having at least 80% sequence identity with it.

[0229] As is clear from the above description, in addition to the specific polypeptide and nucleotide sequences mentioned herein, the uses of their variants or derivatives and fragments are also covered.

[0230] Regarding the proteins or peptides of this invention, the terms "derivative" or "variant," as used interchangeably herein, include any substitution, variation, modification, replacement, deletion, and / or addition of one (or more) amino acid residues to the sequence, provided that the resulting protein or peptide retains the desired function (e.g., in the case where the derivative or variant is an antigen-binding domain, the desired function may be the ability of the antigen-binding domain to bind its target antigen (e.g., a variant of an antigen-binding domain that binds GLP1R retains the ability to bind GLP1R); in the case where the derivative or variant is a signal transduction domain, the desired function may be the ability of that domain to perform signal transduction (e.g., activate or inactivate downstream molecules); in the case where the derivative or variant is a transcription factor (e.g., FOXP3), the desired function may be the ability of the transcription factor to bind target DNA and / or induce transcription; or when the derivative or variant is a safety switch peptide, the desired function may be the ability of that peptide (e.g., when a molecule binds to it) to induce cell death). Alternatively, the variants or derivatives referred to herein are functional variants or derivatives. For example, a variant or derivative may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the functionality compared to the corresponding reference sequence. A variant or derivative may have a similar or the same level of functionality compared to the corresponding reference sequence, or may have an increased level of functionality (e.g., an increase of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%).

[0231] Typically, amino acid substitutions (e.g., 1, 2, or 3 to 10 or 20 substitutions) can be made, provided that the modified sequence retains the desired activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogs. For example, variants or derivatives may have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the activity or ability compared to the corresponding reference sequence. Variant or derivatives may have similar or identical levels of activity or ability compared to the corresponding reference sequence, or may have increased levels of activity or ability (e.g., an increase of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%).

[0232] Proteins or peptides can also have amino acid residue deletions, insertions, or substitutions, resulting in silencing alterations and producing functionally equivalent proteins. Intentional amino acid substitutions can be made based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilic properties of the residues, as long as the intrinsic function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups and similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine, and tyrosine.

[0233] For example, conservative substitutions can be made according to Table 1 below.

[0234] Table 1

[0235] Derivatives can be homologs. As used herein, the term "homolog" refers to an entity that shares some degree of homology with wild-type amino acid sequences and wild-type nucleotide sequences. The term "homology" can be equated with "identity".

[0236] Homologous or variant sequences may include amino acid sequences that share at least 70%, 75%, 85%, or 90% identity with the subject sequence (preferably at least 95%, 96%, 97%, 98%, or 99%). Typically, variants will include the same active site, etc., as the subject amino acid sequence. Although homology can also be considered based on similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of this document, homology is preferably expressed based on sequence identity.

[0237] Homology comparisons can be performed visually, or more commonly, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percentage of homology or identity between two or more sequences.

[0238] Homology, or percentage of sequence identity, can be calculated on continuous sequences; that is, one sequence is aligned with another, and each amino acid in one sequence is directly compared with the corresponding amino acid in the other sequence, one residue at a time. This is called "gap-free" alignment. Typically, this type of gap-free alignment is only performed on relatively short sequences of residues.

[0239] While this is a very simple and consistent approach, it doesn't account for the fact that, for example, in a pair of otherwise identical sequences, an insertion or deletion in one nucleotide sequence can lead to codon misalignment in the following sequence, potentially resulting in a significant decrease in the percentage of homology when performing a global alignment. Therefore, most sequence alignment methods are designed to produce optimal alignments that account for possible insertions and deletions without excessively penalizing the overall homology score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology as much as possible.

[0240] However, these more sophisticated methods assign a "gap penalty" to each gap that appears in the alignment, so that for the same number of identical amino acids, sequence alignments with as few gaps as possible (reflecting a higher correlation between the two compared sequences) will achieve higher scores than sequence alignments with many gaps. Affine gap cost is typically used, which charges a relatively high cost for the presence of a gap and a smaller penalty for each subsequent residue within the gap. This is the most commonly used gap scoring system. A high gap penalty will naturally result in an optimized alignment with fewer gaps. Most alignment programs allow modification of the gap penalty. However, when using such software for sequence alignment, the default value is preferred. For example, when using the GCG WisconsinBestfit package, the default gap penalty for amino acid sequences is -12 for gaps and -4 for each extension.

[0241] Therefore, calculating the maximum homology / sequence identity percentage first requires generating the best alignment and taking into account gap penalties. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST software package (see Ausubel et al. (1999) ibid. – Chapter 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), and the GNEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al. (1999) ibid., pp. 7-58–7-60). However, for some applications, the GCG Bestfit program is preferred. Another tool called BLAST 2 sequencer can also be used to compare protein and nucleotide sequences (see FEMS Microbiol. Lett. (1999) 174: 247-50; FEMS Microbiol. Lett. (1999) 177: 187-8).

[0242] Although the final homology percentage can be measured based on identity, the alignment process itself is not typically based on all-or-nothing pairwise comparisons. Instead, a scaled similarity score matrix is ​​usually used, which assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. A commonly used example of such a matrix is ​​the BLOSUM62 matrix, which is the default matrix in the BLAST program suite. The GCG Wisconsin program typically uses public defaults or a custom symbol comparison table (if provided) (see the user manual for details). For some applications, it is preferred to use the public defaults of the GCG package, or in the case of other software, a default matrix such as BLOSUM62. Appropriately, the identity percentage is determined across the entire reference sequence and / or query sequence.

[0243] Once the software produces the optimal alignment, it can calculate the percentage of homology, preferably the percentage of sequence identity. The software typically performs this as part of the sequence comparison and generates numerical results.

[0244] A “fragment” typically refers to a selected region of a polypeptide or polynucleotide that is of functional interest, for example, a functional or coding segment. Therefore, a “fragment” refers to an amino acid or nucleic acid sequence that is part of (or a portion of) a full-length polypeptide or polynucleotide.

[0245] Such variants, derivatives, and fragments can be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. When insertion is to be performed, synthetic DNA encoding the insertion can be prepared along with 5' and 3' flanking regions corresponding to naturally occurring sequences on either side of the insertion site. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally occurring sequence, allowing the sequence to be cleaved with appropriate enzymes and the synthetic DNA to be ligated into the nick. The DNA is then expressed according to the invention to prepare the encoded protein. These methods illustrate only a variety of standard techniques known in the art for DNA sequence manipulation; other known techniques may also be used.

[0246] Nucleic acid molecules and polynucleotide / nucleotide / nucleic acid sequences as defined herein may contain DNA or RNA. They may be single-stranded or double-stranded. Those skilled in the art will understand that, due to the degeneracy of the genetic code, many different nucleic acid molecules / polynucleotides can encode the same polypeptide. Furthermore, it should be understood that those skilled in the art can use conventional techniques to perform nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule / polynucleotide / nucleotide sequence as defined herein, to reflect the codon usage of any particular host organism in which the polypeptide of the invention will be expressed.

[0247] Nucleic acid molecules / nucleotides / polynucleotides can be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or lifespan of nucleic acid molecules / polynucleotides as defined herein.

[0248] Nucleic acid molecules / polynucleotides / nucleotide sequences, such as DNA nucleic acid molecules / polynucleotides / sequences, can be recombined, synthesized, or produced by any method available to those skilled in the art. They can also be cloned using standard techniques.

[0249] Longer nucleic acid molecules / polynucleotides / nucleotide sequences are typically generated using recombinant methods, such as polymerase chain reaction (PCR) cloning. This involves preparing primer pairs (e.g., approximately 15 to 30 nucleotides) flanking the target sequence to be cloned, contacting the primers with mRNA or cDNA obtained from animal or human cells, performing a polymerase chain reaction under conditions that amplify the desired region, isolating the amplified fragment (e.g., by purifying the reaction mixture using agarose gel), and recovering the amplified DNA. Primers can be engineered to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0250] The nucleic acid molecules / nucleotides / polynucleotides of the present invention may also include a nucleic acid sequence encoding a selective marker. Suitable selective markers are well known in the art and include, but are not limited to, fluorescent proteins—such as GFP. Suitably, the selective marker may be a fluorescent protein, for example, GFP, YFP, RFP, tdTomato, dsRed, or a variant thereof. In some embodiments, the fluorescent protein is GFP or a variant of GFP. The nucleic acid sequence encoding the selective marker may be provided in combination with the nucleic acid molecules herein in the form of a nucleic acid construct. Such nucleic acid constructs may be provided in a vector.

[0251] Appropriately, the selective marker / reporter molecule domain can be a luciferase-based reporter molecule, a PET reporter molecule (e.g., sodium iodide cotransporter (NIS)), or a membrane protein (e.g., CD34 or Thy1.1).

[0252] Nucleic acid sequences encoding one or more selective markers can be separated from and / or from each other by one or more co-expression sites, which enable each polypeptide to be expressed as a discrete entity. Suitable co-expression sites are known in the art and include, for example, internal ribosome entry sites (IRES) and self-cleavage sites, such as those contained in the nucleic acid molecules of the present invention and as defined above. In one embodiment, as described above, this could be a 2A cleavage site.

[0253] The use of selective markers is advantageous because it allows the selection and isolation of cells (e.g., Tregs) from the starting cell population using conventional methods (e.g., flow cytometry) that have been successfully introduced with the nucleic acid molecules, constructs, or vectors of the present invention (enabling the expression of the encoded GLP1R CAR and other modules (e.g., FOXP3 and safety switch peptides)).

[0254] The nucleic acid molecules / nucleotides / polynucleotides used in this invention can be codon-optimized. Codon optimization has been previously described in WO1999 / 41397 and WO2001 / 79518. Different cells differ in their use of specific codons. This codon preference corresponds to the preference for the relative abundance of specific tRNAs in a cell type. By altering codons in the sequence to match the relative abundance of the corresponding tRNA, it is possible to increase expression. For the same reason, by intentionally selecting codons (where the corresponding tRNA is known to be scarce in a particular cell type), it is possible to decrease expression. Thus, additional levels of translational control can be obtained.

[0255] The constructs of the present invention may contain one or more regulatory sequences, such as promoters. A “promoter” is a DNA region that initiates gene transcription. Promoters are located near the gene transcription start site, upstream of the DNA (towards the 5' region of the sense strand). Any suitable promoter can be used, and its selection can be readily made by those skilled in the art. Promoters can be from any source and can be viral or eukaryotic promoters, including mammalian or human promoters (i.e., physiological promoters). In one embodiment, the promoter is a viral promoter. Specific promoters include LTR promoters, EFS (or their functional truncated counterparts), SFFV, PGK, and CMV. In one embodiment, the promoter is an SFFV or a viral LTR promoter. Specifically, the SFFV promoter can be used in the nucleic acid molecules, constructs, or vectors of the present invention to allow transcription of the nucleotide sequence to be initiated. Thus, the promoter can control the expression of the CAR of the present invention. In the presence of more than one nucleotide sequence, each sequence can be operatively linked to the same promoter, such as a nucleotide sequence encoding a CAR, FOXP3, and / or a safety switch.

[0256] The SFFV promoter may contain a nucleotide sequence as shown in SEQ ID NO. 144.

[0257] "Operationally linked to the same promoter" means that transcription of nucleic acid / nucleotide / polynucleotide sequences can be initiated from the same promoter (e.g., transcription of the first, second, and third polynucleotide sequences is initiated from the same promoter), and the nucleotide sequences are positioned and oriented such that transcription is initiated from the promoter. Nucleic acid / nucleotide / polynucleotide operably linked to a promoter is under the transcriptional regulation of that promoter.

[0258] In some embodiments of the invention, the nucleic acid / nucleotide / polynucleotide sequence is contained within an expression vector. As used herein, the term "expression vector" means a construct that enables the expression of CAR peptides and any other peptides, such as FOXP3 peptides or safety switch peptides.

[0259] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. As used herein, for example, some vectors used in recombinant nucleic acid technologies allow entities, such as nucleic acid segments (e.g., heterologous DNA segments, such as heterologous cDNA segments), to be transferred to target cells. Vectors can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technologies include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. Vectors can also be, for example, naked nucleic acids (e.g., DNA). In its simplest form, the vector itself can be the nucleotide of interest.

[0260] The vectors used in this article may be, for example, plasmids, mRNA or viral vectors, and may include promoters (as described above) for expressing nucleic acid molecules / polynucleotides and optional regulators of the promoters.

[0261] In one implementation, the vector is a viral vector, such as a retrovirus, for example a lentiviral vector or a gamma retrovirus vector.

[0262] The vector may also include an additional promoter; for example, in one embodiment, the promoter may be an LTR, such as a retroviral LTR or a lentiviral LTR. Long terminal repeats (LTRs) are identical DNA sequences repeated hundreds or thousands of times, found at either end of a retrotransposon or proviral DNA formed by reverse transcription of retroviral RNA. Viruses use them to insert their genetic material into the host genome. Signals of gene expression are found in LTRs: enhancers, promoters (which may have both transcriptional enhancers and regulatory elements), transcription initiation (such as capping), transcription terminators, and polyadenylation signals.

[0263] Appropriately, the vector may include 5'LTR and 3'LTR.

[0264] Vectors may include one or more additional regulatory sequences that can function pre- or post-transcriptionally. A "regulatory sequence" is any sequence that promotes peptide expression, such as a sequence used to increase transcript expression or enhance mRNA stability. Suitable regulatory sequences include, for example, enhancer elements, post-transcriptional regulatory elements, and polyadenylation sites. Appropriately, additional regulatory sequences may be present in the LTR.

[0265] Appropriately, the vector may include a marmot hepatitis virus post-transcriptional regulatory element (WPRE), which, for example, is operatively linked to a promoter.

[0266] Vectors comprising the nucleic acid molecules / polynucleotides of the present invention can be introduced into cells using a variety of techniques known in the art, such as transformation and transduction. Several techniques are known in the art, for example, infection with recombinant viral vectors, such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, baculoviruses, and herpes simplex virus vectors; direct injection of nucleic acids; and gene gun transformation.

[0267] Non-viral delivery systems include, but are not limited to, DNA transfection methods. Here, transfection includes methods of delivering genes to target cells using non-viral vectors. Non-viral delivery systems may include liposomes or amphiphilic cell-penetrating peptides, preferably complexed with nucleic acid molecules or constructs.

[0268] Typical transfection methods include electroporation, DNA bio-projectiles, lipid-mediated transfection, compressed DNA-mediated transfection, liposomes, immunoliposomes, liposome transfection, cationic agent-mediated transfection, cationic surface amphiphiles (CFA) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

[0269] Although the nucleic acid molecules of the present invention are designed to be used as a single construct and will be contained in a single vector, this does not preclude their introduction into cells together with other vectors (e.g., encoding other polypeptides that may also be expected to be introduced into cells).

[0270] Engineered cells can be generated by introducing nucleic acid molecules, constructs, or vectors as defined herein, using one of a variety of methods, including transduction using viral vectors and transfection using DNA or RNA.

[0271] The cells of the present invention can be prepared by introducing nucleic acid molecules / polynucleotides, constructs or vectors as defined herein into cells (e.g., by transduction or transfection).

[0272] Suitable cells are discussed further below, but cells may be derived from samples isolated from the subject. The subject may be a donor subject or a subject of treatment (i.e., the cells may be autologous cells or donor cells used to introduce cells into another recipient, such as allogeneic cells).

[0273] Cells can be generated through a method that includes the following steps: (i) separating or providing cell-containing samples from the subject; and (ii) Introducing (e.g., by transduction or transfection) a cell-containing sample with a nucleic acid molecule, construct or vector as defined herein to provide an engineered cell population.

[0274] Before and / or after step (ii) of the method, a sample rich in target cells may be isolated, enriched, and / or generated from the cell-containing sample. For example, the isolation, enrichment, and / or generation of Tregs (or other target cells) may be performed before and / or after step (ii) to isolate, enrich, or generate a sample rich in Tregs. Isolation and / or enrichment from the cell-containing sample may be performed after step (ii) to enrich cells and / or Tregs (or other target cells) including CARs, nucleic acid molecules / polynucleotides, constructs, and / or vectors as described herein.

[0275] Samples rich in Tregs can be isolated or enriched by any method known to those skilled in the art, for example, by FACS and / or magnetic bead sorting. Samples rich in Tregs can be generated from cell-containing samples by any method known to those skilled in the art, for example, from Tcon cells by introducing DNA or RNA encoding FOXP3, and / or from in vitro differentiation of induced progenitor cells or embryonic progenitor cells. Methods for isolating and / or enriching other target cells are known in the art.

[0276] Appropriately, engineered target cells can be generated by a method including the following steps: (i) separating or providing a sample rich in target cells from the subject; and (ii) Introducing nucleic acids, constructs or vectors as defined herein (e.g., by transduction or transfection) into a sample rich in target cells to provide an engineered target cell population.

[0277] Target cells can be Treg cells or their precursors or progenitors.

[0278] “Engineered cells” refer to cells that have been modified to include or express polynucleotides not naturally encoded by the cell. Methods for engineering cells are known in the art and include, but are not limited to, genetic modifications of cells, such as by transduction (e.g., retroviral or lentiviral transduction), transfection (e.g., transient transfection—based on DNA or RNA), including lipid transfection, polyethylene glycol, calcium phosphate, and electroporation as discussed above. Any suitable method can be used to introduce nucleic acid sequences into cells. Non-viral techniques (such as amphiphilic cell-penetrating peptides) can be used to introduce nucleic acids. Cells can also be genetically modified, for example, by inserting nucleotide, polynucleotide, or nucleic acid sequences as described herein into the genome using any known gene-editing technology, such as CRISPR, Talens, or zinc fingers.

[0279] Therefore, the nucleic acid molecules described herein are not naturally expressed by the corresponding unmodified cells. In fact, the nucleic acid molecules encoding CARs are artificial constructs, and in one embodiment, the safety switch peptides are artificial constructs such that they cannot exist or be expressed naturally. Appropriately, engineered cells are cells that have been modified (e.g., by transduction or transfection). Appropriately, engineered cells are cells that have been modified (e.g., by transduction or transfection) or whose genome has been modified (e.g., by transduction or transfection). Appropriately, engineered cells are cells that have been modified by retroviral transduction or whose genome has been modified by retroviral transduction. Appropriately, engineered cells are cells that have been modified by lentiviral transduction or whose genome has been modified by lentiviral transduction.

[0280] As used herein, the term “introduction” refers to a method of inserting foreign nucleic acids (e.g., DNA or RNA) into a cell. As used herein, the term introduction includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids into a cell via a non-viral method. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector. Engineered cells can be generated by introducing nucleic acids as described herein using one of a variety of methods, including transduction using viral vectors and transfection using DNA or RNA. Cells can be activated and / or expanded before or after the introduction of nucleic acids as described herein, for example, by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies. Cells can also be expanded in the presence of anti-CD3 and anti-CD28 monoclonal antibodies in combination with IL-2. Where appropriate, IL-2 can be replaced with IL-15. Other components that can be used in cell (e.g., Treg) expansion protocols include, but are not limited to, rapamycin, all-trans retinoic acid (ATRA), and TGFβ. As used herein, “activation” means that the cell has been stimulated to the point of cell proliferation. As used herein, “amplification” refers to the induction of proliferation of cells or cell populations. The expansion of a cell population can be measured, for example, by counting the number of cells present in the population. The phenotype of the cells can be determined using methods known in the art, such as flow cytometry.

[0281] Cells can be immune cells or their precursors. Precursor cells can be progenitor cells. Therefore, representative immune cells include T cells, particularly cytotoxic T cells (CTL; ​​CD8+ T cells), helper T cells (HTL; CD4+ T cells), and regulatory T cells (Treg). Other T cell populations may also be used here, such as naive T cells and memory T cells. Other immune cells include NK cells, NKT cells, tolerogenic NK or NKT cells, dendritic cells, MDSCs, neutrophils, and macrophages. Precursors of immune cells include pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), or more directed progenitor cells, including multipotent stem cells (e.g., HPCs), or cells directed to a lineage. Precursor cells can be induced to differentiate into immune cells in vivo or in vitro. In one aspect, precursor cells can be somatic cells capable of transdifferentiating into immune cells of interest.

[0282] Most notably, immune cells can be NK cells, dendritic cells, MDSCs, or T cells, such as cytotoxic T lymphocytes (CTLs), helper T cells, or Treg cells.

[0283] In a preferred embodiment, the immune cells are Treg cells. "Regulatory T cells (Tregs) or T regulatory cells" are immune cells with immunosuppressive functions that control cytopathic immune responses and are essential for maintaining immune tolerance. As used herein, the term Treg refers to T cells with immunosuppressive functions.

[0284] As used in this article, T cells are lymphocytes, including any type of T cell such as αβ T cells (e.g., CD8 or CD4+), γδ T cells, memory T cells, and Treg cells.

[0285] Appropriately, immunosuppressive function can refer to the ability of Tregs to reduce or suppress one or more of a variety of physiological and cellular effects promoted by the immune system in response to stimuli such as pathogens, allogeneic antigens, or self-antigens. Examples of such effects include increased proliferation of conventional T cells (Tconv) and secretion of pro-inflammatory cytokines. Any such effect can be used as an indicator of the strength of the immune response. A relatively weak immune response to Tconv in the presence of Tregs will indicate the ability of Tregs to suppress the immune response. For example, a relative decrease in cytokine secretion will indicate a weaker immune response, and thus the ability of Tregs to suppress the immune response. Tregs can also suppress the immune response by regulating the expression of co-stimulatory molecules on antigen-presenting cells (APCs) such as B cells, dendritic cells, and macrophages. The expression levels of CD80 and CD86 can be used to assess the inhibitory potency of activated Tregs in vitro after co-culture.

[0286] Indicators for measuring the strength of immune responses, thereby measuring the inhibitory capacity of Tregs, are known in the art. Specifically, antigen-specific Tconv cells can be co-cultured with Tregs, and peptides corresponding to the antigen can be added to the co-culture to stimulate a response in the Tconv cells. The degree of proliferation of Tconv cells and / or the amount of the cytokine IL-2 secreted by them in response to the addition of the peptide can be used as indicators of the inhibitory capacity of the co-cultured Tregs.

[0287] The proliferation of antigen-specific Tconv cells co-cultured with Tregs as disclosed herein can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than that of the same Tconv cells cultured in the absence of Tregs. For example, the proliferation of antigen-specific Tconv cells co-cultured with the Tregs of the present invention can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 90%, 95%, or 99% less than that of the same Tconv cells cultured in the presence of unengineered Tregs. Cells comprising nucleic acids, expression constructs, or vectors as defined herein (e.g., Tregs) can have increased inhibitory activity (e.g., increased inhibitory activity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) compared to unengineered Tregs.

[0288] Compared to corresponding Tconv cells cultured in the absence of Tregs (e.g., in the presence of unengineered Tregs), antigen-specific Tconv cells co-cultured with the Tregs described herein can express at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% fewer effector cytokines. The effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, IFN-γ, IL-4, IL-5, IL-9, IL-10, and IL-13. Suitablely, the effector cytokines may be selected from IL-2, IL-17, TNFα, GM-CSF, and IFN-γ.

[0289] Several distinct Treg subsets have been identified, each expressing different or varying levels of specific markers. Tregs typically express the markers CD4, CD25, and FOXP3 (CD4+). + CD25 + FOXP3 + T cells.

[0290] Treg cells can also express CTLA-4 (cytotoxic T lymphocyte-associated molecule-4) or GITR (glucocorticoid-induced TNF receptor).

[0291] Treg cells are present in peripheral blood, lymph nodes, and tissues, and the Tregs used in this article include thymus-derived natural Treg cells (nTreg), peripherally generated Tregs, and induced Treg cells (iTreg).

[0292] Tregs can be identified using cell surface markers CD4 and CD25 in the absence of the surface protein CD127 or in combination with low levels of expression of that surface protein.+ CD25 + CD127 - or CD4 + CD25 + CD127 低 The use of such markers to identify Tregs is known in the art and is described, for example, by Liu et al. (JEM; 2006; 203; 7(10); 1701-1711).

[0293] Treg can be CD4 + CD25 + FOXP3 + T cells, CD4 + CD25 + CD127 - T cells or CD4 + CD25 + FOXP3 + CD127 - / 低 T cells.

[0294] Appropriately, Tregs can be natural Tregs (nTregs). As used herein, the term "natural Treg" refers to Tregs derived from the thymus. Natural Tregs are CD4+. + CD25 + FOXP3 + Helios + Neurociliin 1 + Compared to iTreg, nTreg exhibits higher expression levels of PD-1 (programmed cell death-1, pdcd1), neurocilitin 1 (Nrp1), Helios (Ikzf2), and CD73. nTreg can be distinguished from iTreg based on the individual expression of Helios protein or neurocilitin 1 (Nrp1).

[0295] Tregs can possess demethylated Treg-specific demethylation domains (TSDRs). TSDRs are important methylation-sensitive elements that regulate Foxp3 expression (Polansky, JK, et al., 2008. European journal of immunology, 38(6), pp. 1654-1663).

[0296] Other suitable Tregs include, but are not limited to, Tr1 cells (which do not express Foxp3 and have high IL-10 production); CD8 + FOXP3 + T cells; and γδ FOXP3 + T cells.

[0297] Different Treg subgroups are known to exist, including naive Tregs (CD45RA). + FoxP3 低 ), Effect / Memory Treg (CD45RA) - FoxP3 高 ) and Tregs that produce cytokines (CD45RA) - FoxP3 低 “Memory Treg” is an expression of CD45RO and is considered to be CD45RO. + Tregs. These cells have increased CD45RO levels compared to naive Tregs (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RO), and preferably do not express or have low levels of CD45RA (mRNA and / or protein) compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs). A "cytokine-producing Treg" is a Treg that does not express or has very low levels of CD45RA (mRNA and / or protein) compared to naive Tregs (e.g., at least 80%, 90%, or 95% less CD45RA compared to naive Tregs), and has low levels of FOXP3 compared to memory Tregs, e.g., less than 50%, 60%, 70%, 80%, or 90% of FOXP3 compared to memory Tregs. Cytokine-producing Tregs can produce interferon-γ and may exhibit lower in vitro inhibitory activity compared to naive Tregs (e.g., less than 50%, 60%, 70%, 80%, or 90% inhibition compared to naive Tregs). Expression levels mentioned herein can refer to mRNA or protein expression. Specifically, for cell surface markers (such as CD45RA, CD25, CD4, CD45RO, etc.), expression can refer to cell surface expression, i.e., the amount or relative amount of the marker protein expressed on the cell surface. Expression levels can be determined by any method known in the art. For example, mRNA expression levels can be determined by Northern blotting / array analysis, and protein expression can be determined by Western blotting, or preferably by antibody staining using FACS to determine cell surface expression.

[0298] Specifically, Tregs can be naive Tregs. As used interchangeably in this article, "naive regulatory T cell, naive regulatory T cell, or naive Treg" refers to a Treg cell that expresses CD45RA (especially CD45RA expressed on the cell surface). Therefore, naive Tregs are described as CD45RA-expressing Tregs. +Naïve Tregs typically represent Tregs that are not activated by peptide / MHC via their endogenous TCR, while effector / memory Tregs involve Tregs that have been activated by stimulation via their endogenous TCR. Typically, naïve Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% more CD45RA than non-naïve Tregs (e.g., memory Tregs). Alternatively, naïve Tregs may express at least 2, 3, 4, 5, 10, 50, or 100 times more CD45RA than non-naïve Tregs (e.g., memory Tregs). The expression level of CD45RA can be readily determined by methods in the art, for example, by flow cytometry using commercially available antibodies. Typically, non-naïve Tregs do not express CD45RA or express low levels of CD45RA.

[0299] Specifically, naive Tregs may not express CD45RO, but can be considered as CD45RO. - Therefore, compared with memory Tregs, naive Tregs may express at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% less CD45RO, or alternatively, compared with memory Tregs, naive Tregs may express at least 2, 3, 4, 5, 10, 50, or 100 times less CD45RO.

[0300] Although naive Tregs express CD25 as discussed above, CD25 expression levels may be lower than those in memory Tregs, depending on the source of the naive Treg. For example, for naive Tregs isolated from peripheral blood, CD25 expression levels may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower than those in memory Tregs. Such naive Tregs can be considered to express moderate to low levels of CD25. However, those skilled in the art should understand that naive Tregs isolated from umbilical cord blood may not show this difference.

[0301] Typically, a naive Treg, as defined in this article, can be a CD4 + CD25 + FOXP3 + CD127 低 CD45RA + .

[0302] As used in this article, low expression of CD127 refers to the expression of CD4 from the same subject or donor. +Compared to non-regulatory cells or Tcon cells, CD127 expression levels were lower. Specifically, compared to CD4 from the same subject or donor... + Compared to non-regulatory cells or Tcon cells, naive Treg cells may express less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of CD127. CD127 levels can be assessed using standard methods in the art, including flow cytometry of cells stained with an anti-CD127 antibody.

[0303] Typically, naive Tregs do not express or express low levels of CCR4, HLA-DR, CXCR3, and / or CCR6. Specifically, compared to memory Tregs, naive Tregs may express lower levels of CCR4, HLA-DR, CXCR3, and CCR6, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% lower expression levels. Naive Tregs may also express additional markers, including CCR7. + and CD31 + .

[0304] Isolated naive Tregs can be identified by methods known in the art, including by determining the presence of one or more of a set of markers discussed above on the cell surface of the isolated cells. For example, CD45RA, CD4, CD25, and CD127 can be used to determine whether cells are naive Tregs. Methods for determining whether isolated cells are naive Tregs or have the desired phenotype can be performed as discussed below with respect to possible additional steps, and methods for determining the presence and / or expression levels of cell markers are well known in the art and include, for example, flow cytometry using commercially available antibodies.

[0305] Appropriately, cells (such as Tregs) are isolated from peripheral blood mononuclear cells (PBMCs) obtained from the subject. Appropriately, the subject from whom the PBMCs are obtained is a mammal, preferably a human. Appropriately, the cells are matched (e.g., HLA matched) with the subject from whom the engineered cells are to be administered or are autologous. Appropriately, the subject to be treated is a mammal, preferably a human. Cells can be generated ex vivo from the patient's own peripheral blood (first party), or in the case of hematopoietic stem cell transplantation from donor peripheral blood (second party), or from peripheral blood of an unrelated donor (third party). Appropriately, the cells are autologous to the subject from whom the engineered cells are to be administered.

[0306] Appropriately, Tregs are part of a cell population. Appropriately, a population of Tregs includes at least 70% of Tregs, such as at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% of Tregs. Such a population may be referred to as an "enriched Treg population".

[0307] In some respects, Tregs can originate from the in vitro differentiation of induced progenitor cells (e.g., iPSCs) or embryonic progenitor cells into Tregs. Nucleic acid molecules or vectors as described herein can be introduced into induced progenitor cells or embryonic progenitor cells before or after differentiation into Tregs. Suitable differentiation methods are known in the art and include those disclosed in Haque et al., J Vis Exp., 2016, 117, 54720 (incorporated herein by reference).

[0308] As used herein, the term "conventional T cell" or Tcon or Tconv (which are used interchangeably here) refers to a T lymphocyte that expresses the αβ T cell receptor (TCR) and possibly a co-receptor of differentiation cluster 4 (CD4) or differentiation cluster 8 (CD8) and does not have immunosuppressive function. Conventional T cells are found in peripheral blood, lymph nodes, and tissues. Appropriately, engineered Tregs can be generated from Tcon by introducing nucleic acids including a sequence encoding FOXP3. Alternatively, engineered Tregs can be generated from Tcon by culturing CD4+CD25-FOXP3- cells in vitro in the presence of IL-2 and TGF-β.

[0309] In another embodiment, the target cell into which the nucleic acid molecule, construct, or vector is introduced is not a cell intended for therapeutic use. In one embodiment, the cell is a production host cell. The cell may be used to produce nucleic acids, such as clones, vectors, or peptides.

[0310] This document also discloses a cell population comprising cells as defined or described herein. It should be understood that a cell population can include both cells of the present invention (which may contain nucleic acid molecules, expression constructs, or vectors as defined herein) and cells not of the present invention (e.g., cells that do not contain nucleic acid molecules, expression constructs, or vectors as described herein, such as untransduced or untransfected cells). Although in a preferred embodiment, all cells in the population may be of the present invention or may contain nucleic acids, expression constructs, or vectors as described herein, cell populations having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of cells of the present invention (e.g., containing nucleic acids, expression constructs, or vectors as described herein) are provided. Furthermore, a cell population may comprise more than one cell type, although in a preferred embodiment, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the cells are of the same type. Specifically, the cell population may contain at least 70%, 80%, 90%, 95%, or 99% T cells, more specifically Tregs. In addition, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the T cells (especially Tregs) may contain nucleic acids, expression constructs, or vectors as described herein.

[0311] Specifically, this document also discloses cell populations comprising multiple cells containing a CAR or a nucleic acid molecule or vector encoding said CAR, the CAR containing an antigen recognition domain that specifically binds to GLP1R. Specifically, this document also discloses cell populations comprising multiple Tregs, the Tregs containing a CAR or a nucleic acid molecule or vector encoding said CAR, the CAR containing an antigen recognition domain that specifically binds to GLP1R. The CAR can be implemented according to any of the embodiments described herein.

[0312] T cell populations (e.g., Treg cell populations) can be "trained" or "reprogrammed" by directly (e.g., via antigen-presenting cells) or indirectly exposed to or contacted with one or more specific antigens (e.g., self-antigens or non-self-antigens), thereby activating or proliferating these cells to those specific antigens (e.g., in the case of Tregs, exhibiting immunosuppressive or immune-tolerant properties to those specific antigens). This can be particularly done in vitro. Specifically, the endogenous TCRs of T cells will bind to those specific antigens, leading to preferential activation and expansion, resulting in a population comprising a larger proportion of T cells activated by those specific antigens (i.e., a higher proportion of cells in the resulting cell population with TCRs capable of binding these specific antigens). Consequently, the diversity of TCRs in the resulting cell population is reduced. Generally, it can be said that the clonality of the cells (or the clonality of the cell's TCRs) has been modified or increased.

[0313] However, this adds an extra step and a layer of complexity to the manufacturing process. Furthermore, it is difficult to control the exact antigens and epitopes to which the TCR is active. Therefore, in some embodiments of the invention, reprogrammed or trained T cells are not used. Thus, in some embodiments, the clonality of the T cells (or alternatively, the TCR clonality or endogenous TCR clonality of the cells) is not modified in vitro. For example, the clonality of the Tregs (or the TCR clonality of the Tregs) is not modified in vitro. Therefore, in some embodiments, the T cell population is not selected to have TCRs capable of binding pancreatic antigens. As used herein, “clonalness” refers to the diversity of antigens that the TCRs within a T cell population can bind. Therefore, increased clonality leads to a decrease in the diversity of antigens that can be bound, and decreased clonality leads to an increase in the diversity of antigens that can be bound.

[0314] Specifically, in some embodiments, T cells are not exposed to or contacted with one or more specific antigens outside the body, for example, through antigen-presenting cells (such as dendritic cells). Specifically, in some embodiments, T cells are not directly (e.g., through antigen-presenting cells) or indirectly exposed to or contacted with one or more specific antigens (e.g., self-antigens or non-self-antigens) outside the body, thereby preventing these cells from being activated against those specific antigens. Specifically, T cells are not exposed to or contacted with one or more pancreatic antigens outside the body.

[0315] A particular method that can be used to "train" or "reprogram" a Treg (especially a Treg's TCR) may involve the following steps: (a) Dendritic cells (DCs) are exposed to interleukin-10 (IL-10) to produce tolerable dendritic cells (tolDCs). (b) Contacting the tolDC with extracellular vesicles (EVs) of pancreatic islet cells to generate antigen-loaded tolDCs; and (c) Contact the Treg cells with the antigen-loaded tolDC to generate the reprogrammed Treg cells.

[0316] The method may also include, for example, expanding the reprogrammed Treg cells in the presence of IL-2. In some embodiments of the invention, Tregs are not prepared by this method, and / or the methods for preparing Tregs (e.g., Treg populations) do not include these steps or any of these steps. In other words, in some such embodiments, tolerant dendritic cells are not used.

[0317] Therefore, relative to the endogenous TCR of T cells, the T cell population (especially Tregs) can be referred to as a polyclonal population, meaning that its TCR is not specific to a particular antigen, but is active against multiple antigens, and / or specific to unknown antigens. TCRs can, for example, be specific to both pancreatic and non-pancreatic antigens. Tregs with polyclonal TCRs can be immune to tolerance to multiple antigens and / or immunosuppressive to multiple antigens, such as both pancreatic and non-pancreatic antigens. It should be noted that the GLP1R-specific CAR described herein will provide antigen specificity for T cells with polyclonal TCRs.

[0318] A pharmaceutical composition is also provided comprising cells or cell populations as defined or described herein, or a vector as defined herein. This vector can be used for gene therapy. Therefore, instead of cells, the vector can be administered to modify endogenous cells in a subject to express the introduced nucleic acid molecule. Vectors suitable for gene therapy are known in the art and include viral vectors.

[0319] Therefore, in a further aspect, the present invention provides cells, cell populations or pharmaceutical compositions as defined herein for therapeutic purposes.

[0320] A pharmaceutical composition is a composition comprising a therapeutically effective amount of a pharmaceutically active agent (i.e., cells (e.g., Tregs), cell populations, or carriers) or composed thereof. It preferably includes pharmaceutically acceptable carriers, diluents, or excipients (including combinations thereof). Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, ed. 1985). The choice of drug carrier, excipient, or diluent can be made based on the intended route of administration and standard pharmaceutical practice. A pharmaceutical composition may contain, as a carrier, excipient, or diluent (or in addition to a carrier, excipient, or diluent), any suitable one or more binders, lubricants, suspending agents, coating agents, or solubilizers.

[0321] "Pharmaceutical acceptable" means that the formulation is sterile and pyrogen-free. The carrier, diluent, and / or excipient must be "acceptable" in the sense that it is compatible with cells or the carrier and harmless to its recipient. Typically, the carrier, diluent, and excipient will be sterile and pyrogen-free saline or infusion media; however, other acceptable carriers, diluents, and excipients may also be used.

[0322] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohols, silicones, waxes, petrolatum, vegetable oils, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, fragrance oils, monoglycerides and diglycerides of fatty acids, petroleum ether fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, etc.

[0323] Cells, cell populations, or pharmaceutical compositions may be administered in a manner suitable for the treatment and / or prevention of the intended disease or condition. The amount and frequency of administration will be determined by factors such as the subject's condition and the type and severity of the subject's disease or condition, but appropriate dosage may also be determined through clinical trials. The pharmaceutical composition may be formulated accordingly.

[0324] The cells, cell populations, or pharmaceutical compositions described herein can be administered parenterally (e.g., intravenously or intrathecally), or they can be administered via infusion techniques. The cells, cell populations, or pharmaceutical compositions can be administered as a sterile aqueous solution, which may contain other substances, such as sufficient salt or glucose, to make the solution isotonic with blood. The aqueous solution may be appropriately buffered (preferably buffered to a pH of 3 to 9). The pharmaceutical composition can be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions can be readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

[0325] The pharmaceutical composition may be contained within cells in an infusion medium (e.g., a sterile isotonic solution). The pharmaceutical composition may be encapsulated in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0326] Cells, cell populations, or pharmaceutical compositions can be administered in single or multiple doses. Specifically, cells, cell populations, or pharmaceutical compositions can be administered in a single, one-time dose. Pharmaceutical compositions can be formulated accordingly.

[0327] Depending on the disease / symptom and the subject to be treated, as well as the route of administration, cells, cell populations, or pharmaceutical compositions can be administered at specific stages of the disease.

[0328] For example, in type 1 diabetes, pancreatic beta cells are destroyed, preventing them from producing insulin. Therefore, the optimal time to administer the cells, cell populations, or pharmaceutical compositions of the present invention is in the early stages of the disease before all pancreatic beta cells are destroyed, in order to maintain at least some functioning pancreatic beta cells (with residual pancreatic beta cell function) and maintain insulin production. Specifically, at the time of administration of the cells, cell populations, or pharmaceutical compositions described herein, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of pancreatic beta cells may be present. Alternatively, prior to administration of the cells, cell populations, or pharmaceutical compositions described herein, less than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of pancreatic beta cells may have been destroyed.

[0329] The pharmaceutical composition may also contain one or more active agents. The pharmaceutical composition may also contain one or more other therapeutic agents, such as lymphodepleting agents (e.g., thymoglobulin, campath-1H, anti-CD2 antibody, anti-CD3 antibody, anti-CD20 antibody, cyclophosphamide, fludarabine), mTOR inhibitors (e.g., sirolimus, everolimus), drugs that inhibit co-stimulatory pathways (e.g., anti-CD40 / CD40L, CTAL4Ig), and / or drugs that inhibit specific cytokines (IL-6, IL-17, TNFα, IL18).

[0330] Depending on the disease / condition, the subject to be treated, and the route of administration, cells, cell populations, or pharmaceutical compositions may be administered at different doses (e.g., cells / kg or cells / subject). In any case, the physician will determine the actual dose best suited for any individual subject, and this dose will vary depending on the specific subject's age, weight, and response. However, typically, for the cells described herein, 5 × 10⁶ cells may be administered per subject. 7 Up to 3×109 One cell or 10 8 Up to 2×10 9 The dose per cell.

[0331] Cells can be appropriately modified for use in pharmaceutical compositions. For example, cells can be cryopreserved and thawed at an appropriate time before being infused into a subject.

[0332] This invention also includes the use of kits comprising the cells, cell populations, and / or pharmaceutical compositions described herein. Preferably, the kit is used for the methods and uses described herein, such as therapeutic methods as described herein. Preferably, the kit includes instructions for use of the kit components.

[0333] The cells, cell populations, and pharmaceutical compositions of the present invention can be found to be particularly effective in treating the following disorders, which are disorders associated with cells expressing GLP1R, or disorders in which GLP1R is located at or near a disease site, particularly disorders in which the immunosuppressive or target-killing activity of the cells of the present invention will benefit.

[0334] The cells, cell populations, compositions, and vectors described herein can be used to treat, prevent, or reduce the risk of diseases or conditions in subjects, particularly those that can be treated with or using CARs. The cells and compositions containing them are used in adoptive cell therapy (ACT). Various conditions can be treated by administering cells expressing CARs according to this disclosure, particularly including Treg cells. As mentioned above, this may be a condition that responds to immunosuppression, and particularly the immunosuppressive effect of Treg cells. Therefore, the cells, cell populations, compositions, and vectors described herein can be used to induce or achieve immunosuppression in subjects. The administered or in vivo modified Treg cells can be targeted by CAR expression. Conditions suitable for such treatment include autoimmune or inflammatory diseases (e.g., type 1 diabetes), or more broadly, conditions associated with any unwanted or harmful immune response. In addition, the cells, cell populations, compositions, and vectors described herein can be used to promote tissue repair and / or tissue regeneration.

[0335] Conditions requiring treatment or prevention include inflammation, or, alternatively, conditions associated with or involving inflammation. Inflammation can be chronic or acute. Furthermore, inflammation can be low-level or systemic.

[0336] The term "target cell" refers to any cell expressing GLP1R, to which the cells of the present invention will be directed to exert their therapeutic effect. In some embodiments, the target cell acts as a marker of the disease site, i.e., attracting the cells of the present invention to provide an immunosuppressive effect. In some embodiments, the target cell is killed or eliminated by the cells of the present invention. As mentioned above, in some embodiments, the target cell will be pancreatic β cells.

[0337] Specifically, the disease or disorder to be treated can be type 1 diabetes. Other diseases or disorders that can be treated with the CAR described herein include, for example, autoimmune pancreatitis and insulinoma. Specifically, the CAR can be expressed in cells with immunosuppressive functions (e.g., CD4+ or CD8+ regulatory T cells, tolerogenic NK or NKT cells, γ-δ cells and immunomodulatory 1 cells (Tr1), as well as other cells that secrete immunomodulatory cytokines (such as IL-10, TGFβ, IL-35, or bimodalin)) to treat type 1 diabetes or autoimmune pancreatitis. The CAR can be expressed in cells with effector functions (e.g., T effector cells, NK cells, NKT cells) to treat insulinoma, for example, by eradicating or killing insulinoma cells.

[0338] Furthermore, the CAR described herein can be used to protect against or prevent rejection of transplanted cells expressing GLP1R (e.g., β-cell substitutes, including allogeneic islet transplants, xenogeneic islet transplants, and stem cell-derived β-cells). Specifically, the CAR can be expressed in cells with immunosuppressive functions (e.g., CD4+ or CD8+ T regulatory cells, tolerogenic NK or NKT cells, γ-δ cells, and immunomodulatory 1 cells (Tr1), as well as other cells that secrete immunomodulatory cytokines (such as IL-10, TGFβ, IL-35, or bimodalin)) to protect against or prevent rejection of transplanted cells expressing GLP1R.

[0339] Furthermore, the CAR described herein can be used to target or homing cells (e.g., cells with effector functions (e.g., T effector cells, NK cells, NKT cells) or cells with immunosuppressive functions (e.g., CD4+ or CD8+ T regulatory cells, tolerant NK or NKT cells, γ-δ cells and immunomodulatory 1 cells (Tr1), and other cells that secrete immunomodulatory cytokines (such as IL-10, TGFβ, IL-35, or bimodalin)) to target cells or target sites expressing GLP1R, for example, to pancreatic islet cells.

[0340] Engineered cells (e.g., Tregs) may be administered to a subject with a disease to reduce, decrease, or improve at least one symptom of the disease, such as hyperglycemia. The at least one symptom may be reduced, decreased, or improved by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the at least one symptom may be completely relieved.

[0341] Engineered cells (e.g., Treg) can be administered to subjects with a disease to slow, reduce, or block the progression of the disease. Compared to subjects who have not received engineered cells, the progression of the disease can be slowed, reduced, or blocked by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, or the progression of the disease can be completely stopped.

[0342] Specifically, the disease to be treated could be type 1 diabetes. As mentioned above, CAR-Tregs specific for GLP1R may be able to be transported to GLP1R expression sites and control inflammation through their bystander effect, thereby slowing the rate of pancreatic β-cell destruction.

[0343] Type 1 diabetes is a chronic autoimmune disease in which the pancreatic beta cells responsible for producing insulin are destroyed by the immune system. This is triggered by both genetic and environmental factors. The destruction of beta cells reduces or eliminates the body's production of insulin and leads to inflammation of the pancreas. Insulin is a hormone required to regulate glucose levels in the bloodstream, and before treatment, subjects with type 1 diabetes will have excessively high blood sugar levels (hyperglycemia). Type 1 diabetes is a serious and lifelong condition. People with type 1 diabetes currently require close monitoring of their blood sugar levels and ingestion of appropriate doses of insulin (e.g., by injection or pump). This therapy is not a cure and must be administered continuously. Over time, irregular blood sugar levels (including large fluctuations in blood sugar levels) can lead to long-term complications such as damage to the heart, eyes, feet, and kidneys, as well as a shortened life expectancy. It should be noted that blood sugar control is particularly poor in younger subjects, especially those aged approximately 16 to 25 years, and the therapy described in this article can therefore be specifically used for this population.

[0344] Type 1 diabetes is a continuous process that progresses sequentially through different identifiable stages at a variable but predictable rate before the onset of symptoms. This is described in more detail in Insel et al., Diabetes Care. 2015; 38(10):1964-1974. The ability to screen for and stage the risk of type 1 diabetes before the onset of symptoms provides an opportunity for early intervention, thereby delaying and ultimately preventing the onset of clinical symptoms.

[0345] Individuals at increased risk of developing type 1 diabetes can be identified through genetic screening. The HLA region on chromosome 6 accounts for approximately 30%-50% of the genetic risk of type 1 diabetes and is associated with HLA class II haplotypes. RB1*0301 - DQB1*0201 (DR3-DQ2) and DRB1*0401 - DQB1*0302 (DR4-DQ8) showed the strongest association. The remaining genetic risk of type 1 diabetes can be attributed to approximately 50 non-HLA genes or loci identified through candidate gene and genome-wide association studies. The highest non-HLA genetic contributions came from the INS, PTPN22, CTLA4, and IL2RA genes. As described in this article, CAR-Treg can be administered prophylactically to individuals identified as having an increased risk of type 1 diabetes but who have not yet developed any signs of disease (i.e., pre-stage 1).

[0346] Phase 1 represents individuals who have developed two or more type 1 diabetes-associated islet autoantibodies (targeting insulin, GAD65, IA-2, and / or ZnT8) but have normal blood glucose levels.

[0347] Phase 2 represents individuals who have developed two or more type 1 diabetes-associated islet autoantibodies (targeting insulin, GAD65, IA-2, and / or ZnT8) but whose disease has progressed to glucose intolerance or impaired glucose metabolism due to loss of functional pancreatic β-cell clusters. Impaired glucose metabolism can be defined as: a fasting blood glucose level equal to or greater than 5.6 mmol / L, or a 2-hour plasma glucose level equal to or greater than 7.8 mmol / L in a 75 g oral glucose tolerance test (OGTT), high glucose levels at intermediate time points of the OGTT (30-minute, 60-minute, and 90-minute levels equal to or greater than 11.1 mmol / L), and / or an HbA1c level equal to or greater than 5.7% (39 mmol / mol).

[0348] Stage 3 represents individuals with typical clinical symptoms and signs of diabetes, including, for example, polyuria, polydipsia, weight loss, fatigue, and diabetic ketoacidosis (DKA).

[0349] The CAR-Treg described herein can be used, for example, to treat subjects in stages 1, 2, and / or 3 of their disease. Alternatively, the CAR-Treg described herein can be used to treat subjects at risk of type 1 diabetes, i.e., before stage 1.

[0350] When a subject is in stage 3 of the disease, CAR-Treg therapy should be initiated as soon as possible to minimize pancreatic beta cell destruction and maximize the function of remaining pancreatic beta cells. This can be determined, for example, by measuring the subject's blood insulin or blood C-peptide levels.

[0351] For example, subjects could be asked to have a minimum post-stimulation C-peptide level of 0.2 pmol / mL at the time of CAR-Treg administration, or a minimum post-stimulation C-peptide level of 0.4 pmol / mL, for example, in a mixed diet tolerance test. These subjects could be considered to have “recently flared type 1 diabetes.”

[0352] For example, when a subject is in stage 3 of the disease, CAR-Treg treatment may begin no more than approximately 24 weeks after symptom onset or diagnosis (e.g., no more than approximately 20 weeks, 16 weeks, 15 weeks, 14 weeks, 12 weeks, 8 weeks, or 6 weeks after symptom onset or diagnosis). Alternatively, CAR-Treg treatment may begin 100 days or less after symptom onset or diagnosis.

[0353] Subjects can be of any age, for example. For example, subjects can be under 30 years old, under 25 years old, under 20 years old, under 18 years old, or under 16 years old. Specifically, subjects can be 8-30 years old, particularly 8-25 years old, 8-16 years old, or 16-25 years old.

[0354] Disease progression and pancreatic β-cell death can be monitored through various methods, for example.

[0355] For example, imaging pancreatic β cells can be used to highlight the integrity of β cells. The CAR-Treg described herein can, for example, maintain or increase the number of pancreatic β cells present in a subject after administration. This can be observed, for example, when a subject receives a reduced dose of exogenous insulin or does not receive exogenous insulin.

[0356] For example, insulin levels in the subject can be monitored. Normal fasting blood insulin levels (blood insulin levels after a subject has fasted (drinks nothing but water) for at least 8 hours) can be considered to be approximately 2 mIU / mL to 20 mIU / mL. The CAR-Treg described herein can, for example, maintain or increase the subject's fasting blood insulin levels after administration, for example, within the normal range for fasting blood insulin. This can be observed, for example, when the subject receives a reduced dose of exogenous insulin or no exogenous insulin at all.

[0357] It can also detect cell-free insulin DNA (cfDNA) and unmethylated insulin.

[0358] C-peptide levels in subjects can be monitored. Pancreatic beta cells first produce a protein called proinsulin. Each proinsulin unit breaks down into one molecule of insulin and one molecule of C-peptide. Both are released when blood glucose levels rise. Insulin and C-peptide are released in equal amounts, but they are broken down in different ways. Therefore, C-peptide can be used as a surrogate marker of beta cell function. The liver breaks down insulin at a variable rate, while the kidneys break down C-peptide at a fairly stable rate. Therefore, C-peptide can be a more reliable measure of insulin production and beta cell function. Under normal fasting conditions (i.e., after fasting for at least 8 hours), C-peptide levels can be considered to be between approximately 0.8 ng / mL and 3.85 ng / mL. The CAR-Treg described herein can, for example, maintain or increase a subject's fasting C-peptide levels after administration, for example, within the normal range for fasting C-peptides. This can be observed, for example, when a subject receives a reduced dose of exogenous insulin or no exogenous insulin.

[0359] In addition, blood glucose levels can be monitored in subjects. Normal fasting blood glucose levels (i.e., after fasting for at least 8 hours) are considered to be between approximately 3.9 mmol / L and 6.9 mmol / L. Alternatively or in addition, an oral glucose tolerance test can be performed and blood glucose levels measured (e.g., using 75 g of oral glucose). Two hours after oral glucose administration, normal blood glucose levels are considered to be below approximately 7.8 mmol / L. At intermediate time points before 2 hours (e.g., 30 minutes, 60 minutes, 90 minutes), normal blood glucose levels are considered to be below approximately 11.1 mmol / L. The CAR-Treg described herein can, for example, maintain or reduce a subject's fasting blood glucose or OGTT blood glucose levels after administration, for example, within the normal range for fasting blood glucose and / or OGTT blood glucose. This can be observed, for example, when a subject receives a reduced dose of exogenous insulin or no exogenous insulin.

[0360] It can monitor the subject's hemoglobin (Hb) and HbA1c levels. HbA1c is produced when glucose binds to hemoglobin and is a measure of the average blood glucose level over the previous 2 to 3 months. Normal HbA1c values ​​are considered to be approximately 4.0% to 5.6% (20 mmol / mol - 38 mmol / mol). CAR-Treg can, for example, maintain or decrease the subject's HbA1c level after administration, for example, within the normal range of HbA1c. This can be observed, for example, when the subject receives a reduced dose of exogenous insulin or not at all.

[0361] Appropriately, the subjects were mammals. Appropriately, the subjects were humans.

[0362] Appropriately, the cells may be engineered Treg cells, and the cell population may be an engineered Treg cell population that has been engineered to express CAR as described herein.

[0363] Appropriately, the CAR may contain an antigen-binding domain capable of specifically binding to GLP1R, i.e., the antigen is GLP1R.

[0364] Methods for treating diseases or conditions relate to the therapeutic use of cells described herein. In this regard, cells may be administered to a subject suffering from a pre-existing disease or condition in order to alleviate, reduce, or improve at least one symptom associated with the disease or condition and / or slow, reduce, or halt the progression of the disease.

[0365] Appropriately, treatment and / or prevention of autoimmune or inflammatory diseases may refer to the administration of an effective amount of cells (e.g., Treg) that reduces the amount of existing medication (e.g., exogenous insulin) required by a subject suffering from the disease, or that enables the discontinuation of the subject's existing medication.

[0366] Prevention of disease or condition relates to the preventive use of the cells described herein. In this regard, the cells may be administered to subjects who have not yet been infected with or developed a disease or condition and / or do not exhibit any symptoms of a disease or condition, in order to prevent the disease or condition or reduce or prevent the development of at least one symptom associated with the disease or condition. The subject may be susceptible to the disease or condition (e.g., pre-stage 1 type 1 diabetes) or considered to be at risk of developing the disease or condition.

[0367] As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural processes of the individual undergoing treatment during the course of clinicopathology. The desired effects of treatment include reducing the rate of progression, improving or alleviating the pathological condition, and alleviating or improving the prognosis of a particular disease, disorder, or symptom. For example, if one or more symptoms associated with a particular disease, disorder, or symptom are reduced or eliminated, the individual has been successfully "treated."

[0368] "Effective dose" refers to the amount that effectively achieves the desired therapeutic or preventative outcome at least within the necessary dosage and time period. An effective dose can be provided in one or more administrations.

[0369] A "therapeutic effective amount" is at least the minimum concentration required to induce a measurable improvement in a particular disease, disorder, or condition. Therapeutic effective amounts, as used herein, can vary depending on a variety of factors, such as the patient's disease state, age, sex, and weight, as well as the ability of chimeric receptors to elicit the desired response in an individual. A therapeutic effective amount is also the amount in which the beneficial therapeutic effect outweighs any toxic or harmful effects of the cell, cell population, or pharmaceutical composition.

[0370] The terms “subject,” “patient,” or “individual” are used interchangeably herein and refer to mammals, preferably humans. Specifically, the terms subject, patient, and individual refer to a person in need of treatment who suffers from a disease or disorder as defined herein.

[0371] In some embodiments of the invention, patients may receive other treatments before, during, or after the treatments of the invention. For example, in some embodiments, patients may be treated with other procedures to treat symptoms associated with a disease or disorder.

[0372] The medical uses or methods described herein may include the following steps: (i) Separate or provide cell-containing samples; (ii) Introducing nucleic acid molecules, constructs, or vectors as defined herein into cells; and (iii) The cells from (ii) are administered to the subject.

[0373] Cells may be Tregs as defined herein. Before and / or after step (ii) of the method, an enriched population of Tregs may be isolated and / or generated from the cell-containing sample. For example, isolation and / or generation may be performed before and / or after step (ii) to isolate and / or generate an enriched Treg sample. Enrichment may be performed after step (ii) to enrich cells and / or Tregs containing CARs, polynucleotides, and / or vectors as described herein.

[0374] Appropriately, the cells can be autologous. Appropriately, the cells can be allogeneic.

[0375] Appropriately, cells (e.g., engineered Tregs) may be administered in combination with one or more other therapeutic agents (such as lymphatic depletion agents, as discussed above). Engineered cells (e.g., Tregs) may be administered simultaneously or sequentially (i.e., before or after) with one or more other therapeutic agents.

[0376] Cells (e.g., Tregs) can be activated and / or amplified before or after the introduction of nucleic acid molecules as described herein, for example, by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies. Amplification protocols have been discussed above.

[0377] After each step of the method, especially after amplification, cells can be washed (e.g., Tregs).

[0378] Populations of engineered cells (e.g., Treg cells) can be further enriched by any method known to those skilled in the art, such as by FACS or magnetic bead sorting.

[0379] The steps of the production process can be performed in a closed and sterile cell culture system.

[0380] The present invention can also provide methods for increasing cell stability and / or inhibitory function, the method comprising the step of introducing a nucleic acid molecule, expression construct, or vector as provided herein into the cell. The increase in inhibitory function can be measured as discussed above, for example, by co-culturing activated antigen-specific Tconv cells with the cells of the present invention, and by, for example, measuring the levels of cytokines produced by the Tconv cells. The increase in inhibitory function compared to unengineered Tregs can be an increase of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%.

[0381] Increased stability of cells (e.g., Tregs as defined herein) refers to increased persistence or survival of these cells compared to unengineered Tregs, or an increased proportion of cells that maintain the Treg phenotype over a period of time (e.g., cells that maintain Treg markers such as FOXP3 and Helios).

[0382] The increase in stability may be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and may be measured by techniques known in the art, such as staining the cell population with Treg cell markers and analyzing by FACS.

[0383] This invention also provides the use of CAR-Treg to reduce or prevent pancreatic β-cell death, for example, in subjects with type 1 diabetes or at risk of developing type 1 diabetes (especially recently diagnosed type 1 diabetes). This can be determined, for example, by measuring the number of pancreatic β-cells, blood insulin levels, and / or blood C-peptide levels in the subject. Any maintenance or improvement in the number of pancreatic β-cells, blood insulin levels, and / or blood C-peptide levels can be considered as a reduction in pancreatic β-cell death or prevention of pancreatic β-cell death. This can be observed, for example, when the subject receives a reduced dose of exogenous insulin or no exogenous insulin.

[0384] Therefore, the present invention also provides the use of CAR-Treg in maintaining or increasing fasting blood insulin levels and / or fasting blood C-peptide levels in subjects, for example, in subjects with type 1 diabetes or at risk of developing type 1 diabetes (especially recently-onset type 1 diabetes). This can be observed, for example, when subjects receive reduced doses of exogenous insulin or do not receive exogenous insulin.

[0385] This invention also provides the use of CAR-Treg for reducing or preventing hyperglycemia, for example, in subjects who have type 1 diabetes or are at risk of developing type 1 diabetes (especially those with recently developed type 1 diabetes). This can be determined, for example, by measuring the subject's fasting blood glucose and / or HbA1c levels. Any maintenance or improvement in the subject's blood glucose and / or HbA1c levels can be considered as reducing or preventing hyperglycemia in the subject. This can be observed, for example, when the subject receives a reduced dose of exogenous insulin or not at all.

[0386] Therefore, the present invention also provides the use of CAR-Treg in maintaining or reducing fasting blood glucose and / or HbA1c levels in subjects, for example, in subjects with type 1 diabetes or at risk of developing type 1 diabetes (especially recently-onset type 1 diabetes). This can be observed, for example, when subjects receive reduced doses of exogenous insulin or do not receive exogenous insulin.

[0387] The CAR of the present invention comprises an antigen recognition domain that specifically binds to GLP1R (e.g., human GLP1R and / or mouse GLP1R), and the CAR may have any of the features of a CAR as disclosed herein.

[0388] Those skilled in the art will also understand that the scFv identified as in this invention can also be used in forms outside the CAR structure. Therefore, in a further aspect of the invention, antibodies or antibody fragments that specifically bind to GLP1R are provided. These antibodies or antibody fragments may, in particular, be scFvs. The antibodies or antibody fragments (e.g., scFvs) may comprise the CDR, VH, and VL sequences or the scFv sequence as defined above with respect to the antigen-binding domain of the CAR. Thus, all sequences have been described above with respect to CDR, VH, and VL, and scFvs may similarly be included within the antibody or antibody fragment in this embodiment.

[0389] Specifically, the antibody or antibody fragment may contain: (i) (a) the VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 152, 2 and 3 respectively, or (b) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 153, 2 and 3 respectively, or (c) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 154, 2 and 3 respectively, and (a) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 156, 161 and 162 respectively, or (b) the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 156, 161 and 163 respectively; (ii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 7, 8 and 9 respectively, and (a) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 156, 161 and 12 respectively, or (b) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 157, 161 and 12 respectively, or (c) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 158, 161 and 12 respectively, or (d) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 160, 161 and 12 respectively; (iii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 13, 14 and 155 respectively, and (a) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 157, 161 and 162 respectively, or (b) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 158, 161 and 162 respectively, or (c) VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 159, 161 and 162 respectively; (iv) (a) as shown in SEQ ID NO: 151, 20 and 21 respectively, or (b) as shown in SEQ ID NO: 152, 20 and 21 respectively, or (c) as shown in SEQ ID NO: 153, 20 and 21 respectively, and as shown in SEQ ID NO: 22, 23 and 24 respectively; (v) (a) the VH CDR1, 2 and 3 sequences shown as SEQ ID NO: 151, 26 and 155 respectively, or (b) the VL CDR1, 2 and 3 sequences shown as SEQ ID NO: 154, 26 and 155 respectively; and (a) the VL CDR1, 2 and 3 sequences shown as SEQ ID NO: 159, 161 and 163 respectively, or (b) the VL CDR1, 2 and 3 sequences shown as SEQ ID NO: 160, 161 and 163 respectively. Alternatively, the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0390] Specifically, the antibody or antibody fragment may contain: (a) VH CDR as shown in SEQ ID No 1-3 and VL CDR as shown in SEQ ID No 4-6; (b) VH CDRs as shown in SEQ ID No. 7-9 and VL CDRs as shown in SEQ ID No. 10-12; (c) VH CDR as shown in SEQ ID No 13-15 and VL CDR as shown in SEQ ID No 16-18; (d) VH CDR as shown in SEQ ID No 19-21 and VL CDR as shown in SEQ ID No 22-24; (e) VH CDR as shown in SEQ ID No 25-27 and VL CDR as shown in SEQ ID No 28-30; Alternatively, the CDR may contain one to three, or more specifically one or two, amino acid sequence modifications in any of the above sequences.

[0391] Antibodies or antibody fragments can be produced by any method known in the art, including recombinant expression in host cells transduced with a vector encoding the antibody or antibody fragment. Suitable host cells include a variety of eukaryotic cells (e.g., yeast or mammalian cells) or prokaryotic cells (e.g., Escherichia coli). Antibodies or antibody fragments can also be prepared by chemical synthesis using techniques well known in protein chemistry, such as solid-phase synthesis or synthesis in homogeneous solutions.

[0392] An N-terminal or C-terminal fusion protein comprising an antibody or antibody fragment as defined herein can be generated, or alternatively, the antibody can be conjugated to another molecule (e.g., a therapeutic molecule or a detectable molecule). Therefore, the invention also includes antibodies or antibody fragments as defined above conjugated to one or more additional molecules, such as immunoglobulins, hormones, growth factors, lectins, insulin, low-density lipoprotein, glucagon, endorphins, transferrin, tags, fluorescent dyes, radioisotopes, or therapeutic molecules (e.g., immunosuppressive drugs).

[0393] Also provided are nucleic acid molecules comprising nucleotide sequences encoding antibodies, antibody fragments, or fusion proteins as defined herein, and vectors and cells comprising said nucleic acid molecules.

[0394] These antibodies, antibody fragments, and fusion proteins / conjugates can be used to detect GLP1R-expressing cells for identification and / or imaging (e.g., islet mass imaging or insulinoma detection). They can also be used to deliver therapeutic molecules to target sites (e.g., GLP1R-expressing cells and / or pancreatic β cells).

[0395] Therefore, in this respect, the present invention provides a method for detecting or imaging cells (particularly pancreatic islet cells) expressing GLP1R, the method comprising incubating the cells with an antibody, antibody fragment, or fusion protein / conjugate comprising an antibody or antibody fragment as defined herein, and determining whether the antibody, antibody fragment, or fusion protein / conjugate binds to the cells. Alternatively, the present invention provides a method for detecting or imaging cells expressing GLP1R in a subject, the method comprising administering to the subject an antibody, antibody fragment, or fusion protein / conjugate comprising an antibody or antibody fragment as defined herein, and measuring or detecting the binding of the antibody, antibody fragment, or fusion protein / conjugate to the cells. Specifically, detection can be performed by fluorescence, wherein the antibody, antibody fragment, or fusion protein / conjugate can be fluorescently labeled. Other detectable labels may also be used.

[0396] Finally, the present invention provides a method for delivering a therapeutic molecule to a target site where GLP1R is present or expressed, the method comprising administering to a subject a fusion protein or conjugate comprising an antibody or antibody fragment of the present invention and a therapeutic molecule.

[0397] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the numbers that define the range. Unless otherwise stated, any nucleic acid sequence is written from left to right with a 5' to 3' orientation; amino acid sequences are written from left to right with an amino to carboxyl orientation.

[0398] Where a numerical range is provided, it should be understood that, unless the context explicitly indicates otherwise, each intermediate value between the upper and lower limits of the range is also specifically disclosed, up to one-tenth of the lower limit unit. Each smaller range between any stated value or intermediate value in the range and any other stated value or intermediate value within the range is covered in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which any limit value is included in the smaller range, neither limit value is included in the smaller range, or both limit values ​​are included in the smaller range, is also covered in this disclosure, but is subject to any specific excluded limit value within the range. Where the range includes one or both of the limit values, the range excluding either or both of these included limit values ​​is also included in this disclosure.

[0399] It must be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise.

[0400] As used herein, the terms “comprising” and “comprises” are synonymous with “including” or “containing” and are inclusive or open-ended, and do not exclude additional unlisted members, elements, or method steps. The terms “comprising” and “comprises” also include the term “composes of”.

[0401] The publications discussed herein are provided solely because their publication predates the filing date of this application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims.

[0402] Specific aspects of this disclosure are described in the following numbered paragraphs, some of which, and combinations thereof, are consistent with the invention as described above.

[0403] 1. A chimeric antigen receptor (CAR) comprising an antigen recognition domain that specifically binds to GLP1R.

[0404] 2. The CAR according to paragraph 1, wherein the antigen recognition domain binds to human GLP1R.

[0405] 3. The CAR according to paragraph 1 or 2, wherein the CAR comprises: The CAR includes an outer domain comprising an antigen recognition domain; a transmembrane domain; and an intracellular domain comprising an intracellular signal transduction domain. 4. The CAR according to paragraph 3 further includes a hinge domain and / or one or more co-stimulatory domains.

[0406] 5. The CAR according to paragraph 4, wherein the hinge domain is selected from the hinge regions of CD28, CD8α, CD4, CD7, CH2CH3, immunoglobulins, or portions or variants thereof, preferably wherein the CAR comprises a CD8α or CH2CH3 hinge region.

[0407] 6. The CAR according to any one of paragraphs 3 to 5, wherein the CAR comprises one or more transmembrane domains selected from the transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or portions or variations thereof, preferably wherein the CAR comprises a CD4, CD28, or CD8α transmembrane domain.

[0408] 7. The CAR according to any one of paragraphs 4 to 6, wherein the co-stimulatory domain is selected from the intracellular domains of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27 or TNFRSF25, or portions or variants thereof, preferably wherein the CAR comprises a CD28 co-stimulatory domain.

[0409] 8. The CAR according to any one of paragraphs 3 to 7, wherein the CAR comprises one or more intracellular signal transduction domains selected from the group consisting of any one of the CD3ζ signal transduction domains or homologs thereof, CD3 polypeptide, syk family tyrosine kinases, src family tyrosine kinases, CD2, CD5, CD28, or portions or variants thereof, preferably wherein the CAR comprises the CD3ζ signal transduction domain.

[0410] 9. The CAR according to any one of paragraphs 3 to 8, wherein the CAR comprises: a CD8α or CH2CH3 hinge domain, a CD28 or CD8α transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signal transduction domain, wherein when the hinge domain is CD8α, the transmembrane domain is CD8α, and when the hinge domain is CH2CH3, the transmembrane domain is CD28.

[0411] 10. The CAR according to any one of paragraphs 3 to 9, wherein the CAR comprises a signal peptide and / or a reporter peptide.

[0412] 11. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain is an antibody, an antibody fragment, or an antibody-derived domain.

[0413] 12. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain is a single-chain antibody (scFv).

[0414] 13. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain comprises: (i) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 1, 2 and 3 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 4, 5 and 6 respectively; (ii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 7, 8 and 9 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 10, 11 and 12 respectively; (iii) The VH CDR1, 2 and 3 sequences shown in SEQ ID NO: 13, 14 and 15 respectively, and the VL CDR1, 2 and 3 sequences shown in SEQ ID NO: 16, 17 and 18 respectively; (iv) VH CDR1, 2, and 3 sequences as shown in SEQ ID NO: 19, 20, and 21, respectively, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 22, 23, and 24, respectively; or (v) The VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 25, 26 and 27, respectively, and the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 28, 29 and 30, respectively; One or more of the CDR sequences in (i) to (v) may optionally include one to three amino acid modifications relative to the CDR sequence, specifically, one or more of the CDR sequences may optionally be modified by substitution, addition or deletion of one to three amino acids.

[0415] 14. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain comprises: (i) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 31 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 32 or a sequence having at least 70% sequence identity with it; (ii) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 33 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 34 or a sequence having at least 70% sequence identity with it; (iii) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 35 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 36 or a sequence having at least 70% sequence identity with it; (iv) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 37 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 38 or a sequence having at least 70% sequence identity with it; or (v) VH domain and VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 39 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 40 or a sequence having at least 70% sequence identity with it.

[0416] 15. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain comprises: (i) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 41 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 41, and the VL domain contains a sequence encoded by SEQ ID NO: 42 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 42; (ii) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 43 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 43, and the VL domain contains a sequence encoded by SEQ ID NO: 44 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 44; (iii) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 45 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 45, and the VL domain contains a sequence encoded by SEQ ID NO: 46 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 46; (iv) A VH domain and a VL domain, wherein the VH domain contains a sequence encoded by SEQ ID NO: 47 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 47, and the VL domain contains a sequence encoded by SEQ ID NO: 48 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 48; or (v) VH and VL domains, wherein the VH domain contains a sequence encoded by SEQ ID NO: 49 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 49, and the VL domain contains a sequence encoded by SEQ ID NO: 50 or a sequence having at least 70% identity with the sequence encoded by SEQ ID NO: 50.

[0417] 16. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain comprises or consists of the following: (i) A sequence as shown in SEQ ID NO: 51 or a sequence having at least 80% sequence identity with it; (ii) A sequence as shown in SEQ ID NO: 52 or a sequence having at least 80% sequence identity with it; (iii) A sequence as shown in SEQ ID NO: 53 or a sequence having at least 80% sequence identity with it; (iv) A sequence as shown in SEQ ID NO: 54 or a sequence having at least 80% sequence identity with it; or (v) The sequence shown in SEQ ID NO: 55 or a sequence having at least 80% sequence identity with it.

[0418] 17. The CAR according to any of the preceding paragraphs, wherein the antigen recognition domain comprises or consists of the following: (i) A sequence encoded by the sequence shown in SEQ ID NO: 56 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 56; (ii) A sequence encoded by the sequence shown in SEQ ID NO: 57 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 57; (iii) A sequence encoded by the sequence shown in SEQ ID NO: 58 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 58; (iv) A sequence encoded by the sequence shown in SEQ ID NO: 59, or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 59; or (v) A sequence encoded by the sequence shown in SEQ ID NO: 60 or a sequence having at least 80% identity with the sequence encoded by the sequence shown in SEQ ID NO: 60.

[0419] 18. The CAR according to any of the preceding paragraphs, wherein the CAR comprises or consists of the following: a sequence shown in any one of SEQ ID Nos: 101 to 105 or 145 to 149, or a sequence having at least 80% identity with it.

[0420] 19. The CAR according to any one of paragraphs 1 to 10, wherein the antigen recognition domain is a ligand of GLP1R.

[0421] 20. The CAR according to paragraph 19, wherein the ligand of said GLP1R is a GLP1R agonist.

[0422] 21. The CAR according to paragraph 19 or 20, wherein the ligand of said GLP1R is an artificial ligand.

[0423] 22. The CAR according to any one of paragraphs 19 to 21, wherein the ligand is derived from GLP1 or glucagon.

[0424] 23. The CAR according to any one of paragraphs 19 to 22, wherein the ligand differs from GLP1 by up to 10 amino acids, for example, by 1 to 5 amino acids.

[0425] 24. The CAR according to any one of paragraphs 19 to 23, wherein the antigen recognition domain comprises or consists of the following: an amino acid sequence of SEQ ID NO: 61 or a sequence differing therefrom by up to 10 amino acids, for example, a sequence differing by 1 to 5 amino acids.

[0426] 25. The CAR according to any one of paragraphs 19 to 24, wherein the antigen recognition domain comprises or consists of an amino acid sequence encoded by SEQ ID NO: 62 or SEQ ID NO: 63 or an amino acid sequence having at least about 70% identity with an amino acid sequence encoded by SEQ ID NO: 62 or SEQ ID NO: 63.

[0427] 26. The CAR according to any one of paragraphs 19 to 25, wherein the CAR comprises or consists of the sequence shown in SEQ ID No: 126 or 150 or a sequence having at least 80% identity with it.

[0428] 27. A nucleic acid molecule comprising a nucleotide sequence encoding a CAR according to any of the preceding paragraphs.

[0429] 28. A vector comprising a nucleic acid molecule as described in paragraph 27.

[0430] 29. The vector according to paragraph 28 further comprises a nucleic acid molecule encoding a FOXP3 polypeptide.

[0431] 30. A cell comprising a CAR according to any one of paragraphs 1 to 26, a nucleic acid molecule according to paragraph 27, or a vector according to paragraph 28 or 29.

[0432] 31. The cell as described in paragraph 30, wherein the cell is a host cell.

[0433] 32. The cell according to paragraph 30, wherein the cell is an immune cell or its progenitor cell or precursor, preferably a T cell or its precursor, or a stem cell.

[0434] 33. The cell according to paragraph 30 or 31, wherein the cell is a regulatory T cell (Treg) or its precursor or iPSC cell, and specifically, wherein the cell further comprises exogenous nucleic acid containing a nucleotide sequence encoding a FOXP3 polypeptide.

[0435] 34. A cell population comprising cells according to any one of paragraphs 30, 32 or 33.

[0436] 35. The cell population according to paragraph 34, said cell population comprising a plurality of cells according to any one of paragraphs 30, 32 or 33, particularly a plurality of T cells according to paragraph 32, and more particularly a plurality of Tregs according to paragraph 33.

[0437] 36. The cell population described in paragraph 35, wherein the clonality of the plurality of T cells (particularly Tregs) is not modified in vitro.

[0438] 37. The cell population described in paragraph 35 or 36, wherein the plurality of T cells (particularly Tregs) have polyclonal endogenous TCRs.

[0439] 38. A pharmaceutical composition comprising cells according to any one of paragraphs 30, 32 or 33, a cell population according to paragraph 34 or 35, or a carrier according to paragraph 28 or 29.

[0440] 39. The cells according to any one of paragraphs 30, 32 or 33, the cell populations according to paragraphs 34 or 35 or the pharmaceutical compositions according to paragraph 38 are used for therapy.

[0441] 40. The cells, cell populations, or pharmaceutical compositions used in paragraph 39, wherein the therapy is an adoptive cell transfer therapy.

[0442] 41. The cells according to any one of paragraphs 30, 32 or 33, the cell populations according to paragraphs 34 or 35 or the pharmaceutical compositions according to paragraph 38, for the treatment or prevention of autoimmune or inflammatory diseases, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration, wherein the cells are Tregs.

[0443] 42. The cells, cell populations, or pharmaceutical compositions used in paragraph 41, wherein the autoimmune or inflammatory disease is type 1 diabetes, for example, recently-onset type 1 diabetes.

[0444] 43. A method for treating and / or preventing autoimmune or inflammatory diseases (such as type 1 diabetes), or inducing immunosuppression, or promoting tissue repair and / or tissue regeneration, wherein the method comprises administering cells (particularly Tregs) as described in any one of paragraphs 30, 32, or 33, a cell population as described in paragraphs 34 or 35, or a pharmaceutical composition as described in paragraph 38, particularly a pharmaceutical composition comprising Tregs. 44. The method according to paragraph 43, wherein the method comprises the following steps: i. Isolate or provide Treg-rich cell samples from the subject; ii. Introduce the nucleic acid molecule of paragraph 27 or the vector of paragraphs 28 or 29 into Treg cells; and iii. Administer Treg cells from (ii) to the subject.

[0445] 45. Use of the cells according to any one of paragraphs 30, 32 or 33, the cell populations according to paragraphs 34 or 35 or the pharmaceutical compositions according to paragraph 38 in the preparation of a medicament for treating and / or preventing autoimmune or inflammatory diseases (such as type 1 diabetes) in a subject, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration, wherein the cells are Tregs.

[0446] 46. ​​A method for preparing cells according to any one of paragraphs 30, 32 or 33, the method comprising the step of introducing a nucleic acid molecule according to paragraph 27 or a vector according to paragraph 28 or 29 into the cells (e.g., transducing or transfecting cells with the nucleic acid molecule or vector).

[0447] 47. The method according to paragraph 46, wherein the cell is a Treg cell, and the method includes isolating or providing a cell-containing sample containing Tregs, and / or enriching and / or generating Tregs from the cell-containing sample before or after the step of introducing the nucleic acid molecule or vector into the cell.

[0448] 48. A cell that can be obtained by the method described in paragraph 46 or 47.

[0449] 49. The cells according to any one of paragraphs 30, 32 or 33, the cell population according to paragraph 34 or 35 or the pharmaceutical composition according to paragraph 38, for preventing or reducing the mortality rate of pancreatic β cells in a subject.

[0450] 50. Cells according to any one of paragraphs 30, 32 or 33, cell populations according to paragraphs 34 or 35 or pharmaceutical compositions according to paragraph 38, for maintaining or increasing fasting blood insulin levels in a subject.

[0451] 51. Cells according to any one of paragraphs 30, 32 or 33, cell populations according to paragraphs 34 or 35 or pharmaceutical compositions according to paragraph 38, for maintaining or increasing fasting C-peptide levels in a subject.

[0452] 52. The cells according to any one of paragraphs 30, 32 or 33, the cell population according to paragraph 34 or 35 or the pharmaceutical composition according to paragraph 38, for reducing or preventing hyperglycemia in a subject.

[0453] 53. Cells according to any one of paragraphs 30, 32 or 33, cell populations according to paragraphs 34 or 35 or pharmaceutical compositions according to paragraph 38, for maintaining or reducing fasting blood glucose levels in a subject.

[0454] 54. Cells according to any one of paragraphs 30, 32 or 33, cell populations according to paragraphs 34 or 35 or pharmaceutical compositions according to paragraph 38, for maintaining or reducing HbA1c levels in a subject.

[0455] 55. The cells, cell populations or pharmaceutical compositions used in any of paragraphs 49 to 54, wherein the subject has type 1 diabetes or is at risk of developing type 1 diabetes, for example, wherein the subject has recently developed type 1 diabetes.

[0456] 56. The cells, cell populations, or pharmaceutical compositions used in any of paragraphs 49 to 55, wherein the subject is not receiving exogenous insulin.

[0457] 57. The cells, cell populations or pharmaceutical compositions used in any of paragraphs 49 to 56, wherein a reduced dose of insulin is administered to the subject compared to the insulin dose administered prior to administration of CAR-Treg.

[0458] The invention will now be further described by way of examples, which are intended to help those skilled in the art to implement the invention and are not intended to limit the scope of the invention in any way.

[0459] Sequence Listing Amino acid sequence of VH CDR1 of SEQ ID NO: 1—A2 GGTFSSYA The amino acid sequence of VH CDR2 of SEQ ID NO: 2—A2 IIPIFGTA The amino acid sequence of VH CDR3 of SEQ ID NO: 3—A2 ARSSAGFDAFDI Amino acid sequence of VL CDR1 of SEQ ID NO: 4—A2 QGLSSW The amino acid sequence of VL CDR2 of SEQ ID NO: 5—A2 DAS Amino acid sequence of VL CDR3 of SEQ ID NO: 6—A2 QQADSFPLT The amino acid sequence of VH CDR1 in SEQ ID NO: 7—A4 GYTFTSYG The amino acid sequence of VH CDR2 of SEQ ID NO: 8—A4 ISAYNGNT The amino acid sequence of VH CDR3 of SEQ ID NO: 9—A4 ARAYFSGDLDY Amino acid sequence of VL CDR1 of SEQ ID NO: 10—A4 QGISNW The amino acid sequence of VL CDR2 of SEQ ID NO: 11—A4 AAS Amino acid sequence of VL CDR3 of SEQ ID NO: 12—A4 QQSYSTPFT The amino acid sequence of VH CDR1 in SEQ ID NO: 13—A9 GYTFTSYG The amino acid sequence of VH CDR2 of SEQ ID NO: 14—A9 ISAYNGNT The amino acid sequence of VH CDR3 of SEQ ID NO: 15—A9 ARVGSSGLLDY Amino acid sequence of VL CDR1 of SEQ ID NO: 16—A9 QDISNY The amino acid sequence of VL CDR2 of SEQ ID NO: 17—A9 DAS The amino acid sequence of VL CDR3 of SEQ ID NO: 18—A9 QQYDNLPYT Amino acid sequence of VH CDR1 of SEQ ID NO: 19—B11 GFTFSSYG Amino acid sequence of VH CDR2 of SEQ ID NO: 20—B11 ISYDGSNK Amino acid sequence of VH CDR3 of SEQ ID NO: 21—B11 AKDSSGYPKYFDY The amino acid sequence of VL CDR1 of SEQ ID NO: 22—B11 SLRGSY Amino acid sequence of VL CDR2 of SEQ ID NO: 23—B11 AKN The amino acid sequence of VL CDR3 of SEQ ID NO: 24—B11 SSRDITTNHVI The amino acid sequence of VH CDR1 in SEQ ID NO: 25—E5 GYTFTSYG The amino acid sequence of VH CDR2 of SEQ ID NO: 26—E5 ISAYNGNT The amino acid sequence of VH CDR3 of SEQ ID NO: 27—E5 ARVGNGGSLDY The amino acid sequence of VL CDR1 of SEQ ID NO: 28—E5 QGISNY The amino acid sequence of VL CDR2 of SEQ ID NO: 29—E5 DAS Amino acid sequence of VL CDR3 of SEQ ID NO: 30—E5 QQFDDLPLT Amino acid sequence of the VH domain of SEQ ID NO: 31—A2 (CDR underlined) EVQLVQSGAEVKKPGSSVKVSCKAS GGTFSSYA ISWVRQAPGQGLEWMGG IIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYC ARSSAGFDAFDI WGQGTMVTVSS Amino acid sequence of the VL domain of SEQ ID NO: 32—A2 (CDR underlined) DIVMTQSPSSVSASVGDRVTVTCRAS QGLSSW LAWYQQKPGKAPELLIY DAS TLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYC QQADSFPLT FGGGTKVEVK Amino acid sequence of the VH domain of SEQ ID NO: 33—A4 (CDR underlined) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYG ISWVRQAPGQGLEWMGW ISAYNGNT NYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYC ARAYFSGDLDY WGQGTLVTVSS Amino acid sequence of the VL domain of SEQ ID NO: 34—A4 (CDR underlined) DIVMTQSPSFVSASVGDRVTITCRAS QGISNW LAWYQQKPGKAPKLLIH AAS SLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYC QQSYSTPFT FGQGTNLEIK Amino acid sequence of the VH domain of SEQ ID NO: 35—A9 (CDR underlined) QIQLVQSGAGVKKPGASVKVSCKAS GYTFTSYG ISWVRQAPGQGLEWMGW ISAYNGNT NYAQKLQGRVTMTTDTSTAYLELRSLRSDDTAVYYC ARVGSSGLLDY WGQGTLVTVSS Amino acid sequence of the VL domain of SEQ ID NO: 36—A9 (CDR underlined) DIQMTQSPSSLSASVGDRVTITCQAS QDISNY LNWYQQKPGKAPKLLIY DASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPYT FGQGTKLEIE Amino acid sequence of the VH domain of SEQ ID NO: 37—B11 (CDR underlined) QIQLVQSGGGVVQPGRSLRLSCAAS GFTFSSYG MHWVRQAPGKGLEWVAV ISYDGSNK YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKDSSGYPKYFDY WGQGTLVTVSS Amino acid sequence of the VL domain of SEQ ID NO: 38—B11 (CDR underlined) SSELTQDPAVSVALGQTVRITCQGD SLRGSY ATWYQQKPGQAPVLVIS AKN NRPSGIPDRFSVSSSGITASLTITGAQAEDEADYYC SSRDITTNHVI FGGGTKLTVL Amino acid sequence of the VH domain of SEQ ID NO: 39—E5 (CDR underlined) QVQLVQSGAEVKKPGASVKVSCKAS GYTFTSYG ISWVRQAPGQGLEWMGW ISAYNGNT NYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYC ARVGNGGSLDY WGQGTLVTVSS Amino acid sequence of the VL domain of SEQ ID NO: 40—E5 (CDR underlined) DIQMTQSPSSLSASVGDRVTITCRAS QGISNY LAWFQQKPGKAPKLLIY DAS TLNAGVPSRFSGSGSGTDFTFTISSLQPEDIGTYYC QQFDDLPLT FGPGTKVDIK SEQ ID NO: 41— Nucleotide sequence encoding the VH domain of A2 GAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAGCTCAGCTGGCTTCGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCA SEQ ID NO: 42 - Nucleotide sequence encoding the VL domain of A2 GATATTGTGATGACTCAGTCTCCTTCTTCCGTGTCTGCATCTGTTGGAGACAGAGTCACCGTCACTTGTCGGGCGAGTCAGGGTCTTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTGAGTTGCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCTAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTGACAGTTTCCCCCTCACTTTCGGCGGGGGGACCAAGGTGGAGGTCAAA SEQ ID NO: 43 - Nucleotide sequence encoding the VH domain of A4 CAAGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTACGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGCCTACTTTTCCGGTGACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 44 - Nucleotide sequence encoding the VL domain of A4 GACATCGTGATGACCCAGTCTCCATCTTTCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAACTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATGCTGCGTCAAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATCTTGCAACTTACTATTGTCAACAGAGTTACAGTACCCCGTTCACTTTTGGCCAGGGGACCAACCTGGAGATCAAA SEQ ID NO: 45 - Nucleotide sequence encoding the VH domain of A9 CAAATCCAGCTGGTACAATCTGGAGCTGGGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACCTGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTCGGGAGCAGTGGCTTGTTGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA SEQ ID NO: 46 - Nucleotide sequence encoding the VL domain of A9 GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCGAA SEQ ID NO: 47 - Nucleotide sequence encoding the VH domain of B11 CAAATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCATTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATAGTAGTGGTTATCCGAAATACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG SEQ ID NO: 48 - Nucleotide sequence encoding the VL domain of B11[[ID= 3]] TCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTTAGAATCACATGCCAAGGAGACAGTCTCAGAGGCTCTTATGCGACCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTCTGCTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGTCTCCAGCTCAGGAATCACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAGTTCCCGGGACATCACTACTAACCATGTGATATTCGGCGGAGGGACCAAGCTGACCGTCCTA SEQ ID NO: 49 - Nucleotide sequence encoding the VH domain of E5 CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTGGGTAATGGTGGCTCCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG SEQ ID NO: 50 - Nucleotide sequence encoding the VL domain of E5 GACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTACGATGCATCCACTTTGAATGCAGGGGTCCCGTCAAGGTTCAGTGGAAGTGGCTCTGGGACAGATTTCACTTTCACCATCAGCAGCCTGCAGCCTGAAGACATTGGAACATATTACTGTCAACAATTTGATGATCTTCCCCTCACTTTCGGCCCTGGGACCAAGGTGGATATCAAAC SEQ ID NO: 51 - Amino acid sequence of the antigen recognition domain comprising the VH sequence and VL sequence of A2 EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSAGFDAFDIWGQGTMVTVSSG GGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTVTCRASQGLSSWLAWYQQKPGKAPELLIYDASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQADSFPLTFGGGTKVEVK SEQ ID NO: 52— Amino acid sequence including the antigen recognition domains of the VH and VL sequences of A4. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTAYMELRSLRSDDTAVYYCARAYFSGDLDYWGQGTLVTVSSGG GGSGGGGSGGGGSDIVMTQSPSFVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGQGTNLEIK SEQ ID NO: 53 — Amino acid sequence including the antigen recognition domains of the VH and VL sequences of A9. QIQLVQSGAGVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYLELRSLRSDDTAVYYCARVGSSGLLDYWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPYTFGQGTKLEIE SEQ ID NO: 54 — Amino acid sequence including the antigen recognition domains of the VH and VL sequences of B11. QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSGYPKYFDYWGQGTLVTVSSG GGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRGSYATWYQQKPGQAPVLVISAKNNRPSGIPDRFSVSSSGITASLTITGAQAEDEADYYCSSRDITTNHVIFGGGTKLTVL SEQ ID NO: 55— Amino acid sequence including the antigen recognition domains of the VH and VL sequences of E5. QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARVGNGGSLDYWGQGTLVTVSSGG GGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKLLIYDASTLNAGVPSRFSGSGSGTDFTFTISSLQPEDIGTYYCQQFDDLPLTFGPGTKVDIK SEQ ID NO: 56 — Nucleotide sequence encoding A2 plus a signal sequence and a tag (the sequence encoding the signal sequence and the tag is underlined). ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCGAAGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAAGCTCAGCTGGCTTCGATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGATATTGTGATGACTCAGTCTCCTTCTTCCGTGTCTGCATCTGTTGGAGACAGAGTCACCGTCACTTGTCGGGCGAGTCAGGGTCTTAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTGAGTTGCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCTAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAACAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTGACAGTTTCCCCCTCACTTTCGGCGGGGGGACCAAGGTGGAGGTCAAACGAACTGTGGCTGCACCATCTGTC GCGGCCGCAGGTTCTGA ACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 57 - Nucleotide sequence encoding A4 plus signal sequence and tag (sequences encoding signal sequence and tag are underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAAGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTACGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGCCTACTTTTCCGGTGACCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGACATCGTGATGACCCAGTCTCCATCTTTCGTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCAACTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCCATGCTGCGTCAAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAGGATCTTGCAACTTACTATTGTCAACAGAGTTACAGTACCCCGTTCACTTTTGGCCAGGGGACCAACCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTC GCGGCCGCAGGTTCTGAACA AAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 58 - Nucleotide sequence encoding A9 plus signal sequence and tag (the sequences encoding the signal sequence and tag are underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAAATCCAGCTGGTACAATCTGGAGCTGGGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACCTGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTCGGGAGCAGTGGCTTGTTGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGTACACTTTTGGCCAGGGGACCAAGCTGGAGATCGAACGAACTGTGGCTGCACCATCTGTC GCGGCCGCAGGTTCTGAACA AAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATCAT SEQ ID NO: 59 - Nucleotide sequence encoding B11 plus signal sequence and tag (the sequences encoding the signal sequence and tag are underlined) ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGCCCAAATCCAGCTGGTGCAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGGCATGCATTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCGAAAGATAGTAGTGGTTATCCGAAATACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTATCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTTAGAATCACATGCCAAGGAGACAGTCTCAGAGGCTCTTATGCGACCTGGTACCAGCAGAAGCCAGGACAGGCCCCTGTACTTGTCATCTCTGCTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGTCTCCAGCTCAGGAATCACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAGGATGAGGCTGACTATTACTGTAGTTCCCGGGACATCACTACTAACCATGTGATATTCGGCGGAGGGACCAAGCTGACCGTCCTAAGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCT GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATC AT SEQ ID NO: 60 - Nucleotide sequence encoding E5 plus signal sequence and tag (the sequences encoding the signal sequence and tag are underlined) [[ID=CAGGTTCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGTTACACCTTTACCAGCTATGGTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAGCGCTTACAATGGTAACACAAACTATGCACAGAAGCTCCAGGGCAGAGTCACCATGACCACAGACACATCCACGAGCACAGCCTACATGGAGCTGAGGAGCCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAGAGTGGGTAATGGTGGCTCCCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGGAGTGCATCCGCCCCAAAGCTTGAAGAAGGTGAATTTTCAGAAGCACGCGTAGACATCCAGATGACCCAGTCTCCATCCTCACTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGCATTAGCAATTATTTAGCCTGGTTTCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATCTACGATGCATCCACTTTGAATGCAGGGGTCCCGTCAAGGTTCAGTGGAAGTGGCTCTGGGACAGATTTCACTTTCACCATCAGCAGCCTGCAGCCTGAAGACATTGGAACATATTACTGTCAACAATTTGATGATCTTCCCCTCACTTTCGGCCCTGGGACCAAGGTGGATATCAAACGAACTGTGGCTGCACCATCTGTC ​ ​ SEQ ID NO: 61 - Amino acid sequence of the antigen recognition domain of the ligand CAR HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG SEQ ID NO: 62 - Nucleotide sequence encoding the antigen recognition domain of the ligand CAR CACGGCGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGAGGAGCAGGCCGCCAAGGAATTCATCGCCTGGCTGGTGAAGGGCGGCGGC SEQ ID NO: 63 — The nucleotide sequence encoding the antigen recognition domain of the ligand CAR, plus the signal sequence and the tag (the sequence encoding the signal sequence and the tag is underlined). ATGAAATACCTATTGCCTACGGCAGCCGCTGGCTTGCTGCTGCTGGCAGCTCAGCCGGCCATGGGC CACGGCGAGGGCACCTTCACCTCCGACGTGTCCTCCTATCTCGAGGAGCAGGCCGCCAAGGAATTCATCGCCTGGCTGGTGAAGGGCGGCGGC GCGGCCGCAGGTTCTGAACAAAAGCTGATCTCAGAAGAAGATCTATCCCATCATCACCATCATC AT SEQ ID NO: 64— Amino acid sequence of human GLP1R MAGAPGPLRLALLLLGMVGRAGPRPQGATVSLWETVQKWREYRRQCQRSLTEDPPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAEGLWLQKDN SSLPWRDLSECEESKRGERSSPEEQLLFLYIIYTVGYALSFSALVIASAILLGFRHLHCTRNYIHLNLFASFILLRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLSCRLVFL LMQYCVAANYYWLLVEGVYLYTLLAFSVLSEQWIFRLYVSIGWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVICIVVSKLKANLMCKTDIKC RLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCPTSSSSGATAGSSMYTATCQASCS SEQ ID NO: 65— Amino acid sequence of mouse GLP1R MASTPSLLRLALLLLGAVGRAGPRPQGTTVSLSETVQKWREYRRQCQRFLTEAPLLATGLFCNRTFDDYACWPDGPPGSFVNVSCPWYLPWASSVLQGHVYRFCTAEGLWLHKDN SSLPWRDLSECEESKRGERNFPEEQLLSLYIIYTVGYALSFSALVIASAILVGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLLSYQDSLGCRLVFL LMQYCVAANYYWLLVEGVYLYTLLAFSVFSEQRIFKLYLSIGWGVPLLFVIPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFIRVICIVVSKLKANLMCKTDIKC RLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKLFTELSFTSFQGLMVAILYCFVNNEVQMEFRKCWERWRLEHLNIQRDCSMKPLKCPTSSVSSGATVGSSVYAATCQSSYS SEQ ID NO: 66— Amino acid sequence of human proglucagon MKSIYFVAGLFVMLVQGSWQRSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAKRHDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRGRRDFPEEVAIVEELGRRHADGSFSDEMNTILDNLAARDFINWLIQTKITDRK SEQ ID NO: 67— Amino acid sequence of human glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO: 68— Amino acid sequence of human GLP1 (1-37) HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO: 69— Amino acid sequence of human GLP1 (7-37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO: 70— Representative connector sequence GGGS SEQ ID NO: 71— Representative connector sequence ETSGGGGSRL SEQ ID NO: 72— Representative connector sequence SGGGGSGGGGSGGGGS SEQ ID NO: 73— Representative connector sequence GGGGS SEQ ID NO: 74— Representative connector sequence GGGGGS SEQ ID NO: 75— Representative connector sequence GGGGGGS SEQ ID NO: 76— Representative connector sequence GGGGSGGGGSGGGGS SEQ ID NO: 77— Representative connector sequence GGGGG SEQ ID NO: 78— Representative connector sequence GGGGSGGGGS SEQ ID NO: 79— Representative connector sequence GGGGSGGGGSGGGGSGGGGS SEQ ID NO: 80— Representative connector sequence GGGGGG SEQ ID NO: 81— Representative connector sequence G6 SEQ ID NO: 82— Representative connector sequence G8 SEQ ID NO: 83— Representative connector sequence KESGSVSSEQLAQFRSLD SEQ ID NO: 84— Representative connector sequence EGKSSGSGSESKST SEQ ID NO: 85— Representative connector sequence GSAGSAAGSGEF SEQ ID NO: 86— Representative connector sequence SGGGGSAGSAAGSGEF SEQ ID NO: 87— Representative connector sequence SGGGLLLLLLLLGGGS SEQ ID NO: 88— Representative connector sequence SGGGAAAAAAAAGGGS SEQ ID NO: 89— Representative connector sequence SGGGAAAAAAAAAAAAAAAAAAAGGGS SEQ ID NO: 90— Representative connector sequence SGALGGLALAGLLLAGLGLGAAGS SEQ ID NO: 91— Representative connector sequence SLSLSPGGGGGPAR SEQ ID NO: 92— Representative connector sequence SLSLSPGGGGGPARSLSLSPGGGGG SEQ ID NO: 93— Representative connector sequence GSSGSS SEQ ID NO: 94— Representative connector sequence GSSSSSS SEQ ID NO: 95— Representative connector sequence GGSSSS SEQ ID NO: 96— Representative connector sequence GSSSSS SEQ ID NO: 97— Representative connector sequence SGGGGS SEQ ID NO: 98—Exemplary connector sequence KLEEGEFSEARV SEQ ID NO: 99—Exemplary connector sequence ILEEGEFSEAGC SEQ ID NO: 100—Exemplary connector sequence X1LEEGEFSEAX2X3, where X1 is K or I, X2 is R or G, and X3 is V or C. SEQ ID NO: 101— Amino acid sequence of a CAR having an antigen-binding domain derived from A2. EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSAGFDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTVTCRASQGLSSWLAWYQQKPGKAPELLIYDASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQADSFPLTFGGGTKVEVKTRFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 102 - Amino acid sequence of a CAR having an antigen - binding domain derived from A4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAYFSGDLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSFVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGQGTNLEIKTRFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 103 - Amino acid sequence of a CAR with an antigen - binding domain derived from A9 QIQLVQSGAGVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYLELRSLRSDDTAVYYCARVGSSGLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPYTFGQGTKLEIETRFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 104 - Amino acid sequence of a CAR having an antigen - binding domain derived from B11 QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSGYPKYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRGSYATWYQQKPGQAPVLVISAKNNRPSGIPDRFSVSSSGITASLTITGAQAEDEADYYCSSRDITTNHVIFGGGTKLTVLTRFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 105 - Amino acid sequence of a CAR with an antigen-binding domain derived from E5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSSTAYMELRSLRSDDTAVYYCARVGNGGSLDYWGQGTLVTVSSGGGG SGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKLLIYDASTLNAGVPSRFSGSGSGTDFTFTISSLQPEDIGTYYCQQFDDLPLTFGPGTKVDIKTRFV PVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRD FAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 106— Wild-type human CD8α hinge domain FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD SEQ ID NO: 107— Amino acid sequence of the modified CD8α hinge domain FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDDFAD SEQ ID NO: 108 is the amino acid sequence of the CD8α hinge used in the CAR used in the examples. FSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY SEQ ID NO: 109 is a nucleotide sequence encoding the CD8α hinge used in the CAR used in the examples. TTCAGCTCTGTGGTGCCCGTGCTGCAGAAAGTGAACAGCACCACCACAAAGCCTGTGCTGAGAACCCCTTCTCCTGTGCACCCTACCGGCACAAGCCAGCCTCAAAGACCTGAGGACTGCAGACCTAGAGGCTCCGTGAAAGGCACAGGCCTGGACTTCGCCTGCGACATCTAT SEQ ID NO: 110— The amino acid sequence representing the human CD8α transmembrane domain at positions 183 to 203 of human CD8α. IYIWAPLAGTCGVLLLSLVIT SEQ ID NO: 111— Amino acid sequence of wild-type CD8α combined hinge and transmembrane domain (transmembrane domain underlined) FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVL LLSLVIT LYCNHR SEQ ID NO: 112— Modified amino acid sequence of the combined CD8α hinge and transmembrane domain (transmembrane domain underlined) FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDDFAD IYIWAPLAGTCGVLL LSLVIT LYCNHR SEQ ID NO: 113—Amino acid sequence of the CH2CH3 hinge domain PCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 114 — Amino acid sequence of the CH2CH3 hinge domain EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 115— Amino acid sequence of CD28 hinge and transmembrane domain (transmembrane domain underlined) IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP FWVLVVVGGVLACYSLLVTVAFIIFWV SEQ ID NO: 116—Amino acid sequence of the CD4 transmembrane domain MALIVLGGVAGLLLFIGLGIFF SEQ ID NO: 117— Amino acid sequence of the signal / leader sequence MALPVTALLLPLALLLHAAAP SEQ ID NO: 118— Amino acid sequence of the wild-type CD8α leader sequence MALPVTALLLPLALLLHAARP SEQ ID NO: 119—Amino acid sequence of the CD3ζ intracellular domain RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 120—Amino acid sequence of the transmembrane and intracellular signal transduction domains of the CD28 combination (transmembrane domains are underlined) FWVLVVVGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 121—Amino acid sequence of the CD28 intracellular signal transduction domain WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS SEQ ID NO: 122— Amino acid sequence of the OX40 signal transduction domain ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI Amino acid sequence of the SEQ ID NO: 123—4-1BB signal transduction domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 124—Amino acid sequence of the ICOS signal transduction domain CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL SEQ ID NO: 125—Amino acid sequence of the TNFRSF25 signal transduction domain TYTYRHCWPHKPLVTADEAGMEALTPPPATHLSPLDSAHTLLAPPDSSEKICTVQLVGNSWTPGYPETQEALCPQVTWSWDQLPSRALGPAAAPTLSPESPAGSPAMMLQPGPQLYDVMDAVPARRWKEFVRTLGLREAEIEAVEVEIGRFRDQQYEMLKRWRQQQPAGLGAVYAALERMGLDGCVEDLRSRLQRGP SEQ ID NO: 126 — Amino acid sequence of a CAR having an antigen-binding domain derived from a GLP1R ligand. HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGTRFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRPPALEWVRSKRSRLLHSDYMNMTP RRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 127 is the amino acid sequence representing amino acid positions 266 to 551 of the human IL-2 receptor β chain. NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 128 is the amino acid sequence representing a truncated and sequence-modified variant (Y510) of SEQ ID NO: 127. NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 129 is the amino acid sequence representing a truncated and sequence-modified variant (Y510 and Y392) of SEQ ID NO: 127. NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV SEQ ID NO: 130 — Amino acid sequence of wild-type FOXP3 (UniProtKB accession number Q9BZS1) MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFL KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPE FLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGP The amino acid sequence of the N-terminal and C-terminal truncated FOXP3 fragment described in SEQ ID NO: 131—WO2019 / 241549. GGAHASSSSL NPMPPSQLQL PTLPLVMVAP SGARLGPLPH LQALLQDRPH FMHQLSTVDAHARTPVLQVH PLESPAMISL TPPTTATGVF SLKARPGLPP GINVASLEWV SREPALLCTF PNPSAPRKDSTLSAVPQSSY PLLANGVCKW PGCEKVFEEP EDFLKHCQAD HLLDEKGRAQ CLLQREMVQS LEQQLVLEKEKLSAMQAHLA GKMALTKASS VASSDKGSCC IVAAGSQGPV VPAWSGPREA PDSLFAVRRH LWGSHGNSTFPEFLHNMDYF KFHNMRPPFT YATLIRWAIL EAPEKQRTLN EIYHWFTRMF AFFRNHPATW KNAIRHNLSLHKCFVRVESE KGAVWTVDEL EF SEQ ID NO: 132— The amino acid sequence of a FOXP3 variant with a mutation at amino acid position 418. MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR E QRPSRCSNPTPGP SEQ ID NO: 133 - Amino acid sequence of the FOXP3 variant having a mutation at amino acid position 422 MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRP A RCSNPTPGP SEQ ID NO: 134—Amino acid sequence of a FOXP3 variant with mutations at amino acid positions 418 and 422. MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVF EEPEDFLKHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHL WGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKR E QRP A RCSNPTPGP SEQ ID NO: 135— Amino acid sequence of an exemplary FOXP3 polypeptide MPNPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPVMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSYPLLANGVCKWPGCEKVFEEPEDFLKHCQA DHLLDEKGRAQCLLQREMVQSLEQVEELSAMQAHLAGKMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTNLEIYHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEFRKKRSQRPSRCSNPTPGPEGRGSLLTCGDVEEN SEQ ID NO SEQ ID NO: 137— Nucleotide sequence encoding an exemplary FOXP3 polypeptide SEQ ID NO: 138—Amino acid sequence of the P2A domain ATNFSLLKQAGDVEENPGP SEQ ID NO: 139— Amino acid sequence of the T2A domain EGRGSLLTCGDVEENPGP SEQ ID NO: 140—Amino acid sequence of the E2A domain QCTNYALLKLAGDVESNPGP SEQ ID NO: 141—Amino acid sequence of the F2A domain VKQTLNFDLLKLAGDVESNPGP SEQ ID NO: 142—Amino acid sequence of the furin protease cleavage site RXXR SEQ ID NO: 143—Amino acid sequence of the furin protease cleavage site RRKR SEQ ID NO: 144— Nucleotide sequence of the SFFV promoter GTAACGCCATTTTGCAAGGCATGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAATAGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGGGCCAAGAACAGATTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGGTCCCCAGATA TGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGGCCGG SEQ ID NO: 145 — Amino acid sequence of a CAR having an antigen-binding domain derived from A2. EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARSSAGFDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSPSSVSASVGDRVTVTCRASQGLSSWLAWYQQKPGKAPELLIYDASTLQSGVPSRFSGSGSGTDFTLTINSLQPEDFATYYCQQADSFPLTFGGGTKVEVKTREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 146 - Amino acid sequence of a CAR with an antigen - binding domain derived from A4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARAYFSGDLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPSFVSASVGDRVTITCRASQGISNWLAWYQQKPGKAPKLLIHAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDLATYYCQQSYSTPFTFGQGTNLEIKTREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 147 - Amino acid sequence of a CAR with an antigen-binding domain derived from A9 QIQLVQSGAGVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYLELRSLRSDDTAVYYCARVGSSGLLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPYTFGQGTKLEIETREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 148 - Amino acid sequence of a CAR having an antigen - binding domain derived from B11 QIQLVQSGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDSSGYPKYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRGSYATWYQQKPGQAPVLVISAKNNRPSGIPDRFSVSSSGITASLTITGAQAEDEADYYCSSRDITTNHVIFGGGTKLTVLTREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 149 - Amino acid sequence of a CAR having an antigen - binding domain derived from E5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYNGNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARVGNGGSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKLLIYDASTLNAGVPSRFSGSGSGTDFTFTISSLQPEDIGTYYCQQFDDLPLTFGPGTKVDIKTREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASCTRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 150 - Amino acid sequence of a CAR with an antigen-binding domain having a ligand derived from GLP1R HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGTREPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKDPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASC TRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 151—The common sequence of VH CDR1 GX 1 TFX2SYG, where X 1 It is Y or F, and X 2 Is it S or T? SEQ ID NO: 152—The common sequence of VH CDR1 GX 1 TFSSY, where X 1 Is it G or F? SEQ ID NO: 153—The common sequence of VH CDR1 GX 1 TFSSYX 2 , where X 1 It is G or F, and X 2 Is it A or G? SEQ ID NO: 154—The common sequence of VH CDR1 GX 1 TFX 2 SY, where X 1 It is Y or G, and X 2 Is it T or S? SEQ ID NO: 155—The common sequence of VH CDR3 ARVGX 1 X2 GX 3 LDY, where X 1 Is it S or N, X 2 It is S or G, and X 3 Is it L or S? SEQ ID NO: 156— The common sequence of VL CDR1 QGX 1 SX 2 W, where X 1 It is L or I, and X 2 Is it S or N? SEQ ID NO: 157— The common sequence of VL CDR1 QX 1 ISN, where X 1 Is it D or G? SEQ ID NO: 158— The common sequence of VL CDR1 QX 1 ISNX 2 , where X 1 It is D or G, and X 2 Is it Y or W? SEQ ID NO: 159— The common sequence of VL CDR1 QX 1 ISNY, where X 1 Is it D or G? SEQ ID NO: 160—The common sequence of VL CDR1 QGISNX 1 , where X 1 Is it Y or W? SEQ ID NO: 161— The common sequence of VL CDR2 X 1 AS, where X 1 Is it D or A? SEQ ID NO: 162— The common sequence of VL CDR3 QQX 1 DX 2 LPX 3 T, where X 1 Is it Y or F, X 2 It is N or D, and X 3 Is it Y or L? SEQ ID NO: 163—The common sequence of VL CDR3 QQX 1 DX 2 X 3 PLT, where X1 Is it A or F, X 2 It is S or D, and X 3 Is it F or L? Example Example 1 — Generation of GLP1R scFv binder and GLP1R specific CAR Materials and methods Formation of scFv binder scFv binders were generated via phage display using the human juvenile phage display library HAL9 / 10. A cell-based panning method was employed, targeting mouse or human GLP1R recombinantly expressed on HEK293T cells. A protein-based panning method targeting native proteins was also used. Thus, these cells were transiently transfected using a vector expressing the recombinant protein and downstream eGFP, with the recombinant protein and eGFP separated by a P2A site. This enabled recognition of the membrane protein in its native conformation. The phage display library or binder from the sequential panning wheel was first incubated with untransfected HEK293T cells to deplete nonspecific binders. Alternatively, the phage display library or binder from the sequential panning wheel was incubated on coated native proteins. The supernatant was then incubated with antigen-transfected cells, washed, and sorted for cells highly expressing GFP. scFv binding to GFP-highly expressing cells (and therefore cells expressing high levels of the target antigen) is eluted from these cells or native proteins using trypsin digestion. The eluted scFv binders are amplified and used for the next round of panning. This process is repeated for three rounds. After the final round, individual scFv binders undergo preliminary screening.

[0460] Screening of scFv binders ( Figure 1 ) First, scFv binders initially obtained through phage display panning were screened to recognize GLP1R expressed on cells. Therefore, soluble scFv was generated in *E. coli* and used to stain HEK293T cells transiently transfected using a vector expressing a recombinant protein and downstream eGFP, with the recombinant protein and eGFP separated by a P2A site. Secondary staining was performed using PE staining of the His tag of the soluble scFv. Flow cytometry was used to measure GFP (antigen expression) and PE (scFv binding) in the cells.

[0461] Pancreatic section staining ( Figure 2 ) Frozen IHC was performed to demonstrate the recognition ability of the selected scFv for islets in pancreatic tissue. Therefore, fresh pancreas from C57Bl / 6 mice was embedded in Tissue-Tek® Cryomold® and rapidly frozen in liquid nitrogen. 5 μm sections were prepared in a cryostat. Autofluorescence of the sections was quenched by incubation in glycine buffer. Blocking was performed by incubation in 2% BSA. A soluble scFv (see above) was used to stain the islets. Continuously, sections were stained with rabbit α-Myc-Tag antibody to recognize the Myc tag of the soluble scFv. Tertiary staining was performed by incubation with FITC-labeled goat α-rabbit antibody. Sections were embedded in Fluoromount-G™ mounting medium containing DAPI, and islet staining was analyzed by fluorescence microscopy.

[0462] Cloning of CAR vectors scFv was cloned into a mouse retroviral vector to generate a GLP1R-specific CAR. The conformation of the CAR construct used is shown in [Figure / Diagram / Illustration]. Figure 7 The CAR has a mouse CD8α hinge and a transmembrane domain (short hinge) or a mouse IgG hinge domain and a CD4 transmembrane domain (long hinge). The sequence of the CD8α hinge is shown in SEQ ID NO: 108 and can be encoded by SEQ ID NO: 109. A retroviral vector containing a second-generation CAR scaffold with LTR side attachments was used for cloning. Cloning was performed by digesting the vector with NcoI / NotI restriction enzymes and subsequently ligating scFv into the CAR backbone. The mouse CAR backbone contains an additional FoxP3 expression cassette that can generate transformed Tregs (cTregs) from CD4+ T cells, but the backbone lacking such an expression cassette is used to generate CAR Teffs or native Tregs (nTregs). The vector also contains Thy1.1 (CD90.1) as a CAR expression marker. In addition, ligand-based CARs were generated by including a mutant GLP1 molecule (see SEQ ID NO: 61) instead of scFv. The CAR conformation is shown in Figure 8 In this context, the term "connector" refers to the mouse CD8α hinge domain. The mutant GLP1 molecule is a GLP1(7-37) peptide with three substitutions (Ala at position 8 is replaced by Gly; Gly at position 22 is replaced by Glu; and Arg at position 36 is replaced by Gly). The mutant GLP1 molecule is resistant to DPP-IV degradation and exhibits increased affinity for GLP1R.

[0463] Production of retroviruses expressing recombinant CARs and transduction of T cells and cell lines. Retroviral particles that allow CAR transduction of T cells were generated in HEK293T cells. These particles were K73-dependent pseudotyped for the mouse construct and VSV-G-dependent pseudotyped for the human construct. For CAR transduction, CD4+ cells were enriched from mouse spleen cells by magnetic bead separation and activated with CD3 / CD28 beads for 2 days, followed by spin transduction with viral particles and protamine sulfate, and cultured for 1 to 4 days. For CAR activation assays, NFAT-GFP reporter mouse T cell hybridoma cells were transduced in a similar manner.

[0464] Hybridoma NFAT activation assay As described above, a mouse T cell hybridoma cell line activated by GFP-expressed reporter NFAT was transduced using a CAR vector. In parallel, HEK293T cells were transfected to express the target antigen GLP1R, either in mouse or human homologous form. Both cell lines were cultured for 24 hours after 48 hours to allow CAR activation. Directly coated (human) GLP1R protein and untreated wells served as controls. Cells were stained against the marker Erb2 to allow differentiation between HEK293T cells and anti-Fab antibody binding to the CAR domain for CAR expression analysis. CAR activation levels reported by GFP and CAR expression were measured by flow cytometry.

[0465] result Many scFvs that bind to GLP1R were identified.

[0466] Figure 1 FACS staining of the exemplary scFv binder on antigen-transfected HEK293T cells is shown. scFv binding was demonstrated by staining with anti-HisPE antibody. Antigen expression was reported by GFP.

[0467] Human antigens are shown in the first row. Mouse antigens are shown in the second row.

[0468] Figure 1 The images show HEK293T cells containing scFvs labeled “SH1989-A2”, “SH1989-A4”, “SH1989-A9”, “HG001-B11”, and “VG002-E5” (referred to as A2, A4, A9, B11 and E5 in this paper, respectively) expressing both human and mouse GLP1R.

[0469] Immunohistochemical staining of frozen C57BL / 6 mouse pancreas sections was shown in... Figure 2 The top row shows DAPI staining, the second row shows staining using scFv, and the bottom row combines DAPI and scFv staining. Figure 2Mouse pancreatic slices showing specific binding of A2, A4, and A9scFv are shown.

[0470] A2, A4, A9, B11, and E5 scFvs and ligands (referred to herein as "Dula") were incorporated into CARs as described above and expressed in mouse hybridoma cells. NFAT was activated by CAR binding to GLP1R and was reported by GFP. Anti-Fab antibody was used for CAR staining of scFv CARs. Anti-GLP1 antibody was used for staining of ligand-based CARs. The left column for each scFv shows the results of mouse hybridoma cells expressing CARs incorporated with short hinges. The right column for each scFv shows the results of mouse hybridoma cells expressing CARs incorporated with long hinges.

[0471] Activation of human GLP1R protein (top row), HEK cells expressing human GLP1R antigen (second row), HEK cells expressing mouse GLP1R antigen (third row), and untransfected HEK cells (control) is shown in the figure. Figure 3 and Figure 4 middle.

[0472] Example 2 — Induction of GLP1R-specific CAR-expressing T effector cells in C57Bl / 6.RAG mice Type 1 diabetes Materials and methods CD4+CD8+ Teff cells from C57Bl / 6 mice were transduced using a CAR incorporating an A9 scFv binder (with a long hinge) and then adoptively transferred to corresponding lymphopened C57Bl / 6.RAG mice (2 × 10⁻⁶). 6 In a study of mice (in cells), potential diabetes induction was observed. Changes in blood glucose levels and body weight were monitored during the experiment. Mice that reached a high degree of diabetes were sacrificed. An overview of the experimental protocol is shown in [reference to...]. Figure 5 middle.

[0473] result During the experiment, unbalanced glucose metabolism was observed in individuals treated with cells expressing a GLP1R-specific CAR, with one animal reaching a blood glucose level of 240 mg / dL. At the end of the experiment, this animal experienced weight loss.

[0474] Example 3 — Administration of T effector cells from BDC2.5.NOD mice and T cells from NOD mice to NOD.SCID mice Mice expressing GLP1R-specific CAR Tregs prevent the induction of type 1 diabetes. Materials and methods The CAR-Tregs from NOD mice were transduced using a CAR-Treg incorporating a ligand-based CAR as described in Example 1, which incorporated a mutant GLP1 molecule (see SEQ ID NO: 61) instead of scFv. These CAR-Tregs (150,000 or 450,000 cells) were adoptively transferred to NOD-SCID mice along with CD4+CD25-Teffs from BDC2.5 NOD mice (150,000 cells, yielding a BDC2.5 CD4+CD25-:NOD CD4+ / CAR ratio of 1:1 (n=8) or 1:3 (n=8)). BDC2.5 NOD mice express a unique T-cell receptor clone, and their CD4+ Teffs, when administered to immunodeficient NOD.SCID mice, induce diabetes. Therefore, the mice were monitored to observe whether NOD CAR-Tregs could prevent the onset of induced diabetes in a lymphopenia model. Changes in blood glucose levels and body weight were examined during the experiment. Mice that reached a high degree of diabetes were sacrificed. BDC2.5CD4+CD25- + BDC2.5 CD4+ / CD25+ cells were used as positive controls (n=3). BDC2.5 CD4+CD25- + NOD CD4+ / CD25+ cells were used as negative controls (n=6). An overview of the experimental protocol is shown below. Figure 9 middle.

[0475] result Compared to the negative control, CAR-Treg delayed the onset of diabetes and approximately doubled survival time, with some mice becoming diabetes-free after 40–80 days. This effect was dose-dependent, meaning the delay was greater with the use of more CAR-Treg. Results are shown in… Figure 10A and Figure 10B In. Figure 10A and Figure 10B In both cases, the lines from left to right represent mice treated with NOD Treg (negative control), CAR-Treg (1:1), CAR-Treg (3:1), and BDC2.5 Treg (positive control).

[0476] Example 4 — Activation and proliferation of mouse Tregs expressing GLP1R-specific CAR Materials and methods Mouse CD4 spleen cells were transduced using A9 CAR (with a long hinge) retrovirus. CAR T cells were labeled with the proliferation dye CFSE and then stimulated for 96 hours with anti-CD3 / CD28 beads, fixed target GLP1R, or control antigen. The upregulation of the early activation marker CD69 in CAR T cells was analyzed by flow cytometry. In parallel, cell proliferation was indicated by the dilution of CFSE signaling.

[0477] result Compared with CAR-Tregs exposed to the control antigen, CAR-Tregs exposed to the target (GLP1R) showed higher expression of the activation marker CD69. Results are shown in... Figure 11 (Left column = beads, middle column = target antigen, right column = control antigen).

[0478] CAR-Tregs exposed to the target (GLP1R) showed increased proliferation compared to those exposed to the control antigen. Results are shown in... Figure 12 (Left column = beads, middle column = target antigen, right column = control antigen).

Claims

1. A regulatory T cell (Treg) comprising a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition domain that specifically binds to GLP1R.

2. The Treg of claim 1, wherein the CAR comprises: a. An outer structural domain, wherein the outer structural domain includes an antigen recognition structural domain; b. Transmembrane domains; and c. Intracellular domain, which includes intracellular signal transduction domains.

3. The Treg of claim 2, wherein the CAR further comprises a hinge domain and / or one or more co-stimulatory domains.

4. The Treg according to claim 3, wherein the hinge domain of the CAR is selected from the hinge regions of CD28, CD8α, CD4, CD7, CH2CH3, immunoglobulins, or portions thereof or variants thereof, preferably wherein the CAR comprises a CD8α or CH2CH3 hinge region; and / or wherein the co-stimulatory domain of the CAR is selected from the intracellular domains of CD28, ICOS, CD134 (OX40), CD137 (4-1BB), CD27, or TNFRSF25, or portions thereof or variants thereof, preferably wherein the CAR comprises a CD28 co-stimulatory domain.

5. The Treg according to any one of claims 2 to 4, wherein the CAR comprises one or more transmembrane domains selected from the transmembrane domains of CD28, ICOS, CD8α, CD4, CD134 (OX40), CD137 (4-1BB), CD3ζ, CD45, CD9, CD16, CD22, CD33, CD64, CD80, CD86, CD154, or portions thereof or variants thereof, preferably wherein the CAR comprises a CD4, CD28, or CD8α transmembrane domain, and / or wherein the CAR comprises one or more intracellular signal transduction domains selected from any one of the CD3ζ signal transduction domains or homologs thereof, CD3 peptides, syk family tyrosine kinases, src family tyrosine kinases, CD2, CD5, CD28, or portions thereof or variants thereof, preferably wherein the CAR comprises the CD3ζ signal transduction domain.

6. The Treg according to any one of claims 2 to 5, wherein the CAR comprises: a CD8α or CH2CH3 hinge domain, a CD28 or CD8α transmembrane domain, a CD28 co-stimulatory domain, and a CD3ζ signal transduction domain, wherein when the hinge domain is CD8α, the transmembrane domain is CD8α, and when the hinge domain is CH2CH3, the transmembrane domain is CD28.

7. The Treg according to any of the preceding claims, wherein the antigen recognition domain is an antibody, an antibody fragment, or an antibody-derived domain, preferably wherein the antigen recognition domain is a single-chain antibody (scFv).

8. The Treg according to any one of claims 1 to 6, wherein the antigen recognition domain is a ligand of GLP1R, for example, wherein the ligand is a GLP1R agonist.

9. The Treg of claim 8, wherein the antigen recognition domain of the CAR comprises or consists of the following: The amino acid sequence of SEQ ID NO: 61 or a sequence that differs from it by up to 10 amino acids, for example, a sequence that differs by 1 to 5 amino acids.

10. The Treg according to any one of claims 1 to 7, wherein the antigen recognition domain comprises: (i) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 1, 2 and 3 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 4, 5 and 6 respectively; (ii) VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 7, 8 and 9 respectively, and VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 10, 11 and 12 respectively; (iii) The VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 13, 14 and 15 respectively, and the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 16, 17 and 18 respectively; (iv) VH CDR1, 2, and 3 sequences as shown in SEQ ID NO: 19, 20, and 21, respectively, and VL CDR1, 2, and 3 sequences as shown in SEQ ID NO: 22, 23, and 24, respectively; or (v) The VH CDR1, 2 and 3 sequences as shown in SEQ ID NO: 25, 26 and 27, respectively, and the VL CDR1, 2 and 3 sequences as shown in SEQ ID NO: 28, 29 and 30, respectively; One or more of the CDR sequences in (i) to (v) may optionally include one to three amino acid modifications relative to the CDR sequence, specifically, one or more of the CDR sequences may optionally be modified by substitution, addition or deletion of one to three amino acids.

11. The Treg according to any one of claims 1 to 7 or 10, wherein the antigen recognition domain comprises: (i) VH domain and VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 31 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 32 or a sequence having at least 70% sequence identity with it; (ii) VH domain and VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 33 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 34 or a sequence having at least 70% sequence identity with it; (iii) VH domain and VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 35 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 36 or a sequence having at least 70% sequence identity with it; (iv) A VH domain and a VL domain, wherein the VH domain contains a sequence as shown in SEQ ID NO: 37 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 38 or a sequence having at least 70% sequence identity with it; or (v) VH and VL domains, wherein the VH domain contains a sequence as shown in SEQ ID NO: 39 or a sequence having at least 70% sequence identity with it, and the VL domain contains a sequence as shown in SEQ ID NO: 40 or a sequence having at least 70% sequence identity with it.

12. The Treg according to any one of claims 1 to 7, 10 or 11, wherein the antigen recognition domain comprises or consists of the following: (i) A sequence as shown in SEQ ID NO: 51 or a sequence having at least 80% sequence identity with it; (ii) A sequence as shown in SEQ ID NO: 52 or a sequence having at least 80% sequence identity with it; (iii) A sequence as shown in SEQ ID NO: 53 or a sequence having at least 80% sequence identity with it; (iv) A sequence as shown in SEQ ID NO: 54 or a sequence having at least 80% sequence identity with it; or (v) A sequence as shown in SEQ ID NO: 55 or a sequence having at least 80% sequence identity with it.

13. The Treg according to any of the preceding claims, wherein the CAR comprises or consists of the following: The sequence represented by any one of SEQ ID No: 101 to 105, 126 or 145 to 150, or a sequence having at least 80% identity with it.

14. A chimeric antigen receptor (CAR) comprising an antigen recognition domain as defined in any one of claims 8 to 12.

15. A nucleic acid molecule comprising a nucleotide sequence encoding the CAR according to claim 14.

16. A vector comprising the nucleic acid molecule according to claim 15, optionally further comprising a nucleic acid molecule encoding a FOXP3 polypeptide.

17. A cell comprising the CAR of claim 14, the nucleic acid molecule of claim 15, or the vector of claim 16.

18. A cell population comprising cells according to any one of claims 1 to 13 or 17, particularly comprising a plurality of Treg cells according to any one of claims 1 to 13.

19. The cell population of claim 18, wherein the clonality of the plurality of Tregs is not modified in vitro, and / or the plurality of Tregs have polyclonal endogenous TCRs.

20. A pharmaceutical composition comprising cells according to any one of claims 1 to 13 or 17, a cell population according to claim 18 or 19, or a carrier according to claim 16.

21. The cells according to any one of claims 1 to 13 or 17, the cell population according to any one of claims 18 to 19, or the pharmaceutical composition according to claim 20, for use in therapy, particularly adoptive cell transfer therapy, or for treatment or prevention of autoimmune or inflammatory diseases, or for inducing immunosuppression, or for promoting tissue repair and / or tissue regeneration, wherein the cells are Tregs.

22. The cell, cell population, or pharmaceutical composition used in claim 21, wherein the autoimmune or inflammatory disease is type 1 diabetes, for example, recently-onset type 1 diabetes.

23. A method for preparing cells according to any one of claims 1 to 13 or 17, the method comprising the step of introducing a nucleic acid molecule according to claim 15 or a vector according to claim 16 into the cells.

24. A cell that can be obtained by the method according to claim 23.

Images